key: cord- - gcpk x authors: koprowski, hilary title: vaccines and sera through plant biotechnology() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gcpk x nan during the last years, more vaccines and sera have become available for prophylaxis or treatment of disease than during the preceding century. yet at the same time, the number of obstacles preventing the use of those biomedical discoveries for the benefit of mankind has risen almost proportionately. stricter regulations for safety and efficacy have led to increased cost of production of biomedicals, but secondly the price to the consumer has risen to a degree that now precludes prophylactic vaccination against some diseases on a global scale. in addition to the extraordinarily high price of new vaccines and sera, the method of administration of these products through syringe and needle presents another burden in their worldwide use. in , i organized a mass vaccination trial with my oral polio vaccine in the then belgian congo. two hundred and fifty thousand individuals were vaccinated in weeks by oral spraying. this became a model for worldwide polio vaccination-a project requiring only one oral administration of a product-to eradicate polio in the world. the global use of vaccine requires that the vaccines be inexpensive and easily maintained and distributed. after considering various alternatives of fulfilling the criteria established for a global approach to immunization, it has become clear that our only choice is the production of vaccines or other materials of biomedical importance in plants. the advantages of plants are quite clear. plants are very inexpensive to grow in mass; some can grow in almost any soil or climate in the world. various parts of plants (leaves, seeds, fruits, and chloroplasts) can also be used as vehicles for the biomedical products. production of biomedicals in plants and their administration to humans and animals also provides an added margin of safety as compared to biologicals produced in animal tissues. early concerns that plants might not be capable of producing biomedicals of animal origin, and that plant-specific glycosylation patterns might functionally alter plant-produced antibodies have proven unwarranted. basic methods to transfer foreign genese into plants are well established. in the first approach, the plant is transfected with a foreign gene in combination with the plant parasite, agrobacterium tumefaciens. in this constitutive approach, future generations of the same transgenic plant species heritably retain the capacity to express the original product. the product is isolated from the extract of leaves, seeds, etc. (table ). in the second approach, the gene is inserted into a plant virus (for example, alfalfa or tobacco mosaic virus), which is used in combination with a foreign antigen to infect plants. here, the final product is harvested from the plant in tandem with the plant virus. in both cases, purification of the vaccine or antibody is a rather simple procedure. the achievements in production of plant-derived vaccines and sera, can be classified in two categories: ( ) vaccines and antibodies for infectious diseases; ( ) vaccines and antibodies for cancer. table shows the list of infectious disease agents against which plant-derived products were made in the biotechnology foundation laboratories, in chronological order, respiratory syncytial virus (rsv), hepatitis b, hiv, rabies, anthrax, diphtheria, sars, and smallpox virus. one of the first vaccines produced in this system was against rsv, which causes disease of newborn children. we produced the vaccine antigen as a fusion protein with the capsid protein of alfalfa mosaic virus in tobacco. immunogenicity was tested in mice, which were either injected with or fed the plant-produced vaccine ( as compared to controls; high-titer antibodies against rsv were also induced. plant-derived vaccines for hiv and anthrax will be presented by dr. alexander karasev. scientists under the leadership of professor andrzej legocki in poznan, poland, have produced a vaccine against hepatitis b in transgenic lettuce. these scientists have shown that vaccines expressed in lettuce leaves can be ground, frozen, dried and powdered without loss of immunogenicity. observation of immune response in mice fed the plant-derived vaccine indicates that two feedings at -month intervals produce optimal immunogenic responses. rabies is a worldwide disease of humans and warmblooded animals. except for the time-honored pasteur type vaccine, all modern vaccines are too expensive for use on a worldwide scale. rabies antibody is essential in treating severe bites but is unavailable because it is not sufficiently lucrative for production by the pharmaceutical industry. rabies in dogs is a major, but not unique, source of infection in southeast asia and in many african countries. an inexpensive bait vaccine for dogs could lead to the eradication of rabies in those parts of the world. to express rabies vaccine in plants, we have used a recombinant alfalfa mosaic virus in spinach leaves. this virus expressed a chimeric peptide containing antigenic determinants of rabies virus glycoprotein, which elicits immune responses; and of nucleoprotein, which increases glycoprotein immunogenicity. the chimeric gene antigen was pcr-amplified and cloned for fusion with the gene encoding the coat protein of alfalfa mosaic virus. tobacco and spinach were the plants used to express the antigen. the presence of rabies virus protein in spinach leaves was checked by western blot and immunogenicity was tested in mice. then volunteers were fed g of spinach extract recombinant virus twice at -week intervals. half of the subjects responded to the oral administration of spinach as indicated by a vigorous booster response after a single injection of commercial vaccine. there is a global crisis in post-exposure treatment of rabies because of worldwide unavailability of the antirabies serum required for severe bites. thus, it was decided on an emergency basis to produce rabies antibody in plants. research conducted by dr. kisung ko, led to the production of a transgenic tobacco plant containing the heavy and light chains of human rabies antibody. the two chains recombined in the plants to produce a complete antirabies antibody, which was as effective as the original antibody in animals, before and after exposure to rabies (table ). two very interesting results emerged from the research with this antibody. first, leaf extract of the transgenic tobacco was found to be virtually free of nicotine and other alkaloids (fig. ) . second, the glycosylation pattern of the plant-derived antibody was considerably different from that of the animal tissue-derived antibody (fig. ) . mannose is the only glycan prevalent in the plantibody, yet the antibody was at least as efficacious as the native antibody produced in animals. table in vivo efficacy of mab p for post-exposure prophylaxis of hamster injected with a lethal dose of coyote rabies street viruses (dr. c. rupprecht, cdc) post-exposure treatment antibody a (iu/animal) vaccine (hdcv) b − + mab p ( ) / c / mab p ( . ) / / mab p ( . ) / / hrig ( ) / / untreated control / / ko et al. [ ] , pnas. a iu: international unit. b hdcv: (imovax, lot mo ) human diploid cell culture rabies virus vaccine, "−" and "+" indicate treatments without or with hdcv, respectively. c number of surviving hamsters/number of hamsters tested. strict guidelines regarding the amount and potency of antirabies antibody for use in post-exposure treatment has enabled calculation-based on the yield of antibody per tobacco plant-of plant acreage needed to produce a given number of antibody international units. if, as expected, kg of tobacco produces - mg of antibody, ha or . acres is needed to produce enough antibody for vaccination of , people. i leave it to accountants to calculate the production cost of such an antibody as compared to antibody produced in animal tissue. one of the many advantages of plants for production of biological products is their ability to express multiple genes in the same plant. vaccines against diphtheria, tetanus, and oral pertussis are the triad injected into infants during their first - months of life. a plan to substitute oral products for injectable vaccines was initiated through production of transgenic tobacco (as a model) and transgenic carrots expressing all three vaccines (table ). plants expressing current diphtheria toxoid have already been produced (fig. ) . the spike protein of sars coronavirus is presumed to be immunogenic. transgenic tobacco and tomato expressing the spike protein were generated (fig. ) . interestingly, only months elapsed between the introduction of the spike construct into the plant and the first observation of sars coronavirus mrna expression by the plant (fig. ) . theoretically, the vaccine could be obtained by growing large numbers of transgenic plants. it is doubtful whether other methods currently available would produce vaccine material against a newly discovered disease in such a short time. after decades of neglect, we are now witnessing a revived interest in immunological approaches to cancer treatment. thus, we sought expression of colorectal cancer anti-gen ga and of the corresponding - a antibody in plants. attempts were also made to express antibodies recognizing epidermal growth factor (egf) which is present at the surface of epithelial tumors in plants. the tobacco mosaic virus-based gene vector (p b) was used for expressing the - ga antigen. nicotiana betthamiana was infected with the construct and checked expression by western blot analysis of the leaf extract. a group of mice were then immunized with the plant-produced - antigen and, for comparison, another group with the same antigen expressed in baculovirus systems. sera obtained from both groups of mice recognized human colorectal cell lines expressing this antigen (fig. ) . a cross reactivity test capable of discriminating the two antigens, that produced in plants and that produced in baculovirus vector, ruled out any "contaminant" basis for the immune response. complement-dependent cytotoxicity assays of sera obtained from mice immunized with the plant-derived - antigen showed lysis of breast cancer cells, but not of antigenically unrelated melanoma cells (fig. ) . the plantderived - antigen also induced antigen-specific proliferation of t cells. using the same technique as for antirabies antibodies, we expressed - -reactive antibody - a in tobacco plants. purified leaf extract of these plants specifically recognized receptors present in human colorectal cancer cells. perhaps the most interesting results obtained from the study of the two plantibodies against rabies and cancer relate to the glycosylation pattern. the plant-derived antirabies heavy chain was fused to kdel (lysineasperagine-glycosamine-leucine) endoplasmic reticulum retention signal. as a result, the anti-rabies plantibody contained abundant mannose, whereas the plant-derived anticancer antibody contained less mannose and more nacetylglucosamine (fig. ) . despite the different glycosylation patterns of the two plantibodies and the difference of both patterns from those of the native antibody, their biological activity was equivalent to each other and to that of the native antibody. antibodies reactive with the epidermal growth factor were recently licensed to treat certain types of epithelial cancers. such antibodies were first developed at the wistar institute many years ago. they have recently been adapted to production in tobacco plants and within the near future might provide an inexpensive therapeutic tool in human cancer. however, plant production of vaccines and sera is not a simple procedure with assured success in each undertaking. one of the most important remaining problems is the low yield of the bioproduct in plants. to date, no magical solution to this problem has been found. codon optimization, careful approaches to harvesting and purifying plant products, use of plant parts such as chloroplasts to increase uptake of the material are but a few potential avenues to help increase the yield of the final product. at present, it is still difficult to produce sizeable amounts of plant-derived products. great strides have been made in our knowledge and expertise in the use of plants as hosts for production of biomedicals. table there are four stages of adopting new ideas the first is "it's impossible" the second is "maybe it's possible, but it's weak and uninteresting" the third is, "it's true and i told you so" and the fourth is, "i thought of it first" as shown in the last table (table ), a segment of the population remains to be convinced that the future of biologicals is linked with production in plants. some people will consider the facts and ultimately opt for acceptance. others will remain "true believers" in harmful effects caused by transgenic plants. their voices may become subdued or even silenced by increasing evidence that the future of global health improvement rests strictly in the progress made on the worldwide use of plants to combat diseases. function and glycosylation of plant-derived antiviral monoclonal antibody key: cord- -xo ruswo authors: new, r.r.c.; moore, b.d.; butcher, w.; mahood, r.; lever, m.s.; smither, s.; o'brien, l.; weller, s.a.; bayliss, m.; gibson, l.c.d.; macleod, c.; bogus, m.; harvey, r.; almond, n.; williamson, e.d. title: antibody-mediated protection against mers-cov in the murine model() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xo ruswo murine antisera with neutralising activity for the coronavirus causative of middle east respiratory syndrome (mers) were induced by immunisation of balb/c mice with the receptor binding domain (rbd) of the viral spike protein. the murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the mers coronavirus (mers-cov). to test the neutralising capacity of these antisera in vivo, susceptibility to mers-cov was induced in naive recipient balb/c mice by the administration of an adenovirus vector expressing the human dpp receptor (ad -hdpp ) for mers-cov, prior to the passive transfer of the rbd-specific murine antisera to the transduced mice. subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the mers-cov resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. the murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of rbd with a human igg fc tag (rbd-fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. the data gained indicate that this dual-route vaccination with novel formulations of the rbd-fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of mers-cov. the middle east respiratory disease syndrome (mers) first emerged in in saudi arabia [ , ] . since then, there have been an estimated laboratory-confirmed cases with deaths, reported from a total of countries in the eastern mediterranean region and from countries elsewhere [ ] . saudi arabia, however, remains the main focus of infection and a disease outbreak in south korea involving cases was traced back to an index case who had travelled from saudi arabia. whilst the incidence of mers cases in saudi arabia peaked in , there are still a significant number of cases reported from the country and in the period september -may , there were cases including deaths with a case fatality rate of . % [ ] . mers coronavirus (mers-cov) is a member of the betacoronavirus genus [ ] and as for other betacoronaviruses, bats may provide a natural reservoir for the virus [ , ] , but high levels of antibodies to mers-cov in dromedary camels [ ] suggest that the dromedary camel is the principal source for animal-to-human transmission of mers-cov [ ] . however, evidence of human-to-human transmission comes from the reporting of outbreaks in countries remote from saudi arabia such as the uk, europe, usa, and china where small outbreaks have also occurred [ ] . mers-cov is an enveloped, positive-sense, single-stranded rna virus [ ] . the virus possesses an envelope-anchored trimeric spike protein which binds to the human receptor dipeptidyl peptidase (dpp or cd ) and gains host cell entry by the fusion of viral and host membranes [ ] . the spike protein comprises an s sub-unit and a membrane fusion s sub-unit. in the coronaviruses, the s sub-units are further divided into n-terminal and c-terminal subdomains and for mers-cov, it is the c-terminal sub-domain that comprises the receptor-binding domain (rbd) [ ] . the rbd also incorporates a receptor-binding motif at its c-terminal and the crystal structures of mers-cov rbd [ ] and of the rbd bound to the extracellular domain of human dpp have been reported [ ] . the rbds of the coronaviruses represent vaccine and therapeutic targets and the rbd of mers-cov as a vaccine antigen has been demonstrated to induce neutralising antibody [ ] and to protect mice transduced with a viral vector expressing hdpp , or nonhuman primates from viral challenge [ ] [ ] [ ] [ ] [ ] . there are significant ongoing efforts to develop vaccines for mers-cov infection, predominantly involving live attenuated viral vectors such as adenovirus, modified vaccinia ankara or measles [ ] to induce anti-viral immunity and some of these vaccines are already in clinical trials. here, we were interested to determine the relative importance of inducing systemic and/or mucosal immunity in vaccination to protect against mers-cov, an infection predominantly of the respiratory tract and lungs. to this end, we have used a dual route immunisation regimen in balb/c mice to induce both systemic and mucosal immunity, to generate rbd-specific murine antisera. initially, we immunised balb/c mice sub-cutaneously (s.c.) with rbd-fc in the mf adjuvant to induce rbd-specific igg. subsequently, we have immunised further groups of balb/c mice by s.c. priming and per oral (p.o.) boosting with the rbd-fc, to induce both systemic igg and mucosal iga responses. to do this, we have used novel formulations of rbd-fc coated onto microcrystals formed from histidine or glutamine and also incorporating calcium phosphate for sub-cutaneous priming [ ] , whilst the formulation for oral boosting comprised rbd-fc in reverse micelles dispersed in a self-emulsifying oil phase, which has been optimised from previous formulations [ , ] . the advantages of these formulations are that they are very stable under extremes of temperature [ ] . furthermore, on translation to the clinic, only one injected priming dose would be required, followed by a p.o. booster dose; the latter could be self-administered in capsule form. we have compared the relative abilities of the two sources of antiserum to neutralise clinical isolates of mers-cov in vitro. to do this, we have used two clinical strains of mers-cov (erasmus medical center or emc and london - :), each of which were derived from severely-ill individuals who had contracted the virus in the middle east in . subsequent sequencing of the polymerase gene from these isolates indicated them to be newly-emerged members of the betacoronavirus genus with a close sequence homology and phylogenetic relationship to the bat coronaviruses hku and hku- [ , ] . mice are not naturally susceptible to mers-cov infection, but susceptibility can be induced by the administration of an adenovirus vector which induces expression of the human receptor (hdpp /cd ) for the virus in vivo for a limited time, providing a non-lethal murine model of the disease [ ] . we have used this transduced mouse model to test the capacity of the antiserum derived from the dual route immunisation to neutralise mers-cov in vivo, by passive transfer prior to challenge with the emc strain and we have demonstrated a significant reduction in viral load in lung tissue in transduced mice. the rbd was synthesised and expressed according to methods adapted from du et al. [ ] . in brief, a single dna fragment containing an in-frame fusion of the coding sequences for the human il signal peptide, the rbd and human igg -fc was synthesised. this was transferred into the plasmid pef-dest (invitrogen) so that the target sequence was expressed as a secreted protein with a c-terminal human igg fc tag. this construct was transfected by cationic transfection into human embryo kidney (hek) cells in suspension (fshek) or adherent hek cells stably expressing the sv large t antigen ( ft), using serum-free media and incubated for - days. small scale purifications of rbd-fc were performed using protein a chromatography. for this, medium from the transfected cells was treated with ammonium sulphate to precipitate the protein, prior to dialysis and resuspension in buffers for binding to protein a beads. the latter were washed and eluted with buffer containing m urea. protein concentration was determined by uv absorbance spectroscopy and purity was estimated by sds-page with coomassie staining and subsequent optical densitometry using a syngene g:box imaging system. the rbd-fc was incorporated on glutamine calcium phosphate (cap) microcrystals for s.c. immunisation, using methodology adapted from [ ] . briefly, aqueous mixtures of rbd-fc with sodium orthophosphate and glutamine were precipitated as cap protein-coated microcrystals (cap-pcmc), by addition to of a fold excess of isopropanol containing dissolved calcium chloride. the resultant suspension contained self-assembled microcrystals comprising a glutamine core with the rbd-fc protein embedded in a thin surface layer of cap (now termed rbd-fc-pcmc). the pcmc were isolated by vacuum filtration and dried to a powder. protein content and integrity was determined by elisa and sds-page. for oral dosing, the rbd-fc was incorporated in mineral oil with added excipients using methodology adapted from [ , ] . the oral formulation comprised rbd-fc with the mucosal adjuvant cholera toxin b sub-unit (ctb), retinoic acid (ra), vitamin d, e and trehalose debehenate (tdb), a synthetic analog of the mycobacterial trehalose dimycolate [ ] and imiquimod, a tlr / agonist [ ] . specific pathogen-free female balb/c mice ( - weeks of age) were obtained from a commercial breeder and used throughout this study. on receipt, mice were randomised for allocation to cages and given free access to food, water and environmental enrichment. mice were fully acclimatised to the animal housing facility for at least five days prior to any procedure. all animal procedures were performed in accordance with uk legislation as stated in the uk animal (scientific procedures) act . the institutional animal care and use committee approved the relevant project licence. naïve mice were randomised for allocation to a treatment group (typically per group) and immunised in one of two regimens: either with a s.c. priming dose followed by two s.c. doses, given at and days after the prime; alternatively, mice received a s.c. priming dose followed by an oral or s.c. booster dose days after the prime (table ) . for s.c. immunisation, mice received . lg of rbd-fc-pcmc in . ml pbs injection volume, whereas for all per oral (p.o.) dosing, mice received lg of rbd-fc in a total volume of . ml mineral oil (mo), by oral gavage. where rbd-fc was administered s.c. in the conventional adjuvants mf or alhydrogel, mf (novartis, us) was used in a : ratio by volume with rbd-fc in pbs, whilst alhydrogel (brenntag biosector, denmark) was used in a : ratio with rbd-fc by volume in pbs. at selected intervals after dosing, mice were blood-sampled from the tail vein for assay of specific antibody titre. at the end of the immunisation schedule, individual mice were terminally anaesthetised for collection of blood by cardiac puncture, then culled prior to removal of small and large intestines for collection of faecal pellets for extraction of iga. titres of rbd-fc-specific antibody in serum samples were determined by elisa. in brief, test sera were bound to microtitre plates pre-coated with rbd-fc and antibody binding was detected with an hrpo-labelled secondary antibody to mouse igg, igg , igg a or iga (bio-rad). a standard curve for calibration comprising the relevant murine ig isotype (sigma) captured with an anti-fab reagent, was included on each plate. plates were developed by the addition of , -azino-bis( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (abts) substrate (sigma) and optical density (od) was read at nm (multiskan plate reader). for assay of antibody in faecal samples, faecal pellets were extracted in supplemented pbs as described previously [ ] . in brief, ml of cold pbs was prepared, supplemented with tablet of complete mini protease inhibitor cocktail (sigma) and ll tween were added. to . g faecal pellets, ml of supplemented pbs was added and left at room temperature for min. samples were vortexed for approximately s, incubated on ice for a further min. and then centrifuged ( , g, min.). supernatants were retained and stored at À °c pending assay. the faecal extracts were assayed for specific igg and iga content, by elisa, as for serum samples. antibody concentrations in all samples were determined from the relevant standard curves using ascent software with fourparameter logistic curve-fitting and reported in ng/ml or lg/ml serum or faecal extract, as appropriate. to determine if the antibody induced by immunisation with to rbd-fc was neutralising for mers-cov in vitro, plaque assays were performed. for this, two strains of mers-cov were used: london - (genbank accession number kc . ) [ ] and erasmus medical center (emc genbank accession number jx ) ( ) . the london - strain was obtained from the national collection of pathogen viruses, phe porton, salisbury, uk and the emc strain was kindly provided by the erasmus university medical center rotterdam, the netherlands. both strains were prepared in serum-free media (gibco) at a multiplicity of infection (moi) of . , equivalent to plaque-forming units (pfu). the murine antiserum for testing was prepared at a dilution range from undiluted to : in pbs. virus was incubated overnight ( °c) with murine antiserum, negative control antibody (nibsc, uk) or media, prior to infection of a confluent monolayer of vero e cells (ecacc, salisbury uk) with ll of the mixture. the neutralising ability of the murine antiserum was tested in duplicate or triplicate at each dilution. after incubation ( h, °c), an overlay comprising a : dilution of carboxymethyl cellulose with serum-free media was added to the cells and incubation continued for a further days ( °c) prior to fixing ( . % formaldehyde) and staining ( . % crystal violet) with enumeration of the number of plaques per ml. mice are not naturally susceptible to infection with mers-cov, since they lack the human dpp receptor. to induce transient susceptibility in balb/c mice, we used an ad construct (oxford genetics) to express the human dpp receptor (ad hdpp ), as previously described ( ) . mice were administered the ad hdpp construct ( .  pfu in ll) by the intra-nasal (i.n.) route under light sedation with inhalational isofluorane and then monitored by serial blood sampling for serum levels of hdpp /cd by elisa (thermoscientific). at peak levels of expression of hdpp (days - ), mice were lightly sedated as before and challenged by the i.n. route with mers-cov (emc strain) at pfu in ll per mouse. mice were weighed prior to challenge on each subsequent day to monitor changes in body weight during infection. to test the in vivo neutralising capacity of murine antiserum raised to the rbd-fc construct, naïve mice (n = per treatment group) were passively immunised by the i.p. route at h. prior to i.n. challenge with the mers co-v (emc strain), as described above. the murine antiserum, pooled from mice who had been primed with rbd-fc pcmc and boosted orally (regimen , treatment group ), was delivered at a dilution of : in pbs and delivered in a total volume of ll per mouse. a further group of mice received a purified polyclonal human igg at a single dose level ( lg/mouse in ll i.p.), which had been raised to inactivated mers-cov. control mice received a non-specific human igg at a single dose-level ( lg/mouse in ll, i.p.). both sets of human igg (specific and non-specific) were raised in a bovine transchromosomal model and purified prior to use. a further group of negative control mice were included, which received pbs in place of either the ad dpp construct or the mers-cov-specific antibody, and were also challenged i.n. with mers-cov (emc strain) at pfu/mouse. to determine the protection afforded by the passive immunisation, pairs of mice from each treatment group were culled on days - after challenge and their lungs were removed and weighed and then rapidly frozen (À °c) prior to the determination of viral load. pairs of lungs from each of mice per treatment group were individually thawed and homogenised in serum-free media ( ml). rna was extracted from ll of each homogenate using the qiaamp viral rna kit (qiagen), following the manufacturers' instructions. real-time pcr was conducted on duplicate ll the amount of virus in tested samples was determined in duplicate using the standard curve and reported as pfu/g lung tissue. all data were analysed using graph pad prism software v. and expressed as mean ± s.e.m. statistical comparisons were made using one-way anova or unpaired t-test. the rbd-fc protein was expressed in both adherent ft and suspension human embryo kidney (hek) cells, but with greater expression in adherent cells (fig. ) . purification of protein from adherent cells with protein a was very effective, yielding protein which was > % pure, with molecular weight of approximately kda (fig. a) . the use of m urea for elution was optimum, as it was sufficient to solubilise the protein without denaturing it, yielding rbd-fc in optimum yield ( . mg/ml) and predominantly in a dimeric form (fig. b) . this method of protein purification was therefore selected for forward use. rbd-fc, formulated for either sub-cutaneous (s.c.) or per oral (p.o.) immunisation, was tested for immunogenicity and the formulations optimised in an iterative approach. initially, a s.c. dosing regimen was used in which rbd-fc was formulated in either alhydrogel or mf to deliver . lg of protein on each of three occasions at , and days. mice were monitored for days after the final boost and igg titre determined ( fig. a) . at day , the total igg titres achieved with rbd-fc in alhydrogel or mf did not differ significantly. to determine if the presentation of rbd-fc in either alhydrogel or mf influenced the ability to develop virus-neutralising antibody, antisera were selected from mice in each immunisation group and tested in a plaque assay for neutralisation of both the emc and london - strains of mers-cov ( fig. b and c) . all four sera gave some neutralisation of viral activity, although at a : dilution, sera and were most potent, against both viral strains. sera and were derived from the treatment group immunised with mf adjuvanted rbd-fc, whereas sera and were derived from alhydrogel-adjuvanted rbd-fc ( fig. a) . based on this pilot data, we subsequently used mf as the conventional adjuvant for rbd-fc, to compare with some novel formulations. having demonstrated to proof-of-principle that the rbd-fc, when delivered in mf can induce a high titre of antibody with neutralising activity, we next investigated how to tailor an rbd-fc vaccine optimally to induce both systemic and mucosal immunity, with the aim also of reducing to a -dose immunisation regimen and increasing functional antibody. for this, we selected novel formulations in which rbd-fc protein was presented as rbd-fc-pcmc for s.c. priming and incorporated into mineral oil (mo) for p.o. boosting. we compared the serum igg response achieved from this -dose dual route immunisation with that induced to rbd-fc delivered in mf in a -dose s.c. regimen (fig. ) . at month after the booster dose, at day , there was no significant difference in the serum igg titres achieved, so that the -dose dual-route immunisation with rbd-fc-pcmc for s.c. priming and incorporated in mo with excipients for p.o. boosting, was just as immunogenic as the -dose s.c. immunisation with rbd-fc in mf (fig. a) . at day , the serum response to rbd-fc in the dual-route regimen was predominantly igg biased, whereas s.c. dosing with rbd-fc in the presence of mf induced both igg and igg a (fig. b) . since dual route immunisation effectively induced serum igg to rbd-fc, it was of interest to determine whether it could also effectively induce mucosal immunity. in this study, the rbd-fc-specific iga response was determined in serum and in faecal pellet extracts from individual animals on day . in this case, the rbd-specific iga responses of mice immunised in the -dose dual route regimen were compared with that of mice immunised by the oral route twice, and with mice immunised by the s.c. route in mf twice, on exactly the same days ( , ) (fig. ) . this comparison showed that s.c. immunisation in mf did not induce serum iga. however s.c. priming with rbd-fc-pcmc with p.o. boosting effectively induced rbd-fc-specific iga and was not inferior to oral priming and boosting in this effect in either serum (fig. a ) or faecal extracts (fig. b ). however mice primed and boosted orally did not develop rbd-specific systemic igg (data not shown). additionally, day sera from mice in all treatment groups were tested for their ability to neutralise either strain of mers-cov in vitro (table ) . from this it can be seen that sera from out of mice in the dual route regimen were fully neutralising neutralisation of mers-cov in vitro. in in vitro for both strains (emc and london - ), when tested at : dilution ( fig. a and b) . in order to test whether the in vitro neutralising activity translated into viral neutralisation in vivo, sera from these mice (highlighted in table ) were pooled in equal aliquots at : dilution to enable a subsequent passive transfer study. in order to design the passive transfer study, it was necessary to define the duration of expression of cd in murine lungs in vivo, following induction with the ad hdpp construct. mice dosed with ad hdpp i.n. at t were culled in pairs and lung homogenates prepared and assayed for cd expression. cd in lung tissue was expressed in a time-dependent manner, with levels peaking at day and declining to day (fig. a) , setting a sufficient window to use the model for the determination of the protection against viral challenge afforded by the passive transfer of mers-specific antibody. to determine the protection afforded by the passive transfer of murine antiserum raised in the dual route immunisation regimen, against infection, susceptibility to mers-cov was induced at t with i.n. administration of ad hdpp to groups of mice. passive transfer by the i.p. route of the pooled serum sample derived from the mice highlighted in table , which had previously been shown to be neutralising in vitro (table ) was conducted days later and mice were challenged after a further h with mers-cov emc . additional groups of mice, which had been transduced with ad hdpp , were passively immunised with a mers-cov specific human igg and a non-specific human igg. at - days after challenge, pairs of mice were culled for the determination of viral load in lungs, which was determined to peak at days p.i. (data not shown). at days p.i., the pooled murine antiserum significantly reduced viral titres in lungs, to the same extent as the specific human igg, and contrasting with the negative control human igg, demonstrating significant in vivo neutralising activity (fig. b ). fig. . a. expression of cd was induced in lung tissue by the administration of ad hdpp ( .  pfu) to mice by the i.n. route at t . subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of cd . the plot shows the time-course of cd expression from to days post-induction. all data points were normalised for background values from control mice. b: content of mers-cov (emc strain) in murine lungs (pfu/g tissue) determined by rt-pcr at day post-infection, (equivalent to day after passive transfer with murine antisera to rbd-fc which had previously been shown to neutralise the emc strain in vitro). mice received either a mers-cov-specific human igg ( lg) or non-specific human igg ( lg) in ll /mouse i.p.; or murine antisera to rbd-fc, which had been pooled from murine donors and which was delivered at : dilution ( ll/mouse i.p.). negative control mice received pbs in place of ad hdpp or antiserum all mice were challenged with mers-cov emc i.n. at pfu/mouse. statistical significance was determined at the p < . level by one way anova and unpaired t-test. no significant differences in body weight were detected between treatment groups challenged with mers-cov, which was attributed to the short time period of the study. mers is a serious endemic respiratory infectious disease for which there is no licensed vaccine, although there are several vaccines in clinical trial currently including adenovirus-vectored delivery of the spike protein and sub-units [ ] , dna vaccines and nanoparticle-delivered sub-unit approaches [ ] . we were interested in determining the advantage of r a vaccine which could induce mucosal as well as specific systemic immunity to the key target, the rbd protein, in order to achieve optimum protective efficacy. vaccination to induce effective immunity at mucosal surfaces, should prime the immune system to respond rapidly to invading pathogens such as mers-cov. previous studies have used adenovirus delivery of the mers spike protein with intra-muscular (i.m.) or intra-gastric (i.g.) delivery to induce neutralising systemic igg, but not iga; further, whilst i.m. delivery also induced specific t-cell immunity, i.g. delivery did not [ ] . others have shown that intra-nasal delivery of a live attenuated adenovirus-vectored subunit vaccines does induce specific mucosal as well as systemic immunity, although translation of this approach to the clinic may raise safety issues [ ] . here, we have relied on novel formulations of a sub-unit protein to enable dual route vaccination (parenteral and oral) to induce mucosal as well as systemic immunity. in this study, we have achieved the expression and purification of a recombinant rbd-fc protein in milligram quantities. we have also demonstrated that when formulated as a sub-unit vaccine, the construct induced murine antibody which effectively neutralised two different clinical strains of mers-cov in vitro. additionally, we have shown that these murine antisera, when passively transferred into naïve mice transduced to express the hdpp /cd receptor, conferred protection against viral challenge in the recipients, with significantly reduced viral loads in the lung tissue of the recipient mice. whilst the use of conventional adjuvants such as mf or alhydrogel to formulate the rbd-fc protein resulted in high titres of specific igg in serum, the mf formulation did not induce specific iga in serum. in order to promote both systemic and mucosal immunity to rbd-fc, we have formulated this protein for injected priming and p.o. boosting, entailing the optimisation of the cap pcmc and of the reverse micelles in oil emulsion, respectively. this has enabled the achievement of a vaccination regimen comprising only two doses and rapidly inducing rbd-fc-specific systemic and mucosal immunity. whilst the pcmc formulation of rbd-fc was as effective as rbd in mf in inducing a primary igg response, we have shown that an oral formulation of rbd-fc in mineral oil with selected immunostimulants was as effective as mf when used as a booster immunisation. additionally, we have shown that non-invasive oral priming and boosting is as effective at inducing a specific mucosal response, measured as specific iga in serum and faeces, as is injected priming with rbd-fc in the pcmc formulation together with oral boosting, leading to the exciting concept of a potential orally-dosed sub-unit vaccine for mers. whilst both alhydrogel and the combination of pcmc and oral formulations are th -polarising, as evidenced by the predominantly igg titres raised to rbd-fc, the influence of mf on the response to rbd-fc was a mixed th- /th effect, with a significant induction of specific igg and igg a. to counter a viral infection, it would be expected that a th response would be most appropriate. however, the fact that neutralising antibody to rbd-fc was raised under either th or th -polarising influences, suggests that either isotype can be protective and primes the immune system sufficiently and would allow for cross-presentation to occur on subsequent exposure to the virus [ ] . in this study, we have not examined the induction of a cell-mediated memory response to the rbd-fc protein, although this will play a significant role in protection against the virus. currently, we are presenting the rbd protein in our formulations with an fc tag, derived from human igg and useful in purifying the protein. the fc tag may contribute additional adjuvantising activity by engaging antigen-presenting cells in the vaccinee [ ] and it may aid mucosal immunity since the fc receptor, an mhc transmembrane protein, is also expressed at mucosal surfaces e.g. in the respiratory tract [ ] . vaccination of the zoonotic host, the dromedary camel, may also effectively curb outbreaks of mers in endemic regions and limit the risk of viral recombination [ ] and significant progress with an orthopox-vectored vaccine for mers has recently been reported [ ] . the potential use of a sub-unit vaccine for mers in camel vaccination could be aided by varying the sequence of the rbd protein [ ] and substituting the human fc tag with an alternative tag recognised by the camel, to design approaches tailored for animal vaccination, bearing in mind that a single dose vaccine would be ideal in this context. however, future work in our laboratory will also address the value of retaining or removing the fc tag from the rbd protein for clinical or veterinary iterations of the vaccine. in this study we have determined vaccine efficacy by demonstrating in vitro and in vivo neutralising ability of murine antisera raised in the dual route two-dose regimen against two virulent clinical strains of mers-cov which have greater than % genome sequence homologyy. in future work, it will be worthwhile to test the efficacy of this approach against other clinical isolates of mers-cov. this is the first report of a dual route dosing regimen applied to a sub-unit vaccine for mers-cov. future development of this approach would require the direct testing of efficacy in the immunised transduced mouse model. as well as giving a direct readout of vaccine efficacy, this will enable the identification of the immune correlates of protection, ready for transitioning this candidate vaccine into more extensive pre-clinical testing and clinical development. the authors declare that all the data supporting the findings of this study are available within the paper. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov origin and 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middle east respiratory syndrome coronavirus cutting edge: mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate the tlr agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type or vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus intracellular recycling and cross-presentation by mhc class i molecules fc-fusion proteins: new developments and future perspectives fc-fusion proteins and fcrn: structural insights for longer-lasting and more effective therapeutics co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants the authors acknowledge with thanks the expert technical assistance of lin eastaugh, louise thompsett and vicky roberts. this work was supported by sbri awards and from innovate uk to rrcn and na, respectively and on independent research commissioned from na and funded by the nihr policy research programme, [ ]. the views expressed in the publication are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health, 'arms' length bodies or other government departments. the authors declare no conflict of interests. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -i dyg n authors: henn, wolfram title: allocation criteria for an initial shortage of a future sars-cov- vaccine and necessary measures for global immunity date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: i dyg n nan institute of human genetics, saarland university, university clinic bldg. , homburg-saar, germany correspondence to wolfram henn (email: wolfram.henn@uks.eu) although healthcare systems around the world currently are fully absorbed with the day-today challenge of slowing down the spread of the sars-cov- virus, ongoing research makes it very likely that a protective vaccine will be developed within a rather short period of time [ , ] . it appears obvious that such a vaccine will not be available ad libitum right from the beginning [ ] , but rather that an initially short supply will meet a huge and desperate worldwide demand. although we currently enjoy impressive examples of individual generosity and altruism in the wake of the crisis, we should not overstate the role of philanthropic actors, as it is still primarily governments that invest in fundamental research. any shortage of vital goods bears severe ethical challenges, as it generates distribution conflicts that can amount to antisocial behavior at the individual as well as at the governmental level, such as bribery, black markets, or protectionist trading practices. if we do not want to face scenarios as epitomized by graham greene in "the third man" for penicillin after world war ii [ ] , we are well advised to equip ourselves with reasonable and transparent criteria for the allocation of a foreseeably scarce vaccine as long as it will not be readily available for everybody. given the unprecedented public attention to the issue, these criteria must be medically adequate, socially fair, transparent, verifiable, and easily understandable for non-experts, in order to bridge thehopefully short but anyway relevant-initial shortage of vaccine supply without creating social discomfort or even unrest. the recent promising data on antiviral therapies or convalescent plasma treatments for already affected patients may at best alleviate the pressure a bit [ ] , yet not resolve the problem as a whole. what is more, it is still unclear how long the postinfection immunity of convalescents will last [ ] . as current data clearly show that covid- mortality is strongly associated with age [ ] , it should be the leading and also easily verifiable medical parameter for the distribution of the expected vaccine during an initial scarcity. however, it goes without saying that a few specific groups of professionals must be preferred as recipients in the interest of all, namely medical and security personnel immediately involved in the fight against the pandemic. the general rule should be that those who are most needed come first, followed by those most in need. accordingly, the following preference list might be reasonable: rank : physicians and nurses in immediate patient care, plus police and comparable public security officers in immediate contact with the general public. high-ranking policymakers or security officers may argue that they are indispensable for societies as well, but as there is no sharp definition for that kind of functional importance, any subjective assignment of professional preference will be sure to diminish the societal acceptance of the chosen ones. rank : documented recipients of organ transplants under ongoing immunosuppressive medication. it might be argued that other patients receiving immunocompromising therapies can be equally endangered, but any openness to other medical indications beyond transplantation will likely give rise to illegitimate attempts of circumventing the restrictions. rank : all other persons, ordered by date of birth from old to young, without any exception, and most notably, irrespective of insurance status. although it is a well-known yet not universally accepted fact that the prosperous and well-insured have access to higher quality healthcare, social inequality in the struggle against a challenge that is obviously equal to all will most likely elicit strong adverse reactions even among otherwise indulgent patients. admittedly, this strict allocation scheme may neglect relevant medical and epidemiological issues such as reduced vaccine efficacy among immunocompromised persons [ ] and among the elderly due to immunosenescence [ ] , as well as the fact that schools and universities are major hotspots of transmission [ ] . however, any granularity in the criteria would render them less transparent and actionable. beyond this ranking, anti-bribery rules will have to be rigidly enforced, and illegitimate vaccine sales pathways such as cryptomarkets [ ] , be they real or fake, vigorously prosecuted. political precautions should also be taken against cross-border buy-outs of vaccine stocks or manufacturers [ ] . after having overcome the expected initial shortage of vaccines, the global community must take appropriate measures to rapidly generate a worldwide herd immunity against sars-cov- through implementing mandatory vaccination programs encompassing all countries and age groups. the who is uniquely experienced and equipped to meet that ambitious challenge, and any political attempt to weaken it in this critical situation is, to say the least, counterproductive. any person left behind for whatever reason will not only be in avoidable peril, but will also put the goal of eradication of sars-cov- at risk [ ] . therefore, the eu and other well-heeled supranational organisations should feel not only morally obliged but also pragmatically well-advised to participate in supplying weaker healthcare systems with sufficient amounts of vaccine and logistic support for their application even in remote areas [ ] . preliminary identification of potential vaccine targets for the covid- oronavirus (sars-cov- ) based on sars-cov immunological studies sars-cov- and covid- : the most important research questions vaccine update: recent progress with novel vaccines, and new approaches to safety monitoring and vaccine shortage fake penicillin, the third man, and operation claptrap covid- : fda approves use of convalescent plasma to treat critically ill patients immune responses and pathogenesis of sars-cov- during an outbreak in iran: comparison with sars and mers clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study influenza vaccination in immunocompromised patients: efficacy and safety vaccines to prevent infectious diseases in the older population: immunological challenges and future perspectives what are the underlying transmission patterns of covid- outbreak? -an age-specific social contact characterization. eclinicalmedicine hidden wholesale: the drug diffusing capacity of online drug cryptomarkets covid- : trump sought to buy vaccine developer exclusively for us, say german officials a classification of the aims of vaccination and its relevance to transgenerational justice challenges for nationwide vaccine delivery in african countries key: cord- - xu hq authors: taylor, deborah r. title: obstacles and advances in sars vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xu hq the emergence of the severe acute respiratory syndrome (sars) that resulted in a pandemic in spurred a flurry of interest in the development of vaccines to prevent and treat the potentially deadly viral infection. researchers around the world pooled their scientific resources and shared early data in an unprecedented manner in light of the impending public health crisis. there are still large gaps in knowledge about the pathogenesis of this virus. while significant advances have been made in the development of animal models, the practicality of their use may be hampered by a lack of pathological similarity with human disease. described here are issues related to progress in vaccine development and the obstacles that lie ahead for both researchers and regulatory agencies. severe acute respiratory syndrome (sars) was first reported as a disease of unknown etiology in guangdong * tel.: + ; fax: + . sars is characterized by fever, cough and flu-like symptoms. severe cases resulted in alveolar damage, interstitial mononuclear cells and heavy fibrin deposition in the lungs. respiratory distress resulted in atypical pneumonia, requiring ventilation for approximately % of patients [ ] . the aim of this review is to describe the advances made in the development of animal models for sars and to identify gaps in scientific understanding that need to be filled. by addressing and possibly overcoming these challenges and making use of the advances made, a safe and effective vaccine may be attainable. the information here may provide the scientific basis for facilitating future regulatory decisions related to the licensing of a sars vaccine. the causative agent for sars is a novel member of the coronavirus family, termed sars-cov [ ] . coronaviruses are large enveloped rna viruses, so named for the radiating spike proteins found at the surface of the virion ( fig. ; [ ] ). there are three groups of related coronaviruses and sars may be a member of a new fourth group [ ] . classification of the sars coronavirus has been controversial, although phylogenetic similarities may place the virus in a subgroup of the group coronaviruses [ ] . human coronaviruses include three members that cause common respiratory infections [ ] . nonhuman coronaviruses include those that cause respiratory infections in birds, and enteric infections in cattle, pigs, dogs and cats [ ] . some of these viruses affect multiple organs, for example, both mouse hepatitis virus (mhv) and feline infectious peritonitis (fip) virus are capable of causing respiratory, enteric, neurologic and hepatic infections [ ] . coronaviruses are positive-sense rna viruses that replicate by a unique mechanism whereby the structural genes are expressed as a nested set of subgenomic mrnas, characterized by shared common ends and a conserved, capped leader sequence [ ] . the nonstructural genes are transcribed from the end as a polyprotein that is processed by viral proteases ( fig. ; [ ] ). proteins are translated from the open reading frame of each mrna [ ] . sars cov has eight open reading frames of unknown function, but has structural proteins found in all coronaviruses that include the envelope (e), the matrix or membrane protein (m), spike (s) and nucleocapsid (n) [ ] . the s protein is glycosylated and required for viral attachment and possibly entry. the nucleocapsid protein coats the viral genomic rna. viruses that belong to group , such as mhv, also contain a hemagglutinin-esterase (he) protein, which is not present in other groups [ ] . the flu-like symptoms and atypical pneumonia, characteristic of sars-cov infection, was also frequently accompanied by lymphopenia [ ] . alveolar macrophages were prevalent in patients with fatal sars [ ] and contributed to the immune-mediated nature of the disease. in situ hybridization showed that viral infection was present in alveolar epithelial cells and viral rna could also be detected the structural genes (spike, envelope, membrane, and nucleocapsid) are located in the end, which includes as many as eight genes of unknown function [ ] . in alveolar macrophages and bronchiolar epithelial cells [ ] . several studies have suggested that the immune system may be impaired by sars cov. t-cell lymphopenia was observed in % of patients observed, with a decline in cd + and cd + cell types [ ] . two weeks after disease onset, th cell-mediated immunity and inflammatory response was noted by the marked elevation of cytokines, ifn gamma, the neutrophil chemokine il- , il- , - and - , but not tnf, il- , - , or - . accumulation of monocytes/macrophages and neutrophils was also observed [ ] . li et al. [ ] noted that a rapid decline of t-cell subsets in the periphery was observed in patients during the acute phase of sars infection, but they observed restoration of t cells during recovery. the presence of proinflammatory cytokines may result from activated alveolar macrophages, suggesting that they may play a role in the pathogenesis of sars [ ] . antibodies to the sars coronavirus were found retrospectively in . % of samples collected in , indicating that the outbreak was not the first time that sars had entered the human population [ ] . most infected patients developed a humoral response to sars-cov and antiviral antibodies (igg and igm) were detected at days post-onset of symptoms [ ] . igm antibodies declined after days and igg antibodies persisted up to day [ ] and antiviral neutralizing antibodies were obtained from convalescent patients [ ] . morbidity rates were greater for older individuals, while children under years of age did not develop the severe disease that was seen in adults [ ] . taken together these data may suggest that the quality of the immune response may play a role in the outcome of virus infection. an additional challenge related to the containment of sars-cov is the lack in identification of its natural host. virus has been detected in wild and domestic animals [ ] . in , the first people to be infected were animal handlers in a food market in guangdong province, china, suggesting a role for zoonotic transmission [ ] . the sars strain observed in animals varies only slightly from the human virus and may represent a recent jump across species. the development of good animal models will not only be useful for identifying the natural host, but will be invaluable for determining correlates of immunity, for testing therapeutics and vaccine development. a remarkable advance in sars research came with the discovery that mice were susceptible to infection with sars-cov [ ] . balb/c mice were infected with or % tissue culture infective doses (tcid ) and by day after infection, yielded and tcid per gram, respectively, from lung tissue. although no clinical disease was observed, mild and focal peribronchiolar mononuclear inflammatory infiltrates were observed upon microscopic examination of the respiratory tract on day [ ] after infection. the presence of these infiltrates may suggest some mimicry with human clinical features, although much milder. the respiratory tracts of the mice were cleared of the virus by day after infection. wentworth et al. found sars-cov in the stomach, intestine, and duodenum, in addition to the respiratory tracts of infected mice [ ] . subbarao et al. also protected mice from infection by passive administration of sars-cov neutralizing antibody from previously infected mice, suggesting that neutralization in vivo is possible [ ] . mice clear the virus by day post-infection, while humans begin to clear the infection by days - [ ] . a small animal model will allow researchers to test therapeutics and vaccines, and because the mice recover from virus infection so efficiently, also identify host factors that contribute to virus resolution. hamsters have also been shown to be a good model for sars-cov infection, reaching similar titers to those seen in mice [ ] . surprisingly, immunodeficient mice can clear a sars-cov infection. mice (c bl/ background) that lack nk-t cells (cd −/− ), nk cells (beige) or those that lack t and b cells (ragl −/− ) cleared the virus by day after infection [ ] . the mice displayed high induction of proinflammatory cytokines, suggesting that the adaptive immune response and nk cells were not required for viral clearance in mice. furthermore, it indicates that the involvement of the innate immune response is important in controlling the virus. it is interesting to speculate that interferon pathways may be important in viral clearance. more evidence for the importance of innate immunity was provided through the infection of stat -deficient mice with sars-cov [ ] . stat is important to the regulation of interferons and stat -deficient mice produced a two log increase in viral titer over control mice. additionally, the mutant mice developed interstitial pneumonia, not seen in the control mice [ ] but not alveolar damage as seen in the lungs of human patients. it is unclear at this time if the observed pathological differences between human and stat -deficient mouse lungs were due to time of sampling or differences in host responses [ ] . domestic cats and ferrets have also been tested for use as a sars-cov animal model. cats and ferrets were inoculated with tcid sars-cov. cats showed no clinical symptoms [ ] , while three out of six ferrets became lethargic and one died. virus was recovered from the lungs of infected cats ( tcid / gram of tissue) and ferrets ( tcid / gram of tissue) [ ] . experiments also showed that horizontal transmission of the virus may have occurred between cats that were housed together or ferrets that were housed together, although the kinetics and mode of transmission are still unknown. the non-inoculated infected ferrets became lethargic, developed conjunctivitis and died at and days post-infection. while the ferrets did not show evidence of pneumonia, they did exhibit hepatic lipidosis and emaciation [ ] . ter meulen et al. showed that ferrets that were infected with sars-cov showed signs of multifocal pulmonary lesions affecting about - % of the lung [ ] . alveolar damage and lymphocyte infiltration was also observed upon histological examination of infected ferret lung tissue. three species of monkeys have been tested for infectivity with sars-cov: cynomolgus, rhesus and african green. african green monkeys supported the highest level of viral replication, yielding a viral titer of approximately tcid /ml from nasal swabs [ ] . some researchers working with cynomolgus macaques reported signs of dis-ease resembling sars after the monkeys were infected with sars-cov [ , ] . from day post-infection, macaques became lethargic, had a temporary skin rash, and one animal showed signs of respiratory distress [ ] . two of the macaques had interstitial pneumonia with lesions present, similar to autopsy tissue obtained from sars patients [ ] . other groups have reported that cynomolgous macaques show limited pathology, mild disease and upper respiratory symptoms [ , ] . there is no apparent explanation for the discrepancy between these groups, possibly virus strain differences or monkey subspecies differences may account for the differences in outcome, but to conclude that non-human primates most similarly mimic human disease is still controversial. vaccine efficacy is measured by the ability of the antigen to raise a protective immunologic response from b and t cells after exposure to the viral agent. ideally, by creating memory within the immune system, individuals will be protected from infection for decades. several veterinary coronavirus vaccines are currently available, but their efficacy is variable. the vaccine for prevention of infectious bronchitis virus (ibv), which infects chickens, is effective [ ] , but the canine and porcine vaccines are only partially effective [ ] . the feline infectious peritonitis (fip) vaccine is actually deleterious to the health of the animal and is discussed in further detail below [ ] . vaccines can be produced by inactivation of the virus, by using an attenuated or weak form of the virus, or by using recombinant forms of viral components. inactivated virus vaccines are relatively safe because they cannot revert back to the live form. they are also relatively stable and may not even require refrigeration. this is important in developing countries and for ease in mobilization during outbreak or emergency situations. however, there are limitations to their use. inactivated vaccines usually require several doses and some are weakly effective at stimulating an immune response. the vaccine to prevent hepatitis a is an example of an inactivated viral vaccine [ ] . live attenuated viral vaccines may require special laboratory development and cannot always be obtained. to reach effective levels, the virus must be capable of robust replication, but must have lost the ability to cause disease. several problems are associated with the use of a live attenuated vaccine. these vaccines must be kept refrigerated or frozen, and have safety issues related to the possibility of reversion to the wild-type form. additionally, they are almost never given to immunocompromised individuals for fears that the attenuated form may cause disease in the absence of an effective immune response [ ] . recombinant dna or viral vectors have been constructed in the lab for use as potential vaccines or to study the tissue tropism of the sars virus [ , ] . the vectors can be used to deliver foreign antigens using attenuated or nonpathogenic organisms. the safety of these types of vaccines [ ] centers around persistence of expression in vivo, possible genomic integration of the foreign dna and possible evolutionary changes that may cause instability of the viral vector. the potential transmission of the viral vector, including its introduction into the environment should also be evaluated. an ideal recombinant vaccine might be engineered to include the inherent ability of the foreign substance to be cleared by drug treatments that are proven safe, such as an antibiotic. antibiotic sensitivity introduced into a recombinant vector may allay fears of future adverse events although these designs may raise additional safety concerns. recombinant proteins can also be used to stimulate the immune response. these proteins are purified from yeast or bacteria and currently used in the manufacture of a licensed hepatitis b vaccine [ ] . the cell substrate used to manufacture all of these vaccines is also a concern so vaccine production must be performed in a well-characterized cell substrate. vero e cells have been used to produce the licensed poliovirus vaccine [ ] and may be appropriate for use in the development of a sars vaccine as sars grows well in vero cells. the fda center for biologics evaluation and research (cber) issued a letter to sponsors using vero cells as a cell substrate for investigational vaccines which can be found on the cber website [ ] . another consideration is the use of fetal bovine serum and bovine derivatives in the growth of cells. bovine tissues may contain the bovine spongiform encephalitis (bse) agent. guidelines on the use of bse-free blood products appear on the fda website [ ] as do guidelines on the use of bse-free bovine derivatives in the production of vaccines [ ] . for viruses that have variable strains, a combination vaccine may be effective. a combination vaccine is one that consists of two or more live organisms, inactivated organisms or purified antigens combined [ ] . this type of vaccine may be useful to prevent multiple organisms or strains of organism. each component must make a contribution to the whole and compatibility of the components is necessary. rna viruses replicate through rna-dependent rna polymerase encoded by the virus. this type of polymerase has no proof-reading mechanism associated with it, which results in a high rate of uncorrected mutations. these mutations may or may not be lethal to virus replication and may even persist, resulting in rapid evolution of the virus. for this reason, many rna viruses have multiple genomic strains, or quasispecies, present at one time in an individual. quasispecies may arise in response to selective immune pressure, thus allowing for escape mutants. the existence of quasispecies during sars-cov infection is just coming to light [ ] and their importance in escape from immune surveillance is still unknown. most coronaviruses are thought to have the ability to recombine due to homologous sequences in the and ends of the mrnas. evidence suggests that the sars-cov originated by recombination between coronaviruses [ , ] and that there was an additional host-species drift [ ] . both large and small animals can be infected with sars-cov, a giant advance for vaccine research. examining infected animal models will provide information that will lead to an understanding of the correlates of immunity. mice have been used to further the understanding of virus neutralization, cytokine upregulation and the minimum requirements for viral clearance, but have yet to show disease that mimics the atypical pneumonia seen in adult humans. while there have been some reports of disease in cynomolgous macaques [ , ] , many groups have not reproduced these findings [ ] . a promising animal model may be the domestic ferret. ferrets show elevated liver enzymes, lymphocytic infiltration and alveolar damage, which has also been observed in humans [ ] . despite the usefulness of these animal models, many challenges lie ahead. first, animal models of sars-cov infection do not mimic human disease. in mice, the virus is cleared in less than week and minor pathology in the lung is observed [ ] . histopathology performed on necropsy samples suggests that lung epithelial cells are involved, although the absence of pneumonia and infiltrating macrophages [ ] is disappointing. stat -deficient mice may prove promising as an animal model that most similarly mimics human disease [ ] . second, in order to test efficacy, large human populations must be tested in areas where the virus is endemic. finally, if sars fails to return, how will vaccine manufacturers test candidate vaccines for efficacy? while the animal rule has been provided for just this type of case, an animal model should mimic human disease in order to be applicable. the final rule was published in the federal register and can be found on the federal register website [ ] . additionally, coronaviruses may induce a short-lived immunity. this may be the reason that humans are subject to multiple infections with coronaviruses that cause the common cold. long-term immunity studies for sars-cov are currently underway. antibody-dependent enhancement (ade) has been observed in vaccinated and wild-type infections of fip. ade is thought to potentiate viral infection through the infection of macrophages. viral entry into macrophages occurs when antibodies bind the virus and attach to macrophages via the fc region of the antibody and its interaction with cell surface expressed fc receptors [ ] . neutralizing antibodies can also be enhancing antibodies if antibody titer is low or is of the igg class [ , ] . because macrophages increase with viral disease, this cell type may provide an abundant reservoir for the virus and thus expansion of the virus in the host. some similarities between fip and sars exist. first, in both cases macrophages can be infected with the virus [ , ] , and in the case of sars, the etiology of disease is contributed by infiltrating alveolar macrophages leading to pneumonitis [ ] . second, the treatment with corticosteroids and/or interferon alpha ameliorates sars disease [ ] , suggesting an inflammatory, immune-mediated disease. while there has been no observation of ade during sars infection, it is worth noting that one coronavirus, fipv, is capable of eliciting ade and in the evaluation of vaccines, we may want to consider this possible outcome. however, the difficulty in testing animal models for ade bears the caveat that if ade is not observed; it has not proved that vaccines are safe with regard to ade in humans. in contrast, if an animal model for ade is developed, we may learn more about the mechanism of sars-induced ade, which may help form the basis for developing guidelines for safe vaccine development. a potential sars vaccine might target the virus specifically through humoral or cell-mediated immune responses. alternatively, therapeutic vaccines may be useful in the treatment of viral infection. spike-specific monoclonal and polyclonal antibodies that neutralize the virus have been developed [ , ] and passive transfer of immune serum into naive mice protected them from infection with sars-cov [ ] . this suggests that neutralizing antibody alone can prevent viral infection. neutralization may not require host recognition of the fc region of the antibody, but the need to develop humanized forms of these types of antibodies may be critical if they are to be considered for use as a treatment. a human monoclonal antibody, derived from a phage display library, was administered to ferrets and protected the ferrets from lung disease and the shedding of virus in pharyngeal secretions [ ] . neutralizing antibodies from convalescent patients have been identified and characterized. usually neutralizing epitopes are located in the spike protein of the virus [ , ] . recent evidence has determined that virus neutralization is sensitive to deglycosylation of the spike protein, suggesting that conformational epitopes are important in antibody recognition [ ] . sars-cov can be efficiently inactivated by ultraviolet (uv) irradiation [ ] . mice immunized with uv-inactivated sars-cov develop humoral and cell-mediated immune responses [ ] . both t cell proliferation and cytokine upregulation was observed after boosting with the inactivated virus. beta-propiolactone-inactivated virus also elicited neutralizing antibodies when administered to balb/c mice [ ] formalin-inactivated sars-cov yielded potent humoral responses in balb/c mice as well [ ] . recombinant viruses may be used to elicit responses to introduced sars-cov genes. the first type of recombinant virus is a defective or non-pathogenic vector that expresses sars-cov proteins. the second type is one that is stimulated to assemble virus-like particles (vlp) in vitro. vlps containing the structural envelope proteins including spike (s), envelope (e) and membrane protein (m) have been assembled by coinfecting insect cells with three baculoviruses expressing one of the three structural proteins [ ] . structural proteins expressed by the live attenuated bovine parainfluenza virus type (bhpiv ) were evaluated for efficacy in hamsters [ ] and african green monkeys [ ] . high titer neutralizing antibodies were obtained after only one intranasal immunization with this vector. single immunization with bhpiv expressing s alone provided complete protection upon challenge with sars-cov [ , ] . recombinant live attenuated modified vaccinia virus ankara (mva) was used to deliver the sars spike protein (rmva-s) into balb/c mice [ ] . neutralizing antibodies were obtained and a reduction in the viral titer was observed after challenge with live sars-cov [ ] . only ferrets that were challenged with sars-cov after vaccination with rmva-s showed enhanced liver disease as demonstrated by increases in alt values and the presence of mononuclear hepatitis upon histological examination [ ] . these data suggest enhanced disease due to vaccination with a sars protein. adenoviruses expressing the s, m or n proteins were used in combination to vaccinate rhesus macaques [ ] . the immunized animals all had antibody responses to the s protein and t-cell responses to the n protein [ ] . highly infectious hiv particles expressing the s protein have been made, primarily to study the host-cell distribution of the putative sars-cov receptor [ ] . additionally, investigating the requirements for viral receptor binding and entry will also enhance our understanding of the requirements for viral control. recombinant hiv particles that express the sars spike protein may provide insight into cell tropism and receptor expression profiles [ ] . another retrovirus, murine leukemia virus, was used to generate infectious particles containing most of the s protein. convalescent serum was able to neutralize infection of the recombinant virus in vero cells [ ] . high cytotoxic t-lymphocyte (ctl) and antibody responses were observed after mice were injected three times with a recombinant plasmid vector expressing the n protein [ ] . mice immunized with a plasmid containing the s protein produced anti-sars-cov igg [ ] and developed neutralizing antibodies and a t-cell mediated response resulting in a six-fold reduction in viral titer in the lungs [ ] . plasmids encoding either the s or s regions of the spike protein elicited antibody production in mice [ ] . neither the s or s antibodies alone were capable of neutralizing the virus; how-ever, cooperatively they enabled neutralization of the virus, suggesting that both regions of the spike protein are important for host-cell viral entry [ ] . the nucleocapsid protein may also stimulate an effective immune response. dna vaccination with calreticulin fused to the n protein generated sars-specific humoral and cellular immunity in c bl/ mice [ ] . calreticulin was used because it was found to enhance major histocompatibility complex class i presentation of fusion proteins to cd (+) t cells [ ] . recombinant viruses may be generated from the fulllength infectious cdna clone of sars-cov [ ] . this clone may provide a source for genetic manipulation of the genome [ ] . once the viral virulence factors are understood, attenuated strains may be obtained by engineered mutation of the virus. vaccine development may proceed through the undertaking of a systematic approach to understanding the correlates of immunity raised by sars-cov. much of the focus has centered towards the humoral response and neutralizing epitopes, but cell-mediated immunity may also be important. ctl epitopes within sars-cov that may be presented by % of the human leukocyte antigen supertypes were identified by advanced bioinformatics [ ] . further characterization of these epitopes, including their recognition by convalescent serum, should advance the understanding of important immunological features in the control of sars-cov. while there has been intense study of sars, there is still much that is unknown about the pathology of the virus. many research questions will need to be answered, thus providing the resources necessary to develop an effective and safe vaccine. to be sure that a vaccine will provide coverage for a potentially variable virus, we need to know what the occurrences of viral quasispecies are and how variable are the important epitopes. what are the viral virulence factors and can we use this information to develop an attenuated strain? can the virus establish persistent infection and can humans be repeatedly infected? we also need to determine the innate, humoral and cell-mediated immune responses in recovered sars patients in order to understand how viral clearance comes about. to understand viral pathogenesis we must know how the immune system mediates disease. what is the role of lymphocytic infiltrates and do alveolar macrophages enhance disease or control infection? if we define the ability of the virus to replicate in monocytes/macrophages can we be assured that antibody-dependent enhancement will not be a problem for a sars vaccine? most importantly, identification of the natural animal host may launch the further development of large and small animal models for correlates of immunity and drug and vaccine screening. development of an animal model that mimics human disease will be the single most important advance in the development of a sars vaccine. development of a vaccine for sars-cov is imperative and research headed in a for-ward direction will enable the public health community to be ready. toronto sars critical care group. critically ill patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome sars-beginning to understand a new virus severe acute respiratory syndrome coronavirus phylogeny: toward consensus identification of a new human coronavirus coronaviridae: the viruses and their replication lung pathology of fatal severe acute respiratory syndrome 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form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice immune responses in balb/c mice induced by a candidate sars-cov inactivated vaccine prepared from f strain assembly of human severe acute respiratory syndrome coronavirus-like particles severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets effects of a sars-associated coronavirus vaccine in monkeys retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein induction of sars-nucleoprotein-specific immune response by use of dna vaccine dna vaccine of sars-cov s gene induces antibody response in mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation kanta subbarao, edward tabor, miriam darnell, robin levis and hira nakhasi are gratefully acknowledged for comments on the manuscript. key: cord- -elhgew x authors: spier, r.e. title: ethical aspects of vaccines and vaccination date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: elhgew x nan published by elsevier science ltd. all rights reserved printed in great britain pii: so - x( ) - - x/ $ + . r. e. spier traditional certainties no longer serve our societies as well as they might. technical developments over a broad range of engineering disciplines have radically changed the nature of the physical world in which we live. our knowledge base has expanded concurrently, enabling us to provide materially based and more convincing answers to questions which baffled our antecedents of years ago. this requires that we review and reconsider the ethical guidelines for behaviour which have been largely founded on philosophies and concepts whose origins may be traced back to between and years ago. an example of the implications of these changes may be seen in the area of vaccines and vaccination which evinces the pressing need to review traditional ethical positions to take the maximum advantage of the potential for animal and human benefit inherent in this prophylactic approach to healthcare. in this paper i examine some of the general ethical issues thrown up by recent advances in the field of vaccines and vaccination. it will touch on issues of the putative autonomy of the individual and way we will have to reassess the cost ((risk * the magnitude of the damage) + cost of manufacture and distribution + surplus) to benefit relationship. issues subtended from the effect of vaccines on the magnitude of populations will be followed by transcultural issues implicit in vaccine testing and delivery. the use of vaccines to obviate behavioural changes (technical fixes), generate transcendental concerns and provide new threats via biological warfare agents will also be treated. it is part of current medical practice when dealing with patients to extol four basic ethical principles: autonomy, beneficence, non-maleficence and justice. however, when we consider issues related to vaccination the principle of autonomy (self-determination; freedom from interference unless the act harms others (j.s. mill); as in un or cfe declarations on human rights) is challenged. while the principle of autonomy may be asailed from a number of different facets such as the competence of an individual to provide informed consent, the rights of a fetus if the pregnant mother decides to be immunized or the implicit implications of the social contract entered into when an individual chases to dwell in a particular society, i will examine in university of surrey, school of biological sciences, guildford, surrey gu xh, uk more detail a further issue involving the expression of social responsibility. it is well known that when a high proportion of a population is vaccinated, those who have not been immunized become protected by the decrease in the level of the pathogenic organism (the herd effect). the question which this poses is, do those who have opted against vaccination have the right to benefit from the expense and the risks of vaccine-induced damage accepted by those who have been vaccinated? not only that, but such unvaccinated individuals pose a threat to the vaccinees as they serve as a reservoir in which the disease can be maintained and propagated. were the society to collectively determine that all its citizens should receive the vaccine then the principle of patient autonomy is infringed. an intermediate position might be to levy a special cash buy-out dispensation to those who refuse vaccination as a contribution to the costs incurred by those who have accepted the risks of vaccine-induced damage. whatever the outcome in any particular society it is clear that vaccination poses a challenge to accepted ethical positions, the resolution of which will be dependent of the degree of social coherence and farsightedness. for much of the last years commercial companies have been required to provide products to the marketplace and society's judgement on the company has been via the acceptability of the product at the price demanded. the function of the company was to accumulate profits for its shareholders and top managers (incentives) and society did not interfere with the way the product was made, nor with the product spectrum on offer. this ethic too, is in need of review. vaccines are a clear benefit to society as a whole. however, there is a risk of vaccine-induced long-term and severely debilitating damage [well recognized for polio vaccines and less well established for any other vaccine cf. swine fever (inactivated influenza) vaccines and guillain-barre syndrome] which is especially poignant when incurred after the vaccination of a -month-old healthy infant. under present conditions the manufacturing company may be sued for damages both actual and punitive (if wilful negligence is proven). but is this the appropriate ethic? may not the society, in accepting the benefit of widespread vaccination, compensate those who suffer damage at a level communsurate with the damage? of course, were the manufacturer culpably negligent in a procedural ethical aspects of vaccines and vaccination: r. e. spier matter, then compensation would also be due from that source. resulting from this liability situation, vaccine manufacturers are reluctant to venture into vaccine projects. also, as the cost of obtaining a product license is estimated to be some $ - million, the implications for the cost of a dose of vaccine is more driven by the cost of the regulatory procedure than the production cost (which rarely exceeds the cost of the bottle plus label). the ethical issue here is whether the regulatory hurdle which has to be overcome is really operating in society's best interests. nothing we eat or do is without the risk of incurring damage. vaccines are not different in that respect. yet the cost of obtaining a license to manufacture and distribute implies that the cost of the vaccine has to be many dollars merely to recoup the costs involved. does this mean that the rich should pay a high price for a vaccine and thus subsidize the provision of cheap vaccines to the poor? or is it the job of the elected representatives of the people to act as purchaser on behalf of the poor and provide the vaccine manufacturer compensation through the tax system or other fiscal dispensation? vaccines for diseases which afflict a few are not a commercial proposition unless communal support is provided. vaccines which lead to a decrease in the need for over-the-counter or prescribed medicaments are also unlikely to be made by commercial concerns. the reluctance of pharmaceutical companies to workup a vaccine for helicobactu pylori which would prevent the recurrence of stomach ulcers and hence stomach cancers is evident from the research programmes of those companies which make anti-ulcer drugs. research on vaccines which would prevent the common cold (caused by a combination of rhinoviruses, coronaviruses and admoviruses) is also conspicuous by its absense. but prophylaxis as an approach to healthcare is also underfunded by society at large. therapeutic research receives over times the funding of prophylaxis; and this is particularly difficult to understand when there are many inexpensive ways of achieving prophylaxis, of which vaccination, which affects the immune system, is but one. other methods of prevention focus on the protection of the immune system to decrease the probability that it will be overwhelmed by an endogenous or exogenous pathogen; such procedures may be termed 'fence vaccines". a new relationship between industry and the community is indicated. the former is now required to recognize that it will be judged as much by the ethicality of its actions as the efficacy of its products. for the pharmaceutical industry, ethics is not an optional extra: it is essential. an ethical argument which is proscriptive of the use of vaccines in the developing and less developed world (containing % of the world's . billion people) is that they will lead to an increase in an already unsustainable population. this will have sequellae via an increase in suffering from malnourishment, population migrations and war. however, recent figures published by unicef refute these projections' and show that the average number of children born to a woman of the developing world throughout the period of her fertility has dropped from . to . between and . this would indicate that more, rather than fewer, vaccines are required; and indeed vaccines protective to the diarrhoeal and respiratory diseases of childhood are on test in the field, while candidate vaccines aimed at controlling malaria languish in laboratory fridges. population may not only be affected by a decrease of infant mortality but also by an increase in the average age of the community. as the level of communicable disease wanes, people live longer and society then has to adjust its working conditions such that there are productive positions for the older people to occupy. it is clearly not practicable to socially provide for protracted retirements, so the emphasis on life-long learning, skill changing, job flexibility and part-time working will become the norms of future social development. is it appropriate to use a technical fix when an almost cost-free change of behaviour will achieve the same effect? such an ethical problem is thrown up by the willingness of our communities to spend billions of dollars to provide therapeutic and prophylactic agents to control the spread and effects of the human immunodeficiency virus (hiv), while the disease would be eliminated were people to engage in safe, condom-protected, intercourse in their pre-or extramarital sexual relationships where the prospective partners had not been thoroughly tested for the presence of serum antibodies to the virus. this provision also applies to the transmission of the viruses which cause hepatitis b, genital herpes, as well as the cancers wrought by the papilloma virus. as a corollary to the practise of safe sex, it might also be expected that gonorrhoeal infections would decline, as would those caused by treponema sllphilis and the yeast candida. many of the food-and water-borne diseases caused by bacteria of the salmonella. escherichia, shigella, listeria, campylobacter and vibrio groups would be eliminated were drinking and washing water to be prepared according to the highest standards prevalent in most developed countries. however, the engineering requirements to achieve this in the short term are daunting. whereas the prospect of the development of orally deliverable vaccines which would provide protection against the diseases caused by the above pathogenic bacteria is a task which may be brought to a successful conclusion within the next decade. a further case where vaccines are used to preclude the expenditure of monies is to protect people from the effects of the diseases of propinquity; typhus and tuberculosis. both of these diseases flourish when people are housed in crowded insanitary conditions. it may be that the vaccination route is cost-effective in monetary terms but this should not be used as a way of avoiding the social improvements which would enhance ethical aspects of vaccines and vaccination: r. e. the dignity of citizens, as well as improving health. spier their infectious disease-causing organisms do not recognize national boundaries. transworld travel for tourism and business is increasing exponentially and with it are opportunities for disease-causing organisms to travel. we are also presented with a situation in which tests of vaccines in developing countries can be effected at considerably less expense than in a developed country. this has led to a series of ethical issues which are exacerbated by the different cultures of the people who may be engaged in the vaccine trials. for example, is it possible to obtain the informed consent of a person who is illiterate and who does not understand the implications of something like a vaccine with which (s)he is totally unfamiliar ? a second issue might be that the removal of blood or tissue for sampling might be regarded as an attempt to capture the spirit of the so deprived individual. additionally, there may be taboos about removal of blood via venipuncture and in some cultures the insertion of needles into bodies may have overtones not foreseen in western cultures. on removal of a sample containing cellular material from the body of an individual (generally a tissue responsible for a pathogenic effect) one obtains the opportunity to work with a highly selected and unique genome. were the genes of that cell to be used to make a pharmaceutical to particularly benefit people in the developed world, what sort of compensation should acrue to the source of the cell line from which the gene was obtained? current thinking by the nuffield ethics committee" would have it that the cell provider has not contributed to the inventive step in the drug or prophylactic development and therefore is not to be compensated. however, if advantage is taken of the uniqueness of the material derived from a person of the developing world then it would be churlish not to recognize this through some financial contribution to the individual and his/her community. one might ask, to what extent is a prophylactic trial in the developing world relevant to the circumstances prevalent in the developed world? are the conditions leading to infection and the challenge organisms relevant? are the people in whom the vaccine is tested likely to respond in an immunologically equivalent manner when the history of the exposure of their immune systems to disease is dissimilar in many ways to a person of the developed world? in the event that there is damage to an individual as a result of exposure to vaccine in a trial, what are the levels of compensation and who pays? and indeed, is it ethical that a person in the vaccine-producing country should enjoy the benefits which have been won at the expense of the risk-taking of a person in less privileged circumstances? to some extent many of these questions may have answers were the developed country vaccine producer to agree up-front to provide cost-free vaccine to all the people of the country in which the vaccine trial had been effected. in that way something of a bargain may be established such that overt exploitation has been subverted into mutual gain. in all such situations it would not be an acceptable ethic to effect trials with placebo controls which did not provide the best possible protection. similarly it would be counterproductive to use vaccines whose safety was an issue and where a less damaging vaccine could be made available, albeit at greater expense. in addition, there is the overriding consideration that if nothing is attempted then there would be a known tally of deaths and disease and our efforts to combat that embody a justification for effecting vaccine experimentation. a definition of the transcendental might be 'that which is outside the cause-and-effect system', where the latter implies that all which exists and the way it interacts is delimited by the energy and matter of its constitution. for example, ghosts, fairies, trolls, spirits, jinn and souls are described in such a way that they perform their tasks with scant regard for the properties of matter and energy, as do the panoply of deities which have been posited as having creative and control capabilities with regard to the affairs of humans. nevertheless, such considerations cannot be obviated when we review the reactions of community members to the production and use of vaccines, particularly when some of the most emphatic proscriptive reactions emanate from the leadership of recognized deitic religions. in kirkpatrick's book on inoculation published in there is the astonishing report of the abreaction of the church to vaccination because, as a result of the possibility of dying from the vaccine of the day (smallpox, occasionally contaminated with syphilis), it may be construed that the vaccinee was indeed seeking to commit suicide, which was sinful. in modern times there were a series of reports in the uk media' wherein the catholic church was seeking to prevail on its susceptible female members to forego vaccination protective against rubella, as the vaccine was prepared from the cells of an aborted human fetus: for human abortion is contrary to the teachings of that church. on a more esoteric plane, it is possible to argue that, through the use of vaccines, mankind has developed a capability which may eventually rid the world of those infectious microoganisms which have been the bane of our struggle to survive. this depletion of the deitic armamentarium may be construed as seeking to deny the deity of one of its controlling effector systems; namely, the threat of divine retribution through the causation of plagues . alternatively, it may be held with equal rectitude, that the deity ordained us to discover and use vaccines as part of its (undisclosed) master plan. consonant with this latter controversy, there are those who assert that it is unnatural to disturb the ways of nature and as vaccines are a creation of mankind, they are not natural and are therefore to be condemned. as this contention rests on the definition of what is natural it is possible to reconstrue this issue totally were we to assert that whatever exists is natural: merely by virtue of its existence. it would follow that there does not exist anything which is artificial (in the sense of unnatural) which implies that all the products of the arts (techniques, crafts, skills) of humans (and animals) are natural and are incorrectly designated as artificial unless the meaning of that term were changed to its more appropriate designation, as works which are the product of humankind's arts (hence arti-ficial; made by art). now that it is practicable to identify a defective gene in an adult, child or even embryo, techniques are in development to repair, exchange or inactivate that gene specifically. these methods imply the existence of 'genetic vaccines'. however, these self-same methods may be used not just to correct defects but to enhance characteristics we may desire to accentuate'. as the word 'disease' is defined as a state of being in which one is not-at-ease, it is not difficult to use the word to describe a situation where a person is not at ease with their height, intelligence, running or ageing, speed etc. this raises the ethical question of the use of genetic vaccines to prevent the disease resulting from such deeply held feelings. there is little doubt that the remediation and/or prevention of situations which cause physical pain is encouraged and applauded by society. the same cannot be asserted where the pain ethical aspects of vaccines and vaccination: r. e. spier may be psychological. nevertheless, this latter pain is just as actionable as the physical pain (indeed it is physical, but of the activities of the brain as opposed to muscle). indeed, not only would we relieve suffering but we might also achieve a human being who is more functionally capable of making a more extensive contribution to society; a feature which is not given to all pain-killing remedies. that such measures might be construed as interfering with some transcendental plan cannot be denied, but the ethic of beneficence might be applied to progress the use of these genetic vaccines in all their manifestations. were we to have an effective orally deliverable contraceptive vaccine' (pregnancy results from the infection of the female by a male spermatozoan) then ethical considerations will be required to determine the way in which such a powerful tool for population control might be used. the contamination of drinking water supplies with a contraceptive vaccine would act counter to the autonomy principle of ethics and could be held to be an affront to the dignity of humanity in denying individuals control over their own fertility. notwithstanding this clear ruling, it is possible to conceive of conditions in which a decrease in population levels is an urgent necessity and the use of an orally delivered contraceptive vaccine might be the only way of achieving that end without recourse to widespread sterilization. as with any other tool, we have to adopt an ethic which permits its appropriate use; what we need to do ahead of time is to discuss and debate what those circumstances might be. anthrax bacteria, botulinum toxin, the plague bacillus (yersinia pestis), measles and influenza viruses have all been proposed as agents of human destruction in the context of international and intranational conflict. both smallpox and measles have been implicated in the decimation of the indigenous populations of the americas during the colonization processes beginning in the s. vaccines protective against these diseases, therefore, become defensive devices which would have to be surmounted by a would-be aggressor. genetic engineering may be used to attempt to enhance the lethality of existing agents or make otherwise benign adding an ethical dimension to industry's external relationships legislators agents lethal. it is unlikely that an increase in lethality can be achieved through the manipulation of the genes which code for toxin structure, for the sophistication of the binding site of these toxins, coupled with the evolution of the enzyme which causes the damage, has probably reached the maximum level of efficacy as a result of the evolutionary process by which it was formed. the addition of new strain of toxin genes to a particular bacterial cell may also be assayed. but the common pattern of bacterial toxins leads to vaccine solutions which are likely to be cross-protective, irrespective of how many toxin genes are compiled in any one bacterial cell. perhaps. of more serious consequence, would be the engineering-out of the epitopes which evoke the immune system to produce toxin-neutralizing antibodies. when this occurs epitopes which heretofore were immunosuppressed become immunodominant'. this would call for the development of a new vaccine which could be made by using the newly engineered binding portion of the toxin molecule to induce neutralizing antibodies to the fully constituted toxin (binding portion plus enzymatic component). this process could be repeated many times. in addition to the manipulation of the toxin system, developments occur in the methods for the dissemination of the agents. while it is possible to conjure scenarios of contaminated water supplies and recognize that the air handling systems of large buildings may constitute a means of agent distribution, the mechanisms for the protection of such seemingly open targets are well in place already; for we do treat water supplies with inactivants and it is recognized that the circulation of air within air-conditioned buildings is fraught with problems if people's natural illnesses are circulated by air-handling equipment devoid of high performance filtration systems. to meet the contingencies of noxious agent distribution the stratagem of 'fence vaccination" may be brought into play. the cycle of measure/countermeasure with which we are familiar in the area of conventional and nuclear weapons has its echoes in the area of biological warfare. that we have a duty to defend the life of our peoples is an ethical principle which few would dispute. in the context of this paper, the pursuit of vaccines to existing and potential biological warfare agents is not just a job done in response to real or perceived threats but should become a mission whose importance ranks so highly that it has to be included with the strategic planning we have to undertake to survive. as in other areas of defensive reaction, we might expect the vaccine production techniques to be enhanced with regard to both the ability to manufacture high quality immunogenic preparations rapidly and also with respect to the engineering of those immunogens. these abilities will have spin-off effects with regard to our ability to produce vaccines protective against the biological agents which pose threats from natural sources, such as influenza viruses whose type changes require us to make immunogenically unique vaccines on a year-by-year basis. we have also to take note of the emergent infectious agents (ebola, hantavirus, hiv) which have achieved a degree of notoriety in recent years"'. our ability to respond to such agents has been slow, cumbersome, disorganized and paltry in relation to the importance of the test situation which these agents proffer. our ethics require that we improve on this performance. vaccine manufacturers must obtain a licence to enable them to market their vaccine products to the wider society. this licence is obtained on the recommendation of a body called a regulatory agency (the fda division of biologics in the usa, the committee for medicines in the uk, etc.). obtaining regulatory agency acceptability" of a vaccine product is a process which may take - years and cost $ - million dollars. as can be seen from figure , the regulatory agency is subject to being influenced by the society which it is set up to serve. in addition to this necessary connection to the regulatory agency, industry may opt to receive from universities and publicly funded research institutes information and materials which enable it to embark on new vaccine projects. this interaction between the private and public sectors is fraught with ethical problems stemming from the difference in the culture of the two types of institution". this has been rendered particularly acute in recent years when attempts have been instigated to make the publicly funded institutions behave as if they were private, profit-making bodies. the insinuation of industrialists into the peer review process for grant applications generated by academics has been one area where the bicameral functionality of the seconded industrialist leads to the funding of conservative research projects which do not compete with the industrialist's undisclosed mission. in this, society is not well served by the monies it sets aside for both academic and publicly funded research. society is not amorphous. it has structure through its institutions. the institutions which affect the way the regulatory process works may be identified as being ( ) the ethical bodies; ( ) the media; ( ) legislatures; and ( ) the educational system (figure ). the ethical bodies include recognized religions whose leaders provide ethical guidance to the members which impinges on the production and use of vaccines, while bearing in mind current social exigencies. there are secular sources of ethical guidelines which are not as well organized and overt. such individuals might recognize that ethics are not guidelines which are provided by the pronouncemnts of a deity but are rather the set points in the control system which modulates human social behaviour with the objective of promoting both the survival of the individual, society and other biotic entities as wealth and the occasion permits'". the media are informed by the religous ethicists as well as picking up on the raw nerve endings of the fears and sensitivities of their fellow socialites. they do not hesitate to evoke the image of the entity created by victor frankenstein at the prospects of the slightest opportunity of affecting a deliberate change to the genetics of the human species. our tradition encourages us to regard monsters as the avenging agents of personal wrongdoing; so such buttons do not require much pressing to evoke negative reactions to vaccine volume number ethical aspects of vaccines and vaccination: r. e. spier the prospects of experiments gone awry. the third effector on the way the regulatory agencies behave is the legislature. it is by law that pharmaceutical products have to be safe, efficacious and be made by a demonstrably consistent process. but how safe is safe? and what is efficacious? and what are the limits by which we define consistency? the answers to each of these questions pose ethical problems. it is recognized that a balance has to be struck between the urgency of the need (if we don't have the agent, people will die) and the side-effects of the product. education is a key feature when we consider how humans behave socially. some attribute the education of children to parents only, and see the education institutions as providing knowledge and capability, but not ethics. others would have us believe that we receive effective training in ethics thoughout our lives from all kinds of sources of which educational institutions are prominent. it is a matter of choice as to whether an individual believes that 'you can't teach an old dog new tricks'. it is a matter of record that modern adults have to relearn their new car's control systems and idiosyncrasies each time they change their car. learning to programme a video, access and use the internet, come to terms with computers, air travel and internationally derived food menues has come to many people late in life, yet they have made changes to their behaviours in the light of these new developments. we are indeed open to lifelong education for which our modern universities are reorganizing. these considerations provide opportunities for industry to accept a new mission. while they have accepted for many years the need for sympathetic public relations (with the media) and for lobbying activities (to affect the laws which receive legislative approval), they have not until now perceived that they have also to meet the challenge of ethics providers. this can be effected at two levels; the one, via the bodies which focus on the generation of ethical guidelines; the other, the educational system from the earliest grades to those who return to education in their third age or later. industrialists will have to recognize that ethics matters. they will have to support institutions which engage in devising an ethics which is wholly compatible with living in a modern world with ever changing technologies and concepts about the nature of life and the way the world works. and they must engage in the promotion of ethics in a society which is hungry for a new ethical synthesis (figure ) . all this has to be done at arm's length. there is no substitute for the realization that we all are members of the community and responsible in some measure for our mutual well-being: sectors cannot profit at the expense of other segments. this concurrence of aims needs a reaffirmation; it is hoped that industry will see its future success through the inclusion of this mission in its project portfolio. introducing fence vaccines the state of the world's children. unicef the analysis of inoculation comprising the history, theory and practice of it: with an occasional consideration of the most remarkable appearances in the small pocks in sickness and in health the sunday times, / / , news section p. which describes a genetically engineered anti-pregnancy oral vaccine based on salmonella evidence of cryptic v epitope(s) on native hiv- virions: immunophysicochemical analysis the coming plague; newly emerging diseases in a world out of balance on the acceptibility of biopharmaceuticals ethical aspects of the university-industry interface ethics as a control system component key: cord- - jikrn authors: borja-cabrera, g.p.; santos, f.n.; bauer, f.s.; parra, l.e.; menz, i.; morgado, a.a.; soares, i.s.; batista, l.m.m.; palatnik-de-sousa, c.b. title: immunogenicity assay of the leishmune(®) vaccine against canine visceral leishmaniasis in brazil date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jikrn leishmune(®) is the industrialized version of the fml-saponin vaccine which has been shown to develop – % protection in vaccinated dogs and – % vaccine efficacy against field canine visceral leishmaniasis (cvl) in brazil. leishmune(®) has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. in the present investigation, healthy seronegative dogs of endemic and epidemic areas of brazil were monitored for leishmune(®)-induced immunogenicity during a -year trial. another group of untreated exposed dogs was also studied in parallel. both groups were seronegative on day . the strong immunogenicity induced by leishmune(®) vaccine was demonstrated by the % of fml-seroconversion, increase in absorbencies, the . % dth positive reactions and increase in skin test size diameters, the average increase in cd + total lymphocytes population in blood ( . %), expected for qs saponin-containing vaccine, the sustained proportions of cd + t cells, and the average increased proportions of cd + b lymphocytes ( . %). the leishmune(®)-induced protection against cvl is demonstrated by the results: . % asymptomatic dogs (at the end of first year) and % healthy survivors (at the end of the second year) among vaccinated dogs, compared to the . % asymptomatic and % survivor dogs (p < . ) monitored in the untreated exposed cohort. in spite of the low vaccine coverage, it was possible to detect a . % (p < . ) reduction in belo horizonte and an . % (p < . ) reduction in araçatuba of the incidence of cvl among vaccinated dogs, when compared to the global incidence of cvl of each town, respectively. our preliminary results support the potential use of leishmune(®) to prevent cvl epidemics. visceral leishmaniasis (vl), a chronic and severe protozoa infection, is fatal if untreated after the beginning of symptoms. the disease is a canid zoonosis (zvl) caused by leishmania chagasi in america and by leishmania infantum in the mediterranean basin and middle east, and an anthroponosis caused by leishmania donovani in africa, india and asia. nowadays there are , new human cases registered annually worldwide [ , ] . the drug resistance and toxicity of chemotherapy, the increase of the disease incidence of immunocompromised subjects, and the difficulties of the epidemiological control, which is based upon sacrificing of seropositive dogs, emphasizes the need for safe pro- [ ] [ ] [ ] [ ] achieving different levels of protection, but no data from field trials of any dna formulation is so far available [ ] . despite the recent intensification in research for a canine vaccine only two second-generation vaccines with native antigens have progressed to phase iii field trials: the fml-saponin [ , ] and the liesap vaccines [ ] . the fml (fucose-mannose ligand) glycoproteic complex [ ] , antigenic for both humans [ ] and dogs [ ] , was formulated, as a second-generation vaccine, with quillaja saponaria saponin and underwent phase i-iii trials becoming the leishmune ® licensed vaccine in brazil [ ] . the fml was immunogenic, immunoprophylactic and immunotherapeutic, in mice and hamsters and canine field trials [ , , [ ] [ ] [ ] [ ] . in the first phase iii dog assay [ ] , deaths and symptomatic cases among placebo-treated dogs ( %) were detected and confirmed by parasite analysis and pcr. no deaths were detected among vaccines and infection was confirmed in / oligosymptomatic dogs ( . %), resulting in % protection and % vaccine efficacy [ ] . in the second field assay [ ] , the infective pressure was higher and years after vaccination, deaths were detected in / ( %) placebotreated and / ( %) vaccinated dogs, resulting in % of protection and % vaccine efficacy. this protection lasted for at least . years and was concomitant with the reduction of the human incidence of the disease in the area [ ] . the fml-vaccine also produced an immunotherapeutic effect when administered to l. donovanior l. chagasi-infected dogs while they were still asymptomatic [ ] . the decrease in the canine and human incidence of visceral leishmaniasis in the vaccinated area [ ] and the maintenance of normal proportions of cd and cd lymphocyte levels in the blood of vaccinated dogs [ ] indicate that dog vaccination with the fmlvaccine reduces dog infectivity to sand flies [ ] . liesap + mdp vaccine, the other second-generation vaccine with native antigens, was recently used in a field assay with naturally exposed dogs of south france [ ] . in this trial any dog showing clinical and/or serological evidence, infection was confirmed (or not) by the presence of parasites in bone marrow cultures and by pcr analysis. after years, the incidence of infection was . % ( / ) in vaccines versus . % ( / ) in placebo-treated dogs ( % vaccine efficacy) [ ] . the authors claimed a vaccine efficacy of % based on the confirmation of infection by very sensitive methods such as pcr or culture, instead of cvl deaths [ ] which did not occur in this area of lower incidence. the fml-vaccine unlike the liesap vaccine revealed protection not only against infection, but also against severe disease and deaths due to cvl [ , ] reducing morbidity and mortality [ , ] which are much stronger criteria of protection [ ] . the fml-vaccine was licensed in brazil, for dog prophylaxis against zvl, under the brand leishmune ® [ ] . dogs vaccinated with leishmune ® (fml-licensed vaccine) are not infectious for sand flies [ ] , as indicated by a complete absence of clinical signs and of parasites in the skin, lymph node and blood pcr-amplified samples. exposed untreated controls on the other hand, were symptomatic ( %) and showed parasites in their lymph nodes ( . %), leishmania dna detected by pcr in blood ( . %) and immunohistochemical reactions in skin ( %) [ ] . leishmune ® is a transmission-blocking vaccine [ ] and when used with increased adjuvant concentration was also effective in immunotherapy on experimental cvl [ ] . recently we described the safety analysis of leishmune ® vaccine performed in a cohort of dogs from brazilian endemic and epidemic areas of canine and human visceral leishmaniasis. the vaccine proved to be tolerable and safe [ ] . in the present investigation we report the immunogenicity assay of leishmune ® , monitored in the same dog cohort, confirming the immuno protective potential previously described for the fml-saponin vaccine [ , ] and disclosing the potential use of leishmune ® vaccine to interrupt epidemics. six hundred healthy dogs from the canine visceral leishmaniasis endemic towns of araç atuba, andradina, valparaíso, guararapes, bauru (são paulo state) and belo horizonte, nova lima, sete lagoas (minas gerais state), brazil, showing previous negative results in leishmania-serology by the immunofluorescent assay [ ] were selected for vaccination with three doses of leishmune ® (fort dodge animal health, campinas, sp, brazil), in a -day interval, through the subcutaneous (sc) route [ ] and a booster in month . on day , before vaccination, from the dogs were excluded due to their positive reaction to the more sensitive fmlelisa assay [ ] . the remaining dogs, seronegative to the fml antigen, asymptomatic and showing good physical condition, became the trial group of this investigation. each of the veterinarians participating in this trial vaccinated dogs with three doses of leishmune ® , making a total of doses. the animals were monitored for their anti-fml igg serum antibody titters by the fml-elisa assay [ ] at days and and in months and , and by their intradermal response to the l. donovani promastigote lysate [ , ] antigen in months and . the serum was collected and intradermal test was carried out before injection of the vaccine booster in month . also, clinical evaluations were performed every months, during the -year period ( ) ( ) ( ) . alopecia, onychogryphosis, cachexia, anorexia, apathy, disseminated ulcers, skin lesions, keratitis, renal failure, loss of weight, lymph node enlargement or diarrhoea were recorded as visceral leishmaniasis symptoms. in vaccinated symptomatic animals, leishmania infection was confirmed either by pcr analysis of lymph node aspirates and/or blood samples [ ] or by direct microscopical observation of leishmania amastigotes in giemsa stained lymph node smears [ ] . the leishmune ® -vaccinated dog cohort included animals ( %) from different breeds and of ( %) mongrel dogs [ ] . all animals were previously vaccinated against distemper, parvovirosis, parainfluenza virus, leptospirosis, coronaviruses, type adenoviruses and rabies. for ethical reasons, veterinarians were not able to keep an untreated and exposed control dog population. for the purpose of comparison, asymptomatic fml-seronegative dogs from another endemic area (jardim progresso, natal, rn, brazil), with similar canine incidence [ ] were included in this study as the exposed untreated group. in this investigation, all manipulations performed on the animals were conducted to ensure minimal animal suffering, as recommended by the nih regulation. each leishmune ® prophylactic vaccine dose [ ] was composed of lyophilized fml antigen adjuvanted with saponin, reconstituted in ml nacl . % sterile saline solution at the moment of vaccination and administered subcutaneously. the fml-vaccine, leishmune ® , is patented: inpi number: pi - ( march ) assigned to universidade federal do rio de janeiro, brazil and is the first second-generation vaccine licensed against leishmaniasis, since th june [ ] . this was determined by injecting dogs intradermally, in the inner aspect of the right hind leg, with . ml of l. donovani freezethawed antigen containing g protein in nacl . % sterile saline solution ( stationary phase promastigotes/ml). the left hind leg received only . ml saline. measure of the increase of intradermal reaction was performed h after antigen injection. indurate areas were marked and each time the values of the saline control were subtracted from the reaction due to the leishmania antigen. reactions showing diameters ≥ mm were considered positive [ , ] . in month after vaccination, pbmc of randomly chosen leishmune ® -vaccinated dogs from araç atuba and andradina were analysed by flow cytometry. three milliliters of blood from the cephalic vein was collected from each dog in heparin-tubes, transported at room temperature and processed h after collection [ ] . for the ex vivo analysis, l of blood was incubated for min at room temperature, with l of each one of the following monoclonal antibodies diluted in facs dil solution ( % fcs-supplemented pbs buffer): anti-thy- (rat-igg bclone ykix . ) ( : ), anti-cd (rat-igg a-clone ykix . ) ( : ), anti-cd (rat-igg a-clone ykix . ) ( : ), and anti-cd (rat-igg -clone ycate . ) ( : ). facs dil solution was used as negative control. after this period, ml of pbs-w (pbs buffer with . % bovine serum albumin and . % sodium azide) were added to each tube and the mixture was homogenised, and centrifuged at rpm, at room temperature, for min. the supernatants were aspirated and pellets homogenised and added to l of anti-rat fitc conjugate ( : ) (serotec, uk) except for the facs dil cell control. at this time, l of the fitc-labelled mouse anti-human-cd (mouse-igg -clone iob a) monoclonal antibody (immunotech co., marseille, france) was used in a direct immunofluorescence procedure. all suspensions were homogenised, incubated for min at room temperature in the dark and treated with ml of the / diluted lysis solution during vortex homogenisation (becton & dickinson, usa). the mixtures were further incubated for min at room temperature in the dark and further centrifuged at rpm for min. supernatants were discarded and the pellet-containing tubes were inverted on to absorbent paper. all these procedures were repeated twice after the addition of ml pbs. the pellets were homogenised carefully and finally fixed with l of . % formaldehyde-pbs. the relative immunofluorescence of cells was counted in a total of , events measured in a becton dickinson facscalibur apparatus and further analysed using windows multiple document interface flow cytometry application (winmdi) version . software [ ] . as control, facs analysis of pbmc of nine normal healthy untreated dogs was also performed. comparison of proportions was carried out using the -test. to test the significance of the differences between groups we used the % confidence interval of the averages. five hundred and fifty dogs previously assayed for leishmune ® safety analysis [ ] were also monitored for the vaccine-induced immunogenicity during a -year trial ( ) ( ) ( ) . another group of untreated exposed dogs were also studied. the differences between the two groups after vaccination were highly significant in all variables (p < . ) ( table ) . both the vaccinated and the untreated groups were seronegative at day , but a strong fmlseroconversion ( %) was detected after complete vaccination on day . by this time, % of the untreated controls developed anti-fml antibodies due to their exposure to natural infection. the vaccine increased, not only the number of dogs with positive table two-year evolution of immunogenicity and incidence of zvl in cohorts of leishmune ® the delayed type of hypersensitivity response (dth) against the leishmanial lysate was positive in % of the vaccinated dogs, in month , and increased along time (table ) disclosing that, as desired for a protective vaccine against cvl, leishmune ® prophylactic vaccine enhances not only the humoral response but also triggers the cellular immune response against the parasite as well. the size of skin test reactions can be used as a measure of potency of a vaccine. the mean ± s.d. diameter of the skin tests at month was . ± . mm (n = ). the skin tests diameters significantly increased until month to . ± . (n = , p < . ), when dth reactions were positive in . % of the vaccines. these results confirm that the natural booster in an endemic area, while sustaining the humoral response of leishmune ® vaccines, contributes to enhance the specific anti-l. chagasi cellular immune response which is known to be responsible for protection against cvl. while both vaccinated and untreated dogs were both healthy at the beginning of the study, the untreated dog group developed a greater number of zvl symptoms along time reaching . % of the cohort by the end of the first year. meanwhile, clinical signs were detected in only . % of the vaccines during the same period (table ) . accordingly, while the cumulative proportions of deaths due to confirmed zvl in the untreated dogs reached % of the cohort, only % of the vaccinated dogs died of zvl during the trial. our results indicate the strong protective prophylactic effect of leishmune ® in seronegative dogs of endemic areas. table summarizes the results of the immunophenotype analysis of pbmc of a randomly selected sample of dogs, collected the great difference in the incidence of zvl disclosed in vaccines and controls (table ) could be partially due to the different infective pressure of the towns where leishmune ® -vaccinated dogs and untreated controls were located. however, when we compared the incidence of zvl in leishmune ® -vaccinated dogs to the incidence of the total dog population of the same town, the vaccine-induced protection was also evident. table shows the official data mean values of canine incidence of zvl during the period - . we show that the zvl incidence in belo horizonte, decreased from . % in the total population [ ] to . % (p < . ) in the leishmune ® -vaccinated dogs, in the first years of the vaccine use. the more striking effect of the prophylactic vaccination was seen in araç atuba, where the incidence of zvl decreased from . % of the total population to . % in vaccines (p < . ), leading to an . % reduction in the zvl incidence and thus confirming the strong protective effect of leishmune ® . the cohort of vaccinated dogs analysed in this investigation was the same previously used for the safety analysis of the leishmune ® vaccine [ ] , which confirmed that the formulation was tolerable and safe. in this investigation, we confirmed the strong immunogenicity of leishmune ® vaccine, previously shown for the fml-saponin vaccine in the field [ , ] indicating that the commercial formulation maintains the characteristics of the laboratory-prepared vaccine. soon after complete vaccination, the percent of seropositivity to fml in dogs treated with the fml-saponin vaccine was % [ ] , while it reached % in leishmune ® vaccines. this indicates the earlier achievement of humoral response induced by the commercial formulation, which is an important indicator of the vaccine potency. the humoral response was also sustained at high levels for months after vaccination, probably due to the natural booster effect of l. chagasiinfected sand flies of the endemic area. conversely, a previous kennel experiment in a non-endemic area, showed in leishmune ®vaccinated unexposed dogs, a decrease in absorbencies from day ( . ± . ) to month after vaccination ( . ± . ) (unpublished results). twelve months after vaccination, a positive dth response to leishmanial antigen was present in % of the fml-saponinvaccinated dogs ( . mm average skin test diameter) [ ] , and in . % of leishmune ® -vaccinated dogs which showed slightly larger diameters of skin tests ( . mm), indicating that beside the antibody response, the induction of the cellular immune response against leishmania lysate was also preserved in the commercial formulation. leishmune ® vaccination induced an increase in cd + total lymphocytes population in blood, also observed after dog immunotherapy with the fml-saponin vaccine [ ] . this was expected for a vaccine containing the qs saponin adjuvant of q. saponaria molina and it was related to its hydrophobic normonoterpene moiety [ , ] but also present in the deacylated saponins of q. saponaria molina [ ] and the deacylated saponins of calliandra pulcherrima [ ] which lack the hydrophobic moieties. evidence of the involvement of cd + lymphocytes in protection against intracellular parasitic infection has increased recently [ ] . cd -defficient mice failed to control leishmania parasite growth [ ] . also, cd specific cells are primed during natural infection or human vaccination and secrete ifn-␥, when re-stimulated in vitro with leishmania antigens [ ] [ ] [ ] . in naturally infected asymptomatic dogs, increased levels of cd + lymphocytes appeared as the major phenotypic feature, as well as in dogs bearing a lower parasite load [ ] , indicating a correlation with natural protection. while no effect was observed after l. infantum lysate vaccination [ ] , an increase in cd + t cells was observed after vaccination with the q. saponaria saponin-containing vaccines: fml-quila ( . %), fml-saponin r ( . %) [ ] , leishmune ® ( . %) [ ] and the l. brasilensis lysate-saponin vaccine ( . %) [ ] . the intense cell proliferation and increased nitric oxide production during in vitro stimulation by l. chagasi soluble antigens, suggests the induction of a potential resistant profile [ ] . the total levels of cd + and cd + lymphocytes are expected to decrease in advanced canine visceral leishmaniasis [ , , , ] with the specific decrease of cd + cells being correlated with dog infectivity to sand flies [ ] . indeed, the average percent value of cd lymphocytes in naturally infected dogs with visceral leishmaniasis was . % [ ] . on the other hand, vaccination against zvl is expected to expand or sustain the cd + t cell levels. the expansion or sustention of cd + t cell levels of dogs treated with l. infantum vaccine immunochemotherapy was % [ ] , with the fml-quila vaccine it was . %, with fml-saponin r . % [ ] and with the leishmune ® vaccine was . % [ ] . sustained cd + t cell were found also in this investigation in dogs vaccinated with leishmune ® , and sustained l. chagasi-specific cd + t cell proportions were found before in dogs treated with leishmune ® after infection [ ] indicating that the commercial formulation maintains the immunogenicity and potency demonstrated by the fml-saponin vaccine in the prophylaxis [ ] and immunotherapy [ ] against zvl. both the leishmune ® prophylactic vaccine, which contains . mg of [ ] and the leishmune ® immunotherapeutic [ ] or the l. brasiliensis [ ] vaccines, that contain mg of the riedel de haen saponin, maintain the normal levels of cd + lymphocytes in vaccinated unexposed [ ] , exposed [ ] or challenged [ ] dogs. higher proportions of cd cells were detected in this investigation in the leishmune ® -vaccinated dogs. the levels of cd + cells were higher in vaccinees, with a mean average that fell outside the ci % interval of the normal dogs. increased cd + b cell levels were also found in dogs vaccinated with the l. brasiliensis-saponin vaccine that uses the same adjuvant included in leishmune ® [ ] and in unexposed dogs vaccinated with leishmune ® [ ] . the increase in total and in leishmania-specific cd + b circulating lymphocytes was synchronous with the induction of an intense humoral response [ ] . also, a positive correlation was found between pbmc proliferation in response to l. chagasi antigen and cd + b cells. this was considered an indication of the major apc function of the cd + cells [ ] . on the other hand, decreased cd + b cell proportions are expected to occur in untreated infected dogs with advanced zvl [ , ] , which also exhibit hypergammaglobulinemia, a hallmark of human and canine visceral leishmaniasis [ ] . these facts suggest that the cd + b lymphocyte population, which increased after vaccination with leishmune ® is involved not merely in the expansion of the total humoral response but in the increase of the specific synthesis of the igg anti-fml antibodies that are related to protection [ ] [ ] [ ] [ ] [ ] and blockage of the transmission of vl in the field [ ] . in this investigation, we have demonstrated the strong immunogenicity of leishmune ® in healthy exposed dogs in epidemic areas, which consequently exhibit a strong and sustained humoral and cellular immune response against the parasite. a classic phase iii trial, with random double-blind selected controls could not be performed because of the ethical restrictions of the veterinarians from these epidemic areas, who refused to include untreated healthy dogs as exposed controls. leishmune ® -induced protection against zvl, is however suggested by the results of . % of asymptomatic dogs (at the end of first year) and % healthy survivors (at the end of the second year) among vaccinated dogs, compared to the . % of asymptomatic and % survivor dogs monitored in an untreated exposed cohort in another endemic area. although a vaccine against zvl is considered an efficient tool for eradication of 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flies is associated with lower proportions of t helper cells despite leishvaccine and leishmune trigger distinct immune profiles, their ability to activate phagocytes and cd + t-cells support their highquality immunogenic potential against canine visceral leishmaniasis immunogenicity of a killed leishmania vaccine with saponin adjuvant in dogs lymphocyte subset abnormalities in canine leishmaniasis the immune response and pbmc subsets in canine visceral leishmaniasis before, and after, chemotherapy evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis visceral leishmaniasis in the german shepherd dog. i. infection, clinical disease, and clinical pathology igg /igg antibody dichotomy in sera of vaccinated or naturally infected dogs with visceral leishmaniasis specific igg and igg antibody responses of dogs to leishmania infantum and other parasites analysis of the humoral response against total and recombinant antigens of leishmania infantum: correlation with disease progression in canine experimental leishmaniasis leishmania infantum-specific igg, igg and igg antibody responses in healthy and ill dogs from endemic areas. evolution in the course of infection and after treatment infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission the most studied first-generation vaccine, composed of total leishmania lysate and bcg, protected against vl in sudan [ ] and against canine visceral leishmaniasis (cvl) in iran [ ] but not in brazil [ ] . lemesre et al. [ ] , using a second-generation vaccine with the culture media of l. infantum containing a -kda excreted protein in formulation with mdp (liesap) obtained protection in beagles in a kennel assay [ ] . regarding the recombinant vaccines, the multicomponent leish- f fusion protein in combination with mpl-se or adjuprime was only immunogenic in dogs challenged with l. chagasi [ ] and l. infantum (mml) [ ] and failed to prevent l. infantum natural infection or the progression of disease in dogs in an open kennel trial [ ] . a few third-generation dna vaccines have been tested in dogs against experimentally induced cvl key: cord- -xcblqg z authors: harmon, shawn h.e.; faour, david e.; macdonald, noni e.; graham, janice e.; steffen, christoph; henaff, louise; shendale, stephanie title: immunization governance: mandatory immunization in global nitag network countries() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xcblqg z international trends currently favour greater use of mandatory immunization. there has been little academic consideration or comparison of the existence and scope of mandatory immunization internationally. in this paper, we examine mandatory immunization in global nitag (national immunization technical advisory group) network (gnn) countries, including countries from every who region and world bank income level classification. we found that although mandatory immunization programs, or mandatory elements within broader immunization programs, are relatively common, jurisdictions vary significantly with respect to the immunizations required, population groups affected, grounds for exemptions, and penalties for non-compliance. we also observed some loose associations with geography and income level. based on these data, we categorized policies into a spectrum ranging from narrow to broad scope. in the assessment report of the global vaccine action plan (gvap), the world health organization's (who) strategic advisory group of experts on immunization (sage) noted a need to understand the variety of ways in which legislation and regulation have been used to advance or undermine the cause of immunization [ ] . given the public health threat posed by low or slumping immunization rates, some countries have implemented, or have considered implementing, mandatory immunization. although there is no globally standardized definition of 'mandatory immunization', it is generally exemplified by requiring certain vaccinations at the individual level to control a vaccinepreventable disease at the population level [ ] . for present purposes, we define it as the governmental imposition of vaccination of an identified group with refusal, if permitted at all, being possible only through a formal 'opt-out' procedure (for example, obtain-ing a medically-indicated exemption due to allergy), independent of whether a legal or economical consequence exists for improper refusal [ , , ] . in this article, we present findings from the national immunization technical advisory group (nitag) environmental scan, a pilot project funded by a small contract from the who's department of immunization, vaccines and biologicals in . it received ethics approval from the research ethics board of the iwk health centre in halifax, nova scotia, canada (ethics approval no. ). the project surveyed global nitag network (gnn) countries on a range of issues relating to their nitags and national immunization programs (nips). we report the binary presence or absence of mandates within nips, and their scope as shaped by the mandated vaccines, applicable population groups, and permitted exemptions. these countries represent a broad range of low, lower-middle, upper-middle, and high-income countries as defined by the world bank, as well as all six who regions. although this kind of landscape analysis has been conducted on a regional level [ , ] , and across high-income settings [ ] , there does not appear to be any recent academic consideration of the content and scope of mandatory immunization on a crossregional international scale. the primary means of data collection in the nitag environmental scan was a secure online survey developed iteratively by the research team, with questions and structure refined through team interactions. the survey, available in both english and french, was piloted for comprehensibility and answerability with the help of five reviewers from three different who regions, all of whom were familiar with nitags. all gnn country members ( as of june ) were invited to participate in the survey by a national representative drawn from the gnn secretariat list; they entered the survey via a password-protected portal. the survey was open from june-september , with three reminders issued by the gnn secretariat. the online survey contained questions in three primary components, as follows: . tick-box questions tabulated quantitatively using simple descriptive statistics; . free-text comments analyzed qualitatively for specific jurisdictional insights and themes; and . requests for legal and/or policy instruments (provided via url or email). in addition to questions around nitag governance and operation, the survey inquired after the existence and content of any mandatory elements of national immunization programs. respondents in countries with mandatory elements were then asked: . what vaccinations were required by law; . what population groups were subject to mandates; and . what grounds, if any, were available for requesting exemptions. wherever possible, answers were corroborated through independent desktop research seeking official (governmental) and peer-reviewed sources. we reviewed government webpages (e.g., ministries of justice, health, public health agencies, and online legislation registries), a range of legal repositories (e.g., vaccine european new integrated collaborative effort [venice], international labor organization's national legislation database [natlex]), un, who global and regional policy webpages, and academic literature accessed through google scholar, westlaw and worldlii. we then supplied results of this research to national experts in the country to verify results and/or seek clarity around certain aspects of the instruments provided. the project received responses from of gnn countries. this represents a response rate of %, a strong majority of the gnn countries worldwide at the time the survey was issued in . of those responding countries, (all respondent countries save nigeria) provided information about mandatory immunization in their country. our sample comprises a broad range of countries in terms of size (geography and population) and government structures (federal and unitary), representation from all six who regions (africa: ; americas: ; eastern mediterranean: ; europe: ; south-east asia: ; western pacific: ), and all world bank income level classifications (low: ; lower-middle: ; upper-middle: ; high: ). as membership in the gnn is voluntary, it is likely that our emphasis on this self-selecting group means that our respondents (and our findings) are indicative of countries for whom immunization is a strong priority. in other words, countries with nitags that have proactively joined the gnn and responded to the survey might have a different profile than those who have not enrolled in the gnn, or who did not respond to the survey invitation. nonetheless, given the scope of our sample, we expect that our findings would likely be repeated across the gnn, and it is not entirely clear what new information could be found by a broader investigation within the gnn absent different and further inquiries. the above highlights several limitations that must be explicitly addressed. first, we acknowledge that, by the end of , there were nitags worldwide meeting gvap process criteria. the sample of gnn-associated nitags ( ) is therefore approximately one-third of all nitags, and our respondents represent % of functional nitags (and % of all countries worldwide). thus, although our data provide an important and interesting snapshot representing all regions, any trends observed in a sample of this size cannot be generalized globally. related to this, the sample does not include any small-country cluster that might share immunization resources and structures (such as the caribbean). inclusion of such clusters could introduce new forms, processes, and observations. a second caveat relates to the limitation on the number of questions we could reasonably pose in the survey and still expect sufficient response rates. this meant that we did not ask respondents about the reasons for the presence or absence of mandatory immunization, as this inquiry would be more amenable to a survey focused entirely on mandates. third, immunization in some countries is governed at the subnational level which means that a single country may exhibit heterogeneity in both their nips and mandates. for example, in canada and the usa, immunization programs are designed and developed, and mandates imposed (or not), at the provincial and state levels respectively. mandatory immunization currently exists in only two canadian provinces (ontario and new brunswick; previously, manitoba also had mandatory immunization). although all us states impose some mandatory immunization, the particular vaccines required and the procedures for enforcement vary widely from state to state [ ] . as such, it is not always entirely appropriate to talk about 'national' programs and mandates, or to draw overly general conclusions (in relation to, for example, significance of geography) from the data. the final limitation is with respect to the fact that vaccination policy is an ever-changing function of government agendas, public health objectives, economic constraints, and practical realities. accordingly, while our data represents (to the best of our knowledge) an accurate picture of mandatory immunization in these countries as of summer , it is likely -particularly in light of the ongoing covid- pandemic -that in at least some jurisdictions, vaccination practice and policy has changed. just over half ( of ) of the responding countries indicated some mandatory element(s) in their nip (table ; note that we were not able to independently verify the existence of mandatory immunization in côte d'ivoire). given the almost pervasive emphasis on autonomy and consent to treatment in the medical setting, this represents a relatively high rate of compulsory treatment. however, these raw data tell only a partial story, for the reality is more complex and nuanced than the numbers convey. in the following sections, we examine more closely the nature and extent of the mandates, taking into account the following factors: before exploring these factors, however, we made several observations from the raw data. for example, country affluence or income level does seem to influence the presence of mandatory immunization (fig. ) . drawing on categories applied by the world bank, high and upper-middle income countries appear more likely to have a mandatory nip element than lower-middle and lowincome countries. for example, seven of the high-income countries ( %), and four of the six upper-middle-income countries ( %) reported having mandatory elements in their nips. conversely, just one of the six low-income countries, uganda, reported a mandatory element. more detailed empirical case studies would be necessary to uncover the policy reasons for the presence or absence of mandates within nips. nonetheless, it may be reasonable to infer that lower-income countries have fewer human and financial resources to undertake, administer, and enforce manda- ywe were unable to verify the legal basis for mandatory immunization in jordan. àat the time of our survey, germany did not have mandatory immunization. however, in march , the measles protection act came into force, mandating measles immunity for certain individuals, including children and health care workers. tory immunization, or that programs are less mature and potentially still evolving. conversely, given the burden of disease in low-income countries and the difficulties that residents thereof sometimes face in accessing healthcare, demand for, and public acceptance of, vaccines may be quite robust, negating the need for mandates. in addition to the economic component, some association was observed between geographic region and the existence of mandates. for two regions -europe and south-east asia -no clear association was observed; mandates existed in exactly half of the respondent countries. the one eastern mediterranean region respondent (jordan) reported mandatory immunization, but no regional conclusion can be drawn from a single response; the similarly small sample of two western pacific countries (china and australia) reporting no mandatory immunization is not helpful. however, mandatory programs appeared to be somewhat less common in the african region, with only two of the six responding countries ( %) reporting a mandatory element. conversely, all five respondent countries from the americas ( %) reported mandatory immunization. this strong response suggests a trend in that region toward mandatory immunization that is not fully explained by income level. although all the respondent countries in the americas were high-or upper-middle income, income status alone is probably not a sufficient explanation because just two of six highincome countries outside the americas reported mandatory immunization. furthermore, countries in the americas appear to have much broader or inclusive mandates. excluding canada and the usa (due to variation across subnational jurisdictions ), respondent countries in the americas required immunization against an average of . diseases, with the next broadest mandates found in south-east asia ( . diseases). respondents from all other regions fell below the total average of . diseases covered (fig. ). all told, there may be something about the approach to immunization, the developmental history of the public health and immunization field, or the politico-legal culture in the americas that makes mandatory immunization more palatable or feasible as a policy option. incentivization of healthcare workers, urbanization/remoteness, public health infrastructure, cold chain issues, and public trust of government or health authorities could also play a role, with potentially profound differences between the americas and other regions. a full explanation for this apparent link would require a more focused empirical case study. for countries reporting the presence of a mandatory element in their nip, we asked which vaccines were required. the responses reveal a great variety of vaccine schedules and a broad spectrum of vaccines that are mandated, with no country mandating all scheduled vaccines (tables and ). for example, belgium mandates just a single childhood vaccination (polio), whereas argentina mandates childhood vaccinations. the average number of mandated vaccines across respondents was . . every mandating country required the polio vaccine, reflecting the historical global burden and long-standing international efforts to eradicate polio [ , ] . measles, bcg, and tdap/dtwp/dtap/td (grouped together in our survey due to their nearly ubiquitous concurrent administration) were the second most commonly mandated vaccines, with every mandating jurisdiction (including vaccine-requiring canadian provinces and all us states) except belgium reporting their inclusion. also of note are the hpv and rotavirus vaccines, which, despite being relatively new, have approximately % of countries reporting their inclusion. in addition to the vaccines listed in table , mandates in some countries included hepatitis a, influenza, japanese encephalitis, typhoid, tick-borne encephalitis, and varicella. of these, the most common mandatory vaccines were hepatitis a and japanese encephalitis, with four and two respondent countries respectively mandating their use. survey participants in countries with mandatory immunization were asked about specific populations subject to mandates (i.e., age [children under and years of age and school-aged children -that yexcludes canada and the usa due to subnational variation in those countries. àthere were no western pacific countries reporting mandatory immunization in our survey. although the mandatory elements in canada and the usa do vary by subnational jurisdiction. the sub-national jurisdictions (provinces and states respectively), which have authority over the implementation of the immunization programs within these districts, have resulted in (at times significant) variation across those countries. however, it should be noted that these subnational jurisdictions appear also to have relatively broad mandates -ontario and new brunswick require immunization against and infectious diseases, respectively, for school entry, with similarthough varying -numbers for us states. . they were also given the opportunity to identify any other population groups subject to mandatory vaccination. finally, they were asked the year in which mandatory immunization was introduced for that specific population. amongst countries with mandatory elements to their nip, children under one year of age and under five years of age were by far the most frequently subject to mandatory vaccination, with of the respondent countries ( %) reporting specific provisions for these groups. mandatory immunization upon school enrolment, and for school-aged children were also highly prevalent. for example, eight of the responding countries ( %) reported requiring immunization for school enrolment and for school-aged children. this likely reflects policy recognition of the importance to individual health of immunization relatively early in life, together with the public health objective of facilitating herd immunity [ ] , particularly in relatively enclosed school environments. with respect to other targeted populations, seven of the respondent countries ( %) reported mandating vaccinations for healthcare workers. respondents from six countries (argentina, belgium, chile, indonesia, maldives, and uganda) indicated that they also require immunization for other specific populations. the most common 'other' category was pregnant women and/or women of childbearing age, although two countries (indonesia and maldives) reported mandating vaccinations (or proof of immunization) for travellers, particularly hajj and umrah pilgrims, and for other, unspecified, 'at-risk populations'. for further specifics on target populations, see table . in rights-conscious societies, many public services will contain some degree of flexibility to account for differences in individual circumstances. as such, an important aspect of any mandatory nip will be the availability and scope of exemptions from the mandate. respondents were therefore asked about the circumstances under which an exemption to any mandatory vaccines will be granted. exemptions can be categorized broadly into medical and non-medical exemptions. medical exemptions are granted to individuals who cannot safely receive a vaccine, usually due to suspected or demonstrated allergic reaction to a vaccine component, or in the presence of immunosuppression. non-medical exemptions include those granted for any other reason; most commonly for religious, philosophical, or other personal objections to immunization [ ] . every respondent country reported allowing exemptions in the event of medical contra-indication (table ) . however, such exemptions are not granted equally across countries. as there is no universally-agreed upon definition of a valid medical exemption for immunization, what constitutes valid grounds in one jurisdiction may not necessarily satisfy another jurisdiction's requirements (even within the same country) [ ] . further, for any given jurisdiction, the presence or absence of non-medical exemptions may also impact how frequently medical exemptions are requested and granted. for example, in california, usa, the proportion of medical exemptions granted more than doubled (from . % to . %) the year after personal belief exemptions were prohibited and the grounds for medical exemptions were broadened [ ] . as shown in table , non-medical exemptions appear far less common in our sample -perhaps unsurprisingly, given that mandatory vaccination is directly undermined by easy access to non-medical exemptions [ ] . in our survey, only canada, indonesia, and the usa reported allowing exemptions for religious, table mandatory childhood immunizations. country kazakh. note canada and the usa are excluded from the table due to significant subnational variability in those countries. key the number ' represents children under one year old. the number ' represents children one year and older, but less than five years old. the letter 's' represents children, of any age, who are attending school. the 'totals' in the bottom row refer to the total number of countries which have mandated for at least one age group the vaccine identified in the column. the 'totals' in the far-right column refer to the total number of vaccines for which each country has erected a mandate. each column counts as one regardless of whether there is one, two, or three checkmarks. the 'other' column signals additional vaccines that are mandated (and reported as 'other vaccines' in the survey). a single checkmark could refer to one or more other vaccines; the actual number is reflected in the figure in the 'totals' row in the far-right column. for details on these additional vaccines, see table . includes dtwp, dtap, td. includes ipv or opv. although most states allow exemptions based on religious or personal belief, five states permit exemptions only for medical contraindication [ ] . to discourage the use of non-medical exemptions, some jurisdictions impose additional requirements before such an exemption may be granted. for example, in ontario, canada, parents must file a sworn statement of conscience or religious belief with a medical officer of health, and must additionally attend an education session on the benefits and risks of immunization prior to obtaining a non-medical exemption [ ] . it is unclear how effective these measures are at reducing the use of non-medical exemptions. the policy objectives advanced by mandating immunization may be seriously undermined if there are no consequences for failure to comply, and no actual enforcement of the mandate. for reasons of survey length, we did not ask direct questions about enforcement dispositions and practices. however, we did pose questions about penalties for non-compliance (table ) . penalties vary considerably between countries. most jurisdictions reported relatively benign, non-compelling, or non-existent sanctions. the approaches taken by these countries largely correspond with the level and level legislative approaches to immunization proposed by the sabin institute [ ] ; that is, mandatory provisions are prescribed either administratively (e.g., as a requirement for school entry) or by law, but such provisions are not supported by serious sanctions, nor by consistent or strong enforcement of those sanctions [ ] . conversely, some respondent countries reported much more severe sanctions. four countries -argentina, belgium, the maldives, and uganda -impose fines for failure to immunize. in uganda, vaccine refusers may additionally face incarceration for up to months [ ]. these countries would likely correspond to sabin institute's level approaches -that is, mandatory provisions are prescribed by law, and sanctions are imposed for failure to comply. again, however, sanctions are distinct from enforcement. regardless of the nature or severity of sanctions for failure to vaccinate, actual enforcement of the mandate is necessary to make the mandate real. our preliminary, albeit incomplete, evidence shows that enforcement varies considerably between jurisdictions, and is not always in compliance with statutory instruction. one respondent, for example, stated that although in their jurisdiction, schools are required to ensure that students are immunized, the penalties for not getting vaccinated are ''not very strict". in other countries, enforcement provisions might be ignored except in the case of an imminent epidemic. our limited data on this issue suggests that significant discretion is exercised in the determination of whether to enforce mandates. this is an important aspect of mandatory immunization that is worthy of further empirical research. this study reveals a spectrum of approaches to mandatory immunization. at one end of the spectrum is a very narrow or largely permissive approach. under this approach, a very small number of vaccines are mandated, or the groups subject to mandatory vaccination is limited, or both. the calculus for setting these very narrow or modest targets in relation to vaccines administered or groups compelled is not clear from the responses given or the instruments examined. this approach could be informed by: national desires to exercise sovereignty in favour of more direct adoption of international policies when otherwise seeking to meet international obligations; national political ambitions to control specific diseases, and to not expend political or economic capital on broader mandates; local disease and cultural conditions that favour a generally permissive or autonomy-privileging approach (i.e., lower social acceptance of vaccination, which would entail expenditure of political capital, or high social acceptance of vaccination, which may negate the need to mandate); or acute or persistent supply-side management issues such as shortages of healthcare workers and stock-outs of vaccines (either nationally or locally). any one or more of these could be in operation in any given jurisdiction, but determining their presence would require detailed investigation. in our data, belgium exemplifies this narrow or largely permissive approach; it mandates just one childhood vaccine -polioand only for newborns. within canada, the province of manitoba previously exemplified this approach; until its restructuring of provincial public health regulations, manitoba required children to demonstrate immunity to measles only (via immunization or natural infection) prior to entry into grade- . [ ] . such a narrow approach may have certain advantages. first, mandating only a few vaccines, instead of many, for a small group of people, instead of a larger group of people, will result in lesser financial (ie, vaccine procurement) and administrative (ie, vaccine delivery and monitoring) burdens on the state. in practice, of course, the financial savings may be modest, especially for countries that publish a relatively inclusive recommended immunization schedule of publicly funded vaccines (as do both belgium and manitoba). in such circumstances, the financial savings may be insignificant, and unlikely to be the driver behind such policies. a more pertinent advantage of this narrow approach may be the avoidance of perceptions of government coercion. mandatory immunization is, by its nature, coercive [ ] ; compelling individuals to receive vaccinations may have unintended negative (political) consequences. many populations, perhaps especially in fragile states, have legitimate concerns relating to trust in their governments and fear of government officials [ ] . attempts by some countries to implement broader mandatory immunization policies have incited some public backlash and increased attention to negative vaccine messages in the media [ ] . mandatory immunization pro- table sanctions for failure to immunize. type of sanction no sanctions reported or found by authors * we were unable to corroborate the sanction for jordan. belgium does also require immunizations for certain other (adult) population groups, but even they tend to be fairly restrictive in nature (for example, requiring tetanus immunization for agricultural workers and animal researchers, as opposed to broader mandates found in other countries such as requiring influenza vaccination for all adults over age or for all pregnant women). currently, all vaccinations in manitoba are voluntary. a true assessment of the financial cost and saving associated with mandates would, of course, go beyond procurement and delivery, and would also have to take into account the costs of subsequent morbidity, hospitalization, and mortality from vaccine preventable diseases. [ ] . ultimately, a narrow or permissive approach to mandates may provide governments with a greater ability to take a balanced and flexible approach to immunization. it may offer an avenue for imposing a strong but otherwise gentle 'nudge' in relation to diseases considered essential to control given local conditions with only minimal encroachment on individual freedoms (thereby maintaining public support). such nudge strategies may include public education campaigns and positive, science-based messaging, and they can be more easily revised for the prevailing situation than can legislated mandates. at the other end of the spectrum is a broad and more inclusive approach to mandates, which requires a relatively large number of vaccinations for a relatively large segment of the population (including specified target groups). as with the narrow approach, this approach was not widely represented in our data. it is perhaps best exemplified by argentina and indonesia, which require and childhood vaccinations respectively. argentina additionally requires vaccines for healthcare workers, women who are pregnant and/or postpartum, and the elderly. indonesia requires vaccines for healthcare workers, women who are pregnant and/or of child-bearing age, some military personnel, and certain travellers. the advantages and disadvantages to a broad or inclusive approach are roughly the inverse of the narrow or permissive approach. essentially, it prioritizes prevention through compelled uptake and coverage, while risking public resistance for government infringements -real or perceived -to bodily or religious integrity. this approach may also be more expensive to administer and deliver, not only because of increased procurement costs, but also because of potentially increased monitoring and enforcement costs. as one might expect, most respondent countries fall between the two ends of the spectrum, though they lean toward the broader or more inclusive approach. it is not clear from the data the extent to which countries falling between these extremes do so as a matter of conscious policy choice. other shaping factors may be vaccine supply issues, national capacity in relation to program delivery, or constitutional conditions. for example, these rather simple characterizations are insufficient to convey the complexity of the approaches taken by countries like canada and usa, where healthcare is a constitutionally devolved and fragmented policy field. in such countries, decisions about mandatory immunization are made largely at subnational levels. this within-country diversity of approach toward mandates can be profound, and generates challenges. for example, it can create uncertainty and stress when residents move from one jurisdiction to another with different immunization schedules and mandatory elements [ ] . to counteract this uncertainty, a robust national vaccine monitoring system seems advisable. however, to date, no such system exists nationally in either canada or the usa, although most states do possess local monitoring databases [ , ] . in addition to the narrow (or largely permissive) and broad (or largely inclusive) continuum, there is a continuum for enforcement which may map loosely but imperfectly on the above continuum. in this regard, approaches on one end can be described as loose (and again permissive), and on the other end as tight (or coercive). the former, exemplified by belgium and certain canadian provinces/territories, applies no substantial consequences for failure to vaccinate. in jurisdictions where few or no vaccines are mandated, and few or no target groups are identified for specific treatment, the loose approach would be expected. in jurisdictions falling closer to the broad/inclusive approach to mandates, tighter or more coercive controls may be expected. in our sample, only uganda legislated the possibility of imprisonment, whereas argentina, belgium, the maldives, and uganda all imposed fines. seven countries (table ) stipulated the possibility of denial of school entry for children. although the latter might be characterized as a positive coercive approach (i.e., forming a gateway to public services, with possible work-arounds), the former are negative coercive measures. a further factor in identifying the character of any given jurisdiction is to assess the actual application or enforcement of sanctions, which was beyond the scope of the project. this project generated data on the status and nature of mandatory vaccination in gnn countries, providing the following insights: obvious diversity occurs in the number of childhood vaccines mandated, ranging from one (belgium) to (argentina), with the average being . per country. every mandating jurisdiction within our survey required vaccination against polio. measles, bcg, and tdap/dtwp/dtap/td immunizations were the second most commonly mandated. children were by far the most common population group subject to mandatory immunization; healthcare workers were second, mandated in over half of our respondent jurisdictions (nine of jurisdictions = %). exemptions from mandates for medical purposes were universal. non-medical exemptions were far rarer (three of jurisdictions = %). sanctions for failure to immunize vary broadly, ranging from no penalty, to loss of access to social services (most particularly admission to school), monetary fines, and incarceration. further, there appears to be some variance between countries as to how strictly immunization mandates are enforced. our findings show the existence of a variety of general approaches to mandatory immunization. this heterogeneity in approaches to nips and the treatment of mandates within them speaks volumes. although differences in disease burdens and healthcare incentives are almost certainly key factors, the differences that exist between countries with respect to legal and healthcare cultures should not be ignored, and these social differences might be more significant shapers of policy choices than actual disease risks and burdens. furthermore, although our data suggest that mandatory immunization plays a significant role in these countries, it is not clear whether the trends noticed here (e.g., wide variety, with the majority of systems occupying the broad or inclusive end of the approaches spectrum) would be reflected worldwide. more detailed and coordinated regional case studies of the situation are warranted to offer a global picture. such investigations could illuminate the extent to which mandatory immunization actually increases immunization rates, an association very difficult to establish [ ] . without robust evidence to support mandates, or differences in policies within mandatory immunization schemes, policymakers are forced to act solely on social, political, or financial grounds, which undermines the notion of evidence-informed policymaking. having said that, a very recent study [ ] did find slightly higher but statistically significant immunization rates for measles and pertussis vaccines in countries with mandatory immunization (i.e., . % and . % higher, respectively). however, the authors acknowledge the inherent difficulty in accounting for the numerous factors that influence coverage rates. therefore, these results must be interpreted cautiously. furthermore, the data and analysis were restricted to european countries (only seven of which mandated vaccination). research from an international perspective in this area is scarce, likely due to the vast heterogeneity in approaches to mandatory immunization worldwide, which poses challenges for generalizability, and by the absence of funding to undertake a suitably robust multi-jurisdictional project. we have highlighted some of the key factors that are relevant to shaping and operating a mandatory nip. these factors are likely to: expand or contract the nip by including or excluding vaccines, target groups, and exemptions; influence the operation and efficiency of the nip by allowing or excluding exemptions, and by managing them in certain ways; and bear on the actual immunization rates achieved by the nip by implementing sanctions (or not), and by enforcing the nip's mandates (or not). countries with mandatory nips -or those considering imposing mandatory elements within their nip -should consciously, cautiously, and collaboratively consider which approach best reflects the political, legal, and healthcare values of their country, as well as their collective public health objectives and risks. ''all authors attest they meet the icmje criteria for authorship." the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. armenia: armenian national advisory committee on immunization (naci) belgium: veerle mertens, belgian superior health council. canada: national advisory committee on immunization (naci) togo: groupe technique consultative pour la vaccination-togo (gtcv-togo). uganda: celia nalwadda, uganda national academy of sciences. uruguay: comisión asesora de vacunaciones. sri lanka: deepa gamage, epidemiology unit, ministry of health strategic advisory group of experts on immunization state vaccination policies: requirements and exemptions for entering school scientific declaration on polio eradication polio working group of the phac committee to advise on tropical medicine and travel. statement on poliovirus and the international traveller medical versus nonmedical immunization exemptions for child care and school attendance medical exemptions to school immunization requirements in the united states -association of state policies with medical exemption rates letters: change in medical exemptions from immunization in california after elimination of personal belief exemptions mandatory infant & childhood immunization: rationales, issues and knowledge gaps national conference of state legislators. states with religious and philosophical exemptions from school immunization requirements immunization of school pupils act. canada legislative landscape review: legislative approaches to immunization across the european region strengthening legal frameworks for vaccination: the experiences of armenia, georgia, and moldova. vaccine public health act, diseases and dead bodies regulation controversies in vaccine mandates. curr probl pediatr adolesc health care world health organization. global vaccine safety blueprint . (draft ) a harmonized immunization schedule for canada: a call to action. paediatr child health (oxford) immunisation registers in canada: progress made, current situation, and challenges for the future progress in childhood vaccination data in immunization information systems -united states mandatory vaccination in europe e the authors wish to thank the who department of immunization, vaccines, and biologicals for the funding of this research, as well as for providing translations into english for the texts of a number of very technical legal documents. they also wish to thank the gnn member countries who participated in the survey, and the specific respondents who answered our queries. key: cord- -m nij v authors: ng, oi-wing; chia, adeline; tan, anthony t.; jadi, ramesh s.; leong, hoe nam; bertoletti, antonio; tan, yee-joo title: memory t cell responses targeting the sars coronavirus persist up to years post-infection date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: m nij v severe acute respiratory syndrome (sars) is a highly contagious infectious disease which first emerged in late , caused by a then novel human coronavirus, sars coronavirus (sars-cov). the virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. the re-emergence of sars-cov remains a valid concern due to the continual persistence of zoonotic sars-covs and sars-like covs (sl-covs) in bat reservoirs. in this study, the screening for the presence of sars-specific t cells in a cohort of three sars-recovered individuals at and years post-infection was carried out, and all memory t cell responses detected target the sars-cov structural proteins. two cd (+) t cell responses targeting the sars-cov membrane (m) and nucleocapsid (n) proteins were characterized by determining their hla restriction and minimal t cell epitope regions. furthermore, these responses were found to persist up to years post-infection. an absence of cross-reactivity of these cd (+) t cell responses against the newly-emerged middle east respiratory syndrome coronavirus (mers-cov) was also demonstrated. the knowledge of the persistence of sars-specific celullar immunity targeting the viral structural proteins in sars-recovered individuals is important in the design and development of sars vaccines, which are currently unavailable. severe acute respiratory syndrome (sars) first emerged years ago as a highly contagious infectious disease, caused by a then novel human coronavirus, termed sars coronavirus (sars-cov) [ ] . the virus spread to countries in a short period of three months, affecting a total of people globally including deaths, a fatality rate of % [ ] . sars-cov is believed to have originated from bats [ ] [ ] [ ] and transmitted to human through intermediate animals such as civet cats [ ] . although no sars cases have been reported since , the re-emergence of sars-cov is of public health concern due to the continual persistence of sars-covs ) and accessory proteins ( a, b, , a, b, a, b, ) proteins [ ] . animal studies have indicated the importance of t cells in the clearance of sars-cov during primary infection and protection from disease [ ] [ ] [ ] . in humans, decreased t cell numbers (lymphopenia) correlated with severe disease, indicating the critical role of t cell-mediated immune response in disease development [ , ] . while sars-specific antibody level in sars-recovered individuals is undetectable at years post-infection, sars-specific memory t cells persisted up to years following recovery [ ] . the long-term persistence of memory t cell immunity could be important in protection against sars-cov re-infection. in this study, the presence of sars-specific t cells was screened in three sars-recovered individuals at and years post-infection. characterization of cd + t cell responses against the structural m and n proteins was carried out, including the determination of hla restriction and the minimal t cell epitope. in addition, crossreactivity of sars-specific cd + t cells against the middle east respiratory syndrome coronavirus (mers-cov) was investigated. a total of peptides were purchased from chiron mimotopes (victoria, australia) at purity above % and their compositions were confirmed by mass spectrometry. the peptides are -mers overlapping by residues spanning the proteome of sars-cov structural (s, e, m, n) and accessory ( a, b, , a, b, a, b, ) proteins. peptides were received in lyophilized forms and diluted at mg/ml in dimethyl sulfoxide (dmso) and then further diluted in rpmi medium (gibco ® ) at working concentrations of mg/ml to mg/ml. three sars-recovered individuals were enrolled from the singapore general hospital, singapore. all participants were diagnosed with sars during the period of according to world health organization's definition of sars [ ] . blood samples were obtained at either or years post-infection. this study was approved by the singhealth centralized institutional review board (singapore). peripheral blood mononuclear cells (pbmcs) were isolated from fresh heparinized blood by density gradient centrifugation using ficoll-paque tm (ge life sciences) and resuspended in aim-v medium (invitrogen) with % pooled human ab serum (aim-v + %ab). cells were either frozen down or used directly for in vitro expansion in the presence of sars peptides, as previously described [ ] . anti-human ifn␥ enzyme-linked immunospot (elispot) assays were performed as previously described [ ] , using the sars peptides arranged in numeric and alphabetic matrix pools (supplementary table ). the positive threshold was set at number of spot-forming units (sfu) per × cells at least twice of that observed in negative control (cells not stimulated with peptides). the peptide responsible for positive elispot results was identified as the common peptide present in both the numeric and alphabetic pools. in vitro expanded pbmcs were incubated in aim-v + %ab medium alone or with peptides at g/ml for h in the presence of g/ml of brefeldin a. anti-cd a-fitc antibody (bd pharmingen) was added for assessing cd + t cell degranulation. positive control consisted of t cells incubated in aim-v + %ab with ng/ml phorbol -myristate -acetate (pma) and ng/ml ionomycin. following stimulation, cells were washed in hank's balanced salt solution (hbss [gibco ® ]) and stained with anti-cd -phycoerythrin(pe)-cy and anti-cd -peridinin chlorophyll protein(percp)-cy . (bd pharmingen) at • c. cells were washed in × phosphate buffered saline (pbs) containing % bsa and . % azide, fixed and permeabilized using cytofix/cytoperm fixation/permeabilization reagent (bd biosciences) according to manufacturer's protocol. intracellular staining using anti-ifn␥-pe (bd pharmingen) was carried out at • c, followed by washing and flow cytometry analysis. hla class i phenotypes of the sars-recovered subjects was determined by pcr amplification and sequencing-based typing method as previously described [ ] and as performed by bgi clinical laboratories (shenzhen, china). epstein-barr virus-transformed lymphoblastoid b cell lines (ebv-lcls) possessing matching hla phenotypes as the subjects were used as antigen-presenting cells (apcs) to determine the hla restriction of cd + t cell responses. restimulation of sars-specific cd + t cells was done using fresh pbmcs from a healthy donor and ebv-lcl consisting of the hla allele restricting the cd + t cell response. specific peptide was added to ebv-lcl at g/ml in r medium and incubated at • c for h, followed by washes with hbss. pbmcs and the peptidepulsed ebv-lcl were irradiated at rads and rads respectively, washed with hbss and added to in vitro expanded t cells in aim-v + %ab supplemented with il- ( u/ml), il- ( ng/ml) and il- ( ng/ml) and co-cultured at • c for days. for mapping of minimal t cell epitope, restimulated shortterm t cell lines were tested with truncated peptides of the -mer peptide by ics. for m minimal epitope mapping, peptides ( - -mers) spanning the m region were tested. for n minimal epitope mapping, peptides ( - -mers) spanning the overlapping region of n and n were used. as it is currently unknown if sars-specific memory t cell responses persist in sars-recovered individuals after years postinfection, pbmcs from a sars convalescent subject (sars subject ) were collected at years post-infection and tested for sars-specific memory t cells. as negative control, pbmcs of a healthy individual with no sars history were also obtained and tested. after in vitro expansion with the mixture of sars -mer peptides of overlapping residues spanning the structural (s, e, m, n) and accessory ( a, b, , a, b, a, b, b) proteins, the pbmcs were subjected to ifn␥ elispot assay using sars peptide pools arranged in alphabetic and numeric matices (supplementary table ). analysis of elispot results was performed with the positive threshold set as the number of spot-forming units (sfu) two times above the mean sfu of unstimulated cells. as shown in fig. , higher frequencies of ifn␥producing sfus were observed for in vitro-expanded pbmcs from sars subject compared to the healthy individual, suggesting the presence of sars-specific memory t cells at years post-infection. these responses were low in frequency since in vitro expansion of pbmcs was required for their detection. this is in agreement with previous reports that reported the decline of memory t cell responses in sars convalescent individuals over time [ , ] . peptides inducing ifn␥ production as identified from elispot were further tested by ics to confirm their abilities to elicit specific t cell ifn␥ response and to define the subset of t cells (cd + or cd + ) involved. a total of sars-specific memory t cell responses were in vitro using a mixture of sars-cov peptides, followed by ifn␥ elispot assay using sars peptide matrix pools of the structural (top panels) and accessory proteins (lower panels). each bar represents the ifn␥-producing response to an individual peptide matrix pool (numeric or alphabetic) in sfu per × cells. the threshold for a positive response was set as two times above the mean sfu of unstimulated cells (neg), as indicated by the dotted line in the right panels. cells stimulated with pma/ionomycin were included as positive control (pos). identified in sars subject and they are specific for structural s, n and m proteins (table ) . three are cd + t cell responses, of which two recognized the s protein (s and s ) and one recognized the n protein (n ). in addition, cd + memory t cell response specific for the sars-cov m protein (m ) was detected. subsequently, pbmcs were obtained from two other sars-recovered individuals (sars subjects and ) at and years post-infection respectively and screened for sars-specific memory t cells using the same method. memory t cell responses specific against sars-cov structural proteins were also found ( table ) . as with that observed in sars subject , n cd + response and m cd + response were found in sars subjects and , respectively. sars subject also possessed a cd + t cell response targeting s . as summarized in table , subject had more sars-specific memory t cells at higher frequencies compared to the other two subjects. it was noted that subject had more severe disease presentation (supplementary table ), which could be related to the more robust t cell responses detected. however, the number of subjects recruited in this study is too small to draw a conclusion to this correlation. the knowledge that sars-cov structural proteins are highly immunogenic in eliciting protective and immunodominant t cell responses is well-established [ ] [ ] [ ] . the cd + t cell epitopes identified here, which are specific against s (s protein residues - ), s (s protein residues - ), s (s protein residues - ) and n (n protein residues - ), have been previously reported from a cohort of sars-recovered patients at year post-infection, suggesting the immunoprevalence and dominance of these responses in convalescent sars patients [ ] . here, the identification of t cell responses against sars-cov structural s, n and m proteins at and years post-infection suggests the long-term persistence of these responses. the cd + t cell response present in sars subject and , which is specific for sars peptide m corresponding to residues - of the structural m protein, was further characterized. using t cells from subject , the m cd + t cell response was determined to be restricted by the hla-b* allele. as revealed by ics, m restimulated cd + t cells exhibited cd + ifn␥ + response at . % when stimulated with m peptide (fig. , left panels) . additionally, cd a expression of t cells induced by m peptide was determined to be . % (fig. , right panels) . the increase in cd a expression, a marker for t cell degranulation and target cell-killing function via the perforin-granzyme pathway [ ] , indicates that the memory t cells were capable of degranulation and likely to exhibit target cell-killing function upon activation by m peptide. table summary of t cell responses in sars-recovered subjects at or years post-infection, identified from screening by elispot and confirmation by ics. percentages of t cell responses represent that of cd + or cd + t cells over total t cell population after in vitro expansion in the presence of sars peptide mixtures. hla class i molecules preferentially bind and present peptides of - amino acids to be recognized by hla receptors on cd + t cells during t cell activation [ ] . since the m peptide is a mer peptide, the identification of the position and minimal number of amino acids within the m region, known as the minimal epitope, capable in eliciting the m cd + t cell response was carried out. to do so, truncated peptides within the m region ranging from -to -mers were tested for their abilities to induce ifn␥ secretion by m -restimulated t cells. as shown in table , the -mer peptide, m - , corresponding to residues - of m protein, was most efficient in inducing the cd + t cell response, resulting in the highest percentage of ifn␥-producing cells of . %. this -mer also represents the minimal epitope of m cd + t cell response, as the removal of either the n-terminus histidine (h) residue (m - ) or the c-terminus leucine (l) residue (m - ) completely abolished ifn␥ production ( table ) . in a study involving sars convalescent patients at year post-infection, cd + t cell response against residues - of the m protein was present in % of study subjects, but the minimal epitope and the hla-restriction of this response were not determined [ ] . the m minimal epitope (residues - ) identified in present study lies within this reported region. other t cell epitopes, both cd + and cd + , within the sars-cov m protein have also been reported [ , ] . in another study looking at sarsspecific memory t cell responses in sars-recovered individuals at years post-infection, . % of them presented t cell responses to m peptides [ ] , further supporting the role of m protein in eliciting dominant cellular immunity during sars-cov infection. the sars-cov n protein is capable in inducing immunodominant t cell responses in sars-recovered individuals and these responses were shown to be involved in disease protection in animal models [ , ] . in our previous study performed at years post-sars, several sars-specific t cell epitopes within the n protein were reported [ ] . in sars subject at years post-infection, a hla-b* -restricted memory cd + t cell response targeting the n peptide, corresponding to residues - of n protein, was detected. to determine the minimal epitope of the n cd + t cell response, truncated peptides were tested for induction of cd + t cell response using pbmcs from sars subject previously collected at years post-infection. truncated peptides consisted of -to -mers within the overlapping residues between n and n peptides, as the n peptide is also capable of inducing table summary of percentage cd + ifn␥ + responses in sars subject induced by truncated peptides within m region. t cells used were obtained from sars subject at years post-infection. results of positive peptides (m , m - , m - , m - , m - ) and selected negative peptides (m - , m - , m - ) are shown. percentage cd + ifn␥ + t cells shown represents the percentage of ifn␥-producing cd + t cells in the total t cell population (after gating the cd + cells) present in the short-term t cell line obtained by restimulation using m peptide. the minimal epitope is indicated in italics. peptide table summary of percentage cd + ifn␥ + responses in sars subject induced by truncated peptides within n region. t cells used were obtained from sars subject at years post-infection. percentage of cd + ifn␥ + cells shown represent the percentage of ifn␥-producing cd + cells in the total t cell population (after gating the cd + cells) present in the short-term t cell line obtained from restimulation using n peptide. the minimal epitope is indicated in italics. the response (data not shown). it was found that -mer peptide, n - , corresponding to residues - of the n protein, was most efficient in inducing n t cell response of . % ( table ) . deletion of n-terminal threonine (t) residue and c-terminal phenylalanine (f) residue from n - led to decrease in percentages of ifn␥-producing cd + t cells to . % and . %, respectively. this indicates that residues - are the minimal epitope of the n cd + t cell response. in previous study using bioinformatics netmhcpan algorithm, the predicted minimal epitope for the n response was determined to be amino acids at position - [ ] , which is within the -mer region identified in current study. thus far, no other studies have reported the identification of the n cd + t cell epitope. having characterized two cd + t cell responses at and years post-infection, the same donor was recalled at years postinfection to determine the persistence of these responses. pbmcs collected from the same individual were expanded in vitro using m and n minimal peptides (m - and n - ) and tested for cd + ifn␥ + responses. as shown in fig. (left panel), when cells were stimulated with m - and n - peptides, cd + ifn␥ + t cell responses of . % and . % were observed respectively, suggesting the persistence of these sars-specific memory t cells up to year after infection. cd a expression at . % and . % were also observed when cells were stimulated with m - and n - peptides, respectively (fig. , right panels), indicating degranulation of t cells upon peptide stimulation. however, cd + t cell responses detected in sars subject at years post-infection (table ) were undetectable at years post-infection (data not shown). a novel human coronavirus, mers-cov, first emerged in [ , ] . like sars-cov, mers-cov is a betacoronavirus which causes serious and sometimes fatal lower respiratory tract infections and extrapulmonary manifestations [ , ] . contrary to sars-cov which is a lineage b betacoronavirus, mers-cov belongs to lineage c [ ] . to investigate if sars-specific m and n cd + t cells can cross-react with m and n peptides of mers-cov, sequence alignments were done to identify corresponding m and n minimal epitopes of mers-cov ( fig. a and b) . when m and n -restimulated t cells were stimulated with mers-cov m minimal epitope peptide (hlkmagmhf) and n minimal epitope peptide (tksfnmvqaf), no cd + ifn␥ + responses were observed (fig. c) , indicating the inability of these sars-specific t cells to be activated by mers-cov peptides. therefore, t cell immunity against sars-cov is highly specific and m and n cd + t cell responses are unlikely to provide cross-protection against mers-cov infection. this is expected as mers-cov is distantly related to sars-cov and is more closely related to other bat coronaviruses [ ] . nonetheless, sequence alignments revealed that the m and n minimal epitopes are fully conserved between human and zoonotic strains (fig. a and b) , including civet sars-cov sz , bat sl-covs rp and rf , and the bat sars-cov rs which is capable of utilizing both human and bat ace receptors for cell entry [ ] . hence, it is likely that the sars-specific m and n cd + t cells can confer cross-protection against infections of these zoonotic sars-cov and sl-cov strains. there are currently no reports on the persistence of memory t cells in sars-recovered individuals beyond years post-infection, therefore, the longevity of sars-cov cellular immunity is unclear. in this study, it was demonstrated that sars-specific memory t cells persist in three sars-recovered individuals at and years post-sars in the absence of antigen. all memory t cells detected were specific against sars-cov structural s, n and m proteins. two immunodominant cd + t cell responses specific against m (m ) and n (n ) proteins were further characterized by defining the minimal epitope and hla restriction. these cd + t cell responses continued to persist in a sars-recovered subject up to years post-infection. the persistence of t cell responses suggests that sars-recovered patients could be protected from re-infection. peptides of the replicase protein, which comprises / of the sars-cov proteome, were not included in memory t cell screening in the current study due to limited amount of sars subject pbmcs obtained. however, based on current literature, the sars-cov replicase protein is less immunogenic compared to structural proteins [ ] . it was also noted that the availability of three convalescent individuals is a significant constraint of this study. in future studies, the conclusions drawn here could be substantiated by including more sars-recovered subjects. in a phase i clinical trial involving a sars dna vaccine encoding the s protein, sars-specific t cell responses were observed in vaccinated individuals, suggesting that the s protein is sufficiently to induce t cell responses [ ] . in line with this, results in current study showed the long-term persistence of t cell responses targeting the s protein, as well as other structural proteins including m and n proteins. this provides evidence for 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disease severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial this work was supported by an a*star bmrc grant ( / / / / ) awarded to y.-j. tan. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . . key: cord- -j f eso authors: liao, qiuyan; ng, tiffany w.y.; cowling, benjamin j. title: what influenza vaccination programmes are preferred by healthcare personnel? a discrete choice experiment date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: j f eso objectives: this study examined the relative importance of factors relating to vaccine characteristics, social normative influence and convenience in access to vaccine for determining decision making for seasonal influenza vaccination (siv) among healthcare personnel (hcp), aiming to optimize existing influenza vaccination programmes for hcp. methods: a discrete choice experiment (dce) was conducted in hcp working in public hospitals in hong kong. the dce was designed to examine the relative importance of vaccine characteristics (vaccine efficacy and safety), social normative influence reflected by the proportion of hcp colleagues intending to take siv, and convenience in access to vaccine indicated by vaccination programme duration, vaccination location, vaccination arrangement procedure and service hours in determining influenza vaccination choice among hcp. mixed logit regression modelling was conducted to examine the preference weight (β) of factors included in the dce for determining vaccination choice. results: vaccination probability increased with increase in vaccine efficacy (β = . for per % increase), vaccination location changing from “designated staff clinic” to “mobile station” (β = . ), vaccination arrangement procedure changing from “by appointment” to “by walk-in” (β = . ), but decreased with the increase in probability of mild reactions to vaccination (β = − . for per % increase). conclusion: vaccine safety was judged to be more important than vaccine efficacy for determining vaccination choice. arranging vaccination service by walk-in and implementing mobile vaccination station should be considered in future siv programmes to compensate for the effect of perceived low vaccination efficacy and concerns about vaccine safety to promote siv uptake among hcp. healthcare personnel (hcp) have a significantly greater risk of seasonal influenza infection compared with general adults working in non-healthcare settings [ ] . work absenteeism among hcp increased significantly during influenza season compared with non-epidemic periods [ ] , leading to a substantial economic loss [ ] and potential staff shortage for healthcare during influenza seasons [ , ] . continued working with influenza-like illnesses among hcp was common, which can increase the spread of influenza to vulnerable patients [ , ] . there were also reports that influenza virus infections in hcp were associated with nosocomial outbreaks [ , ] . seasonal influenza vaccination (siv) can significantly reduce the risk of seasonal influenza virus infection among hcp, reduce work absenteeism and also confer the protection onward to patients and their families [ , , ] . the world health organization recommends that hcp should be vaccinated against influenza each year and free siv has been provided to hcp in many locations [ , ] . however, despite great efforts on promoting siv among hcp, uptake rates remain unsatisfactory in the us [ ] , and widespread low in most european countries [ , ] , australia [ ] and also in hong kong [ , ] . psychological determinants of siv uptake among hcp, including perceived effectiveness of siv for preventing influenza virus infection, concerns about vaccine safety, and perceived personal risk of influenza virus infection have been consistently identified by observational studies [ ] [ ] [ ] [ ] . however, interventional studies focusing on addressing these psychological factors through active educational campaign to promote positive attitudes towards siv indicated mainly small effect sizes for promoting siv uptake among hcp [ ] [ ] [ ] . a systematic review suggested that interventions that additionally combined components addressing the contextual factors relating to vaccination could be more effective in promoting siv uptake among hcp [ ] . these context factors included normative influences on vaccination uptake (e.g., attitudes and siv uptake among colleagues and employers) and convenience of access to influenza vaccine (e.g., time, location and procedure of access to vaccine). however, it is not clear which components are more important to determine resource allocation and optimize an influenza vaccination programme for promoting siv uptake in hcp. discrete choice experiment (dce) is a commonly used methodology for optimizing medical interventions [ ] . in respect of a vaccination programme, dce decomposes a vaccination programme into several important attributes such as vaccine efficacy and vaccine safety which can be further characterized by attribute levels (e.g., a vaccine efficacy of % or %). the attributes and attribute levels are then used to construct a series of choice sets each comprising - alternative hypothetical vaccination programmes and participants are asked to choose a preferred programme within each choice set. through analyzing participants' trade-off between attributes and attribute levels in a series of choice sets, dce enable examination of the relative importance of the selected attributes and attribute levels for determining vaccination preference. one recent study suggests that dce is a valid method for predicting real-world influenza vaccination decision [ ] . several studies have used dce to examine factors that determine preference for influenza vaccination [ ] [ ] [ ] [ ] . however, none were conducted among hcp. in addition, all of these studies mainly chose attributes related to the characteristics of influenza vaccines [ ] [ ] [ ] [ ] and only two additionally included attributes of social normative influence (e.g., doctors' recommendation and others' opinions) [ , ] in the dce. the contextual factors related to convenience in access to influenza vaccine were generally overlooked. this study used dce to examine the relative importance of attributes related to vaccine characteristics, social normative influence and convenience in access to influenza vaccine for determining hcp's preference for siv. the final aim was to identify the optimal siv programme for promoting siv uptake among hcp. development of the dce questionnaire involves choosing attributes, defining attribute levels and constructing choice tasks. the attributes included in a dce was limited to those most important for hcp in their decision for vaccination against influenza to improve accuracy and reliability in the elicitation of preference. before conducting this dce, we conducted a questionnaire-based longitudinal survey to identify important determinants of siv uptake among hcp in hong kong [ ] . in that study, attitudes towards siv was the strongest factor that influenced uptake of siv among hcp, which were mainly assessed by measuring participants' perceived effectiveness and safety of siv. therefore, vaccine efficacy and probability of vaccine reactions were included as attributes in the dce. social normative influence (other people particularly colleagues' opinions and vaccination uptake) was also suggested to be important determinants of siv uptake in hcp [ ] . therefore, a third attribute, the proportion of colleagues intending to take siv was included in the dce. this indicates how much acceptable siv is in the community of the target population which is an important attribute for influenza vaccination decision [ ] . furthermore, contextual factors including time, vaccination location (e.g., on designated or mobile sites) and procedure (vaccination required by appointment or walk-in) that affect convenience in access to siv were reported to be important reasons for refusing siv among the participants in the survey (data not reported). since the contextual factors are important for characterizing a vaccination programme, they were also included in our dce to provide important information for how to optimize the vaccination programme for hcp. after choosing attributes, the levels of each attribute were defined based on the principle that the attribute levels should be realistic for real-life situation and meaningful for policy making. the final attributes and attribute levels chosen for this dce were: vaccine efficacy covering four levels ( %, %, % and %) with the lowest level represents a poorly-matched vaccine strain and the highest represent a well-matched vaccine strain, probability of mild vaccine adverse events (mild flu-like symptoms) comprising four levels ( %, %, % and %), program duration comprising two levels (level : the ordinal fall immunization season and the first month of influenza season in hong kong (october-january); level : extending the programme duration to the end of influenza season (october-march)), vaccination location (level : designated staff clinic; level : mobile station), vaccination arrangement procedure (level : by appointment; level : by walk-in), service hours for vaccination administration covering four levels differed by whether vaccination service was provided during lunch time ( pm- pm) and late afternoon ( pm- pm) (level : am- pm, pm- pm; level : am- pm (also opens at lunch time); level : am- pm, pm- pm; level : am- pm (also opens at lunch time)), and proportion of colleagues intending to take siv (four levels: %, %, % and %) ( table ) . these attributes and attribute levels can generate a total of       = scenarios with each representing a hypothetical influenza vaccination programme. it is not realistic to present all these hypothetical programmes to participants. several alternative approaches are available for choosing a subset of scenarios from the pool such as orthogonal design and d-efficient design [ ] . while recent research suggests that, a d-efficient design is more flexible and can improve the precision of parameter vaccination service hours am- pm; pm- pm am- pm (also opens at lunch time) am- pm; pm- pm am- pm (also opens at lunch time) proportion of colleagues intending to take siv % % % % estimates by minimizing the covariance between parameter values than an orthogonal design, using a d-efficient design requires some prior knowledge of the parameter values and using more complex simulation procedure with specific software (e.g., ngene) [ ] . in comparison, orthogonal design is easier to implement and is suitable when prior knowledge of the parameter values is not available and when researchers are mainly interested in the main effects of each attribute level [ ] . therefore, this study used a fractional factorial designed based on orthogonal arrays [ ] to choose hypothetical programmes from the pool. the chosen hypothetical programmes were then used to construct eight choice tasks, each presented participants with two alternative hypothetical programmes and participants were asked to choose the programme they preferred: ''programme a", ''programme b", or ''neither a nor b" (an opt-out option). one additional choice task which purposively presented a logically superior programme b than programme a was additionally included for rationality test before the eight main choice tasks in the dce questionnaire. the rationality test was aimed to identify participants who were ''irrational" or potentially have difficulties to understand the main choice tasks. the questionnaire was tested for comprehensibility and difficulty before being used in the formal survey. one example choice task is shown in s . in hong kong, the fall influenza vaccination campaign starts in october each year and all hcp are recommended to receive siv before the winter influenza season that typically peaks in january-march annually [ ] . hong kong's healthcare system operates along a dual track that comprises a public sector that dominates the secondary and tertiary care, managing~ % of all hospital admissions, and a private sector that complements the public sector to provide mostly ambulatory primary care services [ ] . this study only recruited hcp who worked in hospitals of the public sector and eligible to receive free siv [ ] . participants who participated in our previous questionnaire survey on determinants of siv uptake in july -april [ ] and gave consent for re-contacting were invited to complete the dce in july-september . subjects for the previous questionnaire survey were recruited using convenience sampling through sending email, telephone calls or poster advertisement at public hospitals of hong kong. hcp who worked in a public hospital, being able to fluently communicate with english, cantonese or mandarin, were eligible to participate in the study. each participant was invited to complete a - min in-person or web-based dce questionnaire whichever they found convenient. all participants gave informed consent before participating in the study. the study received ethical approval from the institutional review board of the university of hong kong/hospital authority hong kong west cluster. participants' elicited preferences were analyzed using a mixed logit regression model to allow heterogeneity as well as correlation between the choices from each participant [ ] . after testing the linear continuous effects of the chosen attributes, vaccine efficacy and risk of vaccine adverse events were treated as continuous variables while other attributes including programme duration, vaccination location, vaccination arrangement procedure, vaccination service hours and proportion of colleagues intending to take siv were treated as categorical variables. for the categorical attributes, a dummy variable was created for each attribute level excepting for the reference level. the preference weight (b) for each attribute and attribute level was calculated by the mixed logit regression model. the statistical significance of a coefficient is set to p-value less than . , indicating that individuals differ between one attribute level and the other in making stated choices. to investigate the willingness of participants to trade off an attribute to achieve an improvement in one level of the other attribute (i.e. the compensatory effects between attributes), we calculated the ratios of the coefficients of more important attributes relative to less important attributes. finally, vaccination choice probabilities (expected mean uptakes) were also calculated to convey dce results into easily understood information to policy makers, which was calculated by taking the exponent of the total utility for vaccination given specific attribute levels divided by the exponent of utility of both vaccination and no vaccination [ ] . we first calculated the expected mean uptake rate for the base case defined as the programme with the least preferred attribute levels of vaccine characteristics ( % vaccine efficacy and % risk of vaccine adverse effect), % of colleagues intending to take siv and contextual factors of existing siv service-a programme duration of six month with vaccine being provided at a designated staff clinic by appointment during ordinal office hours ( am- pm; pm- pm). then we calculated the change in vaccination uptake rate with one level change in a particular attribute compared with the base case while holding levels of other attributes constant. this would inform the relative importance of manipulating different attributes in affecting vaccination choice. all statistical analyses were conducted using the stata . (statacorp llc, - ). a total of hcp completed the dce. demographics of participants who completed the dce are shown in table . overall, of the participants, most were female; the main occupational category was nurse or nursing assistant; and more than one half had an educational attainment of tertiary or above and had frequent contact with patient ( the relative importance weights (preference weight coefficients) of the selected attributes and attribute levels of an influenza vaccination programme for determining participants' preference for influenza vaccination are shown in table . it shows that except for programme duration, all attributes are significantly associated with participants' preference for choosing influenza vaccination. participants' preference for choosing influenza vaccination increased with the increase in vaccine efficacy, vaccination location changing from ''designated staff clinic" to ''mobile station" and vaccination arrangement procedure changing from ''by appointment" to ''by walk-in", but decreased with increase in probability of vaccine adverse events, extending opening hours from '' pm" to '' pm", and knowing that % or more of their colleges intending to take siv. excluding participants who ''failed" in the rationality test (i.e., choosing alternative a) only slightly changed the coefficients but did not change the overall conclusion (s ). based on the estimated preference weights (table ) , the compensatory effects between attributes were calculated for vaccine efficacy, vaccination location, vaccination arrangement procedure and probability of vaccine adverse events. it shows that % increase in probability of vaccine adverse events (reduction in vaccination preference weight = * . = . ) can be compensated by % increase in vaccine efficacy (change in vaccination preference weight = * . = . ) and changing the vaccination arrangement procedure from ''by appointment" to ''by walk-in". changing the vaccination location from ''designated staff clinic" to ''mobile station" can compensate the negative effect of % increase in probability of vaccine adverse events. changes in expected mean vaccination uptake rate with the changes in vaccine efficacy, probability of vaccine adverse events, vaccination location and vaccination arrangement procedure were calculated because these attributes were significantly associated with how to optimize a siv programme for hcp in the model. it shows that in the base case, the expected mean vaccination uptake rate is around . %. expected mean uptake rates can increase to . % if vaccination arrangement procedure changes from ''by appointment" to ''by walk-in", to . % if vaccine efficacy increases from % to %, to . % if probability of vaccine adverse events decreases from % to zero (by changing the effect of vaccine adverse events from À .  = À . to zero on total utility for vaccination), and to . % if mobile vaccination station is implemented (fig. ) . in this dce, we found that vaccination probability increased with the increase in vaccine efficacy and decrease in probability of vaccine adverse events. this finding is consistent with existing studies based on questionnaire surveys [ ] [ ] [ ] [ ] . adding to existing literature, the dce further measured the relative importance of vaccine efficacy to vaccine safety in determining hcp's preference for influenza vaccination. based on their preference weights, it is suggested that the decline in vaccination probability due to a % increase in probability of vaccine adverse events can be compensated by at least % increase in vaccine efficacy. this means that vaccine safety is at least two times more important than vaccine efficacy in determining vaccination probability, suggesting a loss aversion phenomenon in personal vaccination decision [ ] . however, this finding was different to that of a dce survey on dutch public preference for influenza vaccination during hypothetical influenza pandemic which reported vaccine effectiveness was the most important attribute determining vaccination preference followed by vaccine safety [ ] . the inconsistent conclusions between the two studies may be due to the different characteristics between seasonal influenza and pandemic influenza of which the former is usually perceived to be mild and severity is not a significant predictor of vaccination uptake [ ] while acceptability for vaccine risk can become greater during influenza pandemic of greater severity [ ] . for the current coronavirus disease pandemic, acceptability of the vaccine risk, if any, could also depend on the perceived severity of the disease by the time when the vaccine is available in the target population [ ] . among the contextual factors, procedure of vaccination arrangement had the strongest positive effect on vaccination preference. merely changing the procedure of vaccination arrangement from ''by-appointment" to ''by walk-in" can compensate the negative effect of~ % increase in probability of vaccine adverse events on vaccination probability. the procedure that requires individuals to book an appointment for their vaccination (the 'opt-in' procedure) beforehand has been suggested to be a barrier for taking siv [ ] . the ''walk-in" procedure represents an appointment by default without constraining time to receive the vaccination services. this can greatly increase the convenience in access to vaccination services and thereby increase vaccination probability [ , ] . changing the vaccination location from a ''designated staff clinic" to a ''mobile station" was another factor that can significantly increase vaccination probability. previous studies also indicated that providing on-site vaccination was associated with greater siv uptake among hcp [ , ] . however, although others argued that extending vaccination delivering period from the conventional fall immunization season to cover the whole influenza season and extending the office hours of vaccination administration could increase vaccine access and thereby promote siv uptake [ ] , our study found no significant effect of extending the vaccination programme period and a negative effect of extending office hours of vaccination administration on vaccination probability. this indicates that individuals who miss vaccination at the traditional immunization period are unlikely to catch up their vaccination uptake during main influenza season which is deemed to be late for protecting against influenza infection. for timing of vaccination, extending the vaccination service hours to late afternoon ( : pm to : pm) significantly reduced participants' preference for taking influenza vaccination. this reflects that participants may have concerns over receiving influenza vaccination in late afternoon. potential concerns may include perception of insufficient day time to observe the vaccine side effects and being unwilling to put extra burden to their colleague who provided vaccination services but actual concerns await further exploration. notifying a greater percentage of colleagues intending to take siv did not increase vaccination probability but instead, knowing that % or above of their colleagues intending to take siv reduce preference for taking siv. this seems contradictive with existing social norm theories which propose that perceiving greater approval of an intervention in peers can improve acceptance of the intervention [ , ] . however, it is consistent with ''freerider" behaviours in vaccination decision [ , , ] . individuals may avoid vaccination risk when perceiving that the vaccination coverage is sufficiently high to generate herd immunity [ , , ] , a phenomenon that unvaccinated individuals can get indirection protection from others' vaccination uptake. if this explanation is true, participants may have overlooked main risk source of influenza transmission from patients instead of their colleagues. it may also reflect the influence of some psychological roots such as reactance or individualism of anti-vaccination atti-tudes on vaccination decision [ ] . some individuals tend to reject consensus views on vaccination and dislike follow others to make their vaccination decision [ ] . if true, this may be a challenge for implementing the mandatory influenza vaccination policy in hcp [ ] . another possibility is that peers' behaviours may have an implicit [ ] or direct effect on actual vaccination behaviours [ ] in real life rather than on the stated vaccination preference which thereby cannot be assessed in a dce. however, this finding may not be generalized to people's vaccination decision in a severe pandemic when disease severity can become the dominant determinant on vaccination choice but ''free-rider" behaviours remain possible when vaccine safety is perceived to be more uncertain [ ] . an expected mean vaccination uptake rate of % for the base case calculated using the estimated preference weights of the attributes in the dce was approximately the same as the observed vaccination uptake of . % in hcp from our separate questionnaire survey [ ] , representing an indirect validation of the dce findings. increasing vaccination efficacy from % to % or reducing probability of vaccine adverse events from % to zero could increase vaccination uptake rate to over %. however, promoting vaccination efficacy and reducing vaccine side effects can meet technical and contextual barriers such as how accurate the scientists can estimate the main circulating influenza strain in an influenza season and vaccination production technology. instead, our study suggests that simply changing the vaccination delivery procedure from providing by appointment to by walk-in can increase vaccination uptake rate to over %. implementing mobile vaccination stations may further promote vaccination uptake. this study has several limitations. first, our study only recruited hcp in public hospitals in hong kong. therefore, the findings may not be applicable to hcp working in other healthcare settings particularly those working in the private sector for whom free siv may not be completely subsidized. second, participants were those who gave consent to be re-contacted in our previous siv survey and thereby may be those who were more interested in siv. however, post-hoc analysis indicates no significant difference in past-year siv uptake rates between those who participated and those who did not participated in the current dce. third, despite multiple ways had been used to recruit hcp of different occupational categories [ ] , only a small proportion of our participants were doctors. however, our previous survey found that doctors generally had more favourable attitudes towards siv and greater intention to take siv than nurses, nursing assistants and other hcp [ ] . this means that our study findings mainly reveal the relative importance of main determinants on siv choice among hcp who should be the main target population of future influenza vaccination programme. fourth, the proportion of hcp colleagues intending to take siv along may not be a sufficient attribute to reflect the social normative influence on vaccination decision. future dce should consider including opinions from department head or employers for siv. furthermore, although both textual and graphical formats were used to illustrate most attributes and attribute levels, some participants may still have difficult to understand the choice tasks. despite this, excluding the minority of participants who failed in the 'rationality test' did not change the conclusions of our study. vaccine safety was more important than vaccine efficacy for determining vaccination probability in the context of seasonal influenza. changing the vaccination arrangement procedure from by appointment to by walk-in can compensate the negative effect of a % increase in probability of mild reactions to vaccination or a % decline in vaccine efficacy, resulting in a vaccination uptake rate of over % in hcp. implementing mobile vaccination stations could further increase vaccination uptake. this research was funded by the health and medical research fund of the food and health bureau of the hong kong sar government (reference no. ). the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. incidence of influenza in healthy adults and healthcare workers: a systematic review and meta-analysis increases in absenteeism among health care workers in hong kong during influenza epidemics cost of sickness absenteeism during seasonal influenza outbreaks of medium intensity among health care workers medical care capacity for influenza outbreaks disruption of services in an internal medicine unit due to a nosocomial influenza outbreak impact on employee productivity from presenteeism and absenteeism: evidence from a multinational firm in sri lanka 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attitudes: a -nation investigation mandatory influenza vaccination and religious accommodation for healthcare workers: lessons from recent legal challenges. vaccine factors affecting intention to receive and self-reported receipt of pandemic (h n ) vaccine in hong kong: a longitudinal study supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -si g dih authors: he, yu; wang, mingshu; chen, shun; cheng, anchun title: the role of capsid in the flaviviral life cycle and perspectives for vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: si g dih the arthropod-borne flaviviruses cause a series of diseases in humans and pose a significant threat to global public health. in this review, we aimed to summarize the structure of the capsid protein (cp), its relevant multiple functions in the viral life cycle and innovative vaccines targeting cp. the flaviviral cp is the smallest structural protein and forms a homodimer by antiparallel α-helixes. its primary function is to package the genomic rna; however, both steps of assembly and dissociation of nucleocapsid complexes (ncs) have been obscure until now; in fact, flaviviral budding is nc-free, demonstrated by the subviral particles that generally exist in flavivirus infection. in infected cells, cps associate with lipid droplets, which possibly store cps prior to packaging. however, the function of nuclear localization of cps remains unknown. moreover, introducing deletions into cps can be used to rationally design safe and effective live-attenuated vaccines or noninfectious replicon vaccines and single-round infectious particles, the latter two representing promising approaches for innovative flaviviral vaccine development. the emergence or reemergence of flaviviruses, including dengue virus (denv), zika virus (zikv), yellow fever virus (yfv), japanese encephalitis virus (jev), west nile virus (wnv), and tickborne encephalitis virus (tbev), has posed an enormous threat to global public health in recent decades. most of these viruses are arthropod-borne viruses (i.e., arboviruses) that are transmitted to vertebrate hosts through the bites of infected mosquitoes or ticks, causing diseases in animals and humans. among them, denv is the most important human pathogen, with approximately million infections every year [ ] . flaviviruses are a group of enveloped positive-sense rna viruses. the viral shell is formed by copies of glycosylated e and m proteins, which are anchored to a lipid bilayer; within the lipid bilayer, the core of the viral particle is the nucleocapsid complex (nc), which consists of capsid proteins (cps) and viral genomic rna (vrna) [ ] [ ] [ ] [ ] [ ] . the viral genome is approximately , nucleotides in length and contains a single open reading frame encoding a polyprotein of approximately amino acids. after cleavage by the viral ns protease (with cofactor ns b) and host signalase, the polyprotein generates three structural proteins (c, prm, and e) and seven nonstructural proteins (ns , ns a/ b, ns , ns a/ b, and ns ) [ ] . over the past decades, great advances have been made to understand flaviviruses, but the cp-related processes, viral encapsidation and uncoating are still unclear. although vaccines for yfv, jev, and tbev have obtained successes and progress has been made in denv vaccine development, there are still some aspects that are less than satisfactory, and there are still no available vaccines for wnv and zikv. vaccines play a key role in preventing flavivirus infection and controlling viral spread, considering that there is no specific treatment available for the diseases caused by flaviviruses; thus, vaccine development is a consistently important issue in flavivirus research. the mature cp is approximately amino acids in length. although the amino acid sequences of cps are not very conserved among the flavivirus genus (fig. a) , similar structures and properties are shared by all flavivirus cps. approximately one-quarter of the residues are basic and mainly cluster at the n-terminus and cterminus; this property is in accordance with their function in the vrna package. moreover, there is a characteristic region predominately composed of hydrophobic residues in all flavivirus cps, called the ''central hydrophobic domain" (fig. a ) [ ] , which has been proven to play an essential role in viral assembly [ ] . the structures of the denv, wnv (kunjin subtype), zikv and jev cps have been resolved by different methods [ ] [ ] [ ] [ ] [ ] [ ] . all the determined structures reveal that cp is dimeric (fig. b) and that each monomer contains distinct a-helices (termed a -a , they are connected by loops), but its n-terminus (approximately aa) is intrinsically disordered [ , ] . the homodimers are connected mainly by extensive hydrophobic interactions and hydrogen bonds [ , , ] . all flavivirus cp homodimers contain at least two layers of contact interface. the a -a interface forms the bottom of a hydrophobic cleft (fig. b) that is crucial for viral association with biological membranes [ ] and lipid droplets (lds) (fig. c ) [ ] . the longest helix pair, a -a , is at the bottom of the homodimer and forms an interface with a high density of positive charges, and this interface is proposed as the vrnabinding site (fig. c) [ , ] ; the a -a pairs also affect protein stability and overall conformation [ ] . the n-terminus and a -helix of cps are very flexible among the flavivirus genus, which may help them to adopt different conformations for various physiological processes [ , ] . the a of zikv cp is much shorter than that of other flavivirus cps but has a unique long pre-a loop. the orientation of the a of denv cp is different from that of others [ ] . the third layer of the contact interface on the top of the dimer structures, which is formed by antiparallel (wnv and jev) a -a [ , ] or (zikv) a -a with pre-a -pre-a loops [ , , ] that occlude the a -a hydrophobic cleft and form a closed conformation, except the denv cp dimer. by contrast, the a and a -helices of the denv cp dimer form an open conformation without pairing, exposing the a -a hydrophobic cleft (fig. b) . however, it should be noted that the only structure of the denv cp was solved by nmr in solution, which is relatively less stable than the structures of the zikv, wnv and jev cps in crystals. a subsequent study confirmed that the denv a -a interface is in conformation exchange in the free state, interconverting between an open and a closed state regulated by the flexible n-terminus [ ] . whether other flavivirus cps also employ the same mechanism for different biological processes is still unclear. in addition, tetrameric and hexameric arrangements are also observed in the wnv [ ] and zikv [ ] cp crystal structures, respectively. it was assumed that the functional cp requires a highly organized arrangement, and major mutations that transform its structure abrogate its functions [ ] . unexpectedly, the research team of w. mandl showed that tbev cp tolerated large internal deletions, and the generated mutants could be divided into three groups according to their viability in cell culture [ , , ] . for the first group, mutant rna could generate viable infectious viruses, albeit viral growth was correspondingly decreased [ ] . for the second group, the viral production of mutants carrying large deletions was completely or nearly abolished, but pseudorevertants could easily be restored by spontaneous second-site mutations in the capsid gene [ ] . for the third group, viral infection spread was irreplaceably abolished by deletions that were too large; these mutants became noninfectious replicons that secreted only subviral particles (svps) [ , ] . consistent with tbev cp, it was demonstrated that yfv cp, with nearly aa in the nterminus or aa in the c-terminus, was still functional in rna packaging [ ] . similar properties were also observed in wnv; when even or residue deletions were introduced into cp by reverse genetics technology, these mutants were still viable [ ] . these results together suggest the remarkable flexibility of flaviviral cps in both structure and function. however, all of these viable mutants or pseudorevertants displayed attenuated viral growth kinetics in cell culture compared with those of the wild type, without influences on rna replication and translation, but producing substantial amounts of svps [ , , , ] . post-translation, cp anchors in the er membrane via an internal signal peptide sequence (known as the anchor) at its c-terminus. the anchor spans the er membrane and directs the translocation of the downstream prm protein. cleavage of the c-prm junction employs a coordinated two-step proteolytic process by viral ns b and signalase at the two termini of the anchor, and the signalase cleavage of prm in the lumen remains inefficient until ns b cleavage occurs [ ] [ ] [ ] [ ] [ ] [ ] . the lack of polar residues in the c-region of the anchor sequence indicates that it is not a typical signal sequence, and a pqaqa mutant of the c-region with polar residues dramatically uncoupled the coordinated proteolytic process [ ] . however, the pqaqa mutant displayed impaired growth kinetics because enhanced signalase cleavage of anchor-prm increased the assembly and release of svps at the expense of infectious virion production [ , ] ; for yfv, the pqaqa mutant is even lethal for viral production [ ] . this finding indicates that sequential cleavage at the c-prm junction facilitates infectious particle assembly. in addition, the anchor also contributes to e protein stability to prompt assembly of infectious particles [ ] , and a cleavable anchor in context is more efficient than separate mature cp for particle assembly [ , ] . interestingly, a noncleavable c-anchor could not be utilized for packaging the mvev replicon, but that of yfv and zikv could, suggesting that the accessibility of the anchor cleavage site to the ns b protease is virus specific [ , , ] . only mature cps are packaged into virions in flavivirus infection; surprisingly, a recent study reported that unprocessed c-anchor proteins could be packaged into denv pseudoviruses [ ] . in summary, the sequential cleavage of the c-prm junction is pivotal for efficient flavivirus assembly, and this process is controlled by sequence elements in the cp anchor. flavivirus cp is involved in vrna replication in a special manner. although rna sequences are not conserved among divergent flaviviruses, there are several conserved rna secondary structures in the -terminus of the cp-encoding sequence that are necessary for vrna replication. ( ) the aug codon of cp starts from the stem loop b (slb) structure, and a partial -upstream aug region (uar)-flanking stem (ufs) is located in the cp coding sequence [ ] . ( ) the downstream aug region ( dar) and ( ) cyclization sequence ( cs) [ ] [ ] [ ] . ( ) a stable hairpin known as capsidcoding hairpin (chp) is surrounded by the dar and cs [ , ] . ( ) the downstream cs pseudoknot (dcs-pk) element contains sequences complementary to a region in the utr [ ] [ ] [ ] . therefore, these cis-elements must be taken into consideration when studying cp functions or constructing subgenomic replicon tools and inserting markers between the viral utr and cp coding sequence. although current studies on flaviviruses have shown that the flaviviral assembly process does not exhibit a necessary step occurring in the cell nucleus, it has been well demonstrated that many mosquito-borne flavivirus cps partially localize in the cell nucleus [ ] [ ] [ ] [ ] [ ] ; in the cytoplasm, in addition to localizing in the er, denv cp and zikv cp have also been demonstrated to accumulate on lds [ , ] , but the link between the functional importance and the subcellular distribution of cps is still unclear. flavivirus cps localized in the nucleus independent of viral infection, and overexpression of only the capsid gene resulted in its nuclear localization and colocalization with nucleoli [ , ] . although the molecular weight of flavivirus cps is relatively low, they are actively transported into the nucleus but not by simple diffusion [ , ] . interestingly, the nuclear localization signals (nlss) of flavivirus cps are heterogeneous [ ] [ ] [ ] [ ] [ ] [ ] . it has also been demonstrated that the nuclear localization of flavivirus cps is functionally correlated with virus production [ , , ] . it is worth stressing that the key sites of the predicted nls are usually basic residues, which play a role in rna binding for cps. however, it is still to be solved to uncouple the correlation of nucleolar localization and rna association in present studies. both the denv and zikv cps were observed accumulating on the surface of lds in infected cells [ , ] . it can be speculated that the cp accumulation on lds may be a common feature in flavivirus infection, but further verification is needed. experimental evidence indicated that the hydrophobic a -helix and the positively charged n-terminal region participate in the interaction with lds [ , ] , but these regions are also responsible for er membrane association and rna binding, respectively. thus, studies to define the significance of ld-associated cps during viral infection are complicated, although the numbers of lds and viral replication affect each other [ ] . it is possible that lds may serve as a reservoir of cps in early infection and are then mobilized to the er membrane for particle morphogenesis when needed [ ] . however, the functional significance of cp accumulation on lds still deserves further study. one of the most important roles of cps in the viral lifecycle is the formation of the nc, and the cp dimer is believed to be the building block for nc assembly [ ] . previously resolved cryo-em structures of both mature and immature denv virions revealed that the core lacks a well-formed protein organization [ , ] . according to a recent -Å-resolution structure of immature zikv, the nc core was observed close to the transmembrane domains of the e and prm proteins [ ] . the nc core of immature kunjin virus (kunv) was also shown to be positioned asymmetrically with respect to the glycoprotein shell [ ] . therefore, these results indicate that the icosahedral axes of the cp shell may not coincide with the axes of the dominant outer glycoprotein shell, thus causing poor reconstruction of the cp shell. to assemble the nc, cp must bind to vrna. based on the structures of flavivirus cp dimers, the positively charged a -a interface is proposed as the main rna binding site [ ] . in fact, in vitro studies have shown that the n-terminal region of cp also binds to rna [ , ] , and the n-terminal region is essential for viral particle assembly [ ] . the cp dimers showed no specificity toward different nucleic acids in vitro [ , ] ; however, only positive-sense vrna is packaged in flaviviral virions, and a study in vitro showed that the denv cps and zikv cps associate with vrna at specific sites rather than in a random manner [ ] . the encapsidation signals for flavivirus genomes have not been identified to date; thus, the specific mechanism of nc formation remains unclear. the nucleocapsid-like particles (nlps) can be assembled in vitro but are larger than the authentic nc, which may be attributed to cellular or viral components participating in nc formation in vivo [ , ] . the formation of cp-only particles is unlikely due to the highly positive charge of the cp dimer, which is neutralized by the packaged genome [ ] . in cells, the nc may be assembled in a near-neutral environment within the er membranous compartment, which is physically linked to vesicles and contains the prm-e proteins [ ] . it is believed that viral encapsidation and nc incorporation into viral particles is a coordinated process, supported by the fact that the nc has never been isolated from infected cells. thus, the nc assembly process and the nc structure are still elusive. previous structural studies observed a clearly low-density gap between the nc and lipid bilayer [ , , , ]; in addition, unlike in coronavirus and hepatitis b virus, the cytoplasmic sides of the flaviviral prm and e proteins on er membranes are extremely short and are devoid of a distinct nc-binding domain [ ] . these traits suggest that the nc and outer glycoproteins may not directly interact. however, recently determined structures at higher resolutions showed that the nc is asymmetrically positioned with respect to the outer glycoprotein shell in both immature kunv [ ] ; however, nc is still positioned concentrically with the outer glycoprotein shell in mature virions [ , ] , suggesting a rearrangement of the nc during viral maturation. according to these structural studies, a model for flaviviral budding was put forward: the ncs interact with glycoproteins at the beginning of viral budding and form an immature virion with an eccentrically positioned nc core; during viral maturation, the conformational rearrangements of outer glycoproteins possibly result in loosening of nc-glycoprotein interactions and release of the nc core to the center [ ] . recent studies have also proposed a general model for flavivirus virion assembly. in this model, the transmembrane protein ns a plays a central role in orchestrating virion assembly [ ] . svps are routinely generated as a byproduct in flavivirusinfected cell cultures; secreted particles contain only the m and e proteins and lipid membrane but lack the nc core. similar recombinant svps can be produced by coexpressing the prm and e proteins in cell culture. the assembly and budding of these particles occur in an nc-independent manner; however, these particles undergo the same maturation and transport process as whole virions [ ] . two distinct sizes of recombinant svps were observed in tbev; however, smaller svps (approximately nm in diameter) were far more prevalent than the larger svps (approximately nm in diameter) [ ] . a structural study revealed that the orga-nization of the e protein is different in svps than in the whole virus [ ] , but svps show hemagglutination and fusion activities similar to those of whole virions and have been proven to be excellent immunogens with protective capability [ , , ] . in viral infection, how the nc is dissociated and how vrna is released to enable protein synthesis are still unknown steps in flavivirus biology because there is little research data for this process to date. tracing the fate of denv cp after viral entry, we found that cp was degraded after internalization by a ubiquitin proteasomedependent process; however, mutational analysis revealed that denv cp ubiquitination occurs at noncanonical residues but not lys residues [ ] . a deletion mutation study of zikv cp using an infectious clone indicated that the a -helix may also affect nc uncoating steps [ ] . these data indicate that both viral and host factors are possibly involved in the viral nc uncoating process; however, as one of the most understudied viral processes in flaviviruses, further attention is urgently needed to clarify the molecular mechanism of the flaviviral uncoating process. due to increasing transnational communication and climatic and socioeconomic changes, emerging and reemerging flaviviruses have caused an ever-growing threat to global public health; a recent example of this issue is the rapid and wide spread of zikv throughout the americas since [ ] . there is currently no efficient medical treatment against these important viral diseases; thus, safe and efficacious vaccines are of great importance for preventing viral infection and spread. in many respects, flavivirus vac- cine development has historically achieved success, such as the most successful live-attenuated vaccine yf- d and many other live-attenuated or inactivated vaccines against yfv, jev and tbev infection [ ] . however, concerns about safety have been raised regarding adverse reactions observed with yf- d and inactivated jev vaccines [ , ] . in addition, there are still no available vaccines for human against wnv and zikv, and the only licensed denv vaccine is also less than satisfactory for its efficacy and safety [ , ] . hence, innovative vaccine development must meet the stringent need for safety, efficacy and cost effectiveness and face the challenge of the complicated pathogenesis of some flaviviruses [ ] . manipulation of reverse genetics technology has identified cp as a target for flaviviral vaccine development; both live-attenuated and new-style replicon vaccines can be generated by introducing different sizes of deletions into the cp gene of the viral genome (fig. ) . we summarize these reports here, and the potential benefits are discussed. as mentioned above, the flaviviral cps display significant flexibility in structure and function. introducing different sizes of deletions into cp resulted in various degrees of attenuation of viral replication in vitro. do deletions in cp also affect flaviviral virulence in vivo? assessing the % lethal dose (ld ) and the % infective dose (id ) of tbev dc mutants in mouse models, it was found that a small deletion mutant (cd - ) exhibited virulence indistinguishable from that of the wild type, but a viable mutant with a large deletion (cd - ) in the first group was highly attenuated, with a much higher ld than the wild-type virus (! . pfu versus . pfu) [ ] ; the pseudorevertants c(d - /du - ) and c(d - /q l) were equally attenuated (ld ! . pfu) but more infective than the mutant cd - (id of pfu versus . pfu), so a larger attenuation index was achieved (up to . , calculated from the ratio of the ld /id ) [ , ] . more importantly, upon inoculation with any of the mutants, all the seroconverted mice survived a challenge with a lethal dose of wild-type virus; in other words, the % protective dose was equal to the id , indicating that all of these mutants elicited a protective immunity and are ideal and highly efficient immunogens [ , , ] . in addition to tbev, denv dc mutants that were tested in vivo also showed significant attenuation in suckling mice and efficiently induced high antibody titers in adult mice [ ] . additionally, dc pseudorevertants of wnv did not cause disease but could induce protective immunity even at doses of - ffu [ ] . interestingly, a study showed that a cd - mutant had abolished zikv infectious virion production that was then restored by adaptive mutations (prm-e k and ns b-e g) only in bhk cells but not in other cell lines (indicate complex interactions that apparently occur between structural and non-structural proteins during virus replication and/or assembly), making this live virus function like a single-round infectious particle (srip) in vivo and safely inducing strong immunity protection against vertical transmission in mice [ ] . these data indicate that engineering deletions into the capsid gene of flaviviruses via reverse genetics is a feasible approach for generating efficient live-attenuated vaccine candidates. the flaviviral live-attenuated vaccines are characterized by their high efficiency in seroconversion and protection in animals. the classical live-attenuated vaccines were generated by continuous passaging of the virus in cell culture or animal tissue to accumulate various mutations, finally achieving the aim of significant attenuation but immunogenicity in vivo. the admirable example is the yf- d vaccine, which was developed by passages of the asibi strain in mouse embryo tissue and chicken embryo tissue, which markedly reduced its viscerotropism and neurotropism [ ] . however, this traditional method is usually time consuming; it is unpredictable whether the obtained viruses are attenuated and still can elicit protective immunity. in addition, because multiple mutations spread throughout the viral genome, it is toilsome to clarify the molecular mechanism responsible for the attenuated phenotype, limiting the assessment of the risk for spontaneous reversions to a virulent phenotype. however, using reverse genetics tools, we can rationally design the viral genome to obtain a desired phenotype in a relatively short time, when the desired phenotype and its significance for safety and immunogenicity is well understood, along with the specific molecular determinants behind them. introducing deletional mutations into the flaviviral capsid gene on the basis of a well-understood molecular mechanism is a unique way to design and generate live-attenuated vaccine candidates, and this approach has several benefits. ( ) the successful application of this approach in tbev, denv and wnv, in addition to the relatively conserved structures of cps among flaviviruses, suggests that this approach can be a universal approach for different flaviviruses, although there are possibly intricate differences in the length and location of deleted mutations. ( ) with regard to safety, the deleted mutations are very stable, especially large deletions, and it is almost impossible to revert to a wild-type sequence. although risks can arise from intermolecular rna recombination events between the genomes of closely related viruses, there are few reports on flaviviruses; thus, rna combination is not a major risk because of the very low frequency [ ] . in addition, cp deletions in combination with other well-understood mutations, such as point mutations in the e protein and other different principles, further improve the safety [ , ] . ( ) c-deleted viruses contain authentic structural proteins (prm/e) and nonstructural proteins, so they induce only specific immune responses, which should be considered more advantageous than chimeric viruses [ , ] . in addition, c-deleted viruses are highly immunogenic, which is a general advantage of live vaccines; with only a single-dose vaccination, protective immunity can be elicited [ , ] . in summary, introducing deletions into the flaviviral capsid gene is a promising approach for novel flaviviral live-attenuated vaccine development (summarized in table ). although live vaccines are highly efficacious, there are still risks of rare vaccine-associated diseases, especially in immunodeficient individuals. to address this issue, limiting the infectivity of a vaccine by disabling its viral encapsidation process is an elegant solution. this goal can be easily achieved by introducing very large internal deletions into cp to generate noninfectious dc-replicons, therefore opening a new approach for flaviviral vaccines. a selfamplifying dc-replicon that is able to produce highly immunogenic svps may be delivered as rna, dna, or virus-like particles (vlps) [ ] . as a proof of concept, this approach was first verified with tbev [ ] . a -residue deletion in tbev cp (d - ) irreversibly abolished infectious virion formation but maintained rna replication and promoted svp production. by engineering supplementary mutations that uncouple the coordinated cleavage events in the c-prm junction (see previous section), an idealized mutant c (d - )-s that liberates significantly more svps was created. mouse experiments showed that this replicon mrna vaccine (delivered by a gene gun) induced a broad humoral and cellular immune response comparable to that of a live vaccine and protected mice from challenge; even a single immunization induced long-lasting (more than year) and high titers of neutralizing antibodies [ ] . then, a dna-based noninfectious dc replicon for wnv was also tested in mice, and it induced a high-quality immune response by intramuscular injection and effective protection of animals, similar to those of the virus or infectious dna, while the magnitude was relatively lower [ ] . therefore, these studies demonstrated the enormous potential of flavivirus dc replicons as a new type of nucleic acid vaccine. nevertheless, in the early study of nucleic acid vaccines, booster immunizations are often necessary for providing sufficient protection against virulent strain challenges due to the low delivery efficiency of dna and rna in vivo. to overcome this barrier, one advisable alternative is viral delivery, which delivers replicon rna via vlps [ ] . the dc replicon rna can be packaged into vlps by trans-complemented cps to form srips (or pseudoviruses). true to the srip name, these particles can invade cells and release the replicon rna to the cytoplasm, followed by initial rna replication but inability to form infectious virions because of the lack of a functional copy of the structural gene in the genome [ ] [ ] [ ] ; however, the cells infected by srip-packaged dc replicon rna should produce a mass of svps. to date, different expression systems have been used to package the dc replicons of wnv [ ] , yfv [ ] and denv [ , ] to produce srips. the viral delivery approach of the dc-replicon was first applied for yfv and wnv [ ] , the dc-replicons can be easily packaged into vlps, and the titer reached approximately srips/ml [ ] . a single-dose intraperitoneal immunization of mice with  wnv srips completely protected the mice from a lethal challenge with a virulent wnv strain [ ] . in a subsequent study, a more efficient and safer cultivation system was developed for the production of second-generation wnv srips, which was named replivax wn [ ] . single immunization with replivax wn elicited strong protective immunity against wnv disease in mice and hamsters, and durable protective immunity in hamsters lasted for months [ , ] ; in mice, wnv-specific igg antibody responses and vigorous and specific cd + and cd + t cell responses were detected after immunization and induced a th biased immune response, similar to live-attenuated vaccines and different from inactivated vaccines [ , ] ; importantly, longlived plasma cells secreting wnv-specific igg antibodies and cd + memory t cell responses were detected at months post immunization [ ] . furthermore, in comparison with that of liveattenuated vaccines and inactivated vaccines in mice and hamsters, the immunogenicity of replivax vaccines (for wnv and yfv) was comparable in terms of both the magnitude and durability of the response [ ] . based on the replivax pseudoviruses, different single-cycle chimeric flavivirus vaccines were generated for jev, denv , and tbv by replacing the native prm-e genes of cdeleted replicons with those of other flaviviruses [ , [ ] [ ] [ ] [ ] . all of these constructs revealed good capacity to induce a potent and durable immune response in animal models, and it is important that their immunogenicity was not significantly affected by preexisting immunity against the vector backbone [ , ] . there is also an investigation of an srip-producing dna vaccine. this vaccine is based on a c(d - ) replicon cdna of kunv, and a separate but complete cp is encoded in reverse orientation controlled by a second cytomegalovirus (cmv) promoter in the same plasmid. after transfection, both the replicon rna and cp are produced in the cell, therefore forming secreted srips that deliver the dc replicon to adjacent cells, and both dna-transfected and srip-infected cells contain the dc replicon, which produces svps, resulting in an enhanced immune response [ , ] . both dc replicon nucleic acid vaccines and their derived srip strategy appear promising for innovative flaviviral vaccine development, especially srips because of their great capacity to provide protective immunity in animal models after only a single immunization, and they can be conveniently propagated and produced at high titers in a stable cell line; moreover, they are reliably safe without causing spread infection. however, srips suffer from a high cost of production and storage and are difficult to purify [ ] . currently, the issues of mrna instability, inefficient in vivo delivery and the difficulty of manufacturing are no longer barriers in the widespread implementation of mrna vaccines. on the contrary, mrna vaccines have several beneficial features over inactive and live-attenuated viruses, as well as dna-based vaccines, such as being safer, being more efficient and having the potential for rapid, low-cost scalable manufacturing (for a review, see [ , ] ), so dc replicon mrna vaccines are also very promising alternatives to conventional flaviviral vaccines. in addition to having safety and efficiency similar to those of srips, they are more suitable for meeting the challenges of rapid development and large-scale deployment for emerging virus vaccines [ ] . over the past decades, although we have made important advances in our understanding of key steps in the flaviviral life cycle and their application to development of novel vaccines, many aspects of the viral life cycle remain obscure. flaviviral cp plays a crucial role in viral assembly. we have learned about structures, posttranslational cleavage and maturation, and the subcellular distribution of flaviviral cp; however, how the nc is assembled and dissociated, how the nc should be incorporated into viral particles and the process of nc uncoating are not well explained to date. perhaps structural studies on the nc in the future will aid in our understanding of these aspects. due to the lack of specific treatments for flavivirus-related diseases, vaccines are the most powerful measures to prevent flaviviral infection and viral spread. although vaccines have achieved success with respect to yfv, tbev and jev, there is still room for improvement concerning safety, efficiency, and low cost. as a viable approach, c-deleted genome-generated live-attenuated viruses are efficient and convenient approaches with several advantages over traditional methods for attenuation of viruses, but safety concerns for live viruses still exist. by contrast, the noninfectious dc replicon displays significant potential. both viral delivery (srips) and nonviral delivery (nucleic acid vaccine) of table attenuated flaviviruses generated by c-deletion. deletion size a minimal protective dose animal model reference tbev c(d - ) . pfu mouse [ ] tbev c(d - /q l) and c(d - /du - )~ pfu mouse [ ] wnv c(d - /d e) and c(d - )~ pfu mouse [ , ] denv c(d - ), c(d - ) nd b mouse [ ] zikv c(d - ) + prm-e k + ns b-e g ffu mouse [ ] a the deleted amino acids in capsid proteins are highlighted with underscores. b nd: not determined. the dc replicon have potential, and mrna vaccines of the dc replicon are a particularly promising alternative approach [ , ] . however, to meet the need of public health emergencies caused by novel flaviviruses (suggested by zikv), the mrna vaccines of the dc replicon can be rapidly put into use, which is also an advantage. the global distribution and burden of dengue structure of dengue virus: implications for flavivirus organization, maturation, and fusion visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of immature west nile virus structure of the immature dengue virus at low ph primes proteolytic maturation the . angstrom resolution cryo-em structure of zika virus a conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein spontaneous mutations restore the viability of tick-borne encephalitis virus mutants with large deletions in protein c solution structure of dengue virus capsid protein reveals another fold dynamics of zika virus capsid protein in solution: the properties and exposure of the hydrophobic cleft are controlled by the alpha-helix sequence west nile virus core protein; tetramer structure and ribbon formation crystal structure of the capsid protein from zika virus crystal structure of the japanese encephalitis virus capsid protein structural insight into the zika virus capsid encapsulating the viral genome dengue virus capsid protein usurps lipid droplets for viral particle formation maintenance of dimer conformation by the dengue virus core protein alpha -alpha '= helix pair is critical for nucleocapsid formation and virus production partial intrinsic disorder governs the dengue capsid protein conformational ensemble flavivirus immunization with capsid-deletion mutants: basics, benefits, and barriers capsid protein c of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence mimicking live flavivirus immunization with a noninfectious rna vaccine functional requirements of the yellow fever virus capsid protein helices alpha and alpha of west nile virus capsid protein are dispensable for assembly of infectious virions ns b- proteinasemediated processing in the yellow fever virus structural region: in vitro and in vivo studies signal peptidase cleavage at the flavivirus c-prm junction: dependence on the viral ns b- protease for efficient processing requires determinants in c, the signal peptide, and prm mutagenesis of the ns b-ns -mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing mutagenesis of the signal sequence of yellow fever virus prm protein: enhancement of signalase cleavage in vitro is lethal for virus production inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes a flavivirus signal peptide balances the catalytic activity of two proteases and thereby facilitates virus morphogenesis role of capsid anchor in the morphogenesis of zika virus increased expression of capsid protein in trans enhances production of singleround infectious particles by west nile virus dna vaccine candidate production and characterization of vaccines based on flaviviruses defective in replication a single-dose live-attenuated zika virus vaccine with controlled infection rounds that protects against vertical transmission e denv pseudoviral particles with unprocessed capsid protein are assembled and infectious viral rna switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization long-range rna-rna interactions circularize the dengue virus genome essential role of cyclization sequences in flavivirus rna replication the ' and ' downstream aug region elements are required for mosquito-borne flavivirus rna replication rna secondary structure in the coding region of dengue virus type directs translation start codon selection and is required for viral replication the capsid-coding region hairpin element (chp) is a critical determinant of dengue virus and west nile virus rna synthesis novel cis-acting element within the capsid-coding region enhances flavivirus viral-rna replication by regulating genome cyclization overlapping local and long-range rna-rna interactions modulate dengue virus genome cyclization and replication composition of the sequence downstream of the dengue virus ' cyclization sequence (dcs) affects viral rna replication intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus nuclear localization of japanese encephalitis virus core protein enhances viral replication specific interaction of capsid protein and importin-alpha/beta influences west nile virus production detection of dengue virus core protein in the nucleus. i. a monoclonal antibody to dengue virus reacts with the antigen in the nucleus and cytoplasm nuclear localization of dengue virus core protein detected with monoclonal antibodies multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection nucleolar protein b interacts with japanese encephalitis virus core protein and participates in viral replication functional correlation between subcellular localizations of japanese encephalitis virus capsid protein and virus production the disordered n-terminal region of dengue virus capsid protein contains a lipid-dropletbinding motif isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particles cryo-em structure of the mature dengue virus at . -a resolution structure of the immature zika virus at a resolution flaviviruses have imperfect icosahedral symmetry rna binding properties of core protein of the flavivirus kunjin uncoupling cis-acting rna elements from coding sequences revealed a requirement of the nterminal region of dengue virus capsid protein in virus particle formation genome-wide associations of flavivirus capsid proteins. biorxiv in vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue- virus composition and three-dimensional architecture of the dengue virus replication and assembly sites immature and mature dengue serotype virus structures provide insight into the maturation process dengue ns a protein orchestrates virus assembly e a structural perspective of the flavivirus life cycle two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus molecular organization of a recombinant subviral particle from tick-borne encephalitis virus recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system dengue virus genome uncoating requires ubiquitination zika virus outbreak: 'a perfect storm a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available viscerotropic and neurotropic disease following vaccination with the d yellow fever vaccine, arilvax Ò new developments in flavivirus vaccines with special attention to yellow fever recent advances in human flavivirus vaccines attenuated dengue viruses with deletions in capsid protein derived from an infectious full-length cdna clone characterization of west nile virus live vaccine candidates attenuated by capsid deletion mutations the yellow fever d vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines a trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination nucleic acid-based infectious and pseudoinfectious flavivirus vaccines humoral and cellular immune response to rna immunization with flavivirus replicons derived from tickborne encephalitis virus immunogenicity of west nile virus infectious dna and its noninfectious derivatives trans-packaged west nile virus-like particles: infectious properties in vitro and in infected mosquito vectors packaging the replicon rna of the far-eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system a rapid and quantitative assay for measuring antibody-mediated neutralization of west nile virus infection construction and characterization of a second-generation pseudoinfectious west nile virus vaccine propagated using a new cultivation system production of pseudoinfectious yellow fever virus with a two-component genome highly efficient production of a dengue pseudoinfectious virus enhanced production of infectious particles by adaptive modulation of c-prm processing and c-c interaction during propagation of dengue pseudoinfectious virus in stable cprme-expressing cells replivax wn, a single-cycle flavivirus vaccine to prevent west nile disease, elicits durable protective immunity in hamsters characterization of the replivax platform for replication-defective flavivirus vaccines repeated in vivo stimulation of t and b cell responses in old mice generates protective immunity against lethal west nile virus encephalitis immunogenicity of replivax wn, a novel single-cycle west nile virus vaccine construction and evaluation of a chimeric pseudoinfectious virus vaccine to prevent japanese encephalitis construction and characterization of a single-cycle chimeric flavivirus vaccine candidate that protects mice against lethal challenge with dengue virus type efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue /yellow fever viruses single-dose vaccine against tick-borne encephalitis single-round infectious particles enhance immunogenicity of a dna vaccine against west nile virus selfamplifying mrna vaccines mrna vaccines-a new era in vaccinology the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -z o t mz authors: chinsangaram, jarasvech; mason, peter w.; grubman, marvin j. title: protection of swine by live and inactivated vaccines prepared from a leader proteinase-deficient serotype a foot-and-mouth disease virus date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: z o t mz abstract previously, we demonstrated that a genetically engineered variant of foot-and-mouth disease virus (fmdv) serotype a lacking the leader proteinase-coding region (a -llv ) was attenuated and induced an immune response that partially protected cattle from fmd. in this study, a -llv was tested in swine as a live or chemically inactivated vaccine. animals vaccinated with chemically inactivated a -llv or wild-type (wt) virus in oil adjuvant developed high levels of neutralizing antibodies and were protected from fmd upon challenge. animals vaccinated with live a -llv did not exhibit signs of fmd, did not spread virus to other animals, developed a neutralizing antibody response and antibodies to nonstructural protein d, and were partially protected from fmd. animals given a similar dose of chemically inactivated a -llv in the absence of adjuvant developed a poor immune response and were not protected from fmd, indicating that limited replication was responsible for the improved immune response found in animals vaccinated with live a -llv . the results demonstrate the potential of a -llv as a live-attenuated vaccine as well as a safe source of antigen for chemically inactivated vaccines. foot-and-mouth disease (fmd) is a debilitating disease of cloven-hoofed animals including swine and cattle. the disease is highly contagious and characterized by fever, vesicular lesions (mainly on the feet, but also on the mouth, nose, and sometimes teats), as well as abortions, and neonatal deaths. infected animals rapidly spread the virus via aerosol to others, resulting in a dramatic loss of production by the herd. fmd outbreaks are so devastating that countries which do not have the disease restrict the importation of animals and animal products from countries where the disease is present. when fmd is found, infected and exposed animals are slaughtered and ring vaccination surrounding the affected area may be implemented'.'. vaccines prepared by chemical inactivation of fmdv have been used to successfully control fmd . however, there is an association of disease outbreaks with incomplete chemical inactivation and escape of usda virus from vaccine manufacturing facilities .'. furthermore, this conventional vaccine induces relatively short-lived immunity and some vaccinated animals can develop a carrier state following contact with fmdv .'. over the last yr, a number of alternative approaches in fmd vaccine development have been examined. one of the approaches we have examined is the liveattenuated vaccine. a vaccine based on stably attenuated fmdv could provide improved safety over the existing product?. moreover, work on other viral diseases has shown that live-attenuated vaccines can induce longer lasting immunity, present a greater antigenic mass, as a result of limited replication, than inactivated vaccine, and if delivered by the natural route can induce mucosal immunity'. fmdv is an aphthovirus in the family picomaviridae'". the single-stranded positive-sense rna genome is surrounded by an icosahedral capsid composed of four structural proteins, vpl, vp , vp and vp . the rna genome contains a long translational open reading frame that codes for a polyprotein which is processed by virally encoded enzymes into mature structural and nonstructural proteins. the fmdv leader (l) protein, a papain-like proteinase"-' , is associated with viral virulence ,' .' . in infected cells, the l proteinase autocatalytically cleaves itself from the proteinase-deficient fmd vaccines: j. chinsangaram et al. were immunoprecipitated with and dpv serum from swine vaccinated with; lanes - , ; lanes - , bei-inactivated a -ic (no. [ ] [ ] [ ] ; lanes - , control swine (no. and ) . lane , immunoprecipitation with bovine convalescent serum. lane , immunoprecipitation with dpv serum from swine no. . the products were examined by sds-page on a % gel. viral polyprotein' and shuts off cap-dependent host protein synthesis by cleaving eif- g, a component of the cap-binding complex, eif- f' - . as a result, fmdv, which initiates translation by a cap-independent mechanism, does not need to compete with host mrnas for use of the cellular translation machinery. as a part of our program to identify attenuated vaccine candidates, we have genetically altered the genome of fmdv seroxpe al by deleting the l proteinase coding region . in contrast to attenuated viruses selected by serial passage which are genetically unstable', deletion of the l protein coding region makes it highly unlikely that the leader proteinasedeficient virus (a -llv ) will revert to virulence. our previous studies have shown that a -llv replicates more slowly than parental virus (a -ic) in baby hamster kidney (bhk) cellsn and is attenuated in cattle ' . after aerosol inoculation, a -llv was only found localized in the respiratory bronchioles and did not produce clinical signs in cattle, in contrast to a -ic which caused classical fmd including fever and vesicular lesions"'. furthermore, we have demonstrated that a single subcutaneous vaccination with live a -llv induced a partially protective immune response in cattle against a severe challenge by intradermal inoculation in the tongue with virulent cattlepassaged fmdv'. it has been shown that cattle and swine do not necessarily res ond to fmd vaccines or infection in a similar fashior$e' . differences in disease susceptibility of cattle and swine has also been suggested by a recent fmd outbreak in taiwan where the swine population was devastated, but no significant disease in cattle was reportedz . in the present study, the safety and efficacy of live or inactivated vaccine preparations of a -llv were tested in swine. proteinase-deficient fmd vaccines: j. chinsangaram et al. baby hamster kidney (bhk) cells (strain , clone ) were used to propagate virus stocks and for plaque reduction neutralization (prn) assays. secondary lamb kidney (lk) cells (provided by dr c. house, foreign animal disease diagnostic laboratory, usda, plum island, usa) were used to produce antigen for radioimmunoprecipitation (rip). parental wild-type (wt) fmdv al (a -ic), a -llv , and a virulent cattle-passa ed g strain of fmdv al were described previously". - . the a -ic and a -llv viruses were purified by sucrose-gradient centrifugation and chemically inactivated by treatment with binary ethylenimine (bei) as previously described"'. sixteen - kg yorkshire cross or yorkshire/ bluepoint gilts were divided into four vaccine groups of three animals each and two groups of two control animals. these animals were housed in two separate rooms. the first room contained one group of animals vaccinated subcutaneously with pg of live a -llv (lo p.f.u.) and two control animals. the second room contained three groups of animals and the remaining two control animals. each group of animals in the second room was vaccinated either subcutaneously with i i i i i i i i i days postvaccination pug of bei-inactivated a -llv or intramuscularly with pg of bei-inactivated a -llv in mineral oil ( :l marco umontanide ) or pg bei-inactivated a -ic in the above adjuvant. at days postvaccination (dpv), all animals were combined into a single room and challenged with live fmdv by inoculating a single control animal intradermally in the snout and by a combination of intradermal (coronary band and heel bulb) and skin scarification (coronary band) on one foot, with a total of x lo infectious units of virulent cattle-passaged fmdv type a . rectal temperature data and clinical signs, including lameness and vesicular lesions were recorded daily. temperature of over °c for two or more consecutive days was considered to constitute a fever. at the time of necropsy, the number of digits affected by the disease were recorded as a score for each animal (see table i ). serum samples were collected every week and used for prn and rip assays. rip was performed using cytoplasmic extracts from [ s] methionine-labeled fmdv-infected lk cells as antigen . serum samples were screened for antibodies to fmdv structural and nonstructural proteins at : dilution by rip and the precipitated products were analyzed by sds-page on % gels. serum samples were serially diluted and assayed for neutralizing antibodies by prn in bhk cells". neutralization titers were reported as the log of the serum dilution yielding a % reduction in p.f.u. (prn,"). intramuscular vaccination of bei-inactivated virus in adjuvant (both induced high neutralizing antibody titers (table i) and these animals had antibodies to the viral structural proteins as detected by rip (figure ) . neutralizing antibody was first detected in these animals at dpv and plateaued at - dpv (figure ). subcutaneous vaccination of bei-inactivated virus in the absence of adjuvant (no. - ) induced a lower neutralizing antibody response (table ) the titer appeared to decline slightly over time ( figure ). this latter group of animals did not develop antibodies to the viral structural proteins that could be detected by rip (lanes - in figure ). as expected all animals figure immunoprecipitation of viral proteins with serum from swine vaccinated with live or bei-inactivated a -llv in the absence of adjuvant. cytoplasmic extracts from fmdv-infected lk cells radiolabeled with [ s:jmethionine were immunoprecipitated with and dpv serum from swine vaccinated with; lanes - , live al -llv (no. - ); lanes - , bei-inactivated al -llv in the absence of adjuvant (no. - ) ; lanes - , control swine (no. and ). lane , immunoprecipitation with bovine convalescent serum. lane , immunoprecipitation with dpv serum from swine no. . the products were examined by sds-page on a % gel. given inactivated viruses did not produce any detectable antibodies to nonstructural proteins, including d (figures i and , table ). in contrast to animals given inactivated virus preparations, animals vaccinated with live a -llv ( no. - ) developed antibodies to nonstructural protein d and the structural proteins at dpv (lanes - in figure ) . the presence of antibodies to a viral nonstructural protein is indicative of virus replication in these animals. however, these animals did not show any clinical signs of fmd including lameness, fever or any apparent lesions on the feet or mouth. in addition, the two control animals (no. and ) which were housed in the same room with swine no. - for days did not seroconvert or show any signs of fmd, indicating that there was no spread of virus from a -llv -vaccinated animals (table , lanes - in figure ). following commingling of all animals in a single room, one control animal (no. ) was infected with virulent cattle-passaged al virus on the snout and one foot. vesicular lesions were observed on the foot and snout at and days postchallenge (dpc), respectively, and vesicles developed in the following days on all four feet of this animal, and temperatures over °c persisted from two to four and from seven to dpc [ figure (c) ]. all the remaining control animals (no. , , and ) and vesicular lesions at dpc; one of these animals (no. ) also developed a vesicle on the snout. animals vaccinated subcutaneously with bei-inactivated a -llv in the absence of adjuvant ( no. - ) were not protected from challenge (table l) , although all three animals had a delayed appearance of vesicles ( - dpc) as compared to control swine (no. , , and ) . animals vaccinated subcutaneously with live a -llv (no. - ) were partially protected from challenge (table i) . these animals did not develop a fever [ figure (a)], had a delayed appearance of vesicles ( - dpc), and only one animal in this group (no. ) developed vesicles on all four feet (although with a low score; see table ). no fevers [ figure (table i) were found in any of the animals vaccinated with bei-inactivated a -ic (no. [ ] [ ] [ ] or a -llv (no. [ ] [ ] [ ] in adjuvant for the period of observation ( days). sera collected dpc was evaluated to determine the extent of challenge virus replication in the exposed animals. the results revealed a strong boost in neutralizing antibody titers in animals vaccinated subcutaneously with either live or bei-inactivated a -llv and no increase in neutralizing titers in animals vaccinated intramuscularly with bei-inactivated a -llv or a -ic (figure , table ). moreover, rip analyses revealed that only the latter two groups of animals failed to develop antibodies to the nonstructural protein d following exposure, indicating that replication of the challenge virus was absent or limited in these animals (figure ). in this study, we have shown that a leader proteinasedeficient derivative of fmdv serotype al can be safely administered to swine, and that vaccinated swine produce neutralizing antibodies and are partially protected from virulent virus challenge. replication of live a -llv in swine was confirmed by the detection of antibody to nonstructural protein d, and presumably improved the immune response relative to that observed in animals vaccinated with inactivated a -llv by the same route (subcutaneous vaccination) in the absence of adjuvant. in spite of the evidence of virus replication, vaccination with live a -llv did not produce signs of disease and a -llv did not spread to control animals. these results together with our previous results in cattle (see section ) demonstrate the safety of a -llv as a live-attenuated vaccine candidate for livestock. however, an extensive study involving animals of various ages and immune status is required to unequivocally address the safety of this type of vaccine candidate. the severity of disease judged by lesion score and rectal temperature correlated well with the neutralizing antibody titer measured at the day of challenge (table i) . animals vaccinated with bei-inactivated a -ic or a -llv in an oil adjuvant developed high titers of neutralizing antibody and were all protected against challenge. thus, as expected, the deletion of the l proteinase coding region did not alter the antigenic structure of the virion. animals vacci- vaccine volume number nated with live a -llv had lower levels of neutralizing antibody as compared to animals vaccinated with bei-treated virus in the presence of adjuvant and were partially protected from virulent virus challenge, as determined by the absence of fever, delayed lesion appearance, and low lesion score. animals vaccinated with bei-inactivated a -llv in the absence of adjuvant displayed low or non-detectable neutralizing antibody titers at the day of challenge and although lesions appeared later in these animals than in control animals, by the end of the challenge period, no difference in disease severity was noticed between these two groups. subcutaneous vaccination of live a -llv was chosen for this study to allow a comparison with our previous study in cattle*. in addition, this route of inoculation was selected based on a study in cattle indicating that subcutaneous was better than intramuscular vaccination in terms of ability to induce neutralizing antibodies (unpublished observation). subcutaneous inoculation in cattle induced neutralizing antibody titers (prn,,,) of . , . and . and the animal with the highest titer ( . ) was protected against challenge, the animal with the prnto . developed a fever but no pedal lesions, and the third animal (prn," . ) developed fever and mild lesion?. in the present study, we showed that vaccination of swine by the same route induced similar neutralizing antibody titers, and these swine were partially protected from challenge (animals did not develop fever and displayed a low lesion score). since the immune response to live a -llv given by the with dpc serum from swine no. - , , and , respectively. lane , immunoprecipitation with bovine convalescent serum. lane , immunoprecipitation with cl dpv serum from swine no. . the products were examined by sds-page on a % gel. subcutaneous route was superior to that seen in animals given the same antigenic mass of inactivated a -llv by the same route, these studies demonstrated that replication of live a -llv virus in these animals was responsible, in part, for the increased immune response. problems associated with the conventional fmd vaccine have led us to search for safer vaccine candidates. the development of attenuated fmd vaccines by passage in unnatural hosts to obtain viruses which are both innocuous and yet able to induce a protective immune response has proven difficult ,z . in addition, viruses which are selected by this procedure can revert to virulence'. we have taken the approach of deleting the entire l coding region of fmdv to produce an attenuated virus suitable for use as a live vaccine. this virus has a deletion of a complete coding region which significantly reduces the risk of reversion to virulence as compared to attenuated viruses produced by conventional means. m.oreover, we have demonstrated that this virus can be used as a source of antigen in traditional bei-inactivated vaccines; since a -llv is non-pathogenic and does not spread between animals, its use in vaccine production could reduce the risk associated with current vaccine manufacture. the results of this study, in conjunction with our previous result?, suggest that vaccination of cattle and swine with live a -llv is feasible. however, the limited replication of this attenuated virus resulted in an increase in antigenic mass which was insufficient to induce a completely protective immune response. additional studies are currently ongoing to further demonstrate sifety, improve efficacy, and more completely understand the host response to attenuated virus infection. foot-and-mouth disease: world-wide impact and control measures a study of the potential economic impact of footand-mouth disease in the united states. us government printing office biochemical identification of viruses causing the outbreaks of foot-and-mouth disease in the uk subtyping of european footand-mouth disease virus strains by nucleotide sequence determination the carrier state in foot-and-mouth disease -an immunological review evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate immunization against virus disease: in virology putative papain-related thiol proteases of positive-strand rna viruses: identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, c(-, and coronaviruses antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus identification of the active-site residues of the l proteinase of foot-and-mouth disease virus identification of the critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease the foot-and-mouth disease virus leader proteinase gene is not required for viral replication pathogenesis of wild-type and leaderless foot-and-mouth disease virus in bovines a second protease of foot-and-mouth disease virus leader protein of foot-and-mouth disease virus is required for cleavage of the ~ component of the cap-binding protein complex proteinase-deficient fmd vaccines the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of emergency vaccination against foot-and-mouth disease: rate of development of immunity and its implications for the carrier state development in footand-mouth disease vaccines natural adaption to pigs of a taiwanese isolate of foot-and-mouth disease virus. the veterinaty record sensitivity of cell cultures, cattle, mice, and guinea-pigs for detection of nineteen foot-and-mouth disease viruses genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice receptor binding site-deleted foot-and-mouth disease (fmd) virus protects cattle from fmd inactivation of virus antigens for vaccine preparation with particular reference to the application of binary ethylenimine portraits of viruses: foot-and-mouth disease virus. lntervirology we thank marla zellner for expert technical assistance and the plum island animal caretakers for assistance with the animals. key: cord- - wzht authors: kweon, chang-hee; kwon, byung-joon; lee, jae-gil; kwon, geon-oh; kang, yung-bai title: derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: wzht the field isolate of porcine epidemic diarrhea virus (pedv) was serially passaged in vero cells. the cell passaged pedv, designated kpedv- , was tested for its pathogenicity in the neonatal pigs, immunogenicity and safety in the pregnant sows. the result indicated that kpedv- at the rd passage revealed reduced pathogenicity in the neonatal pigs. pregnant sows inoculated with the attenuated virus showed increased immune responses by elisa. in addition, delivered piglets were protected from challenge of wild type pedv. the safety test in pregnant sows indicated that all inoculated animals farrowed the average numbers of litters of piglets. the results of this study supported that the attenuated virus derived from serial passage could be applied as vaccine for protecting suckling piglets against pedv infection. porcine epidemic diarrhea virus (pedv), a member of coronaviridae, is the etiological agent of enteropathogenic diarrhea in swine [ ± ] . although the clinical symptoms of pedv infection are similar to transmissible gastroenteritis virus (tgev) infection, pedv has a wider variety of clinical signs in pigs [ ] . propagation of pedv in vitro was rather limited until vero cells were found to support the growth of virus in the presence of trypsin [ ] . in this study, we described the derivation of an attenuated strain of pedv, as a potential vaccine candidate, through cell adaptation. vero cells obtained from atcc (vero c ) were regularly maintained in alpha-mem supplemented with % fetal bovine serum, penicillin ( unit/ml), streptomycin ( unit/ml) and amphotericin ( . g/ ml). the isolate of pedv was originated from a neonatal pig and plaque puri®ed in vero cells. the isolate, designated kpedv- , was passaged in ± % monolayers of vero cells in alpha-mem with . % yeast extract, . % tryptose phosphate broth (tpb) and ± mg of trypsin as described [ , ] . sequential passages of the virus were normally conducted in roller culture. each passage level of virus was stored at À c or freeze-dried with equal volume of stabilizer ( . m vaccine ( ) ± - x/ /$ -see front matter # elsevier science ltd. all rights reserved. pii: s - x ( ) - lactose, . m khpo , . m k hpo , . m monosodium glutamate, % gelatin). the ®nal stock of the pedv was characterized in two ways. firstly, the culture supernatant was subjected to direct centrifugation at ,  g for h and the pellet was resuspended to / of initial volume for morphological identi®cation by transmission electron microscopy. secondly, the virus infected cells were subjected to reverse transcription polymerase chain reaction (rt-pcr) to detect speci®c pedv sequences. three primers for rt-pcr were selected from the sequences information of membrane protein (m) gene of duarte et al. [ ] . p ( mer); '±ccccagtactgttat-tgacgtataaac± ' (position ± ), p ( mer); '±gtttagactaaatgaagcactttc± ' (position ± ) for pcr and p ( mer); '± gccataaagtttctgtttagactaa± ' ( ± ) as the primer for synthesis of complementary dna, respectively. the extraction of rna and rt-pcr were conducted according to the instructions of commercially available kit (stratagene). pcr reactions were performed in the conditions as described previously [ ] . after ampli®cation, the pcr products were cloned into puc vector for sequencing using sequenase version . (usb, usa). in addition, the presence of adventitious virus such as porcine parvovirus (ppv), japanese encephalitis virus (jev), hog cholera virus (hcv) and other cythopathogenic viruses were examined as described previously [ ± ]. for the detection of attenuation, viral stocks at passages were tested in days old piglets. from . tcid /ml to . tcid /ml of cell adapted pedv was inoculated into suckling piglets intramuscularly or through the oral route. in order to compare the pathogenicity, pedv isolate before cell adaptation was prepared from the small intestines of neonatal piglet. the intestine was ground in phosphate buered saline (pbs, ph . ). the % suspension was then ®ltered using a . -mm membrane ®lter (acrodisk, gelman) and further diluted to -, -and -fold in pbs. four groups of ®ve piglets were orally fed with the suspensions of diluted stock of intestine. the animals were observed for clinical symptoms of diarrhea and mortality in the inoculated animals was observed for days. three pregnant sows ± weeks prior to farrowing were tested for the detection of immune responses. pigs were inoculated intramuscularly with the attenuated viruses at the titer of at . tcid /ml. two pregnant sows remained as uninoculated control. after two weeks, second inoculations of same titer were followed. each paired serum before and after inoculation was collected at two-week interval. the collected sera and colostrum at delivery were tested for the presence of antibodies by elisa. after delivery, suckling piglets of days old were orally challenged with or ld of wild pedv. the clinical signs of diarrhea and mortality of challenged piglets were observed for two weeks. for the preparation of antigen, the cell adapted pedv was concentrated with polyethylene glycol (peg, mw ) as described [ ] . the peg treated viral solution was then precipitated and resuspended at / th of original volume with ten buer ( . m tris, . m edta, . m nacl, ph . ). the puri-®ed virus was then used for the elisa. the procedures for elisa were basically the same with a previous study [ ] . brie¯y, the dilution of antigen and second antibodies were adjusted to the optical density (od) around . (a ) using the negative porcine sera. usually each well in -well microplate (costar) was coated with ± mg of protein in mm carbonate buer (ph . ) at c overnight, followed by the blocking with % skim milk at c. the / diluted porcine sera in pbs with . % bovine serum albumin (bsa) and . % tween (pbst) were reacted at c for min and then washed extensively with pbst three times at -min intervals. the reacted plate was washed again at the same condition and incubated with -fold diluted horseradish peroxidase (hrp) labelled anti-porcine igg (kpl) for h at c. the plate was developed in o-phenylenediamine (opd) at room temperature for min. the reaction was stopped with m h so before measuring od at nm. a total of pregnant sows were inoculated intramuscularly with ml of the virus containing . tcid /ml. twenty-three pregnant sows received one injection ± weeks prior to farrowing. in another farm, pregnant sows received two injections at ± week intervals before farrowing. the average number of the litters were compared with the data of uninoculated pregnant sows at the corresponding farm during same period of time. the sequence accession number for m protein of attenuated kpedv- is genebank accession number afo . the pedv was continuously passaged in vero cells. sequential passage of virus regularly conducted every ± days postinfection in cells. the supernatant was harvested and used for next inoculation in vero cells up to passages. however, cytopathogenic eect (cpe) in vero cells was not so clear that the culture supernatant of virus infected cell was subjected to morphological and genetic characterization. when the culture supernatant of virus infected cell was examined by the transmission electron microscopy, characteristic shape of coronavirus with diameter of ± nm was possible to identify (fig. ). in addition, the com-parision of m gene of cell passaged virus showed the . % in nucleotide and . % in amino acid identity with previously reported pedv strain (fig. ) . no adventitious viral contaminations were detected in the ®nal stock of pedv. the pathogenicity of attenuated virus was tested in the -day-old piglets before taking colostrum. six separate litters of piglets and litters of piglets were inoculated intramuscularly with virus of . and . tcid /ml, respectively. in this experiment, all the inoculated piglets failed to show signs of diarrhea and symptoms related to pedv infection. in order to avoid any possible eects from the maternal immunity through the colostrum and detect the potential pathogenicity of the attenuated virus, eight piglets of days old were infected orally with ml of virus stock, which contained virus of . tcid /ml, and were arti®cially fed with dairy milk. although three piglets showed signs of anorexia and mild signs of diarrhea in two or three days after inoculation, nevertheless, the signs seemed to be transient. in fact, all piglets recovered in the next ± days. however, in the groups of piglets fed with wild virus before cell passages, all the piglets developed symp-toms of watery diarrhea in ± days, and the mortality reached up to ± %, depending on the dilution of virus within one week as shown in table . when the collected sera were tested for the presence of the antibodies by elisa, all inoculated sows showed the rising elisa titers (fig. ) . on the other hands, the antibody titers of control pigs decreased at the time of delivery. in addition, colostrum at delivery showed higher or similar level of antibodies of corresponding sows. after challenge exposures mortality of piglets were compared with uninoculated control. although the mortality of piglets after challenge with ld of wild pedv was reduced to % compared to % in control litter, all piglets survived in the litters after challenge experiment with ld of virus compared to % in control (table ) . however, mild signs of diarrhea were also detected in one litter of piglets days after challenge, but they recovered the next day. the safety test of the attenuated virus in pregnant sow was conducted in two separate farms. one farm has not had any history of epidemic diarrhea in last few years and another farm had the experience of pedv outbreak in the previous year. a total of pregnant sows were inoculated once or twice before farrowing. as shown in table , all the inoculated sows farrowed the same average numbers of litters of uninoculated control group without any clinical problems. in this study, we investigated the attenuation of pedv through serial passages in vero cell cultures and its prophylactic eect in pregnant sows. after serial passages in vero cells, the growth of virus was rather trypsin-independent and the detection of cytopathic eect (cpe) was rather variable, depending on clones of vero cell lines (data not presented here). since the spf or gnotobiotic piglets were not used in this experiment, it might not reasonable to ®gure out the exact dierences in pathogenicity between the wild and the attenuated virus. nevertheless, when compared with the wild pedv, the animals inoculated with the high passage level of virus did not show any severe signs of diarrhea or death in piglets, supporting attenuation. it is known that pedv replicates mainly in the villi of small intestines [ ] . like attenuated tgev, the replication of attenuated pedv may be limited to the small portion of intestine with short duration of secretion compared to virulent virus [ ] . in fact, the detection of signs of diarrhea from piglets inoculated orally with attenuated virus delayed at least by two days and lasted one day compared with the signs from piglets inoculated with wild virus. when we tested the immunoprophylactic eect in pregnant sows, it was demonstrated that the vaccinated swine resulted in reduced piglet mortality after challenge experiment ( ± % in vaccinates compared with ± % in controls), indicating that the attenuated pedv could induce the status of immunity in pregnant pigs, providing protection in piglets like other enteric disease in swine. previously, we found that pedv infections were related to more than % of diarrheal cases in the neonatal pigs, thus causing a considerable losses in pig industry [ ] . in fact, ®eld application of attenuated virus as vaccine resulted in the overall reduction of mortality of neonatal pigs ( ± fig. . immune responses of pregnant sows inoculated with cell attenuated kpedv- strain. animals were inoculated twice at -week intervals and serum samples were tested by elisa. table survival of piglets from attenuated kpedv- vaccinated sows (v) and unvaccinated control (c) after challenge exposure % compared with before vaccination) in the farms having pedv outbreak [ ] . nevertheless, it is worthy to note that the ecacy of protection was rather complicated and con¯icting according to the challenge dose like other enteric diseases. although the pregnant pigs inoculated with attenuated virus showed the increased immune status by elisa as compared to uninoculated control, it is dicult to explain the exact relation to mucosal immunity for protection. since it was well con®rmed that the mechanisms of the passive immunity are extensively related to the presence of iga and igm antibodies [ ] , further experiments, including detection of antibody secreting cells of iga and igm, may give the practical information for immunoprophylaxis against pedv. an apparently new syndrome of porcine epidemic diarrhea some characteristics of a new porcine coronavirus and detection of antigen and antibody by elisa experimental infection of pigs with a new porcine enteric coronavirus cv porcine epidemic diarrhea virus as a cause of persistent diarrhea in a herd of breeding and ®nishing pigs propagation of the virus of porcine epidemic diarrhea in cell culture isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction porcine parvovirus: properties and prevalence of a strain isolated in the us procedures for identi®cation of arthropod borne viruses a new in vitro method (end) for detection and measurement of hog cholera virus and its antibody by means of eect of hc virus on newcastle disease virus in swine tissue culture. i. establishment of standard procedure morphology of bovine viral diarrhea virus cell adaptation and serological survey on porcine epidemic diarrhea virus (pedv) infection in korea use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection field trial of attenuated porcine epidemic diarrhea virus (kpedv- ) as vaccine development of an elispot for the detection of antibody secreting cells against the porcine epidemic diarrhea virus (pedv) in dierent tissues we thank joong-won park's technical assistance for the electron microscopy. we also appreciate the help of dr. t.w. molitor of university of minnesota, usa for his review and comments on this manuscript. this study was supported by the research grant from rda, ministry of agriculture, republic of korea. key: cord- -c aw jkz authors: privor-dumm, lois; vasudevan, prarthana; kobayashi, kana; gupta, jaya title: archetype analysis of older adult immunization decision-making and implementation in countries date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: c aw jkz the global population of adults over years of age is growing rapidly and is expected to double by . countries will face substantial health, economic and social burden deriving from vaccine-preventable diseases (vpds) such as influenza, pneumonia and herpes zoster in older adults. it will be essential that countries utilize several public health strategies, including immunization. understanding the different approaches countries have taken on adult immunization could help provide future learnings and technical support for adult vaccines within life-course immunization strategies. in this study, we describe the priorities and approaches that underlie adult immunization decision-making and implementation processes in high-and-middle-income countries and two territories (“ countries”) who recommend adult vaccines in their national schedule. we conducted an archetype analysis based on a subset of two dozen indicators abstracted from a larger database. the analysis was based on a mixed-methods study, including results from key informant interviews in six countries and a landscape review of secondary data from countries. we found four distinct archetypes: disease prevention-focused; health security-focused; evolving adult focus; and, child-focused and cost-sensitive. the highest performing countries belonged to the disease prevention-focused and health security archetypes, although there was a range of performance within each archetype. considering common barriers and facilitators of decision-making and implementation of adult vaccines within a primary archetype could help provide a framework for strategies to support countries with similar needs and approaches. it can also help in developing context-specific policies and guidance, including for countries prioritizing adult immunization programs in light of covid- . further research may be beneficial to further refine archetypes and expand the understanding of what influences success within them. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. the global population of adults over years of age is growing rapidly and is expected to double by . countries will face substantial health, economic and social burden deriving from vaccine-preventable diseases (vpds) such as influenza, pneumonia and herpes zoster in older adults. it will be essential that countries utilize several public health strategies, including immunization. understanding the different approaches countries have taken on adult immunization could help provide future learnings and technical support for adult vaccines within life-course immunization strategies. in this study, we describe the priorities and approaches that underlie adult immunization decision-making and implementation processes in high-and-middle-income countries and two territories ('' countries") who recommend adult vaccines in their national schedule. we conducted an archetype analysis based on a subset of two dozen indicators abstracted from a larger database. the analysis was based on a mixed-methods study, including results from key informant interviews in six countries and a landscape review of secondary data from countries. we found four distinct archetypes: disease prevention-focused; health security-focused; evolving adult focus; and, child-focused and cost-sensitive. the highest performing countries belonged to the disease prevention-focused and health security archetypes, although there was a range of performance within each archetype. considering common barriers and facilitators of decision-making and implementation of adult vaccines within a primary archetype could help provide a framework for strategies to support countries with similar needs and approaches. it can also help in developing context-specific policies and guidance, including for countries prioritizing adult immunization programs in light of covid- . further research may be beneficial to further refine archetypes and expand the understanding of what influences success within them. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. Ó elsevier ltd. all rights reserved. older adults are a heterogeneous group in the second half of life [ ] . studies demonstrate that vaccine-preventable diseases (vpds), including influenza, pneumonia and herpes zoster, account for a substantial portion of premature death and disability in older adults [ , ] . vpds also have the potential to cause disability that may lead to additional issues, such as declines in functional ability and quality of life [ ] . the economic burden of vpds is also substantial [ , [ ] [ ] [ ] [ ] [ ] [ ] . the global population of adults over the age of is growing rapidly and is expected to double by [ ] . although adults are expected to live longer, they are not necessarily living in good health [ ] . to be prepared for this demographic change, countries must establish plans and infrastructure that support healthy aging. aging populations will impact countries' economies, social security policies, and health systems, as well as affect many aspects of daily living for both the individual and broader society [ ] . at a national level, the consequences of an aging population extend beyond the health sector and solutions must be viewed across the life course [ , ] . it will be essential that countries utilize several strategies to ensure that their older populations age in a healthy manner, including adult immunization [ , ] . as they have for children, vaccines have the potential to significantly reduce burden of disease and disability, dependence, healthcare costs, and more in older populations [ ] [ ] [ ] [ ] [ ] [ ] . given the imminence of a growing, worldwide adult population, anticipation of increased health costs has spurred multiple global, https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. regional and national calls for action for policymakers and practitioners to prioritize adult immunization programs and improve uptake [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many high-and middle-income countries have adopted influenza vaccines [ ] , however few have adopted more than one vaccine for older adults or have comprehensive adult immunization strategies. countries that have adopted adult vaccines appear to have taken different pathways to policy adoption [ ] [ ] [ ] [ ] [ ] . further, to be prepared for healthy aging, countries must think holistically. they will need systems and an appreciation for prioritizing health in older adults to support delivery of vaccines and other crucial interventions [ , , ] . particularly in the context of covid- , preventing further strain on the health system is paramount [ ] . the global vaccine action plan [ ] and the immunization agenda [ ] call for a life-course approach to immunization, which some countries have begun to implement. despite similarities in disease burden, demographic profile, and geographic proximity, these countries have taken different approaches and achieved different results in adult vaccine adoption. to understand those differences and explain the factors influencing country decision-making and uptake, we conducted an archetype analysis. this analysis aims to describe the country priorities and approaches that underlie adult immunization decision-making and implementation. by characterizing groups of countries by features other than disease burden, geography or demographics, the analysis seeks to support global efforts to address country needs in strengthening processes for vaccine decision-making and implementation; facilitating sharing of best practices amongst countries with similar characteristics; and providing evidence, system or advocacy support to help countries succeed within their specific context. the archetype analysis does not replace the need for individual country strategies, but groups needs in a way that enables the global community to provide meaningful support across a broader group of countries. thirty-two countries and two territories (herein referred to as countries) were selected for analysis. countries selected had high proportions of older adults, were geographically diverse, and represented a range of potential archetypes based on their adult vaccine adoption status, financing models, degree of health system centralization, and vaccine coverage. all countries were included in a literature review and data abstraction. six countries (argentina, australia, canada, germany, japan, and the united kingdom (uk)) were selected for further qualitative research to provide additional depth in a diversity of contexts, approaches and performance. the insights gained in each case country helped characterize archetypes. each case country, to some degree, prioritizes adult vaccination and recommends and finances one to three adult vaccines, but varies in terms of government and/or healthcare system centralization and adult vaccine coverage rates. the united states (us) was not selected as a case study country, due to ivac's working knowledge of the american public immunization system and the abundance of publicly available peerreviewed articles and government documentation on older adult immunization. the study plan was reviewed by the johns hopkins bloomberg school of public health institutional review board and deemed to be non-human subjects research. domains (table ) were identified as part of a framework of potential barriers and facilitators for adult vaccine decisionmaking: country characteristics, adult vaccine/aging policies and decision-making, health immunization systems, uptake, and stakeholders and champions. these domains were the subject of key informant interviews conducted in six case countries and a concurrent landscape analysis of the countries. a series of indicators were subsequently identified, informed by previous research [ ] , an adult vaccine situational analysis [ ] , and the case study interviews. a team of abstractors conducted the indicator research. we searched peer-reviewed and grey literature, including government and professional society websites, disease burden and vaccine introduction and program status databases, reports from countries, the world health organization (who), non-governmental organizations (ngos), and media articles. we reviewed minutes from national technical advisory groups on immunization (nitags) as well as official recommendations and policies for both key informant interviews were conducted in in argentina, australia, canada, germany, japan and the uk. informants included vaccine experts, government and former government officials, healthy aging advocates, economists, and civil society. respondents were selected using a snowball approach. one-hour face-to-face interviews were conducted by - people using an interview guide. for each respondent type -technical respondents, economic respondents and health advocacy-focused respondentsunique guides were prepared to ensure the focus of the questions was on their area of expertise. a combination of open-ended questions and probes were used. topics included respondent background; country health priorities; key players and stakeholders; robustness of the process; drivers of decisions and uptake. we used scales of very important, moderately important, not important, and don't know/not sure to ascertain respondents' perceptions about drivers or degree of agreement with certain statements and to map responses. interviews were transcribed and entered into atlas.ti for mac os x (a qualitative data analysis software) to conduct a thematic analysis. no identifiers were included to keep the identities of respondents confidential. descriptive categories of their function (e.g., technical, economic, civil society organization, etc.), were included instead. based on the case study findings, we selected indicators that most differentiated countries and created a scoring of - to rate each country, with meaning that it fit the criterion well and that it did not fit the criterion and/or there was no data available to assess fit ( table ). each indicator was ascribed a score based upon a qualitative description. following validation, we found some of our scoring was insufficient in describing qualitative nuance and added an intermediate score of . to one indicator and . to four indicators. we scored all countries on indicators related to decision-making ( indicators) and implementation ( indicators). each country received a score and a ranking for both domains (i.e., decision-making and implementation). we recorded the scores in a table, organizing countries by pneumococcal vaccine coverage (high to low). we mapped all countries on a grid using graph pad prism version . . we used the case study insights to identify characteristics that could describe the primary driver of a country's approach to decision-making and implementation (the ''archetype"). based on the qualitative insights, we found four distinct archetypes and placed each country into one archetype. although countries could fit in more than one archetype, we selected the archetype that most closely fit each country's primary driver. thus, country performance does not define each archetype's description. we transcribed each country's data into individual country profiles, providing the qualitative description associated with each quantitative score. we asked respondents to indicate their level of agreement (agree/disagree) with each score. we requested the respondents to identify missing data or inaccurate scores, that if corrected, could substantially move a country's score higher or lower. if there was disagreement, we asked respondents to provide an explanation and publicly available source(s) that support the change they suggested. we reached out to - experts per country, including a ministry of health representative, wherever possible. we provided a slide set describing the project, including objectives, methodology and the archetype map to enable countries to provide meaningful input. countries that responded are listed (table ). thirty of the countries analyzed had more than % of their total population adults over years of age. twenty-four countries had over % of their population over years of age. high-income countries and upper middle-income countries, had older populations than lower-middle and low-income countries (table ), but the age of populations did not predict the number of vaccines adopted for their adult populations nor uptake of influenza or pneumococcal vaccine (fig. ). one hundred and twenty key informant interviews were conducted in the six case countries, including a range of respondents covering health, immunization, government policy, aging and economics. overall, respondents in australia and the uk had the greatest degree of confidence in their country's ability to make decisions about new vaccines for adults and implement programs. the uk respondents attributed their country's ability to make decisions on the broadest variety of factors and only the influence of advocacy and bringing a variety of perspectives into decision-making was ranked lower. respondents in canada expressed a high degree of confidence in their country's ability to make decisions but reported lower confidence in their government's level of priority for adult vaccines, access to providers and financing. germany had similar perceptions to canada in that the decision-making process was strong, but respondents questioned the ability to implement, ease of access and the variety of advocacy efforts. argentine and japanese respondents had lower composite ratings on their perception of their country's capability to make decisions and implement adult immunization than the other countries but rated their country's commitment to adult health and the variety of perspectives highly. argentine respondents also rated their nitags as capable. both argentine and japanese respondents had less confidence in ease of access, financing, surveillance for adults. we also noted varying approaches to how cost-effectiveness (c-e) data were used both amongst case study countries and other countries that the respondents described. a few countries, including the uk, used c-e thresholds in their adoption decisions. other countries considered c-e data but did not use it. in some countries, childhood vaccination was viewed as a higher priority, particularly for funding. some respondents in germany even went as far as to state that putting adult health before child health would be unethical. in japan, we saw no evidence of c-e studies used in their nitag decisions, although safety studies were a requirement (fig. ). similar to decision-making, factors influencing implementation varied by country (fig. ). in the case countries, we saw table scoring. early or late adopter of adult vaccines = no or late decision-making and adoption of either pneumococcal or herpes zoster vaccine = follower in decision-making and adoption of at least one vaccine = leader in decision-making and adoption of one or more vaccines country-specific policy requirements of manufacturers = multiple = one = none disease burden surveillance = no surveillance = some surveillance (mostly of flu and pneumococcal disease) = national surveillance of flu, pneumococcal disease, and herpes zoster nitag's prioritization of health security in decision-making = small to no priority on health security, or no evidence available = nitag considers health security, but it is not a main driver of decisions = nitag considers health security as a main driver of decisions nitag's utilization of cost-effectiveness (c-e) data in decision-making = small to no focus on c-e, or no evidence available = nitag considers c-e data, but it is not a main driver . *= nitag mostly considers c-e data as a main driver = nitag considers c-e data as a main driver nitag has adult vaccine working group(s) = such working groups . * = nitag has such working groups, but is involved in other recommending bodies where government is engaged = such working group = multiple such working groups (as part of a broader vaccine-specific working group or a standalone) public policy -pneumococcal vaccination for older adults = pcv or ppsv not recommend, unknown if considered by nitag = pcv or ppsv considered by nitag, but not recommended . * = pcv or ppsv was recommend by nitag, but not yet implemented = pcv recommended by nitag public policy -herpes zoster vaccine (hzv) for older adults = hzv not recommended, unknown if considered by nitag = hzv considered, but not recommend by nitag . *= hzv recommended by nitag, but not yet implemented = hzv recommended by nitag publication of health aging strategies = no healthy aging strategy publicly available = aging strategy available at the sub-national or national level . *= sub-national or national aging strategy available that mentions adult immunization, but is over ten years old = national aging strategy available that mentions adult vaccines publication of national immunization strategies = no immunization strategy publicly available = only pediatric immunization strategy publicly available = national immunization strategy published and covers both pediatric and adult vaccines vaccine financing -level of public financing (for each vaccine) = older adults must pay out of pocket = vaccine covered by private insurance, requires co-pay, or limited coverage is provided in certain geographic areas or at-risk populations . *= mixed system of payment (covered) = vaccine is fully funded by the government for all vaccine registry (for pediatric and adult populations) = countries were assessed on their adult vaccine implementation and decision-making. decision-making was scored upon indicators and implementation upon indicators. each indicator was ascribed a quantitative score, as described above. recommendations were not universally implemented (particularly in canada and germany). the exceptions were the uk, which has a very centralized immunization program, and australia, which was decentralized but took a more centralized approach to monitoring and promotion. access to immunization was a factor reported to influence older adult immunization uptake. lower uptake may be due to limited mobility or lack of awareness of the need for adult vaccines. in some countries, such as germany and japan, respondents stated that vaccines were still viewed as something only for children. additionally, health system complexity was reported as an important factor contributing to access, and ultimately uptake, with some respondents describing receiving vaccination as an older adult as an overly cumbersome process. in canada, respondents' general perception was that there are not many places or providers of adult vaccination. restrictions on who could administer vaccines was also reported as a perceived barrier. improvements in access were described in japan, where nurses can now vaccinate; in france, where pharmacists can offer influenza vaccines; and in the uk, where pharmacists were allowed to administer influenza and pneumococcal vaccines. despite some countries expanding their range of adult vaccine providers, respondents noted that uptake does not always correspondingly increase. this gap offers a role for advocacy efforts in improving uptake. the influence of advocacy can be difficult to assess and is sometimes subjective. nonetheless, we used the number of national advocacy organizations as a proxy for influence in database countries. in case countries, we were able to gather insights. each respondent was asked about advocacy efforts in their country, yet there was a wide range of level of familiarity of country efforts. australia is a clear leader and uses a variety of approaches to advocate for adult immunization. australian respondents described influential advocacy efforts that impacted both the national adult vaccination decision-making and implementation processes. for example, in the country citizens, government, medical societies, and health aging specialists have organized large groups to advocate for better recommendations and financing as well as influence uptake and equal access to vaccines. in japan, respondents were not familiar with any major advocacy effort, although could describe small-scale patient-advocacy groups or activism against the government regarding vaccine injuries. based on the findings from the case studies, ten indicators emerged as descriptors of countries' older adult immunization decision-making capacity, specifically whether countries: were early adopters; had country-specific barriers such as requirements for technology transfer (brazil, india, japan, korea); preferences for indigenous product (china, indonesia, brazil, india, russia); halal vaccines (malaysia); safety study requirements (japan); or c-e requirements (uk, netherlands); prioritized surveillance on vpds in older adults (emerging as a factor in many of the countries concerned about health security); were prompted in their vaccine decisions by health security concerns; considered c-e a major driver in vaccine decisions; had working groups in their national immunization technical advisory groups (nitags) specific to adult vaccine issues; adopted pneumococcal conjugate vaccine (pcv) ; adopted herpes zoster vaccine (hzv); published a national healthy aging policy (including whether there is a mention of vaccines); and included adult vaccines in their national immunization policy. for implementation and uptake, indicators that emerged included whether there was government financing for influenza vaccines, pcv and hzv; centralized vaccine registries for children and adults; coverage data for all three vaccines; advocacy (measured by the number of advocacy organizations); documented influence of organizations or leaders; access (easy to get vaccinated by expanded list of providers or lack of bureaucracy or cumbersome process); equity focus; centralization of the adult vaccine program; and centralization of the health system. few countries that had strong decision-making also had strong uptake and vice-versa. further, the scoring table shows that countries varied significantly in the indicators that drove their performance on adult vaccine decision-making (table ) and implementation (table ). to determine how composite scores corresponded to performance, we ordered countries based on their pneumococcal vaccine uptake and compared that to rankings of the composite score on both decision-making and implementation/uptake. countries with the highest coverage of pneumococcal vaccines did not always perform the highest on decision-making, with the exception of australia, us, uk, and canada. we did a similar exercise for influenza vaccine uptake and found that top ten countries with highest influenza coverage were similar to those with the highest decision-making and implementation scores. the us had the highest ranking, compared to other countries, and scored points when assessed for the robustness of policies and decision-making. australia (score: . ), the uk (score: . ) and italy (score: ), had the next set of highest scores. countries with the least robust policies and decision-making were denmark, table countries responding to validation survey. received survey results responded with feedback the results of the archetype analysis was shared with experts representing all countries. countries responded and participated in the validation process. india, peru, the philippines, switzerland, and saudi arabia (all ranking last; scoring: ) (see table ). when assessed for the promotion of implementation and uptake, the uk (score: out of a possible ), new zealand ( ), and australia ( ) had the highest rankings compared to other countries. in contrast, russia (score: ), peru ( ), and india ( ) had the lowest ranking (see table ). based on a synthesis of case study data, we found four distinct archetypes with unique characteristics: ''disease preventionfocused"; ''health security-focused"; ''evolving adult focus"; and ''child-focused and cost-sensitive" (fig. ) . some countries could belong to multiple archetypes, but we selected the archetype most closely aligned with primary driver of approach. we also noted that the highest performing countries in terms of both decision-making (australia, us, uk, canada, italy, netherlands, germany and mexico) and implementation (uk, new zealand), australia, us, korea, canada, italy and norway) belonged to either the ''disease prevention-focused" or ''health securityfocused" archetype (see fig. ). disease prevention-focused: countries in the ''disease prevention" archetype included canada, france, germany, netherlands, uk, and us. these countries valued the use of data and process. most countries' nitags in this archetype had considered most of the adult vaccines and performed fairly high on decision-making. we noted use of their own disease burden/impact evidence in decision-making, as well as use of other countries' data. most of these countries had their own adult surveillance and formal adult vaccine working groups on their nitag. the uk and the netherlands also placed high importance on economics. most countries considered economics, but it was not a primary driver. germany, and to a lesser extent the us, considered economics, but disease burden was the primary driver in decisions. there was significant variation in implementation performance. reasons varied within this archetype, and included lack of national adult registries, equity focus, sufficient advocacy and centralization. health security-focused: the ''health security" archetype was the largest of the four and also the most diverse in terms of performance. it included argentina, australia, china, greece, hong kong, italy, japan, mexico, new zealand, saudi arabia, taiwan, and turkey. the characteristic of this archetype is that outbreaks (h n in australia and argentina; pneumonia in japan), vpd threats (due to migration or in refugees), and natural disasters (tsunami in japan) were viewed as an important country motivation for action. we saw wide variation of performance within this archetype, with australia, italy, new zealand and mexico performing highest and china, japan, hong kong, taiwan and greece lagging behind. degree of centralization and registries contributed to performance. australia, for example, reported that data use was strengthened during the h n outbreak in . in argentina, surveillance and epidemiologic response was also strengthened as a result of an h n outbreak. it also led to strategies to establish mass vaccination centers to improve access to vaccines more widely. in , the great east japan earthquake struck northeastern japan and destroyed the local healthcare system. at the time, pneumococcal vaccine coverage in japan was only % and many healthcare workers feared the occurrence of pneumonia outbreak at evacuation shelters [ ] . to avoid future outbreaks, pneu-mococcal vaccine was provided free of charge in the three prefectures most affected by the earthquake. in , they reached record levels of uptake with miyagi and iwate at %, and fukushima at % coverage [ ] , leading to significant reduction in deaths attributed to pneumonia in the area [ ] , and eventually to the ministry of health, labour, and welfare's decision in to include pneumococcal vaccine to the routine immunization schedule for adults aged and older. the national coverage increased from % in to % in [ ] . evolving adult focus: countries in the ''evolving adult focus" archetype include belgium, brazil, colombia, ireland, korea, spain, sweden and malaysia. these countries had moderate to strong systems and decision-making ranged from weak (denmark, malaysia) to moderate (korea, belgium). many of the countries in this archetype lack a strong nitag for adult vaccine decisions, but exhibit some elements of the ''disease prevention" archetype. some have healthy aging policies or immunization strategies, but only brazil has both. some countries were early adopters for some adult vaccines, including pcv in ireland and belgium, and hz vaccine in uae and norway. belgium has published an adult immunization strategy recently [ ] . financing for recommended adult vaccines, varies as well. although vaccines were recommended in some countries, they were not always publicly financed or financed for risk groups. child-focused and cost-sensitive: finally, some countries, including russia, peru, india, switzerland and the philippines remain ''child-focused and cost-sensitive" in their public markets. the countries in this archetype have not prioritized adult immunization programs and have generally lacked focus on decisionmaking for older adults. we found no adult vaccine working groups on the three vaccines analyzed (influenza, pneumococcal and herpes zoster) or policies around adult immunization at the time of our study. in terms of implementation, these countries may require patients to pay out of pocket for adult vaccines (russia, india) although there were some exceptions for influenza vaccine like the philippines and switzerland (although in switzerland vaccines were covered through insurance). we did not find a focus on implementation. these five countries have no registries or policies around adult health or adult immunization. additionally, advocacy for adult immunization was not strong and, in most cases, seemed to have little influence on the outcomes of the government. lastly, for most countries in this archetype, there is a cost-based argument: given limited resources, public investments in child health and vaccines are prioritized, as they are seen as necessary to further national economic development (russia, peru, india, philippines). we were able to validate our data and scoring in of countries ( %) ( table ) . where there was disagreement and documented sources, we corrected data and in five instances, adjusted scoring to reflect a more accurate picture of the parameters which had to do with timing of decisions or policies, the importance of c-e data in decision-making, nitag working groups, and a mixed financing system. importantly, the act of classifying countries into a primary archetype has its own set of limitations. firstly, there is a level of subjectivity that comes with archetyping, which we have tried to ameliorate by taking an evidence-based approach. secondly, archetyping elevates a certain set of common characteristics above others and there are variations between and within countries that get under-accounted. countries may not fit perfectly in an archetype and may actually belong to other archetypes simultaneously; we therefore classified countries into primary archetypes, according to the indicator that they scored the highest on. our analysis was also limited by data availability and quality. in some cases, a zero score was given due to a lack of data or clarity on an indicator. we tried to address this through the validation process, but little additional information was provided. additionally, each indicator's scoring was based only on data that was publicly available through october (unless otherwise provided by experts during validation) and may be impacted by timing. changes in scoring of - points is unlikely to impact score mapping, but larger changes may shift archetype categorization. archetypes can be useful in identifying factors that are most influential in improving decision-making and implementation for adult vaccines in the context of a country's approach and priorities. this will enable various countries to learn from experiences amongst countries within the same archetype. there has been some use of archetypes, mainly in health technology assessment [ ] [ ] [ ] [ ] [ ] , but experiences have not yet been widely documented. the archetype describes the primary driver of decisions and implementation as well as country priorities or approach rather than performance. there can be significant variation in perfor- mance within an archetype, which we saw in this exercise. top performing countries belonged to two different archetypes, which were ''disease prevention-focused" (us, uk, canada) and ''health security-focused" (australia, italy), suggesting that there are multiple ways to achieve adoption and uptake. it is likely that countries within the ''evolving adult focus" archetype could improve their performance by learning from countries in the ''disease prevention" or ''health security" archetypes. the improvements needed within the ''child-focused" archetype may require both stronger advocacy as well as additional data. while we provide broad-based archetypes, we also saw different approaches within the archetypes; this is important as each country will need to take lessons based on what works for them. understanding the success factors of the highest performing countries within a particular domain (e.g., decision-making) can also help other countries move up their performance in the context of their structure and priorities. the countries in the ''disease prevention" archetype all value data and its use, albeit to different levels and strategies can focus on data use and advocacy for disease prevention. at the one extreme of this archetype is the uk, where data use permeates through the process of both decision-making and implementation. decisions are evidence-based, with little influence of advocacy or champions. on the implementation side, there was also strong use of data with information on the immunization status of individual patients, which is all mapped to their general practitioner (gp). for countries who will consider a centralized approach, the uk provides a good model. however, many countries have decentralized systems, including the us and canada, who also have strong use of data for decision-making. they also have greater degrees of advocacy, which could perhaps also ensure certain groups have a voice in the process. use of data for implementation in decentralized settings may also benefit from learnings from table for score descriptors. generally, the higher the score the better that country meets the indicator; scores either mean the country doesn't meet the indicator or no data were found. countries that have centralized registries in a decentralized system (e.g., australia, although a different archetype). health security concerns provide countries with an opportunity to overcome a wide array of issues, from financing to barriers that may slow down decisions (e.g., tech transfer requirements as seen in brazil or requirements/strong preference for local products such as in china or india, requirements for local studies as in japan, etc.) and may be a motivator to strengthen surveillance, use of data, communications and platforms. the current context of covid- highlights likely opportunities to leverage the importance of developing a strong system. in doing so, it is important for countries to consider how all people, including older adults and/or marginalized populations such as migrants or refugees, can access vaccines. for example, in the us, adult immunization access increased as a result of the influenza an h n pandemic, with states granting pharmacists the authority to vaccinate against influenza [ ] . also, during that pandemic, a study demonstrated that american indian and alaska native persons have a higher risk for death from the h n influenza [ ] supporting prioritization of seasonal influenza vaccines amongst native americans that year [ ] . we saw wide variation of performance within this archetype and note that while emergencies may provide motivation to take action, other table for score descriptors. generally, the higher the score the better that country meets the indicator; scores either mean the country doesn't meet the indicator or no data were found. contextual factors must be taken into account. degree of centralization of adult immunization is one important factor that influences success of implementation. registries and stronger use of data are an important element in both ''health security" and ''disease prevention" archetypes, but the motivator to get them done may be different -in times of emergency, data are essential, but it is prior to an emergency that data are needed [ ] . after the severe acute respiratory syndrome (sars) epidemic, china was challenged to improve its public health emergency management systems (phems) [ ] . the system was significantly improved within ten years after the outbreak [ ] . more recently, china was able to contain covid- within the country [ ] , but the virus has caused more than . million cases and , deaths worldwide and continues to spread as of early april [ ] . there is now more than ever a strong need for systems to detect risk, transparently communicate the risk, and have infrastructure in place to address both existing and emerging infectious threats [ ] . engaging civil society and building accountability mechanisms can help motivate action more urgently. this current pandemic provides a window of opportunity to build urgency for the need for adult immunization and systems to deliver them. efforts, however, must not focus solely on covid- , but on the platforms needed to build strong health systems for all ages, including older adults. in countries that have taken action in the face of an emergency this message will resonate; for others, particularly without the resources or capacity to address older adults, different approaches may be needed to build focus on the needs of this group. in countries with an evolving adult focus, a strategy could be to emphasize nitag strengthening and sharing experiences with the ''disease prevention" and/or ''health security" archetypes. this will be particularly important should a vaccine become available for covid- and in considering vaccines that can address vpds in an older population now. in countries who follow other countries' recommendations, stronger global guidance is needed to emphasize preparedness, highlight the importance a growing adult population, and the consequences of doing nothing. in countries that need to improve uptake, access, financing, registries, and monitoring are all important. one factor that correlates to uptake is expansion through pharmacists [ ] , although with covid- , perhaps other ways of delivery that don't require contact with people could be considered. additional provider-focused strategies could be implemented to improve uptake of current vaccines, such as financial incentives to vaccinate older adult patients. proactive outreach (e.g., actively sending reminders to patients to get their vaccines) may also be important post-pandemic to remind patients of vaccines' importance. finally, even in the ''child-focused and cost-sensitive" archetype there may be opportunities to take advantage of synergies and address immunization through broader issues such as universal health coverage and antimicrobial resistance. central to these opportunities is building the links between child and adult health, and economic development [ ] . the economic pressure placed on countries through covid- may make adult immunization seem unreachable and studies that capture the broader value of vaccines may help in justifying investments [ , ] . stronger global guidance synthesizing healthy aging, universal health coverage, antimicrobial resistance and immunization recommendations and agendas can also be helpful. importantly, strong individual champions, coalition building across the vaccine and aging communities, and more resonant messaging could all help elevate routine older adult vaccination as a priority [ ] . a critical, momentumbuilding milestone would be when global institutions, like who, commit to leading the coordination of implementation [ ] , empowering countries to think about implementing older adult immunization in a more timely manner. combining two separate analyses, this graph plots countries' performance in implementation and decision-making as a point estimate, overlaid with each country's primary archetype denoted by a symbol (within an archetype, countries share similarities around vaccine decision-making; (see table ). countries are plotted according to their adult vaccine implementation score (see table ) on the x axis and their adult vaccine decision-making score (see table ) on the y axis. for example, the uk scored in implementation and . in decision-making, and is plotted at ( , . ) . some countries share the same coordinate, with two names next to a single point. countries with the highest implementation and decision-making scores appear in the top right corner of the graph. primary archetype (disease prevention focused; health security focused; evolving adult focus; or cost-sensitive and child focused) is designated by the color of the country's name and the symbol overlaid each point. most countries (n= ) belong to the health security primary archetype. within and across all four archetypes, vaccine confidence plays a role as both a facilitator and barrier of countries' performance. although fear of side effects, spread of misinformation, lack of provider recommendation, and reduced belief that vaccines are valuable are common reasons for hesitancy across all ages, a recent review of the literature yielded few insights into describing how adult vaccine hesitancy varied from that for children [ ] . this is an area of further research that may help explain country uptake and better address implementation gaps. specifically, once the research gap is more clearly described, tailored communication and outreach strategies could be developed that encourage older adults to seek immunization services. countries take different approaches to adult immunization. drivers and facilitators of primary adult immunization archetypes should be considered when developing global guidance for countries. experiences and lessons learned should be shared within archetypes to improve performance of countries falling behind on decision-making or implementation. the results of this study may inform strategies in countries with similar contexts and priorities. further research may be beneficial to further refine archetypes and expand the understanding of what influences success within an archetype. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: lpd has received grants from glaxosmithkline, merck and pfizer and an honorarium from pfizer. the other authors declare no competing interests. the world report on ageing and health: a policy framework for healthy ageing global, regional, and national disability-adjusted life-years (dalys) for 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time to realize an unfulfilled potential national vaccine advisory c. a pathway to leadership for adult immunization: recommendations of the national vaccine advisory committee: approved by the national vaccine advisory committee on focusing on the implementation of st century vaccines for adults european geriatric medicine society (eugms) and the world association for infectious diseases and immunological disorders (waidid) first mexican consensus of vaccination in adults adult vaccination: a key component of healthy ageing -the benefits of life-course immunisation in europe a global review of national influenza immunization policies: analysis of the who/unicef joint reporting form on immunization adult immunization policies in advanced economies: vaccination recommendations, financing, and vaccination coverage variation in adult vaccination policies across europe: an overview from venice network on vaccine recommendations, funding and coverage challenges in adult vaccination vaccine myopia: adult vaccination also needs attention what isn't measured isn't done -eight years with no progress in aboriginal and torres strait islander adult influenza and pneumococcal vaccination a blueprint for improving the promotion and delivery of adult vaccination in the united states the world health organization. the global vaccine action plan the world health organization. immunization agenda : a global strategy to leave no one behind evidence-to-policy gap on hepatitis a vaccine adoption in countries: literature vs. policymakers' beliefs situational assessment of adult vaccine preventable disease and the potential for immunization advocacy and policy in low-and middle-income countries an activity report of physicians of the department of general medicine, juntendo university school of medicine against the great east japan earthquake relationship between public subsidies and vaccination rates with the -valent pneumococcal vaccine in elderly persons, including the influence of the free vaccination campaign after the great east japan earthquake pneumococcal vaccine (ppsv ) advies -basisvaccinatieschema development of archetypes for non-ranking classification and comparison of european national health technology assessment systems different paths to high-quality care: three archetypes of topperforming practice sites segmentation of seven asia-pacific health technology assessment (hta) agencies into different evolutionary hta archetypes integrating pharmacies into public health program planning for pandemic influenza vaccine response deaths related to pandemic influenza a (h n ) among american indian/alaska natives - states prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the public health emergency management system in china: trends from evaluation of the effectiveness of surveillance and containment measures for the first patients with covid- in singapore coronavirus resource center early response to the emergence of influenza a (h n ) virus in humans in china: the central role of prompt information sharing and public communication covid- : too little, too late? pharmacy-based interventions to increase vaccine uptake: report of a multidisciplinary stakeholders meeting life-course immunization: building the consensus for adult vaccination in ifpma generation of political priority for global health initiatives: a framework and case study of maternal mortality characterizing global vaccine hesitancy: how adult and pediatric hesitancy may differ glaxosmithkline (gsk) biologicals, belgium provided funding for the archetype analysis. merck and gsk provided funding for the landscape analysis and case studies. the authors thank nina martin, geervani daggupati, nobutoshi nawa, so yoon sim, dexter waters, and gatien de broucker, for their valuable contributions; members of the international council on adult immunization (icai) for their review and input; the in-country experts who participated in case study interviews and provided input in the validation process; and haley budigan for her careful proofreading. key: cord- -jdinf od authors: thindwa, deus; garcia quesada, maria; liu, yang; bennett, julia; cohen, cheryl; deloria knoll, maria; von gottberg, anne; hayford, kyla; flasche, stefan title: use of seasonal influenza and pneumococcal polysaccharide vaccines in older adults to reduce covid- mortality date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jdinf od nan sars-cov- that causes covid- has emerged as a pandemic with all continents now reporting cases, most of them community acquired [ ] . many covid- infections cause pneumonia and some are fatal, predominantly among older adults [ ] . co-infection with other viruses or bacteria, particularly those that similarly cause inflammation of the respiratory tract would likely enhance the risk for severe covid- disease. such disease enhancing coinfections have been frequently reported for respiratory pathogens [ ] [ ] [ ] , most notably so for the influenza pandemic [ , ] . vaccinating older adults at elevated risk of severe covid- disease against vaccine preventable diseases may therefore not only help to reduce the strain on the healthcare system from those diseases during a pandemic, but also alleviate some of the potential covid- mortality due to co-infecting pathogens [ ] . vaccines that can prevent respiratory tract infections in adults and particularly in older adults, either through direct protection or indirectly through high coverage childhood immunisation programmes, include vaccines against seasonal influenza, streptococcus pneumoniae, measles, bordetella pertussis and haemophilus influenzae type b (hib). measles, pertussis and hib vaccines are already included in almost all routine infant immunisation programmes globally and have largely eliminated the targeted pathogens as a risk to the older adult population through indirect protection [ ] . hence, they have limited scope for use in older adults in order to limit covid- morbidity and mortality. pneumococcal conjugate vaccine (pcv), either -or -valent, is used in three-quarters of routine infant immunisation programs globally; in countries that use pcv, the burden of adult pneumococcal disease due to pcv serotypes has also substantially decreased [ ] . these considerations mean there is a relatively small preventable disease burden in countries routinely using pcv in children. as vaccine costs are relatively high and there is no current world health organisation recommendation regarding pcv use in adults, pcvs are not considered further here. two vaccines that target a large burden of the remaining respiratory disease in older adults are seasonal influenza vaccines and -valent pneumococcal polysaccharide vaccine (ppv ). these vaccines are only included in routine adult immunisation in some countries and even there with only moderate coverage [ ] . we conducted a non-systematic review of the published, pre-print and grey literature to evaluate whether vaccination of older adults with seasonal influenza vaccine or ppv could help reduce covid- mortality. the world health organization recommends seasonal influenza vaccine use for pregnant women as well as older adults (> yrs), health care workers and persons with specific chronic illnesses (particularly hiv) [ ] . in , % of countries globally had established a seasonal influenza vaccine programme that targets older adults, hardly any of them are in low or middle income countries [ ] . seasonal influenza as a risk factor for covid- could be an important consideration in tropical climates and the southern hemisphere, and potentially during future waves of covid- in the northern hemisphere [ ] [ ] [ ] . inactivated influenza vaccine effectiveness varies markedly by season from about % in seasons with a poor match to the circulating strains to up to % for closer matched seasons [ , ] . hence, influenza vaccination could prevent % to % of influenza infections and thereby potentially a similar percentage of influenza-attributable covid- morbidity and mortality (table ) . a number of studies to date have tried to assess the percentage of influenza-attributable covid- morbidity and mortality. some reported the occurrence of co-infection of covid- inpatients with influenza viruses, although the proportion of co-infections varies by study, from no influenza coinfection identified to more than % of pcr positive covid- patients being currently or having been recently infected with influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] . whether this co-occurrence is co-incidental, or indeed influenza contributes to the clinical severity of covid- presentation is not yet clear. however, to date there is no evidence that would suggest clinical manifestations in covid- patients with influenza co-infection differ from those without co-infection [ ] . ppv targets of the over serotypes that are responsible for most adult pneumococcal disease. in countries with an infant pcv program, about - % of invasive pneumococcal disease (ipd) in older adults is caused by serotypes that ppv is effective against [ ] ; in countries without an infant pcv programme this percentage is likely about % higher (details see appendix). ppv is recommended for routine use in older adults in most high-income countries, but rarely in low and middle-income countries [ ] . it provides short-term protection against ipd caused by vaccine serotypes in healthy older adults with a pooled efficacy across targeted serotypes of about % [ ] [ ] [ ] . however, ppv 's effectiveness is much lower among high risk groups including the immunocompromised [ ] [ ] [ ] , who may be at particular risk for severe covid- . consequently, ppv use in older adults could prevent up to - % of pneumococcal disease and thereby potentially pneumococcal-attributable covid- morbidity and mortality ( table ) . the extent of pneumococcal-attributable covid- morbidity and mortality is largely unknown. pneumococci have been identified as a major source for often fatal secondary bacterial infections during pandemic and seasonal influenza infections. estimates for the proportion of pneumococcal co-infections among pandemic influenza deaths range from about % during the h n pandemic to more than % during the pandemic [ , [ ] [ ] [ ] [ ] . few studies so far have tried to identify bacterial co-infections among covid- cases, and those that did found very few bacteria and only a limited number of cases with pneumococci [ , , ] . this may be due to the empirical treatment with antimicrobials for the majority of severely ill suspected covid- patients, or because bacterial infection plays little role in the severity of covid- disease [ , ] . however, elevated procalcitonin levels, a sensitive but not very specific biomarker for bacterial infections, have been reported in % of severe and % of fatal covid- infections, but largely absent in covid- infected persons with less severe outcomes, which may suggest some role for bacterial coinfection [ , ] . attending a vaccination clinic during the covid- pandemic will likely come with an excess risk of sars-cov- infection. this risk may be small, particularly if physical contact reducing interventions are implemented. to illustrate the potential magnitude of such excess risk, we assume a reasonably high covid- burden scenario: contact reducing interventions can be implemented and upheld to substantially slow covid- spread but not contain it, so that after months and in the absence of a covid- vaccine herd immunity will end the outbreak [ ] . assuming a basic secondary attack rate of r = . and that contact-reducing interventions spread the covid- infection risk equally across that -month time period, the increase in risk for covid- acquisition attributable to the vaccination clinic visit would be roughly . - . %, depending on the effectiveness of transmission-reducing measures during the vaccination visits (details see appendix) [ ] . this implies, that if either seasonal influenza vaccine or ppv reduces covid- morbidity and mortality by a similar amount or more, their benefit on covid- alone would outweigh the risk associated with the vaccination visit in this scenario, while also preventing morbidity from influenza and pneumococcal disease. both seasonal influenza vaccine and ppv can prevent a substantial burden of targeted disease and mortality among older adults and adults at-risk. despite a potential collateral reduction in influenza and pneumococcal circulation due to contact reducing interventions, in countries where the covid- pandemic coincides with the season of high risk for pneumococcal and/or influenza disease, vaccination at high coverage will have added benefits: minimising the number of pneumococcal and influenza hospital admission reduces the resources needed to care non-covid- patients and minimises the risk of health-care acquired covid- infection. for influenza, the similarity of symptoms with covid- cases also suggests that vaccination will increase the specificity of syndromic covid- surveillance and interventions. similarly, maintaining high vaccine coverage of existing pcv and live attenuated influenza vaccine programmes in children reduces the associated disease burden in older adults through herd effects, and will further enhance benefits for limiting covid- risks. is probably relatively small, although at present we cannot exclude the possibility of either preventing a considerable amount of covid- related mortality (table ). this uncertainty highlights the importance for detailed monitoring and additional studies where possible, in both high and low income settings. the proportion of vaccine preventable covid- morbidity and mortality could be assessed, for example, by post mortem examinations or test-negative casecontrol studies [ ] . in summary, where already in routine use among older adults and/or adults at-risk, maintaining both seasonal influenza and ppv at high coverage have the potential to not only reduce the burden of the targeted diseases but also prevent a proportion of covid- morbidity and mortality, if they can be delivered while minimising the risk for sars-cov- transmission. however, for countries who previously decided that seasonal influenza vaccine or ppv programmes for older adults are not a priority, there is currently little evidence to encourage implementation of either during the covid- pandemic solely for the purpose of reducing covid- mortality. attributed over the number of contacts during the infectious period e.g. . transmissions during the two days before symptom onset and self-isolation, with contacts per day before isolation). then the probability for sars-cov- infection during that vaccine clinic visit is p = -( - %) #contats * % = . to %. hence, the excess absolute risk for sars-cov- infection over the pandemic baseline risk is p*( - %) and corresponds to a relative risk increase over the pandemic baseline risk of . to . %. coronavirus disease (covid- ) estimates of the severity of coronavirus disease : a model-based analysis vaccine trialist group. a role for streptococcus pneumoniae in virus-associated pneumonia bacteriology and histopathology of the respiratory tract and lungs in fatal asian influenza viral and bacterial interactions in the upper respiratory tract the role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(h n )pdm interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness immunization in the context of covid- pandemic. geneva: world health organisation burden of streptococcus pneumoniae and haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for - world health organisation. who | data, statistics and graphics | vaccine introduction slides could enhanced influenza and pneumococcal vaccination programs help limit the potential damage from sars-cov- to fragile health systems of southern hemisphere countries this winter? vaccines against influenza who position paper a global review of national influenza immunization policies: analysis of the who/unicef joint reporting form on immunization potential impact of co-infections and co-morbidities prevalent in africa on influenza severity and frequency: a systematic review influenza-associated hospitalization in a subtropical city influenza in tropical regions efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis seasonal flu vaccine effectiveness studies | cdc the clinical characteristics of pneumonia patients coinfected with novel coronavirus and influenza virus in wuhan precautions are needed for covid- patients with coinfection of common respiratory pathogens early release -co-infection with sars-cov- and influenza 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epidemics in african settings: a mathematical modelling study risks and benefits of sustaining routine childhood immunisation programmes in africa during the covid- pandemic analysis proposals for test-negative design and matched case-control studies during widespread testing of symptomatic persons for sars-cov we would like to thank adam l. cohen the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the discussions in this paper. the pserenade project aims to understand the impact of pcv / on invasive pneumococcal disease (ipd) incidence and serotype distribution using ipd data contributed by surveillance sites across the world. in settings with mature pcv / infant immunization programmes, the estimated proportion of ipd in older adults (≥ years of age) attributable to ppv serotypes is - % for countries using pcv in infants and - % for countries using pcv . excluding st (ppv -minus-st ), these proportions drop to - % and - % respectively. because effectiveness against st has not been observed in all countries, the proportion of ipd in older adults attributable to ppv -minus-st is used here as a more conservative estimate of preventable disease. in the time period prior to pcv use in children, the estimated proportion of ipd in older adults attributable to ppv -minus-st serotypes was about % higher; this can be a proxy for countries without an infant pcv programme.however, most of the data included comes from high-income countries and may not be entirely representative of the serotype distribution in countries without a pcv programme, which are mostly low and middle income. assume that contact reducing interventions spread the risk of covid- out roughly equally over a month period. this scenario is in line with model predictions for sars-cov- spread in the presence of a % reduction in contacts for the duration of months. assuming an r of . and a duration of infectiousness of week means that about % of the population will have been infected at the end of the pandemic and that at any one time about % of the population is infectious. further, assume that an adult attends a vaccination clinic and makes a total of - contacts (hereby may reflect a scenario of effective measures to avoid contacts) related to that visit and that the probability of infection per potentially infectious contact is about % (r key: cord- -ipa wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ipa wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [ ] high-dimensional cell subset immunophenotyping through cytof, [ , ] and vaccine induced metabolic changes [ ]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [ , ] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [ ] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [ ] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [ ] [ ] [ ] [ ] , ] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [ ] [ ] [ ] [ ] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [ ] [ ] [ ] [ ] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [ ] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a unit change in x i Ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. ) [ ] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [ ] , mycobacterium leprae [ , ] , human immunodeficiency virus [ ] , and measles [ ] [ ] [ ] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain % of the inter-individual variability in humoral response [ , [ ] [ ] [ ] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [ , ] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [ ] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [ , ] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr (rs ) and tlr (rs ) genes [ ] . for the rubella vaccine and tlr gene, a tlr gene snp rs was associated with rubella-specific gm-csf production [ ] . our recent mumps vaccine study has identified and replicated tlr snps associated with a % decrease in antibody titer, and a tlr snp associated with a % increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr / adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th /th cytokine secretion [ ] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [ , ] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) , rabies, smallpox, and dengue viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sex-based differences in humoral immune responses are observed through various age groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [ , ] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [ ] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [ , ] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of older (ages - ) and younger (ages - ) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced > . -fold higher a/h n -specific hai antibody titers in women than men across both age groups [ ] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of elderly subjects (ages - ) demonstrated significantly higher rates of seroconversion in females than in males [ ] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of older adults (ages - ) [ ] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il- ra, crp, gm-csf, and il- ) to be more highly expressed in females than males after influenza vaccine [ ] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [ ] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [ , , ] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [ ] [ ] [ ] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [ , ] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [ ] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il- b, mcp- , tnf-a, and serum il- ) known as ''inflamm-aging" [ , [ ] [ ] [ ] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons years of age and older [ ] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as b adjuvant system aimed to improve and preserve with age zoster-specific cd + t cell responses [ ] . a tlr agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th responses to influenza vaccine in older adults [ ] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [ ] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [ ] [ ] [ ] [ ] , , ] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [ ] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to % accuracy [ ] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, % of adults and nearly % of children and adolescents are now overweight or obese [ ] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [ ] [ ] [ ] [ ] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [ ] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [ , ] . for example, obese individuals demonstrate decreased activation of influenza-specific cd + t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [ ] . given only moderate seroprotection of influenza and other vaccines in obese older adults [ ] , and the importance of developing improved influenza vaccines [ ] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [ ] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [ ] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [ , , ] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [ , , ] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [ ] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il a and il snps [ ] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf , and il snps haplotypes [ ] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [ ] , and relationships between mmr vaccine administration and snps in ifi l, cd , scn a, a, and tmem (ano ) genes [ ] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [ ] [ ] [ ] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology . ," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology . "-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology . ," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take > months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h n , h n , h n , h n , h n ) within weeks [ ] and can be safely administered to individuals with egg allergies [ ] . the ebola outbreak in liberia, sierra leone, and guinea in provides an example of the need to rapidly develop vaccine candidates [ ] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [ ] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [ , ] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [ , ] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [ ] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [ ] [ ] [ ] [ ] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [ ] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [ ] and is being evaluated in several clinical trials (nct , nct , nct ), while other inactivated vaccines are in preclinical development [ ] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct ) [ ] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct , nct ). rna-based vaccines [ ] and a variety of subunit and viral vector-based vaccines are also in development [ , , ] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [ , [ ] [ ] [ ] [ ] [ ] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [ ] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf -adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf adjuvanted influenza vaccine (fluad Ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [ ] [ ] [ ] or the highly effective as adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [ , ] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [ ] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r ai , and contract no. hhsn c (n ai ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u ck ). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of 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falko g. title: an inactivated west nile virus vaccine derived from a chemically synthesized cdna system date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nyyunoha a cdna comprising the complete genome of west nile virus (wnv) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. the synthetic wnv, produced by transfection of in vitro transcribed rna into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. no differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. there were also no significant differences in virulence in mice following intranasal challenge. after immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. in addition, the synthetic approach turned out to be very accurate, since the rescued wnv genome contained no undesired mutations. thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. this demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research. west nile virus (wnv) is a mosquito-borne, neurotropic member of the genus flavivirus, family flaviviridae, and has been identified in africa, europe, the middle east, south and central asia, oceania (subtype kunjin), and most recently north america (reviewed in [ ] ). in the u.s. wnv activity in human, bird, companion animals or mosquito has been reported since to the centers for disease control (cdc) from almost all states. besides wnv, the genus flavivirus comprises a number of medically important pathogens including japanese encephalitis virus (jev), yellow fever virus (yfv), tick borne encephalitis virus (tbev) and the four serotypes of dengue virus (denv) [ ] . the flavivirus genome is a positive-polarity, single-stranded rna molecule of about , nucleotides (nt), which functions as mrna for translation of the viral proteins. genomic rna is infectious when introduced into susceptible cells by transfection [ ] . for replication and pathogenesis studies, reverse genetic systems have been established for several members of the genus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these systems comprise one or two plasmids encoding cdna of viral genomic sequence under control of bacteriophage promoters allowing transcription of full-length infectious rna in vitro. for yfv [ ] , den- [ ] , den- [ , , ] , den- [ ] , tbev [ , ] , kun [ ] , mve [ ] and wnv lineage i [ ] and ii [ ] , cdna comprising the full genome was stably cloned into bacterial expression plasmids, whereas in other reports [ , , , , ] cdna was split in two fragments, each integrated in individual plasmids, from which cdna can be fused together before rna transcription. an alternative approach was applied to construct a jev infectious clone, in which the viral coding sequence was put under the control of a eukaryotic promoter and split by introns to circumvent instability during propagation in bacteria [ ] . conventional generation of such cdna clones requires the production of an initial virus stock, viral rna isolation, reverse transcription, pcr amplification of subfragments and engineering into the final transcription units. these approaches are sometimes hampered by low fidelity of reverse transcriptase or sequence variations in the starting isolate, which may lead to undesired alterations of the genomic sequence. as a consequence, in most reports in which the viral cdna clones or generated viruses were analyzed by sequence analysis, nucleotide variations were detected compared to the published sequence of the parent virus [ , , , , , , ] . in , a landmark publication proved the feasibility of de novo synthesis of a poliovirus by biochemical synthesis precluding any preformed components. the viral cdna encoding the . kb genome was assembled from overlapping oligonucleotides and yielded infectious virus after transcription of genomic rna and inoculation into cell lysates [ ] . taking advantage of the rapid progression of gene synthesis technology (for review [ ] ), we intended to adopt such a synthetic approach to produce a flavivirus cdna system for the generation of a synthetic wnv seed virus for use in vaccine development. in this study we report the generation of a fully functional wnv virus from a completely synthetic source. the whole , -nucleotide wnv genomic sequence was generated by gene synthesis without using natural viral templates. the production and characterization of the resulting west nile virus, which fully matched the sequence of the in silico designed viral genome, confirms the feasibility and accuracy of the synthetic flavivirus reverse genetic system. wnv wild-type virus strain ny -flamingo - was obtained from centers for disease control (cdc, atlanta) corresponding to genbank accession #af . this sequence information was also used as template for in silico design for de novo synthesis of the genomic cdnas. the cell lines vero (atcc ccl- ), bhk (atcc ccl- ) and c - (eceacc .p. # d ) were obtained from the american type culture collection or european collection of cell cultures and grown in dubecco's modified eagle's medium (dmem) or tc-vero media (baxter). tc-vero is an animal protein-free medium based on dmem/ham's f medium. six dna fragments corresponding to wnv strain ny -flamingo - (genbank accession #af ) were generated by chemical synthesis (geneart, regensburg, germany). plasmid p tl-ab carried dna corresponding to wnv genomic sequence nt - , plasmid p tl-cd to nt - , plasmid p tl-ab to nt - , plasmid p tl-cd to nt - , plasmid p tl-ef to nt - , and plasmid p tl-gh to nt , - , . three silent mutations at positions , , and were introduced to knock out an ecori site and to create an styi site as described previously [ ] . for assembly of plasmid pwnvsyn- tl (containing the third of the wnv coding sequence) an asci and bsai cut fragment of p tl-ab was ligated to a bbsi and paci cut fragment of p tl-cd (fig. b) . the ligation product was amplified using high fidelity pcr (kod, invitrogen). following digestion with bamhi and xbai, the fragment was cloned into the low copy plasmid vector pbr -pl (derived from plasmid pbr engineered to contain a matching polylinker between the unique restriction sites ecor and eagi resulting in the partial deletion of the tet r gene) yielding pwnvsyn- tl. this plasmid contains the two-thirds of the wnv coding sequence and was generated in three steps: (i) an asci and bsai cut fragment of p tl-ab was ligated to a bsai and paci cut fragment of p tl-cd. this fragment was amplified by high fidelity pcr and integrated into a commercially available ppcrscript vector (clontech). (ii) a btgzi and asci cut fragment of p tl-ef was ligated to a paci and btgzi cut fragment of p tl-gh. the resulting tl-efgh ligation product was amplified by pcr using and flanking primers, digested with spei and xbai and integrated in pbr -pl, leading to plasmid pbr - tl efgh (fig. b) . (iii) the final pwnvsyn- tl was generated by introduction of the tl-abcd fragment (derived from ppcrscript tl-abcd) into pbr - tl-efgh, taking advantage of the unique restriction enzymes spei and bamhi (fig. c) . all plasmids were amplified in bacterial strain hb (promega) and purified with commercially available systems (omega and qiagen). electroporation of bacterial cells was carried out using a genepulser apparatus (bio-rad) with settings of . kv, f and . sequence analysis was carried out using a xl genetic analyzer (abi) using bigdye terminator v . cycle sequencing kit (abi). pwnvsyn- tl was linearized with xbai followed by mung bean nuclease digestion to remove single stranded nucleotide overhangs in order to generate the correct end of the wnv coding sequence. the plasmids pwnvsyn- tl and pwnvsyn- tl were then digested with sphi and the full-length sequence was generated by ligation (t ligase; new england biolabs) via the sphi sequence overhangs of the and parts. the ligated dna fragments were extracted with phenol-chloroform twice, precipitated with ethanol and resuspended in nuclease free water. rna was transcribed at • c for h from ligated template dna by t polymerase transcription, using t megascript kit (ambion). the integrity of rna transcripts was analyzed in % agarose gels containing % formaldehyde. for rna transfection, subconfluent vaccine-certified vero cells were collected with trypsin, washed twice in serum free tc vero medium (baxter) and twice in ice-cold pbs buffer. aliquots of approximately × cells were resuspended in l of ice-cold pbs, mixed with transcribed rna and transferred to . gene pulser cuvettes. cells were electroporated with three successive pulses using a genepulser apparatus (bio-rad) with settings of . kv, f and . to visualize intracellular expression of wnv proteins, cells were infected or transfected. two days later, cells were fixed with acetone-methanol ( : ). cover slips with fixed cells were dried, rehydrated with phosphate-buffered saline and treated with a polyclonal mouse anti-wnv serum ( : dilution) obtained after immunization of mice with a formalin-inactivated whole virus vaccine preparation. bound antibodies were visualized with fluorescein isocyanate-conjugated anti-mouse immunoglobulin ( : dilution; jackson research laboratory). vero or c / cells grown in cm tissue culture flasks were infected with either wnvsyn or wnvwt stock at an moi of . . the inoculum was removed after h, and ml of fresh medium was added. at various time points ( , , , , , and h) . ml of medium was removed. the infectious virus titer of wnv containing samples was determined by a tcid assay. in brief, serial -fold dilutions of virus containing supernatant were inoculated in -well microtiter plates seeded with vero cells. after incubation for days at • c and % co , the plates were screened under a light microscope for the presence of cpe in individual wells. from the number of cpe positive wells per dilution step, the tcid was calculated according to the poisson formula by means of an in house calculation software program. viral rna was extracted from supernatant containing viral material corresponding to × tcid by trizol extraction. rna was precipitated with ethanol and the rna pellet was resuspended in l of nuclease-free water. one l of rna was used for cdna transcription using superscript iii cdna synthesis kit (invitrogen) and primers binding in the end of the ns coding region, the ns b coding region and the noncoding region. for the generation of inactivated whole virus vaccines, the wnvsyn and wnvwt stocks were amplified on bhk cells to serve as prime/boost antigen in animal studies. the wnvsyn preparation (designated cag ) as well as wnvwt preparation (designated cag ) was prepared in the same manner. ten roller bottles of bhk cells were infected with a moi of . . for better virus yields ph was adjusted to . after h of virus adsorption. after days of growth the supernatant was harvested and cleared through a low spin centrifugation step at rpm. the cleared supernatant was treated with formalin (final concentration . %) for h. next, ml of the inactivated virus was loaded on ml of a % sucrose cushion per centrifugation tube (beckman, sw tubes). after h centrifugation with , × g the supernatant was discarded and resulting pellets were pooled in tris buffered saline (tbs). an aliquot of the resulting vaccine preparations was subjected to a safety assay to exclude any possible remaining infectivity. in order to normalize the administered amounts of antigen given in immunogenicity studies, the antigen content was determined in a wnv-specific antigen elisa. the lethal dose (ld ) was determined in female -weekold balb/c mice. groups of six mice were infected intranasally with × , × , × and × tcid of wnvsyn or wnvwt, respectively. survival of mice was recorded for a period of days after infection. the -fold virus dilutions were titrated shortly after challenge and were used to calculate the ld values using the computer program graph pad prism . protection was determined after immunization of female -week-old balb/c mice by subcutaneous injections of formalininactivated wnvsyn or wnvwt vaccines in a volume of l in tbs containing . % al(oh) . mice were challenged intranasally with l of pbs ( . % human serum albumin) containing × tcid wnvwt virus. survival was monitored over a period of days after challenge. for neutralizing antibody determination, serum samples were serially diluted with cell culture medium in twofold steps. the serum dilutions were mixed at a ratio of : with a virus stock suspension adjusted to × tcid , incubated for ± min at room temperature and transferred (eight replicates per dilution) to a -well microtiter plate seeded with vero cells. the plates were inspected under a light microscope for the presence of cpe after incubation for days at • c and % co . the neutralizing titer was calculated by counting cpe negative wells and by usage of the formula nt-titer = (v/ ) × e((nneg/ ) + . ) whereas nneg is the amount of negative wells and v represents the dilution of the sera in the neutralization mix. for each assay a defined serum positive control was measured and the titer of the viral material was titrated. for detecting infectious viral material in formalin-inactivated wnv antigen preparations, vero and c / cells were seeded in five cm tissue culture flasks and inoculated with individual preparations corresponding to ml of the infectious yield from which the preparations were derived. after a day incubation period at • c and % co , supernatant of each flask was titrated by tcid and ml supernatant of each flask was carried onto fresh vero and c / cells. after a -day observation period supernatant of each flask was titrated by tcid . the respective antigen preparations were classified as safe, when no cpe was detectable in individual flasks and no viral material was detected in both tcid assays. the amount of wnv antigen in respective samples was determined by means of an elisa double sandwich system. briefly, -well microtiter plates were coated by overnight incubation at - • c with an anti-wnv igg polyclonal serum raised in guinea pigs. after subsequent washing steps, serial twofold dilutions of a standard west nile virus production material (wnv peak pool) with defined antigen amount, a control sample and individual samples were applied to the microtiter plate which was then incubated for h at • c. after subsequent washing steps a mouse anti-wnv polyclonal serum was applied to the wells and incubated for h at • c. after washing, the wells were incubated with horseradish peroxidase-conjugated donkey anti-mouse igg (jackson immuno research laboratories) for h at • c. after subsequent washing steps, substrate (o-phenylenediamine/h o ) was added, and the enzyme reaction was stopped after min at • c by the addition of . m h so . the absorbance at nm was measured with an elisa plate reader (bio-tek, winooski, vt, usa) and the antigen content was calculated (kc software; bio-tek) by means of the standard curve derived from the dilution steps of the wnv peak pool standard material. all animal experiments were reviewed by the institutional animal care and use committee (iacuc) and approved by the austrian regulatory authorities and were conducted in accordance with austrian laws on animal experimentation and guidelines set out by the association for assessment and accreditation of laboratory animal care (aaalac). animals were housed in facilities accredited by the aaalac. all experiments with infectious virus were carried out under biosafety level conditions. experiments were approved by the baxter internal biosafety committee and by the austrian ministry of health (bmfg- / -iv/b/ / ). for the construction of a bipartite infectious clone, six contiguous cdna fragments encoding the genome of the lineage i wnv strain ny were chemically synthesized and integrated in bacterial expression plasmids (see section ) according to the cloning strategy outlined in fig. . three silent marker mutations were introduced (see also [ ] ) allowing the discrimination of the synthetic virus from the corresponding wild-type isolate (see table ). the six synthetically generated wnv subfragments were ligated stepwise, resulting in two plasmids with corresponding parts of the complete genomic wnv sequence. for this purpose, either unique restriction sites in the wnv sequence were used, or -where appropriate -asymmetric restriction sites were generated in the plasmid vector backbone adjacent to the wnv fragments. cleavage of these asymmetric sites created overhangs in the wnv sequence by which corresponding fragments could be fused together. following this strategy, two plasmids were generated, containing either the third (nt - under control of a t promoter) or the two-thirds (nt - , ) of the wnv genomic sequence, designated as pwnvsyn- tl or pwnvsyn- tl, respectively. each of the cloning steps was evaluated by complete sequencing of the cdna insert and no undesired sequence alterations were observed. further, in the final two plasmids no nucleotide alterations were found with the exception of the intended silent marker mutations. to analyze the functionality of the cdna system, rna transcripts corresponding to the entire genome of wnv were generated. full-length wnv cdna templates were obtained by ligation of pwnvsyn- tl and pwnvsyn- tl. genome length viral rna was transcribed and the integrity of rna transcripts was analyzed in % agarose gels containing % formaldehyde. an rna band of approximately , nucleotides was obtained, indicating the presence of wnv full-length rna (data not shown). to characterize the ability of the transcribed rna to replicate and to be translated after introduction in host cells, viral protein expression was examined by immunofluorescence (if) staining. the wnvsyn rna was electroporated into vero cells which were subjected to indirect if staining days later (fig. ) . viral protein expression was monitored with a specific polyclonal mouse anti-wnv antibody and a fitc-conjugated second antibody (see section ). cells infected with moi . of wnvwt and were used as staining control. wnvsyn-transfected and wnvwt-infected vero cells exhibited wnv protein expression in approximately % of all cells. as expected, viral antigen staining is mainly confined to perinuclear regions of the cells (fig. ) . immunofluorescence staining is only detectable from replication-and translation-competent viral templates and could not be shown in replication-deficient mutant viral genomes [ , ] thus proving the replication and protein expression capacity of the synthetic wnv genome. in order to further analyze the genotypic and phenotypic properties, a stock of the synthetic wnv was produced. confluent vero cells were transfected as described above and upon onset of cytopathic effect (cpe) after days, cell culture medium was harvested and the virus titer was determined on vero cells, yielding a titer of . × tcid /ml. overlapping dna fragments which cover the whole wnvsyn genomic coding region were amplified by pcr after cdna transcription of isolated viral rna. sequencing confirmed that the rescued viral material contained no mutations compared to the in silico designed wnv genome and the presence of the engineered nucleotide changes proved the identity of the synthetic virus. in addition, in order to show if staining behavior in vero cells not only after transfection of rna, cells were infected with moi . of wnvsyn and processed for if as described above. as expected, the wnvsyn virus stock gave rise to a similar staining pattern as seen for the wnvwt stock (fig. d) . in order to analyze the growth properties of wnvsyn and wnvwt, one step growth curves were carried out. susceptible mammalian (vero) and mosquito (c / ) cells were infected with a moi of . . viral titers, determined at the time points indicated in fig. , demonstrate that in both cell types the growth kinetics of wnvsyn match exactly those of the wild-type virus. in addition, plaque morphology ( fig. a and b) and cpe (not shown) were comparable to the wild-type control. virus grown in vero cells peaked at h posttransfection but declined from day on correlating with the onset of cpe of the infected cells from day on (fig. c) . growth kinetics in the mosquito cells was delayed as observed by others [ , ] , reaching equal titers compared to vero cells at day postinfection (fig. d) . taken together, these data indicate that wnvsyn and the corresponding wnvwt isolate are indistinguishable with respect to replication and infectivity in both tested cell lines. in addition, virulence of wnvsyn and wnvwt were compared in cohorts of -week-old balb/c mice. for this purpose mice were infected intranasally with virus dilutions corresponding to × to × tcid per animal. survival was monitored for days postinfection and ld values were calculated. similar mortalities of infected mice induced by the two wnv viruses were observed ( table ). the lethal dose for wnvsyn and wnvwt was . and table virulence of wnvsyn and wnvwt after intranasal application in balb/c mice. . log tcid , respectively. the experiment was repeated once and similar results were obtained. following the demonstration that wnvsyn exhibits indistinguishable biological properties compared to the wnv wild-type isolate, the protective efficacy of experimental vaccines derived from both viruses was analyzed. for this purpose, groups of ten mice were immunized twice with decreasing doses of formalininactivated, alum-adjuvanted whole virus vaccines derived from the viruses (see section ). quantification by elisa of vaccine preparations prior to formulation and adjuvantation confirmed the presence of equal amounts of antigen in the respective dosage groups. further, western blotting confirmed equivalent amounts and protein patterns in the two antigen preparations (fig. b) . the predominant band in these preparations is the envelope antigen (e) migrating in the kda range, the fainter bands representing the pre-membrane (prm) and the dimeric membrane (m) proteins (see also [ ] ). two weeks after the second vaccination wnv-specific neutralizing antibodies were determined by a microneutralization assay. serum analysis demonstrated high neutralizing antibody levels in both vaccine preparations (see fig. a and table ). mice were then challenged intranasally with a lethal dose ( × tcid ) of wnv wild-type virus. vaccination with both preparations resulted in a high degree of protection in vaccinated mice. complete protection was achieved using doses as low as nanograms of the wnv antigens while % of the non-vaccinated controls died. the vaccines clearly induced a dose-dependent protection correlating with nt titers (table ) . reverse genetics systems of positive-sense rna viruses allow, for instance, for mutagenesis procedures and generation of chimeric viruses and thus are invaluable tools for live vaccine development and for studying the biology of those viruses (see e.g. refs. [ , ] ). usually the starting material for the generation of seed viruses for vaccines or such reverse genetics systems are virus stocks derived from a biological source. this virus is plaque-purified, viral rna is isolated, transcribed into cdna which is amplified by pcr and in turn gets assembled into a dna copy of the viral genome. these approaches bear the risk of introducing mutations selected via plaque purification steps. to minimize this type of mutations we chose to generate a reverse genetics system using a different approach, independent of preformed viral rna components and animal sources. the feasibility of generating such systems by chemical synthesis of dna was proven previously, for instance, by the generation of poliovirus [ ] , bacteriophage x [ ] or h n spanish influenza virus [ ] , and sars-like coronavirus [ ] . on the basis of these studies, we report for the first time the generation of an , nucleotide long synthetic genome of a member of the family flaviviridae. sequence data from genbank referring to lineage i west nile virus strain ny were used as template for in silico design of the cloning strategy. rna viruses replicate their genome with an error prone mechanism (for reviews see [ ] ), resulting in a multitude of distinct but related nucleic acids forming a quasispecies [ ] . sequencing of a virus genome (usually cloned by plaque purifications prior to sequence analysis) consisting of millions of molecules, results in a 'consensus' sequence, representing the majority genotype having defined biological properties. biological properties may change, for instance, when pressure imposed by the host selects for changes of the genomic sequence, visible as a new 'consensus sequence' in the sequence analysis. in all of the cloning and propagation steps no mutations changing the wild-type consensus sequence were introduced by pcr using synthetic templates of verified nucleotide sequence proving the accuracy of this approach. thus the synthetic progeny virus was biologically indistinguishable from its natural parent. experimental inactivated vaccines derived from wnvwt and wnvsyn were highly immunogenic in animals. both vaccine preparations induced comparable levels of neutralizing antibodies and led to similar protection results. only in the low dosing groups of the protection study differences were observed that can be explained by the experimental conditions and the inherent inaccuracies of the biological system rather than by genetic differences in the two viruses. in addition, both virus stocks were indistinguishable concerning their virulence in mice. progress in synthetic biology raises biosecurity concerns. the possibility to synthesize pathogens without need for natural sources, for instance the viruses on the select agents list [ ] , results in the expansion of the potential availability of select agents (defined as biological agents and toxins regulated by the us select agent rules that have the potential to pose a severe threat to public, animal or plant health). the us government has developed guidance that addresses this issue [ ] . wnv -although a biosafety level virus -is endemic in large parts of the world and does not belong to the select agents. however, flaviviruses belonging to the tick-borne encephalitis virus complex are on this list. construction of infectious flaviviruses, involving dna synthesis, cloning, assembly into larger units, in vitro transcription and transfection steps, is a complex task and can be done in a professional environment only. a recent review on synthetic viruses discusses the dual use concerns in more detail [ ] . for vaccine manufacturing, the most important advantage of using primary seed virus stocks derived by gene synthesis is the exclusion of potential contamination with unknown and known adventitious agents -including the transmissible spongiforme encephalopathy agents -which maybe co-isolated from animalderived viruses or their host cells. furthermore, this approach renders passaging, plaque purifications and other steps to achieve satisfactory purity of seed viruses from animal sources unnecessary. our study demonstrates the feasibility of generating the flavivirus wnv in a completely synthetic approach. synthetic biology is therefore a valuable alternative to obtain viral seed stocks free from the adventitious agents that might accompany recovery from vertebrate or insect cells. emerging flaviviruses: the spread and resurgence of japanese encephalitis. west nile and dengue viruses flaviviridae: the viruses and their replication functional cdna clones of the flaviviridae: strategies and applications a stable full-length yellow fever virus cdna clone and the role of conserved rna elements in flavivirus replication infectious cdna clones of langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence identification of a major determinant of mouse neurovirulence of dengue virus type using stably cloned genomic-length cdna characterization of infectious murray valley encephalitis virus derived from a stably cloned genome-length cdna synthesis and characterization of an infectious dengue virus type- rna genome (new guinea c strain) completion of kunjin virus rna sequence and recovery of an infectious rna transcribed from stably cloned full-length cdna construction of infectious cdna clones for dengue virus: strain and its attenuated vaccine derivative, strain pdk- infectious rna transcribed from stably cloned full-length cdna of dengue type virus molecular and functional analyses of kunjin virus infectious cdna clones demonstrate the essential roles for ns a in virus assembly and for a nonconservative residue in ns in rna replication infectious cdna clones of tick-borne encephalitis virus european subtype prototypic strain neudoerfl and high virulence strain hypr infectious clone construction of dengue virus type , strain jamaican , and characterization of a conditional e mutation infectious cdna clone of attenuated langat tick-borne flavivirus (strain e ) and a deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice infectious rna transcripts from fulllength dengue virus type cdna clones made in yeast construction of a full length infectious clone for dengue- virus western pacific, strain transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation infectious cdna clone of the epidemic west nile virus from new york city infectious japanese encephalitis virus rna can be synthesized from in vitro-ligated cdna templates an infectious clone of the west nile flavivirus a new strategy in design of +rna virus infectious clones enabling their stable propagation in e. coli chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template synthetic viruses: a new opportunity to understand and prevent viral disease functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses proteolytic activation of tick-borne encephalitis virus by furin kunjin virus replicons: an rnabased, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications preclinical and clinical development of yfv d-based chimeric vaccines against dengue, west nile and japanese encephalitis viruses the test-tube synthesis of a chemical called poliovirus generating a synthetic genome by whole genome assembly: phix bacteriophage from synthetic oligonucleotides characterization of the reconstructed spanish influenza pandemic virus synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice rates of spontaneous mutation viral quasispecies addressing biosecurity concerns related to the synthesis of select agents. nih we thank helga savidis-dacho and her team for performing the animal experiments, kathrin janecki, marie-luise zips and petra cech for expert technical assistance and the geneart team for providing the cloning strategy and the six genomic plasmids. key: cord- - l w p authors: custers, jerome; kim, denny; leyssen, maarten; gurwith, marc; tomaka, frank; robertson, james; heijnen, esther; condit, richard; shukarev, georgi; heerwegh, dirk; van heesbeen, roy; schuitemaker, hanneke; douoguih, macaya; evans, eric; smith, emily r.; chen, robert t. title: vaccines based on replication incompetent ad viral vectors: standardized template with key considerations for a risk/benefit assessment date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: l w p replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express various antigens as a basis for preventive or therapeutic vaccine development. a replication incompetent adenoviral vector based on human adenovirus type (ad ) has been evaluated in several clinical trials. the brighton collaboration viral vector vaccines safety working group (v swg) was formed to evaluate the safety and features of recombinant viral vector vaccines. this paper reviews the biological features of the ad vectors, including tabulation of safety and risk assessment characteristics of ad vector-based vaccines. substantial information on immunogenicity, clinical safety, biological characteristics and manufacturing are reported. in the ad vector, deletion of the e gene, rendering the vector replication incompetent and providing space for transgene insertion, is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. these vaccines are manufactured using the e -complementing per.c ® cell line, a continuous, human cell-line that can be cultured in serum-free medium in a suspension to high cell densities, providing an effective and flexible system for high-yield manufacturing. ad vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains. safety data are compiled in the ad vaccine safety database version . , with unblinded data from ongoing and completed clinical studies for a total of participants in ebola, hiv, malaria, rsv and filovirus ad -based vaccine programs. overall, all ad -based vaccines have been well tolerated, with no significant safety issues identified from the available data in the current ad vaccine safety database. evaluation of ad -based vaccines to further characterize the safety profile is continuing, with more than , participants vaccinated as of st july (cut-off date). extensive evaluation of immunogenicity in humans shows strong and durable humoral and cellular immune responses. clinical trials have not shown meaningful impact of pre-existing immunity to ad on vaccine immunogenicity, even in the presence of ad neutralizing antibody titers or ad -targeting t cell responses at baseline. the first vaccine, against ebola virus, that makes use of the ad vector, received marketing authorization from ec on st july , as part of the ad .zebov, mva bn filo vaccine regimen. new developments based on the ad vector are underway, including a covid- vaccine, which is currently in clinical evaluation. the brighton collaboration (www.brightoncollaboration.org) v swg is an established collaboration who aim to enhance the science of vaccine safety research (http://cms.brightoncollaboration.org: /public/what-we-do/setting-standards/casedefinitions/process). the brighton v swg uses a standardized template to describe the key characteristics of novel vaccine vectors, compiled from the latest research, to facilitate scientific discourse among key stakeholders ( ). the adenovirus type (ad ) wild type virus was first isolated in from an anal specimen of a -month-old male child ( ) . as described in that study, different isolates were obtained from anal and throat swabs from different children, some of whom experienced mild selflimiting enteric infections. although, none of the illnesses could etiologically be associated with the isolated adenoviruses, it suggests that wild type ad can, presumably, cause asymptomatic or minor illness ( ) . human ad has been considered to be a low-prevalent adenovirus due to the low frequency of ad neutralizing antibodies in various populations compared with human adenovirus type . for example, a seroprevalence study of the human adenovirus serotypes known at the time showed that several serotypes from particularly subgroups b and d, including ad , were rare in a belgian population ( ) , suggesting that vectors (rad) derived from these serotypes might be useful alternatives to ad -based vectors for vaccine development, since for ad -based vectors it was shown that their high prevalence hampered their clinical use ( ) ( ) ( ) ( ) ( ) . more extensive seroprevalence and immunogenicity studies showed that while all of these vectors exhibited low seroprevalence, ad -based vaccine candidates were the most immunogenic in animals ( ) . further studies have shown that, depending on geographical location, %- % of people tested have neutralizing antibodies against ad . however, neutralization titers are low to intermediate compared with those observed for other adenovirus types ( - ). adenovirus genomes are linear, non-segmented double-stranded dna molecules with inverted terminal repeat (itr) sequences at each end. the vector system for replication-incompetent ad vaccine vectors consists of an adaptor plasmid and a cosmid ( ) . the adaptor plasmid contains the left end of the genome containing the left itr and the packaging signal. it also contains a transgene expression cassette in place of the e region and a ~ . kb fragment downstream of the e region to enable homologous recombination with the cosmid. the cosmid contains the majority of the ad genome, spanning from the pix sequence to the right itr, with a deletion of the e region and a modified e open reading frame (e orf ). transfection into a suitable packaging cell line (hek cells, per.c ® cells) and subsequent homologous recombination of the adaptor plasmid and cosmid results in the generation of a replicationincompetent e /e -deleted ad vector. packaging cell lines like the hek and per.c ® cell lines contain the e region of adenovirus serotype (ad ). because within the adenoviral life cycle, e protein k and e protein orf form a complex that is pivotal for high-level lategene expression, the e -orf sequence of ad is replaced by the corresponding sequence from ad in the vector. this modification has previously been shown to be necessary to allow for the efficient production of rad virus on ad e -complementing cells ( ) . finally, compensation for the loss of e is not needed since the e proteins are not essential for adenoviral growth in vitro but are involved in down regulating cellular immunological response mechanisms in an attempt of the adenovirus to escape the host immune system ( ) . most adenovirus serotypes use the coxsackievirus-adenovirus receptor for attachment to the target cell ( , ) . in contrast, ad has been reported to utilize cd as its primary cellular receptor ( , ) , but more recent reports indicated only a limited interaction between ad and cd , even showing evidence of a role for αvβ integrin for efficient transduction of epithelial cells, or interaction with sialic acid ( , ) . these data suggest that receptor usage by ad might be host cell-type dependent in vitro ( , ) . target cells in vivo in the natural host are not known, but ad virus can infect a variety of cell types in vitro. detailed studies dissecting the attachment, internalization and intracellular trafficking of adenoviral vectors have shown that ad , amongst others, accumulate in the late endosome to a larger extent and trigger innate immune pathways differentially compared with ad -based vectors ( ) . whether and how these differences may translate into differential profiles of adaptive immunity against the vaccine antigen is not known. ad vector-based vaccines are manufactured using the e -complementing per.c ® cell line, a continuous, human cell line capable of supporting the manufacturing of replication incompetent adenoviral vectors ( ) . one of the key strengths of this cell platform is that the cells can grow in suspension in serum-free media to very high cell densities. cell counts of million cells/ml, with a high percentage of viable cells, can be reached within days of cell culture. janssen has taken advantage of the ability to grow the per.c ® cell line at high cell densities in a so called "intensified process". cell-specific yields are in the same range as is generally achieved with other adenoviral vectors and e -complementing cell lines, therefore, due to the higher cell densities yields per volume unit are higher. the complete manufacturing process has now been scaled up to , l allowing manufacturing at a commercial scale. while lyophilized vaccines are generally more heat-stable than non-lyophilized alternatives ( ), liquid vaccine formulations may have several advantages over lyophilized vaccines, including ease of manufacture, packaging, and simple administration procedures ( ) . for ad -based vectors, progress in formulation development has allowed for long-term storage of product at - °c, enabling product distribution through existing vaccine supply chains. assessments of robustness during storage, handling and distribution conditions have shown that recombinant ad vectors can be maintained stable under frozen conditions or at - °c, and, furthermore, showed to be stable in-use with a syringe/needle, also when subjected to agitation or temperature excursions ( ). ad -based vaccines have been extensively tested in completed and ongoing clinical studies from multiple clinical programs ( figure ) the the brighton collaboration v swg authors declare that they have no known competing ad was rendered replication incompetent by deletion of early region (Δe ). a partial deletion of non-essential early region (Δe ) was made to create enough space in the genome for the transgene expression cassette (inserted in e region) also further attenuating the vector. • in gene therapy experiments n.a. • in any other special populations n.a. the ad vector is replication incompetent in non-e complementing cells and, as such, will be replication incompetent when administered to nonhuman species. in addition, insertion of foreign antigens is not expected to change the host-range of the vaccine vector. what is the risk of reversion to virulence or recombination with wild type virus or other agents? recombination of ad vaccine vectors with wildtype viruses would require sequence homology and presence of both the genome of ad vaccine vectors and wild-type adenoviruses to be present in the same cell(s). nonclinical biodistribution studies show that adenoviral vector dna of ad vaccine vectors did not distribute widely, as the vector dna was primarily detected at the site of administration in the muscle, the draining lymph nodes and, to a lesser extent, to the spleen. dna of intramuscularly administered replicationincompetent ad vaccine vector and wild-type adenovirus dna are unlikely to co-locate in the same body compartments. in the unlikely event that recombination occurs between vaccine vector and wild-type adenoviruses, the virulence can maximally be equal to the wild-type adenovirus already present in the tissue. the majority of the theoretical homologous recombination products are replication incompetent or attenuated forms of the ad . reversion of ad virulence by recombination is therefore highly unlikely. furthermore, reversion of virulence due to nucleotide mutations is impossible since deletion of the e gene from the ad vector cannot be restored by random mutations and or indels. recombination with other viruses has not been described and is considered highly unlikely due to the limited biodistribution and absence of sequence homology and replication. the dsdna genome of ad virus is relatively stable when compared to rna viruses. genetic stability of the vector is confirmed during manufacturing and upscaling by extended passaging and/or genetic stability testing. ( ) what is the potential for shedding and transmission to humans or other species? vector shedding is limited and transmission of the ad vector is highly unlikely in view of: i) the vector is replicationincompetent, and thus, allows only for one-time transduction of the target cell, ii) the limited shedding as observed in clinical studies, iii) the limited biodistribution profile as observed in nonclinical studies, and iv) the very low probability of regaining replication-competence through recombination with co-infecting wild-type virus. biodistribution studies in rabbits have shown that vector dna is not widely distributed, and clearance has been observed indicating that the vector is unlikely to persist in the tissues following intramuscular injection. in nature, wild-type adenovirus is known to be able to cause persistent infections. whether the ad vector can persist for longer time in humans is unknown. this will depend on the design of the antigen and the antigenic diversity of the pathogen. the transgene is inserted in the e region at the site of the e -deletion. the e region is deleted to render the vector replication incompetent and together with a deletion in the e region provide space for ( ) insertion of a transgene expression cassette. . . how is the transgene expression controlled (transcriptional promoters, etc.)? in general, expression of the antigen is regulated using the long human cmv immediate-early promoter, which is thought to be active in most mammalian cells, and an sv -derived polyadenylation sequence. the expressed antigen is not part of the viral particle and, as such, it is not expected that the phenotype of the vector nor the pathogenicity (the vector is replication incompetent) of the vector are altered. . . is the vaccine replicationcompetent in humans or other species? the ad vector is replication incompetent. . . what is the risk of reversion to virulence or recombination with wild type or other agents? see . . genetic stability of the vaccine vector is confirmed during manufacturing and upscaling by extended passaging and genetic stability testing. . . what is the potential for shedding and transmission to humans or other species? see . . the vaccine is replication incompetent and is unable to establish a productive infection. persistence/latency is not expected as the vaccine misses the e and e genes that code for proteins involved in countering the host immune system. in addition, nonclinicalbiodistribution studies of the vaccine have shown that the ad vaccine vector is cleared from vector positive tissues (see . .). . . does the vaccine replicate in the nucleus? the vaccine is replication incompetent. the vector genome (linear ds-dna) travels to the nucleus of the host cell where antigen expression occurs, in the absence of vaccine vector replication. . . what is the risk of integration into the human genome? see . . ( - ) . . list any disease manifestations caused by the vaccine in humans, the strength of evidence, severity, and duration of disease for the following categories: n.a. • in healthy people n.a. • in immunocompromised people n.a. • in neonates, infants, children n.a. • during pregnancy and in the unborn n.a. • in any other special populations n.a. see . . the ad vector is expected to have the same cell tropism as the wild-type ad virus. ( , ( ) ( ) ( ) . . what is known about the mechanisms of immunity to the vaccine? in general, the immune responses to the vaccine antigen encoded by the vector are characterized by a rapid increase in binding and in most cases neutralizing antibodies. in addition, induction of nonneutralizing, functional antibodies with effector functions, like antibodydependent cellular phagocytosis (adcp) and antibody-dependent cellular cytotoxicity (adcc), have been observed. induction of cellular immunity (both cd + and cd + t-cells) is also observed. . what is known about the effect of pre-existing immunity, including both natural immunity and repeat administration of the vector or the vaccine, on 'take', safety or efficacy in any animal model or human studies using this vector? data acquired to date, in more than , vaccinated human participants, have not revealed impact of preexisting vector immunity on the vaccine insert specific humoral or cellular ( , ) response. repeated administration with the ad vector leads to an increase in antigen specific humoral responses and a maintenance of cellular responses. with more than , participants vaccinated overall, no safety issues have been identified. . . . is the vaccine transmissible in humans or other species (including arthropods) and/or stable in the environment? the vaccine is replication incompetent, so no vaccine transmission is expected (see also . .). . . are there antiviral or other treatments available for disease manifestations caused by the vaccine? the vector is replicationincompetent; thus, no disease manifestations are expected besides local and systemic reactogenicity. therefore, there is no benefit for antiviral treatment. the ad vector showed a limited distribution following intramuscular injection in rabbits and clearance of the vector was observed, indicating that the vector does not replicate and/or persist in the tissues following intramuscular (im) injection. the ad vector did not induce any adverse effects in multiple glp repeat-dose toxicity studies in rabbits (and rats), irrespective of the transgene insert used. . . for replicating vectors, has a comparative virulence and viral kinetic study been conducted in permissive and susceptible species? (yes/no) if not, what species would be used for such a study? is it feasible to conduct such a study? not applicable, since the ad vector is replication incompetent . . does an animal model relevant to assess attenuation exist? not applicable, since the ad vector is replication incompetent . . does an animal model for safety including immuno-compromised animals exist? not applicable, since the ad vector is replication incompetent . . does an animal model for reproductive toxicity exist? not applicable, since the ad vector is replication incompetent the general (repeat-dose) toxicity studies conducted with the replication incompetent ad vector in rabbits (and rats) have not revealed any effects on male sex organs that would impair male fertility. in addition, the general (repeat-dose) and/or developmental and reproductive toxicity studies did not reveal any evidence of impaired female fertility nor did not indicate harmful effects with respect to reproductive toxicity in female rabbits. . . does an animal model for immunogenicity and efficacy exist? most mammalian species studied to date have shown induction of insert specific immunity after administration of ad based vaccines, dependent on the studied disease. several efficacy animal models exist -the most studied animal models have been mice, rabbits, ferrets, cotton rats and several nonhuman primate (nhp) species. . . does an animal model for antibody enhanced disease or immune complex disease exist? no . . what is known about biodistribution in animal models or in humans? biodistribution studies have been conducted in rabbits. following intramuscular administration, the ad vector did not widely distribute as vector dna was primarily detected at the site of injection, draining lymph nodes and (to a lesser extent) the spleen. over time, the number of animals with positive tissues and/or the vector copy number present in those positive tissues declined, indicating clearance of the ad vector. . . what is the evidence that vector derived vaccines will generate a beneficial immune response in: • small animal models? mice, cotton rats, rabbits and ferrets have been shown to ( , , , , , ( ) ( ) ( ) ( ) ( ) develop immune responses against a variety of vaccine inserts. • nonhuman primates (nhp)? nhp have been shown to develop protective immune responses against a variety of vaccine inserts. ( , , , , , , , , , ) • overall, no safety concerns were identified in elderly after vaccination with adeno-based vaccines. (cut-off: st july ; active only, estimate based on the study randomization ratios). no safety concerns were identified. • pregnancy and in the unborn in humans? the most recent aggregate review of pregnancy exposure data was performed in september ; this analysis of the current experience with pregnancies after exposure to the ebola candidate vaccines (ad .zebov, ad .filo) in female participants or partners of male participants did not reveal a safety concern. serious complications or saes during pregnancy were reported in out of a total of pregnancies reported in female study participants. none of these serious complications / saes were considered causally associated with the study vaccines by investigators or low risk; pregnancy is an exclusion criterion for all ad -based vaccine studies except study (described below). pregnancy tests prior to vaccination and the use of adequate contraception was mandatory for all female participants of childbearing potential. pregnant women are being enrolled in the ongoing ebola vaccination study in drc (drc-eb- /ebl ). ( ) the company. no apparent concernable pattern of aes is emerging from this review. no congenital malformations were reported to date in foetuses or newborns. spontaneous abortion was the most commonly observed sae ( out of pregnancies) with an incidence of . %, which is within the range of expected spontaneous abortion rates during the first trimester of gestation, even when considering that spontaneous abortion incidences vary significantly depending on geographical areas and individual risk factors (e.g. age, previous abortions) . • in any other special populations. not applicable . . what is the potential for shedding and transmission in risk groups? there is no significant risk for shedding and transmission of ad vectored vaccines across the risk groups (e.g. immunocompromised) who have received the vaccine vector. also designated v ) recombinant vesicular stomatitis virus pseudotyped with ebola zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment four newly recognized adenoviruses replication-deficient human adenovirus type vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity safety and immunogenicity of a replication-incompetent adenovirus type hiv- clade b gag/pol/nef vaccine in healthy adults vaccine-induced immunity in the test-of-concept step study: a case-cohort analysis human adenovirus-specific t cells modulate hiv-specific t cell responses to an ad -vectored hiv- vaccine decreased preexisting ad capsid and ad neutralizing antibodies increase hiv- infection risk in the step trial independent of vaccination phase safety and immunogenicity evaluation of a multiclade hiv- candidate vaccine delivered by a replication-defective recombinant adenovirus vector comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d international epidemiology of human pre-existing adenovirus (ad) 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the interception of hpv -and hpv -induced infections and disease ad /mva therapeutic vaccination with tlr stimulation in siv-infected rhesus monkeys evaluation of a mosaic hiv- vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase / a clinical trial (approach) and in rhesus monkeys (nhp - ) first-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype env vaccine (ipcavd ) characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype hiv- env vaccine in healthy adults (ipcavd ) assessment of the safety and immunogenicity of novel vaccine platforms for hiv- prevention: a randomized trial our efforts to develop a vaccine and identify therapies for covid- immune responses to novel adenovirus type and modified vaccinia virus ankara-vectored ebola vaccines at year safety and immunogenicity of novel adenovirus type -and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial induction of hiv- -specific mucosal immune responses following intramuscular recombinant adenovirus serotype hiv- vaccination of humans safety and preliminary efficacy of cs-based recombinant adenoviral serotype and serotype malaria vaccine candidates in prime-boost vaccination of healthy adults undergoing subsequent experimental p. falciparum sporozoite challenge (abstract) adenovirus transmission: national center for immunization and respiratory diseases, division of viral diseases perspective on adenoviruses: epidemiology, pathogenicity, and gene therapy new insights into stability of recombinant adenovirus vector genomes in mammalian cells progress in human gene therapy episomal vectors for gene therapy guideline on nonclinical testing for inadvertent germline transmission of gene transfer vectors conjunctivitis and enteric infection with adenovirus types and : responses to primary, secondary and reciprocal cross-challenges acute meningoencephalitis caused by adenovirus serotype adenovirus infections in immunocompetent and immunocompromised patients adenoviruses in the immunocompromised host quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors new drug on the horizon for treating adenovirus division of viral diseases viral mutation rates a two-dose heterologous prime-boost vaccine regimen eliciting sustained immune responses to ebola zaire could support a preventive strategy for future outbreaks safety and immunogenicity of a -dose heterologous vaccine regimen with ad filo ebola vaccines: -month data from a phase randomized clinical trial in safety and immunogenicity of a -dose heterologous vaccination regimen with ad .zebov and mva-bn-filo ebola vaccines: -month data from a phase randomized clinical trial in uganda and tanzania packaging capacity and stability of human adenovirus type vectors ad and ad vaccine vectors induce potent and cross-reactive antibody and t-cell responses to multiple filovirus species recombinant low-seroprevalent adenoviral vectors ad and ad expressing the respiratory syncytial virus (rsv) fusion protein induce protective immunity against rsv infection in cotton rats ebovac-salone: lessons learned from implementing an ebola vaccine trial in an ebola-affected country similar epitope specificities of igg and iga antibodies elicited by ad vector prime, env protein boost immunizations in rhesus monkeys recombinant adenovirus serotype (ad ) and ad vaccine vectors bypass immunity to ad and protect nonhuman primates against ebolavirus challenge adenoviral vector type encoding zika virus (zikv) m-env antigen induces humoral and cellular immune responses and protects mice and nonhuman primates against zikv challenge correction: a prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with ebolavirus and marburgvirus species in non-human primates protective efficacy of adenovirus/protein vaccines against siv challenges in rhesus monkeys a very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus low seroprevalent species d adenovirus vectors as influenza vaccines the th immune response to plasmodium falciparum circumsporozoite protein is boosted by adenovirus vectors and with a homologous insert alternative serotype adenovirus vaccine vectors elicit memory t cells with enhanced anamnestic capacity compared to ad vectors adenovirus vectorbased prime-boost vaccination via heterologous routes induces cervicovaginal cd (+) t cell responses against hpv oncoproteins immunogenicity without efficacy of an adenoviral tuberculosis vaccine in a stringent mouse model for immunotherapy during treatment vaccination with adenovirus serotypes , , and elicits higher levels of innate cytokine responses than adenovirus serotype in rhesus monkeys durability and correlates of vaccine protection against zika virus in rhesus monkeys adenoviral vaccine safety database v . . we thank the additional v swg members and participants for their support and helpful comments, and patrick zuber of the world health organization for bringing together many of the coauthors for completing the first draft of this template and for his support of the v swg.we would also like to thank wouter koudstaal (independent scientific writer) and kerstin luhn, ☐ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:jerome custers, maarten leyssen, frank tomaka, esther heijnen, georgi shukarev, dirk heerwegh, roy van heesbeen, hanneke schuitemaker, and macaya douoguih, are current employees of janssen pharmaceuticals and potentially hold stock in j&j. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -oc zd h authors: pagliusi, sonia; ting, ching-chia; khomvilai, sumana title: quality vaccines for all people(): report on the th annual general meeting of the developing countries vaccine manufacturers' network, – th october , bangkok, thailand date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: oc zd h the developing countries vaccine manufacturers network (dcvmn) assembled high-profile leaders from global health organisations and vaccine manufactures for its th annual general meeting to work towards a common goal: providing quality vaccines for all people. vaccines contribute to a healthy community and robust health system; the ebola outbreak has raised awareness of the threat and damage one single infectious disease can make, and it is clear that the world was not prepared. however, more research to better understand emerging infectious agents might lead to suitable vaccines which help prevent future outbreaks. dcvmn members presented their progress in developing novel vaccines against dengue, hpv, chikungunya, cholera, cell-based influenza and other vaccines, demonstrating the commitment towards eliminating and eradicating preventable diseases worldwide through global collaboration and technology transfer. the successful introduction of novel sabin-ipv and oral cholera vaccine in china and korea respectively in was highlighted. in order to achieve global immunisation, local authorities and community leaders play an important role in the decision-making in vaccine introduction and uptake, based on the ability of vaccines to protect vaccinated people and protect non-vaccinated in the community through herd immunity. reducing the risk of vaccine shortages can also be achieved by increasing regulatory convergence at regional and international levels. combatting preventable diseases remains challenging, and collective efforts for improving multi-centre clinical trials, creating regional vaccine security strategies, fostering developing vaccine markets and procurement, and building trust in vaccines were discussed. the developing countries vaccine manufacturers' network (dcvmn) convened its th international meeting, co-organised by queen saovabha memorial institute (qsmi) and bionet asia, under the auspices of the thai red cross society. nearly professionals working in research, development, manufacturing and supply of vaccines, from countries, gathered in bangkok. dcvmn is the largest alliance of corporate manufacturers, supplying over vaccine types in various presentations to immunisation programmes, and contributing significantly to global public health efforts to eradicate polio, eliminate and control the spread of known and emerging infectious diseases around the world. local health authorities and global health organisations represented at this meeting included united nations international the dcvmn to improve quality of life for every child, family and community globally. prof. s. khomvilai invited dr. j.-m. okwo-bele, director at who, to speak on the achievements and challenges of the global vaccine action plan (gvap). j.-m. okwo-bele highlighted the achievements of global immunisation: polio eradication is close, with only two countries currently reporting wild poliovirus; measles vaccination has reduced the disease by over % from to [ ] ; regional meningitis a epidemics have been eliminated in africa, with half of the population vaccinated [ ] ; in all, around million deaths have been averted by vaccination [ ] . still, challenges remain. limitations in vaccine affordability and supply, failures in healthcare integration, disruption of immunisation by geopolitical conflict and poor-quality health data have resulted in stagnant coverage and repeated failure to reach immunisation goals. for instance, in , . million children missed their rd dose of dtp in the eastern mediterranean region, mostly in security-compromised countries [ ] . solutions expected from manufacturers include measures to assure the controlled temperature chain (ctc) where cold-chain facilities are lacking, implementation of improved delivery technologies for ease of use in the field, and assistance in reducing risk of shortages. the monitoring and accountability framework of gvap identifies and responds to immunisation challenges. a. batson, chief strategy officer at path, provided an overview of the developing vaccine markets. over the past decade, demand for higher-value vaccines has grown. more antigens are incorporated into multi-valent combination vaccines (pentavalent vaccine replacing dtp, and mmr/mr replacing measles vaccine) and new, complex vaccines are being introduced. a robust vaccine development pipeline is emerging from both industrialised and developing countries. from the latter, the serum institute of india's meningitis a vaccine reached million people in [ ] , and it is anticipated that japanese encephalitis vaccine from chengdu institute, china, will reach million people by . dcvmn members play an essential role in global vaccine supply, contributing to improving health and driving new markets. they are diverse in terms of ownership (state or private), scale, portfolio and market (local providers or emerging new multinational companies, fig. ). it is important to understand their role and plan for anticipated changes in global versus national financing and procurement of vaccines. s. chunsuttiwat from the ministry of public health, thailand, discussed health security and sustainable development goals (sdg). thailand introduced universal health insurance coverage (uhc) in , and reached . % insurance coverage in . regionally, the association of southeast asian nations (asean, representing % of the world's population [ ] ), has initiated collaboration for regional vaccine security and self-reliance, seeking to understand vaccine needs from asean countries' perspectives, and to identify areas for cooperation. the challenge of harmonising regulatory pathways to improve access to vaccines in developing countries was discussed by m. ward, from who. increasing regulatory convergence at regional and international levels can be achieved by adopting common standards and practices for vaccine regulation, without requiring harmonisation of laws [ ] . such convergence and/or harmonisa- dtp = diphtheria + tetanus + pertussis combination vaccines. tion needs to be implemented consistently and intentionally to succeed. who's role in promoting access to quality medical products is to develop norms and standards and promote regulatory convergence and harmonisation. through who initiatives, benefits and challenges can be identified, efficient collaboration among regulators fostered and in-country regulatory decisions supported. collaboration should replace duplication. h. deehan from unicef briefed the audience on prevention and remediation strategies to manage vaccine supply constraints. unicef's procurement and supply of vaccines to countries needing assistance with vaccination programmes has increased significantly over the years. since , more than % of doses procured annually have been from suppliers in developing countries. recent supply constraints have included bcg , yellow fever vaccine, and ipv vaccine to support the polio endgame strategy. early and transparent communication with manufacturers and close monitoring of stock levels within countries is required to allow prompt mitigation actions and ultimately reduce risk of interrupted supply. deehan invited dcvmn members to work with unicef to achieve sustained global supply of affordable vaccines of assured quality. m. malhame, head of market shaping for gavi, presented gavi's recent accomplishments and activities. pentavalent, pneumococcal and rotavirus vaccines have been steadily introduced in a growing number of countries, surpassing gavi expectations. the volume of vaccines from dcvms continues to grow, although the value of vaccines procured from dcvms remains relatively low. to secure global supply, gavi has fostered procurement from multiple countries and manufacturers ( fig. a and b ). in terms of activities, gavi introduced two major changes in their co-financing policy in : (i) linking co-financing to prices for all countries in transition phases and (ii) developing payment plans to help countries get out of default. gavi's strategic goals for - include a vaccine goal: to accelerate equitable uptake and coverage of vaccines, a health system goal: to increase effectiveness and efficiency of immunisation delivery as a part of strengthened health systems, a financing goal: to improve sustainability of national immunisation programs, and a market-shaping goal: to shape markets for vaccines and other immunisation products [ ] . with these goals, gavi hopes to immunise million additional children around the world by the end of . d. rodriguez, from the paho revolving fund for vaccine procurement took a moment to celebrate the americas region's success, in , as the first region to eliminate rubella. this was enabled by the commitment of member countries to regional goals, the paho revolving fund and vaccine manufacturers. he then outlined challenges faced with vaccine shortages and strategies to overcome these. latin american populations are under the threat from yellow fever virus because vaccine demand exceeds the number of doses available annually by - million [ ] . to avoid supply shortages, countries need concrete demand planning, secured budgets for vaccine purchases and careful stock management. prevention of shortages starts at country level, while paho acts regionally to facilitate timely and sufficient supply, and vaccine manufacturers worldwide are essential for assured global supply. d. steele, bill & melinda gates foundation, emphasised the importance to address global challenges for diarrheal disease vac-cines. international partnerships with globally funded product development partnerships and dcvms have already delivered two vaccines for the prevention of leading causes of diarrheal mortality, demonstrating the power of partnerships. first, a cholera vaccine was developed through partnership with vabiotech, shantha biotechnics, and the ivi [ ] . the vaccine is who prequalified and is widely utilised in countries with endemic cholera. second, a rotavirus vaccine was developed in partnership with bharat biotech, indian department of biotechnology and path [ ] . the vaccine is licensed in india for introduction in early and a dossier is ready for who prequalification. c. egerton-warburton elaborated on the goal of the global health investment fund (ghif): to invest in quality by supporting late-stage clinical trials and early-stage commercialisation of vaccines, drugs, diagnostics and devices required for control of infectious diseases in low-income countries, as to the world bank classification . an example is the investment in the eubiologics manufacturing facility for oral cholera vaccine, prequalified by the who in december , testifies the impact of financial support to start up manufacturers. egerton-warburton noted that new and innovative financing mechanisms, which complement the newlyproposed revolving fund at unicef, may be required to support immunisation in non-gavi countries. while a large portfolio of new vaccines is under development by manufacturers in developing countries, the conference focused on a few novel and relevant results, due to time limitations. a. precioso, director of clinical trials and pharmacovigilance at butantan, reviewed the clinical development of a live, attenuated dengue vaccine. the tetravalent vaccine is expected to protect against all dengue serotypes, inducing both humoral and cellular immune responses, and requires only one or two doses to provide life-long immunity. phase i clinical trials in the us by nih showed that one dose of vaccine was safe and immunogenic in both flavivirus-naïve and seropositive adults. butantan is currently finalizing a phase ii clinical trial, and ethical and regulatory approval for a phase iii trial in brazil is underway [ ] . h.t. pham, ceo of bionet-asia, outlined the development of a recombinant pertussis vaccine in response to the resurgence of pertussis around the world. bionet has constructed new proprietary bordetella pertussis strains which produce non-toxic, genetically-inactivated pertussis toxin (rpt) [ ] . in a phase i/ii clinical trial, the rpt-containing vaccines were safe and immunogenic in healthy adults, and elicited significantly higher anti-pt titres than the conventional dtap vaccine. another phase i study will soon evaluate rpt vaccine with an epi-cutaneous delivery system [ ] . l. shi, ceo of shanghai zerun biotech, summarised the progress made in recombinant hpv vaccine development. he reviewed the strong causal relationship between hpv infection and cervical cancer. zerun's bivalent hpv ( / ) recombinant vaccine candidate, produced in the yeast, pichia pastoris, is currently in a phase iii clinical trial, and is expected to be on the chinese market by , and to obtain who pq thereafter. in addition, a novel recombinant hpv vaccine candidate based on escherichia coli expression systems, developed by another manufacturer in china, is also in phase clinical trials, as previously reported [ ] . l. castello-branco, scientific director at fundaç ão ataulpho de paiva (fap), presented an overview of the bcg vaccine (moreau sub-strain). this vaccine was developed in brazil in the s and used as an oral vaccine in the national program on immunisation until [ ] . genetic data shows that the sub-strain is similar to the japanese and russian sub-strains [ ] . safety and efficacy of intradermal bcg-moreau vaccine has been demonstrated in large clinical trials. it is the only bcg administered to babies born to hiv positive mothers in brazil and it is licensed for use for intravesical therapy of bladder cancer [ ] . fap is now expanding the manufacturing capacity to supply bcg globally. g. liao described development of the sabin ipv at the institute of medical biology, chinese academy of medical sciences (imbcams). to accelerate eradication of circulating polioviruses from live polio vaccines, who launched an initiative to develop ipv. imbcams started sabin-ipv development . a successful phase iii clinical trial was completed in march , and the world's first sabin-ipv vaccine was launched on the chinese market in july . imb-cams is planning to increase production capacity from the current million to million doses per annum, to develop a combination vaccine and to obtain who pq to enable global supply [ ] . k. ella, bharat biotech, presented their chikungunya vaccine, an inactivated, whole-virion chikv derived from an indian isolate [ ] . chikv was established to high titre in vero cells. pre-clinical toxicity studies demonstrated safety with times the dose proposed for phase i testing. phase i dose ranges were based on a comparative study of protective correlates in convalescent patients and data from immunogenicity testing in laboratory animals. a phase i study is proposed in india. vaccine methodology is protected by patents in several countries. m. bottazzi, from sabin vaccine institute, reviewed novel technologies for vaccines against neglected tropical diseases. seventeen tropical diseases, including leprosy, trematodiasis, leishmaniasis, dengue, chagas disease, filariasis and helminth infections are highly prevalent among the poor, and are endemic in countries, affecting . billion people [ ] . the sabin vaccine institute focuses on product development partnerships to develop effective, lowcost vaccines. the human hookworm vaccine initiative illustrates these efforts against a leading cause of maternal and childhood anaemia, which afflicts million people. two candidate vaccines have been manufactured and are currently being tested in brazil and gabon. the institute is seeking partnership with manufacturers from developing countries to advance the product and clinical development of various candidate vaccines. the eubiologics oral cholera vaccine (ocv) was presented by s. park. eubiologics has a contract manufacturing agreement with ivi for production and supply of cholera vaccines. since , investments from green cross corporation, ghif and others have supported the project. the goal in was to maintain a milliondose global stockpile, and thereafter, to gradually increase this to million doses annually, financed through gavi. the stockpile is expected to aid global response to outbreaks, improve overall availability and supply of ocv, attract new manufacturers and incentivize new developments. clinical trials were completed in august [ ] . this vaccine was approved in in korea. a prequalification dossier has been approved by who demonstrating the impact of partnerships among manufacturers. delivery of ocv was discussed by j. [ ] , and is now using the recombinant technology platform to produce, potentially, the world's rd commercialised hpv vaccine. finlay is developing a -valent pneumococcal vaccine. verez noted that, as % of the world's infants are not yet vaccinated against pneumococcal disease, it may be useful to have an affordable valent vaccine that protects against regional disease strains, while waiting for higher valency vaccines to become accessible to middle and low-income populations. head-to-head studies may help understand the impact of multivalent vaccines compared to regionally relevant formulations. leroy asked how a manufacturer such as innovax could accelerate who pq and how dcvmn members could support their clinical development. huang stated that the dcvmn is able to break barriers between countries and facilitate collaboration and knowledge sharing; it is a matter of calling for such an initiative. harmonisation of the quality of clinical trials and dcvmn's role in calling for convergence between regulators was encouraged. a. precioso commented that dcvmn has several initiatives, including workshops on clinical trial management to harmonise practices and support who prequalification of vaccines. dcvmn needs time to develop such complex initiatives, and collaborations between companies can contribute, by enabling joint-clinical development and international multicentric clinical trials. d. curry (centre for vaccine ethics and policy), whose expertise lies in ethical and legal requirements for and oversight of clinical trials, suggested that one area of support to dcvms might be development of ethical frameworks to guide clinical trial design, conduct and assessment. a discussion on vaccine security was moderated by j. kim, director general of ivi, with h. deehan of unicef, m. makhoana of biovac, n. premsri and t. mahmoud as contributors. unicef and gavi are steadily increasing vaccine funding and procurement for developing countries in response to increasing demand. h. deehan noted that, while sourcing vaccine from multiple suppliers helps prevent global vaccine shortages, it also creates fierce competition, and noted the difficulty of finding the balance in having an ideal number of suppliers and fostering a sustainable, competitive market. kim turned attention to the recent ebola outbreak in africa, which was brought under control without a vaccine [ ] . he posed a question on how manufacturers can prepare for re-emergence. m. makhoana agreed that future outbreaks may come, and emphasised that the understanding of the infectious agent must precede product development. vaccines then need to be part of broader strengthening of health systems. ultimately, responses tackling african diseases may need to come from within africa. kim queried vaccine security in asia in the context of the regional threats with bird flu, sars and mers . n. premsri related thailand's philosophy of self-reliance, with the ideal to have capacity to produce essential vaccines and an adequate stockpile from multiple regional manufacturers in order to respond to outbreaks or shortages. t. mahmoud described vaccine-supply challenges in saudi arabia, where the objective of high vaccination coverage is undermined by vaccine shortages and lack of expertise in vaccine manufacturing in the region. technology transfer with the appropriate partners and a competent team of manufacturers will accelerate efforts to secure high vaccination coverage regionally. the discussion on developing markets was moderated by j. chu, senior director of vaccine markets at the clinton health access initiative (chai). he noted that vaccine markets are unique: it requires a high investment for manufacturers, and the lengthy development times, can often result unintentionally in supply monopoly. changes in regulatory standards can cause disruption and delays in lot release and registration. despite the challenges, dcvmn members have been contributing significantly to increasing access to vaccines globally. in , the human-vaccine market was estimated at us$ billion and it is forecasted to reach us$ billion by [ ] . to seize the opportunities and compete successfully in this rapidly growing global market, manufacturers need to continuously innovate. m. malhame (gavi) raised the balance between affordability and sustainability of vaccine supply, explaining that gavi evaluates any new vaccine that has passed phase ii clinical trials for inclusion in the vaccines' portfolio financed by gavi. this evaluation is data-driven, and includes parameters such as disease burden, feasibility and cost-effectiveness of disease prevention. however, she noted that coordination and alignment between global stakeholders need to improve and ensure development of the most needed vaccines. she also commented that the high costs of vaccine development, relative to those of drugs or diagnostics, could be overcome in part by technologies to improve manufacturing processes. k. ella (bharat biotech) shared insights on building successful international partnerships and balancing private and public contributions. privatisation of manufacturing has resulted in stable vaccine supply in many countries, and private enterprise can enhance supply of public-sector. l. shi (zerun institute, walvax) discussed corporate motivation to enter international markets while also serving the needs of domestic markets. walvax is prioritizing product-portfolio investments to allow supply of vaccines internationally. large manufacturing capacity is required to satisfy both domestic and international markets and many chinese companies have this capacity, thus the world is likely to see more vaccines from chinese manufacturers in future. f. lobos described sinergium's journey into the vaccine market in south america. sinergium was a spin-off from a veterinarian vaccine manufacturer in . it established technology transfer agreements with large multinationals, primarily to satisfy the national needs for influenza, pneumococcal and hpv vaccines. importantly, partnerships with government accelerated their progress: technical support was received from the local regulatory agency in building a world-class facility complying with international standards; also, a multi-year governmental supply contract secured stability for the company. both local and international partners were critical for rapid success. now, with modern facilities and equipment, the company aims to expand partnerships with dcvms. s. inglis, director of nibsc, moderated the panel that included s. jadhav (serum institute of india), a. batson (path), p. carrasco (iaim), and k. sampson (apaci) on trust in vaccines. s. jadhav described the experiences of the serum institute of india, one of the largest and most successful manufacturers from emerging countries, with who-prequalified vaccines. still reluctance to purchase products from developing countries on the basis of perceived higher reactogenicity or preference for known brands is a barrier to acceptance of new products. measles vaccine was prequalified by who in , however unicef did not immediately take it up. serum institute supplied more than % of the measles-containing vaccines used to achieve measles and rubella eradication in latin america. for the introduction of meningitis a vaccine in african countries, community leaders were informed about benefits, resulting in high acceptance and very successful prevention of disease. the lesson is that industry needs to invest considerable effort into informing stakeholders of their products and intentions. a. batson noted that vaccine hesitancy is often specific to a country or situation, rather than a global issue, and strongly agreed that engaging the local community leaders can have significant impact on uptake. interestingly, mobile phones have proven useful to advocate for vaccines for teenagers, such as hpv vaccines. k. sampson discussed the confidence in influenza vaccines, where there is a risk that the strains that make up the vaccine may not fully match those causing disease, due to antigenic drift. he noted that asia has a large ethnically and socio-culturally heterogeneous population and a vaccine may not suit all patients. decision-making on influenza vaccination is driven by whether it is likely to protect the vaccinated individual, and also, whether is it likely to help protect others through herd immunity. ready availability of disease data that can be communicated to medical professionals would assist in highlighting value. p. carrasco elaborated further on the role of health workers delivering vaccines, noting that they build patient trust by imparting their knowledge of the vaccines and vaccination, but also inspire confidence when they clearly trust the intervention themselves. educational programs, organised by professional associations, need to provide health professionals with the right information about why and how vaccines are used, in their own language. new tools, such as smart phones, are within reach of most health workers, even in low-income countries. the dcvmn annual general meeting gave rich insights into advances in vaccine technologies and development of new vaccines, and provided valuable opportunity for vaccine manufacturers, regulatory authorities and international organisations to have face-to-face interactions. to achieve global immunisation, local authorities and community leaders play an important role in the decision-making in vaccine introduction and uptake, based on the ability of vaccines to protect vaccinated people and protect non-vaccinated in the community through herd immunity. reducing the risk of vaccine shortages can be achieved by increasing regulatory convergence at regional and international levels. combatting preventable diseases outbreaks remains challenging, and collective efforts for improving multi-centre clinical trials including various populations, creating regional relevant vaccine manufacturing and stockpiling security strategies, fostering stable vaccine markets and procurement, and building trust in vaccines are recommended approaches. the dcvmn members will continue to collaborate with one another, with partners and with regulators towards the goal of improving global access to immunisation, through sustainable supply of vaccines to match demand, as well as greater capacity to rapidly respond to outbreaks. the aim is to provide high quality vaccines for all people. who fact sheet meningococcal conjugate vaccination in burkina faso: analysis of national surveillance data gavi alliance sets out opportunity to save up to six million lives through immunization. gavi pr of media centre of world health organisation. immunization leaders call for increased political support for immunization in pakistan. media centre of world health organisation eliminating epidemic meningitis as a public health problem in sub-saharan africa. a public health breakthough regulatory convergence sought for global pharma market about gavi. gavi's business model: working together for healthy vaccine markets the yellow fever initiative: providing an opportunity for a lifetime safety and immunogenicity study of a killed bivalent (o and o ) whole cell oral cholera vaccine, shanchol, in bangladeshi adults and young children as young as year of age team science and the creation of a novel rotavirus vaccine in india: a new framework for vaccine development clinical evaluation strategies for a live attenuated tetravalent dengue vaccine construction of bordetella pertussis strains with enhanced production of genetically-inactivated pertussis toxin and pertactin by unmarked allelic exchange needle-free and adjuvant-free epicutaneous boosting of pertussis immunity: preclinical proof of concept dcvmn executive committee group. vaccines, our shared responsibility rio de janeiro: an oral vaccine against tuberculosis-review genome sequence of mycobacterium bovis bcg moreau, the brazilian vaccine strain against tuberculosis bcg immunotherapy for bladder cancer-the effects of substrain differences safety and immunogenicity of inactivated poliovirus vaccine made from sabin strains: a phase ii, randomized, positive-controlled trial chikungunya virus and prospects for a vaccine the disease next door. foreign policy a randomized, non-inferiority trial comparing two bivalent killed, whole cell, oral cholera vaccines (euvichol vs shanchol) in the philippines stockpiling oral cholera vaccine hepatitis e vaccine: who position paper ebola and its control in liberia world preview : outlook to . vaccines market to we are grateful to all speakers and moderators whose contribution made the conference possible. we thank corporate partners for supporting dcvmn with unrestricted educational grants: the merck group, temptime corporation, ge healthcare, bioengineering, gea, bosch, ompi (stevanato group), alfa wasserman, pall. this conference was partly supported by a grant from the bill & melinda gates foundation, grant no. opp . we are grateful to m. dennehy for editorial support. the authors are employees of the respective indicated organisations, and have no conflict of interest to declare. dcvmn international did not provide any financial support to speakers or moderators to participate at this meeting. key: cord- - x j authors: vergison, anne title: microbiology of otitis media: a moving target date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: x j abstract the microbiology of acute otitis media (aom) is linked to the nasopharyngeal commensal flora. this respiratory ecosystem undergoes various selective pressures, such as antibiotic consumption and vaccine use. socio-economic conditions also influence the bacterial composition of the nasopharynx. streptococcus pneumoniae, non-encapsulated haemophilus influenzae, moraxella catarrhalis, and group a streptococcus are the leading causes of bacterial aom worldwide. this paper will discuss the causes and consequences of recent shifts in the underlying microbiology of aom. otitis media (om) is a very common childhood disease and a major concern for paediatricians. in a prospective, -year study performed in the usa, over three quarters ( %) of children who completed the investigation experienced at least one episode of acute om (aom) by the age of years and % had suffered from at least three episodes [ ] . the centers for disease control and prevention has estimated that om accounts for more than million physician visits per annum in the usa [ ] . furthermore, in a recent multinational survey, paediatricians reported that they saw at least one patient with om per day [ ] . aom is often preceded by a viral upper respiratory tract infection (urti) -the most common infectious illness in the general population [ ] , and a very common illness in children. in a prospective, -year study following children aged from months to years, a total of urtis were recorded, with aom reported in ( %) children in the course of the viral infection, i.e. . episodes of aom per child per year [ ] . the underlying microbiology of infectious diseases is known to change in response to environmental factors, such as vaccination and antibiotic consumption. for example, the epidemiology of bacterial meningitis has changed in countries where haemophilus influenzae type b [ ] , group c meningococcal, and pneumococcal conjugate vaccines have been introduced, with a dramatic reduction in the incidence of bacterial meningitis overall. however, non-vaccine serotypes of streptococcus pneumoniae now account for a more significant proportion of the disease in the countries * tel.: + ; fax: + . e-mail addresses: anne.vergison@ulb.ac.be, anne.vergison@huderf.be. where the seven-valent pneumococcal conjugate vaccine (pcv ; prevnar tm /prevenar tm ) is widely used [ , ] . furthermore, s. pneumoniae isolates with reduced susceptibility to penicillin were recovered from human infections in the late s in australia and new guinea [ ] . since then, penicillin non-susceptible s. pneumoniae strains have spread all over the world and their prevalence has dramatically increased in various countries [ ] . moreover, s. pneumoniae accumulated multiple resistance determinants in some strains and serotypes [ ] [ ] [ ] , and modified the epidemiological landscape in some regions, including the usa [ ] . although in most regions, penicillin remains active against s. pneumoniae despite increased minimal inhibitory concentrations and can be used safely to treat pneumococcal infections other than meningitis, some multiresistant strains have been described in infections such as aom [ ] . a recent report from the usa presented nine clinical failures in aom due to s. pneumoniae resistant to amoxicillin, oral cephalosporins, macrolides, clindamycin, and co-trimoxazole, and required tube placement for drainage and the use of levofloxacin, a drug which is not licensed for paediatric use [ ] . in another recent us study, an increased proportion of severe mastoiditis cases was observed, mostly due to multiresistant serotype a s. pneumoniae [ ] . at the beginning of the th century, group a streptococcus (gas) was the most common pathogen leading to complications in aom, but it is now rare in the western world. a 'new' triad of aom pathogens has emerged in the last century -s. pneumoniae, non-encapsulated h. influenzae (often called non-typable h. influenzae [nthi]), and moraxella catarrhalis -all of which are commensal bacteria found in the human nasopharynx. this review provides some insight into the microbiology of aom in an era of antibiotic resistance and pneumococcal conjugate vaccine use. aom is a multipathogen disease, and can be caused by a number of different viruses and bacteria. viruses alone are found in only % of cases, while co-infection with bacteria is seen in % of cases [ ] . among the viruses, coronavirus, respiratory syncytial virus, and adenovirus are most commonly associated with aom [ ] . s. pneumoniae and h. influenzae are by far the most common bacterial pathogens in aom, being recovered in up to % of cases. m. catarrhalis is usually the third most frequent bacterium isolated ( - %) and gas makes up - % of cases, although the incidence of gas infection differs between countries, depending on when the study was performed, and whether severe cases of aom were included ( fig. ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the diagnosis of aom is difficult as a number of symptoms, for example pain, fever, conjunctivitis, and headache, are shared with other infections of the upper respiratory tract. furthermore, diagnosis in young children is hampered by the child's inability to describe their symptoms and the likelihood that they will be distressed and experiencing pain. for a clinical diagnosis of aom, the key criteria that should be met are a history of acute onset of signs and symptoms (fever, distinct otalgia that precludes nor-mal activity, or sleep), with signs of middle ear infection (a cloudy, bulging, or clearly immobile eardrum with red colouration of the eardrum and the presence of fluid in the middle ear or otorrhoea) [ , ] . some clinical signs have been associated with particular otopathogens (e.g. conjunctivitis is associated with h. influenzae, while more severe cases of aom are more often caused by s. pneumoniae) [ ] [ ] [ ] . however, accurate identification of underlying pathogens is not possible purely on clinical grounds. in the large, prospective finnish trial, severe tympanic membrane findings (bulging tympanic membrane or spontaneous perforation) with concomitant high fever had a % positive predictive value and a % negative predictive value for a s. pneumoniae aetiology of the aom episode. the presence of a purulent conjunctivitis gave a positive predictive value for h. influenzae aom of % with an % negative predictive value. no useful predictors were found for m. catarrhalis aom [ ] . even in the absence of definitive clinical signs for the identification of the underlying otopathogen(s), most guidelines do not advocate the systematic use of tympanocentesis, in which fluid is collected from behind the eardrum and analysed to identify the infectious organisms involved and perform antibiotic susceptibility testing. if an antibiotic treatment was to be prescribed, it would be chosen on empirical grounds based on local epidemiological data. however, in selected cases (antibiotic treatment failure and complicated aom), it is essential to identify the causative otopathogen accurately and determine its antibiotic susceptibility. tympanocentesis will, therefore, be recommended in order to ensure the most effective treatment course [ , , ] . all-cause aom incidence peaks between the ages of and months, with one study reporting a peak incidence at months of . aom events/ child months (fig. ) [ ] . in this study, nthi showed a distinct pattern of incidence compared with s. pneumoniae and m. catarrhalis. there was an increase in the incidence of nthi aom after the age of months, peaking at months ( . aom events/ child months). moreover, h. influenzae was associated with recurrent aom. it was recovered in middle ear fluid (mef) in % of first aom episodes compared with % of all subsequent om episodes, and when only the first aom episodes were considered, no peak in the incidence of h. influenzae aom could be demonstrated in children over year of age. despite the use and availability of antibiotics and appropriate medical access, aom can often lead to recurrences and, in rare cases, severe intratemporal (facial paralysis, labyrinthitis, and acute petrosistis, which are extremely uncommon) and intracranial complications, such as mastoiditis, meningitis, intracranial abscesses, and sinus thrombosis. although mastoiditis has become infrequent in industrialized countries (incidences from . to / , childyear) [ ] [ ] [ ] , it is still a common belief that antibiotic treatment should be prescribed in aom to prevent its occurrence. the prevention of mastoiditis by systematic antibiotic treatment of aom has never been established [ , ] . many factors can account for variations in the incidence of mastoiditis in different countries: socio-economic and living conditions, antibiotic prescribing rates, exhaustiveness of the epidemiological surveillance systems, and differences in complication rates by pathogens [ , , ] . indeed, not all bacterial otopathogens have the same propensity to cause complications of aom. gas is associated with the most frequent and severe complications, such as mastoiditis (table ) , while severe complications of h. influenzae are uncommon and those of m. catarrhalis infection are rare [ ] . as previously discussed, evidence suggests that s. pneumoniae is more common in severe episodes of om [ ] , while nthi is more commonly associated with recurrent om (rom) (fig. ) [ , ] . one recent study assessed the underlying microbiology of rom (defined as three acute episodes in the previous months or four in the past months) and aom treatment failure (defined as persisting signs and symptoms of aom after ≥ h of antibiotic therapy or within days of completing an antibiotic treatment course) in us children following the widespread introduction of pcv in [ ] . although there was a slight increase in the proportion of s. pneumoniae isolates present during the - season, h. influenzae was the most frequently isolated pathogen ( % of all isolates across three respiratory seasons, [ ] [ ] [ ] [ ] in this difficult-to-treat patient group during a time of increasing and widespread use of pcv (fig. ). difficulties encountered in the treatment of om are not only due to the existence of antibiotic resistance in otopathogens, but are also attributable to the biofilm nature of bacterial om infections. table the risk for development of mastoiditis following aom caused by different bacterial otopathogens [ ] . in natural environments, the majority of bacteria exist as a biofilm (a structured community of microorganisms embedded within a polymeric matrix that is attached to an inert or living surface) rather than in a planktonic state. in contrast to planktonic bacteria, biofilm bacteria are characterized by slow rates of cell division and a tolerance to very high concentrations of antibiotics. biofilm infections are, therefore, difficult to treat effectively with currently available antibiotic agents, which rely on the rapid metabolic and divisional rates of planktonic bacteria for their mode of action. the presence of bacterial biofilms in om was first suspected owing to the persistence of infection despite treatment with antibiotics and the absence of positive cell culture specimens. definitive evidence for the biofilm nature of om has been provided by a number of studies. for example, one study used polymerase chain reaction techniques to detect h. influenzae dna and mrna in mef from children with chronic om with effusion. the presence of the short-lived mrna molecules, even in the absence of positive culture specimens, indicated the presence of viable bacteria in these specimens [ ] . additionally, the three major bacterial pathogens of om have been proven to form biofilms in vitro and in vivo [ ] [ ] [ ] [ ] [ ] , while one study has reported the direct detection of bacterial biofilms on middle ear mucosa biopsies from children with chronic om [ ] . antibiotic use results in the selection of strains resistant to antibiotics. this was demonstrated in vitro for s. pneumoniae by alexander fleming shortly after he discovered penicillin [ ] . more recently, the correlation between antibiotic consumption and resistance was demonstrated in a european study comprising countries. outpatient antibiotic use was correlated with resistance for all antibiotic-pathogen combinations, and more specifically for s. pneumoniae [ ] . the nasopharynx constitutes a wide reservoir where resistant bacteria (s. pneumoniae but also streptococcus viridans, h. influenzae, and m. catarrhalis) can easily be selected whenever antibiotic selective pressure is applied [ ] . antibiotic resistance in s. pneumoniae and h. influenzae has become a major public health issue and a european union priority for research and action. it is expected that the prevalence of chronic obstructive pulmonary disease will increase in the coming years in europe, and both s. pneumoniae and h. influenzae have major infectious roles for this condition [ ] . similarly, the world health organization (who) has set antimicrobial resistance contain-ment as a research priority, particularly regarding s. pneumoniae [ ] . all three major otopathogens cause antibiotic resistance concerns. penicillin and multidrug resistance in s. pneumoniae has already been described above. in europe, dual erythromycin and penicillin non-susceptibility varies widely between countries, from less than % to more than % [ ] . while amoxicillin resistance in m. catarrhalis is universally seen in approximately % of the strains, it is still a limited occurrence in nthi (a mean of % ␤-lactamase production in one international study) [ ] . however, some regions, such as france, the usa, japan, and other southern asian regions have high rates of amoxicillin resistance in nthi [ , ] . several mechanisms cause this resistance, the most common being ␤-lactamase production, which is detected in most laboratories. however, other resistance mechanisms are increasingly being described in france and japan, which confer additional resistance to amoxicillin-clavulanate, cefuroxime, and sometimes to third-generation cephalosporins, and which are usually not investigated in routine microbiology [ , ] . in a recent study conducted in japan, only % of the nthi strains isolated from children with urtis were amoxicillin susceptible and % were ␤-lactamase producers; the others were also resistant to amoxicillin-clavulanate and to a various degree to cephalosporins [ ] . the introduction of conjugate vaccines that impact on the commensal flora creates 'epidemiological niches' for alternative potential pathogens that are not included in the vaccine. the introduction of pcv in the usa resulted in rapid shifts in the microbiology of om [ , , , ] . pcv vaccination was followed by rapid replacement with non-vaccine s. pneumoniae serotypes in the nasopharynx of vaccinated children and their siblings and, as a result, the proportion of aom caused by vaccine serotypes has fallen and disease caused by non-vaccine serotypes and other pathogens, such as nthi, has risen. for example, in one us study, significant increases in the percentage of aom cases due to non-pcv pneumococcal serogroups occurred between and (from % to %, respectively; p < . ). however, no decline was observed in the penicillin non-susceptible s. pneumoniae strains [ ] . as mentioned previously, multidrug-resistant replacement serotypes may arise [ ] , stressing the need for reduced antibiotic prescribing in order to maximize the benefits of the vaccine in eliminating the most prevalent antibiotic-resistant serotypes. additionally, the trend for an increase in persistent aom and aom treatment failure attributable to h. influenzae observed from to in a single us study centre [ ] was also reported in three us centres for the period - [ ] . om is a common disease that affects approximately three quarters of children before their third birthday. currently, s. pneumoniae and nthi are responsible for approximately % of all bacterial aom cases, with s. pneumoniae generally causing more severe episodes, and nthi responsible for recurrent episodes. the underlying microbiology of om, which is inherently linked to the nasopharyngeal commensal flora, is changing over time in response to various selective pressures, such as vaccine use and antibiotic consumption. consequently, continuous monitoring of the 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pneumoniae: epidemiology and patterns of resistance european academies science advisory council. tackling antibacterial resistance in europe who policy perspectives on medicines. containing antimicrobial resistance antimicrobial resistance in streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and group a beta-haemolytic streptococci in - . results of the multinational grasp surveillance program haemophilus influenzae and moraxella catarrhalis from patients with community-acquired respiratory tract infections: antimicrobial susceptibility patterns from the sentry antimicrobial surveillance program (united states and canada, ) clinical bacteriology and immunology in acute otitis media in children epidemiology of haemophilus influenzae strains collected in in france and in vitro assessment of their susceptibility to antibiotics remarkably high prevalence of fts i gene mutations in haemophilus influenzae isolates from upper respiratory tract infections in children of the sapporo district changes in frequency and pathogens causing acute otitis media in - acute otitis media due to penicillin-nonsusceptible streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine i would like to thank philippe lepage and pierre smeesters for the critical proofreading of this manuscript. a.v. has received speaker fees from glaxosmithkline, sanofi and wyeth, consultant fees from glaxosmithkline and wyeth, and research fees from wyeth. key: cord- -ua xoak authors: bennet, rutger; hamrin, johan; wirgart, benita zweygberg; Östlund, maria rotzén; Örtqvist, Åke; eriksson, margareta title: influenza epidemiology among hospitalized children in stockholm, sweden – date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ua xoak background: influenza remains a common reason for the hospitalization of children. there is a need for long term studies that are also population based. we describe the epidemiology of severe influenza in a defined population – . method: retrospective study of annually collected data of virologically confirmed influenza in hospitalized children – years living in the catchment area ( , children). we gathered information about comorbidity and complications from case records, and compared influenza a, b and a(h n )pdm with respect to these factors. results: a total of children with influenza were hospitalized. the mean rate remained unchanged at . – . per , children per year. there were two major outbreaks: influenza a(h n ) in – and the a(h n ) pandemic in – . the proportion of children with influenza b increased from % during the first half of the study period to % during the second half. the highest admission rate was found in children < months of age, per , . children with influenza b were older than those with influenza a. comorbidity was found in %, complications in %, and % needed intensive care management. the mortality rate was . / , children. conclusion: influenza remains an important reason for the hospitalization of children, especially during the first years of life. the increasing proportion of influenza b may have to be considered when recommending influenza vaccines. influenza is common among children, especially before years of age [ ] . we here describe the influenza a, b and a(h n )pdm epidemiology in the - -year-old population of northern stockholm during seasons, / - / , and its implications for the immunization of children. in stockholm, children with certain chronic diseases are eligible for free seasonal influenza immunization. this was introduced in and originally included children with chronic pulmonary or heart disease, but after the pandemic was expanded to include also chronic liver or renal diseases, immunosuppression, diabetes mellitus, extreme obesity (body mass index > ), neuromuscular disease affecting breathing capacity, and children with multiple dysfunctions/handicaps. during the pandemic, adjuvanted monovalent h n vaccine (pandemrix ® , glaxosmithkline biologicals, rixensart, belgium) was offered to all children from months of age. however, except during the pandemic season, when % of all children accepted the offer, the vaccine uptake has been low (< %), perhaps due to fear of side effects or unawareness of the severity of influenza [ ] [ ] [ ] . in order to increase the knowledge base for vaccination recommendations, in addition to data that can be obtained from administrative databases, we here provide population-level influenza burden data based on observed, virologically confirmed hospitalizations. this is a retrospective study of annually gathered information about influenza in children (< years hospitalized at astrid lindgren children's hospital, stockholm, sweden during - . the hospital is a tertiary referral center with surgery and a pediatric intensive care unit (picu) with resources for extracorporeal membrane oxygenation (ecmo), but only children resident in the catchment area were included in the study. we obtained population data from the stockholm area database at statistics sweden (www.scb.se). during the study period, the number of children in the primary catchment area increased by %, from , to , . the number of person years for a specific age group during a certain year was considered equal to the number of children on december . we extracted information about risk factors, complications and intensive care management from the hospital charts. from we have also been able to extract information from the vaccine register in stockholm county [ ] . risk factors were neuromuscular disease, chronic lung disease, immunosuppression, and chronic conditions such as kidney or liver disease, or inborn errors of metabolism. if a child had both neuromuscular and chronic lung disease, the factor judged most important for the clinical course was counted. recurrent wheezing/"asthma" in children younger than two years and uncomplicated preterm birth (> gestational weeks) were noted but not considered risk factors. we counted sinusitis, tracheitis and presumed bacterial pneumonia (with or without empyema), but not otitis media as focal complications. chest x-ray was always performed when pneumonia was suspected. if perihilar infiltrates and/or hyperinflation were present, a diagnosis of bronchitis or bronchiolitis was made. bacterial and viral pneumonia were differentiated mainly by c-reactive protein (crp) levels, with a cut-off value of mg/l. neurologic complications were seizures; either primary or secondary (in children with underlying neurologic disease), and encephalitis confirmed by electroencephalography. other complications included a few rare conditions such as myocarditis. dehydration alone was not considered a complication. as a routine, children admitted from our pediatric emergency ward with respiratory symptoms or fever without localizing signs, including febrile convulsions, are examined for viral etiology. during the winter season samples are primarily investigated for respiratory syncytial virus (rsv) and influenza viruses. if they are found negative, viral investigation is extended to other respiratory viruses (adenovirus, bocavirus, coronavirus, enterovirus, human metapneumovirus, parainfluenza virus - , and rhinovirus) [ ] . all cases in this report were virologically confirmed by the laboratory. influenza virus was detected with immunofluorescence and viral isolation prior to october , when these methods were replaced by real-time polymerase chain reaction (rt-pcr) [ , ] . before the switch to rt-pcr the diagnostic sensitivity of the three methods was evaluated in samples at our laboratory [ ] . the rt-pcr analysis has since then been improved (in and by the inclusion of new probes for influenza a(h n )pdm and in by the redesign of the influenza b probe). rsv has since been diagnosed with a rapid point-of-care test, with negative specimens further investigated for both rsv and influenza with rt-pcr. the chi-square test, the mann-whitney u-test, and exact clopper-pearson binomial confidence intervals were employed as appropriate. for multivariate analysis, we used a generalized linear model with the logit function in statistica ® v. , with influenza subtype (non-pandemic a or b) as the dependent variable, and age and presence or absence of risk factors and complications as independent variables. there were children ( % male) with confirmed influenza during the study period. had non-pandemic influenza a, had influenza b, and had influenza a(h n )pdm , out of whom belonged to the - pandemic. during all winter seasons there were influenza epidemics with varying severity. all but two influenza epidemics occurred during rsv epidemics after january and there were then always more children with rsv during a given week (fig. ). the two largest outbreaks - and - peaked before january and preceded the rsv epidemics. influenza b was present in all but two seasons and dominated during three seasons, all of which occurred during the second half of the study period. co-infections (with any simultaneously circulating respiratory virus) were identified in % of the patients. this is a minimum figure, since some children, for instance those with a positive point-of-care test for rsv, were not examined for additional viral etiology such as influenza. when the period comprising the first eight seasons was compared with the second period, excluding the pandemic and thus also comprising eight seasons, there were significantly more cases of influenza b during the latter period (p < . ). however, the overall rate of influenza cases in children < years remained unchanged at . ( % c.i. . - . ) and . ( . - . ) per person years, respectively. the cumulative age distribution is shown in fig. . children with influenza b were older (as also shown in table ) than those with influenza a. influenza a(h n )pdm is presented separately for the pandemic year - and the post pandemic years, since the pandemic patients were significantly older (p < . ). the yearly incidence rates in different age groups varied considerably, with median (range) for children < years ( - previously known risk factors were found in / ( % , table ), the most important being neuromuscular disease ( cases) and chronic lung disease ( cases). the frequencies of recurrent wheezing (not included among risk factors) and preterm birth were comparable to their occurrence in the general child population at % and . % ( / ), respectively. complications were seen in / ( %). most were related to the respiratory tract where pneumonia dominated ( ) out of which half ( ) were considered bacterial in origin. other bacterial superinfections, such as sinusitis (including periorbital cellulitis) were diagnosed in children. approximately % of the children had a blood culture taken, which was positive in ( streptococcus pneumoniae, staphylococcus aureus, streptococcus pyogenes and neisseria meningitidis). neurological complication was the second most common with encephalitis ( ) and primary or secondary seizures ( ). risk factors and complications were more common among children with influenza b than in those with influenza a in the univariate analysis (p < . ), but when included with age in the multivariate model, only age remained significant (p < . , table ). children with recurrent wheezing had a low rate ( / ) of picu admission. their median duration of hospital stay was days, which is equal to that of other children without risk factors. intensive care admission was required for / children ( %); / ( . %) without risk factors and / ( %) with neuromuscular or chronic lung disease (p < . ). two children were treated with ecmo. six children ( . / person years) died, < year, - years, and > years old. three of these were previously healthy, whereof two had an influenza b infection. the causes of death were encephalitis, myocarditis and bacterial pneumonia in the previously healthy children, and non-specific complications in the three children with co-morbidity. this is a report of children hospitalized for influenza a or b in a defined population in the northern stockholm area - , covering the pre-pandemic period, including the - outbreak, the pandemic, and four post-pandemic seasons. there was a strong seasonal variation and the epidemiologic pattern was often in accordance with what has been seen in other countries, such as the large outbreak of a(h n ) in - [ ] . with the exception of the influenza a(h n )pdm pandemic with a peak in late autumn , the epidemic peaks occurred during winter and coincided with rsv, with co-infections detected in %. as we have reported earlier, rsv and influenza co-infections do not have a more severe course or more late wheezing, but tend to follow the expected clinical course of the rsv infection [ ] . most population based long-term studies are from the north american continent, but one study from finland covers a year period up to [ ] . there have been both single center and multicenter studies [ , ] . some were population based whereas others represented tertiary centers without a defined population. many of these studies have covered the periods before the pandemic and have later been extended to also include the pandemic year [ , ] . several studies described the pandemic and a few have included the first post pandemic season [ , ] . no study has like ours covered both pre-and post-pandemic seasons, when the novel influenza a(h n )pdm continued to circulate. our findings are in accordance with these studies, all pointing to the fact that influenza in children is still a problem. neuromuscular disease and chronic lung disease were the dominating risk factors. in our setting recurrent wheezing/"asthma" and uncomplicated preterm birth were not important risk factors. bacterial superinfection, mostly pneumonia, was the most common complication. in most studies the distinction between bacterial and viral pneumonia has not been discussed. in our study % were considered to be viral [ ] . neurologic complications, such as seizures and encephalitis, were the second most common category. intensive care was needed for % of all children, which is in agreement with most other studies [ , , ] . the need for intensive care was highest ( %) in children with risk factors. the case fatality rate noted by us was similar to that reported from other western countries [ ] . our age distribution and incidence rate calculations are similar to, although in the lower range, of what has previously been reported [ ] . % of the children were below one year and % below five years of age, resulting in an incidence of / and / , respectively. the highest rate, / , was observed among the youngest (< months) infants, as in a recent study from the usa [ ] . an important part of our study is the comparison of different influenza types. in several studies comparisons have been done between seasonal influenza and (h n )pdm [ , ] . we studied influenza a(h n )pdm and the pandemic year separately from the following years, because of the marked difference of age distribution. during the pandemic, children were older in accordance with what has been presented from several other centers [ , , ] . during the following seasons, however, the average age was actually somewhat lower than in children with other influenza a types. one contributing factor could be the high vaccine coverage ( %) with the adjuvanted vaccine (pandemrix) during the pandemic in sweden, which has been shown to give protection for at least two subsequent seasons [ ] . in addition to the emergence of pandemic influenza, also the burden of influenza b has come into focus in recent years [ ] . we found influenza b especially during the second half of the study period. previous epidemiologic studies used different methods for estimating the number of influenza cases, such as excess of cases with influenza-like illness during influenza seasons, virus isolation, and rapid methods like immunofluorescence and pcr [ ] . at our laboratory immunofluorescence and viral isolation were replaced by the more sensitive pcr in october . to what extent this has contributed to our observed increase of influenza b can be discussed. the initial pcr system had a higher sensitivity for influenza b compared to immunofluorescence and virus isolation, while the sensitivity for influenza a was lower. the influenza a pcr has since then been continually improved, and in a review by mahoney et al. it is stipulated that well optimized rt-pcr assays have - % higher sensitivity than virus isolation [ ] . one limitation of our study is its retrospective nature. however, the same pediatricians have been responsible for the care of the infected children during the entire period. the same level of care has been provided from the picu with availability of ecmo treatment during the entire period. furthermore, in our study "retrospective" means yearly reviews and documentation of the previous season as a preparation for planning of the following season. our study is a summary of these reviews. another important factor, as mentioned, is the introduction of new tests that could be more sensitive. on the other hand, the use of rapid tests precluded the detection of coinfections -found in more than % in an earlier study from our hospital [ ] . the effect of influenza vaccine in children has been the subject of several reviews. in contrast to the known effect of trivalent influenza vaccine (the only one used during the studied period except for the pandemic year) in healthy children > months, less is known about its effect in younger children and in those with risk factors. during the - pandemic when % of the swedish childhood population was vaccinated with a monovalent adjuvanted vaccine an effect of % was demonstrated [ ] . as also found in an english study, the protective effect was still present in children with comorbidity, albeit smaller than in children without risk factors [ ] . even though the immunization of children with risk factors is free, few parents (< %) take the opportunity to vaccinate their children. the reason for the low uptake of the vaccine is probably multifactorial. in a recent questionnaire study in the usa two factors were highlighted: fear of side effects and the belief that influenza is not a serious threat to children [ ] . in conclusion, our population based study demonstrates that influenza in young individuals is still a major problem, especially but not exclusively in those with risk factors. we hope to be able to use this information to encourage more parents to immunize their children. there was a significant increase of influenza b during later years, primarily affecting older children with risk factors. the reason for this increase is probably multifactorial, but it points to the need for increased protection against influenza b, e.g. through the development of the quadrivalent vaccine. it may be important to note that in our study half of the children who died had no risk factors and that influenza b was the cause in / cases. none. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis effectiveness of the monovalent as -adjuvanted influenza a(h n )pdm vaccine against hospitalization in children because of influenza long term effectiveness of adjuvanted influenza a(h n )pdm vaccine in children pediatric provider vaccine hesitancy: an under-recognized obstacle to immunizing children development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses respiratory virus infections in stockholm during seven seasons: a retrospective study of laboratory diagnosis multistate surveillance for laboratory-confirmed, influenza-associated hospitalizations in children wheezing following lower respiratory tract infections with respiratory syncytial virus and influenza a in infancy incidence of influenza-related hospitalizations in different age groups of children in finland: a -year study incidence, complications, and risk factors for prolonged stay in children hospitalized with community-acquired influenza burden of seasonal influenza hospitalization in children comparison of children hospitalized with seasonal versus pandemic influenza a complications and associated bacterial coinfections among children hospitalized with seasonal or pandemic influenza the burden of seasonal and pandemic influenza in infants and children risk factors for mechanical ventilation in u.s. children hospitalized with seasonal influenza and pandemic influenza a influenza-associated pneumonia in children hospitalized with laboratoryconfirmed influenza the burden of influenza hospitalizations in infants from influenzaassociated pediatric deaths in the united states vaccination of healthy children against seasonal influenza: a european perspective children with asthma hospitalized with seasonal or pandemic influenza the burden of influenza b: a structured literature review influenza and the rates of hospitalization for respiratory disease among infants and young children molecular diagnosis of respiratory virus infections age-specific effectiveness of an oil-inwater adjuvanted pandemic (h n ) vaccine against confirmed infection in high risk groups in england key: cord- -d x wii authors: chang, chia-yin; hong, willy w.l.; chong, pele; wu, suh-chin title: influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of sars-cov and its implication for subunit vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: d x wii the spike (s) protein of severe acute respiratory syndrome coronavirus (sars-cov) is important for vaccine development. a truncated s protein of the tw strain, s(tr ) ( kda), carrying three s fragments (s – , s – , and s – ) was investigated to study the influences of intron and exon splicing enhancers to improve s(tr ) protein expression in mammalian cells. our results showed that s(tr ) protein expression with the use of an base-pair intron addition increased by . -, . -, and . -fold in vero e , qbi- a cells, and cho/dhfr− cells (dihydrofolate reductase [dhfr] gene deficient cho cells), respectively. using the exon splicing enhancers, including a bidirectional splicing enhancer (bse) or an exon splicing enhancer derived from the eda alternative exon of the fibronectin gene (eda ese), were also found to increase s(tr ) protein expression in cho/dhfr− cells by . - and . -fold. nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e s(tr ) protein expression in in cho/dhfr− cells. our studies also demonstrated the s(tr ) protein was mainly as the endo h-sensitive glycoprotein ( kda) expressed in vero e , qbi- a, and cho/dhfr− cells. however, only a minor form of the endo h-resistant glycoproteins (∼ kda) was detected in cho/dhfr− cells. taken together, our results indicated that intron had a better enhancing effect on s(tr ) protein expression than exon splicing enhancers, and the expression of ∼ kda s(tr ) glycoprotein was enhanced by the intron addition into the expression vector construct. results of the present study can provide an optimal strategy to enhance sars-cov s protein expression in mammalian cells and may contribute to the development of sars-cov subunit vaccine. severe acute respiratory syndrome (sars) is an infectious viral disease that caused by a newly emerged coronavirus (sasr-cov) [ ] [ ] [ ] . sars-cov is a large rna virus that contains four structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins. the s protein is responsible for receptor binding and membrane fusion, and is the most important vaccine target. the s protein can be divided into putative s (residues - ) and s (residues the sars-cov s protein has been the main target for sars-cov vaccine development. as previously reported, intranasal or intramuscular inoculation of highly attenuated modified vaccina virus ankara (mva) that expressed full-length s protein could induce neutralization antibody production and elicit protective immunity in balb/c mice [ ] . another report had indicated that recombinant attenuated parainfluenza type vector that expressed s instead of e, m, and n protein could induce neutralizing antibody and provide protective immunity against sars-cov challenging, which suggested that s was the only significant protective antigen among all sars-cov structure proteins [ ] . moreover, the sars-cov s protein fragments: residues - (receptor-binding domain) [ ] , residues - [ ] , and residues - that fused with thioredoxin (trx) or glutathione-s-transferase (gst) [ ] were found to induce neutralizing antibody production in immunized animals. these results suggested that the s protein is an excellent vaccine target, since it could induce strong neutralization antibody response and provide protection against virus challenging in immunized animals. in this study, the mammalian cell expression of a truncated s protein, s tr , which contains receptor binding region [ ] and an exposed domain (residues - ) [ ] that can induce neutralization antibody production was investigated. the highly glycosylated nature of s protein render mammalian cell to be a suitable expression system, since it have the advantage of carrying out complex posttranslational modification. however, the recombinant protein expression level of mammalian cell is much lower than other systems, while sufficient protein production is required for vaccine development. therefore, several different strategies for improving s tr protein expression in mammalian cells were investigated in this report, including intron addition and the application of exon splicing enhancers. as the intron and its splicing out was known to enhance each step of rna metabolism, including transcription, poly(a) tail addition, mrna exportation, translation, and translated protein stability [ ] , hence we first studied the intron-enhancing effect on s tr protein expression. on the other hand, the exon splicing enhancers (eses) are known to be specific binding sequences for serine/argininerich proteins (sr proteins) that relate to the control of alternative splicing [ ] . it has been demonstrated that some sr proteins, after binding to ese, could assist the exportation of attached mrna [ , ] or enhance mrna translation [ ] . two eses, bidirectional splicing enhancer [ ] and the ese of fibronectin eda exon (eda ese) [ ] , were selected, and analyzed for their influence on s tr protein expression. results of the present study can provide an optimal strategy to enhance sars-cov s protein expression in mammalian cells and may contribute to the development of sars-cov subunit vaccine. qbi- a cells (qiagen) were grown in minimum essential medium (mem) (invitrogen) supplemented with % horse serum (invitrogen). vero e cells (atcc crl- ) were grown in mem supplemented with % fetal bovine serum (fbs) (invitrogen). cho/dhfr− (dhfr deficient) cells (atcc crl- ) were maintained in minimum essential medium alpha medium (mem␣) with ribonucleosides and deoxyribonucleosides (invitrogen), supplemented with % fbs. all growth mediums were supplemented with units/ml penicillin/streptomycin (p/s). cell lines were routinely maintained at • c with % co . three pcr obtained fragments of sars-cov tw s gene: d (nucleotide - ), d (nucleotide - ), tm (nucleotide - ) were ligated into pcdna /to/mychisa (invitrogen) by bamhi/ecor (d ), ecori/hindiii (d ), and hindiii/noti (tm) restriction enzyme sites to generate ps s both d and tm pcr fragments were preceded by gggggcgggggtg-gaggtggtggc, which could be translated into eight glycine residues within s tr protein. ig-chain leader sequence obtained from pcr using psectag a as template was also ligated into ps s by aflll and bamhi sites to generate ps. the bp intron of pires (clontech) that obtained (bases - ) by pcr amplification with primers ivs-f: gcttaagcaggtaagtatcaaggtta caag and ivsr: gggtaccacctgtggagagaaaggcaaagt was inserted into ps by aflll and kpni sites to generate pis. two dna fragments containing ig leader sequence following by bidirectional splicing enhancer (bse) (gacgacgaggatgaagag) (ig-bse) or eda ese (gaagaagac) (ig-eda) was obtained by pcr using sig-f, gggtaccgccaccatggagacagacac as forward primer, and sig-bse-r, gggatccctcttcatc-ctcgtcgtcgtcaccagtggaacctggaa or sig-eda-r, gggatccgtcttcttcgtcaccagtggaac-ctggaa as reverse primers. ig-bse and ig-eda were ligated into ps or pis by kpni and bamhi sites, and plasmids psbs, pses, pisbs, pises which contain bse or eda ese in the open reading frame of s tr were generated. constructs with eda ese placed in the -utr of s tr was also generated. three dna fragments with the sequence of eda ese added either in front, after, or at both side of intron were obtained by pcr amplification by different combination of following primers: eda-ivs-f: gcttaaggaagaagaccaggtaagtatcaag-gttacaag; eda-ivs-r: gggtaccgtcttcttcacct-gtggagagaaaggcaaagt, ivs-f and ivs-r. the three dna fragments were ligated into the -utr of s tr in ps by aflll and kpni to generate peis, pies, and peies. pcr fragment with ig leader sequence proceeding with eda ese sequence (eda-ig) was also obtained by pcr using eda-sig-f: gcttaaggaagaagacgc-caccatggagaca gacac and sig-r: gggatccgt-caccagtggaac as primers. the dna fragment eda-ig was ligated into ps by aflll and bamhi sites to generate pes. for transient expression experiments, cho/dhfr−, veroe , and qbi- a cells was transfected by exgen in vitro transfection reagent (fermentas). following the manufacturer's instructions, g dna was used for cells that had been grown to - % confluency in six-well plate. after h, the cells were harvested and analyzed by sds-page and western blot. after separating on sds-polyacrylamide gel ( or %), proteins were electroblotted onto a nitrocellulose membrane (millipore). r -ae , rabbit serum raised against sars-cov s, m, and e peptides, was used as primary antibody ( : dilution). the alkaline phosphatase-conjugated affinipure goat anti-rabbit igg (jackson immunoresearch) ( : dilution) was used as secondary antibody in the detection. total rna extraction from transfected cells was performed using trizol reagent (invitrogen) according to the manufacturer's instructions. first strand cdna was synthesized by oligo(dt) primers and superscript ii rt (invitrogen). real time rt-pcr was conducted by using sybr green (applied biosystem) analysis where ng cdna of each sample was used as template. the transcripts containing region i (nucleotides - ) and region ii (nucleotides - ) in s tr gene were monitored for quantitative comparison through real-time rt-pcr. the primers used for regions i and ii analysis were s -forward: gccac-catgcatacgtttgg; s -reverse: cccatgggtt-tagaaacagc; s -forward: ttcatgggttgtgtc-cttgc; and s -reverse: ccaatgccagtagtggtgta. the primers for ␤-actin, the internal control used in real-time rt-pcr analysis, were b-forward: gtttgagac-cttcaacacccca and b-reverse: tgccaatggtgat-gacctggc. the real-time rt-pcr analysis was performed on an abi prism sequence detection system and the quantification of relative rna amount was calculated by the ct method. each sample was analyzed in triplicates and the level of total rna transcript was normalized to the ␤-actin control. cho/dhfr− cells were transfected with g ps and pis vectors per × cells. actinomycin d ( g/ml final, calbiochem) was added to the medium h post-transfection. at each specified time, the total rna was extracted using trizol reagent and analyzed by real-time rt-pcr as described above. elongation rate of transcription was determined by using helascribe nuclear extract in vitro transcription system (promega). the in vitro reaction was initiated by adding ribonucleotides and terminated using stop solution according to the manufacturer's instructions. and then, total rna was extracted with phenol:chloroform:isoamyl alcohol ( : : ) and reverse transcribed as cdna using specific reverse primers for region i and region ii, and analyzed by real-time pcr. the amounts of rna transcript containing region i or region ii separately detected by real-time rt-pcr with respect to time were plotted as the transcription curves. the time difference of the initial appearance of rna between region i and region ii was obtained from the transcription curves. the elongate rate (nucleotide/s) was calculated as the number of the time difference of the initial appearance of rna divided by the nucleotide distance between region i and region ii. the cell lysates were diluted in . % sds and % ␤mercaptoethanol and denatured at • c for min. pngase f (new england biolab) digestion was carried out in mm sodium phosphate (ph . ), % np- at • c for h. endo h digestion (new england biolab) was performed in mm sodium citrate (ph . ) at • c for h. enzyme treated samples were analyzed by % sds-page and western blotting. three different constructs of sars-cov tw strain s protein were expressed in cho cells: (i) the full-length cdna (s fl ; ps fl ) and (ii) the cdna of a longer truncated form (s tr , pls) (iii) the cdna of a shorter truncated form (s tr , ps) (fig. ) . the s tr construct contains four s protein fragments: d (s - ), d (s - ), d (s - ), and tm (s - ) while s tr construct lacks of d fragment. the results showed that s tr instead of s fl and s tr could be expressed in cho/dhfr− cells, the s tr protein was recognized by rabbit anti-sars-cov polycolonal serum (r -ae ), giving a protein band around kda in western blots (fig. ) . in order to investigate the influence of intron addition on s tr expression in mammalian cells, pis was constructed by adding an bp intron in the -utr of s tr (fig. a) , and both ps and pis were transient transfected into three mammalian cell lines: cho/dhfr− cells, vero e cells, and cells (fig. b) . cell lines transfected with pis construct had higher s tr expression than those transfected with ps construct (fig. b) . the intensity of intron enhancing effect in the three cell lines was about . -, . -, and . - fold in cho/dhfr− cells, vero e cells, and qbi- a cells, respectively (fig. c ). s tr was mainly expressed as ∼ kda protein in the three cell lines (fig. b) . however, a weak ∼ kda band could also be observed in cho/dhfr− cells that transfected with pis (fig. b) . to further investigate the intron-dependent enhancement in s tr expression, total rna transcript, in vivo rna stability, and the rna elongation rate were measured for the intron-containing (pis) and non-intron containing (ps) constructs. total rna transcripts obtained from the pistransfected cho/dhfr− cells were % higher than that from the ps-transfected cho/dhfr− cells (fig. a) . therefore, the intron-dependent s tr expression in mammalian cells correlated with a higher level of rna transcript accumulation in vivo. to further investigate the mechanism(s) of how the intron possess the ability to enhance rna transcript accumulation, we further measured the in vivo rna stability between the pis-transfected and ps-transfected cho/dhfr− cells. after treating with actinomycin d, the total rna transcripts were extracted at each specified time to measure rna decay quantified by real-time rt-pcr analysis. the results indicated that the rna transcripts obtained from pis-tranfected cho/dhfr− cells as compared to that from ps-tranfected cho/dhfr− cells exhibited similar rates for rna decay (fig. b) . these two rna transcripts exhibited half-lives of ∼ h. furthermore, the difference of the initial appearance time between region i and region ii for the introncontaining construct was . s ( . s for region ii and . s for region i) (pis construct in fig. c) . the difference of the initial appearance time between region i and region ii for ps construct was . s ( . s for region ii and . s for region i) (fig. c) . the corresponding rna elongation rate, as calculated as the difference of initial appearance time divided by the nucleotide distance ( nucleotide residues between region i and region ii), was . nucleotide/s for the intron-containing construct and . nucleotide/s for the intronless construct. therefore, a higher elongation rate was observed for the intron-containing construct, indicating that a higher rna elongation rate accounts for, or at least in parts, the increased rna transcript accumulation for the introndependent s tr protein expression in mammalian cells. the enhancing effects of two chosen exon splicing enhancers, eda ese and bse, on the mammalian cell expression of s tr protein were investigated. either eda ese or bse was first inserted into the open reading frame of s tr gene, right behind the ig signal sequence (psbs and pses). a bp intron was further added in the -utr of s tr to generate pisbs and pises (fig. a) . expression vectors of ps, pis, psbs, pses, pisbs, and pises were transfected into cho/dhfr− cell and the expression of s tr proteins were analyzed by western blotting (fig. b) . the results indicated that bse or eda ese alone was able to enhance s tr protein expression without the use of intron. the enhancing effect of eda ese was . -fold better than bse as quantified on the relative intensity of ∼ kda protein band (fig. c) . however, when either bse or eda ese was coupling with an intron (pisbs and pises), the s tr expression levels were slightly lower than pis (fig. c ). approximately % (pisbs) and % (pises) reduction in s tr expression was observed when compared to the construct only containing the bp intron (pis) (fig. c) . therefore, bse and eda ese could not work with the bp intron to enhance s tr expression. to further investigate whether the combination of intron and ese may work differently as ese located in -utr instead of orf, eda ese was added either in front (peis), after (pies), or on both sides of the bp intron (peies) in -utr of s tr (fig. a) . at the mean time, a plasmid with only eda ese placed in the -utr of s tr (pes) was used as a control (fig. a) . expression vectors of ps, pis, peis, pies, peies, and pes were transient transfected into cho/dhfr− cells, and the s tr protein expression efficiency of each construct was determined by western blot analysis (fig. b) . similar to the result of inserting eda ese in the s tr open reading frame, eda ese sequence alone (pes) resulted in ∼ . -fold increase in kda s tr expression as compared to ps (fig. b and c) . however, the ∼ kda s tr expression by peis, pies, and peies were approximately %, %, and % lower than pis (fig. c) . the results sug-gested that neither of these constructs with ese have better enhancing effect in than s tr protein expression than adding intron alone. the predicted molecular weight of s tr was . kda, however, when pis was transient transfected into cho/dhfr− cells, the major expressed s tr was at molecular weight around ∼ kda, whereas minor amount of ∼ kda s tr glycoproteins could also be detected (fig. ) . since the s tr protein sequence contains putative n-link glycosylation sites, the increase in s tr protein molecular weight might result from the addition of n-glycan during post-translational modification processes. in order to biochemically demonstrate the glycosylation pattern of s tr expressed in cho cells, peptide n-glycosidase f (pngase f) and endo h treatment were used for the following investigation. when pis transfected cell lysate was treated with pngase f, both ∼ and ∼ kda bands were digested into a single ∼ kda band, which represented the predicted unglycosylated molecular weight of s tr protein (fig. ) . this indicated that the ∼ and ∼ kda proteins were indeed glycoproteins. additionally, when the pis transfected cell lysate was digested with endo h, the ∼ kda band was disappeared while the ∼ kda band remain unaffected (fig. ) . therefore, s tr was mainly expressed as endo h-sensitive form (∼ kda) while a minor part of endo h-resistant form could also be produced. although the s gene of sars-cov contains no introns, the expression of its truncated form, s tr , in mammalian cells was greatly enhanced by the intron addition ( fig. b and c). this finding consists with a previous report, which stated that the expression of naturally intronless gene, such as c-jun, could be enhanced by intron addition [ ] . intron and its spliced out processes are highly related to every step of rna metabolism: transcription, poly(a) tail addition, mrna translocation into cytoplasm, and translation, and intron works to improve each above-mentioned step [ ] . we discovered that, no matter it was cho/dhfr− cells, vero e cells, or cells, an bp intron from pires vector could significantly improve s tr expression. however, the intensity of intron enhancing effect on s tr expression was different, . -fold increase of ∼ kda s tr expression can be observed in cho/dhfr− cells, . -fold in vero e cells, and . -fold in cells (fig. c) . similar result was reported that the intensity of intron enhancing effect on cat gene expression is quite diverse in different cell lines [ ] . the intron-dependent enhancement of s tr protein expression in cho/dhfr− cells was further investigated by measuring total rna level, in vivo rna stability, and rna elongation rate in this study. the results indicated that the intron-dependent s tr protein expression in mammalian cells correlated with a higher level of total rna accumulation as determined by quantitative rt-pcr (fig. a) . the increased rna levels by the intron-containing construct is not due to the in vivo rna stability (fig. b ) but from the increase of rna elongation rate (fig. c) . although other mechanisms may also account for the increased rna transcript by intron addition such as pre-mrna or mature mrna stabilization, poly(a) tail addition, nucleocytoplasmic translocation, translation efficiency [ ] , our present study has demonstrated that the increase of rna elongation rate accounted for, or at least in parts, the intron-dependent s tr protein expression in mammalian cells. notably, the used of an intron could result in the production of a higher glycosylated s tr (∼ kda) protein in cho/dhfr− but not in or vero e cells (fig. b) . it might be that intron did not directly lead to the formation of ∼ kda s tr protein, instead, higher s tr protein production in cho/dhfr− cell might spontaneously leads to the production of ∼ kda s tr protein. the higher ∼ kda s tr protein expression efficiency in cho/dhfr− cells among others might result from the difference in endoplasmic reticulum quality control strictness in the three cell lines. when exon splicing enhancers (ese), such as bse or eda ese, was placed right behind the ig signal sequence of s tr , the results showed . -fold (bse) or . -fold (eda ese) increase of s tr expression (fig. c ). when the position of eda ese was changed into the -utr of s tr gene, . fold increase of s tr expression can be observed (fig. c) . the eda ese was reported to increase luciferase expression by . -fold through its interaction with sf /asf, a shuttling sr protein that capable of recruiting ribosome to mrna [ ] . it was also reported that the shuttling sr proteins, such as sf /asf, act as transportation adaptors and assist mrna exportation into cytoplasm by interacting with tap/nxf protein [ ] . both bse and eda ese were reported to interact with shuttling sr proteins such as g and sf /asf, and the interaction might lead to the observed s tr protein expression enhancing effect [ , ] . better s tr protein expression enhancing efficiency of eda ese ( . -fold) than bse ( . -fold) was observed (fig. c ). it is possible that the different enhancing ability of eda ese and bse is due to the different binding preference to different sr proteins, such as g , sf /asf, and sc , of the two sequences. the result indicated that although bse and eda ese themselves had enhancing effect, the s tr expression vector coupling bse or eda ese with an intron resulted in reduced s tr expression compared to the construct with intron alone. the suppression of intron enhancing effect was unaffected by the position of eda ese, in orf or -utr of s tr gene. since the presence of bse in adenovirus e a mrna was found to activate the usage of a weak splice site [ ] . other eses such as drosophila doublesex (dsx) repeat element (dsxre) was also reported to activate the recognition of a weak, sex-specific -splice site [ ] . therefore, it was possible that the presence of bse or eda ese might activate certain cryptic splice sites within s tr sequence, and therefore resulted in reduced s tr expression due to aberrant splicing. the s tr protein, with putative n-linked glycosylation sites in its sequence, is a highly glycosylated protein. the majority of mammalian cell expressed s tr protein has molecular weight around kda, and the ∼ kda glycoproteins were sensitive to endo h treatment (fig. ) . surprisingly, a minor band represented ∼ kda glycoprotein, which was found to be resistant to endo h treatment, was also detected in cho/dhfr− cells while transient transfection of intron containing construct, indicating a different n-glycan pattern that favor golgi complex processing (fig. ) . the main question is why did the major glycosylation form of s tr protein lie in ∼ kda instead of ∼ kda? one of the possible answers might be the extensive quality control process of nascent protein in er [ ] . enzyme or chaperones in er would recognize the properties of non-native protein, such as abnormal exposure of hydrophobic regions, unpaired cysteine residues, and the tendency of aggregation, and result in the er retention of a given protein [ ] . although the truncated nature of s tr renders it easily expressed than its full-length counterpart, linking different s protein fragment together might result in the disruption of nature structure or creation of new structures that cannot be approved by er quality control system. nevertheless, discovering of endo h-resistant s tr suggested that it is possible to improve s tr glycosylation into more complex state. it has been reported that lowering the temperature during protein production can increase correct folding and secretion [ ] . furthermore, small molecular chemical chaperons have been discovered, such as glycerol, dimethylsulphoxide and trimethylamine n-oxide [ , ] , which can carry out nonspecific, folding-promoting effect by stabilize native or native-like structure and preventing protein aggregation [ ] . it will be quite interesting to know whether those molecules are able to increase glycosylation complexity of s tr proteins that produced by amplified cell clones. moreover, culture conditions such as the availability of glucose and glutamine [ , ] , the concentration of ammonia [ ] , the supply of oxygen [ ] , and the percentage of serum [ ] can also affect the complexity of attached n-linked glycans on produced recombinant proteins. therefore, optimization of the culture condition for stable cho cell clones can also be carried out to improve the production of fully glycosylated s tr proteins. the s tr protein contains receptor binding region and residue - of sars-cov s protein that was know to induce neutralizing antibody production in experimental animals, and is therefore an appropriate vaccine candidate. this study provides several possible ways for improving truncated s protein, s tr , production in mammalian cell and may contribute to the development of successful mammalian cell based recombinant sars-cov vaccine. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome molecular modelling of s and s subunits of sars coronavirus spike glycoprotein synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein characterization of a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity receptorbinding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies identification of an antigenic determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies how introns influence and enhance eukaryotic gene expression listening to silence and understanding nonsense: exonic mutations that affect splicing splicing factors srp and g promote the nucleocytoplasmic export of mrna sr splicing factors serve as adapter proteins for tap-dependent mrna export a novel role for shuttling sr proteins in mrna translation identification of a bidirectional splicing enhancer: differential involvement of sr proteins in or splice site activation a novel bipartite splicing enhancer modulates the differential processing of the human fibronectin eda exon analysis of the stimulatory effect of splicing on mrna production and utilization in mammalian cells coupling of transcription with alternative splicing: rna pol ii promoters modulate sf /asf and g effects on an exonic splicing enhancer binding of the drosophila transformer and transformer- proteins to the regulatory elements of doublesex primary transcript for sex-specific rna processing quality control in the endoplasmic reticulum processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive chemical chaperones correct the mutant phenotype of the delta f cystic fibrosis transmembrane conductance regulator protein glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation effects of glucose starvation and puromycin treatment on lipid-linked oligosaccharide precursors and biosynthetic enzymes in chinese hamster ovary cells in vivo and in vitro metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of chinese hamster ovary cells effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms dissolved oxygen concentration in serum-free continuous culture affects n-linked glycosylation of a monoclonal antibody characterization of changes in the glycosylation pattern of recombinant proteins from bhk- cells due to different culture conditions this work was supported by the national science council grant nsc - -e- - and the national health research institutes, taiwan. key: cord- -p wjtjb authors: iyer, arun v.; pahar, bapi; boudreaux, marc j.; wakamatsu, nobuko; roy, alma f.; chouljenko, vladimir n.; baghian, abolghasem; apetrei, cristian; marx, preston a.; kousoulas, konstantin g. title: recombinant vesicular stomatitis virus-based west nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west nile virus strain lsu-ar date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: p wjtjb vesicular stomatitis virus (vsv) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. we constructed recombinant vsvs specifying either the indiana or chandipura virus g glycoprotein and expressing the west nile virus (wnv) envelope (e) glycoprotein. mice were intranasally vaccinated using a prime (indiana)-boost (chandipura) immunization approach and challenged with the virulent wnv-lsu-ar . ninety-percent ( of ) of the vaccinated mice survived as compared to % of the mock-vaccinated mice after wnv lethal challenge. histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against wnv. extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of cd (+) cd (+) ifnγ(+) t cells in vaccinated, but not mock-vaccinated mice. similarly, vaccinated mice developed robust e-glycoprotein-specific cd (+) t cell immune responses as evidenced by the presence of a high percentage of cd (+) cd l(low) ifnγ(+) cells. in addition, a sizeable population of cd (+) cd (+) cells was detected indicating e-specific activation of mature t cells and cd (+) cd (+) cd (low) t regulatory (t reg) cells were down-regulated. these results suggest that vsv-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against wnv infections. west nile virus (wnv) was first isolated more than years ago from a febrile patient in the west nile province of uganda [ ] . wnv is a positive-sense rna virus belonging to genus flavivirus in the falviviridae family [ ] . the lipid-bilayer membrane of the nascent virus contains molecules of the envelope (e) and premembrane (prem) proteins organized into asymmetric non-neurological clinical manifestations include rhabdomyolysis [ , ] , pancreatitis [ ] , hepatitis [ ] , myositis, orchitis [ ] , chorioretinitis [ ] and cardiac dysrhythmias [ ] . typically, less than % of patients suffer from west nile neuroinvasive disease (wnd) including west nile meningitis (wnm), encephalitis (wne) and acute flaccid paralysis (poliomyelitis-like syndrome, wnp) [ ] . among wnd cases, an estimated - % of the patients had wne resulting in an estimated % case fatality. additionally, - % of mortalities in humans could be attributed to wnp [ ] . the absence of effective treatment against wnv infection has encouraged vaccine development. a variety of different approaches have been employed to produce wnv vaccines including inactivated virus, subunit and dna-based vaccines. most of these vaccines appeared to be highly immunogenic, and in some cases protected against wnv-infection in experimental animals [ ] . recently, recombinant viruses expressing wnv antigens have been shown to induce strong immune responses and protection against wnv challenge in animals. specifically, a recombinant live canarypox-vectored vaccine expressing the prem protein and the e glycoprotein induced strong immune responses in horses and cats [ ] [ ] [ ] [ ] , that appeared to be partially protective [ ] . other viral-vectored vaccines that elicited protective immune responses in mice include a lentivirus vector based vaccine (trip/se wnv ) [ ] , and a measles virus-vectored vaccine [ ] . recombinant yellow fever virus (yfv) has also been used to express wnv prem and e proteins based on the extensive safety record of the yfv attenuated vaccine [ , ] . a yfv recombinant vaccine (chimerivax tm ) has shown good immune responses in hamster, mice, non-human primates and humans [ ] [ ] [ ] . a phase ii clinical trial with chimerivax tm -wnv is currently underway [ ] . vsv is an enveloped, negative strand rna virus belonging to the rhabdoviridae family. natural vsv infections of humans are rare causing at most mild flu-like illness [ ] . vsv infectious viruses can be efficiently recovered by a reverse genetic approach that utilizes multiple plasmids expressing vsv genes. this methodology has enabled the rapid construction of recombinant vsv viruses expressing a variety of viral and bacterial antigens for vaccine purposes including influenza virus, bovine diarrhea virus, cotton-tail papillomavirus, human immunodeficiency virus, simian immunodeficiency virus, respiratory syncytial virus, hepatitis c, measles virus, ebola virus, lassa fever virus, marburg virus, severe acute respiratory syndrome virus (sars), and herpes simplex type- virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recombinant vsvs have been also constructed and tested as vaccines for bacterial pathogens including mycobacterium tuberculosis and yersinia pestis [ , ] . vsv-vectored vaccines have been administered via intranasal, intramuscular and subcutaneous routes and have been shown to elicit robust mucosal and systemic humoral and cellular immune responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we constructed recombinant vsvs expressing the wnv e glycoprotein. a prime-boost approach was employed utilizing two different recombinant vsvs expressing either the indiana or the chandipura g glycoproteins for priming and boosting immunizations, respectively. intranasal immunization of mice conferred high protection against lethal challenge with the virulent wnv strain wnv-lsu-ar [ ] . neuronal necrosis was observed in mockvaccinated but not in vaccinated mice. these results suggest that vsv recombinant vaccines expressing the wnv e glycoprotein may be efficacious intranasal vaccines for animal and human use. baby hamster kidney cells (bhk- ) were obtained from the american tissue culture collection (atcc). these cells were grown using dulbecco's modified minimal essential media (dmem) supplemented with % fetal bovine serum (fbs) and appropriate amounts of antibiotics. the west nile virus envelope (e) gene was obtained by first producing a cdna of the e gene from the wnv-lsu-ar strain, and subsequently cloning this gene into the pcdna . plasmid (invitrogen, inc.) after pcr amplification. the e gene was further amplified by pcr from this plasmid using primers that introduced unique noti and bamhi sites at the and using wne-flag-not-i ( -gacgacgcggccgc-atgtttaactgccttggaa tgagc- ) and wne-flag-bamhi ( -gcagcaggatccagcgtgcacgttcacgg agagg- ) primers. noti and bamhi sites are italicized. the fragment was then cloned into plasmid p xflag-cmv- (sigma) placing the flag epitope coding sequence downstream and in-frame with the e glycoprotein sequence. all recombinant plasmids were confirmed by restriction endonuclease digestion and dna sequencing. bhk- cells were transfected with the wnv e- xflag plasmid using lipofectamine (invitrogen) as suggested by the manufacturer. e glycoprotein was detected at h post-transfection using anti-flag (sigma) and anti-west nile rabbit polyclonal antibody (abcam). for immunofluorescence assay (ifa), cells were washed twice with phosphate buffered saline (pbs) and fixed with ice-cold methanol. cells were then washed with pbs and wells were blocked with % bsa and % goat serum in tbs (tris-buffered saline) for h. mouse anti-flag antibodies (sigma) in blocking buffer and rabbit anti-wnv antibodies were added to respective wells at a : dilution and incubated for h at room temperature. cells were then washed six times with tbs and the secondary antibody alexa fluor ® goat anti-mouse igg and goat anti-rabbit igg (invitrogen) were added to the respective wells at the same dilution. cells were incubated in dark for h. finally, cells were washed six times with tbs and observed under a fluorescence microscope. plasmid clones that efficiently expressed the wnv e gene were used as the template for pcr amplification of the gene, while at the same time introducing unique xhoi and nhei sites at the and ends of the gene fragment using -xn -xho-i ( -ccgcggctcgagatgttt aactgccttggaatgagc- ) and -xn -nhe-i ( -gacgacgctagcggatcactac ttgtcatcgtc- ) primers. xhoi and nhei restriction sites are italicized. this dna fragment was cloned into the pvsv-xn -in and pvsv-xn -ch transfer vectors. cells were infected with recombinant vaccinia virus expressing t polymerase (vtf - ) at a multiplicity of infection (moi) of for h. subsequently, bhk cells were co-transfected with pbs-n, pbs-p, pbs-l and pvsv-xn containing the wnv e gene and recombinant virus was recovered as described in detail previously [ , ] . control viruses having no exogenous inserted genes were also produced using pbs-n, pbs-p, pbs-l and the pvsv-xn (empty vector). anti-flag and anti-wnv-e antibodies were used to detect expression of the e glycoprotein by ifa in vsv-infected bhk cells. viral isolates expressing high amounts of the wnv e glycoprotein were selected through multiple rounds of plaque purification. viral titers were determined and stocks were stored at − • c for vaccination studies. all animal studies were carried out after the appropriate approvals were obtained from the lsu institutional animal care and use committee (iacuc) and bsl biosafety committee. four groups of ten -week-old female balb/c mice (harlan, in, usa) were used in this study. each individual mouse was identified with an ear tag (national band and tag company, ky, usa). group i (vaccine group): these animals were mildly anesthetized by inhalation of - % isoflurane and l dose of vaccine containing pfu of the vaccine (rvsv-in-wnv e) was administered intranasally using a l pipette ( l per nostril). animals were boosted with the rvsv-ch-wnv e at days post-vaccination using the same technique. one mouse from this group was not included in the fluorescenceactivated cell sorting (facs) analysis due to sample preparation problems (n = ). group ii (control for vaccine group): control group animals were vaccinated in the same way as described above with the exception that they were inoculated with l of uninfected cell culture supernatant. these animals were boosted at days post-vaccination with uninfected cell culture supernatant. animals belonging to groups i and ii were humanely euthanized at days post-boost. spleens were collected in eppendorf tubes containing rpmi and processed by flow cytometry for intracellular cytokines and cell-surface markers associate with memory t cells, regulatory t cells and cytotoxic t cells among others. for serology, animals were bled by the sub-mandibular route (cheek bleed) using golden-rod lancets (medipoint, ny). animals were bled on days post-vaccination and days post-boost. blood was collected in becton dickinson microtainers with serum separators (becton dickinson). these animals were treated exactly in the same way as groups i and ii until the boost stage. at days post-boost, these animals were transported to the animal biosafety level- (absl- ) facility for acclimatization. blood was collected at days postboost (before challenge). animals were challenged intraperitonially with pfu of wnv-lsu-ar and observed - times a day for days. animals showing severe neurological symptoms (like ataxia and hunching posture) were humanely euthanized and dead animals were surgically processed immediately (thoracic and abdominal cavities opened up and placed in % formalin jars) for pathological studies. serum samples were inactivated by incubation at • c for min. serial two-fold dilutions of the serum were incubated with equal volumes of pfu of wnv-lsu-ar at • c for h. serumvirus mixtures were then added to vero cell monolayers in -well plates in triplicates and the plates were incubated for another hour. plates were then overlaid with dulbecco's modified minimum essential media (dmem) containing % methyl cellulose and % fetal bovine serum. plates were incubated at • c for h and then fixed with % formalin in phosphate buffered saline (pbs). plates were washed three times with pbs and stained with . % crystal violet. plaques were counted and the highest dilution of serum resulting in reduction of % of the plaques was noted. mouse splenocytes were adjusted to cells/ml. one-hundred microlitre aliquots of splenocyte suspension was incubated with appropriately diluted concentrations of antibodies for min at room temperature. cells were washed once with pbs and fixed with x bd stabilizing fixative buffer (bd biosciences) in distilled water. cells were kept protected from light at • c and flow cytometric acquisition was completed within h of staining. polychromatic ( parameters) flow cytometric acquisition was performed on a lsr ii becton dickinson instrument having three lasers ( nm blue laser, nm red laser and violet laser) by using fitc, pe-texas red, apc, apc-cy and pacific blue as the available fluorochrome parameters. single-stained controls for each fluorochrome were used for setting flow cytometry compensation. monoclonal antibodies including cd fitc (a r , ebioscience), cd l pe-texas red (mel- , invitrogen), cd apc ( c , bd biosciences), cd apc-cy (gk . , bd biosciences) and cd a pacific blue ( - . , bd biosciences) were used. at least , events were collected by gating on cd + t cells and those data were analyzed using flowjo software (treestar inc.) version . . . to test cd + or cd + t lymphocytes subsets for ifn␥ production, intracellular cytokine flow cytometry (cfc) assay was employed in response to each wnv peptide pool stimulation as described previously [ ] . briefly, processed splenocytes were resuspended at × cells/ml in complete rpmi- with % fcs, and stimulated with different wnv peptide pools at a final concentration of g/ml of each peptide pool. peptide pools ( - mers with - amino acids overlap) derived from the wnv e glycoprotein were based on the wnv-ny e amino acid sequence (nih biodefense and emerging infections research resources repository, niaid, nih). the peptide array was divided to generate two peptide pools. peptide pool (pp ) was made of peptides - and peptide pool (pp ) was composed of peptides - . for positive controls, pma ( ng/ml, sigma) and ionomycin ( g/ml, sigma) were used. negative controls had no antigen or mitogen stimulation. brefeldin a ( g/ml, sigma) was added to cultures after the first hour, in a -h incubation period. following stimulation, cells were stained for cell-surface markers with directly conjugated mabs to cd fitc (h . f , bd biosciences), cd l pe-tr, cd apc-cy and cd a pacific blue for min at room temperature and washed with dpbs/bsa wash buffer. cells were then fixed and permeabilized by using cytofix/cytoperm (bd biosciences), washed twice in perm buffer (bd biosciences), and stained with intracellular mabs. ifn␥ pe (xmg . , bd biosciences) and/or cd apc (mr , ebiosciences) were added to cells and incubated at room temperature for min. single color and isotype-matched control antibodies were used to confirm staining specificity. after washing, cells were resuspended in % paraformaldehyde in pbs and stored in the dark at • c. data were acquired within h of staining using a lsr ii instrument (bd immunocytometry system) and facsdiva software (bd immunocytometry system). for each sample, , events were collected by gating on cd + t cells. data analysis was performed using flowjo software. gated cd + and cd + t cells were further analyzed for its cytokine production. positive cytokine responses were determined based on the percentage of cytokine responses obtained above background responses (unstimulated medium control) in each experiment. tissues (brain, lung, liver, bilateral kidneys, heart, spleen, skull, and vertebra) were collected from the mice, euthanized or after dead, and fixed by immersion in % neutral buffered formalin. the skull and vertebra were decalcified in % formic acid for days. all sampled tissues were routinely processed into paraffin, and - m sections were cut for hematoxylin and eosin staining (h&e). h&e sections of the nasal olfactory epithelium and bulb in the skull and four sections of the spinal cord including two consecutive anterior cervico-thoracic and two consecutive lumbar-sacral posterior sections in the vertebrae were examined under the light microscope. graphical presentation and statistical analysis of the results were performed by two-tailed student's paired t-test using the graphpad prism . (graphpad software inc., sandiego, ca). expressions of cd l, cd , cd and cd between immunized and mock challenged mice were determined by nonparametric mann-whitney t-test. for all statistical analysis, results were considered significant if p < . . mouse survival analysis was done with graphpad prism . (graphpad software inc., sandiego, ca) using the gehan-breslow-wilcoxin test. the wnv-lsu-ar strain was isolated from a dead blue jay (cyanocitta cristata) in louisiana in . recently, the entire genome of this strain was sequenced and phylogenetically compared to full wnv genomes deposited in genebank [ ] . the e gene was amplified from viral rna using specific primers as described in section and cloned into plasmid p xflag (sigma) placing the entire open reading frame of wnv e in-frame with the xflag coding sequence resulting in the addition of the xflag amino acid sequence immediately after the last carboxyl terminal amino acid of the e glycoprotein. the p xflag-e plasmid was transfected into baby hamster kidney cells (bhk- ) and e glycoprotein expression was detected at h post-transfection using anti-flag monoclonal antibody. the anti-flag antibody detected e glycoprotein expression in xflag-e transfected bhk cells, while mock-transfected bhk cells failed to react with the anti-flag antibody. to construct recombinant vsvs expressing the e glycoprotein, the e gene was amplified with primers engineered to have unique xhoi and nhei restriction sites at the e and termini, respectively. the amplified e gene (with the xflag coding sequence) was cloned within the unique xhoi and nhei restriction sites of plasmids pvsv-xn -in and pvsv-xn -ch containing the indiana and chandipura g glycoprotein gene, respectively (fig. a) . recombinant vsv was recovered after co-transfection of pvsv-xn -e with three other plasmids encoding the vsv polymerase subunits (p and l), and the nucleocapsid (n), purified by filtration and extensively plaque-purified. the appropriate insertion of the wnv gene within the vsv genomes was confirmed by direct dna sequencing of viral rna after rt-pcr amplification of specific cdna regions. wnv e expression was readily detected by indirect immunofluorescence assay (ifa) using anti-flag monoclonal antibody in recombinant vsv-infected bhk cells, while wnv e was not detected in mockinfected bhk cells (fig. b) . cell lysates from bhk- cells infected with recombinant vsvs expressing the wnv e glycoprotein were tested for e glycoprotein expression in western immunoblots. anti-flag antibody readily detected major protein species with apparent molecular masses of approximately - kda, respectively in agreement with previous reports (fig. c) [ , ] . four groups of -week-old balb/c mice (harlan, in, usa) were used for the vaccine-challenge experiments. all four groups of mice were vaccinated by intranasal administration of pfu of pvsv-xn -in-e recombinant virus at day and boosted with pvsv-xn -ch-e ( pfu) days post-vaccination ( fig. a) . mice in groups i and ii were processed for immunological analyses (see section ), while groups iii and iv were challenged with pfu of wnv-lsu-ar administered intraperitoneally. mice in the challenge groups were observed for days for clinical signs including ruffled fur, ataxia, hunching posture, lethargy and mortality. vsv-e vaccinated and boosted animals exhibited % survival, while only % of the mock-vaccinated animals survived wnv-lsu-ar challenge (p = . ) (fig. b) . vaccinated animals appeared to have mild clinical signs post-challenge including mild fur ruffling, but recovered quickly to a full healthy status. in contrast, mockvaccinated animals exhibited severe clinical signs post-challenge including high degree of fur ruffling, ataxia, lethargy and eventually death. post-mortem histopathological examination revealed that none of the vaccinated mice showed any central nervous system (cns) pathology as compared to mock-vaccinated animals, which exhibited severe neuronal necrosis and lymphoplasmacytic perivascular cuffing (fig. ) . the single mouse in the vaccinated group that died at days post-challenge had suppurative rhinitis which may be suggestive of bacterial infection. mild suppurative inflammation was also observed in the visceral pleura and subpleura of three mock-vaccinated mice that died before days post-challenge (not shown). there were no significant histopatho-logical abnormalities within other tissues examined. in a separate set of experiments mice were vaccinated via the intramuscular route and challenged with a different strain of wnv (wnv-ny ) weeks post-boost. the vaccine efficaciously protected % of the vaccinated mice (not shown). the ability of mouse sera to neutralize wnv-lsu-ar strain was tested in a standard plaque reduction neutralization test (prnt ). vaccinated animals developed strong neutralizing antibody responses against the lsu-ar at days after primary vaccination. specifically, of mice developed prnt cd expression in cd + t cells is intimately involved in the polyclonal activation of immature b cells [ ] . therefore, we compared the expression of cd in both vaccinated and mockvaccinated mice after in vitro stimulation with pma/ionomycin followed by facs analysis (see section ). these experiments revealed the presence of a significantly higher population of cd + cd + ifn␥ + t cells in vaccinated mice compared to mockvaccinated mice (mean value . % versus . % in vaccinated and mock-vaccinated mice respectively, p < . , fig. a and c), as also indicated by the observed differences in their mean fluorescence intensities ( fig. b and d) . antigen-specific cytokine responses were determined in all vaccinated and mock-vaccinated mice. specifically, wnv-e specific t cell responses were measured using cytokine flow cytometry (cfc) to determine ifn␥ responses. overall, of vaccinated mice had detectable ifn␥ responses (ranged from . to . %) in splenic cd + t cells. cd + t cell positive ifn␥ responses were absent in any of the vaccinated mice. both peptide pools (e amino acids - ) and (e amino acids - ) appeared to contain t cell epitopes, however, peptide pool contained dominant t cell epitopes. one of the mice developed antigen-specific ifn␥ responses against both the wnv-e peptide pools. none of the mock-vaccinated mice had any detectable ifn␥ responses above background levels. cd l is a lymphocyte homing marker that is generally associated with extravasation of activated t cells to peripheral sites of inflammation. generally, increased percentages of cd + t cells were present in the vaccinated mice compared to the mock-vaccinated mice (mean . % and . % for vaccinated and mock-vaccinated mice respectively, p = . ) (fig. a) . cd + t cell subsets in all vaccinated mice had lower cd l expression compared to mockvaccinated mice (p = . ) (fig. b) . to further characterize the cells responsible for inducing cytokine responses, antigen-specific cytokine positive cells were determined. a significant population ( . %) of the ifn␥ positive cells was memory cells (cd + cd l − ) (fig. c) . cd is an early activation marker indicative of the presence of antigen-specific stimulation of mature t cells [ ] . cd up-regulation of activated cd + t cells was detected in all the vaccinated mice following antigen stimulation compared to mock-vaccinated mice (mean . % versus . % in vaccinated and mock-vaccinated mice, p = . ) indicating e-specific stimulation of mature t cells in vaccinated animals (fig. d) . initial determination of cd + t cell percentages in splenocytes revealed no significant differences between vaccinated and mock-vaccinated mice (fig. a) . cd , the ␣-chain of the il receptor, in combination with cd , the ␣-chain of the il receptor, were used to define the relative abundance of t reg cells within the population of conventional t cells [ ] . analysis of cd + cd + cd low cells revealed that vaccinated mice had a significantly lower population of these cells (mean . %) in comparison to the mock-vaccinated mice (mean . %) (p < . ) (fig. b and c) . vsv-vectored vaccines have shown exceptional promise for protecting animals and humans against different viral and bacterial pathogens. a vsv-vectored vaccine expressing the wnv-e glycoprotein was constructed and found to efficiently protect mice after intranasal administration against lethal wnv challenge. the salient features of this vaccine study are: ( ) a prime-boost intranasal vaccination approach with recombinant vsvs expressing the wnv e glycoprotein produced robust cd + ifn␥ + t cell responses; ( ) this vaccine approach produced strong neutralizing titers against the wnv; ( ) vaccinated mice were protected against lethal challenge and they were free of neuronal necrosis, while unvaccinated mice there was no statistically significant difference observed between these two groups. (b) representative dot plots showing the gating strategy for t reg cells derived from spleenocytes. cd + t cells were first gated and plotted for cd and cd . cd + cd + cd low t cells were defined as t regs. (c) percentage of cd + cd + cd low t reg cells. a statistically significant difference (p < . ) was observed between wnv-vaccinated and mock-vaccinated mice. exhibited severe neuronal necrosis and inflammation in the brain. these results suggest that a prime-boost vsv-vectored intranasal vaccine approach induces strong humoral and cellular immune responses that protect mice against wnv-induced neuronal necrosis. mucosal surfaces constitute the natural route of vsv infections. vsv is primarily a veterinary viral pathogen that infects cattle, horses, sheep and other animals. vsv infects animals via transmucosal and transcutaneous routes [ ] . vsv may also be transmitted through sandflies, blackflies and mosquitoes [ , ] . the vsv g glycoprotein is a potent immunogen and also serves important functions in virus-entry and virus-induced cell fusion [ ] . recombinant vsvs expressing a variety of viral and bacterial antigens have been constructed. vaccine studies with these recombinant vsvs have showed that intranasal and intramuscular administration of the rvsvs were safe and efficient in inducing protective humoral and cellular immune responses against a variety of pathogens [ ] . of particular interest is the ability of the vsv-vector system to elicit strong humoral and cellular immune responses via the intranasal route [ , , , , , ] that can be substantially easier to administer than intramuscularly injected vaccines. in these vaccine studies, although the "empty" vsv vector elicited robust humoral and cellular immune responses against vsv, these responses did not contribute to protection against a variety of pathogens indicating that specific immune responses against the expressed transgene were primarily responsible for protection [ , [ ] [ ] [ ] , ] . we constructed rvsvs that expressed the wnv-e glycoprotein and either the vsv indiana g glycoprotein, or the chandipura vesiculovirus g glycoprotein. this pair of rvsvs was used in a prime-boost-vaccination approach to maximize humoral immune responses against the wnv-e glycoprotein expressed by both viruses, while minimizing the anamnestic immune response against the vsv vector targeted predominantly against the g glycoprotein. this is largely accomplished because the chandipura g and the vsv-indiana g glycoproteins are approximately % different in their amino acid sequences [ ] . recombinant vsvs are known to non-specifically incorporate certain other viral and cellular glycoproteins into their virions without adversely affecting viral infectivity [ ] . the insertion of the foreign e gene into the vsv genome did not adversely affect viral replication and infectivity, because rvsv containing the e gene replicated to similar titers with those of the vsv control virus that did not have a foreign gene inserted within their genomes (not shown). moreover, rvsv-e isolates were stable, since multiple serial passages of virus stocks in bhk cells did not affect e glycoprotein expression and genomic stability (not shown). although it is unclear whether the wnv e glycoprotein is inserted into vsv envelopes, these results suggested that rvsv-e were stable retaining wild-type levels of viral replication and infectivity. recombinant vsv-e expressed wnv-e glycoprotein to high levels in bhk cells and the expressed e glycoprotein appeared to be fully glycosylated as evidenced by the apparent molecular mass of approximately - kda in sds-page in agreement with published reports [ , ] . based on the known strong immune responses generated by vsv, especially when administered via the intranasal route, we devised an experimental vaccine protocol to vaccinate mice through the intranasal route using a prime-boost strategy. this prime-boost-vaccination approach resulted in % ( of ) of the mice surviving lethal challenge with the wnv-lsu-ar virulent strain. the single mouse from the vaccinated group of mice that died late in the experiment ( days post-challenge) appeared to die from wnv-unrelated causes, since histopathological examination showed severe suppurative rhinitis but no histological abnormality in the brain. therefore, the rvsv-e prime-boost intranasal vaccination protocol was highly efficacious in protecting mice against wnv infection. similar results were obtained in a different experiment in which mice were vaccinated via the intramuscular route and challenged with wnv-ny strain instead of the lsu-ar weeks post-boost. in this experiment % of the mice survived indicating that intramuscular immunization may also provide protective immune responses against wnv infection. primary wnv infection is thought to result in local replication of the virus in peripheral organs and viremia that ultimately results in virus invading the cns. wnv mortality is thought to be largely caused by replication of the virus in the cns tissues of animals and the resultant immunopathological damage of cns tissues. accordingly, unvaccinated mice showed obvious clinical signs of neurological disease such as ataxia, hunching posture, lethargy and hindlimb paralysis. histopathological examination of brain tissues showed neuronal necrosis, perivascular cuffing, and microgliosis. in contrast, only a few vaccinated mice developed mild clinical signs such as mild ruffled fur, but recovered quickly. importantly, none of the vaccinated mice exhibited any neuronal necrosis. the interaction of cd on b cells with cd (cd l) on cd + t cells results in t cell mediated activation of b cells resulting in immunoglobulin class switching, somatic hypermutation and proliferation [ ] [ ] [ ] . accordingly, cd + cd + ifn␥ + t cells were up-regulated in vaccinated but not control mice indicating generation of t cell mediated b cell activation. the specificity of this response is not discernable, since it may be due to either or both vsv and wnv antigens. however, strong neutralizing antibody titers were also produced against wnv indicating the induction of especific humoral immune responses. this result is in agreement with previous reports showing that other vsv-vectored vaccines induced strong humoral immune responses against different vsvexpressed antigens. specifically, recombinant vsvs expressing either the respiratory syncytial virus f glycoprotein [ ] , or rvsv expressing the severe acute respiratory syndrome (sars) corona virus (sars-cov) produced high antibody titers against the f glycoprotein and sarc-cov spike (s) glycoprotein, while strong immune responses against the vsv virus was noted [ ] . the wnv e glycoprotein contains multiple predicted and experimentally verified cytotoxic t cell (ctl) epitopes [ ] [ ] [ ] [ ] . the availability of a library of overlapping peptides derived from the wnv e glycoprotein allowed the elucidation of antigen-specific cellular immune responses. peptide pool composed of the first peptides averaging - amino acids each generated stronger cellular cd + ifn␥ + t cell responses in in vitro proliferation assays, in comparison to peptide pool , which represented the carboxyl terminus-half of the wnv e glycoprotein. peptide pool contains the experimentally verified ctl epitope rsycylat (e - ) while peptide pool contains the ctl epitope ialtflav (e - ), both of which have been shown to confer protection against lethal wnv-challenge in mice [ , ] . in vitro stimulation of lymphocytes from vaccinated mice revealed the presence of antigen-specific ifn␥ responses specifically in cd + cd l low t cells. cd l (lselectin) mediates adhesion of resting lymphocytes to peripheral lymph nodes. typically, high expression of cd l (cd l hi ) reveals entrapment of lymphocytes within lymph nodes, while low cd l (cd l low ) cell-surface expression (the result of t cell activation) is indicative of lymphocyte extravasation to sites of inflammation [ ] . splenocytes from vaccinated mice had significantly lower expression of the cd l marker on e-specific ifn␥ + cd + t cells revealing activation and extravasation of these cells to peripheral sites, potentially involved in killing virus-infected cells prior to transmission to the cns. cd is an early activation marker that is absent in resting lymphocytes [ ] . the up-regulation of the cd + cd + e-specific t cell responses in vaccinated versus mock-vaccinated mice provides additional evidence for the stimulation of t cells. accordingly, cd + cd + e-specific population of t cells was up-regulated in vaccinated versus mock-vaccinated mice indicating the generation of activated memory cd + t cells. it is unclear whether the observed cd + t cell memory responses confer long-term immunity against wnv infection. t regs are known to play important roles in down-regulation of anti-self immune responses [ ] , and to suppress proliferation and cytokine production of effector t cells [ ] . typically, during viral infections, up-regulation of humoral and cellular immune responses causes down-regulation of t reg activation. typically, t regs express the foxp and cd markers. the il- receptor cd marker expression is inversely correlated to foxp expression and cd low cd + cells have been shown to be positive for foxp [ , ] . consequently, the cd + cd low population was used to define t regs. as expected, there was a negative correlation between the relative population of t reg cells (cd + cd + cd low ) and antigen-specific ctl responses in the vaccinated mice. however, the specificity of this immune response cannot be discerned, since it most likely is caused by both vsv and e glycoprotein antigens. a variety of experimental vaccine approaches have been reported to generate protective humoral and cellular immune responses against flaviviruses and specifically wnv. the relative role of humoral versus cellular immune responses has been extensively debated in the literature. certain studies have suggested that a strong humoral immune response evidenced by the production of high titer anti-wnv titers is necessary and sufficient to protect mice from cns infection, while other reports have argued that a cellular immune response characterized by a robust anti-wnv cd + t cell responses is necessary for protecting and clearing brain tissues from wnv [ , , ] . one report has argued that ctl-immune responses may result in exacerbated immunopathology in brain and cns tissues at infections with low wnv titers ( pfu) [ ] . in our experiments, wnv pfu were inoculated intraperitoneally. vaccinated mice had no evidence of neuronal necrosis suggesting the cd + t cell responses conferred protection and virus clearance. it is probable that both humoral and cellular immune responses generated against the wnv e glycoprotein prevented the virus from entering cns, potentially arresting the virus at peripheral sites. alternatively, if some virus escaped peripheral immune surveillance, it is possible that ctls cleared the virus from brain tissues before it could cause significant damage and resultant immunopathological manifestations. in summary, the vsv-e-vectored vaccine appeared to elicit robust humoral and cellular immune responses that efficiently protected mice from wnv lethal challenge. intranasal vaccination is second only to oral vaccination with regard to the relative ease of administration and patient compliance issues rendering this approach attractive for human use. recently, single-cycle vsvvectored vaccines have been shown to generate robust immune responses against a number of viral pathogens including hiv, ebola, marburg, lassa, influenza, avian influenza, hepatitis c and rsv viruses [ , , , [ ] [ ] [ ] [ ] [ ] , ] . based on these results, it is expected that single cycle vsv-wnv vaccines would be also efficacious. additional improvements in attenuating vsv can be made by providing more than one viral protein in trans through complementing cells, as well as engineering additional mutations that are known to attenuate vsv. a neurotropic virus isolated from the blood of a native of uganda flaviviridae: the viruses 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high levels of hepatitis c virus glycoproteins this work was supported by nih:ncrr p rr project. kgk and ai were supported by the division of biotechnology and molecular medicine (biommed), lsu school of veterinary medicine. bp was supported by the facs immunology core laboratory of the tulane national primate research center (nih:ncrr p rr ). the following reagent was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: peptide array west nile virus gene e, nr- . we gratefully acknowledge dr. john k. rose (yale university, school of medicine) for providing us with the vsv reverse genetics systems and ms. li huang and other biommed staff for technical assistance. key: cord- -b tj x authors: giersing, birgitte k.; vekemans, johan; nava, samantha; kaslow, david c.; moorthy, vasee title: report from the world health organization’s third product development for vaccines advisory committee (pdvac) meeting, geneva, – th june date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b tj x abstract the third meeting of who’s product development for vaccines advisory committee (pdvac) was held in june , with a remit to revisit the pathogen areas for which significant progress has occurred since recommendations from the meeting, as well as to consider new advances in the development of vaccines against other pathogens. since the previous meeting, significant progress has been made with regulatory approvals of the first malaria and dengue vaccines, and the first phase iii trials of a respiratory syncytial virus (rsv) vaccine candidate has started in the elderly and pregnant women. in addition, pdvac has also supported vaccine development efforts against important emerging pathogens, including middle eastern coronavirus (mers cov) and zika virus. trials of hiv and tuberculosis vaccine candidates are steadily progressing towards pivotal data points, and the leading norovirus vaccine candidate has entered a phase iib efficacy study. who’s immunization, vaccine and biologicals (ivb) department is actively working in several pathogen areas on the recommendation of pdvac, as well as continuing horizon scanning for advances in the development of vaccines that may benefit low and middle income countries (lmics), such as the recent licensure of the enterovirus (ev ) vaccine in china. following on from discussions with who’s strategic advisory group of experts (sage) on immunization, pdvac will also look beyond licensure and consider data needs for vaccine recommendation and implementation to reduce the delay between vaccine approval and vaccine impact. who's pdvac was established by the department of immunization, vaccines and biologicals (ivb) in , following a review of who's process for strategic priority setting for vaccines. the need for a group to advise who specifically on vaccine product development was highlighted, to accelerate vaccine availability and ensure accessibility of vaccines to low and middle income countries (lmics). pdvac's remit is to advise on the product development strategy of vaccine candidates at phase ii of clinical evaluation or earlier, and to report its proceedings to the who's principal committee on immunization policy recommendations: the strategic advisory group of experts on immunization (sage). the pdvac committee has a critical role in assessing the evolving vaccine development landscape and in helping to define where and how who can be most impactful, according to three criteria: likelihood of a product emerging from the pipeline, as defined by probability of technical and regulatory success, and the extent of awareness, activity and investment in a given area, a clear role for who with perceived added value for engagement in the pathogen area. typically, who engages in a pathogen area by working with a broad set of key vaccine development stakeholders to develop consensus on pivotal clinical trial design, vaccine roadmaps, or guidance documents on desired vaccine properties, referred to as preferred product characteristics (ppcs). ppcs define who preferences for the properties of vaccines to be used in lmics that are - years from licensure, and inform target product profiles in use by manufacturers and funders for vaccines. pdvac also encourages developers to be aware of the process and requirements for who prequalification (pq). who prequalification is a service to unicef and other un agencies that purchase vaccines once they have been licensed, to determine the acceptability, in principle, of vaccines from different sources for supply to these agencies. it aims to ensure that diagnostics, medicines, vaccines and immunizationrelated equipment and devices for high burden diseases meet global standards of quality, safety and efficacy, and are appropriate for use in lmics contexts in order to optimize the potential benefit of these interventions [ ] . the third pdvac meeting was held in geneva from - th june . dr. jean-marie okwo-bele, director of ivb, opened proceedings with a synopsis of the significant milestones in vaccine development in the nine months since the previous meeting in september : the first dengue and malaria vaccines have been licensed or achieved the equivalent of licensure, respectively, the first rsv vaccine candidate has entered phase iii studies in the elderly and pregnant women, the most advanced hiv vaccine candidate has met its endpoints in the interim analysis of a phase ii study, and preparations to commence an efficacy study are underway, who convened the mers-coronavirus r&d community, and a phase i clinical study is now underway (nct ), ebola virus vaccines are under review and have progressed to the point of consideration for licensure in record time, there are co-ordinated efforts to develop a zika virus vaccine as expeditiously as possible. a pdvac working group has overseen the development of a zika virus vaccine target product profile (tpp), and developed regulatory considerations towards phase i and emergency use authorization. in addition to these significant advances in vaccine development, the uk government published in may the report on 'tackling drug-resistant infections globally' that it commissioned in collaboration with the welcome trust [ ] . the report highlights the urgent need to reduce reliance on currently available antimicrobials, without which today's , deaths per year from drug resistant microbes is forecasted to increase to million, by . the cost in terms of lost global production due to infections that are not controllable due to antimicrobial resistance (amr) is estimated to be $ trillion by if no action is taken [ ] . the development of vaccines against pathogens that are currently controlled by antimicrobials has become an imperative, as they have the potential to reduce the prevalence and spread of drug resis-tance, as well as to reduce the use of antimicrobials more broadly [ ] . the decade for vaccines' global vaccine action plan (gvap) mid-term review, required an assessment of progress against objectives since its inception in , and strategic planning to achieve the stated targets within the remaining years. part of pdvac's remit is to review the vaccine development pipeline and consider the priority activities for ivb, within this context. during the remaining timeframe of gvap, a number of vaccines could reach licensure, and who needs to ensure early engagement with policy makers regarding potential vaccine implementation, as well as alignment with gavi's vaccine investment strategy. to facilitate information sharing, and tracking of progress within the global vaccine development community, the who has established and maintains an online 'vaccine pipeline tracker' in which information regarding all current clinical studies in several different pathogen areas can be found [ ] . in addition, landscape analyses for pathogens from the meeting have been collated within a special issue of the journal 'vaccine' and all are available through open access [ ] . these documents are authored by independent subject matter experts and review the status of vaccine candidate development, as well as assessing possible pathways to regulatory approval. pdvac reports progress on the global vaccine development pipeline to who's strategic advisory group of experts (sage) on immunization. at the meeting in april , advances in the development of interventions (vaccines and monoclonal antibodies) for respiratory syncytial virus (rsv) were presented for information. the reports from the october and april sage meetings are available online [ , ] . much of the discussion focused on the need to better understand the key factors for early for implementation, as well as safety and efficacy data to support the assessment of a vaccine for policy recommendation. as emerging vaccines are likely to require new vaccination platforms, such as maternal immunization, or visits outside of the current vaccination schedule, such as for the recently licensed malaria vaccine rts,s, cost-effectiveness data informing their optimal use and potential impact must be generated in line with conventional clinical data required for regulatory approval, to minimise the delay between vaccine licensure and uptake [ ] . the goals of this third pdvac meeting were to revisit the pathogen areas where there has been significant progress to report since recommendations from the meeting, as well as to: review status of vaccine development in new pathogen areas where there has been significant vaccine development progress, or where there is significant disease burden but r&d has stalled, refine the workplan and strategic directions for ivb in specific pathogen areas, identify cross-cutting issues that accelerate vaccine development or prepare for policy decisions, where appropriate, consider how to better align pdvac's vaccine development activities and strategies with other areas of research, to inform the vaccine development community regarding steps to be considered beyond vaccine licensure, and who processes for vaccine policy recommendation. the gvap is a -year strategic framework derived from the decade of vaccines collaboration [ ] to prevent millions of deaths by through more equitable access to existing vaccines for people in all communities. within this framework is a specific objective that supports research and development of innovations that will maximise the benefits of immunization, with indicators for progress towards development of hiv, malaria, tuberculosis and influenza vaccines. the gvap has just completed its midterm review stage, and following the recommendation from sage [ ] , the gvap assessment will highlight advances made in these areas. these four pathogen areas are standing agenda items for discussion at pdvac. in , mycobacterium tuberculosis (mtb) killed . million people ( . million of whom were co-infected with hiv) and is now the world's most deadly infectious disease [ ] . approximately , cases/annum are multi-drug resistant (mdr) or extensively drug resistant (xdr) and some strains are untreatable. in , six million new cases of mtb were reported to who, fewer than two-thirds ( %) of the . million people estimated to have contracted the disease. this means that % of new cases were not detected or reported. a vaccine is imperative to achieving the end tb goals [ ] , particularly through reaching the population who are undiagnosed and continue to transmit disease. as such, the tb vaccine development community has turned its focus to the development of vaccines targeted to adolescents and adults as the age-groups with highest burden of active disease and the source of mtb transmission. modelling studies suggest that prevention of pulmonary disease in this population from primary infection and from reinfection or reactivation of existing infections is the most effective strategy to prevent mtb infection and disease in infants and children [ ] . the most advanced vaccine candidates are targeting this indication, including current neonatal bcg replacement candidate vaccines that are also undergoing evaluation as a booster in later life. several of these candidates are in proof-of-concept clinical studies and are approaching key endpoints through prevention of infection or disease, or prevention of disease due to reinfection in this these target populations in the next - months [ ] . with this in mind, pdvac recommended that who prioritize and facilitate consensus building with respect to the development of strategic goal(s) and ppc(s) for vaccines targeted to adolescents and adults, in the first instance. there are several candidates and platforms in the pipeline that target this goal in this population, as well as other important target populations [ ] . pdvac acknowledged the significant need for development for these vaccines in parallel, as well as continued efforts to understand the biological mechanism of disease to support the immunological rationalization of candidates. the pox-protein public private partnership (p ) consisting of sanofi, glaxosmithkline (gsk), bill & melinda gates foundation, the us military hiv research program (mhrp), and the hiv vaccine trials network (hvtn) have been collaborating with the us national institutes of allergy and infectious disease (niaid) to optimize and assess the efficacy of the alvac/heterologous prime boost approach, following the demonstration of partial efficacy in the rv trial in thailand [ ] . the interim data from a phase i/ ii study (hvtn ) met its humoral and cellular immunological 'go' criteria, exceeding the rv responses against sub-saharan clade c antigens. extrapolation of these responses to those observed with rv , suggest that the optimised vaccine could offer at least % protection following a month booster. based on these data, a randomised placebo controlled phase iib/iii efficacy trial (hvtn ) enrolling subjects was initiated in late in south africa, and will evaluate alvac (clade c) prime/ bivalent recombinant gp protein with mf adjuvant as a heterologous boost, as well as the effect of a booster at months [ ] . futility analyses will be undertaken early in the year followup period. correlate of protection studies and assessment of crossreactivity to other regional clades are included in the study design. discussions with the south african medicines control council (mcc) are ongoing, and licensure in south africa could be as early as . other vaccine candidates are in development, including janssen's heterologous prime boost approach with ad /gp , currently undergoing dose regimen selection in phase i/iia trials. antibody-mediated prevention using broadly neutralizing, potent monoclonal antibody (bnmabs) approaches are also undergoing phase i/iia clinical evaluation. the niaid/vaccine research centre's vrc broadly neutralising mab is the most advanced candidate which has been shown to neutralise cd binding of % of viral isolates. hvtn /hptn and hvtn /hptn are phase iib studies to evaluate the efficacy of vrc in reducing acquisition of hiv- infection in high risk populations in the americas and sub-saharan africa, and started enrolment in . if shown to be effective, administration of vrc could be positioned as a long-acting supplement to increase effectiveness of anti-retrovirals. pdvac commended the advances in hiv vaccine development, and requested to be kept informed about progress with hvtn . currently, there are no known intentions for global studies with the p candidate vaccine, or to seek who prequalification. pdvac encourages the p partners and the south african hiv vaccine development community to keep who fully informed about progress with the trial. concerns were expressed regarding the lack of follow-on studies in thailand, given that the initial landmark rv trial was performed there. despite the substantial reduction over the last years (over % for global malaria mortality in children aged < years), mainly due to greater investments in malaria control, the who estimates there were million malaria cases in , % of which were in africa. of the , people who died from the disease in , % reside in africa [ ] . given the increase in multi-drug and insecticide resistance, there remains an urgent need for a vaccine to combat malaria. as reported in the pdvac meeting summary [ ] , the european medicine's agency (ema) provided a positive scientific opinion, indicating a favorable assessment of the risk-benefit balance of rts,s/as from a regulatory perspective. in october , two advisory bodies to who, namely sage and the malaria policy advisory committee (mpac), recommended pilot implementation studies of the -dose schedule of the rts,s/as vaccine in - distinct epidemiological settings in sub-saharan africa, at sub-national level, covering moderate-to-high transmission settings, with three doses administered to children between and months of age, followed by a fourth dose - months later. the intent of these pilot studies is to assess: the feasibility of providing all four doses of rts,s to the target age group through existing health services; the impact of rts,s on child mortality; whether there are any safety issues, particularly evidence of any causal relationships between rts,s administration and either meningitis or cerebral malaria (both signalled in the phase iii trials), whether introduction of the vaccine impacts positively or negatively on existing country immunization programs and on the use of currently recommended malaria control measures. in , the malaria vaccine technology roadmap was updated to include licensure of vaccines targeting plasmodium falciparum and plasmodium vivax by , with protective efficacy of at least % against clinical malaria, and that reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria parasite infection [ ] . the vaccine candidate pipeline is robust, and includes novel antigens and platforms [ ] . second generation vaccines are expected to provide higher protection than rts,s in the longer term. optimised tools are needed to measure incremental improvements and predict potential cost effectiveness of new candidates. the development of controlled human malaria infection (chmi) models, efforts to harmonize elements of clinical trial design and standardization of various assays continue. pdvac stressed the importance of the development of nd generation malaria vaccines in parallel to the pilot implementation program for rts,s, and proposed that the current version of the vaccine roadmap be updated, potentially in , in light of the rts,s pilot implementation. in , pdvac noted that development of universal influenza vaccines will be challenging and protracted, particularly due to the lack of a regulatory pathway for novel antigens that operate through induction of t-cell immunity. rather, pdvac recommended that there be a focus on the definition of, and the collection of data to support implementation of 'improved' seasonal flu vaccines that would offer more immediate impact in lmics. pdvac advised who to develop strategic public health goals and ppcs for improved seasonal influenza vaccines, and to provide guidance on data requirements that would be needed to establish improved performance of such vaccines. a working group has been established, and has proposed a draft statement of unmet public health need: 'safe and well-tolerated influenza vaccines that are effective at preventing severe influenza illness, that provide protection beyond a single year, and that are programmatically suitable for use, are needed for low-and middleincome countries.' draft -and -year strategic goals for development of influenza vaccines that induce broader and more durable protection against severe illness caused by influenza a strains have been developed. these strategic goals and the draft ppc for nextgeneration influenza vaccines were presented at the upcoming eighth who meeting on development of influenza vaccines [ ] . pdvac reaffirmed the value of ppcs based on the two different approaches. there is a public health need to develop improved performance of currently available seasonal vaccines to offer protection over multiple seasons, and against drifted strains, with a view to generating shorter timelines to achieving availability and access in lmics. as part of this effort, it will be necessary to define the criteria needed to demonstrate clinical benefit, and additional data requirements to support policy recommendations. efforts to develop 'universal' vaccines that target conserved antigens, or conserved components of antigens, should continue in parallel, with a focus on identifying correlates of protection to support a regulatory pathway for this novel class of vaccines. diarrheal disease remains the second leading cause of death in children under years of age. although mortality has declined over the past four decades, morbidity has not declined significantly, despite improvements in water and sanitation and benefits from oral rehydration therapy. there are nearly . billion cases of diarrheal disease every year, many with acute and chronic effects such as growth stunting and cognitive impairment. these long term sequelae significantly impact quality of life and economic potential, and are estimated to affect one-fifth of children globally. in , pdvac recommended that who expand its remit to include support for enteric vaccine development, particularly against enterotoxigenic escherichia coli (etec) and shigella. one of the main objectives of the planned who engagement in this area will be to ensure that the design of the phase iii efficacy study, including definition of primary/secondary endpoints and long-term follow up, and the data generated, will be relevant to support a policy recommendation from sage. another key objective is to develop a who preferred product characteristics document which outlines who preferences, including considerations for development towards a potential combined vaccine. several vaccines are in development, with two etec candidates and seven shigella candidates currently in clinical studies. for etec, the most advanced vaccine is etvax adjuvanted with dmlt, which is being developed for both a pediatric and traveller's indication. a phase i/ii dose escalation, age de-escalation study in children is currently ongoing in bangladesh, with intent to further age deescalate into week-old infants in late . in parallel, a phase iib study in travellers is planned to begin in . based on an encouraging phase iib immunization and challenge study and additional positive protection studies in non-human primates (nhps), an adhesin-based subunit etec vaccine (fta) is moving forward with an accelerated clinical program designed to move a complete multi-valent vaccine into descending age field trials in . the most advanced shigella candidate is trivalent shigella killed whole cell (tswc) composed of formalin-inactivated s. flexneri a, s. flexneri a, and s. sonnei, expected to offer coverage across about % of isolates. a phase i study has been completed and a challenge trial with s. flexneri a prototype will begin in , followed by a study that will assess co-administration with etvax. both etvax and tswc are being developed for oral administration. other promising shigella vaccines in early stage clinical testing include two live attenuated vaccines, wrrs and shigetec. wrrs is in a descending age study in bangladesh, while shigetec, which is a combination shigella-etec combination vaccine, will begin a phase i study in early . three subunit approaches for shigella are also in phase i/ii studies; the prototype s. flexneri a bioconjugate vaccine (flexyn a), invaplex and the generalized module for membrane antigens (gmma). one of the critical strategic issues is whether to prioritize the licensure and approval of an etec vaccine, or to focus on the development of a combination with shigella that will likely delay the timeline to vaccine availability. epidemiologic data suggest that both intra-and inter-country disease heterogeneity is likely to exist and this may drive vaccine preferences, and presentation optimization. these data are critical to inform decision-making by country policymakers. for this reason, development of who derived preferred product characteristics for etec and shigella vaccines, alone and in combination is needed. in april , plos released a collection on 'the global burden of norovirus & prospects for vaccine development' [ ] , which includes the most current estimates on global norovirus disease burden of over , deaths in low resource countries, and a global economic burden of more than $ billion [ ] . recent molecular analyses of samples from the community based longitudinal birth cohort mal-ed study suggest that norovirus is the most common diarrheal pathogen in the first year of life, and the second most common in the second year of life. there are vaccine candidates in development, including three strategies to develop a combination vaccine against other enteric pathogens. however, only one candidate, which is composed of two vlps based on the gi.i and gii. norovirus genotypes, has entered clinical studies, a phase iib study began recently [ ] . the advent of cell culture methods for norovirus will facilitate many advancements, including the optimization of a neutralization assay and enable the assessment of antisera against this vaccine to block binding of a diverse genotypes. in addition, in response to the pdvac recommendation to consider incorporating norovirus surveillance within the who global rotavirus surveillance network, a survey of the capability and capacity at representative global sites has been performed to support a pilot study proposal. the recently published epidemiology and burden of disease data indicate that norovirus fulfils the pdvac criterion of unmet public health for a vaccine in lmics. however, the ability of the candidates in the pipeline to offer protection over the range of circulating and emerging viral genotypes, and therefore the duration of protection of these vaccines, is currently unknown. it is conceivable that the vaccine will need to be periodically re-formulated, to include emerging genotypes. in addition to infants as a priority target population, adults and particularly the elderly are at risk, requiring the potential need for two vaccine formulations and/or presentations. fortunately, at the current time, development of a norovirus vaccine that may offer efficacy in the context of low and middle income countries is proceeding with investment from the private sector, however an assessment of vaccine programmatic suitability and applicability to prequalification is needed, prior to phase iii trials to ensure the vaccine is appropriate for use in lmics, assuming it is demonstrated to offer coverage over circulating genotypes within lmics. rotavirus is the leading cause of severe diarrhea among all children below years of age worldwide, causing - % of severe diarrheal hospitalisations, and is associated with significant mortality, with the latest mortality estimates at , deaths in [ ] . the introduction of the live-attenuated oral rotavirus vaccines, rotateq and rotarix, in has had significant direct and indirect impact in countries where they are in use, including saving lives and reducing hospitalizations. however, in gavieligible and lmic countries in asia and africa the vaccine effectiveness is lower, with protective efficacy observed from to % against severe rotavirus diarrhea over the first year of life. waning of protection has also been observed in these settings, with lower protection rates ( - %) in the second year of life. in comparison, in high-income countries protection is higher ( - %) and persists into the second year of life. thus, despite the enormous success of the live oral rotavirus vaccines, several challenges and issues remain such as the lower protection in gavi-eligible and lmic countries in africa and asia, together with the high cost of available vaccines. despite an overall acceptable safety profile, the intussusception rate seems to be slightly increased by vaccination (occurrence - / oral rotavirus vaccine recipients) in high income countries. several new oral, live-attenuated vaccines, composed of alternative strains, are in mid-to late-stage clinical development. the current who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines [ ] would be applicable for these next generation oral, live-attenuated vaccines. of these new oral rotavirus vaccines, rotavac c (developed by bbil) is the only vaccine currently licensed for use in children, having been approved for use in india in . this vaccine is available on the private market in india and staged roll out in public health system is planned in four states in india. another live rotavirus vaccine is being evaluated in a randomised placebo controlled trials in india (nct ) and in niger (nct ). efforts are underway to develop non-replicating rotavirus vaccines (nrrv) as second generation rotavirus vaccines, which may avoid the risk of intusseseption. the most advanced candidate is p -vp ⁄ , a trivalent truncated vp ⁄ of rotavirus genotypes p [ ] , p [ ] and p [ ] , currently in phase ii clinical testing with a parenteral route of administration (nct ). for both nrrvs and additional oral, live-attenuated vaccines in development, pdvac encouraged the rationalization of target product profiles for these new candidates, to clearly articulate the distinguishing/advantageous features over the existing vaccines, i.e. cost, safety, efficacy in lmic, stability, breath of protection, etc. the potential for any of these vaccine candidates to be included in combination with other emerging enteric vaccines will clearly be advantageous and should be encouraged and explorations of combination with ipv could be considered. clostridium difficile is the leading cause of healthcare-associated diarrhoeal disease in the high-income countries, and is strongly associated with increasing age and frailty, immunodeficiency and in particular, modification of the normal flora through antibiotic use. the results of infection range from asymptomatic carriage through mild infection to severe diarrheal disease, with complications including pseudomembraneous colitis and toxic megacolon. in the us alone, it is believed to have caused approximately . million infections and , deaths in [ ] . current interventions include antibiotic treatments, but their use can trigger relapse on withdrawal. data on the burden of disease in lmics is lacking, however hospital based studies in india, thailand and south korea suggest that the c. difficile infection is widespread, and global (douce, manuscript in preparation). there is a correlation between toxin neutralising antibody in human serum and disease protection; antibodies against toxin a are associated with protection against acute diarrhea, whilst immune responses to toxin b appear to be effective against severe disease and relapse. toxin-mediated disease is recapitulated in the syrian golden hamster, which is the standard preclinical model for demonstration of proof of concept. currently there are three vaccines in clinical development. a toxoid vaccine candidate (containing toxins a and b) recently completed a phase ii study in healthy adults and demonstrated induction of high levels of neutralizing antibodies [ ] and a phase iii study has been initiated. a genetically modified, detoxified whole cell vaccine has also completed phase ii, alhough results have not yet been reported. in phase i, the vaccine was shown to be safe and induced toxin-specific neutralizing antibodies that were sustained for months [ ] . the third candidate is an adjuvanted recombinant protein encoding binding domains of both toxins, and the results of a phase i trial has been reported [ ] , and a phase ii study has been completed. passive immunity by administration of a monoclonal antibody is also in phase iii evaluation (nct and nct ). pdvac agreed that the role for who in facilitating c. difficile vaccine development is not clear given the lack of data regarding the disease burden in lmics. however, it would be useful to understand the potential effectiveness of a vaccine in low resource contexts, and pdvac raised the possibility of testing existing samples from the gems and mal-ed studies for the presence of c. difficile. in addition, it would be helpful to assess the impact that these vaccines many have on reducing the use, and cost of antibiotics, and to consider this in the value proposition for vaccine decision-making. h. pylori is a highly motile, gram-negative bacterium that infects the mucus layer lining the stomach. infection typically occurs in childhood, although symptoms and clinical disease develop in only a minority of infected individuals during their lifetime. h. pylori is associated with gastritis, which causes several pathologies including gastric peptic and duodenal ulcer disease. most significantly, long term infection can result in gastric adenocarcinoma (ga) in later years of life; - % of ga cases are due to h. pylori infection. ga is the rd leading cause of death due to cancer, globally ( , deaths in , . % of all cancers) [ ] . the global prevalence of h. pylori is believed to be approximately % with the highest mortality rates in east asia and eastern europe. the route of transmission is poorly characterised but the oraloral route appears to be a common mechanism, as well as vertical transmission from mother to child. if untreated, most h. pylori infections are sustained for life, and % of those infected are thought to develop an associated pathology. if diagnosed, h. pylori infections are currently treatable with combination antimicrobial therapies. however antibiotic resistance is increasing, with % of patients in some countries currently failing first treatment and % failing two rounds of therapy. antimicrobial treatment offers no protection against reinfection. the choice of indication for an h. pylori vaccine is challenging: a prophylactic vaccine would likely need to be given to children in the first few years of life (to reach the maximum number of the target group while uninfected) but would need to offer long term protection to demonstrate clinical benefit against ga. an effective therapeutic vaccine however could be given at almost any age and would ideally be given by the th decade of life, prior to the peak of ga development which typically occurs from years of age. the most advanced candidate is a urease toxin fusion approach and has completed phase iii trials in children, in china, and demonstrated . % efficacy against natural acquisition of infection [ ] . however, protection appeared to wane to % over - years and next steps for this vaccine are not clear. several other candidates are in preclinical development with one close to phase i studies. pdvac concluded that the burden of h. pylori is significant, and that a vaccine that is able to protect against infection, with sufficiently long duration of protection, would be of public health benefit. therapeutic candidates are currently too upstream in development for there to be a role for pdvac. maternal immunization is increasingly considered as a strategy to prevent maternal and/or neonatal disease. this approach has been proven to protect against maternal and neonatal tetanus and has been in place for decades. who recommends influenza and pertussis vaccination of pregnant women to prevent disease in mothers and newborns, respectively. however, for the first time there are now vaccines in development, specifically indicated for immunization of pregnant women as the target population. respiratory syncytial virus vaccines are most advanced in this area followed by group b streptococcal vaccines. since the pdvac meeting, a special journal issue dedicated to the issues regarding the maternal immunization vaccination strategy has been published and a great deal of work is underway to strengthen the maternal immunization platform [ ] . due to the advanced stage of rsv vaccine and monoclonal antibody development, rsv was presented to sage for information in april . rsv causes . million episodes of lower respiratory infection (lri) annually in children and approximately , deaths, % of which are in lmics [ ] . recently updated estimates for rsv acute and severe lri (community based and hospitalized) disease and deaths will be published by the rsv global epidemiology network (rsv-gen) in early . in addition, the pneumonia etiology research for child health (perch) study will present and publish results on the etiology of severe and very severe pneumonia in hospitalized infants and children in sites in africa and asia. preliminary data analyses indicate that rsv was the leading pathogen in infants with severe pneumonia in this study. there are four rsv intervention strategies currently in development: ( ) maternal immunization to enable passive transfer of maternal antibodies to the foetus in utero, ( ) birth or early infant passive immunization with a long-acting monoclonal antibody, ( ) active pediatric immunization and ( ) vaccination of the elderly. the most advanced maternal immunization candidate begun phase iii efficacy testing in late following the demonstration of induction of palivizumab-competing antibodies (measured by elisa) in women of childbearing age (pmid: ) and pregnant women. this efficacy trial has a group sequential design and will enroll - participants in a randomised placebo controlled trial across multiple sites in both the northern and southern hemispheres, and is expected to take - years to complete. monoclonal antibody development for the prevention of rsv in pediatrics is the next most advanced, with an extended half-life candidate (medi ) that has been shown to be more potent in vitro than the currently licensed palivizumab. one dose may offer protection for up to months. a phase iib clinical study in infants born at - week gestation is planned, and the fda recently granted fast-track designation for this product. since the palivizumab patent recently expired, who in collaboration with the university of utrecht will develop a 'biosimilar' of palivizumab and reduce costs for lmic markets through high yield production and a novel financing plan [ ] . the estimated price is $us per child for the full month dose series and the first market authorization is expected in late . pediatric rsv vaccine candidates are the least advanced, however two adenovirus-based approaches have entered the clinic since the last pdvac meeting. a chimp adenovirus (chad) candidate is currently in phase i testing in adults, to be followed by age de-escalation into seropositive, and ultimately seronegative infants. ad is also being evaluated as a heterologous primeboost regimen, currently in phase i testing in adults. a number of pediatric vaccine candidates developed by the laboratory of infectious diseases, nih are in phase i trials in infants and children. of note, a vaccine containing a deletion of the m - gene showed evidence of diminished replication, enhanced immunogenicity, and asymptomatic 'boosting' (anamnestic response) following naturally acquired rsv infection (pmid: ). two vaccine candidates are in clinical development for the elderly with a post-fusion f-based adjuvanted nanoparticle in phase iii efficacy testing, with data expected in early . pdvac fully supported the following sage recommendations and called for who and partners to develop plans to support global policy-making for rsv maternal immunization as well as passive immunization with long-acting mab, following licensure. particular areas of emphasis include: ( ) rsv surveillance to determine seasonality and age-stratified rsv disease burden and community morbidity and mortality, especially in africa and south-east asia ( ) assessment of the long term effects of rsv interventions and the potential impact of vaccination on reducing recurrent wheeze, which, if demonstrated, would substantially increase the costeffectiveness and impact of rsv preventive interventions ( ) generation of cost-effectiveness and impact data. sage also emphasized the need for strengthening of the maternal immunization platform in collaboration with the influenza, tetanus and pertussis vaccine communities, along with preparations for potential country introductions of rsv vaccine. there is an urgent need to establish a who prequalification pathway for monoclonal antibodies, which does not currently exist. as a rsv vaccine or extended half-life monoclonal ab may become available in the next years, it will also be imperative to initiate early discussions with financing bodies, and to align with the gavi vaccine investment strategy (vis) to avoid delay in achieving the potential major public health impact of rsv immunization if recommended for use by who. globally, gbs remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first days of life. intrapartum antibiotic prophylaxis (iap) for women at risk of transmitting gbs to their newborns has been effective in reducing the young infant gbs disease burden in many high income countries, but iap uptake is limited and difficult to implement in lmics. immunization of pregnant women with a gbs vaccine represents an alternative pathway to protecting newborns and young infants from gbs disease, through prevention of gbs colonization and transplacental antibody transfer to the fetus in utero. pdvac prioritized gbs in and encouraged who to engage on developing guidance on the development pathway for gbs vaccines, including development of a ppc guidance document and a vaccine roadmap. in april , who convened its first consultation on gbs vaccine development [ ] . the focus was on gbs maternal immunization development programs targeting lmic with the ultimate goal of reducing global newborn and young infant deaths. the major knowledge gaps about the disease burden characterization were identified. recent data suggesting that gbs is an under-reported cause of stillbirth may have profound implications on the estimate of the global public health impact of a future gbs vaccine. the relationship between gbs colonization and prematurity should also be clarified. disease surveillance in hic also suggest an important residual unmet medical need, despite implementation of iap. two major pharmaceutical companies are currently developing a multivalent polysaccharide conjugate vaccine, based on the available evidence of an association between trans-placental maternalfoetal transfer of antibodies targeting polysaccharides of the gbs envelope, acquired as a consequence of natural exposure, and a reduced risk of invasive infant disease. a vaccine incorporating five of the eleven described gbs serotypes is predicted to cover over % of the global circulating serotypes, but the risk of serotype replacement is unknown. an alternative approach is targeting surface expressed proteins, in an attempt to confer broad protection across all serotypes. epidemiological studies evaluating the role of maternal antibodies acquired following natural exposure will determine whether a protective threshold at birth can serve as an acceptable vaccine-induced correlate of protection. until additional epidemiological and immunological data are available, estimating vaccine efficacy against invasive gbs disease in neonates and young infants in a double blind placebo-controlled vaccine trial remains the gold standard for generating the evidence required to determine potential public health impact and inform policy decision-making. pdvac endorsed the consensus-based prioritization of future activities including the development of a ppc and vaccine development technology roadmap. efforts should be made to raise awareness of the burden of gbs disease and potential public health value of a gbs vaccine, particularly in countries that lack local epidemiological data. as with rsv, efforts must be made to leverage and strengthen the maternal immunization platform by alignment with other vaccines that are administered in pregnancy, including the brighton collaboration's considerations for safety monitoring through the global alignment of immunization safety assessment in pregnancy (gaia) [ ] . antimicrobial-resistant infections currently claim at least , lives each year across europe and the us alone, but amr affects many hundreds of thousands in other areas of the world [ ] . in european countries, more than % of bloodstream staphylococcus aureus infections are caused by methicillin-resistant strains (mrsa), with several of these countries seeing resistance rates closer to %. emerging resistance to treatments for other diseases, such as tb, malaria and hiv, have enormous impacts in lower-income settings, and by , the death toll due to amr infections in africa is predicted to be approx. , , per year. as mentioned above, each year almost . million cases of drugresistant tb are reported, and these are extremely costly to treat; an mdr case costs - -fold more to treat than drug a sensitive case, while an xdr case is - -fold more expensive [ ] . the who estimates that approximately $ billion per year is required to support tb care and control efforts in lmics. this is significantly more than the current investment in tb vaccine development programs. the o'neill review on antimicrobial resistance estimated that by drug-resistant infections could be claiming million lives per year and at an economic cost to the global gdp in excess of $ trillion. at the sixty-eighth world health assembly in may , a global action plan to tackle antimicrobial resistance, including antibiotic resistance, was adopted [ ] . its goal is to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines -including vaccines -that are quality-assured, used in a responsible way, and accessible to all who need them. the amr global action plan (gap) is based on work streams ranging from national plans, stewardship of antibiotics, encouraging r&d through developing new business plans and assessing environmental drivers. one work stream focuses on vaccines to prevent amr. the who gap workstream on vaccines to prevent amr is based on three complementary approaches: increasing the use of existing vaccines; developing vaccines against high burden diseases currently treated systematically with antibiotics; and prioritizing the development of vaccines for diseases where antibiotic resistance is significant. these three approaches, and the challenges associated with implementing them are summarised below: a). increasing use of existing vaccines: while it is logical that increasing the use of existing vaccines would reduce infections and result in reduced use of antibiotics, it is not always clear which vaccines, in which populations, would have the greatest impact on reducing antibiotic use and potentially amr, and should therefore be prioritized. for example, it has been shown that the use of pcv- in children results in a roughly % reduction in antibiotic-resistant strains of s. pneumonia, and the use of pcv- reduces outpatient antibiotic purchase, leading to the suggestion that global pediatric coverage with pcv could prevent million days of antibiotic use in children annually. however, it is thought that the bulk of pneumonia, and antibiotic use for s. pneumonia infections, is in older adults. this would suggest that demonstrating efficacy of pneumonia vaccines to reduce antibiotic use in older adults and an expanded use of these vaccines in that population group where very few countries have a vaccination policy may have a substantial impact on reducing amr. other existing vaccines which could impact antibiotic use include pertussis, haemophilus influenza, neisseria meningitides, typhoid, as well as influenza which, although not directly susceptible to antibiotic treatment, does result in bacterial super-infections and accounts for up to % of excess (winter-related) antibiotic prescriptions in some countries. in order for a rational evidence-based policy on expanding the use of existing vaccines a prioritization exercise needs to be performed, taking into account the disease burden in different populations, the antibiotic use associated with that disease burden, and an evaluation of how many days of antibiotic use would be avoided with each dose of vaccine administered. this exercise is particularly challenging since in most of the world antibiotics are taken in response to a symptom, rather than an identified infection. this means that, for example, preventing salmonella typhi-induced infection with vaccines may have minimal impact on antibiotic use for severe diarrhea. the prioritization exercise therefore needs to consider not only the disease burden, but the symptom burden and the proportion of that burden due to the vaccine-preventable infection. b). developing vaccines for diseases that are consistently treated with antibiotics, where amr is not currently an issue, but where the vaccine could reduce antibiotic use. one such example is group a streptococcus (gas). while gas is not directly associated with antibiotic resistance (there is little evidence of resistance to date) it has a high disease burden and is a source of extensive antibiotic use. in addition, it is thought that vaccine development is feasible. however to date there has been no significant effort from industry, possibly because of the weak market assessment since it can be treated with antibiotics, and such treatment is cheap. however the indirect costs from such antibiotic use, increased environmental exposure to antibiotics and expansion of amr have not been considered. taking these costs into account may contribute to the value proposition for developing and using such a vaccine. other such candidates could include group b streptococcus, and m. catarhalis and non-typeable haemophilus influenza, both responsible for otitis media which is another source of significant antibiotic prescription. while including potential impact on reduction of antibiotic use, prioritization of these candidate vaccines also needs to consider technical feasibility, whether the antibiotic use is appropriate, and whether alternative non-antibiotic approaches may make vaccine use less attractive. for example: while there are over million cases of otitis media per year in under - year olds, for which antibiotic treatment is usually prescribed which could justify development of a vaccine, most otitis media resolves and new guidelines recommend limiting antibiotic treatment. another example is urinary tract infections which are frequent in elderly patients and a cause of significant antibiotic use, yet there is little supporting evidence that these infections could be effectively reduced by vaccination. a prioritization exercise is therefore required for vaccines that are considered technically feasible, takes into account the potential amr impact, and therefore could contribute to the cost effectiveness of the vaccine if they were developed. to achieve this, the evaluation of impact on amr is recommended to be included in the review of vaccine conducted by pdvac. c). the third and most challenging approach is developing vaccines against pathogens that are frequently antibiotic resistant and becoming increasingly difficult to treat, the so-called eskape pathogens [ ] . this list includes staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumonia, as well as clostridium difficile and tuberculosis. there are numerous challenges in this approach. the first is that although infection with these may result in significant morbidity, the current global disease burden of many of these infections remains relatively low so prophylactic vaccination of the entire population would not be cost-effective. tuberculosis is however an example with significant disease burden and rapidly expanding multi-drug resistance. secondly many of these infections are associated with ageing, where immune decline may make immune interventions poorly effective, or are associated with penetrative medical interventions where the time available to induce a protective immune response may be insufficient. and finally, despite significant efforts to make effective vaccines against some of these has so far proven to be difficult. of these, tuberculosis appears to have the greatest global burden and public health impact, and tb vaccines are also the subject of extensive research. the amr gap activity in this area, to be conducted by ivr is firstly to promote tb vaccine research and development through facilitation of preferred product characteristics and the establishment of a roadmap for vaccine use, and highlighting the impact that the vaccine will have on antibiotic use and antibiotic resistance. additional activities involve monitoring the state of development of vaccines against the pathogens that are becoming antibiotic resistance, and facilitating their development. gas is a ubiquitous human pathogen that causes a broad disease spectrum, from mild to severe, the most serious of which is rheumatic heart disease (rhd). rhd affects approximately million people globally, of whom million experience heart failure and an estimated , die. gas is also a major cause of invasive disease, with a case fatality rate of - % in high income countries, and as high as % in lmics [ ] . on the milder end of the spectrum, gas causes approx. million cases of pharyngitis per year, resulting in - % of cases being treated with broad spectrum antibiotics, rather than penicillin ( % of cases), to which gas is universally susceptible. this extensive use of unnecessary and inappropriate antibiotics increases the likelihood of amr emergence against antibiotics that are used to treat a range of pathogens. previous human challenge studies, as well as preclinical animal models suggest that it is feasible to develop a vaccine against gas, and since the previous pdvac meeting, phase i studies for one candidate has been initiated in adults, and two additional candidates are expected to enter phase i studies in the next months. despite this encouraging progress, significant debate remains as to the appropriate indication and optimal clinical endpoints, and the regulatory pathway for a vaccine to prevent or reduce rhd is unclear. in addition, there is a perception that increased prescription of penicillin would be an equally as effective and a significantly more cost effective method of reducing conditions that result from gas infection. these issues are likely major stumbling blocks in incentivising investment in gas vaccine development. gas has been prioritized by pdvac previously, with a recommendation to develop a business case for both a global market, and also specifically for lmics which would focus on prevention of severe outcomes in resource poor settings. despite significant effort, it has been very difficult to engage stakeholders in this activity. on the recommendation of pdvac, who convened a consultation in december to examine the value proposition for gas vaccines, considering its potential impact across both high income and lower income settings -including the consideration of how current antibiotic treatment practices may increase amr, as well as to investigate the perceived regulatory obstacles. s. aureus is a bacterium that is found as both an asymptomatic colonizer of the skin and nares of human hosts, as well as a frequent cause of human disease. it causes a spectrum of clinical manifestations of varying severity, and is the most commonly isolated pathogen from skin and soft-tissue infections, septic arthritis, pneumonia, endovascular infections, osteomyelitis, catheter/other foreign-body infections, septicaemia, and toxic shock syndrome. methicillin-resistant s. aureus (mrsa) has been documented to be emerging at a rapid and increasing rate since the antibiotic was first introduced in , and hospital-associated mrsa (ha-mrsa) clones are now recognized to be the leading cause of nosocomial infections both in the united states and around the world, in high income as well as lmics. the emergence of communityassociated mrsa (ca-mrsa) in the past several decades is of concern, as is the emergence of highly resistant vancomycin-resistant s. aureus (vrsa). to date, active and passive immunization approaches have been based on increasing the concentration of opsonic antibodies to single surface antigens, and all have failed to demonstrate protection. antigenic variation, the multiple invasion pathways and lack of a surrogate of protection all present significant obstacles to vaccine development. following the failure of single antigen vaccine approaches, most development efforts are now focused on multiple antigens, and a number of candidates are in preclinical development. one multi antigen approach, comprised of antigens including two capsule polysaccharides, clumping factor a and a manganese transport protein, is the most advanced [ ] . current efforts are also focused on further characterizing the immunopathology and immunity of s. aureus infections to identify new antigenic targets, and developing more representative preclinical models in which opsonising and/or neutralising immune responses are measured. to date, none of the vaccine candidates in development have contemplated target populations or indications that are prevalent in lmics. focus has been on development of a vaccine that will protect against life-threatening s. aureus infections in high income countries, but it is hoped that such a vaccine would also protect against all s. aureus infections including more commonly encountered skin and soft tissue infections, and therefore be applicable in lmic contexts. since the pdvac meeting, a new global health sector strategy on sexually transmitted infections has been developed for - and adopted by who member states at the th world health assembly. within this strategic framework, sti vaccine development was highlighted as key need for future sti control [ ] . in addition, the global roadmap for vaccines against stis has been updated and included in the who special issue on pipeline vaccines published in vaccine [ ] . currently, the only sti vaccine candidates that are undergoing or approaching clinical development are against herpes simplex virus (hsv) and chlamydia trachomatis, and as such discussion was limited to these pathogens. hsv is the leading cause of genital ulcer disease, and a particular concern for lmics as it increases both acquisition and transmission of hiv infection. hsv type and type disease burden estimates were recently updated [ , ] , and it is estimated more than half a billion people live with genital hsv infection, worldwide. pdvac previously recommended that improved global estimates of neonatal herpes burden be generated, and assessment of available data has recently been completed with preliminary estimates of > , new cases globally, an incidence rate of approx. / , births, which is concerning because of a case fatality rate of % (looker, submitted) . the incidence is likely to be under-estimated in lmics where hsv infection rates are highest and poor healthcare infrastructure means that neonatal herpes cases are likely to be undetected, but primary data are lacking. ongoing evaluation of hsv infection as part of the child health and mortality prevention surveillance (champs) network will help to address this burden gap. at the meeting, the advance of therapeutic vaccine candidates for hsv- was highlighted, and the role of these types of vaccines in modulating the interaction between hsv and hiv acquisition was discussed as an important consideration for these vaccines in lmics. in consideration of this, and with who support, a systematic review/meta-analysis of hsv- and risk of hiv acquisition including studies will inform modelling of the potential impact of an hsv- vaccine on hiv incidence, and is expected to be published in late . a review of biological mechanisms of hsv-hiv interaction and implications for vaccine development has also been drafted. the pipeline for therapeutic vaccines remains robust, with candidates in clinical development, the most advanced of which now has data demonstrating significant reductions in hsv shedding ( %) and days with genital lesions ( %) over months [ ] . in response to these positive data, niaid has formed an hsv working group to propose desired characteristics for therapeutic and prophylactic vaccines for hsv, including indication, priority target populations, clinical trial endpoints, and safety and efficacy criteria. this document could form the foundation for a who consultative process to generate a guidance document on preferred product characteristics (ppc). pdvac encouraged who to actively collaborate and support development of ppcs for hsv vaccines. chlamydia trachomatis is a gram-negative bacterium that can infect genital, ocular and lung epithelium. it includes three sets of serovars: -serovars ab, b, ba, or c -cause ocular trachoma, which can lead to blindness -serovars d-k -cause sexually transmitted infection resulting in urethritis, cervicitis, pelvic inflammatory disease (pid) (and associated infertility, ectopic pregnancy, and chronic pelvic pain), neonatal pneumonia, and neonatal conjunctivitis -serovars l , l and l -cause lymphogranuloma venereum c. trachomatis can ascend to the upper genital tract and cause pelvic inflammatory disease (pid), which can in turn lead to long-term sequelae including tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. other adverse outcomes of chlamydia include preterm birth, neonatal conjunctivitis and pneumonia, and increased hiv risk. currently management is through screening programs in some high income countries that are not feasible in resource constrained settings, where most cases are likely never diagnosed. who estimates that there were million new cases of chlamydia in [ ] with most cases among adolescents and young adults. the global burden of chlamydia-associated pid, infertility and other sequelae has not been well characterised and estimates of the proportion of infertility presumed to be associated with genital infection (e.g., have a fallopian tube etiology) in africa are outdated [ ] , but are thought to be approx. - % in women seeking fertility care. there are several vaccine candidates currently in preclinical development, with a subunit vaccine based on the chlamydial major outer membrane protein (momp) and live-attenuated (plasmid-deficient) approaches being the most advanced. the momp candidate entered phase i clinical testing in late , and a phase i study with the live attenuated candidate will commence in . the intended goal of a chlamydia vaccine is to decrease upper genital tract sequelae, however pid is challenging to use as clinical endpoint as it is difficult to definitively diagnose and the causes of pid are multi-factorial (typically the result of c. trachomatis in / of cases). the chlamydia vaccine community is seeking guidance and consensus building on clinical endpoints for clinical studies, including evaluating the potential role of biomarkers, radiologic, and other measures of upper tract ascension, infection, inflammation and damage. improved global burden of disease data and vaccine impact modelling on long term sequelae are also needed to define the investment case for these vaccines. pdvac commended the progress towards the first vaccine study against chlamydia since the s and look forward to discussing the path ahead once the early clinical data are available. this section refers to vaccines that have been licensed, or are approaching licensure in some areas of the world, but are currently limited in their use outside any single who region. in some instances, the vaccines may have the potential of offering broader public health impact by expanding approval and use in other geographical regions, and pdvac is seeking to understand the perspective in this regard. ev is one of the most common causes of hand-foot-andmouth disease (hfmd). sporadic ev outbreaks have occurred globally since it was first isolated in , but from the late s a series of large hfmd epidemics caused by ev have been reported in the asia-pacific region. in china alone, . million cases were reported between and , of which ( . %) were fatal [ ] . children less than years of age have the highest risk of disease, and although infection is unusually mild and selflimiting, severe infections can result in neurological and cardiopulmonary complications, and death. several ev vaccine candidates are in development, and it was stated at the meeting that the chinese national regulatory authority has licensed two ev vaccines, with another in progress. the first licensed vaccine was developed by the institute of medical biology, chinese academy of medical science, and has been approved for use to prevent ev disease in - month olds, based on a phase iii study that demonstrated % efficacy over month [ ] . sinovac has also licensed in activated vaccine, with supportive phase iii data in - month olds [ ] . beijing vigoo is in the process of licensing its inactivated ev vaccines in china (nct ). all three vaccines are adjuvanted with aluminum hydroxide. given that ev outbreaks occur in other areas of the world (recently reported in spain [ ]), further discussions are warranted in the international health community about how to assess the role of the chinese vaccines during outbreaks outside china. in the wake of the - ebola outbreak, various strategies were proposed to avoid such crises from reoccurring. key to improving r&d preparedness and response is determining which pathogens are likely to be the greatest threat, creating consensus with respect to product development strategies and coordinating global funding for complementary r&d efforts going forward. to tackle these questions, and at the request of its member states, who convened a broad global coalition to develop the r&d blueprint [ ] as a sustainable platform for accelerated r&d, with two complementary objectives: to develop (and implement) a roadmap for r&d preparedness for known priority pathogens, and to enable roll-out of an emergency r&d response as early and as efficiently as possible the main approaches underpinning the improvement of preparedness within the r&d blueprint include: as reported in the meeting summary, a consultation to initiate work towards a mers cov roadmap was held in december with aims of defining the key basic and applied research activities, identifying the priority technologies and capacities to support vaccine development, and finally understanding the financing/procurement opportunities. following this meeting, a draft roadmap was developed and underwent public consultation prior to finalisation and publication [ ] . it is well accepted that the extraordinary rate of ebola virus vaccine development was as a result of unprecedented collaboration and co-ordination of global vaccine r&d activities, and the availability of a number of candidate vaccines that could enter clinical phase evaluation [ ] . in the face of another pheic so soon following ebola virus disease (evd) outbreak, the global vaccine community is rallying, and reflecting on lessons learned from the experience in west africa only years ago. at the time of responding to the evd emergency, the availability of well-characterised pre-clinical models and robust data was essential for the comparative evaluation and selection of candidates to move into clinical studies. novel recombinant viral vector platforms, in combination with recombination proteins have been validated by evd experience and it could be argued are now less risky for development of vaccine against future pathogens, but manufacturing feasibility and scale-up capabilities still need to be confirmed for the most novel platforms. critically, sustainable public sector push and pull investment mechanisms beyond the initial emergency response phase need to be created, to incentivise manufacturers to engage in the long term commitment to developing and licensing vaccines that may only be used in outbreak or emergency scenarios. pdvac noted that a target product profile for a second generation ebola virus vaccine is under development that will likely cover ebola zaire, ebola sudan, and marburg filoviruses, and will need to demonstrate longer duration of protection. this tpp will provide guidance about whos preferences and minimally acceptable criteria for vaccines in this area. during the discussion it was clarified that who tpps include minimally acceptable criteria, whereas preferred product characteristics specify only preferences. the status of zika virus epidemiology and the understanding of its pathogenesis and associated sequelae are evolving so rapidly that publications on these issues are almost immediately out of date. pdvac's role has been to oversee a working group that has developed a target product profile (tpp) for use in an emergency, or future outbreak scenario. the tpp was made available for public consultation, after which subject matter experts, global regulators, developers and manufacturers were convened to discuss the regulatory considerations for developing a vaccine with the characteristics described in the tpp. the finalised tpp and position paper are publically available [ ] . in addition to reviewing the status of vaccine development against pathogens, pdvac considered a number of cross-cutting issues that could better integrate and therefore facilitate product development efforts for vaccines and other interventions. in addition to the significant morbidity and mortality that drives the development of vaccines against pathogens for which vaccines are currently not available, the who estimates that there are approximately . million deaths per year in children under from vaccine preventable diseases [ , ] . one of the reasons for this striking immunization gap is the cost and logistical challenges of delivery of these vaccines, over and above the cost of their manufacture. the remit of who's immunization practices advisory committee (ipac) is to provide strategic advice on immunization practices, tools, and technologies intended to improve the delivery of immunization programs at the country level. it oversees the recently formed delivery technologies working group (dtwg) composed of public health organizations, funders and procurement agencies as well as vaccine developers to evaluate r&d in novel delivery technologies and devices, for example the microarray patch, and compact, pre-filled auto-disable injection technologies (cpad). of particular focus for this group is the development and evaluation of a framework to analyze high-level trade-offs between important variables such as development, procurement and supply chain costs, coverage, efficacy, and safety in order to facilitate investment decisions by product developers, vaccine manufacturers, global policy makers, in-country decision makers and procurement agencies. this framework is referred to as total systems effectiveness (tse). the intent of this delivery technology working group is to offer a platform for discussion and guidance regarding vaccine preferences for lmics, early on in development, so that ultimately the vaccine is suitable for programmatic use. the dtwg reports directly to ipac, but has potential overlap with activities that are overseen by pdvac, particularly in consideration of second generation vaccines or new vaccines that may be developed with an alternative presentation to that of a needle and syringe. pdvac was supportive of the dtwg and encouraged continued communication between vaccine development and device/delivery technology development to identify potential opportunities for novel combination product development. there are several pathogen areas where mab are being developed as vaccine-like interventions, as their single dose regimen and long half-life render them amenable for lmics contexts, where they could offer significant public health benefit. candidates for rsv and rabies are approaching licensure within the next years, and a who procedure for who prequalification is urgently needed to avoid delay implementation. this gap has been recognized and will be addressed. the scope of pdvac overlaps with several other research agendas such as gvap, amr, new delivery technologies and development and consolidation of maternal immunization platforms. the pdvac research agenda needs to be clearly communicated, and pdvac and ivb will strive to be well-informed of efforts in other research areas, to help shape and align strategy where appropriate. future pdvac meetings will consider these potential overlaps in more detail, as well as how pdvac and ivb can facilitate development of integrated product development approaches. since its inception in , pdvac has reviewed the pipeline and vaccine development status of different pathogens. pdvac will continue to review new pathogen areas as candidates progress into clinical studies, providing that who engagement will likely facilitate the use of vaccines to reduce disease burden in lmics. one such pathogen is cytomegalovirus (cmv), which is a leading cause of congenital infections worldwide, resulting in - % of infants developing permanent sequelae including hearing loss and neurodevelopmental disabilities. there are little data on cmv infection in lmics, but a recent systemic review suggests that birth prevalence ranges are higher in lmics than in europe and north america [ ] , and several clinical trials of vaccine candidates are ongoing. as such, an assessment of cmv vaccine development will be undertaken by pdvac . with rsv, tb, hiv and enteric candidates approaching pivotal data points, understanding what data are needed to support earlier policy implementation and outcomes will be key, as well as understanding the potential impact of vaccines within the broader control strategy -including diagnostics and other preventatives -for these pathogens. vaccine impact modelling, and understanding the composite set of cost drivers through to vaccine delivery will be important. interaction with who's ipac and pq teams will increase going forward, to strengthen the link between product development and programmatic requirements. under the recommendation of pdvac, who will seek to broaden its role to support development of value propositions for vaccines against pathogens for which there is a poorly defined business case, for example gas and hsv. raising awareness of lmic disease burden and requirements/procedures for access to lmics markets may help to incentivise financing development of these vaccines. of key consideration may be the potential for these vaccines to reduce the emergence of amr, and pdvac recommended that this be considered as a criterion in future landscape analyses and ppc guidance documents. pdvac and who will continue to align activities with the priorities within the who r&d blueprint. pdvac is aware that several other organizations which are responsible for emergency preparedness have been through a process to prioritize their r&d agendas, with some commonality and some complementarity to the pathogens listed in the who blueprint. in this arena, pdvac will continue its horizon scanning role, and will advocate for commitment to product development of vaccines for emerging diseases to progress through robust preclinical proof of concept to generation of phase i data, as a minimum. pdvac strongly recommends the collaboration with other groups to co-ordinate advocacy and funding for vaccine development to prepare for the inevitable future emergencies. procedure for assessing the acceptability, in principle, of vaccines for purchase by united nations agencies tackling drug resistant infections globally: final report and recommendation how can vaccines contribute to solving the antimicrobial resistance problem? who vaccine pipeline tracker moorthy vasee s, who product development for vaccines advisory committee (pdvac) pipeline analyses for pathogens mind the gap: jumping from vaccine licensure to routine use global vaccine action plan (gvap) impact and cost-effectiveness of new tuberculosis vaccines in low-and middle-income countries status of vaccine research and development of vaccines for tuberculosis vaccination with alvac and aidsvax to prevent hiv- infection in thailand report from the world health organization's product development for vaccines advisory committee (pdvac) meeting who global consultation on universal influenza vaccines the global burden of norovirus & prospects for vaccine development the vast and varied global burden of norovirus: prospects for prevention and control global, regional, and national estimates of rotavirus mortality in children < years of age who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines current status of clostridium difficile infection epidemiology defining the optimal formulation and schedule of a candidate toxoid vaccine against clostridium difficile infection: a randomized phase clinical trial a phase , placebo-controlled, randomized study of the safety, tolerability, and immunogenicity of a clostridium difficile vaccine administered with or without aluminum hydroxide in healthy adults safety, immunogenicity and dose response of vla , a new vaccine candidate against clostridium difficile, in healthy volunteers efficacy, safety, and immunogenicity of an oral recombinant helicobacter pylori vaccine in children in china: a randomised, double-blind, placebo-controlled, phase trial brewinski isaacs and a. sobanjo-ter meulen advancing maternal immunization programs through research in low and medium income countries global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis moorthy vasee s. who consultation on group b streptococcus vaccine development: report from a meeting tb vaccine research & development: a business case for investment clinical relevance of the eskape pathogens invasive group a streptococcus infection among children development of a multicomponent staphylococcus aureus vaccine designed to counter multiple bacterial virulence factors the global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps global estimates of prevalent and incident herpes simplex virus type infections in global and regional estimates of prevalent and incident herpes simplex virus type infections in global estimates of the prevalence and incidence of four curable sexually transmitted infections in based on systematic review and global reporting the pill, chlamydia and pid hand, foot, and mouth disease in china, - : an epidemiological study an inactivated enterovirus vaccine in healthy children efficacy, safety, and immunogenicity of an enterovirus vaccine in china a roadmap for mers-cov research and product development: report from a world health organization consultation on a path to accelerate access to ebola vaccines: the who's research and development efforts during the - ebola epidemic in west africa meeting report: who consultation on considerations for regulatory expectations of zika virus vaccines for use during an emergency global, regional, and national causes of child mortality in : a systematic analysis systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries we gratefully acknowledge the pathogen specific landscape analyses that were prepared by the following individuals: this report contains the collective views of an international group of experts, and does not necessarily represent the decisions or the stated policy of the world health organization, nor of the u. s. government.bsg is an employee of the u.s. government. this work was prepared as part of his official duties. title u.s.c. § provides that 'copyright protection under this title is not available for any work of the united states government'. key: cord- -nmttgxlq authors: garcía, leidy y.; cerda, arcadio a. title: acceptance of a covid- vaccine: a multifactorial consideration date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nmttgxlq nan acceptance of a covid- vaccine: a multifactorial consideration the individual decision process of whether or not to vaccinate against covid- is multifactorial and must be considered in the design of any vaccination campaign. specifically, the willingness to vaccinate against sars-cov- depends on: (a) availability, i.e. the actual existence of an effective vaccine and its country of origin; (b) access to the vaccine, which could be limited by individual or governmental budgetary restrictions to finance preventive public health measures; (c) perceived health risk, which depends on the intensity and severity of side effects and covid- prevalence; (d) information on benefits, risks and access pathways; (e) previous experience with other vaccines and exposure to diseases, as this affects risk perception; and (f) sociodemographic factors including age, education level, gender and more. in fact, the preliminary results of a chilean case study on covid- vaccine perception in the country, with a nationwide representative sample of individuals, shows that % are willing to vaccinate, a relatively high proportion and slightly lower than the rate found by garcía and cerda [ ] , . % for chile. it must be said that the chilean case is unique, as the infection rate per million inhabitants is one of the highest on earth ( . ), but the lethality rate is the lowest ( ), both of which have opposite effects on vaccination intention. this illustrates that circumstantial conditions such as the pandemic context, specifically disease prevalence in a particular population can also impact vaccination intention. in fact, feleszko, wojciech and lewulis [ ] show that there are important differences between countries in the percentage of individuals who declare a positive intention towards vaccination against covid- , which varies between % and %. these differences could reflect individual preferences, influenced by the plethora of factors mentioned above. this is why each country must design its own vaccination strategy based on the characteristics of its population and prevalence, considering a holistic vaccination design that effectively considers all the factors that influence vaccination decisions. according to paakkari [ ] , schaffer et al. [ ] and harrison [ ] , a transparent educational and communicative campaign is needed, one that considers interaction between health policymakers in a way that allows people to value the personal and social benefit of being vaccinated against covid- , reducing hesitation. therefore, it is necessary for each government to take the public health measures necessary for its culture, its perceptions and the demographic characteristics of its population. the same international applicability framework must also be considered to design the vaccination campaign based on information, education and awareness towards social benefit. contingent assessment of the covid- vaccine flattening the curve of covid- vaccine rejection-a global overview vaccine confidence in the time of covid- covid- : health literacy is an underestimated problem planning for a covid- vaccination program the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -xvtt fz authors: gao, lulu; yu, shouyi; chen, qing; duan, zhaojun; zhou, jie; mao, chen; yu, dexian; zhu, wenchang; nie, jun; hou, yunde title: a randomized controlled trial of low-dose recombinant human interferons α- b nasal spray to prevent acute viral respiratory infections in military recruits date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xvtt fz the military population has a high disease burden of acute viral respiratory infections in china. to assess the efficacy and safety of a low-dose recombinant human interferon α- b (rifnα- b) nasal spray in preventing acute viral respiratory infections in military population, we performed this randomized controlled trial. the results showed that application of the rifnα- b nasal spray had the benefits in prevention of infections caused by influenza a virus, influenza b virus parainfluenza viruses – and adenovirus species b. however, no benefit was seen in preventing respiratory syncytial virus. no severe adverse events were reported. therefore, the rifnα- b nasal spray was effective and well tolerated for preventing common viral respiratory infections in the military recruits. acute respiratory tract infections are flourishing in closed and crowded environments. military recruits are prone to outbreaks of acute respiratory tract infections because of their crowded living conditions in barracks, stressful work environments, frequent travels and exposure to novel strains of respiratory pathogens [ ] [ ] [ ] . over % of acute respiratory tract infections are caused by viruses, such as influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, adenovirus and coronavirus. though most of viral respiratory infections usually represent mild, selflimited clinical manifestations, they are leading causes of morbidity in certain groups or populations (e.g., children, military population) and remain a heavy burden of disease [ , ] . in history, the famous "spanish influenza" initiated from army recruits in [ ] . in , the novel a/new jersey/ (hsw n ) influenza virus caused severe respiratory illness in soldiers at fort dix [ ] . adenoviruses have been the most important cause of febrile acute respiratory disease in us military recruit populations [ ] . in china, a comprehensive surveillance systems for influenza in military population showed that the average incidence of influenza was . episode per person-year from to [ ] . viral respiratory infections have become one of the most actual health problems in military population. therefore, it is necessary to seek reasonable and effective measures to prevent outbreaks and epidemics of acute viral respiratory infections among military population. interferons (ifns) are a family of cytokine mediators that are critically involved in alerting the cellular immune system to viral infections of host cells [ ] . the previous studies have suggested that high dosage of intranasal interferons can prevent respiratory infections caused by viruses, such as influenza virus, rhinovirus, coronavirus and respiratory syncytial virus [ ] [ ] [ ] [ ] [ ] . however, the major problem is higher frequency of local side effects such as mucosal irritation, dry mucous membranes, blood-tinged mucus and nasal mucosal erosion [ ] [ ] [ ] . recently, yuance medicine company (beijing, china) has developed a low-dose recombinant human interferon ␣- b (rifn␣- b) nasal spray in order to reduce adverse reactions. to evaluate the efficacy and safety of this new nasal spray in preventing acute respiratory infections in military population, we performed this randomized, placebo-controlled, double-blind trial. from november to december , we did a randomized, placebo-controlled, double-blind, multi-center trial in the military trainees from recruit training units in three geographically distinct cities in china (guangzhou city and foshan city in guangdong province, liuzhou city in guangxi province). candidate subjects were male recruits who aged - years, and finished army physical fitness examination. they were not admitted to the study if any of the following criteria were present: ( ) on regular medical treatment or took other medications within two weeks, ( ) history of serious allergies (e.g., asthma, urticaria, and eczema), ( ) history of autoimmune disorders, ( ) psychiatric disorders, and ( ) acute or chronic illnesses. all candidates were observed two weeks before enrolling into the study, in order to screen eligible subjects. a total of trainees from three areas enrolled the trial initially. after the screening had been completed, eligible recruits remained in the subject pool. all participants understood the implications of the study, and provided informed consents. the study was conducted during the basic training period of new military recruits at recruit training units located in different districts. there were - new recruits in each independent training unit. all the trainees lived in barracks during a three-month training. eight to recruits shared a quarters room about m . participants in the experimental group received the rifn␣- b nasal sprays, the metered spray device delivered . ml ( × iu of rifn␣- b) per spray into each nostril and throat, that was a total of × iu of rifn␣- b for each administration. the spray delivered twice daily after breakfast and supper respectively for five consecutive days. the control group was given placebo in the manner identical to the experimental group. the placebo contained components which were similar to the drug except rifn␣- b. participants were given instructions on when and how to use the sprays. each nostril and pars laryngea pharyngis should be sprayed with breathing deeply. the rifn␣- b nasal spray and placebo were stored at - • c at health clinics of training units. all subjects were followed up and observed for clinical signs and manifestations of respiratory infections for days. local symptoms (e.g., sore throat, dry pharynx, cough, nose running, sneezing, nose congestion), systemic symptoms (e.g., malaise, myalgia, headache, nausea, abdominal pain, diarrhea) and axillary temperature were recorded daily during the observed period. severe adverse reactions (such as allergy, epistaxis, and nasal mucosa erosion) or complications should be reported immediately. once the adverse reactions had occurred, subjects would stop the experiment and be given suitable treatments. assuming a % viral respiratory infections attack rate in the group that received placebo and a % attack rate in the group that received rifn␣- b nasal spray (based on the data obtained from our preliminary studies) and assuming that sufficient data would be collected for % of the cases to be included in the accordingto-protocol population, we calculated that a sample of recruits per group would provide more than % power to demonstrate the superiority of the rifn␣- b nasal spray at a significant difference level of . (two tailed). a list of random numbers allocating to the each spray canister was determined via computer-generated randomization. the generation of randomized numbers and labeling of the spray canisters were performed by the third party (national institute for viral disease control and prevention, china center for disease control and prevention) of this study. participants were sequentially allocated to the treatments in the order in which they were recruited, i.e., the first person who was eligible for inclusion was given spray number , the second one spray number , and so on. when allocated, each participant's name was added to the label details on the spray container. the sequence was concealed until the data were analyzed. both participants and researchers were blind to group assignment. once accuracy of the data were confirmed, the database would be forwarded to the statistician who, only at this time, was supplied with the randomized list. case report forms (crf) consisted of subject demographics data, medical history, respiratory infection symptoms, adverse reactions including local reactions and systemic reactions and concomitant medications from the time that the spray was administered until days later. participants were given a diary crf to record the spray administration, clinical signs and manifestations of respiratory diseases, adverse effects they might have experienced, and other medications they might have taken. observers visited the participants daily and recorded severe adverse events, including high fever (axillary temperature, ≥ . • c), allergy, epistaxis, nasal mucosa erosion and hemafecia. serum samples for assessment of viral respiratory infections were collected on the days and day after the administration. elisa (enzyme-linked immunosorbent assay) kits (shenzhen sciarray biotech co. ltd) were used to test igm antibodies against adenovirus species b (adv), respiratory syncytial virus (rsv), influenza a virus (flu-a), influenza b virus (flu-b), and parainfluenza viruses - (piv - ). coating antigens used in the kits were prepared from adenovirus (strain adenoid ), respiratory syncytial virus (rsv long strain), influenza a virus (h n , strain a/texas / ), influenza b virus (strain hongkong / ), parainfluenza virus type (strain vp ), parainfluenza virus type (strain greer) and parainfluenza virus type (strain c ). the operating procedures of elisa were according to the manufacturer's instructions. briefly, l of : diluted serum specimens was applied to each well of the microtiter plate, the positive and negative standards (provided by the manufacturer) and blank control (dilution solution) were running with each plate to ensure accuracy, then the plate was incubated for min at • c. the specimen was removed and the plate was washed four times by washing solution. anti-human igm (-chain specific) conjugated to horse radish peroxidase was added and incubated for min at • c. the plate was washed four times again. tmb ( , , , -tetramethylbenzidine) solution was added to each well and incubated for min. the reaction was stopped by adding l stop solution ( m h so ) to each well and the optical density (od) value at nm was determined with an elisa reader. serum specific-igm antibodies were defined as positive if od values were greater than two fold negative standard. we compared the positive rates of the viral igm antibodies between two groups. the subject whose serum igm against any of five viruses was positive on the initial administration (day ) was excluded for serological analysis. the recruit whose antibody was negative on day , positive on day after the administration, was considered as having a recent infection with the corresponding virus. serum specimens were collected from participants and detected for antibodies against three viruses (flu-a, flu-b and piv - ), among which of specimens were detected for antibodies against five viruses (adv, rsv, flu-a, flu-b and piv - ). all study data were checked for range and consistency, and were double entered into databases. data of antibodies were entered into microsoft excel , while crf and adverse events data were entered into epidata . . all analyses were performed by using the spss . statistical software package. a descriptive statistical analysis was carried out to compare the baseline characters between two groups. infection rates and adverse events were analyzed by test. for the statistical analysis of body temperature parameters, a repeated measures anova model was used. the level of statistical significance was established as p < . . meanwhile, intention-to-treat (itt) and per protocol (pp) analysis were performed as the assessment. parameters for assessment of benefits or harms of the intervention included: the rate at which events occur in the control group (the control event rate, cer), the rate at which events occur in the experimental group (the experimental event rate, eer), relative risk (rr), absolute risk reduction (arr), relative risk reduction (rrr), and number needed to treat (nnt). measures of rr, rrr, arr and nnt were determined as these following formulae: rr = eer/cer, arr = cer-eer, rrr = arr/cer, nnt = /arr [ ] . the research protocol was followed to the tenets of the declaration of helsinki and the guidelines for good clinical practice. the clinical trial (protocol number l ) was officially approved by state food and drug administration, pr china in april , and also approved by ethical committee of southern medical university. of recruits screened, recruits were eligible for the trial criteria. they were randomized to receive either treatment sprays (n = ) or placebo control sprays (n = ) twice daily for days. during the trial, recruits were lost to follow-up, among which, in the experiment group and in the control group. another subjects dropped off the study due to they were afraid of adverse events psychologically and quit the trial by themselves. subjects who completed the study are shown in fig. . all subjects were male, recruited from different provinces, such as guangdong, guangxi, hubei, liaoning, shandong. the mean age of subjects was . ± . years (range - years). the differences of age, educational level and other demographic characteristics were not statistical significance between two groups (table ) . compliance rates between two groups were not statistically significant different, which the experimental group was . % and the control group was . % (p = . ). the positive rates of igm antibodies against the viruses in recruits whose were negative before the intervention are summarized in table (pp analysis) and table (itt analysis). igm positive rates of anti-adv, anti-flu-a, anti-flu-b, and anti-piv in the control group (cer) were significantly higher than that in the experimental group (eer) (p < . ) during the observational period. although positive rate of anti-rsv igm in the experimental group was higher than that in the control group, no significant difference was found. values of rr were less one and their % confidence intervals ( % ci) did not cross one except for rsv that the % ci included one ( table ). the results indicate that the risk of developing viral respiratory infections is less in the treatment group than that in the control group. the pp analysis exhibited that rrrs (i.e., protection rates) of the rifn␣- b against adv, rsv, flu-a, flu-b and piv - were . % ( % ci: . table the detection of specific serum igm antibodies to five viruses in the intention-to-treat analysis. ( % ci: . - . ), respectively ( table ). the results showed that the pp analysis was consistent with the itt analysis. to sum it up, protective efficacy of the rifn␣- b against four viruses arranged in descending order was flu-a, piv - , flu-b, adv. however, there was a % certainty that the rifn␣- b had no effect for rsv because the % confidence intervals for the rr, rrr and nnt extended from a negative number (treatment may harm) to a positive number (treatment may benefit) (tables and ). no participants withdrew from the trial due to intolerance of the spray. none of the participants were found to have allergy, high fever, nasal mucosa erosion or hemafecia during the follow-up observational period after administration. we found some flu-like symptoms including cough, sneeze, nose congestion and nose running were slightly higher in the drug group than those in the control group in the period of administrating the nasal sprays, particularly during the second to fourth days of the experiment, however, the differences were not significant (p > . ). the incidence rates of epistaxis in the treatment group were low, with from . % ( / ) to . % ( / ) during the period of administration, although the occurrences were significant higher in the treatment group than that in the control group on the third day ( . %, / , versus . %, / , p < . ) and the fourth day ( . %, / , versus . %, / , p = . ). the occurrence of dry pharynx was significantly higher in the treatment group than that in the control group during the whole drug administration period ( . - . % in the treatment group, . - . % in the control group). average incidences of dry pharynx and epistaxis were higher in the experimental group (dry pharynx . %, epistaxis . %) than those in the control group (dry pharynx . %, epistaxis . %) (p < . ) during the followup period ( table ). the peak of these symptoms was found during the first days, thereafter declined. both of the experimental group and the control group had high rates of myalgia ( . %, . %), arthralgia ( . %, . %). these symptoms happened after high intensity training and without significant difference between the two groups. viral respiratory infections are caused by a variety of viruses, among which there are approximately known ones including a vast number of serotypes, and undergo frequent changes in antigenicity [ ] . although several vaccines against respiratory viruses to prevent the infections have been proved useful in military populations [ , [ ] [ ] [ ] , progress has been extremely slow. furthermore, not all kinds of viral etiological respiratory infections have been available for specific prevention and still have unknown pathogens. in the recent decade, several novel respiratory viruses which caused serious illness have been identified, such as human metapneumovirus (hmpv), new sars-coronavirus (sars-cov) that associated with severe acute respiratory syndrome (sars), h n flu virus and novel h n virus, which increase the difficulty of the immunological prevention for viral respiratory infections. in the past years, researchers found that ifn-␣ showed an inhibitory effect for sars-cov in vitro and in vivo [ ] [ ] [ ] . therefore, it suggests the potential benefits of ifn-␣ in preventing and controlling viral respiratory infections in specific population groups and in epidemic period. the common viral respiratory infections possess similar clinical signs and symptoms without special clinical manifestations, explicit diagnosis only depends on pathogen examination. although isolation of virus from patients is the strongest evidence for confirming viral respiratory infections, the diagnostic procedures are complex and time-consuming because of the wide range of viruses, especially in a large sample epidemiological study. in addition, respiratory viruses are frequently detected in respiratory tract secretions samples from healthy people [ , ] . the determinations antibodies against respiratory viruses by comparing acute and convalescent serum specimens in infective people may sometimes be helpful to confirm a specific causative infection. serum specific igm against virus is a sensitive indicator of a recent onset of viral infection, thus, we used specific serum igm as surrogate outcome variables to evaluate the preventive effects of the rifn-␣ b for acute viral respiratory infections in this study. the rr, rrr and arr are common parameters used in reporting randomized clinical trials and epidemiological field trails. the rr is a ratio of the probability of the event rate occurring in the exposed (or experimental) group (eer) versus a non-exposed (or control) group (cer). in interventional trials, if the treatment arm is effective in preventing disease then the rr will be less than one, and vice versa. the rrr is the percent reduction in events in the eer compared with the cer. in other word, the rrr presents the percentage of the risk that has been reduced by the intervention in the control group. the arr is the arithmetic difference in the event rate between treatment group and control group. in interventional trials, if the treatment arm is effective in preventing disease then arr will be positive quantity, on the contrary, if the treatment arm is harm, arr will be negative. rr and rrr can be used to quantify the relative magnitude of the protective (treatment) effects. the arr can be used to measure the absolute difference in event rates between two populations. therefore, arr is considered as a more intuitive measure than rr and rrr. these years, the number needed to treat (nnt) has become a widely used index for interpreting the magnitude of treatment benefits or harms. the nnt is the inverse of the arr. it represents the expected number of persons who must be treated with an intervention in order to prevent one additional adverse outcome event (or, depending on the context, to expect one additional beneficial outcome), compared to the expected event rates under the control. that is to say, the smaller the nnt, the more effective the treatment. therefore, besides rr, rrr and arr, we used the nnt to express the size of efficacy of the rifn␣- b in the present analysis. in our study, serological detection exhibited that serum positive rates of igm antibodies against the five viruses were higher in the control group than those in the experimental group on the th day of the administration. in itt analysis, highly antiviral effects for flu-a, flu-b and piv - were seen, all with the rr of < and the rrr (i.e., protective rates) of > %. a protective effect also was seen for adv, with the rr of < and the rrr of . %. however, there were wide range of % confidence intervals of the rr ( . - . ) and rrr ( . - . %), which indicated the low reliability. no certain effect was found for rsv. although the rr was less than and rrr = . %, the % ci ranges of rr ( . - . ) and rrr (− . % to . %) were very wide and included null values (rr % ci included , rrr % ci included negative). because of small size samples for detecting serum adv antibodies and rsv antibodies and less people of anti-adv and anti-rsv igm positive in both groups and a very wide confidence interval of protection rates in adv and rsv, we considered that the sample of this study was not enough for assessing preventive effects of the rifn-␣ b for these two viruses. in the itt analysis of the study, nnt values were ( % ci: . - . ), ( % ci: . - . ) and ( % ci: . - . ) for flu-a, flu-b and piv - infections respectively, which meant , and people need to administrate the rifn-␣ b to prevent one infection with relevant virus. the nnt for adv was ( % ci: . - . ), which indicated that the benefits of preventing adv infection with the rifn-␣ b may be less than above infections with any three viruses above. the nnt for rsv was ( % ci: − . to . ), which indicated no effect in preventing rsv infection among recruits with the rifn-␣ b nasal spray. itt analysis is a method of analysis for randomized trials in which all subjects randomly assigned to one of the treatments are analyzed together, regardless of whether they completed or received that treatment or not. on the other hand, pp analysis is a method based only on those patients who complete the entire treatment protocol. in this study, similar results were observed for subjects in the itt and pp populations, which indicated no significant missingness and protocol deviation. on the whole, the intranasal rifn␣- b with relative low dose and short term administration ( . × iu daily for five days) was well tolerated. most of the clinical features reported were comparable between the control and the experimental groups. except epistaxis, no other known severe adverse events (such as allergy, high fever, nasal mucosa erosion and hemafecia) of the intranasal interferon were reported during the trial. the rrs of dry pharynx and epistaxis were . ( % ci: . - . ) and . ( % ci: . - . ), respectively, which indicated that dry pharynx and epistaxis might be linked to the interferon. however, the incidence of epistaxis was low and the clinical signs were mild and transitory in present study. the results are the same as our previous study [ ] and the risk is much lower than that appraised and summarized in evidence-based medicine (or = . , % ci = . - . ) by jefferson and tyrrell [ ] . in summary, this randomized controlled trial suggested that the recombinant human interferon ␣- b nasal spray can be used to prevent common acute viral respiratory infections caused by flu-a, flu-b, piv - and adv and was generally well tolerated among military recruits. however, the limitations of this trial may be found on sampling and sample size. all subjects enrolled were healthy and young male recruits, so it may be difficult to extrapolate the conclusion to other populations. the sample was not large enough for evaluating the effects for adv and rsv infections which had relative low incidence in the army recruits. the efficacy of preventing viral respiratory infections by the rifn␣- b nasal spray should be evaluated further in different population groups, such as children and the elderly, and more samples should be involved in the further study. swine influenza a outbreak clinical presentations for influenza and influenza-like illness in young, immunized soldiers reflections on the swine flu vaccination program current research on respiratory viral infections: fourth international symposium large epidemic of adenovirus type infection among military trainees: epidemiological, clinical, and laboratory studies the social history of medicine: beyond the local a doubleblind, placebo-controlled study of the safety and immunogenicity of live, oral type and type adenovirus vaccines in adults influenza surveillance on the army population interferon and their application in the diseases of the lung the efficacy of intranasal interferon alpha- a in respiratory syncytial virus infection in volunteers prevention of experimental coronavirus colds with intranasal alpha- b interferon treatment of respiratory syncytial virus infection with recombinant interferon alfa- a could preventive intranasal interferon lower the morbidity in children prone to respiratory illness? intranasal interferonalpha b for seasonal prophylaxis of respiratory infection tolerance of one-month intranasal interferon intranasal applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose human tolerance and histopathologic effects of long-term administration of intranasal interferon-alpha glossary of evidence-based terms treatment of respiratory virus infections role of u.s. military research programs in the development of u.s.-licensed vaccines for naturally occurring infectious diseases effectiveness of the - influenza vaccine among u. s. military basic trainees: a year of suboptimal match between vaccine and circulating strain vaccination policies in the military: an insight on influenza severe acute respiratory syndrome-related severe coronavirus is inhibited by interferon-alpha anti-sars virus activities of different recombinant human interferons in cell culture system preventive and therapeutic effects of recombinant ifn-b nasal spray on sars-cov infection in macaca mulata a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection upper respiratory virus detection without parent-reported illness in children is virus-specific a field trial for evaluating the safety of recombinant human interferon ␣- b for nasal spray vaccines for the common cold (protocol for a cochrane review) this study was funded by national high-tech r & d program ( program), grant number aa from ministry of science and technology of the people's republic of china.we thank institute for viral disease control and prevention of china cdc for providing and dispensing the spray agents to be tested, and we gratefully acknowledge the cooperation and assistance of the healthcare staff members at the clinical trial sites. key: cord- -i o rs authors: pagliusi, sonia; ting, ching-chia; lobos, fernando title: vaccines: shaping global health() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: i o rs the developing countries vaccine manufacturers’ network (dcvmn) gathered leaders in immunization programs, vaccine manufacturing, representatives of the argentinean health authorities and pan american health organization, among other global health stakeholders, for its th annual general meeting in buenos aires, to reflect on how vaccines are shaping global health. polio eradication and elimination of measles and rubella from the americas is a result of successful collaboration, made possible by timely supply of affordable vaccines. after decades of intense competition for high-value markets, collaboration with developing countries has become critical, and involvement of multiple manufacturers as well as public- and private-sector investments are essential, for developing new vaccines against emerging infectious diseases. the recent zika virus outbreak and the accelerated ebola vaccine development exemplify the need for international partnerships to combat infectious diseases. a new player, coalition for epidemic preparedness innovations (cepi) has made its entrance in the global health community, aiming to stimulate research preparedness against emerging infections. face-to-face panel discussions facilitated the dialogue around challenges, such as risks of viability to vaccine development and regulatory convergence, to improve access to sustainable vaccine supply. it was discussed that joint efforts to optimizing regulatory pathways in developing countries, reducing registration time by up to %, are required. outbreaks of emerging infections and the global polio eradication and containment challenges are reminders of the importance of vaccines’ access, and of the importance of new public-private partnerships. the developing countries vaccine manufacturers' network (dcvmn) is the world's largest vaccine-industry alliance. in , corporate members are working to provide high-quality vaccines, and contribute to global health initiatives, ensuring uninterrupted vaccine supply to countries, to advance eradication of polio and facilitate response to emerging infectious diseases (eids) or outbreaks like the zika outbreak [ ] . and pharma from pakistan, hll biotech, green signal bio pharma and zydus cadila from india, and instituto biologico argentino (biol) from argentina -the th member. he emphasized the importance of collaboration and partnerships among members for achieving sustainable supply of high-quality, affordable vaccines and to accomplish global health initiatives. one such initiative is the switch from trivalent (topv) to bivalent oral polio vaccine (bopv) and eventually inactivated polio vaccine (ipv), in order to contain polioviruses better and, ultimately, eradicate polio. j. lemus, minister of health in argentina, began with the reminder that vaccines and potable water are the most effective ways to reduce morbidity and mortality globally. the role of the ministry of health is to protect the argentinian population and promote healthy living. the h n outbreak in argentina underscored the value of local vaccine manufacture. he also emphasized the importance of supporting international supply, and affirmed the argentinian government's and vaccine manufacturers' commitment to work with paho to provide high-quality vaccines globally. m-p. kieny reviewed evolving collaborative innovation in vaccine development. historically, one scientist, like edward jenner, could develop a vaccine. then came small groups, such as louis pasteur and colleagues. innovation subsequently became the work of a large team, sometimes spanning several corporations. now, after decades of intense competition for high-value markets, collaboration with developing countries has become critical, and involvement of multiple manufacturers as well as public-and private-sector investments are essential. the -year meningitis a project and the -year malaria vaccine development illustrate the efforts for developing vaccines for african epidemics. the accelerated ebola vaccine development in required urgent collaboration between manufacturers, regulatory authorities and funding agencies to respond to and contain the emerging outbreak [ ] . following the registration of the first dengue vaccine in , the most advanced dengue vaccine, a collaboration between butantan and national institutes of health (nih) [ ] , encourages partnerships with dcvms and public-sector entities. full participation of dcvmn members in future innovative collaboration will require increased development capability, novel production processes and regulatory skills, along with equal partnerships, justified investments and national regulatory agencies (nras) capabilities. she concluded that commitment to public health as a joint responsibility is essential. h. sigman, chairman of group insud and sinergium welcomed meeting participants. he introduced sinergium's technology transfer capabilities, providing an example of partnership between public and private sectors, and confirmed the importance of collaboration among vaccine manufacturers. the director of paho, c.f. etienne, presented via video. paho has undertaken to make national health programs sustainable and affordable, encouraging improvements in quality and availability of vaccines from both private and public sectors. rubella was eliminated from the who americas region in , and it is now the first region to be declared free of measles [ ] . she thanked dcvmn members for their contribution to national immunization programs. paho is committed to continue collaboration with dcvmn members and welcome innovative suggestions, to ensure that the americas have sustainable access to life-saving vaccines. i. danel, deputy director from paho, outlined achievements of public health goals in the americas, including extension of the reach of national immunization programs, new vaccine introductions, strengthening of regulatory pathways and improving financing and forecasting mechanisms. elimination of measles from the americas is a result of countries working together successfully. this was made possible by timely supply of measles-rubella (mr) and measles-mumps-rubella (mmr) vaccines by dcvmn members. in , dcvm's vaccines represented % of the total volume of million doses procured through paho's revolving fund. paho is constantly seeking alternative sources of vaccines and adopting strategies to enable manufacturers to find new opportunities. she encouraged dcvmn members to get involved in fast-track development of vaccines, to enhance sustainability of supply and to introduce required vaccines. k. owen from bmgf reviewed the number of deaths averted through immunization by [ ] , then discussed the challenge of creating stable vaccine supply in developing countries. stable supply requires high-quality manufacturing capacity, committed financing, strong regulatory systems and predictable demand. bmgf's efforts have focused on partnering with manufacturers, stimulating procurement, and optimizing regulatory pathways in developing countries, to reduce registration time by up to %. challenges around predictability remain. a recent example of challenge is the transition in demand from opv to ipv. it is essential that dcvms have strategies to sustain their business in a competitive market. r. bright presented barda's role in tackling emerging and reemerging infectious diseases. the large number of potential eids and unpredictability of emergence requires multi-hazard strategies and investment in a broad spectrum of vaccine platforms. emerging threats require a rapid and coordinated response which integrates diagnostics for early detection, therapeutics and vaccines. collaboration between multiple groups to develop and evaluate vaccines and new supply and funding models are becoming increasingly necessary to respond efficiently. there are currently companies developing zika vaccine, including dcvmn members bharat, butantan, and sinergium. he urged dcvms who are working on zika candidate vaccines to meet and foster possible partnerships. f. kristensen provided an overview about the new coalition for epidemic preparedness innovations (cepi) [ ] . cepi aims to prioritize, stimulate, finance, coordinate and advance vaccine development against eids with epidemic potential, especially where market incentive alone will not achieve this. the ebola response showed that it is possible to advance the clinical development of vaccines in an emergency. manufacturing capability and capacity for vaccines is a bottle-neck during crises, thus the effort will focus on driving vaccine manufacturing pipelines, while also funding diagnostics and therapeutics. cepi seeks multi-year donor contributions of up to one billion dollars between and from those aligned with the strategic objectives and mission. the operating principles are: equitable access, cost coverage and shared risks and benefits. funding from cepi will be available to vaccine producers or consortia with a track record of bringing vaccine candidates to human clinical trials. the founders of cepi are the governments of india and norway, the bill and melinda gates foundation, wellcome trust and the world economic forum (wef). cepi was, in fact, launched at the wef meeting in davos, in january . j. kalil discussed emerging and re-emerging diseases in tropical regions. butantan is developing dengue and zika vaccines, currently in phase iii clinical trials, and shows results comparable to the nih vaccine [ ] . development of a zika candidate vaccine was undertaken as an urgent response to the recent outbreak in south america. the ideal situation would have been to develop a combination vaccine against both zika and dengue. butantan is willing to collaborate with other companies developing zika vaccines to test candidate vaccines in an endemic area. m. malhame reviewed the gavi - strategy, which was informed by lessons of previous periods. it includes fostering of innovative products, further strengthening of collaboration though engagement with industry, clearer inclusion criteria for vaccines and improved, consistent market analysis and forecasting. securing multiple vaccine suppliers has a positive impact on the effectiveness of immunization programs. gavi's new strategy has three priorities: . taking a long-term view of markets to help countries and manufacturers prepare for transition to self-financing. . aligning product innovation priorities across market-shaping partners to match needs. . adopting a higher tolerance of risk in markets that require it. lastly, m. malhame extended gavi's gratitude to dcvmn members contributing to the supply of vaccines to poor countries. gavi will continue to shape healthy vaccine supply globally. s. rautio noted that more than % of the total unicef procurement of $ . billion was dedicated to vaccines in . a total of . billion doses of vaccines were procured by unicef, reaching % of the world's children. % of the doses were sourced from dcvms at a total value of $ million, providing an important market opportunity for dcvms. vaccine security is the sustained, uninterrupted supply of affordable vaccines of assured quality. to achieve it, accurate forecasting, availability of funding and appro-priate contracting with a diverse supplier base is critical. therefore, continued engagement with dcvms is essential. beyond securing expanded programme on immunization (epi) vaccines, unicef is engaged in supply of pipeline vaccines, during outbreaks or health and humanitarian emergencies, and improving access to affordable new vaccines in middle-income countries (mics). unicef supports the development of healthy markets through leveraging its procurement, transparency on pricing and building procurement capacity in countries. pentavalent vaccine is an example of a market, which is now considered healthy, years after procurement of the first dose. j. fitzsimmons provided an overview of paho's technical cooperation strategies with countries in the americas, including considerations for national decision-making on introducing new vaccines in national immunization programs. paho's sustainability model for immunization consists of national vaccine forecasting, national budgetary financing, legislation and the revolving fund mechanism which together facilitate access to vaccines. after four decades of experience, this model has proven to be effective: the americas enjoy high immunization coverage for traditional and new vaccines. the success of the paho revolving fund has been enabled by the high-level commitment from member countries, its business model and the high-quality vaccines supplied by dcvms and other suppliers (fig. ). a panel discussion moderated by j. chu, from chai, focused on management decisions that can contribute to increased success and greater commercial viability of vaccines. vaccine manufacturing is a critical component of global health, but it comes with risks. one example is the decline in success rate of clinical trials for pharmaceuticals, including vaccines, over the past years [ ] . both scientific and management factors contribute to four risk categories: -large potential return/high feasibility e.g. innovative respiratory syncytial virus (rsv) and group b streptococcus (gbs) vaccines; -small potential return/ high feasibility; -large market opportunity/ low feasibility; -small market opportunity/ low feasibility (fig. ) and emphasized that a balanced portfolio is critical to ensure that companies remain viable. m. datla said it is increasingly important to choose the vaccine portfolio wisely. biological e, for example, takes pride in supporting epi needs of india as well as gavi countries. there are also few follow-on vaccines and limited new product introductions, so many companies end up with similar pipeline. both under-capacity and over capacity of supply influence sustainability for customers and manufacturers respectively. pentavalent vaccine is a prime example of over-capacity, as the demand is now divided among a larger group of manufacturers. reduction in market shares will challenge manufactures' sustainability as their business models depend on reasonable market shares. mics have unique characteristics, such as size of birth cohort, ability to procure, local registration requirements and reluctance to join pooled procurement. however, they are seeking vaccines at gavi prices posing another challenge to manufacturers' sustainability. high-income countries have different barriers to entry, requiring heavy investments, due to the need for more clinical trials. access to funding for vaccine development is also getting increasingly difficult as it takes about years for a vaccine to reach the market, and most investors prefer faster and higher returns. j. khalil noted that partnerships help to reduce the financial risks. butantan has a history of forging successful partnerships with multinational companies with the primary objective of domestic supply. access to hpv vaccines was only possible after local government enabled a technology transfer agreement. butantan now prioritizes innovative products and products for valuable markets. if manufacturing capacity exceeds demand, lower pricing would be possible. m. malhame explained how gavi assists suppliers to understand the competitive and commercial risks involved by publishing strategic demand forecasts and roadmaps, and analyzing how countries and donors select products. companies also need to develop their own strategy. gavi also analyzes the cost of administration and calculates the weighted average prices of vaccines. there is a need to expand the conversation from price per dose to cost per child immunized. s. rautio commented that unicef's focus is on security of supply. unicef hosts a conference for vaccine manufacturers annually at which market forecasts are provided. unicef works closely with ministries of health in various countries to estimate demand. no supplier, however, should rely only on unicef; similarly, no market should be reliant only on one supplier. j. fitzsimmons commented that another risk for manufacturers is the different product requirements in different markets, such gavi and paho markets. networking opportunities, such as this meeting, offer a forum to discuss these issues and develop a more common target product profile relevant across public-health markets. g. widmyer acknowledged that bmgf is in a unique position to shape global health. while financing agencies seek the lowest pricing, suppliers need support for vaccine development. bmgf has a diverse portfolio, funding projects on various diseases. agendas may overlap, as is the case where investment in a manufacturer may result in excess capacity available for gavi markets, in addition to the domestic market. companies must focus on financial viability. the foundation is keen to engage with suppliers seeking investment who can show a portfolio that reflects a strategic and balanced vision. trust and transparency in interactions are critical. j.l. di fabio opened the session on regulatory convergence efforts to facilitate registration and improve access to health products. p. aprea, director of biologicals evaluation and control at the national administration of medicines, food and technologies, argentina (anmat), remarked that convergence is currently the biggest challenge for regulatory authorities. they require confidence, clarity and competitiveness, and must apply regulatory d. decina presented the draft who good regulatory practices (grp) guidelines [ ] for nras. the guideline is intended to assist regulators and regulated organizations, irrespective of resource levels. it provides a framework for improving practices and principles on which regulatory systems can be established. key concepts are impact analysis and stakeholder consultation. the nine major principles: legality, impartiality, consistency, proportionality, flexibility, effectiveness, efficiency, clarity and transparency are important for sharing information. she presented the types of international regulatory cooperation that help to avoid redundancy in regulatory work, for example, reliance or mutual recognition. a. porrás shared the regional approach to regulatory harmonization used by the pan american drug regulatory harmonization (pandrh) network. the technical cooperation approach for regulatory system strengthening (rss) in the americas encompasses three work streams: . facilitating development of contextspecific national regulatory systems; . promoting regulatory convergence and harmonization; . supporting efficient use of resources by leveraging the work of others. the program relies on peer-assessment of nras using standardized indicators of regulatory capacity, and the adoption of institutional development plans based on identified gaps. participation implies commitment to undergo assessment. assessment results are shared with participating nras, and aggregated data is published online on the regional platform on access and innovation for health technologies (prais [ ] ). she concluded that this effort represents a new trend towards convergence in the regulatory systems in the americas. n. dellepiane discussed how manufacturers can contribute to dialogue on global regulatory convergence. she reviewed existing approaches to regulatory harmonization, including reliance within regulators' networks, provision of mutual support, and alignment of requirements, procedures and standards among regulatory agencies. continued reliance and mutual recognition of nras through networking initiatives, improving technical and scientific expertise through joint review activities and partnerships between nras remain important. alignment of nras, by agreeing and following a common list of essential documents to fulfil countryspecific requirements for registration files may also foster regulatory convergence. she encouraged dcvmn members to collect data related to various registration approaches and contribute to the dialogue between regulatory groups and industry. j. iyer presented the view from access to medicine foundation on the role of manufacturers in promoting access. their goal is to gather insights into how pharmaceutical companies improve access to medicines, by ranking corporations based on criteria such as research and development capacity, affordability, manufacturing and supply. an access to vaccines index [ ] has been developed to help understand how companies make vaccines more accessible in low and middle-income countries. vaccine manufacturers can assess, monitor and track progress based on the analysis provided by the index and benchmark their international business strategy. d. atherly presented a study by path's center for vaccine innovation and access on the dynamics of vaccine uptake in developing-country markets. the project goal is to accelerate the development and introduction of products with high health impact. path collaborated with dcvmn member chengdu institute of biological products (cdibp) on development and introduction of japanese encephalitis (je) vaccine. this collaboration helped to obtain who prequalification, the first whoprequalified vaccine from china. from to , production capacity increased by %. approximately million children outside of china will be vaccinated by cdibp's je vaccine. it is envisioned that by , eight additional countries will introduce je vaccine as part of the national immunization program. y. lim from chai discussed the risks and potential of future vaccine markets. she advised companies to understand the market and existing competition, then compare and select candidate products based on potential risks and returns. the resultant optimized and balanced product portfolio should be responsive to changes in vaccine market dynamics. j. fournier-caruana gave an update on the who global action plan for containment of polioviruses (gapiii, [ ] ) and the containment certification scheme (ccs) endorsed by the strategic advisory group of experts (sage) in and october [ ] , respectively. eradication of poliovirus is a global endeavour and can only be achieved by engaging all stakeholders. the globallysynchronized switch from trivalent (type , , ) opv to bivalent opv (type , ) in april [ ] started the poliovirus type- containment period to eliminate vaccine-derived type poliovirus (vdpv ) infection. however, there have been delays: poliovirus production facilities, research facilities and repositories should have demonstrated implementation of gapiii, and national authorities for containment (nacs) should have certified containment. this has not been realized in many countries due to lack of national regulatory frameworks to implement containment. who and other stakeholders are supporting national containment certification efforts. unicef's role in the endgame, outlined by a. ottosen, includes securing the supply of polio vaccines as well as synchronized withdrawal of topv and roll out of bopv. from december to march , million doses of opv (topv and bopv) were delivered in a timely manner for planned activities. towards topv cessation, however, perceptions of risk increased among both suppliers and programme managers. an industry survey emphasized the necessity for involving suppliers from early stages of decision-making for final opv cessation to allow for integration into their strategies and ensure sufficient supply. the recent polio outbreak in nigeria required million vaccine doses. dcvms provided a large quantity through flexibility, stockpiles and response capacity. due to a shortage of ipv, unicef is not currently able to meet the who recommendation of at least one ipv dose in all countries. therefore, in october , sage recommended a fractional-dose ipv (fipv: one-fifth of the normal ipv intramuscular dose delivered intradermally) [ ] , and catch-up on ipv vaccination when it becomes available. currently countries do not have access to ipv [ ] . s. boyle from bmgf elaborated on ipv use and challenges, and the rationale behind the switch from topv to bopv. since , no indigenous transmission of type- wild poliovirus has been detected, while the type- component in the topv makes up more than % of the vaccine-derived polioviruses, and causes % of vaccine-associated paralytic polio cases [ ] . further, it also interferes with immune responses to type and polioviruses. in response to the shortage of ipv, who has recommended two doses of fipv delivered intradermally at and weeks, along with routine bopv as well as campaign immunization. ipv supply constraints are expected to remain an issue until . bmgf priorities include reliable supply of bopv, introducing and maintaining ipv in routine immunization, and assessing the potential of new mucosal-immunity enhancing adjuvants and more genetically stable opvs. b. giersing presented the role of who's initiative for vaccine research (ivr) in accelerating vaccine development and access to vaccines in lmic. challenges around new vaccine development include investments being driven by high-income countries, lack of robust regulatory pathways and differences in health priorities among stakeholders. since , pathogens, including rsv, were evaluated by ivr. ivr also evaluates new delivery strategies. an example is the microarray patch (map, [ ] ) for administration of mr vaccines. the map requires minimal training, is light-weight and thermo-stable, yet provides the same efficiency as traditional vaccine. ivr wishes to provide neutral evidence on the value of new technologies to overcome challenges in vaccination. d. zehrung reviewed new vaccine delivery technologies, including maps for ipv or mr, blow-fill-seal tubes for rotavirus, integrated reconstitution packaging and intradermal delivery technology. new delivery technologies are likely to reduce commodity and system costs, resulting in greater access and health impact worldwide. m. zaffran reported on the current situation of the global polio endgame strategy. in , the lowest number of cases of polio was reported [ ] , with cases in pakistan and afghanistan. four new cases of wild-type in nigeria and vdpv events caused concern. restricted access to conflict zones contributed to lack of surveillance and access to vaccines. in april , who announced the switch from topv to bopv, removing type- opv. by the end of august , countries had introduced ipv [ ] . priorities for the next six months are to interrupt transmission in the three endemic countries, maintain active surveillance in security-and ipv-deprived countries, and containment to maintain a polio-free status. the th dcvmn annual general meeting provided an occasion for professionals in the vaccine industry and global health organizations to discuss challenges, innovations, risks and gain deeper understanding of regulatory practices and convergence opportunities. the zika outbreak and polio endgame are reminders of the importance of vaccines, but also the importance of collaboration, collective effort and global partnerships. the dcvmn aims to foster networking opportunities to support members in shaping global health. ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations. media centre of world health organization outbreak news. regional office for africa of world health organization clinical evaluation strategies for a live attenuated tetravalent dengue vaccine la región de las américas es declarada libre de sarampión. paho/who regional office for the americas the vaccine alliance. results & evidence new vaccine coalition aims to ward off epidemics the productivity crisis in pharmaceutical r&d good regulatory practices: guidelines for national regulatory authorities for medical products. who working document qas/ . regional platform on access and innovation for health technologies. prais. paho/who regional office for the americas human anthelminthic vaccines: rationale and challenges access to vaccines index who global action plan to minimize poliovirus facility-associated risk after type-specific eradication of wild polioviruses and sequential cessation of oral polio vaccine use. gap iii. who/polio/ . . global polio eradication initiative polio today. global polio eradication initiative replacing trivalent opv with bivalent opv. vaccines and diseases. immunization, vaccines and biologicals -conclusions and recommendations inactivated polio vaccine: supply update. unicef supply division. unicef preparing for the withdrawal of all oral polio vaccines (opvs): replacing trivalent opv with bivalent opv. frequently asked questions opportunities and challenges in delivering influenza vaccine by microneedle patch cessation of use of trivalent oral polio vaccine and introduction of inactivated poliovirus vaccine worldwide we are grateful to all speakers and moderators whose contribution made the agenda invaluable. we thank corporate partners for supporting dcvmn with unrestricted educational grants: merck (milliporesigma), temptime corporation, ge healthcare, bioengineering, gea, bosch, ompi (stevanato group), applikon biotechnology, alfa wasserman, munters. we are indebted to the experts who graciously served as rapporteurs of specific sessions: d. kristensen, s. sobti, and j. hendriks. we thank s. villasenor for administrative assistance and m. dennehy for editorial support. this conference was partly supported by a grant from the bill & melinda gates foundation, grant no. opp . the authors are employees of the respective indicated organizations, and have no conflict of interest to declare. dcvmn international did not provide any financial or travel support to speakers or moderators to participate at this meeting. n. dellepiane is a consultant, partly with dcvmn. key: cord- -ouykx g authors: puig-barberà, j.; díez-domingo, j.; arnedo-pena, a.; ruiz-garcía, m.; pérez-vilar, s.; micó-esparza, j.l.; belenguer-varea, a.; carratalá-munuera, c.; gil-guillén, v.; schwarz-chavarri, h. title: effectiveness of the – seasonal influenza vaccine in preventing confirmed influenza hospitalizations in adults: a case–case comparison, case-control study date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ouykx g introduction: we estimated influenza vaccine effectiveness (ive) to prevent laboratory-confirmed influenza-related hospitalizations in patients years old or older during the – influenza season. methods: we conducted a prospective case-control study in five hospitals, in valencia, spain. study subjects were consecutive emergency hospitalizations for predefined conditions associated with an influenza-like illness episode < days before admission. patients were considered immunized if vaccinated ≥ days before influenza-like illness onset. cases were those with a real time reverse transcriptase polymerase chain reaction (rt-pcr) positive for influenza and controls were rt-pcr positive for other respiratory viruses. adjusted ive was estimated as × ( − adjusted odds ratio). to account for indication bias we computed adjusted ive for respiratory syncytial virus related hospitalizations. results: of eligible hospitalized patients, ( %) were influenza positive and considered cases, and ( %) were positive for other respiratory viruses and considered controls. adjusted ive was % ( % confidence interval, – %). by subgroup, adjusted ive was % ( – %) for those with high-risk conditions, % ( – %) for those ≥ years of age, and, % ( – %) for those ≥ years of age with high-risk conditions. no influenza vaccine effect was observed against respiratory syncytial virus related hospitalization. conclusion: influenza vaccination was associated with a significant reduction on the risk of confirmed influenza hospitalization, irrespective of age and high-risk conditions. yearly seasonal influenza epidemics are associated with excess morbidity and mortality [ ] . vaccination against influenza is considered the most effective strategy for preventing influenza [ ] . as a consequence of antigenic drift, influenza vaccines are to be a produced every year [ ] . despite this achievement, vaccine effectiveness varies from season to season and can be very low one in four influenza seasons [ ] . this is due to the unpredictable antigenic distance between vaccine's and the circulating strains [ ] . as a consequence, evidence on influenza vaccine effectiveness has been difficult to obtain and is disputed [ , [ ] [ ] [ ] . the reappraisal of the evidence on influenza vaccine effectiveness is possible by the availability of reverse transcriptase polymerase chain reaction (rt-pcr) to diagnose influenza infection [ ] . rt-pcr has allowed the development of the test-negative approach for measuring influenza vaccine effectiveness. in test-negative case-control studies, cases are rt-pcr positive for influenza, and controls those negative for influenza. this approach has been advocated for its practicability, comparability between cases and controls, and the use of laboratory confirmed outcomes [ , ] . various authors have used the test-negative casecontrol study [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . under conditions of concurrent circulation an appropriate test-negative control group are patients testing positive for other respiratory viruses, ensuring similarity on quality of sample collection and specificity of outcomes [ , , ] . using a prospective case-case comparison approach, we have estimated seasonal influenza vaccine effectiveness (ive) to prevent laboratory confirmed influenza-related hospitalizations in adults. during the - influenza season, we performed a prospective case-control study in five hospitals in valencia, spain. the five hospitals provided care to , inhabitants years of age or older. the influenza season was defined by the weeks with positive specimens for influenza on enrolled patients. it began on december (week ) and ended on march (week ) . patients with confirmed influenza by a rt-pcr test were considered cases and patients with an rt-pcr confirmed infection for other respiratory viruses were considered controls. influenza vaccines were offered free of charge to health district inhabitants older than months of age with high-risk conditions and to years old or older. three vaccine formulations were used. subunit trivalent non-adjuvanted vaccine (influvac ® , abbot-solvay, illinois, usa; batch numbers v , v , v ) offered to subjects less that years of age, virosomal trivalent subunit vaccine (inflexal ® -v, crucell, leiden, the netherlands; batch numbers , , ) offered to subjects years old or older, and an mf tm -adjuvanted trivalent subunit vaccine (chiromas ® , novartis vaccines and diagnostics, massachusetts, usa; batch numbers , , , ) offered by licensure requirements to those years old or older. subunit trivalent non-adjuvanted was offered in the five health districts included in the study; virosomal vaccine was used in two, and the mf tm -adjuvanted vaccine was used in three. the strains included in the influenza vaccine for the - season were a/california/ / (h n )-like, a/perth/ / (h n )like, and b/brisbane/ / -like [ ] . we established an active surveillance system. full-time field researchers identified, monday to saturday, patients who were hospitalized, coming from the emergency department, in the previous - h. patients whose indications for admission were any of a predefined set of conditions, described as possibly associated with a recent influenza infection [ ] , were invited to participate. patients were excluded if institutionalized, not permanent residents, with reported egg allergy, had been hospitalized in the previous days, or if they had had a previous laboratory confirmed influenza infection. patients were included if they reported an ili episode, defined as at least one of these four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of these three respiratory symptoms (cough, sore throat, shortness of breath), sudden onset was not a requisite for inclusion [ ] , less than days preceding their arrival at the emergency department. the ethics research committee of the centro superior de investigación en salud pública (csisp) approved the study. all study subjects gave written informed consent before enrollment. a nasopharyngeal and a pharyngeal swab were obtained from each included patient. samples were introduced into vials with viral transport medium and kept at − • c until sent to the reference laboratory. four multiplex real-time rt-pcr/pcr qualitative amplifications were performed: multiplex # for influenza virus type a [ ] and influenza virus type b [ ] ); multiplex # for coronavirus, metapneumovirus, and bocavirus [ ] [ ] [ ] ; multiplex # for respiratory syncytial virus (rsv), adenovirus and parainfluenza virus [ ] ; and multiplex # for rhinovirus [ ] . negative results for viruses were only considered if human ribonucleoprotein gene amplification was positive. laboratory procedures to prevent pcr contamination were followed and a series of multiplex assays # to # negative controls without sample nucleic acid were included in all runs. information was obtained on age, sex, indications for inclusion, hospitalization date, time elapsed from symptoms onset to swabbing, presence of major underlying medical conditions, long-term treatments, contact with children, smoking habits, occupation, number of physician encounters in the last three months, number of hospitalizations in the last year, prescription of antivirals, intensive care unit admission, death in hospital and length of stay. functional status, measured by barthel index [ ] , was obtained in study subjects years old or older. social class was assigned according to occupation [ ] . influenza vaccination status was obtained by asking the patient if he or she had received the current season's influenza vaccine, on which month, and if the vaccine had been administered at least two weeks before the onset of symptoms. in addition, vaccination status was independently ascertained by a researcher blinded to patient characteristics, who consulted valencia's population-based vaccine information system. a patient was considered immunized with the - influenza vaccine if the vaccine was registered as administered or more days before the date of ili onset or if the patient recalled the month when the vaccine was administered and if it had been administered more than two weeks previous to current ili episode onset. information related to the administration of the - seasonal influenza vaccine, the a(h n ) pandemic vaccine and previous -valent polysaccharide plain pneumococcal vaccinations was obtained from the vaccine information system. ive was defined as × ( − adjusted odds ratio [or]) [ , ] . the adjusted or was obtained using a logistic regression model using stepwise background selection of the variables, with a criterion of p < . to remain in the model, starting with a fully saturated model, including being immunized with current season's vaccine, sex, age (in years of age intervals), socioeconomic class, number of high-risk conditions, obesity (imc ≥ ), smoking antecedents, number of physician encounters in the last three months, hospitalizations in the last year, pneumococcal vaccination, antiviral prescription, epidemiological week and time from symptoms onset to swabbing. we defined four groups for ive estimation: (a) all cases and controls enrolled (overall group); (b) all cases and controls with high-risk conditions regardless of age; (c) cases and controls years old or older and (d) cases and controls years old or older with high-risk conditions. to validate our estimates, we computed ive against rsv-related hospitalization following the same design and analysis strategy followed for influenza-related hospitalization, but in this instance cases were those positive for rsv and controls those positive for the other respiratory viruses, including influenza. the significance in differences in the distribution of covariates, between cases and controls, was estimated using the chi-squared, or fisher's test, for categorical variables; and the t-test, or kruskal-wallis test, for continuous variables; p < . was considered significant. all probabilities were -tailed. all analyses were performed with stata . (statacorp, college station, tx). we identified eligible patients, complied with all inclusion criteria, ( %) were positive for influenza and considered cases; ( %) were positive for other respiratory viruses and considered controls (fig. ) . swabs were performed days or less after onset of symptoms in % of study subjects. time from onset to swabbing was similar between cases and controls, median was in both instances four days, p = . . hospitalization rate associated with any of the respiratory viruses assessed was per , years old or older. by age group, hospitalization rate associated to respiratory viruses was , , , and per , - , - , - , and years old or older, respectively. influenza-related hospitalization rate was , , , and per , - , - , - and years old and older. type and number of virus identified were: h n pdm , ( %); rsv, ( %); rhinovirus, ( %); coronavirus, ( %); influenza b, ( %); parainfluenza virus, ( %); mixed infections, ( %); h n , ( %); and human metaneumovirus, ( %). including mixed infections, influenza accounted for % (n = ) of all respiratory viruses identified. influenza subtypes identified were h n pdm (n = ; %), influenza b (n = ; %), and h n (n = ; %). influenza viruses circulated concurrently with rsv, rhinovirus and coronavirus (fig. ). there were no differences regarding emergency admission diagnoses between cases and controls; with the exception of heart failure, that was % in cases compared to % in controls (p = . ) ( table ). in % of influenza patients the presenting complain was not for a respiratory condition (table ) . the duration of symptoms previous to admission was evenly distributed between cases and controls, p = . (table ). there were no differences in the percentage of sudden onset of symptoms, malaise, myalgia, headache, sore throat or shortness of breath; the only significant difference was the frequency of fever in influenza cases, p = . (table ) . when compared to controls, cases were younger, had fewer high-risk conditions, were of higher social class, more frequently smokers, and consulted their general practitioners or had been hospitalized in fewer occasions (table ). there were no differences between cases and controls aged years or more in their barthel index scores (table ) . when restricting the comparison, between cases and controls, by the presence of high-risk conditions, the differences that remained significant were age, -valent pneumococcal vaccination, and having been vaccinated with the previous or current season influenza vaccines (table ) . when restricted to those years old or older, age and influenza vaccination with the previous or current seasonal influenza vaccine remained as significant differences between cases and controls ( table ). compared to . % cases, . % controls were immunized with the influenza seasonal vaccine; p < . (table ) . controls also had more often been vaccinated with the -valent pneumococcal polysaccharide vaccine (p = . ), seasonal influenza (p < . ), and pandemic (p = . ) vaccines (table ) . when high-risk conditions or age were taken into account, no differences were observed in the percentage of cases and controls vaccinated with the pandemic vaccine, and only in those with table cases and controls a characteristics and vaccination history by group: overall, highrisk conditions, and years old or older. high-risk conditions, irrespective of age, there was a difference (p = . ) in pneumococcal vaccination ( % cases vs. % controls) ( table ) . this difference was not observed between cases and controls years old or older ( table ). according to the presence of high-risk conditions or age, the percentage of controls who had been vaccinated with the vaccine compared to cases remained significantly higher (table ) . current influenza season vaccination was highly associated with previous influenza-seasonal vaccination (p < . ) and age or older (p < . ). adjusted vaccine effectiveness to prevent confirmed influenzaassociated hospitalization was % ( %ci, - %) ( table ) . for the subgroup analysis, in those with high-risk conditions influenza vaccine effectiveness estimate was % ( %ci, - %); for those years old or older, it was % ( %ci, - %); and for those years old or older with high-risk conditions it was % ( %ci, - %) ( table ). the overall adjusted or of rsv-associated hospitalization of seasonal influenza vaccination was . ( %ci, . - . ); for those with high-risk conditions it was . ( %ci, . - . ); for those years old or older, . ( %ci, . - . ); and for those years old or older with high-risk conditions, . ( %ci, . - . ). subjects vaccinated experienced a risk of influenza-related hospitalization two times lower compared to the unvaccinated. the vaccine preventive effect was specific for influenza. three recent systematic reviews of studies reporting influenza vaccine efficacy or effectiveness [ , , ] reach the conclusion that evidence on ive to prevent influenza in older adults is scarce, elusive or non-existent. osterholm et al. [ ] looked for studies published between january and february with outcomes confirmed by rt-pcr or viral culture, as estimates based on serologic outcomes [ ] overestimate inactivated vaccine efficacy [ ] . they identified only two observational studies in older adults [ , ] . talbot et al. [ ] studied three consecutive seasons ( - ), using a test-negative case-control design, and reported a pooled adjusted ive of % for influenza-related hospitalizations; however, these estimates were only significant when pooled over three seasons. more recently, castilla et al. [ ] conclude that ive for preventing laboratory-confirmed influenzarelated hospitalization in adults years of age or older, during the - influenza season, is % to %. influenza vaccine effectiveness depends closely on the match of the vaccine strain to the circulating strain [ , ] . during the - influenza season, % of hospitalized subjects with confirmed influenza had been immunized with the seasonal vaccine. this was in clear contrast to what was observed during the - autumn pandemic wave, when, in presence of a good match between the circulating and the vaccine strain, vaccine failures were rare [ ] . the percentage of vaccine failures observed during the - influenza season can be interpreted considering that % of specimens collected in europe showed a reduced activity against the a/california/ / vaccine virus strain [ ] . we tried to minimize selection bias by an enrollment strategy based on an active surveillance system, the use of broad eligibility requirements for inclusion, completeness of inclusion, and enrolling subjects without previous knowledge of their vaccination or case-control status. we reduced classification bias by the use of two independent sources to ascertain vaccination, performing rt-pcr for influenza diagnosis, and by the case-case comparison. patients' recall is considered a valid source of influenza vaccination status [ ] , but is limited by recall bias and uncertainty on date of vaccine administration, or type of vaccine administered. record of vaccination reliably indicates immunization, but absence of record is not informative. we estimate electronic vaccine information system sensitivity as %, and specificity as %, during the - autumn pandemic wave [ ] . with the data collected in the present study, and using a capture recapture method [ ], completeness of ascertainment was % for electronic vaccine information system, % for patient recall, and % for both sources. we aimed to reduce classification bias considering a study subject as vaccinated or non-vaccinated adding the information provided by both sources. rt-pcr is the preferred diagnostic test for influenza [ ] ; but, case status misclassification may contribute to underestimation of ive because of false-negative rt-pcr results [ , , ] . to maximize rt-pcr sensitivity, we included patients with onset of symptoms seven or less days before hospitalization. pcr positivity is, with a similar swabbing strategy, % and %, at and days after symptoms onset [ ] . a non-differential misclassification of true positives as negatives cannot be ruled out and underestimation of vaccine effectiveness is to be expected if a test-negative design is used. this was minimized by case-case comparison [ , ] . although nasopharyngeal aspirate is considered the best specimen for detection of influenza viruses [ ] , we opted for pharyngeal (throat) and nasopharyngeal swabbing to reduce patients discomfort and performance easiness. in children nasal swabs are comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses, except rsv [ ] . in adults, swabbing has been used to study respiratory virus disease [ , ] , and ive [ , , , , ] . we obtained a yield of positives similar to other studies on hospitalized adults [ , ] , and the timing of the epidemic wave and types and subtypes we identified were consistent with those reported by spain's surveillance system [ ] . swabbing is a reliable and convenient alternative to obtain specimens for rt-pcr testing [ ] , and accounting for days elapsed from symptoms onset to swabbing, should limit the effect of misclassification of true positives as negatives [ , ] . the case-case analysis approach design assures comparability of controls to cases [ , ] . in a case-case comparison approach, cases and controls should mainly differ in the exposure (and its correlates) associated to the outcome of interest [ ] . all this is even more plausible if influenza and other respiratory viruses cocirculate concurrently (fig. ) . . . impact of age as a confounder and age-related protection due to previous exposure age effect was taken into account by adjustment, and by performing an analysis restricted to those years old or older. vaccine effectiveness could be explained in the elderly by acquired protection due to distant exposure to similar h n strains. we consider this pre-existing protection bias in our results as debatable. first, we observed the third h n pdm wave, this repeated circulation levels exposure to h n pdm over the age range [ ] . second, age-specific h n pdm influenza-related hospitalization rates were in our population two to seven times higher in the years or more age group. third, seroepidemiology studies [ ] [ ] [ ] have described the persistence of protective antibody titers against h n pmd only in a small fraction of subjects years old or older [ ] [ ] [ ] . fourth, when t cell epitopes are compared between h n pdm and seasonal h n , % and % for cd + and cd +, respectively, are conserved [ ] , hence a less dependent on age protection for severe episodes should be expected in those years old or older [ ] . fifth, vaccine effectiveness did not differ when age was considered. the main weakness of our study was the number of influenzarelated hospitalization. although we were able to assess adjusted ive on large groups, this was done with broad confidence intervals, and we did not attain a sufficient number of cases to provide robust ive estimates by virus strain or vaccine type. we report ive estimates with a low probability of bias and the current vaccines provided a significant health benefit. any single ive study results are difficult to generalize. variability of the factors involved, such as circulating strains, vaccine types and composition, match between vaccine's and circulating strains, population characteristics, and outcomes measured are limitations to generalizability. future studies should be planned, after taking into 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vaccine effectiveness in europe (i-move) multicentre case-control study vigilancia de la gripe en españa, temporada - . (desde la semana / hasta la semana / ) incidence of pandemic influenza a h n infection in england: a cross-sectional serological study serologic survey of pandemic (h n ) virus, guangxi province high frequency of cross-reacting antibodies against pandemic influenza a (h n ) virus among the elderly in finland preexisting immunity against swine-origin h n influenza viruses in the general human population the authors thank the staff of the hospital la plana, in vila-real; arnau de vilanova, in valencia; la ribera, in alzira; san juan, in alicante; and hospital de elda, in elda. as well, we thank all the study participants and their families.we wish to express our recognition to prof juan garcía-de-lomas for his support and contribution on the laboratory methods section. we also acknowledge the dedication and commitment of researchers in the field begoña escribano-lópez, verónica alcarria-garcía, ester huet-trujillo, Ángela lópez-doménech, montserrat cano-armenteros m and consuelo calvo-mas.funding: the study was funded in part by contract code grt between sanofi-pasteur and centro superior de investigación en salud pública (csisp). sanofi-pasteur did not participate in the design or conduct of the study, collection, management, analysis, or interpretation of the data, writing the report, and the decision to submit the report for publication. key: cord- -wfaqim d authors: modjarrad, kayvon title: mers-cov vaccine candidates in development: the current landscape date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: wfaqim d middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than people, resulting in more than deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of mers-cov have created an urgent need for effective public health countermeasures, paramount of which is an effective means of prevention through a vaccine or antibody prophylaxis. despite the relatively few number of cases to-date, research and development of mers-cov vaccine candidates is advancing quickly. this review surveys the landscape of these efforts across multiple groups in academia, government and industry. middle east respiratory syndrome (mers-cov) was first isolated in september from a patient in saudi arabia who presented two months earlier with severe acute respiratory infection and acute renal failure [ ] . retrospective testing of samples in jordan identified earlier cases from a nosocomial outbreak in april [ ] . although the majority of mers-cov cases (∼ %) have occurred in saudi arabia, other countries have confirmed imported or autochthonously transmitted cases ( fig. ) [ , ] . the most recent and largest outbreak outside of saudi arabia occurred in south korea in may [ ] , raising concern for an eruption of regional outbreaks or accelerated global spread, similar to the phylogenetically related severe acute respiratory syndrome coronavirus (sars-cov) that killed nearly a thousand people a decade earlier [ ] . although the definitive host for mers-cov has not yet been established, closely related coronaviruses have been isolated from bats across wide geographic areas [ ] [ ] [ ] . mounting evidence has strongly implicated dromedary camels as the intermediate animal reservoir, as serological surveys throughout the middle east and north africa have demonstrated them to have a high prevalence of mers-cov binding or neutralizing abs [ ] [ ] [ ] [ ] . additionally, outbreak investigations have suggested epidemiologic linkage between farm camels and human cases [ ] . mers-cov is a spherical, enveloped, single-stranded, positive sense rna beta-coronavirus [ , ] . its genome contains a replicase * tel.: + . locus at the end and codes for structural proteins toward the end. the most immunogenic of the viral proteins is spike (s), a trimeric, envelope-anchored, type i fusion glycoprotein that interfaces with its human host cognate receptor, dipeptidyl peptidase (dpp ), to mediate viral entry [ , ] . s comprises two subunits: s , which contains the receptor-binding domain and determines cell tropism; and s , the location of the cell fusion machinery. although dpp has a broad tissue distribution, most of the clinical manifestations of mers-cov can be attributed to its localization to the lower respiratory tract [ , ] . much like other coronaviruses, mers-cov can also cause significant dysfunction of the gastrointestinal, cardiovascular, renal, and neurologic systems. mers-cov is distinct, though, in its tendency to cause greatest harm to older individuals with concurrent comorbidities of one or more of these organ-systems [ , ] . despite past efforts to develop coronavirus countermeasures in response to the sars-cov pandemic, there are currently no prophylactic or therapeutic interventions of proven efficacy for mers-cov or any other coronavirus infection. although combination treatment with ribavirin and interferons were shown to improve clinical outcomes in mers-cov-infected non-human primates (nhps), treatment was initiated very soon after viral challenge (∼ h) and results have not been replicated in humans [ ] . in fact, no experimental interventions have demonstrated appreciable benefit in acutely ill patients in a consistent or controlled manner. rapidly scaled treatments based on naturally occurring neutralizing antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have demonstrated mortality reductions for other respiratory infections and may hold promise for mers-cov as well [ ] . their development, however, is limited by logistical challenges, local technical capacity, and donor supply. supportive management, adapted from guidelines developed for sars-cov, has thus far been the mainstay of mers-cov treatment. the global will to develop a coronavirus vaccine faded in the aftermath of sars-cov pandemic but has since gained renewed momentum in the face of the current mers-cov outbreak. previous approaches to coronavirus vaccine development were broad and included whole-inactivated and live-attenuated viruses, recombinant vectors and protein subunits, as well as dna and rna based platforms [ ] . most developers based their immunogen designs on the s surface glycoprotein, the primary target for neutralizing antibodies during any natural coronavirus infection. a number of preclinical and clinical studies showed that the sars-cov s protein subunit, and specifically the rbd at its core, could serve as a dominant target for neutralizing antibodies in mice, non-human primates, and humans [ ] . s , therefore, became the basis for a number of promising sars-cov vaccine candidates. the s protein subunit and rbd have also been the basis for several mers-cov vaccine candidates (fig. ) [ ] [ ] [ ] [ ] [ ] . resolution of rbd crystal structures alone or in complex with the dpp receptor [ , , ] have informed the design of immunogens that have been expressed either as recombinant protein fragments or conjugates to the fragment crystallizable (fc) region of human antibodies. both types of constructs, in formulation with aluminum salt or oil-in-water adjuvants, have elicited neutralizing antibodies of high potency across multiple viral strains. despite their demonstrated immunogenicity in animal models and anticipated safety in humans, rbd or s -subunit based vaccine candidates are limited in their epitope breadth. although the coronavirus genomes are not as variable as other rna viruses, the rbd is the most mutable region, containing mutation sites that define antibody escape variants [ , ] . thus, vaccine candidates that elicit a more diverse antibody repertoire as well as a robust cellular immune response may offer the advantage of broader and more durable protection. full-length s used as an immunogen could at least increase the breadth of the antibody response; however, it has been difficult to express, and may require additional work to produce a stable soluble trimer of the s ectodomain. investigators at the university of maryland, in collaboration with novavax, inc., have overcome this problem through the development of s rosettes that are stable and immunogenic in murine models [ ] . vaccines that mimic natural infection, such as live-attenuated viruses or recombinant viral vectors, may elicit even more robust immunity. live attenuated viruses have historically been among the most immunogenic platforms available, as they have the capacity to present multiple antigens across the viral life cycle in their native conformations. although a live-attenuated mers-cov has yet to be tested, one has been constructed and has the potential to be protective [ ] . however, manufacturing live-attenuated viruses requires containment in a biosafety level or facility. additionally, live-attenuated viruses carry the hazards of inadequate attenuation or reversion to wild type form and causing disseminated disease, particularly in immunocompromised hosts. given that moderately immunocompromised adults with co-morbidities such as diabetes mellitus and chronic kidney disease have suffered the most severe mers-cov disease, these individuals may comprise a target population for immunization, thus making a live-attenuated virus vaccine a less viable option. replication competent viral vectors could pose a similar threat for disseminated disease in the immunosuppressed. replication deficient vectors, however, avoid that risk while maintaining the advantages of native antigen presentation, elicitation of t cell immunity and the ability to express multiple antigens. to date, two recombinant vector platforms-modified although replication deficient vectors are relatively safe and immunogenic, their ability to deliver genetic material for expression could be impeded by pre-existing or developing immunity to the vector itself. one way to overcome this limitation is by administering different vectors in a so-called prime-boost immunization regimen. as this strategy has been effective for other pathogens, it is likely that the same success could be recapitulated for mers-cov. the use of more than one type of platform or antigen in a single vaccine also increases the likelihood of inducing a broad repertoire of antibodies with diverse mechanisms of viral neutralization. one vaccine regimen developed at the us national institutes of health is based on full-length s dna and a truncated s subunit glycoprotein and has elicited neutralizing antibodies in mice directed at both the s -within and outside the rbd-and s subunits. immunization with these constructs also protected nhps from severe lung disease after intra-tracheal challenge with mers-cov [ ] . a dnaonly vaccine, expressing multiple antigens, has also been developed by inovio pharmaceuticals and geneone life science inc. and has been advanced to a phase i first-in-human trial. each of the vaccine candidates that have been mentioned is being developed for prophylactic use. however, as the total number of cases (> ) [ , ] and reproductive rate (∼ . ) of mers-cov are both relatively low [ ] , it will be difficult to define the target populations for vaccination that would support the investment in manufacturing and advanced product development. also, with such low incidence and lack of robust animal models, it would be difficult to achieve a vaccine efficacy result that would be sufficient to support licensure. human mabs, on the other hand, could be used without as much discrimination in an outbreak setting for post-exposure prophylaxis and early treatment. the advantages of mabs over polyclonal antibodies (administered through convalescent plasma or hyperimmune globulin) are their higher potency, greater specificity, more extensive pre-licensing evaluation and consequently improved safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic level detail for the design of better immunogens. however, the timeline for mab development may be longer and potentially cost more than some vaccines. despite requirements for greater upfront investment, several groups have developed highly potent mabs that are currently being advanced through pre-clinical stages of testing (fig. ) . some have been isolated from immunized animals (mice/humanized mice/nhps) [ , ] , while others have been identified from either an antibody human phage library [ ] [ ] [ ] [ ] or memory b cells of infected and recovered human survivors [ ] . almost all of the mabs that have been reported target the spike rbd. it is likely that mabs directed at other sites on the spike glycoprotein have been recovered but are not as potent neutralizers. most of those that have been published bind to recombinant spike with picomolar affinity and neutralize mers-cov pseudovirus at a half maximal inhibitory concentration (ic ) of . mcg/l or less. additionally, some have demonstrated protective efficacy in pre-and post-exposure prophylaxis animal models [ , ] . the successes thus far in isolating potent and protective mabs may prove useful for therapeutic development where the target population is well defined. it may prove more challenging to advancing these products to licensure and fullscale production at affordable costs for the purpose of prophylaxis in as of yet undefined populations. the vaguely defined epidemiology of mers-cov has complicated the design and implementation of appropriate public health countermeasures. most transmission events have occurred either in the setting of household clusters or nosocomial outbreaks [ ] [ ] [ ] [ ] [ ] [ ] . it is also likely that the virus has been introduced multiple times into human populations from a large zoonotic reservoir, i.e. dromedary camels. given the broad distribution and ownership of camels in the arabian peninsula where most cases have occurred, a targeted vaccine campaign may prove difficult. as the outbreak in the republic of korea revealed, patients and workers in the same healthcare facility as an infected patient are at high risk for secondary acquisition. an optimal strategy may be to use vaccines in conjunction with stringent infection control practices in hospitals where mers-cov cases are being treated. the epidemiologic link of mers-cov between bats, camels, and humans presents an opportunity for a veterinary vaccine to interrupt the transmission cycle. a successful precedent for this so-called "onehealth" approach toward mitigating human disease with a veterinary vaccine exists in the example of equivac ® , a hendra virus vaccine developed solely for horses [ ] . although hendra virus is even more rare than mers-cov, it is highly fatal with no treatment other than intensive supportive management. in , a protein subunit vaccine was licensed and rolled-out in australia, where all outbreaks of the virus occurred. since that time, the incidence in horses has fallen precipitously and no human cases have been detected [ ] . a similar strategy may be applicable to mers-cov; however, a veterinary vaccination in this context would be deployed solely for the sake of protecting humans, as the virus causes only mild upper respiratory illness in camels. safety and reduction in viral shedding would have to be demonstrated in immunization, challenge and transmission studies of camel or camelid populations, one of which has shown efficacy [ ] . one of the primary challenges to developing countermeasures to mers-cov is the lack of an appropriate animal model that recapitulates the natural history of human disease. much of the difficulty originates from the absence of the virus's cognate dpp receptor. one group approached this problem by successfully transducing mice with an adenoviral vector expressing human dpp [ ] . although more relevant than a standard murine model, transient transduction of the desired protein may result in inconsistent tissue expression of adenovirus antigens. agrawal et al. made an important advance with the development of a transgenic mouse model that demonstrated productive, disseminated mers-cov infection [ ] . although rhesus macaques do not manifest full clinical disease, they develop a transient lower respiratory infection that can be quantified and evaluated by computed tomography. investigators at the nih rocky mountain laboratories (rml) and integrated research facility (irf) have also independently been developing potentially lethal marmoset models that could be used for the evaluation of vaccines, mabs and therapeutics [ ] . as mers-cov vaccines-both active and passive-are developed and tested, not only will more relevant animal models be required, but there will also be a need for a more detailed understanding of the epidemiology, immunology, and pathogenesis of the virus. in the aftermath of the west african ebola virus epidemic and in the face of the current zika virus outbreak, the global health community has coalesced around the realization that a multi-faceted plan is required to quickly and efficiently respond to global public health emergencies. the world health organization is currently developing a blueprint by which that preparation and response can follow, with mers-cov highlighted as a case study. although mers-cov still causes relatively few cases in a limited geographic distribution, its high case fatality and sudden outbreak in in korea have proven it to be a pathogen of public health concern. the concentration of the epidemic to saudi arabia also raises the specter of international spread every year during hajj, one of the largest mass gathering events in the world. ultimately, the development of a safe and effective vaccine for mers-cov may not yield its greatest benefit for the current epidemic but for the knowledge gained in creating a platform for combating coronaviruses as a 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east respiratory syndrome coronavirus infections hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections hendra virus vaccine, a one health approach to protecting horse, human, and environmental health hendra virus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection key: cord- -cvv f authors: yule, terecita d.; roth, mark b.; dreier, kimberly; johnson, anthony f.; palmer-densmore, melissa; simmons, kris; fanton, robert title: canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: cvv f abstract inflammatory changes following infection are central to the clinical manifestation of disease. however, information regarding such changes in animal disease is limited. in canine parvovirus infected puppies we measured the levels of acute phase proteins and changes in leukocyte phenotypes and cell trafficking by flow cytometry. these parameters correlated with conventional assessment of clinical disease in a vaccine efficacy study. seropositive (cpv- ) -week-old puppies given three doses of a cpv- containing vaccine developed significant antibody titers and remained healthy after experimental infection with cpv- b. unvaccinated controls developed clinical signs and shed virus. importantly, acute phase proteins became elevated, and lymphopenia, neutropenia and modulation of neutrophil-cd were detected in controls but not in vaccinates. type (cpv- ) was described as a new canine pathogen in l; however, the new strains cpv- a *' and cpv- b quickly emerged. today cpv- a is most common in europe while cpv- b represents cu % of current field isolates in the united states'. in puppies this virus replicates in mitotic cells'j.' and is transmitted by the fecal-oral route causing a fulminating enteric disease. initial infection most likely occurs in the tonsil, however, the virus rapidly spreads to other lymphoid tissues and to crypt cells in the gut'. crypt cell death and erosion of the enteric lining results in the vomiting and diarrhea characteristic of this disease. protection in very young puppies is mainly by passive transfer of maternal antibody to offspring. however, as maternal antibody declines, puppies become highly susceptible to infection. susceptibility to parvovirus infection often coincides with the time that puppies are separated from the dam, significantly increasing their risk of exposure'. clinical signs resulting from experimental challenge with the cpv- strain are typically mild", and not representative of the virulent disease observed after natural infection with the cpv- b strain that is more prevalent in the united states". lymphopenia, usually evaluated by a manual differential cell count can be highly variable, and neutropenia, commonly described in natural cases of infection , is inconsistently observed after experimental challenge'. central research diyision, pfizer inc., eastern point road, groton, ct , usa. *to whom correspondence should be addressed. (received july ; revised october ; accepted october ) vaccine volume number / of these parameters and reduction of viral shed have proven useful in assessing vaccine efficacy. our understanding and ability to evaluate clinical disease constantly changes as technologies and reagents are developed, for example, flow cytometry is being utilized more frequently in diagnostic situations because cell specific, quantitative data regarding cell populations can be obtained . when combined with an automated total white cell count this information may yield more powerful, less variable analysis of cell surface proteins and leukocyte trafficking. similarly, the acute phase response is well known as a nonspecific response to pathogenic injury . despite the nonspecific nature of this response, recent evidence has shown that the stimulus for production of acute phase proteins by the liver is a tightly regulated response to cytokines such as interleukin- (il-l) and il- which are released soon after infection m . in addition, the productlon of specific antibody reagents has fostered the development of quantitative assays increasing the accuracy and specificity of detecting acute phase reactants. thus, elevated levels of one or more of these pro-inflammatory proteins may be predictive of clinical infection and disease. elevated levels of il- , u-l acid glycoprotein (a-l ag), and serum amyloid a (saa) have been described in association with acute as well as chronic diseases"-*'. the impact of vaccination on these parameters after infectious challenge has not been examined. in this study we evaluated whether measuring levels of acute phase proteins and investigating changes in leukocyte phenotypes by flow cytometry would complement conventional clinical assessment of a vaccine efficacy study. the association of these parameters with the major clinical signs of parvovirus induced disease in vaccinated vs nonvaccinated animals is described for seropositive puppies given a cpv- vaccine followed by experimental infection with cpv- b. specific emphasis was placed on measuring cpv specific neutralizing antibodies and two acute phase reactants, a- ag and saa. changes associated with leukopenia were characterized using flow cytometric analysis. six-week-old mongrel puppies (alder ridge farms, bwef inc., lakewood, pa) with maternally derived cpv- a serum neutralizing (sn) titers ranging from ~ - were used. fifty puppies were randomized into two treatment groups (n= ) based on sex, litter, and date of birth; each group had a similar range of sn titers. puppies were vaccinated subcutaneously at , , and weeks of age with an experimental cpv- parvovirus modified-live vaccine having a titer > logs tcid,, ml-'. the parvovirus component was delivered in combination with distemper-adenovirus type -parainfluenza, killed corona virus, and leptospira canicola-icterohaemorrhagia bacterin (pfizer, inc., lincoln, ne). a placebo consisting of earles balanced salt solution (biowhittaker, walkersville, md) was given subcutaneously to the control group in order to simulate the status of nonvaccinated puppies in the field. serum samples were collected prior to the first vaccination and then at l- week intervals for antibody titers. serial twofold dilutions of heat inactivated serum samples were made in -well microtiter plates. samples were incubated with cpv- a ( - tcid,j virus for min at °c. dog kidney cells (atcc-ccl ) ( x lo per well) were added and incubated for three more days at °c. each plate contained a virus and cell control. to visualize virus infected cells, the monolayers were fixed with % acetone, then reacted with a monoclonal antibody (mab) ( :loooo) specific for the major capsid protein (vpl), followed by fluorescein conjugated anti-murine igg (kierkegaard and perry international, gaithersburg, md). wells were considered positive for virus if three or more than three cells in a focus were fluorescent. the endpoint neutralization titer was determined as the reciprocal of the final serum dilution where one or less than one of two wells had less than three positive cells. the cpv- b was obtained from the national veterinary services laboratory, ames, ia. the challenge dose for this study was determined previously and given orally and intranasally ( .* tcid,, per dose) at weeks of age. clinical scores were based on clinical attitude [normal= , anorexia= , depression= , moribund (euthanasia)= , death (between observations)= ]; vomiting (food only= , mucus and food= , mucus only= ); dehydration (slight=l, severe= ); and quality of feces (formed= , nonformed= , formed with mucus= , formed with blood= , formed with blood and mucus= , nonformed with mucus= , nonformed with blood= , nonformed with blood and mucus= ). significant clinical scores were calculated as the mean plus s.d. of day scores for all puppies. puppies were observed for days post-challenge, however, clinical signs and rectal body temperatures were scored only during the first days. moribund animals were humanely euthanized during the observation period by the attending veterinarian. fecal samples were collected daily for days postchallenge and stored at - °c. to isolate replicating virus the samples were prepared as clarified, % (w/v) fecal suspensions in tissue culture medium. duplicate, tenfold dilutions of the suspension were mixed with feline kidney cells (atcc-ccl ) ( . x lo cells per well) in -well microtiter plates and incubated for - days at °c. cell monolayers were fixed and stained with major capsid protein specific mabs as described above. virus titer was calculated using a spearman-karber equation**. log,, titers . tcid,, ml-' were below the level of sensitivity of this assay, due to fecal sample toxicity, and scored as no virus shed. peripheral blood leukocytes (pbl) in edta were collected on days - , , , , , post-challenge. each sample was divided into two aliquots; one was submitted to a commercial laboratory (roche diagnostics, burlington, nc) for total leukocyte count and cell differential analysis, and the other was processed for flow cytometry. pbl were washed free of serum with flow buffer; hank's balanced salt solution (biowhittaker) without phenol red, mg*+ or ca +, and containing . % sodium azide, and . % bsa (fraction v, sigma chemical co, st. louis, mo). pbl were resuspended to % of the original volume in flow buffer. cells were reacted with optimal dilutions of unconjugated murine mabs specific for canine leuko-cytes . . antibodies specific for the following canine leukocyte subsets were used: cd ' t-cells (lsm . and gm-l* neutrophilsl monocytes (dh b) (vmrd, pullman, wa). specific binding was detected using fluorescein (fitc) conjugated goat f(ab)', anti-murine igg and igm antibody (biosource international, camarillo, ca) previously adsorbed with canine spleen acetone powder (sigma). control wells contained unstained cells or cells reacted with the fitc antibody alone. erythrocytes were removed with facs@ lysing solution (becton-dickinson, mountain view, ca) and cells were fixed using % p-formaldehyde in flow buffer and stored at °c. data was acquired on ungated cells using a facstar plus (becton-dickinson flow cytometer and analyzed using the pc lysys @j (becton-dickinson) software. logical gates were drawn around lymphocyte, monocyte, and neutrophil populations based on forward and side light scatter profiles. then using side scatter vs fluorescence intensity, markers for fluoresence intensity were set based on the negative control. samples were discounted when erythrocyte lysis was incomplete and significant contamination of the lymphocyte gates prevented accurate analysis. absolute lymphocyte counts from flow cytometrically analyzed leukocytes were calculated using the total leukocyte count obtained commercially multiplied by the percentages derived from flow cytometric analysis [pan-t cells or (cd + plus cd + t cells), plus b cells]. these percentages were compared to the cell counts obtained by the manual differential obtained commercially. a commercially available canine specific radial immunodiffusion kit (developmental technologies, frederick, md) was used according to the manufacturer's specifications. control standards or test serum/ plasma samples ( ~ ) were pipetted into precut wells in a gel impregnated with canine a- ag antibody. after h at room temperature in a humid chamber, the area within the precipitin rings was measured using a bioimage@ gel scanner (millipore corporation, bedford, ma). the quantity of a- ag in test samples was extrapolated from control standards included on each plate. the level of saa was quantitated by a sandwich elisa using two mabs with specificity to different epitopes on canine saa. saa was captured onto wells of an elisa plate with one mab and detected with a biotinylated second mab; amplification was strepavidinhorseradish peroxidase. the antibodies and assay were developed in the laboratory of dr thomas l. of saa in the standard was determined by sequence analysis and amino acid composition because several proteins copurify with canine saa, rendering standard protein determinations inaccurate. sensitivity of the assay ranged from - pg ml-'. inter and intra experimental variation was < %. peripheral blood lymphocytes drawn pre-challenge (days - , ) from all puppies were used to establish normal values for all hematological measurements. upper and lower cutoff values for significance were determined as the group mean * s.d. differences between groups were evaluated using fisher's exact test (p-values for two-sided tests) or l-tests on square root transformed values (p-values were based on cochran and cox's approximation because of differences in group variances)' . statistical correlations were evaluated by the spearman rank method for nonparametric measurementsz . seroconversion after vaccination in seropositive puppies was determined as a fourfold or more increase in sn titer from the previous timepoint. prior to challenge all vaccinated puppies seroconverted, however, this occurred at different timepoints during the three dose vaccine regimen ( figure their sn titers prior to first vaccination were : and continued to drop (range % : at the time of the second vaccination) before a fourfold increase in titer was observed after the third vaccination. post-challenge, sn titers in those three puppies were among the lowest ( : - : ). all puppies in the control group challenged with cpv- b developed very severe signs consisting of vomiting, diarrhea, depression, anorexia, and dehydration in contrast to mild symptoms typically elicited by a cpv- or cpv- a experimental challenge. the normal mean clinical score on day was . f . . elevated clinical scores were observed beginning day and peaked (mean . * . ) on day post-challenge ( figure ,?a) . fourteen puppies died or were euthanized during the observation period. in contrast, occasional observations of mild gastrointestinal upsets resulted in elevated clinical scores given to some vaccinated puppies. however, these signs were not accompanied by depression, anorexia, dehydration, or virus shed. fever was not a consistent finding. elevated temperatures . "c were observed in / vaccinates and controls on a single day. only one puppy in the control group experienced three consecutive days with fever. none of the vaccinated puppies shed detectable levels of replicating virus at any stage of the day observation period ( figure zb) . in contrast, nonvaccinated controls shed virus beginning on day , with peak titers at day post-challenge; surviving puppies still shed significant levels of virus day post-challenge. there days post cpv- b challenge elevated a-l ag (two-to fivefold) and saa ( - fold) were observed in ( figure a) and ( figure b) nonvaccinated controls, respectively, beginning day and peaking at day post cpv- b challenge. normal mean plasma levels of a- ag and saa were * pg ml-' and . h . pg ml-', respectively, a- ag levels were elevated in three vaccinates on day or post-challenge. similarly, saa levels were elevated in two vaccinates prior to challenge. low but significant saa values were observed in three vaccinates on sporadic days post-challenge, but these values did not coincide with clinical signs, virus shed or hematologic changes. one puppy that died on day did not have elevated levels of either acute phase reactant. clinical scores significantly correlated (pco. ) with both a- ag (r= . ) and saa (rz . ) on day and similarly on day . using cell specific antibodies and flow cytometric analysis, a mean of % of leukocytes in peripheral days post cpv- challenge blood were positively identified as t-cells (cd + and cd@, pan-t), b-cells, monocytes, and neutrophils when compared to the absolute leukocyte count obtained conventionally. this is consistent with the fact that antibodies for natural killer cells, basophils, and eosinophils were not available commercially. lymphopenia (a % drop in initial lymphocyte count) was identified in puppies in the control group and in of the vaccinated puppies, primarily on days and post-challenge ( figure a ) by flow cytometric analysis of peripheral blood. lymphopenia was due to loss of both t cells and b cells, but was not selective for cd ' or cd ' t cells because cd :cds ratios remained constant (data not shown). by compari-son, using the manual differential cell count, / control and vaccinated puppies were lymphopenic post-challenge (not shown). neutrophilia (a % increase in prechallenge absolute neutrophil counts) was observed in a total of / nonvaccinated controls between days and , and in f vaccinates between days and post-challenge by both flow cytometric and conventional differential counts. the percent neutrophilia for controls ( ) was higher than for vaccinates ( / ; p=o.o ) on day . neutropenia was not observed in any vaccinate during the day observation period. however, in (manual differential) compared with (flow cytometry) nonvaccinated controls, neutrophilia was followed although flow cytometric differential cell counts were less variable on a day to day basis, more timepoints were obtained manually accounting for the discrepancy between the two methods. post-challenge, a novel change occurred in the pattern of cd expressed on neutrophils when compared to lymphocytes. normal cd expression was equally bright and homogeneous on neutrophils and lymphocytes as determined by the ratio of mean fluorescence intensity of neutrophils to lymphocytes (nonvaccinates=l. f . ; vaccinates= . f . ), and is illustrated in figure az . however, beginning days post-challenge, the intensity of cd expression on neutrophils gradually decreased over a - day period changing from a bright homogeneous pattern to a smear of cells ranging from dim to bright, and in some animals became completely negative. this change is illustrated in a representative histogram from a representative control puppy in figure b . the ratio of mean channel fluorescence intensity between neutrophils and lymphocytes dropped in controls ( . * . ) while in vaccinates remained constant ( . & . ). this was not a global downmodulation of all cell surface proteins on neutrophils because expression of the protein bound by gm-l antibody, present on % of neutrophils, remained constant throughout the study (figure c and d) . to illustrate the change in expression of cd on neutrophils, the percentage of neutrophils whose cd levels were below that on lymphocytes was recorded at each timepoint for every animal (figure ). significant differences in cd levels between neutrophils and lymphocytes was demonstrated for all control and one vaccinated animal beginning day . neutrophil cd expression in nonvaccinated controls is statistically different from vaccinates (p=o.oool) on days and post-challenge. the reduced level of cd on neutrophils on day negatively correlated and was statistically significant (pco. ) with clinical scores (r= - . ) and virus shed (r=- . ) on day post-challenge. these studies clearly demonstrated that information obtained by measuring nonspecific products of inflammation or leukocyte trafficking by flow cytometry can effectively complement traditional methods of vaccine efficacy assessment. it was crucial to the success of these studies to determine whether these parameters correlated with traditional signs of the clinical disease. thus, it was necessary for the cpv b challenge virus used in this experiment to elicit severe clinical signs allowing clear association of each nontraditional parameter with clinical signs and virus shed. elevated a- ag and saa levels occurred at the same time that puppies developed clinical signs and virus appeared in feces. furthermore, the quantity of a- ag or saa measured in individual samples correlated with the severity of clinical signs and virus shed. peak a- ag levels coincided with the drop in total leukocytes on day . only one puppy did not have elevated levels of either a- ag or saa post-challenge. this puppy died days post-challenge and showed other signs of infection (i.e. lymphopenia, virus shed, and a drop in neutrophil-cd expression). the other two puppies had a delayed response to virus challenge. they did not show clinical signs until days and post-challenge, and virus shed did not begin until day , at which time saa levels became elevated. thus, in these instances, elevated production of acute phase proteins closely followed the appearance of virus shed in feces and development of clinical signs. when everything is considered, these data demonstrate that after cpv- b challenge, elevated production of a- ag or saa, as well as changes in cd neutrophil expression, correlate with and provide quantifiable indicators of virus infection and clinical disease. lymphopenia and neutropenia are important hematological changes described for natural infections of canine parvovirus, and a strong correlation between disease outcome and severity of neutropenia has been observed'*. in experimental infections lymphopenia is most commonly documented while neutropenia is inconsistently demonstrated**. in both instances, laboratory assessment of these changes by manual differential count is inherently variable and subjective. this study demonstrated that by using antibodies and flow cytometric analysis to positively identify crucial cell populations, a more accurate, less variable depiction of leukocyte trafficking can be obtained. with flow cytometric analysis lymphopenia was documented for the majority of controls and none of the vaccinates. furthermore, we were able to demonstrate that neutropenia followed neutrophilia in a significant number of controls and this pattern was not observed in the vaccinated animals. thus, this methodology more accurately portrayed the clinical picture most commonly seen during natural infections. in studies of natural infection, the direct or indirect effects of cpv on neutrophils were not examined because the neutropenia was attributed to exhaustion of neutrophils as a result of secondary bacterial infec-tions . the observati ons made in the present study are significant, therefore, because they demonstrate that cpv infection produces phenotypic and trafficking changes in neutrophils before neutropenia occurs. these data suggest that impaired neutrophil function occurs as a direct or indirect effect of cpv infection. cpv may be the predisposing factor for secondary bacterial infections in puppies. the changes in appearance of cd on canine neutrophils is puzzling. cd is most commonly found on a subset of t cells and, on those cells, participates in cell signalling during antigen recognition °. canine and related carnivores are unique compared to other mammalian species because % of neutrophils as well as the t cell subset express high levels of the cd protein". there is no known function for cd on neutrophils, and the significance of a change in level of neutrophil cd expression in this disease is unknown. however, the changes in cd expression and cell trafficking of neutrophils observed after cpv challenge implicate a role for these cells in pathogenesis of cpv disease. the significance of these changes should be emphasized considering that alterations in cd expression were observed only after viral infection, and that vaccination protected from these effects. it is also noteworthy that among the parameters evaluated in this study, the loss of neutrophil-cd expression was one of the most consistent findings and occurred in all the nonvaccinated controls, and only in one vaccinate. several possibilities could account for the loss of cd and for the neutrophilia/neutropenia pattern observed in the controls. one possibility is a direct effect of cpv infection on bone marrow neutrophil precursor maturation. changes in homeostasis may have resulted in export of immature neutrophils into circulation . in support of this claim is the neutrophilia present at the time that cd -neutrophil expression was low, however, the visual cell differential analysis did not reveal an increase in immature (band) neutrophils to account for the observed neutrophilia, and neutrophilia was observed in several vaccinates in the absence of any cd modulation. thus, the changes in neutrophil-cd phenotype and trafficking may be an indirect effect of virus infection. destruction of gut crypt cells and release of lps and other bacterial products are known to induce il-l, tnf-a (tumor necrosis factor-alpha), il-& and y-interferon . . il-l is known to induce neutrophilia and in synergy with tnf will induce changes in vascular endothelium resulting in up or down regulation of cell receptors responsible for cell margination '-"'. il- is a neutrophil chemotactic factor which will activate and recruit neutrophils to sites of tissue damage"', perhaps by drawing on bone marrow reserves of mature neutrophils as has been postulated for this disease'r ' . cd -levels on mature bone marrow derived neutrophils has not been examined, but may be present in lower quantities than on circulating neutrophils, accounting for the change in cd expression we observed. regardless, the kinetics of the neutrophil-cd modulation and trafficking argue that it is the virus and not secondary infections that effect these changes. furthermore, these changes may be a clue indicating pathogenic insult to neutrophil function. vaccination with the experimental cpv- vaccine effectively stimulated a sn response in all puppies. since this response was measured against the cpv- a strain, it supports previous data indicating that cpv- contains all the neutralizing epitopes of cpv- a". . it is important to note that seroconversion occurred at different points in the three dose regimen and did not necessarily correlate with pre-existing titer. three puppies did not sero- vaccine volume number / convert until after the third vaccination and in one of these puppies, the sn titer did not increase after experimental infection with cpv- b. nonetheless, this animal remained normal, and did not exhibit any hematological changes, nor was virus shed in the feces. this observation is significant because this individual may represent a minor population of sn low responders, and suggests that local and cell mediated immunity may be sufficient to develop significant immune protection to cpv . in summary, by studying the inflammatory events resulting from parvovirus infection we have identified quantitative nontraditional parameters that will complement traditional methods of evaluating vaccine efficacy. in so doing, we successfully demonstrated that puppies vaccinated when they were cpv seropositive were protected from the inflammatory and clinical consequences of parvovirus infection after a virulent cpv- b challenge. natural history, and variation of canine mink, and feline parvoviruses the global spread and replacement of canine parvovirus strains natural variation of canine parvovirus rapid antigenic-type replacement and dna sequence evolution of canine parvovirus canine parvovirus type-a-an evolving pathogen of dogs replication of the parvovirus mvm. . dependence of virus multiplication 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enteritis : clinical, haematological and pathological features of experimental infection canine parvovirus enteritis : pathogenesis requirement for association of ~ "~ with cd in antigenspecific signal transduction in t cells monoclonal antibodies specific for canine cd and cd define functional t-lymphocyte subsets and high-density expression of cd by canine neutrophils pathogenesis of canine parvovirus- in dogs: haematology, serology and virus recovery anti-interferon gamma antibody protects mice against the generalized shwartzman reaction interferon gamma, a mediator of lethal lipopolysaccharide-induced schwartzman-like shock reactions in mice lnterleukin increases the binding of human b and t lymphocytes to endothelial cell monolayers e. ef a/. lnterleukin acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat properties of the novel proinflammatory supergene "intercrine cytokine family comparison of systemic and local immunity in dogs with canine parvovirus gastroenteritis the authors wish to thank annika weber in the laboratory of dr t.l. mcdonald for performing the assays key: cord- -gptjqti authors: ball, christopher; forrester, anne; herrmann, andreas; lemiere, stephane; ganapathy, kannan title: comparative protective immunity provided by live vaccines of newcastle disease virus or avian metapneumovirus when co-administered alongside classical and variant strains of infectious bronchitis virus in day-old broiler chicks date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gptjqti abstract this study reports on the simultaneous administration of live ndv or ampv subtype b vaccines alongside two live ibv (massachusetts-h and b-cr ) vaccines in day-old maternal-antibody positive commercial broiler chicks. in the first experiment, chicks were divided into four groups; one unvaccinated and three groups vaccinated with live ndv vg/ga-avinew, live h + cr , or vg/ga-avinew + h + cr . in the second experiment, live ampv subtype b vaccine was used in place of ndv. clinical signs were monitored daily and oropharyngeal swabs were taken at regular intervals for vaccine virus detection. blood was collected at dpv for serology. chicks from each group were challenged with virulent strains of m or qx or ampv subtype b. for ibv, after days post challenge (dpc), tracheal ciliary protection was assessed. for ampv, clinical scores were recorded up to dpc. for ndv, haemagglutination inhibition (hi) antibody titres were assayed as an indicator of protective immunity. in both experiments, ciliary protection for ibv vaccinated groups was maintained above %. the protection against virulent ampv challenge was not compromised when ampv, h and cr were co-administered. ndv hi mean titres in single and combined ndv-vaccinated groups remained above the protective titre (> log ). both experiments demonstrated that simultaneous administration of live ndv vg/ga-avinew or ampv subtype b alongside h and cr vaccines does not interfere with protection conferred against ndv, ibv or ampv. newcastle disease (nd), caused by an avian paramyxovirus serotype (now designated as orthoavulavirus ) [ ] , and infectious bronchitis (ib) caused by an avian coronavirus [ ] , are known to give rise to severe diseases in chickens, with disastrous economic losses and welfare concerns [ , ] . nd and ib have been important and common chicken diseases worldwide since and respectively. in the last three decades, publications from both our and other laboratories have highlighted the importance of ampv [ ] , which causes a drop in egg production and quality, and swollen head syndrome in chickens [ , ] . infectious bronchitis virus (ibv), avian metapneumovirus (ampv) and newcastle disease virus (ndv) are respiratory rna viruses that primarily infect the tracheal epithelium of chickens [ ] . a number of countries have introduced the use of live and inactivated ndv, ibv and ampv vaccines [ ] . simultaneous vaccination with multiple live viral vaccines is carried out in poultry for a number of reasons. these include the avoidance of inducing stress in chicks through excessive handling, and to reduce vaccination costs [ ] . traditionally, ndv and single ibv massachusetts live vaccines are given to day-old chicks at the hatchery or in the farms, mostly by spray. however, as the disease and losses caused by variant ibvs increase [ ] [ ] [ ] , growing numbers of producers have been including a b vaccine in the day-old vaccination programme. all three diseases (ibv, ampv and ndv) are now routinely controlled in the poultry industry through the use of live attenuated vaccines [ , ] , with long lasting antibody titres recorded for inactivated ndv vaccinations [ ] . reports since the s have described how ibv vaccines can impair the efficacy of ndv vaccines [ , ] , potentially leading to production losses [ ] . gelb vaccine in broiler chickens [ ] . with the introduction of ampv in s, the possibility of heterologous co-vaccination of ndv + ampv and ibv + ampv at day-old was investigated [ ] [ ] [ ] [ ] [ ] . these publications showed that simultaneous administration of ampv vaccine alongside ndv or ibv vaccine were compatible, with protection against virulent ibv, ndv or ampv remaining uncompromised. however, in these studies, only a single vaccine strain of ibv (massachusetts) was used. with the emergence of variant ibvs, inclusion of a variant ibv alongside massachusetts for dayold vaccination has become a common practice [ , , ] . recent work has shown that ibv vaccines can undergo genetic mutations following inoculation into the chicken host [ , ] . as persistent mutations can alter strain characteristics [ ] , it is preferable to monitor potential changes. furthermore, it will be beneficial to establish if simultaneous vaccine administration has any role in nucleotide mutations occurring in the ibv s gene and subsequent amino acid changes. in contrast to ibv vaccine strains, prolonged persistence or genetic mutations in ndv or ampv vaccine strains have not been reported in chickens. to date, the impact of co-administration of live ndv or ampv alongside classical and variant live ibv vaccines in ibv-ampv-ndv maternal antibody positive birds has not been investigated. with the increased use of co-vaccination strategies in commercial broiler chicks, it is important to understand how such vaccinecombinations may impact protection against ibv, ampv or ndv challenges. this study reports the results of two experiments where live ibv vaccines of massachusetts and b were coadministered alongside a live ampv subtype b or live newcastle disease vaccine in commercial broiler chicks. chicks: day-old commercial broiler chicks were obtained from a commercial hatchery, and raised in the university of liverpool isolation facilities. chicks were reared on deep litter with water and feed provided ad libitum. no in-feed or in-water antibiotics were used throughout the study. all experimental procedures were conducted according to uk legislation on the use of animals for experiments, and were granted ethical approval by the university of liverpool ethics committee. vaccine strains: commercially available live ibv mass type strain h (bioral, boehringer ingelheim, lyon, france) and ibv variant b type strain cr (gallivac Ò ib , boehringer ingelheim, lyon, france), ndv vg/ga-avinew strain (avinew Ò neo, boehringer ingelheim, lyon, france) and ampv subtype b (nemovac Ò , boehringer ingelheim, lyon, france) vaccines were administered during this study. all vaccines were prepared prior to administration using sterile distilled water, to allow for ml of vaccine per chick to be administered according to manufacturer's instructions [ , ] . challenge strains: virulent virus strains of ibv (m , . ciliostatic dose (cd) /ml; qx, . cd /ml) and ampv (subtype b; . cd /ml) were used for challenge. all challenge strains have been propagated in our laboratory and used in several previous studies [ , , ] . pcr and bacterial culturing confirmed an absence of ndv, ampv, avian influenza virus (aiv), infectious laryngotracheitis virus (iltv), infectious bursal disease virus (ibdv), fowl adenovirus (fadv) and mycoplasmas in all inocula [ ] [ ] [ ] [ ] [ ] [ ] . experiment : chicks were randomly divided into four groups ( chicks per group) and kept in separate isolation units. at day-old, each chick was inoculated via oculo ( ml) and nasal ( ml) routes with the following; vg/ga-avinew (group a), mass + b (group b), vg/ga-avinew + mass + b (group c) and sterile h o (group d). chicks were observed daily for clinical signs [ ] ; mild = coughing, head shaking and depression of short duration, severe = gasping, coughing and depression, accompanied by ruffled feathers. mortality rates and lesions following necropsy were recorded. at dpv, chicks from each group were separated into two further groups (a , a , b , b , c , c , d and d ), with chicks per sub-group. chicks from a , b , c and d were challenged with ml of virulent m , and chicks from a , b , c and d were challenged with ml of virulent qx via oculo-nasal route. chicks were observed daily for clinical signs as previously described, with mortality rates and post mortem lesions recorded. at , , , , and dpv, oropharyngeal (op) swabs were collected from five chicks in each group. at , , dpv, five trachea and kidney samples were collected from each group. at dpc, five trachea and kidney samples were collected and processed as previously described [ ] . blood was collected from ten chicks at dpv and eight chicks at dpv for the detection of ibv and ndv antibodies. experiment : day-old broiler chicks were randomly divided into four groups and kept in separate isolation units, with chicks per group. at day-old, each chick was inoculated via oculo ( ml) and nasal ( ml) routes with the following; ampv subtype b (group a), mass + b (group b), ampv subtype b + mass + b (group c) and sterile h o (group d). chicks were observed daily for clinical signs as described above. at days post vaccination (dpv), chicks from each group (a-d) were further split into twelve groups (a -a , b -b , c-c , d -d ), with five chicks per group. chicks from a , b , c and d were challenged with ml of virulent m , and chicks in the a , b , c and d groups were challenged with ml of virulent ampv b via oculo-nasal route. chicks from a , b , c and d were sham-inoculated with sterile distilled water. all birds were observed daily for clinical signs, with ibv challenge groups scored as above, and ampv challenge groups scored as follows: = no nasal exudate, = mild exudate, = unilateral excessive exudate, = bilateral excessive exudate [ ] . at , , , , and dpv, oropharyngeal (op) swabs were collected from five chicks in each group. at , , dpv five trachea and kidney samples were collected. at dpc, five trachea and kidney samples were collected and processed as previously described [ ] . blood was collected from ten chicks at dpv and eight chicks at dpv for the detection of ibv and ampv antibodies. evaluation of tracheal protection following m challenge: at days post challenge (dpc), five chicks from each group were humanely killed by wing vein injection of sodium pentobarbital. tracheas were removed from each chick and processed for ciliary and percentage protection assessment as previously described [ , ] . briefly, rings from each bird were examined, with a maximum possible ciliary score of indicating no protection (no cilia beating). the mean ciliary score for each bird was calculated and percentage protection for each group was calculated. measurement of antibody levels: commercial elisa kits [ibv (idexx, maine, united states) and ampv (biochek, er reeuwijk, netherlands)] were used according to manufacturer's instructions, with a titre result higher than (ibv) or (ampv) indicating a positive protection titre. haemagluttination inhibition (hi) was utilised to determine ibv m and ndv protection status as previously described [ ] . ibv, ndv and ampv detection by rt-pcr: swabs were pooled and dipped as a single sample per group, per day in a sterile bijou containing . ml of eagles serum-free minimum essential medium with glutamine, streptomycin [ mg/ml] and penicillin [ iu/ml], which was stored at À °c until required. rna was extracted from the swab and tissue samples using the phenol chloroform method [ ] and subjected to rt-pcr for ibv, ndv and ampv [ , , ] . positive amplicons were purified and sent for bi-directional commercial sanger sequencing (source bioscience, nottingham, uk). ibv and ampv viral load in trachea and kidney: total rna was extracted from each group's five trachea and kidney samples using the rneasy plus mini kit (qiagen, manchester, uk). quantification of viral rna was determined by quantitative rt-pcr (qrt-pcr) for both ibv and ampv [ , ] . the ibv qrt-pcr was performed using the qiagen onestep rt-pcr kit and ng of total rna per reaction. the ampv assay was performed using the qiagen quantitect xrt-pcr no rox master mix and ng of total rna per reaction. ct values were converted to log of relative equivalent units (reu) of viral rna, using previously determined standard curves for ibv and ampv [ ] . sequencing analysis: ibv partial-s gene sequences were trimmed to bp, analysed in chromaspro v . . (http://technelysium.com.au/), and blast searches were conducted to confirm the isolate identity. alignments were carried out in mega [ ] , using clustal w [ ] . single nucleotide polymorphism (snp) and insertion and deletion (indel) detection was carried out in mega , following alignment to sequenced vaccine strains. snps were characterised as non-synonymous (ns) if it led to an amino acid change, and synonymous (s) if it led to no amino acid changes. insertions and deletions (indels) were defined as the insertion or deletion of a nucleotide that altered the sequence length. the d s /d ns ratio, to identify positive or purifying selection pressure for each group was calculated per day. this was done using the nei-gojobori method [ ] where < . indicated the recovered strains to be under positive selection pressure, and > . indicated strains to be under purifying selection [ , ] . positive or purifying selection was considered significant at p < . . translated amino acid variations were also identified and variations that resulted in a change in hydrophobicity were noted according to the kyte and doolittle scale [ ] . statistical analysis: statistical analysis was conducted using one-way analysis of variance (anova), followed by turkey's test to examine the differences between pairs of means. differences were considered to be significant when p < . . analysis was carried out using spss statistics . following day-old vaccination, in both ibv-vaccinated groups, mild respiratory signs were recorded from to dpv. after m or qx challenge, groups that received no ibv vaccination had mild signs throughout the experiment. the ibv-vaccinated groups, which were m -challenged or qx-challenged showed mild signs for a total of two days, at - dpc. ciliostasis results showed that all ibv-vaccinated groups (b , b , c , c ) have high protection against both ibv challenge strains, ranging from to % (c - %; c - . %; b - %; b - . %). the ndv hi titres in groups that had not received an ndv vaccine were below log (table ) , whereas the groups that received ndv vaccine singly or in combination with ibv were . and . respectively. a significant increase (p < . ) in titre was seen for group a (ndv vaccinated) when compared to group c (combined vaccination) at dpv. when the same sera were assayed against ibv hi antibodies, ibv-vaccinated groups showed an increase in hi log titre at dpv when compared to dpv (table ) . furthermore, there was a significant increase (p < . ) in titre for both groups a and b when compared to group c for the ibv ( / ) antigen. there was a stronger response towards the / compared to the m hi antigen for all ibv vaccinated groups (group b - . and . ; group c - . and . ). the mean ibv elisa antibody titre at day-old was . (± ), indicating the presence of maternally derived antibodies (mda). by dpv, antibody levels declined to below the detectable titre ( ) in all groups (group a - . , group b - , group c - . and group d - . ). despite being below detectable levels, the two groups receiving ibv vaccines (b and c) had higher antibody titres compared to the ibvunvaccinated groups (a and d). ibv rna was detected at all sampling time points ( , , , and dpv) in groups b and c, and was absent from the ibvunvaccinated groups (a and d). the mass-type vaccine was detected up until dpv in group b and dpv in group c. after this time, only the b-type vaccine was identified in all subsequent samples in both ibv-vaccinated groups. all mass-type identified strains had partial-s nucleotide similarity to the original vaccine, ranging from . to . %, whereas the b-type had . - . % relatedness to the cr vaccine strain. ndv was not detected at any time. total snp counts remained low, with the exception of samples from groups b and c at dpv, which contained greater total snp counts (n = ) ( table ). this translated to amino acid (aa) changes in group b and in group c. all changes in blike samples were from hydrophobic to hydrophilic properties, and the majority ( %) of changes in the mass-like samples were hydrophilic to hydrophobic. the average d s /d ns ratio was . for b strains and . for mass strains, indicating that the mass genotype was significantly under purifying selection pressure (p < . ). no indels were present for any of the sequences analysed. . . . molecular detection of ibv and ndv from trachea and kidney tissues up to dpv: ibv vaccinal strains were recovered from both trachea and kidney tissue in group b at and dpv, whereas all sampling days were positive in group c (table ) . no trachea samples were ibv-positive at dpv and no mass-type strains were recovered in the kidney past dpv. we did not detect ndv in any samples. sequence similarity to the ibv vaccine strains were between and %, with a single b exception of % at dpv. the majority of strains recovered from the kidney and trachea ( . %) had a d s /d ns ratio of over . , demonstrating that the majority of partial s nucleotide variations had no effect on the translated amino acid composition. the exceptions were the two b-like strains from group c kidney samples at dpv. this group had an average of five non-synonymous changes per strain, which translated to three hydrophobicity changes within all recovered strains. ibv m challenge at dpc: ibv was detected in the trachea of group d (non-vaccinated), and in the trachea and kidney of group a (ndv vaccinated) ( table ). ibv vaccine strains were detected in groups b (mass vaccinated À % similarity) and c ( b vaccinated À % similarity). the majority of variations led to changes in the translated amino acids (n = ; average d s /d ns ratio = . ), however only one change in hydrophobic properties (hydrophilic to hydrophobic) was identified from a sample identified as highly similar to the virulent strain (group a ; trachea). ibv qx challenge at dpc: virulent qx strains were detected in the trachea and kidney of group d (non-vaccinated), with - % nucleotide similarity to the virulent strain. however, only tracheal samples were ibv-positive for group a (ndv-vaccinated) ( table ). vaccinal strains were detected in groups b ( b; - % similarity) and c ( b and mass; % similarity). the minority of nucleotide variations caused an amino acid change (n = ; average d s /d ns ratio = . ), which resulted in seven hydrophobicity changes (hydrophilic to hydrophobic = ; hydrophobic to hydrophilic = ). vaccine strains were recovered from kidney samples in groups b and c, with the majority of changes in b-like samples being non-synonymous. from a total of amino acid changes, only six caused a change in hydrophobicity. in the ibv vaccinated tracheal samples, viral load reduced from dpv to dpv (group b = . to log reu; group c = . to . log reu), however ibv presence were significantly higher (p < . ) at dpv for both groups (group b = . ; group c = . ) when compared to dpv (fig. ) . at both and dpv, there was a significantly higher tracheal viral load in the ibvvaccination group when compared to the ndv + ibv vaccine group. post challenge, high viral loads were present in the trachea (fig. for the kidney samples at and dpv, groups b (ibv vaccinated) and c (ndv + ibv vaccinated) increased from to dpv ( . to . log reu and . to . log reu respectively) (fig. ) . post challenge (fig. ) , significantly lower viral loads were present in non-ibv vaccinated groups (a - . log reu; a - . log reu; c - . log reu; d - . log reu) compared to ibv vaccinated groups (b - . log reu; b - . log reu; c - . log reu). following m challenge, viral load did not change in group b (ibv vaccinated). the virulent qx strain was detected at a greater level in the control group (group d) when compared to the m challenge control group. however, the viral load in both ndv vaccinated groups (a and a ) remained low (< . log reu) following challenge. up to dpv: all ibv vaccinated groups demonstrated mild clinical signs (prominent snicking and sneezing), starting at and dpv (group b -mass + b; group c -ampv + mass + b respectively), and continuing up to dpv. group a (ampv vaccinated) and group d (control) showed no clinical signs. no groups presented with moderate clinical signs at any point. up to dpc: following ibv m challenge, groups a (ampv vaccinated) and d (control) showed signs from dpc to and dpc respectively. mild signs in groups b and c subsided after dpc. for ampv challenge, greater clinical signs were observed in non-vaccinated birds (group d ), or birds only receiving the ibv vaccines (group b ), compared to the group receiving the combined vaccination (group c ). all signs were cleared from ampv challenge groups by dpc. groups receiving no challenge virus (a , b , c and d ) were absent of clinical signs. all ibv vaccinated groups showed high protection against the m challenge strains ( . - . % protection score). the combined groups (c and c ) demonstrated a similar level of protection percentage ( . and . respectively) when compared to the single vaccination groups (b and b , both . %). the mean serum ampv antibody titre at dpv was . (± . ), indicating the presence of maternally derived antibodies (mda) against ampv (fig. ) . by dpv, antibody levels declined to below the detectable titre for the ibv vaccinated group (group b). however, the ampv vaccinated and combined groups remained at positive titres ( and . respectively). by dpc, only groups b , b , d and d showed antibody levels below the detectable limit. following ampv challenge, groups a (ampv vaccinated) and b (ibv vaccinated) presented with higher titres ( . and . respectively) when compared with group c , which received the combined ampv + ibv vaccination ( . ). the ampv vaccinated-ampv challenged group (a ), and the combined vaccinated-ampv challenged group (c ) had significantly higher (p < . ) titres compared to the non-challenged sub groups a and c . h ( ) we were able to detect both ibv and ampv as early as dpv. however, while ibv was identified until dpv, we did not detect ampv beyond dpv in either single or combined vaccine groups. partial s sequencing of ibv positive samples demonstrated a high similarity to the original inoculum, with identities ranging from . to . % in group b to . - . % in group c. no mass-types were identified after dpv in either group. on aver- fig. . experiment : quantification of ibv viral load in the trachea and kidney at dpv following vaccination with either (group b) h + cr or (group c) vg/ga-avinew + h + cr . data is presented ± standard error margins (sem). significance testing carried out within sampling point of the same tissue type, with differences between groups (p < . ) labelled with different letters. data is presented ± standard error margins (sem). significance testing carried out between groups of the same tissue type, with differences between groups (p < . ) labelled with different letters. the d s /d ns ratio was an average of . for the mass-like strains and . for the b samples ( table ). the two b-positive samples with greater than average snp counts also showed an increase in amino acid changes, and were the only samples with hydrophobicity changes, with the majority (n = / ) being a change from hydrophilic to hydrophobic. up to dpv: ibv vaccinal strains were recovered from groups b and c at all sampling points, with the exception of the kidney at dpv (table ). all detected ibvs had a high partial-s nucleotide similarity with the initial vaccine strain (over . %), with only a single non-synonymous change at all time points for groups b and c. the tracheal sample from group c at dpv had snps, which translated into nine amino acid variations (containing a single hydrophobic to hydrophilic change). the ampv vaccine was detected up until dpv in tracheal samples, with the kidney remaining negative throughout. ibv m challenge at dpc: ibv was detected in the kidney of group b (ibv-vaccinated) and group c (ampv + ibvvaccinated), with samples having a high similarity with the vaccine strain (over %) ( table ). all samples from groups a (ampv vaccinated) and d (non-vaccinated) were positive for ibv, with % identity to the challenge strain. groups not receiving an m challenge were ibv negative and no ampv was detected in birds challenged with m . ampv subtype b challenge at dpc: no ampv was detected in group a (ampv vaccinated) or group c (ampv + ibv vaccinated). the virus was detected in the trachea of birds in group b (ibv vaccinated) and both trachea and kidneys of group d (nonvaccinated). post vaccination in the ibv vaccinated tracheal samples, viral load increased from dpv to dpv (group b = . to . log reu; group c = . to . log reu) (fig. ) . however, ibv presence was significantly lower (p < . ) at dpv for group b when compared to both and dpv. the ibv vaccination group (group b) had a significantly higher tracheal viral load when compared to the combined (group c) vaccine group at both and dpv. while both groups were negative at dpv, viral load in the kidney was significantly higher at and dpv (p < . ) in group b compared to the combined group c. post challenge with ibv m , groups vaccinated with ibv were showed to be negative by qrt-pcr, whereas group a (ampv vaccinated) and group d (non-vaccinated) showed readings of . and . log reu respectively (fig. ) . post vaccination, kidney samples were negative for ibv from all groups at dpv. however, viral load increased from to dpv in all ibv vaccinated groups (group b - . to . log reu; group c - . to . log reu). post challenge with ibv m , groups not receiving an ibv vaccine showed virus presence (group a - . log reu; group d - . log reu), with vaccinated groups having low detection levels (group b - . log reu; group c - . log reu). co-vaccination of day-old broiler chicks with ndv or ampv alongside two different live ibv (mass and b) vaccines does not impair protection conferred against either ibv or ampv challenge. in both experiments, following ibv challenge, mild respiratory signs were observed for the first two days and none thereafter, demonstrating clinical protection conferred by ibv mass h + b cr , with or without ndv or ampv. ciliostasis analysis was also used to confirm ibv protection [ ] , where a higher protection percentage suggests better protection against a virulent ibv challenge. ciliostasis results in the current study highlighted excellent ibv protection against m and qx challenges in groups receiving h + b, or the same ibv vaccines given along with ampv or ndv vaccines. previous work has shown that the combination of mass + b conferred excellent ciliary protection against asian (qx and q ) and middle east (is/ / and is/ / ) ibv strains [ , [ ] [ ] [ ] [ ] . when ibv h and cr vaccine strains are combined with the ndv or ampv vaccine, protection levels remained at or above %. this reconfirms that such combined vaccination did not compromise the protection conferred against virulent ibvs. in addition to the cilia-stopping test, detection of virus and viral load in the trachea and kidney of challenged groups was attempted to demonstrate protection against ibv m and qx. no vaccine viruses were detected in the trachea at dpc. however, both virulent challenge viruses were present, suggesting host clearance of ibv vaccines by at least dpv [ , ] . the co-administration of either ndv or ampv alongside the ibv vaccine caused a lower ibv vaccine virus load in both the trachea and kidney at dpv. however, post-challenge with virulent ibv, the combined ibv with ndv or ampv vaccination had no effect on viral load when compared with ibv vaccination alone. for both experiments, despite relatively higher tracheal viral load, a low reu value was shown in kidney tissue from non-ibv vaccinated groups following m and qx challenge. these results, along with the cilia-stopping test, demonstrated that comparable levels of protection are found when ibv h and cr vaccine strains are given either alone or alongside an ndv or ampv vaccine. control of newcastle disease is of paramount importance in endemic countries. control is normally achieved by the use of live and inactivated vaccines [ ] . haemagluttination inhibition titres were traditionally used to confirm protection against ndv challenge [ , ] , and the mean ndv hi titre in both single and combined vaccinated groups described here were > . log , which is above the protective titre against virulent ndvs [ , , , ] . current work showed that simultaneous vaccination of ndv + h + cr in commercial broiler chicks does not interfere with the induction of protective immunity against ndv. in this study, following challenge with virulent ampv, no clinical signs were found in the single (ampv) or combined (ampv + h + cr ) vaccinated groups, confirming clinical protection against the challenge virus [ , , ] . furthermore, following challenge with the virulent strain, no antigen was detected by rt-pcr from tissue samples in either of the vaccinated groups. the ampv elisa antibody titres in the triple-vaccinated group was lower than the single vaccinated group, echoing findings from previous work [ ] . however, it has been reported that levels of humoral antibodies have no association with clinical protection against ampv [ , ] . it is evident that experimental vaccination of commercial broilers (with ampv and ibv mdas) with ampv subtype b and two ibv strains (h and cr ) had no adverse effects on clinical protection conferred against a virulent challenge. in both experiments, the persistence of vaccinal strains following individual ibv, ndv, ampv and combined (h + cr + ndv; h + cr + ampv) vaccination were monitored at intervals. for ndv and ampv, classical rt-pcrs [ , ] were applied to rna extracted from oropharyngeal swabs, with no detection of the ndv vaccine at any sampling points. previous work has shown detection of ndv vaccine virus in spf chickens for up to four days post vaccination [ , ] . in this experiment, first sampling was carried out at dpv, and the failure to detect ndv may have been due to rapid clearance of the vaccine [ ] . for ampv, it was possible to detect the virus up to dpv using dry oropharyngeal swabs and until dpv from tracheal tissue. while identified the ampv vaccine at dpv in a combined ibv + ampv vaccinated group [ ] , there was no difference between single and combined vaccinated groups in the current study, suggesting that combined vaccination does not impair a host's ability to rapidly clear the ampv vaccine strain. ibv vaccinal strains have previously been shown to persist in chickens for a longer period [ , ] . to further investigate this, the growth kinetics and genetic characteristics of live ibv vaccines used in this study were cross compared with previous reports [ , ] . vaccinal strains were detected in op swabs throughout the current experiment, with the variant b strain dominating at the later ages of the birds. with both experiments, sanger sequencing showed that the genotype of strains recovered using op swabs altered from mass to b after dpv. as reported previously [ , ] , while the mass genotype made up the majority of detections during early sampling days ( - dpv), the b strain became the dominant strain by dpv. the reason for the shifting of genotypes from mass to b is not known, however, previous work has highlighted that different ibv strains can differentially induce host expression pathways, in particular tlr [ ] and ifn-b [ ] . such differences may have played a role in a host's ability to clear the mass vaccine virus at an earlier time point when compared to the b vaccine. further work is required to understand potential underlying mechanisms. at no point did the nucleotide similarity for recovered mass vaccine strains drop below % when compared to the original vaccine sequence. this suggests that under experimental conditions, the vaccine strain underwent minimal alteration following inoculation [ ] . similarly, the b vaccine strain remained high for all but one sampling point ( - % with the exception of % at dpv in experiment ). limited genetic changes were noted in the tissues samples, with - and - . snps per sequence in the mass and b vaccine strains respectively. a higher number of polymorphisms were seen in the b sequences, compared to massachusetts, indicating that the persistence of this particular genotype may contribute to greater genetic variations within local virus populations [ , , ] . however, the d s /d ns ratios suggest purifying selection for both genotypes [ ] , and therefore evolutionary constraint [ ] . previous work implied b amino acid variations do not follow a consistent pattern over time [ ] , which was also witnessed in the current study. no persistent alteration was witnessed from any tissues in any of the vaccination programs. in conclusion, this study indicates that the combined vaccination consisting of ndv (vg/ga-avinew) or ampv (subtype b) alongside two strains ibv (massachusetts -h and b -cr ) does not impair protective immunity against the globally important ibv, ndv or ampv strains that were 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infectious bronchitis vaccine viruses following single or dual in vitro infection of tracheal organ cultures a -year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china immunopathogenesis of infection in spf chicks and commercial broiler chickens of a variant infectious bronchitis virus of economic importance the use of fta Ò filter papers for diagnosis of avian influenza virus detection and differentiation of newcastle disease virus (avian paramyxovirus type ) longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using typespecific polymerase chain reactions diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus (infectious laryngotracheitis virus) evaluation of fta Ò paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus hexon based pcrs combined with restriction enzyme analysis for rapid detection and 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attenuated turkey rhinotracheitis virus vaccine. . the use of the attenuated strain as an experimental vaccine comparison of viral shedding following vaccination with inactivated and live newcastle disease vaccines formulated with wild-type and recombinant viruses a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles differential innate immune responses induced by classical and variant infectious bronchitis viruses in specific pathogen free chicks infectious bronchitis virus: dominance of arkdpi-type strains in the united states broiler industry during the last decade. revista brasileira de ciência avícola rapid selection in chickens of subpopulations within arkdpi-derived infectious bronchitis virus vaccines likelihood models for detecting positively selected amino acid sites and applications to the hiv- envelope gene genetic diversity and purifying selection in west nile virus populations are maintained during host switching variation in the spike protein of the /b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -b zlaqt authors: kim, denny; robertson, james s.; excler, jean-louis; condit, richard c.; fast, patricia e.; gurwith, marc; pavlakis, george; monath, thomas p.; smith, jonathan; wood, david; smith, emily r.; chen, robert t.; kochhar, sonali title: the brighton collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (rna and dna) vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b zlaqt nucleic acid (dna and rna) vaccines are among the most advanced vaccines for covid- under development. the brighton collaboration viral vector vaccines safety working group (v swg) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of nucleic acid vaccines. this will facilitate the assessment by key stakeholders of potential safety issues and understanding of overall benefit-risk. the structured assessment provided by the template can also help improve communication and public acceptance of licensed nucleic acid vaccines. the brighton collaboration (www.brightoncollaboration.org) was launched in to improve the science of vaccine safety [ ] . the brighton collaboration formed the viral vector vaccines safety working group (v swg) in october to improve the ability of key stakeholders to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when viral vector vaccines are licensed [ ] . one of the tools developed by the v swg is a standardized template describing the key considerations for benefitrisk assessment of viral vector vaccines, to be completed by the vaccine developers/sponsors, ideally subsequently peer reviewed by the v swg and published. the information on the template can facilitate communication of otherwise complex and highly technical data among key stakeholders (some of whom may lack subspecialized training in biotechnology) and increase the transparency, comparability, and comprehension of essential information. the template has been used for the standardized riskassessment of several new viral vector vaccines [ ] [ ] [ ] , including some targeting ebola. the who global advisory committee on vaccine safety (gacvs) endorsed the use of the template for other new candidate ebola vaccines ''as it is a structured approach to vaccine safety" [ ] . in , the development of vaccines for covid- is appropriately occurring with unprecedented speed [ ] . the pace and volume of development make a deliberate and systematic approach that is accessible and understandable to a diversity of stakeholders all the more important. several dna and rna vaccine candidates are among the most advanced covid- vaccines in development. the brighton collaboration v swg has therefore developed a specific template for nucleic acid vaccines that the coalition for epidemic preparedness innovations (cepi) and other key stakeholders will use to evaluate and communicate the benefit-risk of vaccines using these nucleic acid platforms. see supplementary material for definitions and additional guidance for completing this template. dna vaccines have been under development since the early s. they comprise a bacterial plasmid dna expressing an immunogen of interest under the control of a eukaryotic promoter. this results in the de novo synthesis of the immunogen in the vaccine recipient and the stimulation of both b-and t-cell immune responses. dna vaccination was a highly promising approach to vaccination with relatively straightforward construction of the vaccine and ease of large-scale manufacture. some are licensed for veterinary use and some have undergone clinical trials in humans, but to date none are licensed in humans. due to the very low immune response in humans with simple naked plasmid dna, research has focused on methods to enhance the response, including optimizing codon usage, optimizing the formulation for improved uptake of the dna, optimizing the route or method of administration, or the co-administration of dna encoding immune stimulatory molecules. the use of dna to prime an individual followed by a heterologous vaccination with the same antigen in an alternate format, e.g., a viral vector, is producing promising results. due to the uniqueness of dna as a vaccine and the approaches being used to improve their immunogenic effect, vaccination with dna presents a unique set of safety issues [ ] . the proposed revision of the who guidelines on dna vaccines lists the approaches being employed to enhance the immunogenicity of a dna vaccine [ ] . rna vaccines are a more novel approach. an rna vaccine is typically a messenger rna molecule that encodes the immunogen of interest; some rna vaccines employ self-amplifying rna that directs its own replication within the host cell thus expressing more of the immunogen. self-amplifying rna vaccines typically link the antigen-encoding rna to an rna replication cassette derived from an rna virus. none have been licensed for use in either humans or animals, but several have shown promise in animal models and one is currently undergoing phase i clinical trials [ ] . in contrast to a dna vaccine, an rna vaccine is translated directly within the cytoplasm of the cell without the need to be transported into the nucleus for transcription; thus there is no concern regarding insertional mutagenesis. similar to a dna vaccine though, the de novo intracellular synthesis of the immunogen of an rna vaccine stimulates both b-and t-cell responses. due to the greater lability of rna compared with dna, more care has to be given to their formulation. more data are required on rna vaccines safety profile [ , ] . rna and dna vaccines have, in theory, a distinct advantage of rapid development and deployment, especially in the context of an emerging pandemic, because the only requirement for construction of any particular vaccine is the nucleic acid sequence of the immunodominant antigen(s) of the target pathogen. the v swg intends that this template focuses on key questions related to the essential safety and benefit-risk issues relevant for the intrinsic properties of the vaccine components. we recognize that there are many other aspects of manufacturing, quality, and implementation that can play an important role in the safety of a vaccine, but we have chosen to keep some of those issues out of scope for the template in order to summarize information that is the most useful to the most stakeholders. the latest version of the template can be accessed on https:// brightoncollaboration.us/v swg/. vaccine developers are encouraged to complete the relevant templates for their vaccine candidate platform or vaccine candidate and collaborate with the v swg. the draft templates would be shared for review by the v swg and submitted for publication. similarly, updates to the templates by the vaccine developers should be submitted to the brighton collaboration website for v swg review (see table ). please read these instructions before you complete the nine sections. send questions to: brightoncollaborationv swg@ gmail.com the first section entitled ''authorship" should include your name and the latest date completing the form. if you are working with someone else to complete this form, their name should be provided as well. if you are updating the form, please provide the updated date. these co-authors will be included in the final published template in vaccine once reviewed and approved by the v swg and in subsequent wiki updates on the v swg website. sections - collect information regarding the basic vaccine information (section ), the target pathogen and population (sections ), characteristics of transgene and expression, (section ), delivery and administration (section ), toxicology and nonclinical (section ) and human efficacy and other important information (section ). depending on the vaccine, some sections may be redundant or not applicable, for example if the section is for a dna vaccine but the template is being completed for a rna vaccine. in cases of redundancies, an answer may simply refer to the answer in a previous section. answer questions by responding in the column entitled 'information.' if you have any comments or concerns regarding the question or your answer to the question, note these in the 'comments/concerns' column. finally, please provide references in the 'reference' column. more than one reference can be used per question. you can simply write the first author's last name, first name initials, and year of publication (e.g. lewis mh, ) in the ''reference" column here, but please provide the full citation for the reference at the end of the form. unpublished data are acceptable, though we do wish for you to include the source and contact information. sections and have column titles that differ from preceding sections intended to provide a summary assessment of adverse effects and toxicity of the vaccine. please summarize adverse effects and toxicities as requested and rate the risk in the following fashion: none, minimal, low, moderate, high, or unknown. if there is insufficient data for use of the platform in humans to accurately make these assessments, please state so in response to the questions. when completing information on adverse effects in section , please provide as many details as possible based on the brighton collaboration guidelines for collection, analysis and presentation of vaccine safety data in pre-and post-licensure clinical studies [ ] . if a literature search was conducted to complete any of the sections (strongly encouraged), please add the following information in the reference(s) column: ( ) time period covered (e.g., month/year to month/year); ( ) medical subject headings (mesh) terms used; ( ) the number of references found; and ( ) the actual references with relevant information used. for prior published templates, please search pubmed for ''brighton collaboration v swg". the findings, opinions, conclusions, and assertions contained in this consensus document are those of the individual members of the working group. they do not necessarily represent the official positions of any participant's organization (e.g., government, university, or corporations) and should not be construed to represent any agency determination or policy. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the brighton collaboration: addressing the need for standardized case definitions of adverse events following immunization (aefi) the brighton collaboration viral vector vaccines safety working group (v swg) live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment rvsvdg-zebov-gp (also designated v ) recombinant vesicular stomatitis virus pseudotyped with ebola zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment global advisory committee on vaccine safety the covid- vaccine development landscape a comparison of plasmid dna and mrna as vaccine technologies guidelines for assuring the quality, safety, and efficacy of dna vaccines. proposed revision of annex of who safety and immunogenicity study of -ncov vaccine (mrna- ) for prophylaxis sars cov- infection (covid- ) mrna vaccines -a new era in vaccinology mechanism of action of mrna-based vaccines guidelines for collection, analysis and presentation of vaccine safety data in pre-and post-licensure clinical studies d if so describe also consider the safety impact of multi-dose delivery methods, the use of multi dose vaccine vials, and any special considerations for disposal. * stability is considered here in the context of any relevant intrinsic characteristic of the vaccine deemed relevant for safety purposes. for example, among the risks that who, fda, and ema list for the use of dna vaccines is the hazard of integration into recipient's chromosomal dna with the resulting risk of insertional mutagenesis or spreading of antibiotics resistance genes. the probability of chromosomal integration increases if the introduced pdna has been linearized, and this is the reason that regulatory authorities require the plasmid preparation intended for vaccination or gene therapy to contain a high percentage of supercoiled material (usually > %). the percentage of supercoiled material is also used as a criterion of dna vaccine stability at different storage temperatures.d key: cord- -zs m cp authors: kwong, jeffrey c.; stukel, thérèse a.; mcgeer, allison j.; manuel, douglas g. title: appropriate measures of influenza immunization program effectiveness date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zs m cp groll and thomson's evaluation of the effectiveness of ontario's universal influenza immunization campaign used per capita cases of laboratory-confirmed influenza. we argue that these data are susceptible to various biases and should not be used as an outcome measure. laboratory data are traditionally used to identify the presence of influenza activity rather than to identify levels of influenza activity. a better measure of viral activity is the proportion of influenza tests positive; whereas the weekly proportion of tests positive was relatively consistent, a marked increase over time in the numbers of laboratory-confirmed cases paralleled an increase in the number of tests performed. regardless, for evaluating universal influenza immunization program effectiveness, other established and available measures employed in previous studies describing the epidemiology of influenza should be used instead of laboratory data. in their evaluation of ontario's universal influenza immunization campaign, groll and thomson state that there is a lack of high-quality influenza outcome data in ontario, so instead they examined the effectiveness of the program using per capita cases of laboratory-confirmed influenza [ ] . these laboratory data are traditionally used by public health agencies to identify the presence of influenza activity -based on exceeding case or proportion positive test thresholds -and to characterize circulating strains, but there are good reasons why they are not used to identify levels of influenza activity. the most important reason that per capita cases is a suboptimal outcome measure for evaluating the effect of the immunization program is that it is susceptible to ascertainment bias [ ] . heightened awareness of influenza and other respiratory infections have led to increased requests for testing. given the increased attention in recent years to both interpandemic and pandemic influenza, as well as emerging diseases such as the severe acute respiratory syndrome (sars), it is not surprising that more tests for influenza may be ordered by physicians and public health professionals in their diagnostic work-up of patients with acute respiratory illnesses and investigations of respiratory outbreaks, respectively. evidence for increased testing over time is illustrated in fig. . this figure plots weekly surveillance data (obtained from the same source as groll and thomson) for influenza a and b for ontario from to , and compares the number of tests performed for influenza (using viral culture or direct antigen detection) with the number of cases of lab-confirmed influenza. annual peaks corresponding to influenza season are apparent for both number of tests performed and number of cases. it is also fairly evident that number of tests performed for influenza have increased over time, with a sudden increase coinciding with and persisting since the sars outbreak that occurred in ontario in the spring of [ , ] . the increase in numbers of laboratory-confirmed cases seen over time parallels the increase in number of tests performed. a better measure of viral activity is the proportion of influenza tests positive (the number of cases of lab-confirmed influenza divided by the number of tests performed). this is illustrated in fig. , which compares the number of cases of lab-confirmed influenza with the proportion of tests for influenza that were positive. again, annual peaks corresponding to influenza season are evident for both number of cases and proportion of tests positive. however, the peaks for the proportion of tests positive are fairly consistent over time, varying between . and . , whereas the peaks for the number of cases increase dramatically over time, from less than to over cases per week. the relative consistency of the proportion of tests positive over time coupled with the increase in lab-confirmed cases suggests that the increase in lab-confirmed cases is attributable to more tests being performed. on an unrelated note, there was an error in fig. in groll and thomson's paper: the y-axis title should be cases per , , population and not per , population. for december , there were cases of influenza reported out of a population of , , as of july . this would be a monthly incidence of per , , cases and accurately reflects the data in the graph. that only cases of influenza per , people are identified during months of influenza activity, when the actual rates of disease approach per people, further raises concern that any laboratory-derived measures of influenza activity are vulnerable to ascertainment and sampling biases. groll and thomson did examine potential ascertainment bias but -considering the level of concern and importance to the study findings -they did not go far enough. they should have disclosed more information about laboratory testing and performed more analyses to examine bias. better still, they should have used other established and available measures employed in previous studies describing the epidemiology of influenza, such as hospitalizations, mortality, emergency department use and ambulatory physician visits. incidence of influenza in ontario following the universal influenza immunization campaign a dictionary of epidemiology identification of severe acute respiratory syndrome in canada public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto mount sinai hospital, canada douglas g. manuel institute for clinical evaluative sciences, canada * corresponding author at: institute for clinical evaluative sciences, room g key: cord- -a ia kxf authors: bellinzoni, r. c.; blackhall, j.; baro, n.; auza, n.; mattion, n.; casaro, a.; la torre, j. l.; scodeller, e. a. title: efficacy of an inactivated oil-adjuvanted rotavirus vaccine in the control of calf diarrhoea in beef herds in argentina date: - - journal: vaccine doi: . / - x( ) - sha: doc_id: cord_uid: a ia kxf abstract we have assessed the potency of an inactivated oil-adjuvanted rotavirus vaccine in beef herds in argentina. two different vaccine trials were conducted. in a small-scale experimental trial, involving pregnant cows ( vaccinated and eight unvaccinated controls), a significant increase in neutralizing antibody titres against different serotypes of bovine rotaviruses was found in both the colostrum and serum of vaccinated cows compared with that of unvaccinated controls. seven days after birth, half of the calves born to vaccinated dams or to control cows were challenged with live virulent virus whereas the other half of both groups were left in contact with the infected calves in order to mimic a natural field challenge. although no statistically significant differences in the rate of protection were observed among the different groups of animals, a larger number of vaccinated calves were protected in comparison with their controls, particularly where animals in contact with infected calves were concerned. secondly, a large-scale field trial was carried out in beef herds involving a total of vaccinated pregnant cows. in farms morbidity and mortality in calves from vaccinated cows were compared with historical data from the previous years at the same locations. in the other six herds, control groups were used to compare data of the same year: cows were vaccinated and were left as controls. taking into account the previous and current incidence of diarrhoea, morbidity and mortality were significantly reduced in of the beef herds tested. vaccine effectiveness was also evident in farms where other enteropathogens such as cryptosporidium and coronaviruses were present, together with rotavirus. diarrhoea in newborn calves is a syndrome in which several causative agents (acting separately or associated in different ways) may be involved. surveys carried out in different countries have shown that rotaviruses, coronaviruses, enterotoxigenic escherichia coli, salmonella spp. and cryptosporidium are the pathogens chiefly responsible for outbreaks of calf diarrhoea - . the control of diarrhoea caused by different pathogenic microorganisms should be possible by the development of preventive strategies through the use of appropriate vaccines , in addition to the general measures currently used, such as management, nutrition and chemotherapeutics. to be effective, these vaccines must be designed rationally and based on the impact that the different enteropathogens have in a definite geographical region; therefore single agents or appropriate combinations of them should be components of these vaccines. other relevant factors in achieving effective vaccination are the type of vaccine (live attenuated or inactivated virus) the formulation, and the route and timing of immunization(s). calves can be protected against rotavirus and enterotoxigenic e. coil by passive immunization, taking advantage of the lactogenic immunity stimulated by maternal vaccination or directly by feeding calves with hyperimmune colostrum obtained from vaccinated dams ' . in contrast, attempts to stimulate active immunity in newborn calves by oral vaccination with live attenuated virus have been unsuccessful so far ' . with regard to the formulation of the vaccine, the most relevant factor is which type of adjuvant to use. when the target for vaccination is the pregnant cow, it has been demonstrated that oil-based adjuvants are more effective than alhydrogel to enhance colostrum and milk antibody titres, at least against e. coli, rotavirus and coronavirus °, . in argentina, neonatal diarrhoea is responsible for important economic losses in beef and dairy herds; morbidity can reach - % whereas mortality ranges between % and %. rotavirus is the major agent associated with diarrhoeal problems in argentinian beef herds . for that reason, after several years of epidemiological studies, it was decided to develop and test an inactivated oil-adjuvanted vaccine with the aim of controlling diarrhoea in beef and dairy herds in argentina. we now report the results of two trials performed with the experimental vaccine. the first trial was a challenge experiment performed in confinement and involving cows; the second was an open field trial involving vaccinated cows in different beef herds. the results showed that the oil-adjuvanted rotavirus vaccine tested was effective in the control of calf neonatal diarrhoea in argentina. the following rotavirus strains were used throughout this work: strain t . this was isolated from a diarrhoeic calf in argentina ( ) and was adapted to grow in ma- cells; it was cloned four times by limited dilution after the sixth passage. according to its antigenic characteristics this strain was classified as serotype (see table ). strain t . this strain, isolated from a diarrhoeic calf in argentina (in ), was adapted to grow in ma- cells and was cloned three times. it was selected for this work because it is one of the few local strains that does not cross-react with the bovine prototype strain, uk. strain uk. this was kindly provided by dr d.r. snodgrass (moredum research institute, edinburgh, scotland, uk). it was passaged times in ma- cells, cloned three times and classified as serotype (ref. ) . strain b . this bovine strain, isolated in iowa, usa and kindly provided by dr g. woode (texas, usa), was passaged nine times in ma- cells and cloned three times. like the local strain t , this strain did not cross react in neutralization assays with the ncdv prototype strain ~ . hyperimmune antisera were prepared in seronegative guinea pigs by injecting intramuscularly #g of the corresponding purified virus emulsified with an equal volume of freund's complete adjuvant. two more injections were given subsequently with the same quantity of virus but mixed with freund's incomplete adjuvant. sera were collected one week after the last inoculation and monitored by elisa and neutralization assays. the assays were carried out as described by gerna et al. ~ briefly, pl virus, diluted with medium to give focus-forming units (f.f.u.) per well, were mixed with # twofold serial dilutions of the antisera to be tested. mixtures were incubated for min at °c and inoculated on top of confluent monolayers of ma- cells growing in -well microtitre plates. the plates were incubated for - h. after fixation with acetone, cells were stained by the ipa technique . the neutralizing titre of each serum was expressed as the reciprocal of the highest dilution that gave % reduction in the number of the stained cells compared with control wells. homologous control viruses were included in the test for each serum. the vaccine was produced with the local rotavirus strain, t , the virus was grown in monolayers of ma- cells. after complete cytopathic effect, cellular fluids containing debris and virus were clarified by centrifugation ( for rain), inactivated with . % formalin ( h at °c), and emulsified with an equal volume of oil adjuvant comprising a mixture of % marcol and % montanide (trademarks of seppic, france). the innocuity of each inactivated antigen preparation was tested by observation of any posible cytopathic effect after three serial passages in ma- cell monolayers. thirteen pregnant hereford heifers were vaccinated twice subcutaneously with ml of the oil vaccine and days before expected delivery. eight heifers remained as unvaccinated controls. calves were nursed by their mothers throughout the experiment; on the seventh day of life half of the calves of both groups were injected orally with tcidso of the t strain. the uninoculated calves were kept in close proximity to those inoculated in order to evaluate the possible occurrence of natural transmission of challenge virus, as might happen in the field ('field exposure'). calves were examined daily for days and were considered diarrhoeic if animals excreted loose or watery faeces for at least days . daily faeces samples were examined for rotavirus, coronavirus, cryptosporidium, enterotoxigenic e. coli and salmonella as described by snodgrass et al. a. cows were bled before first and second vaccination, at calving and at days after delivery. colostrum and milk samples were collected at calving and at , and days postcalving. rotavirus-neutralizing antibodies were assayed in each sample, as described previously . neutralizing antibodies against the uk and t strains (serotype ), the b strain and the t local strain, were determined in colostrum (first milking). in addition, neutralizing antibodies against the t strain were determined in the milk on various days post-calving. seventeen beef herds were selected to perform the field trials, the criterion being presentation of calf diarrhoea problems during ~> years before this study. the beef herds selected for the vaccine trial were located in the provinces of buenos aires and c rdoba, comprising a considerable area of the principal breeding region of the country. these herds had previously been part of an extensive epidemiological study (involving a total of herds) performed by our laboratory to determine the aetiology of the high incidence of neonatal diarrhoea in this region (r.c. bellinzoni et al., unpublished results) . in herds the results were compared with historical controls and in the other six herds approximately two-thirds of the cows were left as unvaccinated controls. a total of cows were vaccinated once, as described above, month before the onset of calving. in four of those herds, unvaccinated cows were separated from the vaccinated animals. in the other two herds, both groups were mixed; in this case, the animals in the unvaccinated group were selected randomly. all the cows involved in the experiment were correctly identified and each one was feeding its own calf. as shown in table , in the vaccinated group only two of seven challenged calves, and none of those kept in close contact with them, developed diarrhoea, whereas in the control group, three of four inoculated animals and two of four uninoculated animals developed diarrhoea. virus excretion did not differ in the inoculated calves from both groups (vaccinated and unvaccinated), but differences were observed in close-contact controls ( table ). all rotaviruses isolated from positive faeces showed identical genomic rna patterns to the t strain used for vaccine production and challenge (data not shown). the results shown in table suggest a higher rate of protection in vaccinated animals compared with unvaccinated controls; however, because of the small number of animals involved, no significant differences were noted on statistical analysis of the data. other difficulties with this type of experiment are, first, assessment of the correct dose of virus to be used for the challenge, and second, the furthermore, management of the herds was not disturbed by participation in the trial. the experiment was carefully controlled by trained veterinarians in close cooperation with farm personnel. during weekly visits to the herds, information about diarrhoeic animals was recorded. the trial lasted until each calf was days old. in all cases, diarrhoea was defined as loose or watery faeces, a situation that usually called for treatment of the diseased animals. in some herds, faecal samples were taken from diarrhoeic calves the year before the trial ( ) and during the present trial ( ), and were examined for rotavirus, coronavirus, cryptosporidium, salmonella and etec as described previously . inability to ensure that all the unchallenged animals kept in close proximity to the infected calves received a pathogenic dose of the virus . coronavirus, cryptosporidium or etec were not detected in any of the diarrhoeic samples analysed. aspects of the immunological status of vaccinated animals were also analysed. as shown in figure , compared with controls, vaccinated cows showed significantly higher neutralizing antibody levels against rotavirus in serum, colostrum and milk until at least days after calving. in addition, the titres of colostrum neutralizing antibodies were sufficiently high to neutralize other strains of rotavirus as well. as can be seen in table , colostrum from cows vaccinated with a strain belonging to serotype also showed neutralizing activity at similar titres against heterologous serotypes. table summarizes the results obtained from the large-scale field trial. in the case of herds a-f, morbidity and mortality data obtained from the last years were compared with data obtained during the current trial. no significant differences were detected in the incidence of diarrhoea and mortality in the contemporary control vaccine, vol. , june aln farms a-f, data relating to the incidence of diarrhoea and mortality in calves born from vaccinated cows were compared with data obtained from an unvaccinated control group as well as with data from the last years at the same farms. in two farms (e and f), controls were mixed (m) with vaccinated animals; in the other four farms (a-d), control animals were separated (s) from the vaccinated areas. in farms c_,-q =, data were compared only with the data of the last years at the same farms; bmean of the last years; centeropathogens detected the year before vaccination: r, rotavirus; co, coronavirus; cr, cryptosporidium; as, controls kept apart from vaccinated animals; m, control and vaccinated animals kept in close proximity groups ( . and . % respectively) when compared with the data of the last years ( and . % respectively) suggesting that, in general, the epidemiological situation did not change significantly in those herds. thus (apart from herd f which showed a low previous incidence of diarrhoea and mortality), vaccination of the cows led to an important reduction in the incidence of diarrhoea and mortality ( . and . %, respectively). in four herds (a-d), vaccinated and control animals were kept apart and in the other two (e, f), controls were mixed with vaccinated animals. in one case (see table , herd f) the incidence of diarrhoea and mortality in the control group was lower than that previously recorded. however, in the other herd kept under similar conditions (table , herd e) the values for the control group were similar to the mean of the last years. thus it is not possible from these results, to reach any conclusions about the effect of maintaining vaccinated animals together with, or separated from, non-vaccinated animals. in the remaining herds (table , herds g-p) the incidence of diarrhoea and mortality in calves born to vaccinated animals was compared with the data of the previous years. the results show again that in of those herds (g-o) diarrhoea and mortality in calves from vaccinated cows ( . and . % respectively) were far less than previously recorded in the same herds ( . and . %, respectively). in nine of the herds involved in this study, the presence of other enteropathogens was studied the year before vaccination. in addition to rotaviruses, coronavirus and/or cryptosporidium was detected. in three herds (table : ll, n and p) animals were sampled during the year of the trial and the same pathogens were found (data not shown). in this context, it should be emphasized that, in eight of the nine herds in which other enteropathogens as well as rotavirus were present, the vaccine was also effective in decreasing morbidity and mortality due to diarrhoea. a growing body of evidence ' indicates that in order to guarantee adequate protection of newborn calves against enteropathogens it is necessary to supply them with continuous and sutticient amounts of neutralizing antibodies against the specific agent. this can be achieved by immunization of the dams, before delivery, with inactivated oiladjuvanted vaccines that will induce the production and excretion of rotavirus antibodies in colostrum and postcolostral milk ' . in this study an oil-adjuvanted inactivated vaccine was assayed in two different experiments. the main purpose of the preliminary small-scale trial, which involved pregnant cows and was conducted with the animals confined and under careful veterinary control, was to evaluate the immune response of such cows after vaccination. the efficacy of this vaccine in protecting calves from experimental challenge was also assayed in this trial. in concordance with previous reports . , the results of this experiment show that the level of neutralizing antibodies against homologous and heterologous rotaviruses in sera and colostrum was significantly enhanced on vaccination of the pregnant cows. under the conditions of this trial the incidence of diarrhoea in calves born from vaccinated cows was lower than those born from unvaccinated controls. an interesting observation was the finding that all four uninoculated control calves were actively excreting rotavirus, indicating that the natural exposure used in this experiment was a valid method of challenge. this finding is important because the dose of challenge virus used in a direct inoculation probably would not be representative of the field conditions in which spontaneous infections are continuously occurring. in this regard, difficulties (overwhelming or inadequate doses of virus) in evaluation of the efficacy of immunization by direct inoculation of calves, have been reported , . the results of the small experimental trial showed that the experimental vaccine was able to induce, in the milk and sera of vaccinated cows, an adequate level of neutralizing antibodies against homologous and heterologous rotaviruses; in addition, the results of the challenge were encouraging. it was therefore decided to test the vaccine in a large .field trial in order to evaluate the protection afforded by the vaccine against circulating field rotavirus. in the field trial the efficacy of the vaccine was assessed by two different approaches: by comparing the incidence of diarrhoea and mortality between vaccinated and unvaccinated groups of animals and by comparison of these data with previous values of diarrhoea and mortality (mean of the last years) at the same farms. the use of previous data to compare the results of vaccination can be problematical because of the possibility that the incidence of the various enteropathogens may change from year to year; on the other hand, when data are compared with contemporary controls that are in close contact with the vaccinated group, the incidence of diarrhoea and mortality in these controls may be underestimated because of herd immunity . in order to avoid this problem, control and vaccinated groups can be separated during the trial. the experiment shown in table was designed to test the comparison methods. as judged by the results obtained, it was clear that the vaccine was effective in protecting calves born from vaccinated dams independently of the method used for comparison (historical or actual values of incidence of the disease). it is likely that the higher level of protection against rotavirus infection of calves born to vaccinated dams, compared with those born to control cows, was acquired through colostral immunity. all the herds selected for this work had experienced problems with calf diarrhoea during at least the three years before the trial. nine of the herds studied were sampled the year before the trial and, besides rotavirus, coronavirus and/or cryptosporidium were detected; in three of those herds the same enteropathogens that had been found the year before were detected again at the moment of the trial (see results) , suggesting that there were no significant changes in the epidemiological situation at the trial locations. this observation is relevant because the vaccine was equally effective in herds where rotavirus alone, or where rotavirus and the other two enteropathogens, were present. these results are in contradiction to those reported by snodgrass who found that, in two herds in which rotavirus was associated with cryptosporidium, rotavirus vaccination was effective in diminishing the excretion of rotavirus but not in controlling diarrhoea. the reason(s) for such discrepancy are at present obscure and further work with a larger number of herds and animals is necessary to elucidate this problem. however,judging by the results obtained in this work, it is tempting to speculate that under the particular conditions of cattle breeding in our country, rotaviruses could be the agents that play a major part in determining diarrhoea in newborn calves. in this regard, it is likely that biological, nutritional, environmental or management factors could influence the persistence of a given pathogen in the field, or could well influence the degree of pathogenicity of a given infectious agent or different combinations of agents . the results of this work have confirmed and extended the results of snodgrass and colleagues regarding the effectiveness of an oil-adjuvanted inactivated rotavirus vaccine in controlling (in this case) diarrhoea in beef herds in argentina. these data, obtained from vaccinated cattle in different herds, represent the largest field trial so far for this type of vaccine. pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea infectious agents associated with neonatal calf disease in south western idaho and eastern oregon aetiology of diarrhoea in young calves microbiology of calf diarrhoea in southern britain vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity evaluation of a combined rotavirus and enterotoxigenic escherichia coli vaccine in cattle passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or nonimmunized cows intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody laboratory experiments or viral vaccination of calves against rotavirus or coronavirus induced diarrhoea. zentralbl. veterinaermed passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin g antibody secretion in milk evolution des anticorps anti-rota dans le lait de vaches traitees en fin de gestation soit par le vaccine anti-rota complet, soit par i'adjuvant seul incidence of rotavirus in beef herds in argentina serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization antigenic relationships among some bovine rotaviruses: serum neutralization and cross-protection in gnotobiotic calves serotyping of cell culture adapted subgroup human rotavirus strains by neutralization bovine rotavirus serotypes and their significance for immunization bovine milk immunogtobulins for passive immunity to infantile rotavirus gastroenteritis the authors are indebted to the veterinary surgeons and farmers who participated in this trial and to andros bellinzoni and alvina lorensi for technical assistance. this work was supported by consejo nacional de investigaciones cientificas y t~cnicas (conicet), secretaria de ciencia y trcnica (secyt) and the swedish agency for research cooperation with developing countries (sarec). key: cord- -eeh a t authors: lambert, paul-henri; ambrosino, donna m.; andersen, svein r.; baric, ralph s.; black, steven b.; chen, robert t.; dekker, cornelia l.; didierlaurent, arnaud m.; graham, barney s.; martin, samantha d.; molrine, deborah c.; perlman, stanley; picard-fraser, philip a.; pollard, andrew j.; qin, chuan; subbarao, kanta; cramer, jakob p. title: consensus summary report for cepi/bc march - , meeting: assessment of risk of disease enhancement with covid- vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eeh a t a novel coronavirus (cov), severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in late in wuhan, china and has since spread as a global pandemic. safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of coronavirus disease (covid- ) disease and ease the major economic impact. there has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. however, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. a syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. animal models allowed scientists to determine the underlying mechanism for the former in the case of respiratory syncytial virus (rsv) vaccine and have been utilized to design and screen new rsv vaccine candidates. because some middle east respiratory syndrome (mers) and sars-cov- vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for sars-cov- vaccines. to address this challenge, the coalition for epidemic preparedness innovations (cepi) and the brighton collaboration (bc) safety platform for emergency vaccines (speac) convened a scientific working meeting on march and , of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. this report summarizes the evidence presented and provides considerations for safety assessment of covid- vaccine candidates in accelerated vaccine development. since the identification of a novel coronavirus, sars-cov- , as the cause of pneumonia in patients from wuhan china, a pandemic has erupted, resulting in enormous health care, social and economic disruption to our global society [ ] . as of may , there have been , , cases and , deaths worldwide [ ] . in rapid response to the pandemic, academic and industry scientists from around the world have initiated efforts to develop vaccines and therapeutics for disease prevention and patient management. the coalition for epidemic preparedness innovations (cepi), a global partnership between public, private, philanthropic, and civil organizations, is funding work to develop sars-cov- vaccines using a variety of technology platforms. several vaccine candidates are already in phase studies with others likely to enter the clinic in the next few months [ ] . one of the challenges facing rapid vaccine development for sars-cov- is the need to adequately assure the safety of these vaccines. one such safety concern is disease enhancement syndrome that occurred in the s with inactivated rsv and measles vaccines. vaccinemediated disease enhancement is characterized by a vaccine that results in increased disease severity if the subject is later infected by the natural virus. during early trials with inactivated rsv vaccine, the vaccine did not prevent infection, % of those infected required hospitalization and two children died [ ] . lung pathology in patients showed an unexpected inflammatory response with both neutrophils and eosinophils, evidence of immune complex formation and complement activation in small airways [ ] . scientists later learned that the vaccine caused a similar disease enhancement in animals characterized by immunopathology and a t helper cell type (th ) biased response and antibody responses with poor neutralizing activity [ ] [ ] [ ] . since that time, the animal models have been relied upon to predict safety for new rsv vaccines that are developed. of note, the pathogenesis of rsv disease enhancement is distinct from antibody disease enhancement (ade) which occurs for macrophage tropic viruses, demonstrated most notably for dengue in humans and the coronavirus feline infectious peritonitis virus in cats, and is directly caused by non-neutralizing or sub-neutralizing antibodies leading to more efficient viral uptake via fcγ receptor binding [ ] . since pathology consistent with the rsv vaccine enhanced disease (and perhaps ade) has been demonstrated for some sars-cov- vaccine candidates in animal models, there is also a concern that a similar syndrome could occur in humans immunized with sars-cov- candidate vaccines. therefore, cepi and the brighton collaboration safety platform for emergency vaccines (speac) convened a scientific working meeting https://brightoncollaboration.us/brighton-collaboration-cepi-covid- -web-conference/) on march and , of experts in the field of vaccine immunology and coronaviruses to discuss current knowledge that could form the basis for the assessment of the risk of enhanced disease during sars-cov- vaccine development. this consensus report presents considerations for vaccine developers and can serve as a guide for the development and testing of vaccine candidates to avoid these safety concerns. ultimately, the door to clinical trials is controlled by regulators in the context of the risk/benefit for the entire dataset provided by developers and within the local trial context. microbiology and immunology and pediatrics at the university of iowa, both reviewed their work and that of others in animal models developed for sars-cov- and mers-cov. the lessons from these models can inform the development priorities for useful sars-cov- animal models to address both efficacy and safety. in inbred mouse strains, sars-cov- replicates efficiently in the respiratory tract and can cause pneumonitis, but clinical signs and pneumonia were only observed in old balb/c mice [ ] . subsequent passage of sars-cov- through mouse lungs resulted in the isolation of virus that caused severe disease in both young and old mice [ , ] . this virus was used in many subsequent studies. ferret models of sars-cov- also demonstrate virus replication in respiratory tracts with induction of a neutralizing antibody response but also demonstrated little evidence of clinical disease [ ] . hamsters, in contrast to mice and ferrets, demonstrate high levels of viral replication, develop pneumonitis, and can be shown to have clinical signs of disease [ ] . following the identification of human ace as the receptor for sars-cov- , transgenic murine models expressing human ace receptor (hace ) were developed and shown to develop mild pulmonary disease. of note, these mice also developed lethal viral encephalitis, attributed to viral spread through the olfactory nerve, despite the relative scarcity of hace expression in the brain which may have relevance to sars-cov- disease [ ] . efficacy of several sars-cov- vaccines was evaluated in these models with spike (s) protein based vaccines demonstrating neutralizing antibody and protection against pulmonary replication of the challenge virus in mice and hamsters [ ] . for dna vaccine studies, it was shown that candidate vaccines encoding the s protein conferred antibody mediated protection from challenge in mice and that vaccines encoding the n protein induced humoral and cellular immunity [ , ] . for vectored vaccines expressing sars-cov- proteins, it was shown that viral proteins were expressed in mice, ferrets, and hamsters. in these studies, neutralizing antibodies were elicited by b/hpiv , vsv, rabies, mva and adeno viruses expressing s protein, that protected against sars-cov- replication in lungs of challenged animals. however, one mva vaccine expressing the s-protein did not protect against infection [ ] . in contrast to sars-cov- , inbred mice were found to be resistant to mers-cov, thus infection was studied by creating models that expressed the mers receptor, human dpp (hdpp ). ad -hdpp transduced mice could be infected with mers virus but infection was associated with minimal clinical disease except in immunocompromised mice that developed weight loss after infection. of note, hdpp -transgenic mice developed lethal viral encephalitis with concurrent inflammatory changes on histopathological examination of the lung, similar to hace -tg mice with sars-cov- . subsequently, investigators developed mice "knocked-in" for expression of hdpp and after virus passage in these mice, identified mouse-adapted mers strains that caused more severe disease and increased histopathology with more pulmonary edema than those infected with the original mers strain [ ] . importantly, mice without functional t cells, such as rag -/-and tcr alpha-/-, had delayed viral clearance whereas mice that could not produce antibodies, mumt mice, did not show delay in clearance. similar models were developed by crispr/cas mutagenesis of two residues in the mouse ace molecule, followed by mouse adaptation with serial passage, leading to an ards model of lethal infection [ , ] . taken together this evidence supports the notion that t cells are important in viral clearance for mers [ ] . non-human primate (nhp) models have also been established for both sars-cov- and mers-cov. there was evidence of upper respiratory and lower respiratory tract sars-cov- replication in african green monkeys to a greater extent than in cynomolgus macaques, and least in rhesus macaques, with little evidence of clinical disease in all three species [ ] . of note, consistent with findings in older humans and mice, increased pathology has been documented in aged cynomolgus macaques with sars-cov- wild type infection [ ] . there is some controversy on the disease severity in the mers models with different groups seeing different levels of pathology. this has not been resolved [ , ] . both vaccine efficacy and safety have been studied in animal models with many sars-cov- candidate vaccines. the group of experts discussed how the vaccine models were utilized to characterize the response of specific vaccines and to examine both disease enhancement and antibody dependent enhancement (ade) signals. there is evidence for disease enhancement in vaccinated animals after challenge with live virus in multiple studies with sars-cov- vaccine candidates as summarized in table. we are limiting our comments in this report to data in animal models and not discussing in vitro data except to mention that there is some evidence of ade in human primary monocytes [ , ] . different animal models exhibit different pulmonary pathology but generally characterized by cellular infiltrates including eosinophils. in this summary, we provide an overview of the consensus opinion on vaccine related outcomes in animal models that were of concern for risk of disease enhancement and could guide assessments of sars-cov- vaccine candidates. in murine models, evidence for vaccine related disease enhancement has been demonstrated for inactivated whole vaccine (with and without alum), vectored vaccine expressing n protein (but not seen with vectored vaccine expressing s protein in same report), a replicon particle platform expressing s protein, and a vectored vaccine expressing s proteins. in general, the pathology described included pulmonary infiltrates often with eosinophils observed. th dominant responses were documented in some reports by expression of th driven cytokines [ ] [ ] [ ] [ ] [ ] . in a ferret model, hepatitis was demonstrated in animals vaccinated with a recombinant modified vaccinia virus ankara vaccine expressing s protein and then challenged with virus [ ] although questions have been raised about this study [ ] . [ , ] . non-human primate models have also produced evidence of enhanced disease after sars-cov- vaccine immunization. chinese macaques immunized with a modified vaccinia virus expressing s protein then challenged with sars-cov- did not develop clinical disease, but histopathology showed lung injury. this injury was characterized by decreased wound healing, and increased pro-inflammatory macrophages expressing il- , il- , and ccl [ ] . this report also demonstrated that passively administered anti-s antibody was associated with lung pathology after challenge with the live virus although the mechanism may not be through fc receptor and thus not classic "ade". of note, a second report similarly demonstrates the effect with certain anti-s antibody preparations and without fc involvement [ , ] . the relevance of these reports remains unclear as there are multiple studies with administration of neutralizing monoclonal antibodies to different models that did not induce disease enhancement. other investigators have reported absence of disease enhancement in both hamsters and monkeys immunized with a whole inactivated vaccine although these models differed in a number of ways, most notably by the use of bpl (β-propiolactone) instead of formalin for inactivation of the virus [ , ] . finally, we note that there has not be an agreed upon positive control applied in these animal studies and thus interpretations are hampered.ba animal models with sars-cov- are being rapidly developed by multiple research human ace transgenic mice (hace tg) aged - weeks and - months of age were studied and hace expression was observed in lung, heart, kidney and intestinal tissues. following intranasal inoculation with sars-cov- , weight loss was observed, and viral rna was detected in the lungs as well as in the intestine [ ] . gross pathology demonstrated swollen and enlarged lungs with moderate interstitial pneumonia. histological studies documented an accumulation of inflammatory cells including monocytes and lymphocytes in alveolar interstitium, with thickening of alveolar walls. sars-cov- s protein was detected by ihc in alveolar macrophages and epithelia [ ] . nhp were also infected with sars-cov- with rhesus macaques aged - years inoculated intratracheally and although no fever was observed, weight loss and asthenia were seen on multiple days. viral rna was detected from nasal and throat swabs and to a lesser degree in anal specimens, peaking on days to and lasting until day post infection. one animal was euthanized on day for necropsy and viral rna was detected in multiple organs including cns, skeletal muscle and heart. for the two surviving rhesus macaques, positive neutralization titers were documented by day post infection. there was radiographic evidence of multiple ground glass opacities in the lungs on days , and post infection. microscopically the lung lesions represented an acute interstitial pneumonia characterized by mild to moderate thickening of alveolar septum, increased number of macrophages, degeneration of pneumocytes and an inflammatory cell infiltration. presence of viral antigen was confirmed, predominately in alveolar monocytes and macrophages [ ] . analysis of blood samples showed a decline in counts of total white blood cells, lymphocytes and monocytes with no observed changes in percentages. a decrease in both cd +cd + and cd + cd + t-cell counts was observed. importantly, these hematological findings are similar to those seen in sars-cov- infected patients. this model could serve as a critical tool for detailed studies of pathogenesis and the evaluation of intervention strategies including vaccines. of note, following the meeting another group has confirmed sars-cov- infection in rhesus macaques with viral antigen detected in type i and type ii pneumocytes and diffuse pulmonary alveolar damage noted [ ] . experts agreed that these models and others under development should be utilized to evaluate vaccine candidates for any evidence of disease enhancement as specified in later sections. design barney the sars-cov- s protein structure was solved shortly after its emergence and shows similar structure and mobility as the sars-cov- s [ ] . the timing from first knowledge of sars-cov- to the beginning of the phase study was a remarkable sixty-five days. the advantages of mrna vaccines include ability to create a highly precise type of protein to elicit the correct antibodies, to elicit t cell responses that are th predominant, and the rapidity of manufacturing. of course, disadvantages include the novel nature of both mrna and dna vaccines without any licensed vaccine with either technology to date and lack of experience for mass production. therefore, multiple platforms for sars-cov- are under development that mitigate against some of the potential disadvantages of nucleic acid vaccines. although mrna and dna vaccines elicit t cell responses without adjuvants, adjuvants may be important for subunit and whole cell inactivated vaccines to increase their immunogenicity and drive an immune response that could limit the risk of disease enhancement. multiple sars-cov- vaccines are in development including vectored vaccines, whole cell inactivated vaccines, and recombinant protein vaccines. the experts discussed how the choice of adjuvants will be important for both efficacy and safety with these platforms. dr. arnaud didierlaurent from the centre of vaccinology at the university of geneva presented background on the effects of different adjuvants on animal and human immune responses. several adjuvants are now being used in commercial vaccines or are in clinical development [ ] . oil-in-water emulsions such as mf or as have been shown to increase the breadth of the antibody repertoire, binding affinity and affinity maturation when compared to unadjuvanted vaccines [ , ] in human studies with influenza vaccines, h n vaccine adjuvanted with mf (squalene-based emulsion) increased the levels of h -specific antibody for subclasses igg and igg and the binding to fcγr but not to fcγr when compared to alum adjuvanted vaccines. this demonstrates that the use of an adjuvant can skew the functionality profile of antigen-specific antibodies, with the potential to activate different innate effectors based on their fcγr expression [ ] . use of squalene-based emulsion vaccines for influenza have also been shown to increase cd + t cell response frequencies and crossreactivity. even if pre-existing cross-reactive antibodies are present prior to immunization, such adjuvants could activate naïve b cells and promote the adaptability of memory b cells [ ] [ ] [ ] [ ] . in addition to antibodies, adjuvants can promote cellular responses. human malaria challenge studies provided early evidence that the choice of adjuvants(combined with the malaria antigen rts,s) was critical in achieving optimal protection and highlighted the importance of cellular response [ ] . more recently, studies with hepatitis b surface antigen (hbs) vaccine adjuvanted with as , as , as or alum showed that vaccines formulated with as and as induced the highest antibody levels while as promoted best hbs-specific cd t cell response [ ] . these differences were associated with the magnitude of the initial inflammatory response triggered by the different adjuvanted formulations [ , ] . interestingly, although the level of cd t cell response was lower in the alum group compared to the as group, both adjuvants led to similar memory subset profiles and cytokine production profiles given the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for subunit vaccines in providing antigen-dose sparing, increased immunogenicity, breadth and duration of response, potentially eliciting cross-protection against new cov strains and minimizing the risk of enhanced disease. following the presentations, attendees participated in discussion of the suggested consensus statements and all attendees were asked to comment on the draft statements available online. these comments were reviewed and discussed again on the second day of the meeting and resulted in the summary consensus statement that follows. • sars-cov- has a low affinity for murine ace receptor and murine models will require the use of hace transgenic mice, preferably with a 'knock-in' approach. preliminary data indicate the possibility of infecting hace transgenic mice with demonstration of viral replication and mild lung lesions. mouse adaptation of sars-cov- , as done with sars-cov- , will likely be required to obtain a virus that causes more severe disease in mice. models that develop acute lung injury with some lethality and that mimic the human condition will be important for evaluating vaccine safety. • previous studies from sars-cov- and mers-cov indicated that some vaccines, especially those using whole inactivated virus, could enhance the disease induced in mice challenged with sars-cov- or mers-cov. the lung lesions were highly inflammatory, with a dominance of eosinophil infiltration and occurred in animals despite presence of a neutralizing antibody response and reduced challenge virus replication in the lungs. such studies have not yet been completed for sars-cov . • in mice, this immunopathology was considered a consequence of a dominant th type response to the vaccine antigens. it was not seen after including adjuvants (e.g. cpg) in the vaccine or other vaccine formulations known to drive immune responses towards th . the timing of challenge after vaccination may be critical. it would be of major interest to explore the outcome following challenge at later timepoints when antibodies are significantly decaying. • one should be aware of the potential confounding effect of cell-culture excipients in the vaccine and challenge strain material. it is known that impurities such as fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx in any mouse model if the challenge strain also contains the same excipients. • in these models, characterization of the immune response to the candidate vaccine (e.g., igg isotypes, th markers) may have some predictive value. • other small animal models which can be infected by sars-cov- can be considered (e.g. ferret, hamster). development of small animal models of severe disease will also inform studies of vaccine-enhanced disease. • passive transfer in nhps of human antibodies generated during phase trials, followed by viral challenge could be considered to assess the risk of disease enhancement. • challenge of immunized animals with a closely related heterologous cov strains may assess the risk of enhancement during future outbreaks. • in case of disease enhancement, in-depth studies in animal models may give some indications on the mechanism of immunopathology. they can inform human trial designers on the critical immunological risk markers to be monitored in phase trials. • given what will be the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for sub-unit vaccines in providing increased immunogenicity, breadth of response, dose sparing, duration of response, potentially cross-protection against new cov strains, and possibly minimize the risk of enhanced disease. preference should be given to th -driving adjuvants with an established safety profile in humans. • understanding of the role of cross-reacting antibodies from prior coronavirus infections may have on natural disease caused by sars-cov- or influence the risk of enhanced disease following vaccination may inform vaccine design. • data are needed on whether antibody waning could increase the risk of enhanced disease on exposure to virus in the long term. it was the opinion of the experts that animal data to support clinical development could address: • post-vaccination (neutralizing) antibody responses, and t cell analysis to demonstrate a th response. • post-vaccination challenge data from nhps with careful evaluation for immunopathology and quantitative virology in the animals. • small animal data may also provide important supporting evidence of safety, and hamster, ferret and mouse models are likely to be available for developers. • where possible, immunopathology experiments with a positive control (e.g., formalin inactivated alum-adjuvanted sars-cov- or sars-cov- vaccine) and a mockimmunized negative control will provide best guidance. it was felt that it will be important to establish broadly accepted endpoints and scoring systems to allow comparison of various vaccine candidates. who is working on this issue. • for vaccine constructs likely to induce a predominant th response, the group felt that animal studies should be considered before entering human phase trials in more than one animal species including nhps where possible. it was noted that the absence of a th response does not eliminate the risk of enhanced disease. • since not all studies that have begun or are about to begin will prescreen to determine preimmunization serostatus of participants, although this shall be determined retrospectively, appropriate baseline blood specimens should be obtained and stored. because the virus is spreading rapidly, such specimens will allow assessment of the immune response in both seronegative and seropositive persons as both are likely to be vaccinated. • level of neutralizing antibodies and determination of the relative ratio of binding to neutralizing antibodies will be important to assess the potential risk of enhanced disease. also, detection of initial priming that includes cd t cells and/or a cd th biased response is likely to mitigate the risk of disease enhancement. determination of memory responses will be useful, particularly if sars-cov- continues to circulate. • consideration should be given to the use of post-vaccination sera from vaccinees which could be used for antibody transfer studies in animals to look for enhanced disease and for evidence of cross-protection against other coronaviruses. • monitoring for enhanced disease in immunized participants may require longer follow-up than is usual in phase trials but need not delay phase trials. • investigators on the call requested frequent updating with both preclinical and evolving clinical data that are being developed by the different academic and industrial developers to help in decision-making about the various vaccine clinical trials. creation of a central information hub was encouraged for this purpose. • participants on the call expressed the need for standardization of protocols, data collection forms, critical assays (including reagents) and biobanking of samples from initial clinical trials to allow future re-assay once standards are agreed to and enable comparison of results across trials • the group of experts considers that the demonstration of some disease enhancement with any candidate vaccine after viral 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induced with malaria vaccine, rts,s, is linked to plasmodium falciparum circumsporozoite protein-specific cd + and cd + t cells producing ifn-gamma impact of adjuvants on cd (+) t cell and b cell responses to a protein antigen vaccine: results from a phase ii, randomized, multicenter trial different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans. front immunol systems analysis of human vaccine adjuvants innate transcriptional effects by adjuvants on the magnitude, quality, and durability of hiv envelope responses in nhps all authors attest they meet the icmje criteria for authorship key: cord- -m qisld authors: goldman, ran d.; yan, tyler d.; seiler, michelle; parra cotanda, cristina; brown, julie c.; klein, eileen j.; hoeffe, julia; gelernter, renana; hall, jeanine e.; davis, adrienne l.; griffiths, mark a.; mater, ahmed; manzano, sergio; gualco, gianluca; shimizu, naoki; hurt, thomas l.; ahmed, sara; hansen, matt; sheridan, david; ali, samina; thompson, graham c.; gaucher, nathalie; staubli, georg title: caregiver willingness to vaccinate their children against covid- : cross sectional survey date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: m qisld background more than covid- vaccine candidates are in development since the sars-cov- genetic sequence was published in january . the uptake of a covid- vaccine among children will be instrumental in limiting the spread of the disease as herd immunity may require vaccine coverage of up to % of the population. prior history of pandemic vaccine coverage was as low as % among children in the united states during the h n influenza pandemic. purpose to investigate predictors associated with global caregivers’ intent to vaccinate their children against covid- , when the vaccine becomes available. method an international cross sectional survey of caregivers arriving with their children to pediatric emergency departments (ed) across six countries from march to may , . results % (n = ) of caregivers reported that they intend to vaccinate their child against covid- , once a vaccine is available. a univariate and subsequent multivariate analysis found that increased intended uptake was associated with children that were older, children with no chronic illness, when fathers completed the survey, children up-to-date on their vaccination schedule, recent history of vaccination against influenza, and caregivers concerned their child had covid- at the time of survey completion in the ed. the most common reason reported by caregivers intending to vaccinate was to protect their child ( %), and the most common reason reported by caregivers refusing vaccination was the vaccine’s novelty ( %). conclusions the majority of caregivers intend to vaccinate their children against covid- , though uptake will likely be associated with specific factors such as child and caregiver demographics and vaccination history. public health strategies need to address barriers to uptake by providing evidence about an upcoming covid- vaccine’s safety and efficacy, highlighting the risks and consequences of infection in children, and educating caregivers on the role of vaccination. followed by an open-ended question "why?" or "why not?", with a free text box. the description of each response was categorized into themes using an inductive approach by one author and reviewed for completeness by another author. the entirety of each response was analyzed and if more than one sentiment was expressed in the caregiver's description, the individual response was coded to multiple themes. free text responses that were blank were categorized as no comment. the covid- vaccine development landscape covid- : what do we know so far about a vaccine? final estimates for - seasonal influenza and influenza a (h n ) monovalent vaccination coverage -united states factors in vaccination intention against the pandemic influenza a/h n . eur likely uptake of swine and seasonal flu vaccines among healthcare workers. a cross-sectional analysis of uk telephone survey data when a covid- vaccine is ready, will we all be ready for it? int vaccine hesitancy: an overview planning for a covid- vaccination program fauci says covid- vaccine may not get us to herd immunity if too many people refuse to get it exploring lessons learned from a century of outbreaks: readiness for factors associated with uptake of vaccination against pandemic influenza: a systematic review healthcare workers as parents: attitudes toward vaccinating their factors associated with parental acceptance and refusal of pandemic influenza a/h n vaccine in turkey public perceptions of covid- in australia: perceived risk, knowledge, health-protective behaviours, and vaccine intentions determinants of covid- vaccine acceptance in saudi arabia: a web-based national survey hesitancy towards a covid- vaccine and prospects for herd immunity the french public's attitudes to a future covid- vaccine: the politicization of a public health issue preparing for a covid- vaccine: identifying and psychologically profiling those who are vaccine hesitant or resistance in two general population samples low acceptability of a/h n pandemic vaccination in french adult population: did public health policy fuel public dissonance? the influence of altruism on influenza vaccination decisions factors influencing childhood influenza immunization acceptance of vaccinations in pandemic outbreaks: a discrete choice experiment striking a balance between risk and protection: fathers' attitudes and practices toward child injury prevention acceptability of a/h n vaccination during pandemic phase of influenza a/h n in hong kong: population based cross sectional survey factors associated with refusal of childhood vaccines among parents of school-aged children: a case-control study underimmunization in ohio's amish: parental fears are a greater obstacle than access to care mapping vaccine hesitancy -country-specific characteristics of a global phenomenon multisystem inflammatory syndrome related to covid- in previously healthy children and adolescents associations between health communication behaviors, neighborhood social capital, vaccine knowledge, and parents' h n vaccination of their children understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature vaccine beliefs of parents who oppose compulsory vaccination conceptualization, methodology, software, formal analysis, investigation, resources writing -original draft, writing -review & editing, supervision formal analysis, data curation, writing -original draft, writing -review & editing michelle seiler: investigation, resources, writing -review & editing conceptualization, methodology, investigation, resources, writing -review & editing conceptualization, methodology, investigation, resources, writing -review & editing julia hoeffe: investigation, resources, writing -review & editing renana gelernter: investigation, resources, writing -review & editing hall: investigation, resources, writing -review & editing davis: investigation, resources, writing -review & editing sergio manzano: investigation, resources, writing -review & editing gianluca gualco: investigation, resources, writing -review & editing naoki shimizu: investigation, resources, writing -review & editing conceptualization, methodology matt hansen: investigation, resources, writing -review & editing investigation, resources, writing -review & editing samina ali: investigation, resources, writing -review & editing thompson: investigation, resources, writing -review & editing formal analysis, data curation, investigation, resources, writing -original draft key: cord- - qlr authors: yu, wenzhou; lee, lisa a.; liu, yanmin; scherpbier, robert w.; wen, ning; zhang, guomin; zhu, xu; ning, guijun; wang, fuzhen; li, yixing; hao, lixin; zhang, xuan; wang, huaqing title: vaccine-preventable disease control in the people’s republic of china: – date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: qlr background: china's immunization program is one of the oldest and largest in the world. rates of vaccine-preventable diseases (vpd) are comparable to those in high-income countries. the program's evolution has been characterized by ambitious target setting and innovative strategies that have not been widely described. methods: we reviewed national and provincial health department archives; analyzed disease surveillance, vaccination coverage, and serosurvey data from through ; and, conducted in-depth interviews with senior chinese experts involved early vpd control efforts. results: widespread immunization began in the s with smallpox, diphtheria, and bacillus-calmette guerin vaccines, and in the s with pertussis, tetanus, polio, measles, and japanese encephalitis (je) vaccines. the largest drops in absolute vpd burden occurred in the s with establishment of the rural cooperative medical system and a cadre of trained peasant health workers whose responsibilities included vaccinations. from to , incidence per , population dropped % from . to . for diphtheria, % from . to . for pertussis, % from . to . for polio, % from . to . for measles, and % from . to . for je, averting an average of million vpd cases each year. until the early s, vaccines were delivered through annual winter campaigns using a coordinated ‘rush-relay’ system to expedite transport while leveraging vaccine thermostability. establishment of the cold chain system during in the s allowed bi-monthly vaccination rounds and more timely vaccination resulting in rates of diphtheria, pertussis, measles and meningitis falling over % from to , while polio and je rates fell – %. in the s, progress stalled as financing for public health was weakened by broad market reforms. large investments in public health and immunizations by the central government since has led to further declines in vpd burden and increased equity. during – , the incidence per , population was < . for measles and < . for pertussis, je, meningococcal meningitis, and hepatitis a. from to , the prevalence of chronic hepatitis b infection in children < years fell from . % to . %, a % decline. china was certified polio-free in and diphtheria was last reported in . conclusions: long-term political commitment to immunizations as a basic right, ambitious targets, use of disease incidence as the primary metric to assess program performance, and nationwide scale-up of successful locally developed strategies that optimized use of available limited resources have been critical to china's success in controlling vaccine-preventable diseases. china has one of the largest and oldest immunization programs in the world with over million infants vaccinated each year [ ] . incidence rates of vaccine-preventable diseases (vpd) are similar to those in high-income countries and vaccination coverage is uniformly high. these achievements are the result of enormous effort and evolving responses to new challenges and opportunities over the past years. when the people's republic of china was founded in , the new government faced immense health problems. most of the million population lived in rural areas and in extreme poverty with little access to health care. infant mortality exceeded per births and average life expectancy was only years [ ] . lack of united nations' recognition limited international support and scientific exchange. despite these challenges, the government set ambitious health goals and positioned immunizations as a central to their achievement. prior to liberation, vaccines were prohibitively expensive in china and largely inaccessible except to the very wealthy. in , constitutional principles of the communist party in the shaanxi-ningxia-gansu border region affirmed ''people's rights to freedom from ill health", and in line with this progressive policy, free mass vaccination campaigns against smallpox and cholera were implemented in liberated areas [ ] . this was the first time that large numbers of the rural poor in china benefitted from vaccination. since then, china's public health system has undergone numerous reforms with continued strengthening of vpd control efforts through sustained high-level political commitment to immunizations, strong supportive legal frameworks, increased public finance, dedicated efforts of many scientists and public health professionals and grass-roots health workers, and national scale-up of successful innovative delivery approaches that optimized use of available material and human resources. this article describes china's work on vpd control during to , which has not been widely described. annual vpd incidence from to was obtained from the national notifiable disease reporting system (nndrs). the nndrs is a compulsory reporting system established in for diseases with high epidemic potential including cholera, plague, smallpox, japanese encephalitis (je), meningococcal meningitis, poliomyelitis, diphtheria, pertussis, and measles. data on each case is limited to critical information including name, address, date of birth, date of disease onset, age, sex, and occupation. the nndrs has since been expanded to diseases, including hepatitis a and hepatitis b, tuberculosis, neonatal tetanus, mumps and rubella but the type of data collected has remained largely unchanged. hepatitis b burden, using hepatitis b surface antigen (hbsag) seropositivity as a marker for chronic infection, and hepatitis b vaccination coverage were estimated through national serosurveys conducted in , , and [ ] [ ] [ ] . all three serosurveys relied on random sampling of persons in the disease surveillance point (dsp) surveillance system, a nationally representative sample of rural townships and urban neighborhoods representing approximately % of the total population. vaccination coverage surveys were conducted in selected provinces during - while nationally representative surveys were conducted in , , , , and . all surveys utilized multi-stage probability of selection proportional to population size sampling based on world health organization (who) guidelines [ ] . as a proxy for coverage prior to the s, we searched for references to total numbers of persons vaccinated and total numbers of doses of vaccine produced or administered. a search was conducted for published and unpublished reports on vaccine development and vpd control in china from to , focusing on chinese-language documents not widely available in the published literature, and included manual searches through archived documents at the ministry of health (moh), china center for disease control and prevention, and provincial health departments [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . data on immunization expenditures was collected as part of national epi reviews conducted in , , and . because few reports on vaccination efforts were found prior to the s, we interviewed senior chinese experts who were closely involved in spearheading china's early immunization efforts using a structured open-ended questionnaire. persons interviewed included scientists involved in vaccine research and development; moh officials responsible for development of national policies and goals; and, provincial health staff responsible for planning and implementing vpd control activities. work on immunizations began in earnest as soon as the people's republic of china was founded and more than two decades before establishment of the world health organization's (who) expanded programme on immunizations (epi). in , only four small vaccine manufacturers existed in china (beijing, shanghai, lanzhou, changchun) and developing a secure vaccine supply was a high priority. manufacturing facilities were built in wuhan ( ) and chengdu ( ) to create a network of six regional manufacturers under moh supervision responsible for producing vaccines for the entire country. in , a plan was developed to eradicate smallpox nationwide through free mass compulsory vaccination of the entire population. by the end of , moh announced that over million people (about % of total population) had been vaccinated and smallpox rapidly disappeared from most of the country with the last case occurring in [ fig. ]. building on this success, the state council issued a directive in requiring establishment of a network of epidemic prevention stations (eps) at province, prefecture and county levels, with vpd control a main responsibility. directives on vaccination of children with diphtheria toxoid and bacillus calmette guérin (bcg) were issued in and , respectively, and research was accelerated to develop vaccines against diseases responsible for high mortality including polio, measles, and je. oral polio vaccine (opv) were developed in and was the first attenuated vaccine developed in china. the institute of medical biology, academy of medical sciences was established in kunming, to scale-up opv production. in , moh issued ''measures on implementing preventive vaccination" requiring that all provinces conduct annual winter campaigns to vaccinate children with smallpox, bcg, diphtheria, pertussis, and polio vaccines. in the early of s, combined diphtheria-tetanus-pertussis toxoid (dtp) was introduced. three attenuated measles vaccine strains, beijing- , shanghai- , and changchun- , were developed and introduced in . in contrast to the nationally synchronized smallpox campaigns, campaigns with other antigens were organized at the provincelevel and conducted during colder months since most vaccines were in liquid formulation with limited thermostability and there was no cold chain system. annual meetings were held by moh with provincial health departments and vaccine manufacturers to prioritize limited supplies. to expedite delivery before the vaccines lost potency, a highly coordinated ''rush-relay" transport approach was developed; when vaccines were shipped by the manufacturer, usually by train or truck, the provincial eps was notified by telephone or telegraph when the vaccines would arrive and to mobilize of all forms of transport (motor vehicles, bicycles, pack animals, health staff) to move the vaccines as rapidly as possible to the periphery. local cold storage solutions included use of refrigerators at food processing plants, wells, cellars, burial underground, and wooden boxes built with separate compartments for bottles of frozen water or pieces of ice. usually vaccines were transported from one administrative level to the next within a day and campaigns were completed in a single day. limited vaccine supplies precluded province-wide campaigns, so each province was divided into sections targeted on a rotating basis over a five to six-year period, with each campaign targeting all children under years old. as a result, by school entry most children had received only one or two doses each of diphtheria, pertussis and polio vaccines. while killed vaccines and toxoids could remain potent for up to days, opv and measles were live attenuated vaccines and lost potency within one week, limiting their use to urban areas and impacts. in , a measles epidemic coinciding with rural famine during the great leap forward affected nearly million persons and caused , deaths; during - , epidemic poliomyelitis spread throughout the country with , cases reported [ fig. ]. in , mao zedong's ''june directive" called for renewed focus on rural health. civil unrest at the start of the cultural revolution, however, disrupted vaccine work. during - , outbreaks of poliomyelitis, diphtheria, pertussis and measles started to recur in areas where the diseases had previously been controlled and incidence began to rise, and mass movement of students facilitated transmission of meningococcal meningitis group a resulting in an epidemic in - that affected over million persons and caused , deaths [ fig. ]. in the early s, a number of important public health reforms were instituted that had greatly strengthened vpd control efforts. in , the rural cooperative medical system (rcms) was established with commune-level health stations staffed by a new cadre of part-time peasants (''barefoot doctors") who received three months of training in delivery of basic medical and preventive services, including vaccinations. these grass-roots health workers were responsible for transporting vaccines from county and commune hospitals to their villages and administering vaccinations, which were essentially free with delivery costs covered by pooled rcms funds. vaccine production was increased and the frequency of campaigns were increased with most provinces conducting at least two or three province-wide campaigns each year; live vaccines in the fall and winter and killed in support of the united nations resolution on universal childhood immunization (uci), '' - " coverage goals were included in china's '' th -year plan for national social and economic development, - " setting targets of % percent coverage at province-level with bcg, dpt, opv and measles by months of age by , and % coverage at county-level by . while the uci goals were lower than china's coverage targets and excluded je and meningococcal meningitis, the target age for being fully vaccinated was much younger - months instead of years of age. administering the entire primary series of bcg, dpt, opv and measles vaccines during infancy would require at least six vaccination sessions per year nationwide, ability to delivery vaccinations year-round, and huge investments to extend the cold chain to township levels nationwide. the first coverage survey in china was conducted in provinces with assistance from who in highlighted the magnitude of the challenge [ table ]. to achieve the uci goals, a high-level inter-ministerial leading group consisting of moh, ministry of foreign economic relations and trade, ministry of tv and broadcasting, state education commission, state ethnic affairs commission, and the all china women's federation was formed to oversee planning and progress. april was established as ''national vaccination day" and huge investments were made in cold chain, training, social mobilization, and vaccines. by the early s most of the epi vaccines were lyophilized and more thermostable, enabling delivery to more remote areas. by , annual production had increased to million doses each of opv, dpt, and measles, and million doses of bcg. in , the national people's congress passed a law requiring health authorities at all levels implement a system of planned preventive immunizations that included issuing vaccination certificates to all children and establishing registers to monitor vaccination coverage at township levels and above. with tremendous effort, both '' - " targets were achieved despite large areas of the country still having no cold chain. the first nationwide coverage survey, conducted in to verify achievement of the uci goal documented coverage of % with all recommended bcg, dpt, opv and measles doses by months of age [ table ]. polysaccharide meningococcal meningitis group a and trivalent opv were introduced in and , respectively, and between and the incidence of meningococcal meningitis fell %, from . to . per , while the incidence of poliomyelitis fell %, from . to . per , [ fig. ]. incidences of other target diseases also continued to fall during this period. by , < cases of poliomyelitis were reported nationwide, although low levels of transmission persisted and the target for national elimination by was missed. in support of the world health assembly resolution to eradicate poliomyelitis globally by , the moh issued the '' - national plan for eradication of poliomyelitis", setting targets of < per million by , and zero cases by , with the main strategy supplementary campaigns with opv. during - , million opv doses were administered in province-wide campaigns. in , synchronized nationwide campaigns were approved by the state council targeting all children < years of age with two doses of opv, one dose on december and one dose on january for three consecutive years. the first campaign was conducted in winter / with million opv doses administered and the last case of polio occurred in september . despite epis high profile, financing for immunizations became progressively weaker starting in the s as a result of broad market reforms. farm collectives and the rcms were dismantled, and while epi vaccines were still provided for free, funding for vaccine delivery switched to fee-for-service, usually - rmb paid by parents to the village or township doctor for each dose administered. public health departments were largely left to generate their own operating expenses. in some regions, innovative financing mechanisms were developed to secure additional funding for immunizations, such as the ''epi contract", a lump-sum payment by parents to cover the cost of all epi vaccinations that was pooled and divided between village, township and county-levels to cover delivery costs, as well as an indemnity payment if a child developed a vpd against which he or she had been vaccinated. by , however, central government funds accounted for only % of total immunization expenditures while more than % of expenditures were at village and township levels, resulting in falling coverage and large disparities between more and less developed areas. in , coverage in children - months old with all recommended doses of bcg, dpt, opv and measles had fallen below % in nine ( %) provinces, including seven of the poorer western provinces. despite the challenges financing the immunization program, china was one of only two low-income countries (the other was cuba) to introduce hepatitis b vaccine into their epi when universal infant immunization with hepatitis b vaccine was endorsed by the world health assembly in . at that time, china had the largest disease burden of hepatitis b in the world with approximately % of the population chronically infected. adding hepatitis b vaccine would have tripled government vaccine costs per child from . to . rmb, so market strategies were also used to finance introduction of this expensive new vaccine. the government added hepatitis b to the epi but exceptionally allowed the vaccine cost to be passed to parents. this cost-recovery approach provided a strong incentive to health workers to deliver the vaccine and national coverage with three doses of hepatitis b vaccine in infants increased from % in , to % in , preventing millions of infections and averting hundreds of thousands of deaths. coverage, however, was predictably much lower in less developed areas, particularly the western provinces. requirements for all children to be fully vaccinated, and the regulation on vaccine circulation and immunization that included laws specifying that nationally recommended vaccines be fully funded by the government and administered completely free-of-charge. in , the immunization schedule was expanded to include new antigens (measles-mumps-rubella, attenuated hepatitis a), safer products (acelluar pertussis), and older vaccines that had been excluded from the original epi schedule (je, meningococcal meningitis) and epi vaccine procurement was centralized with moh responsible for procuring all epi vaccines. in , the government launched the new essential public health service package with service categories, including immunizations. government subsidies of rmb per person were provided to village and township-administrative levels to cover the cost of immunization services, and increased to rmb per person in , rmb in , rmb in , and rmb in . currently, all village and townships have the full-time health staff that provide vaccination services to all children at , delivery sites nationwide. there are approximately , epi managers at provincial, prefecture and county levels and , immunization staff at township and village levels. with the majority of china's population now living in urban areas, vaccination has shifted from pulse delivery by village-based health workers to predominantly daily delivery at fixed clinic sites. public health reforms during the past decade have strengthen financing for vaccines and vaccination services resulting in increased and more equitable coverage and historically low vpd burden [ table , fig. ]. in the national survey, coverage of recommended infant doses of bcg, dpt, opv, hepatitis b and measles vaccines was % in of provinces, and % in province, a major improvement from . from to , the incidence of pertussis fell % from . to . per million, measles fell % from . to . per , , meningococcal meningitis fell % from . to . per million, je fell % from . to . per million, and hepatitis a fell % from . to . per , . the last case of diphtheria was reported in . in , china was certified as having eliminated maternal and neonatal tetanus and an all-time low of measles cases were reported nationwide, although numbers of measles cases increased in subsequent years. from to , the prevalence of chronic hepatitis b infection in children under- years old dropped from . % to . %, a % decline. sustained political commitment to immunizations at the highest levels, ambitious targets, strong supporting legal frameworks, a vibrant domestic vaccine industry, and innovative financing and delivery strategies to maximize use of available resources have been key to china's many achievements in vpd control over the past years. immunizations has been a universal right since the founding of the people's republic of china, and have been supported at the highest political levels with passage of laws ensuring access and progressively increasing levels of public finance as the country has developed. goals for universal childhood immunization, smallpox eradication, and poliomyelitis eradication were established in china before comparable un global resolutions and before there was an established cold chain system. selfreliance has been a defining characteristic stimulating the development of innovative strategies to maximize use of limited resources, including mass training of village-based lay health workers to deliver vaccinations, cost-sharing strategies to finance introduction of new vaccines, large-scale social mobilization, use of rapid pulsedelivery approaches to leverage existing vaccine thermostability in areas without cold chain, support for domestic vaccine production, and rapid national scale-up of successful pilots. currently, there are seven state-owned and private vaccine manufacturers in china with annual production capacity of around billion doses, including acellular pertussis, influenza, rabies, yellow fever, japanese encephalitis, hepatitis a and b, rubella, varicella, typhoid, and live and inactivated polio vaccines (ipv). all vaccines recommended in the national immunization schedule are domestically produced. globally, vaccination coverage is stagnating with dpt coverage % since , and % in the african region [ ] . progress toward the global vaccine action plan % coverage target is off-track, and in , nearly million infants were not vaccinated [ ] . many of these children are socioeconomically marginalized, live in fragile or remote settings, and have limited access to health care. a delivery model that relies primarily on nurses and clinical officers to administer vaccinations at fixed sites and provide periodic outreach is costly, places a large burden on parents, and likely to be insufficient to achieve and sustain high vaccination coverage levels in resource-poor countries with large dispersed rural populations [ ] . china has more than half a century of experience successfully using community-based health workers to periodically come to clinics to collect vaccines in cold boxes to take back to their villages for pulse administration increasing rural access, community buy-in, and sustained high coverage levels. more widespread adoption of alternative service delivery approaches may be needed to close remaining coverage gaps. use of vaccine vial monitors that measure cumulative heat exposure and development of pre-filled injection devices further facilitate ease and safety of vaccination by community health workers and could also help address cold-chain challenges in remote areas [ ] . in , menafrivac Ò conjugate meningococcal meningitis a vaccine was the first vaccine prequalified by who for use outside the cold chain in controlled temperature chain (ctc); use of ctc for a mass vaccination campaign in chad would have reduced logistics costs by an estimated % [ ] . in china, in villages where village health workers were provided with hepatitis b vaccine with storage at ambient temperatures at the beginning of hepatitis b vaccine introduction, timely birth dose coverage in infants born at home increased from % to %, with no difference in antibody response compared to newborns who were vaccinated with hepatitis b kept in the cold chain [ ] . recent economic analyses indicate that ctc delivery of the hepatitis b birth dose would be cost-saving in most low and middle-income countries [ ] . disease control has always been the main goal of china's immunization efforts and disease incidence has always been the main metric used to guide development of immunization strategies and to assess immunization program performance. close monitoring of temporal, geographic, and demographic trends in vpd incidence has been critical in china for identifying under-immunized populations and for evaluating the effectiveness of delivery strategies, even when the majority of reported cases are primarily clinically confirmed. in contrast, epis in most low-income countries primarily rely on administrative coverage despite recognized problems with data quality and reliability [ , ] . the - ebola outbreak in west africa has spurred new initiatives to strengthen communicable disease surveillance and more effective use of surveillance data by immunization program managers, including district and sub-district mapping of disease incidence, could strengthen program monitoring and accountability. china's experience with hepatitis b vaccine is an interesting case study on use of cost-sharing to finance the introduction of new vaccines that may be of relevance to middle-income countries ineligible for gavi support. when who recommended universal infant immunization with hepatitis b vaccine in , gdp per capita in china was only us$ . adding hepatitis b vaccine to the infant schedule while allowing health workers to recover the vaccine costs from parents enabled early introduction without external or government financing, and provided a delivery incentive that achieved % coverage nationwide, preventing millions of infections. china's experience suggests that cost-recovery can be an effective interim option for countries to finance the early introduction of expensive new vaccines, particularly if the govern-ment can negotiate lower purchase prices, set caps on allowable charges, and provide subsidies for the poor. china also has much to learn from experience in other countries. challenges include strengthening delivery of immunizations within a larger package of integrated health services, balancing policies that make vaccines affordable against those providing incentives for new vaccine research and development, financing new vaccine introductions, and vaccine hesitancy. for many years, vaccination was one of the few preventive services that village and township doctors were required to deliver and china's immunization program is facing the challenges of navigating integration of immunization with delivery of a much wider range of other services. introduction of new expensive vaccines into the recommended schedule remains a challenge. the current infant schedule requires separate injections and there is urgent need for increased funding to develop and add combined products to the schedule, such as dpt-hepatitis b, dpt-hepatitis b-haemophilus influenzae type b (hib), and dpt-hepatitis b-hib-ipv. finally, while public trust in immunizations remains high, china has not been immune to problems of vaccine hesitancy. widespread internet access has facilitated rapid dissemination of often unfounded claims of harmful effects due to vaccination that have had negative effects on vaccination coverage [ ] . these and other challenges will require new approaches as china's immunization program continues moving forward. this work was supported by united nations children's fund for the literature review and the in-depth interviews of chinese immunization experts (yh - ). a -year history of disease prevention and control in china new china's achievements in health work chinese communist party. constitutional principles of the shaanxi-gansu-ningxia border region viral hepatitis in china. seroepidemiological survey in chinese population (part one) - . beijing science and technology press: beijing epidemiological serosurvey of hepatitis b in china -declining hbv prevalence due to hepatitis b vaccination prevention of chronic hepatitis b after decades of escalating vaccination policy world health organization. the epi coverage survey. geneva: world health organization, expanded programme on immunization regarding the launching of the autumn campaign for smallpox vaccination ministry of health of the people's republic of china. measures for implementing preventive vaccination ministry of health of the people's republic of china. regulations on acute infectious diseases, number ministry of health of the people's republic of china. report of the survey of the third % vaccination coverage target of the national expanded programme on immunizations ministry of health of the people's republic of china. compilation of national immunization documents: s national file ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations. beijing: people's health publishing house state council of the peoples's republic of china. regulations of vaccine distribution and vaccination, number ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations national certification committee for the eradication of poliomyelitis in the people's republic of china. documentation for the certification of poliomyelitis eradication hubei province epidemic prevention station. expanded programme on immunization contract system tested study on financing the expanded program on immunizations in selected regions of china routine immunization services costs and financing in china progress and challenges with achieving universal immunization coverage. who/unicef estimates of national immunization coverage (data as of assessment report of the global vaccine action plan strategic advisory group of experts on immunization. geneva: world health organization the cost structure of routine infant immunization services: a systematic analysis of six countries can thermostable vaccines help address cold-chain challenges? results from stakeholder interviews in six low-and middle-income countries economic benefits of keeping vaccines at ambient temperature during mass vaccination: the case of meningitis a vaccine in chad hepatitis b vaccination of newborn infants in rural china: evaluation of a village-based, out-of-cold-chain delivery strategy costeffectiveness of the controlled temperature chain for the hepatitis b virus birth dose vaccine in various global settings: a modelling study tracking progress towards universal childhood immunisation and the impact of global initiatives: a systematic analysis of three-dose diphtheria, tetanus, and pertussis immunisation coverage the immunization data quality audit: verifying the quality and consistency of immunization monitoring systems loss of confidence in vaccines following media reports of infant deaths after hepatitis b vaccination in china the authors declare no conflicts of interest. key: cord- - pw t authors: kusters, j. g.; jager, e. j.; niesters, h.g.m.; van der zeijst, b.a.m. title: sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus date: - - journal: vaccine doi: . / - x( ) -h sha: doc_id: cord_uid: pw t abstract under laboratory conditions coronaviruses were shown to have a high frequency of recombination. in the netherlands, vaccination against infectious bronchitis virus (ibv) is performed with vaccines that contain several life-attenuated virus strains. these highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. this paper addresses the question of the frequency of recombination of avian coronavirus ibv in the field. a method was sought to detect and quantify recombination from sequence data. nucleotide sequences of eight ibv isolates in a region of the genome suspected to contain recombination, were aligned and compared. phylogenetic trees were constructed for different sections of this region. differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo rna recombinant had occurred. infectious bronchitis virus (ibv) causes considerable damage in the poultry industry by infections of the respiratory tract and the reproductive organs . ibv is the prototype strain of the coronaviridae, a family of positive stranded rna viruses with a genome of about kb. this genome contains the information for three structural proteins and a number of enzymes involved in virus replication (figure ). although vaccines offer protection, they are often made ineffective by the continuous emergence of new serotypes " . serotypespecific protective immunity is thought to be mediated by antibodies to a protein on the surface of the virion, the so-called peplomer protein *-- . the peplomer protein is synthesized as a precursor glycoprotein, which is cleaved into the subunits s and $ , derived from its n-and c-terminal half, respectively a-~°. point mutations are obviously involved in the generation of new serotypes. however, previous studies ~ suggested that rna recombination is also involved in the generation of new antigenic variants. to address the question of the frequency of recombination in the generation of new field isolates the nucleotide sequences in five windows of homologous sequences of the genomes of eight ibv strains have been compared. to make this comparison the sequences of the $ genes of d and d were newly determined. both strains are dutch field isolates and are included in vaccines currently used in the netherlands. the alignment of the sequences suggests that the dutch field strain d , the british field strain / and the japanese field strain kb t result from rna recombination events. the origin of the ibv strains as well as the procedures used for the cloning and sequencing of the peplomer genes have been published previously ' ' . sequence data were processed using the programs of microgenic (beckman instruments, ref. ) . phylogenetic trees were derived from nucleotide difference-matrices using the program of dr j. felsenstein (university of seattle) distributed as part of the phylip package . . the $ sequences the newly determined d and d sequences are based on data from two or more independent cdna clones. with strain d , two nucleotide differences between cdna clones were observed. neither of these differences resulted in an amino acid replacement. from the nucleotide sequences (submitted to the embl genbank and ddbj nucleotide sequence databases), amino acid sequences have been deduced. in figure the amino acid sequences are listed together with those of all other known $ sequences. assuming the conserved arg-arg-x-arg-arg-ser sequence to be the cleavage site between s and $ , the (table i) . however, out of cysteine residues and most glycosylation sites are completely conserved. s k v v n rk l s k v t v -- -- r v z e n m v v -- ----f e k v y s v -- p asg e k v h s v -- -- p asg fq kq s s h i gla qdna miq r d nsf -'f--a d ~ i••dllft••e••glptddaykn•ta•plgflkdla•areyngll•l•pi•taem•tlyt••l•a•mafggitaagaipfa n f a -- a -- v ic- s k f i s dk l g s qk s v v qk am i s m u,~y~da~ana~drl~tgrls~ls~la~ak~aeh~r~s~relat~k~ne~vk~ry~f~gngr/~ltip~napng f y l y t s d i yy s d pi yy k le l yak t ke t g g m ibis m u,h m h '\ m kb / d d q q q ivfihfsytpd~fvnvt---a~vgf~vkpana-~s~yai~pangrgifi~vng--ssyyitardmympra~tagdi~tlts~anyvs v t e ~ ~ v ~ h t e n d t e -- n ~ ~ d l m l t e y ----v d-'d'te gl vt ve ---t s n g v k q m u,h m h,s kb / d d m u m h m s,h kb • d d q q q ll o iii vnkt____.vittfvdnddfdfndel skwwndt___~elpdfdkfnyt___vp ildid s eidr iqgviqglnds lidlekls ilktyikw e d i'e--y --i v n tyd k ee k -- t -- d -- e-- g t i-'r- r. ~ --rsrdf n q ~i v n__~sn ~. ---- the nucleotide and amino acid sequences were compared for possible recombination points. from this comparison we conclude that the japanese field strain kb is probably a recombinant. the $ protein of this strain is almost identical to $ of m , except for the region corresponding to amino acids to ( figure i) where the sequence is remarkably similar to d . this suggests a recombination event with two crossovers in this region. the alignment of the $ genes indicates a second potential recombinant: the british field isolate / . its s gene and most of the $ gene are almost identical to those of strain d . at the ' end of the $ sequence (at the lys- codon) the percentage of identical nucleotides switches from % upstream to % downstream. at exactly the same position the nucleotide sequence of / becomes highly similar to the d sequence: from % identity upstream to % downstream. the nucleotide identity between d and figure d is about the same on both sides of this putative crossover site: and %, respectively. the above comparisons are both time consuming and subjective. a method was sought to test and quantify these results more rigorously. this was achieved by comparing phylogenetic trees for each of five sections (windows) of the $ gene and its flanking sequences (see figure a ). the $ gene was divided into three sections. vaccine, vol. , december in vivo recombination of ibv: j.g. kusters et al. the first section (ii in figure b ) contained the '-part of the $ gene up to the putative kb crossover site (in the codon for ile- in figure ) . a second $ tree (iii in figure b ) was constructed for the part of the $ gene between the kb and / crossover sites (from ile- to lys- ). the '-border of this region did not exactly coincide with the second kb crossover site (in the ser-ll instead of the lys-ll codon) but changing this border had no significant effect on the topology. the third $ tree (iv in figure b ) was calculated for the region from the / crossover site (in the lys-ll codon) until the stop codon on position . sequences upstream and downstream from the $ gene were also used to construct phylogenetic trees. the tree based on s gene sequences (i in figure b ) has the same topology as an $ tree based on region ii in $ . kb has shifted to the d branch in a tree for region iii of $ . region iv of $ yields a tree in which / has shifted from the d to the d branch, while kb is again near m and m . finally a tree (v) constructed from the e gene sequences shows that, compared to tree iv, also strain d has shifted towards the d branch, and therefore may also be a recombinant. with the murine coronavirus mhv a high frequency of recombination was found both in vitro and in mouse brain after infection with a ts mutant of mhv strain a and wild type jhm-virus ' phylogenetic trees constructed from five different windows of the genomes of eight ibv strains were used to detect crossover events. a clear advantage of this method is that, due to its simplicity, large numbers of homologous genes can easily be screened. the data presented in this paper suggest that genomic recombination not only occurs under laboratory conditions but also plays an important role in the generation of new virulent strains; at least three ibv strains, all isolated from outbreaks, are potential recombinants. pre,)ious data ~ already indicated that the dutch field isolate v is also the product of a recombination. considering the limited number of sequences analysed so far, in vivo recombination of ibv seems to occur at a rather high frequency. this has implications for the use of ibv vaccines consisting of several serotypes of live attenuated ibv strains. these vaccines offer the best protection, but they may prove dangerous in the long term by the induction of new variants, not only by point mutations but also through recombination. in this respect vaccination protocols that combine ibv with other live attenuated viruses are equally suspect. they may create ideal conditions for non-homologous recombination which may result in the emergence of recombinant virus species with unwanted properties. the biology and pathogenesis of coronaviruses the classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in britain between and molecular epidemiology of infectious bronchitis virus in the netherlands induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus coronavirus ibv; partial amino terminal sequencing of spike polypeptide $ identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor of ibv strains beaudette and m antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies monoclonal antibodies to the $ spike and membrane proteins of avian infectious bronchitis virus strain massachusetts m t coronavirus ibv: virus retaining spike glycopolypeptide s but not $ is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection eyidence for a coiled-coil structure in the spike protein of coronaviruses coronavirus proteins: biogenesis of avian bronchitis virus virion proteins phylogeny of antigenic variants of avian coronavirus ibv occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands comparison of the spike precursor sequences of coronavirus ibv strains m and • with that of ibv beaudette cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the peplomer protein sequence of the m strain of coronavirus ibv and its comparison with beaudette strains a comprehensive sequence analysis program for the ibm personal computer confidence limits on phylogenies: an approach using the bootstrap highfrequency rna recombination of murine coronaviruses in vivo rna-rna recombination of coronavirus in mouse brain cloning and sequencing of the gene encoding the spike protein of coronavirus ibv evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes the authors are grateful to ms g.a.w.m. kremers and ms k.a. zwaagstra for experimental contributions and to dr j.a. lenstra for advice and help with computer programs. the use of the caos/camm computer key: cord- -rydgncys authors: wu, shuangsheng; su, jianting; yang, peng; zhang, haiyan; li, hongjun; chu, yanhui; hua, weiyu; li, chao; tang, yaqing; wang, quanyi title: willingness to accept a future influenza a(h n ) vaccine in beijing, china date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: rydgncys background: the present study aimed to estimate residents’ willingness to accept a future h n vaccine and its determinants in the general adult population in beijing, china. methods: we conducted a multi-stage sampling, cross-sectional survey using self-administered anonymous questionnaires from may to june, in . the main outcome variable was residents’ willingness to accept a future h n vaccine. logistic regression was used to identify the predictors of vaccination willingness. results: of the eligible participants, . % of beijing residents reported that they had not heard of h n . among those who had heard of h n , . % of the general adult population would be willing to accept a future h n vaccine, and approximately half of them reported ‘i am afraid of being infected by h n ’ and ‘h n vaccine can prevent infections’, and . % reported ‘my daily life is affected by h n ’. the variables that were significantly associated with a higher likelihood of reporting willingness were being younger adults (aged – years: or = . , % ci: . – . ; aged – years: or = . , % ci: . – . ), being farmers (or = . ; % ci: . – . ), being unemployed people (or = . ; % ci: . – . ), living in suburban areas (or = . ; % ci: . – . ), having ≥ children in the family (or = . ; % ci: . – . ), perceived risk in china (or = . ; % ci: . – . ), perceived susceptibility to disease (or = . ; % ci: . – . ), perceived negative effect on daily life (or = . ; % ci: . – . ), perceived effectiveness of vaccination (or = . ; % ci: . – . ), and recent uptake of influenza vaccine (or = . ; % ci: . – . ). conclusions: a great number of beijing residents had doubts about the vaccine’s effectiveness and were not concerned about disease risk, which were the factors affecting willingness to be vaccinated. targeted education programs on disease risk as well as vaccine’s effectiveness are needed to improve the willingness of vaccination for potential h n pandemic preparedness. avian influenza a (h n ) virus was first identified as a novel virus in eastern china in march [ ] . global attention was soon focused on the situation because of the increasing number of new cases and the high rate of death associated with the infections [ ] . as of february , , a total of human infections with the virus, including deaths, have been reported from mainland https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. china during the former four epidemics [ ] . current evidence suggests that this virus has not acquired the ability of sustained transmission among humans, but small clusters of infected cases involving healthcare workers have been observed previously [ ] . phylogenetic analyses have suggested there is a possible pandemic threat from new reassortment of influenza a (h n ) virus, emphasizing the importance of continuous surveillance and protective measures against epidemic spread [ ] . vaccination remains one of the most effective strategies in controlling epidemics, but promoting the vaccination uptake can be a difficult challenge for local governments [ ] . who has recommended several candidate vaccine viruses for the development of h n vaccines for the purpose of pandemic preparedness [ ] . although up to now (as of august ), no h n vaccines are commercially available [ ] , a phase i/ii trial suggests that the h n influenza vaccine was immunogenic and safe in adults [ ] . therefore, vaccination is a critical part of h n pandemic preparedness. beijing had a heavy burden of severe acute respiratory syndromes (sars) in and pandemic influenza a (h n ) in [ ] . during the h n pandemic, pandemic influenza vaccination was first provided to priority populations (e.g., older adults, public servants in key positions, students, teachers and people with chronic diseases) and then other persons in beijing, and it was proven to be an effective strategy in controlling epidemics [ ] . our previous study showed the vaccination coverage rate was relatively low within the general adult population of beijing, and the perceptions of not expecting to contract influenza was the predominant barrier to influenza vaccination [ ] . although beijing has only reported laboratory-confirmed cases of influenza a (h n ) and deaths as of august , a potential threat of h n pandemic has always been in beijing. therefore, preventive measures, including pandemic vaccination policy, should be prepared for possible h n pandemic. understanding the willingness to accept a future h n vaccine and its main related factors may enable policy makers to take measures for future vaccination coverage improvement. although several surveys have been conducted in southern china [ ] [ ] [ ] , only one of these studied a general population while the other two focused on food producers or live poultry traders. considering the diverse epidemic strength [ ] , income levels and healthcare access across china, public willingness to accept a future h n vaccine may vary by region. in the present study, we conducted a large population-based cross-sectional survey to estimate residents' willingness to accept a future h n vaccine and to identify its associated possible factors in the general adult population of beijing at the end of the second epidemic wave. beijing is the capital of china and the largest city in northern china. it is divided into districts, which are classified as urban and suburban districts according to the population density and local economic level. as of the census, the city had a population of nearly million [ ] . the target population was chinese adults living in beijing. the participants were classified into ten subgroups according to residence (urban or suburban) and different age groups ( - , - , - , - and ! years). the formula n = l a  p  ( À p)/d  deff was used to estimate the sample size per subgroup, based on an a error of %, the rate of residents' willingness to accept a future h n vaccine in the general population of beijing (p) = %, maximum permissible error (d) = . p, and the design effect of complex sampling (deff) = . [ , ] . we estimated a sample size of participants per subgroup. regarding subgroups, a no-answer rate of % and a rate of % participants who had not heard of h n , the optimal sample size for the present study was ( participants per subgroup  subgroups  .  . ). in this study, participants were recruited by a multistage stratified sampling approach [ ] [ ] [ ] . initially, three urban districts and three suburban districts were randomly selected to be sampled. from each selected district, five towns or streets were randomly selected. and then five communities or villages were randomly selected in each of these towns or streets. in total, committees or villages were confirmed as the survey locations. to meet the sample size requirement, about participants (about participants in each age group) needed to be selected from each survey location. we conducted the survey from may to june, in , at the end of the second epidemic wave of h n . within each survey location, all the households were randomly numbered according to the address numbers. well-trained interviewers from local centers for disease prevention and control visited the households individually according to the random numbers, and interviewed each adult within the households until a total of residents and about participants per age group were investigated in each survey location. because the family size of beijing residents was ranged from to , approximately to households were randomly selected for interview. before visiting a household, they made an appointment with the family. if all the residents in a household were not available for the first visit, re-visits would be made to the household. during the interviews, participants were asked to complete the questionnaire by themselves or with the help of interviewers if they had difficulty with reading or writing. the survey was carried out using a self-administered, anonymous questionnaire, which consisted of four sections: ( ) demographics information (gender, age, educational level, employment status, living area and number of children within the family); ( ) have you ever heard of avian influenza a (h n )? the response options were 'yes' and 'no'. if the response was 'no', the following questions from sections and were not required to answer. ( ) residents' willingness to accept a future h n vaccine if it is available with the response options of 'yes' and 'no'; and ( ) residents' perceptions regarding h n listed as follows: 'h n will remain in china'; 'h n is a serious disease'; 'i am afraid of being infected by h n '; 'my daily life is affected by h n '; 'h n vaccine can prevent infections' and 'i have accepted the seasonal influenza vaccine in the past year'. the six questions were close-ended, and the response options were 'yes' and 'no'. all the questions were based on evidence in the existing literature [ ] . the study approval was obtained from the institutional review board and human research ethics committee of beijing center for disease prevention and control (approval number: - , approval date: december , ). anonymity of the participants was guaranteed to participants, and agreement and informed consent from participants was required during the surveys. the main outcome variable was residents' willingness to accept a future h n vaccine. weighted analysis was conducted to calculate the standardized proportion of people who would be willing to accept a future h n vaccine, accounting for age and living area (urban or suburban) in beijing population, as reported in the census of beijing [ ] . descriptive analyses were initially performed to generate frequency distributions of the survey variables. differences among the subgroups were tested by pearson's chi-square test. logistic regression models were then performed to examine the factors associated with residents' willingness to accept a future h n vaccine. demographic characteristics of participants and residents' perceptions regarding h n were included as the independent variables. adjusted odds ratios with % confidence intervals ( % ci) for the variables were calculated. all the statistical tests were two-sided, with p value < . considered to be statistically significant. all the statistical analyses were carried out using spss version . (ibm corporation, new york, united states). fig. shows the framework for sample selection. in total, of the participants that we approached completed the survey, yielding a response rate of . %. of the eligible participants, ( . %) participants who had not heard of h n were excluded from the analysis of willingness to accept h n vaccine. finally, a total of participants who had heard of h n were included in the further analysis of willingness to be vaccinated. approximately half of the total participants were female (n = ) and lived in urban areas (n = ). the distribution of age was as follows: - : . % (n = ), - : . % (n = ), - : . % (n = ), - : . % (n = ), ! : . % (n = ). participants who had not heard of h n tended to be farmer, were older, and had lower education than those who had heard of h n (table ) . weighted proportions of participants who would be willing to accept a future h n vaccine after being standardized for age and living area, . % of the general adult population would be willing to accept a future h n vaccine. the standardized proportions were . %, . %, . %, . % and . % in the five age groups ( - , - , - , - and ! years), respectively. regarding the comparison between living areas, the proportions were . % and . % among urban and suburban residents, respectively. table shows perceptions regarding influenza a (h n ). a total of ( . %) participants reported 'h n is a serious disease', ( . %) reported 'h n will remain in china', and approximately half reported 'i am afraid of being infected by h n ' (n = ) and 'h n vaccine can prevent infections' (n = ). only . % of participants reported 'my daily life is affected by h n ' (n = ). . . logistic regression analysis for factors associated with residents' willingness to accept a future h n vaccine as shown in table , after adjustment for the potential confounding variables, the variables that were significantly associated with a higher likelihood of reporting willingness to accept a future h n vaccine were being younger adults (aged high population density makes beijing easy to be threatened by pandemics of sars, influenza, and other emerging respiratory infectious diseases. in this cross-sectional study, we found that a large proportion ( . %) of beijing residents had not even heard of h n at the end of the second epidemic wave. unlike the unforgettable experience of sars in , the emergence of h n had note: ''missing" indicates the number of respondents who did not answer this question. a other employees: factory workers, government employees, and employees from business and service industry. note: missing referred to ''how many people did not answer this question". not caused public panic yet; a previous study also showed this result [ ] . in addition, a study in china showed that the experience of h n pandemic in did not also result huge in society [ ] . this may be explained by the great progress in the mechanism for emergency responses to emerging infectious diseases after the sars outbreak in china. the current mechanism allowed china to successfully deal with the subsequent h n pandemic and h n epidemics [ ] . in a public health emergency, pandemic vaccination remains one of the most effective strategies in controlling epidemics, but promoting the vaccination uptake can be a difficult challenge for local governments [ ] . in this study, . % of the general adult population who had heard of h n would like to accept h n vacci-nation in the future. our estimate was comparable to that of hong kong during the epidemic of h n ( . %) [ ] . however, previous studies have shown that residents' willingness of future h n influenza vaccine uptake varied across different regions and sub-populations: . % among the general population of hong kong, % among the employees of food production of guangzhou and . % among live poultry traders of guangzhou, a city located in southern china [ ] [ ] [ ] . our estimate during the epidemic of h n was also consistent with some findings from a systematic review during the h n pandemic [ ] ( . % in the united kingdom [ ] , % in the united states [ ] and . % in australia [ ] ). moreover, the systematic review found that there was a lot of variation ( . - %) in respondents' willingness to accept table logistic regression analysis for the factors associated with residents' willingness of influenza a (h n ) vaccine uptake in beijing, china. willingness to take h n vaccine in the future h n pandemic vaccine across the ten studies [ ] . factors thought to contribute to willingness to receive the h n pandemic vaccine included personal risk perception, vaccination attitude, communications and information sources, access and demographic variables [ ] . the present study only estimated the willingness to be vaccinated, but not actual vaccination. most estimates of vaccination intention tend to be much greater than actual vaccine coverage estimates during the h n pandemic [ ] . although the beijing government launched mass vaccination campaigns during the second wave of the pandemic, the influenza vaccination coverage rates were relatively low ( . %) and did not increased significantly during the h n pandemic [ ] . for the above reasons, we can infer that the actual coverage of h n vaccination will be much lower than our estimate of . % in emergency mass-vaccination campaigns during an emergency. in this study, we found that younger adults, farmers, people waiting for employment and people living suburban areas reported more willingness to be vaccinated than others. in china, these people are more likely to expose to live poultry including raising backyard poultry and visiting poultry market, and thus perceive higher risk of h n infections [ ] , which may promote their willingness to accept a future h n vaccine. consistent with the results of our study, a study conducted during the h n pandemic suggested that a higher intention to accept future vaccination was observed in families with the present of children, which may be explained by a higher level of concern about pandemic influenza [ ] . our results demonstrated many related factors including severity of the public event, risk of infection, severity of disease, vaccination effectiveness and acceptance of previous vaccination, were predictors of the intention to be vaccinated, and the result is consistent with the previous studies [ , , , ] . in this study, only half of participants reported 'i am afraid of being infected by h n ' and 'h n vaccine can prevent infections', and . % reported 'my daily life is affected by h n '. the results showed that a large part of people in beijing had doubts about the vaccine's effectiveness and were not concerned about disease risk, which would lead to less willingness to accept h n vaccination. therefore, targeted education programs on disease risk as well as vaccine's effectiveness would be an effective strategy to help people make informed vaccination decisions. the present study has some limitations. first, as a crosssectional study using a self-administered questionnaire, selfreported data may have introduced information bias in data collection. second, the current study did not include 'i do not know' option, which could also lead to information bias. nevertheless, a large proportion of the participants who had not heard of h n and thus more likely to report 'i do not know' option were not included in the analysis of willingness to accept a future h n vaccine. in addition, during the survey, very few participants asked the interviewers whether there was an 'i do not know' option. thus, we believed that the participants who might otherwise have chosen 'i do not know' accounted for only a small proportion, if any, of the total participants. third, because the households but not individuals were selected at random in this study, the statistical effect of this further clustering would be to increase design effect and result in inappropriately narrow confidence intervals. fourth, the rates for uptake willingness may differ from season to season because of changing conditions such as mortality rates and epidemic strength, which suggests the need for continued monitoring in beijing. finally, the findings of this study are representative at the level of beijing, perhaps northern china, but considering the diverse epidemic strength, income levels and healthcare access across regions, our observations might not be generalized well to other countries or regions. this study demonstrated that there was a large proportion ( . %) of beijing residents who had not heard of h n . among those who had heard of h n , . % of the general adult population would be willing to accept a future h n vaccine in the future, and a large proportion of them had doubts about the vaccine's effectiveness and were not concerned about disease risk, which were the factors affecting residents' willingness to accept h n vaccination. therefore, health education programs targeted at increasing public perceptions towards disease risk and vaccine's effectiveness are warranted to improve the willingness of vaccination for potential h n pandemic preparedness. human infection with a novel avian-origin influenza a (h n ) virus clinical findings in cases of influenza a (h n ) virus infection epidemiology of avian influenza a h n virus in human beings across five epidemics in mainland china, - : an epidemiological study of laboratory-confirmed case series nosocomial transmission of avian influenza a (h n ) virus in china: epidemiological investigation possible pandemic threat from new reassortment of influenza a(h n ) virus in china vaccines for pandemic influenza world health organization. candidate vaccine viruses for avian influenza a (h n ) china food and drug administration. data query safety and immunogenicity of an inactivated cell culture-derived h n influenza vaccine in healthy adults: a phase i/ii, prospective, randomized, open-label trial estimates of the true number of cases of pandemic (h n ) safety and effectiveness of a h n vaccine in beijing influenza vaccination coverage rates among adults before and after the influenza pandemic and the reasons for non-vaccination in beijing, china: a cross-sectional study willingness of future a/h n influenza vaccine uptake: a cross-sectional study of hong kong community attitudes, practices and information needs regarding novel influenza a (h n ) among employees of food production and operation in guangzhou, southern china: a crosssectional study knowledge, attitudes and practices relating to influenza a(h n ) risk among live poultry traders in guangzhou city cluster of human infections with avian influenza a (h n ) cases: a temporal and spatial analysis release on major figures of the national population census in beijing, china factors associated with the uptake of seasonal influenza vaccination in older and younger adults: a large, population-based survey in beijing, china hygiene behaviors associated with influenza-like illness among adults in beijing, china: a large, population-based survey multistage sampling a cross-sectional study of pandemic influenza health literacy and the effect of a public health campaign public perceptions and reactions to h n in mainland china knowledge, attitudes and practices (kap) related to the pandemic (h n ) among chinese general population: a telephone survey from sars to h n : the mechanism of responding to emerging communicable diseases has made great progress in china acceptance of a pandemic influenza vaccine: a systematic review of surveys of the general public the impact of communications about swine flu (influenza a h n v) on public responses to the outbreak: results from national telephone surveys in the uk intent to receive pandemic influenza a (h n ) vaccine, compliance with social distancing and sources of information in nc why do i need it? i am not at risk! public perceptions towards the pandemic (h n ) vaccine the determinants of pandemic a/h n influenza vaccination: a systematic review human exposure to live poultry and psychological and behavioral responses to influenza a(h n ), china influenza vaccination coverage against seasonal and pandemic influenza and their determinants in france: a cross-sectional survey meta-analysis of the relationship between risk perception and health behavior: the example of vaccination factors associated with uptake of vaccination against pandemic influenza: a systematic review we thank the participants and colleagues from cdcs of dongcheng, tongzhou, xicheng, haidian, huairou, and changping district who interviewed the participants. the opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the institutions with which the authors are affiliated. the authors declare that they have no competing interests. key: cord- - vkag z authors: nakayama, tetsuo; sawada, akihito; yamaji, yoshiaki; ito, takashi title: recombinant measles aik-c vaccine strain expressing heterologous virus antigens date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: vkag z further attenuated measles vaccines were developed more than years ago and have been used throughout the world. recombinant measles vaccine candidates have been developed and express several heterologous virus protective antigens. immunogenicity and protective actions were confirmed using experimental animals: transgenic mice, cotton rats, and primates. the recent development of measles vaccine-based vectored vaccine candidates has been reviewed and some information on recombinant measles vaccines expressing respiratory syncytial virus proteins has been shown and discussed. the measles virus is a member of the genus morbillivirus, the family paramyxoviridae, order mononegavirales and consists of , nucleotides that encode six major structural proteins. the nucleoprotein (n), phosphoprotein (p), large protein (l), and genomic rna constitute the ribonucleoprotein complex (rnp) as transcription and replication units. two envelope spike glycoproteins, hemagglutinin (h) and fusion (f), are present on the surface of infectious particles and execute the process of virus attachment and cell fusion. the m protein contributes to viral assembly and maturation [ ] . measles virus was first isolated in by enders and peebles from a patient and the edmonston strain was further propagated in human kidney or alantoic fluid fibroblastic cells [ ] . the aik-c measles vaccine strain was developed from the wild-type edmonston strain through small plaque cloning in sheep kidney cells and chick embryonic cells at . • c [ , ] . the aik-c strain is one of the candidate vaccine strains for the expanded programme on immunization (epi) to overcome maternally conferred immunity, thereby resulting in a high seroconversion rate in young infants at months of age [ ] [ ] [ ] . over million doses have been administered mainly in japan, and no serious adverse events have been reported in post marketing surveillance. the aik-c strain shows optimal virus growth at • c with small plaques, but extremely poor or no growth at - • c, demonstrating temperature sensitivity (ts). these biological markers have been used to validate vaccine production and some responsible genes were identified [ , ] . the aik-c vaccine strain was developed from the edmonston strain and its full-length sequence was reported, in which amino acid substitutions were found relative to the edmonston wild-type [ ] . the recent development of molecular genomic analysis has allowed reverse genetics to be performed in order to generate the infectious measles virus [ ] . the reverse genetics of the aik-c vaccine strain was explored to investigate the specific genome regions responsible for its unique biological characteristics. f and ha protein expression experiments revealed that leu at position of the f protein was responsible for the formation of small plaques in vero cells and phe at position of the f protein of the edmonston strain induced large plaques in vero cells [ ] . infectious cdna clones having f leu and f phe were generated: a recombinant virus having f leu induced small plaques, whereas large plaques were generated by a virus having f phe. however, this position did not influence the ts characteristics [ ] . the replication and transcription of the measles virus are regulated by interactions between the n, p, and l proteins, and a measles mini-genome system, in which the luciferase reporter gene was inserted between the measles leader and trailer sequences, was developed to investigate transcription and replication activities [ ] . limited or no luciferase activity was observed when the p protein expression plasmid from the aik-c strain was used in the mini-genome assay at • c or higher temperatures, whereas the p expression plasmid from the edmonston strain did not influence luciferase activity at • c or higher temperatures. the p protein of the aik-c strain was responsible for the ts characteristics and pro at position of the p protein was a critical point for the ts phenotype based on the findings of a mini-genome assay using the chimerical p expression plasmids constructed from the aik-c and parental edmonston strains. a recombinant measles virus having pro at position of the p protein was generated, and showed the ts phenotype. however, a recombinant measles virus having leu at position of the p protein did not [ ] . currently available vaccines are categorized into two types: live attenuated and inactivated vaccines. inactivated vaccines principally induce humoral antibodies, whereas both humoral and cellular immune responses are induced by live attenuated vaccines. therefore, a live attenuated vaccine has clinical benefits in addition to strong immunity: a basically single or two-dose immunization provides long-term immunity with a relatively lower cost. clinical adverse events are more frequently observed with live vaccines than with inactivated vaccines because of the growth of the vaccine strain in the body. numerous difficulties are associated with developing a new live vaccine by conventional procedures through consecutive passages in primary cell cultures. immunogenicity and safety profiles are confirmed in experimental animal models. final availability is established in a large-scale phase iii clinical trial; however, the number of participants can be limited. many effective live attenuated vaccines are known to be effective and safe through a long history of clinical use. therefore, currently available live vaccine-based vectored vaccines are also expected to be theoretically safe and immunogenic. although a number of recombinant virus vectors derived from the vaccinia virus ankara strain, poxviruses, adeno-associated viruses, and human parainfluenza virus type iii have been developed, some virus vectors were not derived from the licensed vaccine strains, without clinical experience [ ] [ ] [ ] [ ] [ ] . there is no predictable biomarker relevant to the vaccine safety in experimental animals. thus, large-scale clinical trials should be conducted for these vectors in order to assure the safety of the vector. virus vectors derived from currently available vaccines are theoretically considered to be safe and effective. among the currently licensed vaccines, the yellow fever vaccine has been used as a live attenuated vaccine-virus vector for the development of recombinant japanese encephalitis, dengue, and west nile virus vaccines [ ] [ ] [ ] . the pre m + e region of the yellow fever vaccine was replaced by that of the other flaviviruses. of these, the yellow fever vaccinebased japanese encephalitis virus vaccine (chimerivax-je) has been licensed. the live attenuated measles vaccine is a more popular than the yellow fever vaccine. it induces efficient humoral and cellular immunity and its long clinical use has guaranteed its safety [ ] . in addition, the measles virus theoretically replicates in the cytoplasm and there is no evidence to suggest that the genome is integrated into host dna. recent developments in the reverse genetics of the measles virus have allowed recombinant measles virus cdna to be artificially manipulated, thereby, contributing to a better understanding of the mechanisms underlying virus replication, transcription, and pathogenesis. the live attenuated measles vaccine has been investigated for the recombinant virus vector besides the yellow fever vaccine. several strains are now available throughout the world and attenuation mechanisms are supposedly different for each strain [ ] . two groups are now extensively working on recombinant measles vaccine candidates using licensed measles vaccine strains from dr. naim, berna biotech [ ] [ ] [ ] [ ] , dr. tangy, pasteur institute [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and others [ , ] . their construction strategies, targets, and experimental models are summarized in table . they targeted the unmet needs of the vaccines against hiv, sars corona, west nile, dengue, nipah, chikungunya, and hepatitis c viruses. through conventional strategy, effective live or inactivated vaccines were not developed against these targets. the targeted genome regions were inserted at the n/p, p/m, or h/l junctions. most popular insertion site is at the p/m junction. when a heterologous gene was inserted at the n/p junction, amounts of mrna of the p gene decreased, causing poor virus growth. mourez et al. [ ] reported a different strategy by which the f and h genome regions of the measles virus were replaced by the chimeric dna fragment of the simian immunodeficiency virus (siv) gp genome fused with the cytoplasmic region of measles f protein genome, which interacted with the m protein. the immunogenicity of the recombinants was examined in transgenic mice expressing human cd with the deletion of the ifn-␣/␤ receptor or non-human primates. there is no proper experimental animal for evaluating immune responses for measles. transgenic mice depleted of ifn systems are not appropriate to investigate the immunogenicity following administration of recombinant measles-vectored vaccine candidates. in our preliminary experiments, cotton rats (sigmodon hispidus) were susceptible for measles virus infection. measles virus genome was detected from regional draining lymph nodes and virus was isolated. neutralizing antibodies were produced in cotton rats three weeks after immunization of aik-c measles vaccine strain [ ] . the rsv vaccine has been anticipated for many years, among the many unmet needs of vaccines. rsv is one of the most common causes of lower respiratory tract infections in infants and young children worldwide. the peak age for serious rsv infections is less than months and most children have had an rsv infection by two years of age. serious complications have been reported especially in newborn babies born prematurely with lung diseases, or those with congenital heart diseases. rsv also causes lower respiratory tract infections in the elderly and immuno-compromised hosts, as well as in the pediatric population [ , ] . the formalin-inactivated rsv vaccine (fi-rsv) was developed after the discovery of rsv. however, it failed to induce protective immunity and resulted in a paradoxical tragedy of increasing the severity of subsequent rsv infections [ ] . since then, many challenges were performed. the classical biological selection of cold-adapted or ts mutants or reverse genetically engineered vaccine candidates were not appropriately attenuated, and subunit vaccines of the fusion (f) or glycoprotein (g) did not provide longterm protective immunity [ ] . the reasons for the serious outcome of fi-rsv were extensively investigated, and fi-rsv was found to induce a skewed th response. a balanced th and th response is essential for the development of a rsv vaccine [ ] . therefore, livevectored vaccine candidates have been investigated using human parainfluenza, bovine parainfluenza, and sendai viruses [ ] . the measles vaccine-vectored rsv vaccine has also been reported. mok et al. [ ] reported recombinant measles virus cdna with the insertion of rsv f at the p/m junction and epstein-barr (eb) virus gp protein at the h/l junction. the infectious virus was recovered and induced neutralizing antibodies against rsv but not against ebv. the experimental design for the construction of recombinant cdna from the aik-c strain and immunogenicity are shown in fig. . the asc i restriction enzyme site was introduced by adding the ggcgcg sequence at the genome position of the p/m junction. the r and r sequences were added, and a cloning vector was constructed from nucleotide position of the sac ii site and at of the ecot i site. the open reading frames of the heterologous virus genome encoding the protective antigen were cloned at the restriction enzyme sites, nco i and not i. they were introduced into full-length infectious cdna using sac ii and pac i sites. they were then transfected into t cells together with helper plasmids expressing the measles n, p, and l proteins under the control of t rna polymerase. infectious virus particles were rescued through blind passages in vero cells at . • c in % co . recombinant measles viruses expressing the fusion (f) or glyco (g) proteins of rsv subgroup a (mvaik/rsv/f or mvaik/rsv/g) were developed. these viruses induced cross protective neutralizing antibodies (nt) against rsv subgroups a and b in cotton rats. the infectious virus was not recovered from the lung tissues of the cotton rats immunized with recombinant viruses after the challenge with rsv subgroup a, but the protective effects were not sufficient after the challenge with rsv subgroup b [ ] . although no detectable rsv was recovered from immunized cotton rats after the challenge, very mild inflammatory pneumonitis was observed. recombinant measles viruses expressing rsv m - or np (mvaik/rsv/m - and mvaik/rsv/np) were generated, together with mvaik/rsv/f. no detectable neutralizing antibody against rsv was induced in cotton rats immunized with recombinant measles virus expressing m - or np, whereas high titers of neutralizing antibody were induced after immunization with those expressing f or g protein. mvaik/rsv/m - and mvaik/rsv/np induced strong ctl responses, increasing the number of cd + ifn-␥ + cells in spleen cells stimulated with the np, m , and f peptides, or inactivated rsv antigen [ ] . no infectious virus was recovered from lung homogenate following the challenge. a histological examination of the lung tissues demonstrated a significant reduction in inflammatory reactions without alveolar damage, despite negative for neutralizing antibodies. both neutralizing antibody and ctl responses are essential for the protection from infection and mitigating the inflammatory responses. when considering a clinical usage of recombinant measles vaccine candidates expressing rsv antigen in young infants, growth inhibition of vaccine virus was supposed because of maternally conferred immunity. when cotton rats were immunized beforehand with different doses of aik-c vaccine strain, a significant antibody response was demonstrated after immunization with mvaik/rsv/f in cotton rats with pa titer ≤ . considering the protection for newborn or young infants in clinical setting, adult immunization of recombinant virus (mvaik/rsv/f) would be expected before pregnancy. but, the experimental model is hardly developed because of different immunological system in rodents. due to its clinical use for several decades, the measles vaccine is considered to be safe and immunogenic, thereby providing longterm memory, and maybe a promising platform for developing a new vaccine. several issues remain to be resolved: the acceptability of dna-engineered vaccines in public, clinical guidelines for safety profiles, and regulatory guidelines for the manufacture of these vaccines. measles virus measles vaccines studies on the modification of the live aik measles vaccine i. adaptation of the further attenuated aik measles virus (the strain of aik-l ) to chick embryo cells development and characteristics of live aik-c measles virus vaccine: a brief report serological effects of edmonston-zagreb schwarz, and aik-c measles vaccine strains given at ages - or - months immunization of and month old infants with aik-c, edmonston-zagreb, eningrad- and schwarz strains of measles vaccine l comparison of aik-c measles vaccine in infants at months with schwarz vaccine at months: a randomized controlled trial in ghana molecular cloning and complete nucleotide sequence of genomic rna of the aik-c strain of attenuated measles virus rescue of measles viruses from cloned cdna leucine at position of the aik-c measles virus vaccine strain fusion protein is responsible for reduced syncytium formation the phosphoprotein of attenuated measles aik-c vaccine strain contributes to its temperature-sensitive phenotype modified vaccinia virus ankara as antigen delivery system: how can we best use its potential? pox viral vaccine approaches novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques evaluation of the replication and immunogenicity of recombinant human parainfluenza virus type vectors expressing up to three foreign glycoproteins safety and immunogenicity of intranasal murine parainfluenza virus type i (sendai virus) in healthy human adults chimeric live, attenuated vaccine against japanese encephalitis (chimerivax-je): phase clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactive japanese encephalitis antigen a live, attenuated recombinant west nile virus vaccine development of sanofi pasteur tetravalent dengue vaccine attenuated measles virus as a vaccine vector genetic characterization of measles vaccine strains induction of neutralising antibodies and cellular immune responses against sars coronavirus by recombinant measles viruses recombinant measles virus-hpv vaccine candidates for prevention of cervical carcinoma recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (hiv- ) induce cellular and humoral immune responses recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses protection from sars coronavirus conferred by live measles vaccine expressing the spike glycoprotein immunogenicity of a recombinant measles-hiv- clade b candidate vaccine measles vaccine expressing the secreted-form of west nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of west nile virus infection pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses live attenuated measles vaccine expressing hiv- gag virus like particles covered with gp v v is strongly immunogenic a chimeric measles virus with a lentiviral envelope replicates exclusively in cd +/ccr +cells a recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge broadly neutralizing immune responses against hepatitis c virus induced by vectored measles viruses and a recombinant envelope protein booster analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model association between respiratory syncytial virus outbreaks and lower respiratory tract deaths of infants and young children respiratory syncytial virus infection in adults progress in understanding and controlling respiratory syncytial virus: still crazy after all these years evaluation of measles vaccine virus as a vector to deliver respiratory syncytial virus fusion protein or epstein-barr virus glycoprotein gp aik-c measles vaccine expressing fusion protein of respiratory syncytial virus induces protective antibodies in cotton rats recombinant measles viruses expressing respiratory syncytial virus proteins induced virus-specific ctl responses in cotton rats key: cord- -mhx e y authors: fang, xinkui; zhang, shikuan; sun, xiaodong; li, jinjin; sun, tao title: evaluation of attenuated vsvs with mutated m or/and g proteins as vaccine vectors date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: mhx e y vesicular stomatitis virus (vsv) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. in experimental animals (primates, rats, and mice), vsv has been shown to lead to neurotoxicities, such as hind limb paralysis. the virus matrix protein (m) and glycoprotein (g) are both major pathogenic determinants of wild-type vsv and have been the major targets for the production of attenuated strains. existing strategies for attenuation included: ( ) deletion or m r substitution in the m protein (vsvΔm or vsvm r, respectively); ( ) truncation of the c-terminus of the g protein (gΔ ). despite these mutations, recombinant vsv with mutated m protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant vsv with truncated g protein at high dose. thus, a novel recombinant vsv (vsvΔm -gΔ ) as well as other attenuated vsvs (vsvΔm , vsv-gΔ ) were produced to determine their efficacy as vaccine vectors with low pathogenicity. in vitro studies indicated that truncated g protein (gΔ ) could play a more important role than deletion of m (Δm ) for attenuation of recombinant vsv. vsvΔm -gΔ was determined to be the most attenuated virus with low pathogenicity in mice, with vsv-gΔ also showing relatively reduced pathogenicity. further, neutralizing antibodies stimulated by vsv-gΔ proved to be significantly higher than in mice treated with vsvΔm -gΔ . in conclusion, among different attenuated vsvs with mutated m and/or g proteins, recombinant vsv with only truncated g protein (vsv-gΔ ) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. these properties make vsv-gΔ a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease. vesicular stomatitis virus (vsv) is a single-strained negativesense rna virus that has been widely used as a vector for vaccine development [ , ] . recombinant vsv can accommodate large and multiple foreign genes in its genome that are expressed at high levels [ ] and confer advantages over other rna viral vectors. due to its potent capabilities in triggering cellular, humoral, and mucosal immunities in animals, even after a single administration, recombinant vsv has been studied as a vaccine vector not only for preventing vesicular stomatitis disease in livestock [ ] , but a number of human pathogens including: influenza virus, ebola virus, marburg virus, human immunodeficiency (hiv) virus, severe acute respiratory syndrome (sars) virus, and hepatitis c virus [ ] [ ] [ ] [ ] [ ] . however, vsv is a notoriously infectious agent that not only produces acute disease, such as vesicular lesions in cattle, swine and horses, but neurotoxicity in experimental animals, including primates and mice [ ] [ ] [ ] . therefore, modifications are needed to improve the safety of vsv before it can be applied clinically as a replication competent vector. vsv genomic rna is transcribed into five capped and polyadenylated mrnas by the viral rna-dependent rna polymerase. the mrnas encode five structural proteins: nucleocapsid protein (n); phosphoprotein (p), which is a cofactor of the rna-dependent rna polymerase (l); matrix protein (m); and attachment glycoprotein (g) [ ] . m and g proteins are both primary pathogenic determinants of vsv [ , ] . the m protein is a multi-functional protein involved in virus assembly, budding and pathogenesis [ , ] , and capable of inhibiting the transport of host mrnas out of the nucleus significantly inhibiting type i interferon (ifn) signaling [ ] . the g protein is responsible for viral binding to the host receptor and entry of vsv into host cells and its cytoplasmic domain is - x/$ -see front matter © elsevier ltd. all rights reserved. doi: . /j.vaccine. . . considered to play an important role in viral budding and packaging [ ] [ ] [ ] . to date, several strategies have focused on the m or g proteins for the generation of attenuated vsv. it was reported that deletion of methionine residue (vsv m ) or m r substitution (vsvm r) within the m protein can lead to attenuation due to the potent induction of type i ifn [ , ] . another attenuation strategy is dependent upon truncation within the c-terminal region of the g protein (vsv-g ) that significantly impacts viral budding and packaging efficiency [ ] . in vivo, however, vsv m protein mutant proved to be only moderately attenuated in experimental infections [ , ] , whereas there is currently no information available if recombinant vsv with truncated g protein is safe or not when animals challenged with high dose of the mutant virus. the current study developed a novel recombinant vsv (vsv m -g ) with mutations in both m and g proteins, including deletion of methionine in the m protein and a truncation of amino acids in c-terminal region of the g protein. it was hypothesized that vsv m -g could combine the advantages observed for the m and g mutations to produce a safer and more effective vaccine vector. furthermore, for the first time, different attenuated vsvs with mutated m and/or g proteins were comprehensively compared in vitro and in vivo. based on pathogenicity and capabilities to stimulate potent immune responses, we aimed to identify a suitable recombinant vsv vaccine vector and vaccine candidate for preventing vesicular stomatitis disease. the recombinant vsvs rescued in our study were based on the infectious clone, vsv indiana [ ] . the plasmid pvsv m was kindly provided by prof. john bell (univ. of ottawa, canada). the pvsv xn and helper plasmids, pbs-n, p, l, were provided by prof. glen barber's lab (univ. of miami, usa). the pvsv-g and pvsv m -g were constructed based on pvsv xn and pvsv m , respectively. in the constructs, the wild-type vsv g protein cytoplasmic tail ( aa) was truncated with only one arginine residue remaining in the tail (g ) (fig. ). the forward primer used for polymerase chain reaction (pcr) amplification of g was: -ccggagcgctatgaagtgccttttgtactta- (underlined indicates the mlui restriction site), and the reverse primer was: -cc-ggctcgagcgtgatatctgttagtttttttcatacctagcaggatttg-agtcattatcgggagaaccaagaatag- (underlined indicates the xhoi restriction site and bold residues indicate the two tandem repeat stop codons). to construct pvsv-g or pvsv m -g , vsv g gene of pvsv xn or pvsv m was substituted with g by restriction digestion with mlui and xhoi. all the start/stop signals for viral gene transcription were preserved. a schematic representation of pvsv m -g is shown in fig. . recovery of recombinant vsvs from the infectious clones was performed as previously described [ ] . briefly, co-transfection of vsv constructs (pvsv m , pvsv-g , pvsv m -g ) with helper plasmids, pbs-n, p, and l, was performed into bhk cells infected with a recombinant vaccinia virus (vtf - ) expressing t rna polymerase. at h post-transfection, culture supernatants were collected and filtered through a . m filter into fresh bhk cells. cells were checked daily. if typical cytopathic effect (cpe) was observed - days after vsv infection, supernatants were collected and viruses were plaque-purified in vero cells. individual plaques were isolated and seed stocks were amplified in bhk cells. recombinant viruses were concentrated by ultracentrifugation at , rpm/min for h and frozen at − • c. viral titer was determined by plaque assay performed in vero cells. vsv xn with wild-type g (wt g) and m proteins was prepared from the stock kept in our laboratory. to identify rescued recombinant vsvs, western blot was used to identify typical bands for l, g, n/p, and m proteins using specific convalescent sera from vsv xn -infected mice. in short, confluent bhk cells were infected with vsv m , vsv g or vsv m -g at a multiplicity of infection (moi) of . vsv xn was used as a control. at h post-infection (p.i.), cells were lysed in buffer containing % ␤-mercaptoethanol, . % np- , and % sodium dodecyl sulfate (sds). proteins were separated by % sds polyacrylamide gel electrophoresis (page) and transferred to polyvinylidene fluoride (pvdf) membrane (thermo scientific, rockford, il, usa) in a mini trans-blot electrophoretic transfer cell (bio-rad, hercules, ca, usa). sera from a vsv xn infected mouse was used as the primary antibody at a dilution of : with horseradish peroxidase-conjugated goat anti-mouse igg as the secondary antibody at a dilution of : (santa cruz, ca, usa). target bands were observed using west pico chemiluminescent substrate (thermo scientific) and exposed to kodak biomax mr film (kodak, rochester, ny, usa). attenuation of virus could be characterized by analyzing plaque formation sizes in cells [ ] . the a cell line was used as a type i ifn signaling competent cell line [ ] , whereas vero cells were used as an incompetent cell line [ , ] . plaque formation experiments were set up in these cells to identify the role by type i ifns in viral attenuation. briefly, % confluent a or vero cells in -well plates were infected with optimally diluted vsv m -g ,vsv m , vsv g , or vsv xn and then covered with low melting temperature agar (invitrogen, carlsbad, ca, usa) after rinsing with phosphate buffered saline (pbs). at h p.i., % crystal violet was used to stain vero cells, whereas a cells were stained h p.i. plaques were first scanned with gel imager (tanon- r, tanon, shanghai, china) under bright light and then individual plaques were viewed and photographed under × magnification using a microscope (nikon, tokyo, japan) equipped with a digital camera. the mean plaque size was determined by measuring the area of each plaque in each group using nikon nis-elements br software (nikon). the human prostate cancer cell line, pc , was used as another cell line with competent type i ifn signaling [ ] . at % confluency, pc cells in -well plates were infected with viruses at a low moi of . . after h of absorption, the inoculum was removed and cells were washed three times with pbs, fresh dulbecco's modified eagle's medium (dmem supplemented with % fetal bovine serum) was added, and the infected cells were incubated at • c. aliquots of cell culture supernatants were removed at , , and h p.i. viral titers in supernatants were detected by plaque assays in vero cells. the harvests at h p.i. were also assayed for ifn-␤ level using the human interferon-␤ elisa kit (r&d systems, minneapolis, mn, usa). specific pathogen free (spf) female balb/c mice (∼ g body weight) were purchased from shanghai slac experimental animal company (chinese academy of sciences, china) and divided into five groups ( per group) inoculated intranasally with vsv m -g , vsv-g , vsv m , vsv xn , or pbs under light anesthesia with ketamine-xylazine. the inoculated dose of different vsvs was plaque-forming units (pfu) in l of pbs; the highest dose that could be prepared by ultracentrifugation for vsv-g or vsv m -g . animal body weight losses and survival were monitored every day until days p.i. all animal studies were performed in accordance with a protocol approved by the shanghai jiaotong university experimental animal center. since wild-type vsv possesses neurotropism that could lead to serious neurotoxicities in animals, recovery of vsv in brain tissues was performed to identify the associations of pathogenesis with viral replications in host. in short, spf mice were divided into five groups and were challenged intranasally with vsv xn , vsv m , vsvg or vsv m -g at a dose of pfu, and the control group with pbs. in each group, two mice were sacrificed at and days post-inoculation and their brains were removed for viral detection. tissues were weighed in ml cell frozen vials, quickly frozen in liquid nitrogen and then kept under − • c. for viral assay, tissues were thawed and suspended in ml pbs and then completely homogenized using a dounce homogenizer. the homogenized tissues were centrifuged at , rpm for min and the suspensions were serially diluted in pbs. viral titers were determined by standard plaque assays in vero cells, as described above. plaques were observed and counted after h incubation. immune responses induced by vsv m -g or vsv-g were evaluated in balb/c mice (∼ g each). female spf balb/c mice were divided into three groups and immunized with vsv m -g , vsv-g or pbs as the control. animals in vsv m -g or vsv-g treated groups were further divided into two subgroups (five per subgroup) that were housed in isolation hutches and intranasally inoculated once with viruses at doses of pfu or pfu in l pbs. blood was collected from anesthetized mice by retro-orbital bleeds before and days after vaccinations. the vaccinated mice were then challenged with vsv xn . blood was allowed to clot at room temperature and sera were collected after centrifugation, and stored at − • c for neutralization assays. sera from animals were heat inactivated at • c for min. those from vsv vaccinated animals were serially diluted two-fold in dmem starting at : . for determination of neutralizing antibody titer, l of the diluted sera were then mixed with pfu vsv xn in l dmem. the mixtures were incubated for h at • c before being added to - % confluent bhk cells. cells were incubated at • c for at least - days and checked for cpe. additionally, l sera from pbs treated animals were directly combined with pfu/ l vsv xn and treated as for other sera. for titer determination, the reciprocal of the dilution giving a % inhibition of cpe was recorded. at days after immunization, all mice were intranasally administered pfu vsv xn in l of pbs. animals were observed every day to calculate survival curves and body weight loss, as previously described. plaque areas, ifn-␤ concentrations, and body weight loss among different groups were compared by student's t-test, with a twotailed distribution using the statistical features in microsoft excel (microsoft, redmond, wa, usa). all viruses rescued in the current study were based on the vsv indiana infectious clone [ ] . to construct safer recombinant vsv, vsv m -g was successfully generated that, for the first time, incorporated double mutations in m and g proteins. the novel virus and other attenuated vsv (vsv m , vsv-g ) were identified through western blotting with convalescent serum from mice infected with vsv xn was used as the wild-type vsv control. bands representing vsv structural proteins were identified (fig. ) . it was proved that although length of g protein cytoplasmic domain was critical for viral budding and packaging [ ] , we did find that with only one arginine residue remaining in the g protein cytoplasmic domain, vsv could still be rescued. in comparison with wild-type vsv g protein, a low molecular weight band presumed to be the g was detected (fig. ). viral rna of the different recombinant vsvs (vsv m -g , vsv m , and vsv-g ) were extracted and reverse-transcription (rt)-pcr was used to amplify viral m or g genes. the pcr products were sequenced for mutations occurring in m and g genes (data not shown). two methods were used to assess attenuation of vsv m -g , which included plaque formation size and multi-cycle growth curves in type i ifn signaling competent cells (a , pc ) [ , ] or incompetent cells (vero) [ , ] . the results were compared to those produced by other recombinant vsvs. crystal violet stained plaque sizes were determined for vero or a cells infected with different vsvs (figs. a and a) . in vero cells, the average plaque sizes (n = ) between vsvs with wt g (vsv xn and vsv m ) were similar (p > . ), but both were significantly larger than those containing the g mutation (vsv-g or vsv m -g ) (p < . ; fig. a and b) . average plaque sizes for vsv-g and vsv m -g were comparable. these data indicated that, in type i ifn signaling incompetent vero cells, g but not m was involved in vsv attenuation. in a cells, the average plaque areas produced by vsv xn were much larger than those by vsv m , however, areas by vsv m was also larger than vsv-g (p < . ; fig. a and b) . the smallest and turbid plaques were formed by vsv m -g in a cells and were difficult to detect. based on these results, both g and m assist the attenuation of vsvs in type i ifn signaling competent a cell and the attenuation tendencies as follows: vsv xn > vsv m > vsv-g > vsv m -g . pc was also used in the current study as a type i ifn signaling competent cell line [ ] . to quantify the relationship between amount of vsv-induced type i ifn and viral replication titers, multicycle growth curves were performed with inoculation of viruses at low moi of . . as shown in fig. a , titers of vsv m -g reached the highest levels at h p.i., but only around × pfu/ml and decline thereafter. vsv m also reached the ifn-␤ levels in vsv-treated pc cells were quantified at h p.i. to identify the role by type i ifns. in different recombinant vsv-infected pc cells, induction of ifn-␤ was inversely correlated with viral replication titers. as shown in fig. b , the high levels of ifn ␤ were induced in m vsv-treated cells. in vsv m infected cells, ifn-␤ concentrations reached ∼ pg/ml, but the highest replication titer was ∼ orders of magnitude lower than for vsv xn . although ifn-␤ production in vsv m -g treated cells was as low as ∼ pg/ml, this was still significantly higher than vsv xn (p < . ). ifn-␤ detected in the supernatants of vsv-g treated cells was below the limit of detection for the assay. therefore, viral attenuation showed the following trends: vsv xn > vsv m > vsv-g > vsv m -g . importantly, the fact that m led to attenuation of vsv through the induction of antiviral ifns was proved again in our study. based on the above data, both g and m were shown to be involved in the attenuation of vsv, however, g could play a more important role than m . regardless, the double mutation vsv, vsv m -g , showed the most significant attenuation in vitro. the current study investigated the pathogenesis of vsv m -g in balb/c mice and compared with other attenuated vsv or vsv xn as the wild-type virus control. inoculation of vsv through intranasal has been proven to be the most sensitive way to evaluate pathogenesis by vsv [ , ] . a challenge dose of - pfu wild-type vsv has been previously shown to result in significant mortality in balb/c mice [ ] . therefore, in our studies, spf balb/c mice were inoculated intranasally with different recombinant vsvs at a dose of pfu/ l; the maximum dose that could be prepare for concentrated vsv-g or vsv m -g in a l volume. body weight and survival curve were used as indications of pathogenesis and monitored every day for days p.i. as shown in fig. a , the body weight of naïve mice treated with vsv m -g or vsv-g increased steadily post infection without death, similar to the pbs group. however, in the vsv xn treated group, mice lost significant body weight (∼ %) and neurotoxicities were evident in some animals ( fig. a and c) with all animals dying at around days p.i. (fig. b) . vsv m showed moderate attenuation compared with vsv xn , but infected mice still showed significant loss of body weight with % mortality. wild-type vsv possesses neurotropism in many animals, including primates, rats, and mice that can lead to serious neurological deficits, such as hind limb paralysis. to identify the association of pathogenesis with viral replication, naïve mice were inoculated intranasally with different vsvs at a dose of pfu. viruses isolated from the brains of animals were screened by plaque assay at and days p.i. as shown in table , vsv xn treated mice showed viral titers up to × pfu/g at days p.i and . × pfu/g at days p.i. although much lower than vsv xn , viral titers detected in brain tissues from vsv m infected mice were determined to be . × pfu/g at days p.i., but no virus was detected at days p.i. in vsv m -g and vsv-g treated mice, no virus could be detected in brain tissues at the two time points, which could explain why animal body weight kept steadily increased over the duration of the study in these groups. therefore, this study showed that virulence of different vsvs was closely related to replication levels in the target organ, namely the brain. vsv m was not as safe as those with truncated g proteins in vivo. vsv m -g and vsv-g were attenuated enough as vaccine vectors. recombinant vsvs with truncated g protein (vsv m -g , vsv-g ) have been proved to be significantly attenuated both in vitro and in vivo, whereas the attenuation of the viral vector was often associated with the loss of vector immunogenicity. therefore, an ideal viral vector should retain both essential characteristics to be considered safe and effective. to evaluate protective immunity stimulated by vsv m -g or vsv-g in vivo, spf mice were immunized at different doses and then challenged with a lethal dose of vsv xn . blood was taken before and days post immunization for neutralization antibody assays. as shown in fig. , the vsv m -g immunized group immunized with pfu showed survival of all animals after challenge (fig. a) , however, mice still lost body weight up to ∼ %. at an immunization dose of pfu, % of animals died at around days post-challenge and all animals suffered serious body weight loss up to % and recovered very slowly (fig. b) . in mice immunized with vsv-g at doses of or pfu, the body weights of all animals increased steadily after challenge with no death and significant body weight loss. all animals in pbs control group died at days post-challenge (fig. c) . therefore, these data suggested that vsv-g could stimulate more potent protective immunities than vsv m -g . to further characterize the protective response, neutralization antibodies developed in the vaccinated animals were assayed. as shown in table , no antibody titer could be detected in animals before vaccination and pbs treated mice. it was determined that the antibody titer generated by immunization with vsv-g against the parental, vsv indiana , was significantly higher than those produced by vsv m -g immunization. in vsv-g groups, antibody titers were - at a vaccination dose of pfu and - at pfu. however, in the vsv m -g group with a dose of pfu, antibody titer was only - and all mice with a titer of died post-challenge (fig. b) indicating that the titer was unable to protect animals. moreover, even animals vaccinated at pfu, antibody titer only reached - . based on these results, it could be concluded that vsv-g could stimulate a more potent protective humoral immune response than vsv m -g , which might be due to the extreme attenuation of vsv m -g in vivo. as a result, vsv-g may attain an ideal balance between pathogenesis and stimulating a protective immune response and with the potential as a vaccine vector and vaccine candidate for vesicular stomatitis disease. replication-competent viruses with little or no pathogenesis have been important alternatives for developing vaccines or vaccine vectors due to their capabilities in stimulating potent and comprehensive immunity in vivo. however, safety and efficacy are equally important criteria. traditionally, the isolation of spontaneously attenuated virus in host or serial passages of wild-type virus in a non-natural host were important methods to acquire attenuated viruses. with the development of molecular technology, the construction of recombinant viruses based on reverse genetics, have become common for developing novel viral vectors. vsv is a promising vector for both vaccination and oncolysis, but still has not been applied clinically. one of the main reasons lies in its potential pathogenesis in humans and animals. rearrangement of structural protein genes [ ] and mutations in m or g proteins are existing strategies to generate attenuated vsv that are replication competent. our study focused on the two pathogenic proteins of vsv, m and g proteins, for the purposes of: ( ) making a safer vsv; ( ) table recombinant vsvs recovered from brain tissues of mice (pfu/g). naïve mice were inoculated with recombinant vsvs including vsvxn , vsv m ,vsv-g ,vsv m -g or pbs as a control. the brains of two mice were removed at or days post-inoculation. homogenized brains were assayed for viral titers and the mean values were calculated. nd: not detected. viral titers in mice brain (pfu/g) vsv m was attenuated in vivo. on the other hand, vsv is an enveloped virus that is released via budding from host cell membranes. previous studies have noted that the length of cytoplasmic domain on vsv g, not the specific sequence, was required for efficient viral budding [ ] . the real role of truncated g protein in attenuation of vsv has still not been identified, although it was reported that vsv c-terminal truncated g proteins acquired complex oligosaccharides ∼ -fold slower than for wt g protein and displayed a reduced rate of transport from the endoplasmic reticulum to the golgi apparatus, and presumably to the cell surface [ ] . through in vitro assays in type i ifn signaling competent cells/incompetent cells, both m and g were proven to be important for vsv attenuation. in vero cells, g but not m , was involved in vsv attenuation. recombinant vsv with g was shown to form significantly smaller plaques than virus with wild-type g, whereas plaque sizes between vsv xn and vsv m or those between vsv-g and vsv m -g were similar, no matter whether m protein was mutated or not. however, in a cells with effective type i ifn signaling, attenuation tendencies were demonstrated follow these tendencies: vsv xn > vsv m > vsv-g > vsv m -g . this tendency was also proven in pc cells through multi-cycle growth curve characterization. the replication titers of different vsvs were shown to be inversely correlated with the expression levels of type i ifns. vsv m -g formed the smallest plaque in a cells and lowest titer in pc cells among all vsvs tested in the current study due to the double mutations in m and g proteins. this study developed two main conclusions both in vitro and in vivo studies: ( ) both g and m mutations assisted attenuation of vsv, and ( ) g could play a more important role than m for attenuation. therefore, vsv m was not suitable as a vaccine vector due to its potential pathogenesis in animals and safety is the most important criteria for developing a vaccine or vaccine vector. on the other hand, vsv m may be suitable for virotherapy, as many studies have attempted [ , ] , since, ( ) efficacy is a priority as an antitumor drug and ( ) many types of tumor cells are type i ifns signaling defective [ ] . therefore, as an oncolytic virus, vsv m could replicate selectively and potently in tumor cells, but rarely in normal cells with competent type i ifn signaling. an ideal replication competent vaccine vector should possess a suitable balance between pathogenesis and capability to stimulate immunity effectively. both vsv-g and vsv m -g have demonstrated significant attenuation compared to vsv xn or vsv m , which are considered safe as vaccine vectors. however, we sought to determine which attenuated virus would work better in stimulating potent protective immunities that are critical for their clinical applications. to evaluate protective immunities stimulated by vsv-g and vsv m -g , animals were immunized with different doses of viruses and then challenged with a lethal dose of vsv xn , which is the "gold standard" to evaluate efficacy of a vsv vaccine. as shown in the current study, vsv-g could stimulate more potent protective immunities than vsv m -g . of note, vsv-g treated animals inoculated with a low pfu dose, no deaths or significant body weight losses were observed post-challenge with vsv xn . however, animals in vsv m -g treated group suffered serious body weight loss even when immunized with pfu and some table serum neutralizing antibody titers in mice against vsv indianna after inoculation with vsv-g or vsv m -g . animals were inoculated with or pfu of different vsvs with pbs as a control. sera were collected before and days after inoculation. neutralizing antibody titers against vsv indiana were determined and expressed as the reciprocal of the highest dilution of antibody giving a % inhibition of cytopathic effect. '-': not detectable. animals died when the dose was as low as pfu. the results correlated with neutralization antibody titers stimulated by different vsvs. neutralization antibodies titers in animals vaccinated with vsv-g were shown to be much higher than those with vsv m -g . therefore, although vsv m -g contained the double mutated m and g proteins and was a very safe viral vector, it was not as effective as vsv-g in stimulating protective immunity, possibly due to its extreme attenuation in vivo. in summary, among different attenuated vsvs with mutated m or/and g proteins, recombinant vsv with only truncated g protein (vsv-g ) indicated ideal balance between pathogenesis and capabilities in stimulating protective immune response and could be a promising vaccine vector. however, for the purpose of developing a vaccine candidate for the prevention of a vsv pandemic, these vaccine candidates would need to be evaluated in swine and cattle, which are the natural host of vsv, before its application in the field. in the current study, a novel recombinant vsv was constructed with mutations in both m ( m ) and g (g ) proteins. for the first time, vsv with mutated m and/or g proteins (vsv m , vsv-g , vsv m -g ) were compared to evaluate their potentials as vaccine vectors. the experimental conclusions included: ( ) both g and m contribute to the attenuation of vsv, however, g is likely to play a more important role than m . vsv m -g was determined to show the most significant attenuation in vitro. ( ) virulence of recombinant vsvs with truncated g protein (vsv-g , vsv m -g ) significantly decreased compared with wild-type vsv or vsv m . ( ) vsv-g could stimulate a more potent protective immune response than vsv m -g possibly due to the extreme attenuation of vsv m -g . among different attenuated vsvs with mutated m and/or g proteins, recombinant vsv with only truncated g protein (vsv-g ) displayed an ideal balance between pathogenesis and stimulation of a protective immune response that could be used as a promising vaccine vector. recombinant vesicular stomatitis viruses from dna efficient recovery of infectious vesicular stomatitis virus entirely from cdna clones foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles recombinant vesicular stomatitis (indiana) virus expressing new jersey and indiana glycoproteins induces neutralizing antibodies to each serotype in swine, a natural host properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies evaluating replication-defective vesicular stomatitis virus as a vaccine vehicle vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates rhabdoviridae: the viruses and their replication the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host a proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with ww domains of cellular proteins: implications for viral budding modifications of the psap region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo vsv strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immunecompetent mice attenuated vesicular stomatitis viruses as vaccine vectors ns proteins of avian influenza a viruses can act as antagonists of the human alpha/beta nterferon response homozygous deletion of the alpha-and beta -interferon genes in human leukemia and derived cell lines influenza a virus lacking the ns gene replicates in interferon-deficient systems rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine development sensitivity of prostate tumors to wild type and m protein mutant vesicular stomatitis viruses effects of intravenously administered recombinant vesicular stomatitis virus (vsv(deltam )) on multifocal and invasive gliomas impaired interferon signaling is a common immune defect in human cancer key: cord- - ad fw z authors: monath, thomas p. title: vaccines against diseases transmitted from animals to humans: a one health paradigm date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ad fw z abstract this review focuses on the immunization of animals as a means of preventing human diseases (zoonoses). three frameworks for the use of vaccines in this context are described, and examples are provided of successes and failures. framework i vaccines are used for protection of humans and economically valuable animals, where neither plays a role in the transmission cycle. the benefit of collaborations between animal health and human health industries and regulators in developing such products is discussed, and one example (west nile vaccine) of a single product developed for use in animals and humans is described. framework ii vaccines are indicated for domesticated animals as a means of preventing disease in both animals and humans. the agents of concern are transmitted directly or indirectly (e.g. via arthropod vectors) from animals to humans. a number of examples of the use of framework ii vaccines are provided, e.g. against brucellosis, escherischia coli o , rabies, rift valley fever, venezuelan equine encephalitis, and hendra virus. framework iii vaccines are used to immunize wild animals as a means of preventing transmission of disease agents to humans and domesticated animals. examples are reservoir-targeted, oral bait rabies, mycobacterium bovis and lyme disease vaccines. given the speed and lost cost of veterinary vaccine development, some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. opportunities for vaccine-based approaches to preventing zoonotic and emerging diseases that integrate veterinary and human medicine (the one health paradigm) are emphasized. zoonoses (diseases transmissible from animals to humans) account for approximately % of all infectious pathogens of human beings and % of all emerging infectious diseases [ , ] . the origins of these diseases, and underlying factors (including man-made factors) in their emergence have been the subject of considerable interest [ , ] . certain zoonotic diseases have the potential for pandemic spread by human contagion, such as avian influenza, sars and the middle east respiratory syndrome coronavirus, and others for regional cross-border epizootics, such as yellow fever, venezuelan equine encephalitis and rift valley fever. the cost of a short list of zoonotic disease emergences in the interval between and , including bovine spongiform encephalopathy, sars, highly pathogenic avian influenza, west nile, and pneumonic plague (india), was estimated to exceed $ billion [ ] . of major emergencies that concerned public health (including hurricanes, earthquakes, and terrorist attacks) occurring between and , more than % were zoonotic disease outbreaks [ ] . however, the toll on public health is much greater than that caused by such dramatic outbreaks. it is estimated that different zoonotic diseases are responsible annually for . billion cases of human disease with . million deaths and substantial reductions in livestock production [ ] . animals, including livestock and companion animals, also suffer illness and death following infection with many zoonotic infections, and livestock and poultry are subject to large-scale intentional destruction as a means of preventing human infections, resulting in huge economic losses. wild animals, including endangered species, may also be mortally affected, examples being west nile disease in birds, yellow fever in neotropical monkeys, plague in black-footed ferrets, and ebola in the great apes. vaccines are an important means of prevention and control of zoonotic infectious diseases in humans and domesticated animals. however, the target for vaccination is, in almost all cases, the directly affected species, and there are few practically implemented illustrations of the potential for indirectly preventing human disease by immunizing the domesticated or companion animal sources of infection. the concept of immunizing wild animal reservoirs for the prevention of disease in humans or domestic animals is even more challenging and has received limited attention. moreover, human and animal health divisions of the biopharmaceutical industry are generally separate and segregated, and there is no organized approach to the development of new vaccines indicated for the prevention of spread of diseases from domestic or wild animals to humans. in addition, the major funding sources for research in human and animal diseases tend to be stove-piped into different government agencies, stifling cross-cutting approaches. the purpose of this review is to stimulate the science and policy communities to seek innovative ways to interdict zoonotic diseases by integrating human and veterinary medicine and vaccine development, and by creating new streams of funding aimed at the intersection of human and animal health. these aims are consistent with the one health initiative, which seeks to establish "collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and our environment" [ ] . a framework for considering one health vaccine interventions is provided, as well as a brief review of past and current efforts, including a few successes. it will be obvious to the reader that this is a wide-open field with many opportunities that deserve more attention than they have received. the complexity, timeline, and cost of development of animal vaccines and the regulatory hurdles for product approval are far less than for human vaccines. thus some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. it is obvious that the benefit to human health deriving from vaccination of animals is far easier to justify when there is also a benefit to animal health, the latter often being closely linked to economic value. causative agents of zoonotic diseases, including viruses, parasites, bacteria, fungi, and prions, have extraordinarily varied life cycles and modes of transmission. some may persist between periods of active transmission in soil or invertebrate species. many have silent transmission cycles involving wild animals that have coevolved with the infectious agent and exhibit no signs of disease. some zoonotic diseases occur when a causative agent harbored by a wild animal reservoir jumps species to domesticated animals and thence to humans. others are primarily diseases of domesticated animal species. humans may be infected by direct contact with wild or domesticated animals, or indirectly by ingestion of contaminated milk or meat, inhalation of aerosolized secretions or excreta, fomites, or hematophagous insect or tick vectors. despite this complexity of epidemiological patterns, the opportunities for intervention often boil down to a few simple bottlenecks in the transmission process. for example, milk-borne diseases can be prevented by pasteurization, certain meat-borne diseases by inspection and animal husbandry improvements (e.g., trichinella, bovine tuberculosis), and other diseases avoided by limiting contact with known high risk species (e.g., tularemia, turtle-borne salmonellosis, exposure to bats carrying henipaviruses). where the risk of infection is high, or the resulting disease severe, vaccines may be the most efficient and cost-effective means of prevention and control. alternative methods for control of zoonotic diseases have generally employed trapping, poisoning, or other means of destroying the offending animal reservoir/vector; these methods have a mixed (often negative) record of success and are in any case becoming socially unacceptable. this review focuses on the use of vaccines for animals as an acceptable means of interdicting zoonotic disease in both animals and humans. three major epidemiological frameworks are identified for the control of zoonotic disease by means of vaccination of animals ( table ). the scope of this review encompasses only those diseases for which a strategy targeting vaccination of animals is actually used or is under development. there are many diseases of table frameworks for vaccine development and utilization as a means of preventing zoonotic diseases, and status of approval of existing vaccines for humans and animals. the first major framework includes zoonotic infectious diseases that affect both humans and economically important animals, where wild animals are the source of infection. livestock and humans are dead-end hosts, and neither contributes to the transmission cycle. in this case, vaccines are required to prevent disease in both humans and economically important animals, but do not interrupt transmission of the disease in nature. for framework i diseases, there is an important opportunity to accelerate the development of new vaccines by concurrent veterinary and human product research ( table ) . moreover the affected animal species represents a natural disease model of infection, pathogenesis and immunity that may be useful in testing efficacy and immunological correlates of protection of a new vaccine intended for human use, and thus provide data supporting regulatory approval under fda's animal rule [ ] . a list of framework i diseases and vaccines is shown in table , and an example is given below. west nile virus is a mosquito-borne, single strand, positivesense, enveloped rna virus (genus flavivirus, family flaviviridae), closely related to japanese encephalitis virus. west nile virus is a recognized cause of human disease ranging from mild feverrash-headache syndromes to lethal encephalomyelitis. horses, domesticated geese, farmed alligators (as well as a number of wild birds and mammals) are also susceptible to severe and fatal disease. although recognized as a cause of disease as early as the s, west nile became an increasing problem in the s, with outbreaks affecting humans and/or horses in northern africa, western europe, the eastern mediterranean, the black sea region and the volgograd oblast of russia [ ] . the most dramatic development was the introduction of west nile virus into north america in [ ] [ ] [ ] , the occurrence of large outbreaks in and , rapid spread across the us, and subsequent introduction to the caribbean and south america. horses and a number of wild bird species, notably crows and jays, were affected in addition to humans. between and , a total of , cases of west nile fever and , cases of neuroinvasive west nile disease were reported in the us, with the highest incidence in the middle of the country from texas north to the dakotas [ ] . the incidence of neuroinvasive disease in the us has varied between . and . per , in this interval [ ] . over , horses have been affected since , and in these animals the disease is more severe ( % case-fatality, % of survivors with neurological sequelae) than in humans ( - % case fatality, % of encephalitis survivors with sequelae) [ ] . moreover, the incidence of west nile in horses (∼ per , ) is substantially higher than in humans [ ] . the animal health industry rapidly responded to this veterinary emergency, and multiple west nile vaccines were rapidly developed, including a live vaccine (discussed below), whole virion inactivated, dna, and poxvirus vectored vaccines. the first vaccine for horses was marketed in , only years after introduction of the virus into the us. multiple human vaccine development programs were also initiated, but many of these efforts were discontinued or decelerated due to the high market risk associated with low incidence, a slackening of public concern with the disease, and the uncertain regulatory pathway for vaccine approval. although efficacy of a vaccine for equids is established [ ] , field trials to prove vaccine efficacy in humans would be large, expensive, and difficult due to the unpredictable occurrence of west nile outbreaks. the application of the animal rule to licensing a west nile vaccine, while plausible, has not been adjudicated by the fda with a sponsor. although approaches to developing veterinary and human vaccines against west nile were technologically similar, in only one case was a concurrent development program undertaken by a single company. this fact illustrates the current status of stove-piped and separate animal and human health biopharmaceutical industries. the exceptional case is of interest as a model of how vaccine development for zoonotic diseases could be improved. in , within months of the identification of west nile as the etiological agent of the initial outbreak affecting humans and horses in new york, acambis, a publically traded human-vaccine biotechnology company, applied its platform technology for yellow fever dvectored vaccines to the development of a west nile vaccine, with the intent to develop both vaccines for both horses and humans. this technology involved replacing the gene encoding the yellow fever d vaccine virus' envelope (e) protein with the corresponding gene of west nile virus. this chimeric vector vaccine platform had been previously used by the company to construct a chimeric vaccine against the closely-related japanese encephalitis virus [ ] ; at the time, that vaccine had proven to be highly effective, protecting non-human primates against intracerebral challenge with virulent japanese encephalitis virus [ ] . two new vectors were engineered, one with the wild-type west nile ny strain e protein gene and one with an e gene containing three attenuating mutations [ ] [ ] [ ] . the former was neurovirulent in mice, while the latter was more attenuated and thus deemed more suitable as a human vaccine. the principal question was whether the yellow fever d vector would infect and immunize horses, since yellow fever is a host-restricted primate virus. to find out, studies were sponsored by acambis in early at the colorado state university school of veterinary medicine. horses were vaccinated with the west nile/yellow fever chimeric virus or with yellow fever d vaccine, and viremia and antibody responses were determined [ ] . in addition, vaccinated and control horses were challenged by the intrathecal injection of a high dose of virulent west nile virus, following the same model referred to above for protection studies of the chimeric japanese encephalitis vaccine wherein monkeys were challenged by the intracerebral route. these studies showed that horses inoculated with chimeric vaccine developed neutralizing antibodies against west nile and were protected against a severe intrathecal challenge. a similar development plan was successfully followed for the mutated human vaccine version, using non-human primates as the test host. the veterinary and human vaccine candidates were developed side by side, and the data obtained in both programs were designed to support transition to advanced clinical trials in horses and humans. in , acambis licensed the veterinary vaccine technology to a major animal health company (intervet), and in the same year clinical trial materials were made for human trials. intervet completed development of the veterinary vaccine (prevenile ® ) [ ] , which was approved by usda in , and acambis brought the human vaccine (chimerivax-wn ) into clinical trials in the same timeframe [ ] , subsequently outlicensing the technology to sanofi pasteur. as expected, development of the veterinary vaccine and its progression through the regulatory pathway substantially outpaced the human vaccine; importantly, the veterinary application significantly informed the human vaccine table strengths and limitations of vaccination of animals as a means to control of zoonotic diseases. framework (see table development program in providing useful information on safety, durability of immunity and immune correlates or protection. co-development of the veterinary and human west nile vaccines is as a potential model for other vaccines. there are a few other examples where vaccines were co-developed for animals and humans, including vaccines against venezuelan equine encephalitis and rift valley fever (described below). certain issues arise, however, that may need to be considered in the context of such integrated efforts. first, a safety problem arising during use of a veterinary vaccine could provoke regulatory concerns or a liability problem for the human analog. although a safety issue did arise briefly due to anaphylaxis type reactions in horses, resulting in temporary withdrawal of prevenile ® from the market [ ], there were no repercussions for the human analog vaccine (chimerivax-wn ). this raises the interesting question whether the human and veterinary regulatory agencies are integrating information that might be of value, either during development of similar vaccines for different species or after marketing approval. this is an obvious overlooked area for application of one health principles. second, the immune response to veterinary vaccines generally should differentiate naturally infected from vaccinated animals (diva) on the basis of a laboratory test, which allows compliance with trade restrictions. this is, of course not a concern for regulatory approval of human vaccines, although it can be useful in seroepidemiological and vaccine coverage studies. an example of problems associated with diva is the off-label use of commercial horse vaccine to protect emus against eastern equine encephalitis (eee), since some combination vaccines against eee contain equine influenza antigen and such vaccines elicit cross-reactive antibodies to avian influenza resulting in quarantine. the chimeric west nile vaccine described above potentially allows for diva testing (using responses to the yellow fever nonstructural proteins expressed by the vector) [ ] . a number of important diseases are transmitted between domesticated animals and thence to humans. vaccination of domesticated animals has the potential to protect humans against these zoonoses, either indirectly by interrupting transmission where domesticated animals are amplifying hosts in the transmission cycle or directly by preventing spread from infected animals to humans. a list of framework ii diseases and vaccines is shown in table . selected examples are used to illustrate the role of vaccination of domesticated animals in preventing human disease. brucella spp. are facultative, intracellular gram-negative bacteria, pathogenic for domestic animals and humans. brucellosis, caused mainly by brucella melitensis (which infects sheep and goats), brucella abortus (cattle), and brucella suis (swine), occurs worldwide, with the highest prevalence in the middle east, asia, africa, tropical america, and the mediterranean region [ , ] . the annual incidence of human infections is estimated at , cases but the disease is widely acknowledged to be underreported [ ] . brucella canis, an infection of dogs, occurs worldwide with highest prevalence in tropical america. b. canis disease in humans has been reported, especially in persons handling breeding dogs and in immunosuppressed individuals. human brucellosis is acquired by contact or aerosol spread from infected animals, fomites, or ingestion of unpasteurized milk or undercooked meat. not surprisingly, brucellae survive for long periods in dust, animal excreta, soil, meat and dairy products. wild animals are also affected and can be the source of infection of livestock and humans. in animals, brucellosis causes epididymitis in males and abortion, placentitis, infertility and reduced milk production in female animals. the human disease is protean, manifested by chronic fatigue, relapsing fever, endocarditis, spondylitis, osteomyelitis, arthritis, and meningitis [ ] . prevention of human disease by control of brucellosis in livestock has long been a public health priority [ ] . control of brucellosis relies principally on surveillance, testing, removal of infected animals, import/export animal and animal product control provisions, protection from exposure to wild reservoirs (such as elk, deer, and bison), and vaccination. antibiotic treatment of animals is regulated and discouraged due to the large doses and long treatment required and concern about resistance. old, empirically developed live attenuated vaccines, b. melitensis rev vaccine for goats and sheep; b. abortus s and rb vaccines for cattle; and the oral b. suis s vaccine used widely in china for multiple species, elicit cellular immunity against the intracellular pathogen and are more effective than other types of vaccine [ ] . the rb vaccine (a spontaneous rifampin-resistant rough mutant) is approved in the us [ ] . however, the live vaccines have a number of drawbacks. the latter include lack of the ability to differentiate s and rev vaccine immunity from natural immunity (diva) and interference with surveillance and export control procedures; pathogenicity (especially abortion when animals are vaccinated during pregnancy); antibiotic resistance of the vaccine strains; and modest efficacy. live vaccines used in livestock can also cause illness in humans. vaccination as a stand-alone strategy has rarely been carefully evaluated, in large part due to concerns over the quality of the existing vaccines. however, vaccination is largely credited with elimination of brucellosis in the us [ , ] (the us was declared free of brucellosis in cattle in ) and for control of brucellosis in china [ ] . a recent study in greece showed that a mass vaccination program with the b. melitensis rev vaccine resulted in a decrease in human infections [ ] . a number of new vaccine approaches, including diva vaccines, designed to induce th oriented cellular immunity are under investigation, including safer rationally designed, mutated live vaccines [ ] ; recombinant, invasive escherischia coli [ ] ; recombinant subunit microencapsulated vaccines; and dna vaccines [ ] . experimental b. canis vaccines have been investigated in mice. where brucellosis-free status has been achieved, as in the us, wild animal reservoirs (especially bison and elk) threaten to reintroduce the disease. vaccination with existing vaccines is feasible, but delivery is challenging [ ] . this vero cytotoxin secreting gram-negative bacteria is an important cause of sporadic and epidemic food-borne illnesses of humans, including gastroenteritis and hemorrhagic colitis, with potentially lethal complications (hemolytic-uremic syndrome). cattle and sheep are the principal reservoirs of infection and transmission to humans occurs via food (meat, seeds and vegetables) contaminated with animal feces. undercooked ground beef is a source of infection in approximately one-third of human cases and recalls are a significant economic threat to the meat packing and distribution industry. animals concentrated at feed lots and slaughter that shed bacteria can produce lots of meat with high rates of o [ , ] , but there is considerable variability in the occurrence of contaminations [ ] . in developed countries, various sanitary measures and testing have been instituted to reduce the risk to consumers, but these remain imperfect. vaccination of feed lot cattle has been proposed as a measure to reduce the prevalence and duration of shedding and the risk to consumers. o -specific bacterial extract vaccines containing protective outer membrane proteins have been conditionally approved by usda (manufactured by epitopix, willmar, mn) and fully approved by the canadian food inspection agency (bioniche life sciences, belleville, ont.). feedlot cattle receiving or doses of the bioniche vaccine - weeks apart had - % reduction in colorectal colonization or fecal shedding and significant reduction in magnitude and duration of shedding [ ] [ ] [ ] . hurd and malladi [ ] modeled the impact of vaccinating cattle on human health outcomes. assuming % efficacy of the vaccine and % adoption rate, the model indicated a % reduction in the incidence of e. coli o -related human illness. the model also predicted significant reductions in the number of lots of contaminated ground beef and detection by usda, which would have substantial economic benefit to packers and distributors. vaccine effectiveness under conditions of field use will be highly dependent on adoption rate (vaccine coverage), and in part by whether cattle complete the -dose vaccination series on the proscribed schedule [ ] . an interesting question is: who will pay for vaccination of feedlot cattle? is the economic benefit there for the meat industry, and will vaccination reduce the cost of other preventive measures and testing or will vaccine costs simply be on top of other costs? will government agencies concerned with human health subsidize the cost, without empirical demonstration of a human health benefit? since ground beef only contributes about one-third of human infections, how could vaccines be used as a means of reducing other sources of food contamination? another issue for use of the current vaccines is the role of other enterohemorrhagic e. coli (e.g. o , o , and o ) in human disease. this disease is caused primarily by the gram-negative bacterium, bartonella henselae, transmitted between domestic cats by the agency of cat fleas, ctenocephalides felis. human infection occurs by contact spread from cats, including scratches by claws contaminated with blood or flea feces, or possibly by flea bite [ , ] . the prevalence of infection in household cats in the us is approximately %, but in stray animals it is % and high prevalence rates have been found in developing countries [ , ] . disease in humans is manifest by fever, a papule followed by a pustule at the site of infection and lymphadenopathy. rare complications include meningitis, encephalitis, endocarditis, glomerulonephritis, osteomyelitis, neuropsychiatric abnormalities, and relapsing fever and splenomegaly. human infections in immune-compromised individuals are particularly severe. approximately , - , cases and hospitalizations caused by cat scratch disease are estimated to occur annually in the us. in the late s, heska corporation, an animal heath company in colorado, initiated a program to develop a vaccine for household cats, with the goal of limiting the potential for transmission of the bacteria from cats to humans. unfortunately, the program was not completed and no vaccine is available at present. given the fact that cats rarely become ill with b. henselae, this would have been an unusual product providing protection to pet owners, with marginal if any real benefit to the target species. moreover, it would likely have been extremely challenging to demonstrate a health benefit to humans. rabies is a fatal infection of the central nervous system caused by rabies virus, a member of the lyssavirus genus, family rhabdoviridae. it is estimated that up to , - , cases of human rabies occur annually, and dog bite is the cause of over % [ ] . successful vaccination of dogs and humans against rabies was first demonstrated in by louis pasteur, using crude nerve tissue preparations. however, until the first decades of the th century in developed countries, and continuing in many developing countries today, the ancient practice of dog population reduction campaigns have been the main approach to rabies control, a method that has repeatedly proven to be ineffective. dog licensing and vaccination requirements were introduced in the united kingdom in , and gradually at the local and then state levels in the us beginning in the s, with national requirements attained by [ ] . many successful dog vaccination campaigns have been reported in latin american countries and asia [ ] . some countries have declared eradication of canine rabies, notably the united kingdom in , japan in , the us in , as well as malaysia, singapore, taiwan, hong kong. in , the first world rabies day event, the ultimate vision was promulgated of canine rabies elimination through systematic vaccination. however, rabies remains a significant public health problem, due to absent or incomplete dog vaccination in many areas of the world. more than . million post-exposure treatments with rabies vaccines are given annually, at a cost of over $ billion worldwide [ , ] . in china, due to the low prevalence of canine vaccination, the sales of rabies vaccines for human post-exposure prophylaxis outstrips any other human vaccine, accounting for % of all annual vaccine sales, i.e. million doses costing $ million [ ] . the requirement for human post-exposure vaccination at this scale represents an obvious failure of public health, since it plays no role in containing the spread of rabies in the canine vector. a very high rate of vaccine coverage in the dog population (exceeding %) is required for interruption of the rabies transmission cycle [ ] . the development of oral rabies vaccines has allowed vaccination of free-roaming dogs that could not be restrained and vaccinated by injection, as well as the vaccination of wild animal species that are the source of infection in dogs [ ] . oral bait vaccine for dogs has been successfully deployed in trials in many countries, including turkey, thailand, sri lanka, south africa, and the philippines [ ] ; the world health organization has supported this approach as a supplemental program where there are substantial populations of free-ranging or feral dogs [ ] . significant increases in canine vaccination coverage have been achieved when oral rabies vaccine was added to a program of standard parenteral vaccination. oral vaccination of dogs has been accomplished using commercial baits containing live modified rabies vaccines, such as the street-alabama-dufferin (sad) strain, variant b [ ] and the attenuated copenhagen strain of vaccinia expressing the rabies glycoprotein gene (g protein) [ ] . a more detailed review of oral rabies vaccines is provided below (framework iii). hendra virus disease, a severe and fatal infection of horses and humans in australia, has been noted as an example illustrating one health principles in disease prevention and control [ ] , and is especially relevant now that a new vaccine for horses has been introduced. hendra is a member of the henipavirus genus, family paramyxoviridae. the reservoir hosts are fruit bats (pteropus spp.), and the virus is spread from bats to horses by contact (including respiratory droplets), by food or fomites contaminated with bat urine, or by contact with sick horses. all reported human infections have resulted from contact with infected horses [ ] [ ] [ ] . hendra virus disease was first described in . human and equine cases have occurred in coastal queensland and new south wales and positive bats have been detected in the northern territory. a total of deaths in equids have been reported in outbreaks, with a very high case-fatality rate ( %), and cases ( fatal) have occurred in humans, including horse trainers and veterinarians, all of whom had contact with sick horses [ ] . the disease is manifested by severe systemic illness, respiratory symptoms or acute and relapsing encephalitis [ ] . swine appear to be susceptible to experimental infection. nipah virus, a closely related bat-borne agent in se asia has caused outbreaks of severe and fatal disease in swine and humans [ ] [ ] [ ] . in november , pfizer animal health launched equivac ® hev, an adjuvanted subunit protein vaccine for the prevention of hendra virus disease of horses in australia [ ] . since horses are a major source of contact spread of hendra virus to humans, the vaccine promises to make an important contribution to human health as well. fear of acquiring the disease has also constrained equine veterinary practice in australia [ ] , and the vaccine should mitigate this problem. development of equivac ® hev was a collaborative effort between pfizer and csiro's australian animal health laboratory. however, support for the development program was also provided by human medical researchers in the us, at the uniformed services university of the health sciences supported by the henry jackson foundation for the advancement of military medicine. a provisional approval for limited use of the vaccine was obtained in early , with full approval in november. the vaccine is a soluble, recombinant glycoprotein (g) of hendra virus, the ligand for cell attachment and antibodies to the protein neutralize cell receptor binding of the virus [ , ] . the vaccine protects horses and ferrets against experimental infection [ ] , and appears to cross-protect against nipah virus [ ] . the availability of equivac ® hev should lead to rapid uptake by horse owners in australia. the equine and horse racing industry in australia is large, contributing billions of dollars, and over % of total gross domestic product [ ] . hendra virus in horses is a notifiable disease in all australian jurisdictions; the property where the horse cases are located is quarantined and animals that are infected are euthanized. the occurrence of at least one hendra virus outbreak annually since , and the high lethality of the disease have raised considerable awareness in australia. since all human cases of this zoonosis have resulted from contact with infected horses, vaccination of horses against hendra virus promises to be a highly effective strategy for preventing human cases. rift valley fever is an enveloped, single-strand, segmented rna virus belonging to the phlebovirus genus, family bunyaviridae, occurring in africa, with intermittent extensions to the arabian peninsula. rift valley fever virus causes explosive and economically damaging outbreaks of disease in cattle and sheep, with stillbirth, abortion, and very high mortality of young animals; in adult animals, it causes % mortality in sheep, - % in cattle, and - % in goats [ ] . humans typically develop self-limited nonspecific febrile illness, but - % have a complicated course with hemorrhagic fever syndrome, encephalitis, hepatitis, renal failure, or retinitis, and case-fatality rates in severely ill and hospitalized patients is as high as % [ , ] . the virus is transmitted between livestock and from livestock to humans by the agency of mosquito vectors. in addition, humans commonly acquire infection by contact and aerosol routes when handling, treating, or butchering infected livestock, and the virus can persist in meat for weeks. the ecology of rift valley fever and the reasons behind its periodic emergences have been the subject of intensive study. in brief, the reservoir of infection is aedes mosquitoes, especially ae. linneatopennis, which maintain the virus by transovarial transmission [ ] . the dessication-resistant ova containing virus remain in depressions in the earth ('dambos') that are flooded during the rainy season, with the subsequent emergence of infected adult aedes mosquitoes. infected, viremic, livestock in turn served as source for amplified virus transmission by a variety of mosquito vectors [ , ] . there are no approved vaccines for prevention of rift valley fever in humans, although a number of candidates are in development by academic and government laboratories. vaccination of cattle and sheep represents a strategy for preventing disease in these species, and thereby for interrupting virus transmission to humans. rift valley fever epizootics are to some extent predictable based on rainfall patterns and surveillance of disease in livestock [ ] . surveillance provides opportunities for rapid intervention, particularly with a single-dose vaccine (most likely a live, attenuated vaccine) that would protect against viremia in cattle and sheep and prevent mosquito infection. in addition, routine vaccination of livestock in inter-epizootic periods with a product capable of inducing durable protective immunity is a long range goal supported conceptually by modeling [ ] . however, there are many obstacles to livestock immunization in africa, including access, policy, regulatory approval, cold chain, and commercial viability. additionally, diva requirements are driven by regulations prohibiting export of livestock or meat by countries experiencing rift valley fever [ ] . some of the obstacles to vaccination implementation could be overcome by development of improved vaccines. two old veterinary vaccines, the live smithburn vaccine, developed using techniques of serial passage in mouse brain similar to that applied to the early development of the french neurotropic vaccine against yellow fever [ , ] , and a formalin inactivated vaccine [ ] are commercially available from the onderstepoort institute in south africa. however, the smithburn vaccine, now produced in bhk- cells, is reported to cause teratogenicity and abortion when used in pregnant animals [ ] and is used only in countries endemic for rift valley fever due to concerns about reversion. the inactivated vaccine, also grown in bhk- cell culture, requires multiple doses to be effective and probably has relatively short durability [ ] , making it less desirable for the interventions proposed above. nonetheless, the inactivated vaccine was successfully used to interrupt a rift valley fever outbreak in south african sheep [ ] . indeed, following a large epidemic of rift valley fever in egypt in - , the veterinary serum and vaccine research institute (cairo, egypt) produced a formalin-inactivated vaccine in bhk- cells using the epidemic strain (zh ), which matched locally circulating strains compared to the south african strain [ ] . another inactivated vaccine (tsi-gsd ) developed by the us army for human immunization and grown in diploid fetal rhesus lung cells was tested clinically and shown to be well tolerated and immunogenic. ninety % of the subjects developed a neutralizing titer > , which was shown to be higher than the protective level in a passive immunization-challenge study in hamsters [ , ] . in addition to the requirement for multiple doses for primary and booster immunization, inactivated rift valley fever vaccines have the disadvantage of requiring high biocontainment facilities for manufacturing. in recent years, there has been substantial progress in development of newer vaccines, and some of these vaccine development projects have been collaborations between human and veterinary research groups. additionally, there has been a moderate level of support from the us government because rift valley fever is a credible threat of natural or intentional (bioterrorist) introduction. nevertheless, despite very promising technical results, there has been insufficient support from industry and government to propel any of these new vaccine candidates into use. because of the obvious advantages of rapid onset and durable immunity associated with live vaccines, development of an improved live vaccine has been the focus of research. us army investigators attempted to induce attenuating mutations in two rift valley fever virus strains isolated during the egyptian epidemic [ ] . mp- is a live vaccine that was developed from the virulent zh- strain by passages in mrc- cells in the presence of the mutagen, -fluorouracil, resulting in a temperature sensitive virus with amino acid mutations. attenuation was demonstrated in multiple animal models, and reassortment studies showed that attenuating mutations were redundant and resided in all three gene segments [ , ] . development of mp- was undertaken by the us army medical research institute of infectious diseases with the intention to produce a vaccine for both human and animal immunization. the vaccine was clinically tested in human volunteers and shown to be well-tolerated and highly immunogenic [ ] . army investigators, in collaboration with usda, conducted a number of studies of mp- vaccine in sheep and cows, including neonatal, pregnant and lactating animals. these studies showed that mp- caused a low viremia, but with no attendant clinical signs; there was no virus secretion in milk, and no abortions or teratogenicity when vaccine was given in in mid-to late term pregnancy. mp- was highly immunogenic and protected livestock against virulent challenge [ , ] . however, ewes vaccinated with mp- early in pregnancy showed a low incidence of abortion and teratogenicity, indicating some residual virulence of the vaccine [ ] . to improve genetic stability and safety of mp- , reverse genetic techniques were used to introduce deletions in the s and m rna segments in genes encoding, respectively, nss and nsm proteins [ , ] . two deletion mutants were evaluated for safety and immunogenicity in pregnant ewes [ ] and in calves (morrill jc personal communication, ) with positive results for the nsm deletant, making it an attractive candidate as a veterinary and human vaccine. there were no clear safety signals when ewes were inoculated in early-mid pregnancy. further safety studies are required to rule out the low incidence of abortion/teratogenicity seen with parental mp- in early-term ewes. unfortunately, human trials have not yet been performed with the rationally designed mp- derivative. a third live vaccine designated clone , is a plaque-derived clone of a central african strain of rift valley fever isolated from a human subject, and was found to be naturally attenuated for mice and to have an in-frame deletion of most of the nss gene [ ] . this observation was the basis for modifying the mp- vaccine by ns gene deletion, as described above. once again, a collaboration between the human and veterinary researchers led to a study in ewes, showing that clone was highly immunogenic but did not cause abortions [ , ] . another attenuated vaccine designated r , has been developed by reassorting clone and mp- so that it contains the s segment of clone and the l and m segments of mp- . this strain has attenuation domains from both parental vaccine candidates. a number of live, replicating, and non-replicating heterologous viral vectors expressing rift valley fever g and g glycoproteins and nonstructural proteins have been have been investigated in mice, elicited immune responses and protected against challenge. the vectors included lumpy skin disease (capripoxvirus) [ ] , alphavirus (vee and sindbis) replicons [ ] , newcastle disease virus [ ] , adenovirus, and modified vaccinia ankara. several of these constructs were used to immunize sheep and/or cattle (lumpy skin disease virus, newcastle disease virus, and sindbis replicons) with somewhat variable success. for a more comprehensive review see indran and ikegami [ ] and boshra et al. [ ] . live vectors are a promising approach for new rift valley fever vaccines, particularly veterinary vaccines, but may have problems for homologous boosting in light of anti-vector immunity. various prime-boost strategies have been proposed, as for plasmid dna vaccines (see below), but these would be exceptionally difficult to implement for immunization of livestock in the field, and are thus impractical. subunit protein produced in insect cells, virus-like particles [ ] , and dna vaccines [ ] against rift valley fever are also in early stage development. these approaches have potential advantages of safety and thermostability during storage and distribution, but may require multiple dosing and provide less durable immunity than live vaccines, and thus are less desirable products for framework ii implementation. overall, it remains to be seen which of the many rift valley fever vaccines in development progress to regulatory approval and whether an integrated veterinary and human health policy based on the immunization of livestock in africa together with predictive surveillance, can abort impending outbreaks, and lead to long range control of this important disease. vee is a mosquito-borne single strand, positive-sense, enveloped rna virus belonging to the alphavirus genus, family togaviridae. other medically important members of the alphavirus genus include eastern and western equine encephalitis viruses. there are vee virus subtypes identified by antigenic and genomic analyses, and a number of additional varieties. subtype iab and ic cause epizootic disease in equids and associated zoonotic infections of humans [ ] . during epizootics, horses and donkeys infected with these strains develop high viremias, serve as the primary hosts for infection of mosquito vectors and therefore are the indirect source of human infections acquired by mosquito bite. in contrast, the enzootic subtypes ii-vi, are maintained in nature in cycles involving rodent species and mosquitoes, are not amplified by equid viremic hosts, and cause sporadic illness in humans and equid dead-end hosts. epizootics of ic virus are the result of mutation and selection of virulent equine-competent viruses from enzootic strains, particularly the id variant [ ] . in the - s vee iab viruses caused large epizootics in south america, with associated human epidemics of encephalitis. between and , a series of major subtype iab and ic epizootics occurred in northern south america, and between and the virus spread north to central america, mexico, and texas [ ] . the cumulative economic and medical impact of vee outbreaks between and was devastating, with over , equid and , human cases. some of the vee iab epizootics are believed to have been spawned by the injection of horses with inactivated veterinary vee vaccines containing residual live virus [ ] . this likely occurred in trinidad in and again in nicaragua in , but probably was a widespread problem in the past. between and , vee ic re-emerged in venezuela and colombia, with an estimated equid deaths and over , human cases of which had encephalitis [ , ] . since vee causes an acute incapacitating illness in humans and the virus efficiently infects via the aerosol route, it was developed by both the us and soviet union as an offensive biological weapon [ ] . as part of these programs, vaccines for the protection of military personnel were also developed. in the us, a live, attenuated virus (tc- ) was developed by the us army medical research & development command (usamrdc) by empirical passages of the prototype trinidad donkey (subtype iab) virus in fetal guinea pig heart cell culture [ ] . the development of the live vaccine followed poor experiences with chemically inactivated vaccines; in animal models, only the live vaccine protected against aerosol challenge. however, tc- vaccine has a number of drawbacks as a human vaccine, including failure to immunize about % of vaccinees, and moderate-to-severe reactogenicity in about % of subjects. in humans, it remains an investigational product, used solely for the protection of laboratory workers [ ] , with approximately persons vaccinated since . the tc- virus acquired mutations during the empirical passage series in guinea pig heart cells, but attenuation appears linked to only of these, in the -noncoding region and the e envelope glycoprotein, and these substitutions appear to be subject to reversion in the vaccinated host [ ] . in addition, tc- has been isolated from mosquito vectors during field use, illustrating the potential for secondary spread and mutation and recombination events. an investigational formalin-inactivated tc- vaccine (designated c- ) was also developed by usamrdc and used following tc- priming to seroconvert tc- non-responders. since horses and related species are severely affected during epizootics and are the source of mosquito vectors infecting humans, there is an obvious need for a single dose veterinary vaccine that evokes rapid immunity. us army investigators explored the use of tc- live, attenuated human vaccine for immunization of equids beginning in [ ] , and there was limited field use of the vaccine in colombia in . however, when epizootic vee appeared for the first time in central america (guatemala) in may , and then spread southwards to costa rica and northwards to the us, there was considerable urgency to utilize a vaccine strategy for control of the disease in horses, donkeys and mules. in , the us military responded rapidly to requests for tc- vaccine from guatemala and el salvador. the vaccine had been produced and stockpiled at the merrell national laboratories, swiftwater pa under contract to the usamrdc for the purposes of biological defense. by , over million doses of tc- had been given to equidae in the us, mexico and central america [ ] . collaborative studies were also undertaken by agencies concerned with human and animal health (usamrdc, nih and usda) to fully explore the biology of the vaccine in horses [ , ] , ultimately leading to licensure and commercialized by the animal health industry, both as a live vaccine and then an inactivated vaccine combo with eastern and western equine encephalitis vaccines. tc- vaccine was credited with a rapid curtailment of the - outbreak. the history of vee exemplifies many one health principles, including the prevention of human cases through domesticated animal vaccination, use of a single vaccine product for animals and humans, and an agency (the us army) concerned with human health engaged in both veterinary and human vaccine development, and providing a solution for curtailing an emerging zoonosis. after the large epizootic in the s, tc- vaccine was again deployed during the epidemic in in colombia to create an immune barrier to spread of the virus. recent efforts have focused on development of improved vee vaccines for humans that are less reactogenic and more immunogenic than tc- , can be manufactured in a more acceptable substrate, and have a lower risk of reversion to virulence and of mosquito transmission [ ] . in addition, vaccines that crossprotect against the enzootic vee subtypes are needed. vee id is endemic in colombia, peru, venezuela, and ecuador, and the ie subtype circulates in southern mexico. aguilar et al. [ ] postulated that disease caused by vee is confused clinically with dengue, and that, in endemic areas, up to % of dengue cases may actually be due to vee enzootic subtype viruses. subtype id poses the ever-present risk of mutational change to produce high viremia and epizootic transmission in equids, as happened in the s. v vaccine is a rationally designed vaccine from the epizootic subtype iab genome, with insertion of a pe cleavage-signal mutation combined with an e gene resuscitating mutation. v had a good safety profile and was immunogenic and protective in laboratory animals, including nonhuman primates [ ] . while retaining a degree of neurovirulence for suckling mice, v is not virulent when inoculated intracranially in juvenile monkeys [ ] . v has also been evaluated in horses [ ] . the vaccine was safe and highly immunogenic, with subcutaneous doses as low as plaque-forming units shown to protect horses against challenge with virulent subtype iab virus. unfortunately, v proved to be too reactogenic for humans in a phase trial [ ] , and thus development for both human and veterinary use has stopped. the v virus was subsequently formalin inactivated and has been investigated with adjuvants replacement for the c- vaccine. other live and live vector approaches to improved vee vaccines have been investigated only in laboratory animals, including a chimeric virus constructed from nonstructural genes of sindbis and the structural genes from vee [ ] , vee replicon vaccines, and vaccinia recombinants. none of these approaches have reached advanced development. it is only a matter of time before another vee outbreak emerges in tropical america, and there is a substantial risk of cross-border spread. the prospects for vaccine interventions have diminished with dwindling support for new vaccines and increased concerns for vaccine safety. most zoonotic diseases are maintained in transmission cycles involving wild mammals or birds. however, because of the difficulties in vaccinating specific host species, wildlife immunization as a means of preventing spread to domestic animals and humans has been applied in only a few diseases. some of the barriers to implementing wild animal vaccination include (i) involvement of multiple species in natural transmission cycles; (ii) safety concerns for non-target species; (iii) high reproductive rates and population turn-over; (iv) fastidious feeding behaviors and difficulty in designing effective baits; (v) difficult delivery due to very high or, conversely, very low population densities of the target species; (vi) environmental concerns, and release of genetically modified organisms; (vii) difficulty in designing an effective formulation for oral immunization; (viii) instability of a vaccine or vector under prevailing environmental conditions; and (ix) requirement for low unit cost and government funding for vaccine purchase and delivery. nevertheless, targeted immunization of wild animal reservoirs is a subject of considerable interest for future research, not only for control of infectious agents affecting domestic animals and humans but also for control of wildlife diseases. one example of the latter was the effort to develop a means of immunizing great apes affected by ebola virus in central africa with vaccines previously developed for human use. aside from rabies vaccines delivered in oral baits, which is well-established, wildlife vaccination has had limited success. two promising examples of early-stage vaccine applications are described below (lyme disease and mycobacterium bovis). in addition experimental immunization and protection of prairie dogs (cynomys ludovicianus) using a raccoon poxvirus recombinant oral bait vaccine [ ] , and ballistic vaccination of bison against b. abortus [ ] have been described. plague, a global but localized zoonotic disease with rodent wildlife reservoirs, would appear to be a target of particular interest for future research [ ] . there are many other possible targets for new framework iii vaccines, and future research in this field is encouraged. in the united states, lyme disease is the most common vectorborne disease and the th most common infectious disease overall. it is also a major and increasing public health problem in europe. approximately , cases are reported in the us annually, and the number has doubled in the last years [ ] . however, at a meeting in august, , the centers for disease control and prevention (cdc) reported that the annual incidence of infection is believed to be -fold higher, i.e. , cases. although lyme disease occurs across the country, the incidence is highest in the northeast and north central states. in the us, lyme disease is caused by the spirochete borrelia burgdorferi, which is amplified each spring and summer in a cycle principally involving ixodes scapularis ticks and field mice. mice are persistently infected and represent the reservoir of infection in nature [ ] . b. burgdorferi is passed transtadially to nymphal and adult ticks which infect humans and dogs; these species develop clinical disease but are dead-end hosts. the human disease is manifested by a protean syndrome, starting with a localized skin infection (erythema migrans), and progressing to a multisystem disease variably with lassitude, arthritis, carditis, meningitis and other neurological manifestations [ ] . because of the increasing incidence and geographic expansion of lyme disease, the high incidence of tick exposure, and the difficulty in recognizing and removing attached ticks due to their small size, difficult differential diagnosis, troublesome and potentially severe clinical manifestations and medical controversies over treatment and chronicity of the disease, lyme disease has emerged as a high priority for public health interventions [ ] . vaccination of humans would appear to be a logical and cost-effective means to prevent the disease [ ] , and veterinary vaccines for dogs are widely used and have proven to be modestly effective [ ] . however, whereas safe and highly effective vaccines for humans have been developed, none is available for distribution today. glaxo smithkline's lymerix ® vaccine was approved in , but withdrawn in by the company, principally for commercial reasons, a decision that is lamentable given the increasing incidence of the disease [ , , ] . a new lyme disease vaccine for humans active against both b. burgdorferi and species causing lyme disease in europe developed by baxter bioscience is now in phase ii development, but it is uncertain whether it will reach the market. nevertheless, these vaccines established critical immunological principles; the human vaccines are composed of recombinant ospa protein, the dog vaccines of both ospa and ospc, and work via antibody-mediated mechanisms. ospa is expressed by the borrelia spirochete in the midgut of infected ticks. since the tick vector only begins to transmit borrelia - h after initiating blood feeding, ospa specific antibodies imbibed in the blood meal of a vaccinated host kill the bacteria and block transmission [ , ] . if a similar ospa antibody response could be evoked in the natural reservoir hosts of b. burgdoferi (peromyscus spp. field mice), it may be possible to interrupt the transmission cycle and reduce the prevalence of infected nymphal and adult ticks responsible for human and canine infections. proof of concept was obtained in a field study where peromyscus leucopus mice were trapped and vaccinated by subcutaneous injection of ospa; a reduction in the prevalence of b. burgdorferi in nymphal ticks was seen in the following year [ ] . however, practical vaccine delivery and effective immunization of mice in the wild, requires a thermostable oral bait vaccine matched to the high population density and rapid population turnover of the reservoir hosts, the effects of which are not diluted by non-targeted species that play a role in b. burgdorferi transmission [ ] . two promising live oral vaccine approaches have been investigated in the laboratory: a bacterial (e. coli) vector [ , ] and a viral vector (vaccinia) [ , ] expressing ospa. the e. coli vector contained in an oral bait formulation and ingested multiple times elicited anti-ospa antibodies and protected laboratory and wild p. leucopus mice against needle and tick challenge. a -year field study of the oral bait vaccine, sponsored by cdc, has been performed and results are anticipated with interest. a company, us biologics inc., is engaged in bringing this vaccine to market. the vaccinia technology, which rests on the shoulders of the successful oral bait vaccine against wildlife rabies (see below), has been tested in the laboratory. laboratory mice immunized by gavage with vaccinia expressing ospa were successfully immunized after a single dose and were protected against tick challenge. peromyscus consuming oral bait vaccine were also significantly protected against challenge with infected ticks. although the vaccinia vector looks promising, no commercial endeavor has yet emerged to support development. both the e. coli and vaccinia oral vaccines require specialized formulations in baits that incorporate the vaccine in the bait itself, rather than in a liquid sachet embedded in the bait used for delivery of rabies vaccines. many questions surround the application of an oral bait vaccine targeting the reservoir host, including efficacy of this approach in the field, the high density of baits required, cost and sustainability of local and state funded programs aimed at distributing baits, and the role of species not targeted by the vaccine in lyme disease maintenance cycles. if only partially effective, the risk of acquiring lyme disease may be reduced, but the public would still need to take precautions against tick bite. nevertheless, given the lack of a vaccine for humans, the high level of public concern about lyme disease, the high risk to children, the localized nature of b. burgdorferi transmission allowing geospatially focused control efforts, and the possibility that homeowners may be motivated to play an active role in distributing baits, the idea has appeal. m. bovis is the cause of tuberculosis in a wide array of domesticated and wild animals, and it remains a major veterinary health problem worldwide, causing severe economic losses from livestock disease, death and export restrictions. humans become infected by ingesting raw milk or undercooked meat, or by the aerosol route from infected animals or humans. in developed countries where pasteurization and test-and-slaughter programs have controlled the disease, zoonotic infections are relatively rare, accounting for . - . % of tuberculosis cases [ ] [ ] [ ] ; in developing countries which do not practice these measures, it remains more common, although few data on prevalence exist. wild animals are a major source of infection of domestic livestock [ ] . control measures aimed at control of m. bovis by culling wildlife reservoirs is problematic, with inconsistent results and ethical concerns. vaccination of wildlife is an attractive alternative control measure, especially since the traditional tuberculosis vaccine (bacille calmette-guerin, bcg) derived from m. bovis is effective when orally administered [ ] . examples of wildlife that serve as maintenance hosts of m. bovis and sources of infection in livestock, include white-tail deer in the us [ ] ; wild boar, red and fallow deer in europe [ ] ; badgers in the united kingdom [ ] ; african buffalo (syncerus caffer) in south africa [ ] ; and brushtail possums (trichosurus vulpecula) in new zealand [ ] . brushtail possums have been experimentally vaccinated using oral bcg and shown to be resistant to challenge with m. bovis [ ] . proof of concept has been provided by a field study in an endemic area of new zealand. possums were trapped, manually vaccinated using orally delivered bcg in a lipid matrix formulation, and vaccinated and control animals were recaptured at intervals [ ] . vaccinated animals received - vaccinations during the -year study. at the end of study, the ha study area was depopulated, and all animals assessed for clinical and subclinical m. bovis infections. vaccine efficacy against naturally acquired tuberculosis was - %. the authors concluded that oral vaccination of possums could be a practical strategy contributing to elimination of m. bovis in livestock. although the field study did not demonstrate control via freely consumed bait vaccine, captive possums have been shown to consume vaccine in flavored baits [ ] . rabies is transmitted between specific wild carnivore reservoir hosts, which serve as a source of spill-over infections of other wild carnivores, and infection of domesticated animals and humans. oral rabies vaccine was initially deployed in europe for control of rabies in the red fox (vulpes vulpes), using modified live virus vaccine [ , ] . the concept began in the s with the work of george baer at the cdc, which showed that foxes could be orally immunized with modified live virus [ ] . the live, attenuated evelyn-rokitnicki-abelseth (era) vaccine or street-alabama-dufferin (sad) viruses were employed in experimental and field studies. numerous studies in the s confirmed that captive and wild foxes could be orally immunized with a variety of baits containing vaccine [ ] . in , steck and colleagues initiated a wild fox rabies control program in the swiss alps using oral bait vaccine consisting of chicken heads with vaccine and tetracycline biomarker in a container made of polyvinyl chloride and aluminum foil inserted under the scalp [ ] . the trial demonstrated that % of foxes had ingested bait. over the next years, successful fox rabies control programs were carried out in many european countries, after the late s using baits distributed by fixed wing aircraft and helicopters rather than by ground [ ] , and resulting in elimination of terrestrial rabies in several countries [ , ] . for large scale distribution, the laborious chicken head method bait gave way to commercially manufactured molded or extruded baits of various kinds, consisting of fish meal or bone meal, fat, and a pouch or blister containing liquid vaccine virus [ ] . the vaccines currently used in europe are ( ) sag (e.g., rabigen ® , virbac laboratories, france), a modified live attenuated rabies virus derived from the original sad vaccine and having an additional mutation in the codon for amino acid of the rabies g protein, which increases genetic stability of the virus [ ] ; and ( ) recombinant vaccinia virus (copenhagen strain) expressing the era ® strain rabies g protein (raboral ® , merial corp.) [ ] . the rabies g protein gene has been inserted into the thymidine kinase gene of vaccinia, which results in further attenuation compared to the parental virus [ , ] . duration of oral rabies immunity, at least months in adult red foxes, is sufficient to provide herd immunity and reduce the reproductive rate (r ) to less than . the modified live virus vaccines are more thermolabile than vaccinia, require − • c storage, retain some pathogenicity for non-target species, and pose safety risks to humans exposed inadvertently. consequently, recombinant vaccinia is the only oral rabies vaccine approved for wildlife immunization in the us. this vaccine consists of fishmeal and fish oil bound by a polymer (ethylene vinyl acetate) and containing the vaccine in a plastic pouch held in place by a wax mixture. immunization with vaccinia or modified live virus occurs in the buccal mucosa and tonsils, and vaccines are poorly effective after ingestion [ ] . in one study, consumption by red foxes of a single bait containing vaccinia resulted in protection against virulent rabies in only half of the animals [ ] . this observation suggests that high bait densities and repeated vaccinations are important to effective control in the wild. bait densities distributed in europe generally range between and baits/km , resulting in - % of animals potentially immunized (positive for the tetracycline biomarker) [ ] . in addition to distribution density, feeding habits may also be important, since animals have been observed to pick apart baits and consume only the bait portion. while control of terrestrial wildlife rabies has been successful in parts of europe, it has been more challenging in other areas due to the diversity of carnivores involved in transmission of different rabies virus variants. in the arctic regions, a specific rabies variant is maintained in the arctic fox, with spill-over infections of red foxes, skunks, and raccoon dogs. where vaccination is not practiced in domesticated sledge dogs, these animals may be severely impacted by contacts with rabid foxes, and human dog owners placed at considerable risk. while experimental oral vaccination of arctic foxes has been successful, there is limited experience in the field [ ] . in ontario, canada control of arctic rabies variant in red foxes using oral bait vaccine has been successful, but the virus still occurs as a result of spill-over transmission to skunks, which are not efficiently immunized with recombinant vaccinia vaccine [ , ] . oral bait recombinant vaccinia vaccine has been primarily used to control raccoon rabies, which expanded beginning in the mid- s from enzootic areas in florida northwards and westwards to involve many states in the eastern us, as well as new brunswick and quebec, canada [ , ] . the control program relies on distribution of vaccine baits specific zones of rabies activity, particularly along the appalachian ridge, enhanced surveillance and ring vaccination with evidence of spread of the disease. judged from the absence of spread beyond the zones of vaccine distribution, the program has worked well, despite relatively low prevalence of rabies antibody (approximately %) in sampled raccoons [ ] . it is possible that pre-existing immunity to raccoonpox virus may interfere with immunization with vaccinia [ ] . the economics of large-scale oral vaccination programs in the us have been modeled and are generally favorable [ ] . in addition to control of raccoon rabies, successful use of the vaccine has been made in the control of gray fox (urocyon cinereoargenteus) variant rabies in west texas [ ] . in contrast to raccoons, a higher prevalence of rabies antibody ( %) attributed to vaccination is found in gray foxes. rabies in coyotes (canis latrans) was responsible for epizootic canine rabies in the s and s in parts of the us, and was controlled by an oral bait vaccination campaign, contributing to the elimination of canine rabies by [ ] . skunks remain a problematic species for vaccine control of rabies. skunks are an important spill-over host for the arctic fox, raccoon rabies, and big brown bat rabies virus variants [ ] . although the vaccinia vector vaccine is not sufficiently effective in skunks [ ] , promising results were obtained with a replicationcompetent adenovirus type vector expressing the rabies g protein [ ] . aerial distribution of this vaccine (onrab ® , artemis technologies, guelph) showed high rates of immunization of raccoons, and arctic foxes and modest seroconversion ( - % in different plots) in skunks, probably due to lower rates of bait acceptance [ , ] . onrab ® is approved by the canadian regulatory authorities for control of rabies in skunks, raccoons, and foxes, and is under investigation in the us. there are some notable failures of animal vaccination as a means to preventing zoonotic diseases of humans, and these illustrate some of the limitations of the approach shown in table . japanese encephalitis, a mosquito-borne flavivirus closely related to west nile virus, is endemic in asia, with nearly billion people at risk of infection [ ] . horses and humans are dead-end hosts, and framework i immunization of both is widely practiced in many parts of asia, with a long record of success. pigs are an important domesticated animal amplifying host for infection of rice paddy-breeding culex spp. vectors, resulting in spill-over infections of humans. moreover, infection of pregnant sows can lead to abortion and stillbirth, and infected boars may have reduced spermatogenesis and infertility [ ] . framework ii immunization of swine was previously a major initiative in japan, using live, attenuated vaccines. however, it was exceedingly difficult to vaccinate piglets born in spring and early summer during the narrow window between loss of interfering maternal antibody and contribution to virus amplification. the practice of vaccination was abandoned in favor of re-locating piggeries from areas of culex breeding and biting activity. elsewhere in asia, other obstacles precluded consideration of framework ii vaccination against japanese encephalitis, including the prevalence of small piggeries located near vector breeding sites and of feral swine, and the importance of wild ardeid birds and waterfowl in virus transmission. moreover in many areas of asia, less developed than japan, and undergoing rapid urbanization, the locations of pig holdings are not controlled and are often located near rice paddies and urban centers [ ] . consequently, the focus has long been on human vaccination as the primary means of prevention. this case study exemplifies some of the factors that can limit application of framework ii vaccination: ( ) the need to customize vaccination to the breeding and husbandry practices and timing of domesticated animal targets; ( ) the role of wild animals and feral domesticated animals as additional amplifying hosts in the transmission cycle; and ( ) difficult access given the very large scale and geographic complexity of domesticated animal populations. q fever is caused by the intracellular gram-negative bacterium, coxiellla burnetii and an important worldwide infection of ruminants, which serve as the source of infection of humans, especially where large numbers of animals are concentrated [ ] . q fever is a major occupational hazard of abattoir and farm workers, veterinarians, and persons involved in the handling and distribution of animals or animal products. a dramatic outbreak recently occurred in high-intensity goat farms in the netherlands ( ), with human cases and deaths [ ] . transmission of c. burnetii occurs between direct spread between domesticated animals, and from animals to humans. infected animals shed bacteria in urine, feces, vaginal secretions and products of conception, and in milk. ticks are also a reservoir of bacteria in nature and a source of infection of livestock. ruminants, particularly goats and sheep develop pneumonia, abortion, stillbirth, premature delivery, and delivery of weak offspring, and herds can be affected for prolonged periods, causing significant economic losses [ ] . there are two developmental stages of c. burnetii, a small-cell variant (scv, the extracellular form) and the metabolically active intracellular large-cell variant. the scv is highly resistant to degradation and can persist in the environment for long periods of time. infection of humans is acquired principally by the aerosol route via dust containing spore-like scv forms, with oral routes of infection (ingestion of unpasteurized milk) being secondary. the human disease is characterized by an influenza-like illness, and may be complicated by pneumonia, endocarditis, and (pregnant women) abortion and fetal death. person-to-person transmission occurs, but is rare. approximately % of infected persons may develop chronic infections, with various manifestations. prophylactic vaccines have played a role in the control of q fever in livestock, but the practice is not a reliable means of preventing human infections. in russia, a live, attenuated m- vaccine was used for many years in animals and humans, but causes a persistent infection and has not been considered safe for use elsewhere. in europe, coxevac ® , a formalin inactivated strain rsa /nine-mile phase bacterial form (smooth forms, with complete surface lipopolysaccharide) has been used in goats and cattle and reduced the incidence of shedding, but is not effective in pregnant or chronically infected animals [ , ] . the live and phase vaccines are also not diva, which presents substantial issues for export controls. in australia, an inactivated phase vaccine prepared from henzereling strain is marketed for humans by csl ltd (qvax ® ) [ ] . however, severe reactions occur in persons who have previously been naturally infected with c. burnetii, and skin testing to ensure absence of exposure is required [ ] . clearly, improved vaccines are needed for both livestock and humans, but various attempts at recombinant vaccines have been disappointing. the problem for framework ii vaccination against q fever is due to multiple factors, including imperfect vaccines, limited efficacy of vaccines in parous animals, the difficulty in recognizing the disease and intervening expeditiously, and the rapid and widespread contamination of the environment with scv forms. the last problem is the major reason that livestock vaccination is not a reliable means of protecting humans against exposure. a year study of sheep vaccinated with the phase vaccine showed that the proportion of animals shedding bacteria in feces was markedly reduced after year and then eliminated after years, but c. burnetii was still present in environmental samples [ ] . finally, live veterinary vaccines may pose a risk of inadvertent infection of humans handling the vaccine or vaccinated animals. examples include live brucella and orf (contagious ecthyma) vaccines. while this topic is beyond the scope of this review, it is important in several contexts, and indeed little is known about the risk of human pathogens to animals. immunization of swine and poultry workers against influenza as a means of preventing introduction of human influenza viruses to these animals has been emphasized as a means of preventing emergence of reassortant strains [ ] . immunization of humans to prevent spread of viruses to captive nonhuman primates is often practiced, including vaccines for influenza, hepatitis a and b, and measles. prevention of animal diseases and human diseases by use of vaccines is a well-established principle, and there are potential synergies that can be achieved in concurrent delivery of human and animal vaccines in developing country settings [ ] . for some diseases affecting both livestock and humans there is a clear commercial incentive to develop vaccine products (framework i vaccines) ( table ) . however, such development efforts are generally segregated in the animal and human health divisions in industry and academia, and have separate regulatory pathways. this results in a potential waste of resources and duplicated scientific endeavors. interestingly, when one company (akso nobel), an animal health company, decided in on a strategic move into human vaccine development, it drew on its veterinary scientists to staff the program. as pointed out in this review, there have been isolated successful examples, e.g. a west nile vaccine, of co-development of a vaccine for both veterinary and human indications, an obviously efficient strategy that broadened both the commercial and public health opportunity. future efforts along similar lines should be considered on a case by case basis, depending on medical need, but in general there is value in closer connections between human and veterinary vaccines and regulatory science, and in the application of domesticated animals as models for development of infectious disease and cancer vaccines. several issues related to diva requirements and liability concerns have been mentioned. prevention of zoonotic diseases of humans by means of vaccination of domesticated (framework ii) or wild (framework iii) animals is an attractive but under-exploited concept. an obstacle is that there may be limited commercial incentives (table ) . where a market exists, governments may be the principal customers, as is the case for the approved oral bait rabies vaccines and the reservoirtargeted lyme disease vaccine in development. thus, the public health gains for such an intervention need to be compelling and must offset the cost of development and implementation. the goal is far easier to justify when vaccination also prevents disease in an economically valuable animal species, there is a profit incentive for animal vaccination or a clear social gain from improved animal health, and when the public health spin-off is an added benefit. examples of the latter may include vaccines against hendra and nipah virus diseases, brucellosis, chlamydophila felis, rift valley fever, and venezuelan equine encephalitis. the potential for elimination of a disease through vaccination (employed together with other strategies), as has been demonstrated for brucellosis in the us and terrestrial rabies in some european countries is a compelling economic concept, though applicable in only selected cases. the cost of preventative programs is almost always lower than emergency response programs, as illustrated by the significantly lower cost of oral wildlife rabies programs over contingency actions to control epizootic spread [ ] . the recent announcement of a fold higher incidence of lyme disease than previously believed will lead to a reassessment of the economics of preventive strategies for this disease, including wildlife vaccines. economics represent the key determinant for development and utilization of framework ii and iii vaccine strategies. a low unit cost of such vaccines will always be a requirement. the economic barriers are particularly relevant when considering vaccines for developing countries. on the positive side, the cost of developing a new animal vaccine through licensure is a small fraction, approximately %, of the cost of a typical human vaccine (the latter being $ - million by one estimate [ ] , but often far higher) [ ] . the relatively lower cost and shorter timelines for developing animal vaccines reflects the simpler path to regulatory approval, and is driven by the significantly lower market potential for these vaccines. despite the lower cost of developing new veterinary vaccines, high and middle income countries still pay higher prices until the fixed costs of development are paid off, there is an over-supply of vaccine, or competing products enter the market. to redress the pricing barriers in the case of human vaccines, there has been strong advocacy for new approaches to secure vaccine supply and access for developing countries where the burden of infectious diseases is greatest [ ] . as part of this strategy, emerging market manufacturers provide access to low unit cost vaccines, and such manufacturers of veterinary vaccines could play a substantial role in a public health strategy for animal immunization. indeed all of the principles being applied to human vaccines could be extended to vaccines for animals, particularly if there is both a clear rationale for public health and the "pull" of a potentially expanded market or of guaranteed purchase agreements. up to now public-private financing for developing and distributing veterinary vaccines has represented a tiny fraction of support available for human developing-world vaccines, and has focused on vaccines for livestock as a means of improving animal production and protein supply rather than preventing zoonotic diseases [ ] . given the public health impact of zoonotic diseases described in the introduction to this review and the potentially lower cost of interventions targeting animals vs. humans, there should be a new emphasis on the public health improvements that could result from animal immunization. zoonotic diseases that merit consideration because they occur at high incidence or are poorly controlled, include rabies, zoonotic leishmaniasis, brucellosis, rift valley fever, m. bovis, lyme disease, and several enteric bacterial infections. as mentioned above, the regulatory pathway for animal vaccines is considerably simpler than for human vaccines [ ] . this is due to multiple factors, including less onerous requirements for manufacturing and control of veterinary products, the simpler and far less expensive clinical trial requirements for marketing approval, the ability to challenge animals to demonstrate vaccine efficacy, as well as an established regulatory mechanism for conditional approval allowing commercial sales while still gathering more definitive data. there is no requirement for large, statistically powered efficacy field trials to obtain marketing approval, as is the case for human vaccines. the development of veterinary vaccines is consequently far faster than for human vaccines. for example, west nile vaccine for horses was commercialized years after introduction of the virus into the u.s., whereas the first (phase ) human trial of a west nile vaccine was completed years after introduction. the lower costs and accelerated timeframe for development of animal vaccines represent an important rationale for novel investments in public health, particularly in developing countries. to justify investments in a framework ii or iii vaccine where an economic incentive for animal immunization is marginal and a public health benefit is a goal, it is important to plan for vaccine effectiveness studies showing that vaccination of animals actually reduces human disease prevalence, as was postulated in greece following vaccination against b. melitensis [ ] . such demonstrations would really drive the field forward. thus, while the deployment of tc- venezuelan equine encephalitis vaccine was credited with the curtailment of the human epidemic in - , no controlled study to demonstrate an effect on human disease incidence was actually performed. indeed, studies to confirm the attractive hypothesis that immunization of domesticated ruminants against rift valley fever in africa would prevent intermittent outbreaks of the disease in animals and humans, while leaving the mosquito reservoir of infection intact, would be difficult to design and carry out. because of its discrete epidemiology, hendra virus (albeit a low-incidence disease) presents a unique opportunity to demonstrate the effectiveness of animal immunization on the occurrence of a disease in humans. the increasing problem of emerging infections, the majority of which are the result of spill-over from animals to humans, is a compelling reason to consider novel vaccine interventions, and the collaborations between veterinary and human 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infection: vaccines and guidelines for those at risk four-year evaluation of the effect of vaccination against coxiella burnetii on reduction of animal infection and environmental contamination in a naturally infected dairy sheep flock the importance of including swine and poultry workers in influenza vaccination programs human and animal vaccination delivery to remote nomadic families emergency response to raccoon rabies introduction into ontario how the research-based industry approaches vaccine development and establishes priorities current status of veterinary vaccines assembling a global vaccine development pipeline for infectious diseases in the developing world key: cord- -riotkgj authors: seo, yurim; pacifici, eunjoo title: elements of regulatory dissonance: examining fda and ema product labeling of new vaccines ( – ) date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: riotkgj with the ongoing globalization of the pharmaceutical industry, efforts to harmonize technical requirements of registering drugs and biologics, including vaccines, have produced a number of useful guidelines utilized around the world. however, such efforts have not been extended to the regulatory review process or product labeling. prescribing information and patient information leaflet are two types of such product labeling documents. this study examined the differences in the languages of these documents between the united states (us) and european union (eu). the key documents examined were the u.s. food & drug administration’s (fda) package inserts (pis), u.s. centers for disease control and prevention’s (cdc) vaccine information statements (viss), and the european medicines agency’s (ema) summary of product characteristics (smpcs) and package leaflets (pls). prescribing information and patient information leaflet languages were subsequently organized into ten and seven categories, respectively. comparison of fda pis to ema smpcs showed little harmonization between the two regions, and cdc viss to ema pls revealed even less. each year, regulatory agencies worldwide review new drugs and biologics, including vaccines, for marketing approval within their respective regions. this process is essential, as it not only assesses the safety and efficacy of products but also oversees the product labeling intended for use by healthcare professionals and patients. however, because approval processes are not globally harmonized, a pharmaceutical company seeking approval for its product in multiple regions must prepare and submit separate applications, which may be different in content and format. although efforts by the international council for harmonization (ich) to harmonize technical requirements for registering drugs and biologics have produced a number of useful guidelines that are used around the world, such efforts have not been extended to the regulatory review process or product labeling [ ] . currently, the united states food & drug administration (fda) and the european medicines agency (ema) are considered two of the most prominent regulatory agencies around the world. this study compared vaccine prescribing information and patient information leaflet languages between fda/centers for disease control and prevention (cdc) and ema. in both the united states (us) and the european union (eu), vaccines undergo rigorous regulatory approval procedures to ensure their safety, efficacy, and quality. depending on the product, this process can take anywhere from months to years, where delay in access could pose risks to public health. this is especially important and relevant in the case of vaccines that are developed for highly contagious illnesses or in response to infectious disease outbreaks. in the us, fda is responsible for all drug approvals. when a company files a biologics license application (bla) for a vaccine, the application is reviewed by the center for biologics evaluation and research (cber) [ ] . in the eu, all biologics, including vaccines, must be authorized by ema. there are three available pathways for pharmaceutical product approval in the eu: centralized, decentralized, and mutual recognition [ ] . the centralized route allows companies to submit a single marketing authorization application (maa) to ema that leads to the product's approval in all countries within the european economic area (i.e., the member states of the eu plus iceland, liechtenstein and norway). once submitted, it undergoes review by the committee for medicinal products for human use (chmp) [ , , , ] . all products https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. approved by both fda and ema during the time frame of this study were approved through the centralized procedure. on the other hand, the decentralized pathway allows companies to apply for simultaneous authorization in more than one, but not all, eu member states, as long as the product for which they are seeking approval has not yet been authorized for marketing in any eu nation [ ] . in this process, one country is designated as the reference member state and completes a preliminary assessment. if the other countries are in agreement with the reference state's assessment, marketing authorization will be granted. finally, the mutual recognition pathway allows a company whose product has already been authorized in one eu nation (reference state) to apply for approval in other eu countries (target states) [ ] . the target states rely on the scientific assessment of the reference state to decide whether to grant a marketing authorization [ ] . the application for approval includes extensive safety and efficacy data collected during the pre-clinical and clinical phases as well as the company's proposed labeling, which is the ultimate deliverable of the approval process. based on this information, each agency evaluates the risk-benefit ratio of the vaccine [ , ] . one of the final steps of product approval is the completion of product labeling. regulatory agencies work with the manufacturers to finalize their proposed language, ensuring that the information is appropriate, sufficient, and accurate [ ] . there are two types of product labels -one intended for use by healthcare professionals (prescribing information), and one intended for use by patients (patient information leaflets). vaccine labeling for medical professionals are technical documents used by physicians, pharmacists, and other qualified medical personnel to obtain information regarding the administration, precautions, safety, potential side effects, and efficacy of a vaccine and evaluate whether it is appropriate to administer to a given patient. on the other hand, the labels for patients present information regarding directions, indication, contraindications, side effects, and dosages in a more patient-friendly manner, allowing the patients to weigh the risks against the benefits and make an informed decision. product labeling is often the only vaccine information disseminated to healthcare professionals and patients; hence, it is crucial for it to be accurate and sufficient. discrepancies between regions can lead to unwarranted differences in the understanding and utilization of the product. in the us, package inserts (pi) serve as useful communication tools to healthcare professionals, providing sufficient safety and effectiveness data necessary to administer vaccines appropriately [ , ] . the labels for patients are the patient information through fda and vaccine information statements (vis) provided by cdc. however, federal law requires that patients receiving any vaccine in the us be given the vis rather than the patient information document [ ] . in the eu, the summary of product characteristics (smpc), required by directive / /ec, is the official information source for medical professionals [ , ] . the information in the smpc is used to draft the package leaflet (pl), a document intended for use by patients. it is to be written in clear and understandable language so patients are adequately informed about the safety, effectiveness, and directions for use. with the help of healthcare professionals, patients will use this document to discuss and make decisions about their treatment [ ] . in an effort to develop guidelines for international harmonization, the international council for harmonisation of technical requirements for pharmaceuticals for human use (ich) was created in april in brussels, and has since developed quality, safety, efficacy, and multidisciplinary guidelines [ , , ]. an example includes the common technical document (ctd), which is a harmonized electronic application for new products [ ] . the ctd is comprised of modules through , of which module is region specific, and modules through are the same for all regions. included in module is the prescribing information, such as the proposed product information and labeling, which depicts the essential information on safety, efficacy, and guidelines for use [ , ] . the contents of such approved labeling are an important tool for both healthcare professionals and the general public, because although these products are intended to provide therapeutic benefits, they may also pose harm. this concept is particularly applicable to vaccines, as these products are primarily given to healthy individuals, and their benefits must clearly outweigh their risks. although product labeling should be tailored to the needs of the local population, the core safety and efficacy information included should be the same across all regions, especially when they are derived from the same set of clinical evidence. the objective of this study was to compare contents of the product labeling and patient leaflets between the us and eu to identify where they harmonize and where they differ. a retrospective analysis was performed of all vaccines approved by fda and ema between january , and june , . the regulatory aspects examined for each vaccine were the prescribing information and patient information leaflet languages. prescribing information was extracted from fda's pi and ema's smpc, and the patient information leaflet language from the cdc's vis and ema's pl. the most recently updated versions (as of august ) of all regulatory documents were taken from fda, cdc, and ema websites (https://www.accessdata.fda.gov/scripts/cder/daf/, https:// www.cdc.gov/vaccines/hcp/vis/index.html, https://www.ema.europa.eu/en/medicines, respectively). first, the prescribing information and patient information leaflet languages were assessed for the level of harmonization between the two regions. although the same topics could be found in the documents of both regions, the contents were arranged differently depending on the agency. for example, to describe the intended use of the vaccines, fda used the term ''indications and usage," while ema used ''therapeutic indications." for analysis, this labeling element was simply referred to as ''indication" (supplementary table ). ten prescribing information elements and seven patient information leaflet elements were examined (supplementary tables and ). detailed criteria on the type of information assessed for each element are outlined in supplementary tables and . these criteria must be the same for the corresponding element to be considered harmonized, ignoring any spelling, punctuation, grammatical, or phrasing differences. because the vaccines analyzed in this study were approved over a long period of time ( years), the possibility of change in the level of harmonization over the years was explored. the number of harmonized prescribing information and patient information leaflet elements were organized by year of approval into three groups: - , - , and - . for vaccines whose approval dates in the us and eu did not fall into the same category, fda's approval date was used. the average of the number of elements harmonized was calculated for each time period and analyzed for any trends. from january to june , vaccines were approved by fda and by ema. of these, a total of vaccines were approved by both fda and ema (supplementary table ). of the twelve common vaccines across fda and ema, none had harmonized prescribing information across all ten elements ( table ). the greatest level of harmonization across the elements was six out of ten. the labeling elements with the greatest level of harmonization were pregnancy assessment and pediatric assessment, both of which were harmonized across seven out of twelve vaccines. conversely, warnings & precautions, adverse events, and patient counseling information were not harmonized for any vaccine (tables and ). similar to the prescribing information, none of the twelve vaccines demonstrated harmonized patient information across all seven elements (table ) . for all vaccines, harmonization occurred across only one or two out of seven elements. the element that was most harmonized (observed across all vaccines) was side effects reporting, as both cdc and ema have established national reporting systems. the element use during pregnancy was harmonized in four out of twelve vaccines. the elements disease information, contraindications, side effects, and what to look out for were not harmonized in any products across the two regions (table ). no pattern was observed in the number of prescribing information and patient information leaflet elements harmonized over time (fig. ) . this analysis of prescribing information and patient information leaflet languages demonstrated that certain differences exist between the labeling languages of the us and eu. despite the same clinical data submitted for market authorization application, it is evident that the resulting labeling language is different. for example, same products assessed by each regulatory agency with the same set of clinical data may result in labels with different therapeutic goals [ , ] . hence ema's smpc for a shingles vaccine may state that it is for the prevention of herpes zoster (shingles) and post-herpetic neuralgia, while fda's pi states that it should be used for the prevention of herpes zoster only and explicitly indicates the vaccine is not to be used for the treatment of postherpetic neuralgia. these observed differences indicate the lack of a harmonized process to translate the information in the marketing application to the prescribing information and patient information leaflet. although ich provides guidelines on a harmonized method of application submission, there are currently no harmonized guidelines on the content of labels; hence different agencies around the world may be submitted the same set of pre-clinical and clinical trial data but use different assessment criteria that ultimately lead to divergent labeling language. given the findings of this study, the question arises as to what implications these differences could have for medical professionals and patients. for instance, differences in indication, dosing, and recommended ages for use may lead to inconsistencies in not only y. seo and e. pacifici vaccine xxx (xxxx) xxx the number of doses but also whether individuals receive the vaccine at all. an example of differences in the recommended age range for use is demonstrated by the meningococcal vaccine. in the us, fda sets the age range to months to years of age, while ema's age range is from years of age. moreover, the age ranges for use may be complicated by the recommended vaccine schedules set by the public health authorities of each country. within the age ranges set by ema, each country in the eu can have different recommendations based on the epidemiology and culture of the respective region [ ] . in the us, children to months are able to receive the vaccine if they have certain conditions, including complement deficiency or human immunodeficiency virus (hiv). however, in the eu countries, these children cannot be vaccinated due to the restrictions set by the indication statement. what are the implications of this difference? one of the reasons for ema's decision not to include children ages to months was that antibody persistence was decreased in clinical trial participants who received the vaccine in those age ranges [ ] . in this case, the children in the us who receive the vaccine may be placed at unnecessary risk of injection-related side effects. on the other hand, even this temporary protection provided by the vaccine may be beneficial for children who are at especially high risk of contracting the disease; in this case, the children in the eu who do not receive this vaccine may be faced with greater risk of harm. as such, although the implications of these differences may not be clear, they could be clinically significant. furthermore, an important patient information leaflet element that supplements the patient's understanding of the therapeutic indication is disease information, which describes the disease that the vaccine is designed to prevent. with this information, patients can understand what the disease is, the dangers of contracting the disease, and why it is important to be vaccinated against it. however, there were notable differences in the level of detail for this element between the us and eu. although patients may consult other sources as well, the disease information section of the patient information leaflet remains the primary document to learn about the disease that the vaccine protects against. if this section is not sufficient, patients will not be able to properly make an informed decision as to whether they should be immunized. the dangers of having limited information on the populations at risk is that individuals within this population may not know that they are especially vulnerable to contracting the disease. as such, they may not be fully equipped with the information necessary to make an educated decision. the safety profile for vaccines is especially important and must be clearly established, as these products are generally given to healthy individuals. included in the safety profile is the element contraindications. an example of differences in this element is demonstrated by the shingles vaccine. in particular, one of the contraindications listed by ema is ''active untreated tb." however, the us documents do not include this contraindication. if the vaccine administration to individuals with active untreated tb causes harm, then those in the us may not be adequately informed about this risk. on the other hand, if it is generally satisfactory for tb patients to receive the vaccine, these individuals in the eu may be placed at a disadvantage, as they would be at increased risk of contracting shingles without this vaccine. if vaccine labels were harmonized, everyone, regardless of region, would receive the same comprehensive information necessary to make an informed decision regarding each vaccine. moreover, having harmonized schedules and administration would lead to simplified vaccine delivery. this is particularly relevant for individuals traveling or re-locating from one region to another, as their vaccine needs would remain the same anywhere. however, differences in culture, local terminology, and/or epidemiology may result in labeling language differences. as such, the set of vaccines necessary in one country may not be important in another due to a low disease prevalence, and cultural and terminology disparities may mean that the best labeling language is not identical across all countries. harmonized vaccine approval and administration would be valuable for diseases that impact the global community, as it would lead to faster access to potentially beneficial vaccines. for example, the rapid expansion of the -ncov (covid- ) pandemic demonstrates that a swift global response is necessary, and the development of a safe and effective vaccine is crucial to control such outbreaks. as infectious agents can readily cross national boundaries and regulatory jurisdictions, a globally harmonized approach to vaccine approval and administration would be valuable to protect the health of the populations around the world. the lack of harmonization among global regulatory agencies means that each country implements its own system of review. these differences lead to redundancy and decreased efficiency, as several approaches are taken to demonstrate the same concept. this redundancy may lead to increased cost for drug manufacturers that is passed down to consumers as increased drug prices. having a harmonized approach to vaccine approval would lead to lower cost of vaccines and ultimately greater affordability and access. however, different approaches to regulatory decisions are not necessarily a problem. although redundancy may decrease efficiency, it could increase reliability, as repeated results will confirm the same concept [ ] . moreover, the best approach to a product approval or public health matter is not always obvious. when presented with the same set of evidence, different agencies can have differing opinions and draw different conclusions. ultimately, by presenting divergent opinions, the agencies as a whole are able to explore a variety of different paths and contribute to a more thorough assessment of the issue at hand. one limitation of the study is the small sample size, which limits the ability to detect clear trends. the limited sample size was determined by the number of vaccines approved by the two agencies during the -year period. another limitation is the subjective interpretation of the language and approach used by the two agencies required to identify and align the labeling elements. for example, the us pi may state that the vaccine should not be administered to anyone who experienced a ''severe allergic reaction. . .after a previous dose. . .or any component" of the vaccine, while the eu smpc states that ''hypersensitivity to the active substance or to any of the excipients" is a contraindication to receiving the vaccine. in this case, a severe allergic reaction and hypersensitivity were considered as equivalent, as they refer to the same immunological reaction against the vaccine. overall, this analysis compared clinical labeling languages for several vaccines approved by fda and ema. some of the observed differences have clinically significant implications that could affect downstream patient care. future research could investigate differences between the two regions in vaccination completion rates, disease rates, and vaccination-related injuries. moreover, future work could also evaluate what regulatory processes were implemented by the two agencies and whether they led to the observed language differences. moving forward, the us and eu, as well as other countries, should work together to create uniform prescribing information and patient information leaflet content so that all individuals, regardless of region, could have access to the same clinical information. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. transnational pharmacogovernance: emergent patterns in the jazz of pharmaceutical policy convergence frequently asked questions about the fda drug approval process the european regulatory system for medicines authorisation procedures -the centralized procedure the centralised procedure at the ema the centralised procedure marketing authorisation: the evaluation process vaccines europe. eu regulatory framework for vaccines step : fda drug review food and drug administration food and drug administration. the story of the laws behind the labels food and drug administration. an introduction to the improved fda prescription drug labeling a guideline on summary of product characteristics what is a package leaflet -how to review it? the international council for harmonisation of technical requirements for pharmaceuticals for human use. history ich quality guidelines: an implementation guide priority review drugs approved by the fda and the ema: time for international regulatory harmonization of pharmacueticals? the international council for harmonisation of technical requirements for pharmaceuticals for human use. m : the common technical document australian government department of health. ctd module food and drug administration vaccination schedules in other countries national instruments corp. redundant system basic concepts the authors would like to thank yu chung for providing edits and insightful comments. all authors attest they meet the icmje criteria for authorship. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -vevsgkp authors: alharbi, naif khalaf; padron-regalado, eriko; thompson, craig p.; kupke, alexandra; wells, daniel; sloan, megan a.; grehan, keith; temperton, nigel; lambe, teresa; warimwe, george; becker, stephan; hill, adrian v.s.; gilbert, sarah c. title: chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: vevsgkp abstract the middle east respiratory syndrome coronavirus (mers-cov) has infected more than humans, since . the syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. the virus is believed to be circulating in dromedary camels without notable symptoms since the s. therefore, dromedary camels are considered the only animal source of infection. neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. here, we developed four vaccine candidates against mers-cov based on chadox and mva viral vectors, two candidates per vector. all vaccines contained the full-length spike gene of mers-cov; chadox mers vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tpa) where mva mers vaccines were produced with tpa, but either the mh or f promoter driving expression of the spike gene. all vaccine candidates were evaluated in a mouse model in prime only or prime-boost regimens. chadox mers with tpa induced higher neutralising antibodies than chadox mers without tpa. a single dose of chadox mers with tpa elicited cellular immune responses as well as neutralising antibodies that were boosted to a significantly higher level by mva mers. the humoral immunogenicity of a single dose of chadox mers with tpa was equivalent to two doses of mva mers (also with tpa). mva mers with mh or f promoter induced similar antibody levels; however, f promoter enhanced the cellular immunogenicity of mva mers to significantly higher magnitudes. in conclusion, our study showed that mers-cov vaccine candidates could be optimized by utilising different viral vectors, various genetic designs of the vectors, or different regimens to increase immunogenicity. chadox and mva vectored vaccines have been safely evaluated in camels and humans and these mers vaccine candidates should now be tested in camels and in clinical trials. middle east respiratory syndrome (mers) is caused by a novel betacoronavirus (mers-cov) that was isolated in late in saudi arabia [ ] . the syndrome (mers) is described as a viral infection that causes fever, cough, and/or shortness of breath and to a lesser extent gastrointestinal symptoms such as diarrhea [ ] . severe disease from mers-cov infection can cause respiratory failure and organ failure, and cases can be fatal, especially in patients with co-morbidities such as diabetes and cardiac complications. however, the infection can be asymptomatic or mild in many cases [ ] [ ] [ ] [ ] [ ] . mers-cov has spread to countries and infected more than humans with a mortality rate of % [ ] . dromedary camels, especially juveniles, contract the infection and shed the virus, without notable symptoms of disease; this is now known to have been occurring since the early s [ ] [ ] [ ] [ ] [ ] [ ] . the mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact, which is considered an important risk factor [ ] [ ] [ ] . therefore, camels http [ ] [ ] [ ] [ ] [ ] [ ] . other livestock animals such as sheep, goats, cows, chicken, and horses have proved seronegative in many studies [ ] [ ] [ ] [ ] . further, these animals did not productively contract mers-cov when they were inoculated experimentally [ , ] . therefore, to date, dromedary camels are the only confirmed animal reservoir. there is currently no approved vaccine against mers-cov for camels or humans despite active vaccine research and development. a number of vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models [ ] . viral vectors are potent platform technologies that have been utilised to develop vaccines against malaria, tuberculosis, influenza, hiv, hcv, ebola, and many viral pathogens. these vectors include adenoviruses, poxviruses, yellow fever viruses, and alphaviruses [ , ] , and they are preferred for their ability to induce cellular immune responses in addition to humoral immunity. here, we report development of mers-cov vaccine candidates that are based on two different viral vectors: chimpanzee adenovirus, oxford university # (chadox ) [ ] and modified vaccinia virus ankara (mva) [ , ] . each viral vector was developed by generating two alternative versions, resulting in four vaccine candidates that all encode the same complete mers-cov spike gene (s). the two chadox based vaccines were produced with or without the signal peptide of the human tissue plasminogen activator gene (tpa) at the n terminus. previous studies have shown that encoding tpa upstream of recombinant antigens enhanced immunogencity, although results differed depending on the antigens employed. the tpa encoded upstream of influenza a virus nucleoprotein, in a dna vector, enhanced both cellular and humoral immune responses in mice [ , ] , whereas the same leader sequence resulted in increased humoral sequences but decreased cellular responses to hiv gag [ ] . the two mva based vaccines were produced with either the mh or f poxviral promoter driving antigen expression, both including the tpa sequence at the n terminus of mers-cov spike protein. previously, we reported the ability of the strong early f promoter to enhance cellular immunogenicity of vaccine antigen candidates for malaria and influenza, as compared to utilising p . or mh early/late promoters which resulted in a lower level of gene expression immediately after virus infection of target cells, but higher levels at a later stage [ ] . here, we continue to assess the f promoter in enhancing cellular immunogenicity, and to investigate its ability to impact on humoral immune responses. the four vaccine candidates were evaluated in a number of different regimens in mouse models that showed a single dose of chadox mers inducing higher cellular and humoral immunogenicity than a single dose of mva mers, or equivalent to two doses of mva mers. chadox based vaccines have been tested in different animal models, including camels [ ] , and in human clinical trials and proved safe and immunogenic [ ] . therefore, based on our data, chadox mers can be readily developed for use as a mers vaccine in humans. furthermore, utilising chadox mers for camel vaccination can serve the one-health approach whereby blocking mers-cov transmission in camels is expected to prevent human infections. the spike (s) gene of mers-cov camel isolate (genbank accession number: kj . ) was synthesised by geneart gene synthesis (thermo fisher scientific). the s transgene was then cloned into four shuttle plasmid vectors following in-fusion cloning (clontech). two plasmids contained the s transgene within the e homologous region of chadox , driven by the human cytomegalovirus major immediate early promoter (ie cmv) that includes intron a. one of the chadox shuttle plasmids was designed to include the tpa signal sequence upstream of the transgene sequence while the second plasmid did not contain the tpa. the chadox shuttle plasmids contained the s transgene within gateway Ò recombination cassettes. to construct mva mers, one of the shuttle plasmids for mva was designed to have the upstream and downstream (flanks) of the f l orf as homologous sequence arms. inserting the s transgene within these arms enabled the utilisation of the endogenous f promoter, which is part of the right homologous arm, while deleting the native f l orf. this resulted in the shuttle vector for generation of f -mva mers (f shuttle vector). the mh promoter sequence was subcloned upstream of the s transgene; and this mh -s transgene was then subcloned into the f shuttle vector. this resulted in the shuttle vector for generation of mh -mva mers (f /mh shuttle vector). mh -mva mers contained the mh promoter at the f l locus, however, the endogenous f promoter is intact and located upstream of the mh promoter. the endogenous f promoter could not be replaced with the mh since it is part of the essential upstream orf. the chadox shuttle plasmid, described above, was used to validate the expression of mers-cov spike protein in vitro. an african green monkey kidney cell line (vero cells) was seeded into -well plate to % confluence. then the plasmid dna was transfected into vero cells using lipofectamine Ò (thermo fisher scientific) following manufacturer's instruction. twenty-four hours after transfection, cells were fixed, permeabilised, and immunostained using a rabbit polyclonal anti-mers-cov spike antibody, following standard protocols. dapi stain was used to label nuclei. the chadox mers vaccines were prepared by gateway Ò recombination between the chadox destination dna bac vector (described in [ ] ) and entry plasmids containing the coding sequence for mers-cov spike gene (chadox shuttle vectors explained above), according to standard protocols. chadox mers genomes were then derived in hek a cell lines (invitrogen, cat. r - ), the resultant viruses were purified by cscl gradient ultracentrifugation as previously described [ ] . the titres were determined on hek a cells using anti-hexon immunostaining assay based on the quicktiter tm adenovirus titer immunoassay kit (cell biolabs inc). for mva mers vaccines chicken embryo fibroblast cells (cefs) were infected with mva parental virus that encodes dsred marker instead of the native f l orf and transfected with mva shuttle plasmids containing mers-cov spike gene (explained above) to allow recombination with the mva genome and deletion of dsred marker whilst keeping the f promoter sequence. recombinant mva expressing mers-cov s protein was purified by plaque-picking and fluorescent selection using the sorting function of cyclone robotic module of a moflo flow cytometer (dako cytomation, denmark) as previously described [ ] . f -mva mers and mh -mva mers were confirmed to lack the native f l orf (and the dsred marker), and contain mers-cov s by pcr (identity and purity pcr screening). the sequence of the s transgene amplified from these vaccines was confirmed. the recombinant viruses (vaccines) were amplified in cm monolayers of cefs cells, partially purified over sucrose cushions and titrated in cefs cells according to standard practice, and purity and identity were again verified by pcr. female balb/c mice (harlan, uk) aged - weeks were immunised intramuscularly (i.m.) in the upper leg (total volume ml) with a total of iu of chadox mers with or without tpa or with a total of pfu of either f -mva mers or mh -mva mers. for induction of short-term anaesthesia, animals were anaesthetised using vaporised isofloh. in prime only regimens, mice were vaccinated with chadox with blood samples taken at days post immunisation (d.p.i) or d.p.i. for serum isolation; and spleens were collected at d.p.i. in heterologous prime-boost regimens, mice were vaccinated with chadox mers and boosted with mva mers at d.p.i; mice were bled at d.p.i. splenocytes were harvested for analysis by ifn-c elispot or intracellular cytokine staining (ics) and flow cytometry as previously described [ , ] , using re-stimulation with mg/ml s mers-cov s-specific peptide (vydtikyysiiphsi); for vaccine cellular immunogenicity [ ] ); or mg/ml e and f (g) mva vectorspecific peptides [ ] (for anti-mva immune responses). in the absence of peptide re-stimulation, the frequency of ifn-c + cells, which was typically . % by flow cytometry or less than sfc by elispot, was subtracted from tested re-stimulated samples. lg/ml with capturing antigen (s recombinant protein from mybiosource, ca, usa) were used to coat elisa plates, and standard endpoint elisa protocol was followed, as previously described [ ] . sera were prepared in a -fold serial dilution in pbs/t and then ll were plated in duplicate wells. serum from a naïve balb/c mouse was included as a negative control. goat anti-mouse total igg conjugated to alkaline phosphatase (sigma) and pnpp tablet ( mg p-nitrophenylphosphate, sigma) substrate were used in the assay. mers pseudotyped viral particles (merspp) were produced and titrated using huh . cell line as described previously [ ] . for the merspp neutralisation assay, serum samples were serially diluted in -well white plates (nunc). a standard concentration of the merspp were added to the wells and plates were incubated for h at °c. after incubation, huh . cells ( , cells per well) were added to the plate in duplicates. following h incubation, cells were lysed and luciferase activity was measured. ic neutralisation titres were calculated for each mouse serum sample using graphpad prism. induction of virus-neutralising antibodies was confirmed according to previously published protocols [ , ] . briefly, mouse serum samples were tested for their capacity to neutralise mers-cov (emc isolate) infections in vitro with % tissue culture infective doses (tcid ) in huh- cells. sera of non-immunised mice served as negative control. graphpad prism (graphpad software) was used for statistical analysis and to plot data. all animal procedures were performed in accordance with the terms of the uk animals (scientific procedures) act (aspa) for the project licenses / or / and were approved by the university of oxford animal care and ethical review committee. all mice were housed for at least days for settlement prior to any procedure in the university animal facility, oxford, uk under specific pathogen free (spf) conditions. the spike gene from a camel isolate (camel/qatar_ _ mers-cov isolate, genbank accession number kj . ) was cloned into four shuttle vectors that facilitate homologous recombination with the genome of chadox or mva. four recombinant viral vectors, two chadox and two mva, were derived as described in the materials and methods. chadox based vaccine candidates were generated with or without the signal peptide of the human tissue plasminogen activator gene (tpa). the spike transgene expression in chadox mers vaccine candidates is under the control of the human cytomegalovirus major immediate early promoter (cmv ie) that includes intron a. in mva mers vaccine candidates, the tpa was also inserted upstream of the spike transgene, which was under the control of either the ectopic mh promoter or the endogenous f promoter (fig. a) . all of our mers-cov vaccine candidates contain the same codonoptimized spike transgene. the expression of the newly synthesized transgene was first tested by transfection of an african green monkey kidney cell line (vero cells) with the adenovirus shuttle vector, and immunofluorescence staining of the transfected cells ( fig. b and c) . this was performed to confirm the expression of the codon optimized spike transgene in mammalian cells. the level of transgene expression from the four vaccine candidates was not evaluated in vitro. we have previously reported that differences in mva promoter activity detectable in vitro does not correlate with in vivo immunogenicity [ ] , and that only in vivo expression correlates with the in vivo immunogenicity. to evaluate humoral immune responses to chadox mers with or without tpa, balb/c mice were vaccinated with  iu of chadox intramuscularly. serum samples from and d.p.i. were collected and evaluated by elisa. both vaccine candidates induced a high level of s -specific antibodies (mean endpoint titre (log ) = . with tpa, . without tpa), unlike the control vaccine, chadox encoding enhanced green fluorescent protein (chadox -egfp, mean endpoint titre (log ) = ). these antibody levels were similar between the two candidates (with or without tpa) at day . however, at d.p.i. chadox mers with tpa induced significantly higher s -specific antibodies than chadox mers without tpa (mean endpoint titre (log ) = . with tpa, . without tpa, fig. a ). serum samples from day were selected for merspp neutralisation assay. serum antibodies induced by chadox mers with tpa showed significantly higher neutralisation activity than without tpa (mean titre ic (log ) = . with tpa, . without tpa; fig. b ). in order to confirm that the psuedotyped virus neutralisation assay was producing biologically relevant results, serum samples from mice immunised with chadox mers with tpa were also tested in a neutralisation assay utilising wildtype mers virus. this assay confirmed the neutralisation activity of mouse antibodies (nab) with a median of vnt (virus neutralization test antibody titre; fig. c ). we therefore continued to evaluate chadox mers with tpa in addition to generating mva mers vaccine candidates with tpa. having established the utility of tpa in chadox mers vaccines (referred to as chadox mers in the rest of this report) at increasing humoral responses, spleens were collected at d.p.i. from immunised balb/c mice. splenocytes were processed to evaluate cellular immune responses to chadox mers in elispot and intracellular cytokine staining (ics). peptide s , described by others [ ] , was used to re-stimulate the cells in both assays and elispot data showed a high level of ifn-c secreting splenocytes (median = sfu/ splenocytes; fig. a ). ics data confirmed the ifn-c secreting cd + splenocytes also secreted tnf-a and il- (fig. b ). to evaluate humoral immune responses to heterologous primeboost vaccination, balb/c mice were immunised with chadox were collected and evaluated by elisa and merspp neutralisation assay. at d.p.i. chadox mers induced similar levels of s -specific antibodies and nab as observed previously ( fig. a and b) . at d.p.i. s -specific antibodies were boosted to a higher level (mean endpoint titre (log ) = by chadox mers boosted to . by mh -mva mers or . by f -mva mers); fig. a ) with nab also enhanced to a statistically significant level (mean titre ic (log ) = . by chadox mers boosted to . by mh -mva mers or . by f -mva mers; fig. b ). there was no difference in antibody levels induced using either the f or mh promoter in the mva. at d.p.i. splenocytes were also processed to evaluate cellular immune responses to chadox mers mva mers prime-boost vaccination in elispot and ics as shown in fig. . the t cell responses to mers s were boosted by the mva vaccinations; in the ics experiments, f -mva and mh -mva boosted the percentage of ifn-c + splenic cd + t cells to . and . % respectively (fig. d) whereas the percentage was . % after chadox mers prime in fig. b . the percentage of tnf-a + splenic cd + t cells were also increased by mva boost (comparing fig. b and d) . utilising the f promoter resulted in a trend towards greater cell-mediated immunogenicity ( fig. c and d) . splenocytes were also re-stimulated with mva backbone-specific e and f(g) peptides and evaluated in ics. both mva based vaccines induced similar responses to e or to f(g) pep- tides, weeks after mva vaccination (fig. e and f) . this similarity confirmed the efficiency of vaccine titration, vaccination, and sample processing because responses to each of those peptides are not expected to be different unless there is variation in the doses administered or sample preparation. overall, mva mers vaccines were able to boost the humoral and cellular immune responses to cha-dox mers prime vaccination. there was no difference between the f and mh promoter in the resulting antibody titres after cha-dox prime/mva boost, but there was a trend towards increased cellular immunogenicity when the f promoter was used. to evaluate humoral immune responses to a homologous mva mers prime-boost vaccination, two groups of balb/c mice were immunised with f -mva mers or mh -mva mers and boosted with the same vaccine after three weeks. serum samples from d. antibodies (mean endpoint titre (log ) = . and . respectively; fig. a ). at d.p.i s -specific antibody levels had increased to . and . respectively (fig. a) . the titres of nab (mers pp assay) were also similar for both vaccines (mean titre ic (log ) = . (f -mva mers) and . respectively; fig. b ). utilising different promoters in mva vectors did not result in differences in the induced antibody levels. however, at d.p.i. ifn-c secreting splenocytes induced by f -mva mers were statistically significantly higher than those of mh -mva mers ((median = and sfu/ splenocytes, respectively, fig. c ). both mva vaccines induced similar vector-specific immune responses as expected ( fig. d and e) . vaccines against mers-cov have been developed and tested in a number of animal models (including non-human primates [ ] [ ] [ ] and camels [ ] ) as well as in human clinical trials [ ] . all vaccine candidates focused on the spike antigen because it contains the receptor-binding domain used for cell entry by the virus, against which neutralising antibodies may be induced, and it is conserved. therefore, the improvement of mers-cov vaccines focuses on platform and vaccination regimens rather than antigen selection and optimisation. here, we focused on using the same antigen (transgene) to develop a vaccine against mers-cov, and to assess different vectors, different versions of each vector, and different vaccination regimens. we generated a number of mers-cov vaccine candidates based on the same codon optimized spike transgene and ensured its expression in vitro before we evaluated the humoral and cellular immunogenicity in a pre-clinical balb/c mouse model. chadox based vaccine candidates were produced with or without tpa. the tpa signal peptide was predicted to enhance the humoral immunogenicity of encoded vaccine antigens, based on previous reports [ ] . our data supported this hypothesis and showed a significant increase in the s -specific antibody levels at d.p.i. the level of neutralising antibodies was also increased when tpa was utilised. however, chadox mers without tpa was still a potent vaccine candidate, inducing a high level of both s -specific binding antibodies and mers-cov neutralising antibodies. neutralisation activity of mouse serum antibodies was assayed by using mers-cov pseudotyped viral particles (merspp), an approach used by a number of researchers for other human pathogens such as hiv, influenza, and hcv to overcome the necessity of handling bsl- viruses [ ] . additionally, we confirmed the ability of serum samples from vaccinated mice to neutralise live mers virus. we therefore selected chadox mers with tpa (simply referred to chadox mers) for further evaluation. chadox mers also induced cellular responses for mers s, with polyfunctional cd + t cells detected in the spleen of immunised mice. this supports the potency of the chadox viral vector in inducing t cellular immunity, observed previously in animal models [ , , ] as well as in humans [ ] . following chadox prime/mva boost, mva significantly boosted the neutralising antibody titres to higher levels. no difference in humoral immunity was found when either the f or mh promoter was used. regarding the promoter effect on mva cellular immunogenicity, we have previously reported that utilising the f promoter enhanced malaria and influenza antigens in mva [ ] . here, we again report that f -mva mers induced higher t cell responses than mh -mva mers in a homologous prime-boost mva mers vaccination. all of our vaccine candidates induced humoral (with nab) and cellular immune (with polyfunctional cd + t cell) responses against mers-cov spike antigen. modest effects on immunogenicity of different versions of the vaccines were noted, with the use of the tpa leader sequence in chadox , and the use of the f promoter in mva producing small increases in immunogenicity compared to no leader sequence, or the mh promoter. the protective level of either antibodies or cellular immunity required to counter mers-cov infection in humans or in animal models is not yet defined, despite some efforts [ ] [ ] [ ] [ ] . the ideal vaccine would provide rapid onset of immunity and complete protective efficacy after a single dose, with a long duration of immunity. complete protective efficacy of one dose of chadox expressing the external glycoprotein of rift valley fever virus has been demonstrated in multiple species and it is already known that chadox rvf is highly immunogenic in camels [ ] . to date, the only vaccine against mers to be tested in camels is an mva vectored vaccine [ ] which was protective in hdpp transgenic mice immunised with a homologous prime/boost regimen [ ] but in camels required two doses given both intranasally and intramuscularly to provide partial protection and reduction of virus shedding [ ] . here we find that a single dose of chadox mers is as immunogenic as two doses of mva mers, suggesting that this regimen should be tested for protective efficacy in camels. however if this is not completely protective, administration of mva mers as a heterologous boost should be considered next. in our hands one dose of mva resulted in an endpoint titre of logs, two doses of mva produced . logs, one dose of chadox produced logs, and chadox /mva prime boost produced . logs. if a single dose of chadox mers is not protective and a two dose regimen is required, chadox /mva would be more likely to provide complete protection than mva/mva. chadox mers should now be evaluated for immunogenicity and efficacy in larger animal species, including both camels and humans. scg is a co-founder of, consultant to and shareholder in vaccitech plc which is developing vectored influenza and mers vaccines. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates mers-cov outbreak in jeddah-a link to health care facilities state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus infections in health care workers mers coronavirus neutralizing antibodies in camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia ( ) and australia ( ) and characterisation of assay specificity. euro surveillance: bull eur sur les maladies transmissibles = presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan risk factors for primary middle east respiratory syndrome coronavirus illness in humans human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection mers-cov at the animal-human interface: inputs on exposure pathways from an expert-opinion elicitation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study livestock susceptibility to infection with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding vaccines against middle east respiratory syndrome coronavirus for humans and camels viruses as vaccine vectors for infectious diseases and cancer yellow fever d as a vaccine vector for microbial ctl epitopes: protection in a rodent malaria model a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity clinical applications of attenuated mva poxvirus strain clinical development of modified vaccinia virus ankara vaccines immunization with plasmid dna encoding influenza a virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against h n challenge in mice post-translational intracellular trafficking determines the type of immune response elicited by dna vaccines expressing gag antigen of human immunodeficiency virus type (hiv- ) enhancing cellular immunogenicity of mva-vectored vaccines by utilizing the f l endogenous promoter chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever clinical assessment of a novel recombinant simian adenovirus chadox as a vectored vaccine expressing conserved influenza a antigens preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors single-dose protection against plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron a recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus ankara (mva) protective efficacy of recombinant modified vaccinia virus ankara (mva) delivering middle east respiratory syndrome coronavirus spike glycoprotein poxvirus cd + t-cell determinants and cross-reactivity in balb/c mice effective induction of high-titer antibodies by viral vector vaccines an optimised method for the production of mers-cov spike expressing viral pseudotypes middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge evaluation of candidate vaccine approaches for mers-cov an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels army scientists begin first mers vaccine clinical trial. dod news, defense media activity immunogenicity and efficacy of a chimpanzee adenovirusvectored rift valley fever vaccine in mice characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetics of serologic responses to mers coronavirus infection in humans viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection key: cord- -h ma u authors: gellin, bruce g.; qadri, firdausi title: preparing for the unpredictable: the continuing need for pandemic influenza preparedness date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: h ma u nan preparing for the unpredictable: the continuing need for pandemic influenza preparedness ebola, zika, middle east respiratory syndrome (mers), chikungunya, severe acute respiratory syndrome (sars). the cadence of emerging infectious diseases that travel, transmit and stand ready to spark a public health emergency of international concern seems to be accelerating. these infectious diseases cause significant human suffering, can overwhelm unprepared health care systems, and significantly disrupt societies and economies. it is estimated that a severe pandemic similar to the ''spanish flu" could nowadays cost as much as % of global gross domestic product; the global cost of even a moderately severe pandemic could be as much as us$ billion, or . % of global income [ ] . given the health, social and economic impact of infectious diseases with pandemic potential, there has been much attention to these microbial threats and, not surprisingly, the first question is always, ''is there a vaccine?" but too often we find that the vaccines we need most are not available when we need them most. a number of efforts are focused on better anticipating the vaccines that we may need [ ] [ ] [ ] [ ] [ ] and a global emergency fund has been proposed to support accelerated vaccine development [ ] . the current headlines about zika virus have replaced last year's headlines about ebola. . .and next year's headlines will undoubtedly feature something new. imperfect crystal balls fail to guide us with certainty. but looming in the background is the ever present threat of influenza, the only microbe that causes regular widespread epidemics around the world. not so exotic and all too familiar -casually referred to as the ''flu" -influenza is the virologist's trojan horse, as it readily and rapidly mutates (drift), has the ability to swap genetic elements from influenza viruses with origins in other species (shift) and, as an rna virus lacks repair and self-correcting mechanisms that are the basis of its genetic instability and makes it both dangerous and unpredictable. a reassortment leading to an extremely virulent and readily transmissible pandemic virus is inevitable. of the many things that need to be in place to prepare for and respond to the next influenza pandemic, vaccines -together with the capacity to mount a timely global vaccination effort -are paramount. a pandemic can only be brought under control through a shift in the population from susceptibility to immunity. but as we learned in the influenza pandemic, although our response time has improved, a significant shift in approach is needed if an effective vaccine is to be in place before the next pandemic emerges. with that goal in mind, the science of influenza and of vaccine development needs to be looked at with fresh eyes [ ] . the holy grail is an influenza vaccine that provides a broader and more durable immune response than the natural infection and that can provide population immunity to pandemic influenza viruses before they emerge. a number of efforts are now focused on developing such a ''universal" influenza vaccine; however, the scientific challenges involved are enormous [ , ] . until such a universal influenza vaccine becomes available, global influenza vaccine production capacity needs to be ready to respond when the next pandemic emerges. vaccines obviously need to be safe and effective, but they must also be available and affordable. while vaccine supply is clearly essential for a large-scale vaccination programme, the logistics and supporting policies needed to conduct effective and efficient vaccination campaigns in a range of community settings also need to be addressed and the needs of high-risk and vulnerable populations and of low-and middle-income countries accommodated. ultimately, the success of a vaccination programme will hinge not only on the technical aspects of its implementation, but on the demand for it among the population; this underscores the need to understand the factors that promote vaccine acceptance [ , ] . within this broad framework, and with a focus on the role of vaccines in mitigating the impact of both seasonal and pandemic influenza, who established in the global action plan for influenza vaccines (gap) as ''a comprehensive strategy to reduce the present global shortage of influenza vaccines for seasonal epidemics and pandemic influenza in all countries of the world" [ ] . the papers in this special issue of vaccine, provide a foretaste of the in-depth review to come in november , when who will host the third and final consultation on gap as the programme, as it exists today, comes to close. the background papers prepared for this consultation are available on the who website (http:// www.who.int/influenza_vaccines_plan/news/gap _nov /en/). while this event is advertised as a programme review, in reality as an examination of the progress achieved during the decade, it should also serve to direct the global community on the path forward for the work that remains to achieve global pandemic influenza vaccine and vaccination preparedness. this review, combined with the current rethinking of global preparedness for emerging threats, the review of the pandemic influenza preparedness (pip) framework [ ] , and who's restructuring of its approach to global health emergencies the global action plan for influenza vaccines was structured around three broad objectives that underpin pandemic influenza vaccine and vaccination preparedness: -evidence-based increase in seasonal influenza vaccine use; -increase in influenza vaccine production capacity and regulatory capacity; -research and development for improved influenza vaccines. the articles contained in this issue not only relate to the gap objectives but also reflect the principles and goals of pip and highlight the synergy of these two efforts. pip's pandemic preparedness goal of increasing the access to vaccines and other pandemicrelated supplies by developing countries (and the critical importance of improving the sharing of influenza viruses with human pandemic potential toward that end), will ultimately depend on influenza vaccine production capacity in place when the next pandemic occurs. these articles present a variety of perspectives on the system that will develop, produce, regulate, distribute and evaluate pandemic vaccines and mass vaccination programmes. they have been selected to represent some of the many elements of the system that will be necessary for a pandemic vaccine response -including clinical trials of new vaccines, new vaccine production platforms, national regulatory systems, implementation of policy recommendations for vaccine use, and assessment of the sustainability of the global influenza vaccine manufacturing capacity that gap has supported. progress has already been significant and measurable. one clear example is that global production of seasonal influenza vaccine increased from less than million doses in to nearly . billion doses in [ , ] . yet, there is a pressing need for additional burden of influenza illness studies to provide the essential data that will be looked to by policymakers to assess the role of vaccines in national influenza control strategies. it is the implementation of these policies that will determine demand. without a concomitant increase in global demand for seasonal influenza vaccine, the capacity that will produce the world's pandemic vaccines that gap has stimulated cannot be sustained [ ] . at the end of the day there is the expectation that the efforts and investments made to prepare for a pandemic will result in the timely and equitable availability of a vaccine that is safe and effective, and that can be used in vaccination campaigns before the majority of the global population is exposed to the virus. however, a continuing dedicated effort is needed to ensure that all the elements of the system that will allow this to happen are in place before we need them. because we don't know when that day is, the global community must continue with speed and focus. and, as who develops a new structure to respond for the call to be prepared for all hazards, in its complex organizational chart pandemic influenza preparedness must remain front and center. the inclusive cost of pandemic influenza risk (working paper ) an r&d blueprint for action to prevent epidemics. accelerating r&d and saving lives. geneva: world health organization report from the world health organization's product development for vaccines advisory committee (pdvac) meeting antibiotic resistance threats in the united states the coalition for epidemic preparedness innovations (cepi) establishing a global vaccine-development fund (perspective) the compelling need for game-changing influenza vaccines edu/compelling-need-game-changing-influenza-vaccines> advances in the development of influenza virus vaccines induction of unnatural immunity: prospects for a broadly protective universal influenza vaccine (commentary) global vaccine action plan - . geneva: world health organization sage working group on vaccine hesitancy. how to deal with vaccine hesitancy? global action plan for influenza vaccines. geneva: world health organization pandemic influenza preparedness (pip) framework. geneva: world health organization melbourne: peter doherty institute for infection and immunity global action plan for influenza vaccines a literature review to identify factors that determine policies for influenza vaccination key: cord- - vil k l authors: macdonald, angus j.; cao, long; he, yuxian; zhao, qian; jiang, shibo; lustigman, sara title: rov-asp- , a recombinant secreted protein of the helminth onchocercavolvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv- polypeptide and sars-cov peptide antigens date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: vil k l we studied the adjuvanticity of recombinant onchocerca volvulus activation associated protein- (rov-asp- ) for ovalbumin (ova) in mice. after a single immunization and one boost, rov-asp- exceeded the efficacy of alum or mpl + tdm adjuvants in terms of end-point total igg or igg and igg a anti-ova titres. using the helminth-derived adjuvant, igg isotype responses to ova were of a mixed th /th profile and spleen cell cytokines exclusively th -type. the potent adjuvanticity of rov-asp- was confirmed in mice vaccinated with a -mer peptide from the s protein of sars-cov and an hiv- gp -cd chimeric polypeptide antigen. unusually for a helminth product, the rov-asp- adjuvant augmented not only th but also th responses, the latter property being of potential utility in stimulating anti-viral immune responses. filarial nematodes are long-lived parasites in the human host, for example, onchocerca volvulus worms live up to years. they use a large armoury of immunoregulatory molecules to subvert the protective immune responses of the host and minimize severe pathology [ , ] . some of these helminth-derived modulators of human immune responses have great potential as new therapeutics. for example, es- , a secreted glycoprotein product of the rodent filarial nematode acanthocheilonema viteae, has broadly antiinflammatory properties that inhibit th cytokine production in experimentally induced arthritis in mice [ ] . es- is currently being developed as a novel anti-inflammatory therapeutic [ ] . in another nematode, body fluid from the pig roundworm, ascaris suum, contained potent activity that stimulated il- , which is characteristic of th and regulatory t cells. the parasite products reduced the inflammation caused by experimentally induced delayed type hypersensitivity in mice [ ] . two helminth products have also been reported to act as adjuvants. both are strong inducers of th responses to bystander proteins in a vaccine. in particular, proteins secreted by adult nippostrongylus brasiliensis (a parasite of rodents) were found to be strong inducers of th responses in mice immunized with an unrelated protein, hen egg lysozyme [ ] . similarly, lacto-n-fucopentaose iii, a carbohydrate found on the surface of the eggs of a human parasite, schistosoma mansoni, acted as a th adjuvant for a bystander protein (human serum albumin) when injected intranasally, subcutaneously or intraperitonealy into mice [ ] . activation-associated secreted proteins (asp) of parasitic nematodes are highly immunogenic and have been studied as potential vaccine components, particularly hookworm asps [ ] [ ] [ ] . while conducting experiments designed to evaluate recombinant onchocerca volvulus asp- (rov-asp- ) as a possible vaccine candidate against onchocerciasis in humans, we vaccinated mice with the recombinant protein alone or with alum or freund's adjuvants and measured igg and igg a isotypes that are broadly associated with th and th t cell responses, respectively. rov-asp- stimulated both igg and igg a antibody responses to the protein itself, with a slight th dominance [ ] . since these effects occurred in vaccinated mice in the absence of adjuvant, we asked the question whether the protein could act as an adjuvant to assist unrelated proteins in inducing antibody responses. until the present study, purified products from helminths have been found to be strong inducers of exclusively th responses. the recombinant ov-asp- protein was expressed as a histidine-tagged protein in e. coli (dh ␣) using the ptrchis expression vector (invitrogen, carlsbad, ca). inclusion bodies containing insoluble rov-asp- were treated with m urea at • c overnight, and the urea-soluble rov-asp- was then further purified by preparative sds-page on a prepcell (bio-rad, hercules, ca). the protein-containing fractions were eluted in laemmli buffer ( mm tris, mm glycine, . % sds, ph . ) and analyzed by sds-page. fractions containing the purified rov-asp- were identified by india tm hisprobe-hrp (pierce, rockford, il) chemiluminescent western blotting, and protein concentrations were determined using detergent-compatible protein assay reagents (bio-rad). although the rov-asp- protein at working dilutions tested negative for bacterial endotoxin using a limulus amoebocyte lysate assay (sigma, st. louis, mo), we sent a sample for quantitative lps testing following fda guidelines to cambrex bio science (baltimore, md). testing revealed that there was a residue of . endotoxin units (eu)/g rov-asp- protein. at a dose of g/mouse, therefore, the animals would receive eu per injection which is well below the fda accepted limit of eu/ml for biologicals. however, to definitively exclude any possibility of residual lps in rov-asp- contributing to any adjuvant effects, we batch-treated the concentrated stock solution ( . mg/ml) of rov-asp- three times with lps-removing gel (detoxigel tm system, pierce biotechnology, rockford, il), and adjusted the protein concentration accordingly. repeat testing by cambrex showed no detectable lps in the treated rov-asp- preparation and a single batch was used throughout the experiments presented here. we first used ovalbumin (ova, sigma, grade v) as a model antigen that does not stimulate appreciable antibody responses when injected into mice without adjuvants. the peptide derived from the spike (s) protein of sarsassociated coronavirus (sars-cov), designated cp- [ ] , was synthesized by a standard solid-phase fmoc method in the microchemistry laboratory of the new york blood center. cp- spans amino acids - of the heptad repeat (hr ) region of the sars-cov s protein, which plays an important role in fusion between the virus and target cell membranes [ ] . the flsc polypeptide is a chimera of the full-length hiv- bal gp and the first and second extracellular domains (d d ) of soluble cd joined by a -amino acid linker. this polypeptide faithfully duplicates the structural, functional and antigenic properties of the native gp -cd complex intermediate that arises during hiv replication and enables binding to ccr on target cells [ ] . flsc inhibited binding of hiv- in vitro to target cells expressing ccr [ ] . therefore, the antibodies directed against flsc may block hiv- binding to ccr . a plasmid expressing the flsc protein was kindly provided by dr. a. pinter at the public health research institute with the permission from dr. a.l. devico at the university of maryland. a stable cell line expressing recombinant flsc was established by transfecting the plasmid into t cells using fugene (boehringer mannheim, indianapolis, in) according to the manufacture's protocol. soluble flsc was purified from cell culture medium by lectin chromatography with galanthus nivalis snowdrop agglutinin (sigma-aldrich, st. louis, mo) as described [ ] . the procedures dealing with mice were approved by the institutional animal care and use committee at the new york blood center. we used - -week-old male balbc/cbyj mice (charles river laboratories inc., wilmington, ma) for immunization. the commercial adjuvants, alum (sigma) or mpl + tdm (sigma), were used as controls of our test adjuvant, rov-asp- . mpl + tdm (equivalent to ribi tm adjuvant) is composed of equal amounts of monophosphoryl lipid a (detoxified endotoxin) from s. minnesota (mpl) and synthetic trehalose dicorynomycolate (tdm) in % oil (squalene)-tween -water. per immunization, each animal received g of antigen (ova, sc- or flsc) in . ml sterile, lps-free phosphate-buffered saline (pbs) mixed with one of the commercial adjuvants following the manufacturer's instructions, or with rov-asp- ( g/ . ml pbs). each of the antigen/adjuvant mixtures or adjuvants (as controls) was injected into a group of five mice subcutaneously in the nape of the neck. mice received one boost (for ova) or two boosts (for cp- and flsc) days post-immunization. all experiments were performed twice or more and representative data are shown. mice were bled retro-orbitally prior to vaccination to establish antibody baselines. ten days after the final vaccination, the mice were euthanized and bled. total igg and igg isotype antibody responses to each of the antigens were measured by elisa. ninety-six-well vinyl elisa plates (costar # , bio-rad, hercules, ca) were coated overnight at • c with l/well of ova at g/ml, cp- at g/ml or flsc at g/ml in carbonate coating buffer (ph . ). after washing five times, plates were blocked with l % non-fat milk powder in pbs for . h at • c. mouse sera serially diluted in pbs were added ( l/well), followed by incubation for . h at • c. after extensive washes, goat anti-mouse igg, igg , igg a, igg b or igg antibodies (sigma) were added at : dilution ( l/well for h at • c) for igg isotyping. then, the biotinylated rabbit anti-goat igg and extravidin peroxidase conjugate (both from sigma, : dilution for h at • c) were added sequentially. after the final washing step, l of tmb (sigma) was added and the reaction was stopped with an equal volume of m h so . absorbance at nm was measured on a spectramax elisa reader (molecular devices, sunnyvale, ca). serum antibody titres were determined by measuring the last dilution in the elisa that resulted in × s.d. above the appropriate control treatment optical density (od). serum from each mouse was titrated and the mean titres for each treatment group are presented here. after exsanguination of the mice under anaesthesia, spleens were removed, cut into two pieces with sterile scissors and made into single cell suspensions using sterile glass potter-elvehjem tissue grinders (fisher scientific international, pittsburgh, pa). the cell suspensions were then passed through sterile disposable m cell strainers (falcon brand, bd biosciences, bedford, ma). cells were washed three times in rpmi supplemented with % heatinactivated foetal bovine serum (fbs), mm hepes buffer, . mm l-glutamine, m -mercaptoethanol, u/ml penicillin and g/ml streptomycin (all from sigma). cells were counted for viability (always ≥ %) and cultured in rpmi + % fbs in quadruplicate wells at × per well in round-bottomed -well culture plates (nunc brand, fisher scientific) for h at • c in a humidified % co incubator. spleen cells were cultured with medium alone, the optimal concentration of ova ( . g/ml) or pha or pma + anti-mouse cd (bd biosciences) as positive control stimuli. supernatants were collected after h and assayed for mouse ifn-␥, il- , il- and il- using elisa assays (quantikine brand, r&d systems, minneapolis, mn) according to the manufacturer's protocol. in order to evaluate any possible contribution of the small amount of residual lps in the rov-asp- to any adjuvant effect, we compared the adjuvanticity of working dilutions of batches of rov-asp- that were either untreated (rov-asp- b) or batch treated three times (rov-asp- a) with lpsremoving gel. in fig. , the uppermost lines show that the treated recombinant protein (rov-asp- a) performed better than the untreated protein in augmenting antibody responses to ova in immunized mice. the mean anti-ova igg titre obtained using treated rov-asp- as an adjuvant was , compared with , using the untreated batch and for igg a, , and , , respectively. we could, therefore, rule out lps as a contributing factor to the adjuvant properties of our recombinant ov-asp- protein and, since it was a better adjuvant, we used the lps removing gel-treated batch of rov-asp- in all further experiments. when rov-asp- was used at g/mouse, this protein was much more potent in enhancing antibody production than the commercial adjuvants alum and mpl + tdm (fig. ) . even though the end-point titres using rov-asp- were lower in the experiment shown in fig. than the data in fig. , they were still higher than those achieved using the other adjuvants. the igg anti-ova reciprocal end-point titre using rov-asp- as the adjuvant was , , while the titres obtained using mpl + tdm or alum adjuvants were , and , , respectively. the igg a titres were considerably lower than those of igg and only the rov-asp- adjuvant induced an appreciable anti-ova igg a titre ( , ). there were no detectable igg b, igg or ige antibodies to ova. spleen cells from mice receiving ova in rov-asp- showed an exclusively th cytokine profile (ifn-␥ +++ , il- − , il- − ) when re-stimulated with ova in vitro (fig. ) . in contrast, the response using alum adjuvant was solely th type (ifn-␥ − , il- + , il- + ) in nature. mpl + tdm adjuvant resulted in a mixed, but th -dominated response (ifn- ␥ +++ , il- + ) to the immunizing antigen. we were unable to measure appreciable amounts of il- in any of the supernatants from mouse spleen cell cultures with the exception of the positive control-treated (pma + anti-cd ) cultures. having shown that rov-asp- acted as a better adjuvant than alum or mpl + tdm in stimulating production of antibodies to ova, we then tested if the protein had similar adjuvant potency for antigens derived from human pathogens, namely sars-cov and hiv- . we immunized balbc/cbyj mice as before using the same batch of lpsnegative rov-asp- mixed with g of sars-cov cp- peptide or hiv- -cd flsc polypeptide, instead of ova. all immunized mice were given two boosts this time to optimize the response to the small -mer cp- peptide, i.e. a total of three injections of cp- or flsc with rov-asp- as the test adjuvant or mpl + tdm as a control. using ova as the control antigen, the end-point titres were about , , and , , when r-ov-asp- and mpl + tdm were used as adjuvants, respectively. these total igg titres were approximately times higher than in the previous experiments, suggesting that an additional boost significantly enhances antibody production. the adjuvanticity of rov-asp- for the cp- peptide exceeded that of mpl + tdm judging by end-point igg titres of , versus , , respectively (fig. a) . the anti-flsc end-point igg titres achieved using both adjuvants were equivalent (approximately , , ; fig. b ). the igg isotype responses to the cp- peptide and the flsc polypeptide are summarized in table . the rov-asp- protein stimulated higher igg , igg a and igg b titres than mpl + tdm. igg titres were equally low using both adjuvants. igg titres to the hiv- polypeptide were considerably lower than those to the cp- peptide. mpl + tdm induced a higher igg b titre to flsc than rov-asp- , whereas igg and igg titres were the same using igg a igg a igg b igg a end-point titres are the mean of five mice per group. both adjuvants. the most striking differences between the rov-asp- and mpl + tdm-induced responses were: ( ) the lack of an igg a (th ) response to cp- using mpl + tdm; ( ) an eight-fold higher igg (th ) response to cp- using rov-asp- rather than mpl + tdm as the adjuvant; ( ) a four-fold higher igg a response to flsc adjuvanted by rov-asp- compared with mpl + tdm. in contrast to cp- and flsc antigens, igg b and igg antibodies to ova were not detectable using either rov-asp- or mpl + tdm adjuvants (data not shown). each adjuvant/antigen model performed differently depending on the antigen-th dominant antibodies with cp- and th with flsc formulated with either of the adjuvants. however, with either antigen, the th (igg a) response was always higher when rov-asp- was used as the adjuvant. with flsc as the immunogen, there was a switch in igg a and igg b antibodies between asp- and ribi adjuvants. asp- favoured igg a and ribi enhanced igg b. no ige was detectable using rov-asp- as an adjuvant. igm and iga were not tested. ov-asp- is a member of a family of proteins found in both free-living and parasitic nematodes. the native protein is located in secretory granules of the glandular oesophagus of the infective third-stage larvae of o. volvulus [ ] . the recombinant protein has a predicted molecular weight of . kd and also has angiogenic activity in mice [ ] . a hookworm homologue of ov-asp- has been shown to be a promising vaccine candidate when the yeast-expressed recombinant protein was formulated in quil a adjuvant (brenntag biosector, frederikssund, denmark) in hamsters [ ] . our studies have clearly shown that we have discovered a new helminth-derived adjuvant that was highly effective in eliciting antibody responses in mice immunized with unrelated protein, polypeptide and peptide antigens. furthermore, the parasite protein, rov-asp- exceeded the adjuvanticity of alum or mpl + tdm adjuvants, especially in the induction of the th- associated igg a isotype. in these studies, we used the subcutaneous route of immunization in balbc/cbyj mice-a strain which tends to th responses more than th [ ] . the fact that we can induce th responses in this strain with rov-asp- but not mpl + tdm encouraged us to investigate whether we could obtain similar results in other mouse strains that are not as th -biased. in recent preliminary studies using ovaimmunized c bl/ mice which favour th responses [ ] , rov-asp- adjuvant was associated with a th -dominated (igg a) antibody response to ova but also with considerable igg titres (igg :igg a = . ). the antibody response to ova in balb/c mice was th -skewed (igg ) but, as in the data presented here, there was also a significant igg a component (igg :igg a = . ). thus, the rov-asp- adjuvant augments both th and th antibody responses in mice with the overall balance dependent on the genetic background of the immunized animal. we have used a range of doses of rov-asp- adjuvant; . g/mouse was not significantly active for antibody responses and g/mouse gave adjuvanticity intermediate to the final dose that we chose ( g/mouse). mice tolerated the rov-asp- immunization and there were no overt signs of toxicity. to investigate t cell responses that might be associated with the high antibody titres, we looked at cytokines secreted by spleen cells from mice immunized with ova with adjuvant. recall cytokine responses to ova in mice immunized with ova in rov-asp- adjuvant were strongly th -dominated (fig. ) . it is unclear why rov-asp- adjuvant resulted in an exclusively th cytokine (ifn-␥-positive, il- , il- -negative) response from splenocytes of ovavaccinated mice (fig. ) , but mixed th /th serum antibody responses ( figs. and ) . it is possible that the cytokine profile from the draining lymph node cells might more closely reflect the th /th antibody phenotype or that the antibody and cytokine responses were temporally dissociated. an ability to stimulate a th cellular response is unusual for a helminth protein. ov-asp- is highly expressed in the infective o. volvulus third-stage larva (l ) [ ] , but its precise role in the invasion process is not known. the early human response to first exposure to live l of brugia malayi, another filarial nematode, has recently been shown to be th dominated. numbers of cd + and cd + t cells expressing ifn-␥, tnf-␣ and gm-csf were increased on exposure to l in vitro but not t cells expressing th cytokines [ ] . it is possible that l -secreted asps are involved in the stimulation of the th cytokines. in support of this, we found that rov-asp- is a potent stimulator of th cytokines (ifn-␥, tnf-␣ and gm-csf) from o. volvulus-naïve human pbmcs (unpublished data). the mechanism responsible for the adjuvanticity of rov-asp- is not yet clear, however, from our studies using human pbmc we know that the protein binds to a subpopulation of monocytes. it is possible that rov-asp- exerts a direct activating effect on antigen-presenting cells and we are currently investigating this and the potential role of toll-like receptors in binding rov-asp- . we were not able to detect hiv- neutralizing activity in serum of mice vaccinated with flsc in either rov-asp- or mpl + tdm adjuvants. in a separate study, mice immunized with purified flsc, all developed high antibody titres for the immunogen, but none of the sera possessed neutralizing activities against hiv virus in vitro [ ] . the authors suggested that a major portion of the antibody response against the flsc protein may be directed against immunodominant conformational epitopes unique to the fusion polypeptide that do not mediate viral neutralization. in the present study, serum from flsc + adjuvant-immunized mice also recognized hiv- gp protein in elisa. the mean total igg anti-gp titre obtained with rov-asp- was , , and with mpl + tdm adjuvant, , (data not shown). titres to gp were lower than to flsc since all of the gp epitopes may not be accessible in the flsc fusion polypeptide. for safety and regulatory reasons, we are not currently able to assess neutralizing activity to sars-cov. in conclusion, we have shown that a secreted protein from o. volvulus infective larvae acts as a very potent adjuvant for bystander protein, polypeptide and peptide antigens, exceeding the responses induced by commercially produced alum and mpl + tdm adjuvants. unusually for a helminth protein, the antibody and cellular responses induced had a strong th component. depending on the immunogen, adjuvanting with rov-asp- resulted in a th -type (igg )-dominant antibody response that lacked ige. this immunostimulatory helminth protein may have utility as a human therapeutic. immune regulation by helminth parasites: cellular and molecular mechanisms immune evasion genes from filarial nematodes a novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein es- es- , a filarial nematodederived immunomodulator with anti-inflammatory potential modulation of a heterologous immune response by the products of ascaris suum proteins secreted by the parasitic nematode nippostrongylus brasiliensis act as adjuvants for th responses lacto-nfucopentaose iii found on schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing th -type response cloning, yeast expression, isolation, and vaccine testing of recombinant ancylostoma-secreted protein (asp)- and asp- from ancylostoma ceylanicum progress in the development of a recombinant vaccine for human hookworm disease: the human hookworm vaccine initiative a family of activation associated secreted protein (asp) homologues of cooperia punctata ov-asp- , the onchocerca volvulus homologue of the activation associated secreted protein family is immunostimulatory and can induce protective anti-larval immunity interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type gp -cd receptor complex envelope glycoproteins of hiv- , hiv- , and siv purified with galanthus nivalis agglutinin induce strong immune responses angiogenic activity of onchocerca volvulus recombinant proteins similar to vespid venom antigen the regulation of immunity to leishmania major proinflammatory cytokines dominate the early immune response to filarial parasites analysis of the immunogenic properties of a single-chain polypeptide analogue of the hiv- gp -cd complex in transgenic mice that produce human immunoglobulins we would like to thank drs. james farmer and jinkui niu at the microchemistry laboratory for peptide synthesis. this work was supported in part by a grant from the national institutes of health (r ai ) and by the new york blood center. key: cord- - bheqtk authors: hönemann, m.; martin, d.; pietsch, c.; maier, m.; bergs, s.; bieck, e.; liebert, u.g. title: influenza b virus infections in western saxony, germany in three consecutive seasons between and : analysis of molecular and clinical features date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: bheqtk background: the impact of annual influenza epidemics and prevailing strains varies worldwide and regional. the majority of vaccines used contained two influenza a strains and only one influenza b strain (trivalent vaccine). aim: the aim of the study was to compare laboratory confirmed influenza b cases during three consecutive years with respect to vaccination history, clinical symptoms and molecular virology. methods: partial ha gene sequences were analyzed for lineage determination and complete ha sequence in cases with reported vaccination and in fatal cases. clinical data were retrieved from patient charts. findings: during the / season, influenza b cases were retrieved; in / , and in / . the frequency of yamagata-lineage strains increased from . % to %. no difference was detected in the relative frequency of co-morbidities in season / . . % of the adult patients and . % of pediatric patients were vaccinated against influenza. interpretation: phylogenetically, yamagata strains clustered similarly in / when compared to the previous two influenza seasons. while the relative frequency of influenza b cases differed, the clinical symptoms remained similar. conclusion: world health organization recommendations for the use of tetravalent vaccines that contain two influenza b strains (yamagata and victoria) in addition to the two influenza a strains (h n and h n ) should be implemented in national vaccination guidelines. funding: this research was partially supported by the association of sponsors and friends of leipzig university. influenza b virus of the orthomyxoviridae family [ ] has a segmented single stranded, negative sense rna genome. first isolated in , influenza b virus diverged into two lineages, victoria and yamagata, in the late s with similar clinical [ ] but different phylodynamic properties [ ] . although infections in pigs and seals were observed, there is no known animal reservoir for influenza b [ ] . thus, while influenza a infections can be zoonotic, influenza b virus only circulates in the human population. the narrow host range and slower evolution [ ] are thought to be the main contributing factors to the less frequent occurrence of influenza b epidemics [ ] . in / influenza b predominance was reported in countries throughout the northern hemisphere. it was the leading influenza type in europe and canada [ ] [ ] [ ] and the second most common type after influenza a/h n in the usa [ ] . in germany, after the seasons of / , / and / , it was only the fourth season of the st century with a dominance of influenza b [ ] . as the prevailing lineage of influenza b changes frequently, the world health organization (who) recommended to include both lineages in a tetravalent vaccine in [ ] . the key component of the vaccine is the viral hemagglutinin (ha) that covers the viral surface in a trimeric form. the major antigenic regions are the -loop, -loop, -loop, and helix [ ] and are located on the ha subunit which forms the globular head domain. it is responsible for recognition and binding of sialic acids on the surface of the target cells. the ha subunit is the main component of the ha stalk. although some antibody vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e cross reactivity is observed [ , ] , vaccine efficiency between the two lineages might be reduced in seasons in which the formulation of the trivalent influenza vaccine does not match the circulating strain due to antigenic differences [ , ] . additionally, there is growing evidence that vaccine effectiveness is already waning within a season thus reducing immunological protection even if the vaccine contained the matching strain in the previous season [ ] . the aim of this study was to analyze clinical and molecular features of laboratory-confirmed influenza b cases during three consecutive seasons between and . a comparison between patient characteristics was done in relation to the combined seasons of / and / when appropriate. additionally, molecular epidemiology of the viral hemagglutinin (ha) gene was performed on selected isolates and amino acid changes were mapped to the major antigenic domains of the ha protein. specimens and clinical data. , respiratory samples, including nasal aspirates, supernatants from nasal and pharyngeal swabs, throat rinsing fluid, tracheal secretions, and bronchoalveolar lavage fluids, of patients were tested for respiratory virus infections. testing was initiated at the discretion of the lineage determination. na sequences of a bp-long amplicon of the ha gene [ ] were assessed (bigdye terminator sequencing kit v . and abi genetic analyzer, applied biosystems, foster city, usa). the sequenced region corresponds to nucleotide positions - and - of the complete ha gene of the victoria lineage (b/brisbane/ / ) and the yamagata lineage (b/phuket/ / ), respectively. sequences with nucleotides g , g , g , g , c , g , and a belonged to the victoria-lineage. viruses with nucleotides a , a , a , a , t , t , and g belonged to the yamagata-lineage. phylogenetic analysis. na sequencing of the complete ha was performed according to who guidance [ ] . in cases of low influenza b concentration an alternative protocol generating three instead of two overlapping fragments was used [ ] . phylogenetic trees of the nucleotides large ha coding region were constructed at nucleotide level with the mega software version using the maximum-likelihood method. bootstrap analysis was performed with replicates [ ] . complete ha sequences were submitted to genbank (accession numbers mk -mk ). statistical analysis. statistical analysis was performed using ibm spss statistics for windows, version . (armonk, ny: ibm corp.). continuous values were expressed as mean or median (range) and categorical data as frequencies (percentages). student's t-test or mann-whitney u test was performed to compare means. chi-square or fisher's exact test were performed for categorical variables. all tests were two-tailed. a p-level of < . was considered significant. relative and absolute numbers of influenza types detected between and are shown in table . for season / the maximum number of cases occurred between weeks and of . the peak occurrence in patients below years was in week while it was week for the adult patients (fig. a) . the eleven cases of the season of / occurred table study population and clinical features of influenza b infected cases. (table ) . for the pediatric patients there were no significant differences between season / and - except for the combined rate of comorbidities and risk factors for a severe course of the influenza. however, this could not be attributed to a single analyzed factor. when looking at the adult population, there were no significant differences between season / and the previous seasons except for a higher proportion of female patients and a lower proportion of viral coinfections in / . most differences were noted between the age groups. . % of the adult patients were vaccinated, mostly with the trivalent vaccine, whereas . % of the pediatric patients were unvaccinated. the rate of comorbidities was significantly higher in the adult population. furthermore, the clinical course of the disease was more severe in the adult population as higher rates of icu admissions, invasive ventilation, and fatal outcomes were observed. ha phylogeny. sequence analysis of the whole hemagglutinin gene was performed for victoria and yamagata strains and consistently confirmed the results of the initial lineage determination. included were all strains of patients with a known trivalent (n = ) or tetravalent (n = ) vaccination, randomly selected patients without known vaccination (n = ), and of all patients that had a fatal outcome within days of the influenza b detection (n = ). except for one strain of , all victoria strains originated from the / season and belonged to the a clade. all of the strains were phylogenetically closely related and did not cluster based on vaccination history or fatal outcome (fig. a) . all analyzed yamagata strains belonged to clade . all of the strains were phylogenetically closely related, but strain phylogeny indicates a discrete drift from season / to season / . again, no clustering with regard to vaccination history or fatal outcome was observed (fig. b) . amino acid changes. the detected amino acid changes (aachanges) within the ha protein are depicted in table the influenza season of / was dominated by the yamagata-lineage of influenza b in europe. in germany, it was the largest season in terms of case numbers since the establishment of the nationwide surveillance system in [ ] . large scale epidemics or pandemics are usually caused by influenza a. this is attributed mostly to the broader host range and faster rate of evolution of influenza a [ ] . influenza b infections usually occur with a temporal delay towards the end of an influenza adominated season [ ] . the exceptional impact of influenza b infections in / with regard to public health was therefore surprising. however, seasons with a high proportion of influenza b infections occur infrequently. specifically, in germany, the mild seasons of / and / and the severe season of / have been dominated by influenza b with a share of up to % of all influenza cases. the high severity of the season / with an overall estimate of million excess consultations in germany [ ] may be attributed to several factors. according to the ecdc, the influenza season of / started earlier than most of previous ones [ ] . while this is true for the epidemic phase of the two influenza b heavy seasons in this study, the time frame for isolated cases was comparable. in the season of / the epidemic phase started in week of and lasted until week . in / the epidemic threshold was crossed in week of until week of [ ] . another factor contributing to the severity of the season might be the patient population itself. both the attack rate and the relative illness ratio of influenza b are highest for children and decrease with age [ , ] . however, the major difference that was seen for the season / was the higher proportion of adult patients. the age-subgroups themselves, being pediatric patients and adult patients, were not different in their clinical characteristics between / and the two previous seasons. however, the adult population that was seen had a high proportion of comorbidities and risk factors that might have contributed to a fig. (continued) severe outcome of the influenza disease, as it is known for example for cardiac insufficiency [ ] , additionally to age itself [ , , ] . a shift towards the adult population thus increases the impact on the public health system and may explain the severity of this season with a high number of excess consultations. an interpretation of the presented clinical data, however, needs to be done with caution as there may be a bias towards severe disease courses in a hospital setting. a fatality rate of . % of the adult population of this study can thus not be transferred to the general population. a third potential factor may be the circulating influenza type. the yamagata-lineage of influenza b was predominantly found in the season of / . in contrast to the victoria lineage, which has a faster evolution with nearly each season representing a phylogenetic bottleneck, the biology of the yamagata lineage is different. in long term studies of the ha of the yamagata lineage a long co-circulation of yamagata variants was observed [ , , ] . in accordance with these observations the analyzed strains of this study were phylogenetically closely related. furthermore, the detected amino acid changes were scattered throughout the ha and to the best of our knowledge no substitution is known for increased virus pathogenicity. since , changes in the antigenic sites are dominantly located in the -loop and -helix regions, indicating main targets for immune pressure. with regard to the vaccine strain b/phuket/ / the main antigenic regions were only affected in strains and occurred mainly in the -loop region, followed by the -loop region for the analyzed yamagata strains. in contrast, all analyzed victoria strains had at least one amino acid change in the -loop region with regard to the vaccine strain b/brisbane/ / . it is therefore surprising that the yamagatalineage that circulated in the population for three consecutive seasons was able to cause such a major epidemic. table amino acid changes of the victoria-and yamagata-strains with regard to the respective vaccine strain. amino acids are numbered with regard to the ha protein, and ha as well as ha subunits are indicated. the vaccine strains to which the isolates were compared are given in red for each lineage. isolates from this study are labeled as vaccinated with trivalent vaccine (triangle), vaccinated with tetravalent vaccine (square), randomly selected case without vaccination history (circle), fatal cases (black filled symbols). symbols represent the main antigenic sites, -loop (a; amino acids - , , , , , - ), -loop (b; amino acids - ), -loop (c; amino acids - ) and helix (d; amino acids - ). *for the yamagata-lineage, strains, that were identical at amino acid level, are depicted as a pool and contain patients with the following characteristics: xs; xd; xh, xj, x . a reason for this might be the interplay between the circulating influenza strains causing infections and the choice of the vaccine components or type of vaccine. in the season of / influenza a/h n pdm and the victoria-lineage of influenza b caused the majority of influenza infections. in / influenza a/h n was the main cause of flu disease. when looking at the influenza b component of the broadly used trivalent influenza vaccine there was a switch from b/phuket/ / -like (yamagata-lineage) to b/ brisbane/ / -like (victoria-lineage) in the season / , effectively causing a mismatch in each of the three studied seasons. in / the only strain that was not immunologically covered by recent epidemics or the current vaccine composition was the influenza b yamagata-lineage. an increased proportion of non-immune individuals in the adult population thus may have contributed to the observed shift in the age distribution of the infected patients. cross-reactive antibodies can be isolated from vaccinated individuals [ , ] but the contribution to the immunity against influenza b is not well defined. some degree of lineage crossprotection has been reported upon vaccination with trivalent influenza virus vaccines. however, it seems to be limited to certain age groups and the vaccine efficacy is consistently superior for influenza b strains belonging to the vaccine lineage [ , , ] . the capability of an individual to generate a broad antibody response against epitopes that include regions conserved between the two lineages, e.g. in the ha stalk, seems to be of major importance. thus, the factors influencing lineage cross-protection are complex and not easy to predict as also the immune memory may have a substantial impact on the targeted epitopes [ , ] . in % of the patients with a vaccination history in this study the trivalent vaccine, with an expected moderate level of vaccine efficiency [ ] , was used. the majority of the vaccinated patients were adults. however, a major improvement to influenza disease burden may only be accomplished by extending vaccine recommendations to younger age groups that are immunologically more naïve and thus more susceptible to infections [ , ] . who recommendations for the use of tetravalent vaccines that contain two influenza b strains (yamagata and victoria) in addition to the two influenza a strains (h n and h n ) should be implemented in national vaccination guidelines as it was done in germany as a result of the severity of the / influenza season. the biology of influenza viruses clinical characteristics and severity of influenza infections by virus type, subtype and lineage: a systematic literature review the contrasting phylodynamics of human influenza b viruses the continual threat of influenza virus infections at the human-animal interface: what is new from a one health perspective comparison of the mutation rates of human influenza a and b viruses the evolution of seasonal influenza viruses moderate influenza vaccine effectiveness in a b mismatch season: preliminary results from the early season co-circulation of influenza a(h n ) and b(yamagata): interim estimates of / vaccine effectiveness influneza season. last accessed . update: influenza activity in the united states during the - season and composition of the - influenza vaccine seasonal influenza reports the quadrivalent approach to influenza vaccination evolutionary dynamic of antigenic residues on influenza b hemagglutinin molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination three types of broadly reacting antibodies against influenza b viruses induced by vaccination with seasonal influenza viruses efficacy of live attenuated influenza vaccine in children against influenza b viruses by lineage and antigenic similarity prediction of influenza b vaccine effectiveness from sequence data intra-season waning of influenza vaccine effectiveness simultaneous detection of influenza viruses a and b using real-time quantitative pcr who information for the molecular detection of influenza viruses effect of a single mutation in neuraminidase on the properties of influenza b virus isolates mega : molecular evolutionary genetics analysis version . virological surveillance of influenza and other respiratory viruses during six consecutive seasons from to in catalonia, spain distribution of influenza virus types by age using case-based global surveillance data from twenty-nine countries estimation of influenza-attributable medically attended acute respiratory illness by influenza type/subtype and age influenza vaccine in heart failure: cumulative number of vaccinations, frequency, timing, and survival: a danish nationwide cohort study estimating influenza disease burden from population-based surveillance data in the united states age-and sexrelated risk factors for influenza-associated mortality in the united states between - circulating pattern and genomic characteristics of influenza b viruses in taiwan from to human monoclonal antibodies broadly neutralizing against influenza b virus cross-lineage protection by human antibodies binding the influenza b hemagglutinin effectiveness of seasonal influenza vaccine for adults and children in preventing laboratory-confirmed influenza in primary care in the united kingdom: / end-of-season results influenza a/subtype and b/lineage effectiveness estimates for the - trivalent vaccine: cross-season and cross-lineage protection with unchanged vaccine from original antigenic sin to the universal influenza virus vaccine immune history profoundly affects broadly protective b cell responses to influenza age, influenza pandemics and disease dynamics this research was partially supported by the association of sponsors and friends of leipzig university (vereinigung von förderern und freunden der universität leipzig). the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- - cua jzg authors: albanese, grace a.; lee, dong-hun; cheng, i-hsin n.; hilt, deborah a.; jackwood, mark w.; jordan, brian j. title: biological and molecular characterization of arkga: a novel arkansas serotype vaccine that is highly attenuated, efficacious, and protective against homologous challenge date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: cua jzg almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (ibv) using live attenuated vaccines mass administered by spray at day of hatch. although many different types of ibv vaccines are used successfully, the arkdpi serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. in this study, we examined a different ark vaccine strain (ark ), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. further attenuation of the ark vaccine was achieved by passage in embryonated eggs but arkga p , p , and p (designated arkga after p ) were still too reactive to be suitable vaccine candidates. however, arkga p when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. in addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. the full-length genomes of viruses from egg passages , , , and were sequenced using the illumina platform and the data showed single nucleotide polymorphisms (snps) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. arkga p accumulated the most snps in key genes associated with pathogenicity (polyprotein gene ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. these results indicate that the arkga p vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic ark-type virus. avian infectious bronchitis virus (ibv) is a gammacoronavirus that causes an economically significant upper respiratory tract disease in chickens [ ] . because of its prevalence and infectivity, nearly all commercial poultry in the u.s. are vaccinated for ibv in a serotype-specific manner [ , ] . ibv vaccines are developed by passaging a pathogenic field virus in embryonated eggs until the virus has lost its pathogenicity in chickens. during these repeated rounds of embryo passage, the pathogenic field virus will accumulate mutations that result in an adaptation for replication in embryos. conversely, the outcome of this adaptation is a decreased affinity for chicken tissues and therefore a reduced virulence in https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. abbreviations: ark , arkansas ; arkdpi, arkansas delmarva poultry industry; arkga, arkansas georgia; cas, chorioallantoic sac; c t , cycle threshold; eid , % embryo infective dose; ibv, infectious bronchitis virus; mhv, murine hepatitis virus; nsp , nonstructural protein ; nsp , nonstructural protein ; p, passage; pbs, phosphate-buffered saline; qrt-pcr, quantitative real-time reverse-transcriptase polymerase chain reaction; rt-pcr, reverse-transcriptase polymerase chain reaction; sars-cov, severe acute respiratory syndrome coronavirus; sd, standard deviation; sem, standard error of mean; snp, single nucleotide polymorphism; spf, specific-pathogen free; us, united states; usda, united states department of agriculture. chickens [ ] [ ] [ ] . live attenuated vaccines stimulate both humoral and cellular immunity, resulting in high levels of protection, and can be mass applied by spray [ , ] . immunity resulting from vaccination with live attenuated ibv vaccines prevents replication of homologous virulent challenge virus within only a short time following vaccination [ ] . of the vaccines used in the u.s., the arkansas delmarva poultry industry (arkdpi) serotype vaccine has been shown to be highly variable in its protective ability and is frequently isolated from vaccinated chicks [ ] [ ] [ ] . ideally, when mass applying an ibv vaccine, a high percentage of chicks should be infected with actively replicating virus (vaccine coverage) by days - post-vaccination, followed by a gradual decline in viral load. however, it has been shown that the arkdpi vaccine has an atypical vaccine coverage and replication pattern when mass applied by spray, and previous data from our laboratory suggests that the percentage of chicks infected with vaccine virus by days post-vaccination only reaches - % [ , ] . multiple replication cycles also occur in the bird (indicated by viral load and clinical signs in chicks), resulting in ''rolling" reactions at different time points post-vaccination [ ] . our previous research has shown that to achieve an adequate proportion of infected chicks with arkdpi vaccine and eliminate rolling replication cycles, a x dose is required [ ] . the atypical vaccine coverage and cycling observed following arkdpi vaccination is a product of the multiple minor genetic subpopulations in the vaccine bottle [ ] . it has been previously shown that several serotypes of ibv vaccines contain genetic subpopulations and the subpopulations are often recovered in chickens following vaccination, even though these vaccines show a typical infection and replication cycle and protect from challenge [ ] . with arkdpi, the major population in the vaccine contains multiple, distinct amino acid changes in the spike protein that increase binding affinity in the embryonated egg but decrease binding affinity to mature chicken cells [ , ] . conversely, the minor populations, which have the opposite spike protein binding profile, are more suited to infect and replicate in chickens [ , ] . however, these minor subpopulations are only a fraction of the total genetic population contained in the vaccine bottle. thus, the proportion of infected chicks is very low and the time to reach peak infection and replication is delayed [ ] . for these reasons, chickens do not develop adequate immunity following arkdpi vaccination. although using one of the viral subpopulations with binding affinity for chicken cells directly as a vaccine will induce a protective immune response, these subpopulations cannot be maintained through multiple passages in embryonated chicken eggs, which is required to propagate ibv vaccine. research has been performed to homogenize the arkdpi viral population by adapting it for growth in chicken embryo kidney cells, however this vaccine model has not yet been shown to be commercially feasible [ ] . while arkdpi is the only commercially available ark-type ibv vaccine today, it is not the only ark-type ibv vaccine ever produced. the arkansas (ark ) strain was the first ark-type virus to be attenuated for use as a vaccine. when originally mass applied in the field, it caused a severe vaccine reaction in young broilers, and was therefore discontinued when arkdpi was developed [ , ] . the purpose of this study was to reevaluate the original and additionally attenuated ark vaccine by multiple serial passaged in embryonated eggs as a potential ark-type vaccine candidate. in addition, we investigated the mechanisms of attenuation of this vaccine by sequencing the genome and performing snp analysis during the subsequent embryo passages. this study led to development of a new, more attenuated yet still efficacious vaccine strain designated arkansas georgia (arkga). ark vaccine is no longer produced nor is usda license maintained by any vaccine manufacturer. an archived reference sample of live ark vaccine was obtained from a commercial source and passaged once in -to- days of incubation specific-pathogen free (spf) chicken embryos as described below. the university of georgia egg-passaged virus, now designated arkga, was then used for further experimentation. different egg passages, beginning at egg passage (p ) and going to p , were used in this study for consecutive experiments. a pathogenic arkansas serotype challenge virus from our laboratory was also used in this study. spf embryonated chicken eggs were purchased from charles river laboratories (north franklin, ct) and incubated to -to- days of development for virus passage, titration, and isolation experiments. commercial non-vaccinated broiler chickens were used in the vaccination experiments as described below. arkga was serially passaged times by inoculating -to- day-old spf embryonated chicken eggs in a . ml volume via the chorioallantoic sac (cas) route [ ] . inoculated eggs were incubated at °c for h, at which point the embryos were humanely euthanized, and chorioallantoic fluid was collected for subsequent passage into additional -to- -day-old embryos. embryos were candled daily and mortality determined to be from non-viral origin was discarded. viruses were titrated at different egg passage levels using the following protocol: -fold serial dilutions of the virus were made in sterile deionized water and each dilution was inoculated into five -day-old embryonated spf chicken eggs ( . ml/egg). inoculated eggs were incubated at °c for -days and embryos were examined for ibv-specific lesions. embryo mortality within -h post-inoculation was considered nonspecific and not included in virus titer calculations. virus titers were calculated by the method of reed and muench [ ] and expressed as the % embryo infectious dose (eid ). in addition to titration of embryo passages, vaccine and challenge viruses for the arkga p and p experiments were also titrated following dilution for inoculation into chickens to confirm the inoculation dose. one hundred one-day-old broiler chicks were vaccinated with the arkga p vaccine candidate in a ml spray volume using a commercial vaccine spray cabinet and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in horsfal-bauer isolation units as controls. at , , , , , , and days post-vaccination, all vaccinated chicks were swabbed in the choanal cleft for qrt-pcr analysis of viral load. clinical signs corresponding to ibv vaccine reactions were also recorded on those days [ ] . on day post-vaccination, vaccinated and non-vaccinated chickens were challenged with pathogenic arkansas serotype virus in a . ml eyedrop application, while an additional vaccinated and non-vaccinated chickens were held as non-challenged controls. five days post-challenge, clinical signs were scored and all chickens were swabbed and euthanized for necropsy. tracheas were collected at necropsy for ciliostasis scoring. . . experiment . evaluation of infection and replication of arkga p , p , and p vaccine candidates and protection from challenge . . . trial . arkga p the arkga p vaccine candidate was further attenuated by additional embryonated egg passages, yielding the arkga p vaccine candidate. one hundred one-day-old broiler chicks were vaccinated using a spray cabinet with the arkga p vaccine candidate in an ml spray volume and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in isolators as controls. swabs were taken at , , , , and days post-vaccination to assess viral load and vaccine coverage in chicks, and clinical signs were recorded. the arkga p vaccine candidate was passaged an additional times in embryonated eggs to produce the arkga p vaccine candidate and another vaccination trial was conducted as described in trial . the arkga p vaccine candidate was passaged an additional times in embryonated eggs to further attenuate the virus, producing arkga p . one hundred one-day-old broiler chicks were spray vaccinated with the arkga p vaccine candidate in an ml spray volume and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in isolation units as controls. at , , , , , , , , and days postvaccination, all vaccinated chicks were swabbed in the choanal cleft for qrt-pcr analysis of viral load as previously described. clinical signs corresponding to vaccine reactions were also recorded on those days. on day post-vaccination, vaccinated and non-vaccinated chickens were challenged with pathogenic ark-type ibv in a . ml eyedrop application, while an additional vaccinated and non-vaccinated chickens were held nonchallenged as controls. five days post-challenge, clinical signs were recorded, and all chickens were swabbed in the choanal cleft palate and euthanized for necropsy. tracheas were collected at necropsy for ciliostasis scoring. viral rna was extracted from ll of choanal swab fluid using the magmax- rna isolation kit (ambion inc., austin tx) on a kingfisher flex magnetic particle processor (thermo scientific, waltham, ma) per the manufacturer's protocol. quantitative real-time rt-pcr (qrt-pcr) was conducted using an applied biosystems fast real-time pcr system (life technologies, carlsbad, ca) and the agpath-id tm one-step rt-pcr kit (ambion inc.) per the manufacturer's recommendations. primers and probe for the qrt-pcr were previously published [ ] and consist of a forward primer ibv gu ( -gct ttt gag cct agc gtt- ), a reverse primer ibv gl ( -gcc atg ttg tca ctg tct att g- ) and a taqman Ò dual-labeled probe ibv g probe ( -fam-cac cac cag aac ctg tca cct c-bhq - ). cycle-threshold (c t ) values above the limit of detection for each run (determined by a standard curve) were considered negative [ ] . all positive samples were used to determine the total percent positive for each group. post-challenge viral load data was presented as relative viral load values made between the groups within an experiment and were not absolute virus genome copy numbers. clinical signs were scored based on a method described by jackwood et al. [ ] . scoring was conducted on a scale from to , where = negative, = mild signs, = watery eyes and some mucus in the nares, and = watery eyes, mucus in the nares, and tracheal rales. ciliostasis scoring was conducted by examining five rings approximately mm thick cut from each chicken trachea representing the proximal, middle and distal portion. cilia activity was observed with an inverted microscope (olympus, center valley, pa). scoring was conducted based on the method by cook et al. wherein: , all cilia beating; , % of cilia beating; , % of cilia beating; , % of cilia beating; , no cilia beating. each ring was scored by individuals independently. mean scores per group were calculated and relationships between groups were analyzed statistically [ ] . routine virus isolation techniques were used for detection of ibv challenge virus in -to- days of incubation embryonated spf chicken eggs. briefly, ml of ice-cold pbs were added to the choanal swab fluid to match the stipulations of the u.s. code of federal regulations, title ix ( -cfr) [ ] . pbs from the swabs was filter sterilized and . ml of each sample were inoculated into the cas of embryonated chicken eggs. eggs were candled daily ( - h deaths were discarded) for days and the number of deaths and embryo lesions consistent with ibv infection was recorded. complete genome sequencing was performed on arkga p , p , p , and p to detect changes occurring within the viral genome during attenuation. virus stock was filtered with a . lm syringe filter. viral rna was extracted from samples using the direct-zol rna miniprep kit (zymo research) and treated with dnase i (new england biolabs). the sispa method was used for random amplification of rna as previously described [ ] . complementary dna (cdna) was synthesized using superscript iv (invitrogen/ thermo scientific). double stranded cdna (dsdna) was generated from cdna templates using second strand cdna synthesis kit (applied biological materials inc.). complete genome sequencing at a  depth of coverage was conducted using the nextera xt dna sample preparation kit (illumina) and miseq sequencer (illumina) according to manufacturer's instructions. de novo and directed assembly of genome sequences was carried out using the mira sequence assembler and geneious r program (www.geneious.com). non-synonymous substitutions in the assembled sequence reads were compared to consensus sequence at % of minimum variant frequency using geneious r program. whole genome consensus sequences were entered into the gen-bank database, with accession numbers as follows: ark pathogenic field virus mh , arkga p mh , arkga p mh , arkga p mh , arkga p mh . for arkga p , p , p , and p vaccination trials, viral rna from choanal cleft palate swabs each from days , , and post-vaccination was purified and amplified for sequencing of the s region of the genome. briefly, viral rna was purified using the zymo direct-zol rna miniprep kit (zymo research, irvine ca). s gene sequences were amplified by rt-pcr using the titan one-step rt-pcr system (roche diagnostics, indianapolis, in) and previously published primers: news oligo [ ] and degenerate [ ] . rt-pcr reactions were analyzed on a % w/v agarose gel and bands of the correct size were excised and dna was purified from the gel fragment using the genejet gel extraction kit (thermo scientific, waltham, ma). sanger sequencing was performed by the georgia genomics facility, university of georgia, athens, ga. the s sequences were assembled and compared using the dnastar suite of programs (dnastar, madison wi). the s amino acid sequences of viral rna isolated from vaccinated chickens was compared to the consensus s sequences of the vaccine virus for p , p , p , and p obtained from whole genome sequencing to detect changes in the viral population after replication in chicken tissues. statistical analysis was performed using prism v. . . (graphpad software, inc., la jolla, ca). for experiments and , postvaccination viral load determined by qrt-pcr was compared between all vaccinated chickens within each collection time point via analysis of the mean and standard error of the mean (sem). post-challenge clinical signs, ciliostasis, and viral load between challenge groups were analyzed using an alpha of . with ordinary one-way anova and tukey's multiple comparisons test. . . . arkga p whole genome sequence analysis and comparison with ark pathogenic field virus the reference sequence used for comparison of the arkga passages was the full genome of the ark pathogenic field virus (unpublished sequence). in the arkga p vaccine consensus sequence, mutations occurred in polyprotein a, the spike gene, and in a non-coding region (fig. ) . only the mutation occurring in the spike gene resulted in an amino acid change (table ) . arkga p was titrated in embryos prior to vaccination and was determined to have an eid of x /ml. appropriate dilutions were performed to achieve the desired vaccination dose of x . eid per bird, and this was confirmed by back-titration in embryonating eggs. fig. shows viral load and vaccine infection rate (coverage) post-vaccination with arkga p . viral load in chickens was high by day post-vaccination, and remained constant until -days post-vaccination, when it began to decrease ( fig. a) . arkga p vaccine candidate coverage was % by day post-vaccination and remained constant throughout the course of the experiment (fig. b) . clinical signs were also recorded at these time points and tracheal rales were observed in % of the chicks vaccinated with arkga p vaccine candidate at days post-vaccination, which is consistent with previous reports for ark . titration of pathogenic ark type challenge virus showed an eid of x /ml. challenge virus was diluted to a dose of x . per bird prior to inoculation and confirmed by backtitration in embryos. data collected at five days post-challenge is shown in fig. . all groups showed significantly less clinical signs than the non-vaccinated and challenged group (fig. a) , and all groups had significantly reduced ciliostasis scores compared to the non-vaccinated and challenged group (fig. b) , as expected. relative viral load was also significantly reduced in all groups when compared to the non-vaccinated and challenged group (fig. c ). plotting the ct values from individual samples taken from each group shows that / chickens in the arkga p vaccinated and challenged group were positive by qrt-pcr (fig. d ). virus isolation was not performed for this trial. . . . analysis of the arkga p s sequence isolated from vaccinated birds table shows important amino acid positions that were noted to change in the s gene region during the arkga passages. in the arkga p s sequence, there was no difference in viral sequence between the vaccine and the virus isolated from choanal cleft palate swabs of vaccinated chickens. . . . arkga p whole genome sequence analysis and comparison with previous arkga passages between p and p , all mutations that occurred between the ark pathogenic field virus and arkga p were lost, and a new mutation was gained in a non-coding region of the p consensus sequence (table , fig. ). arkga p was titrated and shown to have an eid of  . / ml. as seen in arkga p , for chicks vaccinated with arkga p , viral load and vaccine coverage were typically high early postvaccination (fig. ) . however, thirty percent of chickens vaccinated with arkga p showed severe clinical signs (rales) on day post-vaccination, which is reduced from the previous trial but still much higher than what would be accepted by the commercial poultry industry. because of the excessive clinical signs seen post-vaccination, the arkga p vaccination trial was ended prior to challenge, and back-titration of the diluted vaccine was not performed. non-coding c ? t none analysis of the arkga p s sequence isolated from vaccinated birds sanger sequencing of the s subunit of the spike gene in postvaccination swabs showed two amino acid positions that changed between the vaccine and the swab viral material (table ) . at amino acid position where there was a lysine present in the vaccine, % of the swabs showed a threonine. at position , the vaccine had a glycine, but % of the swabs sequenced showed a change to aspartic acid. . . . arkga p whole genome sequence analysis and comparison with previous arkga passages in arkga p , the consensus whole genome sequence showed mutations compared to the reference ark field virus sequence. a change in the nonstructural protein (nsp ) region of polyprotein a resulted in an amino acid substitution. a substitution also occurred in the s region of the spike gene. two other snps were noted in non-coding regions; one of which was at nucleotide position , that was maintained from p (table , fig. ). prior to vaccination, arkga p was titrated and determined to have an eid of  /ml. in chicks vaccinated with arkga p , viral load and vaccine coverage were lower at days and post-vaccination than in the p trial but peaked by day postvaccination (fig. ) . clinical signs were reduced to % of chicks with tracheal rales at days post-vaccination, which was less than with p , but was still considered too pathogenic for a commercial poultry vaccine. for this reason, the arkga p vaccination trial was terminated at this point, and back-titration of the diluted vaccine was not performed. in arkga p , the s sequence comparison between the vaccine virus and viral rna from swabs of vaccinated birds showed changes in the same two amino acid positions, and , that were seen in p . in p the frequency of change was increased to % in position and % in position (table ) . . . . arkga p whole genome sequence analysis and comparison with previous arkga passages numerous snps were seen in the arkga p consensus sequence compared to the ark pathogenic field virus. five snps occurred in polyprotein ab, of which resulted in amino acid substitutions. the nsp snp from p was maintained, and two polyprotein ab snps occurred in the nonstructural protein (nsp ) region. in p , snps were seen in s as well as s of the table s amino acid sequence comparison of arkga vaccine virus and viral rna isolated from choanal cleft palate swabs on days , , and post-vaccination. included are mutations recorded for amino acid positions in the s sequence that were maintained to arkga p . arkga passage s amino acid position vaccine amino acid reisolated vaccine amino acid and frequency of change spike gene, although the s snp seen in p was not detected. of note, a snp was detected at nucleotide position , in s that generated a stop codon, resulting in truncation of s by amino acids. additional snps were detected towards the end of the genome, including in the membrane, b, and nucleocapsid proteins ( table , fig. ). arkga p was diluted from a determined eid of  /ml to a vaccination dose of  . eid per bird, as shown by back-titration in embryos. arkga p viral load and vaccine coverage are shown in fig. . viral load in chicks was high soon after vaccination, though coverage was lower than expected on days and post-vaccination. by days post-vaccination, coverage had reach % and peaked at % on day post-vaccination. by days post-vaccination, chickens began to clear the vaccine virus, indicated by reduced viral load and coverage (fig. ) . only % of chicks vaccinated with arkga p showed clinical signs (snicks), which was deemed acceptable for an ibv vaccine. back-titration of diluted ark type challenge virus indicated a dose of  . eid per bird. data from five-days postchallenge with pathogenic ark virus are presented in fig. . all groups showed significantly reduced clinical signs (fig. a) , ciliostasis scores (fig. b ) and viral loads (fig. c ) compared to the non-vaccinated/challenged group. when analyzing the individual viral load values, / of the vaccinated and challenged birds were positive by qrt-pcr. it should also be noted that the arkga p vaccinated/nonchallenged group had / chickens positive for virus (fig. d) . virus isolation post challenge was consistent with the results found by qrt-pcr ( table ). all of the non-vaccinated and nonchallenged group swabs were negative for virus isolation. in the vaccinated/non-challenged group, one of the swabs was found to be positive with an embryo death at -h post-inoculation. all of the other embryos in this set died by -h post-inoculation however, indicating a possible bacterial contamination in that sample. in the vaccinated/challenged group, / of the swabs were found to be positive for ark-type challenge virus. all embryos in the th swab sample died at h post-inoculation, so that sample could not be analyzed. all of the non-vaccinated/challenged bird swabs were positive for ibv. to ensure that virus isolation positives in challenged groups were indeed challenge virus and not residual vaccine, the spike gene of samples from both challenged groups was sequenced. in all instances, sequence matched the arkansas challenge virus, indicating it was not residual vaccine. in the vaccinated/nonchallenged group, no sequence could be obtained from qrt-pcr positive samples. when comparing the s amino acid sequence between the arkga p vaccine and the virus re-isolated from vaccinated birds, multiple changes were seen. the two amino acid changes at positions and seen in p and p were % predominant in re-isolated virus in p . in addition, at amino acid positions and , the p vaccine virus population had two substitutions occur compared to prior vaccine passages. the amino acids in virus isolated from swab material showed a reversion to previous vaccine sequences at these positions ( table ) . in this study, serial passage of the arkga vaccine virus in embryonated eggs resulted in numerous changes to the viral genome. over passages, the arkga virus accumulated snps, of which were located in the replicase and spike genes, which have been attributed to ibv attenuation [ ] . most notably, arkga p had snps in the nsp , nsp , and both s and s gene regions. changes in nsp and nsp , which are part of the viral replicase complex, have been shown to impact viral replication and pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the spike gene, which is the major determinant of cell tropism and plays a role in viral attenuation [ , ] , amino acid changes occurred in the s and s subunits. one mutation in s resulted in truncation of spike by amino acids, likely shortening the cytoplasmic domain. changes in the cytoplasmic tail of ibv and murine hepatitis virus (mhv) have been shown to affect endocytosis signaling and regulation of the levels of spike at the surface of infected cells and may reduce infectivity [ , ] . in severe acute respiratory syndrome coronavirus (sars-cov), a truncation of amino acids from the carboxyl terminus of s resulted in reductions in cell fusion and cell surface expression and faster endocytosis compared to wild type [ ] . as this truncation was only seen in arkga p and not in previous passages, further investigation is needed to fully understand the ramifications of this truncation. additionally, a mutation was detected in the nucleocapsid gene, which may be related to reduced replication efficiency [ ] . the genomic changes seen in arkga over serial embryo passage can be correlated to the changes in performance dynamics seen in vaccinated chickens. during passage of arkga, the virus became more attenuated, showing reduced reactivity in vaccinated broiler chickens. different passages of the arkga vaccine were evaluated for infection, replication, vaccine reaction, and efficacy in broiler chicks. experimental vaccine and challenge trials showed that the arkga p vaccine had suitable infection and replication, and induced adequate protection from challenge, but was too pathogenic, causing a severe vaccine reaction in the majority of chicks. further passages in embryonated eggs reduced the severity of the vaccine reaction to % for p , % for p , and % for p . this further attenuation did not adversely affect infection or replication characteristics of the vaccine, as the relative viral load in chicks did not change throughout the trials. arkga p vaccine coverage was slightly less than expected shortly after vaccination but reached % by day . this may be attributed to the s amino acid changes in position and seen between the vaccine and swab sequences in p , as the s sequences re-isolated from chickens had reverted to the more fig. . experiment . trial . arkga p vaccinated and non-vaccinated clinical signs, ciliostasis scores, and viral loads in chickens post-challenge. clinical sign scores were calculated based on severity where = negative, = mild signs, = watery eyes and some mucus in the nares, and = watery eyes, mucus in the nares and trachea (tracheal rales). ct = cycle threshold. clinical sign scores, ciliostasis scores, and relative viral load were compared between challenge groups using ordinary one-way anova (a = . ) with tukey's multiple comparisons test. the -/-, arkga/-, and -/ark groups each contained chickens. the arkga/ark group contained chickens. pathogenic p sequence. in the arkga p vaccine trial a higher vaccine coverage was seen, indicating that these amino acid positions may have an impact on rate of infection. it is also possible that the changes in the replicase gene and the s mutations impacted the performance of arkga p . infection and replication patterns in all trials were predictable and ''typical" of what would be expected of an ibv vaccine. this stands in contrast to the infection and replication cycles of the arkdpi vaccine, which shows very low vaccine coverage and multiple replication cycles during the life of the bird following spray vaccination [ ] . one explanation for this is in the s portion of the spike gene. the arkga p spike sequence contains a histidine at position , which has been previously shown to significantly increase spike protein binding to chicken tracheal tissues, and an asparagine deletion at position , which has been shown to influence the ability of antibodies to recognize the protein. conversely, the arkdpi s contains a tyrosine at position and an asparagine at position , which may be attributed to its reduced ability to bind and replicate in chicken tissues [ ] . the presence of a minor virus subpopulation capable of inducing a protective immune response in arkdpi results in a very low dose of protective vaccine virus when mass vaccinating chickens and consequently poor protection [ ] . although arkga was found to have subpopulations, as evidenced by the snps observed within each passage, which is quite typical for ibv vaccines [ ] , those subpopulations were able to produce a protective immune response when mass applied. the arkga vaccine at p and p was effective at protecting chickens from a pathogenic ark ibv challenge. clinical signs and viral loads post-challenge were significantly lower than nonvaccinated and challenged groups, and all vaccinated birds passed the ciliostasis test. again, this stands in contrast to previous arkdpi vaccine and challenge experiments that showed that chickens were clearly not protected from challenge after arkdpi vaccination by spray [ ] . the attenuated arkga vaccine described herein is a significant improvement over the current commercially available arkdpi vaccine when comparing infection and replication following spray application and induction of protective immunity following homologous challenge. the arkga (p ) is also genetically distinct, making it possible to distinguish the arkga vaccine from the arkdpi vaccine or pathogenic viruses. further molecular investigation is needed to fully evaluate the amino acid changes seen in pass , but these changes do not seem to impact the effectiveness of the vaccine. in conclusion, the arkga vaccine developed herein is safe when given to -day old broilers by spray, and it induces an efficacious immune response against homologous challenge. the authors declare that there were no conflicts of interest. all experiments in this research were conducted in accordance with animal care and use protocols approved by the university of georgia iacuc committee. grace a. albanese participated in the research and article preparation. dong-hun lee performed the complete genome sequencing and de novo and directed assembly of genome sequences and coronavirus avian infectious bronchitis virus vaccination against infectious bronchitis virus: a continuous challenge. vet micro modification of the avian coronavirus infectious bronchitis virus for vaccine development the early history of infectious bronchitis development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review diseases of poultry data from years of molecular typing infectious bronchitis virus field isolates comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different arkansas delmarva poultry industry-derived infectious bronchitis virus vaccines epidemiological and experimental evidence for immunodeficiency affecting avian infectious bronchitis infectious bronchitis virus field vaccination coverage and persistence of arkansas-type viruses in commercial broilers minimum infectious dose determination of the arkdpi infectious bronchitis virus vaccine delivered by hatchery spray cabinet evaluation of infectious bronchitis virus arkansas-type vaccine failure in commercial broilers different evolutionary trajectories of vaccinecontrolled and non-controlled avian infectious bronchitis viruses in commercial poultry avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination polymorphisms in the s spike glycoprotein of arkansas-type infectious bronchitis virus (ibv) show differential binding to host tissues and altered antigenicity rapid selection in chickens of subpopulations within arkdpi-derived infectious bronchitis virus vaccines kidney celladapted infectious bronchitis arkdpi vaccine is stable and protective report: arkansas , a new infectious bronchitis serotype a new serotype of infectious bronchitis virus responsible for respiratory disease in arkansas broiler flocks a laboratory manual for the isolation, identification and characterization of avian pathogens a simple method of estimating fifty per cent endpoints development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-pcr and correlation with virus detection in embryonated eggs avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus title , code of federal regulations, standard requirements for ibv vaccines use of sequence-independent, single-primer-amplification (sispa) for rapid detection, identification, and characterization of avian rna viruses further development and use of a molecular serotype identification test for infectious bronchitis virus redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de strain of infectious bronchitis virus identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi purification, crystallization and preliminary x-ray analysis of nonstructural protein (nsp ) from avian infectious bronchitis virus the nsp replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome replication of murine hepatitis virus is regulated by papain-like proteinase processing of nonstructural proteins , , and genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication genomic characteristics and changes of avian infectious bronchitis virus strain ck/ch/ldl/ i after serial passages in chicken embryos changes in nonstructural protein are associated with attenuation in avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism sequence changes of infectious bronchitis virus isolates in the . kb of the genome after attenuating passage in embryonated eggs contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion revised the article. i-hsin n. cheng and deborah a. hilt participated in the research. mark w. jackwood and brian j. jordan participated in the study design and in the research and article revision. all authors have approved the final article. this research was supported by the university of georgia research funding and by sponsorship from merck animal health. key: cord- -peqz okh authors: girard, marc; nelson, christopher b.; picot, valentina; gubler, duane j. title: arboviruses: a global public health threat date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: peqz okh a conference on «arboviruses, a global public health threat» was organized on june – , at the merieux foundation conference center in veyrier du lac, france, to review and raise awareness to the global public health threat of epidemic arboviruses, and to advance the discussion on the control and prevention of arboviral diseases. the presentations by scientists and public health officials from asia, the americas, europe and africa strengthened the notion that arboviral diseases of both humans and domestic animals are progressively becoming dominant public health problems in the world. the repeated occurrence of recent deadly epidemics strongly reinforces the call for action against these viral diseases, and the need for developing effective vaccines, drugs, vector control tools and strong prevention programs. declaring a dengue pandemic in the s was a sentinel call to action in the fight against a range of emerging arboviral diseases of humans [ , ] . the past years have seen a dramatic émergence/re-emergence of epidemic arboviral diseases [ , ] . the recent outbreak of neurological disorders and neonatal malformations associated with zika virus (zikv) infection in latin america { }, the yellow fever (yfv) epidemics in angola and brazil with importation to china [ ] , the ever-expanding west nile virus (wnv) epidemic in the americas [ ] , the recent emergence in east africa and global spread of chikungunya virus (chikv) [ ] , as well as the ongoing and expanding dengue virus (denv) pandemic in the tropics and subtropics [ ] have reinforced the call for action in the fight against emerging and re-emerging arboviral diseases. these epidemics underscore the urgency and need for integrated control and prevention of arboviral diseases, especially those transmitted by aedes mosquitoes in urban areas [ , ] . prevention and control strategies currently focused on vector control, including insecticide treatment, environmental management and social mobilization have not been effective in practice. it is widely recognized that no strategy alone can fully address the problem. however, some intervention tools have helped reduce the disease burden. for example, timely access to clinical services and appropriate care can reduce mortality dramatically [ ] , indoor residual spraying (irs) and indoor space spraying (iss) may be effective in reducing mosquito populations and exposure to arboviruses [ ] . in addition, personal protection, clinical diagnosis and management, laboratory-based surveillance and vaccination, can be effective [ ] . vaccines are available to protect against japanese encephalitis and yellow fever [ ] , and the first dengue vaccine, even though limited in its applications, was licensed in [ ] . dr duane gubler (duke-nus medical school, singapore) reminded the audience that the frequency and magnitude of the arboviral epidemics and the extent of their geographic spread have progressively increased over time, accelerating in the past years and now occurring globally in the tropics [ , ] . https://doi.org/ . /j.vaccine. . . - x/ as an illustration, denvs were found in the s in less than endemic countries and only a few thousand cases were reported each year. in contrast, in the virus had become endemic in countries, causing an estimated million yearly infections and million symptomatic cases [ ] . in the s, denv serotypes and could be found only in south-east asia. but in the early s, all four serotypes of denv had dramatically spread to to all regions of the tropics [ ] . similarly, a new strain of chikv emerged in east africa in , spreading to asia and then to the rest of the tropical world in years [ ] . and epidemic zikv emerged in the pacific and spread around the world in only years [ ] . all of these viruses are transmitted by the urban mosquito, aedes aegypti. west nile virus (wnv), transmitted primarily by bird feeding culex mosquitoes, was introduced to the western hemisphere for the first time in , rapidly spreading from the east coast of the usa to the rest of the country and to canada before invading the caribbean, central and south america [ ] . in , , cases of wnv encephalitis in horses and , cases in humans were reported in the usa. wnv is now enzootic in the region. dr joao bosco siqueiras (institute of tropical pathology and public health, goias, brazil) described another dramatic example, that of yellow fever, which is also transmitted by aedes aegypti. the mosquito is widely prevalent in the tropics including tropical america and most countries in subsaharan africa. in - yellow fever emerged and expanded into the south and southeastern parts of brazil, where yellow fever vaccination was not common. then, in - , it emerged in central brazil, infecting many travelers. cases of yellow fever were exported from brazil to europe, peru and the usa. the virus continued to spread in - into the areas of bahia, rio and sao paolo and was detected in municipalities, causing small urban epidemics [ ] . the death toll increased to persons in and in the first half of . in africa, yellow fever spread from angola to the democratic republic of congo in - , and emerged in nigeria and uganda in [ ] . more dramatically, cases were imported from angola to china, which is the first time in history that confirmed yellow fever was introduced to asia [ ]! as outlined by dr duane gubler, the new and worrisome aspect of emerging arbovirus epidemics is that they can occur in urban centers, as was observed with dengue fever, zika, chikungunya and yellow fever. the urban vectors are aedes mosquitoes, primarily aedes aegypti, which has spread around the world in past centuries, and secondarily aedes albopictus, whose spread began in the s [ ] . the emergence and spread of dengue, chikungunya and zika infections actually reflect the growing geographical spread of aedes spp across all continents. the fact that arboviral diseases have become a major threat to urban populations is the result of: ) population growth and urbanization, which results in crowding of humans, inadequate housing, waste management and accumulation of trash, including used automobile tires, plastics, tins, etc, creating ideal ecological conditions for urban aedes populations to thrive; ) the spread of aedes spp mosquitoes around the world and throughout the tropics and subtropics; ) the lack of effective vector control and infectious disease prevention; and ) the globalization of air transport, which facilitates the rapid spread of pathogens and the diseases they cause (more than billion passengers have travelled on air lines in the year ) [ , , , ] . as these trends will continue, epidemic arboviral diseases will increasingly threaten global, national and local political and economic security and could create a global public health emergency, similar to or even greater than the current sar-cov- pandemic. one should not forget that over . billion people live in areas exposed to urban aedes mosquitoes. dr duane gubler also noted that since arboviral diseases represent a major threat to urban populations, it is necessary to reassess surveillance strategies. currently, surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. he suggested that all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions supported by serological and virological laboratory testing. three major arbovirus pathologies should be monitored: systemic febrile syndrome, haemorrhagic disease syndrome, and meningo-encephalitis syndrome. an active laboratory-based syndromic surveillance network is also badly needed. weekly reports by local physicians, as done in brazil, where they have turned out to be most useful, should also be encouraged. however, as discussed by dr elizabeth hunsperger (cdc, atlanta, usa), differential diagnosis in the clinical setting may be difficult, especially outside of seasonal disease, as mild and asymptomatic infections are common. as an example, laboratory testing for dengue is important in order to distinguish the disease from other febrile illnesses such as malaria, leptospirosis or influenza, other arbovirus diseases such as chikungunya, zika, west nile, japanese encephalitis or yellow fever, and even from typhoid/paratyphoid (salmonella typhi or salmonella paratyphi spp bacteria). recently, who has recommended the serotesting of individuals in dengue vaccination settings to screen out those with no evidence of past infection. each of theses objectives requires different test characteristics. standard dengue diagnostics assays include rt-pcr, virus neutralization assays, immunoassays to detect denv ns antigen, and assays to detect anti-denv igms [ref] . the most sensitive and specific assays are rt-pcr and the ns elisa assay. low-cost point-ofcare (poc) rapid diagnostic tests (rdts) are currently not adequate for the clinical and vaccination settings, while elisa, rt-pcr and other strategies with much higher levels of performance are are too slow and costly. the latter tests are needed in public health surveillance and research settings. these diagnostic approaches are instrument-dependent and require appropriate facilities and highly trained technical staff to perform complex diagnostic tests, unfortunately none of which are available in resource-limited areas. dengue diagnostics will greatly benefit from a dengue poc test that meets the who assured criteria (affordable, sensitive, specific, rapid, user-friendly and delivered to those in need) [ ] . note that laboratory diagnosis of dengue can be achieved with a single serum specimen obtained during the febrile phase of the illness by testing for denv nucleic acid, non-structural protein (ns ) and anti-denv igms. the current poc rdts detect both anti-denv igms and iggs as well as the ns antigen. this has the potential to change the current situation in resource-limited areas and improve dengue clinical management. denv viremia occurs for up to days following the onset of fever, and anti-denv igms begin to appear around days after fever onset. detection of denv nucleic acid by rt-pcr is the most sensitive and specific means to confirm acute infection, but it may not necessarily reflect infectious virus as it only detects viral rna, which may persist in biologic fluids after infectious virus is cleared. immunoassays to detect denv ns antigen also provide acceptable levels of sensitivity and specificity. both these diagnostic approaches are instrument-dependent and require appropriate facilities to perform complex diagnostic tests. recently, it was found that apoprotein h (apo h) can be efficiently used to detect bacteria and viruses in blood samples. as described by dr francisco veas (french institute for dvelopment (ird), montpellier, france), beads coated with apo h can readily capture viruses with a glycoprotein-rich envelope such as influenza virus, ebola virus, denv and other arboviruses, or even hcv. the beads are easy to use, the process is very fast and shows high sensitivity: one can readily detect pfu of virus in ml of whole blood. dr rome buathong (bureau of epidemiology, dept of disease contrtol, thailand ministry of public health) reminded the audience that until the brazilian outbreak in - , zikv was a little known arbovirus presenting with a mild dengue-like illness without any major complications [ ] . it initially spread from sub-saharan africa to asia without detection. thus, although the virus was present in thailand, it was not detected until may , when a canadian traveler returning from thailand was diagnosed with the disease. a zikv surveillance program was launched in the country (from january to december ) revealing a yearly peak of zikv infection during the rainy season, occurring - weeks after the peak of dengue disease. a total of confirmed cases of zikv infection was recorded in thailand during this two year period. the age groups most affected were the - followed by the - year groups. a total of cases of zikv infection was recorded in pregnant women, leading to miscarriages and birth abnormalities, including cases of microcephaly. aedes mosquitoes in the country were found to be zikv positive, but other routes of transmission included blood transfusion and sexual intercourse. zikv rna could readily be detected from plasma, saliva and urine of infected patients. sexual transmission of zikv has been documented to occur up to three weeks after onset of illness in the male partner. shedding of the viral rna in semen has been well documented, but only % of semen samples that were pcr positive could be shown to contain infectious virus by cell culture. dr nikos vasilakis (university of texas medical branch) reviewed the explosive emergence of zikv in - in the south pacific and south america, with a focus on brazil where major clinical outcomes in pregnant women were common. the alarming numbers of zikv associated microcephaly and other gestational and neonatal complications brought the virus into the spotlight. increased reporting of guillain-barré syndrome (gbs) and other neurological manifestations was also associated with zikv infections. the first association between zikv infection and microcephaly was reported in in recife, brazil where three women infected with zikv during their pregnancy delivered babies with microcephaly. it was retrospectively discovered that neurological diseases, including microcephaly, in babies born to zikv-infected mothers, had been observed in french polynesia as early as . as reported by dr patricia brasil (oswaldo cruz foundation, rio do janeiro), a prospective cohort study for zikv infection in pregnant women and infants was initiated in rio de janeiro in early . zikv was recovered from the amniotic fluid and placenta of the pregnant women, and also from the cerebral spinal fluid of their babies. microcephaly was observed in %- % of the newborns, while structural birth defects such as hypertonicity, cardiac defects, ophtalmic abnormalities, elbow abnormality, and seizures were observed in up to . % of the newborns. a follow-up of brazilian babies born to mothers infected by zikv during pregnancy showed that up to % had abnormal hearing or other manifestations, and % showed signs of abnormal behavior. at months of age, only % of the babies displayed normal neurodevelopment. dr mauricio lacerda nogueira (faculty of medicine, sao jose do rio preto brazil) talked of the interplay between various flaviviruses. given that brazil is hyperendemic for arboviruses, there was concern that previous heterotypic flavivirus (e.g. dengue) exposure could exacerbate zika disease pathogenicity. denv antibodies (ab) bind to zikv. could they drive greater zikv replication through antibody-dependent enhancement (ade)? the phe-nomenom was initially described for denv, but it has also been found to exist for rabies virus, coxsackievirus b , coronaviruses, and even hiv. to answer the question, a cohort of healthy volunteers in vila toninho, brazil, was followed up from to , looking for evidence for a possible ade phenomenom. in spite of the fact that % of the cohort had denv ab at the start of the study, no significant clinical difference could be observed in zikv infections between denv ab positive and denv ab negative persons. thus, the epidemiological evidence in brazil did not support the hypothesis that previous exposure to denv infection could enhance zikv pathogenicity. by contrast, it has been reported that denv- and denv- ab might be protective against zikv infection. similarly, the presence of yfv ab has recently been associated with a better prognosis of zikv infection in pregnant women. other hypotheses, such as enhanced infectivity of zikv for aedes mosquitoes, have not been confirmed. interestingly, a a v mutation in the e gene of chikv has recently been described which is associated with enhanced infectivity of the virus for aedes albopictus mosquitoes. as discussed by dr nikos vasilakis, the most convincing explanation for the sudden aggressivity of zikv lies in the discovery by yuan et al [ ] in of a mutation in the prm gene of the brazilian strain of zikv, which could strongly contribute to the generation of fetal microcephaly. the mutation was also found in zikv strains from french polynesia, where numerous cases of microcephaly have been recorded, but not in virus strains from africa, where microcephaly has never been observed. dr marc lecuit (pasteur institute, paris), addressing the question of the cellular and molecular mechanisms of microcephaly, reminded the audience that, in contrast with chikv, which does not cross the placental barrier, zikv can readily penetrate and infect placental cells. inoculation of zikv in the brain of mouse embryos triggers an endoplasmic reticulum stress which perturbs the physiological unfolded protein response (upr) within apical cortical progenitor cells that control neurogenesis. sustained endoplasmic reticulum stress leads to apoptosis. thus, it is likely that, in pregnant women, zikv crosses the placental barrier, gets to the brain of the foetus and crosses the blood-brain barrier, then targets apical cortical progenitor cells, which leads to the blockade of upr and the arrest of neurogenesis, and, as an obvious consequence, to microcephaly. microcephaly has not been observed in africa; it appears to be a specific property of the brasilian zikv strain, and is most likely related to the prm mutation identified by yuan et al [ ] . dr michael gaunt (london school of hygiene and tropical medicine) reported a series of experiments done on zikv and denv to identify potential genetic determinants of ade. a amino acid long motif in the denv glycoprotein was identified which might be associated with ade. dr annelies wilder-smith (london school of hygiene and tropical medicine) reviewed the eu research program on zikv (the 'zikaplan'), which oversees institutional global partners with plans to set up a sustainable latin american research preparedness network for emerging diseases. a longitudinal cohort study of , subjects aged - is being followed for three years in different locations in brazil, to further refine the full spectrum and risk factors of congenital zika syndrome, including neurodevelopment milestones, mental retardation, etc, during the first years of life. also to be addressed is whether neurological problems associated with zikv infection, such as meningoencephalitis or acute zika myelitis, are the direct consequence of zikv infection, or are mediated by immune responses. novel zikv diagnostic tests will also be developed. dr louis lambrechts (pasteur institute, paris) reminded the audience that aedes aegypti, which is distributed throughout the tropical and sub-tropical world on all continents, serves as the major vector to transmit urban arboviruses, including the denvs, zikv, yfv, chikv, etc. whereas most female mosquitoes take only one blood meal a day, aedes aegypti females need multiple blood feedings every day, which makes them more efficient at transmitting epidemic disease. the comparison between aedes aegypti and ae albopictus also shows that, although ae albopictus is often more susceptible to infection by denv than ae aegypti, it appears to be significantly less efficient at transmitting the virus. in addition, factors such as the mosquito genotype, its geographical location, and the strain of arbovirus, influence the transmission dynamics of the disease. for example, zikv infection of ae aegypti mosquitoes seems to be influenced by the sequence of the ns protein of the virus. as reported by dr scott ritchie (james cook university, cairns, australia), the use of wolbachia infection of aedes mosquitoes has great potential to control the spread of the arboviruses they transmit. wolbachia-infected male aedes mosquitoes become sterile and, if sterile males are repeatedly introduced in a given area, the resident aedes populations can be suppressed. alternatively, wolbachia-infected male and female mosquitoes can be introduced together, which leads to a progressive replacement of the resident aedes population, which could then show a much reduced capacity at transmitting denv, zikv, chikv, yfv and possibly other viruses [ , ] . the use of wolbachia is recommended by the world mosquito program to eliminate dengue. a trial in australia has demonstrated that, shortly after release of wolbachia-infected aedes mosquitoes, over % of the mosquitoes population at the site became wolbachia-infected, and the transmission of denv was stopped [ ] . similar trials are being conducted in viet nam, indonesia and brazil. the fear that wolbachia infection of ae aegypti would lead to its replacement by ae albopictus seems unwarranted so far, but it requires further studies. dr joao bosco siqueira reported that brazil had suffered from a shortage of yellow fever vaccine. fortunately, the usual dose of the brazilian vaccine contains , pfu of attenuated yfv (strain d), when only , pfu is a sufficient protective dose [ ] . it has therefore, been possible to use fractional doses of vaccine with evidence of protection and relatively no difference in reported adverse events. faced with the spread of yellow fever throughout the country, the brazilian government has updated its vaccination policy and now recommends that the entire population should be vaccinated. as part of this effort, a change in schedule for children was implemented from a single dose at years of age to a dose schedule with the first dose administered at months and a second dose at years. this effort is complicated by inadequate vaccine supply and also false rumors that the vaccine would not be protective. fake news is unfortunately powerful and, as a result, vaccine refusal has been rising in the population! as reviewed by dr in-kyu yoon (international vaccine institute, south korea), dr christopher nelson (sanofipasteur, lyon, france) and dr annelies wilder-smith, major advances in dengue research have resulted in development of new vaccines that show promise for use in dengue prevention, such as sanofi pasteur's recently licensed cyd-tdv (dengvaxia Ò , sanofi pasteur, lyon, france) and six other dengue vaccine candidates in various phases of clinical trials. cyd-tdv is a tetravalent live attenuated chimeric vaccine consisting of a d yfv genome with the pre-membrane (pre-m) and envelope (e) genes from each of the four antigenically distinct denv serotypes. the vaccine has undergone large-scale phase clinical trials in asia and latin america [ , ] , demonstrating increasing efficacy with age and higher efficacy in baseline seropositives. efficacy was highest at preventing severe dengue and hospitalization, and moderate for overall dengue. it also varied with serotype, being higher ( - %) for denv- and - infection, and lower for denv- and - . notably, during the third year of the asian phase trial, an increased risk of dengue hospitalization was seen in children aged - years. efficacy of the vaccine was only % or less in - years old and % in - years old. the vaccine was licensed in with an age indication of - years (up to years of age in some countries). the cyd-tdv vaccine is now licensed in countries, and has been introduced into public immunization programs in endemic areas of the philippines and brazil. the vaccine showed high efficacy and good safety in seropositive persons in the - years age group, but a risk of severe dengue was observed in individuals who were naive for denv infection at the time they were vaccinated. it was hypothesized that cyd-tdv mimics primary infection among individuals with no prior dengue infection (previously dengue-unexposed, seronegatives) and a secondary-like infection among those with prior dengue infection (previously dengue-exposed, seropositives) [ , [ ] [ ] [ ] [ ] . a retrospective case-cohort study was undertakern to analyze vaccine safety among dengue seropositive and seronegative trial participants. this work used anti-ns results from month of follow-up that were obtained using an anti-ns igg elisa developed for this purpose. the data confirmed the substantial benefit of cyd-tdv vaccination in those who are dengue seropositive and aged years or older, reducing symptomatic dengue, hospitalized dengue and severe dengue by~ %. however, in individuals of any age without evidence of prior dengue infection, the vaccine elicited an increased risk of severe dengue, as announced by sanofi pasteur in november . the vaccine should therefore be administered only to dengue seropositive persons, which obviously implies the need for a pre-vaccination screening strategy [ ] . the who sage dengue working group reviewed these data and, in april , the sage committee recommended to vaccinate only those with evidence of past dengue infection (seropositives) or with medical documentation of past dengue infection. these results have implications for other dengue vaccine candidates in clinical development, and for immunization program policy and program implementation. two other dengue vaccine candidates are currently in phase clinical trials: tdv, developed by the us cdc and manufactured by takeda, and tv / , developed by the us nih. tdv is a tetravalent live attenuated chimeric vaccine that uses an attenuated denv- backbone with the pre-m and e genes from each of the other three denv serotypes. the tdv vaccine is undergoing phase trials in brazil, columbia, nicaragua, panama, sri lanka and thailand. preliminary phase data show the vaccine is safe and has good efficacy against denv- and- , but the results for denv- and - are uncertain because there were only a small number of cases of these serotypes [ ] . tv /tv is a tetravalent live attenuated vaccine which includes three denv serotypes (denv- , - , À ) that have undergone attenuation through direct mutagenesis (a base-pair deletion), while the denv- candidate consists of a denv- -denv- chimera. the butantan institute in brazil, which is the sponsor of butantan-dv (tv ), received regulatory approval in december to begin a phase trial in brasil. three other denv vaccines are in less advanced development. they are an inactivated vaccine, a dna vaccine, and a subunit (the e glycoprotein) vaccine. but the difficulties of developing effective and safe dengue vaccines are multiple, as seen in the cyd-tdv experience, and many issues still remain, including: . the vaccine should elicit equal protection against the four denv serotypes, which has been extremely difficult to achieve, in view of competition between strains and/or ade of some strains: the question has been raised of whether it really is a must to develop tetravalent vaccines? . whether antibodies are associated with protection or risk (ade) seems to depend on titer, homotypy versus heterotypy, etc. how can that be understood and controlled? . why does severity of the disease, especially that of the postvaccination adverse events, vary with age? and . what is the feasibility and what will be the cost of a necessary pre-vaccination screening? nevertheless, the substantial public health benefits of even a moderately effective dengue vaccine continue to drive all the efforts at developing dengue vaccines [ , ] , hoping that safety concerns may be adequately addressed. a large number of zika vaccine candidates are under development and have entered clinical trials, including: a mrna vaccine; a dna vaccine that encodes the zikv prm and e genes and is presently entering phase iib trials in the usa, brazil, and several countries in latin america (puerto rico, mexico, panama, peru, columbia..); a purified inactivated vaccine adjuvanted with alum, which was shown to be immunogenic after two doses by the im route in a phase clinical trial; and a chimeric measles virus vaccine that expresses the prm and e genes of zikv. as outlined by dr anna durbin (john hopkins bloomberg school of public health) many hurdles still remain before licensure of a safe and effective zikv vaccine can occur. the transmission of zikv has declined so much that it will be difficult to perform a phase efficacy study. in addition, clinical trial efficacy endpoints for a zikv vaccine are not well established: one can wonder if a vaccine will not have to fully prevent infection in order to prevent microcephaly, a very high bar for any vaccine indeed! dr georges thiry (sas senergues, france) reported the creation of the 'coalition for epidemic preparadness innovations' (cepi), which was launched in january in oslo, london and washington with the aim to prevent future epidemics by the development of new vaccines. three major targets have been selected for the manufacturing and testing of candidate vaccines: mers-cov, lassa fever virus and nipah virus. cepi is responsible for vaccine development from preclinical to phase a trial. several vaccines are being followed up: a lassa-vsv vaccine, developed by iavi; a lassa-dna vaccine; a lassa-measles (mv) vaccine, developed by themis; a mers-cov-mv vaccine, also developed by themis; and a nipah glycoprotein subunit vaccine with adjuvant. most phase studies have taken place in - and phase a trials are expected to follow in - . arboviruses are in the news and at the top of social, political and public health agendas. arbovirus epidemics will increasingly threaten global, national and local political and economic security and create a global public health emergency. not only is there an increased risk of epidemic arboviral diseases in rural areas, but there also is now an increased risk of urban epidemics. dengue epidemics have recently seen their amplitude increase dramatically: in , there were fold more reported cases of denv infection in brazil than during the preceding year, leading to deaths; during the same period, , dengue-infected persons had to be hospitalized in bangladesh, where some deaths occurred, and more than deaths from dengue were reported in the philippines! an estimated million dengue infections occur annually [ ] . the development of vaccines and vector control tools to prevent arbovirus diseases is actively being pursued [ , ] , and the possibility of developing a trivalent vaccine against zikv, chikv and wnv is even being entertained. many hurdles however, remain before licensure of a safe and effective zikv vaccine can occur, as outlined above. similarly, the difficulties at developing an effective and safe denv vaccine are multiple, and many questions still arise. what will be the cost of implementing pre-vaccination screening? whom to vaccinate in places where denv seropositivity in years of age turns out to be very low (only % in singapore for example)? the dramatic emergence and spread of epidemic arboviral diseases has made it necessary to reassess surveillance strategies. all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions and supported by serological and virological laboratory testing. an active laboratory syndromic surveillance system, such as was developed in the past by who for influenza or for poliomyelitis, is badly needed. it is hoped that such a system will be set up rapidly. but we also need more effective mosquito control tools to use in conjunction with vaccines, timely access to clinical serices and appropriate care. only by using an integrated approach to prevention and control will we be able to successfully reverse the trend of emergent epidemic arboviral diseases. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the xxth century dengue pandemic dengue and dengue hemorrhagic fever the global threat of emergent/reemergent vector-borne diseases. in: vector-borne diseases: understanding the environmental, human health, and ecological connections epidemic 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fever virus aedes albopictus and the world trade in used tires, - : the shape of things to come urbanization and globalization: the unholy trinity of the st century the global distribution of the arbovirus vectors aedes aegypti and ae albopictus policy and practice. a guide to aid the selection of diagnostic tests isolation of zika virus from aedes aegypti mosquitoes in malaysia a single mutation in the prm protein of zika virus contributes to fetal microcephaly assessing the epidemiological impact of wolbachia deployment for dengue control using wolbachia for dengue control: insights from modelling the awed trial (applying wolbachia to eliminate dengue) to assess the efficacy of wolbachia-infected mosquito deployments in yogyakarta, indonesia: study protocol for a cluster randomised controlled trial immunogenicity of fractional-dose vaccine during a yellow fever outbreak -final report benefits and risks of the sanofi-pasteur dengue vaccine: modeling optimal deployment dengue vaccine: hypotheses to understand cyd-tdvinduced protection the long-term safety, public health impact, and cost-effectiveness of routine vaccination with a recombinant, live-attenuated dengue vaccine (dengvaxia): a model comparison study effect of serostatus on dengue vaccine safety and efficacy efficacy of a tetravalent dengue vaccine in healthy children and adolescents beyond efficacy: the full public health impact of vaccines estimating the full public health value of vaccination the organizing committee of the meeting included the following scientists: drs duane j gubler, jacques louis, christopher b nelson, mauricio nogueira, valentina picot, usa thisiyakorn, in-kyu yoon, and mrs cindy grasso.moderators and presenters were drs duane gubler, usa thisiyakorn, joao bosco siqueira, mauricio lacerda nogueira, christopher nelson, rome buathong, nikos vasilakis, michael gaunt, patricia brasil, marc lecuit, anna durbin, georges thiry, annelies wilder-smith, louis lambrechts, scott ritchie, in-kyu yoon, and elizabeth hunsperger. key: cord- -zzys e authors: ryan, elizabeth j.; harenberg, anke; burdin, nicolas title: the canarypox-virus vaccine vector alvac triggers the release of ifn-γ by natural killer (nk) cells enhancing th polarization date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zzys e we investigated the mechanism by which alvac activates innate immunity. combining alvac with protein antigens significantly augmented antigen-specific igg a responses; this was dependent on the presence of bioactive interferon (ifn)-γ. immuno-depletion of nk cells prior to alvac immunisation abrogated ifn-γ production indicating that they are the main cellular source of early ifn-γ in vivo. murine bone-marrow derived dendritic cells (bmdcs) cultured in the presence of alvac secreted high levels of the chemokines cxcl and ccl and up-regulated expression of the maturation markers cd , cd and cd . therefore, we conclude that alvac acts as an adjuvant through a mechanism requiring nk cell derived ifn-γ, dc activation and chemokine secretion. adjuvants improve the adaptive immune response to coinjected antigen by activating innate immunity [ ] . in spite of intense research, there is still a limited number of adjuvants available for human use, in particular to enhance the induction of the polarized th response required for optimum protective immunity to viral infection and for cancer therapy [ , ] . vaccination strategies based on the canarypox-virus vector alvac have shown potential in both infectious disease [ , ] and cancer [ ] . alvac, a large dna virus whose replication is confined to avian hosts, has a good safety profile in humans [ ] . despite its attenuated nature, alvac does cause inflammation at the site of injection as evidenced by infiltration of neutrophils [ ] . furthermore, previous reports show that alvac possesses adjuvant activity [ , ] . in this study, we confirm that alvac acts as an adjuvant, enhancing antigen-specific igg a to co-injected protein antigen in mice. intra-muscular (i.m.) injection of alvac increases levels of circulating ifn-␣ and ifn-␥ shortly after injection. alvac enhanced antigen-specific igg a responses to co-injected protein antigen in ifn-␣ receptor knockout (ifn-␣r −/− ) mice but not ifn-␥r −/− mice, indicating the importance of bioactive ifn-␥ in mediating the adjuvant effect of alvac. immuno-depletion of nk cells dramatically impaired the ability of alvac to induce circulating ifn-␥, indicating that these cells are the major producer of this cytokine in vivo. a previous study has shown that alvac triggers tnf-␣ production by human dcs [ ] . as the interaction between nk and dcs is important for the induction of the innate immune response [ ] we investigated the effect of alvac exposure on murine bmdc regarding maturation and cytokine production. we show that under the experimental conditions we used, low levels of apoptosis were detected, and that alvac induces the maturation of dcs (up-regulation of cd , cd and cd expression) and the production of low levels of inflammatory cytokines (il- , tnf-␣ and il- ). however, a marked increase in the secretion of the chemokines cxcl and ccl was observed. therefore, this leads us to suggest that alvac can act as a th polarizing adjuvant by inducing local inflammation [ ] , resulting in dc maturation and chemokine production which in turn causes the recruitment of ifn-␥ secreting nk cells. understanding the mechanism of adjuvant action of alvac, will allow more effective targeting of the desired immune response by taking advantage of the fact that alvac can be manipulated to encode a variety of immunomodulatory transgenes, e.g. gm-csf [ ] or il- [ ] that can further enhance the activation of nk cells or dcs. six to eight week old female balb/c byj mice (charles river, les oncins, france) or /sv, ifn-␣ receptor knockout and ifn-␥ receptor knockout (b + k universal, hull, uk) were used. animals were housed and experiments carried out in an accredited facility under the guidelines of sanofi-pasteur's local ethics committee. in this study we used alvac-hiv, vcp (sanofi-pasteur, marcy l'etoile, france) a recombinant canarypox vector that contains the genes for hiv- envelope gp (strain mn) linked to the trans-membrane portion of hiv- gp (strain lai), the hiv- lai gag gene encoding for the entire gag protein, the sequence of the pol gene encoding the protease, and a synthetic polynucleotide encompassing several known human ctl epitopes from the nef and pol gene products. it also contains sequences encoding the e l and k l vaccinia virus proteins in the c site. alvac placebo (sanofi-pasteur) contains a mixture of mm tris-hcl buffer ph . , virus stabilizer and freeze-drying medium. both alvac and placebo were re-constituted in sterile . % saline prior to injection. recombinant hiv tat protein from the hiv- iiib isolate and glycoprotein b (gb) from cytomegalovirus (cmv) were purified from bacterial culture at sanofi-pasteur. all vaccines used in this study were clinical grade and were verified to be free of lipopolysaccharide (lps) and other impurities. groups of balb/c, /sv, ifn-␣r knockout, or ifn-␥r knockout mice were immunised i.m. in the quadriceps following anaesthesia with imalgene (merial, lyon, france) with either gb ( g/dose), tat ( g/dose), alone or in combination with placebo or alvac ( / human dose). mice were boosted at day and serum and spleen samples taken at day . anti-tat and gb antibody titres and specific cellular responses were then analysed as described below. maxi-sorp elisa plates (nunc, wiesbaden, germany) were coated with g/ml of either tat or gb in pbs overnight at • c. plates were blocked with % non-fat milk (gibco, paisley, uk) at • c for h, after washing serial dilutions of serum samples and standards were added for h at • c. plates were then washed and the detection antibody, goat-anti-mouse igg-hrp (jackson immunoresearch laboratories, inc., west grove, pa) was added for a further hour at • c. plates were washed, and a tetramethlybenzidine substrate solution added (tebu bio-labs, le perray-en-yvelines, france), the enzyme reaction was stopped with the addition of phosphoric acid ( m), and od values at nm were determined. antibody titres were calculated from the linear regression curve of a standard specific serum originating from the same animal species as the samples, present on each elisa plate (softmax software tm , molecular devices, sunnyvale, ca). the titre of the standard had been previously determined in independent experiments. multiscreen hts plates (millipore, bedford, ma) were coated overnight at • c with anti-ifn-␥ capture monoclonal antibody (mab) (clone no. r - a , bd pharmingen, san diego, ca), washed and blocked. responder spleen cells were added at a concentration of × cells/well in rpmi (gibco) supplemented with % fcs (hyclone, logan, ut), penicillin-streptomycin (gibco), mm lglutamine (gibco), and mm ␤-mercaptoethanol (gibco). cells were stimulated in triplicate with g/ml of or mer tat or gag peptides (neosystem, strasbourg, france), medium alone as negative control and pma (sigma-aldrich, st. louis, mo)/anti-cd (bd pharmingen) as positive control. alvac specific immune responses were measured by adding p cells (mouse lymphoblast-like mastocytoma cell line) that had been infected with a multiplicity of infection (moi) of of parental virus (cppp) overnight. cultures were incubated overnight at • c/ % co . the following day, detection using mab ifn-␥-biotin (clone no. xmg . , bd pharmingen), followed by streptavidin-hrp (southern biotech, birmingham, al) was visualized using -amino- -ethylcarbazole (aec) substrate (sigma-aldrich). ifn-␥ producing spots were counted using an automated elispot reader equipped with spot tm software (microvision instruments, evry, france). alvac's ability to induce early circulating ifn-␥ following i.m. injection was evaluated using an in vivo ifn-␥ detection assay as described by the manufacturer (bd pharmingen). briefly, prior to i.m. injection with alvac or placebo groups of balb/c mice were given an intraperitoneal (i.p.) injection with g/dose of a biotin-ifn-␥ mab, serum samples were removed at various time-points and frozen at − • c until analysis of the ifn-␥ mab complex by elisa. in some experiments, nk cells were depleted using antiasialo gm polyclonal antibody (cedarlane laboratories, ontario, canada) i.p. at day − and day at g/dose. control mice received the same dose of non-specific rabbit igg (caltag laboratories, burlingame, ca). the capture ifn-␥ mab with either placebo or alvac were injected as described and serum samples were collected at h postimmunisation, and frozen at − • c until analysis by elisa. spleens were removed from both control and treated animals and the efficacy of the nk cell depletion measured by evaluating the % of spleen cells expressing the nk cell marker cd b by flow cytometry using a specific mab, clone no. dx (bd pharmingen). secretion of cytokines/chemokines was analyzed by elisa using commercially available matched pairs of mabs; murine cxcl and cxcl (r&d systems, minneapolis, mn); murine il- , tnf-␣, il- , ifn-␥, il- p , il- , and human il- and il- p (bd pharmingen, san diego, ca). the limit of sensitivity for detection was pg/ml for all elisa assays. murine ifn-␣ was detected in serum samples using a commercially available elisa kit as instructed (endogen, rockford, il). the detection range for this assay was . - pg/ml. in some experiments cytokine determinations were performed using the cytometric bead array (cba) mouse inflammation kit from bd pharmingen as described by the manufacturer. samples were acquired on a bd facscalibur and analysed with cellquest pro tm software. the detection range for all cytokines by this method was - pg/ml. bone-marrow was flushed from the femurs and tibiae of balb/c byj mice using cold complete rpmi. cells were washed and re-suspended in complete rpmi supplemented with gm-csf ( ng/ml, r&d systems). on day fresh media containing gm-csf was added to the adherent cells. on day , cells were washed, phenotyped by flow cytometry and if > % of the cells were cd c + they were then used for experiments. bmdcs were cultured at a concentration of × cells/ ml in complete rpmi for h in the presence of alvac at a moi of . and , or the equivalent volume of placebo. cells cultured in medium alone were used as a negative control and cells cultured with a toll like receptor (tlr) agonist (sanofi-pasteur) at a concentration of . g/ml, as a positive control. cells were washed, re-suspended in pbs with % fcs and . % nan ; labelled with mabs specific for cd , cd , cd , cd , cd c, cd , cd , and cd , with the appropriate isotype controls (all from bd pharmingen). cells were acquired using a facscalibur (bd) and results analyzed using cellquest pro tm software (bd). cell death analysis of either human peripheral blood mononuclear cells (pbmcs) or murine bmdcs was carried out using the tacs annexin-v-fitc apoptosis detection kit as described by the manufacturer (r&d systems). all results are presented as mean ± s.e.m., unless otherwise indicated. differences between the means of multiple groups in an experiment were analysed using one-way anova analysis of variance. in the case where only two groups are being considered results were analysed using a two-tailed independent variable student's t test. all analysis was performed using statview software (sas, cary, nc). p-values < . were considered significant. groups of balb/c mice were immunised i.m. with either antigen alone (tat or gb) or in combination with placebo or alvac. mice received a booster immunisation after weeks, and were sacrificed weeks later in order to investigate antigen-specific immune responses. titres of antigen-specific igg and igg /igg a were determined by elisa. the levels of gb specific igg observed in the group who received gb and alvac were higher than both the group that received gb alone, and the group immunised with gb and placebo (fig. a) . a similar increase in antigen-specific igg was observed when alvac was co-injected with another antigen, tat (fig. b) . looking at the particular subclasses of igg, the addition of alvac to the antigens tat or gb did not cause an increase in the levels of antigen-specific igg ( fig. c and d) . however, a -fold increase was observed in the levels of anti-gb igg a in mice that received gb and alvac compared to mice that received gb alone (fig. e) . a similar augmentation of tat-specific igg a was observed in the groups of mice that received tat and alvac compared to those that received either tat alone or tat and placebo (fig. f) . these results clearly show that alvac can act as an adjuvant augmenting antigen-specific igg a responses to co-injected antigen. next, we investigated the effect of alvac on the cellular immune response to co-injected antigen. groups of balb/c mice were immunised with tat alone, tat and placebo or tat and alvac and weeks after a booster immunisation the mice were sacrificed and their spleen removed to analyse the cellular immune responses. the spleen cells were restimulated in vitro and the numbers of antigen-specific ifn-␥ secreting cells were determined by elispot (fig. g ). the number of tat-specific ifn-␥ secreting cells in the groups of mice that received tat alone or tat and placebo were not significantly different from background levels (fig. g) . however, tat-specific ifn-␥ secreting cells were detectable in the group that received tat together with alvac. ifn-␥ secreting cells specific for the vector itself (cppp) and for gag (encoded by alvac) were detected in all groups that received alvac (fig. g) . these data show that alvac augments both humoral and cellular type responses to coinjected antigen. two major signalling pathways participate in the innate immune response to viral dna, namely, the nuclear factor-b (nf-b)-dependent pathway leading to inflammatory cytokine secretion (including il- and ifn-␥) and the type i ifn-dependent pathway inducing ifn-␣ and ifn-regulated genes [ ] . therefore, we wished to determine if ifn-␣ and/or ifn-␥ were produced in vivo following an injection of alvac. groups of balb/c mice were immunised simultaneously by the i.m. route with / th of the human dose of alvac and i.p. with a biotin-conjugated anti-ifn-␥ mab, and subsequently the mice were bled at various time-points after immunisation. levels of the ifn-␥:mab complex were determined by elisa. significant quantities of ifn-␥ were detectable in the circulation of mice h after immunisation ( fig. a) . the ifn-␥:mab complex continued to accumulate in the circulation of the alvac immunised mice up to h post-immunisation ( fig. a) . the anti-ifn-␥ antibody is stable for up to h in vivo. using / th of the human dose resulted in less circulating ifn-␥ than / th of the human dose, illustrating the effect is dose-dependent ( fig. a) . in order to detect circulating ifn-␣ groups of balb/c mice were immunised i.m. with / th of the human dose of alvac and then bled at various time-points, and the levels of ifn-␣ in the serum were determined by elisa. a low and transient amount of ifn-␣ could be detected at h in the serum of alvac immunised mice (fig. b) . in summary, alvac immunisation induces a low amount of ifn-␣ and significant amounts of ifn-␥ with very different kinetics. ifn-␣ being early and transient and ifn-␥ appearing later but more sustained. however, as we used an in vivo capture assay to determine the circulating amounts of ifn-␥ (stable ifn-␥:mab complex) and a straightforward elisa approach to detect the relatively labile ifn-␣, we cannot draw any conclusions about the relative importance of the two cytokines in the adjuvanticity of alvac from these data. at various time points serum samples were prepared from these mice and frozen at − • c until analysis. levels of the complex of mab + ifn␥ were determined by an elisa using a standard curve generated using recombinant ifn-␥ that had been pre-incubated with biotinylated mab. results are expresses as mean cytokine concentration ± standard deviation. * p < . , *** p < . and ns = not significant. (b) in a separate experiment groups of balb/c mice ( per group) were immunised with placebo or alvac ( / human dose) at time . serum samples were removed at various time-points and frozen at − • c until analysis. levels of serum ifn-␣ were determined by elisa in triplicate on serum pools from each group. results are expressed as the mean cytokine concentration ± standard error. as we demonstrated that alvac could induce both innate ifn-␣ and ifn-␥ (albeit in different amounts and with varied kinetics), we wished to address the question whether the induction of one or both of these cytokines were crucial for the adjuvant activity of alvac. in order to do this we immunised groups of wild type ( /sv), ifn-␣r −/− and ifn-␥r −/− mice with either gb and placebo or gb and alvac and then evaluated the gb specific immune responses. this approach enabled us to observe if alvac maintained its adjuvant properties in the absence of bioactive ifn-␣ or ifn-␥. anti-gb specific igg levels were similar in all groups of mice irrespective of mouse genotype or vaccine (fig. a) . anti-gb specific igg levels were marginally reduced in the groups that received gb and alvac compared . the animals were boosted at day and serum samples taken at day . anti-gb specific igg (a), igg (b) and igg a (c) titres were determined by elisa. results represented are the mean ± standard deviation of mice per group, *** p < . and ns = not significant. the anti-gb specific igg :igg a ratio was determined (d). a value of indicates that equal amounts of antigen-specific igg and igg a was produced, values greater than indicate that more anti-specific igg than igg a, while values less than one signify that more igg a than igg . with those that received gb and placebo in each of the genotypes (fig. b) . however, a clear augmentation of gb specific igg a was observed in both /sv and ifn-␣r −/− mice that received gb and alvac compared to gb and placebo. in contrast, no significant increase in anti-gb igg a was observed in ifn-␥r −/− mice upon the addition of alvac to the protein antigen (fig. c) . these data indicates that although alvac induces both innate ifn-␣ and ifn-␥ production in vivo, ifn-␣ is dispensable for the augmentation of specific igg a to co-injected antigen. nk cells are a major cellular source of the ifn-␥ produced during the first phase of the immune response to infection [ ] . in order to determine their role in the innate immune response to alvac we depleted nk cells from balb/c mice and then measured the ability of these mice to produce ifn-␥ in response to an i.m. injection of alvac. nk cell depletion was achieved using two doses of a polyclonal anti-asialo-gm ab, one given days before and the second together with the alvac injection. the efficacy of this depletion is illustrated by the lack of cd b (a nk cell specific marker in balb/c mice) expressing cells in the spleens of anti-asislo-gm treated mice compared with mice treated with an isotype control antibody (fig. b) . groups of mice, either nk cell depleted or not, were immunised with placebo or alvac and were given a simultaneous i.p. injection of biotin-anti-ifn-␥. after days, the mice were sacrificed and their serum analysed for the levels of circulating ifn-␥:mab complex by elisa. we found that the induction of circulating ifn-␥ by alvac was significantly reduced following nk cell depletion (fig. a ). this indicates that nk cells fig. . alvac induction of circulating innate ifn-␥ that is dependent upon the presence of cd b + nk cells. (a) nk cells were depleted from balb/c mice using polyclonal anti-asialo gm antibody. mice were injected i.p. with g of antibody at day − and day , rabbit igg was used as an isotype control. on day biotinylated anti-ifn-␥ at g/dose was given i.p. to all animals, and then the various groups were immunised i.m. with either placebo or alvac at / human dose. serum samples were prepared at day and the levels of the circulating ifn-␥:antibody complex were determined by elisa. values shown are the mean ± standard deviation of mice per group, *** p < . and ns = not significant. (b) treatment with anti-asialo-gm ab reduced the % cd b + cells in the spleens of the treated mice from . % (± . ) in the control treated group to . % (± . ) in the anti-asialogm treated group (the average of mice/treatment group ± standard deviation). spleen cells were isolated from mice per group and the expression of cd b determined by flow cytometry using a pe-labelled specific mab. quadrants were determined by the staining of the isotype control antibodies; representative facs plots of per treatment are shown. are the primary source of the ifn-␥ secreted in response to alvac within the first h of immunisation. activated dcs augment ifn-␥ production by nk cells [ ] . therefore, we wished to investigate if the exposure to alvac activates dcs. in order for alvac to act as an effective vaccine vector it must infect the host cells in order to produce the antigens which are integrated in its genome. however, here as we are examining the adjuvant effect of alvac we are interested in investigating the immuno-modulatory effects of exposing cells to alvac without them necessarily becoming infected. in addition in vitro infection protocols using low or no serum conditions and a high moi have been shown to induce apoptosis in the target cells [ ] . preliminary experiments showed that a moi of . to did not induce significant cell death in murine bone-marrow derived dcs (bmdcs) over a culture period of h (data not shown). murine bmdcs were cultured for h at a concentration of cells/ml in complete rpmi supplemented with % fcs alone or with the addition of alvac with a moi of . , the equivalent volume of placebo or with a tlr agonist as a positive control. cells were then stained with fluorescently labelled mabs specific for the co-stimulatory molecules cd , cd and cd and their expression levels were analysed by flow cytometry. the percentage of bmdcs expressing co-stimulatory molecules (cd , cd and cd ) was significantly increased following exposure to alvac compared to medium alone or placebo (fig. a-c) . however, the enhancement of costimulatory molecule expression induced by alvac was weaker than that observed following the stimulation of bmdcs with a tlr agonist (fig. a-c) . nonetheless, these data illustrate that exposure to alvac activates dcs. activated dcs secrete inflammatory cytokines and chemokines to recruit and activate the cells of both the innate and adaptive immune response. in response to alvac low amounts of the pro-inflammatory cytokines il- , tnf-␣ and il- p are produced by dcs, and in general the amounts are -fold less than those induced by the tlr agonist used as a positive control in these experiments (fig. d-f) . however, the levels of the chemokines cxcl and ccl produced by bmdcs following incubation with alvac exceeded or were equivalent to those induced by the tlr agonist ( fig. a and b) . cxcl secretion by bmdc was not detected in response to alvac (fig. c ). in order to reap the optimum benefit from therapeutic immunisation strategies in cancer or chronic viral infections, vaccines will need to be formulated in such a way as to augment the th effector arm of the immune response. in this report, we show that alvac can act as a th polarizing adjuvant in a mouse model (fig. g) . we show that bioactive ifn-␥ but not ifn-␣ is crucial for this adjuvant effect, as antigenspecific igg a responses to co-injected protein are enhanced in ifn-␣r −/− mice but not ifn-␥r −/− mice (fig. c) . immuno-depletion of nk cells revealed that they are an important source of the innate ifn-␥ produced in response to alvac injection in vivo (fig. a) . furthermore, we show that stimulation of murine bmdcs with alvac in vitro can induce dc maturation (fig. a-c) and chemokine secretion (particularly cxcl and ccl , fig. a and b) . additionally, alvac injection resulted in an enlargement of the local draining lymph node (popliteal) following i.m. injection (data not shown). therefore, we can conclude that alvac injection activates the innate immune response enhancing the induction of th responses to co-injected antigen. the recombinant alvac (vcp ) that we use in this study has been shown to be well tolerated in clinical trials [ ] . previous reports have shown that alvac can act as an adjuvant [ , ] . in particular, boudet et al. [ ] showed that purified alvac in either an infectious or non-infectious form had an adjuvant effect when co-administered with the recombinant hiv antigen, gp ; resulting in an increase of antigen-specific igg in both mice and guinea pigs. hutchings et al. [ ] furthered this by showing that this adjuvant activity was common to other poxviruses (nyvac, fowlpox (fp ), as well as alvac). the poxviruses: nyvac, fowlpox (fp ), alvac and to a lesser extent, adenovirus all enhanced the immune response to co-injected recombinant hepatitis b surface antigen (hbsag) [ ] . interestingly, the combination of the non-recombinant alvac vector with hbsag induced the strongest antigen-specific cellular response in the draining lymph node compared to the responses resulting from including the other poxvirus vectors investigated [ ] . two major tlr mediated signalling pathways participate in the response to pathogens, the nuclear-factor b (nf-b)-dependent pathway leading to the secretion of il- and other inflammatory cytokines and the type i ifn pathway which synergises with the nf-b pathway for optimal il- p secretion [ ] . we show that injection of alvac results in an increase in the levels of both circulating ifn-␣ and ifn-␥ ( fig. a and b) . in order to determine if the induction of one or both of these cytokines was crucial for the adjuvant effect of alvac, we immunised wild type ( /sv), ifn-␣r −/− and ifn-␥r −/− mice with gb (cmv) either with or without alvac. both the /sv and ifn-␣r −/− mice that received gb and alvac produced higher titres of gb specific igg a when compared to the groups that received gb and placebo (fig. ) , indicating that bioactive ifn-␣ is dispensable for the adjuvant effect of alvac. a study investigating the immunogenicity of adenovirus vectors demonstrated that although type i ifn signalling is an important component of adenovirus-mediated dc maturation, it was dispensable during the generation of transgene product specific cd + t cell responses [ ] . therefore, it appears that although ifn-␣ is induced by alvac and may well play a role in augmenting the innate immune response, it is not required for its' adjuvant effect. activated nk cells can secrete large amounts of ifn-␥ in response to pathogens or activated dcs [ ] . in order to determine if the ifn-␥ produced in response to immunisation with alvac ( fig. a) was produced by nk cells, we immunised nk cell depleted mice with alvac and then determined the levels of circulating ifn-␥ (fig. a) . the ifn-␥ produced in response to alvac in the nk cell-depleted mice was significantly lower than that observed in mice treated with the isotype control indicating that nk cells are a significant source of the ifn-␥ produced. it has been recently reported that alvac preferentially infects human cells of the monocytic lineage and was infact unable to infect human nk cells in vitro [ ] . our data indicates that in a mouse model, nk cells do play a crucial role in mediating the inflammatory response to alvac. further studies with human cells in vitro or ex vivo are required to determine the role of nk cells in the innate response to alvac following vaccination in man. recently, it has become clear that there is an important bidirectional dialogue between dc and nk cells (reviewed by [ ] ). tlr-dependent microbial stimuli typically associated with th responses confer on dcs the ability to activate nk cells [ ] to secrete ifn-␥ directly or induce nk cells the ability to lyse immature dcs [ ] . the discovery that human nk cells express tlr and tlr suggested the existence of tlr-based nk cell-dc crosstalk [ ] . this might be relevant in explaining how nk cells and dcs can be activated simultaneously by the same pathogen and build an efficient innate immune response, which amplifies the downstream adaptive th response [ ] . a previous report showed that both mature and immature human dcs could be successfully infected with alvac [ ] . many alvac infected immature dcs rapidly underwent apoptosis, and the endocytosis of infected, dead or dying dcs by uninfected immature dcs was observed. concurrently, a sub-population of alvac-exposed dcs matured. maturation was driven by the tnf-␣ secreted following exposure to alvac and partially by the ingestion of infected cell debris [ ] . we investigated the effect of exposing murine bmdcs to alvac in vitro and found that exposure for h to a moi of . (conditions that did not induce a significant amount of apoptosis) did activate dcs as evidenced by their up-regulation of the co-stimulatory molecules cd , cd and cd (fig. ) . additionally, exposure to alvac induced murine bmdcs to secrete significant quantities of the chemokines cxcl and ccl (fig. ). cxcl (ip- ) is chemotactic for t cells [ ] and nk cells [ ] expressing cxcr . while ccl (mcp- ) plays a role in the recruitment of monocytes/macrophages by interacting with ccr . recent studies with cxcl neutralising antibodies or cxcl −/− mice demonstrate that the absence of functional cxcl results in increased mortality and reduced t cell infiltration within the brains of mice infected intracerebrally with a murine coronavirus [ , ] , illustrating the importance of this chemokine in co-ordinating a protective immune response against a viral pathogen. ccl 's interaction with ccr is an important factor promoting early inflammatory responses and effective local antiviral defense [ ] . the ability of alvac to induce dcs to secrete chemokines, such as cxcl may provide the signal required for nk cells to migrate to dc-rich areas bringing them in close proximity enabling direct cell-cell contact, thereby resulting in downstream th responses. we found that there was a significant increase in both size and cellularity in the local draining lymph node following injection with alvac (data not shown). using flow cytometric analysis of the lymph node cells we could not determine a change in the % of any cell type (data not shown). these data in conjunction with previous work by boudet et al. demonstrate that alvac can trigger an inflammatory response, induce the migration of neutrophils to the site of inoculation within h of administration [ ] and enlargement of the local draining lymph node. therefore, we now propose the following mechanism for the adjuvanticity of alvac: alvac causes inflammation, and pmn recruitment to the site of injection [ ] . this, in turn, causes the activation of local dcs (as demonstrated by increased co-stimulatory molecule expression). these cells may then migrate to the local draining lymph node. here, dc secretion of chemokines including ccl and cxcl cause an influx of cells including nk and t cells. the interaction of the dc and nk cells in the local lymph node triggers ifn-␥ secretion by the nk cells that results in an increase in th cells specific for the co-administered protein. when these cells re-circulate to the b cell rich areas of the spleen they enhance isotype switching to igg a. innate immune induction of the adaptive immune response towards the rational design of th adjuvants immunological foundations to the quest for new vaccine adjuvants safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp in a diverse population of hiv- -uninfected adults: aids vaccine evaluation group protocol a safety and immunogenicity of an hiv subtype b and e prime-boost vaccine combination in hiv-negative thai adults recombinant viruses as a tool for therapeutic vaccination against human cancers modulation of the antibody response to the hiv envelope subunit by co-administration of infectious or heat-inactivated canarypoxvirus (alvac) preparations novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion the dialogue between human natural killer cells and dendritic cells comparative studies of avipox-gm-csf versus recombinant gm-csf protein as immune adjuvants with different vaccine platforms intratumoral administration of a recombinant canarypox virus expressing interleukin in patients with metastatic melanoma a type i interferon autocrine-paracrine loop is involved in toll-like receptor-induced interleukin- p secretion by dendritic cells induction and regulation of ifns during viral infections safety and immunogenicity of alvac vcp and recombiwith prolonged highly active antiretroviral therapy dendritic cell maturation, but not cd + t cell induction, is dependent on type i ifn signaling during vaccination with adenovirus vectors the small subset of cd brightcd -natural killer cells is selectively responsible for both cell proliferation and interferongamma production upon interaction with dendritic cells comparative analysis of tropism between canarypox (alvac) and vaccinia viruses reveals a more restricted and preferential tropism of alvac for human cells of the monocytic lineage tlrdependent activation stimuli associated with th responses confer nk cell stimulatory capacity to mouse dendritic cells cpg and double-stranded rna trigger human nk cells by toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production neutralization of the chemokine cxcl reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis cxc chemokine ligand controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells ifngamma-inducible protein (ip- ; cxcl )-deficient mice reveal a role for ip- in effector t cell generation and trafficking monocyte chemoattractant protein- and ccr interactions are required for ifnalpha/beta-induced inflammatory responses and antiviral defense in liver key: cord- -hbd euq authors: søborg, christian; mølbak, kåre; doherty, t. mark; ulleryd, peter; brooks, tim; coenen, claudine; van der zeijst, ben title: vaccines in a hurry date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hbd euq preparing populations for health threats, including threats from new or re-emerging infectious diseases is recognised as an important public health priority. the development, production and application of emergency vaccinations are the important measures against such threats. vaccines are cost-effective tools to prevent disease, and emergency vaccines may be the only means to prevent a true disaster for global society in the event of a new pandemic with potential to cause morbidity and mortality comparable to the spanish flu, the polio epidemics in the s, or the sars outbreak in if its spread had not been contained in time. given the early recognition of a new threat, and given the advances of biotechnology, vaccinology and information systems, it is not an unrealistic goal to have promising prototype vaccine candidates available in a short time span following the identification of a new infectious agent; this is based on the assumption that the emerging infection is followed by natural immunity. however, major bottlenecks for the deployment of emergency vaccine are lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we propose mechanisms to facilitate development of emergency vaccines in europe by focusing on public–private scientific partnerships, fast-track approval of emergency vaccine by regulatory agencies and proposing incentives for emergency vaccine production in private vaccine companies. although progress in medical science has eradicated one infectious disease (smallpox) and threats from other infections such as polio have been reduced by widespread vaccination, new infectious diseases emerge at historically surprisingly high rates-more than one disease per year. there are several explanations. globalisation with its correspondingly increased transport of persons, products and animals can rapidly spread new infectious diseases around the world. furthermore, the condensing of populations with worldwide urbanization and encroachment of humans into new habitats, facilitating close contact with wild animals creates new hazards for transmission of zoonotic infectious agents from animals to man and possibly in the reverse direction, transmitting human pathogens to animals [ , ] . it has been suggested that more than % ( / ) of all new human pathogens in the last century originated from an animal reservoir [ ] . there is an international recognition of the importance of emerging infectious disease in an age of changes, as recently under-lined by the world health organisation: "it would be extremely naïve and complacent to assume that there will not be another disease like aids, another ebola or sars, sooner or later" [ ] . the recent experience with emergence of chikungunya virus in italy underlines these issues. the spread of chikungunya exemplifies how easily a well-known virus from a subtropical region in africa is able to shift vector from one mosquito vector (aedes aegypti) to another (aedes albopictus), disseminate to other climatic zones -including europe -and cause disease in a susceptible population [ ] . the adaptation to a new vector can probably be ascribed to a point mutation in the virus, whereas international travel served as the means of introduction of the virus to the competent vector. a. albopictus have recently become prominent around the mediterranean basin from greece to spain and other arboviral diseases including dengue and west nile virus may use the same vector-possibly causing the next outbreak in europe. climate change may boost this development further by expanding the range of vectors and their capacity to spread disease, together with other activities that transfer potential vectors to new areas. a. albopictus has extended its distribution to europe and the americas as its larvae can be transported in used automobile tyres [ ] . emerging infections have impact not only on the health but also on the economics of the afflicted region. the sars epidemic was estimated to have a direct cost of billion us$ in asia, and the international community was on the verge of a true disaster of even larger dimensions. this blow could have disrupted health care services, and affected societies and economies for years. it was a fortunate coincidence that sars was not transmissible before the onset of the patient's signs and symptoms of disease. hence, the epidemic was contained by traditional measures of disease control such as early case finding, isolation and quarantine. if this had not been the case, the rapid development and use of an emergency vaccine might have been the only feasible measure to prevent further spread. over the last century, vaccines have been shown to be one of the most cost-effective ways to prevent and control diseases. in some situations, such as the re-emergence of smallpox or a new influenza pandemic with a severity comparable to the spanish flu, emergency vaccinations may be the only way to prevent a true disaster for europe and the global society. following the early recognition of a new threat, the current advances in biotechnology, vaccinology (including reverse genetics) and information systems offer us the possibility of developing promising vaccine candidate shortly after the identification of a new infectious agent, under the assumption that this emerging infection is followed by natural immunity. however, additional major bottlenecks for the deployment of emergency vaccines include the lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we address mechanisms to facilitate development of emergency vaccines in europe by focusing on regulatory aspects and proposing incentives for emergency vaccine production in private vaccine companies. the control of poliomyelitis by rapid development of a vaccine shows that it can be done. it exemplifies what is possible with a strong governmental commitment, public demand and dedication. it also underlines the importance of government institutions taking the lead and responsibility in vaccine development. within only one year -from to -double blinded placebo controlled studies were conducted involving , vaccines and , controls receiving placebo. these trials proved safety and efficacy, leading to licensing of the vaccine shortly after [ ] . the fruit of basic immunology and vaccinology research led to the success of the vaccine and can be condensed into four major discoveries. firstly, characterization of the poliovirus and the definition of three serotypes leading to a trivalent vaccine [ ] . secondly, pathogenicity: the discovery that poliovirus causes paralysis [ ] . thirdly, proof of principle: confirmation that neutralizing antibodies protect against disease [ ] and finally, that virus could be grown in cell cultures for mass vaccine production [ ] . the vaccine campaign was a huge success, and was accepted very well by the population, leading to a steep decrease in polio cases in the years immediately following the vaccine's deployment in europe and usa. today, polio is eradicated in most parts of the world and remains present only in a few of the poorest countries. on the other hand, diseases selected for mass vaccination have to be chosen carefully. the attempt to prevent the spread of swine influenza by vaccination, in usa , is a good example of why to think twice before initiating mass vaccination. influenza specialists were worried that an influenza strain isolated from swine might cross the species barrier and cause a repeat of the spanish flu pandemic from . although no human cases were detected, the decision to start mass vaccination was made and more than million people were vaccinated within a few months. however, suspicion that vaccination was increasing the risk of guillain-barré syndrome as a side effect soon stopped the vaccination campaign [ ] . thus, with a strong public commitment and vital basic knowledge about the pathogen, a successful vaccine can be developed within a short timeframe. however, the decision whether to implement a new vaccine needs to be based on solid risk assessment. potential and unknown hazards associated with the early mass deployment of a new vaccine must be weighed against the risk and nature of the disease. vaccine development -in particular for emergency vaccinesneeds a different business plan than the market-driven approach that underlies the pharmaceutical industry [ ] . private vaccine companies cannot be expected to use resources on improving existing vaccines or developing new vaccine candidates for emerging infectious diseases when there is no current market or if the market is too small or diffuse to be economically feasible. hence, it is important that governments find mechanisms and funding to ensure the fundamentals for the success of a vaccine, namely basic and applied research in public health institutions and academia. furthermore, clear communication is necessary between governments and the vaccine industry on which vaccines need to be developed from a public health perspective. the challenge is to find incentives for the vaccine industry to take part in the development of products that currently do not have a clear market. one solution might be public support to public-private research and development of vaccine candidates in their early preclinical/clinical phases or advance assurances of confirmed purchases of certain volumes of vaccines if they make it to licensure. the european union has promoted the concept of public-private partnerships, but this concept has not resulted in important changes so far [ ] . it is important to find new ways to achieve these aims: it will be too late by the time we suddenly need new vaccines against an emerging epidemic. public-private partnerships in particular are necessary to secure vaccine production from laboratory bench through pilot plant to mass scale industrial production. specific contracts between governments and vaccine companies must be in place to secure that private production capacity is available for emergency vaccination production if needed. as mentioned, modern biotechnology has opened novel approaches for the development of new vaccines allowing production to be carried out in only a few months under the best circumstances (see table ). but obtaining data on clinical efficacy for licensure and regulatory approval will be a major bottleneck for making use of the current technology. without some indication of the immune response required for protection, basic efficacy studies will be difficult. without an animal model, they will be all but impossible. that leaves only the possibility of going to human studies without an indication that the vaccine is effective-a step that is highly unlikely, even in a dire situation. this leads us back to the responsibility of governments and international agencies -including the european union -for laying the groundwork. the first vaccines for sars were developed this way, leveraging preclinical work that had already been done on other coronaviruses [ ] . seasonal influenza vaccines are also made this way; working on the assumption that what has proved efficacious in the past against a related virus will prove efficacious in the future table a toolbox for the rapid development, production and deployment of an emergency vaccine. early recognition of an emerging microbial threat identification and characterization of the causative agent rapid understanding of natural history, pathogenesis, molecular biology and epidemiology; building on work in related pathogens as well as ongoing clinical, laboratory and epidemiological studies identification of potential vaccine candidates identification of potential delivery systems and suitable adjuvant to improve immunogenicity and sparing of antigen and dosages production at pilot plant level development and acceptance of correlates of immunity development and acceptance of correlates of safety limited trials in animals and humans based on these correlates as outcome measures fast-track approval of the vaccines enhancing production capacity by public-private partnerships based on risk assessment and defined objectives: implementation of emergency vaccination post-licensure follow-up of emergency vaccination with data accessible in real-time to medicine-and public health agencies as a surrogate for phase iii trials and ensuring development with advance purchase agreements to establish a market. testing novel vaccine candidates also becomes a bottleneck: just any combination of antigen and delivery system may not be effective. toxicity testing -which is designed to catch only acute problems with a vaccine -can be generally applied to most vaccines. in theory, it may be possible to produce basic safety data even before a complete product is available by testing "mock-up vaccines" in which a placeholder antigen or antigens are used [ ] . this would provide information on the safety of the delivery system including adjuvant, which is generally the most reactogenic part of a vaccine. safety testing of a specific product would still be required, but the reactogenicity of the generic components would already be defined and be one less variable to consider in designing the vaccine. however, efficacy studies require animal models of the infection or the disease and this is both time-and money-intensive. in the absence of a defined animal model, the obvious choice is nonhuman primates, as the closest match to humans, but even if this is possible (i.e. the pathogen will infect primates and produce disease) it may not be practical. both primate and non-primate animal research facilities are in short supply. support of animal facilities is likely to pay dividends when a need arises for rapid assessment of new vaccines. this should be relatively easily put into place, by making it an explicit goal, since it is really only an expansion of existing activities by research supporting agencies. certainly, in the united states, the biodefense initiative has led to a significant expansion in capacity. but it cannot be put into place on an ad hoc basis, nor is it likely that the private sector will become involved-the return on investment in possible emerging diseases is highly uncertain. such facilities take years to establish and their benefits are primarily in research and public health: therefore bodies involved in research and public health will need to take the initiative. once a potential vaccine has been produced and some evidence for efficacy and safety produced in animals through accepted correlates, the same data needs to be reproduced in humans. the current procedures were designed with safety as the foremost consideration and rapidity is not a characteristic of the process: it can take - years (occasionally longer) for a new vaccine to pass through clinical trials. under normal conditions, this is a sensible application of risk/benefit analysis with the emphasis on "first, do no harm". by definition, emerging diseases are not a major health risk-until a significant outbreak occurs. thus, clinical assessment is built around gradual steps-first screening for major risks (phase i, trials generally conducted in very small groups) then subsequently screening for less frequent risks (phase ii, in larger, but still small groups). it is not until phase iii studies, which are often large (thousands to tens of thousands), that efficacy data are expected to be produced. and while phase iii studies are large, they still occasionally fail to uncover rare risks, which only emerge after hundreds of thousands of people have been vaccinated, for example, intussusception of the bowel after administration of rotashield, a vaccine to prevent diarrhoea caused by rotavirus infection [ ] . such events are only uncovered during post-licensure pharmacovigilance. in an epidemic situation, however, the risk/benefit balance changes: if morbidity and mortality due to the pathogen is high, then even a vaccine with significant side effects becomes much more acceptable. it is therefore important to develop procedures for alternative pathways of approval. this should be done in close collaboration with regulatory agencies, and be based on accepted correlates of immunity and safety plus [probably] limited human data. while it will ultimately be up to governments with who guidance under international health regulations to decide what constitutes an "emergency" in which it could be invoked, the bare bones of a "fast-track approval" system for new treatments already exists (influenza mock-up vaccines emea) [ ] , and this procedure could be expanded to accelerate vaccine-testing in clinical trials during a public health emergency. in most cases, the greatest amount of time in a clinical trial is devoted to paperwork, to ensure that the trial complies with regulations designed to minimize risk to participants, ensure transparency and provide a paper trail as a shield against future litigation, should things go awry. in diseases with a poor survival chance -aggressive cancers, for example, or anti-retroviral therapy in the early days of the hiv epidemicregulatory agencies tended to be more forgiving. in such a situation, while safety remains a major issue (particularly for preventive vaccines administered to healthy individuals), testing for efficacy assumes greater importance.the demands for faster processing of vaccines can be addressed by the following steps: . an already-defined regulatory framework within which fasttrack clinical trials can be conducted. this should contain rules for priority review and approval, some (limited) protection against liability to open the process to commercial entities (as they are best equipped for large scale production and distribution) and rules for invoking such a process. they may not (perhaps should not) lead to open-ended approval of a product, being instead intended to allow limited release. . rapid access for vaccine developers to the appropriate regulatory authorities within the emea and authorization for regulators to draw on necessary expertise (perhaps in the form of expert panels) to enable assessments to be made quickly. regulations on relaxing approval (perhaps in the form of approval for a limited time) for import of vaccines not currently approved for use in europe. . a process whereby approval can be granted under the understanding that the complete necessary paperwork can be submitted retrospectively-enabling a rapid progress of efficacy trials, as soon as initial data suggests a vaccine is safe, without waiting for collation, submission and approval before progressing to the paperwork for the next step. . a broader acceptance of surrogate data, e.g., correlates of protection and safety, in the early steps. if (for example) animal toxicity studies raise no concerns, it may be possible to proceed directly to combine phase i/ii studies. this would provide human safety data, but at the cost of placing slightly more participants at risk. the payoff would be earlier access to data indicating whether the vaccine is immunogenic and stimulates the desired type of immune response, plus a more rapid assessment of vaccine safety. acceptance of immunogenicity data as a surrogate for efficacy, based on animal models (and it is here that expert panels will be crucial) might allow rapid release of a product from phase iii trials, subject to the study continuing to collect efficacy data. such an approach could shorten vaccine-testing time by months to years. . finally, while it is possible to enter phase i trials with an experimental product, phase ii has stricter requirements and the product tested needs to be closer to, if not identical with, that which will be taken into phase iii trials-which itself will be the final product. some flexibility on vaccine composition would allow a more rapid progress to phase iii. phase iii trials are intended to demonstrate that a product is efficacious. this is never an easy task and for emerging diseases is complicated by the fact that such diseases are, by definition, not endemic. that means that without reliable surrogate markers, efficacy studies can only be done in endemic countries or selected high-risk populations. it can be expected that even in countries where the disease is endemic, people will be reluctant to accept testing of a vaccine for which safety data and approval has been fast-tracked: some form of compensation mechanism is almost certain to be required to encourage manufacturers and the public to participate in a clinical trial. this passes out of the remit of organizations such as emea and into that of international cooperation, which needs to be arranged at the governmental level. the risks of rapidly proceeding into phase iii can be ameliorated by compromising (to some extent) impartiality. to avoid bias, such large studies are normally blinded and results assessed at the end of the study. however, in a situation where large numbers of people are being vaccinated with a "fast-tracked" vaccine safety concerns will be higher than normal. by putting enhanced surveillance into place and assessing data from cohorts within the main phase iii study, data on adverse events possibly associated with vaccination and efficacy of the vaccine could be collected much faster. objectivity could be maintained by maintaining blinding with regard to the study monitors. this approach essentially expands the role of the data safety management board, whose role is normally to oversee the safety of the study and who do review results on an ongoing basis, to cover decision-making on vaccine efficacy. in such a case, they would need to involve the study designers, which may raise issues of conflict of interest. this can be addressed by again involving expert review panels, but that will almost certainly face resistance from commercial developers who would face exposure of their operating procedures. but where there is an overwhelming public interest in rapid assessment of vaccine efficacy, the conventional rules may need to be relaxed and increased transparency is the safest counter to decreased regulatory oversight. in conclusion, new infectious diseases are emerging at a historically high rate. to secure both public health and economic stability in the future effective countermeasures have to be instituted in advance at governmental levels. implementing fast-track approval systems for emergency vaccines by the regulatory agencies, and underpinning public private partnerships to enable production in the absence of a market would be an important step in order to be prepared for a new pandemic. finally, innovative research towards the understanding of vaccine safety and efficacy and leading to shorter development times should be promoted. american association of physical anthropologists meeting perceived vaccination status in ecotourists and risks of anthropozoonoses surveillance and response to disease emergence a safer future, global health in the century. the annual health report who infectious diseases chikungunya: no longer a third world disease the used tyre trade: a mechanism for the worldwide dispersal of container breeding mosquitoes making history: thomas francis m.d. jr. and the salk poliomyelitis vaccine field trial differentiation of types of poliomyelitis viruses; the grouping of strains into three basic immunological types viremia in human poliomyelitis evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis iv. final report of results based on clinical diagnoses cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissues guillain-barre syndrome following vaccination in the national influenza immunization program, united states - why certain vaccines have been delayed or not developed at all european union dg interternal policies of the union a dna vaccine induces sars coronavirus neutralization and protective immunity in mice pandemic vaccines: promises and pitfalls rotavirus vaccines: an overview key: cord- -izdl f i authors: qin, ede; shi, huiying; tang, lin; wang, cuie; chang, guohui; ding, zhifen; zhao, kai; wang, jian; chen, ze; yu, man; si, bingyin; liu, jianyuan; wu, donglai; cheng, xiaojie; yang, baoan; peng, wenming; meng, qingwen; liu, bohua; han, weiguo; yin, xunnan; duan, hongyuan; zhan, dawei; tian, long; li, shuangli; wu, jinsong; tan, gang; li, yi; li, yuchuan; liu, yonggang; liu, hong; lv, fushuang; zhang, yu; kong, xiangang; fan, baochang; jiang, tao; xu, shuli; wang, xiaomei; li, changwen; wu, xiaohong; deng, yongqiang; zhao, min; zhu, qingyu title: immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: izdl f i background: in , severe acute respiratory syndrome (sars) resulted in hundreds of infections and deaths globally. we aim to assess immunogenicity and protective efficacy of purified inactivated vero-cell sars vaccine in monkeys. methods: the cultures of sars coronavirus (sars-cov) bj- strain infected vero cells were inactivated with β-propiolactone. sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated sars vaccine. the purified sars vaccine was analyzed with electron microscope, hplc and western blotting. we immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. antibody titers were measured by plaque reduction neutralization test. the vaccinated monkeys were challenged with sars-cov bj- strain to observe protective efficacy. additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated sars vaccine ( . , and μg/time/monkey) on days and , and the monkeys were challenged with sars-cov gz- strain. we assessed the safety of the sars vaccine and observed whether the antibody dependent enhancement (ade) occurred under low levels of neutralizing antibody in rhesus. findings: the purity of sars vaccine was . % by hplc identification and reacted with convalescent sera of sars patients. the purified sars vaccine induced high levels of neutralizing antibodies and prevented the replication of sars-cov in monkeys. under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with sars-cov. in this preliminary animal trial, no side effects were detected when monkeys were immunized with purified sars vaccine either at normal or large doses. interpretation: the purified inactivated sars vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of sars-cov. the sars vaccine prepared in the study appeared to be safe in monkeys. severe acute respiratory syndrome (sars, also called infectious atypical pneumonia in china) is a new emerging respiratory infectious disease with rapid transmission and high mortality. according to statistics released by who on august , a total of clinically diagnosed cases were reported worldwide, causing deaths, an estimated mortality of . %. a novel coronavirus, sars coronavirus (sars-cov), was identified as the pathogen of sars [ ] , in , some new sars cases were reported in china, which suggest that sars may recure in the future. the current medical strategies mainly replying on non-specific anti-viral and supportive treatment are not sufficient. previous experience in controlling infectious diseases indicates that the most efficient protection against sars infection is mass immunization. effective vaccines for protecting the population are urgently needed. since the discovery of sars-cov as the cause of sars. the inactivated sars-cov has been proposed as one of the prophylactic vaccination approaches against sars. a number of studies about coronavirus vaccines have demonstrated that inactivated vaccines are effective methods, such as infectious bronchitis virus and bovine coronavirus. although, little information about pathogenesis of sars-cov is known, we postulated that it may be feasible to develop an inactivated vaccine for sars. the present study developed the techniques for the preparation of sars inactivated vaccine and investigated the immune response and protective effects induced by the vaccination in monkeys. our data suggest that this vaccine could stimulate monkeys to produce high levels of antibodies which could protect monkeys from sars-cov challenge. these results lay a solid foundation for our early phase human study. sars-cov bj- strain and gd- strain were isolated from autopsy lung tissues of sars patients from beijing and fushan, guangdong province, china in march [ ] . the complete sequences have been deposited in gen-bank, with accession numbers ay and ay , respectively [ ] . vero cells (atcc no. ccl- ) were provided by the national vaccine and serum institute, beijing, china. well-grown vero cells were inoculated by bj- strain at a m.o.i of . and maintained with serum-free mem at • c. the culture was harvested when cytopathic effects reached + [ ] , and passed through . m filter (polypure ® , in- /fin silicone) from pall cor., usa. the filtrate was collected and ␤-propiolactone was added at : (v/v). the shaken mixture was kept at - • c for more than h before incubating at • c for h to inactivate the virus completely. the inactivated virus was concentrated by omega ultrafiltration unit and suspended in pbs ( mm, ph . ). all procedures were performed at biosafety level . the concentrated inactivated virus was purified by gel filtration of sepharose fast flow (ff) (index / , amersham biosciences, sweden) at % column volume with elution buffer pbs ( mm, ph . ). the fractions from the outer void were collected. the eluate containing sars antigen was further purified by ion exchange chromatography. aktaexplorer chromatography system was used, the media was fractogel ® emd dwae(m), and the column was xk / . after being equilibrated by pbs ( mm, ph . ), the gel filtration purified inactivated viruses were loaded, then eluted by pbs containing . m nacl. the first elute peak containing the viral antigen was collected. the effluent was diluted according to the protein concentration of the antigen active peak to prepare the purified sars vaccine. the purity of the vaccine was analyzed with hplc analysis. two hundred microliters of purified sars vaccine was run on tskgel g sw column ( . mm × mm, f ) using . ml/min flow rate to acquire the hplc profile. the antigen content of inactivated sars vaccine was determined by double-antibody sandwich elisa. the rabbits were immunized with sucrose density gradient zonal centrifugation-purified virus antigen to prepare the anti-sars-cov antibody (first antibody), and horseradish peroxidase labeled rabbit anti-sars-cov igg was used as the second antibody. the protein content of the purified inactivated sars vaccine was determined by a modification of the lowry method. approximately l of purified sars vaccine was placed on a copper grid and then allowed to stand for min. after the residual fluid was removed by filter paper, the specimen on the grid was stained with % phosphotungstic acid for min. then the grid was dried at room temperature and observed under transmission electron microscope. sds-page was performed according to laemmli method [ ] . after electrophoresis, the protein band in the gel was transferred to polyvinyl fluoride (pvdf) membrane (millipore), and reacted with convalescent sera of sars patients in blocking buffer (containing % skimmed milk) at • c overnight. after washing, the membrane was incubated with horseradish peroxidase labeled rabbit anti-human igg (bio-rad) for h. the membrane was washed again, and the color was developed. cynomologus macaques (male, - years old, animal center of academy of military medical sciences, beijing, china) were divided into four groups, with five monkeys per group. group was immunized with purified vaccine and adjuvant (aluminium hydroxide, aluminium content: . mg/ml). group was immunized with purified vaccine. group was immunized with unpurified inactivated virus. all three experimental groups were immunized by deltoid muscle injection at g on days , , and . a further control group was injected with supernatants of vero cell culture ( . ml/monkey). all monkeys were blood sampled on days , , , , , and . the sera were isolated from the blood and inactivated at • c for min. the neutralizing antibody titers were then determined by the plaque reduction neutralization test. to observe the protective effect of the inactivated vaccine, monkeys in group (four monkeys, immunized with purified inactivated vaccine) and in the control group (four monkeys) were challenged with ml sars bj- strain (log tcid /ml = . ) by nasal route on day after the prime immunization. the specimens of blood, pharynx swab and stool were collected on days , , , and after the challenge. the neutralizing antibodies in sera were determined, and virus isolation and rt-pcr were also conducted [ , ] . all monkeys were sacrificed on day after the challenge, and lymph node, lung, spleen, liver and kidney tissues were collected for rt-pcr. the pathological changes of lung tissues were observed in slide slices by light microscope. safety evaluation tested the antibody dependent enhancement (ade) of the inactivated sars vaccine. three groups of rhesus (male, - years old, animal center of academy of military medical sciences, beijing, china) (three rhesus monkeys per group) were immunized with different doses of purified inactivated sars vaccine ( . , , g/time/monkey) on days and . three monkeys in the control group were injected with vero cell culture. blood was sampled on days , , and for neutralizing antibody determination. all monkeys were challenged with sars-cov gz- strain on day at the dose of . tcid /ml × and were euthanized on day after challenge. internal organs from the monkey were sampled and the systematically pathological changes were observed. to assess possible side effects of large doses of purified inactivated sars vaccine ( g) in major organs, the monkeys were immunized with the sars vaccine by deltoid muscle injection, and then the hematological, biochemical and pathological changes were observed. neutralizing antibodies in the sera of vaccined monkeys were tested by % plaque reduction neutralization test (prnt ) using vero cells [ , ] . in brief, heat inactivated ( • c min) sera were diluted serially and mixed with sars-cov bj- ( pfu/ . ml), then incubated at • c for min. the mixtures were added to a vero cell flask, and absorbed at • c for min, then covered with nutrient solution (containing agarose, calf bovine serum, streptomycin and dmem), and cultured at • c for h. after being dyed by . % neutral red, the flask was incubated at • c for another - h until the plaque was developed. vaccined monkeys were euthanized under ether anaesthesia. the lung, liver and kidney tissues were sampled and placed in % formaldehyde, followed by conventional dehydration and paraffin embed. then the embedded specimens were sliced (with the thickness of m), stained with hematoxyllin-eosine and observed under light microscope. when cytopathic effects in vero cells inoculated with sars-cov bj- strain reached +, the virus culture was harvested and clarified, then inactivated with ␤propiolactone. after concentrated by ultrafiltration, the inactivated virus culture was further purified by sepharose ff gel filtration chromatography and ion exchange chromatography to achieve higher purity. after two steps of purification, the residual bovine serum albumin and cellular dna in inactivated sars vaccine were less than . and ng/ml, respectively. the ratio of antigen activity/protein quantity increased from . to . u/g after purification. hplc analysis showed that the purity of sars vaccine was . %. the purified components of the inactivated viruses showed good antigenicity and immunogenicity in mice (data not shown). sars-cov-like particles could be observed in purified vaccine under electron microscope. as shown in fig. , the molecular weights of purified vaccine by sds-page analysis were consistent with the molecular sizes of the main structural proteins of sars-cov. western blot analysis showed that the protein bands reacted strongly with the convalescent sera of sars patients. to observe whether the sars inactivated vaccines induced sars-specific neutralizing antibodies, cynomolo- gus macaques were immunized with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine. their sera were sampled at different time to measure neutralizing antibody titers. as shown in fig. , weeks (on day ) after the first boost, large increases in neutralizing antibodies were observed in the sera of all three immunization groups. the neutralizing antibody titers in all immunization groups peaked week after the second boost ( weeks after the prime). antibody levels also increased rapidly after the last boost over a -week observation period. the results showed that the neutralizing antibody levels in the adjuvant-containing purified vaccine group were higher and lasted longer than those in other two groups. the quantity of neutralizing antibody production and the time for antibody level maintenance showed no apparent differences between the purified vaccine group (without adjuvant) and unpurified vaccine group, but the antibody levels in the unpurified vaccine group appeared to decrease somewhat faster. the results showed that both the purified and the unpurified sars vaccines can induce high levels of sars-cov specific neutralizing antibodies in monkeys, thus demonstrating high immunogenicity. fig. . anti-sars-cov neutralizing antibody titers of monkey sera elicited by different inactivated sars vaccine. cynomologus macaques were immunized with adjuvant-containing purified vaccine, purified vaccine, unpurified vaccine and supernatants of vero cell culture ( . ml/monkey) as control by deltoid muscle injection at g on days , , and . all monkeys were blood sampled on days , , , , and . the neutralizing antibody titers were then determined by the plaque reduction neutralization test, and presented as the geometric means (n = ). to observe the protective effect of the purified inactivated sars vaccine on monkeys, the aforementioned two animal groups (purified vaccine group and unvaccinated-control group, with four monkeys per group) were challenged with sars bj- strain by nasal route on day after the prime immunization. the results showed no sars-cov isolated from the monkeys in the immunization group. in the control group, sars-cov was isolated by pharynx swab inoculation in two out of four monkeys on days and after the challenge. rt-pcr amplification of the tissue samples from autopsy of monkeys on day after challenge showed that the lymph node, lung, spleen and kidney tissues from all four monkeys in the immunization group were negative. in the control group, the positive rates of rt-pcr amplification of the lymph node, spleen, lung and kidney tissues were % ( / ), % ( / ), % ( / ) and % ( / ), respectively. the results indicated that inactivated sars vaccine may inhibit the propagation of sars-cov in monkeys. further pathological examinations of the challenged monkeys showed that two out of four animals in the unvaccinatedcontrol group displayed moderate and mild interstitial pneumonia. two out of four from the immunization group were normal, and the remaining two immunized monkeys only showed mild interstitial pneumonia, without any pathological changes in other tissues. the results suggested that purified inactivated sars vaccine had protective effects in monkeys. as shown in fig. , the neutralizing antibody levels in the immunization group peaked between and on day after the last immunization (i.e., on day after the prime immunization), and decreased gradually thereafter. on day after the prime immunization, the neutralizing antibody titers in the sera of four immunized monkeys dropped to range of - . however, the antibody levels in the immunization group increased significantly week after the challenge, indicating that inactivated sars vaccine produced immune memory in monkeys. after rhesus monkeys were immunized with different doses of purified inactivated sars vaccines, hematological and biochemical indexes showed no change in any animal, including all nine monkeys in the immunization group and three monkeys in the unvaccinated-control group (data not shown). no fever was observed in any monkey , , , and h after the injection of the sars vaccine, and the appetite and mental state of all animals maintained normality. to examine whether different antibody levels produced by different doses of vaccines induced a corresponding antibody dependent enhancement, the monkeys in the immunization group and unvaccinated-control group were challenged with sars-cov gz- strain by nasal route on day after the prime immunization (fig. ) . the results showed that no clinical symptoms were detectable in immunized monkeys, and their body temperatures and hemograms were normal. on day after the immunization with different doses of inactivated vaccine, systematic pathological observation under light microscope showed no abnormity in the internal organs of monkeys. histopathology observations indicated that all monkeys in the unvaccinated-control group showed obvious interstitial pneumonia. interstitial pneumonia was also observed in the immunization group, but was obviously milder than in the unvaccinated-control group. among the monkeys in the low dose group, especially those with low levels of neutralizing antibodies, the pathological changes were milder than in unvaccinated-control group. the results showed that neither infection enhancement nor immunopathological exacerbation was observed under low neutralizing antibody titers. for monkeys immunized at higher doses ( g/time/monkey), body temperature, breathing, appetite, mental state and all biochemical indexes were normal (data not shown), and no abnormalities were observed in major organs such as lung, liver, kidney, etc. the above results indicated that the purified sars vaccine prepared in the present study was safe in monkeys. the isolation and propagation of high titers of sars-cov in vero cells provided a solid foundation for the development of inactivated sars vaccines. drawing on previous research, currently available technical platforms [ ] , and results from other inactivated viral vaccines [ ] , we developed concentration and purification techniques for a sars vaccine. our inactivated sars vaccine was produced with standard quality controls and showed high purity and immunogenicity. the vaccine could also induce high levels of neutralizing antibodies. to date, several approaches for developing sars vaccines have been reported, including inactivated vaccines [ , ] , recombinant adenovirus [ ] or vaccina virus [ ] , and dna vaccines [ ] [ ] [ ] [ ] [ ] . these vaccines could induce the production of specific anti-sars antibodies and activation of ctl responses, some could to induce protective immunity in mice. the recombinant adenovirus containing the genes encoding protein s fragments and full-length m and n were also proved to be able to induce protective immunity in monkey. our data and other studies about inactivated sars vaccines showed the highest specific abs titers in mice or monkeys, which suggest the high immunogenicities of inactivated vaccines. unlike their studies [ , ] on inactivated vaccines for sars, our inactivated sars vaccine was produced with standard quality controls. our observations of immunogenicity in monkeys showed that the unpurified inactivated sars vaccine induced almost the same level of neutralizing antibody as the purified vaccine. even so, the unpurified production could not be applied to clinical use. it had a high content of protein impurity that might induce underlying pyrogen fever. in this study it was also demonstrated that the vaccination with adjuvant of aluminium could induce higher antibody production of sars-cov, but clinical safety concerns about possible side effects in humans precluded use of the adjuvantcontaining purified vaccine to evaluate immune protection (efficacy testing). thus, we selected the purified inactivated sars vaccine for a detailed safety and efficacy study. in previous sars vaccine studies of other groups, challenges have been performed when monkeys were at high levels of neutralizing antibodies. this approach is not informative for population immunization. our study challenged monkeys at lower levels of neutralizing antibodies, which is more consistent with a general population vaccination context. the findings from the low neutralizing antibody stage challenges were promising. the results showed that sars-cov could replicate in relevant tissues, specifically in lymph node and spleen. further histopathological examinations of lung tissues showed that, moderate and mild interstitial pneumonia was developed in control monkeys, while no pathological evidence found in immunized monkeys. the dose-response relationship is an important criterion in all studies of efficacy of vaccines. the results of our neutralization tests, rt-pcr, and autopsies showed that higher level of neutralizing antibodies induced by higher dose vaccination could decrease the sars-cov viral load and pneumonic changes compared to control monkeys after challenge. more animal studies are needed to quantify the relationship between vaccination doses and protective efficacy accurately and to establish the thresholds for safety. some rna viruses are known to induce antibody dependent enhancement such as the exacerbation of diseases in immunized populations exposed to dengue virus infections. ade is therefore a significant obstacle in inactivated vaccine development. our study of safety and efficacy focused on the occurrence of ade in sars-cov. all immunized monkeys were challenged after their neutralizing antibodies decreased to low levels, mimicking the situation in immunized populations. the preliminary results showed that no obvious ade phenomena was detected in immunized monkeys after being challenged with sars-cov gz- strain. also, the levels of neutralizing antibodies in all immunized monkeys increased sharply after sars onset, indicating that immune memories were established in the vaccinated monkeys. the monkey is the most widely studied animal model in sars research. although our studies of purified inactivated sars vaccine were based on the monkey animal model, we are unable to conclude that the monkey is the ideal animal for sars vaccine evaluation. in our studies, some monkeys appeared lethargic for several days after inoculation. some developed diffuse alveolar damage similar to that in sars patients but without typical clinical symptoms. other animal models may elicit different reactions to vaccination. in this study, we used neutralizing antibody levels and interstitial pneumonia as two main indicators of the immunogenicity and protective efficacy of purified inactivated sars vaccine. the results indicated that the purified inactivated sars vaccine we developed could induce high levels of neutralizing antibody, protect monkeys after a sars-cov challenge, and be administered safely in monkeys. taken together, these findings provide a solid foundation for further clinical research of the inactivated sars vaccine. continued research on severe immunopathological reactions should be undertaken during future preclinical research and clinical trials. coronavirus never before seen in humans is the cause of sars isolation and identification of a novel coronavirus from patients with sars a complete sequence and comparative analysis of a sars-associated virus (isolate bj ) cleavage of structural proteins during the assembly of the head of bacteriophage t establishment of an analyzing method for a japanese encephalitis virus neutralization test in vero cells development of plaque method for sars virus bj strain determination of the partial polymerase gene sequence of novel coronavirus isolated from lung tissue of sars patients production of purified japanese encephalitis vaccine from vero cells with roller bottles development of vero cell-derived inactivated japanese encephalitis vaccine immunogenicity of sars inactivated vaccine in balb/c mice humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from f strain effects of a sars-associated coronavirus vaccine in monkeys severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments dna vaccine of sars-cov s gene induces antibody response in mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immune responses with dna vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in balb/c mice immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine conflict of interest statement: none declared. key: cord- - sxlkjp authors: ramasamy, r.; yasawardena, s.; zomer, a.; venema, g.; kok, j.; leenhouts, k. title: immunogenicity of a malaria parasite antigen displayed by lactococcus lactis in oral immunisations date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: sxlkjp a putative protective protein from plasmodium falciparum merozoites, msa , was expressed in two different ways on the cell surface of the gram-positive food-grade bacterium, lactococcus lactis. the first display format exploits an lpxtg-type anchoring motif of the lactococcal proteinase prtp to covalently anchor msa to the genetically modified producer cells. in a second display format, msa was fused to the peptidoglycan-binding domain (protein anchor) of the lactococcal cell wall hydrolase acma and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder l. lactis cells, termed gram-positive enhancer matrix (gem) particles. the l. lactis recombinants carrying covalently bound msa were used to immunise rabbits through nasal and oral routes. the highest levels of igg antibodies reacting with near-native msa on merozoites was elicited by oral administration. intestinal antibodies to msa were produced only after oral immunisation. msa -specific t(h)-cell activation could be demonstrated. based on these results, the immunogenicity in oral immunisations of msa , bound non-covalently to non-genetically modified l. lactis gem particles, was compared with msa that was bound covalently to genetically modified l. lactis. these two forms elicited similar titres of serum antibodies. the results illustrate the potential of using non-genetically modified l. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant dna into the environment. nasal and oral vaccination is preferable to injections from the point of view of safety, ease of administration and compliance. mucosal vaccines are an alternative to injectable vaccines, because it is e.g. increasingly more difficult to incorporate new injectable vaccines into the existing paediatric vaccination programmes. in addition, mucosally delivered vaccines activate the mucosal immune system to elicit protective secretory iga antibodies and cellular immunity. this is relevant for protection against viruses, bacteria and parasites that enter the body through mucosal surfaces and cause diseases of the intestinal, respiratory and genital tracts. oral administration of soluble protein antigens generally leads to systemic tolerance [ ] but ingested particulate antigens and microbes in some instances generate mucosal and systemic immune responses [ ] through stimulation of the gut-associated lymphoreticular tissue (galt). both soluble and particle-associated proteins given intranasally can, through uptake by the nasopharynx-associated lymphoretic-ular tissue (nalt), elicit local and distal mucosal as well as systemic immune responses [ ] . therefore, oral or nasal immunisation may also be useful against pathogens that enter the body through routes other than mucosa. attenuated strains of bacterial pathogens such as salmonella typhi, mycobacterium bovis and bordetella pertussis are being developed as vectors for mucosal immunisation, but suffer from the disadvantage that they tend to disseminate in the body [ ] and can be unsafe in immuno-compromised persons. lactococcus lactis is ubiquitous in the human environment and in food. it is used for making cheese and buttermilk, and is generally recognized as safe (gras). l. lactis survives passage through the gastrointestinal tract but does not colonise the gut [ ] . this lactic acid bacterium may therefore provide a safer alternative to attenuated pathogens for mucosal immunisation purposes [ ] . several bacterial or viral antigens have been expressed in l. lactis for use in oral and intranasal immunisations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the advantage in relation to the potency of the elicited immune response of anchoring the antigens to the cell wall of the producing l. lactis cells as opposed to intracellular expression has been demonstrated by others [ , , [ ] [ ] [ ] . therefore, we focused in the present study on the cell wall presentation of the antigen. in all previously described studies on the use of l. lactis in mucosal immunisation strategies, genetically modified vaccine strains were employed. although l. lactis is an innocuous bacterium, its widespread use as a genetically modified strain in vaccinations, especially mucosal vaccinations, may cause undesirable shedding of recombinant dna into the environment with the attendant risk of transfer to other organisms. in order to eliminate this risk we developed a non-genetically modified l. lactis support that allows highly efficient binding of proteins that are fused to a lactococcal peptidoglycan binding domain, the protein anchor (pa). for this purpose, l. lactis cells are pre-treated with an acid that removes surface components [ ] , leaving non-living particles that we termed gram-positive enhancer matrix (gem) [ , ] . the pre-treated and neutralized gem particles are used to bind the antigen-pa fusion that was produced by another source (in trans binding). in this way, the antigen can be presented to the immune system as a bacterial particle that does not contain the recombinant dna encoding the antigen. we report here on the immunogenicity after oral delivery in rabbits of a plasmodium falciparum parasite antigen presented in two different ways on the surface of l. lactis; (i) covalently anchored to the peptidoglycan layer of the cell wall of live genetically modified l. lactis, using an anchoring mechanism based on the lpxtg motif from, in this case, the lactococcal proteinase prtp (cp) [ ] and (ii) non-covalently rebound to non-genetically modified l. lactis gem particles using the pa domain [ ] [ ] [ ] . the p. falciparum protein used was the kda merozoite surface antigen msa [ , ] , antibodies against which have been associated with protec-tion against the clinical symptoms of malaria [ ] and, in some instances, inhibition of merozoite invasion of red blood cells in vitro [ ] . l. lactis-nz and nz were grown in m medium (difco, md, usa) containing % glucose at • c in standing cultures. msa -recombinant l. lactis were grown in the same medium with g ml − of chloramphenicol for selection. msa -recombinant l. lactis were induced for expression of msa variants by adding the culture supernatant of l. lactis-nz , containing secreted nisin a, at a : dilution and culturing overnight [ ] . overnight cultures of a strain of l. lactis-nz lacking the cell wall hydrolase acma (l. lactis-∆acma) [ ] were used to prepare gem particles as described before [ ] . the -nucleotide coding sequence of msa [ ] from the d isolate of p. falciparum cloned in pbluescript iisk+ [ ] , was used as source of msa . the relevant msa sequence was pcr amplified from this plasmid and then cloned into a pnz -based lactococcal vector for the expression of msa under the control of the nisin a inducible promoter p nisa [ ] . plasmid png (fig. ) expressed a -aminoacid (aa) fragment of msa (beginning with the sequence knes and ending with aats, genbank accession number a ) lacking the msa signal -and membraneanchoring sequences. instead it had the signal -and prosequence [nucleotides - in ref. [ ] ] of the lactococcal cell wall-associated proteinase prtp at the n-terminus for efficient secretion, together with a c-terminal anchor sequence (pa) derived from the l. lactis cell-wall hydrolase acma [corresponding to nucleotides - in ref. [ ] ]. the corresponding msa fusion protein msa -pa is termed msa -ncov for reasons of clarity. msa -ncov is attached non-covalently to the cell wall of the producer cells through the protein anchor [ ] , and is also secreted into the culture medium after which it can be rebound noncovalently to lactococcal gem particles [ ] [ ] [ ] . plasmid png ( fig. ) expressed the same -aa fragment of msa as in png , with the signal and pro-sequence of prtp at the n-terminus, but with a cell-wall spanning and covalent anchoring sequence of prtp, cp, at its cterminus [corresponding to nucleotides - in ref. [ ] ]. this msa fusion protein msa -cp is termed msa -cov. the plasmids pnz (negative control), png and png were used to transform l. lactis-nz , which has the nisrk genes in the chromosome needed for the nisin-induced activation of p nisa [ ] , by electroporation [ ] . coomassie blue staining after sds-page was performed to quantitate the expression of msa variants. exponentially growing l. lactis cells were induced for h with a supernatant containing nisin a and the cells were pelleted by centrifugation, washed once in distilled water and resuspended in buffer ( % sucrose, mm tris-hcl ph . , mm edta and mm nacl) containing g ml − lysosyme and units ml − mutanolysin (sigma, usa) and heated to • c for min. then volume of × concentrated laemmli sample buffer was added and the sample was heated to • c for min. msa -ncov, non-covalently bound to l. lactis gem particles, was extracted from gems with sample buffer at • c. ten microlitres of aliquots ( × cells) were analysed by sds-page ( % nupage gels with mops buffer; invitrogen, usa) and stained with coomassie r (g) stain. the intensity of the msa bands was compared with bsa standards analysed similarly by sds-page. ten microlitres of aliquots of l. lactis extracts, prepared and separated by sds-page as described above, were transferred to hybond blotting membranes and probed with a : , dilution of a rabbit antiserum to msa raised against an msa -gst fusion protein [ ] . the blots were developed with . g ml − alkaline-phosphataselabelled goat anti-rabbit igg (h + l) antibodies followed by nbt/bcip as a substrate. for ifa, approximately cells of png (msa -cov), induced to express msa , or l. lactis gem particles with bound msa -ncov, were washed in pbs ( . m phosphate buffered saline, ph . ), and incubated with a : dilution of the rabbit anti-msa serum in pbs with % bsa for h at • c. after washing with pbs, the samples were incubated with g ml − oregon green-conjugated goat anti-rabbit igg antibodies (molecular probes, usa) diluted in pbs with % bsa for h at • c, and then viewed and photographed using a zeiss microscope with incident uv illumination and the zeiss axiovision digital imaging system (zeiss, germany). immunogold electron microscopy was performed on whole mount preparations of glutaraldehyde-fixed msa surface-displaying l. lactis cells and gem particles on formovar carbon-coated nickel grids. these were reacted sequentially with rabbit anti-msa and auroprobe nm gold-labelled goat anti-rabbit igg (amersham, uk). samples were then stained with . % uranyl acetate and examined using a philips cm transmission electron microscope at kv (philips, the netherlands). stocks of l. lactis and l. lactis-png for immunisation were prepared from overnight cultures (the latter strain was induced overnight with nisin a for msa -cov expression) and stored in aliquots of colony forming units (cfu) per ml growth medium containing % glycerol at − • c. the cells remain viable under these conditions and l. lactis-png retains msa -cov on its surface, as demonstrated by immunofluorescence assay (ifa). cultures of l. lactis-png cells, induced for h with nisin a to express msa -ncov, were centrifuged and the supernatant was collected and used as a source of secreted msa -ncov. l. lactis gem particles were incubated with the cell-free supernatant containing msa -ncov at a concentration of × gem particles per ml at • c for min with rotary shaking at rpm. the gem particles were then collected by centrifugation, washed twice in pbs and stored in pbs at − • c until further use at a concentration of particles per ml. l. lactis gem particles retain msa -ncov on the surface under these conditions, as demonstrated by ifa. new zealand white rabbits obtained from the medical research institute, colombo, sri lanka were used for table serum antibody responses to immunisation through different routes with recombinant l. lactis-png cells, which carry covalently bound msa -cov immunogen total serum antibody titres, determined by ifa at serial -fold dilutions, beginning at : , after , , , and immunisations ( , st, nd, rd and th, respectively) are expressed as the negative logarithms to the base ten. absence of a detectable ifa reaction at : dilution of serum is indicated as −ve. the numbers in parentheses in column rd are the corresponding titres of igg antibodies determined by ifa with a specific anti-rabbit ␥-chain antibody. -ii is the serum collected weeks after the third immunisation. if antibody was detected by ifa in the undiluted faecal extract this is reported as +ve. nd: not done; i.m.: intramuscular. experimental immunisations in which the nasal and oral routes of administration were explored with the l. lactis-png (msa -cov) vaccine. the care and use of animals were according to who guidelines (who/lab/ . ). the rabbits were ear-bled days prior to immunisation to obtain preimmune sera. details of routes of immunisation and immunogens are given in table . each group consisted of two rabbits. as a positive control, l. lactis-png (msa -cov) was given intramuscularly (i.m.) to one rabbit. prior to immunisations, stocks of l. lactis (no msa expression, negative control) and l. lactis-png (msa -cov) were thawed, washed and resuspended in buffer at the appropriate concentration for immunisations. intramuscular injections were performed with a total of × cfu in l pbs into both rear hind legs. nasal immunisation involved delivering a total of cfu in l pbs into the two nostrils with a pipette. prior to oral immunisation, the rabbits were deprived of water and food for h. they were then each fed × cfu resuspended in ml of . n nahco , . % glucose and % casein hydrolysate. this dose was repeated for three successive days to obtain reproducible oral immunisation. all immunisations were repeated twice at weeks intervals. blood was collected weeks after each immunisation to prepare sera termed bleeds st, nd and rd, respectively. after three immunisations the rabbits were rested for weeks. serum was then collected (termed bleed -ii) to test the decrease in antibody titres. the rabbits were given a fourth and final immunisation weeks after the third immunisation, and serum collected weeks later (termed th), to confirm boosting of immunity. the fourth oral immunisation, however, consisted of two, and not three, successive days of feeding × cfu. collected sera were stored at − • c until use. new zealand white rabbits obtained from harlan laboratories, the netherlands, were used for immunisations in which the l. lactis gem particles carrying msa -ncov and the l. lactis-png (msa -cov) vaccines were compared. the care and use of animals were according to institutional guide-lines. the rabbits were ear-bled prior to immunisation to obtain preimmune sera. details of the immunisation and immunogens are given in table . each group consisted of two rabbits. prior to immunisations, stocks of l. lactis-png (msa -cov) and l. lactis gem particles with bound msa -ncov were thawed, washed and resuspended in % sucrose for oral immunisations. prior to oral immunisation, the rabbits were deprived of water and food for - h. they were then each fed using a plastic disposable pipette tip with ml containing × cfu l. lactis-png (msa -cov) or × gem particles with bound msa -ncov. each dose was repeated for three successive days to obtain reproducible oral immunisation. altogether, three oral immunisations were given at weeks intervals. rabbits were ear-bled weeks after each immunisation to obtain sera for antibody assays. the sera were stored at − • c until use. all vaccines were administered without additional adjuvants. adverse effects consequent to the immunisations were not observed indicating that live recombinant l. lactis as well as non-living l. lactis gem particles were well tolerated by the animals. titres determined by ifa at serial -fold dilutions, beginning at : , are expressed as the negative logarithms to the base ten. no detectable reaction at a : dilution of the serum is shown as −ve; , nd and rd refer to preimmune serum and serum obtained after and immunisations, respectively; rd igg is the igg antibody titre in the serum after three immunisations, as determined with a rabbit ␥ chain-specific secondary antibody. the detection of typical bunch of grape-like patterns of fluorescence on segmented schizonts of d p. falciparum is based on antibody recognition of near-native msa on the surface of the malaria parasite [ ] . this method was used to determine the msa antibody titre in the sera of immunised rabbits. ifa was performed on acetone-methanol fixed late-stage d parasites as previously described [ ] . tenfold serial dilutions of the antisera in % bsa were used in ifa to determine the titre. the greatest dilution of serum giving a discernible bunch of grapes pattern on late schizonts was taken as the titre. for detection of all antibody isotypes, fitc-conjugated sheep anti-rabbit ig (silenius, australia) or oregon green-conjugated goat anti-rabbit ig with h + l specificity (molecular probes, usa), was used as secondary antibody. for detection of igg antibodies only, a fitcconjugated mouse monoclonal antibody rg- , with specificity against rabbit ␥ chains (sigma, usa), or a fluoresceinconjugated, affinity-purified, rabbit antibody with specificity against rabbit ␥ chains (rockland, usa), was used instead. a total cell extract of l. lactis nz was used as target antigen for immunoblotting with the rabbit sera to determine the anti-carrier response. fresh faecal pellets were collected from the rabbits weeks after the third immunisation and stored at − • c until required. one millilitre of pbs containing % bsa and mm phenylmethyl suphonyl fluoride was then added per mg of faeces and incubated overnight at • c. the sample was vortexed to disrupt all solid material and centrifuged at , × g for min. the supernatant containing extracted antibody was stored at − • c until testing by ifa. fitc-conjugated sheep anti-rabbit ig (silenius, australia) was used as the secondary antibody. the expression formats of msa are schematically drawn in fig. . expression of the msa fusion proteins was analysed in coomassie-stained gels and in western blots. an example of such gels and blots is given in fig. . msa -cov was present as a -kilo dalton (kda) protein in the total cell extract of l. lactis-png (fig. , lane ) . msa -ncov was secreted by l. lactis-png as a kda protein (fig. , lane ) . the secreted msa -ncov was bound to l. lactis gem particles (fig. , lane ) . by comparative protein gel electrophoresis experiments using bsa protein standards we estimated that l. lactis produced approximately ng msa -cov per × cells. the same number of gem particles contained about ng of msa -ncov (data not shown). this corresponds to approximately . × msa -cov molecules per cell for l. lactis-png and about msa -ncov molecules per gem particle. the surface localization of msa -cov on l. lactis-png cells and of msa -ncov on gem particles was confirmed by immunofluorescence with rabbit anti-msa serum (fig. ) and by immuno-electron microscopy (fig. ) . the microscopic observations suggest that surface-exposed msa -cov is mainly found in the region of the newly forming septum in dividing cells and at the poles of the l. lactis-png cells. msa -ncov was more evenly distributed on l. lactis gem particles (figs. and ) . in any case, similar levels of msa seem to be surface displayed by these two presentation formats. the most efficient mucosal route for the delivery of msa was first determined using the live recombinant l. lactis antigen surface display system, i.e. l. lactis-png (msa -cov). new zealand white rabbits were used to examine the oral and nasal routes of immunization. the immunisations, including two boosters, with recombinant l. lactis-png (msa -cov) delivered, per rabbit, a total of and g of msa -cov protein after nasal and oral immunisations, respectively. one rabbit obtained three intramuscular (i.m.) immunisations with l. lactis(msa -cov) as a positive control (totally g msa -cov). immunofluorescence reactivity against the surface of p. falciparum merozoites was used to assay antibodies in sera and faecal extracts, as this measures antibodies reacting with near-native msa protein (see section ). the ifa results of the immunised rabbits are summarised in table . a single immunisation with l. lactis(msa -cov) administered i.m., nasally or orally did not yield anti-msa serum antibodies detectable by ifa. a further boosting immunisation was needed in all cases to detect antibodies. it is apparent that the positive control, i.m. immunisation with l. lactis(msa -cov), led to good serum antibody titres. the titres waned weeks after the third injection but could be boosted to the original levels by a fourth i.m. injection. levels of igg antibodies after three immunisations, as measured with an fitc-conjugated mouse monoclonal antibody against rabbit ␥ chains, were one order of magnitude less but still indicate that igg antibodies constitute the major portion of the antibody response against intranasal immunisation with l. lactis(msa -cov) elicited low titres ( − ) of antibodies after three immunisations. the oral immunisation procedure led to higher antibody titres ( − to − ) than did the intranasal immunisation. antibody levels decreased significantly by weeks but could be boosted to original levels by a subsequent ( th) oral immunisation. the results with the anti-rabbit ␥-chain antibody and the boosting suggest that much of the antibody present in the serum after oral immunisation is igg. msa -specific antibodies were detectable in undiluted extracts of faecal pellets after oral immunisation, but not after i.m. immunisation with l. lactis(msa -cov). based on the results obtained for msa -cov, we concluded that the preferred route for mucosal delivery in rabbits of the msa antigen by live recombinant l. lactis is oral administration. we next determined the capability of non-living non-genetically modified lactococcal gem particles carrying non-covalently bound msa -ncov to elicit systemic msa -specific antibodies after oral administration. a comparison was made with l. lactis(msa -cov). l. lactis(msa -cov) delivered a total of g msa -cov while gem particles delivered g of msa -ncov after three immunisations. the rabbit anti-msa serum antibody responses to the immunisations were characterised by ifa and are summarised in table . the results show that specific antibodies against near-native msa were detectable after two immunisations (titres of − to − ), and that anti-body titres increased after a third immunisation ( − ), in all instances. the antibodies were predominantly of the igg subclass in all cases. the immune response to the carrier may be of influence on the safety of the delivery system in humans. also the delivery route may be of influence on the anti-carrier response. as a first exploration of this issue we examined the anticarrier antibody response in more detail in immunoblots in which a total lysate of l. lactis cells was used as a target for serum from rabbits immunised with the different msa presentation formats (fig. ) . the staining bands in the lanes indicate that l. lactis proteins present at that position in the sds-paa gel reacted with the indicated rabbit antiserum. a pronounced anti-l. lactis protein response was observed after or i.m. administrations of the live recombinant l. lactis vaccine (fig. a ). this response was clearly reduced after oral delivery of the vaccine (fig. b) . the anti-l. lactis response was even more minimized in the rabbit orally immunised with the gem particle vaccine (fig. c ). it is clear from these results that mucosal delivery reduces the anti-carrier response and that the l. lactis gem particles generate less anti-carrier antibody responses than the live recombinant l. lactis cells. we investigated the immunogenicity of the putative protective msa antigen of p. falciparum merozoites in mucosal vaccines. it was also our aim to evaluate the feasibility of a novel bacterial antigen display system that avoids the use of recombinant dna in the vaccine. for this purpose, we compared msa vaccines based on a live recombinant l. lactis surface display system that exploits the commonly used covalent lpxtg-type anchoring cassette [ , , [ ] [ ] [ ] , ] , with a non-living non-genetically modified l. lactis surface display system, the gem particles. the gem particles can be loaded with the antigen when the latter is fused to the noncovalent cell wall binding domain (pa) of the lactococcal protein acma. others have already demonstrated that nonliving recombinant lactococci are as efficient as live l. lactis cells in eliciting antibody responses in mucosal immunizations [ ] . the acma cell wall binding domain pa has been used to display antigens on live recombinant [ , ] or nonrecombinant live l. lactis cells [ ] . here we used acid pretreated l. lactis cells, which we termed gem particles [ ] , for antigen delivery because this pre-treatment enhances the binding capacity for pa fusion proteins [ ] [ ] [ ] . moreover, the use of gem particles in vaccines prevents the dissemination of recombinant dna in the environment. the results of the oral immunisations confirm that non-living l. lactis gem particles are equally efficient in eliciting antigen-specific antibody responses as living l. lactis cells. consequently, the present results show that l. lactis gem particles, which consist of peptidoglycan largely devoid of lipotechoic acid and lacking dna [ ] , known immunostimulants [ , ] , can also efficiently stimulate antigen presenting cells without the use of additional adjuvants. in addition, gem particles elicit less anti-carrier response than live l. lactis cells. not requiring additional adjuvant and a minimal anti-carrier response would be desirable characteristics for any vaccine to be used in human vaccinations. further investigations are warranted to reveal the mechanism of immune stimulation. our results with the recombinant l. lactis delivery system suggest that oral immunisation leads to higher levels of serum antibodies than the corresponding nasal immunisation procedure employed. a similar observation was made by pei et al. [ ] in immunisations with l. lactis expressing the sarscoronavirus nucleocapsid protein. also other investigators observed significant systemic immune responses after oral immunisations with recombinant lactococci [ ] [ ] [ ] [ ] and in some cases protection against a lethal challenge was obtained [ , , ] . protection against infection with p. falciparum can not be evaluated in a challenge model using non-primate animals. the anti-msa serum antibodies generated in the present study in the oral immunisations with both delivery formats, react with near-native antigen on merozoites, as evidenced by the ifa studies on intact merozoites. reaction of the antibodies with merozoite surfaces is related to their biological efficacy, i.e. the inhibition of red cell invasion. in addition, the demonstration of systemic igg antibodies whose levels can be boosted implies that msa -specific t h cells are activated through mucosal immunisation. the detection of msa -specific iga antibodies in the extracts from faecal pellets of rabbits immunised orally is consistent with the observation of others [ , , , , ] . therefore, the find-ings suggest that foreign proteins expressed in l. lactis can also be used in oral vaccination procedures to elicit protective secretory antibodies in the gut. antibody titres achieved by using both l. lactis display formats of msa are better than those achieved by immunisation with plasmid dna [ ] or peptide-diphtheria toxoid conjugates [ ] through the intramuscular and intradermal routes. intramuscular immunisation with synthetic peptide polymers based on msa b-cell epitopes elicited high-titre antibodies against the immunising peptide, which reacted only poorly with near-native msa on merozoites [ ] . while very high titres ( − ) of antibodies reacting with near-native msa is achievable in animals with recombinant msa in intramuscular immunisations, this required the use of freund's adjuvant, which is not acceptable for human use [ ] . adjuvants with fewer side effects that have been tried on humans with recombinant msa , have yielded poorer antibody titres [ ] . therefore oral immunisation with l. lactis msa display formats is a promising method of generating systemic anti-msa antibodies in man for protection against malaria. the use of heterologous proteins anchored through the pa to non-genetically modified l. lactis gem particles is as effective as the use of living recombinant l. lactis cells for eliciting systemic igg antibodies against the protein of interest. the apparent advantages of the gem approach are: (i) it avoids the need for introducing recombinant cells or additional adjuvants into vaccine recipients; (ii) it elicits a weaker immune response against the vector, thereby increasing safety and possibly permitting a greater number of immunisations than feasible with intact l. lactis; (iii) the process more easily permits immunisation with different combinations of antigens on the same particle [ ] . experiments are presently in progress to determine whether proteins absorbed on the gem particles are able to induce antigen-specific systemic tc cells, which could further widen the use of the gem particles in vaccine preparations. oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens differences between bacterial and food antigens in mucosal immunogenicity nasal vaccines genetic marking of lactococcus lactis shows its survival in the human gastrointestinal tract the immune response to lactococcus lactis: implications for its use as a vaccine delivery vehicle protection against tetanus toxin in mice nasally immunised with recombinant lactococcus lactis expressing tetanus toxin fragment c oral vaccination of mice against tetanus with recombinant lactococcus lactis expression of helicobacter pylori urease subunit b gene in lactococcus lactis mg and its use as a vaccine delivery system against h. pylori infection in mice ability of lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines immunogenicity and protective efficacy of orally administered lactococcus lactis expressing surface-bound hiv env induction of partial protection in mice after oral administration of lactococcus lactis producing brucella abortus l /l antigen an inducible surface presentation system improves cellular immunity against human papillomavirus type e antigen in mice after nasal administration with recombinant lactococci expression of sars-coronavirus nucleocapsid protein in escherichia coli and lactococcus lactis for serodiagnosis and mucosal vaccination factors affecting the immunogenicity of tetanus toxin fragment c expressed in lactococcus lactis cell wall attachment of a widely distributed binding domain is hindered by cell wall constituents a novel surface display system for proteins on nongenetically modified gram-positive bacteria mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria nucleotide sequence of the cell wall associated proteinase gene of streptococcus cremoris wg studies on glycoproteins in the human malaria parasite plasmodium falciparum. identification of a myristilated kda merozoite membrane glycoprotein identification of two integral membrane proteins of plasmodium falciparum assessment of the role of the humoral immune response to plasmodium falciparum msp compared to resa and spf in protecting papua new guinean children from clinical malaria antibodies and plasmodium falciparum merozoites quorum sensing-controlled gene expression in lactic acid bacteria molecular cloning and the nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of lactococcus lactis, a muramidase needed for cell separation antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites transformation of lactococcus by electroporation anchoring proteins to lactic acid bacteria receptor binding domain of escherichia coli f fimbrial adhesion fedf can be both efficiently secreted and surface displayed in a functional form in lactococcus lactis cell surface display system for lactococcus lactis: a novel development for oral vaccine lipoteichoic acids: a new class of bacterial antigen innate immune recognition induction of mucosal immune response after intranasal or oral inoculation of mice with lactococcus lactis producing bovine beta-lactoglobulin measurement of specific iga in faecal extracts and intestinal lavage fluids for monitoring mucosal immune responses mammalian cell expression of malaria merozoite surface proteins and experimental dna and rna immunisation antibody and clinical responses in volunteers to immunisation with malaria peptide-diphtheria toxoid conjugates model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies safety, immunogenicity and pilot efficacy of plasmodium falciparum sporozoite and asexual blood-stage combination vaccine in swiss adults we gratefully acknowledge the contributions of the late prof. manthri s. ramasamy to this work and thank s. jayaweera, f. grijpstra and j. bouwer for excellent technical assistance. the authors also would like to thank klaas sjollema for excellent electron microscopy work. key: cord- -x ux authors: roth, bernhard; mohr, hannah; enders, martin; garten, wolfgang; gregersen, jens-peter title: isolation of influenza viruses in mdck pf cells and clearance of contaminating respiratory viruses date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: x ux abstract this paper summarizes results obtained by multiplex pcr screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in mdck pf cells. using the resplexii v . (qiagen) multiplex pcr, positive results were obtained in clinical samples collected during an influenza season in germany. the overall distribution of positive results was influenza a . %, influenza b . %, adenovirus . %, bocavirus . %, coronavirus . %, enterovirus . %, metapneumovirus . %, parainfluenza virus . %, rhinovirus . %, and respiratory syncytial virus (rsv) . %. double infections of influenza virus together with another virus were found for adenovirus b and e, bocavirus, coronavirus, enterovirus and for rhinovirus. these other viruses were rapidly lost upon passages in mdck pf cells and under conditions as applied to influenza virus passaging. clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in mdck pf cells. using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼ ), the positive results for the different viruses turned negative already after or passages in mdck pf cells. these results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in mdck cells. in combination with their superior isolation efficiency, mdck cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses. influenza virus isolation for monitoring epidemic influenza activity and for the selection of candidate vaccine strains has traditionally been conducted by cultivation in embryonated hen's eggs. due to receptor limitations, such egg passaging can cause adaptive mutations of the haemagglutinin [ , ] . these egg-adaptive mutations do not revert on subsequent passage in mammalian cells, and they may alter the antigenic properties of the receptor binding site, which is also a critical binding site for virus inhibiting and protective antibodies [ , ] . in contrast to egg-passaged virus, mammalian cell-grown influenza virus preserves the sequence of the original human clinical sample. during the last decade the worldwide national influenza centres have almost completely changed influenza virus isolation from egg culture to cell culture, mainly using mdck cells. this change to cell culture was stimulated not only by the relative ease of conducting multiple isolations in cell cultures but also by the better antigenic match of mdck-isolated viruses with field strains. increasing difficulties in recovering isolates from embryonated eggs, particularly of h n subtypes, has also contributed to the change to cell culture [ ] . several companies are currently developing cell culture-based influenza vaccines [ ] and the first of those vaccines, produced in mdck and vero cells, have been licensed and distributed as interpandemic trivalent and pandemic h n vaccines. using the conventional, recommended reference viruses, these vaccines still originate from egg-derived virus isolates or the corresponding high-growth reassortants. regulatory concerns, mainly with regard to the introduction of adventitious agents, are raised if candidate vaccine strains are derived directly from uncharacterised and uncontrolled cell lines. collaborative studies have been initiated to investigate the growth and yield of influenza viruses in different cell lines, the efficiency and fidelity of influenza virus isolation, and the suitability for vaccine manufacture of different cell substrates [ ] . growth studies with a wide range of potentially contaminating viruses have been conducted and risk assessments have been made, comparing egg-derived and cell-passaged influenza viruses with regard to the risk of carrying adventitious viruses into vaccine manufacturing processes [ , ] . these assessments indicated that, in comparison to manufacturing in embryonated eggs, the introduction of vero cells increases the risk of transmitting various viruses into the vaccine process, whereas the use of mdck cells reduces the overall risk. due to their limited permissiveness to viral growth, mdck cell exert the same filter effect for human adventitious viruses as do embryonated eggs. here, we assess on the presence of co-isolated viruses in influenza virus isolates recovered from mdck cells. this article provides more specific data about the kind and frequency of coinfecting respiratory viruses in human influenza virus-containing samples and about the fate of such co-infecting viruses during passage in mdck cells. nasal or pharyngeal samples from the / influenza season were provided by a clinical diagnostic laboratory located in stuttgart, germany. these samples from patients with acute respiratory tract infections were obtained by physicians mainly from southern germany and were sent to the diagnostic laboratory in liquid virus transport medium. aliquots of the clinical specimens (with a laboratory number as an anonymous identifier) were sent to novartis vaccines in marburg, germany, by a weekly courier service. during transportation the samples were stored at - • c. directly after receipt of the samples, mdck pf cells were inoculated (details see further below) with sample material. the cultures were harvested after days of incubation, and the cell-free supernatants were aliquoted and stored at ≤− • c until further use. mdck pf suspension cells from novartis working cell bank were cultivated in ml disposable spinner flasks (corning) in cdm medium, a chemically defined growth medium used for cell propagation (mdck cdm, lonza) and passaged at - -day intervals. during those - days the cells grew from an initial seeding density of × cells/ml to densities between . and . × cells/ml. for infections . ml cells were seeded in ml filter tubes (tpp, transadingen, switzerland) at a cell density of . - . × cells/ml. cells in cdm medium were diluted at a / % ratio into mdck pfm medium ("protein-free medium", gibco invitrogen) supplemented with . % of a penicillin/streptomycin solution (sigma) and iu/ml trypsin. to obtain a total culture volume of ml, the added viral inoculum was diluted in . ml infection medium and was pre-diluted by several log steps, starting with a total dilution of at least : . inoculated cultures were then incubated at • c for days in a % co atmosphere in a isf- -w shaker incubator (kuhner, birsfelden, switzerland). for virus harvests the cells were separated by centrifugation ( - × g for min) and the supernatant was recovered. unless used freshly, e.g. for haemagglutination tests and subsequent passaging, aliquots of the supernatant were frozen at ≤− • c. haemagglutination (ha) testing was done with harvested material to define the starting material for the next passage. ha testing was performed in u-bottom microwell plates (greiner bio-one) using l of a serial log dilution in pbs (ph . ) of the test samples and l chicken or guinea pig red blood cells ( . % in pbs ph . ). results were read after min (chicken erythrocytes) or min (guinea pig erythrocytes) incubation at ambient temperature within a temperature range of - • c. two different kinds of red blood cells were used since the actual h n influenza strains did not react with chicken red blood cells. material from the highest log inoculum dilution, which showed a clearly positive ha reaction after the previous passage, was used for the following passage. extraction of viral dna or rna from clinical specimens and culture supernatants was performed with the nucleic acid isolation kit i in the magna pure compact extraction system (roche) or with the qiasymphony ® virus/bacteria midi kit (qiagen) in the qiasymphony robotic system. the resplex ii v . multiplex pcr panel (qiagen) was used according to the manufacturer's instructions. the test applies a rt-pcr (reverse transcription and pcr reaction) by the onestep rt pcr kit (qiagen) in combination with two pairs of specific primers for each target. the enzyme mix contains the omniscript tm and sensiscript tm reverse transcriptase and the hotstartaq tm dna polymerase. the dntp mix contained mm of each dntp. the primer mix consisted of a mixture of individual primers for each viral target, carrying a tail with the target sequence for the superprimers, and the forward and backwards superprimers. results of the multiplex pcrs were read with the liquichip detection system, which consists of microspheres coated with target-specific hybridization molecules and a steptavidin-biotin based fluorescence detection reaction giving an individual fluorescence color pattern for each viral target. result readings were evaluated with the qiaplex mdd-rvo beta software. according to the manufacturer's instructions signals above values of are positive, values below are negative and values between and are considered as questionable results. the method's results are given as counts (median fluorescence intensity, mfi) but the method is not intended or designed to be used quantitatively. the resplex ii v . method is designed to detect different virus species or virus subgroups simultaneously. these pathogens and the target genes used are summarized in table . independent, conventional in-house qrt-pcrs or commercially available pcr methods were used to confirm resplex results with clinical specimens. these methods and according references are summarized in table . the table . of particular interest were those specimens in which a double infection of an influenza virus together with other viruses was detected. in samples positive (and questionable) results for other viruses were found associated with influenza virus. these associated viruses are listed below along with extra remarks about samples that gave questionable results ( - mfi). • adenovirus b and e - samples. these samples were passaged up to five times in mdck pf cell as described in section . in addition, sample (compare table ) was also used for these passages, as it was questionably positive for bocavirus and contained influenza b. one other sample (sample , positive for coronavirus hku in association with influenza virus b in the clinical specimen) could not be cultivated because there was not sufficient material. as shown in table , the only virus that was detectable after (or ) passages was influenza virus; the other contaminating viruses were lost during passage. the table also lists the total dilution of the original sample until passage ( − to − ) and passage ( − to − ). only one sample (see sample in table ), in which no virus could be recovered was passaged at lower dilutions. the order in which the detected viruses are listed in table reflects the counts found in the resplex method. most co-infecting viruses had lower counts than the influenza virus. sample had highest counts for an enterovirus and similar counts for rhinovirus and influenza virus, sample had higher counts for adenovirus than for influenza virus. however, it should be noted that the resplex method is not a quantitative method. in a similar way, samples with positive and questionable multiplex pcr results only for viruses other than influenza virus were also cultivated for or passages in mdck pf cells. as shown in table , only two passages usually were sufficient to eliminate the virus, so that almost all samples tested negative. only three of the viruses detected in the original sample still gave a very weak resplex signal after the second cell culture passage: one coronavirus with a signal just above the questionable level and an enterovirus and one rsv at the questionable level. considering the total dilution from the original sample to the second passage of only × , it is possible that the original sample contained more than viruses and remained (weakly) positive during passages without any virus growth. when tested after the third culture passage (representing a : dilution of the clinical sample, these three samples tested negative by resplex ii, indicating no virus growth and that the weakly positive test results from the nd passage were obviously due to residual virus from the original clinical sample. table shows the results of confirmatory test of clinical specimens using independent, conventional pcr methods. influenza virus reference seeds are produced by who on an annual basis to match drifting influenza strains [ ] . reference viruses are released to vaccine manufacturers after the who recommendations have been published. these viruses are not subject to any specific testing for adventitious viruses. the corresponding vaccine must be manufactured, tested and distributed within only a few months in order to meet vaccination schedules [ ] [ ] [ ] . because of this short timeline, conventional broad spectrum testing of the influenza virus seed for adventitious agents cannot be performed in time, particularly if one considers that months may be needed to prepare virus from an independent source and specific antibodies against the same to neutralise the influenza virus. for conventional egg-derived viral seeds it is commonly assumed and supported by historical safety records, that many adventitious viruses are removed by egg passages. because cell-derived influenza virus isolates are now being considered for use as starting material for vaccine manufacture, information is needed about the behaviour of adventitious viruses during cultivation of influenza viruses in suitable cell substrates. our studies contribute such information for a cell line that is qualified for influenza vaccine manufacture. the result presented here should be seen in context with specifically designed growth studies with a wide range of potentially contaminating viruses, which, along with the results of a systematic literature search on growth of viruses in mdck cells, have been published previously, [ , ] . in those studies a standard amount of infectious units (tcid ) per ml culture was inoculated into mdck cells and the cells were grown for at least days ( days for slow-growing viruses) in cdm growth medium. high dilution passaging was avoided but samples of suspended cells and medium were taken at regular intervals to be tested for the virus, and an adequate amount of fresh medium was added after sampling to maintain cell growth. ( ) none ( ) nt coronavirus ( ) none ( ), coronavirus ( , borderline) none ( ) resp. syncytial virus ( ) none ( ), rsv ( , questionable) none ( ) adenovirus ( ) none ( ) nt human metapneumovirus ( ) none ( ) nt parainfluenza virus ( ) none ( ) nt bocavirus ( ) none ( ) nt three double infections were counted individually. maximum dilution of the original specimen for the nd passages was × rd passage was done : from nd passage supernatant. nt: not tested. table ) hmpv [ ] / growth of mycoplasma hyorhinis and chlamydia trachomatis were assessed. in those studies high virus growth was observed for parainfluenzavirus , sv and herpes simplex virus, slow growth was seen with mammalian reovirus , and questionable results (very low or no growth) were noted for the two avian reovirus. no growth was observed for the other viruses and agents tested. infectious titers declined and were in most cases no longer detectable after - days of cultivation. only for few very stable viruses, such as sv- , virus titers persited longer. based upon those studies, and supported by the results of a systematic literature search (applicable to standard adherently growing mdck cells), mdck suspension cells support the growth of only a limited range of viruses. in this context the relevant viruses are influenza virus, parainfluenza virus, reovirus, and herpes simplex virus. this permissiveness spectrum is very similar to that seen in embryonated eggs [ ] . therefore, like embryonated eggs used in current influenza vaccine manufacture, mdck cells should act as an effective barrier for a wide range of adventitious agents. moreover, mdck cells do not support the replication of many avian viruses. this is of particular relevance if an avian virus contaminant is introduced into the process by prior passaging of the vaccine virus strain in embryonated eggs. the clinical specimens used for our studies were collected during the peak of an influenza season in february and march in order to gain more information about isolation rates in mdck pf cells in suspension culture. the results of those studies will be published elsewhere. considering the selection of specimens, the high percentage of influenza-positive results is not surprising, but a significant number of samples ( / or . %) also tested positive for other viruses, such as adenovirus, bocavirus, coronavirus, enterovirus, metapneumovirus (hmpv), parainfluenza virus (piv), rhinovirus, and respiratory syncytial virus (rsv). except for rsv and piv, the same viruses were also detected as co-infections together with influenza virus. such co-infections together with influenza viruses have also been published previously for rsv [ ] [ ] [ ] , piv [ ] , hmpv [ , ] , and for adenovirus and bocavirus [ ] , although the overall frequency was comparatively low. those previous reports were all based on pcr detection methods, applying a more restricted virus spectrum than the resplex ii method. we were unable to find reports about co-infections of influenza virus with other viruses identified via cell culture isolation methods, although such double-infected study materials have certainly been used in high numbers. this indicates that cell cultures selectively support replication of specific viruses and that, in addition, the virus identification methods used were less specific or less sensitive than pcr-based methods. for the purpose of our studies the resplex ii ® multiplex pcr method was chosen because it combined detection of a wide range of relevant respiratory viruses with simple application (one-tube assay, rapid results from only one test run), and particularly because it could be applied to the available small volumes. currently, limited information is available about the sensitivity and specificity of the resplex ii v . test kit. we have compared the novel resplex iii assay and existing techniques for the detection and subtyping of influenza virus during the influenza season - [ ] . the methodology must necessarily make some compromises, for example, with regard to amplification conditions during the first cycles with specific primers. thus it is not expected that sensitivity will be the same as that of monoplex pcrs. when compared to an in-house quantitative real-time pcr for influenza virus (detection limit - tcid /ml of a fresh influenza virus harvest), the resplexii v . test appeared to be about log step less sensitive. the majority of positive results obtained with the resplexii v . test could be confirmed by other, independent conventional published, in-house qrt-pcrs or commercially available pcr methods which used other target regions of the viral genomes. this applies to all influenza positive samples, of rsv a and b positive samples tested, of adenovirus positive samples, of bocavirus positive samples (including one questionable resplex result), and of positive coronavirus samples (including questionable resplex results). differences were found for parainfluenza virus samples, for which resplex results could not be confirmed; likewise only of rhinovirus samples and of enterovirus samples tested negative in independent pcrs, but were positive with the resplex method. it remains to be determined whether the observed discrepancies are weaknesses of the resplex system or of the other, independent pcrs. however, the manufacturer of the resplex method confirmed certain cross-reactivities between enteroviruses and rhinoviruses, which have conserved utr regions that were used as targets for the pcr primers. since it is known that reovirus may grow in mdck cells [ ] , we also screened many samples with an in-house reovirus qrt-pcr specific for mammalian orthoreovirus - (conserved region of the l inner capsid gene). samples in which no other virus was detected by the resplex method were preferably used for the reovirus pcr. no reovirus was found in of the specimens for which sufficient material was still available. whereas the specific virus growth studies summarized and discussed further above applied cell-culture adapted virus strains, the studies reported here used unadapted field virus strains and technical conditions as applied for influenza virus isolation and passaging. these studies confirmed that isolating influenza viruses in mdck pf cells effectively reduced co-infecting viruses. after only two passages and a − to − total dilution of the original specimen, adeno-, boca-, corona-, entero-, and rhinoviruses were no longer detectable. only influenza viruses were recovered and remained the only detectable virus upon further passage. high dilutions of the inocula and short incubation periods of only days until harvest or the next passage are conditions that are normally applied to effectively grow influenza viruses. these conditions certainly contributed to the rapid loss of the contaminating viruses. only viruses that are present at very high titers and which grow very rapidly without adaptation would be able to survive such passaging. in a second series of passages we also monitored more than specimens that did not contain an influenza virus but were positive for other respiratory viruses. in these specimens interference by competing influenza virus growth was excluded. the culture conditions differed, as lower inoculum dilutions were used. each sample/harvest was diluted : into the culture, which is the lowest standard dilution applied to recover very low-titred influenza virus. also under these conditions positive results for different viruses became negative after only or passages and after a total dilution of the original specimen by a factor of × − to × − . when similar passages were conducted with adherent vero cells ("vero who seed"), several positive samples (adenovirus, rhinovirus, enterovirus, metapneumovirus, and bocavirus) remained positive after passages. however, except for adenovirus, the counts did not increase but dropped (data not shown). these results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passaging in mdck pf cells. in combination with their superior isolation efficiency [ , ] , mdck cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate candidate vaccine viruses. evidence for host-cell selection of influenza virus antigenic variants clinical influenza virus and the embryonated hen's egg serological studies with influenza a (h n ) viruses cultivated in eggs or in canine kidney cell line (mdck) efficacy of inactivated influenza a virus (h n ) vaccines grown in mammalian cells or embryonated eggs direct sequencing of the ha gene of influenza (h n ) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus continuous cell lines as a production system for influenza vaccines current challenges in implementing cell-derived influenza vaccines: implications for production and regulation a risk-assessment model to rate the occurrence and relevance of adventitious agents in the production of influenza vaccines a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus a and b by real-time pcr molecular epidemiological analysis of community circulating respiratory syncytial virus in rural south africa: comparison of viruses and genotypes responsible for different disease manifestations comparison of classic and molecular approaches for the identification of untypeable enteroviruses polymerase chain reaction and sequencing for typing rhinovirus rna human rhinovirus group c infection in children with lower respiratory tract infection human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis the annual production cycle for influenza vaccine egg-based production of influenza vaccine: years of commercial experience vaccine manufacturing: challenges and solutions vaccines against seasonal and pandemic influenza and the implications of changes in substrates for virus production epidemiological profile and clinical associations of human bocavirus and other human parvovirus multiple viral respiratory pathogens in children with bronchiolitis de beenhouwer h. simultaneous detection of human bocavirus and adenovirus by multiplex real-time pcr in a belgian paediatric population impact of human metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza viruses comparison of the novel resplex iii assay and existing techniques for the detection and subtyping of influenza virus during the influenza season comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses the authors would like to thank knut schwarz, marion wellnitz, veronika horn and inge lettermann for their skillful technical assistance with these studies. we gratefully acknowledge confirmatory pcr test results by independent methods that were partly provided by marcus panning, of the virology department of the university clinic in freiburg, germany. key: cord- - g gp authors: poland, gregory a. title: tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: g gp nan tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development in the aesop fable, ''the hare and the tortoise," the tortoise unexpectedly beats the hare in a race. the moral of the story is that the race is not always to the swift. this same moral also appears in the wisdom literature of the old testament book of ecclesiastes : , which more generally concerns the limitations of human wisdom-which is nearly always disregarded by humans themselves. in late december , the world was notified of an unusual cluster of severe respiratory disease occurring in wuhan, china. very soon thereafter, the causative agent was identified as the now-named sars-cov- virus-a betacoronavirus that had crossed the species barrier to infect humans. in the last few months, this virus has circulated worldwide and caused over million identified cases and , deaths as of this writing, and those numbers are certainly an under-estimate. almost immediately, the call went forth that a vaccine was needed. i agree and so does every serious scientist knowledgeable about the issue. there is no question that a vaccine against this virus, and other as-yet-to-come coronaviruses, is imperative to protect human health and to quickly respond to future viral introductions, epidemics, and pandemics. but, alarmingly, scientists began to speak of the promise of a vaccine being available in ''months"-promises that began to circulate in the media almost as quickly as the virus. vaccine development has a long and documented history. in the us, as is true to greater and lesser degrees around the world, vaccines go through both scientific and regulatory pathways. these pathways, informed by science and the past history of successes and failures, are designed to maximize the chances of efficacy and safety. further, these pathways are designed to be deliberate, reflective, evidence-based, and peer-reviewed . . . in short, to maximize the chance that the data generated are robust, interpreted correctly, and lead to safe and effective vaccines when used in the population-at-large. perhaps the fastest a vaccine has been licensed in response to a new human pathogen of public health concern is the example of ebola virus. from the first cases to licensure in the us took some years, although work on a vaccine had started in the s. even the pandemic influenza a/h n vaccine in took over months to produce and distribute, and this was for a vaccine we had decades of experience in producing and testing with annual strain changes. even then, many concerns were raised by the public of an ''experimental and untested" vaccine being foisted on the public. it turns out that perception is important (at least in terms of vaccine uptake), and that human decision-making under conditions of uncertainty is both biased and flawed, particularly under distorting influences such as economic incentives or perceived losses, peer pressure, and wide-spread fear. what does history teach us in regard to vaccine development? first, expect the unexpected. research is non-linear and often presents problems and barriers that are unanticipated. from these we learn (supposedly) and build on both successes and failures for the future. in vaccine development, we need only look back a handful of decades to recall failed vaccines against measles and rsv that used inactivated virus approaches. these vaccines led to antibody enhanced disease (aed) in people who were immunized and later infected with wild virus [ , ] . more recently, despite careful studies through years of preclinical and phase - clinical trials, aed was detected in post-licensure studies of dengue vaccine [ ] . second, rna viruses accumulate mutations that can sometimes circumvent vaccine-induced immunity. for example, influenza viruses mutate so fast that nearly annual strain changes are necessary for influenza vaccines. this occurs despite vaccines containing both h and n protein antigens, rather than depending upon single protein/antigen preparations. third, issues of broad immunogenicity exist. given that this is an rna virus, i believe it is critical that more than one viral antigen be included in the vaccine. while the significance remains unknown to date, researchers have already identified at least one mutation in the receptor binding domain of the s gene [ ] . further mutations could conceivably lead to issues of original antigenic sin with resultant disease enhancement after exposure or to vaccines that simply are not effective into the future. ''s only" vaccines risk these issues, whereas vaccines that include other relevant sars-cov- viral antigens considerably reduce this risk. fourth, decisions must be made regarding how much safety data is needed before initiating first-in-man clinical trials. of concern is the push for starting clinical trials in the absence of completed animal studies. novel phase i vaccines should not be administered to humans prior to completion and evaluation of appropriate animal studies for safety, toxicity, and immunogenicity. rushing through animal studies, using irrelevant or single animal species models, and avoiding non-human primate studies is simply transferring risk from animals to humans in an attempt to rush vaccine development. this may be even more important in studies of novel vaccine antigens, vaccine approaches, and concomitant adjuvants or immunostimulants. fifth, some are beginning to call for human challenge models as a method for quickly moving through vaccine development. this would require extensive discussion and ethical consultation to consider factors such as the lack of known effective treatment, the balance between https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. vaccine ( ) - rushed studies to get quickly to licensure presuppose evidences of safety, efficacy, and benefit. these should not be supposed; rather, the burden of proof lies upon the vaccine developer to demonstrate that those presuppositions are justified. for example, what level of risk are we willing to tolerate to immunize against an infection that may disappear in the next year or two? or that could diminish in severity in the short-to mid-term? or to administer to young children whose risk of both serious illness or death is quantifiably very, very low? this begs the question of how to license a vaccine in the midst of an ongoing pandemic like sars-cov- . might reasonable ''accommodations" be made for such a scenario? several seem worth immediate discussion: -could a vaccine be provided through an eua mechanism for mentally competent adults who meet certain risk guidelines, and in the context of study enrollment and data collection, and enhanced informed consent? -could a vaccine be provided through a revised definition of a compassionate use mechanism in the highest risk subjects after signing waivers of responsibility and enhanced informed-consent procedures? who should be included-perhaps healthcare providers and first responders who share the highest risk of infection as a starting point? -what, if any, animal models might be developed that allow the ''animal rule" to be utilized in an effort to accelerate research and licensure? -if phase i and ii trials are conducted earlier than normal procedure, could a phased initiation of studies from highest risk to lowest risk subjects be utilized? -might one conceive of differential regulatory pathways for vaccine candidates using well-understood antigens, vaccine methodology, adjuvants, manufacture, and routes of administration (tbd) versus those using novel delivery technology and novel antigens or adjuvants? -as mentioned above, human challenge studies have been advanced as a method to rapidly determine efficacy in discussions i have had with other vaccinologists. could this be a viable strategy in accelerating licensure? to date, no ethical framework has been advanced to support such an idea. -what will be the endpoint for determining vaccine efficacyprevention of infection? prevention of severe disease? prevention of viral shedding? other? -will different vaccines and different regulatory pathways be feasible for different members of the population with differing risk:benefit ratios? for example, administering a vaccine to a healthy and robust -year-old with no underlying comorbidities should require an exceptionally high safety and efficacy threshold. might that safety profile be somewhat different (to be defined) in an exceptionally high risk -yearold with multiple co-morbidities? what about for pregnant women or younger but immunocompromised persons? these and other such questions are raised to consider more carefully and thoughtfully how best to approach the development and distribution of a covid- vaccine. under current knowledge and disease severity, a vaccine is urgently needed. but such vaccine development must begin and progress cognizant of the many lessons learned from the past. in addition to safety issues, i raise concern over ''s-only" vaccine approaches for the mid-to long-term control of this rna virus. we need a vaccine-and we need it as quickly as one can be developed-that demonstrates safety and efficacy in adequately powered studies. such an extraordinary event as covid- is an argument for carefully developing a new playbook for how to develop novel vaccines against emerging pathogens in the context of epidemics and pandemics. modern science has the ability to rapidly develop vaccine candidates, but wisdom lies in attending to the many lessons of the past . . . including that of the tortoise and the hare. atypical measles and enhanced respiratory syncytial virus disease (erd) made simple brief history and characterization of enhanced respiratory syncytial virus disease dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age analysis of the mutation dynamics of sars-cov- reveals the spread history and emergence of rbd mutant with lower ace binding affinity vaccine research group, c guggenheim building key: cord- -by albr authors: van ginkel, frederik w.; padgett, justin; martinez-romero, gisela; miller, matthew s.; joiner, kellye s.; gulley, stephen l. title: age-dependent immune responses and immune protection after avian coronavirus vaccination date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: by albr infectious bronchitis virus (ibv) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for ibv. therefore we hypothesized that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response contributing to immune escape and persistence of ibv. to test this hypothesis the ibv-specific immune response and immune protection were measured in chicks vaccinated at different ages. this demonstrated a delayed production of igg and iga plasma antibodies in the , and -day-old vaccination groups and also lower iga antibody levels were observed in plasma of the -day-old group. similar observations were made for antibodies in tears. in addition, igg antibodies from the -day-old group had lower avidity indices than day vaccinated birds. the delayed and/or lower antibody response combined with lower igg avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv field strain challenge. the lack of vaccine-mediated protection was most pronounced in the -day-old vaccination group and to a lesser extent the -day-old group, while the -day-old and older chickens were protected. these data strongly support ibv vaccination after day post hatch. ibv is endemic and currently one of the most important causes of economic losses for the poultry industry and represents a continuous threat for this industry. in the past, it was estimated that with the best possible management of flocks ibv infection will reduce income by approximately % when compared to an ibv-free flock [ ] . there is approximately a % vaccine failure for arkansas (ark) serotype of ibv [ ] , the most prevalent vaccine serotype used in the usa. symptoms of ibv infection include, but are not limited to, wet eyes, swollen face, tracheal and kidney lesions, respiratory disease, reduced weight gain in broilers, decreasing and poor egg quality in layers [ , ] . the existence of various ibv serotypes as abbreviations: ark dpi, arkansas delmarva poultry industry; calt, conjunctivaassociated lymphoid tissue; elispot, enzyme-linked immunospot; hg, harderian gland; halt, head-associated lymphoid tissues; hrp, horseradish peroxidase; ibv, infectious bronchitis virus; eid , median embryo infectious dose; tlr, toll-like receptor. * corresponding author. well as antigenic variants [ ] complicates vaccination programs. since immunity induced by vaccination against a single serotype generally provides insufficient protection against other serotypes [ , ] . mucosal immunity plays a role in the control of ibv in chickens as was demonstrated using ibv-resistant and ibv-susceptible inbred chicken lines [ ] . this combined with the finding of gelb et al. [ ] , in which ocular immunization with the massachusetts connaught strain of ibv only on day or on day plus day followed by challenge with massachusetts provided protection of % and % of the chickens, respectively, while the same spf white leghorns only ocularly immunized on day were % protected [ ] . bsa immunization of , and day old broiler chickens obtained very similar results [ ] . this raises questions pertaining the maturity of the immune system and in particular the mucosal immune system, and the ability of chicks to generate a protective immune response when vaccinated at a very young age. conjunctiva-associated lymphoid tissue (calt) and harderian glands do not fully mature as a lymphoid organ until weeks after hatching [ , , [ ] [ ] [ ] . this combined with the practice of immunizing and boosting for ibv early after hatching may set up the immune http://dx.doi.org/ . /j.vaccine. . . - x/© elsevier ltd. all rights reserved. response for failure to protect. the second ibv immunization on day of age, which by itself is fully protective, does not completely compensate for the premature priming on day [ ] . field studies by de wit et al. [ ] demonstrated a lack of protective immunity when birds were boosted between day through day of age. the percentage of birds in a commercial flock positive for ibvspecific igm antibodies was correlated with vaccine protection and increased with the age of boosting. this data supports the notion that early vaccination and boosting of the ibv immune response may limit induction of protective immune responses to ibv. unlike the study by gelb et al. [ ] , the de wit et al. [ ] study can also be interpreted that maternal antibodies interfere with vaccine delivery during the first weeks of life [ ] . further evidence that the immune response may be limited during the first weeks of life comes from the observation that iga levels are undetectable in plasma the first week of life and igm levels are low [ ] . this indicates that immunoglobulin class switching and production of antibodies is very limited during the first week post hatch and therefore chicks are highly dependent on maternal igy antibodies for protection against ibv, which drops ∼ % during the first week of life [ ] . besides diminished b cell response after vaccination, splenic t cells from one week old chickens are also less responsive to polyclonal activation than that of older chickens. the splenic t cells from day old chicks even produce inhibitory factors for proliferation of mature t cells in vitro [ ] . furthermore, splenic lymphocytes of day old chicken displayed better antigen specific proliferation after oral salmonella exposure than day old chicken [ ] . when measuring gene expression in lung and trachea in and week old birds after avian influenza exposure a reduced expression of immune-related genes was shown and included innate immune response genes in the younger birds [ ] . additional evidence that innate immune mechanisms are diminished in young chickens was demonstrated by a lower salmonella phagocytic index of heterophils during the first few days of life [ ] . thus, early exposure to pathogens or vaccines may induce suboptimal innate and adaptive immune responses. based on these observations we hypothesized that early ibv vaccination, i.e., within the first week after hatching, will generate an immature, poorly protective ibv-specific immune response contributing to ibv immune escape and persistence. therefore, the ability of spf chickens of different age to induce an ibv-specific antibody response and protect against challenge with an ibv field strain was measured. our data indicate that early vaccination is suboptimal for induction of ibv-specific immune responses and immune protection. chickens: specific-pathogen-free (spf) white leghorn eggs were obtained from sunrise farms, inc., catskill, ny, hatched and used in all experiments. all hatched chickens were used for the below outlined experiments regardless of sex. chickens were housed in cages in bsl facilities for the duration of the experiment. food and water were provided ad libitum. all experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal use guidelines. auburn university college of veterinary medicine is an association for assessment and accreditation of laboratory animal care (aaalac)-accredited institution. ibv-vaccination and challenge: spf chickens were ocularly vaccinated with × % embryo infectious doses (eid ) of a live attenuated arkdpi ibv vaccine strain (zoetis, new york, ny) in l pbs, which was expanded in our laboratory. chickens were vaccinated day of age and , , or weeks of age. all groups were challenged ocularly with . × eid of the al/ / ibv field strain days after vaccination sample collection: tears were collected as previously described [ ] . blood samples were obtained by puncturing the brachial vein with a sterile g needle into kendall monoject, edta containing, blood collection tubes (tyco healthcare group lp, mansfield, ma) and incubated on ice. blood samples were centrifuged at × g for min. plasma was collected and stored at − • c until tested. ibv propagation and purification for elisa: ibv was propagated in spf white leghorn embryonated chicken eggs (sunrise farms, inc., catskills, ny) by inoculation on day of embryonation as previously reported [ ] . supernatants were titrated for the ibv virus using the reed and muench method [ ] . ibv was treated with . % ␤-propriolactone for min at • c [ ] . inactivation of the virus was confirmed by injection into embryonated eggs. the inactivated ibv was purified based on a previously published protocol [ ] . the virus was then stored at − • c until used. in order to measure igg (igy), iga and igm antibody levels in plasma and tears of chicken, an ibv-specific enzyme-linked immunosorbent assay (elisa) was developed as previously described [ ] . in brief, elisa plates were coated with ␤propriolactone killed, purified ibv at g/ml in carbonate buffer. the plates were blocked with pbs-bsa ( %) after which the samples were loaded at two-fold dilutions. binding of chicken antibodies was detected using biotinylated anti-chicken-igg, -iga and -igm monoclonal antibodies (southern biotechnology associates, inc., birmingham, al) followed by streptavidin-horseradish peroxidase. the plates were developed using tmb ( , , , -tetramethylbensidine; invitrogen corp., frederick, md) substrate. the highest sample dilution with at least an optical density of . above background level at nm was defined as the endpointtiter. ten to thirteen chickens were analyzed per group. the control group consisted out chicken from each age group for iga and igg, which were pooled in one group of since no differences were observed between the controls. for igm levels in plasma the controls (each group containing chickens except day which had ) were displayed separate for each age group. this was done because significant differences were observed between control igm levels to ibv in different age groups. the avidity index was determined as previously described [ , ] using the above described ibv-specific elisa. plasma and tears were diluted : in elisa buffer and were loaded on ␤propiolactone killed ibv coated elisa plates ( g/ml) [ ] . after overnight incubation of these samples at • c, l of increasing concentrations of potassium thiocyanate ( . , . , . , . , . , . , . m kscn) were loaded into the wells and incubated for min at room temperature. after washing the plates, the detection of ibv-specific antibodies was accomplished as previously reported [ ] . the data were normalized to percent inhibition relative to samples not exposed to kscn. the concentration of kscn to inhibit % of the reactivity of the elisa was defined as the avidity index [ , ] . the od read-out for the kscn inhibition data was curve-fitted using -order polynomial regression analyses in microsoft office excell program. the excell provided formula for the inhibition curve was used to determine the x values of y = . , which are the concentrations of kscn inhibiting % of the elisa reactivity, representing the avidity indices of those samples. all samples were analyzed in triplicates and - samples were analyzed per group. the cranial / of tracheae was collected days after ibv challenge with . × eid of al/ / ibv field strain. the tracheae were formalin-fixed and embedded in paraffin. longitudinal m sections were made and were hematoxylin and eosin (h&e) stained and analyzed for mucosal thickness using aperio scan scope and the image j morphometry program (rsb.info.nih.gov/ij/download.html). to measure the mucosal thickness measurements were made at regular intervals on one tracheal ring (see supplemental fig. ). as stated above, histopathology was analyzed in h&e stained tracheal slides days after ibv challenge. besides mucosal thickness (see supplemental fig. ), deciliation (see supplemental fig. ), goblet cells (see supplemental fig. ) and lymphocytes scores (see supplemental fig. ) of the tracheal mucosa were evaluated blindly and scored through based on severity (i.e., normal, mild, moderate, marked, severe). five chickens were used as positive and negative controls, i.e., one of each age group, and - chickens were analyzed for each age group. a visual depiction of the scoring of these parameters is provided in the supplemental data (supplemental figs. - ) . statistical analysis: data were analyzed using a one-way anova test with newman-keuls multi-comparison test or the t-test using graphpad prism software. groups were considered significantly different when p < . . to determine whether age of ibv vaccination affected the humoral immune response, plasma samples were collected and days and tears days after vaccination with × eid of a live-attenuated arkdpi ibv vaccine strain on , , or days of age. the ibv-specific igg endpoint titers in plasma days after vaccination are significantly lower for day vaccinated birds with a mean of . ± . when compared to day , and vaccinated birds, which means vary between . - . . the -day-old group does not differ significantly from day or later vaccinated bird (fig. a) . the igg ibv-specific plasma levels days after vaccination demonstrate, that the day and old vaccination groups chickens were vaccinated at day , , , or of age. unvaccinated chickens of the different age groups served as negative control. the data was analyzed by one way anova with the newman-keuls post-test. the control group contains data points for each age group, which were pooled in one group (n = ). all vaccinated age groups contained between and chickens for igg and iga. a significant difference is observed at p < . and is indicated by different letters. igm levels in ibv vaccinated birds are depicted by squares (n = ) and the controls by circles (n = , d group n = ). for the igm controls the different age groups are shown separately since significant differences were observed between them. a significant increase (p < . ) of igm levels in ibv vaccinated birds over their control group is indicated by a (*). stayed the same, while igg antibody titers in groups vaccinated on day , and still increased (fig. c) . this shows that early vaccination causes a delay in the ibv-specific igg antibody kinetics. the ibv-specific iga plasma antibody titers are not significantly different between groups, although the day vaccinated group mean antibody titer is the lowest of all groups (fig. b) and at least -fold lower than the next lowest group. unlike the igg antibody titers only the day vaccination group increases in mean iga plasma titer on day post vaccination, while day group stays the same and the day , and groups decline in mean iga titer. thus, only the day group increases antibody titers on day when compared to older birds. thus, the day group displays a delayed response in antibody production compared to older birds. the day and day groups have plasma iga titers to ibv that are comparable to, or higher than, the day and groups on day of the response. unlike the day and day vaccination groups the day and groups are declining on day of the response compared to the immune response on day . this indicates that they are past their peak response on day and possibly even on day based on previous observations [ ] . these data are consistent with a delay in the iga plasma response to ibv in birds vaccinated at a younger age and a non-significant decline in mean iga titers in the -day-old group. ibv-specific igm antibody titers were measured in plasma. the plasma samples analyzed were collected on day post vaccination. this time point was selected based on the literature in which the peak igm response was observed between - days after virus challenge or live virus vaccination [ ] [ ] [ ] . due to the variability of ibv-reactive igm in the controls between different age groups, independent controls were included for each age group. the igm levels in the controls decreased considerably by ∼ week of age after ( days after the day old chick vaccination) which increased one week later and stabilized in older age control groups (fig. e) . the day through day control igm levels were significantly higher than in the day and day age group controls. and the day control was significantly lower than the day control igm levels. all igm titers from the ibv vaccinated age groups were significantly higher when compared to their controls (p < . ) with exception of the day age groups (p = . ). this was due to higher control values, which were ∼ . fold higher than in the age or days old groups. this may reflect an initial peak of natural antibodies induced to ibv before stabilizing. the ibv vaccinated age groups did not differ significantly in igm antibody levels to ibv with exception of the day ibv vaccinated group, which mean ± se was . ± . had significantly lower ibv-specific igm levels in plasma than the day ( . ± . ) day ( . ± . ) and day ( . ± . ) vaccinated birds but did not differ significantly from the day ( . ± . ) vaccinated birds (fig. e) . the day igm antibody titers were also significantly lower than those in the day vaccination group but not compared to the other age groups. in tears the ibv-specific igg response is significantly higher in the day and vaccinated groups than in the day and vaccinated chickens for day of the immune response ( fig. a) . the igg endpoint titer in the day immunized group is even significantly lower than the day immunized group, while the day immunized group is intermediate between the day group and chickens vaccinated at an older age. thus, a correlation between age of vaccination and the magnitude of the ibv-specific igg response in tears is observed on day of the ibv-specific immune response. the day and groups have significantly higher iga anti-ibv responses in tears on day after vaccination compared to the day group. the day group is not significantly different from the day and vaccination groups. the day group is also significantly lower than the day and groups (fig. b) . this is likely due to the day group displaying faster kinetics for iga antibody levels in tears after vaccination, rather than a lower response [ ] . the day group iga response is significantly higher than the day and day groups consistent with a delay or deficiency in the mucosal antibody response when vaccinated at an earlier age. our data indicate there is a delay in antibody production when vaccinated at a younger age. the day vaccination group not only displays a delay but also lower levels of antibody production. to determine whether there are not only quantitative differences between antibodies produced when vaccinated on day but also qualitative differences we compared avidity indices for igg and iga antibodies from plasma and tears generated in day old versus fully matured day old birds days after ibv vaccination. as is illustrated in fig. a ,b a significantly higher avidity index is observed for igg plasma antibodies for the day vaccination group when compared to the day vaccinated birds, while no significant difference is observed for iga plasma antibodies. the same observations are also made for tear igg and iga antibodies (fig. c,d) . ciliated cells and goblet cells are the primary target of ibv in the respiratory tract [ ] . fig. displays the deciliation (fig. a ) and goblet cell (fig. b ) scores days after ibv challenge. ibv challenge decreased ciliated epithelial cells and goblet cell score. ciliated cells were fully protected when vaccinated on day of age or later but not when vaccinated on day of age (fig. a) . the protection of goblet cells increases with the age of vaccination and were fully protected when vaccinated on day of age or older (fig. b) . the lymphocytes score and mucosal thickness were also measured in tracheal samples on day post al/ / ibv field strain challenge as indicators of inflammation. as is illustrated in fig. a , a significant decrease in lymphocyte score was observed in the day and vaccination groups when compared to the day group. the day vaccination group did not significantly differ from the day fig. . the ibv-specific igg and iga response in tears. endpoint titers of ibv-specific igg (a) and iga (b) on day of the immune response were measured by elisa. chickens were vaccinated on day , , , or of age. unvaccinated chickens of the different age groups served as negative control. depicted are the means and standard error of each age group and the control group, and different age groups are as described in fig. (n = - per group) . the data was analyzed by one way anova with the newman-keuls post-test. differences were considered significant at p < . and are indicated by different letters. fig. . tracheal deciliation and goblet cell depletion after ibv challenge. to measure the degree of protection against ibv challenge after vaccination tracheal deciliation and goblet cell depletion were measured. chickens were vaccinated at day , , , or of age and challenged days later. unvaccinated/unchallenged chickens of all age groups served as negative control and unvaccinated/ibv challenge chickens as positive control. trachea were collected days post challenge. depicted are the mean and one standard error. for the negative and positive controls n = for the different age groups n = - . the data were analyzed by one way anova with the newman-keuls post-test. significant difference was observed at p < . and are indicated by different letters. and vaccination groups due to a small increase in lymphocyte score in the latter two groups (fig. a) . the day group displayed the highest lymphocytes score from all vaccination groups, which is consistent with the highest inflammatory response to ibv challenge. this was also supported by a significant increase in mucosal thickness in the day vaccinated group when compared with the day , and groups but not day group, which was intermediate between the day and groups vaccinated at an older age (fig. b ). based on our data, the hypothesis that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response, is confirmed. ibv vaccination on day of age, which is routinely performed in the poultry industry, will not be fully protective and as a consequence the chicks remain vulnerable to ibv exposure. thus, early vaccination perpetuates the ibv problems and is a factor in the estimated $ million or more annual loss to the poultry industry due to ibv infection [ ] . our measurements of mucosal and systemic antibody levels demonstrates a delayed production of igg and iga plasma antibodies in the day , day and of age vaccination groups. iga antibody levels in the day group, unlike igg antibodies, do not recover later in the response. besides delayed igg kinetics, the day group displays also a lower avidity index than the day vaccinated group. lower avidity index is not observed in iga antibodies. the delayed and/or lower antibody response and lower igg avidity index translated in increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv challenge when compared to chicks vaccinated later in life. a lack of vaccine-mediated protection is most noticeable in the day of age vaccination group and to a lesser extend the day vaccination group, while the day and older vaccinated chickens are protected. the igm antibodies specific for ibv were significantly elevated above controls on days of the ibv immune response in all age groups except the day group. the day group was not quite significant (p = . ) because of higher igm levels in the control group. this could be due to an initial surge of natural igm antibodies to ibv in this age group. a significant decline in the day old group is observed when comparing the igm antibody levels to ibv in the control day group. this would be consistent with a drop of presumably natural maternal ibv-specific igm antibodies in these spf chickens in the day control age group. these igm antibodies rapidly increases in the day group after which they stabilize in the older age groups. this indicates that a considerable portion of igm antibodies in plasma from the older vaccination groups reacts with ibv without seeing the virus, indicating these are natural antibodies to ibv, which only increase after day of age. bacterial colonization of the intestinal tract of chickens is established during the first two weeks post-hatch [ ] . this, combined with the observation that probiotics enhance natural antibodies in chicken [ ] , indicates that ibv-specific natural igm antibodies to ibv are possibly generated following intestinal colonization presumably by stimulating b cells, which are the main producers of natural igm antibodies in sera of mammals [ ] . in the ibv vaccinated groups we see a steadily incline of igm ibv-specific antibodies in plasma with age as has been reported by de wit et al. [ ] . only the day age group displays significantly lower igm antibody levels when compared with the day - age groups but not with the day age group. this is consistent with an early in life deficiency or delay in the igm response. the lower avidity index for the igg antibodies in -day-old chicks is an important factor contributing to decreased protection to ibv challenge. increased antibody affinity maturation to virus vaccines strongly correlated with better protection [ , ] . a lack of antibody affinity maturation observed following vaccination against respiratory syncytial virus was due to a lack of tlr stimulation [ ] . this indicates that -day-old birds may be deficient in tlr expression. evidence that this is the case comes from a recent publication [ ] demonstrating, that significantly lower levels of tlr expression in spleen and small intestines were observed when comparing -day-old chicks with -to -week-old chickens. inclusion of tlr activating adjuvants could alleviate the problems of early vaccination by boosting antibody production and affinity maturation in -day-old chicks. neither monomeric plasma nor dimeric tears-derived iga [ ] displays this drop in avidity index for the day vaccinated group (fig. b,d) . although there is a lower level of iga antibodies produced by the day vaccinated birds compared to the older groups, which is consistent with a delay in class-switching in the day old group, it is not clear why the lack of a mature mucosal immune system in the -day-old group [ , , [ ] [ ] [ ] did not result in lower affinity maturation of mucosal iga antibodies compared to the -week-old group. another factor influencing early vaccination is the level of maternal antibodies, an issue not addressed in this study. there exists a linear relationship with the hens' plasma antibody levels and transfer of igy to the chicks' circulation [ ] . chicks were over % protected against ibv challenge on day if they had high levels of maternal antibodies but less than % protected when challenged on day . this protection correlated with local respiratory antibodies and not serum antibodies [ ] . ibv-specific maternal antibodies decreased the induction of neutralizing antibodies following boosting [ ] . despite this inhibition by maternal antibodies of the memory response, low or erratic maternal antibody titers to ibv in broiler flocks are associated with ibv-induced economic losses [ ] . this further supports that protection by maternal antibodies, which are predominantly of the igg isotype, is important to prevent activation of an immature immune system that is not capable generating a fully protective immune response early in life. in several studies ibv vaccination was effective against ibv challenge in both spf chickens and commercial broilers when the initial vaccination was performed on day [ ] [ ] [ ] . however, in these studies day vaccination was followed with a second vaccination two weeks later for optimal protection against challenge, which would have masked the relative poor ibv-specific immune responses after the day immunization. extensive immunization on the day of hatch containing three different live attenuated viruses, which caused severe vaccine symptoms, provided similar protection as two single live attenuated ibv virus vaccines on day of hatch and day of age when challenged with a heterologous virus [ ] . this seems to indicate that induction of cross-protective immunity may be less impaired when vaccinated early in life. however, no direct comparison of the same vaccination protocol was analyzed between these two challenge groups, which makes this data harder to interpret in the context of age-dependent immune responses. a decreased humoral immune response to vaccines early in life as seen in chickens is also observed in humans. neonates are highly dependent upon passively acquired maternal antibodies, since their humoral immune system remains underdeveloped [ ] . these passively obtained antibodies in infants can alter humoral and antibody-dependent immune responses to vaccines [ , ] . in broiler chicks maternal antibodies to pathogens persisted only for ∼ days [ ] . in our study interference of maternal antibodies was excluded. the observation that -day-old and -day-old birds are not fully protected when vaccinated are confirming the importance of pathogen-specific maternal antibodies during this period. in summary we can say that ibv vaccination of chickens on day of age contribute to the ibv problem in the poultry industry by inducing lower levels and/or slower kinetics of antibody production as well as lower avidity igg antibodies. this results in a poorly protective immune response as is demonstrated by subsequent ibv field strain challenge. therefore, it is advisable for the poultry industry based on our data to change their practice from vaccinating chicks on day of age to vaccinating after day of age. infectious bronchitis infectious bronchitis virus field 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heterologous serotypes pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by vaccination programme based on the ma and / serotypes induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d (genotype qx) by maternally derived antibodies and by early vaccination the maturing immune system: implications for development and testing hiv- vaccines for children and adolescents determinants of infant responses to vaccines in presence of maternal antibodies decay of maternal antibodies in broiler chickens we thank the alabama agricultural experiment station for funding this research ala - - . supplementary material related to this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . key: cord- -h wrs h authors: liu, xianglei; drelich, aleksandra; li, wei; chen, chuan; sun, zehua; shi, megan; adams, cynthia; mellors, john w.; tseng, chien-te; dimitrov, dimiter s. title: enhanced elicitation of potent neutralizing antibodies by the sars-cov- spike receptor binding domain fc fusion protein in mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: h wrs h the development of an effective vaccine against sars-cov- is urgently needed. we generated sars-cov- rbd-fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. rbd-fc elicited a higher neutralizing antibodies titer than rbd as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. furthermore, rbd-fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. the cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-sars-cov- antibodies and vaccines without the requirement of live virus infection in bsl containment. moreover, the anti-rbd sera did not enhance the pseudotyped sars-cov- infection of k cells. these results demonstrate that fc fusion can significantly improve the humoral immune response to recombinant rbd immunogen, and suggest that rbd-fc could serve as a useful component of effective vaccines against sars-cov- . the coronavirus disease (covid- ) outbreak has become a global pandemic responsible for over million confirmed cases and over . million deaths worldwide as of aug , . severe acute respiratory syndrome coronavirus (sars-cov- ), the novel betacoronavirus, is the etiological agent of covid- . vaccines are a highly effective strategy in preventing the spread of infectious diseases with advantages of low production and distribution cost; stark reduction in morbidity; and minimal negative long-term effects on overall health. currently, no approved vaccines are available for covid patients although tremendous scientific and industrial efforts have been dedicated. several approaches to the development of covid- vaccines have emerged including dna vaccines, rna vaccines, viral vector vaccines, recombinant subunit vaccines, inactivated and attenuated virus vaccines [ ] . as of aug , , candidate vaccines are undergoing clinical evaluations with more than vaccines in preclinical development stages (draft landscape of covid- candidate vaccines, who). among those, cansino's recombinant adenovirus type- vectored vaccine has achieved the most fast clinical progress as the first-in-human trial vaccine, reporting tolerability and high immunogenicity after days postvaccination [ ] . been demonstrated to be an appropriate immunogen capable of eliciting neutralizing antibodies [ ] . both viruses are phylogenetically close to sars-cov- [ ] , thus supporting the rationale of using the sars-cov- s protein as a subunit vaccine. subunit vaccines contain recombinant antigen proteins with strong immunogenicity capable of efficiently stimulating the host immune system. advantages of subunit vaccines include easy manufacture, low cost, and overall safety, since they do not contain genetic material and have a low probability to induce severe adverse reactions. for example, subunit vaccines are reported to be safer than other vaccines such as virus like particles, inactivated whole viruses and an rdna expressed s protein, which have been shown to induce the cytokine th -type immunopathology in sars-cov [ ] . several subunit vaccines based on the full-length sars-cov- s proteins are under-development, with five candidates in clinical trials. however, the full-length s protein immunogens contain many non-neutralizing epitopes that could result in enhanced infection through the antibody-dependent enhancement (ade) effect, which may occur through the fcγr-mediated internalization of antibody-bound virions in fcγr-expressing cells [ , ] . the risk of ade, observed for sars-cov, mers-cov, hiv- , zika and dengue virus vaccination, has become a major concern in vaccine development [ ] [ ] [ ] . the sars-cov- receptor-binding domain (rbd) is the protruding site of the s protein that mediates viral cell fusion during the initial infection event through binding of the human receptor angiotensin-converting enzyme (hace ) [ , ] . based on its highly homology to sars-cov, sars-cov- rbd is corroborated to contain immune dominant epitopes capable of eliciting antibodies that can neutralize viral infection and block viral entry by competing hace binding. the antigenic regions on sars-cov- were also confirmed by computational studies exploring t cell and b cell epitopes [ ] . compared to the full-length s protein based subunit vaccine, reducing the immunogen to only the neutralizing epitope containing rbd can not only specifically mount neutralizing antibody titers, but could also mitigate the risk of ade, which in most cases are mediated by the non-neutralizing antibodies [ ] . therefore, sars-cov- rbd has been selected as a primary target for vaccine design [ , ] . ravichandran et al recently found that compared to full-length s, sars-cov- rbd immunogen elicited a higher titer of neutralizing antibodies with -fold higher affinity [ ] . yang et al also showed that rbd can induce a potent antibody response in the immunized mice, rabbits and non-human primates [ ] . zang et al (preprint) have showed that the anti-rbd sera from mice did not promote (both pseudotyped and authentic) sars-cov- infection of fcγ receptor-bearing cells [ ] . given those advantages, we chose the sars-cov- rbd as a subunit vaccine. to boost the vaccination-induced antibody response, we fused rbd to the igg fc. the fc fragment can serve as a vaccine adjuvant by promoting cellular and humoral immune responses, probably by facilitating antigen delivery and presentation through interacting with fcγ receptors on antigenpresenting cells. in addition, the fc-fusion can also improve recombinant immuogens solubility and stability and extend their in vivo half-life after injection by interacting with human neonatal fcγ-receptor (hfcrn) [ ] . to stimulate anti-rbd antibody titers, we also utilized a well-accepted, highly effective adjuvant, mf tm . mf has been proven to increase neutralizing antibody production and boost the th and th immune responses. several vaccines containing mf as adjuvants are undergoing clinical trials (phases i-iii). notably, mf -adjuvanted seasonal influenza vaccine (fluad™) has already been licensed, showing acceptable safety and tolerability, and improved immunogenicity [ ] . the gene of sars-cov- rbd domain (residues - ) was synthesized by idt (coralville, iowa), then cloned in frame to human igg fc or ×his tag in the mammalian cell expression plasmid. these proteins were expressed in the expi tm expression system (thermo fisher scientific) and purified by protein a resin (genscript) or ni-nta resin (thermo fisher scientific). the purified sars-cov- rbd and rbd-fc were analyzed by sds-page and western blot (wb). protein concentration was measured spectrophotometrically (nanovue, ge healthcare), μg of proteins were separated by nupage ® - % bis-tris gels (life technologies), protein purity was estimated as > %. for western blot, the separated proteins were transferred to nitrocellulose membranes. after blocking at room temperature using % non-fat milk in pbst for h, the blots were incubated for h at room temperature with nm hace -mfc (mouse fc, sino biological, beijing, china). after three washes, the blots were incubated with anti-mouse igg-horseradish peroxidase (hrp) conjugated secondary antibody ( : , sigma-aldrich) for h at room temperature. the membranes were reacted with enhanced ecl chemiluminescence reagent (millipore, billerica, usa) and exposed by bio-rad detection system. protein deglycosylation mix ii (neb) was used for deglycosylation reaction following manufacturer's instructions. briefly, μg proteins were mixed with deglycosylation mix buffer (non-denaturing reaction) or buffer (denaturing reaction, with an additional incubation at °c for min), then incubated with protein deglycosylation mix ii for reaction ( °c for min, and then °c for h). the deglycosylated proteins were analyzed by sds-page. the mw of enzymes in protein deglycosylation mix ii is approximately , , and kda. genes of hace (origene, rockville, md) and the full length s protein of sars-cov- (codon optimized and synthesized by idt) were subcloned into our in-house mammalian cell expression plasmid and used to construct stable cell line t-ace and t-s, which were cultured in dulbecco's modified eagle's medium (dmem, gibco) with % fetal bovine serum (fbs), % penicillin/streptomycin (p/s) and μg/ml zeocin (thermo fisher). for the determination of t-s cell line, nm hace -fc (sino biological, beijing, china) were incubated with cells for min at o c. cells were washed and then incubated with pe conjugated anti-human fc antibody (sigma-aldrich) for min at o c. bound antibodies were detected by flow cytometry using bd lsr ii (san jose, ca). for the t-ace cell line, nm rbd-his followed by pe conjugated anti-his antibody (sigma-aldrich) were used for analysis. four groups of - week old female balb/c mice (n= ) were immunized twice (day and day ) subcutaneously with rbd proteins ( μg/mouse) with or without adjuvant mf . group was immunized with rbd-fc fusion, group was immunized with rbd-fc fusion in emulsion with mf , group was immunized with rbd in emulsion with mf , group served as a control and was immunized subcutaneously with dulbecco's phosphate-buffered saline dpbs (gibco tm ). sera were collected before (pre-vaccination), after days, and after days vaccination. for evaluation of affinity of rbd and rbd-fc to hace , both proteins were coated on a -well plate (costar) at ng/well in pbs overnight at o c. the plate was blocked using % skim milk for h at room temperature (rt). we then added serially diluted hace -mfc (mouse fc, sino biological, beijing, china) and incubated for h at rt. the plates were washed times with . % tween in phosphate buffered saline (pbst). anti-mouse igg-horseradish peroxidase (hrp) conjugated secondary antibody (sigma-aldrich) was added to the plate followed by incubation for h at rt. after another washes with pbst, the plate was incubated with a , ', , 'tetramethylbenzidine substrate solution (tmb, sigma-aldrich) for min. the reaction was stopped using m h so followed by reading absorbance of each well at nm. for detection of anti-rbd or anti-(s +s ) antibodies in mice serum, the sars-cov- rbd proteins or s +s (sino biological, beijing, china) were coated at ng/well in pbs overnight at o c. after blocking, serially diluted mouse serum were added and incubated for h at rt. for antibody isotyping, the bound rbd-specific antibodies were detected by anti-mouse igg, igm, iga hrp conjugated secondary antibody (sigma-aldrich), respectively. for competitive elisa, ~ nm ( μg/ml) biotinylated hace (sino biological, beijing, china) was incubated with serially diluted mouse serum, and the mixtures were added to rbd coated wells. after washing, bound hace was detected by streptavidin-hrp secondary antibody (sigma-aldrich). the pseudovirus neutralization assay was performed based on previous protocols. briefly, hiv- backbone based pseudovirus was packaged in t cells by co-transfecting with plasmid encoding sars-cov- s protein and plasmid encoding luciferase expressing hiv- genome (pnl - .luc.re) using polyethylenimine (pei). pseudovirus-containing supernatants were collected h later and concentrated using lenti-x™ concentrator kit (takara, ca). pseudovirus neutralization assay was then performed by incubation of sars-cov- pseudovirus with serially diluted mice serum for h at °c, followed by addition of the mixture into pre-seeded t-ace cells. the mixture was then centrifuged at × g for h at rt. the medium was replaced hrs later. after h, luciferase expression was determined by bright-glo kits (promega, madison, wi) and read using biotek synergy multi-mode reader (winooski, vt). the % pseudovirus neutralizing antibody titer (nt ) was calculated using the graphpad prism . the standard live virus-based microneutralization (mn) assay was used. briefly, serially five-fold (start from : ) and duplicate dilutions of mice serum were incubated with pfu of sars-cov- at room temperature for h before transferring into designated wells of confluent vero e cells (atcc, crl- ) grown in -well microtiter plates. vero e cells cultured with medium with or without virus were included as positive and negative controls, respectively. after incubation at o c for days, individual wells were observed under the microscopy for the status of virusinduced formation of cytopathic effect (cpe). the titer of mice serum (nt ) was expressed as the lowest dilution folds capable of completely preventing virus-induced cpe in % of the wells. to test mice serum mediated inhibition of cell fusions, the β-gal reporter gene based quantitative cell fusion assay was used. briefly, t-s cells were infected with t polymerase-expressing vaccinia virus (vtf - ), while t-ace cells were infected with vaccinia virus (vcb r lac-z) encoding t promotor controlled β-gal. two hours after infection, cells were incubated with fresh medium and transferred to °c for overnight incubation. the next day, t-s cells were pre-mixed with serially diluted mice serum at °c for h followed by incubation with t-ace cells at a : ratio for h at °c. then cells were then lysed, and the β-gal activity was measured using β-galactosidase assay kit (substrate cprg, g-biosciences, st. louis, mo) following the manufacturer's protocols. fusion inhibition percentage (sample reading, f) was normalized by maximal fusion (reading, f max ) of t-s and t-ace cells in the absence of inhibitors using this formula: fusion inhibition % = [(f max -f)/(f max -f blank )] × %, in which f blank refers to the od reading of t-s and t incubation wells. fusion inhibition percentage was plotted against serum dilution folds from which ic was calculated in graphpad prism . fcγrii expressing cell lines k (atcc, ccl- ) were used to perform ade assays. briefly, the mouse serum were serially diluted, mixed with sars-cov- pseudovirus, and incubated at ℃ for h. then, the mixtures were added to the pre-seeded plates with k cells. the following infection and culturing steps were carried out as described above in the pseudovirus neutralization assay. pseudovirus infected k or t-ace cells were set as the negative and positive controls, respectively. all experiments were conducted in duplicate, and data were averaged and presented as the mean ± standard deviation (sd). significant differences were determined by one-way analysis of variance followed by tukey's test, using the graphpad prism (version ) package. statistical significance was defined as p < . . recombinant sars-cov- rbd (without fc) and rbd-fc proteins were produced in expi tm mammalian cells, and then verified by sds-page and western blot. rbd-fc showed a homogenous band (fig. a) while the rbd exhibited relatively heterogeneous bands due to varying extents of glycosylation, which was also found by yang et al [ ] . the rbd protein showed a single band upon deglycosylation, confirming the heterogeneous bands resulting from glycosylation (fig. b) . besides, western blot results showed that rbd with different glycoforms can react with hace (fig. s ) . elisa binding to hace further validated the qualities of both rbd proteins (fig. c) . rbd antigens produced in this study were also used for panning against our in-house phage antibody libraries to retrieve high affinity binders [ ] . four groups of - week old female balb/c mice (n= ) were immunized subcutaneously at day and boosted at day with rbd-fc, rbd-fc in emulsion with mf (rbd-fc+mf ), and rbd in emulsion with mf (rbd+mf ) for each group at a dose of μg of proteins per mouse. the fourth group injection was of dpbs, which served as the negative control (fig. d) . on day (pre-immunization), day and day , mouse sera were collected and analyzed for rbd binding, pseudovirus and live virus neutralization, and cell-cell fusion inhibition. anti-rbd sera from each trial group were firstly evaluated for rbd binding as measured by elisa (fig. a) . the anti-rbd antibody in post immune mouse sera were also isotyped by anti-mouse igg, igm and iga antibodies, respectively (fig. s ) . the rbd antibody titers were calculated as the dilution folds that remain % of maximal binding signal (ec ). the recombinant rbd (his tag) was used as the detection antigen to avoid the interference of antihuman fc antibody titers in mice. the impact of his tag on the detection of rbd binding titer is marginal ( figure. s c) . results showed that for the sera collected at and days post immunization, the anti-rbd antibodies were mostly composed of the igg isotype with only marginally detectable igm (fig. s a) and no detectable iga isotype (fig. s b) . the low igm titer detected at day and may correlate to the fact that igm is typically rapidly mounted post infection (within one week) followed by isotype switching into igg isotype [ ] . the lack of iga titer may result from iga usually deriving from mucosa immunity, leading to low titers in sera [ ] . for the igg isotype antibodies, the pre-immunization sera showed no binding to rbd, while the day sera from all three rbd immunized groups exhibited varying extents of binding to the rbd. interestingly, the rbd binding titer elicited by the rbd+m group on day was much less than titers of the rbd-fc (titer : ) and rbd-fc+m (titer : ) groups, indicating the immune stimulation roles of fc fusion. however, for post-boosting sera on day , the rbd binding titers of rbd+mf group was significantly mounted to : . in contrast, on day the titers of rbd-fc (titer : ) and rbd-fc+mf (titer : ) groups were only improved by -fold compared to those of day , indicating distinct humoral response kinetics against rbd and rbd-fc immunogens. interestingly, although to a lesser extent than before receiving the booster, the rbd-fc and rbd-fc+mf groups exhibited . and folds higher titers, over the rbd+mf group (p< . ) respectively, assuring the enhancing role of fc in elicitation antibody response by the rbd immunogen. it is also intriguing that the rbd-fc+mf group exhibited slightly higher titers than the rbd-fc group, probably due to the adjuvant role of mf . interestingly, we also correlated the rbd binding titer to the full-length s ectodomain binding titer for the day sera. the elisa showed that the rbd binding sera also bound to s +s with similar titers (fig. s d) , which suggest that the rbd recognition antibody in the sera can also bind to full length s. hace blocking is a surrogate indicator for anti-sars-cov- antibody neutralizing activity. to preliminarily infer the neutralizing titers of post-immunization mouse serum, we performed the hace competitive elisa, in which serially diluted mouse sera in the presence of the biotinylated hace were added into rbd coated plates. bound hace was detected by the streptavidin-hrp secondary antibody. results showed the three rbd immunogen groups developed discernable hace competitive titers on day compared to the pbs control group; further significantly boosted to : , : , : for the rbd-fc, rbd-fc+mf , rbd+mf groups respectively on day (fig. b) . consistent with the above rbd binding titer, the rbd-fc and rbd-fc+mf groups sera showed . folds and . folds higher competitive titers respectively than the rbd+mf group (p< . ), supporting the role of fc in mounting neutralization titers. the competitive elisa results gave the specific hace blocking titers elicited by rbd immunogens, which presumably predict their neutralization activity [ ] . next we exploited the sars-cov- s pseudotyped hiv- to evaluate the neutralization activity of those anti-rbd sera. sars-cov- pseudovirus was packaged by co-transfecting hek t cells with pcdna . -s plasmid encoding codon-optimized full-length sars-cov- s protein and pnl - .luc.re plasmid containing luciferase expressing hiv- genome. serially diluted mouse sera were pre-incubated with pseudovirus followed by infection of t cells stably expressing hace ( t-ace ). as shown in fig. a , on day all the rbd immunized groups sera showed substantial % neutralizing antibody titers (nt ) compared to the pre-immune sera on day (titer : , : , : ), which were largely boosted to : , : , : for the rbd-fc, rbd-fc+mf and rbd+mf groups respectively. intriguingly, unlike the marginal differences for the rbd binding and hace competitive titers across the sera of the three rbd immunized groups on day , the pseudovirus neutralization titers were significantly distinct with the rbd-fc+mf group showing highest titers, . -fold higher than the rbd-fc group and . fold than the rbd+mf group (p< . ). in addition to the pseudovirus neutralization, we also evaluated the live virus neutralization potency of those mouse anti-rbd sera (day ) by using a microneutralization (mn) assay. in this assay, the cytopathic effect (cpe) of vero e was observed after days incubation with live virus, which was pre-mixed with the anti-rbd sera. the neutralization titer of mice serum (nt ) was expressed as the lowest dilution folds capable of completely preventing virus-induced cpe in % of the wells. consistently, the nt of serum in rbd-fc+mf group ( : ) was higher than those of the rbd-fc and rbd+mf groups ( : ) (fig. b) . these results clearly demonstrated that fc fusion could significantly augment the elicitation of neutralizing antibody titers by rbd immunogens, and the adjuvant mf can further stimulate the antigenicity of the rbd-fc fusion proteins. interestingly, we found that the pseudovirus neutralization titer positively correlated with the hace competition titer (fig. c) , demonstrating the utility of our hace competition elisa in predicting neutralization titers in convalescent plasma therapy and in detecting of the presence of neutralizing antibodies in serological tests during the covid pandemic. to further evaluate whether the anti-rbd sera could prevent sars-cov- s-mediated cell-cell fusion, we established a quantitative cell fusion assay using β-galactosidase (β-gal) as a reporter gene. we constructed t cell lines stably overexpressing sars-cov- s ( t-s) and hace ( t-ace ) respectively (fig. s ) . in this assay, the t-s cells were infected with t polymerase-expressing vtf - vaccinia virus and the t-ace cells were infected with t promotor controlled β-gal expressing vcb r vaccinia virus. therefore, β-gal expression is only allowed after cell-cell fusion, which can be quantified by monitoring β-gal activity. serially diluted mouse sera were pre-mixed with infected t-s cells followed by incubation with infected t-ace cells. as shown in fig. a , the pre-vaccination sera at day and the pbs control mouse sera did not inhibit the cell-cell fusion. however, the day rbd-fc+mf sera showed obvious cell-cell fusion inhibition with a % fusion inhibition antibody titers (ic ) of : , which was . -fold higher than the inhibition titers of the rbd-fc ( : ) and . -fold higher than the rbd+mf ( : ) group (p< . ). interestingly, in this assay, the rbd-fc and the rbd+mf groups did not show significant differences (p> . ). we also observed a nearly perfect correlation of the cell-cell fusion inhibition titer with the pseudovirus neutralization titer (r = . , p< . , fig. b ), which may be attributed to theirmechanism of action. anti-rbd antibodies typically neutralize virus by blocking viral entry. for sars-cov- , virus entry and cell-cell fusion share similar mechanisms. both viral entry and cell-cell fusion are initiated by the s protein binding to the receptor ace , followed by s subunit triggered susceptibility to protease cleavage, which causes s dissociation and conformational change of the s subunit. then, the fusion peptide (fp) in s is exposed for anchoring into host cell membrane and heptad repeats (hr and hr ) establishes the six helical-bundle resulting in the membrane fusion between viral and host cells [ ] . molecules, including antibodies, interfering with any of the above processes can block viral entry as well as cell-cell fusion [ ] . based on their similar mechanisms, the inhibitory activity of antibodies for cell-cell fusion can be a highly relevant predictor of the antibody neutralizing activity. this is further supported by our and others' sars-cov- neutralizing antibodies, which shows both potent inhibition of s mediated cell-cell fusion and neutralization of sars-cov- [ ] . although highly correlated, there are differences between these two assays in terms of different environments on the cell and viral surface such as s protein conformation/ density, accessibility of proteases. due to the high correlation to the virus neutralization, this method allows for high-throughput screening and is therefore well suited for the characterization of cell-cell fusion mediated by sars-cov- or other viruses. in addition, this assay is highly effective in screening potential neutralizing antibodies with fast speed since this assay can be finished within one day without the requirement of in biosafety level facilities while the virus neutralization assays typically require several days which is of particular relevance in the context of the global pandemic, which urgently needs vaccines and candidate antibody drugs. from the mechanism, one can also envision that rbd antibodies showing ace competition is sufficient, but not necessary for virus neutralization and cell-cell fusion, since antibodies disturbing other entry steps, rather than ace /rbd binding, can also neutralize virus and inhibit cell-cell fusion, as exampled by the antibody d [ ] . in this regard, one pertinent result from this study is that we found the extent of the correlation of ace competition elisa titer with the neutralizing titer (r = . , p= . , fig. c ) and competition elisa titer with cell-cell fusion inhibition titer (r = . , p= . , fig. c ) were lower than that of neutralizing titer to cell-cell fusion inhibition titer (r = . , p< . , fig. b ). finally, we evaluated whether the anti-rbd mouse sera can enhance sars-cov- infection of fcγrii expressing k cells [ ] . the results showed that sars-cov- pseudovirus alone cannot infect the k cells (fig. s ) . in addition, treatment with serially diluted (ranging from : to : ) anti-rbd sera did not enhance sars-cov- pseudovirus infection, indicating that the anti-rbd sera may not promote ade. after washing, the binding was detected by hrp conjugated anti-mouse igg antibody. (b) ng of rbd were coated and -fold serially diluted mice serum were added in the presence of ~ nm biotinylated hace followed by pbst washing. for detection, streptavidin-hrp secondary antibody was used. experiments were performed in duplicate and the error bars denote ± sd, n = . statistical significance was defined as *: p< . . safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial the spike protein of sars-cov--a target for vaccine and therapeutic development a highly conserved cryptic epitope in the receptor binding domains of sars-cov- and sars-cov immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection fc receptors in antibody-dependent enhancement of viral infections the potential for antibody-dependent enhancement of sars-cov- infection: translational implications for vaccine development molecular mechanism for antibody-dependent enhancement of coronavirus entry tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor the secret life of ace as a receptor for the sars virus immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of -ncov implications of antibodydependent enhancement of infection for sars-cov- countermeasures receptor-binding domain as a target for developing sars vaccines characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine antibody signature induced by sars-cov- spike protein immunogens in rabbits a vaccine targeting the rbd of the s protein of sars-cov- induces protective immunity immunization with the receptor-binding domain of sars-cov- elicits antibodies cross-neutralizing sars-cov- and sars-cov without antibody-dependent enhancement fc-based recombinant henipavirus vaccines elicit broad neutralizing antibody responses in mice vaccines with mf adjuvant expand the antibody repertoire to target protective sites of pandemic avian h n influenza virus potent neutralization of sars-cov- by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus the effects of secretory iga in the mucosal immune system a sars-cov- surrogate virus neutralization test based on antibody-mediated blockage of ace -spike protein-protein interaction cell entry mechanisms of sars-cov- a human monoclonal antibody blocking sars-cov- infection dengue virus neutralization in cells expressing fc gamma receptors inhibition of cell-cell fusion (a) by mice serum, correlation analysis for fusion inhibition assay with pseudo-nertralization (b) and competitive elisa (c). (a) a β-galactosidase (β-gal) reporter gene-based quantitative cell-cell fusion assay was used, in which t polymerase expressing t-s pre-incubated with mice serum followed by mixing with t promotor controlled β-gal expressing t-ace cells correlation analysis between cell-cell fusion inhibition (ic ) and pseudo-neutralization antibody titers (nt ) for sera of day and day . (c) correlation analysis between cell-cell fusion inhibition (ic ) and competitive elisa (ec ) for sera of day and day . correlation and linear regression analyses were performed in graphpad prism using pearson's correlation coefficients. statistical significance was calculated using the two-tailed test conflict of interest statement. the authors declare no conflict of interest we would like to thank the members of the center for antibody therapeutics doncho zhelev, du-san baek, liyong zhang, and xiaojie chu for their helpful discussions. this work was supported by the university of pittsburgh medical center. key: cord- - kdhf authors: lau, yuk-fai; tang, lay-hoon; ooi, eng-eong title: a tlr ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: kdhf the appearance and spread of the h n highly pathogenic avian influenza (hpai) raise concern of a possible pandemic. current preventive measures include the development of a pre-pandemic influenza vaccine and stockpiling of neuraminidase inhibitors. however, their benefits can be significantly reduced by mutations in the hemagglutinin or neuraminidase resulting in antigenic changes and the appearance of drug-resistance, respectively. drugs that target the innate immune system to achieve a ‘heightened antiviral’ state represent another class of antiviral agents that could contribute to the control and treatment of influenza infection. in this study, pika (a stabilized dsrna) provides broad-spectrum prophylaxis against a number of influenza a viruses. in addition, when pika was admixed with influenza vaccine preparations, including a formalin-inactivated whole-virion h vaccine, significant adjuvanting effect leading to accelerated viral clearance was observed in a murine model. these biological effects appear to be mediated by the ability of pika to promote the maturation of dendritic cells, including up-regulation of co-stimulatory molecules, such as cd and cd , and the induction of various cytokines and chemokines. toll-like receptor (tlr ) was shown to recognize pika in a concentration-dependent manner. the potency and versatility in its activities make pika an attractive candidate for use in an influenza pandemic. the most effective tool for controlling an influenza pandemic is a suitable vaccine. for optimal effectiveness, the pandemic vaccine needs to be based on the causative strain and therefore, vaccine cannot be produced until the pandemic strain is identified. this criterion poses serious constraints in the capability of vaccine manufacturers to prepare pandemic vaccines in advance for stockpiles. in addition, a number of studies have shown that the hemagglutinin (ha) molecules of avian h viruses are poorly immunogenic [ , ] , where up to g of antigen ( times the normal dose of human influenza virus ha) was required to elicit potentially protective responses in a substantial number of subjects [ ] . this need for a larger antigen dose further reduces the availability of the 'limited' pandemic vaccines. in an attempt to increase the immunogenicity of the antigen, adjuvants like alum and mf- have been incorporated into vaccine formulations. although alum may enhance the magnitude of the antibody response to a high dose of antigen, it does not have significant antigen-sparing effect [ , ] . other adju-vants, such as mf or a proprietary oil emulsion adjuvant, have been tested and data indicate that the inclusion of these adjuvants in the vaccine preparation would significantly increase antibody titers [ , ] . given the likelihood that a suitable vaccine will not be available before the onset of a pandemic, initial protection in a pandemic may have to rely on antiviral treatment and prophylaxis. currently available anti-influenza drugs include neuraminidase inhibitors (oseltamivir and zanamivir) or ion-channel blockers (adamantanes). the success of these drugs, both for prophylaxis or therapeutics, is based on the susceptibility of the viruses to these drugs. recently reported studies, however, have shown that a number of h isolates are resistant to these drugs [ ] [ ] [ ] . these drugs or classes of drugs alone may thus be insufficient in protecting the human population from an influenza pandemic. an alternative, antigen-independent approach that could slow down the transmission of the virus and provide more time for vaccine production is thus highly desirable. the toll-like receptors (tlrs) are a collection of receptors that detect conserved molecular components encoded by microorganisms (reviewed in refs. [ , ] ) and stimulation of these receptors by specific ligands leads to the activation of the innate immune cells, such as dendritic cells (dc), macrophages and nk cells [ , ] , through a variety of signaling pathways, such as nf-b transcription factors, c-jun nh terminal kinase (jnk), mitogen-activated protein kinases (mapks), resulting in quantitative and qualitative changes in immunological functions, including antigen presentation. in addition, cytokines, such as ifn-␣, tnf-␣ and il- p , are produced in vivo shortly after tlr activation [ , ] . the production of these cytokines results in antiviral effects observed in a number of infection models. for example, administration of synthetic lipid a analogs (tlr agonists) protects mice against an influenza challenge [ ] . a possible explanation for this anti-influenza effect could be related to the production of type interferons, which induce the activation of ifn-stimulated genes including mx proteins, protein kinase r and ' oligoadenylate synthetase [ ] . the induction of these proteins increases the resistance to the replication of the influenza virus [ ] [ ] [ ] . the present study was designed to investigate the efficacy of pika, which is a synthetic stabilized form of double stranded rna, as a stand-alone agent for its antiviral effect and to evaluate the efficacy of pika as an adjuvant when co-administered with inactivated influenza vaccines, including an h n vaccine. our results show that pika has potent anti-influenza effect when administered h prior to or immediately after influenza virus challenge in mice. the antiviral effect is not strain-specific. when used as an immuno-adjuvant, pika was able to achieve significant antigen-sparing effect. when incubated with immature dc, pika altered the expression pattern of a number of immune genes. in addition, pika was capable of interacting with both human and mouse tlr , leading to the production of nf-b. thus, in the face of a potential influenza pandemic, pika could be a candidate as a prophylactic drug or could be used as an adjuvant to increase the population coverage when pandemic vaccines become available. specific pathogen-free (spf) male balb/c mice ( - weeks old) were purchased from the centre for animal resources (care), national university of singapore. the animal protocol used in this study was reviewed and approved by the institutional animal care and use committee (iacuc) of dso national laboratories, singapore. a/puerto rico/ / (h n ) and the reassortant virus mem , which bears the ha of a/memphis/ / (h n ) and the neuraminidase (na) of a/bellamy/ (h n ) are gifts from professor lorena brown, the university of melbourne. a/ws/ (vr- ) is from the american type culture collection (atcc). the reverse-engineered h n influenza virus was made based on the method described by hoffmann et al. [ ] , using an expression plasmid provided by dr brendon hanson (dso national laboratories, singapore). the ha sequence of a/indonesia/cdc l/ (cy , a clade , subclade virus which is closely related to a/indonesia/ / ) was downloaded from genbank and was synthesized by geneart ag (regensburg, germany). the viruses were grown in the allantoic cavity of -day-old embryonated chicken eggs for days at • c. allantoic fluids were pooled and divided into aliquots, and stored at − • c until use. the - south hemisphere seasonal influenza vaccine, fluvax, was from csl ptd ltd. (victoria, australia). virions of the reverse-engineered h n virus were concentrated by ultra-centrifugation and resuspended in pbs. formalin-inactivated purified whole-virion vaccine was prepared by inactivating the virions with . % formalin for days at • c followed by dialysis in pbs. the total protein concentration in the vaccine was measured by coomassie plus (pierce biotechnology, inc., il, usa). the amount of ha content was estimated to be % of the total protein content. complete and incomplete freund's adjuvant (cfa and ifa) were obtained from sigma-aldrich (st louis, usa). pika was obtained from newbiomed pika pte ltd. (singapore). for intranasal (i.n.) immunization, groups of mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine and xylaxine followed by i.n. administration of immunogens in l. for immunization by the subcutaneous (s.c.) route, the immunogens were administered in l at the base of the tail. in selected experiments, g of pika was given to anesthetized mice i.n. in a volume of l. when given by the s.c. or i.p. route, g of pika was given in l volume per dose. pika contains dsrna that is greater than base pairs in length. three weeks after final vaccination, mice were challenged i.n. with . - . pfu of various strains of influenza virus in a volume of l. mice were sacrificed at various time-points and pulmonary viral titers were determined using previously described methods [ ] . mice were anesthetized by ketamine/xylaxine injection and blood samples were collected from the orbital plexus. sera were collected and stored at − • c until analysis. the titer of immunogen-specific antibodies were quantified by elisa, as previously described [ ] . avidity elisa was carried out using previously described method [ ] . hek cells were maintained in optimem (invitrogen) with % fetal calf serum and were transfected in flat-bottomed -well plates as previously described [ ] . each sample was tested in triplicate. six hours after the stimulation, luciferase and ␤-galactosidase activity in each sample was measured by commercial kits (promega corporation, wi, usa). the relative stimulation of nf-b was calculated by normalizing luciferase activity with ␤-galactosidase activity and was expressed using the readings from unstimulated wells as a baseline. the level of expression of various cytokine genes was determined using a lightcycler (roche, in, usa) using primers described in giulietti et al. [ ] . samples containing g of total rna from the abovementioned dc experiment were used for microarray analysis. piqor immunology microarrays (miltenyi biotech, gladbach, germany) were used. samples were amplified and labeled with different fluorochromes according to manufacturer's instructions. samples from pika-stimulated dc and unstimulated dc were labeled with cy and cy , respectively. after hybridization, the slides were scanned using the imagene software (biodiscovery). each gene was printed in quadruplicate on the array. net signal intensity, data normalization and calculation of the cy /cy ratios were performed by miltenyi biotech using the piqor analyzer software. one million d cells were stimulated with g of pika or g of lps ( :b , sigma-aldrich) overnight. the supernatants were collected and the cytokine protein levels measured using the bioplex protein array system (bio-rad, hercules, ca), in duplicates and against a standard curve according to manufacturer's instructions. the significance of any difference between any two different groups was assessed by the mann-whitney test using prism (graphpad software, ca, usa). reported p values < . are considered significantly different. in this study, the murine influenza model was employed to demonstrate the antiviral properties of pika. in this model, anesthetized mice were challenged with influenza virus i.n. this mimics a total respiratory tract infection and the pulmonary viral titer reaches its peak on day post-infection (pi) and remains at a high level till day pi (data not shown). using this model, groups of five mice were given g of pika i.n. h prior to challenge with plaque forming unit (pfu) of influenza/a/pr/ / . the pulmonary viral titer in the lungs was determined on day pi. as shown in fig. a , mice that received pika treatment at and h pi had the lowest pulmonary viral titer at day and this was significantly lower than the titer in control mice that received pbs (p = . ). in addition, significant reductions in pulmonary viral titers were also observed in mice that received only one dose of pika at h prior to infection. the difference in the pulmonary viral titer between the group that received daily treatment and the group that received only one dose of pika was not statistically significant (p = . ). after demonstrating that pika has an inhibitory effect on influenza viral replication in vivo, it was of interest to determine the optimal conditions for the treatment. to examine this, mice were given different concentrations of pika i.n. and were challenged with pfu of pr virus i.n. the mice were treated at and h and were sacrificed on day . a dose-dependent response was observed (fig. b) , in which mice given g of pika daily had the lowest mean viral titer among all the groups. an inhibitory effect of pika on viral replication was detected at g per dose but not at g per dose (p = . ). to determine whether pika could be used as a treatment option at the time of infection, mice were given pika immediately after infection with pfu of pr virus. as shown in fig. c , this treatment protocol was effective in reducing the pulmonary viral titer and the titer was not significantly different from the prophylaxis group that received pika h prior to infection (p = . ). however, it is apparent that daily treatment is preferable because the group that received only a single dose had a significantly higher titer of virus in the lungs compared to those that received daily doses (p = . ). intranasal administration of pika produced the most significant anti-influenza effect compared to s.c. or i.p. administration of the drug (fig. d ) though the pul-monary viral titers in the treated mice were still significantly lower than the titers of the pbs control group (p = . ). after demonstrating the effectiveness of pika in inhibiting the replication of pr virus in vivo, we sought to determine whether similar inhibition would be observed with different influenza viruses. groups of five mice were given pika treatment and were challenged with pfu of pr (h n ), mem (h n ) and a/ws/ (h n ), as previously described. on day pi, the viral titer of the pika-treated groups were significantly lower than the titers of the pbs group, regardless of the subtype of the challenge viruses ( fig. a -c, p = . , . , . ) respectively. in addition, mice were also challenged with higher doses of virus, or pfu ( and ld respectively) of pr . as shown in fig. a and b, the pika-treated mice had lower pulmonary titers than the pbs control group at these higher challenge doses (p = . ). in addition, when challenged with . pfu of mem , there was a % reduction in viral titer in pika-treated mice compared with the pbs control group (fig. c) . to demonstrate the therapeutic potential of the treatment, groups of five mice were challenged with pfu of pr i.n. and received pika treatment or h pi. on day pi, as shown in fig. d , significant reduction in pulmonary viral titers was observed in pika-treated mice (p = . , . ) compared with the pbs-treated group. therefore, in summary, we demonstrated that pika has the ability to inhibit the replication of several strains of influenza in vivo. although intranasal administration of the drug before the establishment of an infection provided the best protection, substantial viral reduction could still be achieved if the drug were given in the course of an established infection. since pika is an effective adjuvant for hepatitis b vaccine [ ] , we examined whether pika could enhance the immunogenicity of influenza vaccine. we sought to demonstrate that the enhancement of the humoral responses would lead to significant viral reduction in replication of challenge virus. previous studies have showed that when administrated at between . and g per dose, different forms of influenza vaccine could induce robust antibody responses without any external adjuvant [ ] [ ] [ ] with protection against viral challenge [ ] . as shown in fig. a , mice immunized with a single dose of the trivalent vaccine at . g s.c. had robust antibody responses which were comparable to those induced when the vaccine was admixed with cfa. when the vaccine dose was reduced by - fold ( . g) , there was about -fold reduction in the mag-nitude of the antibody responses. to mimic a vaccine shortage situation, a dose of . g, (∼ -fold reduction from the optimal dose) was selected and we evaluated whether the inclusion of pika could compensate for the reduction in antigen concentration. groups of five mice were vaccinated with a suboptimal dose of fluvax vaccine by the s.c. or i.n. route. three weeks after priming, sera were collected from the mice before they were boosted. sera were collected from the mice weeks after boost. as shown fig. . pika acts as a potent adjuvant by enhancing the immunogenicity of the seasonal influenza vaccine. (a) groups of four mice were vaccinated with either or l of fluvax influenza vaccine (containing . or . g of ha from each subtype) in pbs or in cfa by the subcutaneous route. on day , sera were collected and the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. (b) groups of five mice were vaccinated with . l of fluvax influenza vaccine (containing ng of ha from each subtype) in pbs, either by the subcutaneous route at the base of the tail or by the intranasal route. some groups received the vaccine with additional adjuvant as indicated in the x-axis. for those that received pika as an adjuvant, g of pika was admixed with the vaccine prior to administration. on day post-vaccination, sera were collected and the mice received another boost by the same route and sera were collected on day . the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. each dot represents the antibody titer of an individual mouse. the primary and secondary antibody titers are represented by the black and red dots respectively and the lines represent the geometric mean antibody titer of each group. the '*' symbol indicates that the difference between two groups is statistically significant (p < . ). (c) to determine the level of protection mediated by the antibody response, the mice were challenged with pfu of pr intranasally weeks after the boosting and the pulmonary viral titer was determined on day post-infection. closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group of mice. the percent reduction in mean viral titer relative to the pbs control group is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < . ). (d) serum samples were diluted : and the avidity of the antibodies was tested in a urea-displacement elisa. the o.d. of the urea-treated plates was expressed as a percent of the untreated plates. data are represented as the mean and standard derivation of five mice. in fig. b , only two out of the five mice that received the vaccine without an adjuvant by the s.c. route had detectable amounts of anti-ha antibody in the primary response. for the cfa group, four mice had anti-ha antibody whereas all animals in the vaccine with pika group had detectable levels of anti-ha antibody. in the secondary responses, although all mice given vaccine alone developed anti-ha antibody, the antibody titers were significantly lower than those that were given vaccine with pika (p = . ). as for intranasal delivery of vaccine, none of the vaccine protocols was able to elicit detectable anti-ha responses after one dose. when two doses of vaccine were given with pika, the vaccine recipient group showed a low titer of anti-ha response. the response was substantially improved by the addition of pika to the vaccine (p = . ). interestingly, the titer of the sera attained by two doses administered i.n. was comparable to titers achieved by two doses given by s.c. injection (p = . ). in order to demonstrate that the enhanced antibody responses can lead to improved efficacy, vaccinated mice were challenged with pfu of pr i.n. and the pulmonary viral titers were determined days after challenge. as shown in fig. c , mice given two doses of the vaccine showed a % reduction in viral titer compared to the pbs control group (p = . ). although the cfa group had the highest titer of anti-ha responses (fig. a) , the group only showed a % reduction in viral titer, which was not statistically different from the group that received unadjuvanted vaccine (p = . ). on the other hand, the group given vaccine with pika the pulmonary viral titer was determined as previously described. the percent reduction in mean viral titer relative to the group treated with pbs is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < . ). showed the most potent viral reduction, with . % reduction in viral titer compared to the pbs control group, and this difference was statistically significant (p = . ). the group that received pika alone served as control to ensure that the observed reduction in pulmonary viral titer was due to the anti-ha antibody responses, instead of the direct inhibitory effects of pika on viral replication shown in the fig. . the enhancement in viral clearance was also observed in the group that received the vaccine by the i.n. route (fig. c) . mice that received either vaccine alone or vaccine with pika showed a . % and . % reduction, respectively, and the difference between the groups was statistically significant (p = . ). it is interesting to note that, although the group received the vaccine admixed with cfa had the highest antibody titers (fig. b) , however, the mice had higher pulmonary viral titers compared with mice received the vaccine administrated with pika (fig. c ). to investigate this discordance between the two parameters, the relative avidity of the antibodies of the two groups against the vaccine antigen was examined in a urea-displacement elisa. as shown in fig. d , the group received the vaccine with pika had antibodies with higher avidity to the surface glycoprotein of the virus as significantly more antibodies remained bound to the ha when exposed to m urea as compared to those induced with cfa as an adjuvant (p = . ). there is an accumulating body of data showing that h n virus vaccines are generally poorly immunogenic. we therefore proceeded to examine whether pika was an effective adjuvant for an h n vaccine. groups of five mice were inoculated by the s.c. route with a formalin-inactivated h vaccine, with or without an adjuvant, and weeks later, sera were collected to determine the antibody titer against the virus. as shown in fig. a , the whole-virion vaccine was capable of inducing a measurable titer of antibody after one dose. however, with the inclusion of pika, there was a ∼ -fold increase in antibody titer (p = . ), and this was comparable to the titer achieved in the group that received the vaccine with cfa. when challenged with the homologous virus i.n., however, this group of mice was the only group which showed significant reduction in pulmonary titer on day pi (fig. b, p = . ) . the efficacy was further boosted by a second dose of h vaccine with pika. mice in this group showed a further -fold reduction in viral titer (fig. c ). in addition, the vaccine-pika formulation was efficacious in reducing viral replication even when the vaccine was delivered intranasally. in contrast, in the absence of pika, the vaccine administered by the i.n. route failed to induce a significant reduction in viral titer compared with the pbs control (p = . ). in summary, we have demonstrated that the inclusion of pika in two different formations of influenza vaccine can achieve substantial antigen-sparing with robust humoral immune responses, leading to potent pulmonary viral titer reduction in vivo. in an attempt to elucidate the biological basis for the inhibition of viral replication and the adjuvant effects of pika, we sought to identify the receptor(s) that might be involved in these processes and to examine how cells of the innate immune system respond to pika stimulation. using a human tlr -expressing plasmid, we showed that pika is capable of interacting specifically with tlr in a dose-dependent manner (fig. ) . similar results were obtained when the experiment was carried out with mouse-tlr -expressing plasmid (data not shown). next, we investigated the outcome of stimulating dc in vitro with pika given that dcs are the most potent antigen presenting cells (apc), responsible for antigen capture and presentation to naïve t lymphocytes. using primary immature dc culture, d cells, we examined how dc responded to pika stimulation in vitro. pika was added into dc cultures for h following which total rna was extracted from the cells. the expression level of more than immunologically related genes between pika-stimulated and unstimulated samples were compared using microarray technology. as shown in fig. a , pika-stimulated dcs up-regulated expression of a number of cellular activation markers, such as cd and cd compared to unstimulated cells. a number of genes that have important immunological functions were also found to be up-regulated after pika treatment, including irf- (interferon regulatory factor ), il- , il- , il- and il- receptor. many chemokines, such as mip (macrophage inflammatory protein), rantes, mcp- (macrophage chemoattractant protein) and ip- (interferon gamma inducible protein) were also found to have increased expression. quantitative real-time pcr was used to confirm these findings. using cdna from unstimulated dc as the baseline, many genes were up-regulated in a dose-dependent manner (fig. b) . while certain genes, such as inos and il- , were up-regulated to a comparable level in lpsstimulated dc, other genes, such as cd l, mip and ifn-␥, were up-regulated more readily by pika stimulation. to demonstrate that the observed changes in gene expression lead to changes in these cytokines/chemokines, the level of these proteins in the culture supernatants was measured. consistent with the up-regulation of cytokine/chemokines genes, there was an increase in the production of these cytokines/chemokines in the supernatant from pika-stimulated dc compared with those from the unstimulated dc (fig. c ). it has been years since the first case of human infection with a highly pathogenic h n virus was identified in hong kong [ ] and the virus has now spread to many regions, including southeast asia, western china [ ] , africa [ ] , turkey [ ] and siberia [ ] . most human cases to date involve close contact with infected poultry and human-to-human transmission remains limited [ ] . nonetheless, the lack of anti-h immunity in humans, together with the continuous evolution of the virus in close proximity with the human population, threatens a pandemic. should the virus acquire the property of efficient transmissibility between humans, in combination with the high case-fatality rate resulting from infection, this virus has the potential of causing an influenza pandemic with damage on a scale similar to that achieved by the 'spanish' flu pandemic. therefore, there is an urgent need to develop effective prophylaxis and therapeutic strategies in preparation for a possible pandemic. in the current study, using a murine influenza model, we have evaluated the potential of pika for prophylaxis and treatment of influenza infection as well as an adjuvant for influenza vaccine. our data demonstrate that administration of pika intranasally prior to or shortly after an influenza infection can inhibit influenza replication, leading to a significant reduction in pulmonary viral titers. this effect is unlikely to be due to a direct antiviral activity, because pre-incubating influenza virus with pika did not inhibit virus infectivity for embryonated chicken eggs (lynn tang, unpublished data). instead, pika appears to act by stimulating the innate immune system (fig. ) , resulting in the production of several chemokines, cytokines and interferons and these, in turn, mediate the observed antiviral activity. myxovirus resistance (mx) proteins, protein kinase r and ' ' oligoadenylate synthetase are some of the products that have been reported to be up-regulated after type interferon production (reviewed in ref. [ ] ), achieving an antiviral state in the host [ ] . the fact that pika administration results in the stimulation of several antiviral proteins in the host, it decreases the likelihood that viruses will develop resistance through mutation. furthermore, as pika does not target a specific component of the virion for its antiviral activity, it is likely to be effective against multiple strains and subtypes of influenza viruses as demonstrated in this study. this broad-spectrum activity is not limited to influenza a viruses only. a number of studies have shown that tlr-ligands are capable of inhibiting a wide range of viruses, including herpes simplex virus- [ ] , cytomegalovirus [ ] , parainfluenza [ ] , west nile virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] and influenza virus [ , ] . the abovementioned advantages make this novel antiviral approach a compelling alternative to traditional antiviral drugs and vaccines especially since avian influenza viruses have rapidly developed resistance to antiviral drugs [ ] and some mutant viruses can maintain drug-resistance without losing virulence [ ] . in addition, thitithanyanont et al. showed that stimulating dc with poly(i:c) conferred the cells with resistance to the cytopathic effects of h n virus which might be important for the induction of virus-specific immune responses during the infection [ ] . apart from being an effective agent for prophylaxis and treatment, pika can also act as an effective adjuvant. using two different forms of influenza vaccine (split subunit vaccine and whole inactivated vaccine), the magnitude of the humoral responses elicited by the combination with pika was comparable to those formulated in freund's adjuvant. our observation is consistent with the report by shen et al. [ ] which showed that administering fig. . pika induces maturation of dendritic cells, with expression of a wide range of immunological genes. immature murine dendritic cells, d cells, were incubated with g per ml of pika or were unstimulated overnight. (a) total rna was harvested from the cells and converted into cdna. the cdnas derived from pika-stimulated dc and unstimulated dc were amplified and labeled with cy and cy dye respectively. the samples were hybridized overnight to a microarray chip and fluorescence signals were measured by an array scanner. each gene target was printed times on the array and the normalized mean cy /cy ratio and coefficient of variation (cv) of the four replicates were determined by the software. (b) rna was harvested from the dcs and converted into cdna. the expression level of each cytokine gene was determined by quantitative real-time pcr. the expression level was normalized with beta-actin, a house-keeping gene, and data were expressed relative to unstimulated samples. the bars and error bars represent the mean and standard deviation of triplicate samples and are representative of two independent experiments. (c) supernatants were harvested from dcs stimulated overnight with either g of pika, g of lps or unstimulated. fifty microliters of the supernatant was used to test for the presence of various cytokines/chemokines in the supernatants using the bioplex protein array system and were measured in duplicate. the bars and error bars represent the mean and standard error. pika with hepatitis b vaccine boosted the humoral response. we explored the concept further by demonstrating that a substantial antigen-sparing effect could be achieved by incorporating pika into vaccines. using the commercial split vaccine, the antigensparing effect was about -fold ( fig. a and b) . furthermore, we demonstrated that the augmented humoral response led to a func-tional reduction in viral titer (fig. c ). our data also showed that successful immunization could be achieved by a non-parenteral route. with two doses, the magnitude of the response achieved by intranasal immunization was comparable to that achieved by subcutaneous immunization and was associated with a similar level of viral clearance. our finding is consistent with the report from ichinohe et al. [ ] in which they showed that another tlr ligand, ampligen, was capable of enhancing the immunogenicity of a trivalent inactivated influenza vaccine when administrated intranasally. in their study, the enhancement effect was demonstrated using a higher dose of the trivalent vaccine ( g) and in this current study, we extended the observation further by showing that similar enhancement effect can be achieved even when the concentration of the immunogen was reduced to . g ( -fold lower). the antigen-sparing effect of pika might be an important strategy for maximizing vaccine coverage when vaccine supplies are limited. it is of interest to note that although the group that received the influenza vaccine with pika did not have the highest antibody titer, it had the lowest pulmonary viral titer (a -fold reduction compared with the cfa adjuvant vaccine group). the higher avidity of the antibodies induced by the vaccine with pika might allow more effective inhibition of viral replication in vivo which translated into lower pulmonary viral titers as observed in this study. this suggests that in addition to augmenting the amount of antibody produced, pika may also have an impact on the quality of the humoral responses, such as affinity maturation [ , ] . because pika is a stabilized form of double stranded (ds) rna that lacks direct anti-influenza activity, we evaluated its effect on the innate immune system in search of the mechanism underlying the observed activity. dsrna was shown to be a ligand of tlr [ ] and using a transient transfection system, we showed that pika maintained its tlr binding property. incubation of pika with primary immature murine dc in vitro resulted in maturation of dendritic cells, with marked up-regulation of co-stimulatory molecules, such as cd and cd and cytokines with potent immunostimulatory functions such as il- and il- . our observation is consistent with those observed by shen et al. with bmdc [ ] . the maturation process enables dc to act as a potent antigen presenting cells for effective activation of naïve lymphocytes [ , ] , which in turn, leads to a more robust immune response. in addition, a study conducted by perera et al. [ ] showed that a vaccinia virus expressing il- induced superior cellular and humoral immune responses compared to the parental strain. therefore, the induction of il- by pika could also have resulted in a more efficient induction of cd + t cell responses, translating to more robust humoral responses. lung tissue has recently been shown to constitutively express tlr in vivo in mice [ ] and intranasal administration of pika could directly stimulate these tlr- expressing cells, which is perhaps why pika worked most effectively when administrated intranasally (fig. d) . we believe that pika will have similar antiviral activity in humans, because we showed that human tlr- can recognize pika (fig. ) and a number of studies have demonstrated the expression of tlr- in various respiratory cell types, such as nasal epithelial cells [ ] , airway smooth muscle cells [ ] and airway epithelial cells [ ] . in addition, ds rna was found to be the most effective tlr ligand in activating airway epithelial cells in producing il- [ ] . although our experimental data are limited to tlr- , dsrna can also be recognized by rna helicase, rig- and mda , and can induce nf-b activation with type- ifns [ ] . it is possible that pika stimulates several pathways in vivo to achieve its antiviral and adjuvant activities. the knockout-mice, such as tlr k/o, mda k/o and rig- k/o mice, will be useful in shedding more light on the contribution of these receptors to the activity of pika. in summary, although poly(i:c) has been shown to be an effective mucosal adjuvant, its use in clinical trials was associated with side-effects that has limited its usage [ ] . in a recent toxicity trial, pika showed no significant toxicity in mice and a phase clinical study showed that the use of pika as an adjuvant was well-tolerated in humans without significant side-effects (peter brazier, unpublished data). the safety profile, together with the long shelf-life, makes pika an attractive complement to currently licensed antivi-ral drugs. in the early phase of an influenza pandemic, when a well-matched vaccine is unlikely to be available, pika can be used as an antiviral drug for temporary protection of the susceptible population, slowing down the rate of viral transmission and allowing more time for vaccine production. furthermore, global influenza vaccine production is likely to lag well behind the demand for pandemic influenza vaccine, so the inclusion of an effective antigensparing adjuvant such as pika can ensure that limited supplies of vaccine achieve maximum coverage. safety and antigenicity of non-adjuvanted and mf -adjuvanted influenza a/duck/singapore/ (h n ) vaccine: a randomised trial of two potential vaccines against h n influenza safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine safety and immunogenicity of an inactivated split-virion influenza a/vietnam/ / (h n ) vaccine: phase i randomised trial vaccines for seasonal and pandemic influenza boosting immunity to influenza h n with mf -adjuvanted h n a/duck/ singapore/ vaccine in a primed human population antigen sparing and cross-reactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial reduced sensitivity of influenza a (h n ) to oseltamivir oseltamivir resistance during treatment of influenza a (h n ) infection distribution of amantadine-resistant h n avian influenza variants in asia innate immune recognition: mechanisms and pathways type cytokine/chemokine production by mouse nk cells following activation of their tlr/myd -mediated pathways maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway synthetic toll-like receptor agonists stimulate innate resistance to infectious challenge interferons, interferon-like cytokines, and their receptors human and mouse mx proteins inhibit different steps of the influenza virus multiplication cycle host gene influences sensitivity to interferon action selectively for influenza virus interferon-induced antiviral actions and their regulation a dna transfection system for generation of influenza a virus from eight plasmids relative immunogenicity of the coldadapted influenza virus a/ann arbor/ / (a/aa/ / -ca), recombinants of a/aa/ / -ca, and parental strains with similar surface antigens antigenic determinants of influenza virus hemagglutinin. xii. the epitopes of a synthetic peptide representing the c-terminus of ha maturation of immunoglobulin g avidity after rubella vaccination studied by an enzyme linked immunosorbent assay (avidity-elisa) and by haemolysis typing myd -adapter-like) is required for toll-like receptor- signal transduction an overview of real-time quantitative pcr: applications to quantify cytokine gene expression pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika immune responses of mice to influenza subunit vaccine in combination with cia as an adjuvant intranasal immunisation with influenza-iscom induces strong mucosal as well as systemic antibody and cytotoxic t-lymphocyte responses crossprotection against h n influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine human influenza a h n virus related to a highly pathogenic avian influenza virus properties and dissemination of h n viruses isolated during an influenza outbreak in migratory waterfowl in western china confirmation of h n avian influenza in africa h n avian influenza: the turkish dimension h n influenza virus avian influenza a (h n ) infection in humans type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors posttherapy suppression of genital herpes simplex virus (hsv) recurrences and enhancement of hsv-specific t-cell memory by imiquimod in guinea pigs efficacy of s against guinea pig cytomegalovirus infection attenuation of virus-induced airway dysfunction in rats treated with imiquimod effect of interferon-alpha and interferon-inducers on west nile virus in mouse and hamster animal models evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice prophylaxis of acute respiratory virus infections using nucleic acid-based drugs nucleic acidbased antiviral drugs against seasonal and avian influenza viruses avian flu: isolation of drug-resistant h n virus neuraminidase inhibitor-resistant recombinant a/vietnam/ / (h n ) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo high susceptibility of human dendritic cells to avian influenza h n virus infection and protection by ifn-alpha and tlr ligands coadministration of cpg oligonucleotides enhances the late affinity maturation process of human anti-hepatitis b vaccine response enhancement of antibodies to the human immunodeficiency virus type envelope by using the molecular adjuvant c d recognition of double-stranded rna and activation of nf-kappab by toll-like receptor dendritic cell maturation is required for initiation of the immune response development of smallpox vaccine candidates with integrated interleukin- that demonstrate superior immunogenicity, efficacy, and safety in mice detrimental contribution of the toll-like receptor (tlr) to influenza a virusinduced acute pneumonia expression of toll-like receptors in cultured nasal epithelial cells toll-like receptor , , and expression and function in human airway smooth muscle activation of airway epithelial cells by toll-like receptor agonists the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses a phase i-ii trial of multiple-dose polyriboinosic-polyribocytidylic acid in patieonts with leukemia or solid tumors we gratefully acknowledge kanta subbarao (niaid, nih) for helpful discussions and critical reading of the manuscript. this study was supported by future systems directorate, ministry of defence, the republic of singapore. key: cord- -yj m n f authors: wang, keyang; holtz, kathleen m.; anderson, karl; chubet, richard; mahmoud, wafaa; cox, manon m.j. title: expression and purification of an influenza hemagglutinin—one step closer to a recombinant protein-based influenza vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: yj m n f numerous human infections with avian influenza viruses in asia in recent years have raised the concern that the next influenza pandemic is imminent. the most effective way to combat influenza is through the vaccination of the public. however, a minimum of – months is needed to develop an influenza vaccine using the traditional egg-based vaccine approach. the influenza hemagglutinin protein (ha), the active ingredient in the current vaccine, can be expressed in insect cells using the baculovirus expression vector system and purified rapidly. an influenza vaccine based on such a recombinant antigen allows a more timely response to a potential influenza pandemic. here, we report an innovative monitoring assay for recombinant ha (rha) expression and a rapid purification process. various biochemical analyses indicate that the purified rha is properly folded and biologically active. influenza is a highly contagious, acute viral respiratory disease, which causes significant morbidity and mortality worldwide each year [ ] [ ] [ ] . influenza viruses are singlestranded ribonucleic acid (rna) viruses surrounded by a lipid containing envelope spiked with two glycoproteins: hemagglutinin (ha) and neuraminidase (na). these glycoproteins, and ha, in particular, have been recognized as key antigens in the host response to influenza virus in both natural infection and vaccination [ , ] . the viruses are well known for their ability to mutate to circumvent immunity and re-infect the host. an antigenic shift, a major antigenic change of the virus due to the genetic re-assortment of two subtype strains that co-infected a host, can cause an influenza pandemic since the population may have no inherent immunity against the new strain [ , ] . the avian influenza a (h n ) epizootic outbreak and numerous human infections with h n in asia in recent years, and events such as the infection of two nurses attending to avian influenza patients in vietnam (who, march , ) and a possible personto-person transmission in a family cluster of the disease in thailand [ ] , continue to raise concern that the next influenza pandemic is imminent. a proven, effective way to combat influenza is through vaccination of the public using the trivalent vaccine produced in embryonated chicken eggs. in the current process, three influenza strains selected by who/cdc are propagated in chicken eggs, chemically inactivated, and semi-purified. the egg-based technology, however, is unable to respond to a pandemic crisis. vaccine development and production take several months following the identification of potential strains and typically requires the re-assortment with a high yield strain to obtained adequate growth properties [ ] [ ] [ ] [ ] . a minimum of - months is needed to develop an influenza vaccine using this approach. more importantly, the h avian influenza strains responsible for recent epizootic outbreak involving numerous human infections are lethal to chicken eggs used for vaccine production and to the chickens that lay the eggs. if the severe acute respiratory syndrome (sars)-coronavirus outbreak serves as a guide, the next influenza pandemic will likely have global consequences spreading within weeks, if not within days. thus, a system that can rapidly produce new influenza vaccine is needed to prevent or to effectively reduce the impact of pandemic influenza. two new approaches have shown great promise to replace the egg-based technique [ ] [ ] [ ] . one is cell culture-based, and the other is recombinant protein (antigen)-based. the cell culture-based approach involves production of influenza viruses in cell culture followed by the current (egg-based) virus inactivation and purification for the down stream processing. the advantages are: cell cultures are easier to handle and can be scaled up in a short period of time, and the influenza vaccines produced with this approach have been tested in phase i and phase ii clinical trials and were found to be safe and at least as effective as the vaccines produced in embryonated chicken eggs [ ] [ ] [ ] . a limitation of the cell culture-based approach is that the process still requires the production of a high-yielding re-assorted virus. this process also may introduce cell line specific mutations in the genes that can lead to the selection of variants characterized by antigenic and structural changes in the ha protein, potentially resulting in less-efficacious vaccines [ ] [ ] [ ] . additional hurdles include: the production and handling of a dangerous virus requires the availability of a high containment facility; mammalian cells can harbor animal viruses that may lead to safety concerns; the residues from the expressing cells may cause some unknown side-effects since no thorough purification process has been introduced into the manufacturing process. on the other hand, the recombinant protein-based approach involves production of viral antigens such as ha and na in cell culture with recombinant dna technology and utilization of the purified antigens as the active ingredients in the vaccine. the rha influenza vaccines developed using the baculovirus-insect cell expression system has been tested in several phase i and phase ii human clinical trials involving over subjects that demonstrated safety, immunogenicity and efficacy [ ] [ ] [ ] [ ] [ ] . in elderly adults, rha vaccine is equally or more immunogenic than the egg-based vaccine (treanor jj, et al. dose-related safety and immunogenicity of a trivalent baculovirus-expressed influenza virus ha vaccine in elderly adults, manuscript in preparation). interestingly, two h n rha vaccines (derived from two strains of a/new caledonia/ / or a/texas/ / ) provided partial protection against the lethal challenge of a reconstructed highly lethal pandemic influenza virus (also a h n strain) in mice, suggesting that cross protection against drifted strains is definitely feasible [ ] . to meet the challenge of a potential influenza pandemic, however, a reliable expression system and a quick, efficient downstream purification process are needed. in this communication, we reported a rapid process capable to purify rha (h n , a/new caledonia/ / ) from the fermentation to % purity within h with a % overall yield. since all chromatographic media used here are chemically stable and commercially available, the process can be easily scaled up in a gmp facility. various biochemical analyses indicated that the purified rha is properly folded and biologically active. in addition, we also developed a quick, simple analytical assay to monitor the expression of rha in the insect cell fermentation to ensure the rha production. the influenza vaccine strain-a/new caledonia/ / (h n )-was obtained from the cdc. the full-length ha gene (containing the ha and ha genes) from the influenza viruses was cloned using rt-pcr and inserted into a baculovirus transfer vector developed by protein sciences corporation. this specialized vector contained the promoter from the baculovirus polyhedrin gene flanked by sequences naturally surrounding the polyhedrin locus. next, the transfer vector was co-transfected into insect cells with the linearized baculovirus genomic dna (autographa californica nuclear polyhedrosis virus) depleted of the polyhedrin gene and part of an essential gene downstream of the polyhedrin locus. the homologous recombination between the transfer plasmid and the linearized viral dna rescued the virus, resulting in recovery efficiencies of recombinant virus of nearly %. recombinant viruses were then selected by plaque assay. the plaque-derived recombinant baculovirus was then used to create a virus stock by infecting increasingly larger cultures of the proprietary insect cells (expressf+ ® , derived from sf cells) in serum-free culture medium (protein sciences fortified medium). the virus stock was then used to infect insect cells ( . × cells/ml) to produce rha in a -liter applikon bioreactor. the multiplicity of infection (moi) of the virus stock was for the experiment. to monitor the infection process and the expression of ha, ml samples were taken from the bioreactor at various times. one millilitre was used for analyzing the changes in cell density, cell viability and cell size distribution. two millilitres were centrifuged at rpm. the supernatant and pellet were stored separately at − • c to be used for srid, gel and blot analysis. one millilitre was used for hemadsorption analysis. the rest was used for protein purification. to . ml fermentation samples (insect cells uninfected, infected with recombinant baculovirus containing ha genes, and infected with recombinant baculovirus containing a non-ha gene) in a . ml eppendorf tube, . ml of % chicken red blood cells (charles river-spafas, north franklin, ct) in pbs was added and shaken gently for min at room temperature. at the end of incubation, the tube was flipped gently for five times to get a homogenous suspension. then, l of the suspension was pipetted on a glass plate and observed under a microscope (ck , olympus optical co., japan) in three representative view fields. on average, about - insect cells were counted in each field. to reduce the chance of false positives, only the insect cells attached by three or more rbcs were counted as the rbc-bound insect cell. the percentage of rbc-bound insect cells against the total insect cells in each time point was calculated from three fields. at each time point, . ml of fermentation sample was analyzed with an automated cell analyzer (cedex as , innovatis gmbh, germany) for cell density, cell viability and cell size distribution using the procedure described by the manufacture. buffer a: mm sodium phosphate, . mm edta, . % tergitol-np , % glycerol, ph . . buffer b: mm sodium phosphate, . % tergitol, % glycerol, ph . . buffer c: mm sodium phosphate, mm nacl, . % tergitol, % glycerol, ph . . buffer d: mm sodium phosphate, . % tween- , % glycerol, ph . . buffer e: mm sodium phosphate, . % tween- , % glycerol, ph . . buffer f: mm sodium phosphate, . % tween- , % glycerol, ph . . buffer g: mm sodium phosphate, mm nacl, . % tween- , ph . . sanitation buffer: . m nacl, . m naoh. unosphere-q (bio-rad, hercules, ca) column, ø . cm × cm, ml; sp-sepharose fast flow (ge/amersham/pharmacia, piscataway, nj) column, ø . cm × cm, ml; hydroxyapatite type i column (hx-i, bio-rad, hercules, ca), ø . cm × . cm, . ml. the fermentations producing rha were harvested by centrifugation at - h post-infection. the cell pellet ( . g) was extracted with ml of % tergitol np- in buffer a by stirring on a magnetic stirrer at • c for min. the extract was clarified by centrifugation at , × g for min. the supernatant was loaded on q/sp columns (equilibrated with buffer a) in tandem at ml/min. after loading, the columns were washed with ml of buffer a. then, the columns were disconnected. ha was eluted from the sp column with ml of buffer b and ml of buffer c, consecutively. the q/sp columns can be regenerated by washing with column volumes (cv) of sanitation buffer and cv of water and equilibrated with cv of buffer a. the ha fraction in buffer b ( ml) was loaded on a hx-i column at ml/min. the column was washed with ml of buffer b. the ha was eluted from the hx-i column with increasing phosphate concentration (buffers d, e and f). the ha preparation in buffer d was further purified and concentrated by ultrafiltration with a stir cell using a kda mwco regenerated cellulose membrane with buffer g. the hx-i column can be regenerated by washing with cv of water, and equilibrated with cv of buffer c. the rha contents in all preparations were determined with srid assay as described by williams [ ] and manchini et al. [ ] . the assay is based on the diffusion of rha into a % agarose gel containing antibodies against the ha. the interaction between antigen and antibody produced a precipitation ring of which the size was directly proportional to the amount of antigen applied. the diameters of the rings in the srid assays were determined with a measuring magnifier (baush/lomb, - - ). the diameters of the precipitate ring were used to determine the actual concentrations based on standards provided by the center for biologics evaluation and research of fda. for complete deglycosylation, g of purified rha was deglycosylated with units of peptide-n-glycosidase f (pngase f, new england biolabs, beverly, ma, usa) or endoglycosidase h (endo h, new england biolabs) at • c for min as described previously [ ] . for limited deglycosylation, g of rha was treated with . g of trypsin on ice for min. the digestion was stopped by adding × denaturing buffer and boiling for min. then the trypsin treated rhas were deglycosylated with , or units of pngase f or endo h on ice, at • c or at • c for - min. the reactions were stopped by adding × sds sample buffer and boiling for min. the protein species at various deglycosylation stages were resolved on sds-page. vaxigrip influenza vaccine - was purchased from canada drug delivery (nanaimo bc, canada). the purity of rha was measured on sds-polyacrylamide gels stained with coomassie blue using scanning laser densitometry (model , bio-rad, hercules, ca, usa) and peak integration analysis. the total amino acid analysis was carried out with a beckman amino acid analyzer at keck facility of yale university. the n-terminal amino acid sequence analysis was executed at the protein core facility of columbia university. the molecular size of the purified rha was analyzed on a size-exclusion columns (tsk- , . × mm, tosa-haas, japan) at a constant flow rate of . ml/min, using the protein molecular weight markers as the reference (sigma, st. louis, usa) as previously described [ ] . elution buffer: mm sodium phosphate, mm nacl and . % nan . a trypsin resistance assay was carried out by incubating rha for min at • c without or with g/ml tpck-treated trypsin as described by copeland et al. [ ] . for this assay, the denatured ha was produced by boiling rha for min. hemagglutination activity assays were done essentially as described by barrett and inglis [ ] with a . % solution of fresh chicken red blood cells in a u-bottom -well microtiter plate. a critical issue in production of a therapeutic protein using recombinant dna technology is determining when to harvest the fermentation [ , ] . too early, the yield may be suboptimal. too late, the expressed protein may be degraded by a variety of proteases released during the lytic process of infected cells. thus, a rapid and sensitive assay is needed to monitor protein expression and to choose the right harvest time. ha is well known for its ability to bind the sialic acid on the surfaces of red blood cells (rbcs) and agglutinate these cells [ ] . this phenomenon has been success-fully used to detect cells and tissues infected with influenza viruses. to determine whether the insect cells infected with the recombinant baculoviruses containing the ha gene can also agglutinate rbcs, both uninfected and infected insect cells were incubated with rbcs for min and observed under a microscope. in the uninfected insect cell sample, the rbcs (the smaller cells) were scattered around on the slide, and no specific binding of rbcs to the insect cells (the larger cells, about m in diameter) was observed as shown in fig. a . on the other hand, most of rbcs were bound to the insect cells infected with baculovirus containing the ha gene derived from influenza strain a/new caledonia/ / (h n ) (fig. b) . this method also works for expression of the has derived from other strains such as b/jiangsu/ / (fig. c) and a/new york/ / (h n ) (data not shown). to verify that the observed hemadsorption is due to the ha genes and not to the other genes in the baculovirus, insect cells infected with recombinant baculovirus containing a non-ha gene were also incubated with rbcs. no binding of rbcs on insect cells was observed (fig. d) . these observations clearly demonstrate that rbc's binding to the surface of insect cells is ha expression dependent, not infection dependent. the data also support the conclusion that the has table . at the peak of hemadsorption, clusters of - insect cells agglutinated by rbcs have also been observed. later, the binding gradually reduces to - % of insect cells, most likely caused by the breakdown of some infected cells and the loss of cell membrane, as evidenced by the rapid decrease of cell viability. to determine the rha levels in the fermentation at each sampling time, the single radial immunodiffusion (srid) assay has been used, since it is a simple, reproducible technique, and relatively unaffected by other proteins in the crude extract [ , ] . consistently, the rbc binding to insect cells correlated well with the ha levels determined by srid. late ha expression is expected because the ha gene is regulated by the polyhedrin promoter, which is a late stage promoter in baculovirus infection. similar results were also obtained for the expression of other has such as b/jiangsu/ / and a/new york/ / (data not shown). a major challenge in the biotechnology industry is purification of biologically active recombinant proteins [ , ] . an ideal purification process should be mild, efficient and capable of achieving high purity in a short period of time. accordingly, each purification step has been carefully designed to optimize the whole process. as demonstrated in the hemadsorption studies, rhas are expressed, folded and transported to the cell membrane at a late stage. to extract rha from the cell membranes, several non-ionic detergents at various concentrations were tested for their efficiency. the best result was obtained using % tergitol np- . to get a relatively clean extraction, a magnetic stirrer was used to avoid the disruption of cell nuclei and other organelles. the extract was clarified by centrifugation to remove cell nuclei and other debris. the ha monomer of a/new caledonia/ / influenza virus strain consists of amino acids with a theoretical molecular weight of , . and a pi of . . therefore, this rha can be bound on a cation-exchange media like sp using a lower ph buffer and eluted with a higher ph buffer to achieve the primary purification and concentration. the supernatant of the extract was loaded on uno sphere q/sp columns in tandem. the anion-exchange q column acts as a scavenger by binding the negative-charged impurities that may foul the sp column. after loading and washing, the columns were detached and eluted separately. as shown in the chromatogram (fig. a) , about % of the proteins flow through q/sp columns, a small amount of protein elutes from the sp with the ph buffer and the rest bind tightly to either the sp or the q column. as shown on the sds-page (fig. b) , the rha captured on the sp at ph was selectively eluted by a shift to ph . the ph shift resulted in a -fold increase in purity based on densitometry of the sds-page gel. to further purify rha, use of hydroxyapatite type i (hx-i) media was explored. the binding preference of hx-i media is significantly different from the ion-exchange media, and yet the binding and eluting conditions are relatively mild so as to preserve the biological activity of the target protein [ , ] . thus, the ph sp column eluate was loaded on a hx-i column. after washing, rha was eluted from the column with increased phosphate concentration. as shown in fig. c , most rha was eluted in mm phosphate, and there was a small loss in the wash and in the mm phosphate elution. the purity was increased from % to % according to the densitometry of the sds-page. however, there was still a protein contaminant of about kda in the preparation as revealed on lane of fig. c . since the size of rha trimer is around kda, the difference between rha size and this impurity could be explored to remove this impurity. thus, the mm phosphate eluate was further purified with ultrafiltration using a stir cell ( kda mwco). as demonstrated in fig. d , the kda band (lane ) was selectively removed from the retentate. the purified rha migrated on sds-page gel as a single polypeptide (rha) with an apparent molecular weight of approximately kda. on the blot, a small amount of rha and rha (the cleavage products of rha) were also observed, with apparent molecular weights of ≈ and ≈ kda, respectively (data not shown). trace amounts of rha dimers and trimers were also detectable, with apparent molecular weights of ≈ and ≈ kda, respectively. as summarized in table , the process described here can purify rha from the fermentation to % purity within h with a % overall yield. the largest single step loss ( %) is on hx- column. to confirm the authenticity of rha, the purified protein was examined by n-terminal amino acid sequencing and total amino acid analysis (aaa). the n-terminal amino acid sequence matched the predicted one ( cycles were used) and the signal sequence peptide (the first amino acids) of the full-length ha gene was absent in rha. the measured amino acid composition of the purified rha was consistent with the theoretical one (data not shown). the authenticity of rha was further verified by the western blot using a/new caledonia/ / antibody provided by the food and drug administration (fda). the purified rha in . % tween/pbs solution was analyzed using size-exclusion chromatography. it was eluted as a single peak at . min as shown in fig. a , corresponding to a molecular weight around - kda, likely a complex of four to five ha trimers (( - ) × × kda). to test whether the purified rha still retained its native structure, the purified protein was treated with trypsin on ice. as shown in the lane of fig. b , rha was cleaved into only two bands, ha - . kda and ha - . kda. on the other hand, the heat-denatured rha was digested into numerous small fragments. the trypsin-resistance data demonstrate that the rha expressed in insect cells folded properly and retained its native structure after purification. table stepwise mass balance of ha-nc purification by srid assay ha (g/ml) volume (ml) ha (mg) purity a (%) step recovery (%) total recovery (%) since glycosylation may play an important role in the biological function of ha [ ] [ ] [ ] , it is of interest to explore whether the rha produced in insect cells is properly glycosylated. thus, rha was deglycosylated with peptide-nglycosidase f (pngase f) or endoglycosidase h (endo h) and resolved on sds-page. as shown in fig. c , the untreated ha migrated at . kda, pngase f deglycosylated ha at . kda, and endo h treated ha at . kda, respectively. these data indicate that the rha produced in insect cells is indeed glycosylated with n-linked oligosaccharide side chains. about . kda of oligosaccharide chains have high mannose content susceptible to endo h, and . kda of oligosaccharide chains have low mannose residues resistant to endo h. to assess the number of n-linked oligosaccharide chains, the trypsin treated rha was subjected to limited deglycosylation with pngase f or endo h under a variety of conditions. on the sds-page of pngase f treated samples (fig. d) (fig. e) , there are three distinguishable ha bands, . , . and . kda, and one ha band, . kda. the data suggest that there are six n-linked oligosaccharide chains in the ha region, and two of them have high mannose content. there is only one n-linked oligosaccharide chain in the ha region, which is to directly compare with the a/new caledonia antigen present in the egg-based vaccine, the purified rha/ a/new caledonia was formulated either alone into a g/ . ml solution or with g rha/b/jiangsu and g rha/a/wyoming in a . ml dosage (flublØk tm , the expected trade name of protein sciences' rha vaccine). as judged by the hemagglutination assay, flublØk is as active as vaxigrip (a licensed egg-based vaccine manufactured by sanofi-pasteur-aventis) in agglutinating rbcs and preventing them from forming a tight pellet as shown in fig. a . antigen specific srid assay is widely used to determine the concentration of active ingredients in a vaccine. on the srid gel prepared with a/new caledonia/ / antibody (fig. b) , the diffusion ring of flublØk is slightly larger than that of vaxigrip, suggesting that flublØk is equivalent to vaxigrip for the active ingredient of a/new caledonia/ / strain. on the sds-page (fig. c) , the flublØk lane is clean, and only three broad bands are visible, representing the ha, ha and ha of the three strains, respectively. the vaxigrip lane is complicated with the major ha band around kda along with many minor bands. similarly, numerous impurities have also been found in other egg-based vaccines [ ] . all these data demonstrate that the purified rha expressed in insect cells is correctly translated, properly glycosylated and folded and biologically active. the traditional egg-based vaccines have been successfully used for more than years to prevent influenza. they are reliable, effective (if there is a good match), and affordable. however, the production cycle of the egg-based vaccines is lengthy and heavily dependent on egg supply and unable to be developed quickly in response to the urgent need in an influenza pandemic [ ] [ ] [ ] [ ] . to replace or supplement the egg-based vaccines, the new vaccine has to be equally effective, reliable, economical, and capable of being developed and delivered in a short period of time. the work reported here is some important progress toward an alternative influenza vaccine-the recombinant protein-based vaccine. a new analytical method based on hemadsorption has been developed to closely monitor the expression of ha in insect cells. this method plays a critical role in ensuring optimal ha production and in determining the right harvest time, in addition to other harvest parameters such as hpi, cell's morphology and viability. it has also been successfully used at protein sciences corporation to accelerate the screening process for the recombinant baculoviruses used for ha manufacturing. a purification process has been developed to quickly purify the recombinant ha from the bulk harvested from the bioreactor while retaining its biological activity. previously, ha purifications were heavily dependent on affinity chromatography using specific monoclonal antibodies or various lectins [ , ] . such methods are highly selective, but difficult to scale up to commercial levels due to a number of limiting factors: ( ) some of the ligands will leach off the column during the purification and they must be removed from the final product; ( ) it is difficult to regenerate an affinity column after use, and thus the performance declines after each use; ( ) the batch to batch variations in the quality of affinity media make it almost impossible to have a robust purification process from time to time; ( ) most affinity media are too expensive to be used at large scale. on the other hand, all chromatographic media used in the present process are chemically stable (can be regenerated repeatedly), commercially available and relatively inexpensive; thus more suitable to scale up in a gmp facility. if the reagents, columns and ancillary equipment are well prepared in advance, the whole purification can be completed in one full working day, avoiding the overnight storage of intermediate rha preparation and possible inactivation of rha. several factors have made this rapid process possible. first, there is no sample manipulation between purification steps, which makes a quick, continuous purification process possible. second, q/sp is connected in tandem, combining two chromatographic processes into one. third, a chemically different chromatographic media-hx-i is used to differentiate rha from the remaining impurities. unlike ion-exchange media, the adsorption of proteins to hx-i involves both anionic and cationic interaction. the calcium group can interact with carboxylate residues, whereas the phosphate group can bind the basic residues on the surface of the protein. the bound proteins can be eluted by an increasing phosphate gradient or a gradient of calcium, magnesium ions. it is worth to point out that the purification process described here needs to be further optimized for large-scale production, for example, a tangential flow filter should be used to replace a stir cell at the polish step. nonetheless, we believe the strategies described here can also be used to develop a rapid purification process for other recombinant proteins. biologically, flublØk is as active as the egg-based vaccine-vaxigrip as determined by the hemagglutination assay. based on the srid assay, flublØk is equivalent to vaxigrip for the active ingredient of a/new caledonia/ / strain of influenza. in challenge studies, chickens were effectively protected against the h n virus infection after inoculation with the rha of the virus [ ] . moreover, two distantly related h n rha influenza vaccines using the baculovirusinsect cell expression system have also been demonstrated to partially protect mice against the lethal challenge of a recombinant pandemic influenza virus [ ] . in clinical trials, the trivalent rha vaccine (flublØk) stimulates anti-ha antibody production at least as well as, and in the case of h rha, superior to the traditional egg-based vaccine [ ] [ ] [ ] [ ] [ ] . the / influenza season phase iib field trial of flublØk that enrolled healthy subjects aged - further showed that the / / dose was % efficacious in preventing culture positive influenza illness compared to placebo (press release, protein sciences corp., june , ) . furthermore, a recombinant protein-based vaccine, such as the flublØk described here, also has other advantages over the traditional egg-based vaccines. it consists solely of three antigens (proteins) stored in sterile phosphate buffered-saline and without preservatives such as thimerosal (a mercury derivative currently used in the egg-based vaccine), antibiotics or adjuvants. unlike the egg-based vaccines, no live influenza viruses, biocontainment facilities or harsh chemicals such as formaldehyde are used in manufacturing. this may explain why flublØk has shown lower side effects than the licensed vaccines in clinical trials [ ] [ ] [ ] [ ] [ ] . therefore, a reliable, effective, and affordable recombinant protein-based influenza vaccine can be and should be developed to meet the challenge of a potential influenza pandemic. for pandemic preparedness, developing and stockpiling rha influenza vaccines against the present h n strain may be a good option to provide some protection for the first response personnel and the population in the hard-hit areas in the case of a pandemic, and to win the precious time for manufacturing of a more specific influenza vaccine. realities and enigmas of human viral influenza: pathogenesis, epidemiology and control serious morbidity and mortality associated with influenza epidemics serum and nasal wash antibodies associated with resistance to experimental challenge with influenza a wild-type virus receptor binding and membrane fusion in virus entry: the influenza hemagglutinin from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza confronting the avian influenza threat: vaccine development for a potential pandemic probable person-to-person transmission of avian influenza a (h n ) cell-based protein vaccines for influenza the role of cell culture vaccines in the control of the next influenza pandemic next generation flu vaccine boosted by chiron debacle preparing for the next pandemic influvac: a safe madin darby canine kidney (mdck) cell culturebased influenza vaccine a phase i, randomized controlled clinical trial to study the reactogenicity and immunogenicity of a new split influenza vaccine derived from a non-tumorigenic cell line webster rg. immunogenicity and protective efficacy in mice of influenza b virus vaccines grown in mammalian cells or embryonated chicken eggs antigen drift and efficacy of influenza virus vaccines alterations in the hemagglutinin associated with adaptation of influenza b virus to growth in eggs evidence for hostcell selection of influenza virus antigenic variants recombinant baculovirus influenza a hemagglutinin vaccines are well tolerated and immunogenic in healthy adults influenza a virus vaccines containing purified recombinant h hemagglutinin are well-tolerated and induce protective immune responses in healthy adults humoral and cellular immune responses following vaccination with purified recombinant hemagglutinin from influenza a (h n ) virus evaluation of a recombinant hemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults safety and immunogenicity of a recombinant hemagglutinin vaccine for h influenza in human vaccine pathogenicity and immunogenicity of influenza viruses with genes from the pandemic virus single-radial-immunodiffusion as an in vitro potency assay for human inactivated viral vaccines immunochemical quantitation of antigens by single radial immunodiffusion characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases the chaperone activity of bovine alpha crystalline. interaction with other lens crystallins in native and denatured states assembly of influenza hemagglutinin trimers and its role in intracellular transport growth, purification, and titration of influenza viruses production of recombinant proteins: challenges and solutions the state of biopharmaceutical manufacturing insect cell culture for industrial production of recombinant proteins recombinant protein expression for therapeutic applications hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of ph separation between the alpha and beta forms of human antithrombin by hydroxyapatite high-performance liquid chromatography importance of hemagglutinin glycosylation for the biological functions of influenza virus effects of glycosylation on the properties and functions of influenza virus hemagglutinin roles of n-linked glycans in the endoplasmic reticulum morphological and biochemical characterization of influenza vaccines commercially available in the united kingdom production of a recombinant influenza vaccine using the baculovirus expression vector system the glycosylation of the influenza a virus hemagglutinin by mammalian cells. a site-specific study baculovirus-derived rha vaccines protect against lethal influenza infections by avian h & h subtypes key: cord- -yy etfek authors: dwivedi, varun; manickam, cordelia; patterson, ruthi; dodson, katie; murtaugh, michael; torrelles, jordi b.; schlesinger, larry s.; renukaradhya, gourapura j. title: cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: yy etfek abstract porcine reproductive and respiratory syndrome (prrs) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. the efficacy of parenteral administration of widely used modified live virus prrs vaccine (prrs-mlv) against genetically divergent prrsv strains remains questionable. therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of prrs-mlv (strain vr ) with a potent adjuvant to elicit cross-protective immunity against a heterologous prrsv (strain mn ). mycobacterium tuberculosis whole cell lysate (mtb wcl) was chosen as a potent mucosal adjuvant due to its th biased immune response to prrs-mlv. unvaccinated pigs challenged with mn had clinical prrs with severe lung pathology; however, vaccinated (prrs-mlv+ mtb wcl) pigs challenged with mn were apparently healthy. there was a significant increase in the body weight gain in vaccinated compared to unvaccinated prrsv challenged pigs. vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced prrsv neutralizing antibody titers and reduced viremia. immunologically, an increased frequency of th cells, th/memory cells, γδ t cells, dendritic cells, and activated th cells and a reduced frequency of t-regulatory cells were detected at both mucosal and systemic sites. further, reduced secretion of immunosuppressive cytokines (il- and tgf-β) and upregulation of the th cytokine ifn-γ in blood and lungs were detected in mucosally vaccinated, prrsv-challenged pigs. in conclusion, intranasal immunization of pigs with prrs-mlv administered with mtb wcl generated effective cross-protective immunity against prrsv. porcine reproductive and respiratory syndrome (prrs) is a chronic immunosuppressive disease of pigs responsible for huge economic loss to the swine industry worldwide. the economic impact of prrs alone to the us swine producers has been estimated to be approximately $ million annually [ ] . according to the animal and plant health inspection service (aphis) report of january , overall, . % of unvaccinated grower/finisher pigs were positive for prrsv antibodies in the us (http://www.aphis. usda.gov/animal health/nahms/swine/downloads/swine / swine is prrs.pdf). as a member of the arteriviridae family, prrsv contains a positive-sense, single-stranded rna genome [ ] . prrsv causes respiratory distress in pigs of all ages and reproductive failure in sows [ , ] . infection with this virus results in suppression of innate immune response (reduced ifn-␣ production and nk cell cytotoxicity) and a delay in the onset of adaptive immune response [ ] [ ] [ ] . different field isolates of prrsv have been found to carry variable levels of genetic diversity ranging between and % and one of the virulent strains, mn is antigenically highly divergent from the vaccine strain, vr [ ] . currently, modified live and inactivated prrsv vaccines have been licensed for use; however, these vaccines are not always efficacious in preventing prrsv reinfections and transmission. there continue to be questions regarding vaccine safety and efficacy against existing as well as emerging antigenically heterologous prrsv strains [ , ] . therefore, development of a broadly protective prrsv vaccine has been a challenge to prrsv researchers. recently, prrsv experts and researchers collectively agreed that only replicating prrsv vaccines have the most promise in the field and successful protection can be achieved by improving the efficacy of live prrsv vaccines (http://vetmed.illinois.edu/news/ prrswhitepaper.pdf). mucosal surfaces cover the largest surface area in the body and almost % of total immune cells in the body are localized in the mucosa-associated lymphoid tissues (malt) and at mucosal sites. nasopharyngeal malt contains the entire repertoire of immune cells which are strategically located to orchestrate regional immune functions against airborne infections [ ] . a majority of pathogens are transmitted through mucosal surfaces, but some of them cause disease primarily at these mucosal sites (e.g. influenza, prrsv, hiv, rotavirus, etc.). it has been demonstrated that systemic stimulation of the immune system mainly results in systemic protection with low mucosal immune responses. conversely, optimal stimulation of the mucosal immune system generates both mucosal and systemic immunity [ ] . the mucosal immune system possesses strong immunoregulatory mechanisms to dampen inflammationinduced pathology; therefore, mucosal immunization generally results in tolerance in the absence of suitable adjuvants or delivery systems [ , ] . intranasal delivery of live attenuated vaccines against bovine herpes virus- , influenza, and parainfluenza- when administered with adjuvants has proven effective [ , ] . intranasal delivery of influenza vaccine with a potent adjuvant resulted in effective cross protection due to immune responses generated against conserved internal viral proteins [ ] . thus, mucosal immunization may be an attractive method to induce prrsv specific cross-protective immunity. design of an effective mucosal vaccine is not easy because the adjuvant/delivery system used must not elicit any toxicity and should overcome the host-and/or vaccine antigens-induced immunosuppression. heat-killed mycobacterium tuberculosis (mtb) in an oil emulsion has been used extensively for experimental purposes as complete freund's adjuvant [ ] . unfortunately, adverse effects mediated by major cell wall components of mtb such as mycolic acids, peptidoglycan, and wax d preclude use of complete freund's adjuvant in humans and food animals [ , ] . adjuvanticity of various purified individual components of mtb have been investigated [ , ] , including water soluble whole cell lysate (wcl) of mtb [ ] . interestingly, a few of the mtb wcl fractions, such as heat shock protein- (hsp ) [ , ] and pe (pro-glu)/ppe (pro-pro-glu) have been identified to elicit potent adjuvant activity [ , ] . however, the knowledge related to mucosal adjuvanticity of mtb wcl to mucosal viral vaccines is limited. initially, we performed studies to choose a suitable bacterial candidate mucosal adjuvant to use with prrs-mlv. pigs were inoculated intranasally with nine different bacterial preparations belonging to mycobacterium, vibrio, and streptococcus, and selected mtb wcl due to its ability to induce increased th and reduced immunosuppressive responses; the detailed results of which will be published elsewhere. subsequently, prrsv specific immune responses to prrs-mlv+/− mtb wcl in pigs inoculated intranasally was evaluated in detail in a pre-challenge study, which resulted in satisfactory immune correlates of protection mediated through mtb wcl [ ] . the purpose of the current study was to confirm the mucosal adjuvanticity of mtb wcl to prrs-mlv in inducing effective cross-protection to challenge with a genetically divergent and virulent prrsv strain. stable mycoplasma-free marc- cells which support the growth of prrsv [ ] were used to prepare prrsv stocks and to perform immunological assays. cells were maintained in dmem with % fbs (atlanta biologicals, lawrenceville, ga) at • c with conventional large white-duroc crossbred weaned specificpathogen-free piglets at - wks of age were transported to animal facilities of the food animal health research program at the ohio agricultural research and development center, wooster, oh. the swine herd was confirmed seronegative for antibodies to prrsv, porcine respiratory corona virus, transmissible gastroenteritis virus, and porcine circo virus . piglets were bled on arrival, and the sera were tested to confirm the absence of prrsv antibodies. pigs were allowed to acclimate for an additional week before initiation of the experiment. animals were maintained in the large animal bsl- facility under the supervision of a veterinarian. throughout the duration of the study, all animals received food and water ad libitum. all inoculations such as adjuvant (mtb wcl, mg/pig), vaccine (prrs-mlv, × tcid per pig) and challenge (prrsv, × tcid per pig) were administered intranasally. adjuvant and vaccine were inoculated separately into each nostril. all pigs were maintained, samples collected, and euthanized as per the protocol approved by the institutional animal care and use committee (iacuc), wooster, and institutional biosafety committee (ibc), columbus, the ohio state university, ohio. for the primary study, pigs were randomly allocated to one of three groups: group , mock pigs (n = ) received dmem and they were unvaccinated and unchallenged; group , unvaccinated pigs (n = ); group , vaccinated (prrs-mlv+ mtb wcl) pigs (n = ). groups and were challenged with prrsv mn on day postvaccination (dpv) . three pigs each from groups and were euthanized on , , and day post-challenge (dpc). mock inoculated pigs (n = ) were euthanized separately prior to killing of any infected animals. in another study, a total of nine pigs were split into three groups and housed in three separate isolation rooms (n = per group). pigs were inoculated intranasally as follows: group , mock (dmem); group , vaccine (prrs-mlv); group , vaccine with adjuvant (prrs-mlv+ mtb wcl). at dpi- , groups and were challenged with prrsv mn on dpv . infection was allowed to proceed for days at which time all pigs were euthanized. mock inoculated pigs were euthanized prior to the euthanasia of any prrsv challenged animals. blood ( - ml) was collected on dpi and dpc , , , , , , , , , , , and , serum was separated from the clotted blood, and aliquots of serum were preserved at − • c until used in assays. serum was used for evaluation of viremia, viral titer, prrsv serum neutralizing antibody titers, and cytokine production. pigs were monitored daily for respiratory disease, and rectal temperature and body weight were recorded twice a week. blood was collected in acid citrate dextrose solution and processed for isolation of peripheral blood mononuclear cells (pbmc) as previously described [ ] . lung mononuclear cells (lung mnc) from individual pigs were isolated at necropsy as described previ-ously [ , , ] . tracheobronchial lymph nodes (tbln) draining the lungs were collected in dmem, cut into small pieces, and homogenized in stainless steel cellectors. homogenates were washed and the pellet was dissolved in rpmi containing % percoll and centrifuged for min at × g at • c, with no brake. red blood cells in the cell pellet were lysed and the mononuclear cells were washed and resuspended in enriched rpmi [rpmi- , % fetal bovine serum, gentamicin ( g/ml), ampicillin ( g/ml), mm hepes, mm l-glutamine, . mm non-essential amino acids, mm sodium pyruvate, and nm of -me]. necropsy was performed and lungs, tonsils, and tbln were examined for gross lesions. grossly evident pulmonary changes were assigned a score based upon the percent of virus-affected lesions (purple-red colored consolidation) in each lung lobe, and a total score for the entire lung was calculated as described previously [ ] . all the analyses were performed using a standard indirect immunofluorescence assay (ifa) as described previously [ , , , ] . for virus titration, a confluent monolayer of marc- cells in a -well microtiter plate was incubated with a -fold dilution of serum for h. for determining the virus neutralization (vn) titers, serum was heat treated for complement inactivation, diluted two-fold in dmem, and incubated with an equal volume of prrsv mn containing tcid per well for h at • c. one hundred microliter of that suspension was transferred into a -well microtiter plate containing a confluent monolayer of marc- cells and incubated for h at • c in a co incubator. cytopathic effects were examined following fixation with acetone water and addition of anti-pprsv nucleoprotein mab (clone, sdow ; rural technologies, inc., brookings, sd) and alexa- conjugated anti-mouse igg(h+l) secondary antibody, and observed under a fluorescent microscope after mounting with glycerol-pbs ( : ratio). five million pbmc, tbln mnc, and lung mnc were subjected to in vitro restimulation in a -well tissue culture plate in the presence of killed crude prrsv mn antigens (ags) ( g/ml), or recombinant prrsv carboxy terminal amino acid fragment of matrix protein (m ) ( g/ml) [ , ] in enriched rpmi for h at • c. the harvested culture supernatant was analyzed for cytokines by elisa. cells cultured in the absence of any antigens were included as a control, and the amount of cytokines secreted by these cells were subtracted from the respective restimulated experimental well values. the frequency of ifn-␥-secreting cells in pbmc was determined by an elispot assay as described previously [ , ] . briefly, pbmc were plated ( × cells/well) in enriched rpmi in a -well mul-tiscreen plate (millipore, billerica, ma) pre-coated overnight with a mouse anti-pig ifn-␥ mab (bd pharmingen, san diego, ca) at • c. cells were restimulated with killed crude prrsv mn ags ( g/ml) for h at • c in a co incubator. plates were incubated with biotinylated anti-pig ifn-␥ detection antibody, subsequently with streptavidin-hrp conjugate and developed using an insoluble substrate tetramethylbenzidine with h o peroxidase substrate system (kpl inc., gaithersburg, maryland). the frequency of prrsv specific ifn-␥-secreting cells was counted using an aid ® elispot reader system. the background values were subtracted from the respective counts of the unstimulated cells and the immune responses were expressed as the number of ifn-␥-secreting cells per million pbmc. cells stimulated with phytohemaglutinin-p were included as a positive control on every plate. pig sera collected at indicated dpc and culture supernatants harvested after in vitro restimulation of pbmc, tbln and lung mnc were analyzed by elisa for secretion of different interleukin (il) classes, such as th (ifn-␥ and il- ), th (il- ), pro-inflammatory (il- ), and immunosuppressive (il- and tgf-␤) cytokines as described previously [ , ] . flow cytometric analysis was performed to determine the phenotype and frequency of different immune cells in a multicolor immunoassay as described previously [ , ] . briefly, previously isolated cell types (pbmc, lung mnc, and tbln mnc) were treated with % pig serum to block fc receptors. cells were then stained with an appropriate mab which was either directly conjugated to a specific fluorochrome or biotinylated, or with a purified antibody to pig specific immune cell surface markers [cd , cd (southernbiotech, birmingham, alabama), cd ␣, cd ␣/␤, cd c (bd pharmingen), cd , sla class ii (serotec, raleigh, nc), tcr n (vmrd, pullman, wa), foxp (ebioscience, san diego, ca)] or with their respective isotype control mab and labeled cells were treated with streptavidin-conjugated fluorochrome or respective anti-species isotype specific secondary antibody conjugated with fluorochrome. finally, cells were fixed with % paraformaldehyde. for intracellular foxp staining, cells were surface stained for cd and cd as described above and overnight incubated at • c in permeabilization buffer, and stained with fluorochrome-conjugated pig foxp cross-reactive anti-rat foxp mab [ , ] .immunostained cells were acquired using a facs ariaii (bd biosciences, san jose, ca) flow cytometer. analysis was performed to determine different immune cell populations based on the cell surface marker phenotypes: natural killer (nk) cells (cd − cd − cd ␣ + ) [ ] ; t-helper cells (cd + cd + cd − ); cytotoxic t lymphocytes (ctls) (cd + cd − cd + ); cd cd double positive t cells (cd + cd + cd + ) also called as t-helper/memory cells [ , ] ; ␥␦ t cells (cd ␣ + tcr n + ); t-regulatory cells (cd + cd + foxp + ); cd + (myeloid cells); cd + cd + foxp − (activated t-helper cells); dendritic cells rich fraction (cd + cd c + slaii + ) using flowjo software (tree star, inc., or, usa). frequencies of individual lymphocyte and myeloid cell subsets were analyzed from a total , to , events. all data were expressed as the mean value of three, six, or nine pigs ± sem. statistical analyses were performed using a nonparametric "wilcoxon t-test" where functionality was compared between two study groups (unvaccinated vs. vaccinated, and in fig. and table , prrs-mlv vs. prrs-mlv+ mtb wcl), or nonparametric "kruskal-wallis test" followed by a "dunn's test" for multiple comparisons when comparing three or more groups for vn titers and body weight gain using sas software (sas institute inc., cary, nc). statistical significance was assessed as p < . . in this study, pigs were either unvaccinated or vaccinated with prrs-mlv+ mtb wcl and challenged using a virulent heterologous prrsv mn . clinically, unvaccinated pigs developed typical prrs symptoms with fever, mild cough, reduced food intake, and lethargy during first wks post-challenge. vaccinated, mn challenged pigs did not suffer from clinical prrs. the mean body temperature of unvaccinated, prrsv challenged pigs (n = ) until dpc was . • f higher compared to vaccinated pigs. pigs vaccinated and challenged with mn had significant increase in the net body weight gain compared to control challenged pigs (fig. a) . unvaccinated, mn challenged pigs had severe gross lung lesions on both ventral and dorsal surfaces (fig. b) , and the lung lesion scores were significantly higher at dpc and dpc compared to vaccinated challenged pigs (fig. c) . protection in mucosally vaccinated pigs against prrsv challenge was associated with a significantly reduced viral load ( fig. a ) and viral titer (fig. b) at dpc . the circulating prrsv in the blood was less but not statistically significant at dpc in vaccinated pigs, and it was almost cleared by dpc in both the pig groups ( fig. a) . prrsv neutralizing antibody titers in serum at different time points until dpc were analyzed. a significant increase in the vn titers in vaccinated and mn challenged pigs was detected (fig. a) . we found a significant difference in the vn titers in vaccinated pigs at dpc , , and when the titer was compared at each dpc. in mucosally vaccinated, prrsv-challenged pigs, there was a significant reduction in serum il- levels at dpc , , and (fig. b) . similarly, tgf-␤ serum levels were also significantly reduced but at a later stage of challenge (dpc and ) (fig. c) . overall, both the immunosuppressive cytokines il- and tgf-␤ were detected at reduced levels in vaccinated pigs ( fig. b and c). information regarding secretion of cytokines by pig mnc after prrsv restimulation ex vivo is important to understand virus specific memory immune responses. consistent with the reduced lung lesions and viremia, a significantly increased frequency of ifn-␥- table frequency of immune cells in pigs inoculated intranasally with mock (no vaccination and no challenge), unvaccinated (n = ) or vaccinated with prrs-mlv+ mtb wcl (n = ) and then challenged with prrsv mn . secreting cells in pbmc of vaccinated, prrsv challenged pigs at dpc was detected (fig. a) . such a trend was noted for ifn-␥-secreting cells in the lungs at dpc (data not shown) with a concomitant reduction in il- -secretion by lung mnc (fig. c) . the extended clinical protection in vaccinated prrsv challenged pigs was indicated by an increased trend in the secretion of ifn-␥ with a concomitant reduction in il- and tgf-␤ secretion by lung mnc and pbmc at both dpc and (data not shown). evaluation of the frequency of various immune cells at both mucosal (lung and tbln mnc) and systemic sites (pbmc) in vaccinated and virulent prrsv challenged pigs is important for associating cytokine responses. at dpc , a significant increase in the frequency of dendritic cells (dcs) and an increased frequency of th cells, th/memory cells, and ␥␦ t cells were detected in the lungs of vaccinated, virus challenged pigs (table a ). in addition, in both pbmc and tbln mnc, a significant increase in the frequency of activated th cells was detected at dpc (table b and c) . at dpc , a % reduction in the frequency of t-regulatory cells (tregs) in the lungs and tbln in vaccinated, virus challenged pigs was detected (table a and c). in blood and tbln, the frequency of th cells was significantly increased in vaccinated pigs (table b and c). at dpc , a significant decrease in the frequency of tregs in both lungs and blood was detected (table a and b). in addition, a significantly increased frequency of dcs in the blood was detected in vaccinated and prrsv mn challenged pigs at dpc (table b) . to strengthen our data on the superior adjuvanticity of mtb wcl to prrs-mlv, we performed challenge studies in pigs vaccinated intranasally with prrs-mlv in the presence or absence of mtb wcl. prrsv specific vn titers were detected at significantly higher levels at dpc and in pigs vaccinated with prrs-mlv+ mtb wcl compared to prrs-mlv alone (fig. a) . virus neutralizing antibody titers were present at low levels in pigs vaccinated with prrs-mlv+ mtb wcl from dpi (dpc ), but not in pig groups receiving prrs-mlv alone (fig. a) . in support of adjuvant mtb wcl mediated enhanced cell mediated immune (cmi) responses to prrsv, lung mnc of pigs vaccinated with mtb wcl secreted significantly higher amounts of ifn-␥ following restimulation using mn ags (fig. b) . another th response inducing cytokine il- was also secreted at significantly higher levels in a mtb wcl dependent manner in vaccinated pigs (fig. c ). as expected, the immunosuppressive cytokine, il- was secreted at significantly reduced levels in pigs vaccinated with mtb wcl compared those pigs vaccinated without mtb wcl following challenge with prrsv mn (fig. d) . frequency of immune cell populations was also evaluated at both mucosal and systemic sites between these two pig groups to determine the mtb wcl mediated adjuvant effects. in lung mnc, a significant increase in the frequency of th cells, activated th cells, and nk cells was detected in challenged pigs vaccinated with prrs-mlv+ mtb wcl compared to pigs vaccinated with prrs-mlv alone (table a) . in pbmc, a significant increase in the frequency of ␥␦ t cells was detected in prrs-mlv+ mtb wcl inoculated pigs (table b ). as expected, the frequency of tregs was significantly reduced in both pbmc and tbln mnc of pigs inoculated with vaccine with mtb wcl compared to vaccine alone (table b and c) . overall, data from these particular pig groups combination study demonstrated the superior adjuvanticity of the mucosal adjuvant mtb wcl to prrs-mlv. until now, as per our knowledge, no successful attempts have been made to elicit effective anti-prrsv immunity by intranasal delivery of live prrsv vaccine. viruses evade the host immunity by promoting secretion of il- and tgf-␤ which antagonize the protective th immune response [ ] . prrsv induces a strong immunosuppressive response resulting in the delayed onset of cell mediated immune responses [ , [ ] [ ] [ ] . both live and inactivated prrsv significantly increase il- gene expression [ ] . an increased concentration of il- in pig lungs was detected from prrsv-infected pigs for long periods of time [ , ] . nonetheless, expression of both il- and tgf-␤ genes was also increased in pigs vaccinated against prrsv by a systemic route [ ] . in our pre-challenge study, increased secretion of both il- and tgf-␤ in both the lungs and blood of pigs vaccinated intranasally with prrs-mlv without mtb wcl was detected [ ] . in contrast, in both pre-and post-challenge studies when prrs-mlv was inoculated intranasally with mtb wcl, secretion of both il- and tgf-␤ was suppressed. infiltration of tregs into the infected pig lungs contributes to the secretion of high levels of il- and tgf-␤ [ ] . the role of tregs in establishment of chronic persistent hiv, hepatitis c and b viruses, cytomegalovirus, and epstein-barr virus infections has been reported [ ] [ ] [ ] . like in mice and humans, foxp -expressing cd + cd + cells with comparable immunosuppressive properties have been identified in pigs [ ] . studies have been reported on the prrsv mediated proliferation of tregs in infected pigs, indicating their involvement in disease progression [ ] [ ] [ ] . in our study, a consistently reduced frequency of tregs in the lungs, blood, and tbln of pigs vaccinated intranasally with prrs-mlv+ mtb wcl was detected which was associated with reduced secretion of both the immunosuppressive cytokines, il- and tgf-␤. co-ordinated immunosuppressive functions of il- , tgf-␤, and tregs have been reported [ , ] . prrsv increases the expression of tgf-␤ from myeloid cells, and tgf-␤ is essential for the de novo induction of foxp and for the regulation of differentiation and function of tregs in mice, humans, and pigs [ , ] . all the mucosal sites are interconnected by a common mucosal immune system, and immunization at one primary site will stimulate proliferation and migration of antigen-specific lymphocytes, resulting in both mucosal and systemic immunity [ ] . virus neutralizing antibodies play an important role in the clearance of prrs viremia [ , ] . like in natural prrsv infection, the prrsv vaccine also induced delayed neutralizing antibody and cmi responses in pigs [ , ] . however, an increase in the prrsv-vn titers with a concomitant reduction in prrsv load in pigs vaccinated with prrs-mlv+ mtb wcl and challenged with mn was detected in our study. this suggests that reduced viremia detected from vaccinated pigs correlates with increased prrsv specific vn titers. apart from an effective humoral response, a potent cmi response is essential for complete viral clearance. the key cytokine associated with a host cmi response is ifn-␥ produced by nk cells, ␥␦ t cells, th cells, ctls, and th/memory cells [ ] . in our study, reduced clinical prrs and viremia in pigs vaccinated with prrs-mlv+ mtb wcl were associated with increased frequency of ifn-␥-secreting cells and increased levels of activated th cells, memory/th cells, and nk cells in both the lungs and blood. cd cd double positive t cells possess memory, t-helper, and cytolytic properties. they are generally called th/memory cells, and they also secrete ifn-␥ [ , ] . this important t cell subset was associated with protection in pigs vaccinated against pseudorabies virus [ , ] . recently, recombinant bcg based respiratory syncitial virus vaccine induced enhanced recruitment of cd + and cd + t cells into the lungs resulting in increased th cytokine responses and protection [ ] . the in vivo adjuvant effects of the water soluble fraction of mtb wcl containing hsp resulted in rapid and prolonged activation of antigen-specific cd + t cells [ ] . consistent with that in our study, mtb wcl-mediated, increased frequency of cd + and cd + t cells with reduced frequency of tregs was detected. importantly, this was associated with enhanced secretion of th cytokines (ifn-␥) and downregulated secretion of immunosuppressive (il- and tgf-␤) cytokines. the pro-inflammatory cytokine il- is critical for induction of specific adaptive immunity [ , ] , but excess secretion of il- results in inflammation-induced pathology [ ] . in our prechallenge study, increased secretion of il- in the lungs and blood of prrs-mlv+ mtb wcl inoculated pigs at dpv and was associated with enhanced prrsv specific cmi responses [ ] . in contrast, in the current study, reduced gross lung lesions observed in vaccinated, mn challenged pigs were associated with reduced secretion of il- . these data suggest that adjuvant mtb wcl mediated regulated secretion of il- might play a role in inducing a prrsv-specific cmi response. pigs possess a higher frequency of ␥␦ t cells and these cells are considered to be an important innate immune cell at mucosal sites. in addition, pigs have non-mhc class i cytolytic activity against prrsv infection [ ] . in mucosally vaccinated and prrsv challenged pigs, increased frequency of ␥␦ t cells in blood, lungs, and tbln that is mediated by mtb wcl was detected, suggesting a possible protective role played by ␥␦ t cells to prrsv mn in these pigs. in summary, mucosal immunization of pigs using a live prrsv vaccine along with a potent adjuvant has the potential to induce heightened innate and adaptive immune responses, with a concomitant reduction in immunosuppressive responses. thus, based on immune correlates of protection detected against virulent heterologous prrsv mn in pigs intranasally vaccinated with prrs-mlv+ mtb wcl, it may be possible to achieve an effective cross-protective immunity against pprs. our future aim will be to conduct field trials to evaluate the efficacy of our mucosal immunization approach to control prrs. assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states 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a national pork board award (npb # - ) and a usda-nifa prrs cap award ( - - ) to rjg. salaries and research support were provided by the state and federal funds appropriated to ohio agricultural research and development center, the ohio state university. we would like to thank drs. juliette hanson, mahesh khatri, and hadi yassine, and todd root and matthew weeman for their help in animal studies. we would like to thank drs. eric nelson, mike roof, dobos, and belisle for providing reagents. we also thank bert bishop for statistics, and dr. michele williams for editing the manuscript.role of the funding source: sponsors have no role in study design, in the writing of the report,or in the decision to submit the paper for publication. key: cord- -wckqscvm authors: maunsell, fiona p.; donovan, g. arthur; risco, carlos; brown, mary b. title: field evaluation of a mycoplasma bovis bacterin in young dairy calves date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: wckqscvm mycoplasma bovis is an important cause of pneumonia, otitis media and arthritis in young dairy calves, and there is a critical need for improved preventative strategies for this pathogen. we conducted a randomized, placebo-controlled, double-blinded field trial to determine the efficacy of a commercial m. bovis vaccine for the prevention of m. bovis-associated disease in calves. calves (n = ) on florida dairies with a history of m. bovis infection received an m. bovis bacterin or a placebo, administered subcutaneously at , and days of age. one of the herds did not experience m. bovis-associated disease; for calves in the remaining herds, the incidence risk for respiratory disease, otitis media and arthritis from to days of age was . , . and . , respectively. vaccination had no effect on the age at first treatment for m. bovis-associated disease, incidence of respiratory disease, mortality, weight gain, or nasal colonization with m. bovis in the first days of life. in one herd, vaccination was associated with an increased risk of otitis media. there was no association between m. bovis-specific serum antibody titers and morbidity in vaccinated calves. under the field conditions in this study, this vaccine was not efficacious for the prevention of m. bovis-associated disease in young dairy calves. mycoplasma bovis is a significant world-wide pathogen of adult dairy cows as well as intensively reared beef and dairy calves [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past decade, m. bovis has emerged as an increasingly important cause of respiratory disease, otitis media and arthritis in young calves less than months of age [ , , , , ] . clinical disease caused by m. bovis tends to be chronic, debilitating and unresponsive to antimicrobial therapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] . disease outbreaks with high morbidity rates occur [ , , , , ] and can be economically devastating for the affected farm. the costs of infection are primarily associated with intensive treatment of affected calves coupled with culling of animals that are unresponsive to therapy [ ] . m. bovis-associated disease is also important from an animal welfare perspective as it often results in calves that are subject to severe, chronic illness for which the producer or veterinarian can provide only limited relief. there is therefore a critical need to develop improved preventative and treatment strategies for m. bovis-associated disease. currently, control of m. bovis infection in calves focuses on removal of identified or potential risk factors. colonization of the upper respiratory tract of calves with m. bovis often occurs within the first few weeks of life [ , ] as a result of feeding of milk from cows infected with m. bovis or, probably, by direct or indirect transmission from other calves shedding m. bovis in nasal secretions. removal of infected milk from the diet by pasteurization or feeding of milk replacer has been successfully applied to reduce infection [ , , , , ] . breaks in pasteurization have been associated with subsequent infection outbreaks. management practices to reduce stocking density and improve ventilation are examples of changes that can reduce undifferentiated respiratory disease in housed calves and have been recommended for m. bovis control [ ] [ ] [ ] . similarly, control of other pathogens that are involved in the bovine respiratory disease complex is likely to reduce m. bovis infections. management techniques that improve general immune function, such as improving nutritional status and minimizing environmental stress, have also been suggested as beneficial [ , ] . vaccination is a potential strategy to control m. bovis infection, but efforts to develop efficacious vaccines against m. bovis for use in young calves have been problematic. vaccines against m. bovis have afforded some protection from respiratory disease in european field trials [ ] [ ] [ ] . other vaccines have been efficacious against respiratory disease [ , ] and arthritis [ , , ] in experimental challenge studies. however, in some cases vaccination against m. bovis has significantly exacerbated clinical disease [ , ] . in addition, most experimental challenge studies have been performed in calves that are substantially older than the age at which natural colonization with m. bovis is typically first observed. young calves are often colonized by m. bovis within the first few weeks of life [ , , , , ] , during which time the immune system is undergoing rapid changes associated with maturation [ , ] . therefore, agespecific features of the immune system are likely to be important in determining the susceptibility of the young calf to mycoplasmal disease and the efficacy of particular vaccines. there are several bacterin-based vaccines against m. bovis that are currently marketed in the u.s., as well as a number of companies that manufacture autogenous m. bovis bacterins. however, no commercial vaccines are licensed for use in the very young dairy calf, and, to the best of the author's knowledge, no independent studies have been published on their efficacy. the paucity of studies that critically evaluate currently marketed m. bovis vaccines and autogenous bacterins in well-designed, independent efficacy studies in an appropriate age group is a major gap in understanding the potential of currently available vaccines as a management strategy to control m. bovis infections in young calves. in part to address this gap, we conducted a field trial using a commercial m. bovis bacterin that was approved for use in feeder and stocker calves. the objective of this field trial was to determine the efficacy of this commercially produced m. bovis bacterin for the prevention of m. bovis-associated disease (respiratory disease, otitis media, arthritis) and mortality in dairy calves up to days of age. additional objectives were to compare vaccinated and placebo-treated calves with respect to ( ) weight gain from birth to days of age, ( ) rates of nasal colonization by m. bovis, and ( ) m. bovis-specific serum immunoglobulin (ig) concentrations. we studied holstein heifers in three florida herds using a randomized field trial design. the reference population for this study was heifer calves in florida dairy herds with endemic m. bovis infection. the study unit was a holstein heifer calf clustered in one of the three herds in north-central florida. herds were selected based on their willingness to participate and on a history of mycoplasma-associated disease in calves. according to calf health records, at least % of calves were treated for respiratory disease, otitis media and/or arthritis during each of the years preceding the study. calves were enrolled from march to december, . herd a had approximately lactating cows. calves were bedded on sand in individual hutches placed approximately m apart in an open-sided barn with mechanical ventilation. calves were fed unpasteurized bulk tank milk. calves received a modified live virus (mlv) intranasal vaccine against parainfluenza virus type (pi ) virus and infectious bovine rhinotracheitis virus (ibr) in the first week of life. an intramuscular mlv vaccine against ibr, pi , bovine respiratory syncytial virus (brsv) and bovine viral diarrhea virus (bvdv) types and was administered at , and weeks of age. a -way clostridial vaccine was administered at and weeks of age. calves were weaned at approximately weeks of age and turned out into group pens at approximately weeks of age. herd b had approximately lactating cows. the majority of calves were housed in individual elevated metal crates in a concrete-floored open-sided barn with mechanical ventilation. a small proportion of calves were housed in individual hutches on grass. calves housed in metal crates had nose-to-nose contact with neighboring calves. the feeding protocol varied during the study period and included milk replacer and unpasteurized or pasteurized waste milk. the vaccination protocol was similar to that described for herd a. calves were weaned at - weeks of age and turned out into group pens at - weeks of age. herd c had approximately lactating cows. calves were housed on grass in individual hutches placed at least m apart. calves were primarily fed pasteurized waste milk, supplemented with milk replacer when necessary. several failures of pasteurization were documented during the study period through culture of milk following pasteurization. calves received an oral bolus containing antibodies against bovine coronavirus and escherichia coli at the time of colostrum feeding (first defense, portland, me). the vaccination protocol for mlv intranasal pi /ibr and clostridial vaccines was similar to that described for herd a except that the intramuscular mlv vaccine against pi , ibr, bvdv types and , and brsv was administered at and weeks of age. calves were weaned at - weeks of age and turned out into group pens at - weeks of age. all holstein heifer calves that were born during the study period and were considered healthy by the producer at days of age were enrolled in the study. calves were assigned to one of the two groups based on ear-tag numbers, with odd numbers assigned to one group and even numbers to the other group. assignment of odd and even numbers to groups was decided on a per farm basis by a coin flip. one group received a ml dose of a killed, single strain m. bovis bacterin in proprietary oil-based adjuvant that had a conditional license for the prevention of respiratory disease in u.s. feeder and stocker calves at the time of the study (texas vet. labs, inc., san angelo, tx), while the other group received a placebo (all vaccine components except antigen; control group). vaccine or placebo was administered subcutaneously in the neck at and days of age. a ml dose was administered at days of age. the second or third doses were not administered to calves that were sick; however, if the calf recovered within days the dose was administered week after the due date. calves that failed to recover within days remained in the study but missed that dose; missed doses were recorded. the bacterin and placebo were prepared and blinded by the vaccine manufacturer, and the investigators and farm personnel were blinded throughout data collection and analysis. data recorded for each calf included date of birth, ear-tag number, group allocation, dates of vaccine/placebo administration, any missed doses of vaccine/placebo, and date of weaning. the dates of administration of any preventative treatments or other vaccines were recorded for each calf. the primary outcomes of interest were treatment for respiratory disease, otitis media, and arthritis as well as mortality attributed to these diseases. calves were followed from days until days of age, during which time all treatment for clinical disease was recorded by farm personnel using standardized case definitions. farm personnel were trained by the investigators and followed a standardized protocol for health assessment that was prepared by a veterinarian. respiratory disease was defined as fever (rectal temperature ≥ . • f) plus increased respiratory rate or effort and/or coughing and/or nasal discharge; otitis media/interna was defined as ear droop and/or evidence of ear pain (head shaking, scratching or rubbing ear); arthritis was defined as lameness with painful swelling of any joint. sick calves were treated as per normal farm protocols. for each clinically ill calf, farm personnel recorded the type and dose of antimicrobial, the date(s) of treatment, and the reason for treatment. whenever a calf died, farm personnel recorded the cause of death if this was obvious. in most cases, cause of death was verified by field necropsy performed by the investigators. study personnel visited each of the dairies at least once a week to collect calf health data, monitor compliance, and collect samples. because passive transfer of colostral immunoglobulins can influence the immune response to vaccination or to infectious agents, blood was collected from all calves between and days of age for the measurement of total serum protein concentration. a subset of calves from herds a (n = ) and b (n = ) was studied more intensively. these calves were weighed at birth and approximately days of age. weight gain from birth to days was expressed in kg/day. nasal swabs and blood samples were collected weekly until weeks of age and then at days of age. prior to collecting nasal swabs, gross debris was wiped from the external nares using sterile gauze. a sterile rayon-tipped swab with a polyurethane plastic shaft (bbl tm cultureswab tm liquid stuart medium, bd, franklin lakes, nj) was inserted into the ventral nasal meatus to a depth of approximately in. swabs were cultured to detect nasal colonization with m. bovis. serum was analyzed for m. bovis-specific iga, igm, igg and igg by enzyme-linked immunosorbent assay (elisa). swabs were kept on ice during transport and were processed within h of collection. each swab was used to streak the surface of modified frey's agar. all mycoplasma cultures were performed in modified frey's broth and agar medium containing . % (wt./vol.) mycoplasma broth base (frey) (bd diagnostic systems, sparks, md), . % (wt./vol.) dna from herring sperm, % (vol./vol.) horse serum, % (vol./vol.) fresh yeast extract, . % (wt./vol.) glucose, and supplemented with , u/l each of penicillin g and polymixin b and mg/l of cefoperazone, with the final ph adjusted to . - . . plates were incubated at • c in % co and examined at , , and days for mycoplasmal growth. colonies with typical m. bovis morphology were plugged into broth, incubated at • c for h and stored at − • c until they could be confirmed as m. bovis by polymerase chain reaction (pcr). to prepare samples for pcr, l of broth culture was thawed at room temperature then pelleted by centrifugation at , rpm at • c for h. the pellet was resuspended in l of lysis buffer ( mm tris [hydroxymethyl] aminomethane, ph . with . % [vol./vol.] tween and . mm dithiothreitol). samples were incubated at • c for min then cooled to • c. l of clarified sample was used as the dna template in the pcr. as a positive control, broth was inoculated with the m. bovis type strain pg and processed with nasal isolates. sterile water was used as a negative control template. m. bovis was identified by pcr of the housekeeping gene uvrc, as previously described [ ] . pcr products were analyzed by electrophoresis at v for h in . % agarose gels and visualized by staining with ethidium bromide. blood samples were allowed to clot after collection, and then serum was harvested by centrifugation and stored at − • c. whole-cell lysate antigen [ ] was prepared from a l culture of m. bovis type strain pg grown at • c in modified frey's broth. the protein concentration was determined using a colorimetric assay (bio-rad, hercules, ca) and adjusted to g/ml. the antigen was stored in aliquots at − • c and thawed at room temperature when required. the elisa procedure was optimized using standard methodology. microtiter plates (maxisorb f , nunc, kamstrup, denmark) were coated with g per well of antigen in . m sodium phosphate buffer (ph . ) containing . m nacl and . % (wt./vol.) nan (pbs/a), and incubated overnight at • c. plates were then washed three times with pbs/a containing . % (vol./vol.) tween (pbs/t) using an automated plate washer (elx auto plate washer, biotek instruments, inc., winooski, vt), blocked with l per well of blocking buffer (pbs/t containing % [wt./vol.] egg albumin), and stored at • c for a minimum of h or until needed. sera were diluted ( : for igg assay; : for igm and igg assays; : for iga assay) in blocking buffer and l of the diluted serum was added to duplicate wells; plates were incubated at room temperature for h. plates were washed as described above and l of goat anti-bovine isotype conjugated to alkaline phosphatase (bethyl laboratories inc., montgomery, tx) and diluted to : in blocking buffer was added to each well. plates were incubated at room temperature for h and then washed as described above. l of . % (wt./vol.) p-nitrophenol phosphate was added to each well and plates were incubated in the dark at room temperature for h. the optical density (od) in each well was read at a wavelength of nm using an automated plate reader (elx ultra microplate reader, biotek instruments, inc., winooski, vt). for each microtiter plate, the blank was the mean value for two wells coated with antigen and incubated with the conjugated secondary antibody and substrate only. the blank od value was subtracted from each sample well, and mean values for each pair of duplicate tests calculated. a pool of sera from calves with naturally occurring mycoplasmal disease and high m. bovis-specific titers was included on each plate as a positive control; the negative control was a pool of serum collected from the same calves prior to ingestion of their first colostrum meal. the cutoff for a positive result was the average od value (minus the blank) for the negative control sera plus two standard deviations, established over ten assay runs. all elisa data was reported as the average od value at the standard serum dilution for each isotype. within-batch and between-batch assay variabilities were assessed by using the youden plot graphic method [ ] . the elisa values obtained for the lowest, middle and highest dilution of the control serum included on each plate were used to establish target values and control limits to be used for monitoring the consistency of the assay (ten batches). the values obtained at the beginning of a series of assays were plotted against the values obtained for the same standards at the end of the series. if values for the pooled sera deviated more than % from target values, the assay was repeated. a standard field necropsy was performed by a study veterinarian on most calves that died or had to be euthanized. selected calves were not necropsied when a cause of death was obvious and there had been no clinical evidence of m. bovis infection (e.g. a leg fracture that necessitated euthanasia). calves were examined to determine the cause of death and specifically determine the involvement of m. bovis-associated pathology. cases were defined as m. bovisassociated mortality when there had been a history of respiratory disease, otitis media, and/or arthritis, where m. bovis (with or without other pathogens) was isolated from sites of pathology, and where the observed pathology was consistent with mycoplasmal disease. all necropsies included collection of swabs of the palatine tonsils, tympanic bullae and primary bronchi for mycoplasmal culture. additionally, if the animal had previously been diagnosed with respiratory disease, arthritis or otitis media, or if any macroscopic lung pathology was observed, appropriate samples were collected from the lesion site(s) to determine the involvement of m. bovis as well as other viral and bacterial respiratory pathogens. further samples were collected when deemed necessary to determine the cause of death by the veterinarian performing the necropsy. all swabs and fresh tissue samples were transported on ice to the laboratory as soon as possible and were processed within h after collection. when tissue samples for fixation were collected, they were placed into containers of % buffered formalin and submitted to the diagnostic pathology service, college of veterinary medicine, university of florida. samples then were embedded in paraffin and sections ( m) stained with hematoxylin and eosin. histopathology was read by diagnostic pathologists without knowledge of experimental treatment groups. in addition to culture for mycoplasmas (described under nasal swabs, above), swabs for aerobic microbiological culture were processed and isolates identified using routine clinical bacteriological methods. these methods were focused on identifying bacterial pathogens of the respiratory tract other than mycoplasmas, particularly arcanobacterium pyogenes, histophilus somni, mannheimia haemolytica and pasteurella multocida as well as pathogens that may cause septicemia and associated sequelae in young calves. additional diagnostic testing was performed as requested by the veterinarian who conducted the necropsy based on the presumptive diagnosis and any macroscopic pathology. samples were submitted to the florida state diagnostic laboratory (kissimmee, fl) for detection of bovine respiratory viruses when indicated. morbidity due to m. bovis was the major outcome of interest and was therefore used to calculate sample size. at the time the study was initiated, health records indicated that the incidence of respiratory disease, otitis media and/or arthritis in the study herds was at least %. we hypothesized that a reduction in incidence to % would be biologically and economically significant. using these values together with % confidence and % power, and taking into consideration an attrition rate of approximately %, the calculated sample size was calves per group. for the secondary outcomes of interest from a subset of calves in herds a and b, we hypothesized that a reduction in the nasal colonization rate from % to % would be biologically significant. using these values together with the parameters outlined above, the calculated sample size was calves per group. calves were excluded from analyses if clinical signs referable to other organ systems occurred concurrently with respiratory disease, otitis media or arthritis, with the exception of diarrhea without fever of less than days duration. incidence risk rates were calculated as the number of calves treated for the disease of interest divided by the initial number of calves less half the calves that died during the study for reasons other than the disease of interest. categorical outcome variables (incidence of clinical disease [%], incidence of mortality [%], incidence of missed dose of vaccine/placebo [%], cumulative risk of nasal colonization [%]), were compared among groups using chi-square tests; data were analyzed for effects of herd and passive transfer status by stratified mantel-haenszel analysis. when the expected count in any cell was less than , fisher's exact test was used. quantitative outcome variables (post-colostral total serum protein concentrations [g/dl], age at first treatment for clinical disease [days], average daily weight gain from birth to days of age [kg/day]) were compared among groups using independent sample t-tests, and elisa data were analyzed using repeated measures anova. the relationships between average daily weight gain and serological data (post-colostral elisa data, week elisa data) were examined using pearson correlation analyses. a p value of < . was considered significant. all analyses were performed using commercial statistical software (spss . , spss inc., chicago, il). three hundred and twenty-eight calves from herds b and c ( and calves, respectively) were enrolled and were eligible for inclusion in the study (table ) . despite a history of m. bovis infection, herd a did not experience any m. bovis-associated disease a in herd b, one calf was excluded because of concurrent disease and one calf was excluded because a dose of vaccine/placebo was inadvertently missed; in herd c, one calf was excluded because of concurrent disease and one calf was excluded because it received a dose of vaccine/placebo for the wrong group. during the study and therefore is excluded from some analyses, but data from this herd are included where relevant. the incidence risk for clinical respiratory disease, otitis media, and arthritis was assessed from to days of age (table ) . m. bovis-associated respiratory disease and otitis media were major contributors to calf disease in herds b and c. eight cases of arthritis were observed in herd b, and none were observed in herd c. herd a had a much lower overall mortality risk ( . ) than did herds b ( . ) and c ( . ); m. bovis-associated mortality in herd b accounted for the majority of the mortality risk ( . vs. . overall). vaccinated and control groups had equivalent levels of postcolostral total serum protein. a small percentage of calves did not receive their second vaccine/placebo due to illness, and this was not different between groups. although there were no significant differences for calves that missed the third vaccine/placebo when data from herds b and c were combined ( % [ / ] vaccinate vs. % [ / ] control), no control calves in herd b missed the third placebo as compared with % ( / ) of vaccinated calves that missed the third vaccine (p = . ). vaccination was not efficacious in limiting m. bovis-associated morbidity in the young dairy calves in this field trial. vaccination did not influence the age of first treatment for either otitis media or respiratory disease. the overall age for first treatment of otitis media was ± days for vaccinated calves and ± days for control animals. calves in herd c were treated for otitis at ± days (vaccinated) and ± days (control), whereas treatment was at a later age in herd b ( ± days and ± days for vaccinated and control calves, respectively). a similar pattern was observed for respiratory disease, with no difference in the overall age at first treatment between vaccinated and control calves ( ± days, vaccinated; ± days, control). as for otitis media, calves in herd herd a calves were excluded as no morbidity was observed in these animals. "all respiratory" includes calves that were treated for respiratory disease alone or for respiratory disease in conjunction with either otitis media or arthritis. "all otitis media" includes calves that were treated for otitis media alone or for otitis media in conjunction with either respiratory disease or arthritis. mbad = mycoplasma bovisassociated disease. a one control calf with arthritis also had respiratory disease. b combined (herds b and c) analyses were not performed for data that included otitis media because of the interaction between vaccination and herd for this outcome. c required earlier treatment for respiratory disease ( ± days, vaccinated; ± days, control) than did calves in herd b ( ± days, vaccinated; ± days, control). vaccination did not reduce the overall m. bovis-associated morbidity or the morbidity specifically associated with otitis media or respiratory disease. in fact, vaccination was associated with increased incidence of otitis media (p = . ) in herd b, but no differences in the incidence of otitis media between groups were observed in herd c (table ) . for herds a and b, weight gain was monitored from birth to days of age; no significant difference was observed in average daily gain between groups in herd a. similarly, no significant difference was observed in average daily gain between vaccinated ( . ± . kg/day, n = ) and control ( . ± . kg/day, n = ) calves in herd b, where endemic m. bovis disease was present. neither overall mortality rates (vaccinated, % [ / ] ; control, % [ / ]) nor m. bovis-associated mortality rates (vaccinated, % [ / ]; control, % [ / ] ) differed significantly between vaccinated and control calves. in herd b, m. bovisassociated mortality contributed to % ( / ) and % ( / ) of mortality events in vaccinated and control calves, respectively. although m. bovis-associated mortality also occurred in herd c, it was not the primary cause of calf mortality: only % ( / ) of mortalities in vaccinated and % ( / ) of mortalities in control calves in herd c were attributable to m. bovis. nasal colonization was not affected by vaccination; in herd b where endemic m. bovis disease was present, the mean percentage (±sd) of calves with m. bovis-positive nasal swabs at each sampling time was . ± . % for vaccinated calves and . ± . % in control calves. the average number of sampling times that m. bovis was recovered from each calf was also not associated with vaccination (data not shown). irrespective of vaccination status, however, the temporal pattern of colonization (fig. ) observed in calves from herd a (no mycoplasmal disease) was quite different from that observed in calves in herd b (significant mycoplasmal disease). calves in herd a had minimal to no nasal colonization with m. bovis during the pre-weaning period. calves in herd a were moved out of individual hutches into outdoor housing in group lots after the -week samples were collected; at the next sampling period ( weeks of age), the level of nasal colonization was similar to that in the herd that experienced m. bovis-associated disease. in herd b, calves had m. bovis present in nasal secretions as early as week of age, and by weeks of age over % of calves were colonized in the upper respiratory tract. this level of colonization was maintained throughout the sampling period. the serum antibody subclass response in a subset of calves in herds a and b was assessed by elisa (fig. ) . no significant differences between vaccinated and control calves were found in either herd a or b for iga, igm, or igg . however, a serum igg response to vaccination was detected. significant differences (p < . ) between vaccinated and control groups were first evident at weeks of age in herd a (no endemic disease) and at weeks of age in herd b (endemic disease). we then assessed if there was an association between immunoglobulin subclass response and outcomes in calves from herd b. there was no association of any immunoglobulin subclass response with morbidity or nasal colonization rate (data not shown). the m. bovis-specific serum igg titers at weeks of age were negatively correlated with average daily gain for control calves (r = − . , r = . , p < . ), but not for vaccinated calves (r = − . r = . , p = . ). interestingly, there was no significant association between postcolostral total serum protein concentrations and the incidence or duration of treatment for respiratory disease or otitis media in herds b and c (data not shown). similarly, the incidence of m. bovis-specific calf mortality in herds b and c was not associated with post-colostral total serum protein concentrations (data not shown). there was also no association between post-colostral m. bovis-specific iga, igm, igg or igg serum titers and either . no significant differences were observed between vaccinated and control calves in the iga, igm, or igg subclasses. vaccinated calves in herd a at , and weeks of age and in herd b at weeks of age had significantly higher levels (p < . ) of igg than did control calves at those time points. results are presented as mean optical density ± sd. morbidity or mortality for the intensively studied subset of calves (n = ) in herd b (data not shown). the commercial m. bovis bacterin tested in this trial was not efficacious in the prevention of either m. bovis-associated respiratory disease or otitis media in pre-weaned calves in two florida herds with endemic m. bovis disease. the response to vaccination was herd-dependent, and a higher rate of otitis media was associated with vaccination in one herd. our findings are not unique; although other investigators have reported some protection from m. bovisassociated disease by vaccination of older calves [ , , , ] , adverse outcomes following vaccination against m. bovis have also been reported [ , , ] . protection from mycoplasmal respiratory disease by subcutaneous vaccination of calves with killed whole cell bacterins has been reported [ , , , ] . in a study of an apparently efficacious vaccine in young calves, nicholas et al. [ ] vaccinated -week-old dairy calves with a single dose of inactivated saponin-adjuvanated bacterin. calves received an aerosol challenge with live m. bovis weeks after vaccination. vaccinated calves had fewer numbers of m. bovis at colonized sites, fewer body sites colonized by m. bovis, and reduced severity and incidence of clinical disease and lesions as compared to control calves. there was also a significant decrease in body weight gain in control calves compared with vaccinates. additionally, no vaccinated calves and two of seven control calves developed arthritis. vaccinated calves produced a strong igg response prior to challenge, but igg subtypes were not reported. no adverse events associated with vaccination were reported. a killed vaccine against four bovine respiratory pathogens (brsv, pi , m. bovis, and m. dispar) was evaluated for protection against naturally occurring respiratory disease in beef calves [ , ] . calves were vaccinated subcutaneously and received two boosters at -week intervals. in one study [ ] , three groups of beef calves aged , and weeks at the time of first vaccination were used, and calves were followed for months. respiratory disease occurred in a significantly higher (p < . ) proportion of the control calves ( %) compared with the vaccinates ( . %). in a second study [ ] using the same vaccination protocol, m. bovis and brsv were implicated in outbreaks of respiratory disease during the trial period. morbidity due to respiratory disease was significantly reduced in vaccinated calves ( %) compared with controls ( %), and mortality in the vaccinated group was similarly reduced ( % and % for vaccinates and controls, respectively). no adverse effects of vaccination were noted. there are a number of key differences between the studies reported above and our study that may have influenced vaccine efficacy. firstly, the strain of bacteria, the antigen concentration, the method of bacterial inactivation and the adjuvant used are all factors that influence the efficacy of bacterial vaccines, although there are limited data on how these affect m. bovis vaccines in particular. although some of these data are not reported in the above studies, and some are not available for our vaccine (e.g. the adjuvant used is proprietary), it is likely that all these factors varied among our study and those listed above. secondly, calves in the above studies were first vaccinated at a substantially older age than the calves in our study. immune responses of the newborn calf have unique characteristics and undergo rapid changes during the first few weeks of life [ ] . vaccination at , and days of age (as was performed in our study) may not elicit the same type of immune response as vaccination at weeks of age (as in the nicholas et al. [ ] study, above). our vaccination protocol was chosen based on (a) protocols that were being applied on dairies in florida, and (b) the early age of infection that had been observed in previous studies [ ] . thirdly, calves in endemically infected herds in our study became colonized at a very early age, meaning that infection was likely well established before a vaccine-induced immune response could develop. adaptive immune responses that develop after infection are very inefficient at clearing mycoplasmal infections and often result in detrimental chronic inflammatory responses, and this could contribute to vaccine failure when animals are already infected at the time of vaccination. lastly, the challenge load of m. bovis that calves are exposed to can affect the efficacy of vaccination. given the high incidence of clinical mycoplasmal disease ( % of calves treated for m. bovis-associated disease) and the early age of colonization observed in our endemically infected herds, the level of m. bovis challenge that calves were exposed to may have been significantly greater than that of the calves in other vaccine studies [ , , ] . vaccinated calves in one herd in our study had a greater risk of otitis media than did control calves. the risk of otitis media in control calves in herd b seemed substantially less than that in herd c, but examination of calf health records from previous years in herd b showed that the risk of otitis media observed in control calves was similar to that which had been historically present (data not shown). therefore, vaccination seemed to exacerbate clinical otitis media in this herd. there are other reports of exacerbation of clinical disease following m. bovis vaccination [ , , ] . however, the immune mechanisms associated with adverse outcomes after m. bovis vaccination have not been determined. vaccination of calves did stimulate a systemic humoral immune response, with an increase in serum igg being detectable after the third vaccination. a tendency towards th -biased igg -dominated humoral responses has also been reported after infection of calves with m. bovis [ , ] . as igg is a much more effective opsonin for phagocytosis of m. bovis than is igg , an igg response may be relatively ineffective for control of m. bovis respiratory infections [ ] . it is somewhat puzzling that a humoral response to infection was not obvious in control calves in herd b where there was a high incidence of m. bovis-associated disease. statistical comparison of igg responses in control groups in herds a and b was not conducted. however, it appears that in the control group in herd a, post-colostral igg antibody levels continued to decline throughout the study period (see fig. ), whereas in the control group in herd b, they did not decline after weeks of age. this result may reflect continued stimulation of the immune response as a result of the endemic nature of m. bovis in this herd. additionally, m. bovis infection can result in local mucosal antibody responses without eliciting a substantial systemic humoral response [ ] . other investigators have also noted a poor correlation between serum antibody responses and m. bovis infection in individual calves during the first months of life [ ] . the vaccine used in our study was ineffective at preventing upper respiratory tract colonization with m. bovis in calves, even when colonization occurred after a humoral immune response to vaccination was established. with the exception of one calf, nasal colonization was not detected in calves in herd a until weeks of age, whereas a significant increase in serum igg responses to vaccination was evident by weeks of age. this is consistent with other reports on m. bovis vaccines; even where m. bovis vaccines have been associated with clinical benefits, they typically fail to induce an immune response that prevents upper respiratory tract infection [ , ] . our findings are consistent with the idea that protection from m. bovis infection is better correlated with local mucosal immune responses than with serum antibody titers [ ] . total protein concentrations or m. bovis-specific antibody levels in post-colostral serum were not associated with protection from m. bovis-associated disease in calves in this study. however, as colostrum was not pasteurized on these farms, it is possible that some colostrum containing high antibody concentrations to m. bovis may have come from cows with intramammary infection and therefore may also have contained live m. bovis. this could certainly mask any protective effect of passive transfer when assessed on a herd level. further studies are required to determine the efficacy of passive transfer for prevention of m. bovis-associated disease in a controlled setting. to the best of the authors' knowledge this is the first peerreviewed, controlled, independent efficacy study of any of the m. bovis vaccines available in north america. vaccination was not efficacious in preventing m. bovis-associated disease in pre-weaned calves in two endemically infected florida dairy herds, nor was it effective at preventing colonization of the upper respiratory tract in older calves in a third herd. the vaccine did stimulate a systemic igg response that was detectable after the third vaccination, but most clinical disease occurred prior to this response. calves in these endemically infected herds were colonized with m. bovis at a very young age, and it is likely that this represents the greatest impediment to successful vaccination in this age group. the vaccine used in this field trial was formulated for use in much older cattle (stocker and feeder calves) for the prevention of respiratory disease, and the results of this study should not be extrapolated to infer whether vaccination is efficacious in that age group. our findings highlight the importance of targeting vaccines for use in young calves specifically to this age group, and illustrate some of the challenges to the development of an efficacious vaccine against m. bovis for use in very young calves, if vaccination of this age group is even possible. association of mycoplasma bovis with otitis media in dairy calves use of valnemulin in the control of mycoplasma bovis infection under field conditions isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in belgium bulk tank milk analysis: factors associated with appearance of mycoplasma sp. in milk mycoplasmal mastitis in dairy herds mycoplasma bovis: disease, diagnosis, and control mycoplasma otitis in california calves naturally occurring mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves clinical study of the disease of calves associated with mycoplasma bovis infection increased severity of calf pneumonia associated with the appearance of mycoplasma bovis in a rearing herd changes in the bacterial flora of the upper and lower respiratory tracts and 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pneumonia in experimentally vaccinated animals mycoplasmal arthritis and meningitis in calves bovine neonatal immunology effects of age and nutrition on expression of cd , cd , and l-selectin (cd l) on t-cells from neonatal calves species identification of mycoplasma bovis and mycoplasma agalactiae based on the uvrc genes by pcr detection of antibodies to a pathogenic mycoplasma in desert tortoises (gopherus agassizii) with upper respiratory tract disease use of youden plot for internal quality control in the immunoassay laboratory experimental intramammary inoculation with mycoplasma bovis in vaccinated and unvaccinated cows: effect on local and systemic antibody response immune response of cattle to respiratory mycoplasmas characterization of the immune response to mycoplasma bovis lung infection interaction of mycoplasma dispar and mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leukocytes association of seroconversion with isolation of agents in transtracheal wash fluids collected from pneumonic calves less than three months of age the authors thank shelly lanhart for her skilled help in sample collection. for help with the laboratory work, we thank janet stevens, barbara crenshaw, dr. marissa curtis and dr. kelly kirk. we are deeply indebted to the herd owners and managers who agreed to participate in the study and to the calf-rearing personnel who recorded data and vaccinated calves. we thank dr. w.h. (bill) wohler at texas vet lab, inc. for providing the vaccine and vaccine vehicle for this study. this work was funded by the florida milk checkoff. key: cord- -ipoelk h authors: crouch, c. f. title: vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: ipoelk h passive immunity against enteric viral infections is dependent upon the continual presence in the gut lumen of a protective level of specific antibodies. this article examines methods currently used to enhance the titre and duration of specific antibody in the mammary secretions of cows and pigs with particular reference to rotavirus and coronavirus infections. in addition, some of the potential problems to be found in attempting to produce vaccines against these viral infections are outlined neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination ~- . in domestic animals economic losses are suffered, as a result of mortality (ranging between and %), and also veterinary costs and decreased productivity of the survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been found to be primary pathogens in calves ,~ and piglets - . these viruses are most frequently isolated during the period from birth to weaning, and animals of this age have been the most intensively studied because of the frequency and severity of these infections. animals of all ages are, however, susceptible, with subclinical infections apparently common in both adult cows and pigs ,'°. in neonatal calves the incidence of rotavirus and coronavirus associated diarrhoea is similar varying between and % '~'-~ . the situation in neonatal piglets is less clear, rotavirus infections are apparently common .t -tt, w.hilst transmissible gastroenteritis virus (tgev), the prototype enteric coronavirus in swine, is an example of a seasonal cold-weather disease, probably related to both the thermal sensitivity of the virus ~ and the effect of cold-stress on converting subclinical to clinical infections ~ . the pathogenesis of enteric rotavirus and coronavirus diseases of swine and cattle are similar. in contrast to tgev, however, rotaviruses appear to be confined to the alimentary tract, predominantly the small intestine, although there is some evidence in both lambs and piglets for infection of the large intestinal , °. the infections are characterized by diarrhoea and dehydration caused by the functional and anatomical loss of the absorbtive cells of the intestine. the principal site of virus replication has been shown to be the intestinal villus epithelium. the infected cells are lost from the tips of the villi and are replaced with immature crypt cells. generally, there is a dimunition in the number and size of the villi and a progressive replacement of the epithelium with squamous and cuboidal cells which lack a brush bordep - .~. °- . such immature cells have been shown to possess reduced levels of disaccharidases =~. . the loss of the absorptive cells of the intestine is assumed to result in the observed malabsorption syndrome. this is further exacerbated by the decreased ability to utilize dietary lactose, resulting in its accumulation in the large intestine, thereby preventing further absorption of water by exerting an osmotic effect. as a result of the severity of these enteric viral infections during the first few weeks of life, passively acquired antibody is the major source of protection. in calves and pigs there is no selective transfer of immunogiobulins from the maternal to the foetal circulation during the last third of the pregnancy. instead, during the period immediately following birth, maternal immunoglobulin is acquired from the colostrum ofthe dam z ' °. absorption of colostral immunoglobulins by the intestinal epithelial cells is a non-selective process t- lasting - h , s. factors present in colostrum may influence the absorption of immunoglobulins , or help prevent their proteolytic degredation . in addition to immunogiobulins, colostrum and milk have recently been shown to contain functional immunocompetent cells including macrophages and t and b lymphocytes '~°. in contrast to colostral absorption, highly specific mechanisms operate in the colostrum-forming m a m m a r y glands of cattle and pigs causing large amounts of igg (relative to iga and igm) to concentrate in the coiostrum ' ' q ' . lgg passively acquired by the neonate from colostrum persists in the serum for several weeks protecting against systemic infection. in tgev infection of pigs ' and rotavirus infection of calves 'g circulating antibody has been found to be of little value. resistance to these infections appears to be mediated instead by local immunity at the epithelial surface of the intestine. in cattle the selective transfer of igg~ from serum to milk continues throughout lactation, although at a reduced level when compared with colostrum formation. the concentration of all three classes ofimmunoglobulin is significantly reduced ( to -fold) in milk and in consequence igg~ remains the primary immunoglobulin in bovine milk ~. in contrasl in pigs the concentration of lgg~ decreases about -fold during the first week of lactation, whilst that of secretory lga declines only about three-fold, leaving it to become the predominant class of immunoglobulin in swine milld t,' . most adult cattle are seropositive for both rotavirus ,s° and coronavirus s~ antibodies. there is a dramatic decline in these colostral antibody titres during the transition to milld ' "s~- , reflecting this reduction in concentration of immunoglobulins. this partially explains the high incidence of rotavirus and coronavirus infections in calves older than five days, as the titres of passively derived protective antibody decline. despite the presence of one or more common antigens it has been demonstrated that rotaviruses isolated from different species can differ antigenically from each other s - . more recently it has been shown that different serotypes exist within isolates obtained from single species. the existence of at least two different serotypes of porcine rotavirus ° and at least three distinct bovine serotypes ~ have been described. bridger et al have suggested the occurrence ofintermediate bovine rotavirus types , although more work is essential to clarify this situation. some recent isolates possessing the distinctive morphology of rotaviruses have been found to lack the common group antigen. to date, these atypical rotaviruses have been isolated from humans, birds, calves, lambs and pigs ~ . in pigs, preliminary results using two previously characterized atypical isolates , ° have indicated that these are distinct and do not share a common group antigen g ,~'. these observations have been extended by snodgrass et al ga who suggest the occurrence of at least four distinct groups ofrotaviruses based on their group antigen. the significance of the serotypic differences observed between rotaviruses in vitro still needs to be fully assessed in vivo. orbiviruses (also members of the reoviridae) possess many serotypes and require the use of multivalent vaccines . many of the cross-protection studies carried out using different rotavirus serotypes are contradictory and the data inconclusive. for example, in utero vaccination of calves with a bovine rotavirus was found to protect against diarrhoea caused by challenge with human rotavirus serotype , although challenge virus was still shed v . in contrast, one out of three calves was protected against a bovine rotavirus challenge after vaccination with a human serotype or an equine rotavirus tm and this animal shed no detectable virus. furthermore, piglets vaccinated with human rotavirus and challenged with porcine rotavirus were protected against the clinical disease but enhancement of lactogenic immunity:. c f. crouch shed virus ~s. using a more defined challenge system. evidence has been obtained indicating that rotavirus isolates from different animal species and of different serotypes show poor cross-protective properties in vivo tm. this observation has been confirmed and extended by studies in gnotobiotic calves and piglets showing that cross-protection only occurred between rotaviruses of the same serotype, and that even a minor serotype difference could be sufficient to affect cross-protection °,g~. further evidence for a lack of cross-protection between rotavirus serotypes can be obtained from studies of sequential infections, where subsequent rotavirus infections were found to be associated with different serotypes ~ . the situation with coronaviruses is simpler. to date, the coronaviruses isolated from mammals and birds have been grouped into four antigenic classes, where little or no cross-reactivity can be demonstrated between classes tm. tgev is antigenically distinct from bovine enteric coronavirus tm as well as from another as yet unclassified coronavirus causing diarrhoea in pigs (cv ) °. two approaches have been used in an attempt to provide calves with protection against rotavirus and coronavirus infections. the first approach involves oral vaccination with live attenuated virus in order to stimulate active immunity in the calf(scourvax ii, norden laboratories). the incidence of diarrhoea in neonatal calves orally vaccinated with attenuated rotavirus was found to be reduced ~- , but the vaccine was not proven to be effective in blind field trials s - . there are a number of limitations associated with this approach. these include the potential of the vaccine to regain virulence: a high incidence of seropositive adult animals, leading to the possibility of interference with vaccine virus replication by maternally derived (milk) antibodies: and the relative immaturity of the neonate's immune system, the second approach utilizes passive protection produced through lactogenic immunity, stimulated by maternal vaccination. attempts to vaccinate dams using an attenuated live vaccine (calf guard, norden laboratories) have failed to significantly enhance milk antibody titres ", ~ (table ) , whilst in- activated, adjuvanted rotavirus preparations have been found to enhance levels of specific antibody in coiostrum and milk (tables and ) . a number of parameters need to be considered in attempting to optimize the enhancement of antibody production in mammary secretions. dose and form of vaccine. in considering inactivated vaccines, it ~s to be expected that relatively large amounts are necessary to achieve a satisfactory response. further. the process of inactivation may decrease the immunogenicity of some viral polypeptides. table shows that no significant differences in milk antibody titres were obtained following vaccination of cows with rotavirus preparations containing either or elisa units (after inactivation) emulsified in freund's incomplete adjuvant. in contrast, if the same preparations were used. but adjuvanted with aluminium phosphate, the higher dose resulted in a greater antibody response. a similar result using an oil adjuvanted rotavirus vaccine has been previously reported ss. formaldehyde inactivated rotavirus vaccines have been used to successfully enhance milk antibody titres as compared with controls s "- °. other workers have reported increased antibody responses using/~-propriolactone as the inactivating agentol although saif et al found that antibody titres in mammary secretions were at least tenfold greater from cows vaccinated with binary ethylenimine inactivated rotavirus compared with those vaccinated with , -propriolactone inactivated rotavirus . adjuvant snodgrass et a found that oil-based adjuvants were more effective than alhydrogel for the enhancement of rotavirus antibody titres in mammary secretions. this concurs with the data presented in table . most workers have demonstrated a satisfactory immune response following vaccination using oil-based adjuvants. generally freunds incomplete adjuvant (table ). route and timing of vaccination. to some extent the route and timing of vaccination are dependent upon the type ofcattle being farmed. thus the intramammary route used successfully by saifetal , whilst applicable to dairy cattle, may not be practical in beef cows. similarly. from an administrative viewpoint a single vaccination would be preferable to a regime utilizing several doses. the majority of studies have reported a significant increase in rotavirus antibody titres in mammary secretions using either subcutaneous or intramuscular injection of oiladjuvanted vaccines. all such vaccines have also proved to be effective when administered as either single or double doses injected prior to or at parturition . - ( table ) . the efficacy of immune milk as a mechanism for providing passive immunity against rotavirus challenge has been examined by a number of workers(table ). the alactogenic antibody originated from either vaccinated (vacc), control (cont) or normal cows (normal). bcalves were either suckled naturally (suckled) or fed a supplemented diet containing antibody (supp). ccalves were either challenged experimentally (exp) or naturally exposed under field conditions (field). d days after challenge edays after birth, r days after start of experiment. ~cows vaccinated with commercial vaccine nr, not reported results, however, are difficult to compare, due to variations in the feeding regime used for the immune milk and also the challenge systems used. the amount and the timing of the feeding oflactogenic antibody and the dose, virulence and serotype of the virus challenge strain used will all affect the apparent susceptibility of the calf to infection. further, in situations where a field challenge has been used. failure of protection may be due to infection by rotavirus serotypes other than those used in the vaccine, or possibly by other agents capable of causing diarrhoea. generally, these investigators reported either a reduced incidence of rotavirus shedding or diarrhoea or both. in only one study did the feeding oflactogenic antibody fail to significantly affect the incidence or onset of diarrhoea. the majority of animals receiving passive immunity appear to be capable of developing active immunity during this period " , consequently vaccination should lead to elimination of clinical disease rather than a delay in its onset investigation of the immunoglobulin isotypes associated with this protective antibody induced by vaccination in bovine milk and colostrum suggests that igg, plays the major role , . these observations are in agreement with those discussed earlier concerning passive immunity in the bovine. in contrast to the bovine system, evidence suggests that milk or colostral immunoglobulin of the iga isotype is more effective than those of the igg isotypes at protecting piglets against infection by tgev -~°°. high persisting levels of lgg may, however, provide some degree of vaccine, vol. , s e p t e m b e r enhancement of lactogenic immunity:. c e crouch protection against virus challeng~ ~. as a result of these observations` most studies have examined methods for optimising the stimulation of secretory iga antibodies in milk. the origin of tgev-specific iga found in mammary secretions remains somewhat obscure, although there is a good correlation with the presence of an infection in the intestinal tract ~, , °°,' . secretory iga in porcine milk is almost certainly locally produced in the mammary glan& °=-~° . in order to explain this phenomenon, it has been suggested that specificallysensitized iga-secreting lymphocytes may migrate to the mammary gland following initial sensitization in the intestine s~-~°°. such an inter-relationship between the intestinal and the mammary immune systems has also been proposed in rabbits =° and humans ~° . direct evidence for such migration, under the influence of pregnancy-associated hormones, has been obtained in micd ° . a summary of various investigations into the antibody response and efficacy of lactogenic immunity following different vaccination protocols is given in table . reduced immunogenicity in pigs of cell culture attenuated tgev has been described ~° . oral vaccination with a live, attenuated tge vaccine, whilst producing neutralizing antibody, did not stimulate good lactogenic immunity in suckling pigs ~°°,~° .~'°. intramuscular vaccination of sows with live, attenuated tgev leads to the enhancement of specific igg levels in colostrum and milld~,"l higher titres oftgev-specific igg have been achieved using intramammary injection, with an associated increase in the protection provided to suckling pigs ~. these results are supported by the observations of other workers "=-" . feline infectious peritonitis virus (fipv) is a member of the same antigenic class as tgev and consequently the two viruses are serologically related. good levels of cross-protection, associated with high titres of tgev-specific neutralizing antibody have been reported in sows vaccinated orally with fipw ~. in contrast, the results of a more recent study have shown that whilst tgev neutralizing antibodies of the lgg subclass are stimulated in milk and colostrum, the survival rate for suckling pigs was low i". it may be possible to boost the level oflga in mammary secretions. preliminary investigations have revealed that specific secretory lga levels in milk can be enhanced by the parenteral injection, at parturition, of tgev or rotavirus into naturally infected (orally primed) animals "s." . a similar approach also combining oral with parenteral antigen administration has been proposed as a means of providing lactogenic immunity against colibacillosis in pigs t=°. lgg can be induced readily in the mammary secretions of cattle, by intramuscular or subcutaneous injection of adjuvanted immunogen. in pigs however, whilst live, virulent virus is capable of inducing high levels in iga iri milk. it is apparent that the ideal candidate vaccine virus must be sufficiently attenuated to produce only mild or no disease in neonatal pigs, whilst retaining sufficient virulence to infect the intestinal tract of adult swine. more work is essential in the possible use of inactivated vaccines for the boosting of existing iga levels in mammary secretions. these may require prior natural infection of the sow, the incidence of which will vary between herds, with an associated affect upon the efficacy of such a vaccine. further investigation into the variety of strains and serotypes of rotaviruses is of obvious importance, as is the response to vaccination of cattle and swine by rotaviruses or coronaviruses. current data suggests that crossprotection between rotavirus serotypes is limited, although there is little information concerning the specificities of the antibodies induced by vaccination of previously infected animals. such animals naturally exposed to a variety of serotypes may produce a heterogeneous antibody response, capable of reacting with a broad spectrum of rotavirus serotypes. it is apparent that the enhancement of lactogenic immunity through the vaccination of the dam provides a suitable mechanism by which neonatal pigs and calves can be protected against rotavirus and coronavirus infections. the production of truly effective vaccines, however, awaits further work in some of the areas outlined above. acute undifferentiated neonatal diarrhoea in beef calves . occurrence and distribution of infectious agents a-l pathogenic relationships of rotavirus, escherichia colz and other agents in mixed infections in calves pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea pathology of neonatal calf diarrhoea induced by a reo-like virus pathology of neonatal calf diarrhoea induced by 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transmissible gastroenteritis transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglet secretory iga and antibodies to escherichia coli in porcine colostrum and milk and their significance in the alimentary tract of the young pig the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow the induction and characteristics of secretory iga antibodies lidinjanson, ( . and sohi-akerlund, a. antibodyforming cells in human colostrum after oral immunization e hormonal induction of the secretory immune system in the mammary gland immunogenicity and distribution of transmissible gastroenteritis in pigs transmissible gastroenteritis in: diseases of swine immunity in tge of swine after infection and vaccination passive immunity in transmissible gastroenteritis of swine: intramuscular injection of pregnant swine with a modified live-virus vaccine experimental immun,zation of sows against transmissible gastroenteritis experimental immunization of sows with cell-cultured tge virus experimental immumzation of sows with inactivated transmissible gastro'enteritis (tge) virus. car~ j present status of products for use against transmissible gastroenteritis cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses efficacy of vaccination of sows with serologically related coronaviruses for control of transmissible gastroenteritis in nursing pigs passive immunity against enteric viral infections passiveimmunityagainstentericviral infections of piglets intestinal defence of the neonatal pig:-inter-relationships of gut and mammary function providing surface immunity against colibacillosis i would like to thank dr s.d. acres for his permission to include some ofthe data obtained during my employment at vido. key: cord- -jh fdks authors: jiang, yi; cheng, xu; zhao, xiumei; yu, yan; gao, mingyan; zhou, sheng title: recombinant infectious bronchitis coronavirus h with the spike protein s gene of the nephropathogenic ibyz strain remains attenuated but induces protective immunity date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jh fdks infectious bronchitis (ib) is a highly infectious viral disease responsible for major economic losses in the poultry industry. a reverse genetic vaccine is a safe, rapid, and effective method of achieving ib prevention and control. in this study, we constructed the recombinant strain, rh -s /yz, using a reverse genetic system, based on the backbone of the h vaccine strain, with the s gene replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , isolated in china. the results of dwarf chicken embryos, growth kinetics, and viral titration in the embryos demonstrated that the biological characteristics of the recombinant virus remained unchanged. like the rh -infected group and in contrast to the ribyz-infected group, no mortality, clinical signs, or lesions were observed in the lungs or kidneys of young chickens inoculated with rh -s /yz. the viral loads in various tissues, cloacal, and oral swabs was lower in most types of samples, indicating that the rh -s /yz strain was highly safe in chicks. compared to rh vaccination group, when the efficacy of this strain was evaluated against the qx-like ibv strain, better protection, with % survival rate and no disease symptom or gross lesion was observed in the chickens vaccinated with rh -s /yz. increased levels of ibv-specific antibodies were detected in the serum of the rh -s /yz-vaccinated animals days post-vaccination. collectively, our results suggest that the recombinant strain, rh -s /yz, may represent a promising vaccine candidate against qx-like ibvs. infectious bronchitis (ib) was first described as a respiratory disease affecting chicks in the us in as an acute and highly contagious viral disease, and continues to cause major economic loss within the poultry industry worldwide [ ] [ ] [ ] . as a result of the damage to the respiratory system, the clinical signs of this disease include depression, coughing, head-shaking, as well as nasal and ocular discharge [ ] . since the virus causing ib replicates in many non-respiratory epithelial surfaces (e.g., kidney and gonads), nephritis, reduced egg production, and egg quality is also observed [ , ] . the causative agent of ib is infectious bronchitis virus (ibv), a non-segmented, positive-sense, single-stranded rna virus, which belongs to the genus gammacoronavirus, family coronaviridae, in the order nidovirales [ ] . the full length rna genome of ibv is . kb in length and encodes non-structural proteins, four accessory proteins, and four structural proteins (spike [s], matrix [m], nucleocapsid [n] , and envelope [e] ). the s protein is cleaved into s and s as two subunits at the s /s cleavage site. the s protein contains the receptor-binding domain and mediates viral attachment to host cells, whereas s is responsible for membrane fusion [ , ] . errors generated during the genomic replication of ibv and the selective pressure following the use of live attenuated vaccines or multiple infections with different ibv serotypes has led to the emergence of numerous ibv variants [ , ] . in particular, small change as little as % in the amino acid composition of s gene may lead to alteration in cross protection among closely related serotypes [ ] . in china, ibv was first observed during the early s, and outbreaks have since been frequently reported [ ] . the qx-like genotype is thought to have originated during the mid- s and continues to be the predominant strain, whereas the ldt , / , and taiwan subtypes have also recently been frequently isolated in china [ ] [ ] [ ] [ ] . severe nephritis, false layer syndrome, and high mortality are observed in chickens infected with this viral genotype, which represents a major problem in the poultry industry, particularly in china. using a live attenuated or inactivated vaccine is typically considered to be the most cost-effective means of controlling viral infections [ ] . unfortunately, inactivated ib vaccines are not effective if used alone, which could induce little or no protection against egg loss [ , ] , as well as impaired ciliary activity in the trachea [ ] ; thus, birds must continue to be given one or a series of vaccinations with live attenuated ib vaccines to provide broad heterologous protection [ ] . commercial attenuated live vaccines used against ibv in china include the h , ldt , and / strains [ ] ; however, phylogenetic analysis indicates that the qx-like genotype is genetically distant from the strains described above, which may explain the poor cross-protectivity against infection in chickens immunized with these classical vaccines [ , ] . recently, there has been increasing research on live attenuated vaccines against the qx-like ibv strains. different strains of qxlike ibvs were serially passaged in embryonated eggs or primary chicken kidney cells (ck) to obtain vaccines that are less virulent and exhibit high immunogenicity against the qx-like epidemic ibv strains [ ] [ ] [ ] [ ] [ ] . however, in addition to causing tissue damage or secondary bacterial infections in young vaccinated chicks [ , ] , live attenuated vaccines of rna viruses may not be genetically stable or they may be associated with the tendency to revert back to a virulent form [ , ] . moreover, the potential for recombination between vaccine and virulent strains can lead to the creation of new virulent virus [ ] . furthermore, it takes a substantial amount of time to prepare a extensively passaged attenuated vaccine strain, which may not be able to effectively solved the loss problem caused by the rapid variation exhibited by prevalent strains on the flocks. in considering the limitations of the live attenuated and inactivated ibv vaccines, reverse genetic ibv vaccines have been developed in recent years as they display increased safety and efficacy [ ] [ ] [ ] . as reverse genetic technology is primarily based on the h and beaudette strains [ ] [ ] [ ] , both of which are passagegenerated attenuated strains, the mechanism of virus virulence attenuation remains unclear, thereby posing difficulties in the development of attenuated vaccines using this technology. in addition to the function of host invasion and increasing genetic diversity, the ibv s subunit can induce virus neutralizing and cross-reactive antibodies [ ] . therefore, some researchers have created an attenuated ibv vaccine by replacing the s or s genes from other virulent strains using reverse genetic technology to generate protection against strains of the corresponding serotype [ , , ] . in the present study, we constructed a recombinant strain, using the reverse genetic system. rh -s /yz is based on the backbone of the vaccine strain h , and the s gene was replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , which was isolated in china. our results demonstrate that this recombinant strain is safe and provides effective protection in young chickens against qx-like ibv challenge. the h vaccine strain is currently a widely used vaccine strain, created from serial passages of the h strain isolated in . the ck/ch/ibyz/ strain (referred to as ibyz, genbank kf . ) used in this study is classified as a qx-like ibv isolated from a flock presenting with ib symptoms by our research group in in jiangsu province, china. both of these strains were recovered from a full-length clone using reverse genetics, as previously described [ , ] . fertilized specific pathogen free (spf) chicken embryos were obtained from beijing merial vital laboratory animal technology co., ltd. (beijing, china) and hatched in our lab (lab of chicken infectious disease protection and control, poultry institute, chinese academic of agriculture sciences, yangzhou, china). for hatching, one-day-old spf chickens from the spf chicken embryos as described previously were incubated at °c in a relative humidity of - %. all chickens were maintained in isolators under negative pressure, and food and water were provided ad libitum. the ribv rh -s /yz strain used in this study is described in the schematic illustration presented in fig. . the genome rna was synthesized in vitro by t rna polymerase and transfected into bhk- cells, as previously described [ , ] . the cell supernatants were harvested h following transfection and propagated in -day-old spf chicken embryos. the allantoic fluid was harvested for rt-pcr, whole genome sequencing, and was then stored at À °c until further use. to determine the recombinants' pathogenicity in chicken embryos, . ml of rh , ribyz, or rh -s /yz virus (diluted to : in normal saline) was inoculated into the allantoic cavities of five -old-day embryonated spf chicken eggs, respectively. chick embryo lesions were examined for lesions at h postinoculation at °c. to examine the viral growth ability in chicken embryos, a realtime reverse transcription quantitative polymerase chain reaction (rt-qpcr) method was established. according to the sequences of ibv from genbank, primers were designed based on conservative area in the -utr. the upstream primer was -ccgttgctt gggctacctagt- , and the downstream primer was -cgcctac cgctagatgaacc- . the amplification product was cloned to pmd -t vector (takara) as a positive plasmid and its concentration was measured. a gradient dilution of  -  copies/ll of the plasmid was used as template for quantitation test. by plotting the cycle threshold (ct) values against the copies of the plasmid, the standard curve was generated. -day-old embryonated spf chicken eggs (six eggs/group) were inoculated with rh , ribyz, and rh -s /yz at a dose of viral rna copies/ ll, and used for growth curve experiments. the allantoic fluids were collected separately by syringe from the six inoculated embryonated eggs of each group at , , , , , , h per inoculation. rna was extracted from the allantoic fluids using trizol reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions. cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral copies were measured by absolute quantitative method of realtime pcr, which was performed using sybr Ò premix ex taq tm ii (takara, otsu, shiga, japan) on an applied biosystems fast real-time pcr system. the standard curve was plotted against the log of the template copy number. all of the assays were run in triplicate and the copy number of each virus was calculated according to the standard curve. serial -fold dilutions from copies to copies per . ml of virus of the recombinant strain, rh -s /yz, and its parental strains (rh and ribyz) were inoculated into the allantoic cavity of -day-old embryonated spf chicken eggs. for each dilution, . ml of the virus solution was injected into each egg and eggs were used for each dilution. pbs was used as a negative control. after a h incubation, the dead embryos were considered nonspecific deaths and discarded. the embryos were examined for the presence of specific lesions caused by the virus after a -h incubation. dead and live embryos that displayed ibv infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples. the % embryo infectious dose (eid ) of these ribvs was calculated using the reed-muench method [ ] . the chickens were separately housed in isolators under consistent conditions, and food and water was provided ad libitum. a total of one-day-old spf chickens were randomly divided into four groups (n = per group). the chickens in the experimental groups were intranasally inoculated with ll allantoic fluid per chick containing eid of the rh , ribyz, and rh -s / yz strains. the control group (n = ) was inoculated with ll sterile pbs via the same route. the morbidity and mortality was schematic diagram for the construction of the chimeric s gene and production of a full-length cdna of rh -s /yz. (a) replacement of the h s fragment by the corresponding sequence of ibyz for construction of pyzs h s . the plasmids pibyzs and ph s contained the s gene of ibyz strain and h strain, respectively, were constructed during the establishment of reverse genetic system. primers ps f and ps r were used to amplify the s fragment of ibyz and vector fragment, while primers ps f and ps r were used to amplify the s fragment of h strain. by overlapping pcr, the pyzs h s was constructed which contained a chimeric s gene. (b) strategy for the construction of full-length cdna clones of rh -s /yz. ten cdna fragments covering the entire genome of h strain was amplified by rt-pcr. unique bsa i sites were inserted at the junctions between each clone, a unique t start site was inserted at the end of clone tm , and a -nucleotide t tail was inserted at the end of clone tm . the s fragment in the pmdtm plasmid of h strain was replaced by chimeric s gene. by using appropriate ligation strategy, the genomic cdna of rh -s /yz was assembled by in vitro ligation using appropriate restriction sites as indicated. followed-up for days. all experimental groups were monitored daily for clinical signs related to ib infection for days postinfection (dpi), including coughing, sneezing, and tracheal rales. dead chickens were examined for gross tracheal, lung, and kidney lesions. a total of -day-old spf chickens were assigned to four groups (n = per group). the birds were inoculated with ll allantoic fluid containing eid of the rh , ribyz, or rh -s /yz strains, respectively via eye drop and the intranasal route and ll pbs per chick was administered to the control group (n = ) via the same route. the tracheas, lungs, kidneys, and bursa from the inoculated birds per group were harvested at , , , and dpi, weighed, and collected into ml pbs per sample and frozen at À °c. after grinding the samples, the viral rna was extracted using trizol, and the cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral rna copies from each of the different samples were detected by real-time pcr as described above. all assays were run in triplicate and the copy number for each virus was calculated according to the standard curve. after inoculation with eid dose of the rh , ribyz, and rh -s /yz strains, three infected chickens per group were randomly selected to be euthanized by bleeding, and the tissue pathology was examined in the different groups at , , , dpi. the tracheas, lungs, kidneys, and bursas of three dead or other randomly-selected chickens from the experimental and control groups (inoculated with pbs) were collected at or dpi and further processed for histopathology. the samples were fixed in formalin, embedded in paraffin, cut into lm sections, and stained with hematoxylin and eosin. the slides were examined under light microscopy for the presence of lesions. to determine the level of viral shedding of the different recombinants, birds per group were inoculated with ll allantoic fluid containing eid of the rh , ribyz, or rh -s /yz strains, respectively. ll pbs per chick was administered to the control group via eye drop and the intranasal route. . oral and cloacal swabs were collected from each bird into ml pbs at , , , , , and dpi and stored at À °c. after freezing and thawing three times and centrifuging at ,  g for min at °c, relative amount of virus present in ll of supernatants of each sample was quantified by rna extraction, reverse transcription, and real-time pcr as described previously. . . efficacy of rh -s /yz vaccination . . . viral challenge in total, one-day-old spf chickens were divided into three groups of chickens. the three groups were intranasally vaccinated with the rh and rh -s /yz strains at a dose of eid / ll, and the control group received pbs. two weeks post-vaccination, the chickens in each group were challenged with the qx-like strain, ribyz, at eid / ll via eye drop and the intranasal route. any clinical signs, as well as the percentage of morbidity and mortality were recorded for days. the dead chickens were also examined for gross tracheal, lung, and kidney lesions. -day-old spf chickens were divided into three groups (n = per group). after vaccinating with the rh and rh -s /yz strains at a dose of eid / ll, the sera of birds from each group were randomly collected and antibodies were tested using an elisa created in our lab at , , , and days post-vaccination (dpv). elisa plates were coated with antigen of culture supernatants of inactivated ibv rmj , a vero cells adaption strain domesticated from its parental strain of ibyz in our lab. individual chicken sera collected from different groups diluted to : were loaded and the plates were incubated at °c for min. ibv-specific igg was detected with anti-chicken igy (igg) (whole molecule)-peroxidase antibodies produced in rabbits (sigma-aldrich, germany). after incubated with , , , -tetrame thylbenzidine(tmb) substrate for min and terminated with mol/l h so solution, the absorbance at nm was measured using a model microplate reader (bio-rad, usa). graphpad prism software (graphpad software inc., la jolla, ca, usa) was used for the statistical analyses. for the antibody test, growth kinetics, tissue tropism, and viral shedding test, the collected data were analyzed using a two-way anova to determine whether there was a significant difference between the different groups. the significance was considered as follows: significant at p . (*); highly significant at p . ( ** ); and extremely significant at p . ( *** ). characterization of the rh -s /yz strain after being inoculated with the recombinant strains, typical ib lesions in the chick embryos from each group were observed, including amniotic membrane thickening, dwarfing, stunted growth, curling, or death of the embryo (fig. a) . the eid of the virus was calculated according to the reed-muench method. the titers of the rh , ribyz, and rh -s /yz strains were determined to be . eid /ml, . eid /ml, and . eid /ml, respectively (fig. b) . the growth kinetics of the ribvs were assessed by inoculation with copies of virus per egg in eggs from each group. the relative viral load was determined at , , , , , , and h post-inoculation by quantitative real-time rt-pcr. the growth curves of all three strains reached a relatively stable plateau at - h after infection; the level of viral rna for the rh -s / yz strain was significantly higher than that of the ribyz strain for the majority of the time points (fig. c ). the earliest death was observed at dpi in the qx-like strain ribyz group, which virus was highly pathogenic to one-day-old spf chickens; the final mortality of this group was . % (fig. a) . the morbidity of the infected chickens in the ribyz group reached higher than %, and the diseased chickens exhibited respiratory symptoms (e.g., sneezing, coughing, as well as tracheal and bronchiolar rales), and the severe cases presented with additional signs of listlessness, huddling, and ruffled feathers. at necropsy, lesions were detected both in the respiratory and urinary system, including mucus, hyperemia, and hemorrhage in the trachea, as well as swelling and urate deposition in the kidney. edema and congestion were observed in the lungs in % of cases (fig. b) . however, following inoculation with the rh or rh -s /yz strains, the morbidity rates of the chickens was less than %, with moderate respiratory signs of coughing in some of the chickens. all of the infected chickens presented no visible lesions in the kidney and the survival rate was % through dpi in both the rh and rh -s /yz groups ( fig. a and b) . no obvious clinical signs, gross lesions, or death attributable to ibv was observed in the control group. for the chickens inoculated with the qx-like strain ribyz, lesions in the trachea at dpi were characterized as cilia necrosis and loss, epithelial cell congestion, hemorrhage, and lymphoid infiltration (fig. c ). in the bronchial and capillary lumen of ribyz-infected lungs, congestion, hemorrhage, as well as erythrocyte and inflammatory cell infiltration was observed in some severe cases from to dpi (fig. g) . moreover, erythrocyte and lymphocyte infiltration, as well as tubular dilation was observed in the kidney of ribyz-inoculated chickens (fig. k) . similar to the rh group, chickens inoculated with rh -s /yz presented with mild pathology in the trachea at the early infection time points of , dpi. lesions in the trachea consisted of the loss of epithelial cells and cilia, decreased number of secretory cells, and thickening of the mucosa (fig. b and d) . in contrast, there were no lesions observed in the lungs and kidneys of rh and rh -s /yz-infected chickens during the infection period (fig. f , h, j, and l). no histopathological evidence of damage was detected in the bursa of all the groups and no ibv-associated lesions were found in the tissue samples of the control chickens (fig. a , e, i, m-p). the time-dependent levels of viral replication in the different tissues determined by real-time rt-pcr presented in fig. are consistent with the tissue histopathology results. compared to rh and rh -s /yz groups, one day after inoculation with the qxlike strain, ribyz, the viral load in all organs began to rise rapidly, except at dpi in the trachea. the viral load in tracheal samples in the rh -s /yz group increased and peaked at dpi. the level of viral rna was significantly lower than the rh group both at dpi and dpi (fig. a) . the viral load in lung samples from all infection groups increased starting at dpi, and the viral rna in the lungs of the rh -s /yz group was significantly lower compared with both the rh and ribyz groups (fig. b) . the viral load in the kidney of all ibv-inoculated groups was detected at dpi. in contrast to the rh and rh -s /yz groups, the viral growth in kidney of ribyz-infected chickens exhibited an extremely significant increase at dpi and peaked at dpi. the viral of the recombinant ibvs, rh , ribyz, and rh -s /yz in spf chicken embryonated eggs. (c) the multicycle growth kinetics of the recombinant ibvs, rh , ribyz, and rh -s /yz in spf chicken embryonated eggs. all data are presented as the mean ± standard deviation (sd). some of the error bars are too small to be seen. the qrt-pcr was carried out with three replicates. markers of statistical difference were acquired by comparing between rh group and rh -s /yz group. *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates a significant difference at . < p . . rna copies of the rh -s /yz group peaked at dpi, after which they gradually decreased (fig. c) . no viral load was detected in the control samples at any of the time points. in all ibv infection groups, the viral load in both the oral and cloacal swabs generally showed a gradual decline from dpi to dpi. compared with the rh group, the reduction in viral load between or dpi was significantly greater for chickens infected with rh -s /yz than for chickens infected with rh , and the viral rna of the qx-like strain ribyz was lower than that of the rh group at , , and dpi but with no statistically significance (fig. a) . in contrast, the viral load in the cloacal swab of the ribyz group was significantly higher than that of the rh and rh -s /yz groups at dpi. compared with other groups, the viral rna in the cloacal swab of the rh -s / yz group was significantly lower at , , and dpi (fig. b) . no virus was detected in the control group at any time point. the rh -s /yz group exhibited effective protection against challenge with the qx-like strain ribyz. the mortality rate in the rh -s /yz group was %, and no clinical signs or lesions were observed during the ribyz challenge period (fig. a) . in contrast, chickens vaccinated with the rh strain began to exhibit clinical signs (e.g., coughing and nasal discharge) at days post-challenge (dpc), and some chickens showed severe signs of listlessness, hud- fig. . pathogenicity of the ribv rh , ribyz, and rh -s /yz strains. (a) percent survival of spf chickens infected at -day-of age during the -day observation period. no mortality was observed in rh , rh -s /yz, or the control groups. (b) gross lesions on the lungs and kidneys of the infected chickens at or dpi. fig. . histopathological changes in the trachea, lungs, kidneys, and bursa of spf chickens infected with the recombinant rh , ribyz, and rh -s /yz strains at -day-ofage. black arrows in the trachea indicate cilia and necrosis loss. black arrows in the lungs indicate congestion and hemorrhage. trachea samples were collected at , dpi (a-d); lung and bursa samples were collected at , dpi (e-h; m-p); and kidney samples were collected at , dpi (i-l). black arrows in kidneys indicate tubular dilation, congestion, and inflammation. scale bar = lm or lm. fig. . viral load in the different organs at , , , and dpi of spf chickens infected with the recombinant strains rh , ribyz, and rh -s /yz at -day-of age. the bars indicate means ± standard deviations. *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates a significant difference at . < p . . dling, and ruffled feathers, with the mortality rate reaching % during the observation period. at necropsy, the dead animals displayed typical kidney lesions characterized by swelling and urate deposition in the tubules and ureters. in contrast to chickens vaccinated with rh -s /yz, the chickens in the unvaccinated control group exhibited clinical signs at dpc, and a % mortality rate; the gross lesions of the dead birds in this group were similar to that of the rh group (fig. b ). antibodies against the ibvs were measured using an elisa (fig. c) . none of the birds in the rh or rh -s /yz groups showed a positive antibody response at dpv, whereas % of the birds had an antibody response detectable at dpv, with the antibody level gradually increasing during the observation period. however, there was no significant difference in the level of serum antibodies between rh -vaccinated chickens and rh -s /yz-vaccinated chickens. no positive serum antibody response was detected in the non-vaccinated controls. vaccines, particularly live-attenuated vaccines, remain the most effective means of protection against ibv challenge (e.g., the mass serotype strains h , h , ma , and w , which are still widely used in the poultry industry) [ ] [ ] [ ] . however, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the ib endemic in china cannot be controlled [ ] . genetic mutations and immune pressure during the replication process of the single stranded rna virus often results in the emergence of ibv variants [ , ] . to date, at least six genotypes of ibv strains have been identified in china, of which the fig. . viral loads at , , , , and dpi in the oral and cloacal swabs of chickens infected with the recombinant rh , ribyz, and rh -s /yz strains. all data are presented as the mean ± standard error of the mean (sem); *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates significant difference at . < p . . fig. . efficacy of the rh and rh -s /yz vaccines. (a) percent survival of the vaccinated chickens challenged with the qx-like ibv ribyz strain during the -day observation period. no mortality was observed in the chickens vaccinated with the rh -s /yz strain. (b) gross lesions observed on the kidneys of the vaccinated chickens challenged with the ribyz strain at or dpc. (c) the mean antibody od value at , , , and dpv. all data are presented as the mean ± standard error of the mean (sem). qx-type (first isolated in ) remains the most prevalent genotype [ , , ] . the poor relationship between the qx-type strains, which are abundant and display variable virulence in various parts of china, and the mass-type vaccine strains could explain the failure of the mass-type vaccination programs to control ibv in these flocks [ , ] . consequently, the development of novel vaccines against circulating ibv strains in china using local ibv strains is required [ , , ] . optimal vaccination against circulating ibv strains in china requires the development of attenuated vaccines designed from local strains [ ] [ ] [ ] ] . however, due to the particularity of rna virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [ , , ] , which represents a substantial hidden concern to the poultry industry. to reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., beaudette strains carrying the s gene of the h vaccine strain, virulent m strain, or qx-like strain) [ , ] . however, the titer of the beaudette strains carrying the s gene of the vaccine h strain only reaches . ± . eid [ ] , which is lower than that of the h backbone strain. therefore, we aimed to develop a recombinant rh -s /yz strain based on the h vaccine strain that carries the s gene of the ck/ch/ibyz/ strain (a chinese qx-like nephoropathogenic strain) using reverse genetic technology [ , ] . in the present study, we isolated a nephoropathogenic strain (ibyz) in . this strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of ibv emerged in jiangsu province. chickens infected with the ibyz strain showed severe kidney damage and a high mortality rate (fig. b) . the complete sequence has been deposited in the genbank database under the accession number of kf . the s gene sequencing data indicated that the ibyz strain belonged to the qx-like genotype (fig. ) , with a nucleotide homology with the attenuated vaccine h strain of only . %, and amino acid homology of . %. moreover, the s /s cleavage site of ibyz is hrrrr/s, which differed from rrfrr/s of the h vaccine strain. after wholegenome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed ribyz. in addition, another strain used in this study is the live attenuated ibv vaccine, h , and the molecular clone strain, rh , which was constructed using the same method. the spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [ , ] . however, replacing the s gene of beaudette strain by the virulent strains does not make it virulent [ , ] . the s protein can be cleaved into s and s subunits at the s /s cleavage site [ ] , the s subunit is responsible for receptor binding to the host cells [ ] . whether the s gene has an effect on the virulence of the virus is unknown. in this study, the recombinant strain, rh -s /yz, expressed the s gene of the qx-like strain, which retains the s /s cleavage site of h , maintained the ability to replicate and exhibited pathogenicity in embryos. however, providing ibyz s sequences to h did not increase its virulence to that of ribyz. ibvs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [ , ] . the qx-like ribyz strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. in addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points postinfection. the ability of ibv to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of ibv to host cells is dependent on sialic acid on the cell surface being recognized by the s subunit [ , , ] . however, the binding of virus to the host cell is the first step in determining tropism and s is responsible for membrane fusion [ ] . thus, to further explore the different tissue tropism between rh -s /yz and ribyz, we examined the viral loads and histopathology in the tissues of the rh -s /yz-infected group. it was found that rh -s /yz remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the ribyz strain. this finding suggests that both the s and s genes might play an important role in viral tissue tropism as also suggested by others [ ] . another possibility is that the lack of viral replication and damage in kidney following rh -s / yz is due to lower replication ability of backbone except s gene from its parental rh strain. the viral load of the respiratorytype strain, rh in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of ribyz, which indicates that the ability of the virus to attached to trachea epithelial cells of the rh strain was stronger than that of the ribyz strain. however, during the later time points post-infection, the level of the rh strain was significantly lower than ribyz, which suggests that the virulence of ibv depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [ , , [ ] [ ] [ ] . the reduction of the viral load at dpi in the group infected with the ribyz strain may be associated with the necrosis and shedding of the trachea epithelium. moreover, the ability to replication in the different organs of chickens infected with rh -s /yz as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the ribyz strain, which demonstrated that the s subunit was not the only determinant of viral tropism that was consistent with results by others [ , , ] , and the significant difference in viral load was related to differences in the viral backbone. however, when comparing the rh -s /yz and rh strains which carry the same backbone, it was found that the viral rna of rh -s /yz in the lungs and kidneys was significantly lower than that of rh , which may due to the replacement of the ibyz s gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the s protein of coronaviruses. although cryo-em structure of ibv spike protein has been determined, the relation between structure and function of different ibv s genes is unknown. [ ] . in addition to the growth in epithelial tissues observed during the early infection period, ibv can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to ibv control [ , ] . in this study, we detected viral rna in the oral and cloacal swabs isolated from infected chickens at , , , , and dpi by real-time pcr. compared with rh and ribyz, the viral load in both the oral and cloacal swabs of the rh -s /yz group at different time points after infection was significantly decreased. however, while it has been reported that the persistent virus will be re-excreted at the point of lay [ ] , the persistence of rh -s /yz strain in the egg-laying chickens needs to be further verified. in a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rh -s /yz can be considered a safe attenuated vaccine candidate for young chickens. in addition to its role in tissue tropism, the s glycoprotein also has an important function in inducing a neutralizing antibody response [ , ] , and small differences in the s contribute to poor cross protectionby neutralization test [ , ] . to evaluate the protective efficacy of this candidate vaccine, we selected the qx-like virulent ribyz strain to infect chickens at dpv that were vaccinated with either the rh -s /yz strain or rh strain. we found that little protection against ribyz virulent strain challenge in the rh vaccinated group was induced, with a % survival rate and severe clinical symptoms associated with ib. this finding could be explained by differences in the antigenicity between rh and ribyz. no death, clinical signs, or lesions were observed in the rh -s /yz-vaccinated group, indicating that this strain could provide effective protection against ribyz challenge in young chickens. since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [ , ] , the level of specific igg antibodies remains an important standard for evaluating the immune response to an ibv vaccine. in the present study, the humoral antibody level of chickens vaccinated with the rh -s /yz strain was observed to gradually increase compared to the non-vaccinated group. however, although the rh strain was able to induce similar levels of humoral antibodies, it provided poor protection against ribyz, suggesting that protection against ibv infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [ ] [ ] [ ] [ ] . thus, the detection of mucosal and cellular immunity in rh -s /yz-vaccinated animals requires further exploration. nevertheless, some studies have shown that a peptide located near the amino terminal end of s can be recognized by neutralizing monoclonal antibodies [ ] , and the s domain also plays an important role in inducing protective immunity [ , , ] . because the s domain plays an important role in cell tropism [ ] , we also considered that expressing the s subunit would provide greater safety than the entire s gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required. in conclusion, we constructed the recombinant strain, rh -s /yz, which was based on the backbone of the h vaccine strain, and replaced the s gene with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , isolated from china using the reverse genetic system. the safety results showed that this recombinant strain was not lethal to one-day-old chicks and it had not gained the ability to tissue invasion or replication in the kidneys and lungs of infected animals. moreover, the viral load of the chickens was significantly decreased compared with the rh and ribyz strains. furthermore, rh -s /yz was found to provide effective protection against challenge with the qx-like nephropathogenic strain in young chickens. the level of antibody production in the serum of the vaccinated chickens detected by elisa continued to increase after dpi. thus, rh -s /yz may be considered a potential live vaccine candidate for protection against chinese qx-like nephropathogenic ibv infection. all of the animals were cared for in accordance with the animal ethics guidelines and approved protocols, and the experimental protocols were performed with the approval of the animal welfare conceptualization, methodology, software, investigation, validation, visualization, resources, writing -original draft, writing -review & editing, visualization, funding acquisition. xu cheng: investigation, data curation. xiumei zhao: visualization, investigation. yan yu: investigation, software. mingyan gao: investigation. sheng zhou: conceptualization, validation, resources, writing -original draft the long view: years of infectious bronchitis research coronaviruses in poultry and other birds 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thank all members of the poultry institute, chinese academy of agricultural sciences (yangzhou, china) for their contribution to this study. yi jiang designed the study and conducted all the experiments, analysis and interpretation of the data, and wrote the manuscript. sheng zhou helped perform the recombinant ibv construction. xu cheng, xiumei zhao, yan yu, and mingyan gao participated in the whole animal experiment in different groups. xu cheng conducted the experiments involving viral shedding. all authors read and approved the final manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -c qr es authors: wright, edward; mcnabb, suzanne; goddard, trudy; horton, daniel l.; lembo, tiziana; nel, louis h.; weiss, robin a.; cleaveland, sarah; fooks, anthony r. title: a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: c qr es the inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. we analysed serum samples from tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the cvs- envelope glycoprotein. compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of % and sensitivity of . % with a strong correlation of antibody titres (r = . ) were observed with the pseudotype assay. to increase the assay's surveillance specificity in africa we incorporated the envelope glycoprotein of local viruses, lagos bat virus, duvenhage virus or mokola virus and also cloned the lacz gene to provide a reporter element. neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. importantly, the cvs- pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. these results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections. rabies is spreading at an alarming rate in some regions of the developing world [ ] [ ] [ ] . collaborative efforts among human and animal healthcare professionals are required to monitor this situation and allow a timely and proportioned therapeutic response, limiting unnecessary use of valuable vaccines. the combination of rabies awareness campaigns, improved vaccine coverage and disease surveillance has already resulted in the successful elimi-nation of canine rabies in north america [ ] . however, vaccines need to be efficacious enough to elicit a response that will confer protection and diagnostic techniques are needed to ensure that adequate levels of virus-neutralising antibodies (vnabs) have been achieved in response to vaccination. the problem with current tests is that they are not easily used in endemic areas within developing countries because they need to be set up in containment laboratories since highly pathogenic zoonotic viruses are required to perform the assay, or such assays are prohibitively expensive. those that are e.g., elisa-based assays, do not have the sensitivity or specificity of the fluorescent antibody virus neutralisation (favn) assay that is widely used within office international des épizooties (oie) rabies reference laboratories. therefore, in addition to the need for further implementation and development of improved anti-rabies biologicals, improved techniques to assess seroconversion are required before canine rabies is success- the owners were invited to bring their dogs to two sessions. during the first, samples were collected for baseline measurements, a rabies vaccine was administered and a welfare assessment conducted. twenty-one days later (* the revisit to bs village was days postvaccination) the dogs had further samples collected. (c) while all samples were processed using the same protocol, the levels of haemolysis and serum collected were variable but did not affect results. fully eliminated in developing countries as it has been in north america. within the lyssavirus genus, classical rabies viruses (genotype ) are not the only pathogen to cause morbidity and mortality in mammalian populations. clearly, infection by lyssaviruses of the other genotypes ( ) ( ) ( ) ( ) ( ) ( ) can result in a clinical manifestation that is indistinguishable from rabies. the other genotypes are distributed geographically predominantly within african, european and australian bat populations [ ] . recently, additional variants that are more divergent than genotypes - have been isolated suggesting further genotypes may yet exist [ ] [ ] [ ] [ ] [ ] . isolates representing genotypes , and - have been identified in insectivorous, fruit or vampire bats [ ] . mokola virus (mokv, genotype ) along with lagos bat virus (lbv, genotype ) and duvenhage virus (duvv, genotype ) comprises the african lyssaviruses. interestingly, only a few clinical isolates representing these genotypes have been identified to date [ ] . the handful of cases that have been reported has given us a limited understanding of the epidemiology and zoonotic threat that these genotypes pose in their respective hosts. recently, surveillance programs and greater access to serosurveillance techniques have resulted in the discovery of a high seroprevalence against lbv in east and west african megachiroptera [ , ] and a common presence of lbv in south african bats collected for routine surveillance [ ] . these reports and others [ , ] emphasise that the potential for increased incidence levels of rabies and related lyssavirus infections is a concern in africa, mainly because of the lack of awareness of these infections in the population. however, there is also poor accessibility to vaccines and post-exposure treatments for those exposed to these viruses and there are difficulties with undertaking (sero)surveillance measures in many countries within africa [ ] . while the most important factor in reducing rabies prevalence is the implementation of vaccination campaigns, it was highlighted at the recent southern and eastern african rabies group meeting that poor infrastructure becomes a major barrier when attempting to control rabies in africa [ ] . these views are shared by the oie and world health organization (who), that list the development of novel diagnostics as an urgent requirement [ , ] . serological techniques that can be employed to study naturally occurring or vaccine-induced humoral responses to rabies virus infection include the favn assay [ ] , rapid fluorescent focus inhibition test (rffit) [ ] and enzyme linked immunosorbant assay (elisa) [ ] . variations of these assays have been described previously [ , ] . the routinely used favn assay and rffit are the current assays of choice with oie/who reference laboratories but must be performed in bsl /sapo high containment facilities because live virus is handled as part of the assay. while a modified rffit that combines green fluorescent protein (gfp) with live recombinant virus can remove the need for expensive conjugates, work with recombinant virus still requires the use of high containment facilities [ ] . with the recent addition to the above mentioned set of neutralisation assays of the elisa-based method that uses plates coated with whole, inactivated virus, the need for live virus has been eliminated. since both non-neutralising and neutralising antibodies are detected, the level of circulating, protective vnabs alone cannot be determined. there are also issues with low sensitivity when using the elisa. we recently described the use of surrogate viruses known as lentiviral pseudotypes as replacements for live or inactivated whole virus to accurately determine anti-rabies vnab responses in vaccine recipients. the samples tested were taken from vaccinated humans, dogs and cats in the united kingdom (uk) [ ] . here we report the results of the largest virus neutralisation study published to date using the surrogate lentiviral pseudotypes rather than the live native or recombinant rabies virus with field serum samples from tanzanian dogs. we further increase the utility of our pseudotype neutralisation assay for laboratories undertaking vaccine trials and serosurveillance in resource-limited, rabies endemic countries by exploring the use of lacz as a reporter gene and incorporating the glycoproteins of a further three lyssavirus "primary" refers to dogs that had never received a rabies vaccination prior to this study, "booster" refers to dogs that had previously received ≥ rabies vaccination and "no record" means there was no vaccination history available or taken. a one sample was missing on arrival in the uk. b one sample vial was empty on arrival in the uk. genotypes, in addition to genotype , which will allow improved serosurveillance for lyssaviruses other than classical rabies. this report describes a highly sensitive yet flexible platform that can be adapted to allow the evaluation of vaccine and antiviral drugs against highly pathogenic viruses without the need for high level containment facilities or expensive reagents and equipment. dogs enrolled in this study were selected from domestic animals living in four villages (ngarawani, runga'bure, nyamburi and bisarara) within km of the serengeti national park perimeter in the serengeti district, northwestern tanzania (fig. a) . in each case the dog owner's consent was sought before enrolling their dog in the study. this project was part of the annual vaccination campaign undertaken by the viral transmission dynamics project, which works to prevent the spread of diseases through animal populations in the serengeti region. a detailed medical history of each dog enrolled in this study, which included any prior vaccinations, was taken at the first visit. sixty-six and a half percent (n = ) of the dogs had not been vaccinated against rabies prior to this study (referred to as "primary"), . % (n = ) had received at least one rabies vaccine previously ("booster") and there was no vaccination history available or taken for . % (n = ; "no record"; table ). the nobivac ® rabies virus vaccine (donated by intervet schering-plough animal health) was administered to all dogs in a single dose inoculation given subcutaneously. it comprises an inactivated vaccine containing > iu rabies virus (pasteur strain). at the first visit, a blood sample was taken from each dog before the rabies virus vaccine was administered. the second visit was - days post-vaccination, the optimum time to detect an immune response stimulated by the vaccine. the overall study protocol is shown in fig. b . blood was drawn from the cephalic or jugular vein, stored on ice, and processed at the end of each day (centrifugation at rpm for min). serum was prepared, inactivated at • c for min and then frozen for transport. in total, samples were taken from the enrolled dogs, blinded and sent to university college london (ucl) and veterinary laboratories agency (vla) for testing. the oie standard reference dog serum diluted to . international units/ml (iu/ml) with pbs was used as a positive control. rabbit anti-sera raised against lbv isolate rv (n = ) and duvv isolate rv (n = ) were used in neutralisation assays with african lyssavirus pseudotypes. anti-sera to mokv were not available but anti-lbv serum is known to cross-neutralise mokv. one serum raised against uninfected tissue culture supernatant and another against australian bat lyssavirus (rv ) were used in the same panel as the lbv and duvv sera. human embryonic kidney t cells [ ] were used for production of the lentiviral pseudotypes (lyssavirus surrogates). neutralisation assays were undertaken on baby hamster kidney cells clone (bhk; [ ] ). the np human glioma cell line expressing cd and cxcr was used as target cells for hiv- pseudotypes [ ] . live virus experiments were undertaken using a restricted version of the favn that is identical to the existing favn assay [ ] but with serum samples diluted -fold to a final titre of : , roughly equivalent to . iu/ml. standard plasmids and the transfection protocol for lentiviral pseudotype (lyssavirus surrogate) production have been described elsewhere [ ] . additionally, glycoprotein (g) gene sequences from lbv (lbv.sa ; accession number ef ), mokv (mokv. / ra ; accession number gq ) and duvv (duvv.rsa ; accession number eu ) were amplified by pcr using specific primers (detailed in supplementary table s ). pseudotypes containing the hiv- envelope gp gene were generated using psviiienv [ ] . g gene sequence analysis was undertaken using clustalw [ ] and treeview [ ] . infection and neutralisation assays using lyssavirus pseudotypes were performed as previously described [ ] with the following three modifications: ( ) plates were centrifuged at rpm for s after the virus was added and once more following addition of bhk cells; ( ) to ensure the oie standard reference dog serum recorded an ic at a : dilution, approximately × tcid of pseudotype was used; ( ) serum was diluted -fold to a final titre of : . . . pseudotype reporter genes . . .ˇ-galactosidase to enable pseudotype particle detection using the appropriate ␤-galactosidase (␤-gal) substrates, we constructed pcslzw, which contains the lacz gene from pmfg-nls-lacz (a kind gift from dr. yasuhiro takeuchi; [ ] ) cloned into pcsgw (primers detailed in supplementary table s ). where ␤-gal was the reporter protein, ␤-gal-pseudotype-infected cells were detected strong correlation between favn and pseudotype neutralising antibody titres using tanzanian dog sera. (a) neutralising titres achieved with the pseudotype assay increase concordantly with those detected in the favn assay. the distribution, according to vnab titres determined by the (b) pseudotype and (c) favn assays, of sera collected at the first visit from primary (dogs that had not received a previous rabies vaccination-black columns), booster (dogs that had received ≥ rabies vaccination previously-grey columns) or dogs with no vaccination record (white columns) is shown. similar analyses for samples collected at the second visit are shown in (d) and (e) for the pseudotype and favn assays, respectively. the dotted line marks the level of vnab that was achieved by the oie positive control serum. percents of samples with an inadequate or adequate vnab response are given left and right of the dotted line, respectively. vaccination history was available for dogs (first visit: primary n = , booster n = , no record n = ; second visit: primary n = , booster n = , no record n = ). (f) results from the pseudotype neutralisation and favn assays reveal a high degree of concordance (sn: sensitivity and sp: specificity) and a strong correlation (r) between titres. sn and sp are relative to the . iu/ml threshold and r/p values were calculated using pearson's product-moment correlation. arrows indicate vnab titres for which there were no serum samples containing that level of antibodies. using -bromo- -chloro- -indolyl-beta-d-galactopyranoside (xgal; sigma), chlorophenol red-beta-d-galactopyranoside (cprg; sigma) or o-nitrophenyl-b-d-galactopyranoside (onpg; sigma). detection using the x-gal substrate was accomplished by fixing the infected cell monolayer and then staining the cells with substrate buffer ( mm deoxycholic acid, mm potassium ferrocy-nate, mm potassium ferricynate, . mm magnesium chloride and . % (v/v) np ) containing mg/ml x-gal. for cprg and onpg substrates, nuclei were lysed and l of reaction buffer ( mm na hpo · h o, mm nah po ·h o, mm mgcl , mm ␤mercaptoethanol) was added containing cprg or onpg at a final concentration of . and . mg/ml, respectively. this reaction was table distribution of sera according to favn neutralising antibody titre. percentages given are the proportion in that category out of the total number for the column. a samples that were positive by favn assay but negative by the pseudotype assay. inhibited, after - h at • c, by adding stop solution ( m sodium carbonate). gfp and luciferase reporter genes were employed to detect gfp-positive cells, which were visualised using a fluorescent microscope and facscalibur (bd biosciences) and luciferase expression, which was detected using the bright-glo reagent and glomax microplate luminometer (promega). of the dogs enrolled in this study, the mean age was . years and . % were female (table ). in total, samples were taken during both visits (table ) . however, in transport one sample disappeared (# ) and one leaked so no serum remained (# ). of the remaining samples, the volume in was too small to test in the favn assay and a further three samples could not initially be tested in the pseudotype assays because of contamination and toxicity. the remaining samples were tested in both assay types. while the serum process times were kept as constant as possible there were varying degrees of haemolysis and volumes of sera produced varied with each collection (fig. c) . haemolysis did not affect the results obtained with either the favn or the pseudotype assays. sera that failed to achieve an antibody titre of : by the pseudotype assay, the lowest recordable result from the dilutions that were used, were given an arbitrary titre of : for this analysis. the oie positive control serum routinely neutralised % of cvs- pseudotype particles at a dilution of : (in this study the oie mean dilution = . and standard deviation = ± . ) and % of live cvs- at a dilution of . , normally equivalent to . iu/ml, for the favn assay (in this study the oie mean dilution = . and standard deviation = ± . ). the lowest titre a serum sample was given by the favn assay in this study is . iu/ml. any dogs with vnab levels that attained an ic at a dilution of greater than or equal to : (by pseudotype assay) or a titre of . iu/ml (by favn assay) were considered positive (also referred to as "adequate" in this study). when results from the favn and pseudotype assays were compared, we observed a concurrent increase in vnab titres reported by the pseudotype assay as the favn titre increased ( fig. a ). there were discordant samples, classified as negative by the pseudotype assay but positive with the favn assay ( table ) . as a result, the average titre of samples classified as borderline positive by the favn assay ( . iu/ml) is : , a borderline negative result with the pseudotype assay. while these discordant samples reduced the sensitivity of the pseudotype neutralisation assay to . %, the specificity of the assay was % (n = ). the majority of the discrepancies clustered around . iu/ml, the titre achieved using the oie positive control serum and currently used to classify vnab responses as inadequate (levels below that achieved by the oie positive control serum) or adequate (levels greater than or equal to that achieved by the oie positive control serum). twelve of the discordant results ( %) were in the lowest dilution classified as positive by the favn assay ( . iu/ml), one ( . %) fell within the . iu/ml category, three ( . %) in the . iu/ml category and one ( . %) scored . iu/ml by the favn assay (table ). there were no discrepant results achieved using the favn or pseudotype assays and samples from the second visit. prior to vaccination at the first visit, results using the pseudotype assay classified . % of the dogs that had never been vaccinated table titres recorded for discordant sera using the favn and pseudotype assays. . a vaccination history was not available for this dog ("no record"). b serum samples taken from dogs with no rabies vaccination history prior to this study ("primary"). . radial tree and panels showing degree of glycoprotein identity and viral titre of lyssavirus pseudotypes. the envelope g genes from lbv, mokv and duvv were cloned into pi. and titres were compared with that achieved by pseudotypes bearing the cvs- g. identity of the full-length g amino acid sequence was used to construct the tree. viral titres were assessed using gfp carrying pseudotypes and are given in ifu/ml. the branch lengths and scale correspond to the number of amino acid substitutions per site. ("primary") as having inadequate levels of vnabs (fig. b) . this compares to . % as determined by the favn assay (fig. c ). there was a roughly equal split of inadequate vs adequate levels of vnabs ( % vs %) for the dogs that had received one or more rabies vaccination(s) previously ("booster") when tested using the pseudotype assay (fig. b ). the discrepant results described above caused this balance to shift in favour of adequate vnab levels ( % vs %) when the same sera were analysed by the favn assay (fig. c ). in sera from the second visit both assays detected three dogs ( . %) that failed to achieve a level of vnabs that was greater than the oie control serum despite receiving the vaccination (fig. d and e) . none of the three dogs had been given a rabies vaccination prior to this study and their level of circulating vnabs was inadequate at both visits. analysis of all results showed that the titres recorded for each serum by favn and pseudotype assays correlated strongly (r = . , p < . [pearson's product-moment correlation]; fig. f ). we cloned the g gene sequences of lbv, mokv and duvv into our expression plasmid and tested their ability to be incorporated into lentiviral pseudotype particles. using gfp as a marker for infection of bhk cells we observed comparable titres for lbv and mokv pseudotypes as we achieved using cvs- ( . × , . × and . × ifu/ml, respectively; fig. ). the titre recorded for pseudotypes expressing the duvv g was . -fold higher at . × ifu/ml. to test the specificity of these african lyssavirus pseudotypes, neutralisation assays were run with sera raised against lbv (rv ; serum nos. and ) and duvv (rv ; serum nos. , , and ) isolates. neutralising titres with the pseudotypes correlated strongly with the titres observed using the favn assay (table ). anti-lbv sera cross-neutralised mokv in both the favn and pseudotype assays. however, the favn assay failed to detect anti-duvv vnab in serum and and only low levels in serum (ic = ) compared to the pseudotype assay, which recorded ic titres of , and for sera , and , respectively ( table ). the negative control and australian bat lyssavirus (ablv) sera did not achieve an ic against any genotype, in either assay, and no cross-neutralisation of duvv by the lbv sera was observed (table ). to broaden the lyssavirus pseudotype platform, we cloned the lacz gene as a reporter. using a panel of serum samples selected to contain low, medium and high vnab titres, we ran parallel assays and determined that using lacz as the reporter gene, instead of luciferase, did not alter the high correlation (r = . , p < . [pearson's product-moment correlation]; fig. a ) or sensitivity and specificity with respect to the titres achieved using the favn assay. while the flexibility to use different read-outs is a key requirement of any assay to be used in africa, it must also be robust, able to withstand fluctuations in temperatures. therefore, we tested the stability of the pseudotype stocks stored under different conditions. freeze-thaw cycles of cvs- pseudotypes revealed an average decrease of . % in viral titre per cycle, while pseudotypes bearing the vsv or hiv- envelope proteins lost . % and . %, respectively (fig. b) . compared to the cvs- pseudotype stock stored at − • c, aliquots stored at room temperature (average of • c) had a half-life of - weeks, which increased to - weeks for aliquots at + • c. pseudotypes stored at − • c were relatively stable for over months (fig. c) . this stability was similar to that observed with rv (duvv) pseudotypes bearing the vsv envelope glycoprotein and was -fold greater than the half-life of pseudotypes bearing the hiv envelope glycoprotein stored at + • c or room temperature (data not shown). the inclusion of lacz as a reporter gene increased the applicability of our pseudotype system in resource-limited laboratories as it allows the determination of vnab titres without the need for expensive regents or equipment. cells were infected with laczbased pseudotypes and subsequently stained with one of three ␤-gal substrates. punctate blue staining of infected cell nuclei was achieved in the presence of x-gal (fig. , far left panels). we also adapted the assay to allow the ␤-gal colorimetric substrates cprg and onpg to be used (fig. , second left and middle panels). these changes, and resulting vnab titres, were recorded using a microplate reader, reading at nm (cprg) or nm (onpg), or simply by eye. the incorporation of lacz is an additional option to the much used gfp and luciferase reporter genes, which allow a more high-throughput approach but at a far greater expense (fig. , second right and far right panels, respectively). while mass culling of stray dogs has previously been shown to be ineffective in controlling the spread of rabies [ , ] , this practice is once again being adopted by a number of countries. on the contrary, dog vaccination programs have been enormously successful in controlling canine rabies [ ] [ ] [ ] , and improved diagnostics to enable rapid serosurveillance in countries undertaking these programs are therefore important. the assay reported here offers a practical, effective and robust solution for rapid lyssavirus serosurveillance. using lentiviral pseudotypes we can accurately measure the concentration of vnabs and, coupled with the use of lacz as the reporter gene for pseudotype particle production, removes the need for high containment laboratories and expensive equipment or reagents. this allows the assay to be undertaken in laboratories previously unable to use the existing favn. furthermore, the assay is not just restricted to lyssavirus serosurveillance as other highly pathogenic viruses that have been pseudotyped could be incorporated into this platform [ , [ ] [ ] [ ] [ ] . to our knowledge this is the largest study published to date using pseudotypes in a diagnostic format for serosurveillance. of the samples received, titres for three could not be determined using the pseudotype assay: one had low level contamination and two caused cellular cytotoxicity in the assay. these issues were overcome using higher concentrations of antibiotics and increasing the number of bhk cells used -fold, to × per assay. in comparison, there were samples that could not be titrated with the favn assay because of insufficient serum volume. while serum volume varied between samples, there was sufficient volume for each sample to run duplicate pseudotype neutralisation assays. this highlights another major advantage of using pseudotypes as surrogate viruses in neutralisation assays, only small serum vol- fig. . repertoire of reporter genes that can be carried within pseudotypes. we have expanded the range of reporter genes to enable the neutralisation assay to be performed in a much greater number of laboratories than previously possible. lacz-based pseudotypes can be detected with x-gal, cprg or onpg ␤-gal substrates that turn blue, red or yellow in the presence of the enzyme, respectively. gfp appears as green cytoplasmic staining while the oxidation of the luciferase substrate results in light emission that can be detected in a luminometer. [vnab]: virus-neutralising antibody concentration. the arrow indicates zero luciferase activity. umes ( - -fold less than live virus assays) are required for each assay. an additional important benefit of pseudotypes is the flexibility to choose the reporter gene depending on the operator's requirements. for this study, luciferase-based cvs- pseudotypes were used, which allowed high-throughput screening of the samples. this permitted many plates to be analysed in a short period, however, both the luciferase reagent and the plate reader are expensive. in contrast, the reagent for the lacz reporter system is over times less expensive than that for luciferase and times less expensive than the fluorescein isothiocyanate conjugate used in the favn assay. additionally, as neutralisation assays using pseudotypes are safer, not requiring bsl /sapo facilities, the overall cost of using them is many fold lower compared to performing the favn assay, thereby allowing expansion of rabies serosurveillance in resourcelimited laboratories. in addition, since pseudotypes carrying the lacz reporter can be detected using x-gal, cprg or onpg, the flexibility of the assay is further increased. with colorimetric substrates it is possible to record vnab titres by simply looking at the assay plate and recording the colour change. neutralisation assays using pseudotypes with lacz as the reporter can also be used for semi-high-throughput screening when using an eli-spot (x-gal staining) or microplate reader (cprg and onpg staining). however, the flexibility offered by packaging lacz, gfp or luciferase into the pseudotype particle alone makes this assay an attractive option for use in any laboratory undertaking lyssavirus serosurveillance. the stability of pseudotypes reported here also makes this assay suitable for use in countries where the infrastructure and coldchain may be unreliable. if the stocks of pseudotype particles were to be thawed and re-frozen or to be stored at temperatures below − • c, they would still be viable for use in subsequent neutralisation assays. this is a major advantage over live virus, where the virus titre decreases rapidly on freeze-thaw or storage above − • c. however, where possible stocks of pseudotypes should be maintained at − • c and stored in aliquots to reduce the number of times each vial is thawed. a future consideration is to freeze dry the pseudotype particles to avoid cold-chain storage and to increase ease of transport. the number of inadequate versus adequate vaccine responders, detected by both types of assays, was comparable at either visit for collection of serum. however, discordant results were observed between the pseudotype and favn assays, which can partially be explained by the fact that they were all close to the . iu/ml threshold. assay variability with the favn assay means that samples scoring . iu/ml had a variable range of . - . iu/ml if the test was repeated. twelve out of these samples that had a . iu/ml titre in the favn assay might have been classified as negative if re-tested, which would result in a new sensitiv-ity score of . %. conversely, it is also possible that the scores of . - . iu/ml obtained with the favn assay and the corresponding titres achieved using the pseudotype assay could change if re-tested. it is worth noting the dogs that were classified as having inadequate vnab responses using the pseudotype assay, in disagreement with the favn results, were sampled before vaccination in this trial. in addition, out of the samples were from dogs with no history of previous rabies vaccination and had been vaccinated ≥ year before the study began, therefore their levels of vnabs could have decreased over time. one did not have a vaccination history collected during the trial (# ). these discordant results coupled with neutralisation data showing duvv pseudotypes can detect vnab in anti-duvv serum not detected by the favn assay suggests that the pseudotype assay may be more specific and sensitive for detection of seroconversion than the favn assay when a . iu/ml cut-off is used. however, to confirm this observation further studies are required using analogous viral isolates and serum, and larger sample sizes. the introduction of the lbv, mokv and duvv g as binding antigens into our pseudotype platform, coupled with existing cvs- , eblv- and eblv- pseudotypes, means that serum can now be screened to detect vnabs against six different lyssavirus genotypes. this is of particular importance because large proportions of these assays are performed on sera from bats, from which only small volumes of serum are available. at a time when emerging infectious disease outbreaks are becoming more frequent and have the potential to spread faster through international travel, technological advances in infectious disease research are making assays more rapid facilitating more automation. however, countries where the outbreaks are most likely to occur are often unable to utilise these new techniques. we have addressed these limitations by developing an assay that can be used for infectious disease serosurveillance and for monitoring vaccine responses within low-containment laboratories. neutralisation assays based on pseudotypes as a source of target antigen are a useful platform to use at the start of a new outbreak, not only for rabies, but also other enveloped rna viruses such as sars coronaviruses, influenza and ebola viruses. incorporating the lyssaviral envelope protein (g) into the pseudotype platform creates an assay that allows rapid screening and vaccine evaluation to be performed within weeks of the start of an epidemic. preventing the incurable: asian rabies experts advocate rabies control re-evaluating the burden of rabies in africa and asia rabies trend in china ( - ) and post-exposure prophylaxis in the guangdong province rabies surveillance in the united states during the challenge of new and emerging lyssaviruses new lyssavirus genotype from the lesser mouse-eared bat (myotis blythi) novel lyssaviruses isolated from bats in russia bat lyssaviruses (aravan and khujand) from central asia: phylogenetic relationships according to n, p and g gene sequences phylogeny of lagos bat virus: challenges for lyssavirus taxonomy new rabies virus variant in mexican immigrant emergence of lyssaviruses in the old world: the case of africa antibodies against lagos bat virus in megachiroptera from west africa lagos bat virus in kenya epidemiology and control of rabies. the growing problem of rabies in africa rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies rabid bytes. alliance for rabies control towards the elimination of rabies in eurasia development of a fluorescent antibody virus neutralisation test (favn test) for the quantitation of rabies-neutralising antibody a rapid reproducible test for determining rabies neutralizing antibody development of a qualitative indirect elisa for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats rabies human diploid cell vaccine elicits cross-neutralising and cross-protecting immune responses against european and australian bat lyssaviruses a novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison analysis of mutation in human cells by using an epstein-barr virus shuttle system syrian hamster fibroblast cell line bhk and its derivatives establishment of a new system for determination of coreceptor usages of hiv based on the human glioma np- cell line rapid complementation assays measuring replicative potential of human immunodeficiency virus type envelope glycoprotein mutants clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice treeview: an application to display phylogenetic trees on personal computers retroviral-mediated gene transfer into hepatocytes in vivo control and prevention of rabies in animals: paradigm shifts the rabies epidemic on flores island, indonesia ( - ) the pan american health organization (paho) role in the control of rabies in latin america a dog rabies vaccination campaign in rural africa: impact on the incidence of dog rabies and human dog-bite injuries rabies control in rural africa: evaluating strategies for effective domestic dog vaccination hepatitis c virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes a sensitive retroviral pseudotype assay for influenza h n -neutralizing antibodies characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines we thank denise marston for help with dna sequence analysis, nigel temperton for constructive discussion and olivia avdis and the viral transmission dynamics project for assistance with sample collection. we are indebted to tanzania government ministries, supplementary data associated with this article can be found, in the online version, at doi: . /j.vaccine. . . . key: cord- - x y o authors: ren, wenlin; sun, hunter; gao, george f.; chen, jianxin; sun, sean; zhao, rongqing; gao, guang; hu, yalin; zhao, gan; chen, yuxin; jin, xia; fang, feng; chen, jinggong; wang, qi; gong, sitao; gao, wen; sun, yufei; su, junchi; he, ailiang; cheng, xin; li, min; xia, chenxi; li, maohua; sun, le title: recombinant sars-cov- spike s -fc fusion protein induced high levels of neutralizing responses in nonhuman primates date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: x y o abstract the covid- outbreak has become a global pandemic responsible for over , , confirmed cases and over , deaths worldwide. in this study, we examined the immunogenicity of cho-expressed recombinant sars-cov- s -fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a covid- vaccine. we demonstrate that the s -fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day . strong virus neutralizing activity was observed on day in rabbits immunized with the s -fc fusion protein using a pseudovirus neutralization assay. most importantly, in less than days and three injections of the s -fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered covid- patient in a live sars-cov- infection assay. our data strongly suggests that the cho-expressed sars-cov- s -fc recombinant protein could be a strong candidate for vaccine development against covid- . the sars-cov- was first identified in wuhan, china at the end of [ ] [ ] [ ] [ ] [ ] . in only five months, the virus has caused a global pandemic, with over , , confirmed cases and over , deaths worldwide. as a novel coronavirus with no effective treatments or drugs currently available, a vaccine is in dire need of development. several broad approaches to the development of a covid- vaccine have emerged, including dna vaccines, rna vaccines, viral vector vaccines, recombinant subunit vaccines, and dead viral preparations [ ] . among these, an rna vaccine from moderna was the first to reach human trials in early march in the us, followed by cansino's adenoviral vector vaccine which began human trials in china later in the same month. considering that the spike protein is the receptor-binding protein that mediates viral-cell fusion during the initial infection event [ , ] , it has been identified as a primary target for vaccine design. the spike protein has a total of , amino acids, which can be divided into two major domains according to their structures and functions [ , ] . the first half is the s protein, which contains the receptor binding domain (rbd) sequence and is located at the n-terminus of the spike protein [ , , ] . the second half is the s , serving as a trimeric structure that supports the s 's rbd and has a fusion bundle which protrudes out into the host cell's membrane after - - it is triggered by the s coming into contact with ace [ , , ] . due to the fact that most of the neutralizing epitopes are located within the s region, proteins containing the rbd, full-length of s , full-length of s (s +s ) or even a trimeric s approach have been considered as candidates for vaccine development [ ] . in this study, we fused the full length sars-cov- s protein (genbank: qic . , gln -arg ) with the fc region of human igg (genbank: car . , glu -lys ) as our vaccine candidate and expressed the recombinant protein using a stable cho-k cell line. the purified s -fc protein was formulated with different adjuvants and used to immunize different species of animals, such as mice, rabbits, and macaques. besides eliciting high levels of anti-s antibodies in all tested animals, high neutralizing activities against sars-cov- were also found in the anti-sera from macaques. these results indicate that the s -fc fusion protein can effectively induce humoral immune responses in various animals and can elicit high levels of neutralizing antibodies in macaques. ad . (saponin based microemulsion) and ad gold+ (nanoemulsion with synthetic mpl) adjuvants were from advaccine, china. freund's complete adjuvant (cfa) was purchased from sigma, usa. female balb/c mice were obtained from vital river co., china. new zealand white rabbits were purchased and hosted at longan co., china. macaques were generated and hosted at xieerxin biotech., china. - - the mice were group housed, while the rabbits and macaques were caged separately. all the animals were fed with general diet, while the vegetables and fruits were added additionally for macaques. drinking waters for all animals were purified and autoclaved. peroxidase conjugated secondary antibodies were sourced from jackson immunoresearch, usa. cho-expressed sars-cov- s -fc fusion protein and s - his were produced by zhenge biotech., china. immunizations weeks-old female balb/c mice, - weeks-old female nzw rabbits and ~ years old macaques were immunized with cho-expressed sars-cov- s -fc fusion after formulated with adjuvants according to manufacturer's instructions. briefly, weeks old female balb/c mice were immunized with s -fc protein immersed in ad gold + ( . μg on day , , and reduced to . μg on day and intramuscularly). nzw rabbits were also immunized with s -fc protein immersed in ad gold + ( μg on day , , and reduced to μg on day , and intramuscularly). for immunization of macaques, cfa was used to prime the primate at the first injection and ad . was used to boost them ( μg in cfa on day subcutaneously, and μg in ad . on day , , and intramuscularly). the immunization process is shown in table s . blood samples were collected at different time points for measurement of antibody levels and neutralizing titers. all protocols involved the use of animals were approved by the iacuc/ethical committee/ animal welfare. the assay was carried out as described by zhao rq, et al [ ] . briefly, wells of well plate were coated with . μg/ml of sars-cov- s - his protein in pbs, were lysed with lysis buffer (glo-lysis buffer, promega), followed by addition of luciferase substrate (bright-glo luciferase assay substrate, promega). luciferase activity was measured using a glomax microplate luminometer (promega) and wells producing relative luminescence units (rlu) above three times of the mean background value were defined as positive. the % neutralization titer was calculated by probit analysis using the spss software. strain cn was chosen for neutralizing assay. sars-cov- virus titer was determined by microdose cytopathogenic efficiency (cpe) assay. serial -fold dilutions of virus contained samples were mixed with -  vero cells, and then plated in -well culture plate. after - days culture in a % co incubator at . °c, cells were checked under a microscope for the presence of cytopathic effect (cpe). virus titer was calculated by the method of karber [ ] . serum samples were inactivated at c for . h and serially diluted with cell culture medium in two-fold steps. the diluted sera were mixed with sars-cov- suspension of tcid in -well plates at a ratio of : , followed by hours incubation at . c in a % co incubator. -  vero cells were then added to the serum-virus mixture, and the plates were incubated for days at . c in a % co incubator. cpe of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of % protective condition. the s subunit ( q- r) was fused with fc fragment of human igg genetically for ease of purification, constructed in pee . , and expressed by a stable cho cell line. s -fc fusion protein was purified from the culture supernatant using a protein-a column, as shown in fig.s a . the purity of s -fc protein was evaluated by running sds-page gel under reduced and non-reduced conditions. a band between - - -kd was detected as monomer of the s -fc, while the dimer appeared as a single band well above kd molecule weight marker (fig.s b) . one female and one male macaque were immunized with s -fc protein with cfa and ad . . the sera were collected on different days (prior to, day , , , and post the first immunization) and evaluated by elisa against sars-cov- s - his protein. the titers of total antibodies against sars-cov- s proteins were first examined by using hrp-conjugated goat anti-human igg (h+l) secondary antibodies, which react with igm, igg , igg , igg and igg . as shown in fig a, on day , anti-s total antibodies were detected only in the female macaque. on day , good titers of anti-s total antibodies were observed in both macaques. however, on day , there was a % drop of total anti-sars-cov- s antibody titers in the female macaque. one more boost was given on day and the total antibody titers in both macaques increased greatly on day . furthermore, we used hrp-labeled anti-human igg fc and hrp-conjugated mouse anti-human μ-chain isotype-specific secondary antibodies to re-examine the levels of iggs and igm in the sera. as shown in fig. b , only anti-s igg antibodies were seen in the female macaque on day . on day , strong anti-s igg titers were detected in both macaques, while the igm levels were also detectable. interestingly, after one more boost of s -fc on day , only a big increase in anti-s igg titers on day , meanwhile, no significant anti-s igm titers was observed. using the same elisa assay, we examined the levels of anti-s igg and igm antibodies in the plasma samples collected at different days post onset of covid- disease from the recovered patient. as shown in fig. , the igg antibodies can be detected days after onset of disease and the titer increased on day , but reduced significantly on day . no igm was detected. the original purpose of this study was to generate positive control sars-cov- s antibodies for development of sars-cov- vaccine and serological assays. some of the immunizations were done with the help of freund's complete adjuvant (cfa), which is not suitable for human vaccination. however, high anti-s antibody titers were also observed in one female macaque which was immunized with μg of s -fc immersed in . ml of ad . after two im on day and (data not shown). to confirm this observation, later on, we switched the adjuvant from the harsh cfa to ad gold + (nanoemulsion with synthetic mpl) which was designed for human use to study the potential of s -fc as the candidate for human covid- vaccine. five -weeks old female balb/c mice were immunized with s -fc protein as described previously. the sera were collected on day and evaluated by elisa against sars-cov- s - his protein using hrp-conjugated goat anti-mouse igg fc-specific secondary antibodies. as shown in fig. a , all five mice developed strong anti-s igg antibody titers ( , to , ). two rabbits were also immunized with s -fc protein immersed in ad gold + . on day , the bleeds were collected for examination of anti-s antibody titers. similar to - - what been observed in mice, the s -fc immunized rabbits also developed very high binding titers ( , or higher) against the s protein (see fig b) . the virus neutralizing activities of the s -fc immunized sera were examined using either pseudo-virus or live sars-cov- . we compared the neutralizing titers of the sera from s -fc immunized macaques with the ones of recovered covid- patient's plasma samples using live sars-cov- the neutralizing activities of the sera from s -fc immunized rabbits or macaques were also examined using sars-cov- pseudo-virus. briefly, heat-inactivated serum samples were pre-incubated with tcid pseudo-viruses for h at °c. the mixtures were then used to infect ace - t cells. three days later, the luciferase activities of the infected cells were measured. as shown in table and fig. a , immunization of sars-cov- s -fc fusion protein with ad gold + as adjuvant also induced very high neutralizing activities with ic titers > and ic titers around - in both rabbits on day after immunizations. as shown in fig. b , excellent neutralizing titers also observed in sera from the two s -fc immunized macaques on day . however, while the male macaque developed anti-sars-cov- s protein antibodies slower than the female one with lower binding titers, it surprisingly had much stronger neutralizing titer ( ) than the female macaque ( ) at the end. our data clearly demonstrated that the cho-expressed full length sars-cov- s -fc fusion protein can solicit very strong neutralizing activities against sars-cov- in a pretty short time period and could be a good candidate for covid- vaccine development. the spike protein of the sars-cov- has a total of , amino acids and two major domains according to their structures and functions. the first half is an s protein which contains the rbd sequence and is located at the n-terminus of the spike protein [ ] . the second half is the s , serving as the base for the trimeric structure to support the s receptor binding structure in place. since the rbd being located within the s region, we generated a s -fc fusion protein as a candidate for vaccine development. since the sars-cov- s protein is predicted to have at least n-linked glycosylation sites [ ] , we chose mammalian cell cho-k as the expression system to ensure the right glycosylation. cho-k is also one of the most popular cell lines used to make human therapeutic antibody drugs. shringrix, a vaccine based on the recombinant ge protein of varicella-zoster virus, was launched in by gsk for prevention and treatment of shingles with great success [ ] . the primary vaccination schedule consists of two dose of μg/ . ml each at two month apart [ ] . assuming the same vaccination schedule for covid- , with the stable expression level at mg/l of s -fc fusion protein, a , l cho cell bioreactor can easily produce million dose of recombinant s -fc vaccine every two weeks. with the capacity of more than million liters of mammalian cell biorectors in the world, our s -fc may be the only feasible way to make enough covid- vaccines for the entire world within one year. there are many therapeutic biological drugs such as tnf receptor-fc fusion protein (enbrel) that have been used for treatments of amd and other human diseases without any known side-effect caused by the fc region of the drugs so it is safe to use the human igg 's fc as the fusion partner for sars-cov- s . dimerization of s -fc fusion protein via the disulfide bonds formed within the hinge regions of fc may also help to mimic the native d conformation of the binding pocket of oligomerized s protein in vivo. in addition, the human fc domain fused to the c terminal of s protein greatly facilitates the purification of the recombinant protein following the standardized antibody drug manufacturing procedures. using the purified recombinant s -fc fusion protein as immunogen, we demonstrated it not only has very good immunogenicity in mice, rabbits and non-human primates, but also can elicit strong neutralizing activities against sars-cov- infection in vitro. it was reported by dr. f. krammer's group that sars-cov- infections selectively induced anti-sars-cov- s antibodies with igg isotype [ ] . normally, igg only consists % of the total iggs in sera, while % is igg . however, in covid- patients, over % of sars-cov- s antibodies were igg . igg 's half-life is only days, the shortest one, while the other three igg isotypes' are -days long [ ] . igg is also the isotype with the strongest inflammatory activities. together with the quick drop of sars-cov- s antibody titers observed both in human patient and the female macaque in this study, we started a new vaccine study with much smaller dose ( , and μg) and weaker adjuvant, hoping to induce less igg isotype of anti-sars-cov antibodies. we have been using the sars-s - his protein to challenge the s -fc immunized mice which have developed strong anti-sars-cov- s titers, and have not observed any abnormality with the mice. in this study, we established a cho-k cell line that stably expresses the sars-cov- s protein with human igg fc fused to its c terminal. the purified recombinant s -fc fusion proteins formulated with either cfa & ad . (cfa was used to prime the primate at the first injection and ad . , a saponin based microemulsion, which was used to boost) or ad gold + (nanoemulsion with synthetic mpl) were used to immunize mice, rabbits and macaques. beside high levels of the anti-s antibodies elicited, higher neutralizing activities against live sars-cov- virus and/or pseudovirus from the anti-sera of macaques and rabbits. our results demonstrated that the s - fig. anti-sars-cov- s antibody levels in s -fc immunized macaques a) the elisa titers of total anti-s antibodies in macaque sera. sera were evaluated by elisa using hrp-conjugated goat-anti-human igg (h+l) secondary antibodies (p < . , one-way anova; n= ). the horizontal coordinate is time and the vertical coordinate is the elisa titer of anti-s antibodies in the sera. b) the levels of igg and igm in macaque sera. sera were diluted times with normal monkey serum and then diluted times with sample dilution buffer. the samples were examined with either hrp-conjugated goat anti human igg fc-specific secondary antibodies ( : ) or hrp-conjugated mouse monoclonal anti-μ-chain of human igm ( : ). the horizontal coordinate is time and the vertical coordinate is absorbance value at nm. anti-sars-cov- s antibody levels in s -fc immunized mice and rabbits a) serum samples collected from different mice were evaluated with s - his coated plates by elisa using mouse igg-specific secondary antibodies. the horizontal coordinate is dilution and the vertical coordinate is absorbance value at nm. b) serum samples collected from different rabbits were evaluated with s - his coated plates by elisa using rabbit igg-specific secondary antibodies. the horizontal coordinate is dilution and the vertical coordinate is absorbance value at nm. the neutralizing titer of macaque and covid- convalescent patient's sera was examined using live sars-cov- (p < . , one-way anova; n= ). the vertical coordinate is the neutralizing titer. fig. pseudo-virus neutralizing activities of antisera from s -fc-immunized rabbits and monkeys a) the neutralizing activity of rabbit sera was evaluated with sars-cov- pseudo-virus. the horizontal coordinate is serum dilution and the vertical coordinate is the percentage of the neutralization. b) the neutralizing activity of macaque sera was evaluated with sars-cov- pseudo-virus. the horizontal coordinate is serum dilution and the vertical coordinate is the percentage of the neutralization. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. the new england journal of medicine a new coronavirus associated with human respiratory disease in china a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with novel coronavirus in wuhan potential of large 'first generation' human-to-human transmission of -ncov vaccine designers take first shots at covid- structure, function, and evolution of coronavirus spike proteins. annual review of virology the intracellular sites of early replication and budding of sars-coronavirus a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace d models of glycosylated sars-cov- spike protein suggest challenges and opportunities for vaccine development preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies early detection of sars-cov- antibodies in covid- patients as a serologic marker of infection. clinical infectious diseases : an official publication of the infectious diseases society of america on the analysis of psychometric functions: the spearman-karber method emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd . emerging microbes & infections development of adjuvanted recombinant zoster vaccine and its implications for shingles prevention. expert review of vaccines a serological assay to detect sars-cov- seroconversion in humans antibody therapeutics: isotype and glycoform selection cho-expressed s -fc protein is very immunogenic in various animals and can rapidly induce strong antibody production s -fc protein solicits strong neutralizing activities against live virus stable cho cell line expressing mg/l of s -fc and a , l bioreactor can produce million doses of human covid- vaccine every days, making it an accessible and affordable option for worldwide vaccination declaration of interest the authors declare that they are affiliated with up to two of four commercial entities with stakes in the data this work is supported by research grants from beijing science and technology commission, and bill & melinda gates foundation to le sun. the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. we thank dr. qiang gao and his team from sinovac for performing the live sars-cov- neutralizing assay, dr.jianqing xu at fudan univ. for providing the ace -transfected hek t cells, dr. pinliang hu at beijing beyond biotech. for purification of s -fc protein. - -the authors declare that they are affiliated with up to two of four commercial entities with stakes in the data. key: cord- - u x z authors: yang, william h.; dionne, marc; kyle, michael; aggarwal, naresh; li, ping; madariaga, miguel; godeaux, olivier; vaughn, david w. title: long-term immunogenicity of an as -adjuvanted influenza a(h n )pdm vaccine in young and elderly adults: an observer-blind, randomized trial() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: u x z background: this study (nct ) evaluated the immunogenicity and relative protective efficacy of one dose of influenza a(h n )pdm vaccine with or without as (an α-tocopherol oil-in-water emulsion based adjuvant system). methods: four thousands and forty-eight healthy adults aged ≥ years were randomized ( : ) to receive one dose of either the adjuvanted split virion ( . μg hemagglutinin antigen [ha]/as ) or non-adjuvanted ( μg ha) vaccine. hemagglutination inhibition [hi] antibody response was evaluated before vaccination and at days , and (month ). safety of the study vaccines was evaluated during the entire study duration. results: at day , both study vaccines induced hi immune responses meeting the us regulatory criteria in subjects – years (seroprotection rate [spr]: . % [ . – . ]; seroconversion rate [scr]: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group) and > years of age (spr: . % [ . – . ]; scr: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group). the as -adjuvanted vaccine induced higher hi geometric mean titers than the non-adjuvanted vaccine at all time points. at month , only subjects – years of age from both vaccine groups still met the us regulatory criteria (spr: . % [ . – . ]; scr: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group). protective efficacy was not evaluated due to low number of rt-qpcr-confirmed a(h n )pdm influenza cases. through month , serious adverse events (in subjects: in the as -adjuvanted and in the non-adjuvanted group) and potentially immune mediated diseases ( in the as -adjuvanted and in the non-adjuvanted group) were reported. conclusion: a single dose of either adjuvanted or non-adjuvanted influenza a(h n )pdm vaccine induced protective hi antibody levels against the a/california/ / strain that persisted through month in the – years population. mass immunization is considered to be an effective prophylactic method of mitigating influenza pandemic-associated morbidity and mortality [ ] [ ] [ ] [ ] . due to the novel antigenic characteristics of the swine-origin influenza a(h n ) pandemic virus [influenza (a(h n )pdm ] [ , ] , the seasonal influenza vaccines available at the time of the - h n pandemic were unlikely to confer protection against the novel virus [ , , ] . the world health organization (who) encouraged the development and use of adjuvanted influenza a(h n )pdm vaccines [ , ] , with the aim of dose-reduction, antigen-sparing and to potentially provide broader vaccine efficacy against drifted strains through cross-reactive immunity [ ] . based on the experience of developing a pre-pandemic a/h n influenza vaccine utilizing as (an ␣-tocopherol oil-in-water emulsion based adjuvant system) [ , ] that was well-tolerated and highly immunogenic in adults [ ] [ ] [ ] , an as -adjuvanted influenza a(h n )pdm vaccine with . g hemagglutinin (ha) content was developed [ ] [ ] [ ] . this large-scale, randomized study in subjects ≥ years of age assessed whether one dose of as -adjuvanted . g ha influenza a(h n )pdm vaccine elicited immune response that met the us and european regulatory criteria. additionally, noninferiority and superiority of this vaccine protective efficacy versus a non-adjuvanted g ha influenza a(h n )pdm vaccine were evaluated. in this phase iii, observer-blind, randomized study (nct ), adults ≥ years of age were enrolled across centers in the us and in canada between november and december , . they were randomized (allocation ratio : ) to receive one dose of either a monovalent as -adjuvanted . g ha a/california/ / pandemic influenza vaccine or a non-adjuvanted g ha a/california/ / pandemic influenza vaccine. the enrolment stratification was by age ( : : : ; - years, - years, - years, ≥ years). the subjects and study personnel involved in evaluating end points were blinded to the intervention administered. double blinding was not possible because the vaccine preparation required mixing of as and a(h n )pdm antigen from two vials. randomization was performed using a central, internet-based system that balanced groups with respect to center, age strata and previous seasonal influenza vaccination. adults were excluded from enrolment: if they had a history of physician-confirmed a(h n )pdm influenza infection or vaccination, those who received any vaccination other than a seasonal influenza vaccine within days preceding study start, those with confirmed or suspected immunosuppressive or immunodeficient conditions, diagnosed with or undergoing treatment for cancer, and/or with a history of allergic/anaphylactic reactions following previous influenza vaccination. in addition, laboratory screening was performed to exclude those with results outside of protocol-specified normal ranges. the following safety laboratory parameters were tested to evaluate the participants' eligibility: hepatic aminotransferases, total and direct bilirubin, alkaline phosphatase, creatinine, serum urea nitrogen, hemoglobin, hematocrit, white blood cell count and platelet count. active surveillance of influenza-like infections (ilis: defined as fever ≥ . • c/ . • f or new or worsening myalgia accompanied by new or worsening cough or sore throat) was done during study visits and through bi-weekly telephonic contact through day ( months after the initially planned administration of the second study vaccine dose). additionally, the subjects were instructed to contact the study sites if they develop any ili symptoms. once the study site had been notified of a possible ili episode, a visit for nasal and throat swab sample collection was scheduled within days of symptom onset and before initiating any antimicrobial/influenza antiviral therapy. if an ili episode was reported more than days after onset, no swab specimen was collected. written informed consent was obtained from all subjects prior to conducting any study-related procedures. the study was conducted in accordance with the good clinical practice guidelines, the declaration of helsinki and local regulations. all study-related documents were approved by institutional review boards. the influenza a(h n )pdm vaccine was a monovalent, inactivated, split-virion antigen suspension (a/california/ / strain) adjuvanted with as (arepanrix tm , a trademark of glaxosmithkline vaccines) or administered as plain antigen. the h n viral seed for the vaccine was prepared as per who recommendations [ ] . as is an oil-in-water emulsion based adjuvant system containing squalene ( . mg per dose), dl-␣-tocopherol ( . mg) and polysorbate ( . mg). the as -adjuvanted influenza a(h n )pdm vaccine doses were prepared by mixing the a(h n )pdm antigen and as ( : ) from separate multidose vials. . ml of the assigned study vaccine was administered into the deltoid muscle within min after mixing the antigen and the adjuvant. the first co-primary objective of the study was to evaluate hi antibody responses days after vaccination in the as adjuvanted vaccine group based on the center for biologics evaluation and research (cber) and committee for medicinal products for human use (chmp) criteria for pandemic influenza vaccines in adults [ , ] . at least rt-qpcr-confirmed a/california influenza cases were required to evaluate the second co-primary objective on noninferior protective efficacy followed by superiority. as only three rt-qpcr-confirmed a/california influenza cases were diagnosed during the study, descriptive analyses of the influenza attack rate and vaccine efficacy improvement (vei) were computed only for ili and pneumonia cases. the study also assessed whether the non-adjuvanted g ha influenza a(h n )pdm vaccine elicited immune responses that met the us and european regulatory criteria, days after vaccination and whether these criteria were met for either study vaccines at day (in a small subset of subjects) and at day (month ). hemagglutination inhibition (hi) antibody levels in serum samples were assessed at glaxosmithkline vaccines central laboratory using a validated in-house assay [cut-off: ≥ : ] that used chicken erythrocytes, as previously described [ ] . the a/california/ / strain was used as the antigen strain. rt-qpcr was performed on viral rna from the clinical samples as described previously [ ] . viral load values were quantified and the sample was considered positive when the measured viral load was equal to or above the assay cut-off [ ] . serum samples were collected before vaccination (day ), at days , (in a subset of subjects) and (month ) for assessment of humoral immune response and for clinical chemistry and hematology assessments at days , and . the immunological assessment was based on hi antibody seroconversion rates (scr), seroprotection rate (spr) and geometric mean fold rise (gmfr), against the vaccine homologous strain. post hoc exploratory analyses included the assessment of possible correlation of hi antibody response with body mass index (bmi) and with previous influenza vaccination history. further assessments were performed to identify the respiratory viruses isolated from swab samples from ili cases using xtag respiratory viral panel (rvp) fast assay (luminex molecular diagnostics inc., toronto, canada) [ , ] . subjects used diary cards to record the solicited local and general symptoms occurring within days following vaccination and the unsolicited adverse events occurring within days following vaccination. potential immune-mediated diseases (pimds: subset of aes that include both autoimmune diseases and other inflammatory and/or neurologic disorders which may/may not have an autoimmune etiology) and serious adverse events (saes) were recorded throughout the study period. the intensity of all solicited adverse events except fever was graded on a scale of ( - ), grade being those that did not interfere with normal activities and grade being those that prevented normal activities (grade redness and swelling: diameter > mm; grade fever: temperatures ≥ . -≤ . • c. fever was graded on a scale of ( - ), grade being temperatures > . • c. based on clinical judgment, the investigators assessed whether the aes/saes were potentially related/not related to the study vaccine. serum samples for the analysis of clinical safety laboratory parameters were collected at days and . the following laboratory parameters were tested: hepatic aminotransferases, total and direct bilirubin, alkaline phosphatase, creatinine, serum urea nitrogen, hemoglobin, hematocrit, white blood cell count and platelet count. the sample size was calculated taking into consideration the co-primary objectives. overall, evaluable subjects ( for vei evaluation) in each of the two treatment groups (accounting for % and % drop-out rates for the co-primary objectives) was estimated to provide a power of . % to meet the co-primary objectives, assuming %/ % as reference for spr/scr in subjects - years and > years of age, respectively, % vaccine efficacy for the non-adjuvanted influenza a(h n )pdm vaccine, and an attack rate of % in subjects who do not receive any h n vaccine (pass ; one-sided test, one-sided alpha = . %). the scr, spr and gmfr and incidence rates of solicited and unsolicited adverse events were calculated with % confidence interval (ci). the analyses of immunogenicity were performed on the according to protocol (atp) cohort which included evaluable subjects meeting eligibility criteria and adhering to protocoldefined procedures. a cox regression model, including the vaccine group as a fixed effect, age and baseline antibody titer as covariates was used to estimate the vei for the any ili cases and any pneumonia cases (the first event was considered if multiple events were reported by a subject). all statistical analyses were performed using statistical analysis software (sas) version . . a total of subjects were screened, received vaccine, and completed the study through day . the reasons for withdrawals and elimination of subjects from the analyses at different time points are presented in fig. . the mean age of subjects in the tvc at the time of vaccination in the - years age group was . years (range: - years); > years age group was . years (range: - years). overall, . % and . % of subjects in the respective two age groups were female and the majority of subjects were caucasians ( . % and . %, respectively). co-primary objectives: the first co-primary objective was met. a single dose of the as -adjuvanted . g ha influenza a(h n )pdm vaccine elicited hi immune responses in the - years and > years age groups that met the cber regulatory criteria at day ( table ). the chmp criteria were met in the - years and > years age groups (data not presented). the second co-primary objective was not evaluated as only three rt-qpcr-confirmed a/california influenza cases were identified (as -adjuvanted: ; non-adjuvanted: ). secondary objectives: in the day subset (n = ) which received the as -adjuvanted . g ha influenza a(h n )pdm vaccine, the cber criteria were met in the - years age group and > years age group (table ) . at day (month ), the cber criteria were met only for subjects - years of age (table ). subjects > years of age had a ll of the % ci for spr of . %, thus not fulfilling the cber criteria at this time point. at day , a single dose of the non-adjuvanted g ha influenza a(h n )pdm vaccine elicited hi immune responses in subjects - years and > years of age that met the cber regulatory criteria (table ) . only those in the - years age group met the cber criteria at day and at day (month ). at this time points subjects > years of age had a lls of the % ci for spr of . and . %, respectively and lls of the % ci for scr of . % and . %, respectively, thus not fulfilling the cber criteria. the chmp criteria were met at day and day in the - years and > years age groups for both study vaccines. at day , the chmp criteria were met in the - years age group but not in the > years age group for both study vaccines (data not presented). hi antibody gmts in both age groups were higher at all post-vaccination time points for those who received the as adjuvanted influenza a(h n )pdm vaccine compared to those who received the non-adjuvanted vaccine; gmts were generally lower in the > years compared to the - years age group at all time points (table ) . persistence of hi antibody response at day (month ) was observed for both study vaccines, although at lower levels compared to that observed at day (table ) . overall, the immune response against the vaccine homologous strain appeared to decrease with advancing age (fig. /web-appendix table ). post hoc exploratory analyses showed that hi antibody responses were mostly comparable across healthy weight, overweight and obese subjects. no clear patterns emerged due to the modest number of subjects in the underweight category (web-appendix table ). a higher hi antibody was observed among influenza vaccine-naïve subjects, compared with those with previous seasonal influenza vaccination, in terms of hi antibody gmts and gmfrs (web-appendix table ). [ . %] in the as -adjuvanted and non-adjuvanted treatment groups) were reported and were sampled during the study. of these, samples were tested and only three cases ( . %) of a(h n )pdm were confirmed by rt-qpcr (one and two cases respectively). the incidence of ili cases was comparable between the two treatment groups, except through day ( respiratory viruses: rhinovirus, identified from ( . %) nasopharyngeal swabs, was the most frequently determined respiratory virus (table ). solicited adverse events: pain at the injection site was the most frequently reported solicited local adverse event. it was reported for . % and . % of subjects in the - years age group who received the as -adjuvanted and the non-adjuvanted influenza a(h n )pdm vaccine, respectively (p < . ) and for . % and . % of subjects in the > years group who received the adjuvanted and the non-adjuvanted influenza a(h n )pdm vaccine, respectively (p < . ) (fig. /web-appendix table ). additionally, in both age groups, a statistically significant higher percentage of subjects receiving the adjuvanted vaccine reported redness and swelling compared with non-adjuvanted vaccine group (p < . for both). muscle ache (as -adjuvanted/nonadjuvanted: - years: . %/ . %, p < . ; > years: . %/ . %, p < . ), fatigue (as -adjuvanted/non-adjuvanted: - years: . %/ . %, p < . ; > years: . %/ . %, p = . ) and headache (as -adjuvanted/non-adjuvanted: - years: . %/ . %, p = . ; > years: . %/ . %, p = . ) were the most frequently reported solicited general adverse events (fig. /web-appendix table ). in the - years age group, a higher percentage on subjects receiving the adjuvanted vaccine reported joint pain, shivering and sweating compared with nonadjuvanted group (p < . for all). in the > years group joint pain was reported by a higher percentage of subjects receiving adjuvanted vaccine compared with the subjects receiving the nonadjuvanted vaccine (p = . ). in this age group, no statistically significant differences were observed between vaccine groups in terms of shivering, sweating and fever (p > . ). solicited local and general adverse events of grade intensity were reported for ≤ . % of subjects. in the - years age group, the incidence of pain at the injection site, joint pain and muscle aches of grade intensity was significantly higher in the adjuvanted vaccine group compared with the non-adjuvanted group (p < . for pain at the injection site; p = . for joint pain; p = . for muscle aches). the incidence of other solicited symptoms of grade intensity in this age group, as well as in the > years age group, was not statistically significant different between the adjuvanted and the non-adjuvanted vaccine groups (p > . ). reporting of solicited adverse events was higher in the - years age group. unsolicited adverse events: a total of subjects ( . %; as -adjuvanted: table ); subjects in the as -adjuvanted treatment group, - years: . %, > years: . %, and subjects in the non-adjuvanted treatment group, - years: . %, > years: . %. two of these events, intestinal obstruction (as -adjuvanted treatment group) and multiple sclerosis (non-adjuvanted treatment group) were considered by the investigator to be possibly related to study vaccine and were also considered pimds. through day (month ), pimds according to the predefined list of pimd preferred terms were reported, with and in as -adjuvanted and non-adjuvanted influenza treatment groups, respectively. seven fatal saes were reported, and in as -adjuvanted and non-adjuvanted treatment groups, respectively. all were assessed by investigators as not related to vaccination. a detailed description of all fatal saes is provided in web appendix table . overall, samples had laboratory values for the hematological and biochemical parameters outside the normal laboratory reference range at days and . of these, were from subjects in the adjuvanted vaccine group and were from subjects in the non-adjuvanted vaccine group. data from this large, controlled study in adults years of age and older demonstrated that a single dose of as -adjuvanted or non-adjuvanted influenza a(h n )pdm vaccine elicited strong hi immune responses days later that met the chmp and the more stringent cber criteria for pandemic influenza vaccines. the hi antibody response persisted through six months after vaccination for both vaccines, although the cber criteria were met only in the - years age group and chmp criteria in the - years age group. the co-primary objective concerning relative vaccine efficacy against influenza was not evaluated due to the small number of rt-qpcr-confirmed h n / influenza cases. the low number of cases observed may be partially due to the timing of the study; the start of study vaccination followed the peak of a(h n )pdm virus transmission in the us and canada by a week or more (last week of october, ), by which time a(h n )pdm circulation had diminished considerably. published estimates of as -adjuvanted influenza a(h n )pdm vaccine effectiveness against influenza range from . % to . % [ ] [ ] [ ] [ ] . overall, the incidence of ili cases was comparable between the two groups, except in the first days after vaccination ( versus ili cases in the as -adjuvanted and non-adjuvanted treatment groups, respectively). this study was not sufficiently powered to detect statistical significance in this analysis. the data for elderly subjects from the present study are in agreement with observations made in previous studies that one dose of the as -adjuvanted . g ha influenza a(h n )pdm vaccine may be insufficient to meet cber criteria at months in elderly [ ] and two doses of vaccine administered days apart induce longterm persistence of hi antibodies at putatively protective levels [ ] [ ] [ ] . nicholson et al. demonstrated that two doses of a different as -adjuvanted influenza a(h n )pdm vaccine elicited hi immune responses that persisted at seroprotective levels in > % of subjects ≥ years of age, up to six months after vaccination, although at lower levels compared to younger adults (p < . ) [ ] . similar to other observations [ , , , [ ] [ ] [ ] [ ] [ ] [ ] , our results showed that previous seasonal vaccination appeared to negatively influence the strength of the immune response elicited by the influenza a(h n )pdm vaccines, especially in terms of longterm immunogenicity. there are conflicting reports on whether previous seasonal influenza vaccination increases the risk of subsequently contracting a(h n )pdm infection requiring medical attention [ , ] . the effect of bmi on immune response was also studied. consistent with previous trials [ , ] , in the present study, high bmi did not appear to impair hi antibody response shortly after vaccination. however, sheridan et al. reported a decrease in hi antibody titers in obese subjects months after vaccination [ ] , an observation also made in the present study. the reactogenicity and safety profile was in agreement with available data in adults and children [ , , ] . the frequency of solicited local adverse events in this study was higher in the as -adjuvanted versus the non-adjuvanted treatment group and the frequency of solicited adverse events were comparatively lower in the > years age group. previous clinical trials of influenza a(h n /)pdm vaccines [ , , , ] comparing safety outcomes between adjuvanted and non-adjuvanted vaccines reported similar observations, with higher frequency of both local and general adverse events with adjuvanted vaccines compared with non-adjuvanted vaccines. in our study, we did not observe any differences between the two vaccine groups in terms of saes considered as possibly related to vaccination ( in each group). although an imbalance in the number of fatal saes was observed between the adjuvanted and non-adjuvanted group ( versus ), none were considered to be related to vaccination and they all occurred in subjects with a relevant medical history. a gradual decrement in the hi antibody gmts elicited by both study vaccines against the a(h n )pdm vaccine strain in older subjects was observed and this could be attributed to "immunosenescence" [ , ] . a decreasing trend with advancing age was also observed in the frequency of solicited adverse events. a possible limitation of this study was the absence of blood samples collection for assessment of the immune response after day (month ). this period of six months was anticipated to cover the period of transmission of influenza virus during one season. a recently published study enrolling subjects randomized to receive one or two doses of the same adjuvanted vaccine and followed up to months, showed that regulatory criteria were met months after the administration of the last vaccine dose in subjects aged - years receiving either one or two vaccine doses and in subjects aged > years receiving two vaccine doses [ ] . at day (month ) the regulatory criteria were still met only in subjects aged - years who received two vaccine doses. in conclusion, a single dose of either adjuvanted or nonadjuvanted influenza a(h n )pdm vaccines elicited protective levels of hi antibodies against the vaccine homologous a/california/ / strain that persisted up to day (month ) in the - years population. adjuvantation potentially offers the opportunity for antigen-sparing, making this as -adjuvanted influenza a(h n )pdm vaccine a candidate to help meet the demands for the large number of vaccine doses required to mitigate pandemic influenza. caroline gesualdi for clinical study management, janine linden for preparation of the study protocol and related study documentation, dorothy slavin, clinical safety representative, rosalia calamera, clinical data coordinator, karl walravens lab manager and clinical readout. finally, we thank avishek pal (glaxosmithkline vaccines) and adriana rusu (xpe pharma and science) who provided medical writing services and dr. santosh mysore (xpe pharma and science, c/o glaxosmithkline vaccines) for editorial assistance and manuscript coordination. financial disclosure: the study was funded by the us department of health and human services (hhs), assistant secretary of preparedness and response (aspr), biomedical advanced research and development authority (barda) and glaxosmithkline biologicals sa. glaxosmithkline biologicals sa was involved in all stages of the study conduct and analysis. glaxosmithkline biologicals sa also took in charge all costs associated with the development and the publishing of the present manuscript. all authors had full access to the data. the corresponding author had final responsibility to submit for publication. conflict of interest: all investigators received compensation for study involvement and travel related to this study. ping li, miguel madariaga, olivier godeaux and david vaughn are/were employees of glaxosmithkline group of companies and report receiving restricted shares of the company. contributorship: w.y., m.d., m.k. and n.a. contributed to the data collection, data interpretation and critical review of the manuscript drafts. p.l., m.m., o.g. and d.w.v. contributed to the study design, data analysis and interpretation as well as to the critical review of all drafts of the manuscript. trade mark statement: arepanrix is a trade mark of glaxosmithkline group of companies. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . . trial of influenza a(h n ) monovalent mf -adjuvanted vaccine safety and immunogenicity of pandemic influenza a h n vaccines in china: a multicentre, double-blind, randomised, placebo-controlled trial safety and immunogenicity of a pandemic influenza a h n vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the - influenza season: a multicentre, randomised controlled trial an adjuvanted pandemic influenza h n vaccine provides early and long term protection in health care 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influenza a(h n ) monovalent mf -adjuvanted vaccine a rapid immune response to influenza a(h n ) vaccines in adults: a randomized, double-blind, controlled trial the unmet need in the elderly: how immunosenescence, cmv infection, comorbidities and frailty are a challenge for the development of more effective influenza vaccines influenza vaccination for older adults longterm persistence of humoral and cellular immune responses induced by an as a-adjuvanted h n influenza vaccine: an open-label, randomized study in adults aged - years and older we are grateful to the new york medical college, new york for providing the vaccine virus strain. the authors are indebted to the participating study volunteers, clinicians, nurses and laboratory technicians at the study sites. we are grateful to the principal investigators, drs. key: cord- -nb miso authors: zhang, chuan-hai; lu, jia-hai; wang, yi-fei; zheng, huan-ying; xiong, sheng; zhang, mei-ying; liu, xin-jian; li, jiu-xiang; wan, zhuo-yue; yan, xin-ge; qi, shu-yuan; cui, zhiyong; zhang, biliang title: immune responses in balb/c mice induced by a candidate sars-cov inactivated vaccine prepared from f strain date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nb miso the immunogenicity of a candidate-inactivated vaccine prepared from sars-cov f strain was evaluated in balb/c mice. potent humoral immune responses were induced under the elicitation of three times of immunizations at -week intervals with this vaccine, combined with three types of adjuvants (freund's adjuvant, al(oh)( ) adjuvant and cpg adjuvant). titers of specific igg antibodies in three test groups all peaked in the sixth week after first vaccination, but significant differences existed in the kinetics of specific igg antibody levels. the strong neutralizing capacity exhibited in micro-cytopathic effect neutralization tests indicated the specific antibodies are protective. western blot assay further demonstrated the specificity of the induced serum antibodies. severe acute respiratory syndrome (sars) is the first severe viral epidemic we encountered this century. it once spread in more than countries and regions in , severely threatened worldwide public health. the causative agent of sars was confirmed and formally announced to be a novel * corresponding author. tel.: + ; fax: + . coronavirus by the world health organization in april , which established solid bases for us to effectively control and finally eradicate this disease [ ] [ ] [ ] [ ] [ ] [ ] . sars coronavirus (sars-cov) is novel, mutative and highly infectious compared to other known coronaviruses. more than genomic sequences of different sars-cov isolates have been determined, and some important structural and functional proteins have been unscrambled [ ] [ ] [ ] [ ] [ ] . the first outbreak of sars has been controlled worldwide, but many things regarding the nosogenesis, the antigenicity and the immuogenicity of sars-cov still remain unclear, and no specific drugs have been developed by now. in the long term, safe and efficient sars vaccines will play im-portant roles in the prevention of sars from possible future outbreaks. the present study was performed with the objective of determining the immunogenicity of a candidate-inactivated sars-cov vaccine made from f strain in balb/c mice. sars-cov f strain (ncbi/genbank ay ) was isolated from the samples of an onset sars patient in the guangdong province, china in , and was screened out as the vaccine strain [ , ] . vero e cells were cultivated routinely with mem medium containing no bovine serum, followed by infection with f strain virus. when the cytopathic effect (cpe) reached above %, the cell suspension was frozen and thawed three times, and stored at − • c. the titer of virus suspension was measured at about . tcid /ml. large-scale cultivated f strain virus was inactivated by treatment with . % formaldehyde (v/v) for h, and the inactivation efficiency ( %) and antigenicity of the inactivated virus were strictly identified [ ] . after centrifugating at × g for min, the virus supernatant was collected and purified (concentration and gel permeation chromatography) as the immunogen for animal immunization. three adjuvants were employed, namely, freund's adjuvant (including freund's complete adjuvant and freund's incomplete adjuvant, sigma), cpg adjuvant ( g/ml, provided by the molecular immunology institute of the first military medical university, china), and al(oh) adjuvant (self-prepared, . mg/ml). an equal volume of adjuvant was mixed with the prepared immunogen before immunization. the -week-old balb/c mice were fed in groups in a negative pressure isolator. these mice were divided into three test groups according to the adjuvant: group a (immunized with freund's adjuvant vaccine, n = ), group b (al(oh) adjuvant vaccine, n = ), and group c (cpg adjuvant vaccine, n = ). simultaneously, adjuvant controls (injected with adjuvant alone) were set. three times of immunizations were given at intervals of weeks (on d , d and d ). the first immunization was performed subcutaneously with a dose of . ml (the virus protein concentration was . mg/ml before mixing with adjuvant) each mouse. the second and the third vaccination were carried out in celiac way with a dose of . ml, but the vaccine for the third immunization contained no adjuvant. after the first vaccination, batches of blood samples were collected by tail bleeding from the initial day (d ) to day (d ). serum was separated by centrifugation at × g for min and stored at − • c. microtiter plates were coated with inactivated sars-cov dilution (containing . g/ml total virus proteins) overnight at • c. after blocking with % bovine serum in pbst (phosphate-buffered saline containing . % tween- ) at • c for min, the plates were washed five times with pbst. then two-fold serial serum dilutions were added ( l/well) and incubated at • c for min. washing the plates, hrp (horseradish peroxidase)-conjugated antibodies ( : goat anti-mouse igg, or : , goat anti-mouse igm, sino-american biotech) were added ( l/well), and incubated at • c for min. washing the plates with pbst, then opd substrate (o-phenylendiamine, sigma) was added ( l/well) and incubated at • c for min. the reaction was stopped by . m sulfuric acid, and the absorbance at nm (a ) was measured by a microplate reader (biorad, model ). in this assay, normal serum was used as negative control, and a positive antiserum was included in each plate as an inter-plate variability control. antibody titer was defined as the highest dilution of serum at which the a ratio (a of sample/a of negative control) was greater than . . neutralizing antibody titer was measured according to the modified protocol for polio antibodies [ ] . each serum sample was diluted into two-fold serial dilutions with mem maintenance medium, then mixed with an equal volume of tcid active virus and incubated at • c for min. after neutralization, the mixtures were successively added ( l/well) into vero e cell monolayers in microtiter plates; but wells for normal cell control were added into l maintenance medium, and wells for virus control were added into unneutralized virus instead. after that, the plates were incubated at • c in % co incubator, and cell status was monitored by sars-cov cpe every h, until all wells of virus control showed cpe but the cell control remained normal. neutralizing antibody titer was defined as the highest dilution of serum, which protects % of the cultures against cpe. photographs were taken by a phase-contrast microscope. western blot assay was carried out according to the protocol in molecular cloning [ ] . first, sars-cov proteins were separated by sds-page on a % stacking polyacrylamide gel in combination with a % separating gel in a mini-apparatus (bio-rad, mini protean well). the separated proteins in the gel were then transferred to a nitrocellulose membrane in a small apparatus (bio-rad, mini trans-blot) with . ma/cm current for . h. after blocking with % bovine serum in pbst for min, the membrane was incubated with : mouse antiserum dilution or with normal serum as negative control for min at room temperature. washing with pbst, then the membrane was incubated with hrp-conjugated goat anti-mouse igg ( : dilution) for min. washing again, dab substrate (diaminobenzidine, sigma) was added and incubated until brown color developed. a convalescent serum from sars patient was used as a positive control, and incubated with : hrpconjugated goat anti-human igg antibodies (sino-american biotech). igm antibody levels of the five sets of mice sera in the first month were measured by elisa, the results were shown in fig. . results of adjuvant controls all were negative. in test groups, anti-sars-cov specific igm antibodies were induced by the inactivated vaccine. in groups a (immunized with freund's adjuvant vaccine) and c (with cpg adjuvant vaccine), igm antibody levels varied regularly, both reached a peak point on d and went down hereafter. in group b (with al(oh) adjuvant vaccine), igm antibody titers presented a fluctuation between d and d . booster immuniza- . eleven batches of mice serum were sampled on d to d , and were detected by elisa. each sample was two-fold serially diluted. hrpconjugated goat anti-mouse igg antibodies and opd substrate were applied. the specific antibody titers were shown as the reciprocal of the highest serum dilution at which a was two-fold greater than that of the negative control. tions showed no positive effects on the rise of igm antibody titers. eleven batches of serum samples from d to d were measured by indirect elisa (fig. ) . specific igg antibodies were detectable one week after the first immunization in all three test groups, but in very low titers. with the stimulation of the second immunization, especially the third one, igg antibody levels increased rapidly, and all peaked in the sixth week. the maximum titer of groups a (with freund's adjuvant vaccine) and b (with al(oh) adjuvant vaccine) was : , and : , , which was four and eight times of that of group c (with cpg adjuvant vaccine), respectively. after this stage, the specific igg antibody titers of three test groups all went down to a relatively steady level. two sets of mice antisera were examined. these samples were collected on d ( days before the third vaccination) and d ( days after third vaccination) from the mice of group a, which were immunized with freund's adjuvant vaccine. the results were shown in table and fig. . for the serum samples on d , no cpe presented at the dilution of : , % cpe at : , % cpe at : (columns , and in row , table ); the neutralizing antibody titer was calculated to be : . for the samples on d , % cpe was observed at the dilution of : , % cpe at : , and % cpe at : (columns , and in row , table ); the neutralizing titer was : . fig. displayed the neutralization photographs at h with the antiserum collected on d from group a. compared with cell control (fig. a) , positive control showed marked morphologic changes with cpe, the cells infected with sars-cov became refractile and rounded (fig. b) . in neutralization tests, the active virus was first neutralized with serially diluted antiserum, then added to vero e cell monolayers. under the neutralization of : diluted antiserum, cells still kept the same normal morphologic characters (fig. c- and c- ) as the uninfected cells. but under the neutralization of : dilution, the cells presented morphologic changes with about % cpe (fig. c- and c- ). the antiserum sampled on d from group a (immunized with freund's adjuvant vaccine) was identified by western blot. separated proteins from inactivated sars-cov suspension showed two dominant bands (about and kda) and several minor bands in the gel (lane , fig. ) . the antiserum interacted with the electro-transferred virus antigenic proteins, and two strong bands showed on the nitrocellulose membrane (lane , fig. ) . a convalescent serum of sars patient was used as positive control, and also showed two similar bands (lane , fig. ). but no positive reaction was detected with normal serum (lane , fig. ). immunogenicity evaluation is an essential work to the development of viral inactivated vaccines. sars-cov is highly infective and lethal, no ready immune protocols may be directly employed. in this study, sars-cov was % inactivated by treatment with . % formaldehyde (v/v) for h, and the antigenicity was well preserved [ ] . three times of immunizations at two-week intervals were applied. the results showed that sars-cov f strain inactivated vaccine could induce potent humoral immune responses in balb/c mice. our other studies on the evaluation of this vaccine in rats, rabbits, horses and rhesus monkeys (results not shown), also suggested its strong immunogenicity. adjuvants may play active roles in the generation and maintenance of immune responses. freund's adjuvant is commonly used in animal vaccines. al(oh) adjuvant is a safe and recommended adjuvant in human vaccines [ , ] . cpg (cpg motif) is a new immune stimulatory dna sequence, it is likely to be used as a new type of human vaccine adjuvant with low side-effects and high immune potency [ ] . in this study, these three kinds of adjuvants were used combining with f inactivated vaccine, a marked difference existed among them, al(oh) adjuvant and freund's adjuvant exhibited stronger stimulatory efficacy than cpg adjuvant. neutralization capacity is an important index to viral vaccines. from the previous experiences, not all vaccines may elicit high level of neutralizing antibodies [ ] [ ] [ ] . in this study, high titers of neutralizing antibodies were induced in balb/c mice. in neutralization tests, cross neutralization was identified between sars-cov f strain (ncbi/genbank ay ) and z -y strain (ncbi/genbank ay ). from the dynamic variation of specific antibody titers (fig. ) , booster immunizations played important roles in the generation of high level of specific antibodies, but a noticeable titer decrease followed the peak point. thus, the longevity of protective antibodies needs to be determined by further studies. based on the potent antigenicity and immunogenicity, sars-cov s (spike, kda), n (nucleocapsid, kda) and m (membrane, kda) proteins may be selected as targets for sars vaccine development [ ] [ ] [ ] . in present study, the specificity of serum antibodies induced by f strain inactivated vaccine was identified by western blot assay. two dominant bands and several minor bands were exhibited in the gel after sds-page. in electrophoresis, the migration of virus proteins was somewhat blocked, the visual effect of which was the arched front (lane , fig. ). considering the imbalance in migration rates between protein marker (lane m, fig. ) and virus proteins, the actual molecular weight of the two main protein bands should be a little smaller than the ap-parent values, which was estimated at about and kda that was equivalent to n and m protein of sars-cov, respectively. s protein was not detected; it is likely due to the loss in long-term storage after inactivation and in the sample treatment, or together with the relatively low s protein content [ , ] . following the immunochemical reactions with the antiserum, two strong bands exhibited on the membrane (lane , fig. ) exactly at the corresponding position in the gel, whereas no color bands generated on the membrane with normal serum (lane , fig. ) . a convalescent serum of sars patient was used, and the same positive result was obtained (lane , fig. ) , which further demonstrated the specificity of the antibodies induced with the inactivated vaccine produced from f strain. in summary, this study described the safety and immunogenicity of the deactivated sars vaccine tested on c bl/ model. the results indicated that the specific igg antibodies were produced after being immunized with inactivated sars-cov vaccine and the kinetics of the igg-specific antibody was shown well regularity. the antiserum could specifically neutralize the sars-cov. the antibodies against sars-cov proteins (m and n) appeared in the antiserum by western blot assay. our results provided the evidence that the inactivated sars vaccine can produce the specific antibodies and implicated the therapeutic application of the deactivated sars vaccine. recent progress in the studies on sars and its pathogeny a cluster of cases of severe acute respiratory syndrome in hong kong update: outbreak of severe acute respiratory syndrome-worldwide coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china in a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection the genome sequence of the sars-associated coronavirus the chinese sars molecular epidemiology consortium. molecular evolution of the sars coronavirus during the course of the sars epidemic in china isolation, identification and variance of a coronavirus from a imoutting sars case establishment of sars virus vaccine line study on the function in inactivating antigenicity of sars coronavirus by formaldehyde neutralization test for polio antibodies molecular cloning-a laboratory manual workshop summary: aluminium in vaccines the global impact of vaccines containing aluminium adjuvants mechanism of activation of cpg dna protective immunity against herpes somplex virus generated by dna vaccination compared to natural infection immunogenicity and peotective efficacy of a formalin-inactivated rotavirus vaccine combined with lipid adjuvants mucosal immunization with inactivated hiv- -capturing nanospheres induces a significant hiv- -specific vaginal antibody response in mice an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus bioinformatics analysis of sars-cov m protein provides information for vaccine development preparation, characterization and preliminary in vivo studies of inactivated sars-cov vaccine this study is supported by the science fund ( z -e and ) of guangdong province, china. key: cord- -de b nq authors: anraku, itaru; mokhonov, vladislav v.; rattanasena, paweena; mokhonova, ekaterina i.; leung, jason; pijlman, gorben; cara, andrea; schroder, wayne a.; khromykh, alexander a.; suhrbier, andreas title: kunjin replicon-based simian immunodeficiency virus gag vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: de b nq an rna-based, non-cytopathic replicon vector system, based on the flavivirus kunjin, has shown considerable promise as a new vaccine delivery system. here we describe the testing in mice of four different sivmac gag vaccines delivered by kunjin replicon virus-like-particles. the four vaccines encoded the wild type gag gene, an rna-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. the vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the siv gag-pol vaccine providing the optimal performance. these results illustrate that for an rna-based vector the rna sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour. a number of rna-based vector systems have been used in the construction of hiv- and siv vaccines. these include vectors based on negative strand rna viruses [ ] [ ] [ ] , positive strand rna viruses such as sendai virus [ ] , coronavirus [ ] and lentiviruses [ ] , and positive strand rna replicon-based vectors derived from alphaviruses [ ] [ ] [ ] , picornaviruses [ ] , and flaviviruses [ ] . replicon-based vectors exploit the ability of viral non-structural proteins to amplify greatly (in the cytoplasm of infected/transfected cells) the replicon rna, which encodes both the heterologous protein and the viral non-structural proteins. this results in high-level expression of antigens and provides potent adjuvant activity via double-stranded rna replication intermediates [ ] . we have previously described a replicon vector system based on the kunjin virus [ ] , a largely non-pathogenic flavivirus found in northern australia [ ] . kunjin replicon rna can be packaged into viruslike particles (vlps) using a packaging cell line expressing kunjin structural proteins in trans [ ] . the resulting vlp vaccines are capable of inducing potent, long-lived and protective cd t cell responses in several murine systems [ , ] . kunjin replicon vlp vaccines encoding hiv- gag have also been shown to induce cd t cell immunity comparable to that seen after immunisation with recombinant vaccinia [ ] . in contrast to most alphavirus repliconinfected cells, kunjin replicon-infected cells (i) do not show overt cytopathic effects, so both double strand rna-induced "danger signals" and antigen production are maintained for extended periods and (ii) do not produce replication-competent viruses via recombination during vlp manufacture, with recombination in flavivirus systems appearing to be extremely low [ ] . the evaluation of siv vaccines in non-human primates has proved to be an invaluable and enduring model system for testing new concepts in hiv vaccine development [ ] [ ] [ ] [ ] . most siv and hiv subunit vaccines encode gag, as gag proteins are believed to be an important target of protective cellular immunity [ , ] . herein we describe the construction and immunogenicity of four siv gag (pol) kunjin replicon vlp vaccines. the first encodes siv gag using the wild-type nucleic acid sequence (wt). the second encodes siv gag using an rna-optimised nucleic acid sequence (dx). hiv and siv gag contain rna inhibitory sequence (ins) elements, which have been shown to act at several levels to inhibit gag expression; primarily via nuclear retention of rna in the absence of the rev protein. rna-optimisation strategies, which remove these ins elements by changing the codon usage, result in significant increases in protein expression from dna-based expression vec-tors [ ] , and such rna-optimised siv gag vaccines have been used in a number of monkey trials [ , ] . inhibition of nuclear export of gag rna by ins sequences is unlikely to be an issue for the exclusively cytoplasmic kunjin replicon rna, however, ins elements have also been reported to reduce rna stability and translation [ , ] . the third siv gag kunjin replicon vlp vaccine encodes a human codon-optimised siv gag gene (opt). codon optimisation has been used extensively to improve recombinant antigen expression in a number of systems and the process involves altering the codon usage to the one most commonly utilized in a given species [ , ] . the fourth vaccine encodes wild-type matrix and capsid from gag-linked in-frame to reverse transcriptase from pol (gagpol). this approach has been described previously for hiv gag and removes from the gag gene sequences that encode proteins with potentially immunosuppressive properties, whilst retaining sequences frequently recognised by t cells [ ] . although all four vaccines expressed gag protein at comparable levels, they behaved quite differently in terms of immunogenicity, protection and insert stability. the rna-based kunjin replicon vector c ubhdvrep (sp kunrep ) [ ] was used to construct wt, dx and opt vaccines, and sp kunrep [ ] was used to construct the gag-pol vaccine. these two rna-based kunjin replicon vectors are the same, except sp kunrep has two copies of the foot and mouse virus a autoprotease, one upstream of mlui (replacing the ubiquitin gene) and another one downstream of the snabi cloning sites [ ] . the kunjin siv gag wt construct encodes siv mac gag subcloned from p spsp [ ] (catalogue # , nih aids research & reference reagent program, germantown, md, usa) using asci sites. the kunjin siv gag dx construct was generated by subcloning rna-optimised siv mac gag dx [ , ] from pfb-sivgag dx cte neo [ ] using asci/snabi sites. kunjin sivgag opt was constructed using asci/snabi sites and p - (catalogue # , nih aids research & reference reagent program), which encodes human codon-optimised sivmac gag. the kunjin sivmac gag-pol vaccine was constructed by separately pcr-amplifying matrix (ma)/nucleocapsid (nc) and reverse transcriptase (rt) genes from p spsp plasmid (nih aids research & reference reagent program) and joining at sacii. the fragment was then cloned into the mlu i and snab i sites of sp kunrep vector. construction of the hiv- gag vaccine has been described previously and was expressed from the sp kunrep vector [ ] . all gag constructs used in this study-encoded alanine in place of the initial glycine in gag. kunjin replicon rnas were transcribed in vitro using sp rna polymerase and electroporated into tetkuncprme bhk packaging cells as described previously [ ] . briefly, rna ( - g) was electroporated into × packaging cells in suspension and the cells were seeded into a -mm dish and incubated at • c for - days and culture fluid collected at days - . the titre of infectious vlps in infectious units (ius) was determined by infection of vero cells with -fold serial dilutions of the culture fluid. at - h post-infection the cells were fixed with methanol/acetone ( : , v/v) and analysed by immunofluorescence antibody staining using the g monoclonal anti-ns antibody [ , ] and the - f anti-siv gag monoclonal antibody (catalogue # , nih aids research & reference reagent program) and the number of positive cells counted using zeiss axiophot fluorescent microscope (carl zeiss microimaging gmbh, berlin, germany). vero or bhk cells were infected with vlps at moi = or electroporated with - g of kunjin siv gag rna constructs and cultured for days in a -mm dish. the cells were lysed with ripa buffer and cell debris was removed by centrifugation. the protein concentration for each sample was determined using the biorad protein assay (biorad, regents park, nsw, australia). fifteen microgram of total cell protein was separated on a . % gel by sds-page and transferred onto hybond-p membrane (amersham-pharmacia biotech, uk) using the mini trans blot transfer tanks (biorad, regents park, nsw, australia) at • c. the his-tagged recombinant siv mac and hiv- (clade b sf /bh ) gag proteins were expressed from the pet a+ plasmid (merck pty. ltd., vic, australia) under control of t -promoter in escherichia coli c (de ) [ ] and purified using a ni-agarose column. gag proteins were detected using the - f or the ag . vero cells were transfected with kunjin siv gag vlps at moi = and were cultured for h. total rna was collected using trizol reagent (invitrogen) and superscript iii reverse transcriptase (invitrogen) was used to produce cdna. pcr primers that bind to c and e of the kunjin replicon vector backbone were then used to amplify fragments from the cdna using dynazyme ext (finnzyme, diagnostics, espoo, finland); forward primer ggctgtcaatatgctaaaacg , reverse primer tctttctctccgtgaacgtgcat . female balb/c (h- d ) mice ( - weeks) were supplied by the animal resources centre (perth, western australia). mice were vaccinated with the kunjin replicon siv gag vlp vaccines wt, opt, dx, or gag-pol, the kunjin hiv- gag vlp vaccine [ ] or control vlps diluted in rpmi and iu injected by the intraperitoneal (i.p.) route in a final volume of l. interferon ␥ (ifn␥) elispot assays were conducted using or overlapping -mer peptides spanning the entire sivmac or hiv- subtype b gag protein, respectively (catalogue # and , respectively, nih aids research & reference reagent program). the peptides were divided into six pools and the total final concentration of the peptide mix in each pool was g/ml. for kunjin-specific responses directed to ns , gyistrvel (h- k d ) was used (auspep, parkville victoria, australia). ex vivo elispot assays were conducted as described previously [ ] except that multiscreen-ip hydrophobic pvdf membrane microtitre plates (millipore australia ltd., north ryde, australia) and iu/ml of il- (kindly provided by cetus corp., emeryville, ca, usa) was used. the highest cell concentration was . × cells/well in triplicate, followed by four doubling dilutions. spots were counted using a ks elispot reader (carl zeiss vision gmbh, hallbergmoos, germany). mean background spots from wells without peptide were subtracted from the mean number of spots obtained from wells with peptide. cultured elispot assays were performed using splenocytes cultured in vitro for days in well plates ( × cells/well in ml medium) with a single pool of the entire overlapping gag peptides ( g/ml of the peptide pool) or gyistrvel ( g/ml). (culture of splenocytes in the presence of g/ml of the siv peptide pool resulted in excessive responses being detected in naïve mice-data not shown). on day a further ml of medium was added to each well. on day the cells were used in the elispot assay (as described above) starting at . × cells per well followed by four doubling dilutions. groups (n = or ) were compared (using the total peptide response for each mouse) with the non-parametric median test and the monte carlo significance given (spps for windows, version . , , spss inc., chicago, il, usa). a plasmid encoding an egfp-siv gag dx fusion protein was generated by subcloning human rna-optimised siv gag dx from the plasmid pcmvsivgagdx [ ] (provided by dr b. felber) into xhoi/ecori sites of the pegfp c plasmid (catalogue # - . bd biosciences, franklin lakes, nj, usa) to generate pegfp sivgagdx. this plasmid was used to transfect the balb/c-derived a murine lymphoblastic leukemia cells (atcc, tib- ) using the genejammer transfection reagent (stratagene, la jolla, ca, usa). cells were cultured for h and sorted using moflo high-performance cell sorter (dako, glostrup, denmark) for high egfp expression. the sorted cells were cultured in medium [ ] containing g ( g/ml) for - weeks then sorted again for high egfp expression. this procedure was repeated eight times at which point expression had stabilised with > % of cells expressing egfp as assessed by facs after a week of culture under g selection. expression of egfp-siv gag dx fusion protein was also confirmed by western blot using anti-siv gag ( - f ) and anti-egfp antibodies (molecular probes, leiden, netherlands). (we were unable to generate a gag-expressing line using j macrophage cells). groups of balb/c mice were vaccinated twice separated by - weeks with iu of the four different vlps encoding siv mac gag, control vlps, or rpmi by the i.p. route. approximately months after the last immunisation mice were challenged with the a -egfp-sivgag cells ( . × cells/mouse) on the back by the subcutaneous route. hair around the injection site was removed with an electric shaver prior to the challenge to facilitate injection and measurement of tumours. the tumour sizes were measured three times a week and mice were sacrificed when the tumour area reached mm . differences between kaplan-meier survival curves were calculated using the log-rank test (spss for windows). four different kunjin replicon vlp vaccines encoding siv mac gag were constructed; sivgag wt (wt), siv gag dx (dx), siv gag opt (opt), and siv gag-pol (gag-pol) (fig. ) . wt encodes full-length wild-type siv gag, dx and opt encode rna-and codonoptimised versions of the siv gag gene, respectively, and gag-pol encodes wild type matrix and capsid from gag connected in-frame with reverse transcriptase from pol. an hiv- gag construct encoding wild-type hiv gag was used as a positive control (see below) and has the same vector backbone as that shown in fig. [ ] . in vitro transcribed rna derived from each kunjin replicon dna construct was used to manufacture vlps in packaging cells [ ] . each vlp vaccine was used to infect vero cells in vitro and western blotting experiments (using the anti-siv gag - f antibody) illustrated that each vaccine expressed a protein of the expected molecular weight (fig. a) . similar data was produced when kunjin rna (rather than vlps) was used to transfect bhk cells (fig. b) , indicating there was no major differences in expression between monkey and rodent cells. in these experiments no major improvements in protein expression levels were seen for dx or opt vaccines despite rna-and codon optimisation. (no in vitro siv gag particle formation was observed by electron microscopy for these vaccine-data not shown.) when the lysates from siv gag and hiv- gag vlp-infected cells were initially compared side by side in western analyses using the ag . antibody, we were surprised to find that the expression of siv gag appeared to be substantially lower (fig. c ). it emerged, however, that this antibody recognises the siv mac gag protein much less effectively than the hiv- gag protein (fig. d ). siv gag expression was thus probably not in reality substantially lower than hiv gag expression in these kunjin replicon systems. a more extensive analysis of a panel of antibodies used for detecting siv gag proteins also revealed some hitherto unpublished differences in reactivity with gag proteins from different siv strains using different techniques. we have shown the data in table and have added published information for completeness. this information will hopefully allow others to identify more easily the appropriate antibody for future siv gag studies. to compare the immunogenicity of the kunjin siv gag vaccine constructs, balb/c mice were immunised twice with iu of each of the four vlp vaccines. iu represents the titre of vlps expressing gag (see table ), so each mouse received the same number of kunjin vlps encoding gag. two negative control groups were included to account for non-specific responses, one group received no vaccination (control naïve) and the second was vaccinated with vlps not encoding gag (control vlp). immunisations with kunjin vlps encoding hiv- gag was also included. ten weeks after the last immunisation splenocytes were assayed by ex vivo and cultured ifn␥ elispot assays using six pools of overlapping peptides spanning the entire siv gag protein or in the case of hiv gag immunised mice, the hiv gag protein. the use of peptide pools covering gag to measure t cell responses in mice has been described previously [ , ] . ex vivo elispot assays measure immediate effector function and thus tend to measure effector memory t cell activity, whereas cultured elispot assays measure the ability of cells to replicate and form effector cells and thus represents a measure of central memory cell activity [ ] [ ] [ ] . ex vivo elispot assays illustrated that responses induced by the wt vaccine were not significantly higher than the control vlp (p = . ) (fig. a) . dx induced responses that approached significance over control vlps (p = . ), and opt, gag-pol and the hiv gag vaccine induced responses that were significantly higher than the control vlp (p = . , . and . , respectively) (fig. a) . the opt and gag-pol vaccineinduced responses were comparable to those induced by the kunjin hiv- gag vlp vaccine tested under the same conditions (fig. a) . using the cultured elispot assay, wt and opt failed to produce significant responses over the controls (fig. b) . the gag-pol con- struct induced significantly higher responses than wt (p = . ), whereas dx (due to the very high mouse to mouse variation in this group, ranging from to , spots) did not reach significance over wt using the median test (although it did reach asymptotic significance using the mann-whitney test, p = . ). dx and gagpol vaccines also induced responses comparable to those induced by hiv- gag vlp vaccination (fig. b) . pool dominated the hiv- gag responses (fig. b ) as this pool contains the immunodominant amqmlketi epitope [ ] . spaulding et al. identified a region in dengue virus ns , which represents a dominant cd t cell epitope [ ] . we have confirmed that the homologous region ( gyistrvel ) in kunjin virus ns , which contains a substitution (m to l) in the last amino acid, also represents a dominant cd t cell epitope for kunjin replicon vaccines (data not shown). to analyse the levels of anti-vector cd t cell responses for the different vaccines shown in fig. a and b, elispot assays using the gyistrvel peptide epitope (at g/ml) were undertaken. ex vivo ifn␥ elispot analysis showed that responses ranged from ≈ to spots/ splenocytes (fig. c) . cultured ifn␥ elispot analysis showed a range of ≈ - spots/ splenocytes for all the vectors except dx, which gave a large mean response in excess of , spots/ splenocytes (fig. d) . the very large response for dx might be due to the large number of vlps not expressing gag present in the dx preparation (table ). these results illustrate that flavivirus replicon-based vectors, like other vector systems [ , ] , also induce cd t cell responses specific for vector proteins. we and others [ ] have noted that flavivirus vlps can lose expression of certain inserted genes. to investigate to what extent the different siv gag vlp vaccines suffered from this problem, vero cells were infected with vlps and dual-labelled with anti-kunjin ns and anti-gag antibodies. for wt and gag-pol the majority of vlp-infected cells expressed both ns and gag, however, for dx and opt only about % and % of ns positive cells, respectively, also expressed gag (table ) . for the hiv gag vlps this value was > % (data not shown). the dx and opt constructs contained rna-and codon-optimised genes, perhaps suggesting that altering the coding sequence somehow reduces the stability of these inserted genes in the kunjin replicon constructs. the in vitro transcribed rna used to transfect the packaging cells did not contain any detectable truncated replicon rna species (fig. a ) indicating that these insert deletion events likely occurred during vlp manufacture. gene deletion events from flavivirus vectors have not been extensively studied. to gain some insight into the process, rna from wt and dx vlp-infected cells was analysed by rt-pcr using primers flanking the siv gag gene insert. the full-length gene is visible at ≈ bp, with shorter pcr products evident in both wt and dx infected cells. gene deletions in wt and dx appear to be different, with dx showing a major band at ≈ bp (fig. b, lane ) . a band of the same size was also observed in samples from optinfected cells (data not shown). this ≈ bp fragment is actually smaller than that obtained from the empty vector (fig. b, lane ) , suggesting that at least one end of the deletion must lie within the ubiquitin cloning site or the fmdv a protease region (see fig. ). sequencing of the ≈ bp fragment from dx illustrated that the deletion occurred nucleotides past the end of the nucleotide ub sequence and nucleotides past the end of the nucleotide fmdv a sequence (fig. b ). there are no obvious homologies between the sequences either side of the insert deletion site that would suggest strand switching or homologous recombination events. each vlp vaccine was titred on vero cells and cultured for h. ifa was then performed using anti-ns and anti-siv gag ( - f ) monoclonal antibodies and the iu titre calculated for each antibody. the ratio of the titres is represented as a percentage (gag titre/ns titre × ). recombinant vaccinia viruses encoding hiv- antigens have been used extensively as surrogate viral challenge models in mice to assess protective immune responses induced by different hiv- vaccines [ , ] . we determined whether two recombinant vaccinia viruses, one encoding siv mne gag/pol [ ] (kind gift from dr. shiu-lok hu, university of washington, seattle, wa) and the other siv mac gag (eva , centre for aids reagents, national institute for biological standards and controls, uk) could be used in such assays. unfortunately, although they grew well in vitro, both vaccinia viruses replicated very poorly in mice following i.p., intranasal or intravenous inoculations of or pfu of virus (data not shown), making them unsuitable for such assays. a vaccinia virus encoding hiv- gag was run in parallel and replicated efficiently as described previously [ ] . a vaccinia encoding sivmac gag has been reported to replicate in mice [ ] , however, this vaccinia virus has not been made publicly available, and the reason for its better replication in mice is unclear. to develop an alternative challenge assay we generated an a lymphoblastic leukemia line stably expressing siv gag dx fused to egfp (a -egfp-sivgag cells). western blotting illustrated that these cells expressed the gag-egfp fusion protein, which has an expected molecular weight of ≈ kda (fig. a) . these cells were used to challenge animals ≈ months after vaccination with the different siv vlp vaccines. the opt, gag-pol and dx vaccines all provided significantly better protection than both the controls and the wt vaccine (log-rank tests all p ≤ . ) (fig. b) . opt vaccination also provided significantly better protection than dx (p = . ), with no other comparisons showing significant differences (fig. b) . mean tumour growth curves from the same experiment are shown in fig. c . the percentage of mice surviving after challenge with a -egfp-sivgag cells ( , , and % for wt, dx, gag-pol, and opt, respectively, fig. b ) correlated well with the mean ex vivo elispot data shown in fig. a ( , , and spots/ splenocytes, respectively), r = . . a similar comparison with the cultured elispot data (fig. b ) failed to show any correlation, r = . , suggesting that effector memory rather than central memory cell activity had a major role in mediating protection in this model. vaccines against a series of pathogens including hiv/siv are being developed using a number of rna-based viral vector systems. here we describe the behaviour of four different kunjin replicon vlp vaccines encoding siv gag and show that only the gag-pol vaccine (i) induced good levels of both effector memory and central memory t cell responses weeks post-vaccination, comparable to those induced by the previously described hiv- gag kunjin replicon vlp vaccine [ ] , (ii) showed good levels of protection against challenge with a cells expressing siv gag ≈ months post-vaccination, and (iii) displayed high levels of insert stability. wt and dx vaccines induced poor effector memory responses and provided poor protective activity, and opt and dx showed high levels of insert deletion. the potential for genetic instability for many recombinant viral vectors is well described and a range of largely vector-specific strategies have been adopted to limit the problem [ , [ ] [ ] [ ] [ ] [ ] . in the kunjin replicon system deletion of inserted genes appears to occur in packaging cells during vlp manufacture. for constructs with low insert stability the proportion of vlps without inserts increases if the time between rna transfection of the packaging line and vlp harvest is extended, and appropriate reductions of this were immunised twice with iu of each of the indicated kunjin vlp vaccines. there were two control groups, one received iu of a control vlp encoding an irrelevant antigen (control vlp) and the other received no vaccination (control naïve). ten weeks after the second immunisation, the mice were sacrificed and the splenocytes were assayed for gag-specific t cell responses by ex vivo ifn␥ elispot using six pools of overlapping peptides spanning either the siv gag protein for siv gag vaccinated animals, or the hiv- gag protein for hiv- gag vaccinated animals. error bars illustrate the variation in responses to each pool. (b) measurement of gag-specific responses by cultured ifn␥ elispot assays. the splenocytes from the animals described in (a) were also cultured for days with the pooled siv or hiv gag peptides, prior to the elispot assay. (c) measurement of ns -specific responses by ex vivo ifn␥ elispot assays. the splenocytes from the animals described in (a) were also used in an ex vivo elispot assays using the ns cd t cell peptide epitope, gyistrvel. (d) measurement of ns -specific responses by cultured ifn␥ elispot assays. the splenocytes from the animals described in (a) were cultured for days with gyistrvel peptide prior to an elispot assay using the same peptide. time period can result in vlp preparations with substantially fewer empty vlps (unpublished observation). these observations parallel those obtained for west nile replicons where vlps with insert deletions were also rapidly selected during serial passage of vlps in a packaging cell line [ ] . in contrast, we have generated by antibiotic selection several cell lines stably transfected with recombinant kunjin replicon vectors and these could be extensively passaged without loss of reporter gene inserts [ ] , suggesting that insert deletion may be rare once a cell is transfected. one might speculate that insert deletion is associated with the cellular stress that leads to the cytopathic effects, which are observed in the packaging cells during vlp manufacture due to the high level of structural protein expression. conceivably, in such stressed cells replicon rna may gain access to the nuclear rna splicing machinery. rna splicing is well known to be influenced by complex rna structure interactions, with distant rna sequences able to regulate splicing events [ ] . why dx and opt vaccines would be more susceptible to insert deletion remains unclear, but may be associated with the loss of native rna structures. the dx gene was engineered to allow rev-independent expression from dna-based vector systems [ , ] , however, this did not provide significant improvements in antigen expression. the negative influence of siv gag ins sequences on rna stability and translation [ , ] therefore do not appear to be significant in this system. successful expression of wild-type siv gag by other vector systems where mrna is generated in the cytoplasm (e.g. alphavirus replicons and vaccinia) also supports the view that the major influence of ins elements is on rna export [ , ] . the human codon-optimised kunjin opt construct did not result in significantly higher gag expression in vero (monkey) or bhk (hamster) cells, suggesting that trnas are not particularly limiting in these replicon-based expression systems when tested in vitro. why opt and gag-pol might provide slightly improved effector memory responses and wt and opt induce such poor central memory responses when delivered by kunjin replicon vlps remains unclear, but is likely to be a reflection of multiple factors including the number of empty vlps in the vaccine inocula, the level and length of antigen expression in vivo, and the level of "danger signals" induced by the replicating replicon rna. the better memory responses induced by the gag-pol vaccine may be due to the removal of the potentially immunosuppressive gag-encoded proteins [ ] . an alphavirus replicon siv vaccine with these regions deleted also showed improved longevity of t cell responses over full-length gag constructs [ ] . the high (albeit variable) central memory responses induced by dx may arise from increased levels of ifn␣/␤ produced [ ] as a result of the large number of empty vlps that compromised ≈ % of the vlp inocula for this vaccine preparation ( table ) . the a -egfp-siv gag challenge model (fig. ) represents a new model for testing the effectiveness of t cell induction by siv gag vaccines. this model is likely to represent a measure of siv gag-specific cd t cell activity as rejection of this tumour has previously been shown to be cd t cell dependent [ ] . protection from this model also correlated well with the magnitude of ex vivo elispot (effector memory) responses rather than cultured elispot (central memory) responses induced by each of the kunjin siv vaccines. this might be expected since tumours are generally poor at stimulating the differentiation of central memory cells into effectors, so prophylactic protection would rely on effector memory cells [ ] . the relative importance of effector memory and/or central memory cells for prophylactic protection against hiv- /siv has not been extensively studied. some evidence suggests central memory cells are more important than effector memory cells [ ] , whereas others have argued that effector activity early in infection (when effector mem- groups of mice (n = - ) were immunised twice with the indicated vlp vaccines and were then challenged ≈ months later with a -egfp-sivgag cells. animals were euthanased when tumours reached mm . (c) mean growth curves for the tumours from the same experiment. when an animal was euthansed a value of mm was included for that animal in the mean for all subsequent time points. ory t cells would be most active) is important for protection [ ] and for slowing progression to aids [ ] . in summary we describe here a kunjin replicon siv gag-pol vlp vaccine, which showed high insert stability, good induction of effector and central memory responses, and good protection against a model challenge. other siv gag vaccines failed in one or more of these criteria, illustrating that antigen construction and the insert's nucleotide sequences can have unforeseen consequences for the performance of these rna replicon vectors. through the aids research and reference reagent program, division of aids, niaid, nih: p spsp from dr ronald desrosiers; p - from dr stephen dewhurst; monoclonal antibody to hiv p (ag . ) from dr. jonathan allan; and sivmac p monoclonal antibody ( - f ) from dr. niels 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cells during acute simian immunodeficiency virus challenge reduction of viral loads by multigenic dna priming and adenovirus boosting in the sivmac-macaque model gag-specific cd + t lymphocytes recognize infected cells before aids-virus integration and viral protein expression long-term control of simian immunodeficiency virus replication with central memory cd + t-cell preservation after nonsterile protection by a cytotoxic t-lymphocyte-based vaccine inactivation of the human immunodeficiency virus type inhibitory elements allows revindependent expression of gag and gag/protease and particle formation potentiation of simian immunodeficiency virus (siv)-specific cd (+) and cd (+) t cell responses by a dna-siv and nyvac-siv prime/boost regimen boosting of siv-specific immune responses in rhesus macaques by repeated administration of ad hr-sivenv/rev and ad hr-sivgag recombinants psf acts through the human immunodeficiency virus type mrna instability elements to regulate virus expression 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secretion during the acute phase of west nile virus infection over-production of proteins in escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels cytotoxic t cell polyepitope vaccines delivered by iscoms peptide based cytotoxic t-cell vaccines; delivery of multiple epitopes, help, memory and problems a consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (siv) gag/pol dna and recombinant vaccinia virus strain dis elicits effective anti-siv immunity immunogenicity of recombinant adenovirus serotype vaccine in the presence of pre-existing anti-ad immunity induction of multifunctional human immunodeficiency virus type (hiv- )-specific t cells capable of proliferation in healthy subjects by using a primeboost regimen of dna-and modified vaccinia virus ankara-vectored vaccines expressing hiv- gag coupled to cd + t-cell epitopes a cd (+) t-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural plasmodium falciparum infection and disease evolution of epitope-specific memory cd (+) t cells after clearance of hepatitis c virus analysis of murine cd (+) t-cell clones specific for the dengue virus ns protein: flavivirus cross-reactivity and influence of infecting serotype from mice to humans-murine intelligence for human cd + t cell vaccine design immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic t lymphocytes evaluation of replicative capacity and genetic stability of west nile virus replicons using highly efficient packaging cell lines fowlpox virus vaccines for hiv and shiv clinical and pre-clinical trials detection of simian immunodeficiency virus (siv)-specific cd + t cells in macaques protected from siv challenge by prior siv subunit vaccination genetic instability of vaccinia virus containing artificially duplicated genome regions genetic instability of a momlv-based antisense double-copy retroviral vector designed for hiv- gene therapy novel design architecture for genetic stability of recombinant poliovirus: the manipulation of g/c contents and their distribution patterns increases the genetic stability of inserts in a poliovirus-based rps-vax vector system genetically stable picornavirus expression vectors with recombinant internal ribosomal entry sites genesis of sindbis virus by in vivo recombination of nonreplicative rna precursors the spliceosome: caught in a web of shifting interactions retroviral mrna nuclear export elements regulate protein function and virion assembly signals required for programming effector and memory development by cd + t cells therapeutic vaccination against murine lymphoma by intratumoral injection of recombinant fowlpox virus encoding cd ligand is cancer dangerous to the immune system? vaccine-induced cd + central memory t cells in protection from simian aids effector cytotoxic t lymphocyte numbers induced by vaccination should exceed levels in chronic infection for protection from hiv hiv-specific cd + t cells and viremia: who's in control? adenovirus-transduced dendritic cells injected into skin or lymph node prime potent simian immunodeficiency virus-specific t cell immunity in monkeys rev proteins of human and simian immunodeficiency virus enhance rna encapsidation macaque bloodderived antigen-presenting cells elicit siv-specific immune responses functional replacement of the r region of simian immunodeficiency virus-based vectors by heterologous elements visualization of the intracellular behavior of hiv in living cells hiv- and ebola virus encode small peptide motifs that recruit tsg to sites of particle assembly to facilitate egress effects of signal peptide exchange on hiv- glycoprotein expression and viral infectivity in mammalian cells sequences regulating tropism of human immunodeficiency virus type for brain capillary endothelial cells map to a unique region on the viral genome we would like to thank drs. barbara k. felber and george n. pavlakis (human retrovirus pathogenesis section, national cancer institute-frederick, frederick, md, usa) for providing pcmv sivgagdx plasmid. we would also like to thank dr. felber for critical review of the manuscript. the following reagent was obtained key: cord- -xu mvc authors: avirutnan, panisadee; mehlhop, erin; diamond, michael s. title: complement and its role in protection and pathogenesis of flavivirus infections date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xu mvc the complement system is a family of serum and cell surface proteins that recognize pathogen-associated molecular patterns, altered-self ligands, and immune complexes. activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. in this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by flaviviruses, with an emphasis on experiments with west nile and dengue viruses. the complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands [ ] . complement is activated through three distinct pathways referred to as the classical, lectin, and alternative pathways depending on specific recognition molecules [ , ] . classical pathway activity is triggered by c q binding to antigen-antibody complexes on the surface of pathogens or by spontaneous tickover [ ] . the lectin pathway is initiated by mannan binding lectin (mbl) or ficolin recognition of carbohydrate structures on the surface of microbes or apoptotic cells. the alternative pathway is constitutively active at low levels through the spontaneous hydrolysis of c and also serves to amplify activation of the classical and lectin pathways. despite the distinct triggering mechanisms, the classical, lectin, and alternative pathways generate convertase enzymes (c bc a for classical and lectin, and c bbb for the alternative) which cleave c , the central component of the complement system, and expose a reactive internal thioester bond on c b necessary for covalent attachment to target surfaces. the binding of c b back to c b a and c bbb c convertases forms the classical and alternative pathway c convertases, respectively. these enzymes cleave c and promote assembly of c b- membrane attack complex (mac), which lyses pathogens or infected cells. sub-lytic amounts of c b- on a cell surface can activate granulocytes and endothelial cells, whereas soluble c b- independently induces inflammation through cytokine induction [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the release of anaphylatoxins (c a and c a) by the c and c convertases also contributes to the host inflammatory response by promoting chemotaxis of immune cells via the interaction with specific g-protein coupled transmembrane receptors (c ar and c ar) [ ] . deposition of opsonic c and c fragments (c b and c b) on a pathogen facilitates binding and phagocytosis by complement receptors (cr , cr , cr , and crig), a process called opsonization, which helps to clear microbial infections [ , ] . to limit inappropriate activation and potential tissue damage, the complement system is controlled by a group of cell surface and soluble regulators [ ] . negative regulation of complement activation is achieved by several independent mechanisms: (a) proteolytic cleavage of c b and c b by the plasma serine protease factor i in conjunction with one of the membrane or plasma cofactors (membrane cofactor protein (mcp or cd ), complement receptor (cr or cd ), factor h, and c binding protein (c bp) [ ] [ ] [ ] [ ] ; (b) dissociation of the c and c convertases, a process known as decay accelerating activity, which involves decay accelerating factor (daf or cd ), cr , c bp and factor h [ ] [ ] [ ] [ ] [ ] ; (c) mac formation is inhibited by the membrane regulator cd (protectin) [ , ] , the soluble regulator apolipoprotein clusterin (apo-j) [ ] [ ] [ ] [ ] [ ] , and vitronectin [ , ] ; (d) specific protease inhibitors (e.g., serpins and c inhibitor) limit cleavage of c and c by dissociating the classical (c r-c s) and lectin (mbl-associated serine protease (masp- )) pathway serine proteases [ ] . beyond its roles in direct recognition and clearance of microbes, complement activation is critical for generating an efficient adaptive immune response. ligation of complement receptors enhances humoral immune responses [ , ] . binding of the complement split products c d, c dg, or ic b [ ] by cr (cd ) lowers the threshold for b cell activation by cross-linking the b cell receptor with the cd /cd /cr co-receptor complex [ ] . indeed, conjugation of c d to viral glycoproteins increases their immunogenicity up to , fold [ ] [ ] [ ] [ ] , and c −/− or cr −/− mice have impaired humoral responses to t cell-dependent (td) antigens [ ] [ ] [ ] [ ] . additionally, expression of cr on follicular dendritic cells (dc) is required for b cell survival within the germinal center, affinity maturation, and the establishment of b cell memory [ ] [ ] [ ] . in addition, cr (cd ), a type i integral membrane protein that binds c b, c b, and c q, and mbl, also plays a role in establishment of b cell responses [ ] [ ] [ ] . this glycoprotein is expressed on all peripheral blood cells in humans with the exception of platelets, natural killer cells and most t cells [ , ] . in primates, cr expression on erythrocytes contributes to immune complex clearance and transfer of c b-opsonized antigens to splenic and hepatic macrophages [ , ] . in mice, cr is expressed as an alternative splice product of the cr gene and is restricted to b cells and follicular dendritic cells [ ] [ ] [ ] . profound defects in humoral immunity have been observed in cr /cr −/− mice [ , , , ] , with little effect on t cell activity [ , ] . cr /cr -mediated antigen trapping on follicular dendritic cells enhances antigen presentation to b cells, and is required for both primary and secondary humoral responses [ , ] . complement and its receptors can also augment t cell activation. cr and cr can mediate phagocytosis of ic b-opsonized antigens on antigen presenting cells, and thus, may augment antigen presentation. in the absence of complement c , t cell responsiveness to influenza virus, lymphocytic choriomeningitis virus (lcmv), leishmania, and alloantigens are reduced [ , , , ] . correspondingly, c b opsonization augments protein antigen uptake [ , ] and t cell stimulation [ , , ] . covalent c b modification can target antigen to specific mhc class ii containing vesicles [ ] and may increase lysosomal peptide-mhc stability [ ] , and the diversity of t cell epitopes presented [ ] . additionally, a deficiency of c q can lead to suboptimal antigen uptake, impaired dc differentiation and maturation, and reduced t cell responses [ , [ ] [ ] [ ] [ ] [ ] [ ] . dc present exogenous antigen in a mhc class i-restricted manner, leading to the activation of naïve cd + t cells through crosspresentation [ ] . dc uptake of complement containing immune complexes (ic) enhances the efficiency of protein antigen crosspresentation compared to free antigens [ , , ] . however, c q may not be necessary to stimulate t cell priming against pathogenderived antigens [ , ] . to minimize recognition and/or destruction by complement several different families of viruses have evolved strategies to evade or exploit complement to establish infection (reviewed in [ ] [ ] [ ] [ ] [ ] ). complement evasion mechanisms include: (a) use of complement receptors to enhance viral entry or suppress adaptive immune response (e.g., hiv, west nile virus (wnv), measles virus, adenoviruses, herpesviruses, enteroviruses, hepatitis b and c viruses ); (b) expression of viral proteins that directly inhibit complement (e.g., herpesviruses, coronaviruses, and astroviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ); (c) modulation of expression of complement regulators on host cells to prevent complement-dependent lysis (e.g., herpesviruses [ ] [ ] [ ] ); (d) incorporation of human regulators on the surface of virions to protect from complement-mediated virolysis (e.g. hiv, htlv, cytomegalovirus, and vaccinia virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] ); (e) recruitment of soluble complement regulatory proteins to the virion or infected cell surface (e.g., wnv and hiv [ ] [ ] [ ] [ ] [ ] ); (f) expression of viral decoy proteins that structurally or functionally mimic complement regulatory proteins (e.g., poxviruses and herpesviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a single virus may utilize several independent strategies to escape from recognition and targeting by complement and modulate the immune response to establish persistent infection. although complement activation inhibits infection of many viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it appears to have both protective and pathogenic roles in flavivirus infection depending on the specific virus, phase of the infection, and immune status of the host. the genus flavivirus is composed of enveloped viruses containing ∼ kilobase single-stranded, positive-polarity rna genomes [ ] . within this family, several are associated with severe human diseases including dengue (denv), yellow fever (yfv), wnv, japanese encephalitis (jev), and tick-borne encephalitis (tbe) viruses [ ] . a single open reading frame is translated in the cytoplasm as a polyprotein and cleaved by virus-and host encoded-proteases into three structural (capsid (c), membrane (prm/m), and envelope (e)) and seven nonstructural (ns) proteins including ns , ns a, ns b, ns , ns a, ns b and ns [ ] . the e protein functions in receptor binding, entry, and membrane fusion and elicits the majority of neutralizing antibodies whereas prm assists in folding, assembly, and function of the e protein [ ] . viral particles assemble at the endoplasmic reticulum and are released by exocytosis following transport through the trans-golgi network [ ] . flavivirus non-structural proteins regulate viral transcription, replication, and attenuation of host antiviral immune response, including antagonizing the interferon response (reviewed in [ ] ). one non-structural proteins, ns , has been recently shown to regulate complement function (see below). ns is synthesized as a monomer and dimerizes after post-translational modification [ , ] . within the cytoplasm, ns acts as a co-factor for the ns rna-dependent rna polymerase during viral replication [ , ] . however, it is also expressed on the cell surface and secreted as a hexamer [ , ] . ns has been implicated in the pathogenesis of severe denv infection [ ] [ ] [ ] and immune evasion by wnv [ , ] . complement can limit flavivirus infection by stimulating adaptive immune responses. c −/− mice are more susceptible to lethal wnv infection and show greater viral burden and reduced antiviral antibody titers [ ] . infection studies with mice lacking c q, c , or factor b suggest that all complement activation pathways orchestrate protection against wnv infection [ ] . however, each activation pathway appears to exert somewhat distinct protective effects in response to wnv infection. humoral igm responses to wnv likely depend upon activation of c by the lectin recognition pathway. in contrast, both the lectin and alternative pathways appear necessary for efficient t cell priming as c −/− , factor b −/− , and factor d −/− mice exhibited reduced wnv-specific cd + t cell responses [ ] . the t cell defects in c −/− mice may be indirect as depressed igm responses could affect viral opsonization and antigen presentation. flaviviruses also directly trigger complement activation in vitro and in vivo. increasing concentrations of complement or serum neutralize as much as % of a given infectious dose of wnv in cell culture in the absence of antibody [ ] . complement activation by flaviviruses also has been described in vivo. c and c consumption were observed in a mouse model of wnv infection prior to the induction of a specific antibody response [ ] . c catabolism and production of complement split products during secondary denv infection correlate with increased disease severity and development of dengue hemorrhagic fever and shock syndrome, the most severe form of denv infection [ , [ ] [ ] [ ] . complement activation augments antibody-mediated neutralization of several viruses, including influenza [ , ] , hiv [ ] [ ] [ ] [ ] , respiratory syncytial [ , ] , varicella zoster [ ] [ ] [ ] , epstein-barr [ , ] , and herpes simplex viruses [ ] [ ] [ ] . complement also improves antibody-mediated neutralization of flaviviruses. complement augments immune serummediated neutralization of yfv, denv, and kunjin virus [ ] [ ] [ ] and monoclonal antibody-dependent neutralization of wnv [ ] . similarly, the protective efficacy of flavivirus neutralizing antibodies in vivo correlates with igg subclasses that efficiently fix complement [ ] . fc-␥r engagement by antibodies in vitro can paradoxically enhance replication of flaviviruses [ ] [ ] [ ] [ ] [ ] [ ] . this phenomenon, known as antibody-dependent enhancement of infection (ade), is hypothesized to contribute to the pathogenesis of secondary denv infection [ , ] . recent studies indicate that complement can restrict ade. complement minimized ade of wnv and denv infection in fc-␥r-expressing cell lines and primary macrophages [ , ] . experiments with mouse sera deficient in individual complement components indicate that c q is sufficient to restrict ade of wnv infection in vitro. this effect was igg subclassdependent, as c q restricted ade by a human igg isotype-switch variant, but had little effect on igg and igg subclass variants [ ] ; these results correlate with the known affinity of human igg subclasses for c q [ , ] . interestingly, complement-dependent inhibition of denv ade may also require c [ ] . while these studies establish that complement restricts ade by flaviviruses, the precise inhibitory mechanisms at the cellular level remain unclear. recent studies suggest that c q also limits flavivirus ade in vivo. whereas enhancement of wnv infection was not observed after passive transfer of antiviral igg a mabs that bind c q avidly in wild type mice, it was observed in c q −/− mice [ ] . the ability of c q to suppress ade may explain some of the difficulties in consistently observing flavivirus ade in animal models. further investigation is necessary to define the links between complement restriction of ade, fc-␥r specificity, and disease pathogenesis of flaviviruses. in cells that express cr , antibody-dependent complement activation may paradoxically enhance viral infection. complement activation by antiviral igm enhanced wnv infection of macrophages and monocyte cell lines [ , ] . blockade of cr abrogated the complement-dependent enhancement of wnv infection in this model system. thus, under certain circumstances, antibody and complement-dependent opsonization of flaviviruses may increase infection in cr -expressing cells. during severe secondary denv infection, a vascular leakage syndrome occurs with fluid transudation into serosal spaces [ ] . although the pathogenesis of denv infection remains controver-sial and implicates cross-reactive antibodies and effector t cells (reviewed in [ ] [ ] [ ] ), a pathological role for complement activation has been suggested. in early clinical studies, reduced levels of c , c and factor b and increased catabolic rates of c and c q were observed, particularly in patients with severe disease [ , ] . additionally, c breakdown products and anaphylatoxins accumulated in the circulation of severely ill patients and peaked at the day of maximum vascular leakage [ , ] . circulating immune complexes formed by virions and denv-specific antibodies were hypothesized to cause the pathological complement activation [ ] , although only small amounts were detected in circulation [ , ] . one alternative hypothesis is that infected cells express sufficient amounts of denv antigens (e or ns proteins) on their surface facilitating immune complex formation and complement deposition [ ] . indeed, denv-infected endothelial cells activate human complement in the presence of antibodies resulting in c b- deposition [ ] . a subsequent study implicated ns as the key surface viral protein responsible for complement activation [ ] . as soluble denv ns differentially binds to cultured endothelial and mesothelial cells [ ] , high levels of intravascular soluble ns , as observed in denv-infected patients, could promote binding and surface expression of ns on selective cells without a requirement for direct viral infection; this could contribute to tissue-specific vascular leakage that occurs during severe secondary denv infection after recognition by anti-ns antibodies, immune complex formation, and inflammatory damage [ , ] . recent evidence suggests wnv ns has immune evasion function and protects against complement activation by binding the negative regulator factor h [ ] . factor h sustains factor imediated cleavage of c b and inactivates the alternative pathway c convertase (reviewed in [ ] ). co-immunoprecipitation experiments demonstrate that soluble wnv ns binds to factor h, leading to degradation of c b in solution [ ] . additionally, cell surface ns limits c b deposition and c b- mac formation [ ] . thus, secreted or cell surface ns may minimize immune system targeting of wnv by decreasing complement activation in solution and on the surface of infected cells. this data appears to contradict early studies that suggested denv ns might be the key viral protein that triggers complement activation [ , ] . in those studies, ns was termed "non-hemagglutinating soluble complement fixing antigens (scf)" because it has activity in the traditional standard complement fixing test that requires specific antibodies to trigger guinea pig complement [ , ] . subsequent experiments indicate that denv ns does not activate complement efficiently, but instead requires specific anti-ns antibodies for complement consumption and c b- generation ( [ ] and avirutnan et al., unpublished results). additionally, denv ns has been reported to bind to clusterin, a complement regulator that inhibits mac formation [ ] . clearly, more studies are necessary to establish the significance of these findings in the pathogenesis of infection of denv, wnv, and other flaviviruses in vivo. activation of the complement system has a critical role in protection and possibly pathogenesis of infection by different flaviviruses. complement activation primes adaptive immune responses and modulates the effector functions of flavivirus-specific antibodies. recent studies suggest that flaviviruses have evolved novel strategies to limit complement activation. the balance between complement activation and evasion likely helps determine the out-come of a productive infection. a greater understanding of how complement restricts and contributes to pathogenesis of individual flaviviruses may expand strategies for developing therapeutics or vaccines to control infection. complement. first of two parts second of two parts continual low-level activation of the classical complement pathway platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex intracerebroventricular injection of the terminal complement complex causes inflammatory reaction in the rat brain cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo soluble complex of complement increases hydraulic conductivity in single microvessels of rat lung transient perturbation of endothelial integrity induced by natural antibodies and complement the cytolytically inactive 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structure-function analysis of the active sites of complement receptor type decay accelerating activity of complement receptor type (cd ). two active sites are required for dissociating c convertases human c -binding protein. i. isolation and characterization control of the amplification convertase of complement by the plasma protein beta h membrane defence against complement lysis: the structure and biological properties of cd human protectin (cd ), an , - , mw complement lysis restricting factor, inhibits c b- catalysed insertion of c into lipid bilayers incorporation of sp- , into the soluble membrane attack complex (smac, sc b- ) of complement molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein , a constituent of rat testis fluid molecular cloning and characterization of the novel, human complement-associated protein, sp- , : a link between the complement and reproductive systems potent inhibition of terminal complement assembly by clusterin: characterization of its impact on c polymerization clusterin, the human apolipoprotein and complement inhibitor, binds to complement c , c beta, and the b domain of c complement s-protein (vitronectin) is associated with cytolytic membrane-bound c b- complexes vitronectinmediated inhibition of complement: evidence for different binding sites for c b- and c structural and functional aspects of c -inhibitor the role of complement and complement receptors in induction and regulation of immunity regulation of b lymphocyte responses to foreign and self-antigens by the cd /cd complex identification of a , mr membrane protein as the c d receptor (cr ) of human b lymphocytes intersection of the complement and immune systems: a signal transduction complex of the b lymphocyte-containing complement receptor type and cd c d of complement as a molecular adjuvant: bridging innate and acquired immunity enhancement of antibodies to the human immunodeficiency virus type envelope by using the molecular adjuvant c d c d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge fusion to c d enhances the immunogenicity of the e glycoprotein of type bovine viral diarrhea virus disruption of the cr locus results in a reduction in b- a cells and in an impaired b cell response to t-dependent antigen antibody response to a t-dependent antigen requires b cell expression of complement receptors regulation of the b cell response to t-dependent antigens by classical pathway complement markedly impaired humoral immune response in mice deficient in complement receptors and b lymphocyte memory: role of stromal cell complement and fcgammariib receptors dependence of germinal center b cells on expression of cd /cd for survival impaired affinity maturation in cr −/− mice is rescued by adjuvants without improvement in germinal center development identification of the membrane glycoprotein that is the c b receptor of the human erythrocyte, polymorphonuclear leukocyte, b lymphocyte, and monocyte complement receptor /cd is a receptor for mannan-binding lectin complement receptor type (cr cd ) is a receptor for c q expression of c b receptors on human be cells and myelomonocytic cells but not natural killer cells both kupffer cells and liver endothelial cells play an important role in the clearance of iga and igg immune complexes clearance of antidouble-stranded dna antibodies: the natural immune complex clearance mechanism their use in a distribution study showing that mouse erythrocytes and platelets are cr -negative the murine complement receptor gene family. iv. alternative splicing of cr gene transcripts predicts two distinct gene products that share homologous domains with both human cr and cr a molecular and immunochemical characterization of mouse cr . evidence for a single gene model of mouse complement receptors and humoral immune responses in cr −/− mice: enhanced affinity maturation but impaired antibody persistence complement component c promotes t-cell priming and lung migration to control acute influenza virus infection complement component is required for optimal expansion of cd t cells during a systemic viral infection expression of complement receptors and on follicular dendritic cells is necessary for the generation of a strong antigen-specific igg response evidence for an important interaction between a complement-derived cd ligand on follicular dendritic cells and cd on b cells in the initiation of igg responses local synthesis of complement component c regulates acute renal transplant rejection natural antibodies and complement are endogenous adjuvants for vaccine-induced cd + t-cell responses c b covalently associated to tetanus toxin modulates tt processing and presentation by u cells covalent binding of c b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific b cells antigen-bound c b and c b enhance antigenpresenting cell function in activation of human t-cell clones modulation of antigen processing and presentation by covalently linked complement c b fragment b cell receptors and complement receptors target the antigen to distinct intracellular compartments complement c b fragment covalently linked to tetanus toxin increases lysosomal sodium dodecyl sulfate-stable hla-dr dimer production c b complexation diversifies naturally processed t cell epitopes maturation of dendritic cells abrogates c q production in vivo and in vitro complement protein c q induces maturation of human dendritic cells immune modulation of human dendritic cells by complement t cell-dependent immune response in c q-deficient mice: defective interferon gamma production by antigen-specific t cells immune complex processing in c q-deficient mice a novel role of complement factor c q in augmenting the presentation of antigen captured in immune complexes to cd + t lymphocytes cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens antigen-antibody immune complexes empower dendritic cells to efficiently prime specific cd + ctl responses in vivo immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine protective immune responses against west nile virus are primed by distinct complement activation pathways complement contributes to protective immunity against reinfection by plasmodium chabaudi chabaudi parasites hiv and human complement: inefficient virolysis and effective adherence viral mimicry of the complement system role of complement in immune regulation and its exploitation by virus virus complement evasion strategies complement evasion by human pathogens cutting edge: productive hiv- infection of dendritic cells via complement receptor type (cr , cd b/cd ) complement dependent trapping of infectious hiv in human lymphoid tissues decay-accelerating factor (cd ), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses opsonization of hiv- by semen complement enhances infection of human epithelial cells interaction of west nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement complement receptor mediates enhanced flavivirus replication in macrophages immune tolerance split between hepatitis b virus precore and core proteins a function of the hepatitis b virus precore protein is to regulate the immune response to the core antigen virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions cr (cd ) and cr (cd ) complement c receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus b cellmediated infection of stimulated and unstimulated autologous t lymphocytes with hiv- : role of complement mechanism(s) promoting hiv- infection of primary unstimulated t lymphocytes in autologous b cell/t cell co-cultures the human cd molecule is a receptor for measles virus (edmonston strain) hepatitis c virus core selectively suppresses interleukin- synthesis in human macrophages by interfering with ap- activation epstein-barr virus receptor of human b lymphocytes is the c d receptor cr cd is a cellular receptor for group b adenoviruses b lymphocytes in lymph nodes and peripheral blood are important for binding immune complexes containing hiv- detachment of human immunodeficiency virus type from germinal centers by blocking complement receptor type c a and c a(desarg) enhance the susceptibility of monocyte-derived macrophages to hiv infection mechanism of suppression of cell-mediated immunity by measles virus interaction between complement receptor gc qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation functional modulation of human macrophages through cd (measles virus receptor): production of il- p and nitric oxide in association with recruitment of protein-tyrosine phosphatase shp- to cd in vitro and in vivo interactions between the hepatitis b virus protein p and the cellular protein gc qr hepatitis c virus core protein inhibits interleukin and nitric oxide production from activated macrophages inhibition of interferon-mediated antiviral activity by murine gammaherpesvirus latency-associated m protein cd is a cellular receptor for all species b adenoviruses except types and binding of human immunodeficiency virus type to the c b/c b receptor cr (cd ) and red blood cells in the presence of envelope-specific antibodies and complement. national institutes of health aids vaccine clinical trials networks ligation of cr (c b receptor, cd ) on cd + t lymphocytes enhances viral replication in hiv-infected cells identification of gp as the viral glycoprotein mediating attachment of epstein-barr virus (ebv) to the ebv/c d receptor of b cells: sequence homology of gp and c complement fragment c d complement-mediated enhancement of hiv- infection in peripheral blood mononuclear cells identification of a cellular protein specifically interacting with the precursor of the hepatitis b e antigen cd is a cellular receptor for human herpesvirus adenovirus type uses cd as a cellular receptor selective suppression of il- production by human herpesvirus in vitro analysis of complement-dependent hiv- cell infection using a model system epstein-barr virus gp / binding to the b lymphocyte c d receptor mediates adsorption, capping, and endocytosis cr (cd ) and cr (cd b/cd ) mediate infection of human monocytes and monocytic cell lines with complementopsonized hiv independently of cd triggering of complement receptors cr (cd ) and cr (cd b/cd ) induces nuclear translocation of nf-kappa b (p /p ) in human monocytes and enhances viral replication in hiv-infected monocytic cells hcv core protein interaction with gc q receptor inhibits th differentiation of cd + t cells via suppression of dendritic cell il- production human astrovirus coat protein inhibits serum complement activation via c , the first component of the classical pathway antibody-induced and cytoskeleton-mediated redistribution and shedding of viral glycoproteins, expressed on pseudorabies virus-infected cells identification of a novel herpes simplex virus type -induced glycoprotein which complexes with ge and binds immunoglobulin herpes simplex virus immunoglobulin g fc receptor activity depends on a complex of two viral glycoproteins, ge and gi mechanism of complement inactivation by glycoprotein c of herpes simplex virus herpesviral fc receptors and their relationship to the human fc receptors herpes simplex virus type evades the effects of antibody and complement in vivo in vivo immune evasion mediated by the herpes simplex virus type immunoglobulin g fc receptor molecular mimicry between s peplomer proteins of coronaviruses (mhv, bcv, tgev and ibv) and fc receptor identification and expression of a murine cytomegalovirus early gene coding for an fc receptor mechanism of host cell protection from complement in murine cytomegalovirus (cmv) infection: identification of a cmv-responsive element in the cd promoter region altered expression of hostencoded complement regulators on human cytomegalovirus-infected cells human herpesvirus infection increases the expression levels of cd and cd in target cells acquisition of host cell-surface-derived molecules by hiv- decay-accelerating factor (cd ) protects human immunodeficiency virus type from inactivation by human complement antibodydependent complement-mediated cytotoxicity in sera from patients with hiv- infection is controlled by cd and cd mechanisms of resistance of hiv- primary isolates to complement-mediated lysis extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope human immunodeficiency virus type incorporates both glycosyl phosphatidylinositol-anchored cd and cd and integral membrane cd at levels that protect from complement-mediated destruction host cell-derived complement control proteins cd and cd are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) west nile virus nonstructural protein ns inhibits complement activation by binding the regulatory protein factor h direct interaction of complement factor h with the c domain of hiv type glycoprotein hiv glycoprotein and complement factor h interact with each other and share functional as well as antigenic homology human complement proteins c b, c b, factor h and properdin react with specific sites in gp and gp , the envelope proteins of hiv- efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor h and decay accelerating factor (daf, cd ) herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein cd the complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting c convertase activity structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation regulation of complement activity by vaccinia virus complement-control protein the cowpox virus-encoded homolog of the vaccinia virus complement control protein is an inflammation modulatory protein variola virus immune evasion design: expression of a highly efficient inhibitor of human complement functional characterization of the complement control protein homolog of herpesvirus saimiri: arg- is critical for factor i cofactor activities complement regulation by kaposi's sarcoma-associated herpesvirus orf protein the relevance of complement to virus biology the effect of complement depletion on the course of sindbis virus infection in mice role of complement in viral infections: participation of terminal complement components (c to c ) in recovery of mice from sindbis virus infection the role of complement in viral infections. ii. the clearance of sindbis virus from the bloodstream and central nervous system of mice depleted of complement natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity enhancement of neutralizing activity of influenza virus-specific antibodies by serum components the role of the complement system in virus infections fields' virology flaviviridae: the viruses and their replication newly synthesized dengue- virus nonstructural protein ns is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization evidence that the mature form of the flavivirus nonstructural protein ns is a dimer genetic interaction of flavivirus nonstructural proteins ns and ns a as a determinant of replicase function immunolocalization of the dengue virus nonstructural glycoprotein ns suggests a role in viral rna replication dengue virus type nonstructural glycoprotein ns is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns and complement secreted ns of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate e high circulating levels of the dengue virus nonstructural protein ns early in dengue illness correlate with the development of dengue hemorrhagic fever west nile virus nonstructural protein inhibits tlr signal transduction complement activation is required for induction of a protective antibody response against west nile virus infection pathogenetic mechanisms in dengue haemorrhagic fever: report of an international collaborative study the potential pathogenic role of complement in dengue hemorrhagic shock syndrome complement and dengue haemorrhagic fever/shock syndrome neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type infection functional activity of an hiv- neutralizing igg human monoclonal antibody: adcc and complement-mediated lysis complement activation by human monoclonal antibodies to human immunodeficiency virus broad neutralization and complement-mediated lysis of hiv- by pehrg , a novel caprine anti-hiv- polyclonal antibody effect of complement and viral filtration on the neutralization of respiratory syncytial virus role of complement in neutralization of respiratory syncytial virus neutralization of vesicular stomatitis virus (vsv) by human complement requires a natural igm antibody present in human serum complement-enhanced neutralizing antibody response to varicella-zoster virus neutralizing antibody 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protect mice against lethal yf encephalitis flavivirus infection enhancement in macrophages: radioactive and biological studies on the effect of antibody on viral fate flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever monoclonal anti-fc receptor igg blocks antibody enhancement of viral replication in macrophages antibody-mediated enhancement of flavivirus replication in macrophage-like cell lines pathogenesis of dengue: challenges to molecular biology complement protein c q inhibits antibody-dependent enhancement of flavivirus infection in an igg subclass-specific manner infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type and viruses are controlled by complement levels human monoclonal igg isotypes differ in complement activating function at the level of c as well as c q the classical complement pathway: activation and regulation of the first complement component clinical spectrum and management of dengue haemorrhagic fever immunopathological mechanisms in dengue and dengue hemorrhagic fever dengue hemorrhagic fever with special emphasis on immunopathogenesis of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) crossed immunoelectrophoresis for the detection of split products of the third complement in dengue hemorrhagic fever. i. observations in patients' plasma the raji cell radioimmune assay for detecting immune complexes in human sera pathogenesis of dengue: an alternative hypothesis dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis factor h family proteins: on complement, microbes and human diseases partial purification and characterization of a dengue virus soluble complement-fixing antigen molecular size and charge relationships of the soluble complement-fixing antigens of dengue viruses secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein key: cord- -d bxe c authors: yuan, xiaomin; lin, huixing; fan, hongjie title: efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: d bxe c to explore the possibility of developing a vaccine against transmissible gastroenteritis virus (tgev) infection, a recombinant swinepox virus (rspv-sa) expressing a tgev protective antigen has been constructed. immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models. an indirect elisa assay suggested that when mice were vaccinated with rspv-sa, the level of igg against tgev was enhanced dramatically. the cytokine assays were employed and the results indicated that both the th -type and th -type cytokine levels raised after vaccination with rspv-sa in mice models. results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rspv-sa, could % protect piglets from the spv infection, and there was no significant clinical symptom in the rspv-sa treatment group during this experiment. the data suggest that the novel recombinant swinepox virus is a potential vaccine against tgev infection. transmissible gastroenteritis virus (tgev) is a member of coronaviridae, which is the etiological agent of transmissible gastroenteritis. although the virus is capable of infecting swine of all ages, suckling piglets are the most susceptible and have a mortality rate up to % [ ] . tgev is a pleomorphic enveloped virus containing a positive-stranded rna genome and four structural proteins: the spike (s) protein, the integral membrane (m) protein, the minor envelope (e) protein, and the nucleocapsid (n) protein. among which, the spike (s) protein, one of the key structural membrane proteins of coronaviruses, is an attractive target for generating neutralizing antibodies against the virus due to the critical role it plays in the host cell invasion [ , ] . precisely, the s protein mediates the attachment of virus particles to targets via binding of itself to the specific receptors. at the n terminus of the s protein, there are four antigenic sites, a, b, c, and d, which have been shown to be involved in the stimulation of neutralizing antibodies (fig. ) [ ] . previous studies have determined that the a site (which is fully dependent on glycosylation for proper folding) is predominantly responsible for stimulating neutralizing host antibodies [ ] [ ] [ ] [ ] [ ] [ ] . spv is a natural mild attenuated virus and has been widely applied as a vaccine. given that poxvirus-vectors can prevent a great deal of important diseases in both humans and animals it is not surprising that many of these vectors been licensed and used extensively [ ] [ ] [ ] . additionally, spv is a safe vaccine vector as there is no risk of cross-species infection [ ] . therefore, both for biological and clinical practicality, spv is regarded as an appropriate and promising veterinary vaccine for swine, owing to its ability to effectively express foreign genes, its large packaging capacity for recombinant dna, its low cost of delivery and its specific host restriction [ ] . the potential value of spv as a live vector vaccine is being studied extensively. because spv is able to pack large amounts of recombinant dna and to induce appropriate immune responses in vivo, it is a promising candidate for the development of a recombinant vaccine [ , ] . as of yet, pigs are the only known hosts of swinepox virus and therefore may be useful in developing a safe vaccine for clinical application [ , ] . in this study, we constructed a recombinant swinepox virus expressing s-a (a epitope of the s protein) of tgev and characterized recombinant virus replication and expression of the s protein in pk- cells. we further investigated the potential of this approach for use in the vaccination of pigs against tge. type culture collection. swine transmissible gastroenteritis virus (tgev, china strain, shxb) was purchased from the jiangsu academy of agricultural sciences, and the titer was determined as × pfu/ml st cell. tgev convalescent positive serum was purchased from the jiangsu academy of agricultural sciences, and the neutralizing antibodies were used at a dilution of : , . the pusz swinepox virus vector was generated previously [ ] . two primers, sa ( -gcgtcgacatgggtcttggtatga-agcgtag- ) and sa ( -cgggatcctta tagcgtcctgt-tagtttgtc- ) were used to amplify the s-a gene ( bp, km ) from the tgev genome, which was inserted into the pusz plasmid to construct pusz -sa subsequently. the recombinant swinepox virus, rspv-sa, was generated by homologous recombination of wtspv with pusz -s-a as previously described [ ] . pcr and indirect immunofluorescence were employed to analyze the s-a gene expression and the expression of s protein. the replication capacity and genetic stability of rspv-sa were also evaluated by. the generation and screening of the recombinant swinepox virus assays were performed as described previously [ ] . a subconfluent culture of pk- cells was infected with wtspv ( . moi) for h, and subsequently transfected with g of the pusz -sa plasmid using exfect tm transfection reagent (vazyme biotech co., ltd). after h, pk- cells were harvested and lysed by five rounds of freezing and thawing. subsequently, the lysate was used to infect pk- cells grown in a -well plate for further purification of recombinant viruses. . ml of medium with % lmp agarose (dingguo, beijing, china) was added to each well and incubation was continued for five days until plaques became visible under a light microscope. after - days, a second overlay medium containing x-gal was added. the plaques were resuspended in . ml of medium with % fbs. plaque isolation was repeated for - rounds until all plaques in a given well were stained blue. the recombinant spv bearing s-a of tgev was designated as rspv-sa. the rspv-sa genomic dna from the pk- cells infected with rspv-sa was extracted by sds-protease k-phenol. we utilized wtspv genomic dna from pk cells infected with wtspv as a negative control. pcr was performed for min at • c; followed by cycles of min at • c, min at • c, and min at • c. amplifications were performed with dna polymerase (promega, shanghai, china) using primers sa ( -gcgtcgacatgggtcttggtatgaagcgtag- ) and sa ( -cgggatccttatagcgtcctgttagtttgtc- ). indirect immunofluorescence assays (ifa) were performed as described previously [ ] . pk- cells were grown on a -well plate and infected with the wtspv and rspv-sa at × pfu/ml per well. pbs-treated cells were used as a negative control. at h post-infection, cells were washed three times in pbst and fixed with cold methanol for min at − • c. cells were then washed three times with pbst and blocked by pbst with % bsa. preparations were incubated for h at • c with tgev convalescent positive serum ( : in dilution buffer, pbst with % bsa). after three washes with pbst, cells were treated with the rhodamineconjugated secondary antibody (staphylococcal protein a-rhod, boshide, wuhan, china) at a : dilution (diluted in pbs) for min at • c. after a final wash with pbs, all wells were examined by fluorescence microscopy (zeiss, germany). nine six-week-old balb/c mice were randomly divided into three groups ( mice per group), and immunized three times at , , and days with rspv-sa ( × pfu/ml in . ml of pbs) or wtspv ( × pfu/ml in . ml of pbs), the control group injected with pbs. eight one-month-old swine (large white) were randomly divided into four groups ( pigs per group) and were immunized twice at and days with infectious rspv-sa ( × pfu/ml in ml of pbs), inactivated-tgev ( × pfu/ml in ml of pbs), wtspv ( × pfu/ml in ml of pbs) or pbs, each time via three routes: oral, nasal, and intraperitoneal. serum was collected days after the last immunization. twelve one-day-old pigs were randomly divided into four groups for passive immunization experiments ( pigs per group). high titers of antibodies were collected from piglets following the first immunization. mice and swine serum were incubated at • c min to complement inactivated. all experimental protocols involving mice or swine were approved by the laboratory animal monitoring committee of jiangsu province. pk- cell monolayers were infected with wtspv and rspv-sa (moi of ) and incubated for h at • c. extracts, representing approximately × cells, were electrophoresed through an sds- % polyacrylamide gel and the separated proteins were transferred onto a pvdf membrane. after a h transfer, the membrane was blocked with % skim milk in phosphate buffered saline with . % tween- (pbst) overnight at • c. the membrane was incubated with swine convalescent serum ( : dilution) containing tgev for h at • c and washed three times with pbst. immunodetection was performed with staphy-lococcal protein a-hrp at • c. following the secondary antibody probing, the membrane was washed four times with pbst. the membrane was then developed with , -diaminoben-zidine substrate until optimal color development was observed. serum was collected from mice and pigs, and detected the tgev-specific antibodies by indirect elisa. the purified tgev was resuspended in l pbs (ph . ), and used the best titer of virus for coating -well plates, which was determined by titration. samples were then incubated overnight at • c. this incubation was followed by three pbst washes, and blocking with % skim milk (in pbst) at • c for h. serum samples were serially diluted and incubated at • c for h. the samples were set up at the same time and divided into three groups: the tgev positive serum, the negative control serum (spv positive serum) and the blank control (without serum). after three pbst washes, horseradish peroxidase (hrp)-conjugated goat anti-spa igg ( : , diluted in pbst, signalway antibody) was added to each test well. the plates were then incubated at room temperature in the dark for min and then washed three times with pbst. the tmb microwell peroxidase substrate system (tiangen) was used to develop the reaction. samples were developed for min and the reaction terminated with . m sulphuric acid. all assays were performed in duplicate. a microplate reader (bio-rad) was used to measure the reaction product at an absorbance of nm [ ] . evaluation of cellular immunity was performed by detecting levels of ifn-␥ and il- . three mice were sacrificed at days after the first inoculation with wtspv ( × pfu/ml in . ml), pbs or rspv-sa ( × pfu/ml in . ml). the mouse spleen was removed aseptically. splenocytes were isolated, counted, and diluted to a density of × cells/ l. the evenly separated cells were aliquot into -well plates ( l/well). then, l/well of dmem with tcid / l tgev was added to each well. after a h incubation, the supernatants were collected and the mrna of ifn-␥ and il- were probed by rt-qpcr relative to ␤-actin as described previously [ , ] . an assay for neutralizing antibodies was performed as described previously [ , ] . to explore whether mice or swine generated tgev neutralizing antibodies, serum from the pbs, wtspv, inactivated-tgev and rspv-sa treated mice and pig were collected at , , , , days post-primary immunization ( : - : , dilution in a l volume). and sera were mixed with equal volume of tcid /ml tgev and incubated at • c. after . h incubation, we utilized sera treated viruses to infect st cells in -well plates and overlaid cells with agar at • c in a % co atmosphere. cells were monitored daily for three days to detect tgev-specific cpe. the virulent tgev strain shxb ( × pfu/ml in ml) was mixed with ml of the porcine antiserum induced by recombinants rspv-sa, inactivated tgev or pbs, incubated at • c for min, and administered using a gastric tube to -day-old swine born from tgev-seronegative sow. inoculated animals were fed three times per day with formula for newborns that contained ml of the antiserum. at three days following viral infection, small intestine tissue was collected from newborn pigs sectioned for histology. microstructure characteristics were analyzed under the microscope (olympus, cx fs , philippines) [ ] . all data were analyzed using one-way anova and values of p < . were considered significant. to analyse the recombinant virus and confirm the presence of the sa gene, two specific primers were designed to amplify the inserted sa gene. the gene encoding the neutralizing antigen epitopes of tgev is a . kb fragment and the specific fragments was detected in spv-sa as shown in fig. a . the recombinant spv-sa was also confirmed by observing blue foci in plaque assays. rspv-sa and wtspv were determined to be approximately × pfu/ml in ml for both. as shown in fig. b , the ifa demonstrated that the expression of the a epitope of the s protein was present in infected cells. western blot analysis showed a specific band of target protein with a size of kda in the cell lysates infected with rspv-sa (fig. e) . the kda molecular weight is consistent with the predicted size of the sa protein of tgev. according to these data, we suggested that the a epitope of the s protein was expressed efficiently by the rspv-sa virus (fig. ) . to monitor the s-specific antibody titers of mice vaccinated with rspv-sa, an elisa was used. after an initial boost days postvaccination, the s-a-specific antibody titers increased gradually in mice (fig. a) . the antibody titers in rspv-sa-immunized mice were higher at all-time points post-vaccination (p < . , n = ) compared to wtspv or pbs-treated mice. the p/n value of the positive group was greater than . . persistent high levels of neutralizing antibodies (fig. a) were detected in the rspv-sa group with a mean titer of : at days post-inoculation. in mice vaccinated with wtspv and rspv-sa at days postinoculation (fig. b) , the qpcr results showed a distinct variation of il- and ifn-␥ between wtspv and rspv-sa treatment groups. the relative quantity normalized by beta-actin of il- mrna in rspv-sa was . times higher compared to wtspv. the ifn-␥ mrna levels increased . fold in rspv-sa when compared to wtspv. the concentrations of il- and ifn-␥ in rspv-sa-vaccinated mice were significantly higher than the control groups. these results indicate that rspv-sa induces th -type and th -type cytokine responses during cellular immunity. during the vaccination procedure, several poxes of mmϕ could be observed around the injection position at five days post-inoculation in both rspv-sa and wtspv treatment groups, respectively, which was not observed in the inactivated-tgev and pbs treatment groups. this symptom could be disappeared spontaneously in the following days. no pigs developed further symptoms such as fever or severe inflammation, and the spiritual condition and appetite of both treatment groups was considered as good. these two group pigs recovered in days. all group pigs maintained rectal temperatures of . - . • c. from this experiment, we reveal that vaccination with rspv-sa and wtspv is well tolerated by pigs. rspv-sa induced a moderate level of tgev-specific igg as shown in fig. . persistent high levels of tgev-specific neutralizing antibodies are shown in fig. b . the second boost led to of the levels of tgev-specific neutralizing antibodies. persistent high levels of neutralizing antibodies were detected in the rspv-sa group at a mean titer of : in swine (p < . , n = ). all newborn piglets in different groups were fed with the mixture of tgev and the corresponding sera from pigs vaccinated with pbs, wtspv, inactivated-tgev, or rspv-sa. both pbs and wtspv treatment groups developed a very severe diarrhea symptom, significantly losing of weight and appetite, and the mortality rate was up to % in days. meanwhile, in the inactivated-tgev and rspv-sa treatment groups, there was no obvious clinical symptoms and % morality rate observed (fig. . and table ). after the sacrificing of all the piglets, the small intestine tissue from different groups were used for the pathological examination. histological samples from pbs and wtspv treatment groups showed prominent histopathological changes in the small intestine characterized by serious fracture of the small intestine mucosa, epithelial expansion, vacuolar degeneration, necrosis, shedding, and lamina propria congestive edema and hemorrhage. and such pathological changes were not observed during the examination of the samples from inactivated-tgev and rspv-sa treatment groups. all the results indicate that rspv-sa inoculation provides complete protection passive immunity against tgev challenge in pigs (fig. ) . in the present study, we have engineered the swinepox virus to express the a epitope of the tgev s protein. our data showed that this recombinant virus was not only able to induce a strong immune response against the a antigen of tgev in mice and pigs, but also had safety degree and protection efficacy against the virulent homologous tgev infection in pigs. the traditional way to protect swine from tgev infection is to immunize pregnant sows with inactivated or attenuated vaccine, resulting in the production of secretory iga in the colostrum. despite the protection that secretory iga offers piglets, this method does not provide protection after the cessation of breastfeeding. furthermore, inactivated or attenuated viral vaccines still maintain the ability of developing the viral toxicity [ ] . currently, there are various potential vaccines against tgev but many present certain challenges, such as difficulties in the vaccination process, high production cost, low immunogenicity, biological risk and carcinogenicity. the results from our experiments indicate that the rspv-sa vaccine is a very promising candidate in the safety and immunogenicity respect. however, it should be noticed that, as an alive viral vector vaccine, it is possible that the spv per se would interfere the vaccination. to evaluate this possibility, randomly sampled pig sera were tested via the agarose gel diffusion assay, and the results showed the spv positive rate was around %. moreover, there was no report of spv epidemic in these recent years. thus, together with gel diffusion assay, the interfering from spv per se could be considered as minimum. for the first time, we generated a recombinant spv that expresses the neutralizing epitopes (a epitope in protein s) of tgev, and we verified that the s-a was expressed efficiently in our system. the antigen induced neutralizing antibodies against tgev in st cells, and potentiated strong th -type and th -type cytokine responses in our mouse model. these results suggest that rspv-sa induces humoral and cellular immune responses effectively and efficiently. given the th cell secretion of il- , ifn-␥, ifn-␣, and tfn-␤, it is apparent that th cells play an important role in antiintracellular pathogenic infection. because th cells in our study secreted il- , il- , il- and il- , it is likely that these cells were able to effectively stimulate b cell proliferation and hence, igg and ige antibody production (relevant to humoral immunity). we defined ifn-␥ and il- to be representative of the th -type and th -type cytokine secretory capacity, respectively. our results indicate that in our hands, both th -type and th -type cytokine levels increased dramatically. due to the immature immune system, the maternal antibodies from breast milk account for the immune defensive ability of piglets. based on the feasibility and relevant published papers [ ] , we decided to mimic the breast milk from female pig by mixing the anti-serum generated by vaccinating the days old piglets with spv-sa and the normal cow milk, and use it for passive immunity protection test on the days old piglets. in the study, the recombinant spv vectors were capable of protecting neonatal piglets against mortality and severe disease after a challenge with virus. the rspv-sa vector induced high titers of antibodies in swine. however it is worth noting that rspv-sa may enhance the anti-tgev antibody titer in swine as well as the anti-spv antibody, which will likely affect immune efficiency. with respect to clinical value, it is imperative to study this multivalent vaccine. to summarize, we first report that spv can be used as a live vector vaccine when it expresses the s-a protein of tgev. we determined that not only b-cells, but also t-cells were induced successfully. thus, rspv-sa provides thorough protection against virulent tgev challenge in swine. lastly, our data indicate that rspv-sa is a promising vaccine to prevent tgev infection. transmissible gastroenteritis 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adjuvants first insights into the protective effects of a recombinant swinepox virus expressing truncated mrp of streptococcus suis type in mice immune responses and protection efficacy of a recombinant swinepox virus expressing ha against swine h n influenza virus in mice and pigs a novel vaccine against streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing m-like protein joint production of prime/boost pairs of fowlpox virus and modified vaccinia ankara recombinants carrying the same transgene recombinant adenovirus encoding the ha gene from swine h n influenza virus partially protects mice from challenge with heterologous virus: a/hk/ / (h n ) co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) parvovirus evades interferondependent viral control in primary mouse embryonic fibroblasts induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein oral immunization of mice with recombinant lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein the authors of this paper have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -eyejnexx authors: liu, genmei; lv, lishan; yin, lijuan; li, xiaoming; luo, dongu; liu, kang; xue, chunyi; cao, yongchang title: assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eyejnexx infectious bronchitis virus (ibv) as an avian coronavirus is still posing a persistent and imminent threat to the poultry industry worldwide. here we report that transfection of sf cells with a single recombinant baculovirus encoding m and s proteins resulted in the assembly of ibv vlps; this is the first report that s protein plus m protein alone were able to be assembled into vlps for coronaviruses. we further showed that the generated ibv vlps could induce humoral immune responses in a level comparable to that of inactivated ibv vaccine, and more importantly the ibv vlps could elicit significantly higher cellular immune responses than the inactivated ibv vaccine. in summary, the assembly of ibv vlps with m and s proteins provided a simple strategy for generating vlps for coronaviruses, and the generated ibv vlps laid a feasible foundation for the development of an effective vaccine against infection of ibv in the future. avian infectious bronchitis virus (ibv) causes an acute and highly contagious viral disease of chickens, and leads to huge economic losses in poultry industry worldwide. ibv is an enveloped rna virus and its virus particle morphology is spherical with a - nm in diameter. ibv contains four viral structural proteins: membrane protein (m), spike protein (s), envelope protein (e), and nucleocapsid protein (n). the m protein contains a short amino-terminal ectodomain with glycosylation sites, and is - kda with varying degrees of glycosylation. homotypic interactions among m proteins and interactions with the s protein and the other structural proteins are required for virus particle formation. thus, the m protein plays an important role in coronavirus assembly [ ] [ ] [ ] [ ] . ibv m protein is found in the cis-golgi network and cis-golgi complex when expressed alone [ ] , and cannot be released into the supernatant unless ibv e is present [ ] . in contrast, studies of other coronaviruses suggested that the m protein was observed in the golgi area and plasma membranes of a variety of cells when expressed alone [ ] , and can form sedimentable particles and release from infected cells [ ] . the s protein forms projections on the surface of the virion and generates two subunits, s and s , through posttranslational cleavage. the s protein contains epitopes that can induce neutralization, hemagglutination inhibition (hi) and serotype-specific antibodies [ ] [ ] [ ] [ ] [ ] [ ] . obviously, the s protein is the main immunogenic protein and will be a target protein when designing new ibv vaccines. the s protein is transported to the plasma membrane when expressed alone [ ] . incorporation of spikes into coronavirus particles is effected by interactions between the s protein and the m protein and governed by the carboxy-terminal domain of the s protein [ ] . numerous vlps assemble in an in vitro expression system, contain the major structural viral proteins, and mimic the conformation and organization of authentic native viruses without the viral genome [ ] . for ibv vlps, some researchers have reported that e proteins are sufficient for formation, but the efficiency is extremely low [ ] . other researchers have described that interactions between the e and m proteins and the membrane bilayer probably played an important role in vlp formation and virus budding [ ] . nowadays, ibv is controlled using live attenuated and inactivated vaccines, but ibv frequently outbreaks in endemic areas. thus, the development of new vaccines is urgent. in the present study, we assembled ibv vlps containing m and s proteins using a baculovirus expression system and we further evaluated the vlps immune responses in mice and chickens. spodoptera frugiperda (sf ) insect cells were grown adherent in t-flask in the complete grace's insect cell culture medium and incubated at • c. human epithelial kidney cells ( t) were grown in the complete dulbecco's modified eagle's medium and incubated at • c in % co . h strain of ibv was propagated in -day-old chick embryos and inactivated by . % formalin at • c for h. the inactivated h was purified by ultracentrifugation at , × g for h at • c on a discontinuous sucrose gradient of %, %, %, %, and % sucrose. ibv m and s genes were amplified from the total rna extracted from the allantoic fluid of h -infected chick embryos using reverse transcriptase polymerase chain reaction (rt-pcr) and subcloned into plasmid pfastbac tm dual (pfdual) (invitrogen), either individually or simultaneously (fig. ) . the recombinant plasmids were chemically transformed into competent dh bac tm escherichia coli cells (invitrogen). the recombinant shuttle plasmids rbacmid-m, rbacmid-s, and rbacmid-s-m were obtained and identified by pcr using m primers. a total of × sf cells per well grown in -well culture plates were transfected with g purified recombinant bacmid dna mixed with l cellfectinr ii ® reagent (invitrogen) in l in supplemented grace's medium. after incubating the transfected cells at • c for h, the transfection mixture was removed and replaced with complete growth medium, and the cells were incubated at • c. the supernatant was collected through centrifugation when % of cells had cytopathogenic changes. the recombinant baculoviruses rb-m, rb-s, and rb-s-m harvested from the supernatant were propagated and purified times using viral plaque in sf cells. at h postinfection, supernatants from infected sf cells were collected, filtered, and centrifuged at , × g for min at • c. sediments were suspended in phosphate-buffered saline (pbs) plus . mm phenylmethylsulfonyl fluoride (pmsf). next, adherent cells were rinsed twice and collected in pbs plus . mm pmsf, sonicated, and microcentrifuged at × g for min at • c to remove cell debris. the samples were resolved through electrophoresis on %, %, and % sds-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (bio-rad). the expressed proteins were detected with chicken polyclonal sera raised against ibv virus at a : dilution and horseradish peroxidase (hrp)conjugated anti-chicken secondary antibody at a : dilution (ptglab, usa). at h post-infection, the infected sf cells grew on glass cover slips were fixed in % ice-cold methanol at • c and blocked with pbs-tween % bovine serum albumin plus . % triton tm x- . fixed cells were incubated with the primary antibody at a : dilution and with the secondary antibody at a : dilution. m proteins were detected with mouse polyclonal sera raised against m protein expression with t cells and secondary anti-mouse fluorescein isothiocyanate (fitc)-conjugated antibody (ptglab, usa). s proteins were detected with chicken polyclonal sera raised against s protein expression with t cells and secondary antichicken cy conjugated antibody (ptglab, usa). cell nuclei were stained with , -diamidino- -phenylindole. cover slips were visualized under a confocal laser scanning microscope (tcs sp , leica). at - h post-infection, the culture media of infected sf cells was collected, filtered, and microcentrifuged at × g for min at • c to remove cell debris. the supernatant was ultracentrifuged at , × g for min at • c. vlps collected in the pellet were suspended in pbs. to further purify them, the vlps suspension was loaded on a discontinuous sucrose gradient of %, %, %, %, and % sucrose and ultracentrifuged at , × g for h at • c. vlps at the interface between % and % sucrose were collected and pelleted by ultracentrifugation at , × g for . h at • c. vlp-containing pellets were resuspended in pbs and analyzed for the presence of ibv structural proteins using coomassie blue stained sds-page electrophoretogram and western blot. the purified vlps were placed onto carbon-coated, -mesh copper grids for min. sample-containing grids were washed with water, dried with filter paper, and stained with % phosphotungstic acid (ph . ) for min. excess staining solution was removed with filter paper. vlp-containing grids were viewed with a transmission electron microscope (jem- cx, jeol ltd., japan). images were taken at , × magnification. the total protein concentrations of vlp and inactivated ibv were determined using bradford protein assay kit (beyotime, china) with bovine serum albumin (bsa; takara, japan) as standard; and the s protein concentrations of vlp and inactivated ibv were determined using sds-page gel electrophoresis by genesnap and genetools from syngene software with bsa as standard. twenty-four -week-old specific-pathogen-free (spf) balb/c female mice (experimental animal center, sun yat-sen university) without ibv-specific antibody were randomly divided into groups of mice. the mice were housed in positive pressure and immunized times (weeks , , and ) with freund's adjuvant. group mice were immunized with g vlps (s protein) per mouse via subcutaneous injection. group mice were injected with g inactivated h (s protein) per mouse as a positive control. group mice received pbs as a negative control. sera were collected from mice at days , , and after initial immunization for ibv-specific antibody detection. sixty -day-old spf chickens without ibv-specific antibody were randomly divided into groups of chickens. the chickens were housed in individual isolators under positive pressure and immunized times (weeks and ) with oil adjuvant. the immunization program and immunization dose of chickens were the same as the mice did. sera were collected from chickens at days and after primary immunization for ibv-specific antibody detection. the purified h virosomes were used as antigen to detect the ibv-specific antibodies in an indirect elisa. the h virosomes reconstitution with the detergent octaethylene glycol monododecyl ether (c e ) and purification with discontinuous sucrose gradient centrifugation were carried out following the procedures as previously described [ , ] . the secondary rabbit anti-mouse or donkey anti-chicken hrp-conjugated antibodies were used at a : dilution. the optical density value was nm. the neutralizing antibody titer was determined using a neutralization test performed on chick embryos following the procedures as previously described [ ] . serum samples were incubated for min at • c to inactivate nonspecific inhibitors. treated sera were serially diluted twofold and incubated with an equal volume of h with eid at the final concentration for h at • c. l of the incubated sera-viral mixture was injected into -dayold spf chick embryos. the eid of h without serum was used as a negative control. the injected eggs were incubated for days at • c. the neutralizing antibody titer was determined using the reciprocal of the highest dilution of serum that gave % neutralization of eid of virus in chick embryos. spleens were collected from mice on day after the primary immunization. the lymphocytes were isolated from the spleens using mouse × lymphocyte separation medium (dakewe, china). the number of ifn-r and il- secreting cells in the single-cell suspension of splenocytes was determined with a elispot kit (dakewe, china) following the manufacturer's protocol. the splenocytes were stimulated with purified h virosomes at a g/ml concentration, and the spots were counted using the immunospot elispot reader (bioreader , bio-sys, germany). the data between groups were statistically analyzed by using a student's two-tailed t test when only two groups were compared or by one-way analysis of variance (anova) when more than two groups were compared. p values less than . (p < . ) were considered statistically significant. the supernatant culture media and cell lysates from the recombinant baculovirus-infected sf cells were harvested and analyzed using western blot. the results showed that m and s proteins could be expressed in the sf cells (fig. ) . interestingly, when m protein was expressed alone, it could be detected in the sedimentable particles of the culture media ((a), lane ), but s protein could not be detected when it was expressed alone ((b), lane ). more importantly, when s protein was expressed together with m protein, s protein could be detected in the sedimentable particles of the culture media ((c), lane ). the expressions of m and s proteins were further confirmed by immunofluorescence staining analysis. sf cells were first infected with rb-m, rb-s or rb-s-m, respectively, and then stained with m-or s -specific primary antibodies and fitc-or cy -conjugated secondary antibodies. the results showed that both m and s proteins were mainly expressed near the plasma membranes, and m and s proteins were co-localized (fig. ) . the detection of s protein in the sedimentable particles when it was co-expressed with m protein strongly suggested that ibv vlps could be generated using m and s proteins only. sf cells were infected with rb-s-m, and supernatant culture media were harvested and then the sedimentable particles were subjected to sucrose density gradient ultracentrifugation. fractions were collected and analyzed by western blot (data not shown). the fraction collected from the interface between % and % showed the coexpression of m and s proteins, where a distinct protein band of roughly - kda corresponding to the m protein and a kda band corresponding to the s protein ( fig. a and b ). using em analysis, we could see a spherical morphology particle with a diameter of about nm (fig. c ). we investigated the immunological characteristics of ibv vlps both in mice and chickens. ibv-specific antibody in the sera of immunized mice was detected using indirect elisa. the results indicated that, weeks after the primary vaccination (day ), serum igg titers could be detected in vlps and inactivated h groups, and the titers continued to increase following the second and third immunizations (days and ) (fig. ) . the vlps and inactivated h groups had significantly higher igg titers (p < . ) than the pbs group. as for the vlps and h groups, the vlps group, after the first and second immunizations, had lower igg titers than the inactivated h group; and the vlps group, after the third immunization, had higher igg titers than the inactivated h group, but they were not statistically different (p > . ). we further researched the immunogenicity of ibv vlps in chickens. the results showed that, weeks after the primary vaccination (day ), both of vlps and inactivated h groups could not detected serum igg titers, and the differences between these and pbs groups were not statistically significant (p > . ); but following the second immunization (day ), the igg titers of the vlps and inactivated h groups increased (fig. ) and were significantly higher (p < . ) than the pbs group. the differences between vlps and inactivated h groups were not statistically significant (p > . ), although the vlps group had a slightly lower igg titers after the second immunization. antisera from vaccinated mice and chickens were analyzed using a neutralization assay to detect functional antibodies with neutralization activity against the h virus. the ibv-specific neutralization antibodies in mice were detected at days after the primary immunization and in chickens the antibodies were detected at days after the primary immunization. the results showed that vlps and inactivated h groups had statistically significantly higher neutralizing antibody titers (p < . ) than the pbs group (fig. ) . the differences between vlps and inactivated h groups were not statistically significant (p > . ), although the vlp group had a slightly higher neutralizing antibody titer. . neutralizing antibody titers of mice and chickens sera. y means neutralizing antibody titers of mice sera collected on day ; y means neutralizing antibody titers of chickens sera collected on day . virus like particles (vlps) and inactivated h groups had significantly higher neutralizing antibody titers (p < . ) than the pbs group. the groups were not statistically different (p > . ), although the vlp group had a slightly higher neutralizing antibody titer. . . ibv vlps induced significant higher cellular immunoresponses than inactivated h vaccine did in mice cellular immunoresponses were evaluated through detection of the number of cells secreting ifn-r and il- after h virosome stimulation of the single-cell suspension splenocytes from vaccinated mice using the elispot assay. the results revealed that the levels of ifn-r and il- in mice which inoculated with vlps were significantly higher than in those which inoculated with inactivated h and pbs (p < . ) (fig. ). the molecules required for the formation of coronavirus vlps varied in different reports. ho et al. showed that m and e proteins were required for the assembly of human sars coronavirus-like particles in sf cells [ ] ; but m, e and n proteins were required for the formation of sars-cov vlp as demonstrated by siu et al. in vero e cells [ ] and nakauchi et al. in t cells [ ] . liu et al. found that influenza m protein and sars s protein could assemble chimeric sars-cov vlps in sf cell [ ] . for mouse hepatitis virus (mhv-a ), vennema et al. showed that transfection of ost - cells with vaccinia virus encoding m and e proteins resulted in the vlp formation [ ] . these reports strongly suggested that m and e were the minimal molecules required for the coronavirus-like particles regardless of virus types and cell lines used in the studies. in contrast, our results showed for the first time that ibv m and s proteins were efficient assembled into vlps in sf cells with a single recombinant baculovirus encoding m and s proteins. this might not be a complete surprise. as for ibv, corse and machamer reported that while the presence of e protein could enhance the release of m proteins in the sedimentable particles from the culture supernatants, m protein alone was capable of being released in the sedimentable particles from the culture supernatants in ost - cells with a vaccinia-t polymerase expression system [ , particularly fig. ]. in addition, the native envelope trimeric s glycoprotein is not necessary for particle formation but is incorporated into vlps when present [ ] ; and incorporation of spikes into coronavirus particles is mediated by the s protein's carboxy-terminal domain and effected by interactions between the s protein and the m protein [ ] . the showing of ibv vlps formed by m and s proteins demonstrated a simple strategy for production of coronavirus-like particles for coronaviruses. s protein is a main antigen for coronaviruses [ , ] . in line with previous literatures, our results showed that ibv vlps induced humoral antibody responses (i.e., serum ibv-specific igg antibodies and ibv-specific neutralization antibodies) in a level comparable to that of the inactivated ibv vaccines in mice and chickens, and both ibv vlps and inactivated ibv vaccine induced significantly higher humoral antibody responses (p < . ). for the cellular immune responses, the ifn-r and il- were detected using the elispot assay. the results revealed that ibv vlps elicited high levels of ifn-r and il- , but the inactivated h vaccine only induced il- at a significantly lower level (p < . ). the results demonstrated that ibv vlps could elicit both th -and th -type cellular immune responses, but inactivated h only stimulated th -type cellular immune response. previous studies showed that vlps could elicit more effective antibody responses than proteins in their nonnative forms [ , ] . the reason for the results that the inactivated h vaccine used in this study had slightly lower neutralizing antibody titers and a significantly lower level of cellular immune responses than vlps might be that a portion of s molecules lost their native forms during inactivation with formalin. our results demonstrated that the ibv vlps mimic the conformation and organization of authentic native viruses, and their immunogenicities were reserved. in summary, we have successfully generated ibv vlps carrying m and s proteins and shown that the generated ibv vlps could effectively elicit humoral immune responses both in mice and chickens, and more importantly, ibv vlps could induce significantly higher cellular immune responses than inactivated ibv vaccine in mice. as the results taken together demonstrated a simple strategy for generating coronavirus-like particles and provided a candidate vaccine against the infection of ibv for future development. assembly of the coronavirus envelope: homotypic interactions between the m proteins coronavirus structural proteins and virus assembly the molecular biology of coronaviruses protein interactions during coronavirus assembly the e glycoprotein of an avian coronavirus is targeted to the cis golgi complex infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles self-assembly of severe acute respiratory syndrome coronavirus membrane protein generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production coronavirus ibv: virus retaining spike glycopolypeptide s but not s is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection structural proteins of avian infectious bronchitis virus: role in immunity and protection the s glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens immune responses to structural proteins of avian infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction reconstitution of the fusogenic activity of vesicular stomatitis virus functional reconstitution of influenza virus envelopes animal virology assembly of human severe acute respiratory syndrome coronavirus-like particles the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles analysis of severe acute respiratory syndrome coronavirus structural proteins in virus-like particle assembly chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin membrane embedded hiv- envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes this study was supported by the grants from chinese national high-tech r&d program ( program, aa a ) and cooperation project in industry, education and research of guangdong province and ministry of education of people's republic of china (grant no. b ). we thank to dr. george d. liu for critical review and revision of the manuscript. key: cord- -k ugjb f authors: liu, shih-jen; leng, chih-hsiang; lien, shu-pei; chi, hsiang-yun; huang, chiung-yi; lin, chang-ling; lian, wei-cheng; chen, chi-ju; hsieh, shie-liang; chong, pele title: immunological characterizations of the nucleocapsid protein based sars vaccine candidates date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: k ugjb f the recombinant nucleocapsid (rn) protein of the coronavirus (cov) responsible for severe acute respiratory syndrome (sars) was cloned and expressed in escherichia coli, extracted from cell lysates containing m urea, then purified by ni( +)-affinity chromatography. in animal immunogenicity studies, we found that most anti-rn protein antibodies were igg a in balb/c mice vaccinated with rn emulsified in montanide isa- containing the synthetic oligodeoxynucleotide, cpg. in contrast, anti-rn protein antibodies of mice immunized with rn protein in pbs were found to mainly be igg . these results indicated that isa- /cpg-formulated rn protein was dramatically biased toward a th immune response. to identify the b-cell immunodominant epitopes of the rn protein in the mouse and monkey, the reactivities of antisera raised against purified rn proteins formulated in isa- /cpg were tested with a panel of overlapping synthetic peptides covering the entire n protein sequence. three immunodominant linear b-cell epitope regions were mapped to residues – , – , and – of the rn protein. when the reactivities of these peptides were screened with human sera from five sars patients, peptides corresponding to residues – reacted strongly with sera from two of the sars patients. these results indicated that the region around residues – of the n protein is immunogenic in the mouse, monkey, and human. we found that peptides corresponding to residues – , – , – , and – contained murine immunodominant t-cell epitopes. to identify functional ctl epitopes of the n protein, balb/c mice were immunized with peptides containing the h- k(d) ctl motif emulsified in adjuvant isa- /cpg. using an ifn-γ secretion cell assay and analysis by flow cytometry, peptides containing residues – were found to be capable of stimulating both cd (+) and cd (+) cell proliferation in vitro. we also only observed that peptides corresponding to residues – were capable of stimulating ifn-γ production in t-cell cultures derived from peripheral blood mononuclear cells (pbmcs) of macaques immunized with the rn protein emulsified in isa/cpg adjuvant. our current results together with those of others suggest that some immunodominant b-cell and t-cell epitopes are conserved in the mouse, monkey, and human. this information is very important for the development sars diagnostic kits and a vaccine. a newly emerging infectious disease caused by the severe acute respiratory syndrome-associated coronavirus (sars cov) had significant economic impacts in countries affected by the disease outbreak in [ ] [ ] . the lack of efficient treatments and prevention means the possibility exists for sars to return in the future. in addition, high-risk populations such as laboratory and hospital workers need effective sars vaccines for protection. unfortunately, recent reports have shown that inactivated coronavirus vaccines in a cat model might worsen the disease rather than prevent it [ ] . furthermore, the sars virus may cause mild liver inflammation in ferrets, and the damage was much more serious if animals were first given a candidate sars vaccine based on the vaccinia virus [ ] . yang et al. [ ] also found that antibodies against sars cov s protein enhanced the entry of virus in in vitro cell culture studies. therefore, in order to generate safe and effective sars vaccines, we urgently need greater understanding of the immunological responses of vaccine candidates in different animal models. the n protein of the feline infectious peritonitis virus (fipv) has been used as subunit vaccines to induce protective immunity and to prevent the progression of diseases in a cat model [ ] . the literature indicates that immunization of dna plasmids that encode the n protein of the ebola virus and influenza virus elicits protective antigen-specific cytotoxic t lymphocyte (ctl) responses [ ] [ ] [ ] . in avian coronaviruses, n protein-specific t-cell responses were shown to generate protective immunity [ ] . these results indicate that a good n protein vaccine candidate should elicit strong cellular immune responses. the n protein of sars cov is amino acids long and highly conserved ( %) within different isolates, but it shares only - % amino acid homology with the n proteins of other coronaviruses [ ] . in fact, the dna vaccine candidate encoding the n protein of the sars cov fused with calreticulin (crt) was shown to induce potent humoral and cellular immune responses in a mouse model, and inhibited replication of the recombinant vaccinia virus that expressed the n protein in vivo [ ] . we therefore focused on the n protein of the sars cov as the target antigen for subunit vaccine development. with an eye toward clinical applications, we selected two potential adjuvants in clinical used that have been shown to be strong cellular immune response inducers: montanide isa- and the immune-stimulating oligonucleotide, cpg [ ] . isa- and cpg were used to emulsify the rn protein as vaccine candidates for mouse and macaque immunogenicity studies. in the present study, we report that ( ) isa- /cpgformulated rn protein was dramatically biased toward the th immune response; ( ) the immunodominant b-and t-cell epitopes of the n protein were present in both balb/c mice and macaques; ( ) conserved immunodominant b-cell epitopes were identified in mice, macaques, and sars patients; ( ) n protein-specific cd + cells could be induced by isa- /cpg formulated with either the rn protein or synthetic peptides containing the h- k d ctl epitope motif. recombinant sars cov n protein (rn) containing a n-terminal his tag gene was cloned into the prseta vec-tor (invitrogen, carlsbad, ca, usa) for protein expression. the plasmid was transformed into an escherichia coli bl (de )gold (stratagene, cedar creek, tx, usa) host strain and incubated at • c overnight. the expression of rn was induced with . mm of isopropyl ␤-d-thiogalactoside (iptg) for h, then cells were harvested by centrifugation ( × g for min), and cell pellets were re-suspended in ml of homogenate buffer ( mm tris-cl (ph . ), mm nacl, % glycerol, mm sucrose, and mm imidazole). after disruption using a french press (constant systems, daventry, uk) at mpa, cell lysates were clarified by centrifugation ( , × g for min). most of the target protein was extracted from the pellet with m urea in homogenate buffer and loaded into ml of ni-nta resin (qiagen, san diego, ca, usa). the resin was washed with the same buffer, and the target protein was purified by changing the ph value. after further washing with ph . and . homogenate buffers and m urea, the rn was eluted from the resin using ph . homogenate buffer and m urea, then the purified rn was refolded by dialyzation against phosphatebuffered saline (pbs). the purified rn was stored at − • c in pbs with % glycerol for further studies. when analyzing samples by sds-page, l of samples were mixed with an equal volume of the sample buffer ( mm tris-hcl (ph . ), % sds, % -mercaptoethanol, % glycerol, and . % bromophenol blue) and heated in boiling water for min. the samples ( - g protein per lane) were separated on a % sds-page, then electrophoretically transferred from the gel to a polyvinylidene difluoride (pvdf) membrane (millipore, billerica, ma, usa) at ma for min. the membrane was blocked overnight with % non-fat milk in pbs containing . % tween- (pbst) at • c. after washing with pbst, the blot was incubated at room temperature for h with a mouse anti-his antibody ( : dilution, amersham biosciences, new territories, hk). after extensive washing with pbst then incubation for h with horseradish peroxidase (hrp)conjugated goat anti-mouse igg ( : , bethyl laboratories, montgomery, tx, usa), the blot was developed with . mm dab ( , -diaminobenzidine tetrahydrochloride, sigma, st. louis, mo, usa). after development for - min, the blot was washed with distilled water to stop the reaction. six-to eight-week old female balb/c mice were obtained from the national laboratory animal breeding and research center (taipei, taiwan). all mice were housed at the animal technology institute taiwan (atit, miaoli, taiwan) animal facility. monkeys (formosan macaques, macaca cyclopis) were maintained in accordance with institutional animal care protocols of the center of disease control, taipei, taiwan. all of the animal studies were approved by the animal committee of the national health research institutes, taipei, taiwan. peptides derived from the sars cov n protein sequence were synthesized as -mer fragments with -amino-acid overlap that covered the entire n protein sequence of the urbani strain (kelowna, taipei, taiwan); peptides were dissolved in % dmso at mg/ml. two ctl epitopes, qfkd-nvill and vwvategal, produced from the n protein with the babl/c (h- k d ) mice class i binding motif, were predicted by a computer-based program on the web site of the bioinformatics and molecular analysis section (bethesda, md, usa) [ ] . mice were immunized subcutaneously with g of rn protein emulsified in incomplete freund's adjuvant (ifa) or montanide isa- (seppic, paris, france) with g of the cpg oligonucleotide (tcg tcg ttt tgt cgt ttt gtc gtt ttg tcg tt). mice received two booster doses of the immunogen, one each at and weeks later. blood samples were collected every weeks post-immunization. for t-cell analysis, mice were sacrificed at days after the final immunization. in the pooled peptides groups, all peptides (table ) containing h- k d -restricted epitopes were emulsified in ifa or isa- with the cpg oligonucleotide (isa/cpg), and immunized subcutaneously. for the monkey experiments, the g of rn was emulsified in . ml of isa/cpg, and multiple injections were given muscularly at , , and weeks. sera were collected at specified time points. sera were obtained from clotted blood samples by centrifugation and were heatinactivated at • c for min. sera from five convalescent-phase humans were obtained from the center of disease control, taipei, taiwan. sera were collected - days after recovery from the disease based on the clinical diagnosis. all sera were inactivated following government guidelines before transfer to our laboratory. the presence of n protein-or peptide-specific antibodies in the sera was determined by elisa. in briefly, l ( g/ml) of purified rn or peptides were coated in well microtiter plates with . m carbonate buffer (ph . ) by overnight incubation at • c. coated plates were washed twice with pbst and then blocked with % non-fat milk in pbs at room temperature for h. diluted sera from immunized animals were applied to wells at room temperature for h. followed by hrp-conjugated goat anti-mouse igg or hrp-conjugated goat anti-monkey igg (sigma, st. louis, mo, usa), the assay was developed with , , , tetramethylbenzidine (tmb), and the reaction was stopped by adding l of m h so per well. plates were read at nm using an elisa plate reader (molecular devices, sunnyvale, ca, usa). for antibody isotype analysis, biotinconjugated rat anti-mouse igg and igg a (bd pharmingen, san diego, ca, usa) were added to the wells for sera binding, then hrp-conjugated streptavidin was added and developed with the tmb substrate. the antibody titer was defined as the reciprocal of the highest dilution that produced an od -nm value of two-fold higher than the pre-immune sera. on day after the final immunization, mice were sacrificed to harvest their spleens. erythrocyte-depleted splenocytes ( . × cells/well) in -well plates were cultured in vitro with g/ml of pooled peptide or individual peptide that covered all of the n protein amino acid plates for h. ifn-␥-secreting cells were detected with a mouse ifn-␥ secretion assay detection kit (miltenyi biotec., gladbach, germany). in brief, × splenocytes were harvested, and labeled with an ifn-␥ capture antibody for min at • c. afterwards, cells were transferred into • c warm medium for min, then washed twice, and stained with phycoerythrin (pe)-conjugated ifn-␥ detection reagent, and fluorescein isothiocyanate (fitc)-conjugated anti-cd or anti-cd antibodies. after washing, cells were collected and analyzed on a facscalibur instrument with cellquest software. dead cells were gated by g/ml propidium iodide. t-cell epitope mapping was performed in mice and monkeys immunized with purified rn ( and g, respectively) emulsified in isa- plus g of the cpg oligonucleotide. seven days after the second boost, splenocytes from mice or peripheral blood mononuclear cells (pbmcs) from monkeys ( × cells/well) were cultured in rpmi (gibco, san diego, ca, usa) supplemented with % heat-inactivated fetal calf serum, u/ml of penicillin, g/ml of streptomycin, and m -mercaptoethanol with g/ml of pooled or individual peptides at • c for days. concanavalin a (con a) or medium alone served as the positive and background controls, respectively. the supernatant was harvested and assayed for cytokine production. mouse ifn-␥ was quantitated by elisa using the matching anti-ifn-␥ antibody set (r&d systems, minneapolis, mn, usa) in accordance with the manufacturer's directions. monkey ifn-␥ was measured using an elisa kit from biosource international (camarillo, ca, usa) according to the manufacturer's protocol. data were evaluated using student's t-test analysis of variance (anova) to determine the statistical significance of differences between samples. p < . was considered statistically significant. the ability of certain individuals to overcome infection without treatment led to the hypothesis that administration of vaccines against sars may provide effective protection against the disease. like other viruses with spike proteins on the viral surface, coronaviruses have been shown to elicit enhancement of disease through non-neutralizing spike protein-specific antibodies [ ] , so vaccines that elicit t-cell immunity against sars proteins are likely to receive more-favorable clinical attention. in animal coronavirus vaccine development, the n protein has been widely tested as an immunogen for protection against coronavirus infections. inoculation of chickens with plasmid dna encoding the infectious bronchitis virus (ibv) carboxyl terminus of the n protein led to the induction of a specific cytotoxic t lymphocyte population capable of recognizing two distinct ibv strains. in addition, the adoptive transfer of t cells from animals inoculated with ibv to naïve chicks provided cd + ␣␤ specific protection [ ] . a th t-cell line resistant to the murine hepatitis virus (mhv) showed marked increases in ifn-␥ expression with a decrease in il- production when incubated with mhv-infected cells [ ] . infusion of this resistant cell line into susceptible mice led to complete protection against mhv infection. the immunity against mhv was demonstrated to be a process dependent upon cd + , perforin, and ifn-␥ in the absence of cd + t cells [ ] . the generation of igg a antibodies following dna immunization of n protein suggested that there was a significant th -type response [ ] . with the strong potential protective immunity of n protein-based vaccines, we hypothesized that the sars cov n protein would elicit effective immune responses against sars diseases. to aid the downstream purification process, the c-terminal end of the n protein-coding region was tagged with six histidine residues. expression of rn protein could be induced by adding mm of isopropyl ␤-d-thiogalactoside (iptg) into the e. coli cell culture, and the level of expression was observed using an sds-page analysis (fig. a, lanes and ) . the electrophoretic position of the rn protein corresponded to the predicted size of kda. rn proteins were collected as pellets from cell lysates using centrifugation. rn proteins were purified using an imac (immobilized metal affinity chromatography) column and by a ph stepwise gradient containing m urea (fig. a, lanes - ) . purified rn proteins were refolded by dialysis against pbs and could be detected by blotting with an anti-his antibody (fig. b) . we obtained - mg of purified rn protein from l of cell culture, and it represented a % overall yield. as mentioned above, a strong adjuvant that elicits strong cellular immune responses is necessary for the n protein to be effective as a vaccine candidate. different adjuvant formulations induced different immune responses in immunized animals. to assess the immune responses elicited by the rn protein in our balb/c mouse model, rn protein was formulated with either incomplete freund's adjuvant (ifa), a potent adjuvant complex, isa/cpg, or pbs alone. after three immunizations ( g of rn protein per dose), the n proteinspecific antibody responses were measured using elisa, and results are shown in fig. a . the antibody titers were found to be × − , × − , and × − in the isa/cpg, ifa, and pbs groups, respectively. to test whether the strong immune response elicited by the rn protein formulated with isa/cpg was useful in clinical application, we analyzed the subtypes of reactive antibodies from all groups. the isotypes generated in different groups are shown in fig. b ; most of the antibodies were igg found in the pbs group ( . × − ), with less igg a ( . × − ). there were similar igg titers found in both the ifa and isa/cpg groups ( × − ). not surprisingly, the isa/cpg group generated strong and dominant igg a antibodies ( × − ). but the ifa group produced less igg a ( × − ). this result indicated that isa/cpg could drive the immune response toward th , and is consistent with previous results which showed that the synthetic oligodeoxynucleotide cpg motif could skew the host's immune response toward th [ ] and the combination of cpg and isa- also showed the ability to enhance the protective efficacy of a subunit malaria vaccine candidate [ ] . identification of the immunodominant b cell epitopes could be useful for future immunological investigation. to identify b-cell epitopes of the n protein, mice were immunized with rn protein formulated with either ifa or isa/cpg. to systemically analyze the b-cell epitopes, each of the peptide pools (np -np ), consisting of consecutive -mer overlapping peptides of the n protein, was used to screen against anti-n antisera (see table ). in this peptide-elisa assay, we used the rn protein as the positive control and internal standard to normalize the reactivity. since we desired to determine the immunodominant epitopes, mouse sera were diluted to : for epitope screening. when mouse antisera were generated from the group immunized with rn protein formulated with ifa, we found two major reactive peptides pools to be np and np . further individual peptide reac-tivity analysis showed that peptides no. , , , , and contained immunodominant epitopes (fig. a) . in the isa/cpg-immunized mouse group, np and np were the two major reactive peptide pools. with further individual peptide screening, we found the major reactive peptides to be the eighty -mer overlapping peptides that cover the entire sequence of the sars cov n protein were used in an elisa to measure the immunodominant linear b-cell epitopes. the "relative reactivity" of each peptide with the tested sera was calculated using the mean od of each peptide divided by the mean od of peptide no. . samples with ≥ times the "relative reactivity" were considered to be positive. nos. , , , , and (fig. a) . regardless of which adjuvants were used, the results showed that balb/c mouse immunodominant b-cell epitopes were located at the cterminal regions (peptides no. , , , and ). however, sera from balb/c mice immunized with ␤-propiolactoneinactivated sars virus recognized the n-terminal region (residues - ) of the n protein [ ] . these different results could have been due to the nature of the antigens (refolded rn protein versus the inactivated sars virus) and adjuvants used. since the rn protein formulated in isa/cpg provided the best immune responses in the mouse model, we selected this adjuvant for the macaque immunogenicity studies. sera from two immunized macaques both had about a − reactivity titer against the rn protein according to elisa (data not shown). for immunodominant b-cell epitope identification, macaque sera were diluted : to screen the reactive peptide pools. serum from monkey no. recognized the peptide pools np , np , and np . serum from monkey no. reacted with peptide pools np and np . to further identify the reactive peptides, we found that peptides no. and contained the immunodominant b-cell epitopes in peptide pool np , since they were recognized by the antisera from both macaques. also peptides no. , , and in np were found to contain macaque immunodominant b-cell epitopes. peptides no. - and in peptide pool np were shown to be most reactive with serum from monkey no. (fig. b ). from the results described above, the conserved immunodominant b-cell epitopes in mice and monkeys were located within peptides no. (residues - ), (residues - ), (residues - ), and (residues - ). we further studied the b-cell epitopes of the n protein which recognized sars patients' sera. when patients' sera were diluted at : and used to screen the reactivity with the peptide pools, we found that only two patients' sera reacted with peptide pool np and purified np (data not shown) out of five randomly selected patients' sera. with further individual peptide screening, the major reactive peptides were found to be peptides no. and that corresponded to residues - (fig. c) . these results were consistent with previous reports by shichijo et al. [ ] , he et al. [ ] , and guo et al. [ ] who also found that the region around residues - reacted with sera from more than % of sars patients. we summarized all these data in fig. , which shows that immunodominant b-cell epitopes corresponding to residues - and - were conserved in mice and macaques. interestingly, the region corresponding to residues - was found to contain b-cell epitopes in all three species. this region (residues - ) will be useful for diagnostic kit development and as a biomarker for vaccine development as well. murine t-cell epitopes were identified using the in vitro stimulatory effects of overlapping peptides on t cells derived from splenocytes of balb/c mice immunized with the rn protein formulated with either ifa or isa/cpg adjuvants. the amounts of secreted ifn-␥ in culture supernatants were determined by ifn-␥-specific elisa kits. no ifn-␥ was detected in pbs, which served as the control group (data not shown). as shown in fig. a , ifn-␥ production was observed in both ifa-and isa/cpg-immunized groups, and several different peptides were found to be capable of stimulating ifn-␥ secretion. the potential t-cell epitopes were found in the ifa group to correspond to residues - , - , - , and - . in the isa/cpg group, the t-cell- reactive regions were identified to be residues - , - , - , and - . only peptides no. (residues - ) and (residues - ) were found to stimulate ifn-␥ production in both immunized groups. although we could not determine the h- k d -restricted ctl epitope from natural sars cov infections, it seemed likely that residues - can be used as an indicator to study the t-cell response of n protein subunit vaccines. interestingly, peptide no. contains a potential balb/c mouse ctl epitope, yyrratrrv, identified by epitope prediction algorithms. macaque immunodominant t-cell epitope mapping was also performed with pbmcs isolated from two macaques (m and m ) vaccinated twice with the rn protein formulated in isa/cpg. we found ifn-␥ production only in the t-cell culture derived from the pbmcs of m (fig. b) . in further studies, we observed that only peptide no. (residues - , gaiklddkdpqfkdn) was capable of stimulating ifn-␥ production in t-cell culture derived from pbmcs of monkey m . this result could be important information for further sars vaccine evaluation, especially in a macaque infection model. in our human immunodominant t-cell epitope identification studies (des et al. published elsewhere), we also found that peptide no. elicited responses from cd -and cd -positive cells derived from pbmcs of two sars patients with the hla-a class i haplotype. our findings suggest that the rn protein formulated in isa/cpg adjuvant can elicit t-cell immune responses, which may be important in the protective immunity against sars diseases. in addition to the b-cell and th -cell responses, the ctl may play an important role in virus-infected cell clearance. a computer-based program, bimas hla peptide binding prediction program [ ] , was used to predict candidate ctl peptides of n protein. two -mer peptides (qfkdnvill and vwvategal) were identified and synthesized based on the highest score values (table ). these two peptides were mixed and formulated in isa/cpg adjuvant with six other -mer peptides of n protein that were identified to consist of h -k d ctl-binding epitopes. at days after the third immunizations, splenocytes of balb/c mice were harvested, and various peptides were added to the splenocyte cultures for in vitro stimulation. after days, cytokine secretions of il- and ifn-␥ were measured using a cytokine-specific elisa. only peptide no. (pddqigyyrratrrv) was observed to be capable of stimulating ifn-␥ production ( pg/ml) (fig. a) . this result and the t-cell epitope mapping described above confirmed that yyrratrrv is a very potent functional t-cell epitope. in order to analyze the reactive t-cell subsets, we restimulated splenocytes with peptide no. in the presence of u/ml recombinant il- . after h, two-color flow cytometric analysis of t-cell subsets and ifn-␥ staining revealed that the t-cell population was only reactive to peptide no. the isolated splenocytes at × in -well plates were stimulated with g/ml of each peptide. the numbers - and - represent the top two ranking nonamer peptides, respectively (see table ); the peptide order was based on the predicted score. (b) positive wells cultured with corresponding peptides ( . g/ml) at • c for days in the presence of u/ml il- . cultured cells were re-stimulated with the corresponding peptide at • c for h. ifn-␥-secreting cd + and cd + cells were analyzed by flow cytometry. . concentrations of cd + /ifn-␥ + and cd + /ifn-␥ + cells were found to be . and . %, respectively (fig. b) . the lower cd + /ifn-␥ + cell population was perhaps due to peptide being a -mer peptide that might not have the best match to the mhc class i preferred peptide length ( - -mer peptides). in conclusion, we have demonstrated in mouse and macaque models that the rn protein formulated with potential clinically useful adjuvants, isa- and cpg, elicited strong th immune responses and may be excellent sars vaccine candidates. some of human b-and t-cell immunodominant epitopes of the n protein were also found in mice and macaques immunized with the rn protein formulated in isa/cpg adjuvants. the conserved immunodominant epitopes identified in these three species provide very important information for further vaccine design. caution urged on sars vaccines one year after outbreak, sars virus yields some secrets evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (fip) virus nucleocapsid protein against progression of fip protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein dna vaccines expressing either the gp or np genes of ebola virus protect mice from lethal challenge dose dependence of ctl precursor frequency induced by a dna vaccine and correlation with protective immunity against influenza virus challenge cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry characterization of a novel coronavirus associated with severe acute respiratory syndrome generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus cpg oligodeoxynucleotide and montanide isa adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains adoptive transfer of infectious bronchitis virus primed [alpha][beta] t cells bearing cd antigen protects chicks from acute infection resistance of naive mice to murine hepatitis virus strain requires development of a th , but not a th , response, whereas pre-existing antibody partially protects against primary infection mhv infection of the cns: mechanisms of immune-mediated control cytolytic activity induced by intramuscular injection of plasmid dna expressing the nucleocapsid protein of the jhm strain of mouse hepatitis virus into c bl/ mice immunotherapeutic uses of cpg oligonucleotides mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long-lasting immunity sars corona virus peptides recognized by antibodies in the sera of convalescent cases the financial support from national science council of taiwan (grant # svac- - ) for this study is gratefully acknowledged. key: cord- - hto qn authors: schoch-spana, monica; brunson, emily k.; long, rex; ruth, alexandra; ravi, sanjana j.; trotochaud, marc; borio, luciana; brewer, janesse; buccina, joseph; connell, nancy; hall, laura lee; kass, nancy; kirkland, anna; koonin, lisa; larson, heidi; lu, brooke fisher; omer, saad b.; orenstein, walter a.; poland, gregory a.; privor-dumm, lois; quinn, sandra crouse; salmon, daniel; white, alexandre title: the public’s role in covid- vaccination: human-centered recommendations to enhance pandemic vaccine awareness, access, and acceptance in the united states date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hto qn given the social and economic upheavals caused by the covid- pandemic, political leaders, health officials, and members of the public are eager for solutions. one of the most promising, if they can be successfully developed, is vaccines. while the technological development of such countermeasures is currently underway, a key social gap remains. past experience in routine and crisis contexts demonstrates that uptake of vaccines is more complicated than simply making the technology available. vaccine uptake, and especially the widespread acceptance of vaccines, is a social endeavor that requires consideration of human factors. to provide a starting place for this critical component of a future covid- vaccination campaign in the united states, the -person working group on readying populations for covid- vaccines was formed. one outcome of this group is a synthesis of the major challenges and opportunities associated with a future covid- vaccination campaign and empirically-informed recommendations to advance public understanding of, access to, and acceptance of vaccines that protect against sars-cov- . while not inclusive of all possible steps than could or should be done to facilitate covid- vaccination, the working group believes that the recommendations provided are essential for a successful vaccination program. since its first appearance in the united states in february , the novel coronavirus (sars-cov- ) has infected over . million americans and killed over , (as of october , ) [ ] . responses to the virus, including closing venues where person-to-person spread was likely, and requiring the use of masks and physical distancing measures when social contact could not be avoided, have reduced virus spread. at the same time, these protective actions have radically transformed social life and disrupted national and household economies [ ] . as the health crisis continues to linger and a sense of pandemic fatigue starts to take hold, political leaders, health officials, and the general public are seeking solutions [ ] . one of the most promising, if successfully developed and deployed, is vaccines. this technology could provide individual and population-level immunity, and through these the eventual conditions for the resumption of routine social and economic activities [ ] . to facilitate the development and dissemination of such vaccines, the us government has committed over billion dollars (via operation warp speed) with the aim of delivering million doses of a safe, effective vaccine by january [ ] . while this timeline is likely overly optimisticvaccine development, especially against a class of pathogens for which no licensed vaccine currently exists, typically takes - years [ ] -progress is being made. as of october , , vaccines are in preclinical evaluation, are in phase i and ii safety trials, have entered phase iii efficacy trials, and five vaccines have been approved for limited use: two in china, two in the united arab emirates, and one in russia [ ] . despite these promising developments, operation warp speed manifests a key social gap. the program rests upon the compelling yet unfounded premise that 'if we build it, they will come.' past experience in routine and crisis contexts demonstrates that, for a variety of reasons, not all segments of the public will accept medical countermeasures including vaccines [ ] [ ] . a recent poll in the us suggests this is already the case for sars-cov- (covid- ) vaccines. about half of us adults ( %) reported they definitely or probably would accept the vaccine, while % said they would not [ ] . in the same poll, only % of black americans indicated they would definitely/probably accept the vaccine compared to % of white americans. a human factor-centered vaccination campaign is needed to address these issues, but this campaign must be effectively planned and implemented. if poorly designed and executed, a covid- vaccination campaign could undermine increasingly tenuous beliefs in vaccines and the public health authorities that recommend them. at the same time, the broad impacts of a successful vaccination program would be considerable. immediate benefits would include interrupted disease transmission; fewer cases, hospitalizations, deaths, and chronic sequelae; and the beginning of reinstated social and commercial exchanges. longer term effects would include improved institutional capabilities to foster vaccine confidence among diverse communities, enhanced public understanding regarding vaccination's value to society, and heightened public trust in government, science, and public health. the purpose of this article, which is based on a report on the same topic [ ] , is to outline the major challenges and opportunities associated with a future covid- vaccination campaign and to provide empirically-informed recommendations to advance public understanding of, access to, and acceptance of vaccines that protect against sars-cov- . with the current lag time in vaccine availability, vaccination planners and implementers in the us and around the world have the opportunity to exercise foresight and take proactive steps to overcome potential hurdles to vaccine uptake and maximize public acceptance. these steps, however, must be taken now before this critical window of opportunity closes. the research and recommendations presented in this paper are a product of the -person working group on readying populations for covid- vaccine (table ) . this group was convened in april by principal investigators from the johns hopkins center for health security and the texas state university department of anthropology with support from the national science foundation-funded converge initiative [ ] . the purpose of the working group was to develop and disseminate recommendations informed by design thinking and evidence from social, behavioral, and communication sciences, that would support realistic planning for a us covid- vaccination campaign. members of the working group-listed as authors on this paper-included national figures in public health and social science with research, policy, and practice expertise in vaccinology, vaccine hesitancy/confidence, health disparities, infectious disease, bioethics, epidemiology, bioinformatics, public health law, pandemic mitigation, public health preparedness, mass vaccination campaigns, community engagement, and crisis and emergency risk communication. a combination of literature reviews on vaccination, pandemic planning, and health crisis communication; an assessment of current news and social media trends regarding covid- vaccines; and key informant interviews with each working group member focusing on their respective expertise formed the basis of the research presented in this article. this research was refined, and the recommendations were developed, through an iterative process involving the development of draft reports by a core working group, feedback from the entire working group via email and comments provided during a virtual meeting on may , , and subsequent rounds of revisions and feedback (including a second virtual meeting on june , ). the final report from the working group, which forms the basis of the recommendations and best practices below, was finalized on july , . envisioned largely as a biotechnology and logistics challenge, covid- vaccination also poses complex human factors challenges. such challenges have been observed during past emergencies. in , for instance, many americans rejected the h n vaccine due to safety concerns [ ] , despite the fact that the vaccine only involved a strain change (i.e., it was not a new technology) and was fully tested before release. the h n vaccine also amplified perceptions of bias. in los angeles, for example, distrust in public health-resulting from both prior experimentation on blacks (e.g. the tuskegee syphilis study) and long-term discrimination of blacks in health care settings [ ] [ ] [ ] -led local faith-based leaders, radio personalities, and other community representatives to advise black community members to avoid vaccination [ ] . even though the los angeles county health department actively sought to address these concerns, these suspicions coupled with a lack of convenient access to h n vaccines ultimately resulted in many blacks in this community remaining unvaccinated [ ] . despite the existence and importance of such challenges, funding for research on human factors related to vaccine acceptance is not commensurate with its significance for vaccination success [ ] [ ] . this type of inquiry-practical research of a social and behavioral nature on a medical technology-generally falls between the priorities of the national institutes of health ([nih] which rarely funds social science research) and the national science foundation (which does not fund applied public health research). funding from other sources including the centers for disease control and prevention (cdc) and private foundations has also historically been limited. in addition, the existing funding infrastructure is not outfitted for rapid response research during dynamic crises like sars-cov- . while initiatives are underway to develop communities of practitioners and a supportive infrastructure for disaster science in the us, including professional networks, streamlined institutional review board processes, and joint responder-researcher training [ ] [ ] , more progress is needed especially in regards to rapid funding opportunities. in the case of sars-cov- vaccination, for instance, while an nih funding opportunity award that could support research on human factors related to vaccine acceptance was made possible in june , the earliest project start date is september , a full nine months after operation warp speed plans for covid- vaccines to become available [ ] . to ensure a successful covid- vaccination campaign, it is necessary for sponsors to invest in time-critical investigations on human factors related to vaccine acceptance, and for public health authorities and other stakeholders to act on the social and behavioral findings of this research. such efforts include:  reconfiguring existing research investments to include social, behavioral, and communication science. one possibility for this is to set aside a small portion of the operation warp speed budget for research on human factors related to vaccine acceptance. such an approach has been used with great success in the past with other cutting-edge scientific initiatives such as the human genome project and manned space flight [ ] [ ] [ ] .  embeding rapid social, behavioral, and communication science within the covid- response, helping to deliver timely data and empirically based advice. by including social scientists in planning and implementation efforts, their people-centered methodologies and specialized knowledge can be integrated in a timely manner to maximize critical insights [ ] [ ] [ ] [ ] [ ] .  transforming the vaccine research enterprise by involving communities as active partners not passive subjects. traditional "one-sided, top down" approaches to community engagement are not always effective. community partnerships during the west africa ebola outbreak, for example, were necessary to overcome issues of trust and produce needed behavioral changes [ ] [ ] .  applying human-centered design principles (aka "design thinking") to the planning and implementation of the covid- vaccination program. user-focused approaches can result in more usable, acceptable, and effective interventions compared with traditional expert-driven methods [ ] [ ] . such an approach has been very successful in promoting hpv vaccination [ ] . vaccines typically require years of development and testing before licensure. nonetheless, us political leaders have publicly promised to accelerate covid- vaccine development at "an unprecedented pace," with the aim of delivering million doses of a safe and effective vaccine by january [ ] . although the use of new technologies can potentially accelerate vaccine production, public expectations around vaccine availability may not align with the practical realities of vaccine development, licensure, manufacture, and distribution. by failing to deliver sars-cov- vaccines as promised, the us government could frustrate pandemic-weary communities, siphon away trust, and suffer a major loss of institutional legitimacy. this situation is further complicated by public perceptions of the risks and benefits of sars-cov- vaccines. recent polling suggests that increasing numbers of americans plan to reject covid- vaccines, even if they are available and affordable [ , ] . a review of news reports, blogs, and other social media suggests a variety of potential causes for this result, including nonchalance about the disease and concern about vaccine safety. public perception, however, is a moving target. new developments, for example, an emergency use authorization (eua)-a power granted to the food and drug administration (fda) to make unlicensed drugs, vaccines, or other therapeutics available during a public health emergency, provided sufficient evidence that the countermeasure in question "may be effective"-for covid- vaccines, could engender additional uncertainties around vaccine safety due to the public's lack of familiarity with this complex regulatory mechanism. whatever the public's beliefs about vaccine benefits, risks, and supply, they cannot be separated from the current cultural milieu. in the us this is currently characterized by division, partisanship, and eroding public trust in government institutions-including the biomedical and public health agencies tasked with overseeing vaccine development, licensure, and distribution. in relation to the latter, for example, the intellectual independence of the fda has come under scrutiny for its ability to objectively assess vaccine safety and efficacy amid immense political pressure to quickly approve a sars-cov- vaccine [ ] . this complicated social environment poses a distinct and unprecedented complication to all vaccine promotion efforts in the us. amid this increasingly complex social landscape, there are several measures that us public health and healthcare practitioners, political leaders and policymakers, and communication experts can implement to prime the general public for sars-cov- vaccines including:  tempering expectations of vaccines as a "quick fix." because covid- vaccines will not immediately be available to everyone who wants them, and time will be needed to develop immunity (especially given the likelihood of two-dose regimens), communicators must prepare the public to continue implementing a mix of protective actions and harm reduction strategies.  forecasting a range of vaccine possibilities: from best case to worst case scenarios regarding vaccine supply and effectiveness. from a position of openness and transparency, public health communicators should address inevitable roadblocks and bottlenecks at every stage of vaccine testing, licensure, distribution, and administration, and convey to the public how this could affect vaccine availability. in addition, it will be necessary to reframe the dialogue about the value of vaccines, given that future sars-cov- vaccines may be not be the public's hoped for silver bullet. a vaccine, for example, may prevent the most severe disease but not prevent sars-cov- infection. in this scenario, vaccination could keep hospitals from being overwhelmed, prevent declines into frailty after severe bouts of disease, and avert medical bankruptcies that may arise with the longer-term impacts of covid- , but not provide the community immunity necessary to halt the spread of sars-cov- .  persisting in transparency around vaccine safety systems and actively work to protect their integrity. health authorities should focus existing vaccine safety infrastructure on the use of sars-cov- vaccines. in this vein, health authorities should develop a robust system for post-licensure surveillance, including ascertaining background rates of anticipated adverse events prior to vaccine rollout to enable comparison with post-rollout incidence of adverse events. independent oversight of vaccine safety, as occurred during the - h n pandemic, should also be used [ ] .  early on, seeking the counsel and input of communities of color that may have historic reticence towards public health. vaccine promotion efforts should engage these communities early and as frequently as possible. as partners in the task, they must also empathize with legitimate concerns around vaccine safety, medical experimentation, and inequalities in health care [ ] [ ] , while also identifying and sharing salient information that can help assuage unwarranted worry. a profusion of true and false information, which the who recently referred to as an "infodemic" [ ] , is now circulating around covid- . in this crowded information landscape, the veracity of information can be difficult to determine and key messages can be lost. in the us, public discourse on the pandemic currently incorporates a panoply of topics including science, public health, social disruptions, political divisions, and economic fallout [ ] , each of which can be a vehicle for misinformation-information that differs from expert consensus at the time it is shared [ ] . while many reasons exist for this flood of misinformation, including the widespread public adoption of social media platforms as a tool for information seeking, the uncertain nature around covid- as a novel infectious disease, and the presence of disinformation campaigns aimed at deflecting blame and pushing false narratives around the global covid- response [ ] [ ] [ ] , no easy solutions exist to stem the tide [ ] [ ] . regarding covid- vaccination specifically, while the first vaccine is minimally months away from materializing, the topic has already commanded immense public attention and generated its own pool of misinformation [ ] [ ] . this ranges from rumors questioning vaccine safety to more complicated narratives suggesting that future covid- vaccines were created alongside the virus and that major organizations are planning to use a covid- vaccination campaign for financial gain [ ] [ ] . while not the sole factor in determining behavior adoption, effective communication is necessary to address these issues and build public confidence in covid- vaccination [ ] . such communication will require addressing the enduring problem of how to best engage, exchange information, and empower audiences who have diverse beliefs and life circumstances. past communication experience with vaccines has shown the importance of engaging with key audiences to understand their concerns, values, attitudes, perceptions, and beliefs [ ] [ ] [ ] [ ] , and using this understanding to develop messages that resonate [ ] [ ] . messages that do not do this are often ineffective and, worse, can move audiences further away from the desired behaviors [ ] . given the diverse nature of social identities in the us, covid- vaccination communications will need to be tailored to meet the needs of specific audiences including essential workers, parents, groups with high comorbidity rates, and communities of color.  investing in qualitative research to identify specific community concerns and hopes in relation to covid- vaccination. qualitative research can provide insight into "how" and "why" participants feel, think, or behave a particular way [ ] [ ] . such insight, in turn, is the basis for developing more meaningful, trusted, and influential communication strategies [ ] . the current climate of racial, political, and economic division in the us has created a charged environment that necessitates both a fair vaccination campaign and widespread, public recognition of its fairness. an initial test of this will be how limited, initial doses of vaccines are allocated. in past public health emergencies, including the - h n pandemic, allocation strategies have been used to prioritize delivery of medical countermeasures to specific groups like critical health care workers and those who are at particular risk [ ] . pervasive racial biases in the us healthcare system, including lack of insurance and a lesser quality of care for non-white, rural, and low-income populations [ ] [ ] [ ] . such disparities have long-term consequences. black populations in the us, for example, experience increased morbidity and mortality compared to their white peers, sometimes in ways that cannot be accounted for by access to health care and income [ ] . public health authorities will need to anticipate and mitigate public discourse regarding vaccine allocation and distribution along with prejudicial ideas about social worth, explaining that vaccinating individuals residing in the us, regardless of social or legal status, is critical to the public's health as a whole. finally, politicization of the pandemic-both real and perceived-may prime expectations of a partisan-based vaccine allocation and distribution rather than an equitable one. some americans, for instance, perceive the use of masks as a slight against president trump by his detractors [ ] . likewise trump has signaled his preference for having a vaccine available prior to the election (a projection not in keeping with expert assessments), prompting concerns about whether he could turn a potential but inadequately tested vaccine into a campaign tool [ ] . such polarized views of covid- raise concerns about whether vaccine allocation and distribution can and will be judged as fair by the majority of americans. people will judge a covid- vaccination campaign's integrity not simply on biomedical merits, but on matters of fairness and equity-that is, have people received their just portion of health services, and has disease prevention, ultimately, been fairly distributed? past experience suggests the following steps may contribute to a fair process:  the us government taking steps to make the vaccine available at no cost to all americans and publicly pledge that everyone who wants covid- vaccines will get covid vaccines. removing cost as a barrier is among the most significant ways to assure that all individuals benefit from the life-preserving benefits of sars-cov- vaccines, and that the public can have the utmost confidence that public health needs and not economics will determine access. in the time that exists before vaccines are produced it is critical that safe and accessible vaccination sites are identified. this process will require ramping up the use of sites that are already available and accessible, but are used less frequently for vaccination efforts. community pharmacies, for example, are widespread and have been mobilized for past vaccination efforts [ ] . to fully utilize pharmacies in covid- vaccination efforts, however, it will be necessary to address state-level policies that may currently preclude pharmacists from administering these vaccines without standing orders from physicians. other nontraditional, potential vaccination settings that should be considered include grocery stores, senior citizen centers, workplaces, and schools [ ] [ ] [ ] [ ] [ ] . in some cases, it also may be acceptable and feasible to deliver vaccination via home visits by community health nurses when vaccination is bundled with delivery of other preventive health services; this approach has received a strong recommendation in the past from the community preventive services task force [ ] . for marginalized populations, including racial and ethnic minorities, additional consideration must be given to what constitutes a "safe" vaccination site. during the - h n pandemic, for example, mistrust and fear among marginalized communities posed a challenge. latino farmworkers were at greater risk for h n -related morbidity and mortality. however, reports of bullying and harassment within and outside of local healthcare settings led many members of this population to be fearful and hesitate to seek out h n vaccination [ ] . while national patterns may exist, assessments of what constitutes safe vaccination sites for marginalized populations should be conducted at local levels. once vaccination sites are identified, it will be essential for public health authorities to disseminate up-to-date, comprehensible, and trustworthy information about vaccination opportunities. much of this communication work will be done by local and state health departments, which may be challenging in light of budget cuts and strained local public health infrastructure. an additional complication will be the likely complex covid- vaccination environment, characterized by multiple manufacturers, multiple vaccine doses, and differently timed follow-up doses. making vaccines widely accessible is a complex endeavor. past experience suggests that this is possible with proactive, thoughtful coordination and clear communication like the following:  utilizing nontraditional vaccination sites like schools, pharmacies, places of worship, workplaces, grocery stores, health departments, mass vaccination clinics, senior centers, home visits, and others. utilizing these sites, as well as clinical sites that already serve vulnerable or underserved populations (e.g., free/low cost community health care clinics, std clinics, substance use treatment centers) will be important to improve uptake in populations that outreach efforts have failed in the past.  preparing, in advance, all necessary educational materials and training that may be needed for those tasked with vaccination at nontraditional sites. training may include information on how to look up immunization records in state immunization registries, how to safely store vaccines, and how to safely recommend vaccines for targeted populations, keeping in mind any contraindications.  anticipating hesitancy among marginalized populations who may be fearful or wary of seeking vaccination at sites that have historically caused mistrust, and plan to either expand sites to better serve these populations or engage these populations early to earn and build trust. this may require using novel sites to better serve marginalized populations (e.g., places of worship, schools, culturally specific community centers or senior centers, mobile clinics). these nontraditional settings will also require those administering vaccines to be culturally competent. vaccination sites should not be heavily policed or send any signals that they may be somehow unsafe for vulnerable persons.  fostering collaboration among interagency and nongovernment partners to make vaccination available alongside provision of other safety net services. bundling services that address individuals' broader needs during the pandemic (e.g., food security, rent assistance, workforce development) could be a way to build trust, streamline vaccine provision, and enhance more convenient access for community members. the protracted covid- pandemic has placed multiple stresses on the american people: the threat of illness and death, the isolating effects of physical distancing measures, and the uncertainties and hardships associated with disrupted economic and schooling activities. the public's patience is understandably wearing thin. operation warp speed is taking revolutionary steps to develop sars-cov- vaccines as swiftly as possible and, along the way, to inspire hope that relief from the pandemic's multiple burdens is coming. despite vaccination's promise of release from the confines of the pandemic, some members of the us public-including those most at risk of covid- 's impacts-are already reluctant to embrace this public health measure [ ] . likewise, current protests against nonpharmaceutical interventions to the sars-cov- crisis, including criticisms about government over-reach, encroachment on individual freedoms, and a clash of personal values, have the potential to further erode public trust in future sars-cov- vaccines. under these circumstances, bold measures are necessary to instill public trust and to change the reality and the perception that covid- vaccination is a top-down program administered without regard to public sentiment, concerns, or priorities. one potential solution to these issues is the formation of public oversight committees at state and, in large metropolitan areas like new york and los angeles, local levels. governance structures that incorporate public oversight and community involvement have the potential to inspire greater public confidence in, and a sense of ownership over, public health interventions. such "ownership" can fortify the intent to vaccinate and strengthen distribution systems to reach throughout communities, thus helping to assure the fitting and fair use of a public good. this type of community engagement entails the collaboration of affected and at-risk populations with policymakers and practitioners in the generation, implementation, and evaluation of measures to safeguard public health and safety [ ] [ ] [ ] . an accountability mechanism and metrics will be necessary to ensure that allocation is fair, target groups receive vaccine, and underserved populations that have been disproportionately affected during the pandemic are justly attended. while vaccines represent a promising solution to the covid- pandemic, the development of vaccines is only part of the answer. widespread acceptance of vaccines is also needed. this acceptance, in turn, requires more than just making safe and effective vaccines available. it is a complex social endeavor that necessitates deep engagement around the human element, and requires the efforts of us policymakers; federal, state, and local public health officials; private funders; professional and community organizations; university researchers; and nontraditional partners. while the content provided in this article is not all-inclusive of what can, or should, be done to support widespread acceptance of covid- vaccines, the recommendations and best practices outlined here are important for such a vaccination program to be successful. as experts in a wide variety of vaccination-related topics, we fear that unless these critical steps are taken, any future covid- vaccination campaign will be less than hoped for. a worst-case scenario would involve an inability to stop the ravages of the disease and its cascading social and economic effects; further erosion of public trust in government, public health, and vaccine science; and potential threat to other life-preserving and live-enhancing vaccination efforts. that said, a successful covid- vaccination endeavor promises an alternative future: a return to a sense of normalcy, major innovations in vaccine research and operations, and the investment of us society as a whole in making vaccines a public good in which all can share and derive value. establish public oversight committees to review and report on systems affecting public understanding, access to, and acceptance of covid- vaccines usg should sponsor a national panel entity (e.g., nasem) to review, synt best practices for engaging communi allocation, deployment, and commun achieve equity, solidarity, and good h each state (and the most populous cit committee that is demographically re incorporates diverse sectors of societ and faith communities. abbreviations: hcd = human centered design; nih = national institutes of health; nsf = national science foundation; cdc = centers for disease control and prevention; activ = accelerating covid- centers for disease control and prevention. coronavirus disease cases in the us the evidence and tradeoffs for a 'stay-at-home' pandemic response: a multidisciplinary review examining medical, psychological, economic and political impact of 'stay-at-home' implementation in america post-abc poll finds. the washington post department of health & human services. fact sheet: explaining operation warp speed the college of physicians of philadelphia. vaccine development, testing, and regulation coronavirus vaccine tracker. the new york times group on vaccine hesitancy. vaccine hesitancy: definition, scope and determinants understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature widespread declines the shares who say they would get a covid- vaccine on behalf of the working group on readying populations for covid- vaccine. the public's role in covid- vaccination: planning recommendations informed by design thinking and the social, behavioral, and communication sciences converge. covid- working groups for public health and social sciences research the public's response to the medical apartheid: the dark history of medical experimentation on black americans from colonial times to the present more than tuskegee: understanding mistrust about research participation implicit racial/ethnic bias among health care professionals and its influence on health care outcomes: a systematic review pandemics and health equity: lessons learned from the h n response in los angeles county public trust in vaccines: defining a research agenda the need for a multi-disciplinary perspective on vaccine hesitancy and acceptance science during crisis: best practices, research needs, and policy priorities converge. converge training modules department of health and human services. nih director's emergency transformative research awards human genome project information archive - . ethical, legal, and social issues the ethical, legal, and social implications program of the national human genome research institute: reflections on an ongoing experiment national aeronautics and space administration preparedness for a high-impact respiratory pathogen pandemic protecting humanity from future health crises: report of the high-level panel on the global response to health crises will ebola change the game? ten essential reforms before the next pandemic. the report of the harvard-lshtm independent panel on the global response to ebola world health organization. implementation of the international health regulations: report of the review committee on the role of the international health regulations in the ebola outbreak and response community-centered responses to ebola in urban liberia: the view from below implementing a novel community engagement system during a clinical trial of a candidate ebola vaccine within an outbreak setting a design thinking approach to effective vaccine safety communication design thinking in health care user-centered design for developing interventions to improve clinician recommendation of human papillomavirus vaccination trump administration announces framework and leadership for 'operation warp speed cnn poll: most americans would be uncomfortable returning to regular routines today could trump turn a vaccine into a campaign stunt national vaccine advisory committee (nvac). h n vaccine safety risk assessment working group coronavirus disease (covid- ) situation report - coronavirus: the us resistance to a continued lockdown. bbc news defining misinformation and understanding its bounded nature: using expertise and evidence for describing misinformation european external action service strategic communications and information analysis division. eeas special report update: short assessment of narratives and disinformation around the covid- /coronavirus pandemic addressing health-related misinformation on social media weaponized health communication: twitter bots and russian trolls amplify the vaccine debate press freedom critical to countering covid- 'pandemic of misinformation': un chief surge of virus misinformation stumps facebook and twitter the new york times anti-vaccination leaders seize on coronavirus to push resistance to inoculation antivaccination activists are growing force at virus protests. the new york times there isn't a covid- vaccine yet, but some are already skeptical about it the epic battle against coronavirus misinformation and conspiracy theories anti-vaccine movement could undermine efforts to end coronavirus pandemic, researchers warn. nature news social construction of the valuebehavior relation the jossey-bass health series. health behavior and health education: theory, research, and practice a comparison of the theory of planned behavior and the theory of reasoned action vaccine education spectrum disorder: the importance of incorporating psychological and cognitive models into vaccine education preserving civility in vaccine policy discourse: a way forward boomerang effects in science communication: how motivated reasoning and identity cues amplify opinion polarization about climate mitigation policies researchers say that the debate over the coronavirus may become more violent focus group methodology: principle and practice qualities of qualitative research: part i american indian/alaska native and russian/ukrainian communities the impact of social networks on parents' vaccination decisions h n influenza vaccination campaign: summary of a workshop series fair allocation of scarce medical resources in the time of covid- the equitable distribution of covid- therapeutics and vaccines racial bias in pain assessment and treatment recommendations, and false beliefs about biological differences between blacks and whites racial disparities in exposure, susceptibility, and access to health care in the us h n influenza pandemic national research council (us) panel on race, ethnicity, and health in later life understanding racial and ethnic differences in health in late life: a research agenda state-of-healthcare-in-the-united-states/racial-disparities-in-health-care/; californians required to cover their faces in 'most settings outside the home'. the washington post cdc's h n vaccine pharmacy initiative in the united states: implications for future public health and pharmacy collaborations for emergency response safety and acceptability of pneumococcal vaccinations administered in nontraditional settings national survey of pharmacy-based immunization services safety of influenza vaccinations administered in nontraditional settings centers for disease control and prevention. national and state-level place of flu vaccination among vaccinated adults in the united states flu season polio: an american story increasing appropriate vaccination: home visits to increase vaccination rates stigma, health disparities, and the h n influenza pandemic: how to protect latino farmworkers in future health emergencies community engagement, organization, and development for public health practice community participation for transformative action on women's, children's and adolescents' health on behalf of the working group on community engagement in health emergency planning. community engagement: leadership tool for catastrophic health events key: cord- - cfhjuxi authors: vergkizi, souzan; nikolakakis, ioannis title: bacillus calmette–guérin (bcg) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: cfhjuxi this work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining bacillus calmette-guérin (bcg) vaccinated site on the dorsum of the ear in guinea pigs. it is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (ppd or leprosin) despite being responsive to whole mycobacteria. besides, t cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to ppd in the presence of autologous antigen presenting cells. interestingly, addition of as low as % nylon wool adherent cells to these, sharply decreased the proliferation by %. looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the t cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. since bcg induced granulomas resolve much faster than granulomas induced by other mycobacteria such as mycobacterium leprae the present experimental findings add to the existing evidence that intradermal bcg vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in covid- patients. regulatory cells play an important role in the control of immune responses. under optimal conditions, immunocompetent cells help, amplify or suppress the activity of other cells so that any foreign material or invading pathogen are eradicated with minimum damage to the host. lapses of such regulation could lead to excess detriment to the body or susceptibility of the host to the invader. normally, under average conditions of antigenic stimulation, the immunostimulatory and suppressor activities are in equilibrium. what conditions decide which of the two mechanisms should choose to occur depends on the host and the occurring infection and are still under investigation. furthermore, it is now generally accepted that the immune response is a collaborative result involving different populations and subpopulations of cells [ ] . helper function to humoral and cellular immunity has been ascribed to subpopulations of t cells, macrophages, dendritic cells and even b cells [ ] . on the other hand, suppression of the two types of immunity has been thought to be regulated by mainly regulatory t cells (former suppressor t cells), certain populations of monocytes/macrophages and b cells [ ] [ ] [ ] . in a number of diseases that are associated with low cell mediated immunity, t regulatory (suppressor) cells play an important role, either on their own or in combination with other cells [ ] [ ] [ ] . macrophages may be either stimulatory or inhibitory in immunological reactions and exhibit both pathogenic and protective roles [ ] [ ] [ ] [ ] . they not only present antigens to mainly t and b cells but also secrete several cytokines which direct the responses of other immunoregulatory cells. the three major functions of macrophages include degradation of non-self or foreign material including apoptotic or necrotic cells, initiation and enhancement of the immunological activation of lymphocytes and, mediation of suppression [ , ] . macrophages may cause suppression either by helping the generation of other suppressor cells [ , ] or, by releasing immune suppressive factors such as prostaglandins [ , ] and immunoregulatory cytokines such as interleukin- these factors in turn, cause the limitation of extensive tissue damage by diminishing the production of inflammatory mediators that cause specific and unspecific immune reactions [ ] . b cells may also cause suppression under certain immunological conditions. involvement of b suppressor cells was demonstrated in delayed type hypersensitivity responses to antigens such as ovalbumin, , -dinitro- -fluorobenzene and keyhole limpet haemocyanin among others [ ] [ ] [ ] . they may act through a negative feedback by specific antibodies or through the induction of suppressor t cells [ ] . their involvement in autoimmune diseases such as multiple sclerosis through memory cell function has also been stressed [ ] . the granulomas induced by bcg are very different from those induced by mycobacterium leprae (m. leprae) in the guinea pig and have been extensively studied for their immune responsive effects. bcg induces an 'immunological' epithelioid cell granuloma that shows containment, successful killing and degradation. here the mononuclear phagocyte series take the form of epithelioid cells with extensive rough endoplasmic reticulum. on the other hand, m. leprae forms a 'non-immunological' macrophage-type granuloma that shows absence of organization of cells with failure to completely degrade. there is no evidence of epithelioid cell formation but the presence of undifferentiated macrophages that remain loaded with mycobacteria [ ] . the bcg vaccine has been used for nearly a century now for protection against tuberculosis but, it also protects against leprosy at a varying magnitude [ ] . recent interest in bcg was triggered because of its relation to the reduction in the severity and the mortality rate of covid- patients that were vaccinated [ ] [ ] [ ] [ ] [ ] and, this has been associated with trained immunity [ , ] . according to some reports bcg may be an option to enhance immunity of at-risk populations such as the elderly and healthcare workers for covid- disease [ ] [ ] [ ] . the objective of this work was to investigate the immune regulatory mechanism responsible for the induction of an 'immunological' type granuloma in the draining lymph node after bcg vaccination in guinea pigs and their early resolution in contrast to that observed with another mycobacterium, m. leprae. outbred dunkin hartley strain of guinea pigs of either sex were from david hall, newchurch, staffs uk. they were fed on rgp pelleted diet (c.f. dixon and sons, ware, herts) supplemented with cabbage and hay. protocols covering the use of animals were followed strictly according to the legislation for animal research of 'the animal (scientific procedures) act, uk . concanavalin a (con a, pharmacia fine chemicals, sweden) was dissolved in phosphate buffered saline at a concentration of . mg/ml, filter sterilized and stored in aliquots at À °c until use. ppd (tuberculin purified protein derivative, central veterinary laboratory, weybridge, uk) À mg/ml was dialyzed against volumes of pbs at °c for h. it was filter sterilized and stored in aliquots of . ml at À °c. leprosin, a soluble extract of m. leprae was obtained from the clinical research center, harrow, london. live bacillus calmette-guérin (bcg, pasteur strain) was obtained from the pasteur institute (paris). in cell cultures, it was used as such, heat killed ( °c for min) or cobalt irradiated at megarads (co-irr). the m. leprae used was always cobalt irradiated ( megarads) because of legal restrictions on the use of the live form due to its pathogenicity in man. guinea pigs weighing about g were injected intradermally on the dorsum of the ear with  bcg, a live attenuated vaccine or  co-irr m. leprae in . ml saline. autologous peritoneal exudate cells were used as antigen presenting accessory cells. animals were injected intraperitoneally with ml paraffin oil and on the fourth day the washing from the peritoneal cavity was collected and immediately centrifuged at g for min to remove the oil, washed twice with plain culture medium and suspended in complete medium ( viable cells/ ml) for further use. the draining post auricular (pa) and cervical (cer) lymph nodes were collected weeks after immunization in the case of bcg (unless otherwise stated) and after weeks in the case of m. leprae. hematoxylin and eosin staining of histological sections and electron microscopy of the lymph nodes were done as described by narayanan et al. [ ] . they were cut into small pieces in hank's balanced salt solution (hbss), gently teased to release the cells and the suspension of cells was passed through a steel wire mesh to remove the cell debris. cells thus obtained were washed three times with hbss at g and re-suspended ( .  viable cells per ml) in complete medium (rpmi- supplemented with % fetal calf serum, iu/ml penicillin, lg/ml streptomycin, mm l-glutamine and mm sodium pyruvate).  of these cells in ll were cultured in -well round-bottom plates with lg/ml ppd, lg/ml leprosin for h or lg/ml con a for h at °c in the presence of % co . lci h thymidine (amersham international) was added h before harvesting and the uptake was estimated using liquid scintillation counter (packard, berks uk) [ , ] . cells were also cultured in the presence of live bcg, heat-killed bcg or co-irr bcg for five days, pulsed with thymidine and harvested as above. all cell cultures were done in quadruplicates. the results are expressed as stimulation index (t/c) defined as counts/min of h thymidine uptake by proliferated cells divided by the counts/min of unstimulated cells. thus, t/c expresses the extent of proliferation of sensitized t cells relative to nonsensitized, unstimulated cells when cultured with antigen or mitogen. additionally, indomethacin was added in the cell culture to check whether the bcg induced suppression was prostaglandin mediated. indomethacin (sigma uk) was dissolved in ethanol at a concentration of mg/ml, the solution was diluted with excess pbs to lg/ml and filter sterilized. when required, lg/ml of indomethacin was added to cell cultures. to identify which fraction of the cell population of the cervical lymph node was responsible for the observed lack of proliferation, the total cells were separated into adherent and non-adherent fractions using a nylon wool (nw) column, a rapid, single step and cost effective procedure for the separation of high viability t cells that flow out in the non-adherent fraction, from heterogeneous mononuclear cell preparations [ , , ] . briefly, cells in ml complete medium were incubated for min in the nw column that was pre-incubated for h with % fetal calf serum. the nw non-adherent cells that are enriched in t cells were eluted with % fetal calf serum, washed and suspended in complete medium. subsequently, the adherent cells were released by teasing the nylon wool and eluting them out. the non-adherent and adherent cells were suspended separately at concentration  viable cells/ml in complete medium for further use. the non-adherent and adherent cell populations were mixed at ratios of : , : , : , and : in cell culture plates to make a total of  cells per well in ll complete medium. these were cultured in quadruplicates in the presence of lg/ml ppd as the antigen and  irradiated ( rads) autologous pecs as the antigen presenting cells (section . ). cell phenotypes present in the nw adherent fraction of the cervical lymph node cells from bcg injected guinea pigs were identified after staining them with respective antibodies.  nw adherent cells were incubated for min in ll of the following anti guinea pig cell monoclonal antibodies [ , ] : msgp , pan t marker antibodies; ct , putative t suppressor cell marker antibodies (free university amsterdam); msgpm, anti-macrophage antibodies; ct (b cell marker). the cells were washed with pbs and incubated for min with fluorescein isothiocyanate labeled anti mouse igm or igg (dilution : , sigma, uk) and the number of positive cells was estimated on a flow cytometer (facs- , becton-dickinson, rutherford, n.j., usa). to elucidate the relative involvement of either t cells or macrophages (isolated in the nw adherent fraction of the cervical lymph node) in the suppression of cell proliferation, enriched fractions of each of the two populations were prepared by depleting the other using immunomagnetic separation. -  cells from the adherent fraction were incubated with . ml of msgpm or msgp for min, washed and incubated with magnetic beads coated with sheep anti mouse igg (for msgpm) or anti mouse igm (for msgp ) (dynal, oslo, norway), for min at °c. the number of beads added was - per positively labeled cells (section . ). the suspension was diluted - times with hank's balanced salt solution containing % fetal calf serum and placed on a cobaltsamarium magnet (magnetic development, swindon, wilts, uk) that attracted the rosetted cells to the sides of the tube (fig. ) . the unrosetted cells were decanted off and the process was repeated. therefore, cells in the decanted fraction contained either the t cell enriched or the macrophage enriched fraction which were subsequently added separately to the non-adherent t cells ( .  cells adherent cells plus .  cells non-adherent cells). the final cell mixtures were cultured in quadruplicates with lg/ml ppd in the presence of  irradiated pecs as antigen presenting cells (section . ). the effects of injected bcg and m leprae on the stimulation index (t/c) measured in the post auricular and cervical lymph node cell preparations cultured with ppd, leprosin, concavalin a, or bcg: live, heat killed or co-irr were compared using student's t-test for means comparison. since the bcg and m. leprae were injected to different animals the independent samples t-test was applied. depending on the result of the equality of variances levene's test the appropriate degrees of freedom were used. differences in the t/c means were considered significant at the p < . level. the measurements are represented as means ± sd. all statistical analyses were conducted using spss statistical software (ibm inc., chicago, il, usa, version . , ). for non-linear model fitting and graphical presentations sigmaplot for windows . (systat software inc. san jose, california, us) was used. in fig. , granulomas in the draining post auricular lymph nodes formed after injection of live bcg or co-irradiated m. leprae on the dorsum ear are shown. histological sections stained with hematoxylin & eosin showed maximum infiltration at weeks for bcg and at weeks for m. leprae. at these times, the draining cervical lymph nodes showed no granuloma formation but extensive lymphoproliferation. fig. a shows a section of a distinct bcg draining granuloma in the post auricular lymph node consisting of large cells with epithelioid cell morphology surrounded by lymphocytes and fibroblasts. no acid-fast bacilli were detected. fig. b shows an enlarged image of an epithelioid cell in the bcg granuloma with characteristic large nuclei, prominent nucleoli and swollen stacked rough endoplasmic reticulum (arrows). fig. c shows a histological section of an m leprae granuloma. the majority of infiltrating cells are phagocytic macrophages. fig. d shows an enlarged image of a macrophage with extensive vacuolation and degraded, undigested remains of m. leprae (arrow). the above findings were in accordance with the results of previous works [ , ] . fig. b corresponding bar plots are shown in the presence of a non-specific mitogen con a. from fig. a it appears that except for pa lymph node cells with ppd, t/c is much lower for animals injected with bcg than m. leprae (compare bars b with d in both groups and a with c in the second). in other words, the cells from bcg injected animals responded to a lesser extent to specific antigens compared with m. leprae. this is particularly obvious for the responses of the cervical lymph nodes cells. the differences were also confirmed by statistical analysis (table ) . except for the pa cells with ppd, the responses of bcg and m leprae injected animals were significantly different for pa with leprosin (p = . ), for cer with ppd (p = . ) and for cer with leprosin (p = . ). on the other hand, in the presence of con a (graph b), the cells from the bcg injected animals proliferated extensively (y-axis scale in b is x larger than in a), indicating that otherwise the cells were active and responsive. furthermore, addition of lg/ml indomethacin did not enhance vaccine xxx (xxxx) xxx the response of the bcg cervical lymph node cells to ppd (first group, lower of the two stacked bars in b), signifying that the suppression was not prostaglandins mediated. the comparatively elevated responses of m. leprae draining lymph nodes to mycobacterial antigens may be an outcome of the excessive inflammatory infiltration observed in vivo and was not further analyzed due to reasons explained below. also, the lower response of these lymph nodes to con a has been addressed by gupta et al. [ ] . to further examine whether the response of bcg injected animals to antigens changes with harvesting time, the stimulation index (t/c) at two and five weeks after bcg injection ( ) to guinea pigs was compared and the results are presented in table . it can be seen that pa lymph node cells responded to ppd both at two and five weeks after injection (t/c from . to . , and from . to . respectively) and the responses at the two harvesting times were not statistically different (t-test, p = . ). additionally, the data in table show that the responses of the cervical lymph node cells both after two and five weeks remained low (from . to . , and from . to . ) and not significantly different (t-test, p = . ). following the unresponsiveness of bcg induced cervical lymph nodes to soluble mycobacterial antigens, it was thought worthwhile to check the response of lymph node cells of bcg and m. leprae injected animals to whole bcg organisms. differently processed whole bcg bacteria ( ) (live, heat killed or co-irr) were used, representing three different states. the results of the cell proliferative responses expressed as stimulation indices (t/c) are presented in fig. . in each group, the first two bars (a, b) correspond to bcg and the last two (c, d) to m. leprae induced lymph node cells. it is seen that for the bcg injected animals the cells from pa and the cer lymph nodes gave t/c between and , i.e. they responded - times more than the starting population. this is in contrast to the previously observed unresponsiveness to soluble antigens (fig. ) , signifying the presence in the lymph nodes of cells reactive to whole bcg. no significant differences are seen in fig. between the responses of bcg draining pa and cer lymph nodes (compare bars a and b in each group). regarding the response from m. leprae injection, with the exception of pa lymph nodes to heat killed mycobacteria, t/c ranged between . (live, bar c) and . (live, bar d) indicating greater overall proliferation than bcg. statistical analysis (t-test) was conducted to identify significant differences between the t/c of pa or cer lymph node cells for bcg and m. leprae injected animals with live, heat killed and co-irr bcg mycobacteria. the results in table show that bcg induced significantly lower response in the cer lymph nodes than m. leprae (p = . ) as it was also the case with the response to soluble mycobacterial antigens (fig. ) . however, bcg induced higher pa lymph node responses compared to m. leprae in the heat killed form (fig. ) bars a and c in the second group) (p = . ) which contradicts the results on soluble antigens, indicating different table statistical analysis (means comparison, t-test) of the effects of bcg and m. leprae injected in the ear on the stimulation index (t/c) of post auricular and cervical lymph node cells in the presence of ppd or leprosin. proliferative responses expressed as stimulation index (t/c, mean ± sd) from the post auricular and cervical lymph node cells of guinea pigs to ppd, two and five weeks after injection with bcg. responses of bcg and m. leprae draining lymph nodes to the heatkilled form of bcg mycobacteria. particularly, this may be due to the inability of proper digestion and presentation in culture in vitro. more studies on m. leprae induced granulomas were not further pursued because of dearth of supply of this mycobacterium. to elucidate which cells of the bcg draining cervical lymph node caused suppression of proliferation and lowering of the stimulation index (t/c) described above, they were separated into two fractions, the nylon wool (nw) adherent and the nw nonadherent. then, the two fractions were mixed at different ratios and the t/c with ppd was determined using pecs as accessory antigen presenting cells. the results are presented in fig. where it can be seen that the non-adherent cells proliferated up to a t/c of when cultured without the adherent, but with increasing proportion (x) of adherent cells the index decreased exponentially. the relationship is described by eq. ( ) with excellent fitting of the data as indicated by the value of the coefficient of determination (r = . ). t=c ¼ : þ : e ðÀ : xÞ ð Þ the sudden drop of proliferation reaching t/c of about at only % proportion of adherent cells, ==signifies their important role in suppression. to further demonstrate the strong abrogative effect of the adherent cells an experiment was conducted using of .  non-adherent cells, the same number as that in the % mixture (point c'), but in the absence of adherent cells (b'). as it can be seen from the dotted line in fig. , the t/c obtained was . , which is about . times higher than the t/c of . obtained with the same number .  non-adherent cells but mixed with equal number of adherent (point c'). this result clearly signifies the strong suppressive effect of the nw adherent fraction of the bcg draining cervical lymph nodes cells. to elaborate the cell phenotypes that were present in the nw adherent fraction of the bcg induced cervical lymph nodes they were labeled with monoclonal antibodies specific for major cell populations found in the guinea pig lymph nodes. these included msgp (pan, total t cells); ct (t suppressor cells); msgpm (macrophages); msgp (b cells). the proportions of the cell phenotypes in the nw adherent fraction of cells from the cervical lymph node are presented in table . the majority were macrophages (total . %) and b cells (total . %) followed by t lymphocytes (totals . %) which consisted mostly of the suppressor lineage ( %), as they labelled with ct , a putative suppressor cell marker. the % difference of the total ( %) from % can be ascribed to a small population of immature or other cells that did not stain with any of the antibodies. it can be noticed that all pan t cells consisted of suppressor/cytotoxic subtype. differences in the percentages of msgp /ct among animals may be ascribed to inter-subject variation. table statistical analysis (means comparison, t-test) of the effects of bcg and m. leprae injection on the stimulation index (t/c) of post auricular and cervical lymph node cells in the presence of live, heat killed or co-irr bcg. levene's test 'student's' t-test since the results of the phenotypic analysis showed that the nw adherent population of cells from bcg stimulated cervical lymph nodes consisted of t cells ( . %) and macrophages ( . %), it was of interest to further look into their role in suppression. for this purpose, enriched cell populations of (i) t cell enriched and (ii) macrophage enriched were prepared from the adherent fraction by immunobead enrichment (section . ). these were added to the non-adherent ( : ratio) in the final cultures. thus, the total number of cells in the resulting cultures was .  enriched adherent (either t cell enriched or macrophages enriched), .  non-adherent,  pecs and ppd ( lg/ml). in fig. are presented the proliferative responses expressed as counts/min of non-adherent cells alone, non-adherent cells with the t cell enriched fraction from the adherent population and non-adherent cells with the macrophage enriched fraction from the adherent population. although there is large inter-subject variability, in out of the five experimental animals, it is seen that when the t cells or macrophages were present individually in the culture, the proliferative response was enhanced. this is more pronounced with the macrophages enriched culture. therefore, t cells or macrophages individually increased proliferation, whereas their co-existence in the nw adherent fraction caused immunosuppression as shown by the reduction of the stimulation index in fig. . in , narayanan et al demonstrated that intradermal injection of mycobacteria in the ear of guinea pigs caused the formation of granulomas in the draining pa lymph nodes while the cer lymph nodes showed no granuloma but extensive blastogenesis. the bcg draining granulomas were analogous to tuberculoid (immunological) leprosy lesions and the m. leprae draining granulomas analogous to lepromatous (non-immunological) leprosy lesions [ ] . therefore, these became excellent models for studying mycobacteria induced granuloma formation and immune responses in an animal model that was not possible in patients. the present study showed that contrary to m. leprae injected guinea pigs, cells from the cervical lymph nodes draining bcg induced granuloma did not respond in vitro to soluble mycobacterial antigens, ppd and leprosin, though they responded to whole bcg organisms. the observed suppression was neither due to prostaglandins (addition of indomethacin did not increase the stimulation index (t/c), fig. ) nor early harvesting of cells from the lymph nodes. interestingly, these cervical lymph nodes did contain ppd reactive t cells as was confirmed when the latter were cultured with the antigen in the presence of autologous accessory antigen presenting cells. additionally, neither the adherent macrophage fraction nor the adherent t cell fraction individually suppressed the response of the t cells, signifying that their synergistic action was required. in humans, bcg vaccination provides long-term imprinting of suppressor t regulatory phenotypes with low inflammation [ ] . similarly, suppressor macrophages have also been demonstrated in a number of studies involving mycobacteria and other intracellular pathogens [ , ] . their mode of suppression may be through direct contact with lymphocytes or by releasing suppressive mediators [ , ] . it has also been demonstrated that macrophages and t cells may act together in concert to induce suppression of other cell functions [ ] [ ] [ ] [ ] . in the present investigation, the in vitro suppression observed in the cervical lymph node cells was not due to classical tolerance to the antigen, because cells from the adjacent pa lymph nodes showed marked responses. therefore, an immune-controlling regulatory mechanism must operate in the cervical lymph nodes of guinea pigs injected with bcg but not with m. leprae. and, this must boost the early resolution at weeks post injection along with progressive replacement of the infiltrated areas by fibrosis and decrease in the lymph nodes weight, signs indicating recovery. similar self-contained lesions are observed in healthy human controls after bcg vaccination and, tuberculoid but not lepromatous leprosy where the lesions are of 'infiltrating' type and wide spread. it has therefore been implied that the suppressor activity in certain infections such as tuberculoid leprosy was generated as part of well-regulated immune response [ ] . thus, it is likely that the suppressor cells in the bcg induced cervical lymph nodes, as seen in this study, participate in the early resolving of the granulomas by controlling excessive proliferation or inflammation. the immune system is inherently a ''double-edged sword" and must be tightly regulated [ ] . bcg vaccination offers heterologous protection against unrelated pathogens through altered responses to subsequent stimuli, termed ''trained immunity" [ ] . even though it is not clear to what extent guinea pigs can be infected by sars-cov and sars-cov [ , ] sars-cov- has been found to be immunogenic in these animals [ ] [ ] [ ] . this has sparked interest in the use of bcg vaccine for potential new therapeutic uses and treatment of autoimmune diseases [ ] . in the case of the current covid- pandemic, in countries where bcg vaccination is obligatory, number of deaths due to infection, was reduced [ , ] . through an experimental model, the present work provides evidence for an induced regulatory mechanism and highlights the role of bcg in the regulation of a granuloma by the induction of immune suppressor cells. finally, we show experimentally, the generation of an immunoregulatory mechanism that controls and resolves a granulomatous lymph node condition induced by bcg vaccination. further characterization of the cell populations involved should be fascinating although it is practically beyond the immediate scope of this investigation. in this study, we show that two important cell populations namely, regulator (suppressor) t cells and macrophages function together in a process that lead to the 'immunological' character and early resolution of a granuloma in guinea pigs vaccinated intra dermally with bcg. for comparison purposes, results from a 'nonimmunological' granuloma induced in the same manner by another mycobacterium m. leprae are also presented. s.v. contributed the in vivo and in vitro experiments, interpretation of data and writing of the manuscript. i.n. contributed statistical analysis, interpretation of data and writing of the manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. training the trainable cells of the immune system and beyond antigen processing and presentation to t cells monocyte-mediated regulation of cellular 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putative intermediate hosts ace and ace receptors development of an inactivated vaccine candidate, bbibp-corv, with potent protection against sarscov- immunogenicity of a dna vaccine candidate for covid- part of this article is from the phd thesis of dr s. vergkizi conducted at the royal college of surgeons of the university of london. the work was funded by the british leprosy relief association (lepra) and is dedicated to late prof john l. turk and late dr jill curtis, stalwarts in mycobacterial research of that time. key: cord- -jit xeqc authors: liou, jenn-fa; chang, chih-wei; tailiu, jui-jane; yu, chun-keung; lei, huan-yao; chen, lih-ren; tai, chein title: passive protection effect of chicken egg yolk immunoglobulins on enterovirus infected mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jit xeqc the objective of this study is to evaluate the passive protective efficiency of immunoglobulin in yolk (igy) specific against human enterovirus type (ev ). the antibody was raised by intramuscular immunization to white leghorn hens, with inactivated human ev serving as the antigen. the titer and specificity of the antibody were analyzed from purified igy in the egg yolks of immunized hens. results indicate that the titer of igy specific against ev increased from the third week after the first immunization. the content of total igy was ± mg/yolk, with an average concentration of specific igy of . ± . mg/yolk in the eggs from to wk after immunization. the results of the neutralization effect of specific igy in ev -challenged mice demonstrate that the ev -specific igy, either by intraperitoneal injection or oral administration, was able to significantly reduce the morbidity and mortality in ev infected mice pups. researchers have extensively studied passive immunization in both humans and animals using specific antibodies to protect against pathogens. an increase in antibiotic-resistant bacteria and the desire to treat pathogens that do not respond to antibiotics, such as viral pathogens, has prompted many researches to use antibodies as alternative to antibiotics [ ] . igy (immunoglobulin in yolk) antibodies are the predominant serum immunoglobulin in birds, reptiles, and amphibians, and are transferred from serum to egg yolk in the females to confer passive immunity to their embryos and neonates [ ] . the potential of orally administered igy for the prevention and treatment of many pathogens has been studied for many years, for escherichia coli [ ] , helicobacter pylori [ ] , salmonella [ ] , rotavirus [ ] , staphylococcus [ ] , streptococcus mutans [ ] , yersinia [ ] , coronavirus [ ] , and the porcine epidemic diarrhea virus [ ] . this paper evaluates the efficiency of igy against enterovirus (ev ). enterovirus belongs to the human enterovirus a family of picornaviridae. these virions consist of a non-enveloped capsid surrounding a core of single-stranded, positive-polarity rna approximately . kb in size [ ] . since the initial description of ev in [ ] , outbreaks of this virus have been identi-fied periodically in countries throughout the world, including the usa, australia, sweden, japan, bulgaria, hungary, hong kong, and malaysia [ ] . in , an outbreak of ev infections occurred in taiwan, in which children were hospitalized and died [ ] . ev infections are generally mild, like hand-foot-and-mouth disease (hfmd) and herpangina, but occasionally lead to severe diseases such as aseptic meningitis, poliomyelitis-like paralysis, and even fatal encephalitis in neonates [ ] [ ] [ ] . in recent years, some efforts have been made to control ev infections. the most promising antiviral agents for ev are win-group compounds, which have undergone clinical trials [ , ] . in addition, a research group from taiwan has developed two candidate ev vaccines, including a formalin-inactivated whole virus vaccine and a vp expressing dna vaccine [ ] . the efficacy of both vaccine constructs is currently being tested in animal models. several approaches have been attempted in the treatment of viral diseases. researchers tried to block the replication of virus through binding the capsid of virus with some chemicals, such as pyridazinamines [ ] , phenoxyl imidazoles [ ] , or pleconaril [ , ] . however, the treatment of ev with pleconaril was not significant, the binding of pleconaril with capsid was limited under higher viral concentration in vitro [ ] . a specific antibody which could bind with the virus and hence reduce the contact of virus with host cells is an alternative choice. human's immunoglobulin has been applied in the treatment of ev infections in babies whose immune system are not well-developed [ , ] . there is no routine therapy for the treatment of ev infection with human's immunoglobulin so far. however, some animal model of ev challenged mouse has been developed, and provided some information about the protective effects of the neutralizing antibody on ev infection [ ] . compared to traditional antibody production, chicken as bio-factory can produce higher yield of igy antibodies than mammals' igg. a chicken can lay eggs in a year and an egg yolk contains - mg of igy antibodies. this study was subjected to produce igy against enterovirus (anti-ev igy) and evaluated the inhibition effects of specific igy on ev , including in vitro virus neutralization test and in vivo icr mice model. this study also provided an animal model for the application of igy in the cure or prevention of ev . the ev strains and mp . strain was originally derived from a patient with ev encephalitis [ ] , while strain mp was a mouse-adapted enterovirus with increased virulence in mice, and it was generated after four serial passages of the strain [ ] . ev stock virus of strains and mp were grown in rd (rhabdomyosarcoma) cells, which were maintained in dulbecco's modified eagle's medium (dmem, invitrogen, u.s.a.) containing % fetal bovine serum (fbs, invitrogen, u.s.a.), mm of l-glutamine (invitrogen, u.s.a.), iu/ml of penicillin (invitrogen, u.s.a.), and g/ml of streptomycin (invitrogen, u.s.a.). all animals received humane care as outlined in the guide for the care and use of experimental animals and viral challenge (institutional animal care and use committee; iacuc approval no. ). four -month-old white leghorn laying hens, obtained from livestock research institute, council of agriculture, executive yuan, taiwan, were utilized for the production of anti-ev igy. a formalin-inactivated ev of strain was utilized as the antigen. one milliliter of ev antigen ( g/ml, . × pfu) was emulsified with an equal volume of freund complete adjuvant and immunized intramuscularly to chickens at sites in the breast muscle. four booster injections with freund's incomplete adjuvant were given at weeks , , , and after the first immunization. the eggs were collected daily from the first day to weeks after the last immunization, and stored at • c. the egg yolk was separated, pooled, and kept at − • c prior to igy purification, and all egg yolks from each chicken for each week were pooled into one analysis sample. the isolation of igy was carried out as described by akita and nakai [ , ] , with some modifications. the egg yolk was first mixed with one of nine volumes of cold distilled water (acidified with . n hcl to ph . ) and stored overnight at • c. the mixture was then centrifuged at × g at • c for min to obtain the water soluble fraction (wsf). the wsf was collected and filtered to remove solids. the resulting igy-containing wsf was further purified by ultra filtration using amicon ultra- filter (pl- , millipore), condensing the sample to / to / of its original volume. these wsf concentrates were then subjected to the virus neutralization test and mice challenge experiments. the antibody activity of anti-ev igy was determined using the elisa method described by lee et al. [ ] , with some modifications. briefly, microtiter plates were coated with l of inactivated ev antigen ( g/well), while control wells were coated with rabbit anti-chicken igy antibody ( g/ml, sigma c ). the plate was then incubated overnight at • c. after washing with pbs-tween buffer, % bsa blocking was conducted overnight at • c. the wells were then washed with pbs-tween buffer. next, -fold diluted wsf was added to the sample wells ( l/well) for testing. wsf from the same chicken before immunization was used as a control. to generate the standard curve, wells were filled with l serial-diluted pure chicken igy at a concentration from . g/ml to g/ml (promega, g a) and incubated at • c for overnight. after washing with pbs-tween buffer, l of alkaline phosphate-conjugated goat anti-chicken igy (promega, g a) was added to the wells and incubated at • c for h. after washing with pbs-tween buffer, l of disodium p-nitrophenyl phosphate was added to each well as a substrate (sigma, n ) and allowed to react at • c for min. the absorbance was then measured at nm using a microplate reader (multiskan ms: thermo labsystems). the resulting absorbance of standard curves provided a relative measurement of anti-ev igy concentration. for the total igy determination, each well on the microtiter plate was first coated with l of rabbit anti-chicken igy antibody ( g/ml, sigma c ), to which l of , -fold diluted wsf was then added. the following experiments were performed following the same protocol described above. the ev was run on sds-polyacrylamide gel electrophoresis (sds-page) and transferred electrically onto a nitrocellulose membrane. the membrane was divided into parts and soaked in blocking buffer ( % nonfat dry milk in tbst) for min at room temperature, then incubated with anti-ev or non-specific igy for h. after washing times with tbs-t, the membranes were incubated with peroxidase-conjugate goat anti-chicken igy ( fold dilution in blocking buffer for each) at room temperature for h. immunoreactivity was detected by incubating the membranes with . % (w/v) -methoxy- -naphthol in tbs and . % (v/v) h o . the tcid of mp virus and the neutralization titer of anti-ev igy were determined using the method described by yu et al. [ ] . in the tcid tests, each -well × rd cell was added to a microplate well, followed by the -fold serial diluted virus. the % tissue culture infective dose was determined after incubation under % co at • c for days. in the neutralization tests, each l sample of serial diluted anti-ev igy concentrate solution was mixed with l of tcid ev in a microplate well. two hours later, the rd cell suspensions ( × cell/well) were added and further incubated under • c and % co for days. neutralization antibody titers were then identified as the highest dilution of purified igy solution that completely inhibited virus growth. the challenge test of ev of the mouse model in this study was modified from wang et al. [ ] . trials - were designed to test the effect of igy with different neutralization titers on mortality in suckling mice. each one-day-old icr strain mouse was intraperitoneally (ip) inoculated with × pfu of mp virus, which is a mouse-adapted strain of ev . thereafter, igy with different neutralization titers ( , , , and ) was injected ip into the challenged mice - days after inoculation (dpi). the dosage of igy week after immunization mg anti-ev igy was l per mouse. in trials and , the igy treatment dates were - dpi and - dpi, respectively. trials - were used to evaluate the protection effect of anti-ev igy by oral treatment. specifically, trials and used a -gauge feeding tube to orally inoculate mice of - d ( . - . g) and - d old ( . - . g), respectively. the mice were given l of mp virus ( × pfu/mouse) after -h of fasting. for each pup, l of anti-ev igy with a titer of was orally administered either h before or h after viral challenge. all mice were checked daily for their body weight and the syndromes of ev infection, including limb paralysis and abnormal hair coat until weeks after viral infection. morbidity and mortality data was collected for two weeks after the viral challenge. the concentration of total igy and specific anti-ev igy was analyzed by elisa. the content of igy in the wsf of egg yolk was investigated for a period of weeks, from the first week of immunization until weeks after the last booster injection. the average content of total igy in each yolk was ± mg (table ) , which was % of the egg yolk's total protein. after condensation by ultrafiltration, the total protein per egg was ± mg on average, and the purity of total igy increased to %. the content of specific anti-ev igy increased gradually from the third week after the first immunization, as fig. indicates. a variation in immune response among individual hens was noticed from the large standard deviation. the average anti-ev igy concentration reached . ± . mg per yolk between the third to eighteenth weeks, equivalent to . % of the total igy. the neutralization titer of specific anti-ev igy was determined by the cytopathic effect (cpe) of ev on rd cells (fig. ) . the resulting neutralization titers of specific igy obtained from eggs of all immunized chickens ranged from to (data not shown). comparing the neutralization titers to the contents of specific igy in the respective yolks showed no significant cor- b the igy-containing wsf was further purified by ultra filtration using amicon ultra- filter (pl- , millipore), condensing the sample to / of its original volume. these wsf concentrates were then subjected to the virus neutralization test. relation between these two sets of data (r = − . , data not shown). western blotting analysis of the immunoreactivity of anti-ev igy. the ev was run sds-page and transferred onto nitrocellulose filter and probe with ev immunized and non-immunized igy. the ev was detected of ∼ kda molecular mass (fig. a) and anti-ev igy was specifically bound to the proteins witch molecular weight corresponding of ev virus (fig. b ). in the first part of the animal tests, icr mice showed both limb paralysis and abnormal hair growth after ev challenge in the ip model (fig. ) . in trials - was conducted to investigate the effect of ip injected specific igy on curing ip ev -challenged mice (table ) . when one-day-old icr mice were challenged with ev of strain mp with a dose of pfu per mouse, the average morbidity was . ± . % in the control group. the resulting mortality was . ± . % based on the challenged mice, or . ± . % when based on illness in mice injected with only non-immunized igy (table ) . we ip administrated the purified specific igy derived from condensation of the immunized yolk wsf to ev -challenged mice. this administrated was applied continuously for days, from the day after viral infection ( dpi) to the third day post infection ( dpi). the therapeutic effects of igy varied according to the neutralization titers of the specific igy administered. the infection rate was % when the titer of specific igy was , which is same infection rate as in the control group (table ). the treatment of igy with a titer of did not significantly reduce mortality in trial . however, when the igy titers were or higher, the morbidities reduced to %, %, and %, respectively for igy treated groups with titers of , or . only the group of titer (trial ) had some deaths after viral infection and igy injections, with a mortality rate of %. without the treatment of specific igy, the challenged mice showed high morbidities and mortality. in trials and , if the treatment between and ) . c non-immunized igy. # differences in proportions morbidity and mortality were tested with the use of the x statistic (p < . ). time of specific igy began at or dpi, the morbidities increased to % and %, respectively. this reduction in cure effects increased mortalities by % and %, respectively. the mortalities of mice showing illness were % and % in trials and , respectively, indicating a decrease in cure effects of specific igy following the delay of igy administration for one or days. the second part of the animal tests involved oral administration of specific igy to ev orally challenged mice. in trial , a dosage of × pfu mp strain ev was orally fed to pups or days old with a body weight of . - . g. the results indicated that if l of specific igy was orally fed h before the viral challenge, the morbidity and mortality of challenged mice was % and %, respectively. both morbidity and mortality were significantly reduced compared to those of the control group, at % and %, respectively (table ). when the specific igy was given to the pups h after the viral challenge, the infection rate was % with a final mortality rate of %. the morbidity and mortality rates of the specific igy treatment groups were lower than those of the control group. regardless of whether the igy was fed h before or after the viral challenge, virus neutralization by specific igy was effective. comparing the mortality of mice exhibiting infection symptoms, the igy treated groups showed a similar rate to the control groups, which all exceeded %. the igy application in trial was similar to that in trial , changing only the age of challenged pups from -to -days old, with their body weight ranging from . to . g. under this situation, the average infection rate decreased to % in the igy treated group and % in the control group. also, the mortality in the igy treated group among ill pups was lower than that in the control groups. although the average mortality of igy treated groups was as low as %, the control groups also exhibited a lower mortality ( . %) than the control groups in trial . in recent years, pathogen-specific igy has also been demonstrated to be effective in the passive protection of human's diseases, such as staphylococcus for holotoxin [ ] , rotavirus for diarrhea [ ] , dental caries caused by s. mutans [ ] , and h. pylori for gastric ulcers [ ] . in this study, we tried to produce the specific igy against human ev . this enterovirus was first reported in the united states in [ ] and has subsequently been reported worldwide [ , ] . in taiwan, some severe cases of human ev are reported every year after the devastating ev outbreak of [ ] . the seriousness of ev infection lies in its complications, which include hand-foot-and-mouth disease, herpangina, aseptic meningitis, poliomyelitis-like paralysis, and fatal encephalitis [ ] . most fatal cases occur in children under years old, and high mortality is correlated with brainstem encephalitis [ ] . the specific igy against ev in the egg yolk increased after the third week after the first immunization, though there were obvious differences among individual hens. this variation in antibody pro-duction, based on the specific igy production, might be the result of differences in individual immune responses [ ] . the origin of antigen also plays an important role in the immune response, as different outer membrane proteins or fimbrial adhesions from the bacteria affected the rate of igy production [ , ] . when the protein antigen contained whole bacteria, or when the virus achieved better specific antibody production, the in vivo neutralization effect of the resultant antibody was also better [ ] . this study used the whole virus as the antigen source to obtain a better igy titer. the total protein obtained from an egg yolk by the water dilution method is around mg per egg. this protein contains few lipids and lipoproteins, and accounts for one-sixth of the total protein of a yolk [ ] . in this study, the purity of igy in wsf was approximately - % (table ). this demonstrates that the water dilution method can easily separate the igy from other egg yolk components, as described previously [ , ] . for the production of igy in trials - , the immunization dosage of antigen used was doubled and the immunization schedule was modified. a longer interval of weeks was applied after the fourth immunization, and the last boost was given on week thirteen. thereafter, the average specific-igy against ev between and weeks increased to . % of the total igy, which is significantly higher than that of the pre-test which is only % of the total igy (p < . ). however, whether or not this modified protocol is beneficial to the production of specific igy remains to be clarified. an analysis of the relationship between the concentration of specific igy with the neutralization titer of the same sample revealed no significant correlation (table ). this indicated that the amount of specific igy estimated by the elisa method was not a good indicator of the neutralization titer of ev virus in this study. previous studies on the effect of antibodies against bacillus spores, botulinum pentavalent (abcde) toxoid and anthrax vaccine also suggest that elisa is not a reliable indicator for the neutralization titers [ , ] . the neutralization titer itself is a better indicator of antibody functionality. we determined the concentration of specific igy by elisa method using the cell lysate from the whole virus particle as the coating antigen. the binding between all viral proteins and specific igy would be counted as part of the igy concentration, whether or not the specific antibody was effective in virus neutralization. kovacs-nolan et al. [ ] studied the epitopes of human rotavirus (hrv) by igy and found that only three out of the five sequential neutralization epitopes on the wa strain subunit protein vp were directly involved in hrv neutralization [ , ] . our results indicate that the therapeutic function was significant when neutralization titer of the anti-ev igy was or higher (table ) . however, further research is necessary to identify the antibodies directly involved in the neutralization of ev . this study includes two panels of animal tests for the ev challenge and igy treatment. in trial , we challenged -day-old mice with a mouse-adapted ev strain mp by intraperitoneally administering a dosage of pfu per mouse, and treated with specific igy of neutralization titer . the results revealed no therapeutic effect. on the contrary, if the neutralization titer value of specific igy was or higher, the protective effects were significant (table ). previous studies also show that this level of neutralization titer is effective for the serum igg of mice [ , ] . compared to the control group, the mice in specific igy treated group not only had a lower mortality, but also had normal weight gains. most of the mice in control group showed a weight loss days after the viral challenge, and continued to lose body weight until they died. less than % of the sick mice in the control group survived the artificial infection of ev at a dose of pfu per mouse. the surviving mice gained some weight despite showing signs of illness. after reaching a body weight of . - . g, they recovered from the infection and returned to healthy status. however, their subsequent weight gains were only - % of the mice in the specific igy treated group (data not shown). some surviving mice also exhibited persistent paralysis of the hind limb and remained paralyzed for the rest of their lives, just like some children infected by ev [ ] . in trials - , the ip injection time of specific igy began from the second day after viral infection and lasted for three consecutive days, before the ev syndrome appeared. in practical situations, children infected by ev will probably only receive the igg treatment after they exhibit the typical symptoms. to determine the critical timing of effective treatment, trials and applied specific igy treatment one and two days later, respectively. clearly, the starting time of specific igy injection was essential in reducing the mortality of infected mice. if igy was given on - dpi, at which point most mice exhibited the symptoms of hind limb paralysis, only % of infected mice survived. it has been reported that on the first day of ev infection, the virus is kept in the small intestine, and then spreads to other organs on dpi. the virus attacks most organs on dpi, including the brain stem, lung, spinal cord, and muscle [ , ] . if we started the specific igy treatment on dpi, which corresponds to the onset of clinical symptoms in children, then the therapeutic outcome was improved. in this case, the survival rate of the ev -infected mice could be increased to %, highlighting the importance of the timing of specific igy treatment. before the on-set of clinical symptoms, specific igy treatment has a better chance of achieving effective therapy. some animal studies use oral feeding of egg yolk powder directly, without any purification of specific antibodies from the yolk [ , ] . these studies prove the possibility of passive protection from the de novo form of yolk powder. in this study, to day-old mice were orally challenged with a viral dosage of × pfu per mouse. challenged mice in the specific igy-treated group showed a lower morbidity rate of % ( / ) than the control group, which showed a morbidity rate of % ( / ) . this indicates that the orally fed specific igy effectively neutralized the viral attack in the gastroenteric duct, thereby blocking the infection of virus in challenged mice. the neutralization function appeared to be effective no matter if it was given h before or after the viral challenge. the mortality among specific igy treated mice was % ( / ), which was significantly lower than that of the control group, at % ( / ). however, the effect of specific igy declined for pups aged to days old with a body weight ranging from . to . g. previous research also shows that the age of mice affects the infection results [ ] . mice more than days old would not be infected by ev after artificial challenge, indicating that the immune system of mice at this age could provide suitable protection. when mice are challenged orally or received the igy through an oral passage, the virus and igy pass through gastroenteric duct and be digested by gastric juice, reducing the viral infectivity of the igy function. this was noticed by the lower morbidity after oral challenge with the same strain of ev and a less protective function of igy among infected mice, compared to the results in trials - , where an infection rate near % and a curing function more than % was obtained. the igy specific to human rotavirus exhibits a passive protection against diarrhea in mice [ ] . although gastric juice had some influence on igy, was still protective when a sufficient amount of igy was provided. oral administration of spray dried yolk antibodies specific against salmonella typhimurium or salmonella dublin is effective in preventing salmonella infections in calves [ ] . igy raised against etec antigen has also been proven effective in controlling the diarrhea of piglets by oral administration [ ] . nevertheless, because the to day-old mice pups in this study could not consume egg yolk, we applied a purified antibody from igy instead. this purified igy, derived from the water soluble fraction of egg yolk, was more accessible to the gastric juice and easily exposed to digestive enzymes. the activity of might be influenced by the change of structure and loss of bioactivity [ , ] . based on the limitations of oral administration in suckling pups, a higher titer of igy or a continuous treatment of igy after viral challenge might achieve greater protection. this possibility is worthy of further research. the results of this study, however, indicate the potential of igy in the prevention of ev infection in young children. several studies have been conducted to evaluate the stability of these antibodies. the igy is not degraded during pasteurization at • c [ ] igy was stable after min in - • c, but was no longer active after min in • c [ ] . so igy technology being new potential market applications in medicine, public health, veterinary medicine and food safety. a broader use of igy technology could be applied as biological or diagnostic tool, nutraceutical or functional food development, oral-supplementation for prophylaxis, and as pathogen-specific antimicrobial agents for infectious disease control. in conclusion, the icr mice ip challenged with mouse-adapted strain mp of ev at a dose of pfu per mouse induced an ev infection resulting in a high mortality rate. however, a survival rate of . % ( / ) was achieved if the challenged mice were ip injected, - dpi for consecutive days, with a purified igy antibody with a neutralization titer of or more. when the challenge was carried out orally with a dose of × pfu per mouse, the lower morbidity in igy treated group proved that the protection could be achieved by the neutralization of virus in the gastroenteric duct, thereby reducing the mortality of challenged mice. this is the first study to evaluate the effect of igy treatment on ev infection, and our positive results indicate that further application in the prevention of ev is possible. in the form of an egg-yolk-added drink, yolk powder tablet, or capsule, it is can potentially be used to prevent the early infection of ev . efficient production of chicken egg yolk antibodies against a conserved mammalian protein phylogen of immunoglobulin structure and function. immunoglobulins of the chicken 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complication disorders of central nervous system antibodies to rotaviruses in chickens' eggs: a potential source of antiviral immunoglobulins suitable for human consumption productivity and some properties of egg yolk antibody (igy) against human rotavirus compared withr abbit igg suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on helicobacter pylori in humans key: cord- -eq obbte authors: kaur, manpreet; rai, anant; bhatnagar, rakesh title: rabies dna vaccine: no impact of mhc class i and class ii targeting sequences on immune response and protection against lethal challenge date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eq obbte rabies is progressive fatal encephalitis. who estimates , rabies deaths and more than million pep every year world-wide. a variety of cell-culture derived vaccines are available for prophylaxis against rabies. however, their high cost restricts their usage in developing countries, where such cases are most often encountered. this is driving the quest for newer vaccine formulations; dna vaccines being most promising amongst them. here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. these strategies include use of signal sequences namely tissue plasminogen activator (tpa), ubiquitin (uq) and lysosomal-associated membrane protein- (lamp- ). tpa, lamp- and their combination were aimed at enhancing the cd (+) t cell and antibody response. in contrast, the uq tag was utilized for enhancing cd (+) response. the potency of modified dna vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. interestingly, the dna vaccines that had been designed to generate different type of immune responses yielded in effect similar response. in conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies. rabies, progressive fatal encephalitis [ ] is caused by rabies virus of genus lyssavirus. majority of rabies cases reported from developed countries involve wild animals like raccoons, skunks, bats, and foxes. however, it is a major health concern in developing countries which account for more than % of all human deaths from rabies [ ] . exposure to rabid dogs is the cause of bulk (> %) of human rabies deaths world-wide [ ] . india has a particularly severe problem, with as many as , human deaths and million people requiring post-exposure vaccination yearly. stray and community dogs cause vast majority of human cases. though potent and safe cell-culture derived inactivated vaccines are available, their efficacy may be compromised by disruption of cold chain storage, poor general health status of the subject, poor vaccination techniques. as a consequence, several approaches are currently being investigated experimentally; out of which dna vaccines appear to be particularly promising as they can induce persistent, cell-mediated and humoral immune responses to antigens isolated from a variety of viral, bacterial, and parasitic pathogens. besides their immunogenicity, dna vaccines offer several other practical advantages. dna vaccines being free from foreign proteins may not cause the various side reactions, which may be observed for conventional vaccines. in addition to the safety, they may have benefits of being inexpensive, overcome the need of time consuming procedures that are needed for purification of subunit proteins, are stable, can be stored and transported at room temperature. in animal models of human disease, dna vaccines have been shown to induce protective responses against hiv, influenza, bovine herpes virus, rabies, leishmaniasis, malaria, herpes simplex virus, and tuberculosis [ ] [ ] [ ] . in addition, human clinical trials have established their safety and potency, further encouraging studies in this direction [ ] [ ] [ ] [ ] [ ] . in this regard, various dna vaccination strategies have shown to provide protection against lethal rabies virus challenge. these strategies relied on the usage of adjuvants like cationic-lipids [ , ] , intradermal injection using gene gun [ ] [ ] [ ] , repeated dna vaccination [ , ] or dna vaccine in association with a single dose of anti-rabies immune serum [ ] for immune enhancement. prophylactic immunization was found to be effective in preventing canine rabies [ , , ] . rabies dna vaccine has also been found to be highly efficient in large size mammals [ ] . for post-exposure treatment, single dose of rabies dna vaccine was found to be as potent as dose regimen of cell-culture vaccine in balb/c mice [ ] . considering these studies, we attempted further improvement in humoral and cell-mediated immune response elicited by dna vaccination by antigen trafficking to various cellular compartments. efficient delivery of antigens to both mhc class i and class ii processing and presentation pathways is required for generating an ideal immune response comprising of both cd + and cd + (cellmediated) and antibody (humoral) immune response. accordingly, this study investigates strategies for targeting glycoprotein antigen to mhc class i and class ii pathways for improving its antigenicity, immunogenicity and protective efficacy. for targeting mhc class ii pathway, we utilized tissue plasminogen activator (tpa) and human lysosomal-associated membrane protein- (lamp- ) signal sequences. tpa-fused antigens are highly expressed secreted proteins with elevated uptake by antigenpresenting cells; and thus, bring about a more generalized activation of the immune system. they have been shown to induce significant humoral and cell-mediated responses [ ] . lamp- is a type of transmembrane protein localized predominantly to lysosomes and late endosomes. antigen trafficking of lamp- -fused antigens to the cellular site of mhc class ii processing and presentation pathway could enhance its presentation to mhc class ii restricted cd + t cells [ ] and thus augment the humoral response. on the other hand, for directing the antigen to mhc class i, signal sequence of ubiquitin a- (uq) was employed. uq-conjugated antigens are trafficked through the proteasome, an organelle that generates short peptides for presentation via the mhc class i pathway [ ] . such uq-conjugated antigens are expected to enhance the cellular immune response. uq-conjugated proteins have been shown to generally undergo rapid intracellular degradation and can elicit cytokine responses in the absence of specific antibody production [ ] . thereby, we explored the potential of targeted dna vaccine encoding the glycoprotein antigen fused to tpa, lamp- or uq to elicit superior immune response in comparison to the unmodified (without target sequence) dna vaccine. baby hamster kidney (bhk)- cells; procured from national centre for cell science (nccs), pune, india were maintained in dulbecco's modified eagle's medium (dmem, sigma) supplemented with % of heat-inactivated fetal bovine serum (fbs, biological industries) and u/ml penicillin (amersham) and g/ml streptomycin (amersham), in a humidified % co incubator at • c. virus pitman-moore (pv- ) strain of rabies virus was propagated on bhk- cells. virus was purified, inactivated with beta-propionolactone (bpl) and used for in vitro re-stimulation assay. the challenge virus standard (cvs- ) strain was propagated and maintained in mice brain. it was titrated on bhk- cells to determine the optimal dose for rapid fluorescence focus inhibition test (rffit) to determine virus neutralization antibodies and for intracerebral rabies virus challenge to determine protection conferred. plasmid ptarget-rab-g [ ] was used as the parental plasmid for construction of all the clones used in this study. the glycoprotein ( . kb) gene was amplified by pcr from ptarget.rabgp plasmid using sequence-specific primers (supplementary table ) and cloned in eukaryotic plasmids bearing address tags; pdnavacc vectors (nature technology corporation, nebraska). the sequences of clones bearing the address tags, pgp-native, ptpa.gp.lamp- (genbank accession number eu ), ptpa.gp (genbank accession number eu ), puq.gp (genbank accession number eu ), pgp.lamp- (genbank accession number eu ) were confirmed by sequencing using abi prism, model , version . (sequencing primers listed in supplementary table ) and designated as represented in the fig. . the respective constructs were processed for the purification of plasmid dna using the endofree plasmid isolation maxi kit (qiagen) according to the manufacturer's instructions. the purified plasmid dna ( - mg/ml) was dissolved in autoclaved milli quartz (mq) water and stored at − • c, until further use. the glycoprotein gene was similarly pcr amplified (see supplementary table for primer sequences) using ptarget.rabgp as template and cloned in pqe expression vector (t expression system). rgp was expressed as a fusion protein with × histidine tag in e. coli sg (prep- ) strain and was purified on a ni + -nta column to more than % homogeneity under native conditions. the rgp was dialyzed against mm hepes overnight and stored in aliquots at − • c. the ability of vaccine constructs to express glycoprotein antigen was studied in vitro in a mammalian cell-culture system. briefly, bhk- cells were cultured and seeded at a density of × cells/ml in a -well tissue culture plate, a day prior to transfection. bhk- cells were subsequently transfected with ng dna complexed with l of lipofectamine (invitrogen) and l of plus buffer (invitrogen). the analysis of expression and localization was carried out h post-transfection. for assessing expression, flow cytometric analysis was carried out, in which bhk- cells were transfected with various dna vaccine combinations. transfected cells were fixed with % paraformaldehyde (pfa) and then permeabilized in . % triton x- in pbs. the cells were then probed with mouse anti-rabies polyclonal sera, diluted in pbs containing . % bsa; followed by staining with alexa fluor labeled secondary antibody (molecular probes) diluted in pbs containing . % bsa and analyzed for fluorescence using cell lab quanta tm sc mpl flow cytometer (beckman coulter inc.). control healthy bhk- cells were also stained. ten thousand cells per sample were analyzed using fl filter ( nm) and percent green fluorescent cells were recorded using quanta sc mpl analysis software version . (beckman coulter inc.). for immunoblotting, total cell lysate was prepared. transfected cells were solubilized using radioimmunoprecipitation assay (ripa) buffer [ mm tris(hydroxymethyl)aminomethane (tris), ph . , mm sodium chloride (nacl), . % sodium dodecyl sulphate (sds), % triton x- , % sodium deoxycholate, mm phenyl methyl sulphonyl fluoride (pmsf)] supplemented with protease inhibitor cocktail (sigma). lysosomal fraction was extracted using lysosome enrichment kit for tissue and cultured cells (pierce) according to the manufacturer's protocol. the presence of lysosomes in different fractions was determined by analyzing the activity of ␤-hexosaminidase [ ] . cell membrane protein fraction was prepared by qproteome membrane protein kit (qiagen) according to the manufacturer's protocol. the presence of cell membrane in the fractions was determined by the associated nadh oxidase activity [ ] . culture supernatant proteins were precipitated by ice-cold acetone. solubilized proteins from the total cell lysate, lysosomes, membranes and cell-culture supernatants were subjected to % sds-page, blotted on to nitrocellulose membrane and probed with mouse anti-rabies polyclonal sera, followed by incubation with goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (sigma) and visualized with nbt/bcip substrate (sigma). all animal experiments were conducted in compliance with the animal ethics committee. four to six weeks old female balb/c mice were used to verify the immunogenicity of the constructs. mice were purchased from nin, hyderabad; and maintained in pathogen free environment at the animal house facility. each group comprised of ten mice. mice were vaccinated intramuscularly (i.m.) with g endotoxin-free plasmid dna in l pbs/animal in the individual groups (dna vaccine or vector control), thrice at three-week intervals. control mice were immunized with only pbs. the mice from each group were bled at days, , and ; sera were prepared and stored at − • c. antigen-specific antibody (igg total) and isotypes (igg , igg a) levels were determined by elisa in the serum from immunized mice. recombinant glycoprotein, expressed in bacterial system ( ng/well) in l of . m pbs was coated overnight at • c [ ] . plates were then blocked with % bsa in pbs for h at • c followed by three washings with pbs-tween ( . %). this was followed by incubation with sera samples for h at • c and washing with pbst. secondary antibodies, anti-mouse igg or its isotypes conjugated with horseradish peroxidase; raised in sheep (santa-cruz) were incubated for h at • c. estimation of the enzymatic activity was carried out using tmb as the substrate. the reaction was stopped with l of m h po and the absorbance was measured at nm, with nm as the reference filter using microplate reader (bio rad). the antibody response generated in a group of vaccinated mice was represented as the geometric mean of the absorbance obtained by pooled serum samples of the animals; the reaction being carried out in triplicates. mouse sera were tested in vitro for the presence of virusneutralizing antibodies with rffit, as described previously [ ] . briefly, sera from mice were heat inactivated at • c for min. l of various sera dilutions were mixed with l of the cvs- strain of rabies virus (containing ffd ) in -well tissue culture plate and incubated at • c, % co for min. after the incubation period, bhk- cells ( × ) were added to each well and the plates were incubated for h, following which they were fixed with chilled acetone and stained with fitc-conjugated antirabies monoclonal antibody (vmrd, usa) for min. the wells were washed thrice with pbs, mounted in glycerol: pbs ( : ), and visualized under fluorescence microscope (nikon, japan). data were expressed as the neutralizing antibody titer that is the mean of the serum resulting in a % reduction in the number of the virus-infected cell foci in the presence of the test serum. rabies reference antiserum of known international units (iu/ml) of rabies virus-neutralizing antibody was included as positive control in the assay. splenic cells were prepared by grinding spleens between frosted slides. erythrocytes were lysed with . m ammonium chloride. remaining spleen cells were washed twice with dmem medium and then were suspended in complete dmem medium supplemented with % heat-inactivated fetal bovine serum and − m -mercaptoethanol. viability was determined by trypan blue exclusion test. splenocytes were cultured in triplicate ( × cells/well) in a -well culture plate (costar), stimulated without antigen or with g/ml of bpl inactivated pv- virus or concanavalin a (cona) ( g/ml; sigma), and incubated at • c under % co and % humidity. supernatants were harvested after , and h and the levels of cytokines were determined. levels of il- , il- , il- , and ifn-␥ were determined using bd opt eia tm kits according to manufacturer's protocol (pharmingen). briefly, -well microtiter elisa plate was coated with capture antibody of the respective cytokines and incubated overnight at • c. plate was aspirated and washed thrice and blocked with l subsequently, the protein samples were resolved on % sds-page under reducing conditions and transferred onto nitrocellulose membrane. presence of rabies glycoprotein was detected using mouse polyclonal anti-rabies hyperimmune sera followed by alkaline phosphatase-conjugated anti-mouse igg. the blot was developed using bcip/nbt as substrate. of % bsa for h at • c. after the incubation period, plate was aspirated and washed thrice and incubated with the harvested supernatants for h at rt. the plate was then aspirated and washed five times; plate was incubated with detector (anti-mouse igg-hrp) for h at rt. following this, plate was aspirated and washed times and incubated with l substrate solution for min in dark at rt. reaction was stopped by adding l stop solution to each well. the absorbance was read at nm using a microplate reader (bio rad) within min of stopping the reaction. the concentrations of cytokines in the culture supernatants were calculated using a linear regression equation obtained from the absorbance values of the standards provided by the manufacturer. each vaccine construct was tested in two independent experiments. for challenge, immunized mice were inoculated with ld of rabies virus cvs- strain intracerebrally days postimmunization. the challenged mice were observed for days for symptoms indicative of rabies virus infection. mice that developed complete bilateral hind leg paralysis, characteristic of the terminal stage of rabies, were euthanized for humanitarian reasons. upon challenge, pbs or vector vaccinated mice died within - days. surviving mice were kept and observed for an additional two to three weeks to ensure that they survived the infection. survivorship rates obtained with the different vaccine constructs were compared. the experimental data were analyzed by sigma plot . and were expressed as means ± standard deviations (s.d.). comparisons between individual data points were made using a student's t-test and levels of significance (p value) were determined. p value < . was considered statistically significant. rv-g based dna vaccine constructs were made wherein the glycoprotein gene was fused to various signal sequences (fig. ) to analyze the influence of signal sequences on immunogenicity and generation of rvna titers. the sequences of insert in native dna vaccine construct (pgp) and modified constructs bearing the target sequences, pt.gp.l, pt.gp, pu.gp, pgp.l were confirmed by sequencing. for assessing the expression of dna vaccine constructs, transiently transfected bhk- cells were subjected to flow cytometric analysis. the percentages of cells stained with the antibody are shown in the figure (fig. a) . the transfected cells were expressing the rabies glycoprotein as indicated by fluorescence recorded as . %, . %, . %, . %, . % for pgp, pt.gp.l, pt.gp, pu.gp and pgp.l respectively; whereas the control cells revealed a low fluorescence signal ( . %) (fig. a) . as the majority of cells showed expression of the rabies glycoprotein, it can be inferred that all the constructs were capable of expressing the protein efficiently in transfected cells. for assessing the localization of dna vaccine constructs, transiently transfected bhk- cells were subjected to subcellular fractionation and subsequently visualization by western blotting. for the same, total cell lysate, lysosomal fraction, membrane fraction and cell-culture supernatant of tranfected cells were resolved on sds-page followed by probing with hyperimmune polyclonal serum from mice immunized with rabies virus. prominent immunoreactive protein bands were observed on the blot corresponding to cell lysate of the all the dna vaccine constructs (fig. b, topmost panel) . there was no corresponding band in cell lysate from vector-transfected or mock-transfected bhk- cells (data not shown). the observed molecular weight of approximately kda was consistent with the expected sizes of glycosylated glycoprotein. analysis of subcellular fraction revealed that the pgp.l construct encoded lysosomal form of glycoprotein (second panel). the pt.gp construct encoded secreted form of glycoprotein (third panel). further, dual tagged construct pt.gp.l expressed both secreted and lysosomal form of glycoprotein (second and third panels). pu.gp construct was exclusively expressed in cell associated form (topmost panel). the native construct encoded membrane associated glycoprotein (fourth panel). expression of glycoprotein in various subcellular fractions was comparable to that from cell lysate. to address the issue if these vaccines could induce efficient humoral response was assessed. groups of balb/c mice were vaccinated intramuscularly with dna encoding either the unmodified or modified antigen. anti-glycoprotein antibody response was estimated by elisa with recombinant glycoprotein (expressed in bacterial system, unpublished data). all the mice sero-converted after priming, however, maximum titers were obtained after second booster both for unmodified as well as modified dna vaccine. for clarity, only the means and standard deviations for each group are shown (fig. ) . all vaccine groups mounted antibody response higher than the unmodified vaccine. the highest antibody response was generated in the pgp.l immunized group (p value < . ), closely followed by pt.gp.l. there was insignificant antibody response in vector and pbs immunized mice. trafficking of glycoprotein through different pathways may affect the type of immune response elicited against it. like pt.gp mediated trafficking of glycoprotein from cytoplasm to secretion pathway, which targets molecules through the endoplasmic reticulum and golgi; may lead to higher induction of th type of immune response. likewise, pgp.l mediated trafficking may drive the glycoprotein through trans-golgi network directly to . the isotype profile of the rv-g-specific igg (black bars) and igg a (gray bars) titers in mice immunized by different protocols. each group of mice (n = ) was immunized respectively by dna, vector or pbs. mice were bled at three weeks after the last immunization and glycoprotein-specific igg and igg a titers were detected by elisa. optical density was measured at nm. data shown represent geometric mean titers and standard deviations for each group of animals. endosomes and then to lysosomes, again influencing the th type response. pt.gp.l may channelize the glycoprotein through either of the above pathways, to affect the th type of immune response. on the contrary, pu.gp is expected to enhance the proteolysis of conjugated glycoprotein mediated by the ubiquitin-proteasome pathway for enhancing the processing and presentation for th type of immune response. to examine such a possibility, serum from mice immunized with pgp, pt.gp, pt.gp.l, pu.gp and pgp.l was assayed by probing with isotype specific secondary antibodies. immunization with all the constructs led to an igg -dominated response (fig. ) indicating the th bias. thus, our results show that addition of signal sequence did not affect the isotype inclination following the immunization and there was convergence of the antibody response, in spite of differential targeting. further, we explored the possibility of enhancement in neutralizing antibodies against glycoprotein when modified antigens were employed for the immunization experiments. rabies virusneutralizing antibody (rvna) titers were assessed by rffit; three weeks post the last immunization corresponding to the time of lethal challenge. the rvna titer in all the groups of immunized mice was > . iu/ml; the minimum titer against rabies as recommended by who. as shown in fig. , the highest geometric mean rvna titer was observed for pgp.l ( iu/ml, p value < . ), followed by pt.gp.l and pu.gp with titer of iu/ml. the neutralizing antibody potential of tpa tagged vaccine was found be the lowest, equivalent to the unmodified antigen based dna vaccine ( iu/ml). in comparison, vector or pbs immunized group did not induce significant neutralizing antibodies. t helper cells (th /th ) play an important role in eliciting both humoral and cellular responses via expansion of antigenstimulated b cells and cd + t cells or ctls respectively. the levels of some cytokines which may play key roles in the induction of protective immune responses against rabies virus were studied as parameters of polarization of immune response. th cytokines (il- , il- , and ifn-␥) and th cytokine (il- ) were measured from splenocytes from immunized mice by elisa at , and h after re-stimulation with inactivated pv- virus. il- production substantially increased on immunization with pgp.lamp- ; . pg/ml i.e., ∼ fold higher as compared to the response from control (splenocytes from pbs immunized mice) was observed (p value < . ). all the constructs exhibited significant il- and ifn-␥ production. there was no significant increase in the cytokine levels of mice immunized with vector or pbs. il- production also strongly increased in case of pgp.lamp- , ∼ fold superior than the control group (p value < . ). the cytokine profile is summarized in fig. . the ability of these dna vaccines to elicit protective responses in immunized mice was assessed by intracerebral challenge with ld of virulent rabies virus cvs strain. for controls, vector and pbs immunized mice were also challenged. the lethality of the challenge was confirmed by death of all the mice in the vector and pbs immunized group within - days post-challenge. groups of vaccinated mice that developed significant levels of virus-neutralizing antibodies also survived rabies virus challenge. the protection conferred by dna vaccines was found to be significant (p value < . ). all modified antigen groups apart from pt.gp conferred higher protection than the unmodified dna vaccine, with % and % protection levels conferred respectively. surviving mice did not show any signs of rabies virus infection. kaplan-meier curves for survival of dna vaccine immunized mice are summarized in fig. . a variety of cell-culture derived vaccines are available for prophylaxis against rabies [ ] . however, the high cost of the vaccination therapy along with the risk of developing anaphylactic, neuroparalytic or encephalitic side reactions limit their therapeutic application. these facts indicate the need of more faithful candidate vaccines which must be capable of inducing strong immune response to protect from infection. more importantly, the candidate immunogen must be able to induce a strong th immunity as it has been established that th ; that is, the humoral immune response plays a predominant role in induction of protective immunity against rabies virus [ ] [ ] [ ] . rabies virus glycoprotein is the main antigen responsible for inducing the production of rabies virus-neutralizing antibodies and for conferring immunity against lethal rabies infection. out of the various strategies being employed for enhancing the immunoprophylactic potential of vaccination strategy, dna vaccines have been the most promising. in an effort to develop an optimal dna vaccine against rabies virus, this study was aimed at evaluating the immune enhancement potential of different antigen targeting strategies to selectively improve responses mediated by cd + and cd + t lymphocytes and by antibodies, induced after intramuscular immunization with dna plasmids. addition of target sequences like tpa, lamp- , uq have been employed for vaccination against various pathogens including sars coronavirus [ ] ; dengue virus [ ] ; orthopox virus [ ]; influenza a [ ] ; mycobacterium [ ] . the signal sequences would target the heterologous protein to different sites targeting the model; for (i) high expression and secretion by fusing with tpa and a more generalized activation of the immune system for induction of significant humoral and cell-mediated responses [ ] (ii) lysosomal degradation by fusing with lamp- and class ii presentation [ , ] ; (iii) wider and enhanced immune response by fusion with tpa and lamp- and (iv) cytoplasmic degradation by the proteasome by fusing with ubiquitin and class i presentation [ ] . classically, the transmembrane (tm) region is excised from the dna vaccine immunogen, such that it can be secreted into extracellular milieu. targeted dna vaccines based on immunogens with deleted tm have been successfully employed for vaccination against tumours [ ] . nevertheless, wang et al. found that hemagglutinin (ha) proteins from different serotypes of influenza a virus elicits contrasting response to full length and truncated transmembrane forms [ ] . further, rath et al. reported that tm domain along with a secretion signal of rv glycoprotein was required for eliciting highest level of neutralizing antibodies. they inferred that tm domain is critical for proper folding of protein otherwise the critical epitopes may get disrupted [ ] . gupta et al. also reported that dna vaccine encoding rabies virus glycoprotein lacking transmembrane domain though enhances antibody response but does not confer protection [ ] . therefore, we retained the tm domain in our dna vaccine constructs and utilized full gene for targeting strategies. thus, different plasmid dna constructs were made-pgp, the unmodified constructs and modified constructs including p.gp.l (n terminal tpa and c terminal lamp- ), pt.gp (n terminal tpa), pu.gp (n terminal uq), and pgp.l (c terminal lamp- ). transient transfection of bhk- cells with all the plasmid dna constructs revealed expression of rabies glycoprotein by flow cytometric analysis. majority of the cells were found to express glycoprotein as seen by the fluorescence monitored by cell sorter. thus, dna vaccine constructs were capable of efficiently expressing the glycoprotein. distribution of chimeras was analyzed by subcellular fractionation and immunoblotting. total cell lysate of transfected bhk- cells of all the constructs expressed glycoprotein at approximately kda. the observed high molecular weight of rv-g expressed in bhk- cells could be due to the influence of host factors on glycosylation [ ] . morimoto et al. showed both bhk and murine neuroblastoma (mna) cell lines, transformed with the same retroviral expression vector encoding rv-g cdna, show different patterns of glycosylation of the expressed rv-g [ ] . rrv-g expressed by bhk cells was highly glycosylated and sialylated in comparison to mna expressed rrv-g, indicating that the glycosylation and sialylation of rv g is dependent on the cellular conditions in which rv-g is produced. analysis of subcellular fractions indicated that glycoprotein along with the targeting sequences was suitably recognized by mammalian cells and directed towards the respective pathway. from flow cytometric and immunoblotting analysis of transfected cells, it can be inferred that there was efficient recognition and expression of dna vaccine immunogens in the mammalian system. the signal sequences successfully directed the glycoprotein to respective cellular locations, with comparable levels of expression as of total cell lysate. vaccination of mice with all rv-g plasmid dna constructs led to the generation of anti-rv-g antibodies. all the modified vaccines elicited higher anti-rv-g antibody levels than the unmodified one. the highest antibody response was observed with pgp.lamp- . the generation of rvna is the most important adaptive immune system response for conferring protection against rabies. therefore, to compare the utility of the rv-g plasmid dna constructs, rvna response elicited by each construct was determined by rffit. the neutralizing antibodies were more than . iu/ml, which is the minimum titer recommended by who. the highest rvna titer was elicited by pgp.lamp- which is also supported by an enhanced antibody response by elisa, in comparison to other rv-g constructs. the effectiveness of the constructs to induce th /th type of immune response was indirectly evaluated by determining th (igg a) and th (igg ) antibody isotypes. we found a strong igg response in all the dna constructs. even though igg a antibodies were produced, the ratios of igg /igg a were consistently more than one, thus emphasizing on the th bias. presence of both types of immune responses may be due to the presence of more than one type of antigenic sites in the glycoprotein immunogen. it is worth noting that differential targeting for enhancing th and th responses yielded in effect a similar response. the increase in antibodies to dna vaccine may reflect an effect of the antigen on the t helper cell response needed to promote differentiation of naïve b cells into antibody secreting plasma cells. this was assessed by cytokine profiling of splenocytes immunized with signal sequence tagged glycoprotein based vaccine or only vector; upon in vitro stimulation with inactivated pv- virus. we found that all the cytokines analyzed could be detected from the splenocytes of dna vaccine immunized mice, with a pronounced enhancement in the level of il- and il- in the pgp.lamp- immunized group. for other cytokines, namely il- and ifn-␥, similar levels of cytokines were observed for all the four groups, with the level being several folds in comparison to the splenocytes from control group. antigen binding to the t cell receptor (tcr) stimulates the secretion of il- , and the expression of il- receptors il- r. the il- /il- r interaction then stimulates the growth, differentiation and survival of antigen-selected cytotoxic t cells via the activation of the expression of specific genes. il- facilitates production of immunoglobulins made by b cells and induces the differentiation and proliferation of natural killer cells. il- , produced mainly by macrophages and dendritic cells, is quickly induced by viral infections or by vaccination stimuli. il- strengthens the non-specific immune responses by activating nk cells to produce ifn-␥ and in synergy with ifn-␥, drives the differentiation of cd + t cells into th cells, more adapted to the control of viral infections. various groups of immunized mice when challenged with cvs virus showed higher protection as compared to a vehicle control. high titers of rvna and protection conferred in dna vaccines might be due to the possibility that modified immunogens led to the expression of rv-g with appropriate folding and better accessibility of epitopes to immune system, critical for generating rvna titers. in spite of similar magnitude of immune response generated, protective efficacies against viral challenge varied. the unmodified and secreted forms of vaccines were found to be inferior in inducing protection against viral challenge. xiang et al. also reported that secreted form of vaccine did not confer significant protective immunity [ ] . protection against rabies virus is mainly mediated by neutralizing antibodies [ ] ; subtle differences in the conformation of the secreted protein, not readily detectable by conventional biochemical methods, might select a different repertoire of neutralizing antibodies with lower avidity to the full length g protein present on the surface of viral particles, thus being less able to prevent the spread of virus [ ] . interestingly, pt.gp.l, pt.gp, pu.gp, pgp.l dna vaccine combinations designed to generate different types of immune responses yielded in effect similar data. a probable explanation for this could be that the tagged antigens evoke similar levels of immunity and act to enhance survival via the same primary protective mechanism. we observed that ubiquitination of antigen for mhc class i targeting also enhanced the igg antibody and cd + mediated cytokine response. thus, we infer that the peptides generated by proteasomal degradation could also be presented by mhc-ii. while, there is no specific information of how protein processing in transfected cells occurs in vivo, different mechanisms have been postulated. they include direct priming by somatic cells, direct priming by antigen-presenting cells, or cross priming of antigen-presenting cells. activation by cross priming appears to be the most probable immune mechanism which occurs following intramuscular vaccination that could be shared by the tpa, lamp- and uq vaccines [ , [ ] [ ] [ ] [ ] . cross priming may occur via exit of exogenous antigens from the endocytic compartments and its processing in the cytosol, recycling of mhc-i molecules through endosomal/lysosomal pathway and transfer of processed peptides to the endosomal compartments. it is well known that cd + t-cell stimulation can result from endocytosis of exogenous peptides or proteins followed by antigenic processing via mhc class ii pathways [ ] . lamp- targeting of antigen has been reported to increase the number of immunogenic peptide epitopes that activate cd + t cells, thus inducing a broadened immune response in comparison to untargeted antigen [ ] . recent studies have also demonstrated that exogenous proteins or peptides, possibly complexed to heat shock proteins, can be taken up by antigen processing cells, processed through the mhc class i pathway, and ultimately stimulate naïve cd cells [ , ] . thus, via cross-priming mechanisms, secreted fusion proteins expressed from tpa plasmids, membrane bound fused proteins expressed from lamp- or peptides released from cells transfected with the uq constructs could induce both cd + and cd + t-cell populations. several researchers have applied targeting strategies and reported conflicting results with different antigens and different infectious systems. successful targeting was demonstrated for several pathogens including human papillomavirus [ ] , influenza a [ ] ; mycobacterium [ ] ; but not for all the constructs tested against malaria [ ] . thus, a tagged dna vaccine may represent an 'ideal' immunogen for generating protective immune response, nevertheless; the antigen dependence of immune strategies has to be considered for successful vaccination against any pathogen. further, optimization of immunization doses, routes, schedule, adjuvant supplementation and a greater understanding of the immune mechanisms responsible for producing protective immunity in response to dna vaccination should facilitate the creation of further improved rabies dna vaccination strategies. rabies re-examined world survey of rabies: no. for the year . geneva, world health organization rabies: a new look at an old disease genetic immunization: a new era in vaccines and immunotherapy first human trial of a dna based vaccine for treatment of human immunodeficiency virus type infection: safety and host responses cellular cytotoxic response induced by dna vaccination in hiv- -infected patients induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine excellent safety and tolerability of the human immunodeficiency virus type pga /js plasmid dna priming vector vaccine in hiv type uninfected adults phase i clinical trial safety of dna-and modified virus ankara-vectored human immunodeficiency virus type (hiv- ) vaccines administered alone and in a prime-boost regime to healthy hiv- -uninfected volunteers sustained protective rabies neutralizing antibody titers after administration of cationic lipid-formulated pdna vaccine dna vaccination of mice against rabies virus: effects of the route of administration and the adjuvant monophosphoryl lipid a (mpl r ) gene gun particle-mediated vaccination with plasmid dna confers protective immunity against rabies virus infection rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of dna, inactivated virus, or recombinant vaccinia virus vaccines rabies dna vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers immunization of dogs and cats with a dna vaccine against rabies virus immunization of dogs with a dna vaccine induces protection against rabies virus post-exposure dna vaccination protects mice against rabies virus canine rabies dna vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody field trials of a very potent rabies dna vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions rabies dna vaccine in the horse: strategies to improve serological responses postexposure therapy in mice against experimental rabies: a single injection of dna vaccine is as effective as five injections of cell culture-derived vaccine immunogenicity of dna vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences dna vaccine encoding human immunodeficiency virus- gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response enhancing dna immunization dna vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity development of rabies dna vaccine using a recombinant plasmid cortisone-evoked decrease of acid-galactosidase, glucuronidase, n-acetyl-glucosaminidase and arylsulphatase in the ileum of suckling rats breaking down the wall: fractionation of mycobacteria dna vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response a rapid fluorescent focus inhibition test (rffit) for determining rabies virus-neutralizing antibody rabies virus glycoprotein. ii. biological and serological characterization oral immunization and protection of raccoons (procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens dna vaccines against dengue virus based on the ns gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice targeting the vaccinia virus l protein to the cell surface enhances production of neutralizing antibodies immunization with plasmid dna encoding influenza a virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against h n challenge in mice evaluation of the anti-tuberculosis activity generated by different multigene dna vaccine constructs the enhanced immune response to the hiv gp /lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway hiv- p gag encoded in the lysosome-associated membrane protein- as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses regulating protein degradation by ubiquitination dna vaccines encoding full-length or truncated neu induce protective immunity against neu-expressing mammary tumors hemagglutinin (ha) proteins from h and h serotypes of influenza a viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized ha dna vaccines comparison of rabies virus g proteins produced by cdna-transfected animal cells that display either inducible or constitutive expression of the gene immune responses to nucleic acid vaccines to rabies virus use of mouse anti-rabies monoclonal antibodies in post-exposure treatment of rabies antibodies i post exposure treatment of rabies in vivo priming by dna injection occurs predominantly by antigen transfer mhc-i class restricted ctl responses to exogenous antigens priming of cytotoxic t lymphocytes by dna vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes dna vaccines: immunology, application, and optimization alternative processing of endogenous or exogenous antigens extends the immunogenic, h- class-i restricted peptide repertoire capture and processing of exogenous antigens for presentation on mhc molecules characterization of mhc class ii-presented peptides generated from an antigen targeted to different endocytic compartments receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class i antigen presentation via two distinct processing pathways multiple antigen-specific processing pathways for activating naive cd + t cells in vivo characterization of hpv- e dna vaccines employing intracellular targeting and intercellular spreading strategies targeting antigen to mhc class i and class ii antigen presentation pathways for malaria dna vaccines inactivated pv viral antigen and rabies reference antiserum were kindly provided by dr. v. srinivasan, indian immunological ltd. the authors acknowledge dr. shardul solanke (national biotechnology center, ivri, india) for transfection and anuj kumar sharma (school of biotechnology, jnu, india) for flow cytometry studies. special thanks are extended to dr. subhash chandra (cornell university, ny) for vital inputs in the study. this work was supported by department of biotechnology, government of india. manpreet kaur is recipient of senior research fellowship from csir, government of india. supplementary data associated with this article can be found, in the online version, at doi: . /j.vaccine. . . . key: cord- -y smbtd authors: crouch, c. f; oliver, s; hearle, d. c; buckley, a; chapman, a. j; francis, m. j title: lactogenic immunity following vaccination of cattle with bovine coronavirus date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: y smbtd abstract in order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between and weeks before calving. this was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. the overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. no abnormal local or systemic reactions were observed as a result of vaccination. it is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection. neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination [ , , ] . in calves, economic losses are suered as a result of mortality, which can reach up to %, and also veterinary costs and decreased productivity of survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been shown to be primary pathogens in calves [ , ] . although this syndrome is most overtly associated with young animals, subclinical infections are also common in adults which may therefore act as reservoirs for reinfection [ ] . passive immunity against enteric viral infections is dependent upon the continual presence of a protective level of speci®c antibody in the gut lumen. this can be achieved by allowing the neonate to ingest colostrum or milk containing these speci®c antibodies from its dam (lactogenic immunity). although most adult cattle are seropositive for both rotavirus and coronavirus antibodies [ ] , during the transition from colostrum to milk production there is a dramatic decline in antibody titres which partially explains the high incidence of infection in calves older than days. successful rotavirus vaccines aimed at increasing both the titre and duration of speci®c antibody in both colostrum and milk have been developed [ ] , however, similar success has not been reported with vaccines targeted against bovine coronavirus [ ± ] . this paper reports the ability of a new single dose vaccine to signi®cantly increase both the level and duration of coronavirus neutralising antibodies in the serum and milk of vaccinated cattle. bovine coronavirus was grown in mammalian cell culture. for antigen production, cell culture supernatant was harvested and clari®ed by low speed centrifugation. the virus was then inactivated by treatment with . m binary ethyleneimine for h at c. finally, virus was concentrated up to -fold using a hollow ®bre ®ltration system with a , molecular weight cut-o. bovine coronavirus antigen levels were determined by quantitative elisa. immulon removawell strips (dynex technologies) were coated with an optimal concentration (previously determined by titration) of a sheep polyclonal anti-bovine coronavirus antibody diluted in . m carbonate±bicarbonate coating buffer of ph . . after incubation at c for h, the wells were blocked using pbs containing . % bovine serum albumin and % tween for min at ambient temperature. ml of pbs containing . % tween was added to each well followed by ml of sample, standard or control. after incubation for h at c on a shaker incubator the wells were washed three times using pbs containing % tween . ml of optimally diluted mouse monoclonal antibody speci®c for the virus haemagglutinin was then added to each well, followed by incubation for h at c with shaking. after washing, ml of an optimal concentration of a horseradish peroxidase -conjugated goat anti-mouse igg antibody (nordic immunologicals) was added to each well and incubated with shaking for a further h at c. after washing, bound conjugate was visualised using o-phenylenediamine dihydrochloride (sigma) as the chromagen. after stopping the reaction using . m sulphuric acid, the optical density ( nm) was proportional to the amount of bovine coronavirus antigen in the sample. full quantitation up to a range of antigen units per ml was achieved by including a set of six standards in each assay. the vaccine comprised of rotavirus and e coli k antigens from rotavec k (schering plough animal health) plus inactivated bovine coronavirus antigen and was adjuvanted using a mineral oil based adjuvant. the aqueous and oil phases were prepared separately and combined prior to emulsi®cation using a silverson homogeniser. twelve maiden heifers of mixed breeds that had not previously been treated with vaccines containing coronavirus were housed indoors, bedded with straw, although access was given to a grass paddock for exercise. the animals were fed hay, silage and a standard cattle ration (quinns of baltinglass), water was available ad libitum. the health of all the animals was monitored by daily observation throughout the study. all the animals were bled days prior to vaccination, serum prepared and the coronavirus antibody titre determined. the animals were ranked by descending antibody titre, and allocated into four groups of three animals each based on random assignment of one animal from each third of the ranking. animals in groups to were immunised with ml of the appropriate vaccine. group , vaccine a containing . vaccinated animals received a single ml injection intramuscularly into the neck. all animals were bled before vaccination, , and days post vaccination, heifers in groups and were also bled at , and days post vaccination. blood samples were stored for h to allow clotting to occur, the serum separated by centrifugation, and stored at À c prior to testing for bovine coronavirus (bcov) antibodies by virus neutralisation and haemagglutination inhibition. these were carried out on four farms, two (studies a and b) were beef suckler herds and two (studies c and d) were dairy herds. a double blind trial design was used. thirty cows on each farm were paired according to their expected calving dates and then randomly allocated to either vaccinate or placebo treatment groups ( cows per group). animals were included in the study on the basis that their expected calving dates fell between and weeks later than the day of vaccination. animals were excluded only if unhealthy or where they were known to have been previously vaccinated against coronavirus. all animals received a single injection of ml intramuscularly in the neck. animals in the vaccinate group were immunised with a preparation containing antigen units of coronavirus per dose, whilst animals in the control group were vaccinated with a saline/oil emulsion placebo containing none of the vaccine antigens. rectal temperatures were taken from all animals either days before vaccination (studies a and b) or immediately before vaccination (studies c and d), approximately h after vaccination and days after vaccination. on all sites, injection sites were examined for local reactions day, days and days after vaccination. calves from the beef suckler herds (studies a and b) were allowed unrestricted access to their dams for suckling. calves from the dairy herds (studies c and d) were fed twice a day by hand with approximately . l of colostrum or milk from their own mothers for at least the ®rst days of life. blood samples were collected from all cows at intervals from pre-vaccination to post-calving as indicated in the results section. in studies a and b, milk samples from the dam and blood samples from the calf were collected at day intervals from the day of calving (post-suckling) to days post-calving, whilst in studies c and d samples were only taken on the day of calving (post-suckling) and three days post-calving. blood samples were processed as described above. colostrum or milk samples were stored at À c prior to testing for the presence of bcov neutralising antibody. prior to testing, all sera were thawed, heat-treated at c for min, sub-aliquoted and stored frozen at À c until required. colostrum and milk samples were thawed, centrifuged at  g for min and the heavy liquid fraction removed aseptically by a pipette to a clean sterile container. sterile rennet solution was then added to a concentration of % v/v and incubated for h at c. the mixture was then centrifuged at  g for min and the upper liquid fraction (the whey) aseptically removed by pipette and dispensed into subaliquots of at least . ml before storage at À c. bovine coronavirus neutralising antibody responses were quanti®ed using a modi®cation of a standard plaque reduction assay [ ] . viral plaques were visualised by haemadsorption using % (w/v) rat red blood cells and the virus neutralising (vn) titre de®ned as the % end point following challenge with plaque forming units of bcov. bovine coronavirus haemagglutination inhibition (hai) antibodies were quanti®ed as follows. test sera were ®rst pre-treated to remove non-speci®c agglutinins and inhibitors by incubation with an equal volume of % (w/v) rat red blood cells overnight at ± c followed by treatment with . % (w/v) potassium periodate for min at room temperature. standard hai methodology was used, doubling dilutions ( ml sample + ml pbs containing . % bovine serum albumin) of test samples were carried out in`v' bottomed microwell plates. an equal volume of bcov antigen containing four haemagglutinating units was then added and incubated for h at room temperature. ml of . % (w/v) rat red blood cells was then added and the test plates incubated overnight at ± c. the haemagglutination antibody titre was de®ned as the reciprocal of the highest dilution giving % inhibition of agglutination, allowing for the pre-treatment dilution. all animals possessed some pre-existing antibody to bovine coronavirus as measured by both hai (range log ± and vn (range log x ± x ). no de®ned increase in antibody titre could be detected by either hai or vn in animals immunised with the lowest dose of bcov ( . antigen units). however, animals receiving r antigen units of bcov antigen showed an increase in mean hai antibody titre by day . peak hai titres of log x and log x were observed either days post-vaccination with antigen units per dose, or days post-vaccination with antigen units per dose, respectively. these animals were maintained for days and showed a steady drop in titre of approximately log x per month from day (fig. a) . the vn antibody response was similar but peaked earlier at day with titres of approximately log x x however, while vn antibody levels declined between days and in cattle receiving antigen units (fig. b) , those obtained from animals receiving the higher dose of units did not decline until after day . only the group immunised with antigen units of bcov still possessed a vn titre r log above the pre-immunisation titre at days post-vaccination (fig. b) . the sentinel group showed no increase in antibodies to bcov by either hai or vn during the study. as a result of this study, a bcov antigen load of antigen units per dose was selected for further study on the basis that it was likely to produce an optimal antibody response in terms of antibody titre and duration. vaccines for the cow/calf studies were therefore formulated at this level. the vaccine was well tolerated and no signi®cant local or systemic side eects were observed. the antibody results from these studies are shown in fig. . since virus neutralisation is the key parameter involved in protection by lactogenic immunity, antibody titres were only determined by vn. with the exception of study b, the serum vn antibody levels of the placebo groups did not change signi®cantly during the course of the experiment. in study b however, % ( / ) of placebo treated animals showed an increase in serum vn antibody levels of log x ± x during the experiment suggesting that`wild type' bcov was circulating within the herd. after immunisation, serum vn antibody levels in the vaccinated groups showed a signi®cant p` x increase in serum vn antibody levels compared with those observed in the placebo control groups. mean serum vn antibody levels were increased by at least log x by days post-vaccination and these levels were typically maintained until calving. associated with this increase in serum vn antibody levels, ®rst day colostrum vn antibody was also signi®cantly higher log x ± x in the test vaccine groups compared with the placebo control groups. similar dierences were maintained in milk antibody throughout the period of study (up to day on the dairy farms and up to day on the beef suckler farms). the smallest increase occurred in study b where sero-conversion amongst the placebo treated animals was observed (fig. b) . serum taken from calves allowed to suckle vaccinated dams showed that levels of circulating vn antibody were up to ten-fold higher than those obtained from calves suckling placebo-vaccinated cows. the eect of the interval between vaccination and calving on the increase in milk vn antibody titre can be seen in table where data from studies a and b have been combined into ®ve sets, each set comprising cows (three vaccinate and three placebo cows from each study) with mean vaccination/calving intervals of , , , and days, respectively. for placebo treated animals, irrespective of the vaccination/calving interval, day colostrum possessed vn titres of ap- proximately log x , falling rapidly to levels of approximately log x (background) in day milk. in contrast, for the vaccinated cattle, day colostrum possessed vn titres of approximately log x , with a much slower reduction in antibody level, only approaching background levels in -day milk. the duration of the milk antibody response seen in the vaccinated animals where the vaccination/calving interval was days was less marked than where this interval was longer. this re¯ects a reduced response to vaccination seen in approximately % of the vaccinates in the -day set. bcov antigen input into the formulated vaccine was measured by elisa, since measurements based on infectivity do not quantitate total antigenic mass, only viable virus particles and therefore could not be used post-inactivation. the elisa procedure used was primarily targeted towards the viral haemagglutinin which has been shown to contain a key virus neutralising epitope [ ] . antigen levels could therefore be tracked throughout the formulation processes. the results of the dose response study indicate that in the presence of pre-existing antibody, relatively large amounts of immunogen (> antigen units) are needed to stimulate a signi®cant increase in bcov serum antibody titre. there appear to be some minor dierences in the pattern of the antibody responses determined by either hai or vn in that the hai responses peak later than the vn responses. this may re¯ect dierences in antibody anity or sub-class requirements between the two methods of antibody measurement. in terms of neutralising antibody, the major eect of increasing antigen content appears to be in the duration rather than the magnitude of the response. with a vaccine dose containing antigen units of antigen the response was rapid, reaching a plateau approximately days post-vaccination which was maintained until at least days post-vaccination. with a dose containing antigen units of antigen, the virus neutralisation response was equally rapid and of similar magnitude, although in contrast declining rapidly to days post-vaccination. selection of the correct antigen load therefore appears critical in ensuring an optimal window in terms of the interval between vaccination and calving. an additional advantage in using a higher antigen dose was also demonstrated in cow / calf study b where elevated antibody responses were still observed even in the face of circulating infection. this suggests that vaccination could still play a signi®cant role in enhancing overall herd immunity even in an active disease situation. fig. . mean bcov vn antibody results in sera and milk from vaccinated (q) and placebo treated (q) cows and sera from their respective calves. studies were carried out on four farms, two beef suckler herds (a and b) and two dairy herds (c and d) and samples were obtained at vaccination (v), calving (c) or at the number of days indicated after the event. error bars denote % con®dence limits. the main source of antibody in bovine mammary secretions is serum igg . this is selectively transferred from serum throughout lactation, albeit at a reduced level in milk compared to colostrum [ ] . the initial serology data therefore suggested that with a bcov antigen load of approximately antigen units, successful vaccination could take place at any time between and weeks pre-calving. this hypothesis was supported by the observations on the magnitude of the antibody titres found in the colostrum and milk of cows vaccinated at dierent times pre-calving with a vaccine containing antigen units of bcov. signi®cant responses in colostrum and milk were observed at both ends of the vaccination window, although not all cows vaccinated approximately days pre-calving appeared to show an optimal response. individually, there was a large variation in the levels of speci®c bcov antibody present in both vaccinate and placebo control groups throughout the study. this variability was particularly noticeable in colostrum and milk samples, and presumably arose from the dierential levels of pre-existing bcov antibodies observed in the animals. although this would be expected to adversely aect the statistical testing of the results, signi®cant dierences p b x between test and control groups were found on the majority of days tested. this contrasts strongly with previously reported studies wherein increases in bcov antibody titre in serum or milk following vaccination were either minimal or non-existent [ ± ]. the reasons for the improved responses observed with the test vaccine are related both to the concentration of bcov antigen present and the use of an oil adjuvant, which have generally been reported to be highly eective in the enhancement of rotavirus antibody titres in mammary secretions [ ] . the magnitude of the antibody response observed following a single vaccination may be the result of the use of pre-primed (as a result of previous infection) cattle. bovine coronavirus is ubiquitous and in the ®eld situation it is extremely dicult to ®nd a seronegative individual. this observation is supported by the data presented in this study where a total of heifers and cows, with no previous bcov vaccination history, were tested prior to treatment and all were found to have substantial levels of pre-existing antibody. a major decline in speci®c antibody levels present in mammary secretions was observed in the ®rst few days after calving. this is consistent with previously reported research [ , ] , which showed that the switch from colostrum to milk was associated with a decrease in immunoglobulin concentration. enhanced antibody levels are maintained for to days post-calving, although as bcov speci®c antibody levels decrease calves will become more susceptible to sub-clinical infection. such infections will stimulate active immunity and hence confer long term protection upon the calf. in the beef suckler situation, where the calf is allowed to suckle naturally from its dam, it is relatively easy to ensure the continual presence of protective antibody in the gut lumen. in the dairy situation, typically the calf is allowed access to colostrum but is prevented from suckling milk and thus fails to ensure the presence of protective antibody in the gut lumen beyond the ®rst few days after birth. a modi®ed feeding regimen is therefore required whereby colostrum table the eect of the interval between vaccination and calving on the magnitude of the bcov vn antibody response in mammary secretions mean log bcov vn antibody titre in colostrum/milk following vaccination at the speci®ed time before calving feeding is continued as part of the calves' diet for at least the ®rst weeks of life. the levels of speci®c antibody present in the colostrum samples from the dairy herds (studies c and d) showed a marked drop in titre over days, this was associated with the switch from colostrum to milk but may be further exacerbated by a dilution eect related to the larger volumes of milk produced by dairy cattle compared with beef cattle. these antibody levels were however signi®cantly higher than those in the placebo treated group and equivalent to those seen in milk from beef cattle at days post-calving. continued feeding of colostrum pooled from collections obtained during the ®rst days post-calving should therefore provide an enhanced level of protection to the calf over this time period. it is accepted that by analogy with rotavirus infections in the calf [ ] and transmissible gastro-enteritis virus infections in the piglet [ ] , lactogenic immunity should protect against bcov infection in the calf [ ] , and this has been con®rmed experimentally (crouch c.f., unpublished observation). there are however sig-ni®cant problems in assessing the ecacy of vaccines where protection is based on boosting lactogenic immunity. this is a result of the number of variables that interact to eect the apparent level of protection obtained. such variables include the management system for the target animal, the titre of speci®c antibody achieved in both colostrum and milk, the duration of the enhanced antibody response, the volume and timing of antibody ingested and the nature (amount and pathogenicity) of the challenge. of these, the key parameters able to be in¯uenced directly by the design of the vaccine are the titre and duration of speci®c antibody achieved in colostrum and milk. this report indicates that a single dose of coronavirus vaccine administered to the pregnant heifer to weeks before calving is capable of signi®cantly increasing the titre and duration of speci®c antibody present in colostrum and milk. work is currently underway to con®rm the minimum level of speci®c antibody required to protect the calf from challenge and thus establish the duration of immunity. acute undifferentiated neonatal diarrhea in beef calves . occurrence and distribution of infectious agents can pathogenic relationships of rotavirus, escherichia coli, and other agents in mixed infections in calves pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhea pathology of neonatal calf diarrhea induced by a reo-like virus pathology of neonatal calf diarrhea induced by a corona-like virus prevalence of rotavirus and coronavirus antigens in the faeces of normal cows radioimmunological (ria) and enzymimmunological (elisa) detection of coronavirus antibodies in bovine serum and lacteal secretions vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic vaccination of dams with a combined rotavirus± coronavirus vaccine to protect newborn calves against diarrhea antibody levels in milk of vaccinated and unvaccinated cows against organisms of neonatal diarrhoea incidence of diarrhoea and of rotavirus-and coronavirus-shedding in calves, whose dams had been vaccinated with an experimental oil-adjuvanted vaccine containing bovine rotavirus and bovine coronavirus new polyvalent vaccine against intestinal infections in newborn calves enhancement of passive immunity with maternal vaccine against newborn calf diarrhea preliminary studies of a bovine coronavirus (bcv) antigen responsible for neutralization monoclonal antibodies to bovine coronavirus glycoproteins e and e : demonstration of in vivo virus neutralizing activity synthesis and distribution of immunoglobulins comparison of dierent antigen preparations as substrates for use in passive hemagglutination and enzyme-linked immunosorbent assays for detection of antibody against bovine enteric coronavirus passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunised or nonimmunised cows antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus enteric vaccines for farm animals and horses we thank k. webb, s. binnie and i. darby for their technical assistance in the preparation of the trial vaccines and dr. h. specter for performing the statistical analysis. key: cord- -h xjudm authors: nyon, mun peak; du, lanying; tseng, chien-te kent; seid, christopher a.; pollet, jeroen; naceanceno, kevin s.; agrawal, anurodh; algaissi, abdullah; peng, bi-hung; tai, wanbo; jiang, shibo; bottazzi, maria elena; strych, ulrich; hotez, peter j. title: engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: h xjudm abstract middle east respiratory syndrome coronavirus (mers-cov) has infected at least patients and caused deaths since its first appearance in , yet neither pathogen-specific therapeutics nor approved vaccines are available. to address this need, we are developing a subunit recombinant protein vaccine comprising residues – of the mers-cov spike protein receptor-binding domain (rbd), which, when formulated with the addavax adjuvant, it induces a significant neutralizing antibody response and protection against mers-cov challenge in vaccinated animals. to prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant mers s - protein in chinese hamster ovary (cho) cells. to accomplish this, we transfected an adherent dihydrofolate reductase-deficient cho cell line (adcho) with a plasmid encoding s - fused with the human igg fc fragment (s - -fc). we then demonstrated the interleukin- signal peptide-directed secretion of the recombinant protein into extracellular milieu. using a gradually increasing methotrexate (mtx) concentration to μm, we increased protein yield by a factor of . the adcho-expressed s - -fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected hek t cells. in addition, hcd /dipeptidyl peptidase- (dpp ) transgenic mice vaccinated with addavax-adjuvanted s - -fc could produce neutralizing antibodies against mers-cov and survived for at least days after challenge with live mers-cov with no evidence of immunological toxicity or eosinophilic immune enhancement. to prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension cho cell line. with over deaths and confirmed cases since its original appearance on the arabian peninsula in [ ] , middle east respiratory syndrome (mers) coronavirus (mers-cov) has emerged as an important global pathogen and potential pandemic threat. there remains a critical need for a vaccine targeting mers-cov [ ] , and the newly established coalition for epidemic preparedness innovation (cepi) has now designated research and development for the mers-cov vaccine as a global priority [ ] . recently, phase i studies of dna-based vaccines against mers-cov showed that % of vaccinated volunteers generated antibody against mers cov [ ] . however, to date there is no licensed dna vaccine for humans due in part to questions about their long-term safety, and their ability to induce high titers of protective or neutralizing antibodies relative to recombinant protein-based vaccines [ , ] . a lead candidate for such a protein-based vaccine is the receptor-binding domain (rbd) of the mers-cov spike (s) protein. the mers-cov rbd plays an essential role in host receptor binding, membrane fusion, and cell entry [ ] [ ] [ ] , thus making it an ideal vaccine target. moreover, focusing on the rbd component, rather than the full-length s protein, reduces the likelihood of eosinophilic-or antibody-dependent immune enhancement [ , ] . expressed and purified as a recombinant fusion protein with the human fc fragment, the addavax(mf -like)-adjuvanted rbd (residues - ) of mers-cov has been shown to elicit high neutralizing antibody titers in both mice and rabbits [ , [ ] [ ] [ ] [ ] [ ] . these antibodies displayed potent neutralizing activity against almost human and camel mers-cov strains, including those with amino acid mutations in the rbd region of their spike proteins [ , , ] . in a complementary approach, recombinant rbd mutants representing different human and camel virus isolates were all able to elicit broad-spectrum neutralizing antibodies against a wide range of human and camel mers-cov strains [ ] . antibodies against the rbd were able to block the binding of the rbd to the mers-cov's cellular receptor, dpp [ ] , and thus block the viral entry into permissive human cells [ ] . most importantly, recent studies found that these neutralizing antibodies were indeed associated with protection, when vaccinated ad -hdpp -transduced mice and hdpp -transgenic mice were found to be immune against lethal mers-cov challenge [ , , ] . although the vaccine antigen has been produced at small, laboratory scale in a transient hek cell system [ ] , little effort has been put forth to develop and scale up of mers-cov rbd suitable for future vaccine manufacture. therefore, we have now engineered a stable cho cell line suitable for producing this mers-cov protein vaccine antigen. the codon-optimized dna sequence encoding the human igg fc-fused s - and the signaling peptide of interleukin- (il ) residing at the n-terminus were synthesized and cloned into pjet . using xbai and noti restriction sites (genescript) (pjet . _il _s - -fc). sequences encoding signal peptides derived from igk light chain (igk), human serum albumin (sa), and azurocidin (azu) were incorporated into the mers s - -fc by touchdown pcr with ultramer oligomers (table , supp. table ). the pcr products were gel purified using a qiaquick pcr purification kit (qiagen) and subsequently cloned into the poptivec-topo vector (invitrogen), followed by escherichia coli top transformation. ampicillin-resistant transformants were selected on lb agar plates containing lg/ml of ampicillin and subsequently grown in lb broth. plasmid dna prepared from isolated colonies were sequenced. to construct the expression plasmid with the il signal peptide, pjet . _il _s - -fc was digested with xbai and noti restriction enzymes, and the gene cassette was gel purified. the expression plasmid poptivec was digested with the same enzymes and gel purified, followed by ligation with t dna ligase. adherent, dihydrofolate reductase (dhfr)-deficient cho cells (adcho) (atcc Ò crl- tm , cho-dhfr) were cultured in iscove's modified dulbecco's medium (imdm, gibco) supplemented with mm l-glutamine (gibco), % fetal bovine serum (fbs, gibco) and . mm sodium hypoxanthine/ . mm thymidine (h/t, gibco). to establish s - -fc-expressing adcho cell lines, x cells were transfected with lg plasmid dna with different signal peptides using lipofectamine Ò (invitrogen), according to the manufacturer's instructions. all plasmid dnas were linearized with ahdi prior to transfection. stable transfectants were selected by culturing in selective medium (same medium as previously described without h/t supplementation). cells were passaged every days after reaching a maximum cell density of .  viable cells per ml (split ratio : ). to investigate the effect of the different signal peptides on protein expression, conditioned medium from each transfection was collected for quantitative analysis. for gene amplification, transfected adcho cells were grown to confluence ( days) in the selective medium supplemented with mtx. the concentration of mtx was increased gradually during each passage ( days per passage) of adcho cells ( nm > nm > nm > nm > nm > nm > nm > nm > nm > nm, [ ] ). the recombinant mers s - -fc was loaded onto a hitrap protein-a hp column (ge) at a flow rate of ml/min. the column was washed with x pbs (ph . , amesco) for column volumes (cvs) and eluted with cvs of - % elution buffer ( mm citric buffer ph . ), followed by cvs of % elution buffer. the elution fractions ( . ml) were collected in tubes containing . ml of m tris-hcl (ph . ) to elevate the ph of the eluted protein to ph . . sds-page and western blotting analysis were performed with rabbit-anti-mers-cov rbd ( : , [ ] ) and rabbitanti-bovine (fab) -biotin ( : , sigma) antibodies to identify the s - -fc protein in the elution fractions. the peak fractions containing the mers s - -fc protein were pooled and concentrated to mg/ml with amicon ultra- centrifugal filter unit (mwco kda) and buffer-exchanged into mm tris-hcl and mm nacl (ph . ). the protein secondary structure was predicted from circular dichroism (cd) spectra. samples for cd experiments were prepared in mm citrate phosphate at a concentration of . mg/ml. cd spectra were recorded with a jasco j- s spectrophotometer, scanning from nm to nm at nm/min with a bandwidth of nm and response time of s. experiments were performed using one quartz cuvette with a path length of . cm, keeping a constant temperature of °c. the average value was determined after five scans, and the spectrum of the matching 'buffer alone' sample served as the control. the secondary structure was predicted using the cdpro software by comparing with three reference sets (sp , sdp and smp ) and using two data fitting programs (contin and cdsstr). a real-time protein melt experiment was performed using protein thermal shift tm dye (thermo fisher scientific) in an applied biosystems viia realtime pcr system (thermo fisher scientific), yielding a fluorescence table overview of tested signal peptides in the adcho expression system. signal peptide signal peptide sequence ref. profile specific to purified mers s - -fc. to evaluate ptm, the purified protein was treated with peptide-n-glycosidase f (pngasef), o-glycosidase and neuraminidase in a nondenatured form and subsequently analyzed by gel electrophoresis. a co-ip assay was carried out to analyze the interactions between adcho-expressed mers s - -fc and human dpp (hdpp ) receptor in huh- cell lysates, using mers s - -fc expressed in hek t cells [ ] as a positive control and hek t-expressing sars-cov rbd-fc as a negative control. the huh- cell lysates (  /ml in ml lysis buffer containing . % n-decyl-d-maltopyranoside-phosphate-buffered saline [ ] ) and sars-cov rbd-specific g mab (anti-sars-rbd, lg/ml, [ ] ). flow cytometry analysis was carried out to quantify the binding between mers-cov rbd and hdpp -expressing huh- cells. cells were incubated with s - -fc ( lg/ml), expressed either in adcho cells or hek t cells, for min at room temperature, followed by addition of fitc-labeled anti-human igg antibody (abcam) for min. cells were then analyzed by flow cytometry. to detect binding between mers-cov rbd and hdpp protein, -well elisa plates were pre-coated overnight at °c with ll of lg/ml purified s - -fc protein, expressed either in hek t cells [ ] or in adcho cells, and blocked with % fatfree milk at °c for h. serial dilutions of hdpp protein (histagged, ll/well) were then added to the plates and incubated at °c for . h, followed by four washes with pbs in . % tween- (pbst). subsequently, the plates were incubated with mouse anti-his primary antibody ( : , sigma) at °c for . h. after four washes with pbst, horseradish peroxidase (hrp)-conjugated anti-mouse igg antibody ( : , ge healthcare) was added to the wells and incubated at °c for min. finally, plates were washed with pbst, and binding was visualized by adding the colorogenic substrate , , , -tetramethylbenzidine (tmb, sigma). the reaction was stopped after min by adding n h so , and absorbance at nm (a ) was measured on an elisa plate reader (tecan). detection of the binding between mers-cov rbd and rbdspecific neutralizing mabs was performed following a protocol similar to that described above, except that the plates were precoated with lg/ml of purified s - -fc proteins, followed by sequential incubation with serially diluted mouse mab (mers-mab ) or human mabs (m -fab, m -fab, and m -fab) [ ] [ ] [ ] and hrp-conjugated anti-mouse igg ( : , for mouse mab, ge healthcare) and anti-human-fab ( : , for human fab-mabs, sigma) antibodies. the binding between denatured mers-cov rbd and the aforementioned rbd-specific neutralizing mabs or rbd-immunized mouse sera was tested by elisa as described above, except that the plates were pre-coated with s - -fc ( lg/ml) protein treated with dithiothreitol (dtt) ( mm, sigma) at °c for h, followed by incubation with iodoacetamide ( mm, sigma) at °c for h to stop the reaction [ ] . after three washes, the elisa was carried out as described above. all in vitro and in vivo studies required the usage of infectious mers-cov (emc/ strain) and were conducted within approved biosafety level (bsl- ) and animal bsl- laboratories at the galveston national laboratory, strictly following approved notification-of-usage (nou) and animal protocols and the guidelines and regulations of the national institutes of health and aaalac. for a ''proof-of-principal" study to confirm that adchoexpressed mers s - -fc is an effective and safe vaccine, two groups of five age-matched cd /dpp transgenic (tg) mice were immunized twice, four weeks apart, via the intramuscular (i.m.) route, with either lg of mers s - -fc formulated with addavax (invivogen) or pbs/addavax only (as control). this immunization protocol was selected because it is optimized for mers-cov rbd proteins [ ] . the addavax adjuvant was chosen because it promoted the rbd-fc protein to generate the highest neutralizing antibodies among several adjuvants tested in our previous studies [ ] . serum specimens were collected at day after the second immunization through the retro-orbital bleeding route to determine the prospective capacity to neutralize infectious mers-cov by using the standard vero e -based micro-neutralization test. immunized mice were subsequently challenged intranasally (i.n.) with x % lethal dose (ld ) ($ tcid ) of mers-cov (emc/ strain), a gift of heinz feldmann (nih, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, the netherlands), followed by daily monitoring for the onset of clinical manifestations (e.g., weight loss and other clinical manifestations) and mortality. three mice from each group were euthanized at day post-infection (p.i.) to assess lung viral loads by vero e -based infection assay and quantitative (q) pcr analysis targeting the upe gene of mers-cov for quantifying infectious virus and viral rna, respectively. additionally, de-paraffinized lung tissues were hematoxylin-and-eosin (h&e)-stained for routine histopathologic evaluations, as described. we continued to monitor the remaining two mice in each group for their overall well-being for a total of weeks until terminating the experiment. all methodologies required to assess the immunogenicity (neutralization antibody titers) and efficacy of mers s - -fc have been previously reported ( [ , ] , supplementary methods). suspension cho (cho dg , gibco, hereinafter termed sus-cho) cells were cultured in cd dg medium (gibco) supplemented with mm l-glutamine (gibco) and . % pluronic Ò f- prior to transfection. transfection was performed by combining lg ahdi-linearized plasmid popti_il _s - -fc and ll of freestyle tm max reagent (invitrogen) in . ml optipro sfm and incubated at room temperature for min, followed by dropwise addition to .  cells in ml of cd dg culture medium (non-selective) according to the manufacturer's instructions. after h, cells were transferred to selective medium (cd opti-cho, invitrogen), supplemented with mm l-glutamine and . % pluronic Ò f- (gibco), and cultivated until cell viability reached %. after selection, stably transfected suscho cells underwent dna amplification by gradually increasing mtx concentration ( - nm) in selective medium. all suspension culture flasks were maintained in a humidified incubator, °c/ % co on a shaker, at a constant rotation rate of rpm. the lm mtx-adapted suscho cell pools from serum-free medium were used for single-cell cloning by limited dilution at . - cells/well. cloning was performed in -well plates (falcon u-bottom untreated), utilizing a cloning medium composed of % hybridoma sfm (clonacell) and % conditioned media supplemented with . x cho acf supplement (clonacell) at °c/ % co for approximately days. conditioned medium from wells with actively growing single colonies was assayed by elisa as follows. a mixture of ll of conditioned medium and ll of coating buffer were added to a -well elisa plate (thermo fisher scientific) and incubated at °c overnight. after washing the plate with pbst, rabbit anti-human igg was used to detect s - -fc protein, followed by a biotinylated goat antirabbit antibody and streptavidin hrp. tetramethylbenzidine (kpl inc., vwr) was added ( ll) before the reaction was stopped with ll m hcl. elisa plates were read on an epoch microplate spectrophotometer (biotek instruments, inc) at nm. clones which gave high absorbance reading were further propagated in -well plates, utilizing a cd opticho medium with mm l-glutamine and . % pluronic Ò f- . conditioned medium from -well plates was also screened by elisa for confirmation, and the highest expressing clone was selected and expanded by passaging in shake flasks ( °c, % co in air on an orbital shaker platform rotating at rpm). clonal cell lines and heterogeneous suscho cell pools were collected daily (up to days) and counted using the acridine orange (ao) and propidium iodide (pi) nuclear staining dyes (nexcelom bioscience), which enter live cells and dead cells, respectively. conditioned medium from each time point was analyzed by sds-page, and the protein concentration was estimated by densitometry, comparing it to protein standards using the chemidoc tm imaging system (biorad). statistical significance was calculated by student's t test using graphpad prism statistical software. ⁄⁄⁄ indicates p < . . in our previous studies, the human igg fc-fused s - fragment of the mers-cov s protein (genbank afs . ) (hereinafter termed mers s - -fc) had already been expressed in transiently transfected hek t cells [ ] . however, to establish stably expressing cell clones, we transfected an adherent cho (adcho) with a poptivec construct containing an internal ribosome entry site (ires)-driven dhfr gene for selection and copy number amplification, as well as mers s - -fc gene fusion. signal peptides were added to the n-terminal end of the s - -fc gene in order to drive secretion into the culture medium (fig. a) . addition of gradually increasing methotrexate (mtx) to the culture medium of transfected cells resulted in binding to and inactivation of dihydrofolate reductase (dhfr) activity. transfected adcho cells compensated for this reduced dhfr activity by increasing the dhfr copy number in the genome to overcome inhibition by mtx. since the mers s - -fc fusion gene was integrated into the same genetic locus as that of the dhfr gene, the s - -fc gene was amplified, as well, leading to increased production of the protein. in the course of developing the adcho cell line expressing the s - -fc protein, optimization of a purification protocol using hitrap protein-a hp was performed. on the purification chromatogram, two protein peaks were observed in the elution step (fig. b) . denaturing sds-page and western blotting analysis with anti-mers-rbd-specific antibodies confirmed the second peak to be the s - -fc fragment (fig. c) . further analysis using non-denaturing sds-page and western blotting with rb-anti-bovine (fab) antibodies showed that the first peak was mainly contaminating bovine igg (fig. d-e) that originated from the fetal bovine serum (fbs) supplemented in the culture medium. we estimated relative productivity of adcho cells by measuring the ratio of the area of first peak and second peak. different signal peptides have been shown to result in different expression levels in cho cell systems [ ] . therefore, we transfected adcho cells with linearized mers s - -fc expression plasmids with four different signal peptides at the n-terminus. peptides were derived from interleukin (il ), igk light chain (igk), human serum albumin (sa), and azurocidin (azu). conditioned media from confluent monolayers of each transfected cell line were collected, followed by protein purification using protein a to establish yield and estimate relative productivity. in these studies, we found that adcho cells with the signal peptide derived from il showed - % more secretion of s - -fc than cells with other signal peptides (fig. a) . elevated expression levels were achieved by gradually increasing mtx concentration during each cell passage from nm to lm. conditioned medium from transfected adcho cells was collected during the dna amplification process, followed by protein purification using protein a to estimate relative productivity (fig. b) . we observed the expected correlation between the expression of s - -fc and increased resistance to higher levels of mtx. compared to adcho cells with nm mtx, expression of s - -fc was increased -fold in the presence of - lm of mtx (fig. c ). the purified protein was analyzed by circular dichroism (cd) spectroscopy. the mers-cov s - -fc consists mainly of beta-sheet ( . %) and loop structures ( %) with limited helices ( . %) (fig. a) . the secondary structure of the protein starts to unfold at °c. during thermal melt analysis, mers s - -fc had two endothermic transitions: . °c and . °c (fig. b) . a comparison between mers s - -fc expressed from hek t cells and adcho cells was carried out using different page analyses. both proteins appeared to be identical following reduced and non-reduced sds-page. on native page and ief gels, adchoexpressed mers s - -fc had a lower isoelectric point (pi . ) when compared to the protein expressed in hek t cells (pi . ) (fig. c) . after removal of n-linked glycans, the molecular size of mers s - -fc was slightly smaller than that of the n-linked glycosylated form (fig. d) . no change in molecular weight was observed after o-linked deglycosylation and desialylation (reduced sds-page). enzymatic treatment did not affect dimerization of mers s - -fc, as determined by nonreducing sds-page. the high pi (> . ) isomers of s - exhibited a lower shift (between pi . and pi . ) in electrophoretic mobility after n-linked deglycosylation, while no change of band pattern for the protein was seen after treatment with o-glycosidase. after removal of sialic acid through neuraminidase treatment, s - -fc isomers shifted to pi higher than . (fig. d) . three assays, including co-immunoprecipitation (co-ip), flow cytometry, and elisa, were performed to detect the binding of mers-cov rbd to its receptor, hdpp . co-ip demonstrated that similar to the hek t-expressed mers s - -fc protein, the rbd protein expressed in adcho bound strongly to hdpp expressing huh- cells. two clear bands were identified from immunoprecipitated mixture of s - -fc and huh- cell lysates, and these bands were recognized by both anti-hdpp antibody and anti-mers-rbd antibody. in contrast, only one band was identified in huh- cells only and s - -fc protein only samples, and it was reactive with either anti-hdpp antibody or anti-mers-rbd antibody, but not with both antibodies. as expected, sars-cov rbd-fc protein was only recognized by sars-cov rbd-specific mab g (fig. a) . flow cytometry analysis further quantified the binding between mers-cov rbd protein and hdpp receptor in huh- cells. results showed a similar strong binding for all mers s - -fc proteins from adcho and hek t, but not for sars-cov rbd-fc control (fig. b) . the elisa analysis demonstrated a dose-dependent binding between these mers-cov rbd proteins and hdpp protein, while no binding was observed between hfc control and hdpp (fig. c) . the antigenicity of mers-cov rbd proteins was carried out using elisa to test their binding with rbd-specific neutralizing antibodies (mersmab , m , m , and m ), which recognize epitopes at rbd residues f , d , r , w , d , y , r , or w , and demonstrate strong activity to block rbd-hdpp receptor binding and neutralize mers-cov infection [ ] [ ] [ ] ] . similar to the s - -fc protein expressed in hek t, the rbd proteins expressed in adcho bound strongly to mouse mab mersmab and human mabs m , m , and m in a dose-dependent manner (fig. d) , confirming their antigenicity. while these mabs bound strongly to the non-denatured (no dtt) s - -fc proteins expressed in both adcho and hek t, they had significantly reduced affinity to rbd proteins treated with dtt, a reducing agent cleaving disulfide bonds of rbds and thus disrupting a protein's native conformation (fig. e) . these results demonstrate that the neutralizing mabs recognize conformational structures of mers-cov rbd [ ] . transgenic mice expressing the human cd /dpp receptor (hdpp -tg) are a well-characterized animal model with which to evaluate the efficacy of vaccine candidates against mers-cov infection and disease [ , ] . the immunogenicity of the mers s - -fc-based subunit vaccine was verified in hdpp -tg mice. immune sera obtained from immunized mice were tested by elisa for the binding with denatured and non-denatured s - -fc protein or subjected to the vero e -based micro-neutralization assay to quantify their capacity to neutralize infectious mers-cov. to evaluate protective efficacy against viral infection, viral loads and histopathology of the lungs of three mice in immunized and control groups were measured at day after lethal challenge with x ld of mers-cov. the remaining two mice in each group were monitored for morbidity (weight loss) and mortality to determine if this vaccine formulation would sufficiently protect against the disease and lethality caused by mers-cov infection. mers s - -fc induced high titers of rbd-specific igg antibodies, which reacted strongly with non-denatured s - -fc protein. nevertheless, these rbd-specific antibodies had significantly reduced activity with s - -fc treated by dtt (fig. ) . this suggests that the rbd vaccine-induced antibody response was indeed directed towards one or more conformational epitopes. mice immunized with pbs/addavax uniformly failed to elicit any detectable neutralizing antibodies; however, those immunized with mers s - -fc/addavax consistently produced readily detectable titers of neutralizing antibody, ranging from - and - of neutralizing titer (nt)- (nt ) and nt- (nt ), respectively ( table ) . consistent with the readily detectable neutralization antibodies, mers s - -fc/addavaximmunized mice were fully protected against viral infection and disease, as evidenced by the absence of recoverable infectious virus and negligible focal inflammatory responses, if any (supp. fig. ) , within the infected lungs at days post-infection (dpi). importantly, the remaining two immunized mice did not suffer any significant morbidity (weight loss) and survived until dpi when the experiment was terminated. in contrast, pulmonary infectious viruses, albeit low in titers, were detected from all three unimmunized controls, with an average of . ± . tcid /g of mers-cov recovered at dpi, accompanied by mild inflammatory responses. the remaining two control mice suffered profound weight loss prior to succumbing to infection by dpi. taken together, results of this ''proof-of-principle" pilot study indicated not only immunogenicity of mers-cov s - -fc, but also its efficacy and safety in the protection of hdpp -tg mice against lethal challenge with mers-cov. similar to the adcho cell development described earlier, the dna copy number of stably transfected suspension cho dg dhfr-cells was amplified by gradual exposure to increased mtx concentrations of up to lm. the resulting heterogeneous suscho cell pools were subsequently cloned by limited dilution to obtain a monoclonal cell population. elisa analysis was performed on the supernatants from individual clones, leading to the identification of clone b as the highest expressing clone. by comparing the growth curves of clone b to the heterogeneous (non-clonal) suscho cells, we discovered that clone b and heterogeneous suscho cells reached their maximum growth on the seventh day with viable cell counts of  cells/ml and  cells/ml, respectively (fig. ) . through quantitative analysis using sds-page, we determined that the supernatant of clone b expressed approximately mg/l of mers s - -fc, while the heterogeneous non-clonal cell pools expressed about mg/l ( table , supp. fig. ) on the seventh day. the mers-cov rbd subunit fragment s - has been identified to be a critical neutralizing receptor-binding fragment and an ideal candidate for the development of an effective mers-cov recombinant protein vaccine [ ] . our aim here was to optimize expression and purification conditions suitable for pilot scale production of this rbd vaccine candidate. initially, both escherichia coli (bacteria) and pichia pastoris (yeast) expression systems proved well suited for recombinant vaccine production because of low production costs. however, our data showed that e. coli could not produce soluble mers s - , and yeast cells could not overexpress mers s - . thus, these two systems were not considered suitable to advance the mers vaccine candidate into process development and scale up production (see supplementary information for more detail). in previous reports, mers fc-fused s - had been expressed in transiently transfected hek t cells. however, transiently transfected cell lines may give low protein yield and potentially lose their production ability over time in continuously expressing recombinant proteins [ , ] . unlike transient transfection, dna is integrated into cells long-term through stable transfection. despite the fact that the development of stably expressing cell lines is laborious and time-consuming, production with stable cell lines can be scaled up easily and would be suitable for use in manufacturing processes. hence, we generated a stably transfected adcho cell line by transfecting mtxdriven poptivec into cells to produce recombinant mers s - -fc protein. fc-fused gp protein was first constructed in as a potential candidate for aids therapy [ ] . while there is no fda approved fc-fusion vaccine, vaccine development using fc-fusion proteins is active and ongoing. a number of studies have been initiated on the development of vaccines against ebola, hiv, influenza, as well as tuberculosis [ ] [ ] [ ] [ ] . it is noted that adverse side effects with vaccines are likely limited as biotherapeutic fc fusions have been repeatedly shown to be safe and biocompatible in humans. currently, all commercial therapeutics use the fc domain from human igg , although other options, such as igg , iga, and igm are also currently being explored [ ] . furthermore, the fc domain is known to increase plasma half-life and simplify the purification process [ ] . purification of s - -fc was performed using a protein a sepharose column, removing most of the impurities from the culture medium. bovine igg originates from fbs in the culture medium in constant amount and was used as a standard to gauge expression levels of the s - -fc protein. this estimation method allowed us to select the most suitable signal peptide and to evaluate the effect of the mtx-induced dna amplification process. since the poptivec plasmid has no signal peptide, four different signal peptides were subsequently tested at the n-terminus of the s - -fc sequence to drive secretion of the protein, leading to the identification of il signal peptide as the most suitable signal sequence. although signal peptides derived from human sa and human azu have been reported to improve production rates in other adcho systems [ ] , the yield in our hands was lower than that with the il signal peptide. stable and highly productive cell pools were then isolated through a gradual increase in the mtx concentration in the culture [ ] . analysis of the relative productivity of adcho cells indicated an increase in s - -fc in media proportional to the mtx concentration in the medium, reaching a plateau at lm mtx. the biophysical and biochemical characterization of the mers s - -fc protein revealed that is was stable up to a temperature of . °c. after the first major unfolding event at . °c, another unfolding event occurred between and . °c, which could have resulted from destabilization of the ch -ch bond of the fc domain of the recombinant protein [ ] . no change in molecular weight was observed after removal of o-linked glycan on mers s - -fc, which suggested no o-linked glycosylation in the protein. after neuraminidase treatment, the recombinant protein band pattern showed fewer bands and a pi shift, suggesting that sialic acids might contribute to the charge of the protein. interestingly, although the complexities of the multiple protein bands were greatly reduced after glycosidase and sialidase treatments, the pattern representing charge heterogeneity remained, suggesting the existence of additional ptms, such as mannose- phosphate, of the recombinant mers s - -fc protein. due to the charge differences between adcho-and hek texpressed s - -fc proteins, we evaluated the functionality and antigenicity of the target protein. functionality studies, including co-ip assays, flow cytometry analyses, and elisa binding assay, confirmed that the adcho-expressed mers s - -fc protein maintained functionality equal to that expressed in hek t cells in binding the dpp receptor of mers-cov, both of which showed dose-dependent binding with the soluble hdpp protein. in addition, mers s - -fc expressed in either adcho or hek t demonstrated similar dose-dependent binding to rbd-specific neutralizing antibodies, an indicator that both s - -fc proteins could maintain sufficient antigenicity. we further investigated the protective efficacy of adcho-expressed s - -fc protein vaccine in protecting against mers-cov infection in the established transgenic mouse model expressing hdpp (hdpp -tg). by formulating this s - -fc protein with adda-vax all vaccinated animals could produce neutralizing antibodies and survive a live viral challenge for days. taken together, we confirmed the absence of functional, antigenic and immunogenic differences between adcho-and hek t-expressed mers s - -fc proteins. moreover, mouse vaccinations with the rbd subunit vaccines did not appear to elicit eosinophilic or antibody-dependent immune enhancement. although we verified adcho-expressed mers s - -fc protein as an effective vaccine against mers-cov infection, the use of fbs in the growth medium proved unsuitable for a human vaccine antigen [ ] . for both safety and compliance with future regulatory requirements, we therefore developed a stably transfected suspension cho cell line in serum-free medium. the adcho cell development process described here became the foundation for the establishment of the serum-free suspension cho cell line. subsequently, we transfected the poptivec expression plasmid with the il signal peptide into suscho cells and carried out the dna amplification as before. from the heterogeneous cell pools adapted to lm mtx, we isolated clone b , which was the highest expressing clone from a two-cycle screening process. in shake flasks, the growth of clone b was slightly slower than that from the heterogeneous cell pools, but b expressed % more mers s - -fc protein than the heterogeneous cell pool. typically, highly productive cell clones have lower growth rates since a significant portion of resources are used for expression of the recombinant protein [ ] . additional experiments in the transgenic mice and non-human primate models will be needed to further determine the immunogenicity of mers s - -fc protein that produced by suscho. we envision that with a proper production process, the recombinant protein can be scaled up, manufactured, formulated and stockpiled as an efficient countermeasure against future mers-cov outbreaks. table s - -fc expressed from heterogeneous non-clonal suscho cell pools and from the monoclonal clone b . the protein concentration (mg protein per liter of culture supernatant) was determined by sds-page gel analysis (supp. fig. ). who. middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus -a continuing risk to global health security vaccine development against prioritized epidemic infectious diseases inovio reports new positive clinical data on vaccine advances in the 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amplified cho cell line based on the location of amplified genes a recombinant receptorbinding domain of mers-cov in trimeric form protects human dipeptidyl peptidase (hdpp ) transgenic mice from mers-cov infection junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus optimized signal peptides for the development of high expressing cho cell lines mers-cov spike protein: a key target for antivirals evaluation of transfection methods for transient gene expression in chinese hamster ovary cells gene expression in mammalian cells and its applications designing cd immunoadhesins for aids therapy ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice a neonatal fc receptortargeted mucosal vaccine strategy effectively induces hiv- antigen-specific immunity to genital infection adjuvant-free immunization with hemagglutinin-fc fusion proteins as an approach to influenza vaccines apc targeting enhances immunogenicity of a novel multistage fc-fusion tuberculosis vaccine in mice fc-fusion proteins: new developments and future perspectives stabilisation of the fc fragment of human igg by engineered intradomain disulfide bonds a plea to reduce or replace fetal bovine serum in cell culture media advances in mammalian cell line development technologies for recombinant protein production identification of a receptorbinding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction this study was supported through the us-malaysian vaccine development program, funded by the university of malaya, and grants from the nih (r ai - s and r ai ). we thank drs. dimiter s. dimitrov and tianlei ying for providing m , m , and m mabs. the authors are involved in the development of a vaccine against mers coronavirus. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.vaccine. . . . key: cord- - aurua o authors: falchieri, marco; lupini, caterina; cecchinato, mattia; catelli, elena; kontolaimou, maria; naylor, clive j. title: avian metapneumoviruses expressing infectious bronchitis virus genes are stable and induce protection date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: aurua o the study investigates the ability of subtype a avian metapneumovirus (ampv) to accept foreign genes and be used as a vector for delivery of infectious bronchitis virus (ibv) qx genes to chickens. initially the gfp gene was added to ampv at all gene junctions in conjunction with the development of cassetted full length dna ampv copies. after recombinant virus had been recovered by reverse genetics, gfp positions supporting gene expression while maintaining virus viability in vitro, were determined. subsequently, either s or nucleocapsid (n) genes of ibv were positioned between ampv m and f genes, while later a bivalent recombinant was prepared by inserting s and n at ampv mf and gl junctions respectively. immunofluorescent antibody staining showed that all recombinants expressed the inserted ibv genes in vitro and furthermore, all recombinant viruses were found to be highly stable during serial passage. eyedrop inoculation of chickens with some ampv-ibv recombinants at one-day-old induced protection against virulent ibv qx challenge weeks later, as assessed by greater motility of tracheal cilia from chickens receiving the recombinants. nonetheless evidence of ampv/ibv seroconversion, or major recombinant tracheal replication, were largely absent. avian metapneumovirus (ampv) is a major endemic respiratory pathogen of global domestic poultry [ ] , and secondary infections can exacerbate the disease [ ] . ampv belongs to the subfamily pneumovirinae, genus metapneumovirus [ ] of which four subtypes have been recognized (a, b, c and d) [ ] [ ] [ ] . the genome has genes, with genome sizes typically close to . kb [ ] . reverse genetics (rg) systems for subtypes a and c have allowed their rational mutation [ , ] . to date rg has produced viruses with deletions, gene modifications and reporter gene insertions [ , [ ] [ ] [ ] . some studies have considered the replicative ability of recombinants in vivo [ , ] but not foreign viral gene insertion, nor the genetic stability of expanded genomes. avian infectious bronchitis (ibv) is the major worldwide endemic respiratory pathogen of chickens (family coronavirus, genus gammacoronavirus) which, like ampv primarily infects the respiratory tract but can also infect the kidneys, intestine and reproductive system [ ] . ibv epidemiology is characterized by the emergence of virulent variants which evade prevailing ibv immunity. in the last decade the qx genotype [ ] has emerged, which predominantly affects the respiratory tract and kidneys [ ] . it was first recognised in china and has continued to spread to much of europe and asia. in this study, novel qx candidate vaccines based on ampv vectors were constructed and tested. the major ibv surface protein spike s becomes cleaved into the outer, antigenically important s , and inner membrane bound, s section [ ] . previously inoculation of s expressed in baculovirus was shown to induce protection [ ] . the internal nucleocapsid (n) protein can also induce protective immunity [ , ] involving both t and b lymphocyte epitopes [ , ] . here two different qx s genes and a qx n gene were inserted into ampv, both to assess the vector's acceptance of foreign genes, and its suitability for delivery of ibv genes to chickens. three ampv genomes acted as vectors. prior to introduction of s or n genes, a cloning site flanked by a transcription start and stop sequence, was added to intergenic regions of full length (fl) dna genome copies. to aid ibv recombinant optimization, green fluorescent protein (gfp) was initially added to these sites and recombinant viruses recovered; then ampv s and n recombinant viruses were constructed. the two s genes used differed by a nucleotide deletion present in a proportion of the donor virus. recombinant viruses were tested in chickens in two experiments to determine their protective capacity. ibv inoculation of chickens leads to infection of and damage to the tracheal ciliated epithelium [ ] . loss of cilial motility is readily observed [ ] and protection is considered best assessed by the maintenance of motility following challenge [ ] , as also adopted in european pharmacopoeia, ibv vaccine monographs. to assess replication of recombinants, real time rt pcr was performed on material from choanal swabs while induced specific antibody to ibv and ampv was measured by elisa and haemaglutination inhibition (hi). three subtype a ampv viruses were employed as vectors for gfp and later ibv genes. first was a german field isolate (virus a) passaged in vero cells [ ] and found avirulent in turkeys [ ] , second was virus avf which contained an f gene modification found to better induce protection in turkeys [ ] and third was / , a virulent field isolate deriving from a subtype a vaccine and arising in field conditions [ ] . seven versions of virus a [ ] fl cdna were made by site directed mutagenesis, as outlined in fig. . an xho restriction endonuclease (re) site was added to each intergenic region, following the preceding gene stop signal and prior to the downstream gene start signal. a cloning cassette flanked by cut sal re sites, allowing gene insertion and transcription, was ligated into each cut xho re site of the seven fls. after cloning into stb cells (invitrogen, according to manufacturer's recommendation (amr)), the seven fls were cut with sal . gfp genes flanked by xho sites were produced by high fidelity pcr using primers gfp ins+ and gfp ins neg (table ) and these were added to the seven fl cdnas by ligation after xho digestion. colonies containing dna of correct orientation and sequence were selected by sequencing. fls containing gfp were recovered by rg [ ] . gfp expression was assessed by fluorescence microscopy. virus viabilities were evaluated as maximum titres obtained following three vero passages. an ibv qx virus isolated in germany was grown in eggs, titrated in tracheal organ cultures (toc) and stored in aliquots at - • c. as the genome sequence was unknown, a range of ibv genomes were aligned to identify conserved regions within, and flanking, s and n genes. these were used for the design of rt-pcr and sequencing primers, as detailed in table . sequencing of the s gene amplicon revealed two populations, one of which showed a nucleotide deletion (del) ( -uguuugauucugaua- ) between bases and . . . predictive computational comparison of s populations s genes with and without the deletion were compared. the protean program of the dnastar multiple program package (lasergene inc., usa) was used to estimate physicochemical properties, composition of the proteins and prediction of secondary structures. order-disorder prediction used the vl-xt predictor on the pondr server [ ] . prediction of immunodominant helper tlymphocyte antigenic sites from primary sequence data was by analysis of the occurrence of amphipathic fragments using the amphi algorithm [ ] . the predictive algorithms had been previously shown correct in more than % of cases. qx s + ggtttaattccttgtcagtttctcttacttatgg s sequencing qx s + gctgctaattttagttatttagcagatggtgg s sequencing qx s neg cctgaagaggtgctgtcatagc s sequencing qx s + ggcatgattccacgtgatcatattcg s sequencing qx s neg cagtagttttgttggaagtaaaaacaagatcacc s sequencing qx s end neg cgaaccatctggttcaatacaaaatctgc s pcr amplification qx s start+ ccagttgtgaatttgaagaaagaacaaaagaccgacttag s pcr amplification rt qx s neg catctttaacgaaccatctgg s rt amplification s + gctgctaattttagttatttagcagatggtgg s primer for mrna pcr s start xho+ ggtaaattattgctcgaggatgttggtgaagtcactgtttttagtg s amplification adding xhoi sites s stop xho neg gttacgttttgctcgagttaacgcctacgacgatgtgagctattgg s amplification adding xhoi sites sx + taatactggyaatttttcaga s sequencing for gene insertion, xho i re sites were added to s and n gene extremities by rt-pcr using modifying primers s start xho+ s stop xho neg, n start xho+ and n stop xho neg (table ) . ibv genes were inserted into fls a, avf [ ] and fl / [ ] with the cassette at the mf intergenic region. for insertion of a second gene, the cloning cassette was additionally added between g and l genes. after cloning into stb cells, seven recombinant cdnas were produced as detailed in table . recombinant viruses were rescued from fls [ ] then passaged in vero cells to produce sufficient virus for protection studies. viruses were titrated in well plates containing vero cell monolayers. cytopathic effect end points were observed using low power microscopy and titres were calculated [ ] . virus stocks of . ml aliquots were stored at − • c. to verify the transcription of inserted s and n genes in vero cell grown virus, a previously reported protocol [ ] was used, except that primers within those genes were s + and n + for s and n genes respectively (table ). s and n protein expression were assessed using immunofluorescence (if) on ampv recombinant infected vero monolayers, using a polyclonal chicken antiserum (gd) anti qx for s expression and a monoclonal mouse antibody (biozol) for n gene expression. appropriate fitc conjugated antibodies were used to visualize specific s /n proteins (amr). approximately one-day-old spf chickens were divided into seven groups, each containing ten animals. in groups one to four, birds were inoculated by eyedrop with log tcid a full s mf , avf full s mf , a del s mf and avf del s mf respectively. the ampv control group was inoculated with avf. at days post vaccination (dpv), all birds, and half the unvaccinated control (c+) were challenged with log tcid qx ibv strain by eyedrop, while the other controls remained unvaccinated (c−). for sampling, of tracheas and kidneys, half the chickens per group were humanely killed at days post challenge (dpc) and the remainder at dpc. approximately one-day-old spf chickens were divided into six groups of ten birds, of which four were inoculated with log tcid of viruses full s mf , avf full s mf + n gl , avf n mf and avf full s mf virus by eyedrop. the remaining groups acted as controls. at dpv, all vaccinated birds and half the unvaccinated controls (c+) were challenged with log tcid of ibv qx by eyedrop, while the other controls remained unvaccinated (c−). in each group, five birds were humanely killed at dpc and five at dpc and tracheas and kidneys were collected. chickens from each group were bled at dpv for ibv and ampv serology. sera from birds receiving s recombinant ampvs were tested using an ibv qx hi test, while those receiving either recombinant containing the n gene were tested by ibv hi and elisa (biochek, amr). ampv elisa (idexx, amr). ten choanal swabs were collected from all groups at , and days post vaccination (dpv) for ampv real time rt-pcr [ ] to assess recombinant replication. at and dpc in both chicken experiments, tracheas were collected and cut into mm transverse sections. for each trachea, ten sections were collected (three upper, four middle and three lower) and examined under low power microscopy to determine activity of cilia. individual sections were classified as either containing beating cilia or beating being entirely absent. statistical analysis of cilial motility was performed using the chisquare test. a p value < . was considered statistically significant. birds were examined daily for the presence of nasal exudate and generally for overall health. the gfp gene was added to modified fl ampv copies and viruses were recovered. maximum tcid titres per ml of vero cell lysate, following three vero cells passages, were low when the inserted gene was positioned at a gene junction close to the virus leader. hence with gfp placed between n and p genes, the titre was . while in all other positions it exceeded . . at the mf position it was . . for all constructs, strong fluorescence was observed when infected vero cells were viewed by uv microscopy for all constructs as illustrated for recombinant gfp mf (fig. ) . protean suite software analysis predicted s proteins to share the same physicochemical properties and pondr predicted no differences to the disordered region. the protean suite predicted the proportion of alpha helix to remain constant while a difference of beta pleated sheets was evident. the amphi program predicted a t cell epitope present only in the complete s protein, at amino acid positions - . however s containing the deletion possessed two additional predicted t cell epitope regions (amino acids - and - ). virus was rescued from ampv fl cdnas containing ibv genes as confirmed by cytopathic effect typical of ampv on vero cell monolayers. viruses were sequenced to after three passages. rt-pcr of virus mrnas confirmed transcription of the inserted ibv genes (data not shown). expression of ibv proteins was confirmed in all recombinant viruses by if (fig. ) . ibv and ampv antibody responses were not generally detectable (table ). for ibv in experiment , a single bird in group avf del s mf and two birds in group avf full s mf had detectably seroconverted by hi. for ampv elisa serology, two seroconversions were detected in the avf control group (experiment ) and three in the full s mf group (experiment ). except for full s mf , real time rt-pcr showed minimal ampv recombinant replication (table ) . at dpc, sections from all birds challenged with ibv were ciliostatic. at dpc, some sections from birds previously inoculated with ampv qx recombinants showed cilial motility, with those birds given avf full s mf + n gl showing most, followed by avf n mf and with the least for avf viruses expressing only the s gene. in all cases the distribution of beating rings within any group was even. no differences were observed between recombinant ampvs expressing the full and deleted s . comparing ampv vectors with identically positioned inserts, a led to greater cilial recovery than either / or avf. see table for details. significantly increased cilial activity, calculated by comparing each group to the respective positive control, was found for birds vaccinated with a full s mf (p = . ), a del s mf (p = . ), avf full s + n gl (p = . ) and avf n mf (p = . ) and analysis between these groups did not reveal any significant differences. table chickens experiments and : post vaccination virus detection by real time rt-pcr, pre challenge serology and cilial activity post challenge. a del s mf / / / / / n.d. c a full s mf / / / / / n.d. c avf del s mf / / / / / n.d. c avf full s mf / / / / / n.d. no signs were detected after vaccination. in experiment , clinical signs were observed in three birds at dpc. one positive control bird and two avf control birds were humanly killed after displaying lethargy, ruffled feathers and reduced response to external stimuli. gross kidney lesions, typical of qx ibv infection, were detected at post-mortem examination in the same birds. no signs or lesions were seen in experiment . when ibv mf recombinants were used to inoculate one-day-old chickens, many induced ibv protection of the trachea, yet serology and real time rt pcr virus detection indicated poor tracheal replication. surprisingly, recombinants replicating least induced most protection whereby recombinants based on virus a protected best while virus / induced no detectable protection. despite computer analysis predicting t cell epitopes differences concerning the s nucleotide deletion, no protection differences were observed. the primary site of ampv and ibv replication in chickens is the upper respiratory tract, hence an ampv based ibv recombinant might be predicted to be ideal for inducing ibv protection. conventional wisdom suggests that the minimal levels of upper respiratory tract replication detected in our study, led to the observed protection. therefore any ampv recombinant replicating better might yield greater protection. growing worldwide commercial field evidence is indicating that subtype b ampv better infects commercial chickens than subtype a. if a subtype b rg system becomes available, protection afforded by any resultant ibv recombinants will be keenly compared to the current study, which counter-intuitively suggests that recombinant ampvs inducing the best protection replicate least well. if more protection inducing recombinants are found not to appreciably replicate in the respiratory tract or produce generalised seroconversion, it will become necessary to investigate whether replication might occur at another site or alternatively protection may result from a yet unrecognized mechanism. there is already some evidence that, ampv replication in the respiratory tract of turkeys does not imply induction of ampv protection [ ] , protection following live vaccination does not require initial replication in the trachea [ ] and ampv vaccination can lead to apparent protection without ampv seroconversion [ , ] . for the first time, ampv recombinants are reported carrying foreign viral genes. previously ampvs were shown highly stable both in cell culture and during natural passage [ ] as has also been reported for other members of the mononegavirales such as vesicular stomatitis and rabies viruses [ , ] . this contrasts with single strand positive viruses such as ibv and feline calicivirus where minimal passage readily results in consensus sequence mutations [ , ] . furthermore, the recombinant ampv genome carrying more than extra nucleotides (ibv s and n genes) was stable with respect to functionally irrelevant inserted genes, and their presence did not appreciably reduce virus viability, if distanced beyond the phosphoprotein gene. our study suggests that a range of ampv vectored candidate vaccines could be readily prepared. for ibv, this is in marked contrast to conventional attenuation where more than egg passages is typically required. such ampv recombinants would have greater genetic stability in two key respects. firstly instability on simple passage implies that any live vaccine will be genetically heterogeneous. of greater significance is the ability of ibv live vaccines to recombine with other ibv genotypes during co-infection, via homologous recombination, thus potentially leading to new field genotypes. in contrast, ampv has been shown to be stable with respect to multiple passage while the absence (or extremely low frequency) of homologous recombination avoids recombinant generation. the nature of the protective immune response to ibv is not well understood [ ] . while virus avf containing n gene alone or n + s genes together protected tracheas better than avf containing the s gene alone, the expression of the inserted genes would need to be quantified, before conclusions about the relative contribution of each gene to protection, could be drawn. previous studies indicate that the internal n protein is able to stimulate a cell mediated immune response (cmi) [ ] whereas the exposed s protein would be more likely to stimulate antibody production [ ] [ ] [ ] , hence our study adds weight to the notion that both a cmi and antibody response are important in protection. in conclusion, we report for the first time 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methods to demonstrate experimental infection demonstration of loss of attenuation and extended field persistence of a live avian metapneumovirus vaccine sequence data analysis for long disordered regions prediction in the calcineurin family prediction of immunodominant helper t cell antigenic sites from the primary sequence a simple method of estimating fifty percent end points development of a real time rt-pcr assay for the simultaneous identification, quantitation and differentiation of avian metapneuomovirus subtype a and b demonstration of a virulent subpopulation in a prototype live attenuated turkey rhinotracheitis vaccine a live attenuated turkey rhinotracheitis virus vaccine i. stability of the attenuated strain further studies on the development of a live attenuated vaccine against turkey rhinotracheitis the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus highly stable expression of a foreign gene from rabies virus vectors the challenge for the next generation of feline calicivirus vaccines coronavirus ibv: virus retaining spike glycopolypeptide s but not s is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection identification of amino acids involved in a serotype and neutralization specific epitope within the s subunit of avian infectious bronchitis virus monoclonal antibodies to the s spike and membrane proteins of avian infectious bronchitis coronavirus strain massachusetts m there is no conflict of interest. key: cord- -fdp mies authors: belák, sándor title: molecular diagnosis of viral diseases, present trends and future aspects: a view from the oie collaborating centre for the application of polymerase chain reaction methods for diagnosis of viral diseases in veterinary medicine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: fdp mies the emergence and re-emergence of transboundary animal diseases (tads), e.g., foot-and-mouth disease, classical swine fever and the highly pathogenic avian influenza strongly indicate the need for the development of powerful and robust new diagnostic methods. the experiences of an oie-collaborating centre and of two eu project consortia are summarised on the diagnostic application of gel-based pcr, general pcr systems, phylogeny, molecular epidemiology, real-time pcr (taqman, molecular beacons, primer-probe energy transfer), amplification without thermocycling (invader), multiplex pcr, nucleic acid extraction and pipetting robotics, automation and quality control, including internal controls. by following the steps of oie validation, the diagnostic assays are nationally and internationally standardised. the development of padlock probes and microarrays, as well as ultra rapid pcr and sequencing methods is further improving the arsenal of nucleic acid based molecular diagnosis. further trends of diagnostic development are also mentioned, in order to combat tads and other viral infections more effectively in the future. the recent incidences of emerging and re-emerging transboundary animal diseases (tads) lead to very heavy losses all over the world. the outbreaks are associated with huge economic and social impact in many countries. during such a short time as the last months, the office international des epizooties (oie, world organisation for animal health) reported the occurrences of a high number of tad outbreaks, e.g., foot-and-mouth disease on three continents (africa, asia and south america), classical swine fever (africa, asia, and europe), rinderpest (africa, asia), or highly pathogenic avian influenza (africa, asia, and europe). the costs should be viewed both in terms of efforts to bring the disease under control and the consequent loss of livelihoods. concerning the eu, during the last decade the member states have suffered from a rather high number of devastating outbreaks of diseases notifiable to oie. the european commission scientific committee on animal health and animal welfare report (adopted - th april ) has stated: "recent outbreaks of foot-and-mouth disease (fmd), classical swine fever (csf) and avian influenza (ai) have occurred in several member states and resulted in the slaughter of large numbers of animals as well as severe economic consequences". as an example, with regard to the uk fmd outbreak in , the cost to the public sector was estimated at over . billion euro, and the cost to the private sector at over . billion euro. the ethical problem risen under the current eradication strategy and the social consequences of the slaughter of large numbers of animals must also be taken into account when considering the effects of the diseases notifiable to oie: (http://www.oie.int/eng/info/hebdo/a dsum.htm). the national and international organisations, veterinary health authorities, research institutes, diagnostic laboratories and field services make large efforts worldwide to prevent and combat tads. the early warning systems and the rapid and highly specific detection of the agents are major tasks, considering that the timely recognition of such viral infections would prevent the spread of the diseases to large animal populations in huge geographic areas. thus, the development of novel, powerful diagnostic assays is a basic issue today in veterinary research and animal health care. molecular virology offers a range of new methods, which are able to accelerate and improve the diagnosis of infectious diseases in animals and in man. the new assays provide possibilities for a very rapid diagnosis, since the detection of viruses can be completed within several hours or minutes. concerning the direct detection of viruses or viral components, various molecular approaches are introduced, like conventional gel-based pcr, real-time pcr, multiplex pcr, improved in situ hybridisation and in situ pcr. in addition, pcr-robotics, portable pcr machines, improved sample enrichment, amplification without thermocycling, macro-and micro-arrays are under development. as far as the indirect detection of the various viruses is concerned, antibody-detecting molecular methods are under construction, like improved elisa systems, pcr for protein detection (dna tags, proximity ligation), pen-side tests (e.g., dip-stick assays). in order to standardise the new methods worldwide, the steps of oie validation are followed and introduced [ ] . this article is illustrating new trends in the field of molecular diagnosis of viral diseases, summarising the recent achievements of an oie collaborating centre and its eu project partner laboratories. the results of previous reviews are updated; new achievements are shown and discussed with special regard to modern trends in molecular detection of tads and other viral infections in domestic or wild animals and in food safety. by introducing single and nested pcr assays as early as - , two years after the description of the pcr principle, our laboratory was among the first ones to use this technique for diagnostic purposes [ , ] . between and more than diagnostic pcr assays have been developed and applied at our laboratory, to diagnose a wide range of diseases in various animal species. these assays, including precautions and laboratory practices to avoid false positive and false negative results, are summarised in previous general articles and reviews [ , , ] . a great diagnostic power of the pcr techniques is that the nucleotide sequencing and the comparative sequence analysis of the pcr products provide possibilities to determine the genetic relationship(s) of the detected viruses or other microbial agents. by this way phylogenetic trees are constructed, illustrating the genetic relatedness and background of the viruses. the genetic variants are rapidly identified and the routes of virus spreading are promptly traced and recorded in a country or even on much larger geographic areas. this approach, termed molecular epidemiology, provides novel tools to the veterinarians and to the animal health authorities in combating viral diseases. for performing studies of molecular epidemiology, variable genomic regions of the viral genomes are selected, amplified and sequenced. we developed such "phylogeny pcr assays" for the amplification of members of various virus families, like the flaviviridae and the coronaviridae. in the flaviviridae family pestiviruses bovine viral diarrhoea virus (bvdv), classical swine fever virus (csfv) and border disease virus (bdv) were amplified and grouped by amplifying and sequencing various regions of viral genomes, such as ncr, ncr, npro, e and ns [ ] [ ] [ ] [ ] [ ] [ ] . the assays of molecular epidemiology contributed to the detection and identification of bvdv variants in scandinavia and determination of genetic clustering of the virus [ , ] . furthermore, the pcr-based molecular detection and identification of bvdv variants proved to be a useful tool to study the biology and pathogenesis of the virus [ ] . an interesting and practical approach is when the retrospective genetic analysis is applied to determine the identity of virus strains used for vaccine production. in one of our recent studies a virus "pick up" was observed in a commercially produced live attenuated bvdv vaccine. the results of this work emphasize that the contamination of commercially available live vaccines with exogenous bvdv strains is a real risk factor, and unequivocal analysis, including molecular methods, is needed to verify their authenticity [ ] . molecular epidemiology and viral phylogeny provide new possibilities to combat and eradicate infectious diseases. national and international tracing of virus variants during outbreaks helps the work of the veterinary health authorities, allowing to trace the ways of the spread of infection and to identify the various virus variants in the outbreaks [ ] [ ] [ ] . the majority of the diagnostic pcr systems is designed to be highly specific for the detection of the selected target virus(es). as a complement to these highly specific systems, "general", or "universal", pcr systems are also developed at our laboratories. the "general" systems are constructed to detect a wide range of related viruses, e.g., members of an entire virus family, or virus genus. to obtain a wide range of target amplification, the primers are selected from well-conserved regions of the genome. these pcr systems provide very useful tools for the diagnostic virologists. such systems are practical when more members of the same virus family or genus cause the same or very similar clinical picture(s). for example, cattle can be infected with bvdv and with border disease virus (bdv, alias ovine pestivirus), while swine can develop infection with any of the three pestiviruses, classical swine fever virus, bvdv and bdv, frequently showing only mild or innapparent symptoms. in such cases if a "general" pestivirus pcr is available, the diagnostician can directly screen the herds, in order to demonstrate or exclude the presence of any pestivirus, in a single pcr assay (instead of running three separate assays). thus, the positive samples, pre-selected by the "general" pcr, can then be further tested with the virus specific pcr assays, in order to identify the pestivirus, which is causing the infection. the parallel use of "general" and specific pcr assays provides a rapid and effective diagnosis. considering this requirement, a "general" pestivirus taqman assay is used at our laboratory for the simultaneous detection of the three pestiviruses in domestic and in wild animal populations [ , , ] . recently a sybr green real-time pcr system was developed here for the generic detection of coronaviruses [ ] . the real-time pcr assays provide novel rapid means of virus detection in the diagnostic laboratories. several variants of real-time pcr methods and chemistries are used today, e.g., taqman, molecular beacons (mb), scorpion primers, dual probe systems as utilized in the lightcycler ® , dye-labelled oligonucleotide ligation (dol), primer-probe energy transfer system (priproet). compared to the "classical" single or nested pcr methods, the diagnostic application of the real-time pcr assays has certain advantages, such as: (a) faster and higher throughput assays; (b) post-pcr handling of the products is not needed; (c) despite of a non-nested set-up, the real-time-pcr is providing sensitivity close to or equal to traditional nested pcr; (d) the amplified products are detected by measuring fluorescence in the reaction vessel without having to open the system, thus, the risk of contamination is minimised; (e) the result of the pcr is not only "positive" or "negative", but the real-time pcr assays allow quantitative estimation; (f) real-time quantitative pcr is more accurate and less labour-intensive than current quantitative pcr methods; (g) the hands-on time is greatly reduced, compared to traditional detection in agarose gels followed by ethidium bromide staining; (h) the principle of the real-time pcr allows automation of the procedure, and the use of a -well microplate format, without the need for nested pcr, makes it very practical to automate it; (i) diagnosis can be further automated by using robots for dna/rna extractions and pipetting; (j) probes for real-time pcr can be labelled with a number of different fluorophores, which function as individual reporter dyes for different primer sets. thus, real-time pcr technique is very suitable for the development of multiplex pcr systems; (k) lower diagnostic costs. the advantages of diagnostic real-time pcr assays and more details are summarised in previous reviews and reported in recent articles [ , , , [ ] [ ] [ ] [ ] . during the last years we have developed a wide range of real-time pcr assays for diagnostic purposes. the various assays were developed at the oie collaborating centre or in collaboration with our partner laboratories in large diagnostic projects of the european commission (http://www.multiplex-eu.org/, http://www.labonsite.com/). by comparing different approaches, the taqman, but especially the priproet techniques were found robust and highly reliable for diagnostic purposes. this fluorescence resonance energy transfer detection system combines probe-based realtime monitoring of pcr amplification with confirmation of probe hybridization from the melting temperature (t m ) curve. the priproet system allows quantification of the specific amplicon, as the fluorescence emitted from the reporter depends directly on the amount of amplicon formed [ ] . in contrast to priproet, the performance of other methods, like molecular beacons, was rather strongly influenced by the point mutations in the target sequences. we have found that even the taqman and the scorpion primers assays can be fragile to single point mutations, or they demand longer conserved areas for hybridisation. since many of our targets, especially the rna viruses show a high mutation rate, we suggest using first of all the priproet principle (and with some considerations the taqman) for the development of diagnostic real-time pcr assays [ , , ] . for the detection of various viruses a range of real-time pcr assays has been developed in the last years at our laboratory and is used in routine diagnosis ( table ). the majority of these methods is based on taqman or priproet principles. in addition, isothermal amplification methods, like the invader technology, were developed for the rapid detection of viruses in the frame of our eu projects [ ] . some of the very recently developed assays target even viruses, which are important in zoonoses and in food safety, such as noroviruses and hepatitis e virus ( [ ] and, fig. ). in general, the multiplex pcr methods (using multiple primers to allow amplification of multiple templates within a single reaction) are useful for diagnostic purposes, providing the diagnostician the ability to detect more than one infectious agent(s) in a single assay. for example, we analyse a single nasal or rectal swab collected from an animal suffering from a respiratory disease, or from enteritis/diarrhoea syndrome, respectively. by performing multiplex pcr, we seek to diagnose all possible pathogens, which can be considered in this disease. the real-time pcr is very suitable for multiplexing, since the individual probes for the component assays can be labelled with different fluorophores, each of which functions as a specific colour reporter dye for one set of primers. since the fluorescent probes emit at different colour wavelengths, it enables an easy multiplexing of the assays. although the "classical" pcr techniques are also suitable for the development of multiplex systems, the use of "classical" nested pcr for the construction of multiplex assay would be rather complicated, considering the large number of primers required. these might "compete" with each other, as they have to be placed in the same reaction mix of the classical nested pcr. in general, the real-time pcr assays (using only single primer pairs) provide better possibilities for the construction of multiplex systems with multiple target components. considering the diagnostic advantages of this principle, various multiplex pcr assays were developed at our laboratory, partly as own development or in collaboration with eu project partner laboratories (http://www.multiplex-eu.org/ and http://www.labonsite.com/). for example, a multiplex (duplex) real-time pcr assay was developed and is used in routine diagnosis for the simultaneous detection of bovine respiratory syncytial virus and bovine respiratory coronavirus, two pathogens important in the respiratory disease complexes of young calves (fig. ) . we have found that multiplex real-time pcr has the potential to produce considerable savings in time and effort, without compromising the robustness and sensitivity of the virus detection assays [ ] . . . a simple way of complex diagnosis: development of "multi pcr" assays as mentioned above, "primer-competition" may occur during the construction of multiplex pcr systems. the development of multiplex pcr assays in such cases might be very difficult and time consuming. the levels of specificity and sensitivity may strongly drop. our experience is that in such cases it is more advisable not to "force" the co-amplification in the same reaction vessel, but rather to amplify the various viruses side-by-side on a microplate. this system is also "multiplex", regarding that the various viruses are simultaneously detected from a tested clinical sample. we term this approach "multi" pcr. by using automated systems, the multiplex and rapid detection of the various pathogens is achieved very rapidly not only in multiplex pcr, but also in multi pcr [ , ] . in frame of a recent eu project (http://www.multiplexeu.org/) we developed a quantitative real-time pcr assay for the simultaneous detection of all the seven serotypes of fmdv. this method is based on the priproet principle and is targeting the d gene of the virus. the real-time pcr assay was validated for the efficacy to detect all known fmdv serotypes. the test method was linear over a range of at least seven orders of magnitude and the detection limit was below the equivalent of genomic copies of the virus. analysing african clinical samples the method was able to detect fmdv in materials from both cattle and buffalo. a considerable diagnostic advantage is that the priproet method provides a laboratory result much faster than virus cultivation. the rapid detection and diagnosis of a new foot-and-mouthdisease outbreak, including the determination of the serotype of the virus, is accomplished within several hours [ ] . since fmdv, swine vesicular disease virus (svdv) and vesicular stomatitis virus (vsv) cause very similar or clinically identical vesicular lesions in swine, it is a crucial requirement to develop methods for the rapid detection and identification of these three viruses, in order to assure the differential diagnosis. for this purpose, the above mentioned priproet diagnostic pcr was completed by a range of other real-time pcr assays, to detect all the three viruses. by comparing the detection range and applicability of the priproet assays and molecular beacons we found the priproet as a superior tool to detect various variants of svdv. results with different svdv strains and especially those with mutations in the probe region demonstrated the robustness of the priproet system compared to the other real-time pcr assays (molecular beacon, taqman) that usually require perfect probe match with a target of interest. with the priproet system there is a chance to identify phylogenetically divergent strains of svdv, which may appear as negative in other real-time pcr assays. initially the primers and probe used in these experiments were intended for detection by molecular beacons. in spite of the theoretical fitness of the probe we discovered a reduced sensitivity using molecular beacon compared to priproet. this discrepancy in the sensitivity is explained by the fret system. for the priproet system even a low efficient hybridising probe will bring the reporter fluorophore in proximity of the donor enabling release of reporter fluorescence. there is no competition with the stem-loop structure (as in molecular beacons) and there is no need for probe degradation to release fluorescence (as in taqman), which impairs detection of strains with mutations in the probe target region. in addition, the priproet system gives a specific t m for each of the strains, which can reveal mutations in the target. in conclusion, the observation that priproet detects more svdv strains than molecular beacons makes the former superior for analysis of unknown diagnostic specimens. the high sensitivity and specificity of the svdv priproet assay may improve the early and rapid detection of viral nucleic acids of a wide range of svdv strains, allowing reduced turnaround time and use of high-throughput, automated technology [ ] . the use of nucleic acid extraction robots is further accelerating the diagnostic procedures worldwide. the labo-ratories are using various types of the nucleic acid purifying robots. for example, the genovision m extraction robots (biorobot m station, qiagen, norway), utilize magnetic separation of the target molecules. by comparing the results of nucleic acid preparations of the robot with manual procedures, we found the robot more efficient and reliable. in this robot the viral nucleic acids are purified simultaneously from samples and the procedure is finished within . h. the products are clean enough to be amplified directly in the pcr. in addition to high speed, robustness and low labour-input, a further advantage of the robots is the reduced risk of crosscontamination between specimens. by the introduction of special tools laboratory practices and internal controls (mimics) it was possible to reduce the danger of false positivity and false negativity rather soon in the history of the diagnostic pcr, as it was previously reported and summarised [ , , ] . the introduction of the closed and automated systems of the robots provides further insurance today for the safe diagnostic reliability of the pcr-based diagnostic assays. the simultaneous use of the nucleic acid extraction and pipetting robots with the real-time pcr machines provides an automated diagnostic chain. such automated diagnostic chains have been established at our laboratory for the detection of several viruses. by this way high throughput and robust diagnostic assays have been established, with reduced manipulation requirement, less contamination risk and a very rapid diagnosis time, which is shortened from hours to minutes [ ] . the international validation and standardisation of the diagnostic assays is very important today. national and international authorities require rigorous proof that the assays, used in various laboratories, are as reliable as possible and give identical results. international agencies like the oie, the joint fao/iaea division, national research institutions and commercial companies make large efforts to agree on international standardisation. the oie regularly publishes standards for the validation of diagnostic assays (see http://www.oie.int/eng/publicat/en standards.htm). validation and international standardisation of nucleic acid amplification-based diagnostic methods (like pcr) is the major task for these authorities. the usual practice is that the specificity and the sensitivity of the newly developed pcr assays are compared to conventional assays, like virus isolation. the "in house" pcr assays will soon have to be replaced by validated and standardised procedures. the validation, standardisation and quality control of pcr-based diagnostic techniques, which are now in progress, are a major task. our laboratory, in collaboration with other international partners has been actively involved in the validation processes, by following the stages of assay validation as suggested by the oie [ , , , ] . considering these efforts and achievements, the oie designated in our laboratory as "oie collaborating centre for the application of polymerase chain reaction methods for diagnosis of viral diseases in veterinary medicine". by acknowledging this important title, we put large efforts in development of molecular diagnostics and to continue the international standardisation and validation. furthermore, we provide international training programmes in molecular diagnostics. as summarised above, the various real-time pcr assays provide powerful novel means for the very rapid detection and quantitation of targeted viral nucleic acids in clinical specimens. the real-time pcr assays have opened a new area of molecular diagnosis. however, although it has many advantages, a vulnerable side of the pcr-based diagnostic assays is that the detection efficiency is decreased by the high nucleotide sequence variability (mismatches) in the genomes of the various variants of the targeted virus(es). the increasing numbers of mismatches between target and primer sequences result in decreased amplification or even in negative pcr results. the variability of the target nucleotide sequences might be associated with various phenomena, such as mutations, deletions or duplications in the genomes of the sought viruses. similarly, weaker or negative pcr results may occur when trying to amplify the genomes of newly emerging genomic variants of a virus genus. the pcr analysis of emerging new viruses may yield negative results, due to the novel nucleotide sequences in these viral genomes. considering these vulnerable sides of the pcr-based diagnostic assays, there is a high need to develop further approaches of molecular diagnosis. there is a strong tendency today to further increase the number of various methods, in order to strengthen the molecular diagnosis, to reduce the diagnosis time and to improve the diagnostic complexity. we list several examples here, including the development processes of our group and its partners. the padlock probes, adapted to microarray formats, provide novel means of powerful and very complex novel molecular diagnosis. padlock probes are circularizable oligonucleotides useful for highly multiplex genetic studies [ , ] . these probes have the capacity to detect simultaneously thousands of different target sequences in a single multiplex array system. each target serves as template for a padlock probe equipped with a unique sequence (tag) associated to that specific target. circularized probes are amplified with a single universal primer pair and the fluorescently labelled products are then sorted, using the tag sequences, on a microarray [ ] . the application of padlock probes for detection of pathogens is a very recent trend in molecular diagnosis [ ] . we developed a padlock probe package for the simultaneous and rapid detection of multiple viruses in the vesicular disease complex of swine, using a microarray-based read-out. the assay principle was straightforward comprising a few internally controlled reaction steps in a single vessel. run-times were comparable to real-time pcr, but with the benefit that the presence of several viruses and their various serotypes can be analyzed within the same reaction ( fig. a and b ). in conclusion, our multiplex detection system could have immediate implications in more effective screening for viruses causing similar vesicular symptoms in the swine populations [ ] . a simple multiplex real time pcr system was designed recently for the general and simultaneous detection of influenza a-c viruses, i.e., many members of the orthomyxoviridae family, originating from animals and humans [ ] . the assay is performed in a single tube, using broadly targeted primers and probes. the "consort" © computer program (developed by j. blomberg, uppsala university) indicated conserved regions in the nucleoprotein genes, based on comparative sequence analysis of several thousands of influenza virus genome sequences. these regions were used to design a triplex reverse transcription real-time pcr (" qpcr"). it was evaluated with eight influenza a reference strains (h n scotland , h n turkey h n , h n , h n , h n , h n and h n ), serial dilutions of influenza a-c, and synthetic dna targets. the real time pcr assays were able to detect - viral rna copies per pcr assay for influenza a-c, when testing virus isolates and clinical specimens. our study shows that qpcr is a reliable, sensitive, and rational method for detecting and identification of influenza viruses. its generic nature should enable the detection not only of most human influenza viruses, but also of avian and other influenza strains. this wide-range assay provides a powerful novel detection tool for diagnostic laboratories. the broad detection range ensures that the qpcr provides powerful means for the discovery of newly emerging variants of influenza viruses [ ] . we developed a one-step real-time pcr assay, based on the amplification of genomic sequences from the ha gene, for the rapid and simultaneous detection of a broad spectrum of influenza viruses, including highly pathogenic avian influenza viruses. a prototype of real-time pcr system, which uses the superconvection principle ("superconvection qpcr"; alphahelix, uppsala, sweden), was used both for amplification and for cycle sequencing reactions (fig. ) . identification of pathogenicity of aiv (hpai or lpai) including sequence information of ha gene was obtained in less than h: rna purification - min, superconvection qpcr - min, cycle sequencing reactions with superconvection - min and sequencing by capillary electrophoresis (up to b) - min. the wide screening of different subtypes of avian influenza in a single qpcr, followed by rapid sequencing that covers cleavage site of ha gene, will allow monitoring the viral load of influenza strains in wild birds and in farmed poultry. furthermore, the method could provide a very rapid and highly reliable molecular diagnosis in a possible pandemic influenza a scenario [ ] . we participate in the work of a joint research group, which is developing proximity ligation reactions to detect proteins of bacteria and of virus particles via nucleic acid amplification. the principle is that antibodies recognizing viral or bacterial surface proteins are coupled to dna strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents. this method enables the detection of target proteins by ligated dna strands, which are then amplified by real-time pcr. our partner laboratories (olink and svanova, uppsala) reported that detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium (m. merza, personal communication). compared to agelisa, the sensitivity of proximity ligation proved to be higher. thus, proximity ligation of proteins can be of great value for early diagnosis of infectious disease and in biodefense [ ] . at present, we are adapting the proximity ligation technique to the detection of surface antigens of various viruses causing tads, such as fmdv and csfv. the combination of nucleic acid and antigen detection approaches will hopefully yield a more complex, multilateral diagnosis of tads. in summary, the goal of this article was to illustrate some of the recent achievements in the development of molecular diagnosis, with special regard to the achievements and experiences gained at our institute and at our partner laboratories. as it is shown by the examples, the illustrated methods provide novel means for detecting and identifying the viruses and for combating the tads and other viral diseases in a more effective way. molecular diagnosis of animal diseases detection of pseudorabies virus dna sequences by the polymerase chain reaction equine herpesvirus type : detection of viral dna sequences in aborted fetuses with the polymerase chain reaction the molecular diagnosis of porcine viral diseases recent achievements and trends in the molecular diagnosis of bovine viral diseases-a view from the oie collaborating centre for the application of polymerase chain reaction methods for the diagnosis of viral diseases in veterinary medicine genetic identification of pestivirus strain frijters, isolated from pigs, as a border disease virus genetic variability of classical swine fever virus molecular characterization of ovine pestiviruses organization and diversity of the -noncoding region of classical swine fever virus genome genetic heterogeneity of classical swine fever virus in europe genetic analysis of pestiviruses at the end of the genome 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fluorogenic rt-pcr assay for detection of bovine respiratory syncytial virus in clinical samples development of invader squared assay for the detection of african swine fever dna and comparison to pcr based assays development of a rational diagnostic single tube real time pcr for human caliciviruses, norovirus genogroup i and ii development of a novel quantitative real-time pcr assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus a highly sensitive and specific gel-based multiplex pcr assay for the simultaneous and differential diagnosis of african swine fever and classical swine fever in clinical samples development of a real-time pcr assay based on primer-probe energy transfer for the detection of swine vesicular disease virus validation and quality control of polymerase chain methods used for the diagnosis of infectious diseases. office international des epizooties (oie), manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees) parallel gene analysis with allele-specific padlock probes and tag microarrays multiplexed genotyping with sequence-tagged molecular inversion probes diagnostic application of padlock probes-multiplex detection of plant pathogens using universal microarrays microarray-based molecular detection of foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease virus, using padlock probes broadly targeted triplex real-time pcr detection of influenza a, b and c viruses based on the nucleoprotein gene very rapid detection of highly pathogenic avian influenza viruses detection of individual microbial pathogens by proximity ligation thanks are due to all my colleagues at the national veteri- key: cord- -j k oel authors: herrera-rodriguez, josé; signorazzi, aurora; holtrop, marijke; de vries-idema, jacqueline; huckriede, anke title: inactivated or damaged? comparing the effect of inactivation methods on influenza virions to optimize vaccine production date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: j k oel the vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. the first step in the production process is virus inactivation with β-propiolactone (bpl) or formaldehyde (fa). recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. we assessed the effect of inactivation with bpl and fa on different influenza virus strains. the properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (ha) binding ability, fusion capacity and the potential to stimulate a toll-like receptor (tlr ) reporter cell line were then assessed and compared to the properties of the untreated virus. inactivation with bpl resulted in undetectable infectivity levels, while fa-treated virus retained very low infectious titers. hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with bpl-treated virus binding and fusing at a lower degree compared to fa-inactivated samples. on the other hand, bpl-inactivated virus induced higher levels of activation of tlr than fa-inactivated virus. the alterations caused by bpl or fa treatments were virus strain dependent. this data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product. influenza virus is a threatening pathogen, that causes significant morbidity and mortality worldwide. it is responsible each year for epidemics that cause millions of cases of severe illness, and it is also a threat because of its potential to cross species barriers and generate pandemics [ ] . vaccination is the most cost-effective strategy to prevent infection and severe outcomes [ ] . currently marketed influenza vaccines include live-attenuated vaccines, whole inactivated virus (wiv), split vaccines and subunit vaccines (which can be derived from viral particles or recombinant antigens) [ ] . live virus vaccines rely on attenuation; e.g. use of reassortant strains with the backbone of cold-adapted viruses, but equal (or comparable) immunogenicity. for all the other types of vaccines derived from whole viral particles, a crucial step in production is virus inactivation. the general concept behind the inactivation procedure, stated by international guidelines as provided by ema, fda, and who [ ] [ ] [ ] , is that the process must inhibit the replication of the virus without destroying its antigenicity. thus, the inactivation process should cause minimum alteration of the main antigens, which for influenza virus are the viral surface glycoproteins hemagglutinin (ha) and neuraminidase (na). preparation of inactivated influenza vaccines is conventionally achieved by exposure of cell-or egg-derived live virus to betapropiolactone (bpl) or formaldehyde (fa). bpl reacts readily with nucleophiles, resulting in alkylated and acylated products. according to older literature, nucleic acids are the main targets of these modifications, which comprise nicks and cross-links between rna and viral proteins [ , ] . however, recent findings shed new light on the mechanism of bpl inactivation and show that it also alters viral proteins, resulting in loss of ha and na functionality and fusion ability [ ] . the stability of bpl is known to depend on the type of buffer and the ph of the mixture used, suggesting that the amount and nature of modifications in viral components will also be determined by these variables [ ] . in the case of formaldehyde, viral inactivation is achieved by the alkylation of amino and sulphydrilic groups of proteins and purine bases [ ] . as fa crosslinks the viral proteins [ ] , the fusion ability of the virus can be affected. since inactivation is a critical step during vaccine preparation, given that the manufacturing of not only wiv but also split and subunit vaccines depends on effective virus inactivation, the major pharmacopoeias specify quality standards for this process. recommendations in international guidelines regarding inactivation are, however, quite vague, and the establishment of optimal conditions is left to the manufacturers. specifications provided for the inactivation procedure include indications on the temperature of storage and the maximum amount of inactivating agents. nonetheless, the choice of variables such as the concentration of the virus at the time of inactivation, the buffer systems used to dilute the inactivators, the ph of the virus suspension, or the duration of the incubation with the agents remains yet unspecified. furthermore, the guidelines are not clear about whether these parameters are to be optimized on the basis of the vaccine strain, the concentration of the virus or any other criterion. more precise guidelines would be desirable in order to prevent the receptor binding sites and epitopes in the vaccine from being destroyed during the inactivation process. recent studies show that excessive inactivation with fa and bpl may cause unanticipated modifications to the vaccine antigens that result in diminished potency; lower hemagglutination titers and loss of na activity [ , [ ] [ ] [ ] [ ] [ ] . this suggests that chemical inactivation might affect the protein conformation leading to a loss of immunogenicity of the antigenic epitopes of the key surface proteins. the present study presents a systematic evaluation of different inactivation protocols (in accordance with the international guidelines) on several influenza a virus (iav) strains. our aim was to compare the effects of these procedures on the key properties, namely residual infectivity, receptor binding, fusion, and toll-like receptor (tlr ) mediated activation of innate immune mechanisms, and to determine whether these effects are similar for different virus strains. the strains used in this study were a/puerto rico/ / h n (pr ), a/new caledonia/ / h n (nc), a/perth/ / h n (h ), nibrg- a/turkey/turkey/ / h n (h ), and nibrg- a/anhui/ / h n (h ); all vaccine strains with a pr backbone produced through classic reassortment (nc, h ) or recombinant technology (h , h ). all seed viruses were obtained from the national institute for biological standards and controls, potters bar, united kingdom and were propagated in days old embryonated eggs. after inoculation of the virus and incubation for h, the allantoic fluid was harvested and clarified by low speed centrifugation, followed by two rounds of purifications of the virus on a sucrose gradient [ ] . protein and phospholipid determinations were performed on the purified virus using the micro lowry and the bligh and dyer method, respectively [ , ] . all virus preparations were used at a concentration of . nmol/ml of phospholipid for inactivation. the number of virus particles was calculated assuming that each virion contained about , phospholipid molecules. this number was derived using the reported composition ( - % lipid) and dry weight (  À g) of influenza virions and a mean mw for lipids of - g/mol [ ] . this calculation does not take into account a possible contamination of the virus preparations with exosomes which would also be assumed to contain lipids and would presumably co-purify with virus on the sucrose gradient. however, the amount of exosomes is not expected to vary among the virus strains and is therefore neglected here. all procedures and dilutions were performed in hne buffer ( mm hepes, mm nacl, . mm edta, ph . ). three standard inactivation protocols were selected: in the first one, beta-propiolactone % (acros organics, geel, belgium) was used at a final concentration of . % v/v and inactivation was done by overnight incubation (bpl). in the second protocol, % formaldehyde was used at a final concentration of . % v/v and incubation was for h (fa- ). the third protocol also involved the use of formaldehyde at a final concentration of . % v/v but incubation was done for h (fa- ). all incubations were performed at °c under constant stirring. after incubation with either bpl or fa, the preparations were dialyzed in , mwco dialysis tubes against hepes buffer overnight at °c to remove all traces of the chemicals. again, protein and phospholipid determinations were performed on the dialyzed samples, and all samples were normalized to a concentration of . nmol/ml of phospholipids. infectivity of both untreated and treated virus samples was tested by performing a tcid assay on mdck cells. briefly, confluent cultures of mdck cells in -well plates were incubated with ll of two-fold serial dilutions of virus or vaccine at an initial concentration of . nmol/ml of phospholipids at °c in % co for h. cells were then washed with pbs and incubated for days at °c with ll/well of episerf medium (gibco) supplemented with lg/ml tpck-treated trypsin. tcid titers were calculated according to the trimmed spearman-karber method [ ] . hemagglutination assays were performed using two-fold serial dilutions of culture supernatants in pbs in v-bottom plates ( ll/ well). subsequently, ll of % guinea pig red blood cells (harlan, the netherlands) were added to each well. the plates were incubated for h at room temperature and the hemagglutination or the absence of hemagglutination was determined visually for each well. membrane fusion was assessed by measuring leakage of hemoglobin during fusion of virus particles with erythrocytes [ ] . briefly, either guinea pig or chicken blood was diluted : with hne buffer ( mm hepes, mm nacl, . mm edta, ph . ). after centrifugation for min at g, erythrocytes were removed from under the layer of peripheral blood mononuclear cells, washed once again with hne buffer, counted and brought to a concentration of  / ll. virus-induced hemolysis was measured by mixing either live virus or vaccines ( nmol of phospholipid) with red blood cells (  ) and fusion buffers of different ph values, ranging from . to . in a final volume of ml. after min of incubation at °c the suspension was centrifuged at g for min and absorbance of the supernatant was read at nm. autohemolysis (occurring in fusion buffers of different ph values in the absence of virus or vaccine) and maximal hemolysis (in water) were used to set % and % of hemolysis. fusion was calculated as: %hemolysis ¼ à expod À autohemod at same ph maxod À autohemod at same ph % hek-blue tlr cells (invivogen) co-express human tlr and an nf-kb-inducible secreted embryonic alkaline phosphatase (seap) reporter gene that can be monitored using the detection medium quanti-blue tm . hek-blue tlr and hek-null (control, not expressing any tlrs) cells were cultured according to the manufacturer's instructions. . cells/well were plated in a -well plate and stimulated with serial -fold dilutions of untreated (starting dilution : v/v) or inactivated (starting dilution : v/v) influenza virus. initial concentration of stocks of live and inactivated virus was . nmol/ml of phospholipids. after h, ml of supernatant were added to ml of quanti blue. after h of incubation at °c, the plates were read in an elisa reader ( nm). results are expressed as relative activation of cells, in comparison to the activation level obtained upon stimulation with ng/ml of the tlr stimulant r (invitrogen) [ ] . in order to allow comparison of the effects of different inactivation processes, samples of different influenza virus strains (pr , nc, h , h and h ) were purified and diluted so that all the batches had the same virus concentration. since the amount of contaminating ovalbumin might differ among different virus preparations, phospholipid content was chosen over protein content for quantification of virus particles. each of the virus preparations at the desired concentration ( . nmol phospholipid/ml) was then divided in independent batches: of them were inactivated with bpl (bpl), were inactivated with formaldehyde with days incubation period (fa- ) and others were also inactivated with formaldehyde, but with days incubation period (fa- ). the last batch was kept untreated as a control. tcid assays on mdck cells were performed in order to determine the number of infectious particles in the virus samples before and after inactivation. the initial infectivity of the virus strains differed substantially: pr showed particularly high infectivity resulting in a particle/tcid ratio of about , while infectivity of h was low (particle/tcid ratio:  ). infectivity of the other strains was rather similar (particle/tcid ratios: between  and  ). after the inactivation process we observed that all treatments had reduced the virus titers by at least logs. in case of bpl treated virus, pr , h and h were found to have no residual particles in any of the independently treated batches while one batch of nc and all batches of h showed some (though very low) residual infectious particles ( - tcid /ml). fa, irrespective of the incubation period, was unable to completely inactivate the virus samples. residual titers varied from to tcid /ml with nc showing the lowest and h showing the highest titers. in the case of pr and h , we observed slightly improved reduction in the residual infectivity after longer incubation periods with fa, yet this effect was not observed for the other strains. hemagglutinin is an important determinant of the infectivity of influenza a virus. because the role of the hemagglutinin is to bind to the sialic acid receptors on the infected cell, we consider binding as a reflection of a functional protein and therefore of an antigen similar to the one found on the live virus. thus, we assessed the binding capacity of the viral hemagglutinin after the inactivation process. for untreated samples of the different virus strains the binding capacity in hau/ml was rather similar: .  for pr , .  for nc, .  for h , .  for h and  for h . for the inactivated samples the percentage of relative hemagglutinating capacity was calculated taking the obtained value of the untreated samples as %. as shown in fig. all treatments caused a reduction in the binding capacity of the virus. after inactivation with bpl there was a complete loss of binding ability for pr as well as for h , consistent among all batches. for nc, two batches retained a minimal amount of binding ability. h and h were the strains that retained most of the binding ability after inactivation with bpl, consistent among the batches ( - %). fa treatment had less effect on the hemagglutination ability of the virus strains than bpl, independent of the inactivation period. however, the effect of fa varied for the different virus strains. while pr lost almost its entire binding ability, nc retained most of its binding ability after inactivation with fa ( %). h , h and h retained some of the binding ability ( - %) with some variation among the different batches. next to being responsible for binding of influenza virus to a target cell, ha also mediates fusion of the viral with the endosomal membrane during the infection process. retaining this function after inactivation would be a further indication that ha is in its native state. moreover, it has been reported that fusion-active whole inactivated virus vaccines are better inducers of type interferons and allow better induction of cd t lymphocytes [ , ] . fusion ability was measured by incubation of viral or vaccine particles ( nmol of total phospholipids) with either guinea pig erythrocytes for pr , nc and h or chicken erythrocytes for h and h , following a protocol reported previously [ ] . as shown in fig. , all viruses fused with the respective erythrocytes with final rates of fusion varying from % for h and h to % for pr . the optimal ph for fusion was higher for pr (ph . ) than for the other virus strains (ph . or . ). bpl completely inhibited the fusion ability of the virus, except for h where some minimal fusion ability was retained (% %). in contrast, after fa inactivation, most of the virus particles retained some fusion ability. only the fusion ability of h was severely affected by fa. for pr , the inactivation process appeared to change the optimal fusion ph causing the inactivated samples to fuse at a lower ph. also for h , loss of fusion activity after fa treatment was more pronounced at higher than at lower ph. when comparing inactivation with fa for days and days, we noticed a further reduction in the fusion ability of the virus treated for days. this was most noticeable for h and h where we saw a reduction of around % of the fusion ability of the samples inactivated for days as compared to days. after infection, tlrs present on and in antigen-presenting cells of host cells sense the viral components; thus, they form an important constituent of the innate antiviral response [ ] . in particular, tlr is the sensor for the single-stranded rna present in live virus and whole inactivated vaccines; its activation is a good indicator of the ability of the virus to be internalized since the receptor is exclusively located on endosomal membranes [ , ] . in order to determine whether the inactivation procedure would affect the ability of the viruses to attach, fuse and activate tlr in the endolysosomal compartments, we stimulated hek blue tlr cells (expressing a reporter construct upon ligand binding to tlr ) with untreated and inactivated pr , nc, h , h or h . results are expressed as percentage of activation of the cells, compared to the response to a fixed amount of the tlr stimulant r (set as % activation). after h of incubation in a medium containing tpck-trypsin, the supernatants were harvested and presence of virus was determined by hemagglutination assay. tcid was calculated as described previously [ ] . stimulation of hek blue cells with untreated virus showed that the different strains varied largely in their ability to activate tlr and for the different virus optimal activation was observed at different dilutions (fig. , left panels) . inactivation had marked effects on the ability of the viruses to stimulate tlr . no activation was observed at dilutions found optimal for the different live viruses. for inactivated nc, h , and h even at the lowest dilution of : no or very little activation of tlr was found while pr and h showed some activation at low dilutions. bpl inactivation generally preserved the ability to activate tlr somewhat better than fa. thus, all inactivation protocols strongly reduced the ability of the viruses to stimulate tlr ; however, there were differences among the different virus strains. inactivation is a crucial but ill-defined step in influenza vaccine preparation. we therefore intended to systematically assess the effect of common inactivation procedures on different phases of the interaction of inactivated virus particles with cells in vitro. to this end, we used different virus strains in order to reveal possible strain-specific differences and performed the inactivation procedures for all viruses using a single buffer and the same virus concentration to eliminate possible variation, an approach also taken by others [ ] . our results show that, under the conditions used, bpl was quite efficient in inactivating the viral particles while formaldehyde, although reducing the infectious titer by - logs, was unable to completely abolish infectivity. all inactivation methods caused damage to the binding capacity of the viral particles, with bpl causing greater loss than fa. the ability of the virus to fuse with target membranes at low ph was especially affected by bpl treatment, while fa-treated virus still maintained some fusion ability. the stimulation of a tlr reporter cell line was also affected by the treatment, with fa inactivation leading to a greater loss of the ability to stimulate tlr than bpl inactivation. the magnitude of the effects caused by bpl or fa treatment appeared to be virus strain dependent, as different iav strains were affected to varying degrees. from our results, it is clear that chem-ical inactivation impacts on various properties of influenza virus in a treatment dependent and strain dependent way. our study revealed that bpl reduced the infectivity of pr , h and h virus to undetectable levels. nc and h virus showed some (although very low) residual infectivity (fig. ) indicating that these two strains might be more resistant to inactivation with bpl. previous studies show that bpl is capable of complete inactivation of influenza virus; however, the effectivity might vary depending on the incubation time and temperature. a review of the literature suggests that at a concentration of . % bpl is able to completely inactivate the virus if incubation is performed for - h at temperatures above °c (but below °c). however, if the same concentration of bpl is used but incubation is executed at °c like used in our study, an inactivation period ranging from h to a week is needed for complete loss of infectivity [ ] [ ] [ ] . in contrast to bpl, fa was not able to completely inactivate influenza virus in our study. again, there seem to be strainspecific differences in the sensitivity to fa since some strains were more effectively inactivated than others and for some strains (pr , h ) inactivation improved with longer exposure times to fa while for other strains this was not the case. a variety of different methods for fa inactivation has been described in the literature [ , , , [ ] [ ] [ ] [ ] . these methods vary with respect to the exact experimental conditions used, including concentration and incubation time. in some cases, incomplete inactivation is reported, mostly occurring when the inactivation was performed at low temperature [ ] , as was done in this study. inactivation procedures should not affect the immunological properties of the viral antigen. to determine the effects of the different inactivation protocols on the recognition of the virus by haspecific antibodies, we tried to perform a single radial diffusion (srid) assay on the virus samples before and after the inactivation procedure. however, the relatively low concentration of virus used for inactivation (chosen to allow optimal access of the inactivating agent to the viral proteins) was not suitable to render reliable readings. yet, functional properties of the viral ha, namely binding to sialic acid residues on the target cell and mediating fusion of the viral and the endosomal membrane, are also indicative of a func- fig. . effect of inactivation procedures on fusion ability. fusion ability was measured by incubation of viral or vaccine particles ( nmol of total phospholipids) with either guinea pig rbc (a-c) or chicken rbc (d-e) followed by the addition of fusion buffer to reach the desired ph. after min incubation, samples were centrifuged and the supernatants were collected. samples were read at nm on a spectrophotometer. percentage of fusion was then calculated as described previously [ ] . tional and unaltered protein. we therefore investigated the effects of the different inactivation protocols on hemagglutination and on hemolysis as surrogates for binding and fusion, respectively. as all our observations are based on in vitro tests, the correlation with the different vaccine potencies in vivo should be explored in future experiments. the use of single radial immunodiffusion assay as an estimate for protection has been shown to correlate both with in vivo responses [ ] and, more recently, with in vitro results of differently manufactured influenza vaccine [ ] . however, while the srid assay would have been an alternative method to measure ha integrity, it would have given information on antibody binding only. binding as measured in a hemagglutination assay was strongly reduced by bpl inactivation, with only h and h retaining some binding ability. fusion was even more deeply affected as all virus strains completely lost their ability to fuse after treatment with bpl. virus binding to the target membrane is a prerequisite for fusion (at least as measured in the assay used). thus, the loss of fusion ability was to be expected. previous reports have shown that h n and h n viruses had reduced agglutination capacity following bpl treatments [ , , , , ] -although in many cases the inactivation was performed at different temperatures, incubation times or concentrations than used in our experiments. for what concerns the effects of bpl on the fusion ability of iav, some studies show that h n and h n strains were almost completely inhibited in their fusion ability at bpl concentrations ranging from . % to . % [ , ] . however, budimir et al. [ ] managed to inactivate iav with bpl and retain the fusion ability of the virus; yet, the temperature discrepancy with our methods could have led to the observed differences in virus alterations. as for the effect of fa on the binding ability, fa treated virus strains were still able to bind to erythrocytes though with reduced effectivity, except for the pr strain which almost completely lost its ability to agglutinate erythrocytes. literature on the effect of fa on virus binding is contradictory. studies report that fa inactivation damaged the binding ability of the virus at conditions (incubation time, concentration and temperature) similar to those tested in our work [ , ] , while others found little effect of the fa treatment, though using different conditions as per the temperature at which the inactivation process was conducted [ , ] . all fatreated samples almost completely retained their fusion ability. geeraedts et al. [ ] and budimir et al. [ ] reported complete loss of fusion ability with fa treated virus, yet in these experiments iav was deliberately treated with fa at extremely high concentration or prolonged exposure to inhibit their fusion ability. it has been previously reported that the magnitude and the phenotype of the immune response induced by wiv influenza vaccines are superior to those induced by split or subunit vaccines [ , ] . the higher immunogenicity of wiv could be largely attributed to activation of tlr by ssrna present in the viral particles [ ] . furthermore, since the stimulation of an endosomal toll-like receptor depends on viral endocytosis (and thus on ha functionality) [ ] , the decreased stimulation of such a receptor can indicate possible modifications of the surface antigen induced by the inactivation procedure. we therefore investigated the effect of the different inactivation methods on tlr triggering. all inactivation procedures studied markedly reduced and often completely abolished tlr triggering, with effects of bpl inactivation being somewhat less severe than those of fa treatment. this was surprising considering that bpl is supposed to mainly affect nucleic acids while fa is supposed to mainly affect proteins [ ] . loss of tlr triggering did not necessarily correlate with loss of binding and was thus not only a result of less virus reaching the endosomal compartment. to our knowledge, the effect of virus inactivation on the capability of wiv to trigger tlr has not been studied previously. earlier studies demonstrating that wiv is capable of activating tlr were per-formed using much higher concentrations of virus ( mg/ml viral protein as compared to about . mg/ml used in this study) [ ] . in these studies, effects of the inactivation procedure might therefore have been obscured. several studies have reported the negative impact of chemical inactivation with bpl on virus characteristics; however, those studies have focused mainly on h n strains. bpl was found to affect binding [ , ] , fusion activity [ , ] and modify protein residues [ , ] , in line with our findings. though all the studies were in compliance with the established guidelines with respect to bpl concentration ( . %), all used different conditions with respect to buffer, virus concentration, incubation time and temperature etc. this might explain variations in outcome among the different studies. one of the initial effects of bpl addition is a decrease in ph caused by hydrolysis of bpl to b-propionic acid and hydracrylic acid derivatives [ ] ; this could then lead to all the other observed undesirable consequences of bpl treatment on ha and na functions. it is known that influenza virus strains vary in their sensitivity to low ph, with pr being one of the most labile strains [ , ] . our findings corroborate these earlier observations. on the other hand, fa has been linked to incomplete inactivation which can cause outbreaks of virus infections upon vaccination; this was reported for several viruses, such as foot-andmouth disease virus (fmdv) [ ] and venezuelan equine encephalitis virus (veev) [ ] . it has also been shown that temperature is an important factor in virus inactivation by formaldehyde: darnell me et al. [ ] reported that sars-coronavirus could not be inactivated at a low temperature of °c even after days of incubation. however, when using a higher temperature of or °c, formaldehyde could inactivate most of the virus after day. our results contribute to the increasing evidence that the inactivation protocols have to be adapted by virus strain and that many other important factors beyond the concentration of the inactivator itself, such as virus concentration, buffer, incubation time and temperature, have to be considered. novel inactivation protocols, such as uv and gamma radiation [ ] or the use of hydrogen peroxide [ ] , have already been mentioned in the literature but will need thorough testing and standardization before they can be employed in the context of influenza vaccine production. there are currently new emerging technologies to manufacture influenza vaccines that would not require an inactivation process, such as production of iav proteins on in vitro cultures or peptides derived from iav proteins, all showing promising results; yet, egg cultures are currently the cheapest and most efficient way to produce high amounts of vaccines in a relative short amount of time. therefore, until a new vaccine production method that can compete with the egg culture is developed, inactivation will be a standard procedure in vaccine manufacturing. our results are therefore a call for establishment of more detailed inactivation procedures for vaccine manufacturers, and for a search for different and more efficient inactivation methods to be included in the international guidelines. the authors declare that there are no conflicts of interest. pandemic influenza-including a risk assessment of h n advances in the development of influenza virus vaccines traditional and new influenza vaccines the european agency for the evaluation of medical products. note for guidence on harmonisation of requirements for influenza vaccines annex -recommendations to assure the quality, safety and efficacy of influenza vaccines (human, live attenuated) for intranasal administration summary minutes - nd vaccines and related biological products advisory committee meeting effect of formalin, beta-propiolactone, merthiolate, and ultraviolet light upon influenza virus infectivity chicken cell agglutination, hemagglutination, and antigenicity principles of selective inactivation of viral genome. vi. inactivation of the infectivity of the influenza virus by the action of fl-propiolactone treatment of influenza virus with beta-propiolactone alters viral membrane fusion reactions of betapropiolactone with nucleobase analogues, nucleosides, and peptides: implications for the inactivation of viruses inactivation of avian influenza viruses by chemical agents and physical conditions: a review identification of formaldehyde-induced modifications in proteins: reactions with insulin hydrogen peroxide inactivation of influenza virus preserves antigenic structure and immunogenicity effect of inactivation method on the cross-protective immunity induced by whole ''killed" influenza a viruses and commercial vaccine preparations evaluation of different methods of inactivation of newcastle disease virus and avian influenza virus in egg fluids and serum influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling development of a dried influenza whole inactivated virus vaccine for pulmonary immunization a simplification of the protein assay method of lowry which is more generally applicable bligh and dyer" and folch methods for solid-liquid-liquid extraction of lipids from microorganisms. comprehension of solvatation mechanisms and towards substitution with alternative solvents models of structure of the envelope of influenza virus determination of % endpoint titer using a simple formula induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity smallantiviral compounds activate immune cells via the tlr myd -dependent signaling pathway effect of viral membrane fusion activity on antibody induction by influenza h n whole inactivated virus vaccine the role of membrane fusion activity of a whole inactivated influenza virus vaccine in (re) activation of influenza-specific cytotoxic t lymphocytes toll-like receptor control of the adaptive immune responses th cells primed during influenza virus infection provide help for qualitatively distinct antibody responses to subsequent immunization evaluation of the effects of concentration, duration and temperature of incubation with beta-propiolactone on inactivation of cell-derived rabies virus evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation effect of physico-chemical factors on survival of avian influenza virus (h n type) whole influenza virus vaccine is more immunogenic than split influenza virus vaccine and induces primarily an igg a response in balb/c mice superior immunogenicity of inactivated whole virus h n influenza vaccine is primarily controlled by toll-like receptor signalling evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy single-radial-immunodiffusion as an in vitro potency assay for human inactivated viral vaccines influenza vaccine manufacturing: effect of inactivation, splitting and site of manufacturing. comparison of influenza vaccine production processes effect of the b-propiolactone treatment on the adsorption and fusion of influenza a/ brisbane/ / and a/new caledonia/ / virus h n on a dimyristoylphosphatidylcholine/ganglioside gm mixed phospholipids monolayer at the air-water interface whole inactivated virus influenza vaccine is superior to subunit vaccine in inducing immune responses and secretion of proinflammatory cytokines by dcs influenza virus entry surface modifications of influenza proteins upon virus inactivation by b-propiolactone variations in ph sensitivity, acid stability, and fusogenicity of three influenza virus h subtypes stability of infectious influenza a viruses at low ph and at elevated temperature biochemical identification of viruses causing the outbreaks of foot and mouth disease in the uk review of accidents caused by incomplete inactivation of viruses inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov analysis of antigen conservation and inactivation of gammairradiated avian influenza virus subtype h n we would like to thank sarah skeldon, national institute for biological standards and controls, uk, for running srid assays on the virus samples. key: cord- -ngms ie authors: sawada, akihito; komase, katsuhiro; nakayama, tetsuo title: aik-c measles vaccine expressing fusion protein of respiratory syncytial virus induces protective antibodies in cotton rats date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ngms ie abstract respiratory syncytial virus (rsv) is the most common cause of respiratory infection in infants, and no vaccine is available. in this report, recombinant aik-c measles vaccines, expressing the rsv g or f protein of subgroup a (mvaik/rsv/g or f), were investigated as a rsv vaccine candidate. mvaik/rsv/g or f had the original ts phenotype and expressed rsv/g or f protein. cross-reactive neutralizing antibodies against rsv subgroups a and b were detected in cotton rats immunized intramuscularly with mvaik/rsv/f but not mvaik/rsv/g. in cotton rats infected with rsv, rsv was recovered and lung histopathological finding was compatible with interstitial pneumonia, demonstrating thickening of alveolar walls and infiltration of mononuclear cells. when cotton rats immunized with mvaik/rsv/f were challenged with homologous rsv subgroup a, no infectious rsv was recovered and very mild inflammation was noted without rsv antigen expression. when they were challenged with subgroup b, protective efficacy decreased. when cotton rats immunized with mvaik/rsv/g were challenged with rsv subgroup a, low levels of infectious virus were recovered from lung. when challenged with subgroup b, no protective effects was demonstrated, demonstrating large amounts of rsv antigen in bronchial-epithelial cells. mvaik/rsv/f is promising candidate and protective effects should be confirmed in monkey model. human respiratory syncytial virus (rsv) is a member of the family paramyxoviridae in the order mononegavirales. the paramyxoviridae consist of two subfamilies, paramyxovirinae and pneumovirinae [ ] . classified into the genus pneumovirus, rsv is characterized by a non-segmented, negative sense, single-stranded rna genome, and has approximately , nucleotides. all members of the paramyxovirus family are similar in structure and characteristics [ ] . viral particles of rsv are surrounded by a lipid bilayer with two viral glycoproteins, g and f [ ] , involved in the attachment to, fusion with, and entry into cells during infection. g protein is not always required for infection and cell fusion and the expression of f protein alone leads to cell fusion [ ] . rsv was first isolated in and two antigenically different subgroups, a and b, co-circulate [ ] . rsv is the most common cause of lower respiratory infections in infants and young children worldwide, and is responsible for a variety of illnesses, including - % of pneumonia cases and - % of bronchiolitis cases among hospitalized children [ ] . the peak of serious rsv infections is at - months of age and most children experience an rsv infection by two years of age [ ] . the infection causes serious illnesses especially in babies born prematurely and having chronic lung diseases, or congenital heart diseases. rsv also causes lower respiratory tract infections in the elderly, and in immunocompromised hosts [ ] . the global annual morbidity and mortality for rsv are estimated to be million and , deaths, respectively [ ] . a recent study of the immune response to rsv showed the importance of innate immunity in regulating adaptive immune responses [ ] . adaptive immunity is generally considered effective due to neutralizing antibodies (nt) and cellular immune responses for the clearance of viruses are influenced by innate inflammatory responses. secretory and nt antibodies were generated after repeated infections with rsv, although the responses were weak in young infants [ ] . the presence of igg antibodies in the lung has been shown to reduce viral load [ ] . even a natural infection did not provide long-term protective immunity against reinfection in young infants, and a humanized monoclonal antibody against the f protein is available as a prophylaxis against rsv, or for reducing serious diseases in high-risk infants during epidemics [ ] . however, the high medical costs for monthly administration mean that there is a great need to develop an rsv vaccine [ ] . there are several obstacles to developing a rsv vaccine. an aluminium-precipitated formalin-inactivated rsv vaccine (fi-rsv) was developed in the 's, but did not prevent infections [ ] . in fact, symptoms were exacerbated among recipients subsequently infected with rsv. fi-rsv generated only binding antibodies without neutralizing activity because of the denatured f protein, and did not induce cytotoxic t cell lymphocytes (ctl) activity [ ] . several strategies have been adopted to develop subunit vaccines, live attenuated vaccines through conventional methods of cloning or selecting ts mutants, genetically modified-strain by reverse genetics, and vaccinia virus vector-based recombinant vaccines [ ] [ ] [ ] . recently, a method for direct manipulation of the genomic rna of mononegavirales has been established, known as the infectious cdna clone system [ ] . the transcription and replication of minigenome rna are driven by viral proteins, which are coexpressed by plasmids or helper viruses. using this system, the infectious recombinant viruses can be retrieved from the authentic full-size genome cdna [ , ] . these "reverse genetics" techniques are powerful tools not only for basic research into viral properties, such as the characteristics of viral proteins, and mechanisms of replication, transcription and pathogenesis, but also for practical purposes, such as the development of new vaccines and viral vectors. as vector-based recombinant vaccines, human parainfluenza virus type iii (hpiv iii) vector-based, or sendai virus vector-based vaccines have been evaluated [ , ] . current measles vaccines used throughout the world were attenuated from the edmonston strain, classified as genotype a [ ] . the aik-c strain of the measles vaccine was developed in in japan from the edmonston strain, by plaque cloning through passages in sheep kidney cells and chicken embryonic cells at • c [ ] . it shows optimal growth at • c and little or no growth at • c [ ] . the safety and immunogenicity of the aik-c measles vaccine were established through clinical trials [ ] [ ] [ ] [ ] . reverse genetics of the aik-c live attenuated vaccine was performed and in this study, recombinant aik-c mv vaccine strains encoding the rsv g or f protein were constructed, and immunogenicity and protective effects against rsv were investigated in cotton rats immunized with recombinant measles vaccines, expressing rsv g or f protein. the aik-c seed strain for vaccine production was used. wildtype strains of rsv subgroups a and b were isolated in hep- cells from patients. long and wild-type strains were used for the neutralization test (nt) against rsv subgroups a and b. t and hep- cells were maintained in eagle's mem (sigma-aldrich, dorset, uk) supplemented with % fetal bovine serum (fbs). vero cells were maintained in eagle's mem supplemented with % fbs. b a cells are marmoset b cell line, and maintained in rpmi- medium (sigma-aldrich, dorset, uk) supplemented with % fbs [ ] . these media were supplemented with mm l-glutamine, , iu/ml penicillin, and , g/ml streptomycin. genomic rna was extracted from a clinical isolate of subgroup a and b, and the rsv genome was amplified by rt-pcr. the viral rna was first converted to cdna using a cdna primer: -acacgatttgcaatcaaacc- . the rsv g gene was amplified with -gtttccatggccaaaaccaaggaccaa- and -ccaagcggccgctagtttgttgtgttggatggaga- , which amplified bp. the rsv f gene was amplified with -gttgccatggagttgccaatcctcaa- and -tgtggcggccgctaactaaatgcaatattattt- , which amplified bp. the f and g genes were cloned into pmv/ - using two restriction enzymes, nco i and not i (underlined sequences). a schematic diagram of the strategy used for the construction of the recombinant cdna plasmid is shown in fig. . the full length plasmid was divided from two parts as previously reported. the first half contained the n, p, m and f genes from the leader sequence to the pac i site at nucleotide position of the aik-c genome. the second half contained the h and l regions from the pac i site from position of the aik-c genome to the trailer sequence. the full-length cdna, pmvaik, was constructed using these two plasmids [ ] . the cloning vector for the rsv genome, pmvaik/ - , was constructed from positions (sac ii) to (ecot i). the rsv g or f pcr product was digested with nco i and not i and ligated into pmvaik/ - , resulting in pmvaik/ - /rsv/g and pmvaik/ - /rsv/f, respectively. the pmvaik/ - /rsv/g or pmvaik/ - /rsv/f was digested with sac ii and pac i and ligated into pmvaik. then, full-length infectious cdna clones, pmvaik/rsv/g and pmvaik/rsv/f, were constructed. monolayers of t cells in -well plates were infected with the vaccinia virus mvat pol, expressing t rna polymerase. mvat pol was derived from a highly attenuated and host range-restricted vaccinia virus, the ankara strain [ ] . open reading frames of the n, p, and l genes were cloned downstream of the t promoter of pbluescript sk, and the expression plasmids pcian , pciap , and pcial were constructed [ , ] . after h of adsorption, the cells were washed with opti-mem (gibco, grand island, ny, us) and transfected with . g of pcian , . g of pciap , . g of pcial , and . g of pmvaik/rsv with transit-lt reagent (mirus bio corporation, us). after incubation at • c for h, the medium containing the transfection reagent/plasmid complex was replaced with fresh mem supplied with % fbs. the transfected cells were incubated at • c in % co for days. after days, t cells were detached and co-cultured with b a cells. when a demonstrable cytopathic effect (cpe) was observed, the supernatant and cell lysate were harvested and stocked. to examine viral growth, b a cells were infected with mvaik, mvaik/rsv/g, and mvaik/rsv/f (m.o.i. = . ) and the plates were placed at temperatures of , , and • c. the culture fluids were obtained on days , , , and of culture and infective titers were examined and expressed as tcid /ml in b a cells. b a cells were infected with mvaik, mvaik/rsv/g or mvaik/rsv/f at m.o.i. of . in -well plates and cultured for two days at • c. b a cells were collected and subjected to indirect immuno-staining without fixation to detect the surface expression. polyclonal antibodies against rsv raised in goat (abcam, cambridge, uk) were used and the cells incubated for h at • c. the cells were washed extensively with phosphate-buffered saline with . % tween (pbst), and stained with second antibodies against goat igg conjugated with fitc, raised in rabbit (vector laboratories, burlingame, ca, us), and thereafter, mouse monoclonal antibody against mv ha protein (kindly supplied by dr. sato, national institute of infectious diseases, japan) was used and followed by second antibodies against mouse igg conjugated with rhodamine raised in goat (rockland immunochemicals, gilbertsville, pa, us). vero cells were infected with mvaik, mvaik/rsvf, and mvaik/rsv/g and hep- were infected with rsv subgroup a, long strategy for the construction of the recombinant aik-c genome cdnas having rsv protein genes. the recombinant aik-c viral cdnas expressing rsv g or f protein were constructed based on aik-c cdna (pmvaik). pmvaik/ - was constructed for the cloning of foreign genes. the asc i site was introduced by adding ggcgcg after position of aik-c and r and r sequences were added. the nco i-not i fragment of rsv g or f was cloned into pmvaik/ - , designed as pmvaik/ - /rsv. pmvaik/ - /rsv had unique restriction enzyme sites, sac ii and pac i sites, located in the p gene and between the f and h gene. the dna fragments between the sac ii and pac i sites of pmvaik/ - /rsv/g and pmvaik/ - /rsv/f were inserted into pmvaik using sac ii and pac i sites. the recombinant plasmid constructs were designated pmvaik/rsv/g and pmvaik/rsv/f, respectively. strain in a -well plate. culture supernatants were collected and cells were freeze-thawed and total protein of g of supernatants and cell lysate was applied. samples were subjected to western blotting. briefly, after sds-page, proteins were transferred to membrane (immobilon; millipore, danvers, ma, us). membranes were washed with pbst, incubated with an rsv polyclonal antibody raised in goats, washed again, and incubated with a donkey antigoat igg (h + l) conjugated with horse radish peroxidase (hrp). the final reaction was performed with a dab substrate kit for peroxidase (vector laboratories, burlingame, ca, us) used as recommended by the manufacturer. culture medium of vero cells infected with mvaik/rsv/g or f was collected and fractionated through sucrose discontinuous gradient ultra-centrifugation. fraction was obtained at the top of the gradient, % sucrose, fraction between % and % sucrose, and fraction between % and % sucrose. each fraction was electrophoresed and analyzed by western blotting, using rsv polyclonal antibodies and monoclonal antibodies against mv n protein. six-week-old cotton rats were purchased from harlan (indianapolis, in, us) and charles river (usa). five cotton rats for each group were immunized intramuscularly with × tcid of mvaik, mvaik/rsv/g or mvaik/rsv/f. serum samples were obtained immediately before and , , , , and weeks after immunization. cotton rats immunized with mvaik/rsv/g or f were boosted with the same dose after weeks, and serum samples were collected one week after re-immunization ( weeks). neutralization tests (nts) against rsv were performed with the % plaque reduction assay, using long strain and wild-type isolate of subgroup b. briefly, serum samples were serially diluted by : , starting from a : dilution, and mixed with an equal volume of rsv ( pfu) in mem for h at room temperature. the mixtures were inoculated on monolayers of hep- cells in -well plates. plates were incubated for h at • c in % co and then overlaid with mem supplemented with glutamine, antibiotics, % fetal bovine serum and . % agar. after incubation for six days at • c in % co , cells were fixed with % formalin. agar was removed and cells were stained with neutral red. plaque numbers were counted and nt antibody titers were calculated as the reciprocal of the serum dilutions that showed a % reduction of the plaque number. for the particles agglutination (pa) test, gelatin particles were coated with purified measles virus antigen (serodia ® -measles, fuji rebio, tokyo, japan). sera were serially diluted two-fold, starting from a : dilution, and each serum dilution was mixed with an equal volume of gelatin particles to detect agglutination, according to the recommendations of the manufacturer. the pa antibody titers were expressed as the reciprocal of the serum dilution which induced particle agglutination. cotton rats were sacrificed days after immunization with mvaik/rsv/g and f, and samples of liver, kidney, spleen, lung, thymus, and nasal turbinate were obtained to detect the mv genome. the tissues were homogenized, and total rna was table primer and probe sequences for the detection of the mvaik n gene and rsv n gene by taqman real-time pcr. sequences ( table . seven week-old cotton rats were immunized intramuscularly with mvaik/rsv/f or mvaik/rsv/g and, five weeks later, challenged with pfu/ . ml of rsv subgroups a and b. they were sacrificed four days after the challenge and nasal wash, bal, nasal turbinate, and lung tissues were obtained. lung samples were divided into two portions, one for pathological examination, and another for recovering the infective particles and rsv genome. tissues were homogenized and . ml volumes of serial -fold dilutions of homogenized samples were placed on hep- cells and overlaid with mem % fbs and . % agar. plaque numbers were counted after incubation for six days at • c and infectivity was expressed as the number of plaques. rna was extracted from nasal wash, bal, nasal turbinate and lung homogenate. cdna was synthesized and reverse-transcribed real-time pcr was done at position - of the rsv n genome, using the primers and taqman probe listed in table . the rsv genome copy number was calculated by referring to a linear regression assay of serial dilutions of the corresponding plasmid. lungs were inflated to their normal volumes with % formalin and submerged in formalin for overnight fixation. the fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin-eosin, and immuno-staining was performed using four clone blend monoclonal antibodies against rsv p, f, and n proteins (adb serotec, uk), and anti-mouse igg conjugated with hrp. the culture supernatants were harvested on day of the culture and infectivity was examined. both mvaik/rsv/g and mvaik/rsv/f showed tcid /ml at • c, and mvaik/rsv/f grew little at • c. but, however, no infectious virus was detected at • c, and the ts phenotype was maintained (fig. ) . culture medium and cell lysate were examined for the expression of rsv g and f by western blotting and the results are shown in fig. . live vector virus mvaik and rsv were used for the negative and positive controls. rsv g and f proteins were detected in both supernatant and cell lysate infected with mvaik/rsvf, and mvaik/rsv/g, similar to those infected with rsv (fig. , panel a) . culture fluid was collected and fractionated through sucrose discontinuous gradient ultra-centrifugation. fraction was obtained at the top of the gradient, % sucrose, fraction between % and % sucrose, and fraction between % and % sucrose. each fraction was electrophoresed and analyzed by western blotting, using rsv polyclonal antibodies and a monoclonal antibody just before the appearance of cpe, culture media was replaced with serum free medium (vp-sfm). ml of culture medium was harvested and l of pbs was added in plate. cells were freeze-thawed and cell lysate was clarified. as for the western blotting, / of initial supernatants and / of cell lysate were subjected for experiments. they were stained with polyclonal antibodies against rsv. (b) infectious particles were obtained through sucrose discontinuous gradient ultra-centrifugation. fraction was obtained at the top of the gradient of % sucrose, fraction between % and % sucrose, and fraction between % and % sucrose. each fraction was analyzed by western blotting, using rsv polyclonal antibodies and monoclonal antibodies against mv n protein. against the mv n protein. rsv g or f was detected in fraction , and, whereas the mv n protein was detected in fractions and ( fig. , panel b) . accordingly, rsv g or f protein translated from the inserted gene was considered not to be incorporated into mv particles. the recombinant viruses, mvaik/rsv/g and mvaik/rsv/f, were inoculated into cotton rats to confirm the immunogenicity intramuscularly of the inserted rsv g or f protein. five cotton rats were immunized with mvaik, mvaik/rsv/g, and mvaik/rsv/f for each study group and serum samples were obtained before and , , , , , and weeks after immunization. the results are shown in fig. . pa antibodies against mv were detected three weeks after the immunization in all animals. high levels of pa antibody, - × ( : - : ), were maintained until th week in those immunized with mvaik, mvaik/rsv/g, and mvaik/rsv/f. two rats were reimmunized weeks after the first immunization and sera were obtained one week after the reimmunization for each group. pa antibodies increased from . ± . to . ± . × in the mvaik/rsv/g group, and from . ± . to . ± . × in the mvaik/rsv/f group. pa antibodies against mv increased after the reimmunization by four to eight-fold. the results for nt antibodies against rsv are shown in fig. . in the cotton rats immunized with mvaik/rsv/g, nt antibodies against rsv subgroup a were detected one week after immunization but the mean titer began to decrease week after immunization. the mean nt titers against rsv subgroup a decreased to undetectable levels weeks after the immunization. in the mvaik/rsv/f group, nt antibodies against rsv subgroup a were detected one week after the immunization in all animals with a mean titer of . ± . . high titers were observed at the th week with a mean of . ± . . levels of these antibodies were maintained until th week. in this experiment, rsv source of the recombinant mvaik/rsv/f or mvaik/rsv/g was derived from the rsv subgroup a wild type. cross immunity against rsv subgroup b was further investigated. in cotton rats immunized with mvaik/rsv/f, nt antibodies against rsv subgroup b were detected at the rd week with a mean titer of ( . ) and maintained for weeks. however, cross-reactive antibodies against rsv subgroup b were not detected in the cotton rats immunized with mvaik/rsv/g. in the cotton rats immunized with mvaik/rsv/f, nt antibodies against rsv subgroups a and b increased after reimmunization by two fold, but not significantly. as for the rats immunized with mvaik/rsv/g, nt antibodies against rsv subgroup a were boosted from an undetectable level before the reimmunization to . ± . , but those against rsv subgroup b were not detected. the peak response against rsv was observed five weeks after immunization. three cotton rats were immunized with mvaik/rsv/f and mvaik/rsv/g and challenged with the homologous rsv subgroup a (long strain) and heterologous subgroup b (wild-type). no infectious virus was recovered from nasal wash and bal but rsv genome was detected. rsn genome copy number was slightly lower in immunized groups but not significant (data not shown). the recovery of infectious virus and genome copy numbers from lung tissues are shown in fig. . . and . pfu of infectious virus were recovered from mg of lung tissue in two cotton rats of the control group challenged with rsv subgroup a, but no infectious virus was recovered in three cotton rats immunized with mvaik/rsv/f. meanwhile, . , . and . pfu of infectious virus were recovered in cotton rats immunized with mvaik/rsv/g. as for challenge with rsv subgroup b, . - . pfu of rsv was recovered from lung infected with rsv subgroup b in nonimmunized rats. in cotton rats immunized with mvaik/rsv/f, virus titers were slightly lower, . - . pfu but . - . pfu from their lung tissues in the cotton rats immunized with mvaik/rsv/g. there was no significant reduction in rsv n gene copy number. for histopathological examinations, lung tissues were obtained four days after the challenge with rsv subgroups a and b and the results of he staining and immuno-staining against rsv antigens are shown in fig. . the non-immunized rat challenged with rsv subgroup a showed prominent interstitial pneumonia fig. . recovery of rsv infectious virus and genome copy numbers after challenge with rsv subgroups a and b. three cotton rats were investigated in the normal control group, non-immunized group, and group immunized with mvaik/rsv/f or mvaik/rsv/g. animals non-immunized group, and group immunized with mvaik/rsv/f or mvaik/rsv/g were challenged with . × pfu of the homologous rsv long strain and wild-type subgroup b five weeks later. virus infectivity was monitored in lung homogenate, and rsv infectivity is shown as pfu in mg of lung tissue. and g of total rna of lung tissue was used for real-time pcr, and each column represents individual result. (panel ; thickening of alveolar wall, and infiltration of inflammatory mononuclear cells) with rsv antigens in bronchial epithelial cells (panel ). in cotton rat immunized with mvaik/rsv/f showed very mild inflammation (panel ), though most sections were normal, without rsv antigen in bronchial tissue after rsv challenge with subgroup a (panel ). in cotton rat immunized with mvaik/rsv/g, moderate interstitial pneumonia was observed with a small amount of rsv antigen (panels and ). as for the challenge with rsv subgroup b, histological findings in non-immunized rat challenged with subgroup b were similar to the results challenged with rsv subgroup a. the results of immunostaining are shown. large amounts of rsv antigen were detected in non-immunized rat (panel ). small amounts of rsv antigens were fig. . pulmonary histopathology in cotton rats challenged with rsv subgroups a and b. cotton rats were immunized intramuscularly with mvaik/rsv/f (panels , , and ) or mvaik/rsv/g (panels , , and ) and then challenged five weeks later, with rsv subgroup a (panels , , , , , and ) and subgroups b (panels , and ). they were sacrificed four days after the challenge. histological examination was performed by he staining of lung tissues (panels , , , and ) and the results of immuno-staining of bronchiolar regions are shown in panels , , , , , , and . immuno-staining was performed using four clone blend monoclonal antibodies against rsv p, f, and n proteins and anti-mouse igg conjugated with hrp. he staining and immuno-staining of normal control are shown in panels and . detected in mvaik/rsv/f group (panel ) in comparison with mvaik/rsv/g group (panel ). finding of rsv antigens were well correlated with the results of the recovery of infectious virus from lung tissues. inoculated virus would be cleared and demonstrated a mild pathological finding in rats immunized with mvaik/rsv/f. rsv is a clinically important cause of respiratory tract infections, especially among high-risk infants, immunocompromised hosts, and the elderly. despite a serious disease burden, there is no licensed vaccine for rsv. initial efforts to develop a vaccine involved fi-rsv which unexpectedly enhanced the disease in clinical trials in rsv-naive children [ ] . fi-rsv failed to induce a secretory iga response after pareteral administration without inducing a ctl response, which was a serious drawback of the inactivated vaccine. the defeated f protein would not induce th response and the aluminium-precipitated vaccine induced only th response. the allergic reaction to this vaccine would be caused by the th prone reaction [ ] . several subunit vaccines were investigated, but failed to generate effective antibodies. a live attenuated vaccine has the advantage of inducing humoral and cellular immune responses similar to a natural infection. temperature-sensitive (ts) and cold-adapted (ca) rsv vaccine strains have been developed by conventional attenuation methods. over the last years, cautious and deliberate progress has been made toward developing a rsv vaccine using various experimental approaches, including live attenuated strains and vector-based and viral protein subunit vaccine candidates. but the balance between the safety and immunogenicity is a key issue to the development of a live attenuated vaccine, and the (ts) rsv vaccine candidate resulted in insufficient attenuation, causing similar respiratory illness [ ] . based on a vaccine candidate having the ts phenotype, several recombinant vaccine candidates were developed by deletion of the sh gene or ns gene or mutation by reverse genetics. these recombinant rsv vaccines induced sufficient immune response in chimpanzees [ ] . another approach involved the application of reverse genetics to express rsv protein in a recombinant vectorbased vaccine. the first vector-based candidate was evaluated using vaccinia virus. recombinants expressing rsv f or g was highly immunogenic, induced protection in mouse but provided inconsistent protection in chimpanzees [ ] . mva strain of vaccinia-based recombinants expressing rsv g and f protein were immunogenic in rodent but not in rhesus monkey model [ ] . several vectorbased live vaccine platforms were established using hpiv-iii and sendai virus [ , ] . through preceding experiments, the f protein is known to be more effective than g. but there were no experiences for clinical usage and the hpiv-iii-based recombinant vaccine was poorly immunogenic in human clinical trials. in this report, reverse genetics using the aik-c live attenuated measles vaccines were developed. a recombinant measles virus vector-based vaccine was established using the schwartz strain, expressing the west nile virus [ ] . as well as the schwartz strain, the aik-c measles vaccine is a further attenuated vaccine strain having the ts phenotype, and its safety and immunogenicity has been confirmed [ , ] . thus, in this report, aik-c was used for a live virus vaccine-vector. expression of the rsv g or f protein was confirmed by indirect immuno-staining of b a cells infected with mvaik/rsv/g or f with polyclonal and monoclonal antibodies against the f protein. by western blotting, the g or f protein was detected in culture medium and cell lysate of b a cells infected with mvaik/rsv/g or f. the rsv g and f proteins were considered not to be incorporated into mv particles because theoretically they had no binding site for the mv m protein. mv envelop proteins bound the m protein [ ] . the genetic stability of the vaccine candidate was examined and inserted genes for rsv g and f were stable even after passages. the recombinant measles virus (mvaik) triggered an immune response three weeks after vaccination in cotton rats. levels of these antibodies were maintained for weeks. the same was observed after immunization with mvaik/rsv/g or f. to investigate the viral growth, samples of nasal turbinate, lung, thymus, spleen, liver, kidney, and bone marrow were obtained days after immunization, but no infectious virus was recovered. total rna was extracted and rt-real time pcr was performed to detect the measles n gene by real-time pcr. the mv genome was detected only in thymus in cotton rats immunized with mvaik, mvaik/rsv/g, and mvaik/rsv/f (data not shown). infectious virus was recovered from inguinal superficial lymph nodes three days after infection in the previous study [ ] . nt antibody titers against rsv were investigated, using rsv long (subgroup a) and wild-type subgroup b. mvaik/rsv/g or mvaik/rsv/f induced the production of nt antibodies against rsv subgroup a from one week after vaccination in cotton rats. antibody titers were higher after immunization with mvaik/rsv/f than with mvaik/rsv/g. rsv has distinctly different subgroups, a and b. the g or f gene of subgroup a was used in this study. therefore, the cross reaction of nt against subgroup b was investigated. mvaik/rsv/g did not generate nt antibodies against rsv subgroup b, but mvaik/rsv/f induced production of cross-reactive nt antibodies against rsv subgroups a and b. the predicted amino acid sequence of the rsv f protein used in this study exhibited . % homology among f proteins of subgroup a strains and . % in comparison with those of subgroup b strains. the predicted amino acid sequence of rsv g protein has . % homology among subgroup a strains but . % in comparison with subgroup b. thus, f protein was relatively conserved between subgroups a and b but the g protein of rsv was variable and thought not suitable as a vaccine antigen. recently, a humanized monoclonal antibody against the rsv f protein was used for prevention of serious rsv infections in young infants having cardiac and pulmonary disorders, with a low birth weight, or born prematurely. in this study, recombinant mvaik/rsv/g or f was administered intramuscularly and induced sufficient nt antibodies. secretory iga antibodies and ctl response were not examined but it protected against the challenge with homologous rsv subgroup a. in non-immunized cotton rats, . and . pfu of infectious virus were recovered from mg of lung tissue four days after the rsv challenge. but those immunized with mvaik/rsv/f were protected, without recovery of infectious virus from the lung tissues. and they did not demonstrate interstitial pneumonia. cross reactive nt antibodies were demonstrated after immunization with mvaik/rsv/f but its protective effect is not sufficient against subgroup b, demonstrating slightly lower levels (approximately / of non-immunized control) of the recovery of infectious virus. protective effects of mvaik/rsv/g were poor in comparison with mvaik/rsv/f similar to the serological responses. as for the experimental animal models, transgenic mice expressing human cd with the knock out of type i interferon (ifn) receptor gene were used to evaluate the immunogenicity of a recombinant mv vaccine candidate produced using the west nile virus [ ] , sars corona virus [ ] , hepatitis b virus [ ] and hiv [ ] . efficient immune responses were reported, but the ifn system is the most important signal for innate immunity. in the case of the rsv vaccine candidate, innate immunity modified the adaptive immunity, and, therefore, cotton rats without gene manipulation were used in these experiments [ ] . recombinant mv vaccine-based vectors have practical limitation for timing of immunization. in young infants, maternal conferred immunity would interfere with vaccine effects. in field trials, aik-c gave efficient sero-conversion and induction of cellmediated immunity even when the vaccine was given at the age of six months [ , ] . they demonstrated more than % seroconversion rate to overcome maternal conferred immunity and the safety was similarly confirmed, suggesting no evidence of immunesuppression. rsv infection was observed even after six months of age, and, therefore, mvaik/rsv/f would be applicable for six months to provide protective immunity both against rsv and measles especially for developing countries. as for the effective protection against rsv infection, intranasal administration is desired. but we have no experience of intranasal administration of aik-c vaccine, and, in our previous experiments, the recombinant mvaik did not induce serum nt against mv through intranasal administration because of the strict ts phenotype in cotton rat model, having high body temperature [ , ] . therefore, the comparative studies are planning to investigate the immunogenicity and challenge tests in monkeys immunized with mvaik/rsv/f. in conclusion, a new mv vaccine-strain-based rsv vaccine candidate was demonstrated to confer protection against rsv in cotton rats. the xenogeneic recombinant might induce simultaneously protective immunity against backbone-mv and inserted-rsv infections. recombinant mvaik expressing rsv f protein is a promising candidate and protective effects should be confirmed in monkey model, considering the immunization routes. 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b-cell and t-cell responses in cotton rats and confers protection against rsv subtypes a and b temporal and geographical distribution of measles virus genotypes studies on the modification of the live aik measles vaccine. ii. development and evaluation of the live aik-c measles vaccine immunization of and month old infants with aik-c, edmonston-zagreb, leningrad- and schwarz strains of measles vaccine cell-mediated and antibody immune responses to aik-c and connaught monovalent measles vaccine given to month old infants the effect of dose and strain of live attenuated measles vaccines on serological responses in young infants vaccine adverse events reported in post-marketing study of the kitasato institute from to marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus recombinant measles aik-c strain expressing current wild-type hemagglutinin protein non-replicating vaccinia vector efficiently expresses bacteriophage t rna polymerase an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine a role for immune complexes in enhanced respiratory syncytial virus disease evaluation of two live, cold-passaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in human adults, infants, and children recombinant respiratory syncytial virus that does not express the ns or m - protein is highly attenuated and immunogenic in chimpanzees priming and boosting immunity to respiratory syncytial virus by recombinant replication-defective vaccinia virus mva vaccination of infant macaques with a recombinant modified vaccinia virus ankara expressing the respiratory syncytial virus f and g genes does not predispose for immunopathology human piv- recombinant sendai virus (rsev) elicits durable immunity and combines with two additional rsevs to protect against hpiv- , hpiv- , hpiv- , and rsv live measles vaccine expressing the secreted form of the west nile virus envelope glycoprotein protects against west nile virus encephalitis functional aspects of envelope-associated measles virus proteins analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model induction of neutralising antibodies and cellular immune responses against sars coronavirus by recombinant measles viruses a vectored measles virus induces hepatitis b surface antigen antibodies while protecting macaques against measles virus challenge recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (hiv- ) induce cellular and humoral immune responses the cotton rat model of respiratory viral infections pathogenesis and immunity we key: cord- -bdse o authors: okada, masaji; okuno, yoshinobu; hashimoto, satomi; kita, yoko; kanamaru, noriko; nishida, yasuko; tsunai, yoshie; inoue, ruriko; nakatani, hitoshi; fukamizu, reiko; namie, yumi; yamada, junko; takao, kyoko; asai, ritsuko; asaki, ryoko; kase, tetsuo; takemoto, yuji; yoshida, shigeto; peiris, j.s.m.; chen, pei-jer; yamamoto, naoki; nomura, tatsuji; ishida, isao; morikawa, shigeru; tashiro, masato; sakatani, mitsunori title: development of vaccines and passive immunotherapy against sars corona virus using scid-pbl/hu mouse models date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: bdse o we have investigated novel vaccine strategies against severe acute respiratory syndrome (sars) cov using cdna constructs encoding the structural antigens: (s), (m), (e), or (n) protein, derived from sars cov. pbl from healthy human volunteers were administered i.p. into il- receptor γ-chain disrupted scid mice, and scid-pbl/hu mice were constructed. these mice can be used to analyze the human immune response in vivo. sars m dna vaccine and n dna vaccine induced human ctl specific for sars cov antigens. alternatively, sars m dna vaccines inducing human neutralizing antibodies and human monoclonal antibodies against sars cov are now being developed. these results show that these vaccines can induce virus-specific immune responses and should provide a useful tool for development of protective and therapeutic vaccines. the causative agent of severe acute respiratory syndrome (sars) has been identified as a new type of corona virus, sars corona virus (sars cov) [ ] [ ] [ ] . sars has infected more than patients in about months in over countries and caused more than deaths. however, no sars vaccine is currently available for clinical use. therefore, * corresponding author. we have developed novel vaccine candidates against sars cov using cdna constructs encoding the structural antigens; s, m, e, or n protein. in immunized mice, neutralizing antibodies against the virus and t cell immunity against virus-infected-cells were studied, since these immunities play important roles in protection against sars cov and many virus infections. in particular, cd + ctl plays an important role in t cell immunity dependent protection against virus infections and the eradication of murine and human cancers [ , ] . in the present study, the scid-pbl/hu model, which is capable of analyzing in vivo human immune response, was used because it is a more relevant translational model for human cases [ ] . these vaccines induce neutralizing antibody and ctl. this is the first report inducing antibody against sars m. theses vaccines should provide useful tool for development of protective vaccines in human. three kinds of sars cov strains: hku [ ] , tw- and ffm- [ ] and their cdnas were used. s, m, n or e cdna was transferred into pcdna . (+) vector [ ] . pbl from healthy human volunteers were administered i.p. into il- receptor ␥-chain disrupted nod scid mice [il- r(−/−) nod-scid], and scid-pbl/hu mice were constructed [ ] . pcdna . (+) vector, g each, containing sars s, m, n, or e dna was injected i.m. into scid-pbl/hu three times, at an interval of days. neutralizing antibodies against sars cov in the serum from the mice were assayed by use of vero-e cell. ctl activity against sars cov was studied using human cells, expressing sars antigens. ctl activity of human cd -positive lymphocytes in the spleen from scid-pbl/hu was assessed using cr-release assay [ , ] . human monoclonal antibodies were produced from b cell hybridoma using p u myeloma cell and spleen cells from human immunoglobulin transchromosomic mice (km mice) [ ] . the production of neutralizing antibodies against sars cov was observed in the serum from mice immunized with s dna vaccine sars (m) dna vaccine and n dna vaccine induced murine t cell responses against sars [ ] . to analyze the human immune responses, scid-pbl/hu were constructed and human cd -positive t lymphocytes and human b cells and macrophages were replaced in the spleen cells and pec from these scid-pbl/hu mice, as shown in fig. . human neutralizing antibodies were induced from scid-pbl/hu mice vaccines with sarss [ ] and m dna vaccines (fig. ) . titer of neutralizing antibody in the serum from scid-pbl/hu mice immunized with sars (m) dna vaccine was : . in contrast, inhibition of cytopathic effect was observed even in -fold dilution of the same serum. furthermore, b cell hybridoma producing human monoclonal antibodies were established by the fusion of p u cells and spleen cells from human immunoglobulin transchromosomic mice immunized with sars antigens (table ) . specificity is now being studied. the m dna vaccine enhanced the ctl activity against antologous b blast cells transfected with sars m dna but not with sars n dna in scid-pbl/hu mice (fig. ) . on the other hand, sars n dna vaccine augmented the ctl activity specific for autologous b blast cells transfected with sars n dna. we have demonstrated that sars (m) dna and (n) dna vaccines induce virus-specific immune responses (ctl and t cell proliferation) in the mouse systems using type ii lung alveolar t cell lines in clone target models [ ] . gao et al. developed adenovirus based a sars dna vaccine encoding s polypeptide was capable of inducing neutralizing antibody, while another sars dna vaccine encoding n protein generated ifn-␥ producing t cells in rhesus monkeys [ ] . sars s dna vaccine which elicits effective neutralizing antibody responses that generate protective immunity in a mouse model [ ] . however its immunogenicity in humans has yet to be established. therefore, it is very important to evaluate the efficacy of sars dna vaccine in a scid-pbl/hu mice, which is a highly relevant translational model for demonstrating human immune responsiveness. in the present study, sars m dna vaccine and n dna vaccine induced human ctl specific for sars m antigen and sars n antigen, respectively. furthermore, sars m dna as well as sarss dna vaccine induce human neutralizing antibodies against sars cov by the scid-pbl/hu model. antibody against sars m antigen exerted high inhibitory activity against cytopathic effect by sars cov. it was reported that monoclonal antibodies against external domain of m neutralize murine hepatitis corona virus, but only in the presence of complement, and both e and m proteins are required for budding of virions [ ] . neutralizing antibody activity against m protein was not at all eliminated by the inactivation of complement. antibody against m protein might inhibit the growth of sars cov in the cells. therefore, the effect of combination immunization with such sars vaccines (m vaccine and s vaccine) and the specificity of human monoclonal neutralizing antibodies are now being studied. these vaccines are expected to provide useful tool for development of therapeutic vaccines. coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the severe acute respiratory syndrome the development of vaccines against sars corona virus in mice and scid-pbl-hu mice the anti-human tumor effect and generation of human cytotoxic t cells in scid mice given human peripheral blood lymphocytes by the in vivo transfer of the interleukin- gene using adenovirus vector establishment and characterization of human t hybrid cells secreting immunoregulatory molecules production of human monoclonal and polyclonal antibodies in transchromo (tc) animals effects of a sars-associated coronavirus vaccine in monkeys a dna vaccine induces sars coronavirus neutralization and protective immunity in mice coronaviridae: the viruses and their replication this study was supported by grant-in-aid for the science and technology and grant-in-aid for scientific research on priority areas from the ministry of education culture sports, science and technology, japan. this study also supported by a heath and labour science research grant from the ministry of health, labour, and welfare, japan. key: cord- - lsof authors: suzuki, motoi; katsurada, naoko; le, minh nhat; kaneko, norihiro; yaegashi, makito; hosokawa, naoto; otsuka, yoshihito; aoshima, masahiro; yoshida, lay myint; morimoto, konosuke title: effectiveness of inactivated influenza vaccine against laboratory-confirmed influenza pneumonia among adults aged ≥ years in japan date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: lsof background: the effectiveness of inactivated influenza vaccine (iiv) against laboratory-confirmed influenza pneumonia in older adults remains to be established. methods: pneumonia patients aged ≥ years who visited a study hospital in chiba, japan, were prospectively enrolled from february to january . sputum samples were collected from participants and tested for influenza virus by polymerase chain reaction assays. influenza vaccine effectiveness (ive) against laboratory-confirmed influenza pneumonia was estimated by a test-negative design. results: among a total of pneumonia patients, ( . %) tested positive for influenza: were positive for influenza a virus, and two were positive for influenza b virus. the ive against laboratory-confirmed influenza pneumonia was . % ( % confidence interval, . – . %). the ive against influenza pneumonia hospital admission, severe pneumonia, and death was . % ( % ci, . – . %), . % ( % ci, . – . %), and % ( % ci, − . % to . %), respectively. in the subgroup analyses, the ive against influenza pneumonia was higher for patients with immunosuppressive conditions ( . %; % ci, . – . %) than for those without ( . %; % ci, . – %) but did not differ by patients’ statin use status. conclusion: iiv effectively reduces the risk of laboratory-confirmed influenza pneumonia in older adults. influenza is a major public health concern for older adults. influenza infections generally cause self-limited illnesses but can result in severe disease such as pneumonia in older adults with and without underlying conditions. older age is associated with a higher risk of pneumonia and mortality in influenza patients [ ] . based on our recent estimates, the incidence of influenza pneumonia and its related mortality among people aged ! years in japan were and per , persons/year, respectively [ ] . cumulative evidence has suggested that influenza vaccines are effective at reducing the risk of medically attended influenza in children and adults [ , ] . currently, seasonal influenza vaccination is recommended for older adults in more than countries [ ] . however, its clinical benefit has long been discussed because vaccine responses are reduced by an age-related decline in adaptive immunity [ , ] . positive results have been reported from recent meta-analyses: influenza vaccines reduce medically attended influenza by - % [ ] and influenza-associated hospitalization by % in older adults [ ] . however, evidence is lacking for the protective effect of influenza vaccination on influenza pneumonia, including primary influenza pneumonia and secondary bacterial pneumonia. in a study by grijalva influenza pneumonia by . %, although the majority of their patients were people aged < years [ ] . therefore, the influenza vaccine effectiveness (ive) against laboratory-confirmed influenza pneumonia in older adults remains to be established. we conducted this study to investigate the effectiveness of the trivalent inactivated influenza vaccine (iiv) against laboratoryconfirmed influenza pneumonia and its related outcomes in adults aged ! years. we also conducted subgroup analyses to explore differences in ive by patient characteristics, particularly those related to immunosuppressive status. this single-center prospective study was conducted at kameda medical center (kmc), kamogawa, chiba, japan, as part of the adult pneumonia study group-japan (apsg-j) study [ , [ ] [ ] [ ] . the apsg-j study was a multicenter prospective study of adult pneumonia conducted at four community-based hospitals in japan from september to august . to investigate ive, influenza vaccination history was systematically collected at kmc. in this study, pneumonia patients aged ! years who visited kmc from february to january were included. the diagnosis of pneumonia was made by staff physicians according to clinical signs, symptoms, and radiological findings. demographic and clinical information was collected from patients and medical charts. sputum samples were collected from patients at the time of enrollment. if the patient was unable to cough up sputum, sputum was induced with the inhalation of hypertonic saline solution. details of study settings and designs have been described previously [ , ] . gram staining and sputum culture were performed on site. sputum samples were transferred to the institute of tropical medicine, nagasaki university, and tested by in-house multiplex polymerase chain reaction (pcr) assays to identify the influenza virus (a and b) and other viral pathogens (respiratory syncytial virus [rsv], human metapneumovirus, human parainfluenza virus types - , human rhinovirus [hrv], human coronavirus e/oc , human adenovirus, and human bocavirus) [ ] . the detection limits of the multiplex pcr assays were - copies per reaction as reported previously [ ] . influenza virus subtyping was performed for influenza a-positive samples via rt-pcr of the influenza ha genes using previously published methods [ , ] . patients were defined as having laboratory-confirmed influenza pneumonia if their sputum sample tested positive for influenza a or b virus by pcr. influenza pneumonia patients were classified as having influenza-associated bacterial pneumonia if their sputum samples were microscopically purulent (i.e., geckler's classification groups and ) and tested positive for bacterial pathogens by culture or pcr; otherwise, they were classified as having primary influenza pneumonia. a test-negative design (tnd) case-control study was applied to estimate ive [ ] . unlike the conventional case-control design, the tnd does not require non-disease controls; instead, in this study design, researchers collect clinical samples from patients with a specific condition (eg, influenza like illnesses) and classify the patients into cases (i.e., influenza tested positive patients) and controls (i.e., influenza tested negative patients) according to the influ-enza test results. the tnd is less susceptible to bias due to differences in health care-seeking behavior among cases and controls and provides reliable ive estimates [ , ] . recently, tnd studies have been widely used to estimate ive against medically attended influenza and influenza-associated hospitalization [ , ] . in the current study, our primary outcome was laboratoryconfirmed influenza pneumonia. cases were pneumonia patients who tested positive for influenza a or b, and controls were pneumonia patients who tested negative for both influenza a and b. the odds of vaccination were compared between cases and controls, and ive was expressed as ( -odds ratio)  %. our secondary outcomes were ( ) primary influenza pneumonia, ( ) influenza-associated bacterial pneumonia, influenza pneumonia-related hospital admission, ( ) severe influenza pneumonia, and ( ) influenza pneumonia death. in japan, all adults aged ! years are recommended by the ministry of health, labor and welfare to receive one dose of the seasonal influenza vaccine [ ] . the trivalent iiv vaccine was used during the study period ( - , - , and - seasons); the quadrivalent iiv vaccine was introduced in the - season. high-dose or adjuvanted iivs have not been licensed in japan. the compositions of the trivalent iiv vaccines used during the study seasons and their antigenic match status are summarized in supplementary table . influenza vaccination histories were collected from medical records and confirmed by patients and/or their guardians. patients were considered vaccinated for influenza if they had received at least one dose of influenza vaccine in the months before the hospital visit. because the duration from influenza vaccination to the hospital visit was recorded as a monthly data, all patients who had been vaccinated within a month were considered vaccinated in our primary analysis. patients were considered as having unknown influenza vaccination statuses if their influenza vaccination histories were not recorded in medical charts and could not be confirmed by the patients or their guardians; this group was excluded from our primary analysis. patients were categorized into three age groups: - years, - years, and years or older. patient disability status was evaluated using the eastern cooperative oncology group performance status score [ ] . body mass index (bmi, kg/m ) was classified as underweight (< . ), normal ( . - . ), or overweight (! . ). chronic conditions included diabetes mellitus, heart failure, ischemic heart disease, cerebrovascular disease, liver disease, renal disease, neurological disease, cancer, chronic obstructive pulmonary disease, bronchial asthma, and previous tuberculosis disease. immunosuppressive status included cancer, oral steroid use, and immunosuppressive drug use. patients were considered to have severe pneumonia if they required oxygen therapy, mechanical ventilation, or a vasopressor after admission. the period from november to april was considered the influenza season. the characteristics of patients were compared according to influenza infection status (i.e., influenza pneumonia vs. noninfluenza pneumonia) and influenza vaccination status (i.e., vaccinated vs. unvaccinated) using chi-square tests and fisher's exact tests for categorical variables and wilcoxon rank sum tests for numerical variables. ive was estimated using logistic regression models. pre-specified confounding factors were sex, age, the pres-ence of chronic conditions, the presence of immunosuppression, smoking status, the duration from onset to hospital visit, and the period of the study, and all these variables were included in the final multivariable logistic regression models. we also considered the performance status score and bmi category as potential confounders and examined if ive estimates changed after adjusting for these variables. confidence intervals (cis) were adjusted for the residential area level clustering of patients using robust standard errors. we conducted sensitivity analyses as follows: ( ) restricting the analysis to patients who visited during influenza seasons; ( ) excluding patients vaccinated < month prior to hospital visit; ( ) excluding patients vaccinated > months prior to hospital visit; ( ) using patients who were negative for influenza virus but positive for non-influenza respiratory viruses as controls; ( ) using patients who were negative for all viruses as controls [ ] ; ( ) using propensity scores for adjustment; and ( ) including patients with unknown vaccination status using multiple imputation. stratified analyses were conducted to investigate the potential effect modifications by patient characteristics (i.e., sex, age group, underlying condition, immunosuppressive status, and statin use status). stratum-specific ive estimates were compared using a likelihood ratio test (test for interaction). this study was approved by the institutional review board (irb) of the institute of tropical medicine, nagasaki university, nagasaki, japan and the irb of kameda medical center, chiba, japan. anonymized data were used in this study. during the study period, a total of pneumonia patients aged ! years were enrolled in the study. among them, sputum samples were obtained from patients ( %). after excluding patients whose influenza vaccination history were unavailable ( % of patients with sputum samples), a total of patients were eligible for our analyses (fig. ) . among them, ( %) tested positive for influenza virus by pcr: were positive for the influenza a virus, and the other two were positive for the influenza b virus. among the influenza a-positive samples that were subtyped ( % of all influenza a-positive samples), all were positive for the h n strain. non-influenza viruses were detected in patients: hrv was the leading virus detected (n = , %), followed by rsv (n = , %). non-influenza viruses were co-detected in of the influenza-positive patients ( %) and detected in of the influenza-negative patients ( %). bacterial pathogens were co-detected in of the influenza-positive patients ( %). demographic and clinical characteristics were compared between influenza pneumonia patients (i.e., cases) and noninfluenza pneumonia patients (i.e., controls) (tables and ). cases were more frequently found in winter seasons than controls, but other characteristics were similar between cases and controls. among patients, ( %) had been vaccinated for influenza. vaccinated patients more frequently had received home oxygen therapy and had been diagnosed with chronic respiratory obstructive disease than unvaccinated patients, while other characteristics were similar between two groups (tables and ) . after adjusting for confounders, the ive against laboratoryconfirmed influenza pneumonia was . % ( % ci, . ( table ). the change in ive estimates was marginal after additional adjustment for performance status ( . %; % ci, . - . %) or bmi category ( . %; . - . %); therefore, these variables were not included in the final models. the sensitivity analyses showed similar results. ive was relatively higher ( . %; % ci, . - . %) when we used patients who were negative for influenza but positive for non-influenza viruses as controls, but the value was almost identical to the primary analysis when we used patients who were negative for all viruses ( . %; % ci, . - . %). for the secondary outcomes, the ive against primary influenza pneumonia ( . %; % ci, . - . %) was higher than that table . the ive against influenza pneumonia was higher in patients with immunosuppressive conditions ( . %; % ci, . - . %) than in those without these conditions ( . %; % ci, . - %; test for interaction, p = . ). ive did not differ by sex. the point estimate of ive decreased with increased age, but the difference did not reach a statistically significant level (test for interaction, p = . ). patients' chronic conditions and statin use status did not modify ive. iiv effectively reduced the risk of laboratory-confirmed influenza pneumonia in adults aged ! years. ive was higher among patients with immunosuppressive conditions, while statins did not modify ive. to our knowledge, this is the first study that confirmed the beneficial effect of seasonal influenza vaccination against laboratory-confirmed influenza pneumonia in older adults. the benefit of seasonal influenza vaccination in older adults is still debated [ , ] . in this age group, the age-related decline in adaptive immunity results in reduced responses to influenza vaccination [ , ] ; moreover, multiple chronic conditions and frailty may also contribute to weak immune responses [ ] . however, despite an observed lower antibody response compared with that of younger adults [ ] , recent evidence supports the protective effect of influenza vaccination against medically attended influenza in older adults. according to a systematic review by belongia et al, the pooled ive was % ( % ci, À % to %) for the h n strain, % ( % ci, - %) for type b, and % ( % ci, - %) for the h n pdm strain among adults aged > years [ ] . darvishian et al conducted an individual participant data meta-analysis of tnd studies and demonstrated that influenza vaccination is moderately effective against laboratory-confirmed influenza in this age group during epidemic seasons but not during non-epidemic seasons [ ] . on the other hand, evidence is still limited for the beneficial effect of influenza vaccination against influenza-related severe outcomes such as pneumonia. previous studies have estimated the ive against all-cause pneumonia or influenza-related hospitalization in older adults [ , [ ] [ ] [ ] ; however, these studies used less specific outcomes and may have underestimated the true ive [ ] . the tnd study by grijalva et al demonstrated that the overall estimate of ive against hospitalization with laboratory-confirmed influenza pneumonia was . % ( % ci, . - . %) [ ] . however, their study included all age groups, and only % of their patients were aged ! years. in their analysis restricted to patients aged ! years, ive showed a positive effect but did not reach a statistically significant level ( . %; % ci, À . % to %). therefore, the authors concluded that additional studies were needed to establish the ive against pneumonia in older adults. our study targeted this age group and demonstrated that the vaccine effectively reduces the risk of laboratory-confirmed influenza pneumonia by . % ( % ci, . - . %). our ive estimates against influenza pneumonia in older adults may be higher than generally expected values. ive is commonly lower for severe outcomes than for medically attended influenza and is lower in older adults than in children [ , ] . in addition, ive is usually lower for the h n stain than for the h n pdm strain [ ] . however, our estimates are not dissimilar to those of previous reports: in the study by grijalva et al, the ive against influenza pneumonia related to the h n strain in all age groups was . % ( % ci, À . % to . %) [ ] , and in another study conducted during the - influenza season when h n was the dominant circulating strain, the ive against influenza hospitaliza- [ ] . the use of sputum samples in our study may also explain our high ive estimate. identification of influenza from sputum samples may be more sensitive and specific than that from upper respiratory tract samples in diagnosing influenza pneumonia and may provide less biased ive estimates [ , , ] . consistent findings in our sensitivity analyses also support the robustness of our ive estimates. although a higher ive estimate was observed for primary influenza pneumonia, iiv was also effective at preventing influenzaassociated bacterial pneumonia ( . %; % ci, . - . %). this finding is important because influenza-bacterial co-infection increases the risk of severe outcomes [ ] . our finding also suggests that iiv may be effective at preventing influenza pneumonia death; however, the association did not reach a statistically significant level because of the limited sample size. it was unexpected that the ive was significantly higher among people with immunosuppressive conditions ( . %; % ci, . - . %) than among those without ( . %; % ci, . - %). the opposite finding was observed in the study by grijalva et al, which included children and adults (À . % vs. . %) [ ] . this difference might be, at least partially, explained by a lower hiv prevalence in our patients. although seasonal influenza vaccinations have been recommended for adults with immunosuppressive conditions [ ] , only a few studies have evaluated the ive against clinical outcomes among this population [ ] . our finding provides supporting evidence for the current recommendations but needs to be confirmed in future studies. recent studies have suggested that statins may reduce the ive against medically attended influenza among older adults by their immunomodulatory effects [ ] [ ] [ ] [ ] . however, such an effect has not been observed in our study. although the degree of its effect remains controversial, statins are also known to modify the risk of pneumonia and pneumonia-related outcomes [ ] [ ] [ ] . the impact of statin use on the ive may be different according to influenza outcomes. influenza infection is a threat to older adults because of its potential to cause pneumonia and secondary bacterial infections [ ] . the burden of pneumonia is rapidly increasing in highincome countries such as japan because of the aging population [ ] . therefore, the prevention of influenza pneumonia is an important public health measure in controlling pneumonia. the moderate effectiveness observed in our study supports the current seasonal influenza vaccination policy. in japan, the proportion of people vaccinated against influenza among adults aged ! years has been increasing but still remains approximately % [ ] . in addition to improving vaccination coverage, an introduction of newer vaccines such as the more immunogenic high-dose influenza vaccine must be considered [ , ] . on the other hand, it must be noted that only % of pneumonia cases have influenza pneumonia, and thus, the impact of influenza vaccination on allcause pneumonia is limited [ ] . newer multidimensional approaches are needed to reduce the pneumonia burden in the aging population. our study has limitations. influenza vaccination history was not documented for % of our patients. however, our sensitivity analysis using multiple imputations showed very robust estimates. we believe that the exclusion of this patient group did not affect our ive estimates. although all potential confounders were considered, unmeasured confounders may have remained. recently, andrew et al argued that frailty must be considered in estimating ive for older adults [ ] . we have not measured the frailty of our patients but measured their performance status and bmi. we confirmed that the inclusion of performance status or bmi category in the final model did not change the ive estimates. our observation is based on the analyses of older patients aged ! years and there- fore may not be generalizable to younger adults. finally, our sample size was too small to estimate subtype-specific ive. seasonal influenza vaccination is moderately effective against laboratory-confirmed influenza pneumonia in adults aged ! years. considering the increasing burden of pneumonia in an aging population, we must improve influenza vaccination coverage and establish newer approaches. konosuke morimoto reports speaker fees from taisho toyama pharmaceutical, pfizer, and asahi kasei pharma. all other authors declare no competing interests. populations at risk for severe or complicated influenza illness: systematic review and meta-analysis the burden and etiology of community-onset 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systematic review and metaanalysis is statin use associated with reduced mortality after pneumonia? a systematic review and meta-analysis statin use and hospital length of stay among adults hospitalized with community-acquired pneumonia estimated influenza vaccination rates in japan comparative effectiveness of high-dose versus standard-dose influenza vaccines among us medicare beneficiaries in preventing postinfluenza deaths during comparative effectiveness of high-dose versus standard-dose influenza vaccination on numbers of us nursing home residents admitted to hospital: a cluster-randomised trial inactivated influenza vaccines for prevention of community-acquired pneumonia: the limits of using nonspecific outcomes in vaccine effectiveness studies we are grateful to all the adult pneumonia study group-japan contributors. we would like to thank professor koya ariyoshi, dr. eiichiro sando, and dr. tomoko ishifuji for their contribution to the study. we also thank rina shiramizu, kyoko uchibori for performing the pcr and yumi araki for administrative work. this study was supported by nagasaki university and pfizer japan, inc. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.vaccine. . . . key: cord- -biq chyn authors: torres, juan m; ramírez, miguel a; morales, mónica; bárcena, juan; vázquez, belén; espuña, enric; pagès-manté, albert; sánchez-vizcaíno, josé m title: safety evaluation of a recombinant myxoma-rhdv virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: biq chyn we have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (mv) expressing the rabbit haemorrhagic disease virus (rhdv) capsid protein [bárcena et al. horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. j. virol. ; : – ]. administration of the recombinant virus protects rabbits against lethal rhdv and mv challenges. furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. however, potential risks must be extensively evaluated before considering its field use. in this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. results indicated that vaccine administration is safe even at a -fold overdose. no undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. the recombinant virus maintained its attenuated phenotype after passages in vivo. safety evaluation of a recombinant myxoma-rhdv virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease myxomatosis and rabbit haemorrhagic disease (rhd) are considered the major viral diseases aecting the european rabbit (oryctolagus cuniculus ). myxoma virus (mv), the causative agent of myxomatosis, belongs to the leporipoxvirus genus of the poxviridae family [ ] . in its natural host, sylvilagus rabbits in the americas, mv induces a mild benign infection. in european rabbits however, mv causes the systemic and lethal infection known as myxomatosis [ , ] . the disease is endemic in the entire rabbit range in europe since the deliberate release of mv in france ( ) as a biological control agent of wild rabbit populations. immunization of domestic rabbits against myxomatosis is currently achieved using heterologous vaccines based on shope ®broma virus, a less virulent leporipoxvirus, or homologous vaccines based on cell culture-attenuated strains of mv [ , ] . rhd was ®rst reported in the people's republic of china [ ] . the disease spread throughout europe between and [ ] and is endemic since then. infected rabbits usually die within ± h of necrotising hepatitis. rhd is responsible for high economic losses in rabbitries as well as high mortality rates in wild rabbit populations [ ± ] . the etiological agent, rabbit haemorrhagic disease virus (rhdv), is a member of the caliciviridae family [ ] . the rhdv virions are non-enveloped and icosahedral, with capsids composed of a major protein component of kda (vp ). commercial vaccines against rhd are prepared from the livers of experimentally infected rabbits [ ] , since in vitro systems are not available for ecient virus propagation. in the last years, the vp gene has been successfully expressed in several heterologous systems [ ± ] and has been shown to induce full protection of rabbits against a lethal challenge with rhdv. while the currently available vaccines against myxomatosis and rhd have proven eective in the control of these diseases in domestic rabbits, they are not suited to immunize wild rabbit populations, as vaccines need to be delivered individually by conventional veterinary practices, which is not a feasible approach to vaccinate free ranging animals. as a novel approach for wildlife vaccination, we have explored the possibility of developing``transmissible vaccines'' by the use of viral vectors capable of spreading within an animal population. in order to protect wild rabbits against both myxomatosis and rhd, we constructed a recombinant virus based on the naturally attenuated mv ®eld strain [ ] , that expressed the rhdv vp protein [ ] . a linear epitope tag from the nucleoprotein of porcine transmissible gastroenteritis coronavirus (tgev) was included within the recombinant vp protein to allow monitoring the spread of the recombinant virus in the environment. following inoculation of rabbits, the recombinant virus ( vp -t ) induced speci®c antibody responses against mv, rhdv as well as for the tgev tag. administration of vp -t by the subcutaneous, intradermal or oral routes protected rabbits against lethal rhdv and mv challenges. furthermore, the recombinant vp -t virus showed a limited horizontal transmission capacity, either by direct contact or in a¯ea-mediated process, promoting immunization of contact uninoculated animals [ ] . the promising results obtained so far under laboratory conditions suggest the recombinant vp -t could be used in large-scale immunization schemes for the control of myxomatosis and rhd in wild rabbit populations. however, before considering its environmental release, vaccine safety considerations should be extensively evaluated. potential risks with regard to vaccine dose (i.e., accidental administration of an overdose), age, physiological condition (i.e., pregnant does) and immune status of exposed individuals, should be taken into account. biological stability is another important aspect to evaluate in a recombinant virus intended for environmental release. in the present study, we report the safety evaluation under laboratory conditions of recombinant vp -t virus concerning the above mentioned issues. recombinant virus vp -t was propagated in rk- (rabbit kidney) cell line grown in dulbecco's minimum essential medium (dmem) supplemented with % foetal bovine serum (fbs), mm l-glutamine, u/ml penicillin, and mg/ml streptomycin. sirc (rabbit cornea) cells were used for viral titre determination on plaque assay. both rabbit cell lines were obtained from the american type culture collection (atcc). common rabbits (brown coloured) free from anti-mv and anti-rhdv antibodies, were provided by a commercial breeder. these rabbits are routinely used for restocking in the ®eld and from now on will be referred to as``wild rabbits''. groups of eight wild rabbits ( month old, weighing around . kg) free from mv and rhdv antibodies, were inoculated at the back by intradermic (i.d.) or subcutaneous (s.c.) route with dierent doses of the vaccine ( , , pfu of vp -t recombinant virus). rabbits were observed daily for days and clinical symptoms were recorded. weight and temperature determinations were made on each animal until the st day. serum samples extracted from the marginal ear vein of the rabbits on days and after immunization were used to evaluate the serological responses against mv and rhdv, by using an enzyme-linked immunosorbent assay (elisa), as previously described [ ] . antibody titres were de®ned as the reciprocal of the highest dilution giving an a value two-fold over the background level (negative control rabbit sera). groups of eight wild rabbits ( month old, weighing around . kg) were immunosuppressed by treatment with prednisolone ( mg per animal per day) for days before vaccination and days after vaccination. prednisolone treated rabbits were inoculated by i.d. or s.c. route at the back with pfu of vp -t virus. control rabbits were vaccinated but not treated with prednisolone. rabbits were observed daily for a period of days and clinical symptoms were recorded. weight and temperature determinations were made on each animal until the st day. serum samples extracted and days after immunization were used to evaluate the serological responses against mv and rhdv by elisa. antibody titres were de®ned as described above. groups of six pregnant does were inoculated at dierent times of gestation (days , , and ) by s.c. route at the back with pfu of vp -t virus. control does were inoculated at the same days of gestation with . ml of phosphate-buered saline (pbs). animals were observed daily and general clinical symptoms were recorded. no body weight and temperature determinations were performed in order to minimise the handling-induced stress in does, which are specially sensible during gestation. the following reproductive parameters were recorded both at ®rst and second parturition: number of animals born alive per litter; number of animals born dead per litter; number of living animals per litter days postparturition (dpp), and weight of each litter at dpp. two rabbits ( month old, weighing around . kg) were inoculated by i.d. route at the back with pfu of vp -t virus. seven to days postvaccination the inoculation site nodule was extracted, homogenated in pbs, and reinoculated into two new rabbits. this procedure was repeated up to passages. the virus obtained from the last passage was titrated and the eects of inoculating pfu by s.c. in a group of eight rabbits were evaluated as described above and compared with those of the original recombinant virus. serum samples extracted and days after immunization were used to evaluate the serological responses against mv and rhdv by elisa. antibody titres were de®ned as described above. in order to evaluate the genetic stability of vp -t virus after passages in rabbits, dna extracted from the nodules at the inoculation site was analysed by pcr. the oligonucleotides used were mv and mv , which are derived from the mv genomic sequence¯anking the foreign gene insertion site [ ] . the ampli®cation of a . -kb pcr product, instead of the . -kb product obtained from wild-type mv, was indicative of the presence of the inserted vp gene construct. data were analysed using a student's t-test for nonpaired variants. signi®cance was considered when p x x previous work showed pfu was an ecient vaccine dose to ensure horizontal transmissible protection against myxomatosis and rhd, either by direct contact or in a¯ea-mediated process [ ] . to evaluate the eects of administering an overdose of the vaccine, wild rabbits were inoculated by i.d. or s.c. route with dierent doses of vp -t virus ( , and pfu). in order to obtain a semi-quantitative measure to allow graphic representation and objective comparison, the classical myxomatosis symptoms were classi®ed in a ranking of to points (see table ), and the results registered during the observation period were represented ( fig. ) . rabbits inoculated by i.d. route displayed similar clinical signs at all vaccine doses tested. these consisted of a localised primary nodule at the inoculation site and, in some rabbits, scanty secondary skin lesions in the form of small discrete nodules, usually less than . cm in diameter, in face, ears or eyelids. lesions appeared ± days postinoculation (dpi) and completely resolved in all rabbits normally by dpi. none of the infected rabbits exhibited classical severe myxomatosis symptoms like closure of the eyes, generalised oedema, or respiratory syndrome (fig. ) . rabbits inoculated by s.c. route showed similar clinical symptoms but these were consistently groups of eight wild rabbits were inoculated by i.d. route with (*), (q), or (r) pfu. rabbits were observed daily for a period of days and the clinical signs due to virus infection of each animal were ranked from to according to table . milder: there were less secondary nodules, which were slightly smaller and resolved earlier (results not shown). no febrile response or loss of body weight was detected. table shows temperature increases registered from to dpi and from to dpi, as well as the weight increase from day to day . no sig-ni®cant dierences in the increases of body temperature or body weight were observed in recombinant virus-infected rabbits as compared with control rabbits, regardless of virus dose or inoculation route. to evaluate the immune responses elicited by the inoculated rabbits, sera samples obtained dpi were monitored by elisa for the presence of anti-mv and anti-rhdv antibodies. the inoculated rabbits developed high anti-mv and anti-rhdv antibody titres, which increased with the vaccine dose (table ). there was no gross dierence in the antibody titres induced by vaccine administration by i.d. or s.c. inoculation routes. to evaluate the eects of recombinant virus infection on immunocompromised animals, rabbits were immunosuppressed by treatment with prednisolone. treated rabbits were inoculated (by s.c or i.d. route) with pfu of vp -t virus, and clinical signs due to virus infection were compared with those induced in control rabbits, which were vaccinated but not treated with prednisolone (fig , table ). results indicated that administration of vp -t virus to immunocompromised animals was safe (either by i.d. or s.c routes), as prednisolone treated rabbits exhibited only mild clinical symptoms and were all completely recovered by dpi. fig. shows a graphic representation of the symptomatology observed in rabbits inoculated by i.d. route, according to the ranking of myxomatosis clinical signs established in table . after i.d. inoculation, immunosuppressed rabbits exhibited similar local lesions to those observed in control non-immunosuppressed rabbits. lesions appeared at the same time ( ± dpi) in both cases but showed a subtle tendency to resolve later in immunosuppressed rabbits ( ± dpi vs. dpi). results obtained with rabbits inoculated by the s.c route were essentially the same (data not shown). no signi®cant dierences in body temperature increase or body weight increase were observed when immunosuppressed rabbits were compared with control rabbits ( table ). the humoral immune responses elicited dpi in both prednisolone treated and control rabbits were similar. all vaccinated rabbits developed high anti-mv and anti-rhdv antibody titres (table ) . secondary skin lesions in the form of small discrete nodules near the inoculation site, in face, or ears small discrete nodules in eyelids small nodules in genitals, limbs, and other parts of the body severe myxomatosis symptoms like closure of the eyes, generalised oedema, or respiratory syndrome death to evaluate the eects of recombinant virus infection on reproduction, pregnant does were inoculated at dierent times of gestation (days , , and ) by s.c. route. the daily observation of the animals showed a total absence of general clinical symptoms in all inoculated animals. reproductive parameters such as number of animals born alive per litter, number of animals born dead per litter, number of living animals per litter dpp, and average weight of each litter at dpp, for both ®rst and second parturition, have been summarised in table . the overall results showed that recombinant virus infection did not induce any alteration during reproduction. pregnant does infected at dierent days of gestation showed reproductive values being in the expected range for rabbits, and no dierences were observed when recombinant virusinfected does were compared with control does inoculated with pbs at the same day of gestation. the absence of alterations in reproductive parameters was maintained in the following parturition (table ) . furthermore, none of the rabbits born from vp -t virus-infected does showed any symptomatology associated with myxomatosis. the biological stability of the recombinant virus, and therefore its potential to evolve to a virulent state were evaluated by comparing the eects of rabbit infection with``passage '' virus (the same virus stock used in all the experiments reported in this paper), with the eects of rabbit infection with the virus obtained after serial passages in rabbits (passage virus). fig. shows a graphic representation of the symptomatology observed in rabbits infected with either passage or passage virus, according to the ranking of myxomatosis clinical signs established in table . rabbits infected with passage virus exhibited the same mild clinical signs as those infected with passage virus. symptoms appeared ± dpi and completely resolved by dpi in both cases. none of the infected rabbits exhibited classical severe myxomatosis symptoms. table shows temperature increases from to dpi and from to dpi, as well as weight fig. . eects of administering vp -t virus to immunosuppressed rabbits. groups of eight rabbits treated (r) or untreated (q) with prednisolone were inoculated by i.d. route with pfu of vp -t virus. rabbits were observed daily for a period of days and the clinical signs due to virus infection of each animal were ranked from to according to table . increases from day to . no signi®cant dierences in body temperature increase or body weight increase were observed when rabbits infected with passage virus were compared with rabbits infected with passage virus or control uninfected rabbits. the humoral responses elicited by rabbits infected with passage or passage virus were similar. all infected rabbits developed high anti-mv and anti-rhdv antibody titres. the genomic stability of vp -t virus was analysed by pcr using oligonucleotide primers external to the insertion site of the vp gene. after serial passages in rabbits, a product of . kb (the expected size for the recombinant virus) was ampli®ed by pcr with no detection of the corresponding wildtype mv . kb product (not shown), indicating that the vp gene was stably integrated in the mv genome. a number of vaccines are available to protect rabbits against myxomatosis and rhd [ , , ] which are useful for immunizing domestic rabbits. however, control of both diseases among wild rabbit populations remains an unsolved problem of great concern. in this regard it should be noted that the european rabbit plays a key ecological role in mediterranean ecosystems. in addition, rabbits are among the most important small game species in several european countries. immunization of wildlife is dicult to achieve because direct delivery of vaccines to free ranging animals is not possible. the oral route is considered a feasible way of vaccine administration. for example, oral vaccination is being used to control enzootic sylvatic rabies in europe and north america by means of a recombinant vaccinia-rabies vaccine delivered by baiting [ ] . an alternative strategy is the use of`t ransmissible vaccines'', i.e., viral vectors capable of spreading within an animal population. hopefully, the administration of a recombinant vaccine of this characteristics to a small number of captured individuals, would eventually lead to the immunization of a fraction of animals within a given population, which is sucient to reduce the spread of the target disease. this approach might be useful, especially when the distribution, size, and turnover rate of a population precludes capture or baiting techniques as the only means for antigen delivery. the european rabbit is an example of such a population. with this in mind, we have developed a transmissible vaccine against both myxomatosis and rhd based on a recombinant mv-vp virus capable of spreading through rabbit populations [ ] . the results obtained under laboratory conditions suggest the recombinant virus might be eective for wild rabbit immunization. however, since the proposed use of vp -t involves the environmental release of a recombinant virus, considerations regarding safety issues are as important as the potential ecacy of the candidate vaccine. it is for this reason that safety concerns have been at the core of the rational design of the proposed immunization strategy. the biological characteristics of mv make it a good candidate as a vaccine vector in terms of safety considerations. mv exhibits a very restricted host range, infecting exclusively rabbits (both sylvilagus and oryctolagus spp.). the virus has been widely distributed throughout europe, australia and the americas for nearly years with no evidence of infection of other species. thus, the host restricted nature of mv minimises the risk of recombinant vaccine spreading to non-target species in nature. on the other hand, given the current widespread geographic distribution of mv, which is similar to the distribution of rhdv, the ®eld use of a recombinant mv-vp vaccine would normally not involve the introduction of a virus species that does not already exist in a particular area. safety aspects were also considered in the choice of the parental mv strain. it was decided not to use one of the available vaccinal strains, obtained by cell culture-attenuation of virulent mv strains [ ] , as this would involve the release of a new strain to the environment, which might undergo reversion to virulence in nature. instead, we decided to use an attenuated mv ®eld strain which was already circulating among wild rabbit populations. strain was selected from a ®eld survey of mv strains circulating in spain, which were analysed for virulence and transmissibility [ ] . this strain exhibited adequate biological characteristics for the development of a recombinant transmissible vaccine, as it caused a non-pathogenic infection comparable to that of cell culture-attenuated vaccinal strains, yet retaining the capacity of horizontal spreading [ ] . since preservation of the valuable biological properties of strain was of major importance in the development of the recombinant virus, the foreign gene was inserted in the intergenic site between orfs mj and mj a, as recombinant mvs with insertions at this site have been shown to retain overall parental biological characteristics [ ] . moreover, the vp expression cassette was inserted into the mv genome using the tds twostep selection system [ ] . this procedure enables the construction of recombinant poxviruses without any marker genes inserted in the ®nal recombinant viral genome. thus, the recombinant vp -t does not harbour selectable markers such as antibiotic resistance genes, the widespread of which is currently regarded as a major health and environmental threat. considering the potential risks associated with the dna sequence inserted, it should be noted that the vp gene has been cloned in a wide range of heterologous systems[ ± ] and no indication of toxicity or side eects associated to the expression of vp have been reported. previous results indicated that administration of either mv or recombinant vp -t virus to healthy rabbits under laboratory conditions by standardised procedures is safe, as all rabbits exhibited only mild clinical symptoms and rapidly recovered [ , ] . in this report we have extended the safety assessment of the vaccine by analysing the potential table eects concerning vaccine dosage and the possibility of accidental administration of an overdose, the results demonstrated vaccine safety even when a -fold overdose ( pfu) was inoculated (fig. , table ). assessment of vaccine eects in immunosuppressed rabbits was considered relevant, given the incidence in nature of immunocompromised individuals due to infections, environmental or genetic causes. for this reason we assayed the eect of vaccine administration in rabbits treated with prednisolone, a potent immunosuppressor. this treatment induces depletion of circulating eosinophils and mononuclear cells, causing a strong decrease of the t-cell response with only a slight eect on b-cell function [ ] . it is a commonly used procedure for the safety evaluation of veterinary vaccines [ ± ] . results showed that prednisolone treated rabbits exhibited similar symptoms to those observed in control rabbits (fig. , table ). the only remarkable observation was that immunosuppressed rabbits showed a subtle tendency to delay the resolution of local lesions: ± dpi vs. dpi (fig. ) . another important aspect addressed was the eect of vp -t virus infection in reproduction. results showed that recombinant virus inoculation did not alter the reproduction parameters and none of the rabbits born from vaccinated does showed myxomatosisassociated clinical signs (table ). in conclusion, the overall results obtained demonstrate a notable lack of adverse eects attributable to the recombinant virus, regardless of dose, route or life history stage of individuals (i.e., neonate, young, pregnant does or immunocompromised). finally the biological stability of the recombinant virus was analysed. the environmental release of recombinant vp -t virus would involve a certain number of serial passages in its natural host, even when this capability seemed to be limited to only two serial passages under laboratory conditions [ ] . should there be a tendency for the virus to evolve to a virulent state, serial passage in rabbits would cause it to do so. accordingly, the biological stability of vp -t was studied by subjecting the virus to serial passages in rabbits, and the results obtained (fig. , table ) indicated the recombinant virus maintained grossly the same biological characteristics through the passages. thus, the attenuated nature of vp -t seems to be a stable trait. on the other hand, the genetic analysis indicated that the vp gene remained stably integrated in the mv genome after serial passage in rabbits, in agreement with the previously reported results obtained after serial passages of vp -t virus in rk- cell monolayers [ ] . on the basis of the results previously reported [ , ] and those presented in this paper, along with experimental data addressing further safety and ecacy issues (to be published elsewhere), the recombinant vp -t has been subjected to the mandatory risk assessment process relative to the release of genetically-modi®ed organisms. a limited ®eld trial authorised by the spanish competent authorities is in course. this trial will assess the ecacy and safety of the vaccine under controlled ®eld conditions, in the perspective of its use in a large-scale program for the control of myxomatosis and rhd among wild rabbit populations. virus taxonomy: classi®cation and nomenclature of viruses. sixth report of the international commitee for the taxonomy of viruses the european rabbit. the history and biology of a successful coloniser myxoma virus in rabbits protection of laboratory rabbits against myxomatosis by vaccination with ®broma virus etude d'une souche de virus myxomateux modi®e a new viral disease in rabbits hepatitis of viral origin in leporidae: introduction and aetiological hypotheses rabbit haemorrhagic disease (rhd): characterization of the causative calicivirus incidence of viral haemorrhagic disease in wild rabbit populations in spain rabbit haemorrhagic disease: the new scourge of oryctolagus cuniculus impact of viral haemorrhagic disease on a wild population of european rabbits in france the initial impact of rabbit haemorrhagic disease on european rabbit populations in south australia virus taxonomy Ð san diego viral haemorrhagic disease of rabbits: vaccination and immune response molecular cloning, sequencing and expression in escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (spanish isolate ast/ ) recombinant rabbit haemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection oral immunization of rabbits with vp particles confers protection against rabbit haemorrhagic disease two independent pathways of expression lead to self-assembly of the rabbit haemorrhagic disease virus capsid protein protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-rhdv recombinant virus protection against myxomatosis and rabbit viral haemorrhagic disease with recombinant myxoma viruses expressing rabbit haemorrhagic disease virus capsid protein a recombinant canarypox virus protects rabbits against a lethal rabbit haemorrhagic disease virus (rhdv) challenge a single dose immunization with rabbit haemorrhagic disease virus major capsid protein produced in saccharomyces cerevisiae induces protection immunization with potato plants expressing vp protein protects against rabbit haemorrhagic disease virus isolation of an attenuated myxoma virus ®eld strain that confers horizontal transmissible protection against myxomatosis on contacts of vaccinates horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease using a recombinant myxoma virus field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in europe and north america construction of recombinant myxoma viruses expressing foreign genes from dierent intergenic sites without associated attenuation transient dominant selection of recombinant vaccinia viruses immunosuppressive eect of corticosteroids on rabbit's humoral and cellular response contribucio n a la pro®laxis de la mixomatosis del conejo mediante el uso de una cepa homo loga eects of corticosteroids mediated immunosuppression on the distribution of rabies vaccine virus in red foxes orally immunized against rabies immunogenicity and ecacy of a commercial feline leukemia virus vaccine this work was supported by an agreement between the``fundacio n para el estudio y defensa de la naturaleza y la caza'' (fedenca) and the``instituto nacional de investigacio n y tecnologõ a agraria y alimentaria'' (inia). key: cord- -lfn ggg authors: kenner, julie; cameron, fiona; empig, cyril; jobes, david v.; gurwith, marc title: lc m : an attenuated smallpox vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: lfn ggg the frequency of moderate to severe adverse reactions associated with smallpox vaccines currently stockpiled in the us, and the continued threat of bioterrorism have prompted the development of effective vaccines with improved safety profiles. lc m , an attenuated, replicating smallpox vaccine derived from the lister strain of vaccinia, is currently licensed in japan where it was safely used in over , children in the s. it has been shown to have markedly less neurotoxicity than unattenuated vaccines in nonclinical studies. lc m is immunogenic after a single dose, and recent studies in two different animal models have demonstrated protective efficacy equivalent to that of the only fda-licensed smallpox vaccine. this article reviews the history and available scientific literature regarding lc m and provides comparisons to other smallpox vaccines. as efforts intensified internationally to eradicate smallpox in the s, health officials became increasingly concerned about adverse reactions to vaccinia vaccines. officials in countries with a low incidence of smallpox were particularly alarmed by the morbidity and mortality associated with postvaccination encephalitis and encephalopathy. while central nervous system (cns) adverse reactions were generally considered idiosyncratic events, the incidence seemed to vary by strain of vaccinia. this observation was eventually substantiated when the marennikova group of the viral pharmaceutical institute in russia published animal data that characterized over commonly used strains of vaccinia and showed a correlation between the degree of pathogenicity and viral replication in cns and other tissues [ , ] . according to the marennikova report, the ikeda strain of vaccinia was classified as having high pathogenicity, whereas the lister strain used by the world health organization (who) and the uk national health service (nhs) was rated as hav-ing medium pathogenicity, and the new york city board of health strain (nycbh, also manufactured under the trade name dryvax ® ) used in the us was rated as having low pathogenicity. in response to a growing concern about vaccinia-related adverse events (aes) in japan, the japanese ministry of health formed the smallpox vaccine research group (svrg) in [ ] . this organization, comprised of both clinical experts and basic scientists, was charged with providing and collecting detailed information on adverse reactions to vaccinia vaccines and advising the japanese public health authorities. at the time of the marennikova report, the strain of vaccinia used for smallpox vaccination in japan was ikeda, and the rate of encephalitis in japan was approximately per million per year [ ] . the highest risk of mortality was in primary vaccinees, particularly in infants. as one of its first tasks, the svrg investigated the cns toxicity of several strains of vaccinia in mice that were inoculated intracerebrally [ ] . the results from the mouse studies confirmed the greater neurotoxicity of the ikeda strain reported by marennikova and spawned several clinical trials in children comparing ikeda with lister and em- , a strain used in russia. after collecting data from several thousand primary vaccinees, it was found that all of the studied vaccines induced similar take rates, antibody responses, and febrile reactions, but that lister had the mildest local reactogenicity. while no direct conclusions could be made regarding the relative neurotoxicity of the studied vaccines, local reactogenicity was at that time regarded as an indicator of overall vaccinia toxicity, and was used to identify lister as the safest vaccine strain in the trials [ ] . in , the svrg recommended that routine smallpox vaccination in japan be conducted using lister instead of the ikeda strain [ , , ] . to further reduce vaccine-related morbidity and mortality, the svrg recommended that vaccinations be initiated at - months of age rather than at - months, and that only - punctures be administered with a bifurcated needle [ , ] . despite these changes, deaths from vaccine-related encephalitis (table ) continued in japan [ , ] . being at geographic risk of importing smallpox from india, the svrg was reluctant to discontinue vaccinations altogether as other industrialized nations had done in the early s [ , ] . therefore, a decision was made to search for a vaccine that reduced or eliminated cns risks. the search began with a review of study data from two attenuated smallpox vaccines that had undergone considerable development in other nations. the modified vaccinia ankara (mva) strain had been developed in germany in the s by passaging vaccinia ankara in chick embryo explants (cee) in excess of times [ ] [ ] [ ] . the resulting mutant was dramatically modified, having lost nearly % of the original genome [ ] as well as the ability to replicate in many mammalian cells [ ] . although clearly not neurotoxic in nonclinical studies and nonreactive in both normal and dermatologicallyor immune-compromised hosts, antibody response following single vaccination was poor [ , ] . since humoral immune response had become the leading criterion for determining vaccinia protective efficacy, further development of the mva vaccine in japan was abandoned. another attenuated strain, originally called the first revived strain and then later renamed cvi (cv- ) by parker et al. [ ] , had been developed in the us in the early s through repeated passage of the nycbh strain in rabbit testes, cee, and chorioallantoic membranes (cam) [ , , ] . the attenuated cvi- strain (cvi after passages), developed by kempe et al. at the walter reed army institute of research in the late s, was ultimately selected for development based on the mild dermatologic reactions it produced in rabbit and human studies [ ] . similar results were seen during numerous clinical studies where only mild local reactogenicity and fevers without serious adverse reactions were observed [ , , ] . additionally, cvi- appeared to offer a major advantage for dermatologically compromised individuals as kempe et al. confirmed the safety of this vaccine in children with eczema [ ] . nevertheless, two critical findings dampened enthusiasm for the cvi- vaccine. first, the neurotoxicity of cvi- following intracerebral inoculation of monkeys was shown to exceed that of the unattenuated ikeda, lister, and nycbh strains, suggesting a greater human risk for encephalitis with cvi- [ ] . second, it was found that the immune response to cvi- , particularly the neutralizing antibody response, was low when compared to unattenuated lister or ikeda strains, calling into question the protective efficacy of this vaccine [ , , ] . the svrg ultimately decided to sponsor the development of an entirely new attenuated strain. spearheaded by dr. hashizume of the chiba serum institute in japan, the latest technologies and understanding of vaccinia viruses were coupled to develop a variant of lister that did not produce neurotoxicity in animal models, yet maintained neutralizing antibody titers comparable to lister [ , , ] . in addition, cell culture techniques were employed to avoid contamination problems inherent to animal lymph-derived vaccines such as lister and nycbh, which may have contributed to toxicity, and to allow for mass production under controlled conditions. to produce the new strain, lister was initially passaged in primary rabbit kidney (prk) cells at low temperature ( • c) [ ] . at the th generation, clones were selected and evaluated for their ability to grow in vero cells. the clone with the lowest titer of growth, designated lc (lister clone ), was isolated and evaluated for growth in rabbit skin and the cns of rabbits and monkeys, where it compared favorably to lister. a small clinical trial was then conducted, comparing lc to lister in children [ ] . the vaccine was well tolerated and had a reduced fever rate compared to lister. however, a delay in pock formation and resolution was observed in children vaccinated with the attenuated strain. to lessen the chance of autoinoculation complications due to a prolonged pock reaction, a clone of lc that produced small pocks, a feature correlating with diminished growth capacity in mammalian tissues, was selected on cam [ ] . the virus was passaged six more times in prk at low temperature followed by growth on cam. medium-sized ( - mm) pocks were isolated and designated lc mo (lister clone medium pock size on cam original clone). finally, the lc mo clones were passaged three more times in prk cells and grown on cam where small ( . - mm) pocks were selected. the final attenuated clone was called lc m (clone ). formal in vitro comparisons were subsequently carried out to evaluate the replicative capacity of lc m , lc mo, lc , lister, and cvi- strains at , . , and • c [ ] . whereas all strains were able to grow at • c, lc m was unable to grow at temperatures above . • c, and lc and lc mo clones were unable to grow at temperatures above • c. in contrast, lister and cvi- grew at all temperatures tested. the neurotoxic effects of the parent lister strain and the three attenuated strains (lc , lc mo, and lc m ) were assessed by inoculating rabbits with these strains intracerebrally at a concentration of . tcid of each virus [ ] . all of the rabbits inoculated with unattenuated lister developed encephalitis, whereas none of the rabbits inoculated with the attenuated strains developed any adverse neurological symptoms. homogenates of the brains of rabbits inoculated with lc m had low levels of detectable virus, whereas brain homogenates of rabbits inoculated with comparators, most notably with the lister strain, contained higher levels of virus. a follow-up study was performed in cynomolgus monkeys [ ] . in this study, . ml of pfu of lc m or . tcid of lc mo was injected intrathalamically, with one monkey in each group euthanized on post-injection days and to evaluate cns tissue and virus titers. the growth of lc m in cns tissues was observed to be profoundly lower. in addition, fewer pathologic changes were observed in the brains and spinal cords of monkeys injected with lc m . to evaluate the ability of the attenuated virus strains to infect tissues beyond the skin, virus titers were assessed in the blood and brain of normal and immunosuppressed (cortisonetreated) mice [ ] . mice were inoculated intraperitoneally (ip) with . pfu of lister, lc , lc mo, lc m , or cvi- . five mice in each group were sacrificed daily for evaluation of viremia or cns viral growth. when inoculated with the lister or cvi- strain, virus was detected in the brain between days and in both normal and immunosuppressed animals, whereas the lc virus was only identified in the brains of immunosuppressed animals. in animals inoculated with either the lc mo or lc m strain, no virus was detected in the brains of either normal or immunosuppressed animals. however, lc m was detectable in the bloodstream for days in normal mice and days in immunosuppressed mice (fig. ) . lister, lc , lc mo, lc m , and cvi- were also compared for local reactogenicity by inoculating rabbits intradermally and scoring erythematous responses [ ] . the lc virus produced the largest erythematous reaction, followed by the lister strain, lc mo, cvi- , and finally lc m , which produced the smallest erythematous reaction. vaccine protective efficacy was extrapolated from rabbit studies that measured hemagglutination-inhibition (hi) and plaque reduction neutralizing titers (prnt) [ ] . as shown in table , both hi and prnt responses to lc m compared favorably with the responses to the lister strain and supported the clinical evaluation of lc m . in , sufficient doses of lc m and doses of lc mo were provided to health officials in prefectures throughout japan to vaccinate , and children, respectively [ ] . practitioners were instructed to use these vaccines or the standard lister vaccine for routine primary vaccination of children that year (predominantly - year olds, but some infants were included). exclusion criteria included known congenital or acquired heart problems, "wet eczema", known history of severe drug allergies, severe lister and lc mo: inoculated tcid /animal. lc m : inoculated pfu/animal. note: with respect to the units used to report titers, hi titers were done on two-fold dilutions of test sera, while prnt titers were done on four-fold dilutions of the test sera. source: hashizume et al. [ ] . kidney or liver disease, epilepsy, immunocompromise, and a history of convulsion in the past years [ ] . all vaccines were administered using an approximately l dose percutaneously at concentrations of . × to × . pfu/ml via bifurcated needles with - punctures in the upper deltoid region [ ] . within the large cohort of vaccinees, lc mo-and , lc m -vaccinated children were closely evaluated for local and systemic reactions [ ] . take rates were assessed at - days, with a physical exam performed and weeks after vaccination [ ] . since a vesicular or pustular reaction following vaccination had been used since the time of jenner as evidence that a vaccinee has developed protective immunity [ , ] , take rates were defined as a vesicular or pustular reaction at the site of vaccination; local redness or induration alone was recorded as a non-take. data for local and systemic reactogenicity were derived from surveys provided to the parents or guardians of vaccinees, and clinical assessments (objective and queried) were conducted during postvaccination clinic visits. endpoints assessed included fever or other systemic symptoms, lymphadenopathy, local redness and induration, take rates, and assessment of adverse reactions [ , ] . lc m produced take rates equivalent to other smallpox vaccines that had been used in japan (table ) . as with other vaccinia strains, the maximal adverse effects of lc m were reported around the time of peak pock reactions ( - days postvaccination) [ , ] . local induration at the site of vaccination, rates of fever, peak temperature, and fever duration were significantly less than with lister vaccination [ ] . in closely evaluated subjects receiving lc m , there were cases of urticaria, case of eczema vaccinatum (ev), cases of autoinoculation, cases of rash localized around the vaccine site, and benign febrile seizures of unclear etiology [ , ] . although the case of ev was mild and resolved without sequelae, details regarding any pre-existing skin condition in the vaccinated child are not known. several substudies were conducted within this large clinical trial. one involved the removal of excess vaccine at the site of vaccination by alcohol wipes to assess the possible impact on rates of autoinoculation and take rates [ ] . excess vaccine was removed at , , or min postvaccination. results showed that vaccination take rates were high ( - %) and autoinoculation rates were low when wiping the vaccination site within min. in contrast, when the vaccine was allowed to dry for min before wiping, autoinoculation rates were higher ( . %) [ ] . a second substudy examined a lister challenge vaccine administered at various timepoints, ranging from days to months post-primary vaccination [ , ] . japanese public health officials hoped that, by varying the interval between the primary and challenge vaccinations in this substudy and monitoring cutaneous effects, they would be able to gain insight on the speed with which the primary attenuated vaccine elicited an immune response as well as the robustness and longevity of that response. as shown in table , cross-protective immunity against lister challenge vaccination was induced early and in the vast majority of vaccinees (∼ %) [ , ] . findings with lc m were comparable to ikeda/dairen results [ ] . takeuchi reported that when children ( - year olds) were vaccinated with lc m and challenge-inoculated with lister days later, only / developed a small pustule; the remaining children had either no detectable reaction ( . %), or redness/induration or scab at the vaccination site [ ] . these findings suggested that cross-protection against other poxviruses, such as variola, could be mounted quickly following lc m primary vaccination, and be maintained with little variation during the first year post-lc m vaccination (the longest timepoint studied). however, other studies have shown that protection against a primary reaction following revaccination, although proposed historically for this purpose [ ] , is not a definitive measure of likely protective efficacy [ , ] . the relevance of a cutaneous reaction upon revaccination is therefore unclear. humoral immune responses at - months post-lc m vaccination were compared to data generated following vaccination with lc mo, cvi- , or lister vaccines [ ] . hi titers induced by the four vaccines did not differ significantly (table ) . although the numbers of lc m recipients assessed for prnt antibody generation were less than those sampled for hi ( versus ), titers following lc m vaccination appeared comparable to those generated following lister vaccination and superior to titers achieved with cvi- vaccination. although encephalitis had been rarely observed in subjects vaccinated with different strains of vaccinia, the investigators believed that strains with the potential to induce overt neurotoxicity might also produce transient brainwave anomalies. while the use of an electroencephalogram (eeg) to predict postvaccination encephalitis has never been validated, eegs were performed to document possible subclinical neurotoxicity in a cohort of children (aged - ) vaccinated with lc m [ ] . testing was done prior to vaccination, and , , and weeks postvaccination. findings were then compared to historical eeg data from children vaccinated with lister and cvi- (table ) [ ] . the combined clinical and immunological data convinced the svrg that lc m was a safe and effective vaccine. ninety-thousand doses of lc m vaccine were distributed for use in and , with no reports of severe aes [ ] . nevertheless, it is unclear how many of these doses were actually administered as the rapid eradication of smallpox in india led to the discontinuation of routine smallpox vaccination in japan in . shortly thereafter, the last known naturally acquired case of smallpox occurred in africa in . the lc m strain differs from the parent lister strain in a number of biological characteristics as summarized in table . the temperature sensitivity of lc m is due to restricted replication at temperatures above . • c, rather than thermal lability. the stability of the lc m phenotype on cam (small pock size), growth kinetics, and temperature sensitivity have been tested and confirmed by repeated passaging of virus in prk cells [ ] . with regards to replication kinetics, lister and lc m viruses are essentially the same in singlestep growth curves [ ] , suggesting that the small pock size of lc m on cam is not due to restricted intracellular replication but rather to the relative inability of the lc m virus to spread cell-to-cell. analysis of lister and lc m genomes by restriction endonucleases and cross-hybridization has revealed that lc m contains a new restriction site (xhoi), located in a hindiii d fragment near the terminus of the genome [ ] . recombinant studies, in which the hindiii d fragment of lister has been introduced into the lc m strain, have shown that a part of the genome containing the extra xhoi is responsible for small plaque and pock size, and restricted growth in vero cells [ ] . the gene in this fragment, originally called ps/hr for plaque size/host range, and now identified as b r, is related to the regulator of complement activation (rca) gene family in mammals that contain the characteristic short consensus repeat (scr) domains (fig. ) [ , ] . recent analysis of the full sequence of the lister, lc mo, and lc m genomes confirmed the -base deletion in the b r gene of lc m , which introduces a stop codon within the gene [ ] . as a result, while the full-length b r gene encodes for a -residue protein, the altered gene in lc m theoretically would encode for a -residue protein that, if processed correctly, would be secreted from infected cells as a -residue protein. all orthopoxviruses produce four forms of virus particles: the intracellular mature virus (imv), the intracellular enveloped virus (iev), cell-associated enveloped virus (cev), and the extracellular enveloped virus (eev) [ ] . deletion of b r generally results in decreased production of eev [ ] , which is critical for cell-to-cell transmission of virus within the infected host and plays an important role in disease pathogenesis [ , ] . b r is also a target for neutralizing antibodies [ ] , and anti-b r antibodies have been shown to protect mice against lethal infection [ ] . yet recent lethal poxvirus challenge studies in rabbits and mice [ , ] demonstrated that a deletion in the b r gene does not diminish lc m 's efficacy or its ability to induce eev antibodies. a primate study [ ] also suggested that the b r protein is not critical for smallpox vaccine efficacy. hooper et al. were able to show that a dryvax-vaccinated monkey with no detectable b r-specific neutralizing antibodies could survive a lethal monkeypox virus challenge. the serum of this particular monkey was able to neutralize monkeypox and the vv-connaught vaccine strain (derived from the nycbh strain) in prnt assays. it is possible that the protection conferred by lc m is b r-independent and other eev surface proteins may serve as epitopes for neutralizing antibodies [ , , ] . there is also evidence that smallpox vaccines induce cell-mediated immunity (cmi) [ ] [ ] [ ] [ ] [ ] [ ] . hence it is possible that neutralizing epitopes on the truncated b r protein might contribute to the overall protective immune response without being the primary mediator. lc m compared with that of other vaccines. recently it was shown that with prolonged passaging in cell culture, lc m undergoes a phenotypic reversion characterized by the production of plaques that are of intermediate size between wild-type lc m and the precursor virus, lc mo. this plaque-size heterogeneity can be mapped to the b r gene where some variants exhibit point mutations upstream of the known b r single-base deletion. these compensatory mutations correct the observed frameshift and lead to the reconstitution of a full-length b r gene. western blot analysis has confirmed a restored ability of these variants to produce a full-length b r protein. in a severe combined immunodeficiency (scid) mouse ld study, the pathogenicity of two revertant viruses and the lc mo precursor strain was similar. in addition, plaque-purified lc m and a construct of lc m lacking the b r gene were shown to have safety profiles comparable to that of mva in the same animal models and to confer protective immunity in a mouse/intranasal vaccinia (western reserve [wr] strain) challenge study [ ] . the specific clinical consequences that correlate with deleted or mutated genes in attenuated vaccinia strains are only just beginning to be studied. orthopoxviruses are known to use a wide array of immunomodulatory strategies to establish a rapid and ongoing infection within the host [ , ] . these mechanisms target the innate, humoral, and cellmediated immune pathways, using mechanisms as diverse as functional mimicry of host proteins, masking, and avoidance of innate antiviral pathways [ , ] . it is not yet known what immunomodulatory mechanisms are used or altered by the attenuation of lister to lc m . the complete genome sequences of the lc m , lc mo, and lister viruses have been published recently [ ] and studies are underway to compare the genomic and proteomic profiles of this group with profiles of other orthopoxviruses. in , two groups independently showed that vaccinia viruses could be modified to serve as cloning and expression vectors by inserting foreign dna into non-essential regions of the genome [ , ] . since then, numerous studies have demonstrated the utility of recombinant vaccinia viruses (rvvs) as effective vector systems due to their ability to produce robust cellular and humoral immune responses. with a carrying capacity for large dna fragments that rivals other vector systems (e.g., lentivirus, alphavirus, aav, adenovirus), rvvs may be a preferred strategy for the delivery of foreign antigens. both replication-deficient (e.g., mva) and replicationcompetent vector strategies have been employed, with safety being a major distinction between the two approaches. since fever, encephalitis, progressive vaccinia, and myopericarditis sometimes accompany vaccination with replication-competent vaccines, a replicating rvv vector system that is both safe and capable of eliciting a strong immune response is highly desirable. lc mo and lc m are attractive options for live rvvs because they are characterized by temperature sensitivity, restricted host range, and infrequent low-grade adverse events. moreover, lc mo has been found to be more immunogenic than its unattenuated parent, lister, and lc m , so it has generally been the preferred vector system [ ] . table highlights many of table history of lc mo-and lc m -based rvvs as vectors for antigen delivery and protein expression (modified and updated from [ ] the antigen delivery and protein expression applications that have used either lc mo or lc m as a vector system. a recent abstract [ ] showed particularly promising results for an lc m -vectored sars coronavirus (sars-cov) vaccine. it was observed that neutralizing antibody titers to the sars-cov spike protein increased -fold week after vaccination and increased an additional -fold weeks after a booster injection. interestingly, rabbits with preexisting antibodies to the vector (after being pre-immunized with lc m ) still generated an antibody response, suggesting that this vector system might produce vaccines suitable for populations that have undergone prior immunization with smallpox vaccine(s). work by kidokoro et al. [ ] demonstrated that lc mobased vectors provide a robust protein expression system that preserves the functional integrity of the expressed protein. these investigators were able to obtain yields of up to mg/l and purity of - % for the glycosylated measles virus hemagglutinin (h) protein. based on these promising results, further studies to determine the general utility of this lc mo vector for large-scale expression of other proteins are warranted. since animal challenge studies were not conducted during the development of lc m in the s, alternative measures of immunity that had been used to characterize other smallpox vaccines [ , ] were used to evaluate the efficacy of lc m in early clinical trials in japan. these included basic antibody and t-cell assays and monitoring of cutaneous responses to primary vaccination with lc m [ ] . however, current standards for determining vaccinia efficacy rely on more sophisticated measurements of humoral and cmi responses as well as lethal poxvirus challenges in vaccinated animal models [ , , , ] . one example of the latter is the lethal rabbitpox virus (rpxv) in rabbits, which mimics smallpox infection in humans [ ] since it is rapidly and widely disseminated, is highly infectious, and produces high levels of eev. the latter appears to play a prominent role in the pathogenesis of both rpxv and variola [ , ] , making the rpxv challenge model particularly suitable for testing the eev-neutralizing capability of lc m . hence, the protective efficacy of lc m was recently evaluated in the rabbit model using a rpxv challenge [ ] . in this comparative study, rabbits per group were vaccinated by scarification with approximately × pfu of lc m or dryvax, or with phosphate-buffered saline (pbs) as placebo. after days, the groups were divided in two, challenged with low or high intradermal doses of rpxv ( and pfu, respectively), and monitored for days. at days post-challenge, all vaccinated animals had survived, whereas / (low-dose challenge) and / (high-dose challenge) placebo (pbs) animals had died ( fig. ; only the antibody response was characterized at days , , and postvaccination and measured for the ability to bind imv as well as the ability to neutralize both imv and eev of rpxv in prnt assays. antibodies from the lc m vaccinated rabbits had a greater capacity to bind rpxv in an elisa (data not shown) as well as neutralize rpxv imv when compared to antibodies from dryvax-vaccinated rabbits (fig. ) . interestingly, antibodies from lc m -and dryvax-vaccinated animals showed comparable abilities to neutralize rpxv eev. fig. . antibody responses to lc m and dryvax in rabbits. sera obtained from rabbits and days after immunization were tested at a : dilution for neutralization of imv and eev forms of rpv by plaque-reduction assays. each data point represents the mean of serum samples. error bars indicate % confidence intervals. imv neutralization titers elicited by lc m were significantly greater than those elicited by dryvax (p < . , two-sided wilcoxon rank sum test). sera obtained from pbs-immunized animals demonstrated no neutralization. these data are not shown. finally, a plaquing virus assay used to measure virus in different tissues showed there was no detectable rpxv in the lung, liver, or spleen of both groups of vaccinated animals, whereas placebo animals had high titers of virus in these organs. in a second animal model, lc m was compared to dryvax for its ability to protect a/ncr mice from aerosolized ectromelia virus (ectv), the causative agent of mousepox [ ] . this model was chosen since it closely mimics the route in which smallpox might be delivered during a bioterrorist attack [ ] . ten mice per group were vaccinated by scarification with approximately × pfu lc m or dryvax, or with pbs as placebo. forty-nine ( ) days later, all animals were challenged with a dose approximately - times the lethal dose (ld ) of aerosolized ectv. at days following challenge, all vaccinated animals had survived, whereas % of the placebo mice had died (fig. ) . no significant clinical symptoms or weight differences were noted between the lc m -and dryvax-vaccinated mice post-challenge. serum samples were taken from all mice days after vaccination. each sample was assessed for recognition of orthopoxvirus using an elisa with vaccinia western reserve (wr) as the antigen. as was seen in the rabbit study, sera from lc m -vaccinated mice generated higher vaccinia-specific elisa titers than sera from dryvax-vaccinated mice (fig. ) . the rabbit and mouse data from the preceding studies are in agreement with recent work in a mouse-vaccinia wr intranasal challenge model by kidokoro et al. who reported that b r-deficient lc m was able to induce prnt antibodies, and that protection against lethal challenge was equivalent to that of the b r-containing dryvax [ ] . in the same report, these authors determined that, similar to mva and in contrast to dryvax, lc m did not induce mortality in scid mice, despite being given at doses -fold higher than the protective dose in this particular mouse model. fig. . geometric mean titer of vaccinia virus-specific antibodies in mice vaccinated with dryvax or lc m . sera obtained from mice days after immunization were tested for vaccinia virus-specific antibodies by elisa. the bars represent the geometric mean titers from dryvax-immunized animals and lc m -immunized animals. although mice were vaccinated with dryvax or lc m , serum volumes from some animals were insufficient for analysis. error bars indicate % confidence intervals. the geometric mean titer elicited by lc m were significantly greater than those elicited by dryvax (p < . , two-sided wilcoxon rank sum test). sera obtained from pbs-immunized animals did not contain detectable vaccinia virus-specific antibodies. these data are not shown. further confirmation of the protective efficacy of lc m in another mouse-vaccinia wr challenge model was reported by morikawa et al., who demonstrated that protection was equivalent to that of the b r-containing lister and lc mo vaccine strains [ ] . finally, it has recently been reported by saijo et al. that lc m and lister provided protection from disease in monkeys challenged with severe intranasal monkeypox [ ] . japan has retained a national stockpile of lc m since conditionally licensing it in (unconditional licensure was granted in ). the government decided to increase the volume of its lc m stockpile in in response to a resurgence of monkeypox in africa, and again in early in response to terrorist threats in the us and other regions of the world. in anticipation of increased lc m production, employees of the chiba serum institute (manufacturers of lc m until mid- ) volunteered for vaccinations in november and were followed for safety and immunogenicity [ ] . fifty-six ( ) of these were revaccinees (the strain and date of previous vaccination unknown) and were primary vaccinees. lc m was administered using a fivepuncture bifurcated needle inoculation technique, and symptom diaries were kept for weeks. the vaccination site was inspected by medical personnel - days postvaccination. clinical observation records for of the vaccinated workers are summarized in table . vaccination site symptoms were mild (mostly pruritis) and resolved within a week. a phase i/ii clinical trial of lc m is presently underway in the us to evaluate the safety and immunogenicity of lc m . preliminary data was recently presented and will be formally published shortly [ ] . the current us stockpile of smallpox vaccines is comprised of vaccines using the nycbh vaccinia strain, primarily the calf lymph derivatives (apsv "wetvax" and dryvax manufactured in the s and early s, respectively) and one of the more recently manufactured cell culture derivatives (acam ) [ , ] . although well proven with regard to protective efficacy against smallpox, a first-generation vaccine such as dryvax may produce rates of local and systemic adverse reactions that are unacceptable in a post-endemic smallpox era, particularly given the higher rates of immunosuppression and skin disease in today's population. common reactions to dryvax include fever, lymphadenopathy, and lymphadenitis, while more serious adverse events include auto/contact inoculation, generalized vaccinia, eczema vaccinatum, progressive vaccinia, and encephalitis [ ] . concern has also been expressed about the possible presence of adventitious agents in stockpiled dryvax and apsv since calf lymph-perpetuated smallpox vaccines were not screened for mycoplasma, viruses, or prions during manufacturing [ ] . deaths from dryvax vaccination in the us prior to the eradication of smallpox were frequently due to encephalitis, which occurred at a rate of approximately one per million primary vaccinees [ ] . in recent clinical trials evaluating dryvax, myopericarditis, a serious adverse event rarely reported in the past, was found to occur at high rates among primary vaccinees [ , ] . through careful evaluation of vaccinees for both clinical and subclinical symptoms, this reaction was identified in up to / primary vaccinees, prompting the addition of a black box warning to the package insert of dryvax in [ ] . this discovery has further fueled the drive to identify smallpox vaccines with improved safety profiles. initial attempts to improve vaccine safety focused on modernizing the manufacture of dryvax by growing the virus in cell culture and cloning single subpopulation strains of nycbh that had been characterized by protective efficacy and low neurovirulence in animal studies [ , , ] . clinical trials with the acambis-produced second-generation nycbh vaccines, acam and acam (derived from acam and grown in vero cells rather than mrc- cells), confirmed high take rates (∼ %) and immunogenicity [ ] . the quality, progression, and timing of pock reactions were similar to those seen with dryvax controls, as were most local and systemic reactions. apart from local reactions at the injection site, the most common adverse reaction to acam or acam was lymph node or axillary pain (seen in - % of primary vaccinees) [ ] . unfortunately, subclinical or overt myopericarditis was also found to occur at high rates in both the acam and dryvax control groups [ , ] . in addition, one vaccinee given the experimental vaccine experienced a new onset seizure days postvaccination [ ] . in another recent phase ii study, myopericarditis developed in one subject days after vaccination with . × pfu/ml of acam [ ] . ccsv (cell-cultured smallpox vaccine), a version of the nycbh vaccinia strain grown in mrc- cells by dyn-port vaccine company (dvc), was recently evaluated in a double-blinded dryvax-controlled clinical trial conducted in vaccinia naive and non-naive volunteers [ ] . as with the acambis products, rates of adverse reactions to ccsv were similar to dryvax, however, no serious adverse reactions or myopericarditis were reported during the trial. ccsvinduced prnt titers and seroconversion rates were generally lower than those induced by dryvax. given the disappointing safety findings of cell culturederived second-generation vaccines, focus has once again shifted to third-generation vaccines such as lc m and mva (imvamune, bavarian nordic; and mva , acambis) as a potential means of replenishing national stockpiles. the japanese government recently announced that it will significantly expand its national stockpile of lc m , affording protection for million people in the event of a bioterrorist attack [ ] . mva, with its extensive history of safety in humans (> , people have been vaccinated in germany although some with only a single dose of approximately pfu), is presently being reassessed in various clinical and nonclinical studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although the rationale for using this non-replicating vaccine in high-risk populations such as those with immunosuppression or skin disease is compelling [ ] , its efficacy against smallpox is unknown. most nonclinical studies evaluating the protective efficacy of mva indicate that multiple-dose regimens using higher concentrations of approximately pfu are required [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in one study of mice challenged intradermally or intranasally with replicating vaccinia, protective efficacy was observed only after two or three doses of mva had been administered [ ] . lc m is currently the only smallpox vaccine under investigation that is both attenuated and able to replicate in humans, potentially offering protection after a single dose. the available clinical and nonclinical data suggest that lc m merits further study as a potential vaccine for use in the future. however, development of any new smallpox vaccine is complicated by the evolving nature of the field. it is clear that historical data on protective antibody titers from the smallpox eradication era cannot be relied upon. with smallpox no longer an endemic disease, vaccine efficacy can only be inferred from animal challenge studies and corresponding immune responses in humans. the us fda has published specific requirements for proof of efficacy in animal models that every new smallpox vaccine must meet (the so-called "animal rule") [ ] . given the multifactorial nature of the immune response to smallpox vaccines, various markers for both humoral and cellular immunity are being studied with the aim of identifying objective measurements of efficacy [ ] [ ] [ ] [ ] [ ] [ ] . correlates of protection in humans have yet to be defined unequivocally, but may rely in part on the elicitation of neutralizing antibody responses that correspond to those demonstrated to be efficacious in animal models. although the threshold titer of neutralizing antibodies needed for protection is unknown [ , ] , it was recently shown that human vaccinia-specific antibodies passively transferred to nonimmunized macaques offered protection against a lethal monkeypox challenge [ ] . a lack of published data and the varying reagents used in plaquing virus assays make comparison of absolute prnt titers elicited by different smallpox vaccines difficult. however, published results suggest that cell-cultured nycbh vaccines produce immune responses similar to dryvax [ , ] . no recent data on prnt levels elicited by mva or lc m in humans have been published, but findings should be forthcoming from ongoing clinical trials. the duration of humoral and cell-mediated protective immunity afforded by smallpox vaccination remains unknown, yet there is both empirical [ , ] and experimental [ , [ ] [ ] [ ] evidence that some degree of immunity to smallpox persists for several decades postvaccination. in addition, hammarlund and colleagues recently reported that smallpox vaccination as much as years pre-exposure conferred immunity to humans exposed to monkeypox [ ] . no data on the duration of long-term immunity to lc m are currently available, however, immunity duration will be an important consideration for all new smallpox vaccine candidates. while the biodefense-focused quest for a safe and effective smallpox vaccine is urgent, only the eventual outcomes of controlled clinical and nonclinical studies of new smallpox vaccine candidates will ensure their suitability for use in humans should smallpox recur in the future. characteristics of virus strains for production of smallpox vaccines effort to improve smallpox vaccine clinical special edition: vaccination in future-everything about attenuated vaccine vaccination research group research report: ministry of health and welfare special research: postvaccination side effects and research regarding treatment of complications vaccination in japan comparison of virus production in chicken embryo fibroblasts infected with the wr, ihd-j and mva strains of vaccinia virus: ihd-j is most efficient in trans-golgi network wrapping and extracellular enveloped virus release successful 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neutralizing antibodies after revaccination against smallpox human cytotoxic t-cell memory: long-lived responses to vaccinia virus responses to smallpox vaccine multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox we would like to thank the following individuals for their contributions to this manuscript: hiroyuki yokote, munehiro hirayama, and so hashizume for permission to reproduce figures and tables from original lc m research reports; kouji matsushima for helpful discussions concerning the use of lc m as a vector system; catherine bolger for editorial assistance; and timothy corrigan for document translation support. in addition, we would like to express our grati- key: cord- -xb mv authors: lebrun-harris, lydie a.; mendel van alstyne, judith a.; sripipatana, alek title: influenza vaccination among u.s. pediatric patients receiving care from federally funded health centers date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xb mv introduction: during the – influenza season, vaccination coverage among u.s. children was . %. the purpose of this study was to estimate the prevalence of influenza vaccinations among pediatric patients seen in u.s. health centers, and to explore potential disparities in vaccination coverage among subpopulations. funded by the health resources and services administration (hrsa) within the u.s. department of health and human services, these health centers provide primary and preventive care to underserved and vulnerable individuals and families in order to reduce health disparities based on economic, geographic, or cultural barriers. methods: cross-sectional data, analyzed in , came from the most recent waves of the health center patient survey ( , ). the sample consisted of children ages – years receiving care from hrsa-funded health centers. the outcome of interest was self- or parent-reported receipt of influenza vaccine in the past year. multivariable logistic regression was used to estimate the adjusted prevalence rate ratios for the association between demographic characteristics (age, sex, race/ethnicity, poverty level, urban/rural residence, geographic region), health-related variables (receipt of well-child check-up, asthma diagnosis), and influenza vaccination. results: influenza vaccination coverage among pediatric health center patients increased from . % in to . % in . in the adjusted model for , there were few statistically significant differences in vaccination coverage among subpopulation groups, however american indian/alaska native children had % increased vaccination coverage compared with non-hispanic white children (aprr: . , % ci: . – . ) and children living in the south had % decreased vaccination coverage compared with those living in the northeast (aprr: . , % ci: . – . ). conclusions: influenza vaccination coverage among pediatric health center patients in exceeded the national average (as of – ), and few differences were found among at-risk subpopulations. hrsa-funded health centers are well-positioned to further increase the vaccination rate among children living in underserved communities. vaccinations, extolled as one of the greatest public health achievements of the th century, continue to be a critical tool for mitigating death and disease in the united states (u.s.) and globally. for a single-year birth cohort, it is estimated that routine childhood vaccination prevents nearly million cases of disease in the u.s., including over , deaths [ ] . moreover, avoided morbidity and mortality due to vaccination is estimated to save $ . billion in direct costs and $ . billion in total societal costs over the cohort's lifetime [ ] . despite the remarkable outcomes provided by recommended vaccines they remain underutilized among children, due to barriers to accessing vaccination services, vaccination exemptions, and parental vaccine hesitancy [ ] [ ] [ ] . in the u.s. each fall, the centers for disease control and prevention and the american academy of pediatrics recommend https://doi.org/ . /j.vaccine. . . - x/published by elsevier ltd. seasonal influenza vaccination for everyone six months of age and older, excluding those with medical contraindications [ , ] . and each year, public health departments and primary care providers work to implement this recommendation. historically, underserved populations, such as those living below the federal poverty line, and those who are uninsured or underinsured, have lower seasonal influenza vaccination rates [ ] [ ] [ ] , leaving those individuals and their communities more susceptible to influenza and related complications. young children are at particularly high risk for severe influenza complications [ , ] . estimates from the - influenza season based on the national immunization survey-flu indicate that the childhood influenza vaccination rate was nearly %-an increase of almost percentage points from the - influenza season [ ] . while this marks a positive shift, the coverage rate remains below the national benchmark of % [ ] . health care providers caring for the underserved are uniquely positioned to help prevent seasonal influenza infections through vaccination and to increase community immunity in the areas they serve. health centers funded by the health resources and services administration (hrsa) within the u.s. department of health and human services, under section of the u.s. public health service act [ ] provide high quality, accessible, and affordable primary and preventative health care to underserved and vulnerable populations across the u.s. in order to reduce health disparities based on economic, geographic, or cultural barriers. in , hrsa-funded health centers served over million patients, including over . million children ages through years, which represents one in nine u.s. children [ , ] . health centers operate with a sliding scale fee structure for patients, based on their income level. with over % of patients living at or below % of the federal poverty guideline [ ] , the health center program provides a model of low cost, high quality health care that can contribute to improving utilization of preventative health services including seasonal influenza vaccination [ ] . given the program's reach among children within underserved communities, health centers are cornerstones in promoting public health in general and influenza community immunity in particular [ ] . to date, there have been no studies examining influenza vaccine uptake among patients seen in hrsa-funded health centers. only one previous study, published in , has examined pediatric vaccinations in the context of health centers, however it focused on the childhood immunization schedule rather than seasonal influenza vaccinations. that study found few disparities in childhood immunizations, and the authors hypothesized that enabling services provided by health centers facilitated access to timely vaccinations [ ] . in , the advisory committee for immunization practices (acip) recommended universal seasonal influenza vaccinations among those six months of age and older [ , ] . thus, this study sought to examine the rates of pediatric influenza vaccinations among health center patients before and after the acip recommendation. the current study contributes to the literature by examining, for the first time, the prevalence of influenza vaccine administration among children served by hrsa-funded health centers. the strengths of the study include the use of hrsa datasets which are nationally representative of u.s. individuals who receive primary and preventive care from health centers, as well as the analysis of several sociodemographic characteristics to explore potential disparities in influenza vaccine uptake across pediatric subpopulations in underserved communities. based on previous studies which have documented minimal disparities in health care access and utilization among health centers [ , [ ] [ ] [ ] , we hypothesized that we would find few disparities in pediatric influenza vaccinations in these settings. we conducted secondary analysis of data from the health center patient survey, a nationally representative survey of people who receive care from u.s. health centers funded by hrsa. the survey is conducted periodically (every - years) by hrsa's bureau of primary health care and is designed to capture information on patient sociodemographic characteristics, health conditions, health behaviors, access to and utilization of health care services, and satisfaction with care. survey questions are based on other established national health surveys, including the national health interview survey, national ambulatory medical care survey, medical expenditure panel survey, and national health and nutrition examination survey. sample selection is based on a stratified three-stage random sampling design. first, health center organizations are sampled, stratified by funding stream, size, u.s. census region, urban/rural location, and number of care delivery sites per health center organization. then, up to three care delivery sites per health center organization are sampled. finally, individual patients from each site are sampled. patients are eligible for the survey if they had at least one medical visit to the health center site in the past year. surveys are conducted through computer-assisted personal interviews by trained field interviewers. for children ages and under, parents or other knowledgeable caregivers respond to the survey; children ages and over respond to the survey themselves, after parental/caregiver assent is obtained. the health center patient survey was most recently fielded survey, when a total of , patients were surveyed between october and april . among patients who were screened and determined to be eligible, % completed an interview. the previous fielding was the health center patient survey, which included a total of , patients surveyed between september and december ; in this fielding, % of patients who were screened and deemed eligible completed an interview. institutional review board approval for the original data collections in both and was obtained from research triangle international, the organization that administered the survey on behalf of hrsa. for the current study, conducted in , the analytic sample of pediatric health center patients included , children between the ages of and years from the survey, and children from the survey. we examined receipt of annual influenza vaccine, as assessed by parent-or self-reported responses to two questions. the first question asked about influenza vaccination injection: ''during the past months, {have you/has name} had a flu shot? a flu shot is usually given in the fall and protects against influenza for the flu season. the flu shot is injected in the arm. do not include an influenza vaccine sprayed in the nose." the second question asked about influenza vaccination nasal spray: ''during the past months, {have you/has name} had a flu vaccine sprayed in {your/his/her} [ ] nose by a doctor or other health professional? this vaccine is usually given in the fall and protects against influenza for the flu season." for this study, we combined responses to both questions to create a dichotomous outcome measure to capture the receipt of an annual influenza vaccine (yes vs. no). sociodemographic covariates of interest included patient age, sex, race/ethnicity, family poverty level, urban/rural residence, and u.s. census region. we also included two health-related variables, specifically, receipt of a well-child check-up in the past months and lifetime diagnosis of asthma (an indicator of a highrisk population). we examined the rates of influenza vaccination among pediatric health center patients in versus . we also stratified vaccination rates by age group ( - years, - years, - years) to determine whether rates declined with age, as is found in national trends [ ] . we limited subsequent analyses to the sample of pediatric patients, the most recent year of health center data available, to assess potential disparities in receipt of influenza vaccines among this population after the acip issued its recommendation for universal vaccination. we first examined the distribution of sociodemographic and health characteristics for patients, and conducted bivariate analyses and chi-square tests of independence with design-based f statistics to assess the associations between receipt of influenza vaccine and each of the characteristics of interest. we set statistical significance at p < . , and calculated % confidence intervals for each estimate. finally, we conducted multiple logistic regression to examine the independent associations between each covariate of interest and receipt of an annual influenza vaccine. results are presented as adjusted prevalence rate ratios (prrs), which represent the likelihood that children with specific characteristics of interest received an annual influenza vaccine, relative to other children in meaningful reference groups while holding all other correlates constant. about % of the sample had missing income data, therefore we created a separate category for ''missing income" in order to retain these observations. we conducted all analyses using stata/se, version . , and employed weights based on the survey's sampling design to produce estimates that adjusted for the complex sampling design and were representative of the underlying population. the overall rate of influenza vaccination among pediatric patients seen in hrsa-funded health center increased from % in to % in , a % increase over years (fig. ) . increases in vaccination coverage were seen across all three age groups. the largest relative increase occurred among youth to years, from % in to % in , a % increase ( percentage points). in both and , there was an inverse doseresponse relationship between vaccination and patient age, with the youngest age group ( - years) having the highest rates of vaccination and the oldest age group ( - years) having the lowest rates. in , % of hrsa-funded health center pediatric patients in the analytic sample were between the ages of and years, and another % were between and years, with the remainder between and years (table ). there were slightly more male patients ( %) than female patients. the most common racial/ethnic group among pediatric patients was hispanic ( %), followed by non-hispanic white ( %) and non-hispanic black ( %). about % of the sample lived below the federal poverty level. over % of pediatric patients lived in rural areas, and slightly more lived in the west ( %) and south ( %) compared with the northeast ( %) and the midwest ( %). almost % of children had received a well-child check-up in the past year, and about % had a lifetime diagnosis of asthma. in unadjusted analyses, only two characteristics were associated with receipt of annual influenza vaccination among health center pediatric patients in (table ) . specifically, a larger proportion of children living in urban settings received a influenza vaccine than those living in rural settings ( % vs. %, p = . ). in addition, children living in the northeast and the west had higher rates of influenza vaccination than those living in the south and the midwest (range: %- %, p = . ). there were no statistically significant associations between influenza vaccination and child age, sex, race/ethnicity, federal poverty level, well-child check-up, and asthma diagnosis. after adjusting for all covariates simultaneously in the multiple logistic regression, the association between urban/rural residence and influenza vaccines was no longer statistically significant (table ) . however, children living in the south had a % decreased prevalence of influenza vaccine receipt, compared with those living in the northeast (aprr = . , % ci: . - . ). in addition, american indian/alaska native children had a % increased prevalence of influenza vaccine receipt relative to non-hispanic white children (aprr = . , % ci: . - . ). among u.s. children ages - years receiving care from hrsafunded health centers, there was a percentage point increase in annual influenza vaccinations between (prior to the acip recommendation) and , from % to % of patients. the influenza vaccination rate among this traditionally underserved population exceeded the - rate seen among u.s. children nationally ( %) by almost percentage points. the age-related pattern seen in health center pediatric patients, showing lower vaccination rates with increasing age, was similar to the pattern in the general u.s. population [ ] . several subpopulations of health center patients exceeded the national benchmark of % uptake, including young children (ages - years), american indian/alaska native and hispanic children, and those living under the federal poverty level, in urban locations, and in the northeast and west. one possible explanation for the increased influenza vaccinations among these subgroups is that health centers may deliver childhood vaccines through the federal vaccines for children program, which provides free or low-cost vaccines for uninsured, underinsured, and medicaid-insured children, as well as american indian/alaska native children [ ] . another potential explanation is that hrsa implemented new activities during this timeframe to promote continuous quality improvement among its grantees, including publicly disseminating clinical performance data and implementing a pay-for-performance program [ ] . additional studies are needed to assess potential differential impacts of these quality improvement efforts on specific subpopulations. although these results demonstrate a vast improvement in influenza vaccinations among this population within a -year span, there is a continuing need to further increase vaccination rates. plans-rubió's ( ) study of community immunity against influenza viruses suggests that % vaccination coverage is required in healthy persons (and % in high-risk persons) to establish a sufficient network of protection in the u.s [ ] . results of this study indicate that hrsa-funded health centers are effectively providing equitable access to seasonal influenza vaccination to a wide spectrum of medically underserved pediatric patients. health centers have a long history of reducing health care disparities by providing access to primary and preventive care to medically underserved and vulnerable populations [ , [ ] [ ] [ ] . the health center data on influenza vaccinations corroborated this pattern, revealing few disparities with the exception of statistically significant lower rates among pediatric patients in the south ( %, compared with % in the northeast) and significantly higher rates among american indian/alaska native patients ( %, compared with % among non-hispanic white patients). previous studies have identified several institutional factors which may contribute to health centers' ability to provide equitable access to services across a broad range of clinical domains, such as cancer screening, chronic condition management, prenatal care, and well-visit check-ups. these institutional factors include the provision of supportive services (e.g., care coordination, health education, translation, transportation), early and widespread implementation of electronic health records, and recognition as patient-centered medical homes [ , , ] . strengthening access to and quality of primary care broadly through these efforts may translate to increased use of specific services including influenza vaccinations, and fewer missed opportunities for influenza vaccinations. indeed, yue and colleagues ( ) found that adult health center patients who used support services had a percentage point higher likelihood of receiving an influenza vaccine compared with those who did not use support services [ ] . given the demonstrated and potential efficacy of these factors in improving health care provided by health centers, hrsa has invested hundreds of millions of dollars to accelerate and optimize the adoption of health information technology, as well as incentivize the adoption of the patient-centered medical home model of care through its quality improvement awards [ ] . while previous studies have found positive effects of these innovations on access to care, quality of care, and disparities [ , , ] , additional analyses may examine their potential impact specifically on pediatric influenza vaccine uptake. our finding of lower rates of pediatric influenza vaccination among health center patients in the south was consistent with patterns identified using the national immunization survey and the national health interview survey [ , , ] , suggesting opportunities to geographically target interventions and resources to improve the uptake of influenza vaccines in this region. results from this study can be leveraged to inform efforts by hrsafunded primary care associations and health center controlled networks that work with health centers in the south [ , ] . additional research is needed to better understand the underlying factors that lead to lower influenza vaccinations among children seen in health centers in the south. racial/ethnic disparities in childhood influenza vaccination are well established in the literature, although the findings are mixed. studies of young children (under age ) have found that white children have higher vaccination coverage than black or hispanic children [ , ] , while national estimates of all children up to age show that white children have similar vaccination rates to black children and lower rates than hispanic children [ , ] . in contrast, our study found no statistically significant differences between these three racial/ethnic groups among the population of pediatric patients served by hrsa-funded health centers. the lack of disparities found within these settings may be a reflection of the health center program's mission to provide culturally competent, patient-centered, comprehensive primary care regardless of individuals' ability to pay, as well as supportive services that promote access to health care [ ] . in particular, hrsa requires that health centers incorporate cultural competency activities to address the unique needs of the populations they serve. this includes arranging interpretation and translation services for patients with limited english-speaking ability, providing resources and training to staff on delivering culturally sensitive services and bridging cultural differences, and regularly conducting needs assessments to improve service delivery with a particular focus on patient population characteristics that impact health status or health care access/utilization (e.g., social factors, physical environment, cultural/ethnic factors, language needs, housing status). in addition, health centers are directed by governing boards that must be composed of a patient majority; at least % of board members must be patients at the health center and must reflect the population served in terms of demographic factors such as race/ethnicity and gender. we also found that the influenza vaccination rate among american indian/alaska native pediatric health center patients was notably higher than the national average for american indian/alaska native children in - ( % vs. %) [ , ] . researchers using the national health interview survey have also reported higher influenza vaccination coverage among american indian/alaska native children compared with other racial/ethnic groups [ ] , which may reflect clinical recommendations to target vaccination efforts for this subpopulation [ ] . our results showed that about % of pediatric health center patients lived in rural areas, twice the proportion of children living in rural settings in the u.s. in general [ , ] . although rural pediatric patients were less likely to receive influenza vaccinations than urban patients in the unadjusted analysis, there were no differences based on urban/rural status in the adjusted analysis, indicating that health centers are successfully serving areas with lower access to care overall. there are study limitations worth mentioning. first, while the results are generalizable to the population of underserved pediatric patients seen in hrsa-funded health centers, they are not representative of the general u.s. child population. although the findings apply only to a specific subgroup, they represent . million u.s. children ages and under, a not insignificant number. second, because the health center patient survey includes patients of all ages, the sample of pediatric patients is relatively limited, and the small sample sizes for certain subgroups may have limited our ability to detect statistically significant differences. third, although influenza vaccines are recommended for infants starting at months, it was not possible to examine vaccination among health center patients younger than years because of how age was coded in the dataset; therefore we were unable to obtain estimates of influenza vaccination for this age group. in addition, two influenza vaccine doses are recommended for children months through years in their first vaccination season but the health center patient survey does not ask whether children received or doses or whether it was their first time receiving a vaccine, therefore it was not possible to distinguish between the proportion of children with full versus partial influenza vaccination coverage. another limitation is potential recall bias. specifically, parents may not accurately remember details of their child's influenza vaccinations, and children years and over who respond for themselves on the survey may be less reliable in reporting their health care utilization in general and in particular may be less aware that they received an influenza vaccine. under these circumstances, our study may underestimate the prevalence of annual influenza vaccination. additionally, the survey includes limited parental or household information, such as parents' education level or health literacy, so it was not possible to assess the association between several potentially relevant family characteristics and pediatric patients' influenza vaccine uptake. in particular, children ages and over were not asked about their health insurance coverage, so we were unable to examine influenza vaccination rates based on insurance status. however, previous research has cited a positive correlation between state-level medicaid reimbursement and influenza vaccination rates among poor children across three separate influenza seasons [ ] , which may be particularly relevant for improving vaccination rates among health center patients, given that nearly half are medicaid or chip beneficiaries [ ] . similarly, the study did not examine the role of knowledge, attitudes, and practices toward vaccines among parents of pediatric health center patients, which might influence the decision to obtain influenza vaccinations. recent literature illustrates that parental concerns about seasonal influenza vaccine exist, including beliefs that the vaccine causes influenza illness, the vaccine is unnecessary because influenza illness is mild, and the vaccine is not effective [ ] [ ] [ ] [ ] . beyond concerns specific to influenza vaccine, more generalized concerns may influence decisions to vaccinate. concerns over safety and vaccine ingredients, number of vaccines administered, mistrust of the mainstream medical system, and the belief that vaccination should be a personal choice persist, and are often shaped by a parent's social sphere of influence [ ] [ ] [ ] [ ] . finally, the health center patient survey was last conducted in - , so the data are now several years old. however, examining the data is still informative in terms of comparing to national averages and providing a baseline for comparing pediatric influenza vaccine rates in the future. the next wave of the survey is planned to be conducted in summer/fall of , which will provide valuable information to ascertain whether hrsa-funded health centers have continued to increase influenza vaccine uptake among their pediatric patients, and to examine any current disparities impacting subpopulations of interest. furthermore, future research should use these anticipated data to examine how the covid- pandemic may impact patients' perceptions of vaccines and influence vaccine uptake. additional research is needed to further explore patient, family, provider, and organizational factors that may influence influenza vaccination among children receiving care at hrsa-funded health centers. notwithstanding the limitations mentioned above, this study provides the first nationally representative estimates of influenza vaccination rates among pediatric patients receiving care from hrsa-funded health centers, both overall and for subpopulations based on demographic and health-related factors. results reveal opportunities to increase pediatric influenza vaccination in health centers so that all subpopulations can attain and surpass the national benchmark of % coverage. possible strategies to improve influenza vaccine coverage include partnering with state and regional primary care associations, which are tasked with providing programmatic, clinical, and financial training and technical assistance to safety-net providers, to support health centers in developing immunization strategies tailored to their localized communities [ ] . additionally, health information technology can be used to better facilitate important vaccine conversations, including patient and provider reminders and prompts in electronic health record systems and patient portals [ ] [ ] [ ] . other potential vehicles for improvement include parent education, proactive appointment scheduling, and strong provider recommendations [ , ] . findings suggest that hrsa-funded health centers can serve as critical providers in engaging and serving diverse constituencies, reducing disparities in influenza vaccination, and increasing immunity among the nation's most underserved communities. the authors have no financial disclosures. the views expressed in this article are those of the authors and do not necessarily reflect the official policies of the us department of health and human services or the health resources and services administration, nor does mention of the department or agency names imply endorsement by the us government. simply put: vaccination saves lives economic evaluation of the routine childhood immunization program in the united states vaccination coverage among children aged - months -united states vaccination coverage for selected vaccines and exemption rates among children in kindergarten -united states, - school year department of health and human services 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children an assessment of parental knowledge, attitudes, and beliefs regarding influenza vaccination what is 'confidence' and what could affect it?: a qualitative study of mothers who are hesitant about vaccines mott children's hospital. national poll on children's health. mott poll report: do parents have selective hearing about flu vaccine for children vaccine safety: myths and misinformation factors influencing african-american mothers' concerns about immunization safety: a summary of focus group findings association between patient reminders and influenza vaccination status among children using reminder/recall systems to improve influenza immunization rates in children with asthma the impacts of email reminder/recall on adolescent influenza vaccination association between provider recommendation and influenza vaccination status among children the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -sls bsm authors: dean, natalie e.; pastore y piontti, ana; madewell, zachary j.; cummings, derek a.t; hitchings, matthew d.t.; joshi, keya; kahn, rebecca; vespignani, alessandro; elizabeth halloran, m.; longini, ira m. title: ensemble forecast modeling for the design of covid- vaccine efficacy trials date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: sls bsm to rapidly evaluate the safety and efficacy of covid- vaccine candidates, prioritizing vaccine trial sites in areas with high expected disease incidence can speed endpoint accrual and shorten trial duration. mathematical and statistical forecast models can inform the process of site selection, integrating available data sources and facilitating comparisons across locations. we recommend the use of ensemble forecast modeling – combining projections from independent modeling groups – to guide investigators identifying suitable sites for covid- vaccine efficacy trials. we describe an appropriate structure for this process, including minimum requirements, suggested output, and a user-friendly tool for displaying results. importantly, we advise that this process be repeated regularly throughout the trial, to inform decisions about enrolling new participants at existing sites with waning incidence versus adding entirely new sites. these types of data-driven models can support the implementation of flexible efficacy trials tailored to the outbreak setting. the covid- pandemic is a public health emergency, and there is an urgent need for effective vaccines to limit morbidity and mortality. efforts are underway to accelerate all steps in the vaccine development pathway ( ) . large randomized field trials are crucial for determining the safety and efficacy of candidates to inform regulatory decisions ( ) . in these trials, many thousands of eligible and consenting participants across multiple sites are enrolled and individually randomized to vaccine or control. these trials are event driven, where an expected primary endpoint is laboratory-confirmed symptomatic disease ( ) , with infection regardless of symptoms as a valuable secondary endpoint ( ) . selecting vaccine trial sites where disease incidence is highest during the study period can accelerate the accrual of endpoints. mathematical and statistical models are recognized as valuable tools for planning infectious disease clinical trials ( ) . they can be used to optimize design features such as cluster size or to examine the validity of the trial's statistical analysis ( ) . the use of spatially explicit forecast models to select vaccine trial sites was first explored during the - zika epidemic ( ) . these forecast models synthesize available data to make projections about which sites might have the highest future disease incidence. an important value of models is that they standardize projections across locations. trends in raw reported numbers of cases depend heavily on the sensitivity of the underlying surveillance system. case definitions and access to care and testing may vary over time and space. models that integrate many data sources, such as reported cases, test positivity, hospitalizations and deaths, can facilitate more meaningful comparisons across locations. forecasts provide estimates along with the uncertainty associated with those estimates to make best use of the available information. models can incorporate many features to capture the complex dynamics of infectious diseases. incidence is expected to vary widely over time and between locations, as a function of control measures in place, patterns of introduction, seasonality, and other sources of variability. mathematical models naturally account for prior circulation of the virus and any buildup of population-level immunity. areas that have already experienced substantial outbreaks may be less suitable for inclusion, and this would be reflected in projections. models can explicitly capture correlation due to movement between nearby sites or between sites and a common hub ( ) . models can also reflect relevant population-level features associated with expected incidence, such as density, race/ethnicity, age distribution, and educational status. we recommend the use of ensemble modeling, whereby multiple modeling groups prepare independent projections and these are combined to guide decision-making. individual models can be agent-based, compartmental, or statistical, can use different assumptions and data sources, but are all tasked with the same question of which sites are likely to have the highest disease incidence over a moderate time horizon. ensemble modeling has been shown to be more robust for complex systems than specialist models and better able to capture the complete range of possible outcomes ( ) . the strength of ensemble modeling has been shown for diseases like influenza ( ), dengue ( ) and ebola ( ) . ensemble modeling for covid- , like the covid- forecast hub, is similarly more robust ( ) . in addition to using forecast modeling for initial site selection, we propose that modeling be repeated at regular intervals throughout the trial. in the context of outbreaks, trials should be flexible to allow new sites to be added in response to evolving epidemiology ( ) . some sites will have lower than projected incidence during the trial period. for example, local policies or voluntary changes in behavior could effectively reduce transmission, meaning that the site is no longer a "hotspot." the modeling results can guide investigators deciding whether to continue to enroll new participants from existing sites, or to enroll new sites in emerging hotspots. in this paper, we describe a simplified framework for the use of ensemble modeling to guide the selection and continued evaluation of sites for a vaccine efficacy trial, with a focus on the covid- pandemic. individual modeling teams are welcome to contribute to the consensus model. we assume that models would already be built for general public health planning, so they would not be constructed only for this effort, though they may need to be modified. investigators can leverage existing groups or form new groups of modelers. participating modeling teams would be provided with a list of all candidate sites being explored. this list of sites may be based on previous engagement between the trial investigators and potential research partners. for a multi-country trial, this may include several sites per country from multiple countries. participating models must meet a minimum set of requirements. suggested guidelines are the ability to: (i) capture all geographic areas in the candidate list of sites, (ii) disaggregate to at least the first administrative level (e.g. state, province), though finer levels may be preferred for certain planning activities, (iii) project the covid- symptomatic cumulative incidence, i.e. the number of new symptomatic infections of any severity divided by the total population size during a pre-specified period (three months suggested), and (iv) produce a minimum of simulated epidemics. models must also be screened for internal consistency and basic plausibility when compared to historical trends. for each site, each model must generate a probabilistic predictive distribution for quantities of interest, such as the symptomatic cumulative incidence. these are bins of % width centered around whole numbers [ . , . %), [ . , . %), [ . , . %) and so on. the bin that includes % is narrower [ , . %). for each site, probabilistic predictive distributions are aggregated across models using stacking. figure describes a hypothetical model stacking procedure for a target site, per ray and reich ( ) . for simplicity and transparency, each model is assigned an equal weight, which is one over the number of models as done by the covid- forecast hub ( ) . if a participating team has developed more than one model, they must specify which model is primary and will contribute to the aggregate. more complex weighting schemes exist that preferentially weight models that performed best in previous rounds after an appropriate burn-in period ( ) . for each site, we can use the combined predictive distribution to produce summary statistics. suggested summary statistics are: (i) median incidence value, (ii) th percentile incidence value, (iii) th percentile incidence value, and (iv) probability incidence value is [ , . %) (probability of a very small or no outbreak). to present this information in a way that is easy for trial investigators to explore, we recommend reporting stacked projections, summary statistics, and basic information about the sites in an interactive tool, like the r shiny platform ( ) . this allows the end user to sort the table or select rows for closer examination. in this way, they could select a subset of "best rows" and view these together to approximate the formation of a trial. figure is a sample screenshot from such a program (code provided in supplemental materials). by generating a range of possible outcomes, models can capture the stochasticity of future transmission, including scenarios where incidence is much lower or much higher than the median projection. where incidence is highly variable with the potential to be very low, it may be preferable to include a larger number of sites to guard against the chance of accruing no efficacy data. the goal of ensemble modeling is to provide a simple and informative resource rather than a definitive recommendation. investigators will simultaneously consider many operational, political, and scientific factors. to provide context, we describe several other key considerations. to ensure a high-quality trial, sites should have adequate capacity for testing, safety monitoring, active surveillance, and high participant retention. nonetheless, sites with projected high incidence but poorer capacity should not be excluded if there is a potential role for mobile trial teams, as was used in the ebola ring vaccination trial in guinea ( ) . approval for the trial may be at the national or sub-national level, with flexibility to identify the particular target population when investigators are ready to start enrollment. for multi-country trials, investigators must weigh including multiple sites per country against including more countries. on one hand, given the complexity of country-specific procedures for approving clinical research, it may be easier to include multiple sites per country from fewer countries overall. on the other hand, the global community must ensure equitable access to potentially effective vaccines. broad representation also increases generalizability of the trial results, as it can best capture the effectiveness of vaccine candidates in diverse settings. these include variations in population age profile, race/ethnicities, climate, background presence of non-pharmaceutical interventions, and co-circulation of other coronaviruses. including many different geographic locations makes trials more robust to changes in the epidemic. while china was once the center of the covid- epidemic, several treatment trials initiated there were underpowered due to waning transmission ( ) . as other countries adopt more effective control strategies, incidence would likely decline, but it is less likely to wane in all areas, and new sites can also be added. experience with zika in the americas provides a useful counter-example, though, where trials were not possible because incidence dramatically declined everywhere ( ) . if that were to occur, the ensemble modeling process would be useful for assessing trial feasibility. finally, the ensemble modeling process should be evaluated by comparing model projections to subsequently observed data. an evaluation procedure could be conducted prior to each new round of modeling, before investigators want to make decisions about adding new trial sites. this process could assess how well model-projected rankings corresponded to observed rankings of hardest hit sites. where there is a lot of uncertainty in which sites will have highest incidence, as reflected in low correlation, investigators may feel more comfortable making future decisions based on logistical or political considerations rather than purely on model rankings. this evaluation procedure could also be conducted formally after the trial ends to compare model-projected and observed cumulative incidence and observed incidence during the target time periods. these types of reports are very useful for understanding the role of modeling as a tool for real-time decision-making in outbreaks ( , ) . we describe an ensemble modeling procedure to inform site selection for a vaccine efficacy trial planned during an ongoing epidemic. by prioritizing sites with highest projected disease incidence, investigators can accelerate the pace of endpoint accrual. mathematical and statistical models synthesize the best available evidence to guide this planning. we focus on covid- as a motivating example, but the general principles apply to other emerging infectious diseases. we present a highly simplified procedure to reduce the burden on modeling groups to prepare results and potentially enable more groups to participate. for example, models could, but not be required to, explicitly account for the impact of vaccination on transmission dynamics. the assumption is that population vaccine coverage will be relatively low even in large trials, and that the rank ordering of sites is similar in less complex models. this is a recommended minimum structure, but other relevant practical questions will likely emerge that can be explored as add-ons. for example, the modeling results can be used to answer questions about expected duration of the trial as a function of enrollment rates and expected incidence. nonetheless, it is important to remember that projections can be very uncertain, particularly as they depend upon rapidly changing policies and human behavior. thus, we focus on simple output for the purposes of prioritization, acknowledging that other important questions may be difficult to answer precisely. in addition to identifying geographic locations, models could also be used to explore targeted enrollment in sub-populations defined by age, occupation, or other covariates. models could also guide the design of post-licensure observational studies for continued evaluation of vaccine effectiveness. it is a top priority to rapidly evaluate the safety and efficacy of candidate covid- vaccines. data-driven models can help to optimize site selection and contribute to accelerating trials in a setting where every day counts. developing covid- vaccines at pandemic speed design of vaccine efficacy trials during public health emergencies world health organization. an international randomised trial of candidate vaccines against covid- antibody testing will enhance the power and accuracy of covid- -prevention trials simulations for designing and interpreting intervention trials in infectious diseases statistical power and validity of ebola vaccine trials in sierra leone: a simulation study of trial design and analysis preliminary results of models to predict areas in the americas with increased likelihood of zika virus transmission in spread of zika virus in the americas prediction of infectious disease epidemics via weighted density ensembles accuracy of real-time multi-model ensemble forecasts for seasonal influenza in the u.s an open challenge to advance probabilistic forecasting for dengue epidemics the rapidd ebola forecasting challenge: synthesis and lessons learnt ensemble forecasts of coronavirus disease (covid- ) in the u creating a framework for conducting randomized clinical trials during disease outbreaks ring vaccination with rvsv-zebov under expanded access in response to an outbreak of ebola virus disease in guinea, : an operational and vaccine safety report a trial of lopinavir-ritonavir in adults hospitalized with severe covid- demonstrating vaccine effectiveness during a waning epidemic: a who / nih meeting report on approaches to development and licensure of zika vaccine candidates mathematical modeling of the west africa ebola epidemic funding: this work was supported by the national institutes of health r -ai (ned, meh, iml, conceptualization, writing -review and editing. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -hboc xcz authors: roncati, luca; vadalà, maria; corazzari, veronica; palmieri, beniamino title: covid- vaccine and boosted immunity: nothing ad interim to do? date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hboc xcz summary today, coronavirus disease (covid- ) is a global public health emergency and vaccination measures to counter its diffusion are deemed necessary. severe acute respiratory syndrome coronavirus (sars-cov- ), the etiological agent of the disease, unleashes a t-helper immune response in those patients requiring intensive care. here, we illustrate the immunological mechanism to train the immune system towards a more effective and less symptomatic t-helper (th ) immune response, thanks to a subcutaneous vaccine containing lysates of corynebacterium parvum (synonym for propionibacterium acnes), a gram-positive bacterium able to evoke a strong th response. in the - years of last century, an experimental wave of immunological studies attempted to fight cancer by exploiting live or killed bacteria, among which the most investigated were the bacillus calmette-guérin (bcg), an attenuated strain of mycobacterium bovis, and corynebacterium parvum (c. parvum), later renamed propionibacterium acnes and then cutibacterium acnes [ , ] . administered percutaneously or into the neoplastic mass, they proved able to induce the tumor lysis at some extent, and to delay or arrest the cancer growth through the innate immunity potentiation [ , ] . bcg gained approval since and is currently the standard of care for patients with nonmuscle-invasive bladder cancer by means of mucosal instillation, besides to be registered as an antituberculosis intradermal vaccination [ , ] . as of march , bcg vaccine is furthermore in phase iii or iv trials to prevent coronavirus disease (covid- ) among healthcare workers in the netherlands, australia, usa, germany, france, denmark, colombia, mexico, egypt, and south africa [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additional trials in the netherlands, germany, greece, and india are evaluating whether bcg vaccine provides protection against covid- in the elderly and in middle age [ ] [ ] [ ] [ ] ; besides, randomized trials on volunteers over years to test bcg vaccine in this context has been launched in brazil and canada [ , ] . c. parvum is an aerotolerant anaerobic rod-shaped gram-positive bacterium largely commensal and part of the skin flora present on most healthy adults, but also associated to sarcoidosis and juvenile acne, hence its taxonomic renaming [ ] . after an initial registration like immunoadjuvant and immunomodulator, c. parvum was added to the chemotherapy protocol for colon cancer by repeated injections in the form of formalin-killed freezedried vaccine preparation (coparvax ® , wellcome research laboratories, beckenham, uk); however, it was discarded because no partial remission, overall survival or significant benefits were achieved in the treated cohorts of patients [ ] . at that time, one of us (prof. palmieri) had the chance to perform a clinical pilot trial with c. parvum administration into subcutaneous and lymph nodal metastases from lungs, thyroid and breast malignancies, noting local shrinking and colliquative effect in - hours, accompanied by mild symptoms and occasional febrile peaks. in a few cases, very rapid regressions of concomitant herpes infections involving the head, the thorax and the genitals were incidentally observed. by searching on the english biomedical literature, several studies from the past fully support the c. parvum antiviral power on man and animal against, for example, influenza virus, hepatitis b, rabies, encephalomyocarditis virus, herpes zoster and human papilloma virus [ ] [ ] [ ] [ ] [ ] [ ] . in addition, a scientific evidence of c. parvum vaccine protection against a coronavirus (mouse hepatitis virus type ), dating back to murine model by schindler and colleagues, is also reported [ ] . always working on a murine model, teixeira and collaborators proved in that c. parvum enhances the immunogenicity of the hivbr vaccine, a vaccine against epitopes of the human immunodeficiency virus, subtype b [ ] . in the same year, hsu and coworkers discovered short rna sequences from c. parvum similar to ebola virus micrornas, capable to protect the human host by influencing the thrombospondin expression, a multifunctional protein which plays also an initiating role towards cell-mediated immunity in the skin [ ] . palmieri dropped out his clinical trial on bacterial immune stimulation against cancer in , but he followed up with anecdotical compassionate treatments of severe herpes, and others heavy viral infections, such as influenza, mumps, varicella and measles, by subcutaneous injections of c. parvum cultured and killed in our microbiological lab, after signing an informed consent out of a total of patients, males and females, aged between and years (mean age years). during our evolving experience, we modified the subdermal injection formula ( x phenol-killed bacteria in ml saline), adding high molecular weight hyaluronic acid as slow delivery system, having found aspecific virucide properties of this non-sulphated glycosaminoglycan, notoriously able to bind the cluster of differentiation (cd ) receptors present on the surface of activated leukocytes [ , ] . the (t h ), classically directed against extracellular non-phagocytosable pathogens (e.g. helminths), whose main effectors are eosinophils, basophils, mastocytes and b cells (humoral immunity) [ ] . in spite of this, severe sars-cov- infections are associated with marked t c lymphopenia [ ] . during our researches on covid- , we have disclosed that the immune system is forced to mount in critically ill patients a t h response, the only one still mountable in the attempt to counteract the viral load, rather than a t h response, which would keep the infection under control by means of macrophages and t c cells [ , ] . moreover, for the first time in worldwide literature, we have provide evidence that a life-threatening escalation from t h immune response to type hypersensitivity (immune complex disease) in covid- vasculitis takes place, and that the inflamed smooth muscle cells of blood vessels concur to the «cytokine storm» via interleukin (il)- [ ] . therefore, we have proposed that an effective vaccination strategy should be able to prevent or limit the systemic imbalance of t h cytokines [ ] , inducing a protective t h response to be exploited against sars-cov- (fig. ) . among the t h cytokines, there are il- , il- , il- , il- , il- , il- and il- , while il- , il- , interferon-γ (ifn-γ) and tumor necrosis factor-α (tnf-α) are the master t h cytokines [ ] . many researches have ascertained that c. parvum subcutaneous injection is able to induce a strong t h response favoring the production of il- , il- , ifn-γ and tnf-α, in practice as bcg works, and that the characteristic allergic t h response can be counterbalanced by c. parvum vaccination [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; besides, it has been found a natural killers (nk) and dendritic cells activator [ ] [ ] [ ] . in cioffi and colleagues reported that c. parvum protects splenectomized sprague dawley rats from respiratory challenge with streptococcus pneumoniae, without alter the number or activity of lavageable alveolar macrophages, and they hypothesized that c. parvum protection is more likely due to an increased clearance of blood-borne bacteria by the expanded and enhanced reticuloendothelial system [ ] . if we transfer this murine model to man, a t h cytokines release syndrome from activated pulmonary macrophages after c. parvum lysate subcutaneous injection, such as to aggravate a possible superimposed covid- , appears somewhat unlikely. therefore, our long-standing experience lays the foundation to revalue c. parvum lysate as a further surrogate vaccine against covid- , safer and more manageable than an attenuated living bacillus like bcg, in the attempt to prevent or mitigate the cumbersome pandemic morbidity and mortality. the accurate preparation of the lysate is a crucial time to avoid opportunistic bone implant-associated infections or acute septic polyarthritis, whose a single case has been described in after c. parvum instillation for malignant pleural effusion; an episode of prolonged fever from immune system hyperactivation has also been reported [ ] [ ] [ ] . previous efforts to develop subunit vaccines against the most lethal human coronaviruses, for instance the severe acute respiratory syndrome none. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. fig. . illustrative scheme of our vaccination rationale: following sars-cov- entry (point ), the immune system is forced to mount a t h response in those patients requiring intensive care, at the expense of a more effective and less symptomatic t h response, compromised by the viral load (point ). through c. parvum vaccine (point ), it is 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intradermal administration of corynebacterium parvum on the immune response to hepatitis bs antigen increased interleukin- associated with low il- concentration correlated with greater survival rates in mice infected by rabies virus vaccinated against it and immunomodulated with p. acnes role of interferon, antibodies and macrophages in the protective effect of corynebacterium parvum on encephalomyocarditis virusinduced disease in mice immunomodulating and antiviral therapy in herpes zoster treatment of common warts with the immune stimulant propionium bacterium parvum protection of mice against mouse hepatitis virus by corynebacterium parvum propionibacterium acnes enhances the immunogenicity of hivbr human immunodeficiency virus- vaccine on revealing the gene targets of ebola virus micrornas involved in the human skin microbiome in vitro evaluation of antiviral and virucidal activity of a high molecular weight hyaluronic acid cd is the principal cell surface receptor for hyaluronate renin-angiotensin system: the unexpected flaw inside the human immune system revealed by sars-cov- type /type immunity in infectious diseases lymphopenia is associated with severe coronavirus disease (covid- ) infections: a systemic review and meta-analysis signals of th immune response from covid- patients requiring intensive care the «moonlighting protein» able to explain the th immune lockdown in severe covid- type hypersensitivity in covid- vasculitis what about the original antigenic sin of the humans versus sars-cov- ? propionibacterium acnes promotes th and th /th responses in acne patients th and th immune responses to viable propionibacterium acnes in patients with sarcoidosis intratumoral injection of propionibacterium acnes suppresses malignant melanoma by enhancing th immune responses mis , a nontoxic microparticle adjuvant derived from propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial dna promotes cross-priming and th immunity propionibacterium acnes vaccination induces regulatory t cells and th immune responses and improves mouse atopic dermatitis innate, antigen-independent role for t cells in the activation of the immune system by propionibacterium acnes bcg-induced trained immunity: can it offer protection against covid- ? modulatory effect of killed propionibacterium acnes and its purified soluble polysaccharide on peritoneal exudate cells from c bl/ mice: major nkt cell recruitment and increased cytotoxicity a randomized prospective clinical trial of adjuvant c. parvum immunotherapy in patients with clinically localized melanoma (stage i): prognostic factors analysis and preliminary results of immunotherapy interferon production and lymphocyte stimulation in human leucocyte cultures stimulated by corynebacterium parvum the quantity and function of pulmonary alveolar macrophages after splenectomy and corynebacterium parvum interaction of cutibacterium acnes with human bone marrow derived mesenchymal stem cells: a step toward understanding bone implant-associated infection development acute polyarthritis following the use of corynebacterium parvum vaccine (coparvax) for malignant pleural effusion prolonged fever after pleural instillation of corynebacterium parvum (coparvax) key: cord- - ces rn authors: tondella, m. l.; carlone, g. m.; messonnier, n.; quinn, c. p.; meade, b. d.; burns, d. l.; cherry, j. d.; guiso, n.; hewlett, e. l.; edwards, k. m.; xing, d.; giammanco, a.; wirsing von könig, c. h.; han, l.; hueston, l.; robbins, j. b.; powell, m.; mink, c. m.; poolman, j. t.; hildreth, s. w.; lynn, f.; morris, a. title: international bordetella pertussis assay standardization and harmonization meeting report. centers for disease control and prevention, atlanta, georgia, united states, – july date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ces rn abstract an international meeting on bordetella pertussis assay standardization and harmonization was held at the centers for disease control and prevention (cdc), atlanta, ga, – july . the goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the world health organization (who). the primary objectives were ( ) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; ( ) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; ( ) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and ( ) to search for a multicenter process for addressing these priority gaps. presentations and discussions by content experts addressed these objectives. a prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. the major items included: ( ) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; ( ) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; ( ) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; ( ) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; ( ) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; ( ) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and ( ) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines. pertussis (whooping cough) is a highly contagious, acute respiratory illness caused by the bacterial pathogen bordetella pertussis. despite high vaccination coverage, pertussis continues to be a major cause of morbidity and mortality in the united states, with , and , probable or confirmed cases reported in and , respectively [ ] [ ] [ ] [ ] [ ] . among the reportable bacterial vaccine-preventable diseases in the united states with universal childhood vaccination, pertussis is the least well controlled [ ] . pertussis also remains a substantial public health problem in other developed regions, such as the european union and canada [ ] [ ] [ ] [ ] [ ] . studies suggest that there are approximately . mil-ଝ the findings and conclusions in this report are those of the author(s) of each section and do not necessarily represent the entire group of participants or the official position of the centers for disease control and prevention/agency for toxic substances and disease registry. the findings and conclusions in this report have not been formally disseminated by the u.s. food and drug administration and should not be construed to represent any agency determination or policy. lion annual cases of pertussis worldwide, with , deaths [ ] [ ] [ ] . however, the total burden of pertussis is likely underestimated, especially among adolescents and adults, in whom typical pertussis symptoms are often absent making diagnosis difficult [ , , , [ ] [ ] [ ] [ ] . the laboratory diagnosis of pertussis is challenging [ , ] . culture is highly specific but requires a long incubation and sensitivity can be low [ , ] . rapid, sensitive, and specific polymerase chain reaction (pcr) assays have been designed to detect b. pertussis [ , [ ] [ ] [ ] ; however, these assays are not well standardized and problems have occurred with specificity, mainly during outbreaks [ , [ ] [ ] [ ] . serologic testing with b. pertussis antigens, pertussis toxin (pt) in particular, can be sensitive and specific, but the tests are not widely available, have not been fully standardized and no universally accepted serologic correlates for protection are available [ ] [ ] [ ] [ ] . the meeting sessions covered the following topics: global epidemiology of b. pertussis disease; pathogenesis and immunology of b. pertussis; assay standardization and harmonization; serodiagnosis of b. pertussis; current and future b. pertussis vaccine composition; and issues in vaccine evaluation with perspectives from regulatory authorities and vaccine manufacturers. group discussions included serodiagnostics, vaccine evaluation, correlates of protection, and harmonization of assays. a list of action items was developed to identify existing knowledge gaps and to coordinate international efforts to standardize pertussis assays for immunologic and diagnostic use. the meeting was opened by m. lucia tondella, phd (cdc), followed by nancy messonnier, md (cdc), who stressed the importance of having laboratory, epidemiologic and statistical groups working together to facilitate and support global harmonization. dr. messonnier also highlighted the necessity of improving pertussis diagnostics, pointing out that in alone, cdc experienced numerous occasions when problems with diagnostics impeded epidemiologic investigations, resulting in a substantial public health outcry [ ] . cdc's meningitis and vaccine preventable diseases branch has an extensive experience working on standardization and harmonization of assays on a variety of organisms, and this experience can be brought to bear at this meeting to determine the current status of pertussis serologic technologies in key laboratories, to facilitate discussion of the key issues in pertussis serology and to establish consensus on the action plan needed to address these issues. presentations and discussions were summarized below. presenters: james d. cherry, md (david geffen school of medicine, university of california, los angeles) and nicole guiso, phd (institute pasteur, paris, france) in the prevaccine era in the united states, pertussis was a disease with cyclic peaks averaging every . years. the average rate of reported cases was per , population. more than % of reported cases occurred in children younger than years of age. by the s, pertussis was well controlled by immunization, and only - cases were reported per year; % of those cases occurred in infants. at present, more than % of pertussis reported cases are in persons older than years of age. since there has been a modest increase in reported pertussis from < to cases per , population. in recent years ( ) ( ) the reports have increased considerably; however, the number of cases is still approximately -fold less than in the prevaccine era. the cycles of reported pertussis today are essentially the same as they were in the prevaccine era [ , ] . examination of reported pertussis in several countries between and the present found two different patterns. in some countries (argentina, bangladesh, bolivia, brazil, cambodia, ghana, pakistan, peru, sudan, vietnam), there was a reduction in total cases, similar to that seen in the united states between and . a similar pattern was also noted in the united kingdom, where pertussis was reasonably well controlled by vaccination in the s and early s, but then concerns for vaccine safety resulted in a marked decline in immunization and pertussis became epidemic. it was then brought under control in the early s by increased vaccine usage. in italy, where vaccination was not being consistently offered until the efficacy trials of , a marked reduction in pertussis was related to the use of diphtheria and tetanus toxoids and acellular pertussis component (dtap) vaccines. success in the control of reported pertussis in all of the above countries with the exception of italy was secondary to use of doses of dtwp (whole-cell vaccines) in the first year of life. in france, because of high vaccination coverage among children with dtwp vaccine during the last years, pertussis predominantly affects children who are too young to be vaccinated and adults who are no longer protected by vaccine or disease-induced immunity [ ] [ ] [ ] [ ] [ ] [ ] . a survey undertaken in and in a pediatric hospital network demonstrated an increase in the number of children hospitalized with pertussis suggesting the importance of introducing booster dose(s) to prolong vaccine immunity and reduce the exposure to b. pertussis of infants too young to be immunized [ ] . therefore, a fifth dose of acellular pertussis (ap) vaccine was introduced for - -year olds in , followed by the introduction of the so-called "cocoon strategy" in . the current vaccine schedule consists of primary immunization for children aged , and months and boosters at - months, at - years old, and for young adults and healthcare workers in contact with infants [ ] . no dtwp vaccine has been commercially available since . the national hospital-based surveillance (renacoq network) has not shown any resurgence of pertussis. as observed in other countries, cycles of the disease have occurred every - years; however, the number of pertussis cases in did not reach the values observed in the earlier peak years which could be linked to the impact of the - -year booster introduced years previously [ , ] . reported pertussis cases in australia, chile, israel, japan, norway, and poland have increased in recent years. in australia, israel, and norway, the increase followed the introduction of dtap vaccines. possible reasons for the resurgence of reported pertussis are ( ) greater awareness of pertussis; ( ) waning of vaccine-induced immunity; ( ) lessened potency of pertussis vaccines; ( ) genetic changes in b. pertussis; and ( ) the general availability of better laboratory tests for the diagnosis of pertussis. of most importance is a general greater awareness of pertussis. the availability of better laboratory tests (pcr) and single-serum serology for diagnosing adolescent and adult pertussis has also contributed to the increase in reported cases. as evidence also suggests that in general dtap vaccines may have less efficacy in children than the more potent dtwp vaccines [ , , ] , it is plausible that decreased efficacy or duration of protection could possibly contribute to the outbreaks that have been seen in pre-adolescence and adolescence. investigators in the united states and other areas of the world have used various laboratory methods to diagnose pertussis in subjects with prolonged cough illnesses. one of the most commonly used diagnostic markers is high single-serum titers to pt. results of these studies suggest that about % of prolonged cough illnesses are due to b. pertussis infection. however, because not all adults have a pt antibody response following infection, the percentage is likely to be higher [ , ] . in summary, b. pertussis infections in adolescents and adults are very common. rates of reported pertussis may be as much as to -fold lower than actual illness rates, and unrecognized infections (e.g., asymptomatic or with nonspecific symptoms) are likely to be more common than symptomatic infections. today, symptomatic adolescents and adults appear to be the major sources of infection for nonvaccinated children. much of the discussion relating to epidemiology focused on the increase of pertussis in recent years despite adequate or increasing vaccine use in young children. in general, the most likely cause was thought to be greater awareness of pertussis. however, data from australia (presented later in the meeting) suggested that falsepositive laboratory tests contributed to the increase in reported pertussis in at least one setting. discussion included the possibility that in some instances genetic change in the pertussis organism may have contributed to increases. in countries using dtwp, changes in vaccine manufacturers and even lots may have a significant relationship to efficacy. the increase in observed pertussis associated with the introduction of acellular vaccines should be studied more carefully. presenter: erik l. hewlett, md (university of virginia school of medicine, charlottesville, virginia) multiple virulence factors produced by b. pertussis are able to disrupt the normal physiology of the host, causing the disease of pertussis [ ] . this disease consists of a localized infection of the respiratory tract, with secondary systemic effects resulting from the actions of several bacterial products and from the host response to those factors. the molecular basis for the characteristic paroxysmal cough is not known, but is not mimicked by intravenous pt [ ] . one likely role of pt is to impair the innate immune response of the host, causing leukolymphocytosis and blocking the migration of murine macrophages [ ] . reduced expression of l-selectin on the surface of leukocytes from infants infected with b. pertussis has also been demonstrated [ , ] . the leukolymphocytosis and several other effects of pt are mediated by the adp-ribosyl transferase activity of the catalytic domain. the pt binding to its receptor elicits a separate set of responses, known as b-subunit effects. recently it has been shown that pt b-subunit activates signaling through the t-cell receptor, without any contribution from adp-ribosylation [ ] . similarly, pt and an enzymatically inactive (non-adp-ribosylating) mutant stimulate cytokine production and dendritic cell maturation [ ] . evidence indicates that multiple virulence factors from b. pertussis have immunomodulatory effects. in several instances the effects appear to be antagonistic to one another. for example, it has been shown that filamentous hemagglutinin (fha) is both pro-inflammatory and able to induce apoptosis in macrophages [ ] . similarly, adenylate cyclase toxin (act), which is known for its inhibitory effects and cytotoxicity on neutrophils and other phagocytic cells [ ] [ ] [ ] also has pro-inflammatory effects, inducing il- from respiratory epithelial cells [ ] and cyclooxygenase- (cox- ) in cells expressing the integrin, cd b/cd [ ] . the disease process of pertussis appears to be multifactorial, rather than the function of a single toxin/virulence factor. therefore, the ideal immunologic response from the host consists of antibodies directed against multiple virulence factors, resulting in inhibition of bacterial adherence, neutralization of the actions of the toxins and clearance of the infecting organisms. in addition, a cell-mediated component in the host response to immunization and infection has been studied, but the relevant target antigen(s) to which this cellular response is directed and the contribution that it makes to bacterial clearance are unknown [ ] [ ] [ ] . several major issues in pertussis pathogenesis remain unresolved. despite the numerous known biological effects of individual virulence factors, especially in vitro, the in vivo target tissue(s) of these molecules and the mechanism by which the characteristic cough is generated are mysteries. the molecular details of how bvg, the two-component regulatory system, controls production of the virulence factors are not known. in summary, pertussis is a multicomponent disease, which requires more than a simple approach to its recognition, treatment and control. recent analyses of the effects of known toxins and other virulence factors on the immune system have revealed that they are often multifunctional: cytotoxic, stimulatory, inhibitory, pro-and anti-inflammatory. much of the discussion focused on the potential neurophysiologic effects of the multiple virulence factors; animal modeling, including new methods of infection through inhalation techniques; and novel imaging analysis that could help in understanding the pathogenesis of pertussis in humans. recent research studies have shown presence of b. pertussis dna from clinical specimens of children several months after infection. the possibility that the remaining dna could be triggering the cough by an unrecognized mechanism was speculated. presenter: kathryn m. edwards, md (vanderbilt medical center, nashville, tennessee) standardization and harmonization are necessary for several reasons: to assess the immunogenicity of new pertussis vaccines or new combination vaccines that include pertussis antigens, to compare the serologic responses to these new vaccines with those evaluated in earlier vaccine efficacy studies for the purposes of vaccine licensure, to establish serologic criteria for the clinical diagnosis of pertussis disease, and to conduct seroepidemiologic studies to assess the circulation patterns of pertussis in the community. multiple ap vaccines were licensed for use in infants, adolescents and adults [ , , ] . combination vaccines incorporating various antigens into a single injection have been designed. these combination vaccines may be associated with reduced serologic responses to one or more vaccine antigens. therefore, antibody titers induced by these products must be compared with those seen after the separate administration of the individual vaccines before combination vaccines can be considered for licensure. safety and immunogenicity of ap and two dtwp vaccines have been assessed in a single large clinical trial [ ] . although the trial evaluated different pertussis antigens administered in various concentrations, all the vaccines were found to be safe and immunogenic. several of these vaccines were then evaluated in efficacy trials in the european union and africa and were found to be efficacious in preventing culture-confirmed pertussis. since placebo-controlled efficacy trials would now be considered unethical, new vaccines must be evaluated by comparisons with the immunogenicity data of the vaccines evaluated in earlier efficacy trials. thus, the need for standardized assays is critical. standardized serologic assays are also needed to diagnose pertussis disease, particularly in adolescents and adults. a study was conducted to determine population-based antibody levels to three pertussis antigens, pt, fha, and fimbrial proteins (fim), for the purpose of establishing diagnostic cutoff points in adolescents and adults in the united states [ ] . enzyme-linked immunosorbent assays (elisas) were performed on sera from more than us residents aged - years. quantifiable (> elisa units [eu]/ml) anti-fha and anti-fim igg antibodies were common but quantifiable anti-pt igg antibodies were less frequent. an anti-pt igg level of ≥ eu was proposed as the diagnostic cutoff point in subjects years of age and older. application of this cutoff point to culture-confirmed illness yielded a high diagnostic sensitivity ( %) and specificity ( %). anti-pt igg assays with a single serum sample appeared to be useful for identification of recent b. pertussis infection in adolescents and adults. however, these assays must be standardized to remain useful in the diagnosis of pertussis. finally, standardized serologic methods for b. pertussis are needed to assess epidemiologic trends in pertussis disease in the population. this is particularly relevant with the universal recommendations for adolescent and adult pertussis vaccine in many developed countries. how universal vaccination will affect disease burden in the various age groups remains to be determined. in addition, comparisons of seroepidemiologic results from one country to another are dependent on standardized assays. presenters: bruce d. meade, phd (meade biologics llc, hillsborough, nc), dorothy xing, phd (national institute for biological standards and control (nibsc), potters bar, uk) and anna giammanco, phd (department of hygiene and microbiology, university of palermo, italy) important insight on strategies for international standardization of pertussis immunoassays is provided by the results of an international collaborative study performed in [ ] . in that study, participating laboratories were asked to quantify specific antibody to pt, fha, pertactin (prn), and fim in a panel of blinded samples by using those elisas routinely performed in that laboratory. the laboratories employed a variety of procedures, antigens, conjugates and other reagents, and calculation methods. results indicated an overall consistency among the laboratories. a reasonable quantitative agreement was achieved among the laboratories with assays calibrated using the us reference pertussis antisera. this agreement demonstrated that use of a common reference serum had been successful at harmonizing results. to promote a higher level of international standardization of pertussis immunoassays, the highest priority recommendations would be to improve the availability of reference reagents and methods, including international reference sera, a proficiency panel of well-characterized sera, and well-characterized antigens. in recent years, significant progress in the preparation and standardization of international reference sera has been made. the who collaborative study, "evaluation of proposed international reference preparations for pertussis antiserum (human),"was a multicenter collaboration between nibsc, uk; the center for biologics evaluation and research (cber) at the food and drug administration (fda), us; the institut für infektiologie (ifi), germany; and a number of international participants. reference sera from the united states (us reference pertussis antiserum [human] lot and lot for igg-antibodies and lot for iga-antibodies) were prepared by fda, cber more than years ago, and since then have been commonly used as reference standards for pertussis serologic assays [ ] [ ] [ ] [ ] [ ] [ ] . currently, only limited quantities of these sera remain. with an increasing number of vaccine and diagnostic studies, more widely available international standard preparation(s) are needed. the who ad hoc pertussis working group recommended the preparation and standardization of an international human reference antiserum to pertussis antigens to replace the current widely used cber preparations before the supply is exhausted [ ] . it was agreed that as far as possible, continuity with the existing cber reference preparations should be maintained. these new international reference materials were intended for the following uses: ( ) vaccine studies of products in current distribution, as well as those under development; ( ) studies of serologic responses to infection; ( ) epidemiologic surveillance; ( ) future assessment of antibodies to other antigens apart from pt, fha and prn; and ( ) evaluation of new generation vaccines. serum pools were kindly donated by dr. wirsing von könig, ifi, krefeld, germany. they were prepared after recalcification of plasma samples selected from a blood bank during a pertussis outbreak in germany. these sera were then lyophilized at nibsc, resulting in four batches of freeze-dried preparations of ampoules of higher titer and , ampoules of lower titer igganti-pt. preliminary assessment of the freeze-dried materials in nibsc showed that in comparison with the materials before freezedrying, all freeze-dried material maintained their original elisa binding activities. furthermore, comparison of the candidate materials with the us reference sera showed that the dose-response lines did not deviate significantly from parallelism. an international collaborative study to evaluate the candidate preparations was initiated in march . the aims of the study were to ( ) characterize candidate international reference preparations for pertussis antiserum (human); ( ) compare candidate references with the us reference preparations, lots , , and ; ( ) compare candidate reference preparations with other available reference preparations, e.g., in-house preparations; and ( ) define unitage for the candidate preparations for anti-pt, anti-fha and anti-prn, maintaining continuity with widely used reference preparations. samples provided to the participants were the four candidate reference preparations: the three us reference sera plus a negative control serum. all participants were quested to ( ) carry out three independent assays for igg anti-pt, fha and prn by elisas; ( ) use their own methodology, reagents and calculation methods; and ( ) include their in-house references and controls. participants were also encouraged to perform other assays that could be of interest, e.g., igg anti-fim / , iga antibodies to the antigens, ptneutralizing antibodies (cho-cell assay). a total of laboratories worldwide participated in this study, including from europe, from north america, from south america and laboratory from each of asia and australia. all participants were requested to submit their raw results to nibsc, where the statistical analysis will be performed. a study report will be submitted to the expert committee on biological standardization (ecbs) of who. another multilaboratory effort to standardize pertussis serologic assays was the european sero-epidemiology network (esen) project, coordinated by the health protection agency (hpa), communicable disease surveillance center (cdsc), london [ , ] . the aim of the esen project was to coordinate and harmonize serologic surveillance for a variety of infections in europe. age-stratified serum banks of a recommended sample size of individuals were collected by each participant country and one laboratory was selected to serve as a reference laboratory for each disease. all laboratories tested sera from their own country plus a shared panel of approximately serum samples [ ] . the standardization process allowed a comparison of national serosurveys for pertussis [ ] . because of the difficulty with standardization of cell extracts, the esen project emphasized purified antigens, focusing on igg anti-pt assays because of the higher sensitivity and specificity for serodiagnosis [ ] . since most individuals had been exposed to pertussis vaccine or infection, the study focused on an estimation of the incidence of recent infections, as defined by the presence of igg anti-pt antibodies above defined thresholds [ ] . to accomplish this, quantitative assays were required and standardization efforts were based on analytical performance in the assay range near the diagnostic thresholds. in conclusion, the esen project demonstrated that standardization of pertussis igg anti-pt assay was possible [ , ] . appropriate application of a diagnostic laboratory test requires an understanding of the test as well as knowledge of the patient. relevant criteria for the test include test format; specificity and sensitivity; and reference systems. clinical interpretation of a test result concerning b. pertussis infection also requires an understanding of ( ) the distinction between primary and secondary infection; ( ) the differential diagnoses, including other agents; ( ) the kinetics of immune responses to infection; and ( ) an understanding of the information provided by the test beyond that provided by the clinical history alone. the performance of laboratory tests can be displayed by cutoff values as shown in a reporter-operator characteristics (rocs) curve, as well as by positive and negative predictive values. the differential diagnosis for b. pertussis as an agent of prolonged cough includes infectious agents such as adenovirus, respiratory syncytial virus, rhinovirus, human parainfluenzavirus, influenzavirus a and b, mycoplasma pneumoniae, human metapneumovirus, and human coronavirus. several alternate approaches have been used for serodiagnosis. when paired sera were used, one study defined a case if there was % or more increase in antibody concentration or % or more decrease in antibody concentration. for single-sample serology, various cutoff-values have been proposed, such as >∼ eu/ml igg-anti-pt in the netherlands; and > eu/ml igg-anti-pt or igg-anti-pt > eu/ml and iga-anti-fha > in germany. in most diagnostic laboratories, a so-called "grey-zone" is defined for sera that are positive and above a specified threshold but between the diagnostic cutoffs. this "grey-zone" is set in the netherlands and sweden to eu/ml of igg-anti-pt. the sensitivity and specificity of single-sample serologic assays have been determined. one study was done in a population of children - years of age, mostly nonvaccinated (germany), using a cut-off of ∼ eu/ml ( th percentile of population). with a specificity of %, a sensitivity of % was found, which increased to % when iga-anti-fha was added [ ] . another study was done without age limit in a mostly vaccinated population (netherlands) using a cutoff of ∼ eu/ml ( th percentile of population). the study found a sensitivity of . %, whereas a cutoff ∼ eu/ml (∼ th percentile of population) had a sensitivity of . % [ ] . a recent german study evaluated persons in hospital departments, pediatrician's offices, and child-care facilities, who received one injection of ap vaccines. after informed consent, blood was sampled before vaccination, and month, year, years, years and years after vaccination. igg-and iga-antibodies to pt, fha and igg-anti-prn were measured. in this population, when the distribution of igg anti-pt levels prior to immunization was examined, the th percentile was eu/ml, the th percentile was eu/ml, and the th percentile was eu/ml. applying a cutoff of eu/ml to a seroepidemiologic study in a population of persons aged to > years in various countries in the european union resulted in the following percentage of the cohort showing values above the cutoff: netherlands ( . %), finland ( . %), germany (former gdr) ( . %), france ( . %), germany (former frg) ( . %), uk ( . %), and italy ( . %) [ ] . seroepidemiologic studies can also demonstrate the cyclic nature of b. pertussis infections. in germany, a population of - -year-old blood donors was screened in different years. the percentage of this cohort with igganti-pt ≥ eu/ml was as follows for a diagnostic laboratory, it is important to define how the customer will use the serologic information, specifically if it is used for clinical decisions and patient management or if it is used for epidemiologic studies. similarly, clarity is needed regarding whether the definition should include all infections, symptomatic infections, infections with medical resource use, or severe infections. diagnostic serology is also strongly influenced by the time when patients seek medical attention relative to onset of symptoms. in german studies performed among persons with pcr-positive cases, the median number of days of coughing before seeking medical attention was found to be . days for schoolchildren, aged - years, . days for adolescents, aged - years, and . days for adults, aged - years. in france, serologic testing is performed for epidemiologic studies and surveillance. serology consists of measurement of igg anti-pt antibody titers using the reference elisa and purified pt provided by the manufacturers [ ] . a positive case is defined by either a twofold change in the titers between two serum samples obtained at a -month interval or a titer > eu/ml in a single serum sample, only if the serum is collected after more than weeks of cough and years after a vaccine booster. in some french laboratories, an "in house" western blot assay using purified pt, as well as various commercial tests have been used for routine diagnoses. these assays are not validated and have also been responsible for false-negative or false-positive results. for this reason, microbiologists have preferred real-time pcr for routine diagnoses. serology based on measurement of anti-pt titers may be less useful in the future, since children, adolescents and now adults are vaccinated with ap vaccines containing pt. the proportion of laboratory tests performed to diagnose pertussis in france has changed over the years, according to the renacoq database [ ] . in , % of pertussis cases were diagnosed by culture, % by pcr and % by serology, whereas in ; % of cases were diagnosed by culture, % by pcr and only % by serology. in the united states, the massachusetts state laboratory institute has been using a single-serum anti-pt igg eia on patient samples since . other versions of the current assay have been used in the past, including measurements of iga and igm to pt, igg to fha, and anti-pt igg in paired sera. ultimately, all these other assays were discontinued when data indicated that they did not contribute much additional information beyond that obtained from the single anti-pt igg alone. plates are coated with commercially prepared pt. standards are derived from pools of positive patient samples and are calibrated against the us reference lot . a concentration (g/ml) is assigned, calculated from a five-point standard dilution curve. the cutoff point for positivity was established on adults, including blood donors and healthy volunteers with anti-pt igg levels ranging from . to g/ml [ ] . a cutoff point was chosen based on the % upper tolerance limit, such that there is % confidence that % of the population falls below the cutoff. the cutoff point was set at g/ml, which is equivalent to cber units per ml. this is actually quite high relative to what other groups have chosen [ , ] . clinicians are asked to order a serologic assay only for patients who are older than years of age and only when coughing has been present for more than days. specimen volumes have been increasing steadily over the last years, so that in , more than specimens were tested. seropositivity has been fairly constant at approximately % of the submitted samples. many of the challenges associated with the assay arise from manipulations needed to produce a quantitative test result. it may make sense, at least in massachusetts, to consider some modification of the assay to make it more qualitative, simple, and robust, and able to be performed consistently and accurately with less effort than is currently expended. at least two knowledge gaps prevent wider application of the assay. first, the impact on assay results of adolescents and adults having recently received tetanus toxoid, reduced diphtheria toxoid and acellular pertussis (tdap) vaccination is not fully understood. the massachusetts immunizations program contacts every seropositive patient and asks about recent vaccination history. in the last years, since tdap has been available, only two seropositive patients with recent tdap vaccination have been identified. one person was vaccinated the day before blood was drawn for serology, so the antibodies detected were unlikely to have been due to vaccine. in massachusetts, tdap-associated igg may be less of a problem than was originally anticipated. possibly, post-tdap immunization igg levels do not frequently exceed the relatively high assay cutoff value. alternatively, clinicians may not be submitting samples from recent vaccine recipients, recognizing that those individuals are likely to be protected from disease. a second knowledge gap is the uncertain applicability of igg eia results to children younger than years of age. results are considered uninterpretable for that age group, because of the possible presence of vaccine-associated antibody. massachusetts state laboratory institute data on seropositivity by age demonstrate a peak in seropositivity among adolescents and young adults, and also a peak at - years of age. admittedly, no clinical information is available for the - -year-old patients because they are not considered to be pertussis case-patients and are not investigated. in addition, their vaccination status is unknown. nevertheless, the peak in seropositivity that occurs at this age may correspond to the fifth dose of dtap. interestingly, the peak is not very wide, spanning only - years, so the application of the assay could likely be extended down to some ages less than years. australia has a -year experience of using commercial serologic kits for the routine diagnosis of pertussis. iga has been used rather than igg kits because iga is believed to be short-lived ( - weeks) in comparison to igg ( - weeks) , and, in children at least, iga is not produced in response to the vaccine. unusually large numbers of pertussis iga-positive tests were reported to public health authorities during . at the time, only two iga commercial serologic tests were used, elisa kit ( % of laboratories) and elisa kit ( % of laboratories). this prompted a widespread evaluation, comparing various commercial available elisa kits with complement fixation (cf), iga immunofluoresence (if) and iga western blot (wb) kits. ninety healthy adults with no clinical history of respiratory illness in the preceding weeks and negative by cf, if and wb formed the negative group for the evaluation. the positive group consisted of children and adults with a clinical history of pertussis of less than weeks' duration and positive by cf, if and wb. the respective antigens, sensitivity and specificity of each commercial iga elisa kit were as follows: ( ) in , cidms laboratory tested serum samples for pertussis iga using the elisa kit . following the evaluation of various commercial tests, over % of these samples were retested and % were found to be false-positives. the false-positive specimens reacted with the fha antigen band only, suggesting that pt would be a better choice for an elisa antigen. however, the only kit that used pt as an antigen performed poorly, which emphasizes the need for better standardization. in addition, a group of negative samples was tested by the esen igg elisa [ ] using a eu/ml cutoff; samples had igg levels > units, which would have been interpreted as evidence of recent infection when clearly these individuals were not symptomatic. this highlights the need for caution in determining a cutoff for recent infection in adults, particularly in highly vaccinated populations. in general, diagnostic laboratories prefer commercially available assays. two studies so far have evaluated selected commercially available elisas for b. pertussis serology: kösters et al. used paired sera from patients (children), sera from vaccinees, interlaboratory comparison samples, and reference preparations [ ] . schellekens et al. used paired sera from pcr-positive patients (median age years) and control patients with respiratory symptoms (median age years) [ ] . another study with various currently existing serologic tests is ongoing and results are expected to be available in early . in summary, b. pertussis serodiagnosis still suffers from many unsolved problems, such as that diagnostic testing is performed in immunologically non-naive populations with antigens that are components of ap vaccines. generally an anamnestic immune response develops more rapidly than symptoms, and immune responses to vaccine antigens cannot be distinguished from response to infection. problems also exist with serodiagnosis of b. parapertussis infections. serologic interpretation requires knowledge of pertussis immunization, information not typically known by the testing laboratory. reliable, standardized elisa systems are not commercially available at this time. additionally, the clinical course appears to differ between primary and non-primary infections. for most subjects, clinical case definitions for pertussis remain nonspecific. there are currently no suitable serologic tests for recently vaccinated patients, since tests based on nonvaccine antigens have not been satisfactory for diagnosis. finally, when using single-sample serology, population-based cut-offs will need re-verification after any change made in a vaccination schedule. discussion focused on the use of antigens not present in vaccines for serologic diagnosis of pertussis. cherry et al. [ ] showed that patients with vaccine failure responded poorly to the act antigen, suggesting an induced tolerance due to the phenomenon called "original antigenic sin." in this phenomenon, vaccinated patients respond to antigens that they have been primed with and do not respond to new antigens (e.g., act) associated with infection. therefore, it was suggested that nonvaccine antigens may not be as good as pt for serologic diagnosis of pertussis in vaccinated populations. other points of discussion included the potential use of reverse vaccinology approach to identify new antigen candidates to be used in serologic diagnostics and lack of specificity of other antigens other than pt, such as fha and act. the lack of sensitivity and specificity of commercial tests currently available worldwide was broadly discussed. it was pointed out that one commercial igg and iga anti-pt test available in the united states has been standardized to provide adequate sensitivity and specificity for the diagnosis of pertussis in persons older than years of age. presenter: james d. cherry in studies of whole-cell pertussis vaccines, agglutinin titers of : or greater were found to be protective against pertussis when children were exposed within the household [ ] . agglutinating antibodies were thought to be primarily directed against the agglutinogens fim- / , prn and lipopolysaccharide. only two of the nine ap vaccines efficacy trials conducted during the s and s were done in such a way that data relating to serologic correlates could be developed. these trials were centered in erlangen, germany, and stockholm, sweden [ , , , ] . to determine serologic correlates, antibody titers to specific antigens were determined at the time of exposure. both studies indicated that prn was most important in protection and that antibody to fim was next most important. also noted in both studies was an apparent antagonism between antibody to pt and fim. in clinical trials in which children with both mild and severe disease were included in the analyses, efficacy jumped considerably for vaccines that contained prn as well as pt and fha when compared with pt and pt/fha vaccines. in the erlangen trial, the imputed titers at the time of exposure in dtp and dtap vaccine failures were examined. in the majority of the failures, subjects had high levels of antibody to prn, which is difficult to explain in relation to the overall data on serologic correlates. this indicates that there is much yet to be learned about serologic correlates. of interest is the fact that when a dtwp vaccine (evans vaccine) was compared with a two-component dtap vaccine, a three-component dtap vaccine and a five-component dtap vaccine, the greatest efficacy was noted with the dtwp vaccine. this vaccine elicited minimal antibody responses to pt and fha ( and eu/ml, respectively) and high values to prn and fim ( and eu/ml, respectively). discussion centered on diagnosing pertussis serologically using antigens contained in the vaccines and antigens not contained in the vaccines. although still controversial, if acute-phase serum is collected early, titer increases against both vaccine antigens and nonvaccine antigens are useful for diagnosis. further discussion related to cell-mediated responses associated with infection, use of animal models in studies of infection and consideration of additional antigens that might be included in vaccines. presenter: john b. robbins, md (national institutes of health, bethesda, maryland, us) dr. robbins presented evidence supporting the hypothesis, based on the classic article by pittman [ ] , that an inactivated and immunogenic pertussis toxoid (ptox) is both essential and sufficient for a pertussis vaccine [ ] . furthermore, he discussed data suggesting that multicomponent pertussis vaccines include nonprotective antigens [ , ] and may be more difficult to standardize [ ] . us pertussis cases reported to cdc have increased despite a high immunization rate among infants and children [ ] [ ] [ ] , ] . booster doses, starting in adolescence and offered every years, will maintain a high level of immunity in the population, and dr. robbins recommended that a genetically inactivated toxin should replace the chemically inactivated toxoids for mass immunization of infants, adolescents and adults [ , ] . with respect to the goals of the meeting, the importance of assays to measure serum pt igg was emphasized because pt igg is the only reliable assay for serologic diagnosis of pertussis after the acute phase of pertussis has passed [ , ] , and because the concentration of vaccine-induced igg anti-pt correlated with the efficacy of monocomponent ptox [ ] . the correlation indicates that the level of igg anti-pt may predict the efficacy of ap vaccines [ ] . a number of participants raised questions related to the hypothesis that ptox alone is sufficient for a pertussis vaccine. it was mentioned that the study supporting the hypotheses did not include serology. other studies, including serologic ones, showed evidence that addition of fha in the vaccine provides some protection against infection. data (not included in this report) based on extensive investigations in sweden and presented at the meeting in a supplemental presentation by rose-marie carlsson, md, swedish institute for infectious disease and control, contradicted many of the conclusions presented in this session. some participants also questioned the appropriateness of the analogy between diphtheria and ptoxs. the presentation on pathogenesis and immunity as well as the presentation on correlates of protection at this meeting are also at variance with the hypothesis that ptox alone is sufficient for a pertussis vaccine. the discussion was cut short to allow adequate time for consideration of issues related to assay standardization, the main focus of the meeting. some conclusions previously reached by a who-convened working group on the clinical evaluation of new ap vaccines served as an introduction to discussion. one such conclusion was that additional placebo-controlled protective efficacy studies were not feasible since withholding pertussis vaccine was no longer a possibility. relative efficacy studies of adequate size and statistical power also seemed unlikely. the approval of new ap vaccines would have to rely on comparative immunogenicity data. all the ap-containing vaccines that have been licensed thus far in the united states and european union incorporate the same pertussis antigens made by manufacturer-specific processes as were included in at least one of the successful vaccine efficacy trials in infants. therefore, immunogenicity data have been used to link the demonstration of efficacy for the tested ap vaccine to the new ap vaccine even when the total antigen compositions of the two vaccines differ. comparisons of immune responses between the new and reference ap-containing vaccines raise several difficult questions. for example, with no identified immunologic correlates of protection against pertussis, it is not known which antigen(s) elicits immune responses that are important for prevention of clinically apparent infection. it is also not known whether the primary comparison should be in terms of percentages with postvaccination titers above the assay cutoff, on seroconversion rates (which are open to various definitions) or to geometric mean antibody titers. whichever immunologic parameter is chosen for the primary comparison, it is difficult to decide what might constitute a clinically important difference. in addition, the predefined criteria for noninferiority might be met for one or more, but not all immunologic parameters, with unknown implications for protection. because of these uncertainties, the overall judgment of the likely protective efficacy of an ap-containing vaccine should take into account all aspects of immune responses. thus the immunogenicity data should be described in terms of percentages of subjects reaching assay cutoffs, percentages achieving fourfold increments in antibody concentrations (or a similar definition of seroconversion), geometric mean antibody titers and reverse cumulative distributions. the uncertainty regarding the predictive value of these immunologic comparisons supports the importance of postlicensure surveillance programs to assess the effectiveness of ap vaccines against pertussis disease. however, it is very unlikely that vaccinespecific estimates of effectiveness could be generated, since this would require data from a region or country in which the vaccines used all contain the same ap antigens from the same manufacturer. because of the complexities of such programs and the infrastructure needed to generate reliable data on disease, such data are most likely to come from surveillance conducted by public health agencies. in the future, new ap-containing vaccines might include the same range of ap antigens previously shown to be efficacious, but some or all of these may be made by a different manufacturer and/or by a different process. new ap-containing vaccines could also have a different composition of pertussis antigens (in type and/or amount) compared with vaccines that were previously evaluated for protective efficacy. there is no agreed regulatory position on the minimal data that would be required to support approval of these products; however, practical limitations point to the need to consider how immunogenicity data could be best used to provide reassurance regarding likely efficacy. if a new ap-containing vaccine contains only antigens that were included in at least one vaccine evaluated in previous protective efficacy studies then immune responses could be compared between the new vaccine and an approved vaccine that has the most similar ap antigen content. if the new ap-containing vaccine contains fewer antigens, different amounts of antigens and/or additional antigens as those in vaccines previously shown to provide protective efficacy there is a need for a careful justification of all the differences between the new and past efficacious vaccines, which would likely have to be based on nonclinical studies. in particular, the response to the pt component should be fully assessed for efficacy in animal models and by estimating the functional antibody to pt (for example, by using the cho cell neutralization assay). the purity, integrity and functional activity of all antigens should be assessed by physical-chemical evaluation, measurement of residual toxicity (if applicable) and assessment of immunogenicity based on binding and functional assays and protective effects in relevant to begin with a brief overview of pertussis vaccine evaluation in the united states, ap vaccines were first licensed for toddlers in , for infants in , and for adults and adolescents in . serologic responses to ap vaccination have been used as a measure of lot-to-lot clinical consistency, to bridge populations, and to evaluate booster immunizations, new combinations and concomitant vaccination. relevant parameters measured have included geometric mean concentrations as well as percentage of responders. in addition, reverse cumulative distribution curves have proved to be an informative way to display serologic response data. the composition and formulation of ap vaccines vary widely between manufacturers and products, and no globally accepted reference preparations and release criteria have been established; thus, current reference materials and release criteria are productspecific. this causes difficulty in standardization of the laboratory tests for control of these vaccines. the basis of current regulatory approaches is to demonstrate that newly manufactured lots are comparable either to lots shown to have acceptable clinical trial performance or to lots considered equivalent to these clinical trial lots. current routine control tests for ap vaccines include characterization of antigens, assay of immunogenicity, and assay of residual pt bioactivity by the histamine sensitization test and identity testing of antigenic components. a modified intracerebral challenge assay (mica, modified kendrick test) has been used in japan, korea and china as the potency assay for release with a specification ≥ unit/dose; vaccines regulated using this approach have been shown to be effective in controlling pertussis. in other countries, no nationally defined official potency test has been used at present. an immunogenicity test has been used for monitoring consistency of production in routine control procedures with comparison with a clinical trial lot (or equivalent). however, the criteria for evaluating equivalency must be defined and no defined common specification has been set. recent evidence suggests that both antibody and cell-mediated immunity contribute to the protective process to a variable extent. following the decision made at the who ad hoc working group meeting ( , nibsc, uk), international collaborative studies on protection models for ap vaccines were initiated in , and the outcome of these studies was discussed in subsequent who working group meetings. the harmonized procedure for the intranasal challenge assay (inca), defined in , has been shown to be effective for monitoring the activity of different vaccines and to be transferable between laboratories [ , , ] . future work on optimizing experimental conditions to allow calculation of a relative potency is needed and a reference vaccine needs to be agreed upon. this model should be useful for development and characterization of new products or formulations, performing preclinical evaluation or stability and lot consistency monitoring. the current safety tests for ap vaccines include assays for residual active pt and reversibility of detoxification based on histamine sensitization activity, an endotoxin assay and a general toxicity test. the absence of other toxins at significant levels (heat-labile toxin, act, tracheal cytotoxin) has been controlled through process validation. problems associated with the histamine sensitization test include variation in test performance among laboratories and the absence of an internationally defined acceptance criterion for the assay. the influence of significant interactions between pt and other vaccine components over histamine sensitization assay is unknown. the assay could be improved by ( ) defining assay sensitivity by including reference groups; ( ) narrowing the range of permissible sensitivity; ( ) expressing the results in international unit (iu) of pt activity relative to a common reference; and ( ) establishing limits based on a panel of products with a known safety history. in summary, current quality control tests and specifications for ap vaccines are product-specific, and the clinical relevance of this approach remains uncertain. an effective potency assay for new products and formulations should be defined, and suitable reference preparations should be identified. for the current histamine sensitization test, an upper limit for active residual pt should be defined and use of a reference preparation should be encouraged. finally, a specific assay for residual active pt that does not require use of animals should be identified. the challenge in testing of ap-based combination vaccines still remains. for the immunologic evaluation of dtap combinations and coadministrations with dtap-based vaccines, gsk referred back to the original dtap efficacy trials. the cornerstone of comparisons among combined vaccines to their separately administered licensed counterparts is the evaluation of the antibody responses to pt, fha and prn [ ] . noninferiority of the percentage of vaccine responders as well as of geometric mean titers forms the basis for the licensure of new combinations and co-administrations. reverse cumulative antibody distribution curves are also useful for such comparisons. further characterizations of new dtap combinations by gsk involve the evaluation of clinical t-cell responses as well as preclinical evaluations in a mouse lung clearance model [ ] . with respect to vaccine composition and protection, the available data suggest potent dtwp and dtap containing pt and prn induce high levels of protection against whooping cough [ , , , ] . the mechanisms linked to prn-mediated protection relate to the observations that prn expression affects act activity [ ] and induces opsonophagocytic antibodies [ ] . the dtap efficacy trials have been contradictory with regard to the efficacy of dtap (pt + fha without prn). initially the efficacy of dtap was reported to be % in senegal, according to who criteria, but this was later corrected to % since the original diagnostic criteria were different from other trials [ ] . this % efficacy of dtap in senegal probably is similar to the % efficacy observed with another dtap in sweden because of the finding that pt immune responses in senegal are more than . times higher than in western europe [ ] . in conclusion, dtwp and dtap with ≥ components are the preferred options for immunization against whooping cough. dtap has demonstrated efficacy comparable with dtap , although it is uncertain if fim is a protective antigen [ ] . the duration of protection with dtwp and dtap can be estimated at years or more [ ] . to further control pertussis in newborns, adolescents and adults, dtap booster immunizations can be recommended [ ] . sanofi pasteur has been using immunoassays for b. pertussis -igg elisas for pt, fha, prn, and fim antigens -to support the evaluation of vaccine-induced immune responses. the assays used are essentially those identified by manclark et al. [ ] . key reagents are qualified and standardized, coating antigens are highly purified and assessed both by physical chemistry and immunologic methods to ensure purity, antigenicity, and specificity. assay reference sera are bridged to international references, and the performance has demonstrated to remain stable. control sera are shown to be specific and represent suitable ranges across the range of the assay. all components are qualified, with performance panels of sera to ensure consistency. all assays have been fully validated by precision (inter-and intralaboratory), accuracy (using spike recovery), specificity (assessing matrix effects and performing competition studies), linearity/dilutability, limit of detection (lod), limit of quantitation (loq), and robustness. from the perspective of a vaccine manufacturer such as sanofi pasteur, issues and concerns for the task of global standardization should focus on critical reagents and data reduction methods. for coating antigens, consideration needs to be given to the impact of antigen source as well as the criteria for quality, and consumption rate. for references, most laboratories have been forced to develop internal references that are bridged to international standards; guidance is needed on how best to select these internal references and the sustainability of international references to support frequent calibrations. similarly, guidance is needed on the best characteristics for controls and implementation of quality control and assay acceptance criteria. finally, for data reduction, guidance is needed to establish which data reduction systems are most suitable in today's laboratory. chairpersons: freyja lynn, bs (national institute of allergy and infectious diseases, national institutes of health, bethesda, maryland, us) and annette morris, bs, bed (canadian center for vaccinology, dalhousie university, nova scotia, canada) to achieve harmonization of assays for the clinical diagnosis of pertussis and evaluation of immunity postvaccination, several issues will have to be addressed. first, consensus on methodologies, assay validation, and analysis of results is required to facilitate comparisons among data generated in different laboratories. the requirements for a diagnostic assay will likely differ in some aspects from assays used to perform postvaccination immunity evaluation. stability of assay results over time will be dependent on the consistent availability of appropriately characterized reagents and materials. studies are required to determine the effect on assay results of different sources and lots of antigen, conjugate and other reagents. a proficiency panel of human sera is vital to harmonization of methods between laboratories, although creating a panel with sufficient volume for worldwide use will be a challenge. a reference laboratory or a series of connected reference laboratories will be essential as resources for methodology, change control and management and evaluation of the proficiency panel. finally, a repository for the distribution of standardized materials, such as antigens, conjugates, and reference materials, will be crucial for standardization and harmonization of b. pertussis assays. a steering committee should be established for the purpose of identifying and prioritizing the steps toward harmonization, including identification of resources required for implementation. if harmonization is to be accomplished, it must be a global effort with the collaboration of many research, government and corporate organizations. the who is currently evaluating a collaborative study of proposed international reference preparations for human pertussis antiserum. if these are shown to be acceptable, the availability of international reference material will be valuable for promoting harmonization. additionally, the collaborative study data, available mid- , will provide information on comparability of results in participating laboratories and will help point to where priorities should be focused. actionable items to accomplish standardization and harmonization of b. pertussis immunoassays identified during group discussion included the following: ( ) identify a group that will organize, prepare, maintain, and distribute a common pertussis performance serum proficiency panel that can be used to help laboratories to develop their assays and monitor stability; ( ) assemble a working group to assist with the details of the panel; ( ) encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; ( ) focus on key reagents such as reference and control sera; ( ) define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; ( ) develop guidance on quality of other reagents, e.g., pt and other antigens, and methods to demonstrate their suitability; ( ) establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; ( ) create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and ( ) seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines. preventing tetanus, diphtheria, and pertussis among adults: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine recommendations of the advisory committee on immunization practices (acip) and recommendation of acip, supported by the healthcare infection control practices advisory committee (hicpac), for use of tdap among health-care personnel cdc. preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee 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inflammatory cytokines and dendritic cell activation in whole blood: impaired responses in human newborns proinflammatory and proapoptotic activities associated with bordetella pertussis filamentous hemagglutinin phagocyte impotence caused by an invasive bacterial adenylate cyclase bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin inhibition of monocyte oxidative responses by bordetella pertussis adenylate cyclase toxin bordetella pertussis adenylate cyclase-hemolysin induces interleukin- secretion by human tracheal epithelial cells bordetella pertussis adenylate cyclase toxin (act) induces cyclooxygenase- (cox- ) in murine macrophages and is facilitated by act interaction with cd b/cd (mac- ) t-cell immune response assessment as a complement to serology and intranasal protection assays in determining the protective immunity induced by acellular pertussis vaccines in mice a murine model in which protection correlates with pertussis vaccine 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toxin in a single serum sample for diagnosis of infection with bordetella pertussis european sero-epidemiology network: standardisation of the assay results for pertussis pertussis in massachusetts, - : incidence, serologic diagnosis, and vaccine effectiveness ad hoc working group on acellular pertussis vaccines, world health organisation the european sero-epidemiology network the seroepidemiology of bordetella pertussis infection in western europe evaluation of a single-sample serological technique for diagnosing pertussis in unvaccinated children evaluation of an immunoglobulin g enzyme-linked immunosorbent assay for pertussis toxin and filamentous hemagglutinin in diagnosis of pertussis in senegal comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to bordetella pertussis serodiagnosis of pertussis with commercial elisa's determination of serum antibody to bordetella pertussis adenylate cyclase toxin in vaccinated and unvaccinated children and in children and adults with pertussis studies on pertussis immunization comparison of values of antibody to bordetella pertussis antigens in young german and american men a comparative efficacy trial in germany in infants who received either the lederle/takeda acellular pertussis component dtp (dtap) vaccine, the lederle whole-cell component dtp vaccine, or dt vaccine pertussis toxin: the cause of the harmful effects and prolonged immunity of whooping cough. a hypothesis primum non nocere: a pharmacologically inert pertussis toxoid alone should be the next pertussis vaccine immunity to pertussis. not all virulence factors are protective antigens mass vaccination of children with pertussis toxoid-decreased incidence in both vaccinated and nonvaccinated persons who working group on standardisation and control of acellular pertussis vaccines-report of a meeting the rise in pertussis cases urges replacement of chemically-inactivated with geneticallyinactivated toxoid for dtp the diphtheria and pertussis components of diphtheria-tetanus toxoidspertussis vaccine should be genetically inactivated mutant toxins correlation between pertussis toxin igg antibodies in postvaccination sera and subsequent protection against pertussis who working group on the standardisation and control of pertussis vaccines-report of a meeting informal consultation with manufacturers and who ad hoc working group on mouse protection models for acellular pertussis vaccines. national institute for biological standards diphtheria-tetanuspertussis (dtp) combination vaccines and evaluation of pertussis immune responses pertussis antibodies, protection, and vaccine efficacy after household exposure low levels of antipertussis antibodies plus lack of history of pertussis correlate with susceptibility after household exposure to bordetella pertussis role of adhesins and toxins in invasion of human tracheal epithelial cells by bordetella pertussis crucial role of antibodies to pertactin in bordetella pertussis immunity pertussis vaccines priming effect, immunogenicity and safety of an haemophilus influenzae type b-tetanus toxoid conjugate (prp-t) and diphtheria-tetanusacellular pertussis (dtap) combination vaccine administered to infants in belgium and turkey acellular pertussis vaccines and the role of pertactin and fimbriae pertussis vaccines-who position paper serological responses to bordetella pertussis support for the meeting was graciously provided by the centers for disease control and prevention, atlanta, ga. the authors thank barbara slade, md, jackie goolsby, brandy kimble, shoranda ifill (cdc) and logistics health incorporated for their contribution to the scientific and administrative organization of the meeting. we thank lynne mcintyre for critical editing of this manuscript. key: cord- -hwvkvdlk authors: decaro, nicola; desario, costantina; elia, gabriella; campolo, marco; lorusso, alessio; mari, viviana; martella, vito; buonavoglia, canio title: occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hwvkvdlk abstract a total of faecal samples collected from dogs with diarrhoea following canine parvovirus (cpv) vaccination were tested by minor groove binder (mgb) probe assays for discrimination between cpv vaccine and field strains and by diagnostic tests for detection of other canine pathogens. fifteen samples tested positive only for cpv field strains; however, both vaccine and field strains were detected in three samples. eleven samples were found to contain only the vaccine strain, although eight of them tested positive for other pathogens of dogs. only three samples were found to contain the vaccine strain without evidence of canine pathogens. the present study confirms that most cases of parvovirus-like disease occurring shortly after vaccination are related to infection with field strains of canine parvovirus type (cpv- ) rather than to reversion to virulence of the modified live virus contained in the vaccine. canine parvovirus type (cpv- ) causes haemorrhagic gastroenteritis primarily in - -month-old pups [ ] . cpv- , first identified in the late s, was replaced a few years after its emergence by two antigenic variants, cpv- a and cpv- b. those two types are now distributed worldwide [ ] . more recently, a third antigenic variant, cpv- c, was first reported in italy [ ] . subsequently, it was reported in vietnam [ ] , spain [ ] , germany and the united kingdom (decaro, unpublished data) . this new variant appears to be replacing cpv- b in the italian dog population [ ] [ ] [ ] [ ] [ ] [ ] . the cpv variants differ in amino acid changes occurring at residue of the capsid protein, with types a- c displaying amino acids asn, asp and glu, respectively [ , ] . this residue is located in a major antigenic site close to epitope b, i.e., over the shoulder region of the capsid in a region considered to affect viral immunogenicity. concerns have been expressed * corresponding author. tel.: + ; fax: + . e-mail address: n.decaro@veterinaria.uniba.it (n. decaro). that the antigenic differences between cpv type and its variants may decrease the effectiveness of the cpv- -based vaccines [ ] [ ] [ ] ; however, type b vaccines are now licensed in several countries and some investigators recommend their extensive use [ ] . characterisation of the cpv variants was controversial until minor groove binder (mgb) probe technology was applied to obtain rapid and unambiguous identification of the viral type [ , ] . mgb probes are short taqman probes conjugated with molecules that form hyper-stabilised duplexes with complementary dna, allowing reduction in length of the probe and an increase in specificity [ ] . mgb probes are, therefore, an attractive tool for revealing single nucleotide polymorphisms in the capsid protein gene between types a and b and types b and c. since the assays differentiating types a/ b and type b/ c did not discriminate between vaccine and field strains, additional mgb probe assays were developed. we had previously developed an assay to differentiate field strains from type -based vaccines that react as cpv- a, using the type a/ b assay [ ] ; in addition, two assays were developed to discriminate between type b vaccine and field strains [ ] . such assays could be of practical help, since gastroenteritis in pups within week after cpv vaccination occurs frequently in practice (buonavoglia, personal observation) . in such cases, conventional diagnostic tests are able to detect a cpv strain in the faeces of vaccinated dogs, although it is frequently uncertain whether the virus is a vaccine or field strain. in the present study, faecal samples from cases of parvovirus gastroenteritis that occurred - days after cpv vaccination were analysed by the novel mgb assay in order to determine whether the puppy illnesses were associated with virulent or vaccinal cpv types. in such cases, it was especially important to attempt to rule out any involvement of cpv vaccines. a total of faecal samples from dogs that had clinical signs typical of parvovirosis during the first week after cpv vaccination were collected between and and tested subsequently. twenty-two samples were from dogs administered monovalent or multivalent cpv- vaccines and four samples were from dogs vaccinated with cpv- b. the types/brands of cpv vaccine used in the remaining three pups were not reported. those samples included a unique specimen collected in scotland, uk ( / - , courtesy of christopher davies, institute of comparative medicine, university of glasgow) and specimens collected from two italian dogs ( / and / ). as regards the scottish dog, the vaccine used and the day of vaccination were unknown; the two exceptional italian dogs also received vaccines that were uncertain. six of the dogs had been imported to italy from poland (n = ) or hungary (n = ), but vaccine had been administered in italy. in some cases, disputes between dog owners and veterinarians were in progress because of concerns regarding the possible relationship between cpv vaccinations and the subsequent enteric illness. in order to evaluate the post-vaccinal virus shedding in healthy dogs, faecal samples were also tested that had been collected from non-diarrhoeic dogs between days and after cpv vaccination. the specimens were from dogs inoculated with cpv- (n = ) or cpv- b (n = ) vaccines. detection and characterisation of cpv- was obtained using two real-time pcr assays with the mgb probes described previously [ ] . types a/ b and b/ c assays were employed to discriminate between types a and b, and between types b and c. the mgb probe assays were carried out in a -l reaction containing l of template or standard dna (both in duplicates), . l of iq tm supermix (bio-rad laboratories srl, milan, italy), nm of primers and nm of probes. the thermal protocol was done as follows: activation of itaq dna polymerase at • c for min, cycles of denaturation at • c for s and primer annealing-extension at • c for min. all reactions were conducted in an i-cycler iq tm real-time detection system (bio-rad laboratories srl) and the data were analysed with the appropriate software (version . ). specificity, sequence and position of real-time pcr primers and mgb probes are shown in table . discrimination between vaccine and field strains of cpv- was carried out using additional mgb probe assays [ , ] . the type /variants assay is able to differentiate cpv- (vaccine) from the field variants; the sah/field assay also discriminates between cpv- b field strains and vaccine strain sah contained in vaccines duramune ® dappi + lc (fort dodge animal health) and procyon ® dog da ppi/cvl (schering-plough animal health, welwyn garden city, hertfordshire, uk). the /field assay discriminates between cpv- b field strains and vaccine strain cpv- contained in vaccine virbagen ® puppy b (virbac tierarzneimittel gmbh, bad oldesloe, germany). reactions were carried out following the same protocols used for detection and characterisation of cpv- and the oligonucleotides reported in table . molecular methods were used for detection of other viral pathogens of dogs, including mammalian reoviruses (mrv) [ , ] , rotaviruses [ ] , caliciviruses [ , ] , canine adenoviruses [ ] , canine distemper virus (cdv) [ ] , canine herpesvirus [ ] and canine coronavirus (ccov) [ , ] . specimens that tested positive for cpv vaccine strains and negative for viral pathogens were examined for bacterial and parasitic pathogens by standardised methods. for bacterial screening the faecal samples were plated onto macconkey's agar (oxoid s.p.a., garbagnate milanese, italy), whereas detection of the most common enteric parasites was achieved using zinc sulphate flotation. the ziehl nielsen staining was also performed for detection of cryptosporidium spp. results of the diagnostic tests carried out on the postvaccinal faecal samples from diarrhoeic dogs are reported in table . cpv field strains (virulent virus) were identified alone in samples- were type a and were type c [ ] . b [ ] . c [ ] . viruses. thirteen samples were from dogs vaccinated with cpv- , one sample was from a dog vaccinated with cpv- b and one sample from a dog vaccinated with an unknown formulation. a cpv- vaccine strain and a cpv- a field strain were detected simultaneously in the other three samples; two were from dogs administered a type- -based vaccine and one from a dog given an unknown vaccine. eleven samples, including seven and three samples collected from dogs vaccinated with cpv- and cpv- b, respectively, and one sample from a dog administered an unknown vaccine, were found to contain only the vaccine virus. other canine pathogens were detected in of samples, including ccov type i, ccov type ii and isospora canis. the remaining three samples contained only vaccine strains: cpv- (one sample); cpv- b strain sah (two samples). there was no evidence of other viral, bacterial or parasitic pathogens. in the two samples positive for cpv- a ( / ; / ), the co-presence of mrv strains was found that gave a signal only in the nested pcr assay. the mrvs could not be characterised by type-specific rt-pcr assays [ ] . one sample ( / ) was found positive for cdv by a real-time rt-pcr assay [ ] . in order to rule out the vaccine origin of the cdv strain, partial sequences of the haemagglutinin gene were obtained by rt-pcr amplification and subsequent sequence analysis showed that it clustered with field strains of the european lineage [ ] . eleven out of the post-vaccinal faecal samples collected from healthy dogs were found to contain the vaccine virus. eight samples were from dogs vaccinated with cpv- and threes samples were from dogs administered a cpv- b vaccine (data not shown). the onset of clinical signs similar to those of canine parvovirosis is a frequent finding in veterinary practice. often pups become infected with field strains of cpv- shortly before or after vaccination; however, diarrhoea may be a consequence of other viral or bacterial infections, parasitosis or poor management. nevertheless, many veterinary practitioners and dog owners erroneously believe that enteric illness subsequent to the administration of a cpv vaccine results from reversion to virulence of the modified live vaccine (mlv) virus. previously, there were few opportunities to address this issue since vaccinated dogs may shed the mlv vaccine virus in their faeces alone, or concurrently with a virulent field strain. in both instances, conventional diagnostic tests could not provide definitive results. in fact, virus isolation from faecal samples in cell cultures is poorly sensitive [ ] , especially in mixed infections; furthermore, it may allow the isolation of only the most adapted (vaccine) or most abundant (field) strain. unless sequencing of several clones is carried out, pcr may selectively, or more efficiently, amplify either of the viruses, so that the other one remains undetected by subsequent sequence analysis. moreover, the use of conventional tests, e.g., haemagglutination, immunochromatographic test, virus isolation, or pcr, may misdiagnose diarrhoeic dogs as cpv-infected due to the presence of a mlv strain in the faeces. on the other hand, the novel mgb probe assays represent an effective tool for rapid discrimination between vaccine and field strains of cpv- since they are able to detect vaccine and field strains that occur simultaneously in the faeces of vaccinated dogs, even when low titres of vaccine strains are shed [ , ] . reversion of virulence of cpv mlv has been frequently postulated, but never demonstrated, as the attenuation of virulence has proved to be highly stable [ ] [ ] [ ] . in the present study, analysis of faecal specimens collected from dogs that developed gastroenteritis shortly after cpv vaccination confirmed that the diarrhoeas observed were most commonly related to infection with field strains of cpv- . when a vaccine strain was detected in a diarrhoeic faecal sample, it was generally present together with a cpv- field strain, or with other pathogens commonly associated with enteritis in dogs. only three vaccinated dogs lacking evidence of other canine pathogens were found to shed a vaccinal cpv strain in their faeces, e.g., the cpv- b strain sah (two dogs) and a cpv- strain (one dog). even in those cases, diarrhoea with low amounts of vaccine virus in the faeces suggested that the gastroenteritis was probably related to pathogens not detected by our tests (data not shown). noninfectious causes, such as sudden changes in the diet, also may have been responsible for the diarrhoeas in pups. according to previous reports of the progressive replacement of cpv- b by cpv- c [ ] [ ] [ ] [ ] [ ] [ ] , no type- b field strain was detected in the samples tested. another noteworthy finding was the detection of a cdv strain of european lineage in the faeces of one dog that had been infected simultaneously with cpv- a and mrv. since the vaccine formulation (monovalent or multivalent) administered to this dog was not known, the vaccine origin of this strain could not be ascertained. however, sequence analysis of the haemagglutinin gene revealed that the cdv strain had diverged from the onderstepoort, rockborn or snyder hill cdv vaccine strains, and it clustered with field strains of the european lineage, as reported by martella et al. [ ] . in conclusion, since reversion to virulence of the cpv mlv vaccine strains has not been shown to occur, the present study demonstrates that most cases of gastroenteritis subsequent to vaccination are related to infection with cpv field strains shortly before or after the vaccine administration. new enteric viruses in the dog rapid antigenic-type replacement and dna sequence evolution of canine parvovirus evidence for evolution of canine parvovirus type- in italy a novel antigenic variant of canine parvovirus from a vietnamese dog first detection of canine parvovirus type c in pups with haemorrhagic enteritis in spain a canine parvovirus mutant is spreading in italy surveillance activity for canine parvovirus in italy canine parvovirus infection: which diagnostic test for virus? a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs new approaches for the molecular characterization of canine parvovirus type strains characterisation of the canine parvovirus type variants using minor groove binder probe technology comparison of isolates of canine parvovirus by restriction enzyme analysis, and vaccine efficacy against field strains canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine immunogenicity of an intranasally administered modified live canine parvovirus type b vaccine in pups with maternally derived antibodies -minor groove binder-dna probes increase sequence specificity at pcr extension temperatures a minor groove binder probe real-time pcr assay for discrimination between type -based vaccines and field strains of canine parvovirus diagnostic tools based on minor groove binder technology for rapid identification of vaccine and field strains of canine parvovirus type b detection of mammalian reovirus rna by using reverse transcription-pcr: sequence diversity within the -encoding l gene virological and molecular characterization of a mammalian orthoreovirus type strain isolated from a dog in italy identification of bovine and porcine rotavirus g types by pcr matson do. design and evaluation of a primer pair that detects both norwalk-and sapporo-like caliciviruses by rt-pcr nested pcr for the diagnosis of calicivirus infections in the cat detection and differentiation of cav- and cav- by polymerase chain reaction detection of canine distemper virus in dogs by real-time rt-pcr nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs heterogeneity within the hemagglutinin genes of canine distemper virus (cdv) strains detected in italy development of a modified live, canine origin parvovirus vaccine canine parvovirus type- . an evolving pathogen of dogs dog response to plaque variant of canine parvovirus we thank donato narcisi, carlo armenise and arturo gentile for their excellent technical assistance. this work was supported by grants from university of bari, italy: project ex % , "caratterizzazione delle varianti di campo del parvovirus del cane mediante real-time pcr con sonde minor groove binding (mgb)". key: cord- - nxsua authors: paul-pierre, pastoret title: emerging diseases, zoonoses and vaccines to control them date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nxsua abstract vaccination, when available, is undoubtedly the most cost-effective means of preventing and controlling, and even eradicating, infectious diseases. in recent years vaccination has also been used for other purposes in animal health, production and welfare, e.g. immunocastration. vaccination of animals serves many different purposes, such as controlling animal infections and infestations, thus improving animal health and welfare; controlling anthropozoonoses and food poisoning in humans, thereby protecting public health; solving problems associated with antibiotic and anthelmintic resistance; helping to leave food-producing animals free of chemical residues; protecting the environment and biodiversity and ensuring animal farming sustainability. the problem is nevertheless more complex when facing emerging or re-emerging infections particularly zoonotic ones. vaccination, when available, is undoubtedly the most costeffective means of preventing and controlling, and even eradicating, infectious diseases. unfortunately the problem is more complex when facing emerging or re-emerging infections particularly zoonotic ones. for instance canine parvovirosis was a real emergence in animal health [ , ] ; the first step was to vaccinate dogs with a vaccine directed against feline panleukopaenia since the two causative viruses are antigenically nearly identical. this first step was rapidly followed by the development of vaccines, either inactivated or attenuated, specifically directed against canine parvovirosis. some situations are more problematic when facing the outbreaks of diseases caused by viruses showing broad antigenic diversity such as foot-and-mouth disease virus or bluetongue virus; in this latter case it is even more difficult due to the fact that the infection is transmitted by a culicoides vector from the family ceratopogonidae (biting midges). it took two years in northern europe before inactivated vaccines against serotype of bluetongue virus were available [ ] . in northern america, the spectacular spread of west nile virus infection, another vector transmitted disease, in humans and horses, was rapidly followed by the development of several vaccines, including a dna-based vaccine for horses. one solution to be ready to vaccinate in face of an outbreak of a re-emerging infection is to stockpile vaccines as exemplified by foot-and-mouth virus vaccines as concentrated antigens. stockpiling is also envisaged for the possible pandemic of avian influenza h n in humans [ ] (pandemic pre- * tel.: + ; fax: + . e-mail address: pp.pastoret@oie.int. paredness) or to mitigate the risk of bio-agro-terrorisms. for time being it seems more appropriate to combat the h n influenza infection at the animal source to reduce human exposure. to prevent nipah virus (henipavirus) infection in pigs a vaccine has recently been developed but, unfortunately, in countries like bangladesh, humans are directly infected by the reservoir, a fruit bat species. animals may be vaccinated against certain infections not for their own sake, but to prevent human contamination. one of the best example being wildlife vaccination against terrestrial rabies by the oral route using baits [ ] . animal vaccination may also be used to prevent food poisoning in humans; a vaccine against escherichia coli :h has for example recently been conditionally approved for cattle in the united states. the changes following globalisation, climatic change [ , ] , and the opening of previously closed ecosystems, have considerably modified the pattern of endemic (or enzootic) infections/diseases, and contributed to the emergence of new agents that are pathogenic for humans and domestic animals. emerging infections is a collective name for infections that have been identified and taxonomically classified recently. in humans, in the final quarter of the twentieth century, more than such conditions were recognised [ ] . zoonoses are defined as infectious diseases that can be transmitted naturally between humans and wild or domestic animals. these infections are particularly important in the context of emerging infectious diseases of humans as the majority of these are of zoonotic origin; a comprehensive review by cleaveland et al. [ ] identified species of infectious organisms known to be pathogenic to humans, including viruses and prions, bacteria and rickettsia, fungi, protozoa and helminths. out of these, ( %) were classified as zoonotic and pathogenic species were considered to be associated with emerging diseases. of emerging pathogens of this group, ( %) were zoonotic [ ] , the vast majority of which coming from wildlife. wildlife obviously constitutes an important potential of new pathogenic agents for humans and domestic animals [ ] . this paper will mainly focus on viruses, mammals and birds. nowadays, the total number of viruses identified reaches approximately species [ ] , but the likely number could exceed , according to the first estimates. even this number is most probably an underestimate (e.g. a well-known mammal species like human beings harbours at least different herpesviruses, and different herpesviruses have been identified in cattle until now), but if one takes into account the estimated number of , , only % of viruses have already been identified. moreover, it does not take into account their extreme variability, particularly rna viruses, leading to populations of quasi-species. if one considers that there are recognised mammal species and that, for instance, herpesviruses have been isolated from all classes of vertebrates and even from oysters, one must admit that the world of viruses is huge. the use of viral metagenomics will help to identify more viruses [ ] . for mammalians, different species have already been recognised, whereas the expected number of species is estimated to be around ; for mammalian species we are, therefore, nearly at the end of the inventory, since % of the species are already known. the inventory of mammalian species was first established in , when only species were recognised; the same inventory established in contained different species. in , as already mentioned, the complete list of mammal species consisted of species [ ] . this increase in number seems to be paradoxical and even contradictory if one takes into account the extinction of some species during the same period of time. this increase in number can be accounted for when one considers that each phenotype of newly discovered species is listed separately and, more importantly, that the advent of modern molecular technology allows for the discrimination of species according to their genotypes and increasingly detailed comparisons of species limits and evolutionary relationships (taxonomic revision). among mammals, there are species of rodents pertaining to genera. since , new rodent species have been recognised. the rodents therefore compose % of recognised mammal species. this number is particularly important if one takes into account the fact that the order of rodents harbours, and is the reservoir of numerous zoonotic infections. among the most spectacular are hantaviroses [ ] ; some african sciuridae species (funisciurus spp., heliosciurus spp.), are the reservoir of monkeypox [ ] . recently, the introduction of one of these species into the united states nearly provoked an ecological disaster, due to the transmission of the virus to indigenous rodent species (prairie dogs) [ ] . to date, the order of chiroptera contains species, pertaining to genera; new species have been identified since . bats make up therefore . % of the total number of mammal species. this is worrying, since bats have been the source of many emerging diseases, many of them being previously unknown. for instance, insectivorous and frugivorous bats are the reservoir of the archeolyssaviruses, from which all lyssavirus strains derive, even the strains responsible for terrestrial rabies. frugivorous bats are the reservoir of newly discovered viruses such as nipah and hendra (henipavirus), responsible for numerous human fatalities, of the coronavirus responsible for the epidemics of the severe acute res-piratory syndrome (sars) and, most probably, of filoviridae such as the virus responsible for ebola disease in africa. there are approximately , species of birds. in , species were recognised, pertaining to genera [ ] , of which are passerines ( genera) and are non-passerines ( genera). birds, previously dinobirds, descend from dinosaurs and are therefore further removed from us than mammals but they are, nevertheless, reservoirs of zoonotic infections. the recent epidemics of west nile virus infection in the americas is a good example. the problem arising from highly pathogenic avian influenza, particularly its strain h n , is equally worrying, since avian infection, which is already zoonotic, may be responsible for a new human pandemic, similar to the one following the first world war. the virus responsible for this pandemic, which caused more human fatalities than the war itself, was recently reconstituted [ ] [ ] [ ] and is highly virulent when inoculated to non-human primates. it is noteworthy that wildlife biodiversity hot-spots are mainly found in tropical and sub-tropical regions, such as sub-saharan africa, indonesia and south america. through selection, man has created a number of different breeds of domestic animals, e.g. there are approximately recognised breeds of cattle worldwide [ ] , but many of these are on the verge of extinction (less than breeding cows). there is therefore currently a swift erosion of genetic variability in cattle that is really worrying. there are more than recognised dog breeds, showing a remarkable phenotypic and genotypic variability. for instance a survey of the adaptive humoral response of different dog breeds following vaccination against rabies within the british pet scheme showed that there was a significant variation in response between breeds after vaccination [ ] . there obviously exists a large variation of responses between breeds after vaccination which could be used for the selection, marker assisted or not, of good or bad responders to vaccination. poultry lines selected according to their humoral adaptive immune response have already been obtained [ ] . as a matter of fact, the differences in the susceptibility of breeds to some infections or infestations has been well observed by breeders, for instance the "resistance" of the n'dama cattle breed to trypanomosis. the mechanisms allowing emergence or re-emergence of infections are numerous [ ] . a key factor is the extreme variability of viruses (particularly rna viruses) leading to generation of populations of quasi-species, which allows them to easily cross the species barrier. viruses evolve far quicker than their hosts, by several mechanisms: point mutations, deletions, recombination, reassortment and acquisition of cellular genes. moreover, viruses have co-evolved with their natural hosts, often leading to unapparent infections. in animal health, the spectacular emergence of canine parvovirosis in , which resulted in a disastrous epizooty within the dog population worldwide, was the result of a mutation of another parvovirus which is responsible for feline panleukopaenia [ , ] , whereas dogs and cats were living peacefully before, without inter-specific transmission. another source of emergence is the opening of previously closed ecosystems, which leads to new contacts between unrelated species and shows that a species previously unknown to be susceptible to an infection (because of the lack of opportunity to be infected) is in fact fully susceptible. a recent illustration is given by the emergence of bluetongue serotype , in northern europe, transmitted by a culicoides vector culicoides dewulfi previously unknown to be susceptible to the infection [ ] . invasive species or migratory species may also be responsible for emergence, as could be the deliberate or accidental release of foreign species in a new environment, as exemplified by the introduction of monkeypox virus in the united states. other possible sources of emergence include veterinary biologicals, climate change and globalisation with its ts (trade, transport, travel, tourism and terrorism). in animal health, in face of an emergency, there still exist two possibilities, either mass slaughtering of animals or vaccination. unfortunately vaccines are not always available; for instance in face of an outbreak of african swine fever and in the absence of a vaccine, the only solution is to kill the infected animals as quickly as possible, and to destroy the carcasses, in order to avoid the transmission of infection to uninfected premises. anyway, the pigs will die from a disease which provokes nearly % mortality. it is even more true when facing a really emerging disease that moreover is zoonotic such as nipah virus infection [ ] for which no vaccine was available yet, because the causative agent was previously unknown; the only solution is once again to kill and destroy the infected and in-contact animals. a vaccine has recently been developed to prevent nipah virus infection in pigs [ ] ; unfortunately in countries like bangladesh, man is mainly at risk due to direct contact with the reservoir, a fruit bat, or contact with its secretions. in other cases, when re-emergence of a previously well-known infection and when a vaccine is already available one may have the choice; either slaughtering or vaccination [ ] . foot-and-mouth virus infection gives an excellent example. footand-mouth virus is represented by seven serotypes, further divided into numerous sub-types. preventive vaccines are available as highly purified concentrated antigens stockpiled in liquid nitrogen [ ] ; being highly purified, they allow the differentiation between vaccinated or infected animals (even if previously vaccinated) thanks to a companion diagnostic test based on the detection of antibodies directed against non-structural proteins. unfortunately this technology only allows certification of freedom at the herd level. in the two recent outbreaks in united kingdom, the choice was to slaughter the animals. following the dramatic outbreak of foot-and-mouth disease in united kingdom, and to a lesser extend in france and in the netherlands, the european union lightened its regulation and is nowadays more prone to consider emergency vaccination as an alternative to slaughtering. if preventive vaccines are used in an emergency situation they could still be improved by conferring an early onset of protection. whenever viruses are represented by several serotypes, an infection can re-emerge in a previously vaccinated population against another serotype than the wild circulating one. for instance, an influenza pandemic can emerge in a human population with a herd immunity against seasonal flu. in case of highly pathogenic avian influenza (particularly strain h n ), one may choose to kill the birds or to vaccinate depending on the situation [ ] . there are some instances where vaccination is the only reasonable option, particularly when facing arthropod-borne virus infections. among arthropod-borne infections, there are infections caused by viruses represented only by one serotype, such as west nile virus or rift valley fever virus and others caused by viruses presenting multiple serotypes such as bluetongue virus. the majority of animal infections involve only two partners: the pathogen and the host, which has at its disposal its genetic background (natural resistance to infection) [ ] and its immune system. besides these infections or infestations, some vectorial infections are arthropod-borne and mainly transmitted by biting arthropod. these infectious systems are therefore more complex, because the vectors must be competent and able to multiply the pathogenic agent. in northern america there was the spectacular spread of west nile virus infection in humans and horses, rapidly followed by the development of several vaccines for horses, including a dna-based one. vaccination seems to be the only option since the reservoir is to find among birds and the infection is transmitted by mosquitoes. it is therefore nearly impossible to control the infection by other mean than vaccination without highly detrimental effect on the environment [ ] . rift valley fever is expanding its range in africa [ ] . until it was introduced into saudi arabia and yemen in ; rift valley fever tended to be confined to sub-saharan africa. the disease has recently occurred in madagascar. as of july , at least people reportedly died as a result of the infection, and the disease has claimed the lives of thousands of animals since the beginning of the year . since the disease is transmitted by mosquitoes, extreme weather events might create the necessary conditions for rift valley fever to expand its geographical range northwards and cross the mediterranean and arabian seas, with an unexpected impact on the animal and human health of newly affected countries [ ] . once again, vaccination is the best way to prevent the disease; an attenuated vaccine exists for sheep but is still abortigenic and should be improved. an arthropod-borne disease caused by a virus with multiple serotypes is typically bluetongue ( serotypes) and perhaps a new one [ ] . until , bluetongue was only observed in the mediterranean regions of europe and only serotypes , , , , and were involved. the disease appeared unexpectedly in northern europe in and serotype was involved, which is typically a sub-saharan serotype. cattle and sheep herds were fully susceptible to the infection; moreover, the strain involved was particularly virulent in cattle. bluetongue is transmitted by a biting midge, a member of the family ceratopogonidae, the culicoides. in the mediterranean region, the main species involved in the transmission is culicoides imicola, which originated in africa and asia and extended its range towards the north of its previous distribution, probably due to climate change. however climatic change does not seem to be responsible for the extension of the infection in northern europe, since the main culicoides species involved is culicoides dewulfi, a typically nordic species, whose transmission competence was unknown until bluetongue appeared in northern regions; in fact, this potential vector competence had not previously had the opportunity to be expressed due to the lack of bluetongue virus. the biology of the larval stage of culicoides impedes the control of the vector without damaging the environment. the most sensible option is vaccination of domestic ruminants with an inactivated vaccine containing serotype . in developed countries, partly as a result of overproduction, public concern for food security has been replaced by a major concern about food safety [ ] . this concern has increased following the bse (bovine spongiform encephalopathy) crisis. people are concerned about food-borne infections, the presence of drug residues following treatment of food-producing animals and the possible transfer of antibiotic resistance from bacteria causing disease in livestock to those which affect man [ ] . veterinary vaccines may help to solve some of these problems. the best example of a veterinary vaccine used for public health purposes is the vaccination of wildlife against rabies; the primary goal was not to protect wildlife species from rabies but to prevent human exposure and the disease in human populations [ , ] . being considered as products working by natural mechanisms, vaccines, except for some of their excipients, do not need to have an mrl (maximum residue limit) determination associated with a withdrawal period. in fact, since vaccine protection works after a lag period, the use of vaccines intrinsically contains a withdrawal period. veterinary vaccines can be used to prevent food poisoning as demonstrated by the "in ovo" vaccination of poultry against salmonellosis, in order to decrease carcass contamination. more recently a vaccine against escherichia coli :h has been conditionally approved for cattle in the united states. a vaccine against sheep cysticercosis has been developed experimentally and may lead to the development of similar vaccines to control bovine cysticercosis and thus taenia saginata infestation in humans. bacterial resistance to antibiotics is an emerging problem for both the animal and public health sector. several antibacterial vaccines used in veterinary medicine disappeared after the second world war, and were replaced by the use of antibiotics. the resistance to antibiotics in the animal health sector with possible implications for human health, as well as the resistance of several parasites to anthelmintics may lead to the reappearance or the appearance of antibacterial and antiparasitic vaccines. even if other pathways such as the selection of food-producing animals for genetic resistance to diseases are followed, the story of marek's disease in chickens demonstrates that vaccines are often more economical to procure an animal's resistance to pathogens. canine 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sources of zoonotic diseases key: cord- -ah dvnxv authors: cao, weiping; kim, jin hyang; reber, adrian j.; hoelscher, mary; belser, jessica a.; lu, xiuhua; katz, jacqueline m.; gangappa, shivaprakash; plante, martin; burt, david s.; sambhara, suryaprakash title: nasal delivery of protollin-adjuvanted h n vaccine induces enhanced systemic as well as mucosal immunity in mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ah dvnxv sporadic, yet frequent human infections with avian h n influenza a viruses continue to pose a potential pandemic threat. poor immunogenicity of unadjuvanted h n vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. here, we show that protollin, a nasal adjuvant composed of neisseria meningitides outer membrane proteins non-covalently linked to shigella flexneri a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion h n clade a/viet nam / (a/vn/ / ) vaccine in a mouse model. protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal iga responses, and h n -specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade virus a/indonesia/ / (a/in/ / ). detailed analysis of adaptive immunity revealed that protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of b cells, broad-spectrum of cd t cell response. our findings suggest that nasal delivery of h n vaccine with protollin adjuvant can overcome the poor immunogenicity of h n vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade and clade h n viruses and achieve significant antigen dose-sparing. the world has already experienced three influenza pandemics in the th century in addition to the recent pandemic caused by swine-origin h n virus, which has taught us how newly emerging pathogens can be a risk to global populations [ ] . highly pathogenic h n avian influenza viruses continue to be prime candidates for the next influenza pandemic, as they have steadily caused fatal infections in the human population [ ] . so far, direct human-to-human transmission appears to be infrequent; however, the accumulation of mutations may break this genetic barrier and generate an h n virus that is transmissible among humans potentially causing pandemic. the inherently poor immunogenicity of unadjuvanted h n influenza vaccines warranted efforts to explore novel adjuvants and alternate delivery systems to improve immunogenicity and protective efficacy of h n vaccines. intradermal (i.d.), oral and nasal delivery of vaccines represent alternative routes to conventional intramuscular (i.m.) delivery [ ] . unlike i.m. and i.d. routes, oral and nasal deliveries are noninvasive and needle-free that are known to induce mucosal and systemic immune responses [ ] . since degradation of antigens by proteolytic enzymes, poor absorption, need for large doses of antigen, and concerns about tolerance still remain as challenges for oral delivery of non-replicating vaccine antigens [ ] , the nasal route has become an attractive option for vaccination, particularly against respiratory pathogens as the respiratory tract is equipped with pathogen sensing defense mechanisms, has a large surface area for absorption, and is highly vascularized [ , ] . a live attenuated seasonal influenza vaccine, which is delivered intranasally (laiv, flumist Ò , fluenz tm ) (medimmune astrazenica) is approved for healthy individuals of - years of age in the us. however, the absence of correlates of immunity, cold-storage requirement, and potential of genetic recombination with seasonal influenza viruses may limit the use of laiv as a pre-pandemic influenza http vaccine [ ] . therefore, an ideal h n vaccine formulated/adjuvanted for nasal delivery should induce mucosal as well as systemic serological and cellular immune responses that can correlate with protection. protollin, a nasal adjuvant composed of neisseria meningitidis outer membrane proteins (omps) noncovalently complexed to shigella flexneri a lipopolysaccharide (lps) activates the innate immune system via activation of tlr and tlr by omp and lps respectively. protollin has been shown to induce effective systemic as well as mucosal antibody responses in preclinical studies when administered with several viral antigens such as measles, sars corona virus, rsv, and recombinant plague antigen f -v [ ] [ ] [ ] [ ] [ ] . therefore, protollin represents an attractive adjuvant suitable for nasal delivery of pre-pandemic h n vaccines. in this study, we evaluated the immunogenicity and protection against challenge conferred by protollinadjuvanted h n inactivated split vaccine. h n monovalent influenza split vaccines (a/vn/ / and a/in/ / ) were provided by glaxosmithkline vaccines (ste-foy, quebec, canada). protollin from gsk vaccines consists of proteosomes (outer membrane proteins derived from wild-type neisseria meningitidis group b) noncovalently complexed in approximately a : ratio with lipopolysaccharide (lps) isolated from shigella flexneri serotype a. the amounts of protollin were expressed as lg of lps and the amount of h n vaccine was expressed as lg of ha. two virus strains were made by reverse genetic engineering, ha and na from clade a/viet nam/ / (a/vn/ / ) or clade a/indonesia/ / (a/in/ / ) and the remaining six gene segments from a/puerto rico/ / -(pr ). viruses were propagated in -day old embryonated chicken eggs for h. pooled allantoic fluid was clarified by centrifugation, aliquoted, and stored at À °c until use. female balb/c mice (jackson laboratory, bar harbor, maine) weeks of age were anesthetized with intraperitoneal injection (i.p.) of , , -tribromoethanol in tert-amyl alcohol (avertin; sigma-aldrich, st. louis, mo) before nasal inoculation with , , or . mg of h n vaccine, either alone or with mg of protollin [ , ] in a final volume of ml administered slowly drop by drop at ml each per nostril. both the vaccine antigens and adjuvants were mixed prior to immunization. mice nasally administered with adjuvant alone were used as negative controls. four weeks later, mice received a second dose of the same vaccine preparation. one week after boost, mice were euthanized for collection of spleen and bone marrow tissues to assess adaptive immunity. bronchoalveolar lavages (bal) and nasal lavages were also performed at the same time by injecting . ml pbs with protease inhibitors and re-collecting the fluid to measure mucosal antibody response by elisa. mice were bled weeks post-primary and again weeks post-boost to measure hemagglutination inhibition (hi) titers using horse red blood cells. four weeks after boost, mice from each group were challenged with ld of a/vn/ / or a/in/ / viruses. animal were monitored daily for morbidity by changes in body weight and any mouse that lost > % of preinfection body weight was euthanized. animal research was con-ducted under the guidance of the cdc's institutional animal care and use committee. statistical analysis was performed using graphpad prism . software (graphpad software, la jolla, ca). the student's t test was used to analyze differences in antibody isotype between two groups. one-way analysis of variance with bonferroni post analysis was used to analyze differences among treatments. the mann-whitney test was used to determine significance among hi titers. finally, the logrank (mantel-cox) test was used to compare percent survival among groups of mice. all differences were considered statistically significant when the p-value was . . to investigate the adjuvant effect of protollin, mice were nasally administered with , , or . mg of h n vaccine with or without mg of protollin using a prime-booster regime and then challenged with homologous a/vn/ / viruses. all mice immunized with or . mg of h n vaccine alone succumbed to challenge between day and post-challenge, while % of mice that received the highest dose of vaccine ( mg) initially showed significant weight loss but eventually survived challenge ( fig. a and b ). all mice immunized with vaccine plus protollin including mice that received the lowest vaccine antigen dose ( . mg), survived and no significant weight loss was observed ( fig. a and b) . these data demonstrate that protollin significantly enhanced the protective efficacy of h n vaccine. to test whether the protollin-mediated protection extended against cross-clade h n virus, h n vaccine-immunized mice were challenged with the clade virus, a/in/ / . mice immunized with either protollin or vaccine alone ( . or mg) succumbed to challenge ( fig. c and d) . among the mice immunized with mg vaccine alone, % of mice showed % weight loss and eventually recovered and survived. however, protollinadjuvanted vaccine, even at the lowest vaccine dose ( . mg) significantly reduced weight loss and completely protected mice against the lethal challenge ( fig. c and d) . therefore, protollin also enhanced cross-clade protection with significant antigen dosesparing. next, we assessed whether the improved protection conferred by protollin-adjuvanted h n vaccine was due to enhanced antibody titers. mice were immunized as described in fig. and sera were collected at weeks following primary immunization (week ) and booster immunization (week ) to measured hi titers against a homologous a/vn/ / virus as well as heterologous a/in/ / virus. mice immunized with unadjuvanted h n vaccine did not induce detectable antibody titers following primary immunization. however, mice immunized with protollin -adjuvanted h n vaccine ( mg ha) developed low level of antibody titers even after a single vaccination (data not shown). at week , all mice immunized with vaccine plus protollin developed significantly higher levels of hi titers against both homologues and heterologous viruses ( fig. a and c) . however, none of the mice immunized with vaccine alone had detectable hi titers against a/ vn/ / virus ( fig. a) and only a few mice immunized with either or mg ha of unadjuvanted vaccine developed detectable hi titers against a/in/ / virus (fig. c) . furthermore, vaccine alone elicited more igg production than igg a, whereas protollin adjuvantation induced a mixed igg and igg a response with a clear trend towards a t h response (igg a-dominant) ( fig. b and d) . these results indicate that protollin significantly enhanced the immunogenicity of h n vaccine and skewed the immune response towards a t h response. to determine whether protollin increased the frequency of agspecific memory b cells, mice were immunized as described in fig. and the frequency of h n -specific antibody-secreting cells (ascs) in spleen and bone marrow was examined by elispot assay. as shown in fig. a and b, the frequency of h n -specific igg and igm ascs in the spleen increased in mice immunized with unadjuvanted h n vaccine; protollin significantly increased the frequency of h n -specific igg and igm ascs even at the lowest dose ( . mg), although ag-specific igm ascs frequency is much lower than that of ag-specific igg ascs. in bone marrow, the frequency of h n -specific igg ascs was similarly enhanced by the use of protollin as an adjuvant, while the frequency of igm ascs was minimally affected by protollin ( fig. c and d) . together, these data suggest that intranasal administration of h n vaccine with protollin enhanced the overall frequency of ascs with significant dose-sparing effect. next, the activation of vaccine-specific t cells in spleen was assessed in mice immunized with mg h n vaccine with or without protollin. for cd t cells, while the production of il- and ifnc was detectable in mice immunized with unadjuvanted vaccine as compared to the protollin control group, protollin adjuvantation further increased the il- and ifnc production significantly (fig. e ). on the other hand, il- , il- , tnfa, and il- production by activated cd t cells was comparable between protollin alone vs. vaccine group; however, co-administration with protollin and vaccine significantly increased il- , tnfa and il- production from cd t cells. vaccine-specific cd t cells responses were slightly increased upon protollin adjuvantation but this increase was not statistically significant (data not shown). in summary, our data suggest that protollin-adjuvanted h n vaccine enhanced a broad spectrum of the cd t cell activation pathways: t h (ifnc and tnfa), t h (il- and il- ) and t h (il- ). systemic increase in asc frequency by protollin prompted us to assess the extent of homing of ascs back to the local immune induction site. thus, we measured the h n -binding iga by elisa week following booster immunization in the fluids from bronchoalveolar (bal) and nasal lavage from mice that had been immunized as previously described in fig. . the iga secretion from unadjuvanted h n vaccine antigen-immunized mice was barely detectable both in nasal lavage (fig. a) and bal (fig. b ). in contrast, the mice immunized with h n vaccine antigen plus protollin produced a/vn/ / -specific iga at a minimum of fold higher than that of mice immunized with the unadjuvanted vaccine. in addition, protollin significantly enhanced clade a/ in/ / -specific iga secretion both in nasal and bal as compared to vaccine alone ( fig. c and d) ; secretion of igg antibodies was also greatly enhanced by protollin and the reactivity was extended against clade , a/in/ / virus (fig. a-d) . however, the frequency of igm ascs was much lower and minimally affected by protollin (fig. e-h) . these data indicate that nasal administration of protollin-adjuvanted h n vaccine enhanced local mucosal immunity throughout the respiratory tract. next, the activation status of b cells at peak of the primary response (day ) was assessed by flow cytometric analysis. com- pared to mice immunized with vaccine alone, mice immunized with vaccine plus protollin had a higher frequency of b cells participating in gc reaction (fig. a) , higher overall mean fluorescence intensity of cd among gc-participating b cells, and more gc-b cells expressing high level of cd ( fig. c and d) . the plasma cell differentiation was at low level and did not reveal differences among groups (fig. b) . interestingly, protollin alone recruited b cells into gc reaction and induced activation (fig. a) , emphasizing the significant role of innate signaling through tlr and tlr in b cell activation. nonetheless, their recruitment to gc reaction and activation did not result in the production of ag-specific antibodies (fig. ) . we also measured the frequency of the splenic ag-specific igg and igm during the primary response and found that protollin adjuvantation significantly increased the early igg secretion and marginally igm secretion ( fig. e and f) . overall, these data suggest that protollin recruited more naïve b cells at the early phase of the vaccine response. the spread and evolution of highly pathogenic influenza h n virus in birds worldwide and the increasing number of cases of direct transmission to humans leading to fatalities, have raised concern about an imminent h n influenza pandemic [ ] . vacci-nation is unquestionably one of the most cost-effective public health interventions available to protect against such pandemics. however, currently available unadjuvanted h n vaccines administered by i.m. route are poorly immunogenic. new strategies to improve the immunogenicity of vaccines with adjuvants, novel formulations and alternate delivery methodologies to meet the demands of global populations are still urgently needed. in the current study, we evaluated the use of protollin-adjuvanted h n vaccine as a nasally delivered, pre-pandemic vaccine in a mouse model. the mucosal immune system in the respiratory track plays an important role in prevention of influenza virus infection. the upper respiratory tract is equipped with not only the physical and dynamic barrier of mucus as well as both soluble and membrane bound pathogen sensors but also contains nasopharyngealassociated lymphoid tissues (nalt) that are enriched in agspecific mucosal effector cells as well as innate immune cells [ ] . hence, nasal delivery of antigens triggers local mucosal as well as a systemic immune response, which is crucial against respiratory pathogens including influenza [ ] . nasal delivery of split, inactivated influenza vaccine generally requires a mucosal adjuvant to induce strong protective immune responses [ ] . some mucosal adjuvants containing bacterial toxin derivatives, including esherichia coli heat labile enterotoxin (lt) have been clinically eval- . the data is representative of independent experiments ( - mice per group) and error bars represent standard error of the mean (sem). oneway analysis of variance with bonferroni post analysis was used to analyze differences among treatments. the student's t test was used to analyze differences in antibody isotype between two groups. uated with subunit influenza vaccines and ultimately licensed in europe, but soon withdrawn from the market due to an increased incidence of bell's palsy post-vaccination [ ] . clinical trials with proteosome-adjuvanted either monovalent h n or trivalent inactivated vaccines have shown that proteosome-adjuvanted vaccines are well-tolerated, while inducing significantly higher serum hi and mucosal secretory iga titers as compared to unadjuvanted vaccines [ ] [ ] [ ] . preclinical studies using protollin-adjuvanted h n vaccines also showed the similar results [ , ] consistent with this, our data showed that protollin-adjuvanted h n vaccine induced significantly higher levels of mucosal iga antibodies against a/vn/ / virus both in nasal washes and lung, with considerable dose-sparing effect. the breadth of the mucosal iga response was extended against clade virus, a/in / , as a pre-pandemic vaccine formulation, cross-clade immunity is an important and desirable feature, because the current endemic h n viruses continuously evolve in ha antigenicity through antigenic drift and reassortment, thus making it hard to predict which strain will cause a pandemic. protollin also induced cross-reactive igg as well as low, yet detectable levels of igm antibodies. lung-resident memory b cells as well as the mucosal, secretory iga and/or igg antibodies play a crucial role in conferring protection against influenza virus challenge [ , ] . the mice immunized with protollin-adjuvanted h n vaccine were completely protected against lethal challenge with clade and clade viruses. therefore, the protollin-adjuvanted h n represents a novel pre-pandemic vaccine candidate that is efficient in inducing protective mucosal and systemic immunity. the adjuvant properties of protollin have been documented in conjunction with antigens from various infectious agents including measles [ ] , recombinant sars-spike glycoprotein [ ] , respiratory syncytial virus (rsv) [ , ] , and recombinant plague antigen f -v [ ] . the level of serum igg response induced by protollin is generally comparable to that by alum-adjuvanted vaccines and shows a mixed t h /t h response with a t h trend (higher igg a/igg ratio) as compared to unadjuvanted vaccines [ , , ] . consistent with these findings, our data demonstrated that protollin potentiated the immunogenicity of h n vaccine. after a single vaccination, mice immunized with protollin -adjuvanted h n vaccine ( mg) developed detectable levels of serum hi titers. following booster immunization, protollin -adjuvanted h n vaccine significantly increased serum hi titers compared to unadjuvanted vaccine, which coincided with complete protection against homologous virus challenge. the breadth of antibody response was also broadened by protollin-adjuvanted h n vaccine, as they significantly increased serum hi titers against a/in/ / virus compared to the vaccine alone group and fully protected mice against a/in/ / virus challenge. protection against influenza virus infection includes antibodymediated neutralization/blocking of virus and cell-mediated clearance of virus-infected cells. although the antigen-specific cd t cell responses was not significantly enhanced by protollin adjuvantation (data not shown), protollin-adjuvanted vaccines significantly increased the production of cytokines by antigen-specific cd t cells: t h (ifn-c, tnfa), t h (il- ) and t h (il- ). ifnc, as a representative cytokine of t h response promotes overall microbial killing through enhancing phagocytosis and recruiting mononuclear cells into the site of infection, while favoring the production of igg a from b cells [ ] [ ] [ ] . on the other hand, il- along with il- , shapes the t h response and promotes the production of igg isotype [ ] . consistent with the cytokine profiles, the mice immunized with protollin-adjuvanted h n vaccine showed a mixed igg /igg a response with higher production of igg a than igg . il- is typically associated with pathogenesis of inflammatory diseases [ ] . however, the protective role of il- for survival against high dose challenge and severe cases has been recently demonstrated [ , ] . of interesting note, protollin-adjuvanted h n vaccine elicited a sharp increase in il- production as compared to vaccine alone. il- is an anti-inflammatory cytokine produced by natural cd + cd + foxp + regulatory t cells (tregs) as well as antigen-specific cd + and cd + effector t cells [ ] [ ] [ ] . it remains unclear the source of il- in our study, but regardless, the enhanced il- production indicates the fine balance between effector vs. control mechanisms was achieved by protollin. using gene knock-out mice, tlr has been shown to be critical for antigen-specific antibody responses in protollin-adjuvanted rsv vaccine, while myd was required to elicit a balanced t h /t h immune responses. although tlr is required for nanoparticle formation, it is not playing a role in the adjuvanticity of protollin [ ] . in summary, protollin demonstrated a significant antigen dosesparing effect. with protollin-adjuvantation, h n vaccine antigen at the -fold lower dose elicited higher serum hi titers, mucosal antibody responses than the highest vaccine antigen dose ( mg ha/mouse) against both clade and clade h n viruses. these enhanced immune responses culminated in enhanced protection against challenge with lethal doses of h n viruses of either clade or clade . therefore, nasal delivery of protollin-adjuvanted h n vaccines is a promising approach that merits further development to prepare for a h n pandemic. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention or the funding agencies. w.c., j.k., s.s. designed the experiments and interpreted the data; w.c., j.k., a.r. and m.h. did the in vivo experiments; j.b. did the challenge experiment; x.l did the hi assay; w.c., j.k., and s.s. wrote the manuscript. s.g., j.k., m.p. and d.b. edited the manuscript. dsb and mp were employees of the gsk group of companies at the time of the study, own stock options in gsk and are listed as inventors on patents owned by the gsk group of companies. the remaining authors declare no commercial or financial conflict of interest. work was supported by the influenza division, centers for disease control and prevention. . protollin increased the early b cell response at the local lymph nodes and spleen. balb/c mice ( - mice/group) were nasally administered with mg ha h n split virion vaccine antigen with or without mg protollin or protollin alone as a control. a week later, mediastinal lymph nodes and/or spleens were collected and b cells (b + cd À ) were stained. the lymph nodes were analyzed for percentage of gc-participating b cells (b + cd À gl + cd À ) (a), plasma cells (b À cd + ) (b), the mean fluorescence intensity (mfi) of cd (c) as well as % b cells expressing high cd (d) among gc-participating b cells. spleen were harvested and the frequency of a/vn/ / -specific igg + ascs (e) or igm + ascs (f) were measured by elispot assay. the number of a/vn/ / -specific igg + (or igm + ) ascs were normalized against the number of total igg + (igm + ) secreting ascs and presented as % ag-specific igg + (igm + ) b cells. the data is representative of at least independent experiments ( - mice each group) and the error bars represent standard error of the mean (sem). one-way analysis of variance with bonferroni post analysis was used to analyze differences among treatments. two years after pandemic influenza a/ /h n : what have we learned? human influenza a h n virus related to a highly pathogenic avian influenza virus microneedle and mucosal delivery of influenza 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induce stronger immunogenicity in mice and confer higher protection in ferrets than unadjuvanted inactivated vaccines memory b cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection cross-protection in mice infected with influenza a virus by the respiratory route is correlated with local iga antibody rather than serum antibody or cytotoxic t cell reactivity interleukin- and interferon-gamma: the quintessence of a mutual antagonistic relationship functional diversity of helper t lymphocytes cellular responses to interferongamma th and th hypercytokinemia as early host response signature in severe pandemic influenza il- deficiency unleashes an influenza-specific th response and enhances survival against high-dose challenge imbalanced pro-and anti-th responses (il- /granulocyte colonystimulating factor) predict fatal outcome in pandemic influenza the development and function of memory regulatory t cells after acute viral infections the regulatory t cells in antiinfluenza antibody response post influenza vaccination effector t cells control lung inflammation during acute influenza virus infection by producing il- tlr and myd control protection and pulmonary granulocytic recruitment in a murine intranasal rsv immunization and challenge model we thank the members of influenza division, centers for disease control and prevention (cdc) for providing reagents and constructive comments during the course of this investigation. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . . key: cord- -oq o qr authors: ahlquist, paul; schwartz, michael; chen, jianbo; kushner, david; hao, linhui; dye, billy t. title: viral and host determinants of rna virus vector replication and expression date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: oq o qr positive-strand rna viruses have proven to be valuable vectors for delivery and expression of antigens for direct vaccination of animals and vaccine production in plants. however, optimal use of these viruses as vectors for vaccine and other purposes is limited by incomplete understanding of their replication pathways and associated constraints on inserted foreign genes. further insights into rna virus vector design and optimization are emerging from recent advances on the function of viral rna replication factors, the nature of the viral rna replication complex as a membrane-bounded compartment sequestering replication components from competing processes and host defenses, and identification of surprisingly diverse host genes contributing to many virus replication steps. as the premier natural agents for gene transfer and expression, viruses and their derivatives are valuable tools for engineered gene expression in medicine, biotechnology and research. this includes not only dna viruses and the reverse transcribing retroviruses, but also rna viruses, which replicate and express their genes solely through rna intermediates. the largest class of rna viruses are the positivestrand rna viruses, which package messenger-sense, single strand rna in their virion particles. these viruses include many important pathogens such as hepatitis c virus, the severe acute respiratory syndrome (sars) coronavirus, poten-tial bioterrorism agents, and the vast majority of known plant viruses. nevertheless, such positive-strand rna viruses also have beneficial uses, serving as useful expression vectors in both animals and plants [ , ] . among other advantages, such viruses generally have small genomes, high wild type replication and gene expression levels, and lack dna forms to genetically transform host dna. vector derivatives of such viruses can provide high level expression of recombinant proteins and rnas for many purposes including direct immunization of humans and animals, and vaccine production in plants. efficient use of such rna virus vectors presently is limited in part by incomplete understanding of their replication cycle and its constraints. for example, insertion of foreign genes in rna viruses as a payload for vaccine production or other directed expression often decreases the efficiency of viral genomic rna replication and subgenomic mrna expression for reasons that are not well understood. the degree to which viral genomic rna replication is reduced varies with the gene inserted, is often substantial, and is not simply a function of the length of the inserted foreign sequence. such reductions in replicative fitness not only reduce the level of gene expression but usually make the expression vector genetically unstable, since the high levels of rna recombination in such viruses lead to the appearance of deletion variants that have lost part or all of the payload gene, and thereby gained increased replicative fitness that allows them to overgrow the starting vector ( [ ] and references therein). since the mechanism(s) by which foreign genes inhibit viral genome replication are not understood, such results imply the existence of presently unrecognized requirements for viral rna replication. better understanding of such constraints and the overall mechanisms by which positive rna viruses replicate their genomes and synthesize mrnas could greatly increase the utility of these viruses as vectors for protein expression, rna expression, rna silencing and other applications. advances in understanding these viral replication mechanisms also offer targets for improved virus control, better understanding of virus pathology, and other benefits. one virus that is being used as a model to study positivestrand rna virus replication is brome mosaic virus (bmv). bmv is a representative member of the alphavirus superfamily of human, animal and plant viruses. all members of this superfamily share multiple conserved domains in their rna replication proteins and conserved features in their rna replication pathways. bmv was the first rna virus engineered to express foreign genes [ ] , related human and animal alphaviruses have been found to be valuable vectors for gene expression in animal cells [ , ] , and related plant viruses such as tobacco mosaic virus are useful vectors for gene expression in plants [ ] . bmv encodes two large, multifunctional rna replication proteins, designated a and a. a has a c-terminal helicase domain and an n-terminal domain with m gtp methyltransferase and covalent gtp binding (putative guanylyltransferase) activities required for capping viral rna in vivo. a has a central polymerase-like domain and an n-terminal extension that interacts with the helicase-like domain of a. below we discuss selected recent findings on viral rna replication mechanisms from studies of bmv, including the nature of the viral rna replication complex and the role of surprisingly diverse, host-encoded functions in viral rna replication and gene expression. some advances with potentially important mechanistic implications for rna virus vectors have come from the realization that bmv rna replication does not occur in the open cytoplasm but rather in a virus-induced, membrane-bounded compartment [ ] . these findings appear to have relevance for additional positive-strand rna viruses since all such viruses replicate their rna on intracellular membranes, usually in association with vesicles or other membrane rearrangements. the structure of such replication complexes, their interaction with the host, and the processes by which rna templates are recruited and progeny rna products exported appear likely to have significant practical effects on the optimal design and performance of rna virus vectors. the recent bmv results emerged from a combination of genetic, biochemical and cell biology approaches [ ] [ ] [ ] . these findings show that the bmv a rna replication protein plays key roles in directing the form and assembly of the rna replication complex (fig. ) . a localizes to the cytoplasmic face of the perinuclear endoplasmic reticulum (er) membrane, and induces the membrane to invaginate into the er lumen to form - nm vesicles or spherules [ ] . the interiors of these er luminal, membrane-bound spherules, which remain connected to the cytoplasm by a narrow, membranous neck, become compartments or mini-organelles for viral rna synthesis. a is the sole viral factor needed to induce spherule formation. by other interactions (fig. ) , a also independently recruits viral rna templates and a polymerase to these compartments, which become the sites of negative-strand rna synthesis. negative strand rnas then are retained in these compartments and used as templates to synthesize new positive-strand rna for further viral translation and assembly of new infectious virions. thus, these replication compartments concentrate the viral replication factors and rna templates and link successive rna replication steps. viral positive-and negative-strand rna templates in these structures also are protected from nucleases [ ] , suggesting that these compartments also protect potentially double stranded (ds) viral rna replication intermediates from dsrna-induced host defense responses including rna interference and interferon responses [ ] . immunogold electron microscopy and biochemical approaches show that each spherule contains one to a few hun-dred copies of a [ ] . since a self-interacts [ ] , these large numbers of a proteins may form a capsid-like protein shell to direct the formation and membrane envelopment of the spherular replication compartment. structure and assembly of the spherular replication compartment thus appear potentially very similar to those of a budding, membrane-enveloped virion particle. in particular, bmv rna replication complex assembly closely parallels the steps by which the reversetranscribing, replicative cores of retrovirus virions assemble and become membrane enveloped. specifically, the functions discussed above for bmv a, a polymerase, and certain arecognized cis-acting signals on bmv genomic rnas recapitulate the functions of gag (the major capsid protein), pol (polymerase or reverse transcriptase) and rna packaging signals in virion assembly by retroviruses like hiv [ ] . the similarities revealed bridge retroviruses, positive strand rna viruses and dsrna viruses, which also package rna templates and rna polymerase in a protein shell for replication. these and other similarities suggest that all three virus classes use related mechanisms for nucleic acid replication and may have evolved from common ancestors. in addition to virus-encoded factors, most if not all steps in virus infections involve host factors [ ] . such virus-host interactions are crucial determinants of virus host range, replication, and pathology, offer insights to viral and cellular function, and provide antiviral targets. identifying such interactions and the associated host factors thus is a major frontier in virology. an unusual feature of bmv for identifying and characterizing host functions in viral replication is that bmv directs rna replication, gene expression and virion formation in the genetic model yeast, saccharomyces cerevisiae [ ] . the potent approaches of yeast genetics and the large and growing understanding of yeast molecular biology thus can be applied to studying bmv replication and virus-host interactions. interestingly, such yeast genetic studies of bmv replication depend heavily on using engineered virus derivatives as vectors to express selectable, counter-selectable, or screenable marker genes, making colony-level yeast phenotypes dependent on viral rna replication. in recent years, classical yeast genetics have been used to identify host genes that function in controlling bmv translation [ , ] , selecting bmv rnas as replication templates [ ] , activating the viral rna replication complex [ ] , maintaining a lipid composition required for membrane-associated rna replication [ , ] , and other steps. to more globally and systematically identify host factors affecting virus replication, we also have used engineered bmv derivatives and high-throughput approaches to individually assay viral rna replication in each strain of an ordered, genome-wide set of ∼ yeast single-gene-deletion strains, covering ∼ % of all yeast genes [ ] . specifically, we transformed each of the yeast deletion strains with plasmids expressing bmv a, a and a bmv rna replication template with the capsid gene replaced by a luciferase reporter gene. luciferase expression then depended on viral rna replication and rna-dependent mrna synthesis. bmv-directed luciferase expression levels in this entire collection of deletion strains were independently assayed twice, and selected strains were analyzed further with detailed assays for various viral rnas, proteins and activities. this systematic approach identified nearly genes whose absence either inhibited or, in a smaller number of cases, stimulated bmv rna replication and gene expression by -to > -fold [ ] . examples of some of the host genes identified and the effects of their deletion on the accumulation of viral rna replication products are shown in fig. . of the pool of yeast genes identified, several had previously been shown to function in bmv replication, confirming that this approach could identify relevant host genes. yeast genes that were newly implicated in bmv rna replication by this screen included genes in rna, protein or membrane modification pathways, and many genes of presently unknown function. thus, these screens identified many new host factors that affect bmv replication and implicated previously unconsidered pathways in the virus lifecycle. further studies should determine more directly the diverse roles by which these host factors contribute to virus replication and identify additional host genes, such as essential genes not covered by this screen, that contribute to bmv replication. moreover, since even the cellular function of many of these genes is unknown, these virus-motivated studies also should help to illuminate basic cell biology. like the growing understanding of virus-encoded replication functions discussed earlier, developing a basic understanding of the essential ways in which rna viruses interact with their hosts to replicate and express their own genes should provide new insights for using such viruses to optimally deliver, maintain, replicate, and express foreign genes, such as those for vaccine antigens. in these and other ways, basic studies should provide the foundation for translating the full potential of these viruses, most frequently considered as harmful pathogens, into useful tools. replicon-based vectors of positive strand rna viruses making an ally from an enemy: plant virology and the new agriculture the a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus rna replicons in yeast but retained over ( )-fold amplification bacterial gene inserted in an engineered rna virus: efficient expression in monocotyledonous plant cells alphavirus vectors for gene therapy applications alphavirus replicon particles as candidate hiv vaccines a positive-strand rna virus replication complex parallels form and function of retrovirus capsids brome mosaic virus rna replication protein a dramatically increases in vivo stability but not translation of viral genomic rna a brome mosaic virus intergenic rna replication signal functions with viral replication protein a to dramatically stabilize rna in vivo rna-dependent rna polymerases, viruses, and rna silencing interactions between the structural domains of the rna replication proteins of plant-infecting rna viruses host factors in positive-strand rna virus genome replication rna-dependent replication, transcription, and persistence of brome mosaic virus rna replicons in s. cerevisiae a mutant allele of essential, general translation initiation factor ded selectively inhibits translation of a viral mrna yeast lsm p- p/pat p deadenylation-dependent mrna-decapping factors are required for brome mosaic virus genomic rna translation identification and characterization of a host protein required for efficient template selection in viral rna replication mutation of host dnaj homolog inhibits brome mosaic virus negativestrand rna synthesis mutation of host fatty acid desaturase inhibits brome mosaic virus rna replication between template recognition and rna synthesis membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand rna virus rna replication protein systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus we thank all members of our laboratory for insights and helpful discussions. this research was supported by the national institutes of health through grant gm . p.a. is an investigator of the howard hughes medical institute. key: cord- - r ng a authors: graffigna, guendalina; palamenghi, lorenzo; barello, serena; stefania, boccia title: “cultivating” acceptance of a covid- vaccination program: lessons from italy date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: r ng a nan dear editor, in their article, garcía and cerda [ ] found a relatively high vaccine acceptance rate ( . %) and stated that the covid- pandemic has not only had a negative impact on people's health and life behavior, but also on economies around the world. we agree with the authors and we think that a robust vaccination program against sars-cov- would mitigate its huge effects on social, clinical, and economic levels for the global population. however, vaccine hesitancy risks to hamper such effort. in italy, which is among the countries most severely impacted by covid- outbreaks early on, experts have been primarily involved in social and traditional media to disseminate scientific knowledge against covid- vaccine-related fake news [ ] . however, improving health literacy is probably not enough to improve vaccine acceptancy [ ] . citizens need to be engaged in a deep process of psychological acceptance, in order to adopt a socially responsible approach to covid- preventive behaviors [ , ] . a sample of individuals representative of the italian adult population was involved in a cross-sectional survey study in may . participants were asked whether they would vaccinate against covid- , along with questions regarding their attitude towards preventive behaviours. a series of x contingency tables with pearson's chi-squared tests was performed to test variables association. percentage distribution of people willing and unwilling to vaccinate were compared across groups with a z-score test. results of our study showed that % of the participants reported to be unwilling or hesitant towards covid- vaccine. chi-squared and z-tests showed that the percentage of willing people is significantly higher amongst those who agree with the statement ''i have the primary responsibility for preventing the infection by covid- " ( %) when compared with those who disagree ( . %). moreover, our data showed that the percentage is higher amongst those people who agree that preventive behaviours are an act of social responsibility ( . %), when compared to those who don't agree ( . %). conversely, when comparing the percentage of willing and unwilling people between groups of persons that relied on social media to search for covid- information and people who didn't, no significant difference was found, as no difference was found between people who reported being exposed to fake news and those who didn't. our data suggest the opportunity for a shift from ''preaching the pros" of the future covid- vaccine to ''cultivating citizens' engagement" and their partnership with the institutions in the battle against covid- . citizens need to be sustained in growing more confident in their own ability to individually contribute to slow down the pandemic. involving citizens in a more accountable dialogue with public health experts is a priority; this requires a climate of mutual trust [ ] . engaging citizens towards the acceptance of the covid- vaccination is a urgent challenge that the public health community needs to face. in our opinion, developing a covid- public vaccination program should start from considering people's attitudes to engage them in an educational campaign focused on fostering the citizens-science alliance. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. contingent assessment of the covid- covid- -related web search behaviors and infodemic attitudes in italy: infodemiological study covid- : health literacy is an underestimated problem planning for a covid- vaccination program all rights reserved vaccine hesitancy' among university students in italy during the covid- pandemic the science of the future: establishing a citizen-scientist collaborative agenda after covid- . front public heal key: cord- - utk k z authors: tarpey, i.; orbell, s.j.; britton, p.; casais, r.; hodgson, t.; lin, f.; hogan, e.; cavanagh, d. title: safety and efficacy of an infectious bronchitis virus used for chicken embryo vaccination date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: utk k z commercial vaccines for in ovo vaccination have not yet been developed for infectious bronchitis virus (ibv), the major coronavirus in the poultry industry. recombinant ibvs based on the beaudette strain expressing the beaudette spike protein (beau-r) or that from the virulent m strain (beaur-m (s)) were assessed for their potential as prototype vaccines for application to -day-old embryos. pathogenicity was assessed by observing the effect on hatchability, and/or the production of nasal discharge and/or the effects on ciliary activity in the trachea at various time points post hatch. in contrast to commercial ibv vaccines given in ovo, the beau-r and beaur-m (s) strains did not reduce hatchability or cause nasal discharge, and caused minimal damage to the ciliated epithelium of the trachea. the presence of the spike protein from a virulent virus did not increase the pathogenicity of the virus according to the criteria used. assessment of the beaur-m (s) strain for efficacy showed that it protected up to % of chicks against challenge with virulent ib virus (m ) in a dose dependent manner. further egg passage of the beaur-m (s) strain (beaur-m (s) ep ) did not increase its pathogenicity though it did improve its efficacy, based on serology and protection against a virulent challenge. beaur-m (s) ep was more efficacious than beaur-m (s) protecting more birds against virulent challenge and providing a better serological antibody response. beaur-m (s) ep induced a serological response similar to that of a commercial vaccine given at day-old though the commercial vaccine provided slightly higher efficacy. these results are promising for the development of embryo safe efficacious ibv vaccines for in ovo application. infectious bronchitis virus (ibv) is one of the primary causes of respiratory disease in domestic fowl. ibvs primarily and initially infect the trachea though they can also infect the kidney and oviduct and other epithelial surfaces. infection with ibv reduces the performance of broilers and in laying birds drops in egg production and egg quality can occur. live-attenuated vaccines against infectious bronchitis are generally given to chicks at day of age in the hatch-ery by spray, or later in the field by either drinking water or spray. however, as with any live-attenuated vaccine administered via the respiratory tract, which must replicate in order to be effective, vaccination at an early age can damage the epithelial lining of the trachea and depending on the extent of this damage, secondary bacterial infections can occur. for this reason it is important to assess post vaccinal reactions when preparing vaccine strains. vaccination in ovo against marek's disease and infectious bursal disease is commonplace in the usa [ ] . in ovo delivery gives the advantage that all vaccinations can be done prior to hatching as long as the vaccines are shown to be compatible and safe. whilst considerable work has been done on in ovo delivery of marek and infectious bursal disease vaccines, little work has been done on delivering ibv vaccines in ovo. this is due to the fact that most strains of ibv are embryo lethal, both in the - -day-old embryos that are used for producing ib vaccines, and in -day-old embryos. one group has shown that in ovo vaccination with ibv can be successfully accomplished, although the strain used had residual pathogenicity even at low titres [ ] . as uniform mass application of ibv vaccines post hatch can be problematic with regard to obtaining even distribution of spray or ensuring all birds receive the correct dosage from drinking water, delivering ibv in ovo, via which a precise dosage can be achieved, could bring significant advantages to the poultry industry. the beaudette strain is a highly egg-adapted virus of the massachusetts serotype that does not cause damage to the ciliated epithelium of the trachea in vivo [ , ] . previous work has demonstrated that using a reverse genetics approach, the efficacy of the beaudette strain can be improved by substituting the beaudette spike protein with the spike protein from the virulent m strain, also of the massachusetts serotype [ ] . interestingly, although this altered the cell tropism of the virus [ ] , the pathogenicity was not altered. this strain was able to afford significant protection to vaccinated birds against challenge with a virulent m strain. the aim of this work was to determine whether beaudette strain-based ibvs produced by reverse genetics could be used as in ovo vaccines. therefore the safety and efficacy of these strains were investigated. specified pathogen free (spf) white leghorn chicken embryonated eggs (lohmann, germany) were used. all chicks were hatched in a contained environment and transferred to negative pressure isolators for the remainder of the experiments. chick starter crumbs and water were provided ad libitum during the course of the experiments. the beau-r and beaur-m (s) strains were prepared using reverse genetics as described previously [ , ] . both viruses were grown on primary chick kidney cells. the beaur-m (s) was passaged ten times on - -day-old embryonated eggs to give beaur-m (s) ep . two commercial vaccines, nobilis ma , (intervet, referred to as cv in the text) and ib mm (fort dodge, referred to as cv in the text), both of the massachusetts serotype were used. an m strain adapted to chick kidney cells (m -ck) was also used [ ] . the titres of the viruses were established in - day-old embryonated eggs, calculated using the method of reed and muench [ ] and expressed as % egg infectious doses (eid ). viruses were diluted in marek's disease vaccine diluent (nobilis marek diluent, intervet). for the ibv challenge a non-ck adapted massachusetts serotype m strain was given by the oculonasal route ( . ml/bird) at a dose of . eid per bird. for embryo vaccination, eggs on the th day of embryo development were inoculated into the amniotic fluid using a gauge, . cm long needle. virus dilution ( . ml) was inoculated. placebos received . ml of the diluent. eggs were hatched in separate incubators. hatch was assessed after . days of incubation. for day-old vaccination each bird received . ml of the vaccine dilution via the oculonasal route. examination of general health was done daily during the course of the experiments. nasal discharge was assessed by gently squeezing the nares of the chicks and determining if any fluid was visible. assessment of ciliostasis was done as previously described [ ] . chicks were killed by an intravenous injection of barbiturates and their tracheas removed aseptically. ten, mm explants of each trachea were observed microscopically and the ciliary activity of each ring was scored according to the percentage of the cilia beating on the luminal surface. post challenge ciliostasis assessments were done on days and , these days coinciding with the peak replication of ibv in the trachea. serological responses to ibv were studied using an elisa. briefly elisa plates were coated with m antigen and incubated with serial dilutions of test chicken serum for h. after washing a rabbit anti chicken antiserum conjugated to alkaline phosphatase was added for min. after washing a p-nitrophenyl phosphate substrate was added for min. titres were calculated in comparison to known negative and positive controls and a positive response was considered to be ≥ . log . a two-tailed t-test was used to compare ciliary activity and serological responses. p-values of less than . were considered to be significant. thirty-three spf eggs were inoculated with high doses of the beau-r ( . eid ) or beaur-m (s) ( . eid ) vaccines or three graded doses of the commercial vaccine (cv ; . , . or . eid ). a placebo vaccinated group was included. the chicks were allowed to hatch in separate hatchers, live chicks counted at . days of incubation and groups of at least chicks formed when the hatch rate allowed. at days post hatch the birds in each group were examined for nasal discharge and up to five chicks from each group were euthanized, their tracheas removed and assessed for ciliary activity. the remaining chicks were bled and serum collected at weeks then challenged with virulent m ibv. at and days post challenge the remaining birds in each group were euthanized, their tracheas removed and assessed for ciliary activity. one hundred spf eggs were inoculated with a dose of . eid of the beau-r, beaur-m (s) or beaur-m (s) ep vaccine candidates or with placebo. the chicks were allowed to hatch in separate hatchers and live chicks counted at . days of incubation. at , and days post hatch five chicks from each group were euthanized, their tracheas removed and assessed for ciliary activity. a group of one-day-old birds were vaccinated with the eid of the cv commercial vaccine via the oculonasal route. at and days post vaccination five chicks from this group were euthanized, their tracheas removed and assessed for ciliary activity. the chicks were bled and serum collected at weeks then challenged with virulent m ibv at weeks. at and days post challenge birds from each group were euthanized, their tracheas removed and assessed for ciliary activity. sixty spf eggs were inoculated with a dose of . eid of the beaur-m (s) or beaur-m (s) ep vaccine candidates. the chicks were allowed to hatch in separate hatchers and live chicks counted at . days of incubation. at and days post hatch the chicks were examined for nasal discharge. the chicks were bled and serum collected at weeks then challenged with virulent m ibv. at and days post challenge birds from each group were euthanized, their tracheas removed and assessed for ciliary activity. fifty spf eggs were inoculated with the beaur-m (s) ( . eid ), beau-r ( . eid ) or two different doses of the cv vaccines ( . or . eid ) or with a placebo. the eggs were marked, mixed in a single incubator and the hatch assessed by counting the marked eggs remaining. fifty spf eggs were inoculated with the m -ck virus ( . eid ) or with a placebo. the eggs were marked, mixed in a single incubator and the hatch assessed by counting the marked eggs remaining. to determine if the candidate vaccines based on the beaudette strain were safe for embryo vaccination, five experiments were performed in which embryonated eggs were inoculated at day of incubation. in experiments - separate hatchers were used therefore the performance of each hatcher may have influenced the results. in experiments and all eggs were hatched from the same machine to avoid the influence of individual hatchers. in experiments and commercial vaccines were included to determine their effect on the hatchability. in experiment , spf eggs were inoculated with high doses of beau-r or beaur-m (s) or graded doses of the commercial vaccine (cv ). as shown in fig. a , beau-r ( %) and beaur-m (s) ( %) gave slightly lower hatch than the placebo vaccinates ( %). the lowest hatch in the candidate vaccines was in the beau-r group which had four fewer chicks hatch than the placebo group. this reduction in hatch may have been due to the pathogenicity of beau-r but could also been an effect of hatching the different groups in separate hatchers. in contrast to the candidate vaccines, the cv commercial vaccine gave a very poor hatch in all three groups even though ( % hatch), , ( % hatch) or , ( % hatch) fold lower doses than beau-r and beaur-m (s) were inoculated. in experiment , spf eggs were inoculated with fold less beaur-m (s) or beau-r ( . eid ) than in experiment . in addition the beaur-m (s) ep strain was included to determine if egg passage could improve the immunogenicity of this strain without affecting its pathogenicity. inoculation with any of the candidate vaccines caused a lower hatch than the placebo ( % hatch) in this experiment with the beaur-m (s) ( % hatch) vaccine candidate giving the best result (fig. b) . though slightly lower than the placebo vaccinates the difference in hatch with these vaccine candidates may again have been due to the different hatchers used. in the third experiment spf eggs were inoculated with . eid of beaur-m (s) or beaur-m (s) ep . inoculation with beaur-m (s) gave a higher hatch ( % hatch) than the placebo ( %) whilst inoculation with beaur-m (s) ep caused a slightly lower hatch ( %) than the placebo (fig. c) . in the fourth experiment spf eggs were inoculated with beaur-m (s) or beau-r ( . eid ). a second commercial vaccine (cv ) was included at doses of . eid and . eid . to exclude any variability in hatchers all eggs in this experiment were placed in the same hatching unit. beaur-m (s) gave a higher hatch (fig. d, % hatch) than the placebo ( %) in this experiment, whilst beau-r vaccine gave a slightly lower hatch ( %) than the placebo. the cv vaccine gave very low hatchability at both doses ( % at eid and % at eid ). in the fifth experiment spf eggs were inoculated with m -ck virus ( eid ), the donor of the m spike gene inserted into beaur-m (s) [ ] , or with placebo. to exclude any variability in hatchers all eggs in this experiment were placed in the same hatching unit. m -ck gave % hatch in comparison to % hatch in the control group. as the birds were held in isolators during each trial it was difficult to examine some of the clinical parameters associated with ibv (tracheal rales, coughing and sneezing). in experiment in ovo inoculation with beau-r or beaur-m (s) did not cause any morbidity during the post hatch observation period. nasal discharge was not detected in birds vaccinated in ovo with either beau-r or beaur-m (s) ( fig. a) suggesting that these viruses were not pathogenic. in contrast, % to % of the hatched cv in ovo vaccinates had nasal discharge when examined, the occurrence of nasal discharge not being dose dependent. in the third experiment none of the birds vaccinated with beaur-m (s) or beaur-m (s) ep had nasal discharge when tested at and days post hatch (data not shown). overall these results suggest that the beau-r, beaur-m (s) and beaur-m (s) ep vaccine candidates are safe for embryo vaccination and the presence of the spike gene from virulent m did not have an effect on hatchability, nor cause clinical signs post hatch. analysis of ciliary activity post vaccination can be used as a measure of the safety of an ibv vaccine. in experiment , three birds from the cv -vaccinated groups and five in each of beau-r and beaur-m (s) in ovo-vaccinated groups were euthanized days post hatch and their ciliary activity assessed. the results are shown in fig. b . cv inoculated in ovo, although only assessed in three birds, gave very high ciliostasis scores independent of the dose given. ciliostasis scores for beaur-m (s) and beau-r vaccinates were not significantly different from each other though both were significantly higher than the placebo vaccinates (beaur-m (s), p = . , beau-r, p = . ). these scores are acceptable on the grounds of safety [ ] , again suggesting that neither of these viruses were pathogenic and that the inclusion of the m spike gene does not increase pathogenicity. in experiment , a more extensive analysis of the ciliostasis caused by the candidate vaccines was undertaken (fig. c) . in this experiment an egg-passaged vaccine candidate, beaur-m (s) ep was included. the beaur- m (s) ep vaccinate caused the highest levels of ciliostasis of the beaudette based strains, though there was no significant difference between the ciliostasis caused by any of the beaudette vaccine candidates at any time point (data not shown). peak ciliostasis of the beaudette based strains was detected at days post hatch and the ciliostasis levels decreased after this time. the ciliostasis caused by the commercial cv vaccine given at day-old was assessed on days and post vaccination. the highest ciliostasis was detected on day though this was not significantly different to any of the candidate vaccines given in ovo (p = . ). in experiment the serological responses were compared at weeks post hatch (fig. a) . the cv vaccine given in ovo at all doses gave the highest antibody responses and all birds seroconverted regardless of dose. the lowest dose of the cv vaccine gave a significantly higher response than beaur-m (s) (p = . ) and beau-r (p = . ). beau-r gave a lower antibody response than beaur-m (s) but this was not significant (p = . ). sixty percent of the beau-r vaccinates and % of the beaur-m (s) vaccinates had seroconverted at weeks post hatch. in experiment beaur-m (s) ep vaccination in ovo gave the highest antibody response of the beaudette based strains with all birds in the group seroconverting (fig. b) . the response to beaur-m (s) ep was significantly higher than both beaur-m (s) vaccine (p = . ) and beau-r (p = . ) which gave approximately equal responses with only % of birds seroconverting in each of these groups (data not shown). the cv vaccine given at day-old gave the highest antibody response in this experiment with % of the birds responding. however this response was not significantly different to that induced by beaur-m (s) ep (p = . ). in experiment , beaur-m (s) ep vaccination in ovo gave the highest antibody response with % of birds in the group seroconverting (fig. c) . the response to beaur-m (s) ep was significantly higher than vaccination in ovo with beaur-m (s) (p = . ) and only % of the birds vaccinated with beaur-m (s) seroconverted (data not shown). in experiment (vaccination dose for the beaudette-based viruses was eid ) birds were challenged weeks post hatch with virulent m virus and the ciliary activity tested on days and post challenge (fig. a) . for this purpose birds vaccinated with different doses of the cv vaccine were pooled to give a group of birds. based on ciliary activity of % or less being indicative of protection [ ] all of the cv vaccinated birds were protected, % of the beaur-m (s) in ovo-vaccinated birds were protected and % of the beau-r in ovo-vaccinated birds were protected. all non-vaccinated birds had at least % ciliostasis. this result indicates that the expression of the spike protein of virulent m increases the protective capacity of the beaur-m (s) vaccine in comparison to beau-r. in experiment (vaccination dose for the beaudette-based viruses was eid ) birds were challenged at weeks post hatch with virulent m virus and the ciliary activity tested on days and post challenge (fig. b) . all non-vaccinated birds had at least % ciliostasis. beaur-m (s) ep inoculated in ovo protected % of the birds whilst none of the beau-r in ovo vaccinates were protected and only % of the birds vaccinated in ovo with beaur-m (s) were protected. the cv vaccine administered at day-old provided % protection. in comparison to experiment this result suggested that efficacy may be dose and/or time dependent for the beau-r and beaur-m (s) vaccines with better protection given by a high dose ( eid ) with a -week challenge (fig. b ) than a lower dose ( eid ) and a -week challenge. in experiment birds were challenged at weeks post hatch with virulent m virus and the ciliary activity tested on days and post challenge (fig. c) . all non-vaccinated birds had at least % ciliostasis. one of the birds in the beaur-m (s) ep in ovo-vaccinated group had to be euthanized due to a genetic defect. beaur-m (s) ep inoculated in ovo protected % of the vaccinates whilst only % of the beaur-m (s) in ovo-vaccinated birds were protected. overall the results indicated that ibv beaudette strain expressing the spike gene from the m strain was safe for delivery to embryos and had potential as an efficacious vaccine. as the poultry industry is moving towards greater use of in ovo vaccination, the purpose of this study was to determine if ibv vaccines based on the beaudette strain could be delivered via this route. surprisingly, even at high doses, the beaudette based strains tested did not significantly affect the ability of the chick to hatch (fig. a-d) or cause clinical signs post hatch ( fig. a and experiment , data not shown) when compared to vaccination with a placebo. this is in contrast to work of wakenell and sharma [ ] whose ibv in ovo vaccine candidate showed residual pathogenicity at a dose of pfu. as expected the embryo adapted commercial vaccines greatly reduced hatchability ( fig. a and d) and gave clinical signs post hatch ( fig. a) . the reason why the recombinant beaudette strains are safe for in ovo vaccination is unknown though could be associated with the passage history of these particular strains. the parent beaudette virus has been passaged on embryos more than the commercial vaccines and also passaged on ck cells, the precise passage history being unknown. subsequently the recombinant viruses have been passaged further on ck cells. in line with the results of hodgson et al. [ ] , with hatched chicks, we found that that the substitution of the beaudette spike protein with that of the virulent m strain did not increase the pathogenicity of the beaur-m (s) strain over beau-r in -day-old embryos or post hatch. this result suggests that the ability of m -ck virus to reduce hatchability (to % in comparison to % with the placebo in experiment ) is not associated with the spike protein but may be associated with differences in other genes. it is interesting to note that further egg passages of the beaur-m (s) strain did not increase pathogenicity based on hatching criteria. the ciliostasis test is used to determine the damage to the trachea following growth of ibv in this tissue. the beaudette-based vaccine strains caused tracheal ciliostasis following in ovo inoculation but at an acceptable level with respect to vaccine safety [ ] . this indicates that the beaudette strains were able to replicate in the trachea of the in ovovaccinated chicks. in contrast, virtually no tracheal ciliostasis was caused by the beaudette strains following inoculation of chicks by the oculonasal route [ ] , although beaudette does cause ciliostasis when inoculated onto tracheal organ cultures. this suggests that delivery of ibv in ovo enables it to grow better in the trachea than post hatch delivery possibly due to the embryos ingesting amniotic fluid containing the viral inoculum, introducing the virus directly to the trachea. in contrast the commercial vaccine tested caused very high ciliostasis scores post hatch after delivery in ovo (fig. b) . this is in contrast to the results for the commercial vaccine delivered at day-old by nose and eye-drop appli-cation (fig. c) , again suggesting that delivery of ibv in ovo enables it to grow better in the trachea than delivery at day-old. interestingly the spike protein did not appear to affect the amount of ciliostasis caused by the beau-r and beaur-m (s) strains as these strains showed similar results in two experiments (fig. b eid & c eid ). in contrast in a single experiment (fig. c, eid ) the egg-passaged beaur-m (s) ep virus gave higher, but not significantly higher, ciliostasis scores. it is difficult to draw conclusions from a single experiment but this result suggests that egg passage may have increased the ability of the beaur-m (s) ep virus to grow in the trachea. although a serological response against ibv has not been correlated with protection against virulent ibv respiratory challenge, it is a useful criterion with which to assess the immunogenicity of the virus strain in the host. no significant differences were seen in the serological responses induced by beau-r and beaur-m (s) in two experiments ( fig. a and b) and it is of interest to note that there was no dose dependency regarding the serological response or rate of seroconversion, a -fold more virus giving a similar response and seroconversion rate (fig. a versus b) . however, eggpassaged beaur-m (s) ep induced significantly higher responses than beaur-m (s) (fig. b and c) . this response was not significantly different to that induced by a commercial vaccine given at day-old (fig. b) indicating a high level of immunogenicity of this virus. the reasons for the difference in immunogenicity of the egg-passaged virus requires further investigation though one could speculate that as the virus may have replicated more in the trachea, it subsequently induced a better immune response. the efficacy of the beaudette-derived viruses as potential vaccines was studied using a respiratory challenge with the virulent ibv m strain, using tracheal ciliary activity as the criterion of protection. a high dose ( eid ) of beaur-m (s) induced levels of protection comparable to that induced by the commercial vaccine given in ovo (fig. a) , though the commercial vaccine was not safe as determined by the criteria described above. the level of protection ( %) resulting from in ovo vaccination with beaur-m (s) was higher than that induced by beau-r ( %), suggesting a positive effect of the m spike protein. as these viruses appeared to grow equally well in the trachea ( fig. b and c) it can be speculated that the increase in protection observed with the beaur-m (s) was due to the m spike protein which is homologous to the spike protein of the challenge virus. alternatively, as a cytotoxic t cell response is thought to be important in protection against ib challenge [ ] it is possible that the % amino acid difference between beau-r and beaur-m (s) spike proteins [ ] contain relevant ctl epitopes for protection against m challenge. the results presented herein are in agreement with the finding that vaccination with beaur-m (s) given at -days-old gave higher protection against virulent m challenge than vaccination with beau-r [ ] . we observed a dose and/or time dependency of the protection afforded by beaur-m (s) as at a -fold lower dose and challenge at weeks the protection fell from % (fig. a) to % (fig. b ). in contrast, the egg-passaged virus, beaur-m (s) ep , at the lower dose ( eid , fig. b and c) provided - % protection in two experiments suggesting that this vaccine was more adapted to growth in the embryo or was more immunogenic. in summary, vaccine candidates based on the beaudette strain were safe for delivery, by in ovo vaccination, to embryos as determined by their effects on hatchability, nasal discharge post hatch, and ciliary activity. the expression of the m spike protein by beaur-m (s) did not alter the pathogenicity of the ribv on the basis of these criteria. expression of the m spike protein, rather than the beaudette spike protein, and further egg passage of the virus expressing the m spike protein improved the efficacy of the virus. to confirm the potential use of these candidate vaccines in the field, future work will concentrate on the effect of maternally derived antibody on the efficacy of these viruses as vaccine candidates following in ovo vaccination. in ovo vaccination technology chicken embryonal vaccination with avian infectious bronchitis virus the pathogenesis of virulent and avirulent avian infectious bronchitis virus recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m strain remains attenuated but induces protective immunity recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism reverse genetics system for the avian coronavirus infectious bronchitis virus a simple method of estimating fifty per cent endpoint breadth of protection of the respiratory tract provided by different live attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotype avian infectious bronchitis vaccine (live) the carboxyl-terminal -residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection key: cord- -ytnv map authors: bahnemann, hans g. title: inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine date: - - journal: vaccine doi: . / - x( ) -x sha: doc_id: cord_uid: ytnv map viral antigens for human and veterinary vaccines are still inactivated with formaldehyde. this is not an ideal inactivant and the problems of formaldehyde inactivation of vaccines are discussed. vaccines inactivated with aziridines are superior in safety and antigenicity. aziridines inactivate viruses in a first-order reaction and the inactivation rate and endpoint can be determined. the preparation and application of the aziridine compound binary ethylenimine (bei) and the necessary conditions for and controls of the inactivation process are described and discussed. a computer program has been written for assistance in the use of bei for controlled inactivation of viral antigens. a perusal of recent and current literature on the preparation of viral vaccines with inactivated antigen and, in particular, of experimental vaccines of this type shows that very often the inactivation is still obtained with formaldehyde and without the necessary controls. in the preparation of an inactivated viral vaccine the inactivation process is a very important step. the innocuity of the vaccine must be assured before the question of potency can be addressed. it seems worthwhile to discuss the inactivation of viral antigens with reference to a few older (and perhaps forgotten) publications as well as some more recent studies. the procedures and process controls which must be applied in order to assure a safely inactivated vaccine will be described. the example of foot-and-mouth disease (fmd) vaccine preparation will be used as this vaccine is by volume the largest viral vaccine produced at present. for many years most of the viral vaccines with inactivated antigen were prepared with formaldehyde as inactivating agent. the work of sven gard and his collaborators t' with poliovirus during the period of to demonstrated that the inactivation of this virus with formaldehyde was not a linear or first-order reaction. similar results were obtained for the formaldehyde inactivation of fmd virus by wesslrn and dinter in and by graves in . a recent publication stating the linearity of formaldehyde inactivation of fmd virus comes to this erroneous conclusion because the infectivity titration for the inactivation slope was based on final readings of plaque forming units (p.f.u.) at days. this is far too the extended incubation period for formaldehyde treated virus also means that an innocuity test in animals is inappropriate for detecting small amounts of residual infectious virus. when this virus begins to reiglicate with a delay of between several days and weeks, the animal is already beginning to produce antibodies. the new virus will then be neutralized by antibody and the animal has an abortive or subclinical infection. this subclinical infection can be detected by testing the animal for the -virus infection associated antigen (viaa), the viral rna polymerase . in alonso et al. reported a study of cattle exposed to fmd and found that at days postvaccination, of animals vaccinated with formaldehyde inactivated vaccine five animals were positive for viaa. of animals vaccinated with n-acetylethylenimine (aei) inactivated vaccine no animal was positive for viaa. pinto and garland ° later found viaa positive cases also in animals revaccinated with aei inactivated vaccine, but emphasized that the response in these animals was much weaker and only transient. alonso et al. ~ were able to confirm this transient and weak response and also found, with binary ethylenimine (bei) inactivated ai(oh) and oil adjuvant vaccines, a response to viaa only after revaccination. the weak response to viaa is caused by the presence of this antigen in the inactivated virus suspension used for vaccine preparation. however viaa antibodies induced by safely inactivated vaccines are only detected after revaccination and the presence of such antibodies after primovaccination still indicates recent virus replication in the animal. lucam et al. analysed in the fmd vaccination campaigns in france ~ and suspected residual infectivity in some formaldehyde inactivated fmd vaccines, which had passed official innocuity tests. recently beck and strohmaier ~ studied viruses from field outbreaks of fmd in europe by determination of their nucleotide sequences. they found that most of these isolates were related to virus strains in (formaldehyde inactivated) vaccines. this led strohmaier to the opinion that most of the fmd outbreaks in europe in the last years were 'homemade', i.e. were caused by vaccination. he made the recommendation that inactivation of fmd vaccine antigens should be changed from formaldehyde to first-order inactivants or that vaccination should be stopped altogether. the fmd vaccine production regulations in several south american countries have for several years now permitted only the use of first-order inactivants. at least one european country has recently also adopted this position. most of the fmd vaccine production laboratories in these countries apply the aziridine compound ei in the form of bei. one group of laboratories uses diluted ei. the first report of a (bacterial) virus inactivation by ethylenimine, the basic aziridine substance, was published in by raettig and uecker is. hurst in x was of the opinion that vaccines prepared with aei as inactivant were antigenically superior to vaccines inactivated with formaldehyde and would guarantee inactivation of the virus. the antigenic superiority was later confirmed also for vaccines inactivated with be - . ici patented the use of aei for inactivation of microorganisms in °. the first report on the inactivation of fmd virus by aei was published by brown and crick also in . this compound was subsequently used by a leading fmd vaccine production laboratory for many years in the preparation of inactivated antigens. however it did not come into general use because of its patent protection. in uecker reported the linearity of inactivation of bacterial viruses by ethylenimine derivatives and graves and arlinghaus described in the linearity of aei inactivation of foot-and-mouth disease virus . at ambient temperatures aei is not stable and it therefore has to be kept at °c or preferably at - °c. fellowes remarked in , that aei has a low boiling point and very little is left in a biologic preparation at reaction temperatures of °c or above. this observation probably made him use an inactivation temperature of °c instead of the usual °c. the problem of the stability of aei was perhaps the reason for introducing the double dosing regimen for inactivation at °c, i.e. the application of two doses for h each as described by pay et al. . this procedure is still being used with bei by the same laboratory although ei is much more stable . an extended incubation of the antigen at °c damages the antigen, as was shown in comparative inactivations of fmd virus at °c and at °c a. this damage is not due to the inactivant but is probably caused by the action of proteolytic enzymes present in the virus suspension. other laboratories continued to work on fmd virus inactivation by aziridines and in the early s reports on ethylethylenimine, eei zg, ethylenimine, e and binary ethylenimine, bei ° were published. both eei as well as e are difficult to obtain in quantity. for this reason, as well as the ease of preparation and handling, bei is now the preferred inactivating agent for fmd and other veterinary vaccines. the viruses which have been reported as inactivated with bei are given in table i . they belong to a variety of families of viruses with either rna or dna, which makes it very likely that most known viruses would be inactivated by an aziridine. inactivation in vaccine preparation transforms an infectious antigen into a non-infectious one. this transformation step should therefore be done in a well identified intermediate area between the virus-containing and the virus-free area. access to the intermediate area should be limited, and only be possible from the virus-containing side. the antigen must be held in the intermediate area until completion of the necessary control tests (inactivation endpoint and innocuity). the facilities for holding of the inactivated antigen in this area, cold room or cooled storage tanks, should be able to accommodate a volume of at least - weeks production of virus in order to allow termination and if necessary a repeat of the control tests. only after confirmation of the innocuity can the antigen be transferred to the virus-free area and be used for vaccine preparation. the inactivation process should be done under slow agitation in two different vessels, with perhaps one quarter or a third of the time in the first vessel, and transfer of the virus suspension under inactivation in a closed system to the second vessel for the remainder of the time. this procedure is indicated in order to avoid pockets of the virus suspension into which the inactivant did not enter or reinfection from non-inactivated virus on the tank wall above the liquid level at the end of the inactivation period and after hydrolization of the inactivant. the virus suspension should be checked to determine that it is at the desired temperature and is at a ph of ~ . before the inactivant is added. it is also advisable to control the osmolarity of the virus suspension, which for cell culture produced fmd virus is - mosm. the ph and osmolarity of the virus suspension affect the velocity of inactivation. figures and show the results of a study some years ago of the effect of ph and osmolarity on the inactivation rate of ei. the experimental conditions were the same as given previously . it can be seen from figure that an increasing alkalinity reduces the inactivation rate. this is in contrast to formalin inactivation, where the velocity of inactivation was found to increase with increasing alkalinity °. figure shows that increasing osmolarity also slows down the inactivation rate. the ph and osmolarity effects on the inactivation rate are perhaps due to conformational changes in the viral capsid proteins which affect the permeability for the inactivant. the inactivant was called binary ethylenimine or bei because it is prepared from two substances, -bromoethylamine hbr (bea) and naoh, and also to distinguish this preparation from pure ei. bea converts in an alkaline solution to 'binary' ethylenimine. the active substance is the ethylenimine ring as in all aziridine compounds. the bea solution is . m or . g - of a . n naoh solution. the conversion to bei is completed in - min at °c and is accompanied by a ph drop from = . to = . . the formation of bei is indirectly controlled by visualizing this change with the ph indicator fl-naphthol violet (bnv). from a % aqueous stock solution of bnv, . ml is added per litre of naoh inactivation of viral antigens: h. g. bahnemann solution. the colour of the bea solution changes at °c in = min from violet to orange upon formation of bei. the bei preparation should not be used for inactivation if the colour has not changed to orange. the . ra bei preparation contains only . % ei and therefore is much easier to manage than concentrated ei or aei. however it should still be handled with care and prepared in a closed vessel which allows transfer of the bei in a closed system to the inactivation vessel. higher concentrations of bei, such as or m, can be prepared. but the preparation and handling of such bei solutions requires much more attention and precautions as the el concentration is much higher. it is recommended to use the . na-thiosulphate hydrolyses bei. the bei inactivation is stopped by the addition ofa ta sterile na-thiosulphate solution at % of the volume of the bei solution used. it should also be used for hydrolysationt of bei in any spills or for cleaning of the vessel in which the bei solution was prepared. the inactivation process must be accompanied by appropriate in-process controls. this begins with the bei preparation. as described above, the formation of the ethylenimine ring is monitored in a simplified manner. if necessary, a colorimetric determination of the produced bei can be performed using the method described by epstein et al. x , and has been used by czelleng et al. for bei and preaud et al. for ei. it should not be necessary to do this colorimetric determination with each bei preparation. however it is advisable to do so at intervals, as it is also a control for the bea salt. no information is available on the shelf life of bea. some inactivation failures are suspected to have been caused by old bea. the inactivation of viruses with bei is a first order or linear reaction. for each inactivation, the rate has to be determined. this is done by taking samples for infectivity titration during the early part of the inactivation process, at , , , and h for example. the inactivation rate is obtained from the infectivity titres by calculation of the regression coefficient and is used to calculate the inactivation endpoint. the endpoint is the most important parameter to be determined and is a function of the inactivation rate, the reaction time and the volume of the virus suspension. the minimum endpoint is defined as being one log( ) lower than the titre which gives one infectious unit in the total volume under inactivation. for example: one infectious unit in a volume of one litre has a titre of vaccine, vol. , august inactivation of viral antigens: h. g. bahnemann log - (for the usually expressed titre in ml). the minimum endpoint for this volume therefore is log - . for a successful inactivation the calculated endpoint has to be lower than the minimum endpoint. the difference between the calculated and the minimum endpoint, or the dim (difference of inactivation endpoint to minimum) value has to be positive. it is important to determine the dim value for each inactivation process for an assessment of a successful inactivation. after termination of the inactivation each virus suspension has to be tested for innocuity on cell cultures. this can be done by inoculation of at least two roller bottles or similar cell culture vessels and two subsequent blind passages at h intervals. the cell cultures for this test have to be prepared in the virus-free area and the test should be done in the intermediate area. only after determination of the inactivation endpoint and completion of the innocuity test can the virus suspension be considered to be properly inactivated. the major application of bei will be in the preparation of inactivated antigen for vaccines. but there are other areas in which bei can be used. since bei does not react with proteins it can be used for inactivation of adventitious viruses in biological preparations from animal or human tissues or fluids. the bei treatment of bovine serum used for cell cultures has been reported in . the usefulness of this method was later confirmed in by heuschele , who applied it over a -year period for the inactivation of adventitious bovine viral diarrhoea virus in calf serum used in primary or secondary animal cell cultures. bei can also be used for inactivation of viruses in enzyme preparations of animal origin. a commercial trypsin preparation was treated with bei without any loss of activity (unpublished results). it is very likely that other biologicals, like factor viii, could be treated with bei for inactivation of adventitious viruses. pure aziridines are highly toxic and have to be handled with special precautions and extreme care. this high toxicity is the reason for the . m preparation of bei. at this molarity the bei preparation contains only . % el and the vapour pressure at this concentration is low enough that for temperatures under °c no ei will escape into the atmosphere (the boiling point for ei is °c). on the basis of experiments in laboratory animals, aziridines are considered to be carcinogenic substances. according to dermer and ham , however, no cases of human cancer caused by e have ever been reported. fellowes z cites hurst who states that an injection of . mg of aei into rats did not produce any tumours during an observation period of days. the total annual production of fmd vaccine worldwide is probably between and million doses, of which nearly million doses are produced in south america alone. furthermore between % and % of all fmd vaccines are inactivated with bei, which means that about million doses of fmd vaccine are inactivated with bei and applied in cattle annually. the majority of these cattle are revaccinated many times over the years. no increase in the incidence of cancer in cattle after vaccination with bei inactivated vaccines has ever been reported from any country. neither is there any reason to expect any increase, as the residual bei after inactivation is hydrolized with na-thiosulphate. a computer program has been written to assist with the calculations needed for a controlled inactivation of viruses with bei or any other first-order inactivant. the screen menu for the program is given in figure . inactivation of poliomyelitis virus by formaldehyde inactivation of poliovirus by formaldehyde the inactivation of foot-and-mouth disease virus by formalin formaldehyde inactivation of foot-and-mouth disease virus as applied to vaccine preparation formaldehyde inactivation of foot-and-mouth disease virus. conditions for the preparation of safe vaccine partially inactivated poliomyelitis virus: initiation of infection in tissue culture inactivation of poliomyelitis virus by formaldehyde. incubation time in tissue culture of formalin treated virus inhibition of cell-free foot-and-mouth disease virus ribonucleic acid synthesis by antibody the use of virus-infection-associated antigen (via) in the detection of cattle exposed to foot-and-mouth disease the induction of antibodies against viaa in cattle vaccinated and revaccinated with inactivated fmd vaccine la vaccination antiaphteuse en france subtyping of european foot-and-mouth disease strains by nucleotide sequence determination die maul-und klauenseuche ill erfahrungen und folgerungen aus jahren bek~mpfung der maul-und klauenseuche inaktivierung yon bakteriophagen durch athyleniminderivate approaches to the chemotherapy of virus diseases inactivation of o~ fmd virus by binary ethylenimine (bei) binary ethylenimine and foot-and-mouth disease vaccine production in thailand a preliminary vaccine potency trial of a newcastle disease virus inactivated with binary ethylenimine application of agar-<jel diffusion analysis to a study of the antigenic structure of inactivated vaccines prepared from the virus of foot-and-mouth disease inaktivierung yon mikroorganismen durch alkylierende verbindungen. i. untersuchungen zur kinetik der inaktivierung yon bakteriophagen acetylethylenimine in the preparation of inactivated foot-and-mouth disease vaccines comparison of the inactivation and antigenicity of foot-and-mouth disease virus by acetylethylenimine and by combined effect of ultraviolet light and ~-propiolactone some observations on foot-and-mouth disease virus inactivation with acetylethytenimine. eur. foot-and-mouth disease commission, meeting technical committee, te/bingen, , fao, rome pay the inactivation of foot-and-mouth disease virus by ethylenimine and propylenimine tt~e production and application of an oil adjuvant vaccine against inactivation of viral antigens: h. g. bahnemann foot-and-mouth disease in cattle die inaktivierung des maul-und klauenseuche (mks) virus durch athyl&thylenimine und die eignung des inaktivierten virus zur impfstoffherstellung binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production kinetics of inactivation of african swine fever antigen with binary ethylenimine inactivation of bluetongue virus with binary ethylenimine evaluation of an inactivated bovine leukemia virus preparation as an immunogen in cattle inactivation of viruses in serum with binary ethytenimine vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity inactivation of adventitious viruses in serum. art to science an inactivated oil emulsion vaccine for the prevention of porcine parvovirus-induced reproductive failure comparison of pseudorabies virus inactivated by bromo-ethylenimine, ~°co irradiation and acridine dye in immune assay systems rabies virus inactivation by binary ethylenimine: new method for inactivated vaccine production studies of the inactivation of poliomyelitis virus by formaldehyde. the effect upon the rate of inactivation of temperature, ph and concentration of formaldehyde use of ~-( -nitrobenzyl)-pyridine as analytical reagent for ethylenimines and other alkylating agents effect of ethylenimine and formaldehyde inactivation on the immunogenicity of foot-and-mouth disease virus commission on foot-and-mouth disease inactivation of foot-and-mouth disease virus by ethylenimine -kinetics and control, int. foot-and-mouth disease syrup key: cord- -uy f f o authors: nara, peter l.; nara, deanna; chaudhuri, ray; lin, george; tobin, greg title: perspectives on advancing preventative medicine through vaccinology at the comparative veterinary, human and conservation medicine interface: not missing the opportunities date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: uy f f o abstract vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life—resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para- and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it’s melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life-resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it's melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. published by elsevier ltd. the new millennium did not bring the anticipated global internet technology shutdown however, it has brought with and heralded a time of significant change, opportunity and challenges. i and my co-authors goal in this overview are to celebrate, provocate, instigate innovate and activate those in society who are in interested to contributing to the betterment of human and animal health through vaccination. for vaccination to have its greatest chance of working policy-relevant science for sustainable conservation in developing countries needs to address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the wellbeing of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the pre-ventative sciences and medicine lead to sustainable cultural public health and economic changes of the future is never more evident than today. if the article gets the attention of researchers, educators/teachers, funders, policy makers, economists and the general public in both developed and developing countries to become involved in finding collaborative solutions to the conservation crisis than we will consider it a success. vaccination has and remains one of the most cost-effective and safest forms of medicine toward improving health today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives in both rich and poor countries-resulting in the eradication of smallpox and huge reductions in the burden of previously common diseases such as polio, typhoid and measles. immunization is particularly well suited to all countries including those with weak health systems, because it requires relatively less training and equipment and does not depend on skilled diagnosis, long-term drug regimens or extensive medical care. immunization and sanitation remain as the most important public health modality responsible for improving the gnp of developing countries through additional gains in healthier children who are better educated and grow up to impact on their productivity. like schoolchildren, healthier workers have better attendance rates and are more energetic and mentally robust. workers in healthy communities, moreover, need to take less time off to care for sick relatives. body size, which is greatly influenced by one's health during childhood, has been found to have large impacts on long-term productivity. recent economists [ ] have calculated that a -year increase in life expectancy improves labor productivity by %. despite the weakness of health systems in many developing countries, three-quarters of the world's children now receive a standard package of childhood vaccines through the who/unicef expanded program on immunization to protect them against diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis [ ] . these vaccines currently save an estimated million lives a year -almost , lives a day -and protect millions more from illness and permanent disability, thus providing as mentioned above a healthier cohort of people to contribute to the economic development of the nation. the full package of basic vaccines (diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis) costs less than $ per year of life saved in poor countries. "life-years" and "year of life" consistently refer to disability-adjusted life-years (dalys). interventions are generally considered extremely costeffective if the cost per year of life is less than $ . by comparison, antiretroviral treatment for hiv/aids-an intervention that donors widely support in the developing world costs up to five times as much at $ to $ per life-year saved; by way of comparison, in the us and the uk medical interventions are considered cost effective at $ , to $ , per life-year saved [ , [ ] [ ] [ ] . the week during the conference in amsterdam it was reported that "vaccine-preventable deaths reach new low in u.s." as reported in a federal report released tuesday, november , people readily associate the role of veterinarians with private veterinary practice focused on pets and farm animals, but the true dimensions and contributions of veterinary medicine are much broader and reflect expanding societal needs and contemporary challenges to animal and human health and to the environment [ ] . veterinary medicine has responsibilities in biomedical research; ecosystem management; public health; food and agricultural systems; and care of companion animals, wildlife, exotic animals, and food animals. the expanding role of veterinarians at cdc reflects an appreciation for this variety of contributions. veterinarians' educational background in basic biomedical and clinical sciences compare with that of physicians. however, unlike their counterparts in human medicine, veterinarians must be familiar with multiple species, and their training emphasizes comparative medicine. veterinarians are competent in preventive medicine, population health, parsitology, zoonoses, and epidemiology, which serve them well for careers in public health. the history and tradition of the profession always have focused on protecting and improving both animal health and human health [ ] . since , a total of diseases have been eliminated from equine, poultry, and livestock populations in the united states [ ] . the elimination of these livestock diseases, along with outstanding research in animal health, is key to the remarkable gains in the efficiency of u.s. animal production [ ] . partly as a consequence, u.s. residents spend only approximately % of their disposable income on food, whereas residents in other countries pay three or four times more [ ] . although this achievement is recognized to have added billions of dollars to other parts of the u.s. economy, its success in allowing the u.s. public access to a nutritious, affordable, and sustainable food supply -also important for the public's health and well-being -is far less appreciated. the success of the national brucellosis and tuberculosis elimination campaigns has benefited not only the u.s. livestock industries but also human health by substantially reducing these zoonotic threats in animals. additional public health contributions can be attributed to the food safety and inspection service of the u.s. department of agriculture (usda), which has substantially reduced the burden of food-borne illnesses, improved food safety, and eliminated other zoonotic threats. over the years, cdc has worked closely with usda and the food and drug administration to improve the safety of u.s. foods and reduce antimicrobial resistance in pathogens that infect both humans and animals. veterinary scientist and veterinarians within the health and human services serve in many critical capacities. veterinary officers in the commissioned corps work throughout the u.s. department of health and human services and in other federal agencies. most veterinary officers are assigned to the cdc, nih, fda, usda, epa, ogha, ndms and state department. other veterinarians and veterinary scientists function as medical research scientists post-doctoral nih/nci fellows, principal investigators, some specializing in lab animal medicine and providing critical lab animal health infrastructure and support, design of animal models for human disease in most of the hhs institutes. some are part of the hhs national disaster medical services and were deployed as the veterinary medical assistant teams (v-mats) and supported the search and rescue in the world trade disaster. additionally, since , the avma and the college of american pathologists have been working together to create a standard nomenclature that would allow veterinarians, physicians, and other medical professionals to create electronic medical records that use a common language. the systematized nomenclature of medicine, snomed, was initially created by the college for human medicine but has sincethrough the partnership with avma -expanded to include veterinary terms. on july , , the department of health and human services announced it would make snomed available nationally at no charge, a step toward instituting a standardized electronic medical records system. the hhs signed a -year, $ . million contract with the college to license snomed and make it available nationally. the national library of medicine is administering the program. prior to the hhs's agreement with the avma, practitioners in areas of practice nationally on the front line of surveillance would have had to pay a $ to $ annual registration fee to access snomed. the cdc has expanded the role, scope, and influence of veterinarians and veterinary scientists and epidemiologists in public health since its inception in [ ] . early in the history of cdc, veterinarians in the u.s. public health service and the cdc veterinary public health division helped reduce zoonotic diseases, especially rabies and food-borne illnesses [ ] . today, veterinarians serve throughout cdc in positions that address not only infectious diseases but also the entire spectrum of public health challenges: environmental health, chronic diseases, human immunodeficiency virus infection and acquired immunodeficiency syndrome, injuries, immunizations, laboratory animal medicine, global health, migration and quarantine, health education, and bioterrorism. veterinarians contribute as epidemiologists, laboratory scientists, policymakers, researchers, and surveillance experts and in environmental and disease prevention and control programs both domestically and globally. at cdc, veterinarians have participated in the epidemic intelligence service since [ ] . forty-one states now have state veterinary public health officials. in , almost students and faculty attended the first veterinary student day at cdc; in april , cdc will co-host an inaugural conference with the association of schools of public health and association of american veterinary medical colleges. in addition, cdc has been recognized as a world association for animal health collaborating center for emerging and re-emerging zoonoses. the cdc publication, emerging infectious diseases, has highlighted zoonotic diseases in nearly every issue to zoonotic diseases and has devoted an annual issue in each of the previous years. the cdc has provided an important scientific forum for zoonotic disease research and programs both domestically and globally and should serve as a template for the nih, as will be discussed later in this paper (section . ), for moving these highly trained and broad-based medical skill set professionals from a more decentralized setting into a central institute at the nih. benjamin franklin's famous quote, "an ounce of prevention is worth a pound of cure" was actually fire-fighting advice-he founded the first fire fighting organization in philadelphia, its obvious application to medicine, although obvious, has not been a mainstay of heavily invested research and development in human health practices. many of the leading causes of death and disability in the united states can be prevented [ ] . primary prevention can prevent or arrest the disease process in its earliest stages by promoting healthier lifestyles or immunizing against infectious disease [ ] . secondary prevention, by detecting and treating asymptomatic risk factors or early asymptomatic disease, can substantially reduce subsequent morbidity or mortality. the human and veterinary clinician plays a pivotal role in both primary and secondary prevention. health professionals deliver vaccinations, screen for modifiable risk factors such as high blood pressure and high cholesterol, counsel patients about smoking and other behavioral risk factors, provide screening tests for early detection of cancer and other chronic conditions, and advise patients about the benefits and risks of preventive therapies such as postmenopausal hormone replacement therapy. the preventative health care landscape has changed in some regards in the years since the u.s. preventive services task force (uspstf/task force) was first established in to provide advice about prevention for health professionals. prevention became more of an integral component of primary health care [ ] . delivery of clinical preventive services such as immunizations, mammograms, and cholesterol screening has risen steadily over the past two decades (national center for health statistics [ ]). roughly % of employers now include well-child visits, childhood immunizations, screening tests, and adult physical examinations among covered health benefits, compared to less than half that did so in [ ] . interest in prevention grew significantly among the public, clinicians, educators, employers, and policymakers [ ] and health plans and individual clinicians were increasingly being held more accountable for the quality of the preventive care they provide to their patients [ ] . at the close of the th century, health care costs in the united states continued to rise steadily, accounting for . % of the gross domestic product in [ ] , and debate on health care funding for the aging american population intensified. no doubt fueled by the incredibly imbalanced historic spending in preventative healthcare of cents for every cents spent for curative treatment [ ] . numbers are harder to come by for recent years, but given the spiraling costs of treatment since it is likely that this ratio has gone down considerably since then-possibly grossly estimated to be closer to : today. in this environment, preventive services often compete with one another and with diagnostic-and treatment-oriented care for increasingly constrained resources [ ] . while preventive services are often believed to save costs, delivery of most preventive services, with few exceptions (e.g. some immunizations), incurs net costs [ ] . evidence that us society clearly favors the cure (or treat) approach to disease over prevention can be shown in the following ways. first, though there is a shortage of preventive medicine specialists (public health, general preventive medicine, occupational medicine, and aerospace medicine physicians), in the us the number of residents in training in was less than . % of all residents, not sufficient for replacement or to fill the expanding demand for the specialty's skills and talents [ , ] . second, preventive medicine residencies and subspecialties in human and veterinary medicine are generally found in only graduate medical education programs not financed by cms or mainstream academic training programs. third, we believe that our preventive acts are only statistical, whereas our curative acts are certain. this mistaken belief perhaps derives from our sense that we have more control over cure outcomes than prevention outcomes-we think that we do cure, whereas we only facilitate prevention. this notion of doing vs. facilitating is an important one, because if we believe that our curative actions are more effective than our preventive ones then we will more likely act toward the more effective. the editor of the british medical journal, fiona godlee, expressed this well when she states, "because it is acted on healthy people, preventive medicine needs even stronger supporting evidence on benefits and harms than therapeutic interventions" [ ] . thus substantial gaps in the delivery of effective preventive care in the united states remained, however, because clinicians continued to face many of the same barriers that originally spurred the formation of the first uspstf. identifying effective interventions were and are difficult in prevention, where prospective controlled trials are often difficult to conduct. these studies come from the field of epidemiology which has changed remarkably during its growth in the past quarter century. one of those changes has been a mixed blessing of ever-increasing specialization among its practitioners at the cost of the generalist. this phenomenon has shaped the field and a partial explanation for this trend is found in the decline in the availability of training funds not focused on specific and general disease areas. without returning to the training of general conservation-medicine based epidemiologists, the needed trend associations and study designs that are needed to show the economic and public health returns related to preventative practices field will not be realized and in addition lose some of its ability to quickly respond to new and expected emerging public health challenges. conflicting recommendations from different organizations, further exacerbated by the advocacy positions of some groups, leave many clinicians uncertain about what to do. clinicians facing increasing time pressures in practice may question the value of some routine preventive interventions, as may employers and other payers struggling with accelerating health care costs. although more prevention information is reaching the public, the messages conveyed are often inconsistent and increasingly colored by commercial self-interest. clinicians may feel compelled to provide unproven or ineffective services because patients demand them or they fear being sued, but patients may find that insurance coverage for individual preventive services, especially new technologies, is inconsistent. the importance of clarifying what we know and do not know about the effectiveness of specific preventive services is as important in as it was in . although the uspstf was disbanded in with the release of the guide, the need to keep pace with the rapid growth in scientific evidence led to convening a second panel in . the second uspstf was smaller, with only members, eight of whom were primary care physicians. it refined the previous group's methods for reviewing evidence and making recommendations, and expanded the scope of topics. it adopted policies for disclosure of conflicts arising from financial interests, funding sources, or other affiliations. the work of the second uspstf was marked by strengthened ties with both federal and nongovernmental partners, including primary care subspecialty societies. the work of the second uspstf culminated in the publication of the second edition of the guide in , which covered over interventions in areas. by the time the second edition of the guide appeared, the environment for preventive medicine and evidence-based medicine had changed dramatically. managed care organizations, which had emerged as a dominant paradigm for delivering and paying for health care, included some preventive care among basic covered services more commonly than had traditional fee-for-service insurance. at the same time, the heightened competition spurred by managed care brought increased attention to costs and value of treatments with less attention given to prevention. the guide was frequently cited by health plans and systems of care in defending their health maintenance programs and benefits packages, and its recommendations informed many of the health plan employer data and information set (hedis) quality measures developed by the national committee on quality assurance for evaluating health plan performance but not integrated into cost saving practices by the insurance companies. the rapid progress towards universal vaccination coverage in the s and s has slowed in recent years. unicef funding for vaccination fell from $ million to $ . million between and [ , ] . global coverage of the diphtheria, tetanus, and pertussis (dtp ) vaccine has been at around % since [ ] . fifty-seven developing countries have yet to eliminate neonatal tetanus, and , babies died of the disease in . ten developing countries reported cases of polio in june , despite the massive (and largely successful) global effort to eradicate the virus [ , , ] . sixty-two percent of countries, meanwhile, had still not achieved full routine immunization coverage in , with gavi estimating that at least . million additional infants need to be reached to achieve full coverage. there are several factors behind this loss of momentum. although dramatic progress has been made in increasing worldwide vaccination coverage from below % to above %, the task has inevitably become harder now that the easiest-to-reach populations have been vaccinated. because these communities are more elusive, the average cost per vaccination has increased, and it may be that other apparently cheaper health interventions have become more attractive. there are many practical problems impeding vaccine delivery. delivering vaccines to patients requires functioning freezers and reliable transport to move the vaccines from port to clinic; clinics refrigerators (which in turn require a constant supply of energy); good roads and with access to people who need to be immunized; parents who know the value of vaccination; trained medical staff to deliver the dose; and sterile syringes. only % of vaccineimporting countries could guarantee vaccine safety and quality [ ] , while a further study of developing countries found that at least half of injections were unsafe [ , [ ] [ ] [ ] [ ] . the third factor behind the lack of progress in recent years is political. political disruptions have affected coverage in some areas. in somalia and congo, for example, where vaccination rates have fallen rapidly in the past decade, war and social breakdown have impeded public health campaigns, despite "vaccination days" in congo that temporarily halted fighting. gauri et al. have found that the quality of institutions and governance are positively correlated with vaccination coverage [ , ] . politics in the developed world have also played a part. according to a report by the us institute of medicine, in the us vaccine industry was forced to stop offering low-price vaccines to develop-ing countries following congressional hearings that "savaged" the industry for "allegedly subsidizing vaccines for the poor children of the world by charging high costs to us families and taxpayers". as the institute of medicine points out, this move was based on a flawed premise, as the us vaccines would have been developed anyway to protect american children and travelers. public perceptions of vaccination change-as coverage spreads through a community and it reaches a point at which those who are unvaccinated are highly unlikely to catch a disease because herd immunity has set in. at this juncture, it may be more rational for an individual to refuse vaccination in order to avoid any risk of side effects. with the oral polio vaccine, for example, there is a one in a million chance of paralysis, and in societies where mass vaccination has eliminated the disease, the risk of paralysis is greater than that of catching polio itself. what had once been a public and private good is now a public good but a private risk. as more and more people choose to avoid this risk, of course, overall coverage rates decline, and the community is once again exposed to the threat of the disease. public perceptions have been influenced by vaccine scares. controversy and the attendant bad publicity about the safety of vaccines have been abetted by incidents such as the withdrawal of half the us supply of flu vaccines in due to contamination at the manufacturer [ ] . in addition, alarms over the safety of vaccines such as that for measles, mumps and rubella (mmr), which some believe to cause autism, have further fanned the anti-vaccine movement's flames [ ] . in the us, disputes continue to rage about the scientific basis of such claims, but the preponderance of the evidence, according to the us centers for disease control (cdc), says that the mmr vaccine is safe [ ] . in response to these types of controversies in the us, the institute of medicine has called for independent oversight of vaccine safety studies to ensure the fairness and openness of the vaccine safety datalink program, which is overseen by the cdc. as one can see there are many complex factors that have to be considered when bringing vaccination programs into existence. the impact of vaccination of animal diseases on agriculture is typically assessed in quantitative terms-lost revenues; costs of eradication, decontamination, and restocking; and the numbers of affected farms, animals and humans. this approach can be applied universally to all outbreaks in all countries because it normally reflects the hard data supplied by large commercial operations and the estimates by relevant governmental agencies of small farmer impact [ ] . when used exclusively, however, it fails as a barometer, because it does not and cannot factor in the multi-dimensional character of major disease events-and the accompanying societal effects that often get lost when it comes to assessing the damage in developing countries. the quantitative approach must also be interpreted, and cannot be used "as is" for comparing impacts in developed and developing countries. further, while export trade losses in a developing country may be small in terms of the dollar amount, the impact upon its pre-epidemic market share is inevitably greater and more persistent. other impacts such as effects on human health and community stability tend to be more visible and last longer in developing countries, particularly at the village level where animal are husbanded primarily for the benefit of the immediate family, and often in impoverished circumstances [ ] . the consequences of animal diseases in domesticated birds and livestock can be complex and generally go well beyond the immediate effects on affected producers. these diseases have numerous impacts, including: • productivity losses for the livestock sector (e.g. production losses, cost of treatment, market disturbances). • loss of income from activities using animal resources (in such sectors as agriculture; energy; transportation; tourism). • loss of well-being of human beings (morbidity and even mortality rates; food safety and quality). • prevention or control costs (production costs; public expenditure). • suboptimal use of production potential (animal species, genetics, livestock practices). the most direct economic impact of animal diseases is loss of production and/or productivity, and ensuing income losses for farmers [ ] . however, if the economy depends on one or some of the vulnerable products, the impacts can be serious, and local food security can be threatened. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. if the farm economy is diversified or if there are other opportunities to generate income, the impacts can be mitigated. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. the loss of the farmer's "well-being" will generally be lower than the value of the lost product, except where the farmer has few alternatives or is wholly dependent on the affected product, which is quite often the case in developing countries. direct losses are the result of the disease itself (they may be very high when mortality rates are between and %), or from animal health measures (stampingout policies) [ , ] . in vietnam, one of the countries most seriously affected by the avian flu, almost million birds - % of the country's poultry population -had to be destroyed at an estimated cost of us $ million ( . % of gnp) [ ] . the smaller scale producers lost the least in absolute terms, but the most in relative terms, as the outbreak resulted in losses equivalent to upwards of times their daily income (from us $ a day or less). in africa, abortions caused by the rift valley fever virus not only affect birth rates, but also push human consumption of milk downward in the year following an outbreak [ ] . in the dairy farming sector in kenya, it is estimated that losses in milk production accounted for % of all losses caused by an outbreak of foot-and-mouth disease in the s. direct costs are generally well below the indirect costs of animal diseases and are directly linked to the rapid containment of outbreaks: case studies have shown that early detection and the implementation of appropriate measures in the event of an outbreak are essential to help minimize direct losses as much as possible. conversely, inappropriate control and eradication measures are at the root of such endemic situations, which are much more difficult, and infinitely more costly, to keep under control or eradicate. the livestock sector plays a significant role in the economic development of many countries and vaccination can serve as one of the most important means of assuring its health. as such the cost of not developing new and important or properly applied vaccines can have tremendous economic consequences. the production of meat and other animal-based food items generates income, jobs, and foreign exchange for all stakeholders in the animal industries. consequently, an epizootic which could have been otherwise mitigated by vaccination can affect the industry's upstream (inputs, genetic resources) and downstream activities (slaughterhouses, butchering operations, processing, marketing) in terms of jobs, income for the stakeholders in the industry, or market access. a survey by the food and agriculture organization of the united nations (fa ) on avian flu revealed that in the most seriously affected regions of indonesia, % of permanent workers at industrial or commercial farms lost their jobs [ ] . similarly, an outbreak of contagious bovine pleuropneumonia in botswana led to the destruction of more than , animals in the most seriously affected province, and the immediate closure of the export slaughterhouse, which employed persons. owing to the catalyst role of livestock raising in the rural economy as a whole, the costs of the indirect effects of these measures were later estimated to be seven times higher than the costs caused by direct losses [ ] . in vietnam, % of the poorest segment of the population, for which poultry farming accounts for - % of household income, is particularly vulnerable to income losses caused by avian flu. the fao and world organization for animal health (oie) estimate that between one-third and one-half of the populations living in the most seriously affected areas of southeast asia depend on poultry farming for at least a part of their income [ , ] . in france, the leading european poultry producer, it is estimated that farmers affected by the crisis lost % of their income in months (between january and march ). the effects of the production losses are also linked to price variations, which are caused by supply and demand (im)balances. depending on the market, prices can rise sharply (consumer product on the domestic market) or plummet (product banned for export but cleared for consumption on the domestic market, product deemed too dangerous for human consumption or perceived as such). in brazil, where % of products are exported, the price of a day-old chick, an early indicator of a possible change in production, reportedly fell by %. and even in cases where the country is not infected, market uncertainties and the fall in prices prompted the largest producers to cut back production by % this year. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. in / , the rift valley fever outbreaks in east africa seriously affected pastoral economies in somalia, with a decline of more than % in exports (which generate more than % of foreign exchange in "somali land"), following an embargo declared by saudi arabia on all animal products from the horn of africa [ , ] . conversely, the world bank has reported that eradication of certain major diseases to facilitate access to "high value" export markets can provide considerable benefits. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. uruguay is a good example of a country that gained access to a lucrative market after eradicating foot-and-mouth disease. beef exports increased in volume by more than % and in value by % after the oie declared uruguay to be officially foot-and-mouth disease-free without vaccination in . access to the u.s. market (where prices are double those of the domestic market) provides uruguay with additional revenue to the tune of us $ million each year. a medium-term analysis showed that access to "pacific rim" markets would generate additional revenue of us $ million each year, and yet, before the disease was eradicated, uruguay had been spending (only) us$ million to us $ million each year on vaccines to combat foot-and-mouth disease. in this case, control costs would account for less than % of the revenue generated by exports alone [ ] . animal diseases that could be controlled by vaccination can have major effects on food availability and quality for poor communities. it is well known that agriculture plays an important role in the generation of income and jobs in other sectors but the closeness of this interdependence became particularly obvious during recent epizootics. for pastoral societies, animal husbandry contributes directly and indirectly to food security and to nutrition as a source of quality proteins, vitamins and trace elements, traction, and com-mercially tradable products [ , ] . certain diseases could have significant repercussions on food supply and the nutrition of poor communities that do not have readily available substitute products, which could therefore lead to famine (rinderpest for example). poultry meat is the primary animal protein in africa (which has little to begin with) and the indispensable source of discretionary income for the survival of millions of small farmers. the high mortality rates as a result of avian flu, which is extremely pathogenic, and the sanitary slaughter of poultry would therefore have a negative impact on the food available to the entire population, as well as on rural revenue. furthermore, developing or transition countries which generally have poor public health systems are particularly at risk from zoonoses making vaccination against these diseases particularly important to target. in / a major rift valley fever epidemic in egypt resulted in , human cases and fatalities [ , ] . twenty years later, a new epidemic affected over , persons in east africa, and persons succumbed to the hemorrhagic form of the disease. but zoonoses also affected industrialized countries with high health standards as was the case with the bovine spongiform encephalopathy crisis in europe [ ] . food-borne diseases (over have been classified) are a major source of acute gastroenteritis (which costs the netherlands us $ million per year) and the cause of major morbidity with fatalities among children in the third world [ ] . in the specific case of a pandemic, most of the economic loss is caused by the increase in morbidities and fatalities in the human population and its repercussions on the world economy. the most recent estimates suggest that the "spanish" influenza in caused the death of million persons, that is, . % of the population at the time. the most obvious economic losses were the reduction in quantity and productivity of the workforce, and according to the experts, in the case of a pandemic could represent times more than all the other losses combined [ ] . another category of economic impact is linked to individual strategies to avoid contamination-or to survive possible contamination. the example of the severe acute respiratory syndrome (sars) clearly shows the sharp drop in demand in the services sector (tourism, public transport, retail trade, hospitality and food services) resulting from the combined efforts of individuals to avoid any close contact [ ] . based on the experience with severe acute respiratory syndrome in south-east asia, the world bank thinks that an avian flu pandemic could result in a % loss of the world's gross domestic product and cost the world economy us $ billion in the space of year. the losses are difficult to calculate and would undoubtedly be much more significant in light of the extremely high mortality rates in developing countries which do not have good health care systems. the impact of animal diseases on the tourism and leisure sectors could also be quite significant. the negative effect of foot-and-mouth disease in the united kingdom on these two sectors amounted to us $ billion because of restrictions on access to rural areas and represented more than half of the total cost of the disease [ ] . the federation of american scientists' animal health/emerging animal diseases (ahead) project proposed a major program in sub-saharan africa to detect and document the extent of infectious diseases shared by farm and wild animals, and to supply treatment, prevention and control services to remote communities that have previously been neglected by other programs, both national and international. this program, international lookout for infectious animal disease (iliad), was implemented in south africa [ ] . at the core of iliad is the need for a permanent and sustainable regional program of in situ surveillance designed to detect, monitor, treat, prevent and control infectious diseases with the goals of increasing livestock production in remote farming communities, protecting the health of wild species, building indigenous physical and professional resources, and introducing communications and epidemiology information technologies. transmission of infectious diseases is rampant in remote communities in the sub-saharan region, just as they once were in the united states and as they always are wherever poverty and farming co-exist. diseases shared by wild, farmed and captive/bred animals, and by animals and humans, suppress food production, frustrate species preservation efforts and greatly affect public health. detection, prevention and control of these diseases are an essential element in expanding trade, improving nutrition, exploiting ecotourism and ensuring food security. iliad is structured in the investor mode-an international consortium of donor groups providing short-term developmental assistance with program direction and oversight provided by veterinary diagnostic, public policy and epidemiology experts representing the sub-saharan africa partnership members-the renowned onderstepoort veterinary and exotic disease institutes (ovi) and tuskegee university (tu), and fas-ahead. given positive assessments of the benefits of the program after years, national or provincial institutions will integrate some or all of the activities into their official veterinary and agricultural activities. it is difficult to calculate the cost of the public's loss of confidence in animal industries in their countries, or of an importer country towards the veterinary services of the exporter country. animal diseases can have major effects on food availability and quality for poor communities. consumers' obsessive fear of bovine spongiform encephalopathy (mad cow disease), fed by the media and which a good communication strategy could have prevented, would have tremendous social repercussions on a europe still reeling from long term economic repercussions. in italy, the baseless perception of a food risk related to avian flu coupled with low confidence in public health services eventually resulted in a % reduction in the consumption of poultry and eggs. the loss of confidence by an importer country can trigger a lasting embargo and major economic and social repercussions (arabian peninsula embargo on the horn of africa, affected by the rift valley fever virus). loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. animal diseases might also have indirect long-term impacts, affecting deferred productivity. this is the case for example of the reduction in the fertility rate of long-cycle species, the effects of which span periods of - years [ ] . in short, the long-term costs of a slow response are rarely taken into account. economic analyses focus primarily on the effects of the outbreaks and rarely take into account the long-term effects of an endemic situation (characterized by less virulent outbreaks which recur for several years). this is the case of classic swine fever in haiti where recurrent outbreaks reduced the usage rate by %, which for pig farmers meant a loss of revenue of us $ . million per year [ ] [ ] [ ] . with major crisis, long-term impacts would make themselves felt, since the additional costs of financing prevention and control measures would lead to an equivalent reduction in savings and investments. for example, the analysis of the global impact of the avian flu crisis in europe was complicated by outbreaks of foot-and-mouth disease in brazil, the largest global exporter of beef and poultry. it is therefore easy to imagine what the combination of these two events would mean in terms of the upward push of prices of all meats, similar to what occurred in with north american beef and bovine spongiform encephalopathy. the european union, a net importer of beef, especially from brazil, would see an increase in the price of beef in its internal markets stemming from the embargo imposed on brazilian beef because of the foot-and-mouth disease. it must be pointed out that the crises could have a cumulative impact, particularly since they are amplified by the effects of globalization the following example therefore illustrates the ripple, spillover and remote effects: in the united states, where % of oleaginous and cereal production is geared towards animal production. an epizootic which reduces animal production by % would have the immediate consequence of the loss of , jobs, a surplus of . ton in cereals and oleaginous products, a % reduction in world trade and, crises in other producing countries. in , nearly americans out of every , died each year of infectious disease. laurie garrett, author of "the coming plague: newly emerging diseases in a world out of balance", writes that in the postwar environment, powerful medical weaponry (antibiotics, vaccines, water treatment, anti-malaria drugs) gave scientists confidence that they could significantly control and/or eradicate infectious disease from viral, bacterial or parasitical sources. in the late s, the surgeon general of the usa, william h. stewart, said that ". . .it was time to close the book on infectious diseases and pay more attention to chronic ailments such as cancer and heart disease." a measure of that success came towards the end of the s, when the world realized that smallpox had become the first disease to be eradicated from the human species. such halcyon days from the s to the early s are but a memory. by , the numbers were down to per , . the "health for all" accord, signed in , set a goal of the year for eliminating many international scourges. but amid all this optimism, the numbers started rising. in , people per , died and we know the rest of the story, . . . or do we? the grandiose optimism rested on two false assumptions; that microbes were biologically stationary targets, that for the most part human and other animal diseases were for the most part limited to those species and geographically sequestered. scientists have witnessed an alarming mechanism of microbial adaptation and change, anything but stationary, microbes and the insects, rodents and other animals that transmit them are in a constant state of biological, ecological flux and evolution [ ] . according to the u.k. centre for tropical veterinary medicine, - % of all the known species of infectious organisms that affect human health (causing a quarter of the world's deaths) can be transmitted by animals. approximately of these infectious organisms are linked to diseases that have only recently emerged, or have increased in severity (and geographic distribution) in recent years. who averages outbreak investigations every year, and around will require an international response. more than new and highly infectious diseases have been identified in the last years. furthermore, known strains of diseases such as tuberculosis, many species of gram positive and negative bacteria as well as many parasites, e.g. malaria, food animal coccidia have developed resistance to various classes of antibiotics, while old diseases have reappeared, such as cholera (in angola, with deaths), yellow fever (new cases recently reported in guinea, sudan, mali, and senegal), plague, dengue fever, meningitis, hemorrhagic fever, measles, mumps, rubella and diphtheria. there are emerging diseases just among marine life, reports the book conservation medicine, and these include tuberculosis in fur seals and chlamydiosis in sea turtles. more staggering than these cited numbers in living animals including humans are those coming out of the microbial genetic sequencing and diversity studies involving the oceans. recent data in this field suggest that the oceans of the world contains approximately ( ) phage particles or virions (cf. million metric tons), much of it turning over once per day and most likely be a regular source for current and future zoonotic and human infections. this vast mutation engine, even if one assumes a minimal mutation rate, generates the equivalent of hundreds of new complete human genomes per day. a -l sample of surface seawater was concentrated; ∼ × ( ) viral particles, the dna once randomly sheared and cloned yielded , fragments for sequencing. data analysis showed that most of the sequences were from previously unknown viruses. approximately . % of the total sequence samples overlapped, suggesting that the marine viral community was highly diverse. a unique mathematical analysis further suggested that approximately ( ) different new viral types may be present. it is obvious from this most recent description that complex and confounding zoonotic interactions can be expected to occur as ecosystems become concentrated and/or diluted during the upcoming environmental and ecological change. this will require a new breed of medical scientists that have foregone the days of super-and sub-specialization and are now grounded in both depth and broad general eco-and bio-medical systems training. few people recognize the broad range of clinical and basic veterinary research and its many important contributions to society in the realms of public health and food safety, vaccination, fertility, drug and vaccine development, surgical techniques and biodegradable materials, space medicine, animal health and welfare, and comparative medicine [ ] . opportunities in veterinary research include comparative studies with animals that shed light on human health problems; the development of tools to better detect, prevent, and control zoonotic diseases (that spread from animals to humans); the establishment of scientifically based policies for the humane treatment of animals; and the development of measures to secure and protect the nation's food supply and farm-animal economy from a potential act of bioterrorism [ , ] . the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-economic problems. the single profession that is most centered on the new paradigm is veterinary medicine. the three major disciplines within veterinary medicine and research -public health, comparative clinical and basic medicine, and animal health -are closely intertwined [ , ] . for example, research in comparative medicine contributes to animal health through the development of preventive medicine and treatment. the study of wildlife diseases contributes not only to wildlife health and conservation, but also to public health because many animal diseases can spread to humans. therefore, collaborative and interdisciplinary research is crucial in translating scientific advances from one traditional discipline to another. however, interdisciplinary research is in many cases hampered by administrative, funding, and cultural barriers between institutions. furthermore, agencies that support veterinary research have specific missions. funding to support proposed interdisciplinary research can be difficult to obtain when it is partially related to the mission of several agencies but does not perfectly fit the mission of any one agency. the future requires the veterinary research community to encourage research funders to develop a long-term interagency strategy for veterinary research [ , , ] . in , rudolph virchow, the father of comparative medicine, stated, "between animal and human medicine there are no dividing lines-nor should there be. the object is different but the experience obtained constitutes the basis of all medicine" [ ] . sir william osler ( - ), considered the best-known physician in the english-speaking world at the turn of the century, called the "most influential physician in history" is quoted as saying "veterinary medicine and human medicine complement each other and should be considered as one medicine." dr. calvin schwabe, a retired uc davis professor of veterinary medicine who pioneered the use of human disease tracking techniques in the study of animal illnesses, a global authority on animal diseases that are communicable to human beings and was an early visionary in a field that today is marked by the emergence of pathogens such as avian influenza, mad cow disease and sars. in , dr. schwabe established the department of epidemiology and preventive medicine at the uc davis school of veterinary medicine-the first of its kind in the world at a vet school. the author of more than publications, he promoted the concept of "one medicine," which attempts to bring the fields of human and animal health care together. to this goal in june of the american veterinary medical association (avma) announced that in partnership with the american medical association (ama) there was an adopted resolution calling for collaboration on a one health initiative. the two national, medical organizations will work collaboratively on areas of mutual medical interest, such as pandemic influenza, bioterrorism risks, biomedical research and will be charged with developing strategies to promote collaboration among the various health science associations, colleges, government agencies and industries. a quote from a ama board member, duane m. cady, md "new infections continue to emerge and with threats of cross-species disease transmission and pandemic in our global health environment, the time has come for the human and veterinary medical professions to work closer together for the greater protection of the public health in the st century," the avma policies supporting the concept of "one health" include: furthermore to strengthen this initiative and give it a renewable funding foundation, it would be recommended to move toward, the creation of: ( ) a specific focus at the national institutes of health (nih) on integrated veterinary research via the roadmap initiativemore specifically to create or combine existing potions of institutes into an institute of comparative medicine (icm); ( ) a joint interagency collaborative programs could also be established to enhance interdisciplinary collaborative research and have either re-routed an/or new congressionally supported intra-mural and extra-mural funding program; and ( ) while working out and introducing the legislative changes for the icm the nih should create a veterinary liaison with all the current institutes like the veterinary-medicine and public-health liaison at the centers for disease control and prevention (cdc) in which a veterinarian, dr. lonnie j. king, was selected to head up the agency's national center for zoonotic, vector-borne, and enteric diseases-a relatively recent creation, the center is dedicated to understanding infectious disease ecology and will help to ensure integration of veterinary and human medical research. the american veterinary medical association, with information provided by many other organizations and institutions, conservatively estimates a current deficit of public health veterinarians (e.g. usda food safety and animal disease control, homeland security, research on domestic and foreign animal diseases, wildlife disease control, laboratory animal care and research) and is expected to increase possibly to , by as the human population increases without intervention. comparably the health resources and services administration (hrsa) in the u.s. department of health and human services (dhhs) released a report in , projecting a shortfall of approximately , physicians in [ ] . if current trends continue, the full time equivalent (fte) physician supply is projected to grow to , by , while demand for physicians will increase to , due to the growth and aging of the u.s. population (physician workforce policy guidelines, ). the report projects shortages will be in greatest in non-primary care specialties. it has been over years since the federal government has allocated funds to increase the number of veterinarians and physicians that graduate each year. to begin to alleviate this problem the veterinary workforce expansion act (s. /h.r. ), which is currently being considered by the united states house of representatives and senate would provide an average of $ million per year for the next years in the form of competitive grants to help increase the number of veterinarians entering public practice. if passed and funded, this bill would allow the nation's veterinary schools and other institutions training public health veterinarians to apply for competitive grants to increase capacity in the form of classrooms, teaching laboratories, research facilities, and administrative space. this bill would be vital to the nation's ability to protect human and animal health, as veterinarians are often the first line of defense for both. the text of the bill can be found at: http://thomas.loc.gov by searching by the bill numbers listed above. a list of co-sponsors to the bill can also be found via that site by selecting the link entitled "bill summary & status". over the past century, humanity has had a devastating impact on the earth's wildlife and ecosystems. we are in fact living through the largest mass extinction since the end of the dinosaurs million years ago. unless effective solutions are found, this new century will see the demise of countless more species and pristine ecosystems, particularly in the tropics. the global society, and what surrounds and influences it, are in profound change. these changes will have very significant impacts on future veterinary medicine and veterinary medical education. there are major demographic, political, environmental, disease, technological, and economic influences, all forcing changes onto society. a few examples illustrate the point [ ] . • with immigration into north america accelerating, combined with a declining birth rate, the ethnic diversity in society will continue to increase, with the associated impact on values. • in , for the first time in history, urban people will outnumber rural people. • political destabilization, inflamed by bio-terrorism and religious fanaticism, is expected to increase. • changes in the atmosphere are causing powerful shifts in the environment (melting of the ice caps, rising sea levels) and in the climate (hurricanes, flooding). • global water shortages, especially in heavily populated areas, will soon approach critical levels. to information and service (http://www.jvmeonline.org/cgi/ content/full/ / / #b #b ). • consumer spending power in emerging economies will go from $ trillion to $ trillion by , but the gap between rich and poor is increasing. how will these changes alter the needs of society? how must academic veterinary medicine adapt to prepare veterinarians to respond to these new needs? clearly, humanity has yet to find a way to live on planet earth in a potentially sustaining manner where by stabilizing flora and fauna remain intact to promote healthy ecosystems diversity for all species including our very ownhumans. for example, it is estimated that the equivalent of six earths would be needed to sustain the current world's population if people everywhere consumed natural resources at the rate we do in the united states. it is interesting to think that vaccination directly or indirectly impacts six of the seven united nations millennium goals. understanding and coping effectively with an emerging crisis may sometimes require the birth of action-oriented "crisis disciplines." conservation medicine: ecological health in practice brings together an impressive group of experts from diverse specialties medicine, veterinary science, conservation biology, epidemiology, parasitology, public health, and others) to examine the links among human health, wildlife health, and ecosystem health and begin to address questions like: • how factors such as climate change, endocrine disruptors, and toxic microalgae affect wildlife and human health; • the importance of biodiversity for human health (as medical models, sources of medicines, factors in the ecology of infectious diseases, and indicators of environmental quality), with a review of biodiversity-related biomedical research projects funded by the national institutes of health from to ; • how the health of rainforest-dwelling peoples depends on such diverse factors as forest integrity, floods, seasonality, community organization, education, gender dynamics, national budgets, and global markets; • how wildlife health relates to environmental security; • the health hazards of ecotourism; • the causes and impacts of emerging infectious diseases of humans and wildlife; • how the health of terrestrial and marine animals and ecosystems are monitored, and descriptions of innovations using stool dna and retrovirus evolution as markers of animal population dynamics, stool hormones to indicate species stress, and animal behaviors as proxies for the health of ecosystems; • how habitat fragmentation and reduced biodiversity can increase the risk of lyme disease infection; • how land use changes such as deforestation and water projects influence the ecology of malaria and other vector-borne infections; • how ecological health and wildlife disease are managed in national parks; • the role of zoos in the recovery and conservation of endangered species; • how reducing the burden of infectious disease among park workers in africa could prevent a devastating epidemic among the world's remaining mountain gorillas; • how efforts to control livestock diseases are affecting wildlife health and ecosystems in botswana; • teaching ecosystem health in an undergraduate medical curriculum. more than one billion poor people in asia and africa are closely linked with animals for their livelihoods and they pay a really high tribute to different animal diseases-one can easily demonstrate that improving animal-health mechanisms at the national and local level, and decreasing this weight of animal diseases, will lead very quickly to the alleviation of poverty for this class of people. the world organization of animal health (oie) brings together chief veterinary officers from countries in an effort to create global standards of animal health and animal welfare robust enough to withstand the daunting challenges of worldwide commerce. with an annual budget of around $ million, it is a significantly smaller organization that its human-health counterpart, the world health organization, which has some $ billion a year expenditure on its programs. last year, over billion food animals were produced worldwide to help feed a population of billion people, resulting in trillions of pounds of animal products distributed worldwide and projections for show that demand for animal products will increase by %, especially in developing countries. this poses unprecedented risks for human safety and will require a closer linking of the two agencies. along with those core concerns, oie also addresses farming practices, analyzes import risks, and mediates bilateral trade disputes among its member countries. among its most important achievements in recent years, has been the eradication of rhinderpest, a centuries-old intestinal disease also known as "cattle plague in some countires." in the early s, rhinderpest wiped out upwards of $ million worth of african livestock, contributing to widespread human famine. in general professional students of veterinary and human medicine are exposed more likely to a more scientifically exposed than a politically exposed culture. animal and human health policies must be science-based the rallying cry that is heard from animal and human health professionals around the world. the second verse of this mantra is often 'science, not politics', as if science is unquestionably 'good' and politics 'evil'. antipathy toward politics is worn as a badge of honor. the crowning moment frequently comes with the proclamation, 'i am a scientist, i want nothing to do with politics. ' the phrase 'science-based' infers that the underlying justification of animal/human health policy is derived from knowledge gathered through the systematic observation of, or experimentation with, phenomena. scientific knowledge implies the compilation and analysis of data by individuals with advanced education in specific disciplines. scientists seek facts, the fundamental truths which explain the world around us. at face value, the 'science, not politics' paradigm has a great deal of appeal. however, the very notion of fundamental truth is illusory, as scientific knowledge changes frequently with new observations and experiments. furthermore, conjecture and refutation characterize the scientific method, with disagreement and debate the recognized features of scholarly pursuit. scientists often reach conflicting interpretations of observational and experimental data. consequently, individual scientists may champion different, even diametrically opposed, sets of ideas and principles, so that any number of alternatives may be justified as 'science-based'. finally, animal health professionals typically consider only the biological and physical sciences as 'true sciences', dismissing the social sciences. politics reflect the human need for organization of authority, whether in public or private life. politics exist whenever two or more people come together. the terms 'office politics' and 'family politics' are recognized as clearly as the collective activities surrounding local or national governance. politics exist even in science, affecting scientific organizations, refereed publications and academe. indeed, politics are inescapable. all public courses of animal/human health action adopted by governments emerge from the interplay of science and politics. the policy-making process is governed by rules and regulations, affected by the organizational culture of the government agencies involved, and constrained by legal authorities, political correctness and resource availability. the animal/human health policy-making process involves consideration of current biological and physical scientific knowledge. policy decisions also consider social science factors including ideologies, economics and public opinion. hence the etiology of animal/human health policy is multifactorial. the current older traditional schools of veterinary and human medicine and future schools of "one medicine" will need to include leadership, economics and local, state, national and international governances courses and better training in mechanisms of public policies and rule making. epidemiologists accept the concept of multifactorial etiology as a basic tenet. we discuss disease in terms of agent, host and environment interactions. the practice of applied epidemiology demands a breadth of knowledge and the ability to work in interdisciplinary teams. the more complex the problem, the greater the demand for additional knowledge and insights. veterinary and human epidemiologists study risk factors for disease in animal/human populations and develop strategies for health promotion and disease control [ ] . unfortunately these are done frequently in a vacuum when in fact the two are inter-related and contributing to the observed and at the time undiagnosed disease spectrum however, animal/human health problems cannot be resolved by consideration of biological and physical factors alone. the veterinary and human (someday to become one and the same) epidemiologist working with field problems soon recognizes the critical role played by people in animal/human health issues. applying epidemiological principles to animal/human disease prevention requires consideration of social issues such as attitudes toward animals, cultural and religious mores, and individuals' willingness and capability to implement the prevention strategies. implementing prevention on a national scale brings additional factors into the equation such as availability of resources, adequacy of veterinary services, and animal health infrastructure, among others. therefore, animal health policy development and imple-mentation require attention to macroepidemiology, the study of all of the economic, social and political inputs which affect the distribution and impact of animal or human disease at the national level [ , ] . the low hanging fruit of yesterdays fields of microbiology, zoonotic and emerging infectious diseases, immunology, antibiotic sensitivity, vaccine development, oncology, anti-viral drugs, public health, ecology, environment, human and animal health, to name a few have been picked. today comparative and interdisciplinary research is critical to translating scientific advances from one discipline or species to another and providing new insights into human health problems. scientific fields such as laboratory animal medicine, pathology, immunology, biophysics, mathematics, bioinformatics, genetics, molecular biology and toxicology, when combined with veterinary medicine, have proven especially relevant to success in biomedical research. veterinarians also have contributed to public health through the care of companion animals. fifty-seven percent of all u.s. households own a dog, cat, or both. in addition, millions of exotic animals, birds, and reptiles are kept as pets [ ] . although pets enrich the lives of humans, they also potentially can threaten public health. veterinarians help educate the public about prevention of zoonoses; vaccinate large numbers of pets for zoonotic diseases, such as rabies and leptospirosis; and reduce the level of ecoparasites that can transmit human diseases and intestinal worms, such as roundworms and hookworms, which can cause serious health problems in humans. the , private-practice veterinarians in the united states form a valuable front line for detecting adverse health events, reducing zoonotic diseases, and delivering public health education. because veterinarians work at the interface of human, animal, and environmental health, they are uniquely positioned to view this dynamic through the lens of public health impact. significant changes in land use, expansion of large and intensified animal-production units, and microbial and chemical pollution of land and water sources have created new threats to the health of both animals and humans [ ] . because animals share human environment, food, and water, they are effective sentinels for environmental, human, and public health problems, including bioterrorism. concerns are increasing about antimicrobial resistance of pathogens, waste and nutrient management, and potential runoffs into streams, rivers, and oceans. food animal and wildlife populations are inextricably linked to some environmental problems. together these have led to creation of a new scientific discipline called conservation medicine and ecosystem health, and veterinarians are assuming a leadership role in the field [ ] . several decades ago, special factors came together to create a new epidemiologic era characterized by increases in emerging and reemerging zoonoses [ ] . humans, animals, and animal products now move rapidly around the world, and pathogens are adapting, finding new niches, and jumping across species into new hosts. in , approximately billion food animals were produced to help feed a world population of . billion persons; the united nations' food and agriculture organization estimates that demand for animal protein will increase by % by , especially in developing countries [ ] . the lessons learned from severe acute respiratory syndrome, west nile virus, monkeypox, and avian influenza are reminders of the need to view diseases globally; integrate animal and public health surveillance, epidemiology, and laboratory systems; and create new strategic partnerships among animal, human, and public health professions [ , ] . veterinarians are essential to the detection and diagnosis of and response to these threats and are integral to first-line defense and surveillance for bioterrorism agents. the convergence of human and animal health drove creation of the newly proposed national center for zoonotic, vector-borne, and enteric diseases. plans are being completed to establish several multidisciplinary state-level zoonosis research and development centers. the veterinary profession has recently gained a important foothold on the health and human services government research institutes at the cdc and evolved in prominence as a member of the health professions and has established its importance and usefulness to human and public health. it is hoped and expected that with the developing visions and challenges outlined in this overview we will see the continued melding of veterinary and human medicine to create new educational programs and tools for developing future leaders for solving globally some of the most challenging public and animal health, ecosystem, and conservation problems of the st century. the value of vaccination world health organization. measles deaths drop dramatically as vaccine reaches world's poorest children the value of vaccination: a global perspective intervention cost-effectiveness: overview of main messages the costs and benefits of front-loading and predictability of immunization. cgd working paper veterinarians in population health and public practice: meeting critical national needs cattle, priests and progress in medicine veterinary medicine: an illustrated history committee on assessing the nation's framework for addressing animal diseases. national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases veterinary medicine and public health at cdc the th anniversary of the veterinary medical corps officers of the u.s. public health service veterinarians and public health: the epidemic intelligence service of the centers for disease control and prevention actual causes of death in the united states primary care: balancing health needs, services, and technology mercer survey of employer-sponsored health plans recommendations to the congressional prevention coalition: nine high-impact actions congress can take to protect and promote the nation's health hedis : the health plan employer data and information set and the health accounts team health spending in : signals of change effectiveness in disease and injury prevention estimated national spending on prevention-united states disease prevention research at nih: an agenda for all. workshop j: prevention strategies, economic realities, and identification of prevention research needs dissecting cost-effectiveness analysis for preventive interventions: a guide for decision makers us graduate medical education editor's choice: preventive medicine makes us miserable immunization in developing countries: its political and organizational determinants progress towards immunization goals : summary presentation of key indicators global market-global 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veterinary science committee on increasing veterinary involvement in biomedical research. national research council. national need and priorities for veterinarians in biomedical research animal agriculture and the global food supply. task force report . ames, iowa: council for agricultural science and technology council on graduate medical education. physician workforce policy guidelines for the u.s. for towards a better map: science, the public, and the media. economic and social research council the general epidemiologist: is there a place in today's epidemiology? misleading media reporting? the mmr story vaccine delays hit chiron's recovery. la times ten great veterinary public health/preventive medicine achievements in the united states advance market commitments: a policy to stimulate investment in vaccines for neglected diseases historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states priorities in developing countries surveillance and monitoring systems for animal health programs and disease surveys committee on assessing the nation's framework for addressing animal diseases, national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases. washington sustainable agriculture solutions (sas)-action report the annual report the annual report understanding and improving health and objectives for improving health. vols an agenda for action: veterinary medicine's crucial role in public health and biodefense and the obligation of academic veterinary medicine to respond investing in health immunization at a glance immunization at a glance world development indicators polio eradication website, global case count measuring the national economic benefits of reducing livestock mortality the primary author wishes to acknowledge and dedicate this paper to the late dr.'s jonas salk and maurice hilleman whose lifetime experiences and contributions are unprecedented in medicine and vaccinology and whom personally encouraged and instilled in me during numerous public and private discussions and meetings the importance of working in and helping to advance the field of vaccinology for the betterment of global human and animal health. key: cord- -grz fe l authors: liu, shengwang; zhang, xiaonan; gong, liyang; yan, baolong; li, chengren; han, zongxi; shao, yuhao; li, huixin; kong, xiangang title: altered pathogenicity, immunogenicity, tissue tropism and ′- kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: grz fe l in this study, we attenuated a chinese lx -type nephropathogenic infectious bronchitis virus (ibv) strain, ck/ch/lhlj/ v, by serial passage in embryonated chicken eggs. based on sequence analysis of the ′- kb region, the ck/ch/lhlj/ v virus population contained subpopulations with a mixture of genetic mutants. the titers of the virus increased gradually during serial passage, but the replication capacity decreased in chickens. the virus was partially attenuated at passage (p ) and p , and was fully attenuated at p . it lost immunogenicity and kidney tropism at p and p , respectively. amino acid substitutions were found in the ′- kb region, primarily in the spike (s) protein. substitutions in the s subunit occurred between p and p and all subpopulations in a virus passage showed the same substitutions. other substitutions that occurred between p and p , however, were found only in some subpopulations of the virus passages. a -bp deletion in the ′-utr was observed in most subpopulations of p and p , and might be related to virus replication, transcription and pathogenicity. the changes described in the ′- kb region of the virus are possibly responsible for virus attenuation, immunogenicity decrease and tissue tropism changes; however, we cannot exclude the possibility that other parts of the genome may also be involved in those changes. coronaviruses belong to the family coronaviridae and the order nidovirales, and are classified into three groups based on the absence of genetic and antigenic relationships between the species of the different groups [ , ] . they are known to cause upper and lower respiratory diseases, gastroenteritis, and central nervous system infection in a number of avian and mammalian hosts, including humans [ ] . the infectious bronchitis virus (ibv) belongs to the group coronaviruses. it primarily causes respiratory disease in domestic fowl, although it also replicates on epithelial surfaces of the alimentary tract, oviduct, and kidney, and is one of the most economically important pathogens in the poultry industry [ ] . ibv has four essential structural proteins: the phosphorylated nucleocapsid (n) protein, and the three membrane proteins spike (s), integral membrane (m), and small envelope (e). although the s subunit of the s protein carries virus-neutralizing and serotype-specific deter-embryonated eggs, recombination between virus subpopulations, and accumulation of mutations in the s region can lead to the formation of attenuated viruses [ ] . the importance of s in determining cell and tissue tropism has been demonstrated for several coronaviruses, such as murine hepatitis virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , porcine transmissible gastroenteritis virus [ , ] , and severe acute respiratory syndrome coronavirus [ ] . in the case of ibv, the s subunit of the s protein determines the serotype of ibv and is responsible for viral attachment to cells. furthermore, it has been shown that s is a major determinant of cell tropism in culture [ ] , and the majority of changes accumulated during the adaptation of ibv to vero cells are in the s gene [ ] . however, differences in one or more other genes are responsible for the highly attenuated phenotype of the beaudette ibv laboratory strain [ ] , and the roles of these gene products in the attenuation process have yet to be determined. in spite of the extensive use of vaccines, nephrotropic ibv outbreaks are frequent in china [ ] [ ] [ ] [ ] . lx -type has been the predominant ibv type in china in recent years [ ] [ ] [ ] , and appears to have become widespread in several countries in europe, causing severe losses to both the layer and broiler industries [ ] . in addition, this type of ibv has increased in recent years in both china and european countries; thus, the development of an efficacious live-attenuated vaccine against lx -type ibv is important. we are developing an ib vaccine by serial passage of the ibv strain ck/ch/lhlj/ v, which represents the lx -type, in embryonated eggs. evaluating the attenuation, the growth of viruses in embryos, the efficacy in poultry populations, and the changes in molecular characteristics after serial passage were the primary focus and objectives of the present study. in a future study, practical considerations regarding the development of such a vaccine will be examined. we used a virulent ibv strain, ch/ck/lhlj/ v, which was previously isolated during an outbreak of ib in at a broiler farm in the heilongjiang province in china. clinical signs and lesions observed during the outbreak included nephritis and mortality. the virus was isolated from the kidney of a dead broiler using -dayold embryonated specific pathogen-free (spf) chicken eggs. the ibv strain was identified by means electron microscope examination, reverse transcriptase-polymerase chain reaction (rt-pcr) and sequencing of the entire s protein gene, as described previously [ ] . an analysis of the molecular characteristics showed that this virus exhibited limited homology (not more than % of amino acids) to genotypes representing the vaccine strains h and w [ ] . phylogenetic analysis showed that this virus was an lx -type [ ] . the virus stock for this study was produced by inoculating the virus into embryonated spf chicken eggs via the allantoic cavity and collecting the infectious allantoic fluid h post-inoculation. the allantoic fluid was clarified by centrifugation at × g for min and filtered with a teflon membrane. in addition, three ibv strains, ck/ch/ldl/ ii, ck/ch/lxj/ i and ck/ch/lshh/ i, were used as references for the pathogenicity study; their backgrounds and types were reported previously [ ] . fertile white leghorn spf chicken eggs, and white leghorn spf chicks were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. the birds were maintained in isolators with negative pressure, and food and water were provided ad libitum. fifty-one-day-old white leghorn spf chickens were used to assess the pathogenicity of the ck/ch/lhlj/ v strain. five groups of chickens were kept in isolators with negative pressure. at the age of days, groups - were inoculated intranasally with . ml per chick containing . - . median embryo infectious doses (eid ) at passage level of strains ch/ck/lhlj/ v, ck/ch/ldl/ ii, ck/ch/lxj/ i and ck/ch/lshh/ i. group was mock-inoculated with sterile allantoic fluid and served as a control ( table ). the chicks were examined daily for signs of infection for days after inoculation. tracheal swabs and blood samples were collected from all birds in each treatment groups at , , , and days post-inoculation. serum was stored at − • c until elisa testing was performed. the tracheal swabs were used for virus recovery attempts in embryonated chicken eggs. the ch/ck/lhlj/ v strain was serially passaged times by inoculating -day-old spf chicken eggs by the allantoic cavity route as described previously [ ] . inoculated eggs were incubated for - h at • c in an egg incubator (heraeus, germany). the chorioallantoic fluids were harvested and stored at − • c or used directly for subsequent passage. at every th passage starting with passage , the virus was examined for viability by inoculation of two to three additional eggs for days and observation of the embryos for clinical signs consistent with ibv infection. in addition, these selected passages were examined by negative contrast electron microscopy (jem- , ex) for the presence of coronavirus, and by reverse transcriptase-polymerase chain reaction (rt-pcr) and sequencing [ ] to verify the virus type. passage (p ) for the pathogenic strain, and p , p and p were examined in more detail. the viruses of these four passages were propagated once in -day-old embryonated spf chicken eggs as described for field isolates [ ] , to obtain titers of - eid per . ml. before use, viruses from the allantoic fluids of inoculated eggs were confirmed by negative contrast electron microscopy and by rt-pcr and sequencing. one hundred fifteen, -day-old spf white leghorn chicks were housed in different isolators and divided into five groups. groups - had birds each, and group included birds. chickens in groups - were inoculated with p , p , p and p , respectively, by oculonasal application at days of age with a dose of log . -log . eid per chick (table ). birds in group were mock-inoculated with sterile allantoic fluid and served as the control. five birds from each group were killed humanely days post-inoculation. the trachea and kidney were collected for virus titration. blood samples from birds in each group were collected at , , , and days post-inoculation. the serum was stored at − • c for elisa testing. the chicks were examined daily for signs of infection for days after inoculation. ninety, -day-old spf white leghorn chicks were housed in different isolators and divided into five groups. groups - , and the positive control group, had birds each, and the negative control group included birds. chickens in groups - were inoculated with p , p and p , respectively, by oculonasal application at days of age with a dose of log . -log . eid per chick (table ) . birds in the positive and negative control groups were mock-inoculated with sterile allantoic fluid. at days post- a ten chicks per group. b dose per chick, l. c two procedures were used for virus recovery after challenge. first, lesions in embryos that had been inoculated with pooled samples (tracheal swabs) were observed. second, rt-pcr using oligonucleotide primers n(+) and n(−) on rna recovered from allantoic fluid of the same eggs was conducted. the results from the two procedures were identical. d number seroconverted/number inoculated. e days after challenge. f ck/ch/lhlj/ v passage (p ) was used for the virulence study. inoculation, birds in groups - and in the positive control group were challenged by oculonasal application with . eid / . ml of pathogenic ch/ck/lhlj/ v virus, while the birds in the negative control group were mock-inoculated again with sterile allantoic fluid. ten birds each from groups - and the positive control group, and birds from the negative control group were killed humanely days post-challenge. the trachea and kidney were collected for virus recovery. blood samples from birds in each group were collected at , , , and days after challenge. the serum was stored at − • c for elisa testing. the chicks were examined daily for signs of infection for days after inoculation. the virus stocks used for the pathogenicity study, and the tissue samples of tracheas and kidneys collected days post-inoculation from experiment , were used for rt-pcr amplification and virus titration. tissue samples were homogenized individually and rt-pcr was conducted using primers n(+) and n(−) as described previously [ ] . virus titrations were performed in -day-old embryonated chicken spf eggs via the allantoic cavity route of inoculation, and titers were expressed as % (median) embryo infectious doses (eid ) [ , ] . serial l -fold dilutions were used for titrations. at each dilution, five embryos received . ml inoculum. the embryos were candled daily and examined for one week; those showing characteristic ibv lesions, such as dwarfing, stunting, or curling of embryos, were recorded as infected by ibv. the swab samples taken in the pathogenicity study from each group at each time point were pooled, and the tissue samples of tracheas and kidneys collected days post-challenge from experimental were homogenized individually for virus isolation. for virological examination, the pooled samples were clarified by centrifugation at × g for min and filtered with a teflon membrane. for virus isolation from the trachea and kidney, individual samples the morbidity and mortality were those of p , p and p vaccinated chickens after challenge. c number seroconverted/number inoculated. d two procedures were used for virus recovery after challenge. first, lesions in embryos that had been inoculated with individual tissue samples (trachea or kidney) were observed. second, rt-pcr using oligonucleotide primers n(+) and n(−) on rna recovered from allantoic fluid of the same eggs was conducted. the results from the two procedures were identical. e days after challenge. sequence and position of the oligonucleotides used in rt-pcr. oligonucleotide sense a sequence ( - ) gene position in genome b ibv- + tattgattagagatgtgg s - s oligo − cataactaacataagggcaa s - ibv- + gcttcttgagaa (t/c) were homogenized, diluted : with pbs, clarified by centrifugation at × g for min and filtered with a teflon membrane. the filtered samples were inoculated into at least four spf embryonated eggs via the allantoic cavity ( . ml per egg). the eggs were candled daily to record embryo mortality, and allantoic fluid from two of the inoculated embryos was collected h post-inoculation for rt-pcr amplification. after days, the remaining embryos were chilled at • c and examined for characteristic ibv lesions such as the dwarfing, stunting, or curling of embryos. embryo mortality recorded in the first h post-inoculation was considered nonspecific. samples were considered negative if the embryos did not show lesions after three blind passages of -day duration. a positive sample was recorded if the specific lesions were observed and the rt-pcr amplification was positive. serum samples were assayed using a commercial total antibody elisa (idexx corporation, westbrook, maine, usa) according to the manufacturer's instructions. each sample was usually tested in triplicate. serum-to-positive ratios (s/p ratios) were calculated as described previously [ , ] . individual serum titers were calculated from these s/p ratios, evaluated as positive or negative, and expressed as od nm values according to the manufacturer's instructions. the strategy for cloning the - kb region of ck/ch/lhlj/ v strains was described previously [ ] . briefly, four fragments spanning the - . kb region of the ibv genome were obtained by rt-pcr from each of the four virus passage levels. the sequences and locations of the primers used in this study are in table . briefly, viral rna was extracted from l of allantoic fluid from p , p , p and p virus stocks using trizol reagent (invitrogen, grand island, usa) according to the manufacturer's instructions. rna was air-dried for - min, re-dissolved in l rnase-free water and stored at − • c until use. reverse transcription (rt) was performed with m-mlv reverse transcriptase (invitrogen, grand island, usa) using the reverse primer n(−). rt procedures were performed using l of rna in a l reaction volume as previously described [ ] . each cdna fragment was amplified from rt products by pcr as previously described [ ] . pcr products were purified from agarose gels using a dna extraction kit (boehringer mannheim, germany) and sequenced directly or cloned into the pmd- t (takara, dalian, china) vector following the manufacturer's instructions. rna extraction, cdna generation, pcr amplification and gene fragment cloning and sequencing were independently conducted times for each of the four passages, p , p , p and p . the viral stocks used were from independently inoculated embryos. in total, clones of each gene fragment were selected and sequenced for each of the ck/ch/lhlj/ v passages. five clones were selected for sequencing each time. sequences were compiled and orfs were determined using the gene runner program, version . (http://www.generunner.com). nucleotide and amino acid sequences for the - . kb fragments were assembled and aligned using the megalign program (dnastar). the genomic sequences of the - kb region of ibv ck/ch/lhlj/ v p , p , p and p have been submitted to the genbank database and have been assigned the accession number fj and fj to fj . in the pathogenicity study, all four ibv strains produced typical ib-induced disease. all chicks exhibited respiratory clinical signs at about - days post-challenge with all four ibv strains. clinical signs included tracheal rales, watery eyes, nasal mucus, and sneezing, similar to those caused by other ibv strains with affinity for the respiratory tract [ ] . all ibv strains caused death - days post-challenge; however, strain ck/ch/lhlj/ v caused the highest mortality. gross lesions of the dead chickens were mainly confined to the kidneys. the kidney parenchyma of the affected birds was pale, swollen and mottled; tubules and urethras were distended with uric acid crystals. hemorrhagic lesions of the cecal tonsil and respiratory tract were also observed in some of the affected chickens. the clinical signs of the inoculated birds tended to disappear gradually after days of challenge. no clinical signs and gross lesions were observed in the negative-control group (data not shown). all of the challenge ibv strains could be detected in the trachea at days post-challenge by the recovery of the virus using -day-old embryos and subsequent rt-pcr; however, strains ck/ch/lhlj/ v and ck/ch/ldl/ ii could be detected in the trachea of the birds at day post-challenge. the virus was not detected in the trachea of the unchallenged negative-control birds. as summarized in table , most of the chicks challenged with the four ibv strains showed no seroconversion at days post-challenge, but antibodies were detected by elisa in most of the birds after days postchallenge. nine-day-old embryonated eggs were used to determine the growth ability of p , p , p and p in vitro. equal doses ( eid ) of each virus at each passage level were used to inoculate three -day-old embryos. the inoculated embryos were incubated at • c, and allantoic fluid was harvested at h for virus titration. based on movement and the extent of bleeding, curling, and dwarfing, all of the inoculated embryos were determined to be infected but alive after h. eid was determined for each sample. the titers of ck/ch/lhlj/ v increased gradually from p to p , indicating an increase during serial passage in spf embryonated chicken eggs (fig. ) . clinical signs, mortality and gross lesions were used to assess the attenuation of ibv ck/ch/lhlj/ v p , p , p and p using spf chickens. as summarized in table , all birds given p and p , and % of birds given p by oculonasal application showed overt disease, as did the birds challenged with virulent ck/ch/lhlj/ v in the pathogenicity study, in comparison with p -inoculated and negative control chicks. clinical signs were observed from day or , to day post-inoculation. eight chicks inoculated with p and two chicks inoculated with p died during the experiment. gross lesions of dead chicks were mainly confined to the kidneys, and were similar to those in the birds in the pathogenicity study. in addition, mucous exudate was observed at days post-challenge in the tracheas of all birds inoculated with p and p , while only one of the chickens in the p -inoculated group exhibited a respiratory lesion. chickens in both the p -inoculated and negative control groups showed no clinical signs, death or gross lesions. based on the clinical response, mortality and gross lesions, we can conclude that ibv ck/ch/lhlj/ v p and p were partly attenuated, but p was fully attenuated by serial passages in embryos. two criteria were used for evaluating altered immunogenicity of selected ibv ck/ch/lhlj/ v passages. first, serum igg antibodies specific for ibv ck/ch/lhlj/ v were measured with an indirect elisa test. as shown in table and fig. , none of fig. . humoral immune responses in spf chickens inoculated with ibv ck/ch/lhlj/ v passages evaluated by indirect elisa. ten chickens were tested in each inoculated group at , , and days after inoculation. dashes show the s/p ratios, calculated as described in section . serum samples with s/p ratios equal or above the dashes were considered positive, and those below were considered negative. the serum sample s/p ratios of chickens in the negative control group were all below the dashes and are not indicated in figures. the chickens inoculated with the four selected ck/ch/lhlj/ v passages showed seroconversion at days post-inoculation. at days post-inoculation, %, % and % of the p -, p and p -inoculated chickens showed seroconversion, respectively. however, none of the p -inoculated chickens showed seroconversion at that time. all chickens inoculated with p and p showed seroconversion from days on, and only % and % of the p -and p -inoculated chickens, respectively, showed seroconversion at days post-inoculation. fewer than % of the p -inoculated chickens showed seroconversion at days postinoculation. however, almost all chickens inoculated with p , p and p showed seroconversion at that time point. second, the vaccination-challenge test was used to evaluate the immunogenicity of ibv ck/ch/lhlj/ v after serial passage in spf embryonated eggs. as summarized in table , none of chickens inoculated with p or p showed clinical signs or death after challenge with the virulent p level strain, indicating good clinical protection provided by vaccination with p or p . however, % of the p vaccinated chickens showed clinical signs, and of vaccinated chickens died after p challenge. parallel to the clinical protection results, vaccination with p and p also offered good trachea and kidney protection against virulent p challenge, although one of the p -vaccinated chickens was positive for virus recovery in the trachea after p challenge. in contrast, more than % of the p vaccinated chickens were positive for virus recovery from both tracheas and kidneys after challenge with the virulent p strain, indicating poor trachea and kidney protection after p vaccination. based on these results, the partly attenuated p and p viruses were considered capable of stimulating systemic immunity in chicks; however, immunogenicity against the fully attenuated p decreased as expected. viruses were identified by rt-pcr at days post-inoculation in both the tracheas and kidneys of the p -, p -, p -and p inoculated chickens. all trachea samples, and kidney samples from p -and p -inoculated chickens, were positive by pcr; however, only one kidney sample from p -and p -inoculated chickens was positive, respectively. all trachea and kidney samples were titered at that point, and as shown in fig. , viral titers steadily decreased with passage. p had almost the same titer in the trachea as p ; however, the titers of p and p in the trachea were lower than those of p and p , as expected. it is puzzling that viral titers in the kidneys rapidly decreased and were eventually lost when the corresponding virus adapted to embryonated eggs. compared to the virulent p virus, p showed a lower titer in the kidneys, while p and p had already lost kidney tropism after serial passages in spf embryonated chicken eggs (fig. ) . the ibv ck/ch/lhlj/ v strain was propagated in embryonated eggs and passaged times. to investigate whether nucleotide and/or amino acid sequences changed during passage, p , p , p and p were chosen for rt-pcr amplification and sequence analysis of the - kb region. all rt-pcr products were analyzed, and a single band of expected size was visible after ethidium bromide staining of the products on a . % agarose gel. twenty independent clones of each fragment from four independent rt-pcrs were selected and sequenced, so the sequence profiles represented all genetic diversity within the populations of the viral rnas from a given passage. as summarized in table , no sequence changes were observed in orf a or gene (orf a and orf b) after passages. only one to two nucleotide mutation(s) occurred in the orf b, c (e), m and n genes after passages, and all amino acid substitutions in those regions occurred between p and p . however, most nucleotide mutations were found in the s region between p , p , p and p and all of the nucleotide mutations resulted in amino acid substitutions. significantly, a majority of the sequence changes in the s region of the s protein occurred between p and p . most of the sequence changes in the s region were observed between p and p , however. in addition, a single base mutation (g → t) was observed in out of clones of p , and this mutation was located at the -terminal region of the s gene, at nucleotide from the aug start codon of the s gene. this caused a frameshift that would result in a c-terminally truncated product, if synthesized. the truncated product would contain residues instead of the residues of the normal s protein (fig. ) . furthermore, the s proteins of p , p and p all contained the mutation for truncated s. to investigate whether serial passage of ibv ck/ch/lhlj/ v in embryonated eggs can cause sequence changes in the untranslation region ( -utr), the -utr sequences of p , p , p and p were amplified and sequenced. surprisingly, a -bp deletion, located nucleotides downstream of the stop codon of the n protein gene, was found in the -utr of % and % clones of p and p , respectively, as shown in fig. . interestingly, both the deleted and non-deleted -utr sequences of the secreted offspring viruses were detected in the respiratory tracts of the chickens inoculated with ibv ck/ch/lhlh/ v p at days post-inoculation (data not shown). nucleotide and amino acid changes in the region of ibv ck/ch/lhlj/ v and embryo-passaged derivatives. position (nt) a nucleotide change codon change amino acid change ibv ck/ch/lhlj/ v is a nephropathogenic ibv strain of the lx type that is highly pathogenic to spf chickens, with % morbidity and approximately % mortality. lx -type ibv has been one of the major types of ibv circulating both in china and european countries in recent years [ , [ ] [ ] [ ] [ ] . however, both experimental infections and field results have shown that available commercial vaccines provide poor protection against the lx -type ibv [ ] . hence, we selected the lx -type ibv ck/ch/lhlj/ v strain for attenuation by serial passage in spf chicken embryonated eggs. coronaviruses, including ibv, have been shown to exist as a mixture of genetic mutants within an isolate [ ] [ ] [ ] . this is also the case for the ibv isolate ck/ch/lhlj/ v. based on sequence data from cloned rt-pcr products, % of the sequences in the low-level virus populations (p ) showed a -bp deletion upstream of hypervariable region (hvr ) of the s gene compared to the other sequences in the same population. however, this deletion was not observed in all of the s sequences in the high-level virus populations (p , p and p ). we cannot determine if this reversion of the s gene sequence was due to a recombination event or the selection of a subpopulation in the process of serial passage in chicken eggs. in addition, a stop codon was observed in % of the s protein sequences of p at residue due to a mutation changing a glu codon to a stop codon, resulting in an s protein with amino acids missing at its carboxy-terminal end. all sequences in p , p and p showed this mutation. this indicates that this region is not necessary for virus formation. the above mentioned s gene sequence heterogeneity indicated the presence of different proportions of subpopulations in ck/ch/lhlj/ v. most genetic changes occur in the s gene during adaption to the host [ , , ] . however, until now, it was not clear if mutations or the selection of a fit subpopulation was responsible for the changes observed when coronaviruses were attenuated or adapted to a particular host system [ ] . routinely, ib attenuated vaccines are developed by multiple passages (generally or more) of a field isolate, in embryonated domestic fowl eggs, until the desired blend of non-pathogenicity and immunizing capacity has been achieved. the mutations that cause the attenuation of pathogenicity are not known. we found amino acid substitutions between pathogenic ck/ch/lhlj/ v p and its embryo-passaged, attenuated derivative p . of these substitutions, were in the s protein ( substitutions each in the s and s regions). importantly, a common substitution at residue in the s protein (his to tyr), found in the d and tw / ibv strain and related to antibody attachment [ ] , was also found in the s protein of ck/ch/lhlj/ v after passages. this substitution was maintained for p and p , indicating that this residue was likely to be important for virus pathogenicity. however, other amino acid substitutions in the s protein were not observed between the m vaccine strain and m challenge strain [ ] , the / pathogenic and / attenuated strains [ , ] , the tw / pathogenic and tw / attenuated strains, the tw / pathogenic and tw / attenuated strains, the tw / pathogenic or tw / attenuated strains [ ] , or the arkdpi strain [ ] . investigations with other coronaviruses have shown that the s protein is a determinant of pathogenicity; however, the replacement of the s protein gene of the apathogenic beaudette strain with that of the pathogenic m strain resulted in a recombinant virus that was still not pathogenic [ ] . thus proteins other than, or in addition to, the s protein must affect pathogenicity. our findings were consistent with this result. we found that nearly all of the amino acid substitutions in the s subunit were between p and p , and other substitutions were between p and p . the virus was still pathogenic to chickens after passages and it was fully attenuated after passages in embryonated eggs. the structural e gene in sars-cov is a virulence factor, and a sars-cov that lacks the e gene is attenuated in vitro and in vivo [ ] . in this study, a synonymous mutation in the e gene was found between p and p , and three substitutions in the a, m and n proteins were also observed between p and p . nonetheless, the relationship between those substitutions and virus pathogenicity is unknown. a single mutation, tyr his, in mouse hepatitis virus mhv-a nsp resulted in attenuated virus pathogenesis in mice [ ] . sequence changes in the -two-thirds of the genome, which contains two overlapping replicase genes, were not investigated in this study and should be further studied. an additional orf, detected within the -utr of several ibv isolates, and with the potential to encode hydrophobic proteins is referred to as orf [ ] . it was also observed in the ibv ck/ch/lhlj/ v strain. in other coronaviruses, such as porcine transmissible gastroenteritis virus (tgev) and fcovs, a relationship between gene and virus virulence has been observed [ ] , although it is difficult to compare the hydrophobic proteins of these coronaviruses directly with that of ibv. until now, the corresponding protein and its ibv function have not been clear. however, it is hypothesized that the sequences in the ibv -utr are involved in regulating viral rna replication and transcription [ ] . in addition, sapats et al. [ ] reported that shorter forms of the -utr in the australian n / , q / , and v / strains are associated with a decrease in virulence. huang and wang [ ] found a -bp deletion in the -utr immediately downstream from the n protein at passage of strain tw / , that is not present in the pathogenic parent. in this study, we observed a longer ( -bp) deletion at the same position in the -utr in most subpopulations of p and p ( % and %, respectively), indicating that not only this deletion, but also the size of the deletion may be correlated with ibv attenuation. in addition, both the deleted and non-deleted sequences in the -utr of the offspring viruses were detected in the respiratory tracts of chickens inoculated with p at days postinoculation, implicating that this deleted region is not necessary for viral replication in the chicken respiratory tract. although multiple passage of a field isolate in embryonated domestic fowl eggs is the usual method for development of ib attenuated vaccines, not all ibv strains stimulate the immunity after serial passages. the beaudette strain is apathogenic in chickens after many serial passages in embryonated chicken eggs [ ] . in addition, this embryo-passaged virus is considered to be poorly immunogenic [ ] , and consequently, has never been used as a vaccine strain. this is also the case with the ibv ck/ch/lhlj/ v strain. the immunogenicity of the virus has been gradually decreased by serial passage in embryonated eggs. the ck/ch/lhlj/ v p did not confer immunity to spf chickens when compared to p , p , p and the negative control. the reduced immunogenicity of the attenuated virus may correlate with its reduced replication efficiency and infectivity in chickens. the s glycoprotein induces protection against virulent challenge, and several epitopes that induce virusneutralizing antibodies have been mapped within the s protein [ ] [ ] [ ] [ ] [ ] [ ] . these epitopes showed the importance of inducing the ctl response in primary infections and neutralizing the antibody response against secondary exposure to the same virus [ , ] . in this study, amino acid substitutions were observed in the abovementioned epitopes of s protein, and no substitutions were found in the epitopes residues of the s protein. in addition, n is another important protein that induces protection in ibv. in this study, a substitution (tyr → the) was observed in the n protein between p and p viruses. this substitution was located in an identified epitope in the ibv n protein that induces a t-cell response and protection [ , ] . the amino acid substitution at residue (thr to ile/ala) in the m protein, which was observed to be related to antigenicity and/or virulence of ibv strains h /h , tw / and arkansas [ , ] , was not observed in our ck/ch/lhlj/ v strain. the ibv strains, as a group, infect a large range of epithelial surfaces, literally from the top to the bottom of the chicken. isolates differ in their extent of replication in non-respiratory tissues, and some produce clinical disease in non-respiratory tissues, most notably the kidney and proventriculus. ck/ch/lhlj/ v is a nephropathogenic strain; however, it lost kidney tropism after serial passages in embryonated eggs. using a reverse genetics system, the s glycoproteins for a group coronavirus (mhv), a group coronavirus (tgev) and a group coronavirus (ibv) were demonstrated to be involved in the tropism of these coronaviruses [ ] [ ] [ ] [ ] [ ] , ] . for the tgev, several amino acid changes at the nterminus of the s protein resulted in the loss of enteric tropism [ , ] . in this study, an amino acid substitution was found at residue (leu → phe) between p and p ; however, loss of kidney tropism of ck/ch/lhlj/ v occurred between p and p . the only amino acid difference between p and p was at residue ; however, this residue is not likely to be a determinant of tissue tropism of ck/ch/lhlj/ v, because some viral subpopulations in p and p showed this change. several other substitutions were found in s and other proteins in this study. further investigation by reverse genetics and animal studies is needed to verify the exact function of substitutions. in this study, the titers of the embryo-adapted ibv ck/ch/ lhlj/ v strain increased gradually with the serial passage in embryonated eggs, indicating that the virus had a high replication capacity in vitro; however, its capacity for in vivo replication decreased dramatically. the n, m and e proteins of ibv play a role in viral replication and assembly [ ] . it is difficult to conclude that the decreased replication of the virus in vitro in this study was due to substitutions in m and n proteins. a point mutation in the coronavirus hcov- e and the arterivirus eav nendou (nsp ) resulted in a lack of viral genome replication and transcription, indi-cating that this rnase mostly affected viral production [ ] [ ] [ ] . it is unclear whether this is the case for ibv ck/ch/lhlj/ v. the embryo-adapted ibv strains appeared to contain a mixture of genetic variants, and selection and mutations occurred in the viral populations during the passages in the embryos. in the process of serial passage, almost all of the amino acid substitutions in s proteins occurred between p and p , and all the subpopulations in the virus passages showed those substitutions; however, other substitutions were found between p and p and only parts of the subpopulations in the virus passages showed those substitutions. the exact roles of different subpopulations in changes in virus replication, pathogenicity, antigenicity, immunogenicity and tissue tropism are unknown; we have not succeeded in isolating the different subpopulations from the virus population by limited dilution (data not shown). understanding the molecular mechanism of ibv attenuation, tissue tropism and immunogenicity changes is important, because not only is this virus of economical importance to the poultry industry, but it also shows antigenic and biological similarities and differences to other coronaviruses. although it is reasonable to conclude that some of the few sequence changes described in this study in the - kb region are responsible for virus attenuation, decrease in immunogenicity and tissue tropism changes, we cannot conclude that they are the only predictors for these changes. we also cannot completely exclude the possibility that other parts of the genome are responsible for the observed changes, because ib coronavirus has a large genome ( . kb) . in addition, similar to other reports [ , , , ] , we found that none of the sequence changes were shared by all pathogenic ibv strains and their attenuated derivatives, indicating that there may be many factors and pathways that affect virus replication, pathogenicity, antigenicity, immunogenicity and tissue tropism. a comparative sequence ananlysis to revise the current taxonomy of the family coronaviridae the molecular biology of coronaviruses coronavirus 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vivo clearance of infectious bronchitis virus localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism differences in virus receptor for type i and type ii feline infectious peritonitis virus major genetic marker of nidoviruses encodes a replicative endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle key: cord- -zpi uis authors: roberts, anjeanette; wood, john; subbarao, kanta; ferguson, morag; wood, david; cherian, thomas title: animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting – august , london, uk date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zpi uis abstract severe acute respiratory syndrome (sars) emerged in the guangdong province of china in late and spread to countries. by the end of the outbreak in july , the cdc and who reported cases with a . % case fatality rate. the disease was caused by a previously unrecognized coronavirus, sars-cov. drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. available data suggest that vaccines should be based on the the kda viral spike protein, s, the only significant neutralization antigen capable of inducing protective immune responses in animals. in the absence of clinical cases of sars, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the united states food and drug administration's “animal rule” would apply to licensure of a sars vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. this report summarizes the recommendations from a who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on – august in south mimms, uk, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate sars vaccines. severe acute respiratory syndrome (sars) is a severe respiratory illness caused by the sars coronavirus (sars-cov) [ ] . the disease emerged in the guangdong province of china in late [ ] and spread to countries mostly within asia, although europe and north america were also affected, notably toronto, canada. the epidemic was finally controlled by july through strict implementation of prevalence for sars-cov antibody, although they had no history of sars-like disease. sars-cov-like viruses that were isolated from civets and raccoon dogs had more than % homology with human sars-cov, with major differences found in orf , whose deletion has been suggested to represent a sign of adaptation to humans [ ] . only four amino acid residues in the receptor glycoprotein ace binding domain of the viral spike protein differ between the human epidemic sars-cov strains and civet strains, but they cause more than a -fold difference in binding affinity to the ace molecule [ , ] . although a high prevalence of sars-like coronaviruses were found in chinese horseshoe bats [ , ] , their great genetic diversity makes it difficult to identify which one might be the ancestor of sars-cov and to decide with certainty whether bats indeed are the animal reservoir of the virus. sars-cov infection exhibits a wide clinical course characterized mostly by fever, dyspnea, lymphopenia and lower respiratory infection, often with concurrent gastrointestinal symptoms including diarrhea [ , ] . pathology in sars patients has been associated with diffuse alveolar damage, epithelial cell proliferation and multinucleated giant cell infiltrates of epithelial or macrophage origin, suggestive of syncytium-like formation in the lung. the virus can be recovered from peripheral blood mononuclear cells, respiratory secretions, stools, urine and even sweat (for a review, see [ ] ). sars vaccine development efforts were initiated very rapidly after the identification of the etiologic agent, even though the immune correlates of protection were not known. research efforts to identify protective antigens and to develop animal models were undertaken in parallel with efforts to develop candidate vaccines [ ] , drawing on experience with animal coronavirus vaccines and using several vaccine strategies, including inactivated virus vaccines, purified subunit vaccines, plasmid dna and viral vector-based vaccines as well as virus-like particles. much effort has been made to identify appropriate animal models for sars-cov replication and pathogenesis. several research groups have shown that mice [ , ] , ferrets [ ] , hamsters [ ] and nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] support replication of sars-cov with varying degrees of associated disease. these animal models were used for the evaluation of candidate vaccines, and the common conclusion that has emerged from the evaluation of several vaccines is that the kda viral spike protein, s, is the only significant neutralization antigen [ ] [ ] [ ] [ ] and the only one to elicit protective immunity in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . the s protein can be divided into two domains by analogy with other coronavirus spike proteins: an n-terminal s domain, which contains the receptor-binding site and neutralization epitopes and a cterminal s domain which forms the membrane-anchored stalk region and contains a putative fusion peptide followed by two heptad repeats predicted to form a six-helix coiled-coil bundle [ ] . in the absence of clinical cases of sars, candidate vaccines will have to be evaluated for efficacy in animal models. the united states fda "animal rule" states that, when efficacy studies in humans are not feasible, vaccines may be approved based on animal data alone, provided the pathophysiological mechanism of the disease is reasonably wellunderstood as is its prevention or reduction by the vaccine. moreover, the protective effect of the vaccine should be demonstrated in more than one animal species expected to react with a response predictive for humans. the endpoint of animal studies should be clearly related to the desired benefit in humans (i.e. enhancement of survival or reduction in major morbidity), and the data generated should allow selection of an effective dose in humans. at the present time it is uncertain whether the "animal rule" would apply to licensure of a sars vaccine. however, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. scientists at the who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on - august in south mimms, uk, discussed many aspects of research pertaining to the use of animal models in vaccine development including available animal models, suitability of the various models, correlates of protection, critical components of potential vaccines, and the potential for disease enhancement in vaccinated animals following exposure to sars-cov. in addition, standardization of antibody assays and the establishment of a who international standard for sars-cov antibody were also discussed. this report endeavors to summarize the recommendations from this meeting, based on consensus agreement. recommendations for use of each animal model are given in section below. correlates of protection, an overview of vaccine development, and observations pertaining to potential disease enhancement are summarized in the following sections - . in selecting animal models for vaccine evaluation, it is important to remember the principle underlying the so called "animal rule", where data from more than one animal species is often required: each animal species should contribute something different to our understanding of disease and protection. at this time, no single model offers a direct reproduction of what was seen in humans with sars. pathology (including pneumonitis, alveolar edema, and diffuse alveolar damage (dad)) in humans is probably the most difficult element to reproduce in an animal model. attention also should be given to the reduction of viral shedding because this would likely correlate with decreased risk of further spread of the disease among humans. in using animal models to study aspects of sars-cov infection, it must be emphasized that the kinetics of viral replication and appearance and resolu-tion of pathological findings are much more rapid in animal models than in humans. whichever animal model is employed, special consideration should be given to the presence of co-existing pathogens, the age of the animals and the route(s) of infection. a sufficient number of time points and large enough number of animals should be used to allow statistical evaluation. the strain of sars-cov used also could be of importance. it should be emphasized that different species may prove useful for studying different aspects of sars-cov. whereas vaccines or antivirals may be addressed in many models, pathogenesis is best evaluated in those animal models for which immunological tools and reagents are available for detailed analysis of the immune response to the vaccine. this includes inbred mice and rhesus and cynomolgus macaques. it may actually be worthwhile to enhance the virulence of a sars-cov isolate by serial passages in an animal model to produce a challenge virus stock for vaccine studies that would elicit more reproducible disease in the animals. if a highly virulent host-adapted virus were to become available, such as a mouse-adapted or a monkey-adapted sars-cov strain, demonstration of the capacity of vaccines to protect against challenge with these more virulent strains would provide an almost ideal animal model. different models may also need to be employed to evaluate pathogenesis versus immunogenicity. for pathogenesis studies in animal models, mortality is not required as a readout. it would be ideal to develop animal models with comparable levels of mortality to that seen in humans (∼ % overall), including the increased mortality at increased age (∼ % > age ). the optimal result would be to demonstrate efficacy of vaccines or antivirals in sars-cov animal models that mimic human morbidity and mortality and that show protection without vaccine-associated immunopathology. inbred mouse strains (balb/c, c bl/ , svev, stat −/−) support sars-cov replication and can develop pneumonitis ( s), but pneumonia and clinical symptoms are only observed in older balb/c mice [ ] . the mouse model is suitable for immunogenicity and efficacy studies of vaccines. prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in stat −/− mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs. specific pathogen-free (spf) animals must be used. their age can be either - weeks or over months and should be specified. the number of animals included must be sufficient for statistical analysis, and should include mock-infected controls. a variety of sars-cov strains has been tested in mice, including urbani, frankfurt, hku- , tor- , and the mouse-adapted sars-cov strain ma- . these were inoculated by the intranasal (in) route under light anesthesia [ , ] , using a dose of tcid in l/mouse. critical time points for specimen collections are days (peak titer) and post-infection (p.i.) for quantitative virology, days and p.i. for the study of interstitial pneumonitis and dad in aged mice (inflate lungs with % neutral-buffered formalin), and day for resolution. golden syrian hamsters are an excellent animal model because they demonstrate high levels of sars-cov replication and develop pneumonitis. hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies [ ] . in contrast with balb/c mice, in which the virus is detected only in the respiratory tract and is cleared by day p.i., hamsters demonstrate a longer duration of viral shedding from the upper respiratory tract, some transient viremia, spread of the virus to liver and spleen and, most significantly, inflammation of respiratory tissues [ , ] . spf animals older than weeks of age should be used in sufficient number for statistical analysis and study design should include mock-infected animals. the animals can be housed in pairs or by three if the experiment is to last less than - weeks. reserve space should be available to separate the animals in case of fights. males and littermates tend to fight less. virus strains that have been tested in hamsters are urbani, frankfurt and hku- . virus should be administered by the in route under light anesthesia, using a dose of tcid in l/hamster. as an outcome of efficacy studies, quantitative virology should be preferred over quantitative pcr. for pathology studies, one can grade pathology as none, mild, moderate or severe as per roberts et al. [ ] . critical time points for specimen collection are days or p.i. (peak viral titer) and (clearance) for quantitative virology studies; and days p.i. (consolidation and pneumonitis) and or (resolution) for pathology studies (lung). there is evidence from one study that ferrets support sars-cov replication and develop pulmonary lesions [ ] but according to another study, the animals remain asymptomatic, in the presence of sars-cov replication [ ] . in view of these conflicting data, the ferret model needs to be further studied to determine its optimal utility for vaccine efficacy and immune prophylaxis studies. additional studies are needed to define the extent of biological variability of the model and the possible role of co-pathogens that may contribute to the variability observed between different laboratories. animals aged months or older should be used. although not well documented, more consistent viral replication, pathology and clinical symptoms seem to be observed in older animals. the animals should be screened for viral co-pathogens: aleutian mink disease, respiratory viruses, hepatitis viruses, and others. the route of inoculation may be in or it, but not iv. the dose of virus (strains tor- , hku- ) sufficient to ensure reproducibility of infection in all animals is likely to be pfu or more/ferret. again, quantitative virology is preferred over qrt-pcr. for pathology studies, the same recommendations apply as for nonhuman primate studies (see below): slides should be shared between pathologists to develop a scoring system. regarding specimen collection of respiratory tissues, further studies are needed to establish how much variation occurs in samples from different lobes of the lungs. critical time points are day p.i. for quantitative virology and days - p.i. for pathology studies (pneumonitis). nhps support sars-cov replication and develop pneumonitis with a variable degree of clinical symptoms depending upon the species employed. no single nhp species is preferred at this time. the number of animals in a given study needs to be large enough to account for animal-toanimal variability: a sample of or animals is not sufficient. in view of the cost of the experiments, challenge studies should be limited to those vaccine candidates that are most promising, using larger sample sizes ( - animals/group) and avoiding animals with free-range periods in life if possible. immunological responses are best studied in species for which microarrays and reagents for identifying immune components are available, such as rhesus or cynomolgus macaques. however, the limited viral replication observed in cynomolgus macaques might be a disadvantage in selecting this species for studies. other recommended nhp species are the common marmoset and african green monkeys (agms, chlorocebus aethiops sabeus). the country of origin may play an important role and should be specified, e.g. philippines (cynomolgus macaques, macaca fascicularis); china (rhesus, macaca mulatta); brazil (marmosets, callithrix jacchus), etc. prior to the experiment, the animals should be housed indoor to limit exposure to potential co-pathogens. they should be screened for parasites (strongyloides, pneumonyssus simicola (lung mites)) and for possible viral co-pathogens (retroviruses, respiratory viruses, adenoviruses). the sars-cov strains tested in nhp models are hku- (cynos), pumc (rhesus) and urbani (marmosets and agms). these were inoculated by the respiratory route (in, it) at a dose of pfu or more/nhp. here again, quantitative virology is preferred over qrt-pcr. for pathology studies, it would be an obvious advantage that laboratories share pathology slides for review by different pathologists in order to develop a scoring system. specimens of respiratory tissues should be collected, but further studies are needed to establish how much variation occurs in samples from different lobes of the lungs, as was done and reported for african green monkeys (agms) [ ] . critical time points are days - p.i. for quantitative virology in cynomolgus macaques and agms, and later than day p.i. for rhesus macaques of chinese origin. for collection of tissues for histopathological analyses, days - p.i. are optimal for cynomolgus macaques and agms, and later than day p.i. for rhesus macaques. due to limitations of immunological reagents (including microarray assays now available), research may be limited to rhesus and cynomolgus macaques. data on other animal models are insufficient for consideration for use in sars-cov vaccine and antiviral evaluations [ ] [ ] [ ] . any additional model other than the four listed above (section . . to section . . ) would require thorough characterization including viral replication data and histopathological analysis of sars-cov-infected and mockinfected animals of the same age and gender. viral replication and histopathological data in any new animal model should be reminiscent of at least some aspect of sars in humans. although all the correlates of protection from sars associated disease have not been identified in human infections, neutralizing antibodies are present in convalescent human serum. antibodies to sars-cov spike (s) protein have been shown to prevent virus entry and neutralize virus infectivity in vitro [ , ] . prophylactically administered monoclonal antibodies and passively transferred sars-cov hyper-immune sera have been shown to prevent sars-cov infection and associated disease following sars-cov challenge of naive mice and hamsters [ , , [ ] [ ] [ ] . monoclonal antibodies administered therapeutically (i.e. post-infection) also have been shown to limit viral replication and reduce associated disease in hamsters [ ] . although cell mediated immunity may have a protective role in viral clearance or resolution of disease, work in animal models shows that antibody alone is effective for prevention and treatment of sars. thus, mice immunized with live-recombinant vaccines expressing the sars-cov spike protein, using rabies virus [ ] , vesicular stomatitis virus (vsv) [ ] , adenovirus (ad ) [ , ] or attenuated vaccinia virus mva [ , ] as a vector, as well as mice immunized with dna vaccines expressing the s gene [ , ] , developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. similar findings were reported after mucosal immunization of hamsters and agms using a bovine parainfluenza virus type (bpiv ) vector expressing the sars cov s gene [ , ] . several whole inactivated virus and recombinant protein candidate vaccines also have been developed and shown to elicit a neutralizing antibody response that provided protection against infectious challenge [ ] [ ] [ ] [ ] [ ] [ ] . in addition, passively administered sera from vaccinated animals prevented sars-cov infection upon subsequent challenge of naïve mice, demonstrating that antibodies induced by these vaccines did confer protection [ , ] . the neutralizing antibody titer that is necessary to achieve protection in humans exposed to sars-cov is, however, still not known. it was recommended that when evaluating vaccine efficacy in future animal experiments, the challenge virus should be administered at two different time-points, once when postimmunization neutralizing antibody titers are high, and later when neutralizing antibody titers have waned or are low. it also was suggested that viral titers and pathology should be evaluated at two different time points. specific times points for sample collection are given for each animal model in section above. previous observations of disease enhancement have been reported for human viral pathogens and shown to be due to antibody-mediated enhancement of virus entry (for reviews see [ , ] ). enhanced disease and mortality have been observed in kittens immunized against or infected with a type-i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection [ ] [ ] [ ] . aggravated fip is apparently a result of enhanced viral entry into macrophages mediated by sub-neutralizing antibody levels [ ] . children vaccinated with inactivated respiratory syncytial virus (rsv) vaccines developed serious disease on subsequent exposure to rsv [ ] [ ] [ ] . individuals exposed to one of the four serotypes of dengue virus developed severe disease when subsequently infected with a second, different serotype [ , ] . enhanced disease following rsv vaccine or dengue infection occur by different mechanisms than fipv. in view of such examples of enhanced disease following infection in a vaccinated host, there has been heightened concern that a similar phenomenon could occur with sars-cov vaccines. it was highly recommended, therefore, that known mechanisms of disease enhancement observed with other viruses and especially with other coronaviruses should be examined in sars-cov infections, especially in vaccinated animals. although none of the studies to date have shown enhanced respiratory disease following sars-cov challenge in previously immunized animals, further studies in this area are warranted in view of some of the available in vitro data. antibodies against human sars-cov isolates were shown to enhance the entry of pseudo-typed viruses expressing the civet sars-like cov-spike protein into a human renal adenocarcinoma cell line ( -o) . enhancement was only demonstrated at the level of entry, but not of replication [ ] . this phenomenon was seen with pseudo-typed lentiviruses expressing sars-cov spike protein of civet sequence specificity, but not with pseudo-typed viruses expressing spike proteins of human sars-cov isolates. it also was not observed with human isolates of sars-cov. the role of enhanced viral entry, as observed in these in vitro studies, has not been related to any known component of human disease or infection in vivo. however, given that sars-cov may replicate, albeit poorly, in human pbmcs [ , ] , in vitro experiments looking for antibody-enhancement of sars-cov replication in human cells (e.g. macrophages and b-cells) should be performed. several groups have studied sars-cov infection in animals in the presence of neutralizing and sub-neutralizing levels of sars-cov anti-sera or anti sars-cov s-protein monoclonal antibodies, but no evidence of enhanced respiratory disease has been observed. however, foci of hepatic necrosis were noted following sars-cov challenge in mva-sars-s immunized ferrets [ ] . although these findings are worrisome, several questions were raised regarding the significance of the observation. the mva-sars-s vaccine used in these experiments was poorly immunogenic in the ferrets. the question of whether there could have been any co-pathogen in the animals was raised. it also would be important to know if the observed phenomenon depends on the mva vector or on the animal model. it was strongly urged, therefore, that the experiment be repeated in ferrets. additional experiments, in nonhuman primates and hamsters, looking for evidence of enhanced respiratory and hepatic diseases upon vaccination and challenge were also encouraged. several candidate sars vaccines that are at various stages of pre-clinical and clinical development are being developed worldwide. in china alone, three companies have been given regulatory approval for the clinical evaluation of a candidate sars vaccine. it is important, therefore, to be able to compare data from each of the candidate vaccines, which, in turn, requires international standardization of the immunological assays used for the evaluation of these vaccines. the accepted method of international standardization is to employ a who international standard (is), which allows comparison of results from different laboratories [ ] . this is essential for establishing international requirements for vaccines, diagnostics or therapeutics. an is is prepared from material bearing a close resemblance to the samples being assayed; the material is distributed in glass ampoules with high precision and reproducibility and then freeze dried. it is important that a sufficient number of ampoules ( - ) be prepared so as to provide for about years of use, and that the activity of the contents remain stable over this period. the process of establishing a who is involves an international collaborative study, in which the candidate is is compared with other samples. if the results of the tests are suitable, the candidate is assigned a provisional arbitrary unitage, a report is distributed for approval by study participants and for eventual approval by the who expert committee on biological standardization (ecbs). the preparation, storage and distribution of over % of is have been undertaken by the national institute for biological standards and control (nibsc) at south mimms (uk), which is a who laboratory for biological standards. nibsc has developed in the past several who iss for calibration of the antibody response against virus vaccines, including iss for antibodies against dengue, hepatitis a virus (hav), [ ] hepatitis b virus (hbv), measles, [ ] polio, rabies, [ ] rubella, and smallpox. a candidate standard against human papilloma virus- (hpv- ) is under evaluation. the corresponding is's were used for a variety of antibody assays including virus neutralisation (vn), haemagglutination-inhibition, single radial diffusion, enzyme-linked immunoassay and radio-immunoassay. antibody iss are most useful in epidemiological studies and in clinical trials. their use allows correlates of immunity and potency requirements of prophylactic and therapeutic products to be expressed in international units (ius). data from several collaborative studies demonstrate that use of an is generally reduces the level of variability between assay results. however, there may be problems in using iss due to the complex array of antibody populations in each serum and the different sensitivity of different assay systems. examples of potential problems can be found in hbv and parvovirus b studies [ ] , which showed that different assay kits gave different results even when the is was included in the assays. another issue is the degree of antigenic homology between the viral antigen used for the preparation of the is and the virus used in the assays. in a jev collaborative study, a candidate antibody is, which had been prepared from the sera of vaccinees immunized with an inactivated vaccine that was antigenically different from some of the viruses used in vn assays, demonstrated that the response to at least some inactivated vaccine is strain specific and the candidate is was consequently not established by the who ecbs. whether a panel of monoclonal antibodies to sars-cov could be used to prepare an is is an attractive alternative which should be explored. significant progress with standardisation of sars-cov antibody assays had been made in china with the development of a national antibody standard. in order to develop the national standard, sera were collected from convalescent sars patients who were found to have sars-cov vn antibody titers ranging from undetectable to : . one serum sample was selected for further evaluation based on crossreactivity with four sars-cov strains and on western blot analysis. this serum was freeze dried in . ml aliquots and was then assessed for stability by vn assays. the chinese standard was assigned a vn titer of . with % confidence limits of . - . . a further important development in china was the preparation of human immunoglobulin for treatment of sars patients. national guidelines have been prepared for collec-tion of plasma, quality control testing and standardisation of assays. three chinese manufacturers have been licensed for preparation of sars immunoglobulin. the source material was plasma from convalescent patients at more than days after infection. all were in good health and their plasma tested negative for blood-borne agents. plasma samples were processed by a combination of cold ethanol fractionation and ultrafiltration. in september , three lots of igg were produced and assayed by nationally-agreed procedures. the stock of immunoglobulin currently available is sufficient to treat patients. monoclonal antibodies have not yet been considered for treatment purposes in china. the importance of assessing immunogenicity of candidate sars-cov vaccines using vn assays is well acknowledged, but the variety of vn tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. it is therefore desirable to establish an antibody is to serve as a basis of comparison in all vn assays. the most important activity at this time is to obtain a suitable source of antibody. a number of options can be considered, such as convalescent human sera, post-immunization human sera, monoclonal antibodies or hyperimmune animal sera. as an example, the availability of a suitable source of serum from convalescent patients in hong kong needs to be explored, although antibody levels in these individuals are probably quite low by now. it also would be important that other assays than vn be included in the collaborative study, and that the impact of sars-cov strain variation be examined by using different sars-cov strains and/or sera with different specificities. the centralized facility for aids reagents (cfar), which is based at nibsc, could be a suitable model for a sars-cov repository [ ] . the cfar was established in to support aids vaccine research and it is now eu-funded [ ] . there are currently reagents available including peptides, recombinant proteins, human sera/plasma, monoclonal and polyclonal antibodies, expression systems, cdna clones and viruses. a comparison can be drawn between sars-cov and hiv vn assays. currently, there are several different hiv neutralization assays formats under consideration and a lack of agreement on the most suitable assay. the cfar is supporting a joint who/eu project (neunet) to evaluate and standardize hiv vn assays in an international collaborative study. in the usa, a sars-cov repository has been established on behalf of the american type culture collection (atcc) in order to meet the needs of biodefence and the threat of emerging infections [ ] . the type of reagents stored includes viruses, peptide arrays, monoclonal antibodies and proteins. it is hoped that an active collaboration can be established between niaid and nibsc in order to meet the expanding needs of the sars research community. based on the discussion at the meeting, the following recommendations were made with respect to standardization of the immunological assays for sars vaccine evaluation: . a who repository for sars-cov reagents ought to be developed. collaboration between niaid and nibsc is recommended to achieve this goal. . consensus must be reached for the reagents to be given priority in the repository. needed. . the most suitable source of antibody for the is is convalescent human sera, but post-vaccination human sera could also be used. . a protocol for an international collaborative study aimed at validating the is should be developed and distributed to prospective participants. . collaborative study participants should be asked about their assay capabilities, e.g. number of sera, virus strains handled, etc. . . . the proposed is collaborative study should include a core set of antibody preparations to be distributed and assayed in each laboratory (e.g. monoclonal antibodies, animal sera, other human sera). . tests should be conducted using the same strain of sars-cov in each laboratory, but the different genetic lineages of sars-cov should also be represented in the study. biosafety issues associated with sars-cov vaccine development stem from the reports of laboratory-acquired infections in china. sanofi pasteur has adopted bsl practices and bsl equipment (e.g. class or microbiological safety cabinets with respiratory protective equipment) for the preparation of sars-cov vaccines. of note is the fact that sars-cov appears to be quite resistant to normal methods of virus decontamination (jf saluzzo, personal communication). who has developed guidance, both general [ ] , and specific for handling sars specimens [ ] . the rapid success in the development of immunogenic and protective vaccines against sars using a variety of platforms is encouraging, but should be tempered with concerns about the possibility of enhanced disease following exposure in vaccinated individuals [ ] . concerns mainly stem from reports of enhanced disease in fipv-immunized or -infected kittens [ , ] , from observations that antibodies elicited against certain coronaviruses mediate antibody-dependent enhancement of viral entry [ ] , and from the observation of inflammatory foci in liver tissue following sars-cov challenge in mva-sars-s vaccinated ferrets [ ] . candidate sars vaccines will need to be evaluated in more than one animal model. they also will need to be thoroughly evaluated for the duration of the antibody response they induce, as well as for the breadth of their protective effi-cacy against different strains of sars-cov. the implications of the sequence heterogeneity among sars-cov strains are difficult to test at this time because the most divergent strains (civet sars-like viruses) have not been recovered in culture. validation and international standardization of immunological assays for the evaluation of candidate sars vaccines are essential to compare data across different trials. this requires the establishment of international standards for sars-cov antibody and a repository for sars-cov reagents, with an international collaborative study to validate the iss. the establishment of the repository by who in collaboration with nibsc and niaid was recommended. severe acute respiratory syndrome who investigates china's fall in sars cases who says sars outbreak is over, but fight should go on summary of probable sars cases with onset of illness from identification of a novel coronavirus in patients 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transmissible vaccines have the potential to revolutionize infectious disease control by reducing the vaccination effort required to protect a population against a disease. recent efforts to develop transmissible vaccines focus on recombinant transmissible vaccine designs (rtvs) because they pose reduced risk if intra-host evolution causes the vaccine to revert to its vector form. however, the shared antigenicity of the vaccine and vector may confer vaccine-immunity to hosts infected with the vector, thwarting the ability of the vaccine to spread through the population. we build a mathematical model to test whether a rtv can facilitate disease management in instances where reversion is likely to introduce the vector into the population or when the vector organism is already established in the host population, and the vector and vaccine share perfect cross-immunity. our results show that a rtv can autonomously eradicate a pathogen, or protect a population from pathogen invasion, when cross-immunity between vaccine and vector is absent. if cross-immunity between vaccine and vector exists, however, our results show that a rtv can substantially reduce the vaccination effort necessary to control or eradicate a pathogen only when continuously augmented with direct manual vaccination. these results demonstrate that estimating the extent of cross-immunity between vector and vaccine is a critical step in rtv design, and that herpesvirus vectors showing facile reinfection and weak cross-immunity are promising. vaccines have had a wide range of positive impacts on the health of human and animal populations. in many cases, however, the full potential of vaccination cannot be realized because it is difficult or impossible to vaccinate a substantial proportion of the host population. particularly challenging scenarios for efficient vaccine delivery include human diseases in regions with poorly developed public health infrastructure and diseases of wild animal populations. an important consequence of the challenges associated with vaccinating large proportions of wild animal populations is that we may be missing opportunities to eliminate or reduce reservoir populations of diseases that occasionally spill over into human populations (e.g., ebola, rabies; [ ] [ ] [ ] [ ] [ ] ), or that provide the raw material for full scale host shifts into the human population (e.g., sars; [ ] ). this problem is particularly pressing in light of evidence suggesting that the incidence of such host-shifts is increasing due to the greater prevalence of humans in regions with high levels of wildlife diversity [ ] . one promising new technology for dealing with the challenges associated with these difficult-toimmunize animal populations is the development of transmissible vaccines. in the most general sense, transmissible vaccines are vaccines that can spread from one individual to the next, with the benefit being that for every individual that is immunized directly, additional individuals are immunized indirectly. one way in which a transmissible vaccine can be developed is through the process of attenuation [ ] . although attenuation can be accomplished in many ways, the goal in all cases is the transformation of the original viral disease into a benign yet transmissible vaccine. this approach has been used to develop transmissible vaccines both unintentionally, as with the oral polio vaccine [ , ] , and intentionally, as with myxoma virus in rabbits [ ] [ ] [ ] . however, there are limitations and risks associated with transmissible vaccines produced through attenuation. for instance, because attenuated vaccines are built from the pathogen itself, the ability of the vaccine to spread between hosts is bounded by the transmissibility of the pathogen. more worrisome is the possibility of reversion to wild type virulence, as has been observed in the oral polio vaccine [ , , ] . for this reason, and the constraints imposed by the process of attenuation itself, transmissible vaccines developed through attenuation will generally be only weakly transmissible. despite these limitations, recent theoretical work has demonstrated that weakly transmissible attenuated vaccines could be highly effective tools, particularly against infectious diseases with relatively low transmission rates [ ] . an alternative approach to designing a transmissible vaccine relies on recombinant genetic engineering rather than traditional attenuation. specifically, rather than weakening the pathogen itself through a process of attenuation, recombinant transmissible vaccines (rtvs), also called transmissible recombinant vectored vaccines [ ] , are developed by inserting one or more pathogen genes with antigenic activity into the genome of a benign but transmissible vector organism. infection with the modified vector, now termed the vaccine, exposes the host immune system to pathogen antigens and prompts a pathogen-specific immune response (fig. ) . this approach has been used to develop a transmissible vaccine against sin nombre virus within deer mice [ ] , and is now being used to develop a transmissible vaccine against ebola within mice and nonhuman primate models [ , ] . in principle, rtvs offer several advantages over vaccines produced by attenuation. for instance, evolutionary reversion in a rtv is likely to produce the benign vector organism rather than the virulent pathogen itself. also, because the transmission rate of the vaccine is related to the transmission rate of the vector rather than the pathogen, there are fewer limitations on the vaccine transmission rate. however, because a rtv necessarily integrates components of both disease and vector, it may struggle to spread through a host population where substantial cross-immunity to either the disease or vector already exists [ , ] . this reduction in effectiveness is important in cases where the vector organism is already present in the population, or where vaccine-reversion produces free vector. for these reasons, it is currently unclear whether rtvs that generate cross-immunity can serve as effective tools in the battle against infectious disease. the limitations imposed by cross-immunity have focused research efforts on vectors thought to largely circumvent existing host immunity [ ] . cytomegalovirus (cmv), for example, is a type of herpesvirus that can evade an existing immune response, in part by down-regulating the presentation of mhc antigens [ , ] . rtv's that use cmv as a backbone are seemingly capable of reinfecting hosts that have had previous exposure to the vaccine, and superinfecting hosts with previous exposure to the cmv vector from which the vaccine was built [ , ] . these studies suggest that under natural conditions, cmv-based vaccines may experience little or no cross-immunity with the vector, and therefore largely escape the detrimental consequences of competition with the vector. for rtvs in general, levels of cross-immunity are likely to lie somewhere between the extremes of perfect host exclusion, in which infection with the vaccine or vector always precludes infection with the other, and the complete absence of cross-immunity. our goal is to establish bounds on the role of cross-immunity in hindering the effectiveness of a rtv. we begin by evaluating the ability of a rtv to facilitate pathogen control when the vector is absent from the host population. this first scenario also applies to the extreme case for which cross-immunity with the vector is absent. we compare this baseline case to the other extreme case where cross-immunity is perfect and the vector is circulating in the population or likely to be introduced through sporadic reversion. we direct part of our analysis towards current rtv designs which envision a vaccine that, when administered to a small number of 'founder' animals in a population, will spread autonomously and sufficiently to vaccinate the remaining population. we use a mathematical model to address the following specific questions: ( ) can a rtv autonomously eradicate and/or protect a population that has previously experienced vector infection? and ( ) if autonomous vaccination by a rtv is impossible, can augmenting the vaccine with direct, manual vaccinations make a rtv an effective tool for the control of infectious disease? our results indicate that supplementation of a rtv through direct vaccination is critical for disease control if reversion to the vector form is likely and vector-vaccine cross-immunity is present. our results identify key criteria that must be satisfied for a rtv to be an effective tool in infectious disease management, and outline quantities that must be estimated during the process of vaccine development to evaluate these criteria. ideally, it would be possible to engineer a rtv that, when introduced into the host population, would spread autonomously and sufficiently to eradicate an existing pathogen or prevent future infection by a pathogen not yet present. if the vector used to construct the vaccine remains absent from the population, or exhibits no cross-immunity with the vaccine, this ideal situation can be realized. specifically, if vaccine-infected individuals transmit the vaccine to more than one additional unvaccinated individual (r ,v > ), and the vaccine's r is larger than the pathogen's (r ,v > r ,w ), then a host population is cleared of an endemic pathogen and/or protected from future infection (si, section ). in some cases, however, it will be impossible to engineer a rtv that exhibits no crossimmunity with its corresponding vector. in the extreme case where the vector and vaccine share perfect cross-immunity, model analyses show that the vector will always outcompete the vaccine and drive the vaccine to extinction (si section ). this occurs regardless of the initial number of vaccinated individuals, and is a direct consequence of the cross-immunity between the vector and vaccine, and the vaccine's reduced rate of transmission relative to the vector (r ,v < r ,c , fig. ). the reduction in transmission of the vaccine relative to its vector renders the vaccine unable to repel the spread of its associated vector should reversion occur, while perfect cross-immunity ensures that the vector will drive the vaccine to extinction by imparting vaccine-immunity to a substantial portion of the population. these results suggest that the viability of autonomous vaccination depends critically on the degree of crossimmunity between the vector and vaccine. in light of this result, we next investigate the extent to which vaccine transmission, when coupled with a steady program of direct vaccination, can facilitate pathogen control even when the vaccine and vector share perfect cross-immunity. in most cases, sporadic reversion events will inevitably introduce the vector into an otherwise vector-naïve population, possibly resulting in significant competition between the vaccine and its associated vector. a simple strategy for protecting the rtv against the threat of competition with its vector is to manually vaccinate individuals at a rate sufficient to drive the vector to extinction. model analyses show that if the vector and vaccine share perfect cross-immunity, any vector incidence in the population will decrease to zero if the fraction of newborns vaccinated (σ) satisfies given that direct vaccination meets or exceeds expression ( ), spontaneous reversion events will be unable to spread through the population. if vaccine supplementation exceeds the critical value in expression ( ), a pathogen-naïve population is protected from a pathogenic threat and a pathogen-infected population is cleared of the pathogen when the fraction of newborns vaccinated satisfies ( ) therefore, if it is possible to supplement the vaccine at a rate that exceeds both of the critical values defined by expressions ( ) and ( ), the threat of pathogenic disease in a vector-naïve population will be eliminated, as will be the potential for interference from competition with the vector (si, section ). though pathogen prophylaxis can be achieved with lower levels of direct vaccination, supplementing at the maximum of expressions ( ) and ( ) is not too small (fig. s ). even if the vector organism is endemic in the host population prior the vaccination program, pathogen control can still be achieved with supplemental direct vaccination. in the absence of cross-immunity between vector and vaccine, a pathogen-naïve population is protected against the threat of an invading pathogen when a minimal fraction, , of newborns are vaccinated [si, sections , ] . if, on the other hand, vector infection imparts perfect vaccine immunity, a fraction ( ) of newborns must be continually vaccinated (si, section and fig. ). the necessary vaccination effort for prophylaxis depends critically on the relative r ′s of the vector and pathogen. eq. ( ) shows that the critical level of supplementation is unaffected by vectorvaccine cross-immunity as long as the vector is less transmissible than the pathogen (r ,c < r ,w ). given perfect cross-immunity and fixed ratio , the benefit in vaccination effort that occurs when increasing the vector's r plateaus when r ,c = r ,w (fig. , top right panel) . by comparison, a traditional, non-transmissible vaccine requires a threshold fraction of newborns to be vaccinated to prevent a pathogen from spreading through the population. thus, if the vector organism has reached endemic levels in the population, has a larger r than the pathogen, and imparts vaccine cross-immunity, the use of a rtv reduces the rate of direct vaccination required for prophylactic protection from a pathogen by a factor ( ) eq. ( ) indicates that, should the vector become endemic in the host population, substantial savings in vaccination effort are still realized as long as the ratio of the vaccine's transmissibility relative to its vector is not too small. again, the ratio of the vaccine's r relative to the vector's is a key quantity for gauging a vaccine's potential when competing against its associated vector. to our knowledge, this ratio has not been measured in any empirical experiments involving rtvs, despite its fundamental importance for predicting the vaccine's effectiveness in pathogen control when vector-vaccine cross-immunity is present (figs. and s ). if both the vector and the pathogen are endemic to the population prior to the administration of the vaccination program, the use of a rtv results in a substantial reduction in the fraction of new-borns that must be vaccinated to eradicate the pathogen relative to a nontransmissible vaccine (figs. and s , si: section ). as before, the role of vector-vaccine cross-immunity has a limited effect on the vaccine's effectiveness as long as the vector is less transmissible than the pathogen (r ,c < r ,w , fig. s ). fig. s shows that the ratio is most influential when the vector is more transmissible than the pathogen (r ,c < r ,w ). even when achieving the levels of continuous, manual vaccinations necessary for pathogeneradication exceed the logistics or budget of the vaccination program, a rtv can still greatly reduce the pathogen incidence in vector-infected population relative to that achieved by a non-transmissible vaccine (fig. ). our mathematical results demonstrate that recombinant transmissible vaccines (rtvs) can be powerful tools for the management of infectious pathogens under some circumstances. for instance, if infection with the vector does not confer vaccine immunity to hosts, or if the vector is absent from the population altogether, our results show that a rtv can autonomously sweep through a host population and eradicate an endemic infectious pathogen or provide prophylaxis against a future epidemic. alternatively, if the vector organism is already circulating within the host population and vector infection imparts perfect vaccine cross-immunity, the benefits of a rtv are more modest, and require that autonomous vaccination be supplemented with continuous direct vaccination. the level of direct vaccination that is required, and the benefits that a rtv provides relative to a nontransmissible vaccine, depend critically on levels of cross-immunity between vector and vaccine, the likelihood of evolutionary reversion to free vector, and the relative transmission rates of vector, vaccine, and pathogen. unfortunately, because these key parameters are largely unknown in natural infection scenarios, it is challenging to predict how well rtv's currently under development [ , , ] will work. although reliable estimates for key model parameters are not yet available, the biology of the transmissible vaccines currently under development is promising. for instance, the rtv's being developed for sin nombre virus and ebola use cytomegalovirus (cmv) as a vector [ , ] . available evidence suggests that reinfection and superinfection by cmv is possible, such that cross-immunity between vector and vaccine may be weak or absent [ , ] . in addition, at least within the lab, it is possible to engineer a genetically modified herpesvirus similar to cmv that does not revert, suggesting it may be evolutionarily stable, at least over relatively short time periods [ ] . finally, at least under some circumstances, cmv is highly transmissible [ ] , suggesting that it could be a useful vector against even infectious pathogens with relatively high r . in short, the biology of the rtv's currently being developed provides reason for optimism, but without accurate estimates for the strength of cross-immunity, and rates of reversion and transmission, their likely efficacy remains uncertain. recombinant transmissible vaccines have the potential to revolutionize control of infectious disease but their successful engineering and deployment will require estimating key parameters governing their epidemiology and evolution. a more accurate description of the population dynamics of a rtv and vector that potentially compete and coevolve will require a model that allows intermediate levels of cross-immunity. however, the predictions of such multi-strain models are highly sensitive to assumptions made regarding the presence of strain competition within a co-infected host, the rate of strain re-infection, as well as the level of cross-immunity between strains [ ] . once estimates of these specific parameters are found, our theory allows the potential advantage provided by vaccine transmission to be quantified. because rtv's carry with them the risks associated with the release of any genetically modified organism [ , ] , their use should only be entertained in cases where the advantages can be quantified and demonstrated to be substantial relative to a traditional, directly administered vaccine. to quantify the effectiveness of a rtv-based vaccination program, we developed a mathematical model that describes the spread of a rtv in a population that is susceptible to the associated vector and an infectious pathogen. the model consists of ordinary differential equations based on a standard sir framework that tracks transitions between classes of individuals that describe infectious and immune states of hosts in the presence of the vector, vaccine, and pathogen. the vaccine is administered by continual and direct vaccination of a fraction of newborns, and can also transmit between individuals resulting in indirect vaccinations. state variables are notated x i,j , where infection and immunity status are described by the subsets i, j ⊂ {Ø, c, v, w}; subscripts c, v, and w refer to the vaccine vector, the recombinant transmissible vaccine (rtv), and the pathogen respectively. susceptible individuals are denoted x Ø,Ø because they are not currently infected and have no immunity. individuals may be infected by, or recovered from, the vaccine vector, the rtv, or the pathogen; the current infection statuses are indicated with a c, v, or w respectively in the i set: x cw,Ø , for example, describes individuals infected with both vector and pathogen but no immunity, whereas x Ø,cw describes individuals that are currently free of infection, but have recovered from both the vector and pathogen. standard mass action terms describe disease transmission; infectious agent i ∈ {c, v, w} has transmission rate β i , and recovery rate δ i . individuals are born at a constant rate b, a fraction σ of which are vaccinated at birth (and placed in class x v,Ø ), and die at rate d. we assume disease-induced mortality is negligible and can be ignored. because a rtv is engineered by inserting a foreign, non-beneficial antigenic region into a vector organism, it is likely that the rtv's ability to transmit between hosts will be reduced relative to the vector organism; we incorporate this key biological constraint by assuming β v < β c . for simplicity, we assume that a host infected with the vaccine recovers at the same rate as a host infected with the vector (δ v = δ c ). defining the basic reproduction number of infectious agent i ∈ {c,v,w} as , these latter two assumptions imply r ,v < r ,c . cross-immunity between infectious agents is assumed to be either perfect or absent altogether. in the scenarios that describe the vector circulating in the population, we assume perfect cross-immunity exists between the vector and vaccine, as well as between the pathogen and vaccine, but that cross-immunity between the vector and pathogen is absent. these assumptions allow us to describe the epidemiological dynamics of vector, vaccine, and pathogen using the following system of ordinary differential equations (odes): where i c and i w are defined as the total number of vector infected and pathogen infected individuals, the biological interpretations of the model variables and parameters are described in tables and , respectively. a flow diagram of the model is presented in fig. . current rtv research is focused on vector organisms that impart little or no vaccine crossimmunity upon host infection [ , ] . we use the subsystem consisting of equations a, c, d, h, k to describe cases in which the vector does not impart vaccine immunity to hosts, and the full model to describe cases in which vector/vaccine cross-immunity is perfect. by exploring these two extreme descriptions of cross-immunity, our model captures the limits of a broad range of rtvs. refer to web version on pubmed central for supplementary material. pathogen genes are inserted into a benign vector organism, resulting in the expression of pathogen antigen(s) on the viral vector, now termed the vaccine. infection with the vaccine exposes the host immune system to pathogen antigen(s), and prompts a pathogen-specific immune response. r ,c , r ,v , and r ,w represent the basic reproduction numbers of the vector, vaccine, and pathogen, respectively. because the vaccine is produced by inserting non-beneficial foreign genes into the vector organism, the vaccine r is likely bounded above by the vector's r (r ,v < r ,c ). the rtv outperforms a non-transmissible vaccine, even when the vector is circulating in the population and vector/vaccine cross-immunity is perfect. the "vector absent" panes describe scenarios in which the vector remains absent from the host population, or when vector/vaccine cross-immunity is absent. when the vector is endemic, cross-immunity is perfect, and is constant, the benefits of increasing vector transmission plateau when the vector's transmission approaches the pathogen's. in each case, parameters were set to r ,w = , b = , d = . , δ c = and δ w = . ebola virus disease outbreak in west africa transmission dynamics and prospects for the elimination 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can be found, in the online version, at https:// doi.org/ . /j.vaccine. . . . key: cord- -a tvcgqj authors: moore, george e.; ward, michael p.; kulldorff, martin; caldanaro, richard j.; guptill, lynn f.; lewis, hugh b.; glickman, lawrence t. title: a space–time cluster of adverse events associated with canine rabies vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: a tvcgqj electronic medical records of a large veterinary practice were used for surveillance of potential space–time clustering of adverse events associated with rabies vaccination in dogs. the study population was , dogs vaccinated in hospitals in us metropolitan areas during a -month period. using a scan statistic for population rate data, significant space–time clusters were identified involving the atlanta and tampa/st. petersburg areas during a -month period. separate spatial–temporal analyses of these cities using coordinates for individual address coordinates identified one significant patient cluster (p = . ), associated with a . km-radius area in atlanta ( adverse events in dogs; . %) from november through february . this percentage of adverse events was significantly increased after adjustment for host-related factors and the number of concurrent vaccinations. traditional post-marketing surveillance of veterinary vaccines relies on veterinarians or owners to voluntarily report suspected reactions to manufacturers or to the us department of agriculture (usda), the federal regulatory agency for animal vaccines. this type of surveillance provides case (numerator) information only, and it is often characterized by underreporting and variability in report quality [ , ] . trends in reports of vaccine-associated events (vae) may be related to the immunogenicity of the vaccine, improper administration of a vaccine, a veterinarian's or owner's perception of an association between the event and vaccination, and/or the inclination of the veterinarian or owner to initiate a report. severe or life-threatening vaes are more likely to be reported voluntarily and may also be more likely to be investigated. clustering of adverse events or disease can serve as an indicator of a potential association between an adverse event and vaccine administration [ , ] . clustering of health events should be considered in the dimensions of both time and space, and clustering can occur in time-and-space without being apparent in either dimension alone. clusters of vaes in time may occur with the administration of a newly marketed vaccine or a new batch/lot of routinely used vaccines. clusters of vaes in space may occur if the geographic distribution of a new vaccine or lot is not homogenous among veterinary hospitals. such clustering may also appear with changes in vaccination techniques by hospital personnel or with changes in adverse event reporting policies. the "best" situation in which to investigate whether or not a suspected adverse reaction is linked to vaccination has been described as one where there is a clearly defined population with a record of all vaccinations, all instances of the disease event of interest (potential adverse effect), and the possibility of linking these back to individuals [ , ] . practice consolidations and improved medical informatics have increased the likelihood and availability of such resources in human and veterinary medicine. a large privately-owned veterinary practice, banfield, the pet hospital ® , currently provides primary health care to more than two million dogs and cats each year in more than locations in states, treating approximately , patients daily. banfield uses a single fully computerized (paperless) veterinary medical record system at all locations. this proprietary system has standardized codes for > different clinical signs, > laboratory tests, > diagnoses, and > procedures or treatments. electronic records from all hospitals are uploaded weekly to a central data facility for quality assurance audits and data warehousing. the banfield practice database has been recently used to identify patient characteristics associated with increased risk for adverse events within -day post-vaccination in pet dogs [ ] . these events were consistent with immediate-type hypersensitivity reactions, and increased risk was associated with body weight less than kg, age approximately one to years of age, and being surgically neutered. dogs receiving multiple vaccines at one encounter were also at increased risk of vaes. because rabies vaccination of dogs is commonly required by local or state authorities, rabies vaccine is often administered alone or concurrently with other vaccines to pet dogs. patient risk factors and/or practice vaccination protocols may therefore influence the occurrence and potential clustering of reported vaes. the purpose of this study was to determine if practitioner-diagnosed adverse events occurring within days of canine rabies vaccine administration are clustered in space and time. the electronic medical records of banfield, the pet hospital ® , were searched to identify all dogs that received rabies vaccine alone or in combination with bordetella vaccine, coronavirus vaccine, multivalent distemperadenovirus-parainfluenza-parvovirus-leptospirosis vaccine, giardia vaccine, or borrelia vaccine between january and december . all vaccines were produced by one manufacturer (fort dodge animal health, fort dodge, ia), except for bordetella vaccine (biocor animal health inc., omaha, ne). records were excluded if the dogs concurrently received vaccine(s) and an injectable heartworm preventive since the latter product may also induce a hypersensitivity reaction. information extracted from each patient record included home address, date of birth, breed, sex, neuter status, weight, vaccine received, date of vaccination, and hospital location. vae were defined as any coded diagnosis of "vaccine reaction", "allergic reaction", "urticaria", "anaphylaxis", "cardiac arrest", "cardiovascular shock", or "sudden death", if the diagnosis occurred within days of vaccination. diagnosis validation was performed through a record review for clinical signs and treatments [ ] . for this study, banfield hospitals were grouped into units of or more hospitals located within a miles radius; units were designated as metropolitan-hospital-groups (mhg). dogs were included in the study only if they were vaccinated at one of these hospitals. patient addresses (street, city, state, and zip code) were geocoded to determine longitude and latitude coordinates using an address reference dataset and geocoding software (streetmap usa and arcgis v. , esri, redlands, ca). addresses that could not be geocoded were assigned the longitude and latitude coordinates of the centroid of their zipcode. cases were defined as dogs that experienced a vae within days following rabies vaccination, and non-cases were vaccinated dogs not diagnosed with a vae. data sets of the mhg populations were evaluated separately for spatial, temporal, and spatial-temporal clusters of accumulated vae. clusters, a geographically bounded group of events of sufficient size and concentration to be unlikely to have occurred by chance [ ] , were identified using the space-time scan statistic. this statistic, defined by a cylindrical window composed of a circular geographic base and height corresponding to time [ ] , imposes overlapping circles of different location and size on the map, each of which is a potential cluster. the scan statistic calculates the expected number of cases within the scanning window based on either a poisson (rate data) or bernoulli (case-control data) distribution. for analyses of the large mhg populations, the distribution of vaes was assumed to be poisson. scanning for clusters included spatial and temporal dimensions ranging from up to % of the study area and/or study period. the selected time precision for temporal and space-time analyses was month, e.g. calculated vae rates per month. data sets were scanned for clusters with only increased rates of vae occurrence (equivalent to a one-sided statistical test) in time, space, and space-and-time. a likelihood-ratio test statistic was calculated, based on the maximum likelihood function, for each cluster of adverse events identified. its distribution and corresponding p-value were obtained by monte-carlo simulation-randomly generating replications of the data set under the null hypothesis of spatial and temporal randomness. the rank of the maximum likelihood from the real data set was compared with the maximum likelihoods from the random data sets. if this rank is r, then p = r/( + simulations). the p-value was an indicator of the evidence for a real cluster in the test statistics calculated. in addition to the most likely cluster, any other identified cluster was reported if the associated p-value was statistically significant and if it did not overlap with the most likely cluster. analyses were adjusted for patient sex (male, female), neuter (intact, neutered) status, age, weight, and number of vaccines received concurrently. age categories were - months, > months- . years, > . - . , > . - . , > . - . , > . - . , and > . years. weight categories were - , > - , > - , > - , and > kg. if mhgs were within a cluster identified by the crude and adjusted poisson model analyses, the mhg study population was analyzed by a bernoulli model using the point locations of the geocoded patient addresses of cases and non-cases. analyses were performed with and without those addresses assigned to zipcode centroids to assess for possible bias introduced by this method. this was useful to compare the sensitivity of the poisson model assumption for aggregated data to that of the bernoulli method. if space-time clusters were identified in the bernoulli model, cases in the cluster were compared to cases located outside the identified cluster in the same city for differences in patient factors (sex, neuter status, age, and weight) and number of concurrent vaccinations. comparisons of categorical variables were made by chi-square and fisher's exact test. all space-time analyses were performed using satscan software version . (www.satscan.org); other statistical calculations were performed with stata statistical software. a type i error of . was used. thirteen metropolitan areas were identified with or more hospitals and were designated mhg; a total of banfield hospitals were located in these groups (fig. ) . during the -month study period, rabies vaccination (alone or with other concurrent vaccinations) was given to , dogs; there were vaes diagnosed ( . %; % ci: . - . %). in the unadjusted poisson model for all mhg, the most likely temporal cluster for vae involved the -month period of september through february , but the identified cluster was not statistically significant. during this time period, there were observed cases and the calculated expected number of cases was . (ratio = . ; p = . ). in a purely spatial analysis, a single statistically significant cluster that included both atlanta and tampa/st. petersburg was identified, with observed cases and . expected (ratio = . ; p = . ). in time-and-space, a significant cluster of vae was also identified; the spatial window was not altered but the cylindrical window was reduced compared to temporal analyses alone ( table ) analyses of mhg populations with adjustments for patient covariates did not alter the space-time dimensions of the most likely cluster, although the adjustment for the number of concurrently administered vaccines caused the greatest reduction ( . %) in the ratio of observed-to-expected cases ( table ). analyses of geocoded address locations for cases and noncases using a bernoulli model to further define space-time clustering in atlanta and tampa/st. petersburg was performed separately for each mhg. significant space-time clustering of vae was not detected in tampa/st. petersburg using the bernoulli model, but a significant (p = . ) cluster of vae was identified in atlanta (table ) . this space-time cluster involved vae in dogs vaccinated in a -month time period (vae percentage = . %; % ci: . - . %) (figs. and ). in comparison, the atlanta case and study populations for the months outside this time period were and , , respectively (vae percentage = . %; % ci: . - . %). in analyses excluding dogs with addresses that could not be geocoded, a significant space-time cluster was not identified in tamp/st. petersburg (p = . ), whereas a significant cluster identified in atlanta covered the same month period and similar area as detected in the full analysis (p = . ). in atlanta, patients within the identified vae cluster compared to patients outside the cluster were not significantly different in sex (p = . ), neuter status (p = . ), age (p = . ), or weight (p = . ); nor were they significantly more likely to be geocoded to a zipcode centroid (p = . ). patients within the cluster, however, were significantly more of the banfield hospitals in the atlanta mhg, contributed patients to the identified cluster. two hospitals contributed . % ( / ) of the cases and . % ( / ) fig. . zip-code-based rates for canine rabies vaccine-associated adverse events (vae) during a -month period for atlanta, ga. inset is state map of georgia. of the non-case population in the cluster, but this difference was not statistically significant (p = . ). supportive evidence for the contribution of cases by these hospitals was indicated when space-time cluster analysis, using a poisson model with hospital-based populations and vae rate data adjusted for number of concurrent vaccinations, identified a significant (p = . ) cluster of vae involving the same hospitals during a -month period of september through february . case and non-case data coordinates were based on geocoded addresses for individual dogs. scan window was ≤ % of the geographic area and ≤ % of the study period. a there were no other statistically significant clusters identified. b llr: log likelihood ratio statistic. this study demonstrated the utility of an electronic medical record database for surveillance of space-time clustering of canine rabies vaccine-associated adverse events. the practice database provided information of post-vaccination events in the exposed population, allowing analysis of group and individual-based data for the study of vae. investigations of increased rates of vaes in space or time should consider the crude event rate and rate changes after adjustments for important covariates [ ] . separate cluster analyses in this study showed that the greatest change in the ratio of observed-to-expected cases occurred after adjustment for the number of vaccines administered per visit. this covariate did not fully explain the vae increase in the detected cluster, indicating further investigations are warranted to determine other potential risk factors associated with cluster events. the veterinary practice database used in this study did not have an entry field for vaccine serial or lot number, an important covariate in vae inquiries. the national distribution pattern and period of use for different lots of vaccines in the veterinary practice was also unknown. in a recent study of human vaccines, using a convenience sample, % of the vaccine doses in each lot were estimated to be used within - months of distribution [ ] . although a -month maximum scan window was used ( % of total study period), the most likely cluster involved only a -month period and this time period may have encompassed the use period for a specific lot of vaccine. the temporal window and primary involvement of two hospitals in the most likely cluster may also indicate changes in vaccination technique or in the personnel preparing or administering the vaccines. factors related to vaccine administration or aftercare have been demonstrated to impact the risk of vaes [ ] . hospital protocols for standardization and quality assurance are established in many practices, and banfield personnel administering vaccinations are instructed on appropriate vaccination techniques. the scan statistic was selected as a detection tool for clusters because it adjusts for heterogeneous population densities and confounding variables, searches for clusters without pre-selected bias of their size or location, takes multiple testing into account, and specifies the location of a cluster if the null hypothesis is rejected [ ] . conceptually, the space-time scan statistic provides a cluster detection test that both identifies and evaluates the statistical significance of specific clusters [ ] . the test however has low power for clusters with noncircular patterns, such as extending along a long and narrow river or highway. analyses in this study used assumptions for two different discrete probability distributions, bernoulli (binomial) and poisson. with spatial-temporal information available for each patient (cases and non-cases), the bernoulli distribution could have been applied in all analyses; however, the poisson model provided the capability of adjusting for covariates and is much less computationally intensive for larger datasets. in analyses for rare or uncommon events, the poisson model is a very good approximation to the bernoulli model and produces slightly conservative p-values [ , ] . spatial analysis with geocoding for point data was facilitated in this study by the use of patient addresses. aggregated data, e.g. individual hospital populations, identified a most likely spatial-temporal cluster in atlanta that was similar to the analysis with individual patient addresses. methods are being developed in geographic information systems for geomasking of patient addresses to protect human subject confidentiality [ ] . these techniques can include movement of each address point in a limited but randomized direction and distance before analyses and mapping. although diagnoses were selected by practitioners from the available codes in the software, computerized databases are dependent on coded outcomes and some codes may be nonspecific. standardized case definitions for vaes are not available in veterinary medicine and are only currently being addressed in human medicine [ ] . due of the large number of hospital locations contributing data, the impact of reporting biases or misdiagnoses that might be potentially introduced by a few individuals is reduced. nevertheless, validation of automated codes for health outcomes has been advocated [ ] . the clinical signs recorded in patient medical notes supported the coded diagnoses when reviewed in a random sample of this study population. analyses to detect potential clusters of health events can be considered hypothesis-generating [ ] . cluster detection indicates a concentration of events that are unlikely to have occurred by random chance, but other possible causes must be evaluated through additional investigations. further epidemiological studies using more intensive methods or more detailed inquiries into patient records may be requested by regulatory agencies, vaccine manufacturers, or hospital administrators. an advantage of the population composing this practice database is its open-cohort structure, contributing cases and controls from the same population for a 'nested' study, e.g. the bernoulli analysis. further investigations into potential clustering of vaes based on diagnostic codes should also address standardization of coding by health care providers. the vaccine adverse event reporting system (vaers) vaccine-associated adverse events monitoring the safety of vaccines: assessing the risks investigation into a cluster of infant deaths following immunization: evidence for methanol intoxication vaccine safety surveillance using large linked databases: opportunities, hazards and proposed guidelines use of the case-control approach in vaccine evaluation: efficacy and adverse effects adverse events diagnosed within days of vaccine administration in pet dogs methodology of enquiries into disease clustering. london: london school of hygiene and tropical medicine a spatial scan statistic applied spatial statistics for public health data tracking vaccine lot lifecycles using reports to the vaccine adverse event reporting system (vaers) evaluating cluster alarms: a space-time scan statistic and brain cancer in procedures for geomasking to protect patient confidentiality addressing the need for standardized case definitions of adverse events following immunization (aefi) this research was supported by the centers for disease control and prevention, grant no. r ci . the authors would like to thank the many banfield, the pet hospital veterinarians who generated the clinical data and case records which formed the database for this study. key: cord- -nsj dh authors: chakraborty, chiranjib; agoramoorthy, govindasamy title: india’s cost-effective covid- vaccine development initiatives date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nsj dh nan the creation of cost effective vaccine is fundamental for the effective mitigation of the deadly covid- pandemic. india is moving to achieve this target to meet the high demand to produce the cheapest vaccine against covid- . india also harbors over . billion people and many cannot afford a costly vaccine. in addition, millions of people who live across over world's poorest countries will also expect the affordable low-cost vaccine. india has a long history of vaccine production and the haffkine institute for example has been recognized by the who as a prequalified vaccines producer before the country got institute of india has long history of producing vaccines against tetanus, influenza, rabies, measles, and mumps. it's currently collaborating with codagenix to develop a vaccine, including live-attenuated vaccine against covid- . besides, it has a partnership with codagenix, a new york based firm specialized on vaccines and the oxford university to produce the covid- vaccine. india also has a tie up with the oxford university to produce the oxford covid- vaccine or chadox ncov- . the serum institute has announced that it will produce the vaccine at low cost and it's registered for phases ii and iii clinical trials (nct , clinicaltrials.gov). india has exported complex vaccines such as the penta-valent rotavirus vaccine before. what's unique about india is that it has the expertise for low-cost per-unit vaccine production of vaccines. due to the low cost vaccine making history, new products against covid- will be of great use in over low-income countries worldwide benefiting millions of people who cannot afford expensive vaccines. india has manufactured the oral polio vaccine and distributed freely across the country as part of the polio eradication initiative by the who. recently, india has requested the world trade organization to waive some provisions in the international agreements that regulate intellectual property rights, to speed up efforts to contain the covid- pandemic and to make sure low-income countries such as india are not left behind. india is currently finalizing the electronic vaccine intelligence network or evin to provide real-time information on the stock and storage details on vaccines nationwide. the government has formed an expert committee to advice on the priorities of vaccine distribution throughout the country. few months ago, the who has praised india's vaccine production capacity in a meeting of covid- . it's time for the developing world to collaborate with india to produce and distribute the cost-effective covid- vaccine as soon as possible. no potential conflict of interest was declared by authors. writing-original draft, c.c; editing, c.c, g.a; all authors have read and approved the final version of this manuscript. madhavi y. vaccine policy in india vaccine development and deployment: opportunities and challenges in india india's vaccine deficit: why more than half of indian children are not fully immunized, and what can and should be done key: cord- -eeg k a authors: detoc, maëlle; bruel, sébastien; frappe, paul; tardy, bernard; botelho-nevers, elisabeth; gagneux-brunon, amandine title: intention to participate in a covid- vaccine clinical trial and to get vaccinated against covid- in france during the pandemic date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eeg k a introduction the world is facing the covid- pandemic. the development of a vaccine is challenging. we aimed to determine the proportion of people who intend to get vaccinated against covid- in france or to participate in a vaccine clinical trial. methods we conducted an anonymous on-line survey from the th of march to the th of april . primary endpoints were the intention to get vaccinated against covid- if a vaccine was available or participate in a vaccine clinical trial. results three thousand two hundred and fifty nine individuals answered the survey; women accounted for . % of the respondents. according to their statements, . participants ( . %, % ci . - %) will certainly or probably agree to get vaccinated against covid- . older age, male gender, fear about covid- , being a healthcare worker and individual perceived risk were associated with covid- vaccine acceptance. vaccine hesitancy was associated with a decrease in covid- vaccine acceptance. one thousand and five hundred and fifty respondents ( . % % ci . - . %) will certainly or probably agree to participate in a covid- vaccine clinical trial. older age, male gender, being a healthcare worker and individual perceived risk were associated with potential acceptance to participate in a covid- vaccine clinical trial. vaccine hesitancy was associated with refusal for participation in a covid- vaccine clinical trial. conclusions nearly % and % of the survey respondents were respectively likely to accept vaccination or participation in a clinical trial against covid- . vaccine hesitancy will be the major barrier to covid- vaccine uptake. coronavirus: the severe acute respiratory syndrome-coronavirus (sars-cov ). the world is now facing a pandemic, more than millions of people have become infected worldwide, cases are described in more than hundred countries, and more than , people died [ ] . in , the world health organization identified ten threats to global health [ ] . among these threats, vaccine hesitancy, the risk of a global influenza pandemic, and the risk of emergence of highthreat pathogens such as middle-east respiratory syndrome and/or severe acute respiratory syndrome were identified. since this statement, covid- had emerged. developing covid- vaccines is a crucial challenge, and several candidates are currently under basic development [ ] . some of them will be tested in phase i trials in the weeks to come [ ] . the time it takes to develop a vaccine is estimated to be or . years as different steps are necessary during the clinical development of a vaccine [ ] . recruitment of volunteers in a vaccine clinical trial is a real challenge [ ] , and some trials are stopped due to difficulties in recruitment. vaccine hesitancy may also have an impact on recruitment in covid- vaccine clinical trial [ ] . after its clinical development, a covid- vaccine will also face the challenge of acceptance by the general population in a post-crisis context. . the impact of the current pandemic on the intention to participate in a covid- vaccine clinical trial and on the intention to get vaccinated against covid- vaccine is not obvious. this is a particular concern in france, which has been shown to be the leader-country of vaccine hesitancy [ ] . the aim of this study was to evaluate the intention to get vaccinated against covid- among the general population and healthcare personnel, in the context of the current pandemic. we conducted an anonymous online survey (lime survey®) from the th of march to the th of april among adult general population and adult patients. the survey was proposed to individuals via social networks (facebook, twitter), shared by e-mail, on the website of the university hospital of saint-etienne (france), and in centers for covid- diagnosis and in medical centers. we developed a standardized questionnaire based on a literature review. the questionnaire addressed: ( ) demographical characteristics (age, chronic medical conditions), ( ) fears about covid- , ( ) history of vaccination against pandemic h n influenza and seasonal influenza, ( ) intention to get vaccinated if a covid- vaccine was available, ( ) vaccine hesitancy. according to the who strategic advisory group of experts on immunization, vaccine hesitancy refers to delay in acceptance or refusal of vaccines despite availability of vaccination services. vaccine hesitancy is complex and context specific, varying across time, place and vaccines. it is influenced by factors such as complacency, convenience and confidence [ ] . we evaluated participants' self-reported vaccine hesitancy according to the who definition using three previously adapted questions: "have you ever refused a vaccine for yourself or a child because you considered it as useless or dangerous?" "have you ever postponed a vaccine recommended by a physician because of doubts about it?" "have you ever had a vaccine for a child or yourself despite doubts about its efficacy" [ ] . if a participant answered yes to one of these proposals, he or she was considered to be "vaccine hesitant". the questionnaire included items to be answered on a -level likert scale including a "don't know" option to evaluate intention to participate in a clinical trial and to get vaccinated if a vaccine was available. we combined survey responses into two categories (strongly agree/agree, don't know/disagree/strongly disagree) and ran ordinal regression models to examine demographic and attitudinal factors predictive of respondents' willingness to get vaccinated against covid- . to identify suitable candidate variables for regression models, we first conducted univariate analysis using a chi-squared test. candidates that were significant at p< . in univariate analyses were then included in a multivariable regression model. data were analyzed using spss version . . the protocol complied with the data privacy laws of the national commission for informatics and civil liberties and was approved by the institutional review board with the number during the study period, , people opened the web links for the online survey, according to their statements, , participants ( . %, % ci . factors associated with covid- vaccine acceptance are displayed in table . in multivariable analysis, older age, male gender, fear about covid- , be healthcare workers and individual perceived risk remained associated with covid- vaccine acceptance. vaccine hesitancy was associated with lower acceptance of a covid- vaccine. one thousand and five hundred and fifty two respondents ( . % % ci . - . %) will certainly or probably be willing to participate in a covid- vaccine clinical trial. among the . men, ( . % % ci . - . %) will probably accept to participate in a covid- vaccine clinical trial, this proportion is significantly greater than women ( . % % ci . - . %, p< . ). the percentage of potential participants in a covid- vaccine clinical trial was . % ( . - . %) in the - years age group, and . % ( % ci . - . %) in the - years age group. healthcare workers are more prone to participate in a vaccine clinical trial than non-healthcare workers ( . % vs . %, p< . ). factors associated with covid- vaccine clinical trial acceptance are displayed in table . in this online survey, we observed that nearly three quarters of the respondents would accept a vaccine against covid- although % of the respondents were qualified as "vaccine hesitant". moreover, around a half of the respondents would accept to participate in a clinical trial for a covid- vaccine. concerning the intention to get vaccinated, our results are similar to the results of the longitudinal coconel study conducted by the observatoire régional de santé provence alpes côte d'azur [ ] and to results of investigations conducted in the usa [ , ] . similar to coconel, we observed that men were more prone to get vaccinated than women. women accounted for the vast majority of our study respondents, suggesting that in real-life settings, covid- vaccine acceptance could be greater. in addition, older individuals are more prone to get vaccinated in both studies, this is probably due to a greater perceived risk of getting infected and developing a severe disease in older people. we analyzed factors associated with perceived risk of sars-cov- infections, and with fear about covid- . male gender and older age were not associated with perceived individual risk but were associated with fear about covid- (data not shown). in the same vein, healthcare workers were more prone to get vaccinated or to participate in a vaccine clinical trial than the others. we hypothesized that healthcare workers perceived a greater risk to get infected. healthcare workers are particularly vulnerable and accounted for % of the infected people in italy, and in greece [ , ] . on the contrary, expecting a low infection risk is associated with a lower willingness to get vaccinated [ ] . seventy-eight percent of healthcare workers considered themselves at-risk to get infected, however after adjustment on age, gender, and comorbidities, being a healthcare worker was not associated with perceived risk to get infected, but with fear about covid- (data not shown). around the half of the respondents will accept to participate in a covid- vaccine clinical trial. in the context of a clinical trial, men were also more prone to participate. fears about covid- were not associated with willingness to participate in a clinical trial. however, individuals who considered themselves at-risk for covid- infection were more prone to accept to participate in a clinical trial for a vaccine. outside the pandemic context, self-perceived risk for a disease was not the main motivation to participate in a vaccine clinical trial [ ] . moreover, older age was associated (except in the group over years) with willingness to participate in a covid- vaccine clinical trial, this observation contrasts with a previous study about willingness to participate in a vaccine clinical trial [ ] . this observation suggests that in the pandemics context, individuals are more prone to participate in a clinical trial for a vaccine. we also observed that a part of respondents who will certainly accept to participate in a vaccine clinical trial will not certainly get the vaccine if available. further studies are needed to explore indepth the differences in barriers and motivations to participation in a vaccine clinical trial, and to the acceptance of the vaccine. next steps will be the construction of multi-component interventions to facilitate recruitment of volunteers in clinical trials, and to increase covid- vaccine acceptance. communities should be involved in the development of these interventions. the rush to develop a covid- vaccine may jeopardize public confidence in scientists [ ] . the proportion of french people favorable to a covid- vaccine can appear low, but we have to put this in perspective with the fact that the vaccine would be a new vaccine, and that vaccine coverage against h n pandemic influenza was only . % in france [ ] . however, a greater proportion of respondents to our survey declared they had been vaccinated against h n pandemic influenza, so this observation may suggest that the respondents are more pro-vaccine than the general population in france, and more often healthcare workers. we observed a similar prevalence of vaccine hesitancy than in a recent study in france, which identified a prevalence of % [ ] . one limitation of our work may be the use of social media to recruit study participants. social media users who use platforms, such as facebook and twitter, as health information sources are more prone to get vaccinated against seasonal influenza in the united states of america [ ] . furthermore, a great number of healthcare workers answered the survey and we observed that healthcare workers were more prone to get vaccinated or to participate in a vaccine clinical trial independently of the perceived risk to get contaminated. however, vaccine hesitancy also affects healthcare workers [ ] [ ] [ ] . in our study sample, vaccine hesitancy affects around % of the healthcare workers and % of the non-healthcare workers. our work suffers from limitations. first, our sample is not completely representative of the french general population, and the great amount of hcws may overestimate the proportion of individuals with intentions to get vaccinated. in a recent report, in european countries, the proportion of individuals with intention to get vaccinated against covid- was . %, but in france, it was only % [ ] . this survey was also conducted on line but a priori in a representative panel of . individuals per country. secondly, we did not precise in our survey, the type of clinical trials, and intention to participate may change between early and later phases clinical trials [ ] . in conclusion, during the pandemics, around % of the french people would agree to get vaccinated. due to the burden of the disease, and the potential natural immunity, it may well be possible that this proportion would be enough to obtain a herd effect [ ] . antecedents of vaccine hesitancy may affect vaccine acceptance even in a context of a pandemic due to an emerging pathogens. around fifty percent will agree to participate in a covid- vaccine clinical trial; we can hope that vaccine trials would not be stopped because of recruitment difficulties. co-authors have no conflict of interest to declare for this work. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: dr amandine gagneux-brunon on the behalf of all co-authors covid- map. johns hopkins coronavirus ten health issues who will tackle this year n developing covid- vaccines at pandemic speed the covid- vaccine development landscape human challenge studies to accelerate coronavirus vaccine licensure public interest in medical research participation: differences by volunteer status and study type barriers and motivations for participation in preventive vaccine clinical trials: experience of clinical research sites the state of vaccine confidence : global insights through a -country survey who | improving vaccination demand and addressing hesitancy vaccine hesitancy in the french population in , and its association with vaccine uptake and perceived vaccine risk-benefit balance a majority of vaccine skeptics plan to refuse a covid- vaccine, a study suggests, and that could be a big problem planning for a covid- vaccination program covid- : protecting healthcare workers is a priority cov- infection in healthcare personnel with high-risk occupational exposure: evaluation of sevenday exclusion from work policy future pandemics and vaccination: public opinion and attitudes across three european countries the rush to create a covid- vaccine may do more harm than good social media use and influenza vaccine uptake among white and african american adults vaccine hesitancy among general practitioners: evaluation and comparison of their immunisation practice for themselves, their patients and their children discrepancies between general practitioners' vaccination recommendations for their patients and practices for their children vaccine hesitancy and self-vaccination behaviors among nurses in southeastern france once we have it, will we use it? a european survey on willingness to be vaccinated against covid- herd immunity and herd effect: new insights and definitions key: cord- - l nixc authors: wong, j.p.; christopher, m.e.; viswanathan, s.; karpoff, n.; dai, x.; das, d.; sun, l.q.; wang, m.; salazar, a.m. title: activation of toll-like receptor signaling pathway for protection against influenza virus infection date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: l nixc this study aims to evaluate the antiviral role of nucleic acid-based agonists for the activation of toll-like receptor (tlr) signaling pathways, and its protective role in respiratory influenza a virus infections. tlr- is expressed on myeloid dendritic cells, respiratory epithelium, and macrophages, and appears to play a central role in mediating both the antiviral and inflammatory responses of the innate immunity in combating viral infections. influenza viruses can effectively inhibit the host's ability to produce interferons, and thereby suppress the immune system's antiviral defence mechanisms. poly iclc is a synthetic double stranded rna comprising of polyriboinosinic-poly ribocytidylic acid (poly ic) stabilized with l-lysine (l) and carboxymethylcellulose (c). poly iclc and liposome-encapsulated poly iclc (le poly iclc) are tlr- agonists and are potent inducer of interferons and natural killer cells. intranasal pre-treatment of mice with poly iclc and le poly iclc provided high level of protection against lethal challenge with a highly lethal avian h n influenza (hpai) strain (a/h n /chicken/henan clade ), and against lethal seasonal influenza a/pr/ / [h n ] and a/aichi/ [h n ] virus strains. the duration of protective antiviral immunity to multiple lethal doses of influenza virus a/pr/ / virus had been previously found to persist for up to weeks in mice for le poly iclc and weeks for poly iclc. similarly, pre-treatment of mice with cpg oligonucleotides (tlr- agonist) was also found to provide complete protection against influenza a/pr/ / infection in mice. rt-pcr analysis of lung tissues of mice treated with poly iclc and le poly iclc revealed upregulation of tlr- mrnas gene expression. taken together, these results do support the potential role of tlr- and tlr- agonists such as poly iclc and le poly iclc in protection against lethal seasonal and hpai virus infection. the innate immune system plays a central role in the detection of invading viral pathogens, and responds by activating inflammatory and antiviral defence mechanisms to combat the viral agents. to evade these defence machanisms, the influenza viruses are known to circumvent the potent antiviral interferon action by inhibiting the interferon regulatory factor (irf- ), and the general interferon signaling pathway [ ] . the viruses achieve this in part through their double stranded rna (ds rna) binding protein called nonstructural protein- (ns- ) [ ] . by doing so, influenza viruses can replicate unabated in the host's respiratory tract. during influenza viral replication, single stranded (ss rna) and ds rna are intermediate molecules which are recognized by the toll-like receptors (tlrs) which are expressed by cells of the innate immunity including dendritic cells, natural killer cells and macrophages [ ] as well as on respiratory epithelium and elsewhere. toll-like receptors are transmembrane signaling proteins which are designed to specifically recognize various proteins, carbohydrates, lipids and nucleic acids of invading microorganisms. when activated, they trigger immune and inflammatory responses to respond to these infectious agents. there are four known toll-like receptors which are found in mammalians cells and which recognize nucleic acids. these are tlr- , tlr- , tlr- and tlr- and these are found in the endosomal membranes [ ] . since these nucleic acid-recognizing tlrs regulate the induction of type i interferon and other antiviral gene functions, they are rationale targets for antiviral drug development. previous study had demonstrated that tlr- contributes to the immune response of respiratory epithelial cells to influenza and ds rna [ ] . the molecular mechanism of this immune enhancement is generally believed to be mediated through mitogen-activated protein kinases, phosphatidylinositol -kinase/akt signaling and the tlr- associated adaptor molecule trif [ ] . this paper outlines the antiviral activity of two tlr agonists and examines their applications in inducing protective antiviral immunity against influenza viruses, including seasonal and avian strains. poly iclc is a synthetic ds rna condensed with poly-l-lysine and carboxymethylcellulose, and is a known tlr- agonists [ ] . the x-ray crystal structure of the poly ic:tlr- receptor signaling complex has been determined [ ] . since tlr- activation by ds rna results in induction of type i interferons, it follows that poly iclc, when delivered in liposomes to the endosomal membrane location, may strengthen the host antiviral defence against influenza virus by priming the interferon levels and, therefore, reversing the interferon knockdown by the viruses. in addition, oligonucleotides containing unmethylated cpg motifs, a tlr- agonist [ ] , are evaluated as stand alone immunomodulating antiviral agents. poly iclc, and oligonucleotides containing unmethylated cpg motifs used in this study were supplied by oncovir inc. (washington, dc) and oligos etc inc. (wilsonville, or), respectively. the method of preparation and lipid compositions of liposomeencapsulated poly iclc have been previously described [ ] . tlr- primers were procured from applied biosystems (abi, streetsville, on). mice were pre-treated with two intranasal doses of poly iclc, le poly iclc ( mg/kg body weight), sham liposomes or pbs given at h apart. at , , and h post-drug treatment, mice were euthanized by cervical dislocation, and the lungs were aseptically removed and homogenized in trizol. total rna was extracted from lung tissues by trizol method (invitrogen). one g of rna was used for dnase i (fermentas) digestion before doing rt (reverse transcription) to make st strand cdna. rt was performed using high capacity cdna rt kit with random primers (abi). thermal profile for the rt step was as follows: • c for min (hold); • c for min (hold); • c for s (hold). real-time pcr was performed using taqman probe-based detection (abi) that was run on an abi fast system. taqman gene expression assay (abi) consists of two unlabelled pcr primers and a fam dye labelled taqman mgb (minor groove binder) probe. the pcr step was as follows: • c for s for enzyme activation, cycles of denaturation at • c for s, annealing and extension at • c for s. ␤-actin was used as an internal control to normalize the expression of target genes. comparative ct method for relative quantitation was used to calculate fold changes. serial dilutions of different inputs of target and reference genes were performed to verify that efficiencies of target and reference are approximately equal (data not shown). data analysis was performed using software v . (abi). poly iclc and cpg oligonucleotides (cpg odn), whether unencapsulated or encapsulated in liposomes were administered intranasally into - -week-old ( g body weight) balb/c female mice. for the prophylaxis of influenza virus infection, groups of mice ( - animals per group) were given two doses of free or liposomal poly iclc ( mg/kg body weight) h apart by the intranasal route. the volume of the inoculum used was l. at various days post-drug treatment, the mice were intranasally challenged with either ld of influenza a/pr/ / , or with - ld of influenza a/h n /chicken/henan viruses. the animals were then monitored daily for symptoms of infection, body weights and survival. at day post-infection, the number of mice which survived the virus infection in each group was recorded. the survival patterns of the control and treated mice were graphed using the log-rank test (graphpad prism version . , san diego, ca). the survival of the various test groups were compared to the control groups using the log-rank test. differences were considered significant at p < . . the effect of poly iclc and le poly iclc on the tlr- expression in the lungs of mice intranasally pre-treated with these drugs were determined by rt-pcr (fig. ) . both poly iclc and le poly iclc were found to up-regulate tlr- mrna expression in the lung tissues (fig. ) . increased tlr- expression was detected as early as h post-drug administration, and was still pronounced at h postdrug administration. in contrast, sham liposomes (empty liposomes without poly iclc) did not have any effect on tlr- expressions in the lungs. the magnitude of tlr- up-regulation was slightly higher in the poly iclc group for the , and h time points compared to the le poly iclc, whereas le poly iclc tlr- upregulation was higher compared to the poly iclc for the h. the differences between poly iclc and le poly iclc on tlr- expression were not significant statistically. similar increases in tlr- expression were also observed in the poly iclc and le poly iclc groups in the spleens of mice but the increases were lower in magnitude and shorter in duration compared to the levels obtained in the lungs (data not shown). using established lethal influenza infection murine models previously described [ ] , the protective activity of poly iclc and le poly iclc against the various influenza a strains were determined by assessing the survival rates at day post-infection with ld dose of the influenza viruses ( table ). the results summarized show that both poly iclc and le poly iclc were highly effective in the protection of mice against both seasonal and avian influenza viruses (table ) . pre-treatment of mice with poly iclc or le poly iclc were found to be more efficacious than pretreatment with exogenously administered ifn-␣ or ifn-␥ against influenza a/pr/ / . the data from these studies suggest that the prophylactic protection provided by poly iclc and le poly iclc is broad-spectrum, effective and protects against h n , h n and h n strains. /kg body weight) , sham liposomes and pbs were analyzed using qrt-pcr as described in section . results were normalized to ␤-actin as endogenous control, and are shown as fold increases relative to the pbs control group. error bars represent standard error of the mean. the duration of protection provided by poly iclc and le poly iclc was previously reported [ ] . le poly iclc provided % protection against ld influenza a/pr/ / when pre-treatment was given for up to days prior to virus challenge. in comparison, pre-treatment with poly iclc was % protective when given up to days prior to virus challenge, and it provided % protection when given at days prior to virus challenge [ ] . the longer duration of le poly iclc compared to poly iclc was assumed to be attributable to liposome delivery and gradual release of the poly iclc from the liposomes. this remains to be confirmed. in a study evaluating the efficacy of cpg odn against influenza, mice were given g of a cpg odn days prior to infection with a lethal dose of influenza a/pr/ / (h n ) (fig. ). using this model, mice pre-treated with cpg odn and liposome-encapsulated cpg odn were completely protected ( % survival rates at day postinfection) against the otherwise lethal virus challenge. studies will be carried out to determine whether the incorporation of cpg odns in liposomes would confer a longer window of protection compared to unencapsulated cpg odns. the results presented affirm the antiviral role of nucleic acidbased tlr agonists such as poly iclc and cpg odns for the broad-spectrum protection against various seasonal and avian influenza a viruses. the activation of the tlr signaling pathway results in the stimulation of both innate and adaptive immune responses and may be partially responsible for the antiviral state against influenza infections. influenza viruses are highly effective and adaptable infectious agents which can overcome antiviral therapy by developing drug resistance, and by overcoming the host's immune defence mechanisms. conventional anti-influenza drugs target the virus structure, and they are susceptible to the emergence of drug resistance. animal studies suggest that activation of tlr signaling pathway by tlr agonists provides protection not only against influenza virus infections, but also respiratory syncytial virus [ ] , sars-cov [ ] , and hepatitis b [ ] , among others. the activation of the toll-like receptor signaling pathway may represent an effective broad-spectrum strategy against these viruses. this approach targets the cells of the innate immune system and hence, is less likely to result in drug resistance. in addition, it provides an effective means to enhance and/or restore interferon production during viral replication. in summary, tlr agonists may represent an affective and broad-spectrum antiviral strategy to combat influenza viruses which are unpredictable and ever changing. activation of interferon regulatory factor is inhibited by the influenza virus ns protein transcriptional signaling by double stranded rna: role of tlr involvement of toll-like receptor in the immune response of lung epithelial cells to double stranded rna and influenza a virus structural basis of toll-like receptor signaling with double stranded rna antiinfective applications of toll-like receptor agonists nucleic acid-based antiviral drugs against seasonal and avian influenza viruses evaluation of immunomodulators, interferons, and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections toll-like receptor signaling inhibits hepatitis b virus replication in vivo key: cord- -hqgwj vs authors: fehr, daniela; holznagel, edgar; bolla, stefania; hauser, beat; herrewegh, arnold a.p.m.; horzinek, marian c.; lutz, hans title: placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: hqgwj vs abstract a modified live virus vaccine against feline infectious peritonitis (fip) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. the vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (fcov) at the time of vaccination. although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with fip were found to be rt-pcr positive for fcov in plasma and showed changes in blood parameters consistent with early stage of fip. it is concluded that vaccination can protect cats with no or low fcov antibody titres and that in some cats vaccine failure was probably due to pre-existing infection. feline infectious peritonitis (fip) is a normally fatal disease of cats caused by infections with feline coronaviruses (fcov) which are antigenically related to a respiratory coronavirus strain of man (hcv e), transmissible gastro-enteritis virus (tgev) of swine and canine coronaviruses '. in switzerland, infections with fcov in domestic cats are widespread. about % of the cattery cats and % of all cats with access to outdoors were found to be seropositive . five to % of these develop lethal fwt. certain cat populations seem to be more susceptible to fip. young cats are especially prone: % of all fip cases affected cats younger than months of age and % cats younger than year?. a genetic disposition in certain breeds and in cheetahs was described -', and cats living in multiple-cathouseholds such as catteries or cat shelters and cats with access to outdoors are more likely to get exposed to fcov and develop fip than animals from single-cathouseholds'. clinical signs include the effusive or the non-effusive, granulomatous form of fip; both can also appear together. characteristic laboratory findings in fip ; accepted december ) are anaemia, neutrophilia, lymphopenia, increase of total serum protein, hyperglobulinemia and decreased albumin . in , a low virulent fcov type, called feline enteric coronavirus (fecv), which caused only mild gastrointestinal and respiratory diseases mainly in kittens, was described'. antibodies to these fecv and the virulent fip-causing viruses (feline infectious peritonitis virus: fipv) do crossreact. these authors formulated the hypothesis that most of these seropositive cats are actually infected with fecv and that fipv is just a mutant of fecv which has the ability to infect macrophages. at this time, no molecular or immunological differences are known between fecv and fipv which can explain the difference of virulence between these coronaviruses". therefore, it appears to be justified to generally designate them as fcov and to consider every fcov infection in cats as a potential risk ,". several observations point out the important role of the cell mediated immunity (cmi) in fip patho-genesis - , but the detailed immune mechanisms for controlling fcov infection remain unknown. under experimental conditions humoral immunity does not lead to protection. on the contrary, after experimental fip infection seropositive cats develop fip after a much shorter incubation period than seronegative control cats '- . this antibody dependent enhancement (ade) is thought to occur when virus-antibody complexes are formed and bound to the fc receptors of macrophages. macrophages are then more efficiently infected by the fc receptor-mediated endocytosis than by the virus alone + . when we initiated this study, this modified live virus vaccine (primucell fip@) was already commercially available in the usa, but many questions concerning safety and efficacy under field conditions were still unanswered. the safety of the vaccine was confirmed under experimental and field conditions , but vaccinated cats showed ade when challenged with a high dose of heterologous virus strain . the efficacy of the vaccine was assessed only under experimental conditions. with this trial the safety and efficacy of the vaccine was evaluate under field conditions in two high risk populations. a preliminary report has been presented at the fecv/fipv-workshop in davis, ca in and published in the proceedings". the study was performed as a placebo-controlled double blind assay. neither the investigators, nor the cat owners, knew which of two colour coded vials contained the vaccine. the code was not opened to the investigators, veterinarians and cat owners, until all cats terminated the month observation period. two populations with a high risk for fcov infection and fip were included in this trial. the first population consisted of cats from catteries with fip problems. in all of these catteries, fip cases had occurred in the last months prior the beginning of this trial either in the cattery itself or in kittens which had been re-homed to new owners. we expected that some of these cats had been already exposed to fcov. the second population consisted of cats < months of age, which were vaccinated by veterinarians in switzerland. as already mentioned, this ge group is more susceptible to fip than older cat?,-. the cats of each population were further subdivided into two groups, vaccine and placebo, respectively, which were comparable regarding age, sex, breed and living conditions. only clinically healthy cats older than weeks of age were vaccinated and pregnant queens were excluded from the study. in week and - weeks later the cats were vaccinated intranasally with either the coded vaccine or the placebo. after the vaccination the two coded groups were kept separately for h to prevent spread of the vaccine virus to cats of the placebo group. in both populations a blood sample was collected before vaccination (week ) and tested for felv and fcov-antibodies. in cattery cats only, haematology and clinical chemistry were done in week , and and in cats each of the vaccinated and of the placebo group, cd +/cd +-t-cells were measured in week , and . fiv-tests were carried out in week in the cattery cats. of sick cats, a blood sample was collected and haematology, clinical chemistry, felv and fcov-antibodies were determined. the modified live virus vaccine was developed by gerber et a .' briefly, fipv-df was attenuated in cell culture passages on the norden laboratories feline kidney (nlfk) cell line. passages - were propagated at °c. the th passage was exposed to ultraviolet irradiation. the vaccine has been shown to induce iga antibodies in the mucosa and to stimulate the cell mediated immune response '. the serial of the vaccine used in this study was a commercial batch (serial number ) with a titre of .' tcid,,. the placebo consisted of supernatant of non-infected nlfk cell culture. the vaccine and placebo were provided by the manufacturer in identical vials coded with coloured labels. the code was not broken to the veterinarians and the cat owners until all cats had finished the months observation period. the characteristics of the cattery cats and young pet cats are summarized in table . animals of the placebo and the vaccine groups in both, the cattery cats and the young pet cats, did not differ significantly with respect to age, sex, breed and living conditions. antibody titres to fcov were measured by indirect immunofluorescence using pd- cells of swine origin infected with tgev as antigen. plasma dilutions of : , : , : and : were tested. plasma samples of all cats were examined for circulating feline leukemiavirus (felv) p antigen and plasma samples of the cattery cats were also examined for antibodies to feline immunodeficiency virus (fiv) by indirect immunofluorescence using fiv-infected fl- cells as antigen . o samples with positive fluorescence results were subjected to western blotting for confirmation '. in cattery cats cd +/cd +-t-cells were measured by flow cytometry as described *. of all cats dying of fip, ~ of plasma samples taken at the time of first vaccination were retrospectively examined for presence of fcov-rna by polymerase chain reaction (pcr) . the mean of the haematological and clinical chemistry parameters between the vaccine and placebo group were analysed for significant differences by the mann-whitney u test, changes of laboratory values obtained from different cats over time were examined by the wilcoxon test. frequencies of fcov antibody titres in the placebo and vaccine group were compared using the x test. to determine differences in the frequencies of fip in the vaccine and placebo group, the exact test of fisher was performed . the results are presented separately for the cattery cats and the population of the young pet cats. the side-effects reported after the vaccination in the cattery cats are summarized in table . during the - months of observation, cats of the vaccine group and of the placebo group died due to various causes. five cats of the vaccine group and six cats of the placebo group died due to non fip-related causes. all cases, except one cat of the vaccine group which died months after the vaccination with liver problems and two cats of the placebo group which died and months after vaccination due to an accident and joint problems in a -year-old cat, respectively, were submitted to necropsy and fip was excluded. fip cases occurred in six catteries. the characteristics of all cattery cats which died of fip are summarized in table . some of these cats, though clinically healthy, showed changes in blood parameters at the time of vaccination. to our knowledge, the safety of the vaccine in breeding cats has not been investigated so far neither under experimental nor under field conditions. therefore, all data collected from queens which had kittens after the vaccination are summarized in table . no differences were found between the parameters evaluated. with respect to the laboratory parameters no differences were found between those in the vaccine group and the placebo group at the different time points (haematology, clinical chemistry, cd +/cd +lymphocytes). however, in both the vaccine and placebo groups, changes in some of the laboratory parameters were observed at the different time points. both groups showed a decrease in albumin in weeks and compared to week and an increase in plasmaprotein in week compared to weeks and (pco. ) (data not shown). at the beginning of this trial, all cattery cats had tested negative for felv and fiv-antibodies, but . % and . % of the cats in the vaccine and placebo group showed fcov antibody titres of or higher. the frequency of the fcov titres in cats of the vaccine and placebo group at different time points after vaccination (weeks , and ) is shown in figure . there was no statistically significant difference in the distribution of the fcov antibody titre in the vaccine and. placebo group at the different time point, but the vaccine group as well as the placebo group showed a transient increase of titres in week compared to week (pco. ) followed by a decrease in week compared to week (pco. ). retrospectively, plasma samples collected from cats at the time of first vaccination, which were stored frozen, were submitted for rt-pcr for fcov (table ) . of plasma samples tested, three were positive. the side-effects reported in the population of the young pet cats are summarized in table . the observation period in this population was between and months. the health condition of the cats at the end of the observation period is summarized in table . thirteen cats of the vaccine group and cats of the placebo group died from fip. all, except one in each group, were confirmed by necropsy (table ) . two cats in the vaccine group were already ill at the time of (table ). of samples tested, were found positive. in one cat shelter with high fip incidence, cats were vaccinated (placebo cats, vaccine cats), of which cats developed fip (placebo , vaccine ). the frequency and distribution of antibody titres to fcov at the time of first vaccination is presented in figure . more than % of these clinically healthy young cats had already been exposed to fcov in the first year of life. the distribution was identical in the vaccine and placebo group. domestic shorthair cats showed statistically significantly lower fcov antibody titres than pure-bred cats of the same age (p<o.ool) (figure ) . cats of the placebo group, which showed a titre of or higher, had a significantly higher risk for developing fip in the next months ( . %) than cats with a titre of or lower ( . %) (pzo. ) (figure ) . though seven cats of the vaccine group and six of the placebo group were felv positive at the time of first vaccination, three more cats of each group tested positive during the observation period. one of these felvpositive cats of the vaccine group and three of the placebo group died due to fip which was confirmed by necropsy. the aim of this study was to evaluate the efficacy and safety of a modified live virus vaccine in a double-blind study under field conditions in two cat populations with higher risk for fip. not all cats infected with fcov develop lethal fip and the incidence of fip in a cat population can hardly be predicted'. in this study, the placebo and vaccine group were indistinguishable regarding distribution of age, sex and living conditions and no statistically significant difference in laboratory parameters (haematology, clinical chemistry, serology, cd +/cd +-lymphocyte subsets) were observed at the time of first vaccination. therefore, cats in the vaccine and placebo group had about the same probability to become infected with fcov and to develop fip, and differences in the frequency of fip cases or changes in laboratory parameters must be attributed to the vaccine. the vaccinated cats did not show an accelerated form of fip (tables and , figure ). one cattery cat aged . months was euthanized days after vaccination, but this cat, though clinically inconspicuous, showed growth retardation compared to litter mates, anaemia (pcv: %), increase of total serum protein and viremia with fcov at the time of first vaccination. two young pet cats were euthanized and days after vaccination. cat , which died days after vaccination had shown chronic nasal discharge and anorexia and had been treated with antibiotics and corticosteroids the last weeks before vaccination. the fcov antibody titre was high ( ) at the time of vaccination and in retrospect this cats was positive for fcov in the rt-pcr. cat , which died days after vaccination had also been anorectic for some time and had been treated by the veterinarian. this cat too had a high titre at the time of vaccination ( ) and was pcr positive. these two cats were vaccinated by mistake and should not have entered the study in the first place. they remained in the study for the sake of completeness and because our study represent the real situation in the field where cats with obvious clinical signs may be vaccinated by mistake. it can be argued that in these three cats vaccine induced acceleration of fip may have happened. however, if the incidence of fip cases during the first days after vaccination were compared in vaccinated vs placebo cats, there is absolutely no difference in that cats in the vaccine group and cats in the placebo group died during this time. in the population of the cattery cats where prevalence of fcov antibodies was especially high ( %) the average time of death after vaccination was . weeks in the vaccine group and weeks in the placebo group. from this, the lack of side-effects and effect on fertility it was concluded that the vaccine is safe. as expected, most of the cattery cats showed antibodies to fcov, indicating prior infection. not only the vaccine group, but also the placebo group showed an increase of titres in week compared to week and a decrease in week compared to week (figure ). increase of antibodies could be explained by vaccination in the vaccine group, but not in the placebo group. as animals of the vaccine and placebo groups were separated for h after the vaccination, spreading of the vaccine virus to cats of the placebo group seems unlikely. during the weeks between vaccination and the collection of a second blood sample, antibody titres increased in cats of catteries, where fip cases occurred. a similar rise of antibody titres was observed in cattery cats where no fip cases were seen during this time period. it is well known, that stress can influence the immune system. therefore, we speculate that the stress caused by the handling during vaccination and the collection of blood samples led to a weakening of the cm and to a reactivation of a persistent fcov infection and an increase of the antibody titres. surprisingly % of the clinically healthy pet cats younger than months showed antibodies to fcov (figure ) . pure-bred cats showed higher titres and were more frequently seropositive for fcov than domestic cats (figure ) , which is probably due to different living conditions. pure-bred cats are normally raised in multiple-cat-households with close contact of the cats to each other, whereas domestic cats are often kept in single-cat-households or in small groups. when vaccination was performed according to the recommendations of the manufacturer, the vaccine showed no overall efficacy in these cattery cats. in view of the fact that in all catteries fip cases had occurred during the last months prior this study, it has to be concluded that some of the cattery cats, which later died of fip during the observation period, had already been vaccine volume number field trial of an fip vaccine: d. fehr et al. infected with virulent fcov and were vaccinated during the incubation period. this conclusion is also supported by the facts that all cats showed antibodies to fcov, that some cats showed changes in blood parameters consistent with fip (anaemia, low serum albumin, high serum protein, high bilirubin), and that retrospectively some cats were rt-pcr positive for fcov at the time of first vaccination (table ) . vaccination itself did not inauence laboratory parameters such as haematology, clinical chemistry or cd +/cd +-lymphocyte subsets (data not shown). in the population of the young pet cats no reduction of fip cases in the vaccinated cats was observed in the first days after infection ( figure ). however, after this time point until the end of the observation period, the vaccinated cats showed significantly less fip cases in one cat shelter the incidence of fip was % in the vaccine group (six out of cats) and % in the placebo group (nine out of cats). it can be concluded that the exposure to fcov was extremely high for these cats. as these animals were vaccinated after they had already been housed in the shelter for some time, it can be concluded that most of the vaccine failures in this population were due to previous infection with fcov. if cats with high fcov antibody titres at the time of first vaccination ( or higher) were excluded and only cats with titres of or lower at the time of first vaccination were examined in our study, vaccination significantly decreased the incidence of fip (p=o.o ) ( figure ). this observation suggests that low antibody titres are related with some degree of immunity, probably through a strong cell-mediated immune response via the thl pathway , and presumably with a lower virus load compared to cats with titres of or higher. this interpretation would also be in agreement with the finding that high titres (in the placebo group) are associated with a higher risk for the development of fip (figure ) . it can be concluded that vaccination of cats, which were already infected with fcov, cannot prevent or alter the course of the disease. however, vaccination of cats with no or low antibody titres seems to be efficient. these findings confirm the results of a placebocontrolled trial in which seronegative animals were vaccinated, before they were entered into a cat shelter with fip problems . to increase the efficacy of the vaccine changes in husbandry and management conditions may be initiated that lead to a decrease in coronaviral load in the kittens ,". under experimental conditions, vaccination of fcov naive cats was also found to reduce the incidence and the severity of infections with the low virulent viruses called fecv . since every fcov infection has to be considered a potential risk for fip ,", reducing these infections by vaccination may also help to reduce the fip incidence. cats of the placebo group with a titre of showed a significantly higher risk for developing fip in the following months ( . %), than cats with lower titres ( . %) (pzo. , figure ). this finding and the observation that many of these cats were pcr positive at the time of first vaccination suggest that they were already infected with fcov and were vaccinated during the incubation period. this would explain why vaccination showed a low efficacy in the young pet cats. however, it is difficult to apply the classic term of an incubation period to fip. according to pedersen and co-workers more virulent mutants of fcov capable of infecting macrophages can emerge spontaneously any time during an fcov infection . as mutations in viruses occur randomly, it can be concluded that a high virus load increases the probability that such mutants arise. a functioning immune system can control the virus load at a low level. therefore, all factors which suppress the immune response, may increase the virus load and the genesis of virulent mutants. thus, a cat can develop fip many months or even years after an fcov infection. in conclusion, the use of a live modified vaccine against fip was found to be safe in high risk populations under field conditions. efficacy was clearly shown in cats with low fcov antibody titre, but overall was rather low in high risk populations. to optimize its efficacy, changes in management and husbandry should help to prevent fcov exposure prior to vaccination. our laboratory for their help as well as to the cat breeders, veterinarians and cat owners that participated in this trial. cornell vet. , , scott, f.w., corapi, w.v. and olsen, c.w. evaluation of the safety and efficacy of primucell-fip vaccine. feline hlth top. , , fehr, d., holznagel, e. and bolla, s. et a/. evaluation of the safety and efficacy of a modified live fipv vaccine under field conditions. feline pratt. , , pedersen, n.c. feline infectious peritonitis and feline enteric coronavirus infections. part i, feline enteric coronavirus. feline pratt. , , the biology and pathogenesis of coronaviruses antigenic homology among coronaviruses related to transmissible gastroenteritis virus das feline immunschwlchevirus in der schweiz: klinik und epidemiologie im vergleich mit dem leukbmie-und dem coronavirus a study of naturally 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hypersensitivity skin response associated with feline infectious peritonitis in two cats evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity immunologic phenomena in the effusive form of feline infectious peritonitis early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive fipv vaccine monoclonal antibodies to three epitopic regions of feline leukemia virus p and their use in enzyme-linked immunosorbent assay of ~ felines t-lymphotropes lentivirus (ftlv): experimentelle lnfektion und vorkommen in einigen laendern europas. kleintierpraxis specificity assessment of feline t-lymphotropic lentivirus serology detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr two types of murine helper t cell clones. i. definition according to profiles of lymphokine activities and secreted proteins thl and th cells: different patterns of lymphokine secretion lead to different functional properties vaccination against naturally occurring fip in a single large cat shelter the potential use of a modified live fipv vaccine to prevent experimental fecv infection experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd the study was supported by a grant from pfizer animal health inc. the authors thank drs alasdair wiseman and serge martinod from pfizer animal health inc. for their assistance. we are indebted to the technicians of key: cord- - b mt a authors: garcía, leidy y.; cerda, arcadio a. title: contingent assessment of the covid- vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b mt a the covid- pandemic has not only had a negative impact on people’s health and life behavior, but also on economies around the world. at the same time, laboratories and institutions are working hard to obtain a covid- vaccine, which we hope will be available soon. however, there has been no assessment of whether an individual and society value ​​a vaccine monetarily, and what factors determine this value. therefore, the objective of this research was to estimate the individual’s willingness to pay (wtp) for a hypothetical covid- vaccine and, at the same time, find the main factors that determine this valuation. for this, we used the contingent valuation approach, in its single and double-bounded dichotomous choice format, which was based on a hypothetical market for a vaccine. the sample used was obtained through an online survey of n = individuals from chile. the main results showed that the wtp depends on the preexistence of chronic disease ([formula: see text]), knowledge of covid- ([formula: see text]), being sick with covid- ([formula: see text]), perception of government performance ([formula: see text]), employment status ([formula: see text]), income ([formula: see text]), health care ([formula: see text]), adaptation to quarantine with children at home ([formula: see text] and whether the person has recovered from covid- ([formula: see text]. according to our discrete choice model in double-bounded dichotomous format, it was concluded that the individuals’ wtp is us$ . (ci: . - . ; p < . ). this implies a social valuation of approximately us$ , million, corresponding to . % of the gnp per capita. format, which was based on a hypothetical market for a vaccine. the sample used was obtained through an online survey of n = individuals from chile. the keywords: covid- ; sars-cov- ; vaccine; acceptance; health economics; contingent valuation the severe acute respiratory syndrome coronavirus (sars-cov- ) as a pandemic coronavirus disease (covid- ) has had a negative impact on people's health and life and on economies around the world. for this reason, laboratories and institutions are working hard to obtain a covid- vaccine, which should be available in the future [ ] . such a vaccine is important in reducing mortality and the health costs of treating the disease. the vaccine is expected to be available free of charge to at least the poorest people with financing from the governments of each country, while the richest people could voluntarily seek vaccines in private clinics. however, the question arises of how much society and the individual value this vaccine. therefore, considering the adverse effects and high costs of the global pandemic generated by this syndrome, the aim of this research is to provide more information about the individual and social assessment of the vaccine. this assessment is important because it would yield useful information for the implementation of public policies aimed at improving health services. specifically, in terms of public health, governments should be designing potential vaccination campaigns against covid- ; however, due to logistical aspects and costs, in a first stage vaccination could target high-risk, socioeconomically vulnerable individuals , while other groups could paid for their vaccines. additionally, the individual and social assessment of the vaccine that we conduct in this paper, would help corporate research and development (r&d) laboratories to have an approximation of the expected benefits if they manage to develop the vaccine. furthermore, the acceptance rate of payment for a possible vaccine illustrates people's willingness to be vaccinated, so our findings would provide elements for discussion about the anti-vaccine movement. for this, it is important to understand the factors or variables that affect consumer demand and the decision to pay for a vaccine. this is addressed in this article through the estimation of a probabilistic model of the willingness to pay (wtp) for the vaccine. therefore, the objective of this research is to estimate an individual's wtp for a covid- vaccine and, at the same time, find the main factors that affect this valuation. the method used to estimate the wtp is the contingent valuation approach (cva), in its single and double-bounded dichotomous choice format. the double-bounded dichotomous choice format presents greater statistical efficiency by better estimating the variance and allows obtaining more precise confidence intervals for the mean of the wtp [ ] . cva is widely used in medical and health literature, which is based on a hypothetical market for a vaccine. specifically, wtp has been estimated for vaccines against diseases such as ebola [ ] , hepatitis b [ ] , chikungunya fever [ ] , dengue [ ] and diseases caused by meningococcus b [ ] and human papillomavirus [ , ] . there are no studies yet for covid- . the importance of the wtp for the vaccine is related to the model of provision and reliability of medical care. this study considers the chilean case, where individuals assume a significant part of the cost of preventive health care, including vaccines. specifically, chile has a mixed health system made up of public health insurance with groups according to health care coverage (ranging from % to %) and a private system where members pay according to the contracted plan and the type of hospital or clinic they wish to access. all workers must contribute to the health system with the equivalent of % of their taxable income and can choose to pay it to the public or private system. the indigent have free care in the public system. regarding vaccination, there is currently a national vaccination plan in chile that covers some vaccines for high-risk groups, while the rest of the population pays % of them. in the future, this could be replicated for covid- , due to the high costs that mass vaccination plans usually entail. the information was collected through a self-applied online questionnaire. we used a mixed sampling process (snowball and convenience sampling), but under an active recruitment system that allows access according to the demographic characteristics required to search for population representativeness. the target population were people years of age or older, with a medium and higher income level, who according to their health insurance would eventually have to finance the prevention costs of covid- , both in the public and private health systems (this target group corresponds to . % of the chilean population). people with lower incomes were not considered because they receive state aid. the survey was answered by individuals between april and may , . it is worth mentioning that chile is a high-income country according to the world bank [ ] because the it has a gross national income (gni) per capita of us$ , , where . % of the population has internet access [ ] and % uses social networks [ ] . the theory indicates that the wtp depends on individual preferences, income, attitude and perception towards the vaccine (as "good"), and the sociodemographic characteristics of the individual and their family [ ] . therefore, the questionnaire was structured in three sections that allowed capturing these aspects. in the first section, we asked about perception and individual context, previous chronic diseases, self-perception of the risk of contagion, and the general context of the pandemic, among others ( items). in the second section, the potential attributes of the vaccine and the context of contagion risk were presented; that is, we described the contingent market and asked about the wtp and the protest responses of individuals who are not willing to pay due to economic or moral reasons ( items). in the third section, the respondents were sociodemographically characterized according to age, gender, educational level, income, household composition, employment status, and health system ( items). the statistical validation of the instrument was performed with the traditional reliability indicators of the qualitative scales used. the cronbach's alpha indicator was . , indicating that there was internal consistency of the items of the perception scale (qualitative). the wtp measures the change in well-being as a result of the hypothetical acquisition of the covid- vaccine, and is expressed mathematically as follows: , where is a matrix of variables or =´+ characteristics of the individual observable under the hypothetical scenario with vaccine, is composed of the covariates in the scenario without vaccine (current scenario), such that: ; is the vector of = parameters that indicates the dependence of the wtp on the exogenous variables (x); and is the difference of the random components in both scenarios: which has a normal distribution, we used the relationship between wtp and its determining variables (x) to predict the probability of payment for the vaccine. this was obtained by comparing a given initial value with others that were above or below. these values were defined by the payment vector, such that: where and are the upper ( ) and lower ( ) limits of the wtp, and epsilon ( ) is the threshold of change between these that define the range of the wtp. it should be noted that wtp values were obtained from an open-format pre-survey of individuals. with these values, the distribution model of the payment ranges with equal selection areas was applied, assuming a normal distribution [ ] . this is an iterative technique that allowed us to find the minimum mean square error of the design of the payment vector, which in our case was limited to four initial values. with these values, the upper and lower payment vector of each of them were obtained at the same time, as the average of the contiguous values, which are presented in table and are used in equation ( ). the estimation of the wtp was made through a probabilistic model in which the dependent variable ( ) is a dummy that takes the value of if the individual is willing to pay and otherwise, depending on t he value of the assigned payment vector and the covariates that allow controlling for factors that may affect the wtp. thus, the probability of paying is: , where is a ( finite category selected by the individual under the hypothetical scenario, is a specific threshold for that category, is the probability of paying, and represents the standard normal density function, that is, we (.) defined a probit model. the estimations were made with the maximum likelihood method under the assumption that the errors distribute normally. through the analysis of the marginal effects we could identify the covariates or predictors of wtp for the covid- vaccine, which are determined as: these are the derivatives of the probability of paying a fixed value, given a change in a continuous explanatory variable ( ). subsequently, with the estimation of the vector ( ), the average value of the wtp per individual was obtained. the previous dichotomous method was applied to estimate two types of different formats: simple and double [ , ] . the simple format only considered the first response of the individual on whether ("yes" or "no") a given amount of money is paid that represents the value of the good. with this format, lower rejection rates are obtained, and the possibilities of guessing the response, the starting point bias and the induction of responses are reduced. however, this format has the disadvantage that it is a discrete indicator of the actual wtp and that the selection of the functional form can affect the results [ , ] . the double format solves this problem as the individuals are first asked whether they would pay a given amount, and according to the answer, they are offered a lower or higher value alternative. thus, the wtp is found in four types of possible intervals ("yes-yes", "yes-no", "no-no", and "no-yes"). with (table a ) . regarding the individual circumstances and perceptions of the covid- pandemic, most individuals considered that they were well informed about the disease caused by sars-cov- ( . %), most of them would be willing to pay for a vaccine ( . %), they believed that they will get sick ( . %), and only . % have or have had covid- . most individuals were quarantined at home ( . %), although most would prefer to work outside the home ( . %), which could be explained by the fact that they believed that it is "impossible to work with children" ( . %). regarding the context (table a ) the estimates of the models of discrete choice were made with two different groups of covariates. model contained the most relevant explanatory variables to predict the wtp for the covid- vaccine and model included the same variables as model plus the characteristics of the individual, which in conventional literature tend to be significant [ , , ] . this was done to avoid the statistical problem of omitted relevant variables [ ] . considering the bid vector estimates presented in table valor-p (pr > chi ) . . standard errors in parentheses = + p< . , * p< . , ** p< . , *** p< . . two observations from the sample (n= ) were dropped in the estimation process and were not considered because they were rejection responses. on the other hand, the variables that negatively influenced the wtp for the covid- vaccine were health care ( ), inability to work from home with children and △ - , %, ≤ . ( △ - , %, ≤ . ) covid- recovery . the first variable indicated that having a private health system ( △ - %, ≤ . ) that costs health expenses reduces the wtp for prevention measures by . % ( ). the second ≤ . variable showed that the negative perception of working from home with children, due to preventive or mandatory quarantine, could make the person less exposed to contagion; therefore, the wtp for a vaccine will be less ( . %, ). finally, the third variable indicated that those who have had covid- and △ -≤ . recovered, believe they have obtained greater immunity to the disease and that their risk of dying or worsening is less, so they would be less willing to pay for a vaccine ( %, ). it should be noted that both models have a good statistical fit. the goodness-of-fit, measured as the ability of the estimates to adequately predict the observed data, was high, as the probability predicted by the models was more than % (table ). the chi test indicated that the variables were significant together, that is, it allowed us to reject the null hypothesis that the regression parameters are zero, with a confidence level of % ( . = . ) of the sample, individuals ( . %) indicated that they were not willing to pay for a covid- vaccine (table a ). the self-declared reasons why they would not pay are presented in fig. . this shows that the main reasons for not paying are because they believe that the government should finance the cost of the vaccine ( . %) or they do not have the resources available to do so ( . %). the latter are individuals who may have a positive evaluation of the vaccine, but their budget constraint does not allow them to pay for it. in general, people would be willing to pay for a covid- vaccine, as . % of the individuals answered "yes" to the initial question of whether they would pay. however, this question is ambiguous because there could be people in that group who would only pay a penny. to solve this, the questionnaire had a double dichotomous design, which randomly presents values of the payment vector and reveals the true preferences of the individual. of the sample, % individuals answered that they would pay the initial value and that they would also pay a second value, higher than the first; whereas % answered "yes" to the first value, but "no" to the second higher value. however, the double format model is considered more appropriate in the technique because it is more efficient in estimating the variance of the parameter, therefore, its confidence interval is also more efficient [ , ] . this was evidenced through the difference between the upper and lower confidence interval values, which was smaller for the double dichotomous model ( . %, . , table ). additionally, when extrapolating these results to the population of legal age (over years), approximately million, and discounting the percentage of responses rejecting payment for the vaccine ( . %), it leaves a population of slightly more than million people. this value was multiplied by the estimated wtp that reached $ . per individual, which gives us the social assessment of us$ , million for the covid- vaccine, which represents . % and . % of chile's gross domestic product (gdp) and gdp per capita, respectively. the individual and social assessment of the covid- vaccine is key to defining prevention strategies, and allows visualizing the perceived benefit of the investment that research laboratories could have if they develop a vaccine, which is important considering the current global r&d activity focused on creating one [ ] . this study shows that there is a high individual wtp, with an average of us$ . under the most accurate estimation technique and with a reliability level of . %. these results are similar in magnitude to those found by [ ] for the meningococcal b vaccine (us$ . = au ) in australia. however, the values are higher than those found in other studies conducted with other serious diseases with risk of death [ , , ] . specifically, the evaluation of the covid- vaccine was far superior to that of the hepatitis b vaccine in malaysia (us$ [ ] ), zika in brazil (us$ . [ ] ), dengue in malaysia (us$ . [ ] ) and ebola in indonesia (us$ . [ ] ). this are upper/middle-income economies, except for malaysia, which is a low/middle-income country [ ] -thus, the high economic valuation of the covid- vaccine could be explained because sars-cov- has had a higher contagion rate, has spread more rapidly and has affected all countries in the world [ ] . the country's high per capita income is also a contributing factor. in fact, we found a relatively high vaccine acceptance rate ( . %), considering that other studies have found acceptance ranges that go from . % to . % [ , , ] . on the one hand, there could be a tendency in individuals to accept a payment or to say "yes" when they have less-formed preferences, which according to [ ] tends to occur with health-related goods and services. this could also explain the high approval rate with the payment vector thresholds presented. on the other hand, it could also be that individuals foresee a high risk of getting sick ( . %) and therefore would be more willing to pay, which has already been pointed out by other studies [ ] . this, especially considering that covid- could be perceived as "catastrophic" due to the health costs and the increased risk of death of vulnerable people (older adults and people with pre-existing chronic diseases), according to the results of [ , ] ; in addition to the high social and economic cost of this disease [ ] . furthermore, we precisely demonstrated that one of the main variables that determine the wtp is the pre-existence of chronic diseases, the level of knowledge of covid- and having covid- . thus, both the high approval rate for the vaccine ( . %) and the belief that one will eventually get sick ( . %) demonstrate a positive intention of individuals towards it, even without knowing the details of its real effects on health. this is an important argument against the so-called "vacillation" which, according to [ ] , becomes a rejection of vaccination. in other words, our results indicate that there are fewer people against vaccination in the case of covid- . additionally, we found that perception of government performance in managing the pandemic also influences the wtp. this variable had not been considered in previous studies related to vaccine evaluation, such as those carried out by [ , , ] . this is an important variable because the information provided by the government on the negative effects of covid- and the strategies applied to mitigate the pandemic (such as restriction on mobility or quarantine), are key to educating the population and affect the wtp for the vaccine. in fact, there are studies that indicate that education, information and communication can improve the willingness to vaccinate against respiratory viruses [ ] . therefore, it is important that the government executes credible measures, informs and educates clearly about the impact that sars-cov- contagion generates. it should be noted that the results of this study would be important to consider if the vaccine were to be introduced in a different country in the future, as they provide information to target available economic resources and many countries have budgetary constraints to deal with the sars-cov- health emergency. the results are applicable to countries that have mixed health systems (public and private provision) and that are based on copayments, such as some countries in north america and europe. in addition, considering that income is one of the important factors for the wtp for a vaccine, it is proposed that the government or the authorities in charge of public health carry out free covid- vaccination campaigns, especially for people with lower incomes, leaving private provision to households with higher incomes. this last strategy is endorsed by the literature [ ] . in this study, we found a high social and individual valuation for a covid- vaccine. the average value to pay per individual was us$ . , considering a discrete choice model in double dichotomous format, which implied a social valuation of approximately us$ , million. the variables that positively impacted the wtp were the pre-existence of chronic diseases, knowledge of covid- , being sick with covid- , perception of government performance, employment status, and income. the variables that negatively affected the wtp were belonging to a private health system, non-adaptation to working from home with children (due to quarantine) and having recovered from covid- . these latter variables could be used to define strategies for public health policy intervention to confront the covid- pandemic. additionally, if the vaccine will become a "public good" globally, there would still be costs associated with production and distribution, and the laboratory that develops it should be financially compensated. therefore, the wtp results from this study can serve as a compensation benchmark for vaccine developers. conceptualization, ac; methodology, formal analysis investigation, resources, writing-original draft 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critical review knowledge, attitudes, and behaviors (kab) of influenza vaccination in china: a cross-sectional study in scope and magnitude of private sector financing and provision of immunization in benin, malawi and georgia. vaccine key: cord- - t uxmj authors: lamphear, barry j.; jilka, joseph m.; kesl, lyle; welter, mark; howard, john a.; streatfield, stephen j. title: a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: t uxmj recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. a subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals’ serum, colostrum and milk. thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization. oral administration of vaccines has the potential to greatly cut the cost and increase the safety of vaccine delivery. in the case of human vaccines, avoiding the use of needles reduces equipment costs, removes the requirement for trained medical personnel to supervise delivery and eliminates safety concerns associated with needle disposal. the economic benefits of oral over parenteral delivery are also apparent with animal vaccines, where equipment and labor costs can be substantially reduced. also, in the cases of farmed animals destined for meat markets, carcass quality may be compromised by repeated injections and oral vaccines overcome this concern. subunit vaccines are generally considered to have a low safety risk since they are well defined and do not contain attenuated or inactivated pathogens with the potential for adverse affects. thus, oral delivery of subunit vaccines is a particularly attractive option for safe, inexpensive vaccination programs. however, oral delivery generally requires very high levels of specified antigens to be administered in order to attain efficacy. this is presumably because of degradation of the selected proteins in the digestive tract and only a small proportion of the relatively intact molecules being presented to the immune system in a manner favoring an immunogenic response. several recombinant systems are being utilized to generate large amounts of subunit vaccines, including, for example, the use of yeast to produce the surface protein of hepatitis b. however, despite the development of such recombinant vaccines, the economic production of large quantities of desired antigens is severely limited. recently, certain recombinant plant expression systems have begun to offer a means to produce very large quantities of proteins in a sufficiently concentrated form to make oral delivery feasible for a wider array of antigens. levels of expression have been achieved with various antigens in plants that allow practical oral delivery of a sufficient dose to elicit desired immune responses in humans and target animals (reviewed in [ ] ). the approaches followed to achieve these high levels of expression include the use of tissue-specific promoters to direct expression to tissues well suited to the stable storage of proteins [ ] and the targeting of the expressed proteins to sub-cellular locations conducive to their accumulation [ ] . many plant-based oral vaccine candidates have been tested in animal studies and several responses have been noted, including the generation of serum and mucosal antibodies (reviewed in [ ] ) and raised cytokine levels [ ] . protective efficacy has also been recorded with some of these vaccine candidates in model species trials (reviewed in [ ] ). a few plant-based vaccine candidates have advanced into early phase human clinical trials or target animal trials. among human vaccines, these include those directed against travelers' diarrhea and norwalk virus delivered in potato tubers [ , ] , against hepatitis b virus delivered in lettuce leaves [ ] and against rabies virus delivered in spinach leaves, themselves infected with a recombinant plant virus [ ] . immune responses were observed during these trials, and although there were some reports of nausea, presumably resulting from the administration of up to g of unprocessed, unpalatable plant material, the vaccine candidates were generally well tolerated. in the case of farmed animals, a corn-based vaccine directed against transmissible gastroenteritis virus (tgev) can induce protective immunity in piglets [ , ] . a key issue in producing plant-based oral vaccines is the selection of plant material that both expresses high levels of a chosen subunit vaccine candidate and is also suitable for extensive storage and oral delivery. the chosen plant material must also be ready for direct administration or must be suitable for inexpensive processing into an appropriate form for oral delivery to the target species. much of the work to date on plant expression systems has been conducted using tobacco leaf tissue (discussed in [ ] ). however, tobacco leaves are inedible, and therefore protein extraction is required prior to delivery. several edible options have also been pursued, including the tubers and leaves of certain vegetable crops such as potato and lettuce, respectively [ ] [ ] [ ] . some fruits, such as bananas, are also being considered. however, perishable items are not practical for extended storage and expression levels can vary considerably between, for example, potato tubers taken from a single harvest [ , ] . cereal seeds are particularly well-suited systems for the oral delivery of subunit vaccines. they have low water contents and naturally store proteins over long periods of time without degradation. corn (zea mays) is an especially attractive option because of its intensively studied genetics and the availability of established transformation procedures. several vaccine candidate antigens have been expressed at high levels in corn seed and the proteins are stable when stored in this tissue for periods of at least a year and probably for much longer, obviating the requirement for a cold chain during distribution and storage [ ] . furthermore, the antigen concentration is uniform across a corn grain harvest, facilitating even dosing [ ] . a wide range of processing alternatives have been developed by the food and feed industries to convert corn grain into readily edible forms and pilot-scale processes have been developed that ensure antigens are not degraded during processing [ ] . in the case of farmed animals, such processing is unnecessary since the livestock can consume corn grain directly. here, we focus on the development of a corn seed-based subunit vaccine directed against swine tgev. this virus causes a contagious enteric disease that is particularly severe for piglets. it results in severe diarrhea and vomiting and is associated with high mortality rates among piglets under weeks of age [ ] . tgev is a coronavirus and has a large surface glycoprotein referred to as the spike (s) protein displayed on its surface [ ] . the tgev vaccine candidate assessed here comprises the s protein expressed in corn seed. feeding studies have been conducted with this vaccine candidate delivered orally to piglets. the animals showed a priming of their immune system and were protected against infection [ , ] . here, we extend these swine feeding studies to assess the potential for this oral tgev vaccine candidate to boost immunogenic responses in gilts (young sows) previously sensitized with a commercially available modified live viral vaccine. we focus particularly on the level of antibodies in the colostrum and milk as a guide to whether immunity could be acquired passively by piglets through suckling. the subunit vaccine candidate comprised milled yellow grain corn expressing the s protein of tgev. a single dose corresponded to kg of corn containing mg of the antigen. the placebo for the study comprised kg of non-transformed milled yellow grain corn. a commercially available modified live tgev vaccine (intervet inc., millsboro, de) with a titer of . tcid (tissue culture infectious doses)/ ml was used to prime all animals and to provide booster treatments to a positive control group. this vaccine was administered according to label directions. a total of specific pathogen free gilts of suitable age for breeding were included in the study. they were taken from a low disease incidence herd and were seronegative for tgev at the outset of the study. the gilts were randomized into six treatment groups with from five to eight animals in each group (table ) . duplicate ear tags were used for identification purposes. all gilts in all groups were orally administered the modified live tgev vaccine on the day of breeding ( days before farrowing) and also days before farrowing. they were then administered the tgev modified live vaccine by intramuscular injection days before farrowing. the subsequent immunization regimen for each group is outlined in table . all animals were fasted overnight prior to oral administrations of the corn-based vaccine to test groups. standard lactation gestation rations were administered to all gilts throughout the study. during the period comprising the three administrations of modified live virus to all gilts, the groups were housed together and allowed pen-to-pen contact. prior to days before farrowing gilts were separated into their separate groups and during subsequent vaccine administrations, animals were individually isolated. blood samples were collected from gilts on the day of breeding ( days prior to farrowing), and days prior to farrowing and on the day of farrowing. blood was allowed to clot and was sedimented by centrifugation, so allowing the serum to be collected. tgev neutralizing titers were determined by incubating a specific dilution of tgev with multiple serum dilutions for h at • c. these mixtures were then inoculated onto a swine testicular cell line and the capacity of the serum to interfere with the viral infection was assessed after days. sample titers were calculated using a spearman-karber % endpoint table. colostrum samples of at least ml were collected on the day of farrowing and at least ml milk samples were collected , , and days after farrowing. the samples were sedimented by centrifugation, and the central region was collected. tgev neutralizing titers were determined as with serum samples. for all tgev neutralization data, geometric mean titers were compared and differences in excess of four-fold were considered to be significant. the generation of transgenic corn containing the s protein of tgev has been previously described [ , ] . in brief, sequence encoding the s protein was synthesized with optimal codon usage for expression in z. mays. an n-terminal cell surface targeting signal was included to direct accumulation of the protein to the cell wall. dna encoding the s protein was introduced into immature zygotic z. mays embryos by agrobacterium tumefaciens mediated transformation and selection was imposed for transgenic callus. transgenic plants with sequence encoding the s protein integrated into the nucleus were regenerated, and those expressing the highest levels of the s protein were taken through a plant-breeding scheme to increase and stabilize expression levels. this culminated in a large-scale grain harvest in which the s protein was present at mg kg − , as determined using a sandwich enzyme linked immunosorbent assay [ ] . at this concentration a practical antigen dose of - mg can be delivered in an amount of corn material easily consumed at a single feeding. all animals were seronegative for tgev at the time of breeding. subsequent serum neutralization titers are summarized for each study group in fig. . the modified live virus vaccine, which was administered twice orally and then once intramuscularly resulted in gilts in all groups having similar tgev serum neutralizing titers days prior to farrowing. analysis of serum samples taken from gilts at days prior to farrowing showed that animals that had received the oral corn-based tgev vaccine (groups a-c) had notably higher serum neutralization titers than those that had received no material at this stage (groups d and f). the difference between the test and control groups was significant in all cases except for that of a single administration of corn-based vaccine (day − to group c) over group d. animals that had received the modified live virus vaccine as a single intramuscular boost (day − to group e) responded to an almost identical level to those that had received a single oral administration of the corn-based vaccine (group c). although more oral administrations of the corn-based vaccine appeared to increase the neutralization titer, differences between the treatment groups (a-c) were not significant and none of the treatments induced a significantly stronger response than the intramuscular boost of modified live vaccine delivered to group e. similarly, at the time of farrowing the tgev serum neutralization titers in gilts administered the corn-based tgev vaccine as a boost (groups a-c and f) were raised over those observed with animals that had received the corn placebo (group d). this difference was significant in all but the case of gilts that had received six administrations of the corn-based vaccine (group b) compared to those that had received the placebo (group d). animals given intramuscular administrations of the modified live virus vaccine as a boost (group e) responded similarly to those that received the oral corn-based vaccine, again with two administrations of either vaccine giving almost identical results (groups c and e). gilts administered a boost of the corn-based tgev vaccine only during the second week before farrowing (group f) showed the most marked increase in the serum neutralization titer at the time of farrowing, although differences between the groups that received the corn-based vaccine (groups a-c and f) were generally not significant. interestingly, for groups that received two blocks of booster administrations (a-c and e), in no case did the second set of treatments elevate the serum neutralization titers over those observed with the first set. indeed, neutralization titers appeared to decline with the second set of administrations, although in no case was the drop statistically significant. colostrum and milk neutralization titers are summarized for each study group in figs. and , respectively. each of the groups of gilts that were orally administered the corn-based tgev vaccine as a booster (groups a-c and f) showed a greater level of neutralizing antibodies than did gilts administered two intramuscular injections of the tgev modified live virus vaccine as a booster (group e). however, these differences were not sufficient to be considered significant, and therefore all of the booster treatments with either the corn-based oral vaccine or with the modified live intramuscular vaccine are considered similarly effective. all of the booster regimens with the corn-based vaccine (groups a-c and f) resulted in significantly greater neutralizing antibody levels than those observed among animals that were administered the control corn placebo material (group d). gilts in all groups that received an oral corn-based tgev vaccine boost (groups a-c and f) showed similar levels of neutralizing antibodies in their milk, with levels trailing off steeply between and days after farrowing and continuing to decline thereafter. these levels correspond closely to those observed with the modified live tgev vaccine delivered intramuscularly (group e). with all groups that received a corn-based oral booster treatment (groups a-c and f) the neutralizing antibody titer in milk days after farrowing is considerably higher than for the group that received the control corn placebo (group d). this difference is significant in all cases except that of the group that received two blocks, each of seven consecutive days, of the corn-based vaccine (group a). by days after farrowing differences in the neutralization titers between the placebo (group d) and other groups are not significant. the corn-based tgev vaccine candidate described here shows great potential for expediting the administration of an efficacious vaccine to large herds of swine. the current standard regimen for administrating a vaccine comprises both priming and boosting stages. during the priming phase of the regimen the modified live tgev vaccine is often administered along with other swine vaccines. thus, at this point no reduction in labor costs is achieved through delivering an oral corn-based tgev vaccine separately. however, replacing subsequent injections of the modified live tgev vaccine with oral corn vaccine boosters would clearly save considerable time and effort. the orally administered corn-based tgev vaccine is effective in boosting the serum neutralizing titer response in animals previously sensitized to tgev using the modified live virus vaccine. when administered as a booster to gilts the corn-based vaccine also results in increased levels of neutralizing antibodies in the colostrum and early milk. milk antibodies of the igg class have typically disappeared within h of farrowing, so the neutralizing antibody activities observed in milk samples collected days after farrowing most likely reflect iga levels. protection against tgev amongst nursing piglets has been linked to iga levels [ ] , indicating that the corn-based tgev vaccine is inducing an immune response with the potential to confer protection. in this regard, a potato-based vaccine candidate directed against rotavirus, and assessed in a mouse feeding study, has been shown to confer passive immunity to pups when administered orally to dams [ ] . protective efficacy has previously been demonstrated with a corn-based oral vaccine directed against tgev and administered to piglets [ , ] . the neutralizing antibody levels achieved here in the colostrum and early milk of gilts extends the scope for how this vaccine candidate can be deployed. future studies with this tgev oral vaccine candidate will focus on optimizing the administration reg-imen for maximum responses and on conducting larger scale trials. these will include an assessment of whether the lactogenic immunity observed here results in protection being conferred to piglets. the results presented here successfully demonstrate a commercial application for a corn-based vaccine and indicate that there is great promise for plant-based vaccines that can be easily administered to large farmed animals by oral delivery. plant-based vaccines expression of a synthetic e. coli heat-labile enterotoxin b sub-unit (lt-b) in maize corn as a production system for human and animal vaccines a plant-based multicomponent vaccine protects mice from enteric diseases immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus expression in plants and immunogenicity of plant virus-based experimental rabies vaccine plant-based vaccines: unique advantages delivery of subunit vaccines in maize seed medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants congress symposium on molecular farming: development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli molecular biology of transmissible gastroenteritis virus immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine key: cord- - svm le authors: assink, m.d.m.; kiewiet, j.p.; rozenbaum, m.h.; van den berg, p.b.; hak, e.; buskens, e.j.; wilschut, j.c.; kroes, a.c.m.; postma, m.j. title: excess drug prescriptions during influenza and rsv seasons in the netherlands: potential implications for extended influenza vaccination date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: svm le influenza and respiratory syncytial virus (rsv) infections are responsible for considerable morbidity, mortality and health-care resource use. for the netherlands, we estimated age and risk-group specific numbers of antibiotics, otologicals and cardiovascular prescriptions per , person-years during periods with elevated activity of influenza or rsv, and compared these with peri-season rates. data were taken from the university of groningen in-house prescription database (www.iadb.nl) and virological surveillance for the period – . during influenza and rsv periods excess antibiotic prescriptions were estimated for all age groups. in the age groups – and – years, excess antibiotic prescriptions during periods with elevated rsv activity ( % and % of peri-seasonal rates) exceeded the surpluses estimated during the influenza-activity periods ( % and % of peri-seasonal rates) while for otologicals excess prescriptions were higher for influenza ( % and %) than for rsv ( % and %). among persons of years and older, notably those without medical high-risk conditions, excess prescriptions for cardiovascular medications were estimated during the influenza periods at approximately % (this was also already seen in persons aged – ). our results may have implications for influenza vaccination policies. in particular, extension of influenza vaccination to groups of non-elderly adults and young children may lower excess prescriptions during these influenza periods for all three types of drug prescriptions investigated. in many countries annual influenza vaccination has been recommended for the elderly and persons with high-risk medical conditions. in the netherlands until , the age-threshold for such vaccination was years for non-high-risk groups. in , this threshold was lowered to include all persons aged years and older [ ] . in addition in the netherlands, all high-risk groups are vaccinated, including individuals suffering from chronic conditions, respiratory diseases, cardiac diseases, diabetes mellitus, renal failure, those being immunocompromised and individuals aged less than years of age on chronic salicylates use [ ] . vaccination rates among these high-risk groups under years of age ranged from % to % in the netherlands in [ ] . persons of years and older with a medical indication showed higher vaccination coverages compared to those of the same age without such an indication, at % and % in , respectively [ ] . influenza may cause acute bronchitis and pneumonia in high-risk groups and elderly and may lead to exacerbations of underlying chronic medical conditions such as cardiovascular diseases, asthma and diabetes, potentially leading to hospitalisations and death. prevention of influenza infection by vaccination is of high importance for these groups with an increased risk for complications from influenza infection [ ] [ ] [ ] . although some countries recommend routine influenza vaccination among children aged months to years, clinical data about the impact of vaccination are limited [ , ] . yet, vaccination of all healthy children in this age group could be cost-effective or even cost-saving for some societal settings [ ] . in the netherlands, universal influenza vaccination of such children is in debate, however yet the vaccine efficacy and effectiveness for this specific age group is not considered to be sufficiently demonstrated [ ] [ ] [ ] . therefore, the dutch health council concluded in , not yet to start with influenza vaccination of these groups [ ] . comparable conclusions were made for healthy children aged years and older. although vaccination was shown to be effective for these children, influenza was considered not to cause serious morbidity or mortality in the netherlands in those groups [ ] . next to influenza virus a and b, respiratory syncytial virus (rsv) has been shown to cause similar types of complications, including complications in the respiratory tract. rsv is often recognized as a cause of morbidity and mortality among both children and adults, contributing to a major burden of illness [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . no preventive vaccine is yet marketed for rsv. in general, it is difficult to estimate the individual contributions of influenza and rsv to the aforementioned disease burdens accurately as influenza and rsv co-circulate during winter seasons. for burden of disease often the label influenza like illnesses (ili) is considered to primarily comprise disease due to influenza infections, however known to potentially comprise various other agents inclusive rsv, with the identification of the relative contributions of both viruses being strongly hampered [ ] [ ] [ ] [ ] . in particular, even further viral agents, such as adeno-, parainfluenza-, and corona-viruses may contribute to the burden of ili [ , ] . for exactly analysing the burdens of disease of influenza and rsv separately, which is the goal of our current paper, ili is therefore an inappropriate concept. therefore, we chose to analyse isolates exactly related to the respective causal agents. given the potential complications of influenza and rsv, one may expect excess health-care resource utilization during epidemics [ ] . studies estimating such excess resource utilization have previously been directed to hospital admissions and gp-visits, but there is as yet hardly any information on the association between the occurrence of influenza and rsv and the use of medications among the general population [ ] . drug-use for specific complications of influenza and rsv could certainly temporarily be elevated. in particular, prescriptions for antibiotics, otologicals and cardiovascular medication may be elevated, dispensed for otitis media, cardiac complications, respiratory illness and other pulmonary complications. to address this topic, we investigated the association between weekly reported rsv and influenza isolates in the netherlands and the weekly number of prescription drugs dispensed by dutch pharmacies, in particular those for antibiotics, otologicals and cardiovascular drugs. from the preventive point of view, high influenza-associated drug-use may justify more extended use of preventive measures, such as influenza vaccination. in particular, it may enhance the health-economic profile of extended influenza vaccination for currently yet unvaccinated groups. as such, our research may contribute to discussions as to whether, for example, young children should be vaccinated or whether the age limit of the vaccination program should be lowered further. during - , the dutch working group on clinical virology gathered data from laboratories throughout the netherlands, testing clinical specimens for respiratory viruses, including rsv and influenza a and b. the number of patients who tested positive for rsv or influenza was reported on a weekly basis. in the absence of any drastic changes in recruiting and testing of specimens the weekly time series can validly be conceived as representing the actual time trend, without requiring any corrections to be applied on the data. to exclude weekly random fluctuations a -week moving average was used for presentation ( fig. ) and for defining the specific weeks exhibiting elevated activity for influenza and rsv. two methods for defining weeks with elevated activity of influenza and rsv were applied. the first method defined a week with elevated activity as one with a moving average of more than two times the gross overall average weekly number from week in onwards to week of (week obviously being the st week in january; week generally being considered as the start of the influenza season). following jansen et al., the second method defined the weeks with elevated activity as those weeks from any period of at least consecutive weeks, with each individual week accounting for over % of the season's total number of influenzaor rsv-positive specimens [ ] . from both methods, it appeared that periods consisting of a number of subsequent weeks resulted, rather than individual weeks or short periods of, for example, weeks only. such periods would be expected -and were indeed found -to be between week and week in the next year (the non-summer season) [ , ] . the weeks that contained both influenza and rsv activity were excluded for defining the periods with elevated activity, given the difficulty to separate out the individual influences in these weeks with combined activity. as a result, only those weeks with either elevated influenza or rsv were analyzed. furthermore, a periseason was defined, containing the weeks from week up to and including week of the next year, which did not belong to the influenza or the rsv periods (seasons). the weeks from week up to and including week were labelled "summer". information on drug-use in the population was provided by a university of groningen in-house prescription database (www.iadb.nl). the database iadb.nl contains prescription, demographic and population data of , persons adherent to pharmacies in the north and east of the netherlands. for analytical purposes, the prescriptions were divided over -year age categories. the age category - years was further divided in the ages - and - , to enable analysis of infants separately. for presentation the following categorization was applied: - , - , - , - , - , - , - and +. the exact age of any person was determined every year on the st of october, close to the period in which in the netherlands the invitations for influenza vaccination for risk groups are sent out by the gps, supposedly just prior to the season with increased risk for influenza epidemics from october onwards to may. the annual total population sizes were based on estimates for the st of january by the local authorities in the places where the pharmacies are located. persons who received one of the studied drugs were divided in high-risk and low-risk groups for influenza. the respective populations belonging to both groups were estimated using prescriptions as a proxy, for those medications that are consistent with highrisk indications as specified by the dutch health council [ ] and the dutch gps (http://nhg.artsennet.nl) and that are uniquely prescribed for these indications [ ] . for example, dornase alfa, a drug prescribed for cystic fibrosis, was included to define persons with respiratory diseases. other medications for cystic fibrosis, such as acetylcysteine, are also prescribed for cough and were therefore not included. table lists these drugs specifically. for heart medication, diuretic sulfonamides were included. other diuretics used for lowering blood pressure were excluded, as high-blood pressure is not a high-risk identified condition. furthermore, only calcium antagonists with atc-code c d were included as only these are assumed to have cardiac effects. beta blockers and raas system agents were excluded as these are not exclusively prescribed for cardiac diseases, but also, for example, for high-blood pressure only. for renal diseases, medication for the treatment of hyperkalemia and hyperphosphatemia, and antianemic preparations and sulfonamides were included as these are often prescribed for dialysis or renal insufficiency. to identify immunocompromised patients, immunosuppressives were included, which can be prescribed, for example, for patients after organ transplantation. also hiv medications were included as also hiv-patients are listed as high-risk group. as mentioned, in the netherlands, influenza vaccination is recommended to those at the increased risk of complications based on specific medical conditions. to determine which specific patients should be labelled as belonging to the high-risk group, the recommendations of the dutch health council [ ] and the guidelines of the dutch gps (http://nhg.artsennet.nl, accessed th september ) for influenza and high-risk indications were used [ ] . in particular, persons were labelled belonging to the high-risk group if they had two or more prescriptions from the same group of medications included on the list, on two different dates in the year before the first of october. the latter was supposed to guarantee that the condition would be chronic in that specific year (note that a person's risk status may change from year to year). the population size of the high-risk group was subsequently determined on the st of october by counting the number of high-risk persons in iadb.nl; the rest of the population was assumed at low risk. we specifically directed our analysis to the drug groups of antibiotics, otologicals and cardiovascular drugs, as these drugs may be considered for those complications of both viral infections that have yet been published in the scientific literature [ , , [ ] [ ] [ ] . from iadb.nl, prescriptions of antibiotics (atc-code j ), otologicals (atc-code s ) and medications for the cardiovascular system (atc-code c) were selected. antibiotics are commonly prescribed for the treatment of acute otitis media (aom), in particular for young children in which aom accounts for approximately half of all antibiotics courses delivered [ ] [ ] [ ] . otologicals may also be prescribed for aom, despite that they are not recommended by the dutch college of general practitioners (nhg) [ ] . in addition, more than average numbers of antibiotics may be prescribed for elderly persons with ili during periods with elevated activity, as particularly this group may develop acute respiratory illnesses (for example, pneumonia) as a complication of the viral infection (trimethoprim and nitrofurantoin were excluded from the analysis as they are prescribed primarily for urinary tract infections) [ ] . for antibiotics and otologicals both initial and next prescriptions within the same year were considered. it is well known that influenza-related complications are more prevalent among persons with cardiovascular and other chronic diseases than in persons without such underlying conditions [ , ] . in persons of years and older with high-risk conditions an increased rate of hospitalisation for cardiac problems has been reported [ ] . some further recent studies suggest that there might also be an association between cardiovascular problems and influenza epidemics among groups without any cardiovascular history yet, such as the elderly or even among those aged below years of age [ , ] . for analysing cardiovascular medications, only first prescriptions were considered as our current interest was to investigate whether the viral infections were related to new cardiovascular disease and/or exacerbations of existing -yet untreatedbackground cardiovascular conditions, rather than identify chronic medications for cardiovascular diseases (note that chronic cardiovascular medication use was used as a criterion for assigning persons to the high-risk group). a prescription was defined as a first prescription when a person had not had a prescription for the same drug or a drug from the same subgroup in the year before that specific prescription. after the various periods of elevated activity were determined, the number of prescriptions in the weeks belonging to the influenza and rsv periods/seasons were compared to the number of prescriptions in the peri-season. for comparative purposes also results for comparing with the summer season are presented. this was done in every age group for the total population, the high-risk group and the low-risk group. incidence rates were calculated and corresponding %-confidence intervals were estimated [ ] . the incidence rate (i) was calculated as the weekly number of prescriptions per , person-years: with p i being the number of prescriptions in week i of a season (influenza, rsv, peri or summer), n the number of weeks of that specific period of interest and n the population size (per age group, for high risk or for low risk). the division by is to transfer personweek estimates into person-years (for years instead of had to be used). microsoft office excel was used for processing the data, calculations and graphics. during the study period, the average weekly number of influenza-positive and rsv-positive specimens were . and . , respectively. the first method for defining the periods of elevated activity using two times the average number of isolates per week as a lower limit ( . for influenza and . for rsv) resulted in table , showing the weeks which were labelled as belonging to the influenza, rsv, peri-and summer periods. the second method, defining the periods with elevated activity as at least two consecutive weeks in which each week accounted for over % of the season's total number of rsv-or influenza-positive specimens yielded comparable results (data not shown). hereafter, only for those results where both methods differed, the results of both methods are presented otherwise the results of only the first method are shown. obviously, periods with elevated activity and the peri-season changed from year to year, both in length (the summer season of course always ranged from week up to and including week of the next year). in some years overlap in influenza and rsv activity weeks were seen, in which case weeks were excluded. table person-years during periods of elevated activity per age group for the total population, high-risk group and low-risk group. table excess drug prescriptions for the periods with elevated activity compared to the peri-season, shown as numbers of prescriptions per , person-years (as % of peri-seasonal levels). the number of persons was insufficient for valid estimation. a result not statistically significant. b although borderline significant not shown here for the whole age-group as further -year age-group specific analyses revealed that significance was related only to a significant and clinically relevant surplus for the age category - : . ( . %) and . ( . %) for the total population and low-risk group, respectively. table presents the number of person-years for the influenza, rsv, peri-season and summer periods per age and risk group. for infants and children aged - , the high-risk group was very small with person-years in the influenza period and personyears for the rsv period, and therefore only the figures for both low-and high-risk groups taken together were used for these ages. furthermore, in all age groups the number of person-years in the high-risk group was lower than the numbers in the low-risk group. we also note, as expected, that the older age groups contained relatively more person-years in the high-risk group than the younger age groups. fig. shows the number of positive findings for rsv and influenza per week. the annual influenza and rsv epidemics are clearly seen, as is the overlap in some years. also the number of (first) prescriptions per , persons for antibiotics, otologicals and cardiovascular medication is plotted for the total population. in particular, for antibiotics a clear pattern is visible in which during influenza and rsv activity periods a peak in antibiotic use occurs. additionally, we notice that in the younger age groups amoxicillin was the mostly prescribed antibiotic, whereas from the age group - onwards other antibiotics were mostly prescribed (data not shown). the excess prescriptions per , person-years during the influenza and rsv periods are presented in table , as compared to the peri-season. we noted that both methods used for estimating the activity periods showed similar results. the number of prescriptions for antibiotics was significantly elevated during the activity periods in each age group. for the age groups - and - , excess prescriptions were highest in the rsv periods, in the older age groups surpluses were higher during the influenza periods. for influenza, the low-risk group showed higher excess prescriptions compared to the high-risk group. prescription of otologicals was significantly elevated during the periods of elevated activity, however only in the youngest age groups of infants and children aged - years. in contrast to antibiotics, excess prescriptions were higher during influenza periods than during rsv periods. for first cardiovascular medications, the prescription rate in the rsv periods was lower than in the peri-season, although not significant. during the influenza period, higher prescription rates were found for those aged years and older. when the age group of - was analyzed in -year age categories separately, also a significant difference for the age category - was seen. fig. shows the prescription rates per , person-years and confidence intervals for selected aggregated age groups for the different periods per year separately, as well as for the aggregated years. the incidence rates of antibiotics were increased during the influenza and the rsv periods in comparison with the peri-season (and the summer season). this increase was noticeable for every year. for otologicals, the incidence rate was increased but the confidence intervals were wide and therefore not every year rendered a statistically significant difference during the influenza and rsv periods. for first cardiovascular prescriptions also an increase was noticeable during influenza periods as compared to the peri-season, but again not for every year a significant difference was found. finally, we note from fig. that elevations in the influenza and rsv periods are not necessarily followed by relatively lower levels in the peri-and/or summer seasons, suggesting that surpluses detected are actual extra prescriptions that are not "neutralized" by subsequent dips in prescriptions. statistically significant excess antibiotic prescriptions during periods with elevated activity of influenza and rsv were found for both viruses in all age groups, each year investigated and irrespective of the method used for exactly defining the influenza or rsv activity periods. for otologicals during both influenza and rsv-active periods, statistically significant surpluses were found in young children only. oppositely, excess cardiovascular drug prescriptions were identified in adults and elderly in periods with elevated influenza activity. we generally found a tendency for higher percentages of surpluses in low-risk groups than in high-risk groups. for antibiotics and cardiovascular drugs, this can probably be explained by the fact that individuals belonging to a high-risk group have a higher likelihood of being vaccinated against influenza, lowering the chance of infection and secondary bacterial or cardiovascular complications. this tendency also applied to low-risk elderly as compared to highrisk elderly, despite the fact that this whole group is recommended for vaccination. some choices in our research should be noted. specifically, the lower limit for the weekly moving-averaged number of isolates for weeks to be labelled as active was chosen by two different methods. the first used a cut-off of two times the average number of influenza or rsv isolates per week. this limit was chosen to achieve continuous periods per year of weeks subsequently labelled as active, i.e. to guarantee epidemic periods rather than fluctuations. the second method was used to be in concordance with a previously performed dutch study using the weeks which accounted for % or more of the season's total number of influenza or rsv isolates [ ] . in general, both methods resulted in similar results. by using the influenza and rsv activity periods that excluded weeks of combined activity, possible major influence by the presence of the respective other virus was reduced. however, the exclusion of weeks in which both influenza and rs viruses were active was only necessary in out of the years included in this analysis. also, in methodology excluded weeks were not included in counting person-years, so the effect of excluding those weeks maybe limited. yet, possible influence of any other respiratory virus obviously remains present. however, the impact of these viruses is probably limited, as they may have long periods of marginally increased activity rather than a clear seasonal pattern [ ] . furthermore, complications are expected to be milder compared to influenza and rsv infection [ ] . we compared our data for the activity periods with the periseason, which provided a more conservative estimation of the surpluses than if we would have compared with the summer period. yet, some underestimation of the surpluses may be introduced in this way. we do feel, however, that comparison with the peri-season is most appropriate, as other potential influences concerning the climate and possible other viruses that circulate in non-summer periods are probably comparable between the peri-season and the activity periods. every year, rsv-positive specimens reached a relatively small and intensive peak around week . earlier studies have shown similar tendencies, with rsv isolates peaking every year around the same time [ , , ] . this suggests the presence of potential common strict seasonal factors which might increase both the number of isolates and prescriptions; however probably not invalidating the associations and surpluses found in our analyses, which are truly seen and are in line with other studies [ , , ] . persons were labelled as belonging to the high-risk group based on specific medication profiles. in particular, persons belonging to the high-risk group were selected based on prescriptions that corresponded rather uniquely to the high-risk indications. prescriptions potentially meant for other indications, not labelled as high risk, were consistently excluded. still, it is possible that these selection criteria unjustly labelled individuals as belonging to the high-risk groups. also, some individuals may have been incorrectly excluded and labelled as non-high-risk. finally, one may hypothesize that excess prescriptions are merely shifts in time of extra prescriptions later to be outweighed by dips in prescriptions (sometimes referred to as "the harvesting effect"). visual inspection of our data however did not give any reason to support this hypothesis in our study. additionally, a formal statistical comparison of the number of prescriptions during the peri-seasons and during the first weeks after the active seasons did not show any peak-dip pattern (data not shown). previously, various investigations have been performed on the association between influenza and rsv epidemics, on the one hand, and hospitalisations, mortality and outpatient visits, on the other [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . below, we briefly compare the outcomes, knowing that the validity of making such comparisons between studies is limited due to differences in outcome measurement, statistical models, study period, and health-care system concerned. previously, only one study investigated excess antibiotic use during influenza-activity periods in the general population [ ] . this study did show that otherwise healthy children get more prescriptions for antibiotics during these influenza periods. however, the surplus reported was relatively low compared to our findings [ ] . two other studies estimated the excess antibiotic prescriptions during both rsv and influenza-active seasons, focussing on specific target groups [ , ] . the first study focussed on patients suffering from chronic lung disease, showing the highest surpluses for the youngest age groups [ ] . the latter study showed higher surpluses due to influenza as compared to rsv for those living in nursing homes [ ] . both results are in line with our findings for antibiotics [ , ] . several other studies indicate that, in general, both during rsv and influenza-activity periods, infants and elderly show the highest morbidity and mortality rates [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] . this is certainly in line with our findings on excess antibiotic and otological prescriptions among the youngest age groups and cardiovascular medication surpluses among the older age groups. in contrast to other studies which showed higher morbidity and mortality among the oldest age groups compared with non-elderly adults, our study shows that the elevation in the prescriptions of antibiotics in the oldest age groups ( years of age and older) is not higher than in the two younger age groups ( - and - ). yet, if compared with other adult age groups, a small increase could be seen. for influenza, this slightly deviating finding compared to non-dutch settings, might be explained by the high vaccination coverage among elderly in the netherlands. a recent dutch study showed that rsv-related excess hospitalisations were considerably higher as compared to those due to influenza [ ] . comparably, a study performed for england and wales showed greater excess rates for complications during rsvactive periods among the youngest age groups as compared to influenza; similar rates were found for all other age groups [ ] . five other studies focussing on excess morbidity, mortality and hospitalisation among children confirmed these results, showing that rsv was responsible for higher hospitalisation rates than influenza [ , [ ] [ ] [ ] [ ] . for the netherlands, jansen et al. recently showed excess hospitalisation for cardiovascular complications among the - years old, during influenza-active periods but not during rsv-active periods [ ] . in line with their findings, we showed a significant surplus in first cardiovascular medication prescriptions during influenzaactive periods, but not during rsv-active periods for those aged years and over. elevated hospitalisation and prescription rates during influenza periods in persons aged around years and beyond suggests that influenza may cause cardiovascular diseases or that it may aggravate existing non-diagnosed cardiovascular diseases in older adults. another hypothesis explaining this increase might be that increased cardiovascular problems during influenza periods are related to the increased use of analgesics during those periods to alleviate influenza symptoms [ ] . further research is definitely needed into this topic. thus, in general, our results seem to be comparable with most other studies relating elevated viral activity to the use of health-care resources, morbidity and mortality. all studies consistently show that the highest excess rates for the youngest age groups are mostly due to rsv, whereas those for influenza are seen in elderly. vaccination may prevent part of the excess prescriptions we have found. for example, healthy children are not recommended to be vaccinated against influenza in the netherlands, while their vaccination might prevent part of the surplus prescriptions found for this group. in particular, vaccination may prevent influenza infection and potential subsequent bacterial super infection(s) and thus avert antibiotics prescribed for the prevention and treatment of such bacterial super infections. additionally, reducing the prescription of antibiotics may also be important from the perspective of limiting the development of antibiotic resistance. such reasoning could be an additional motivation for vaccinating yet uncovered groups against influenza [ ] . an effective vaccine against rsv may potentially even prevent more antibiotic prescriptions, especially in young children [ ] . yet the introduction of a vaccine again rsv is not expected in the very near future [ ] . during influenza-and rsv-active periods, elevations in antibiotic prescriptions were identified in all age groups. for otologicals, such an elevation was shown in the age groups of - and - years, both during influenza-and rsv-active seasons. by vaccinating young children against influenza, a part of these prescriptions for antibiotics and otologicals may be prevented. in persons of years and older an elevation of 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viruses in children with acute otitis media primary care management of respiratory tract infections in dutch preschool children influenza-and respiratory syncytial virus-associated morbidity and mortality in the nursing home population association of influenza vaccination and reduced risk of recurrent myocardial infarction influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly epidemiological studies: a practical guide morbidity profiles of patients consulting during influenza and respiratory syncytial virus active periods influenza and respiratory syncytial virus morbidity among - aged group in yunus emre health center mortality associated with influenza and respiratory syncytial virus in the united states contribution of respiratory syncytial virus, influenza and parainfluenza viruses to acute respiratory infections in hospitalization attributable to influenza and other viral respiratory illnesses in canadian children population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease the annual impact of seasonal influenza in the us: measuring disease burden and costs understanding the nsaid related risk of vascular events mark rozenbaum was supported by an unrestricted educational grant of sanofi pasteur msd (hoofddorp, netherlands). key: cord- - s ubbxl authors: drury, georgina; jolliffe, siobhan; mukhopadhyay, tarit k. title: process mapping of vaccines: understanding the limitations in current response to emerging epidemic threats date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: s ubbxl vaccination remains the most successful and effective mechanism of pathogen control. however, their development and deployment in epidemic settings have been limited, and the ebola outbreak in west africa identified several bottlenecks linked to a lack of investment in pathogen research, infrastructure or regulation. shortly after this outbreak, the uk government established the uk vaccine network to ensure the uk is better prepared to respond to pathogens outbreaks of epidemic potential. as part of their work, the network commissioned the creation of a vaccine development tool (http://www.vaccinedevelopment.org.uk/) to serve as a guide to the key stages in vaccine development. the tool also set out to capture the key, rate-limiting bottlenecks in the development of vaccines against emerging infectious disease such that corrective action could be taken, be it through research, funding, infrastructure and policy, both in the uk and internationally. the main research bottlenecks were related to understanding pathogen biology, identification of appropriate animal models and investment in the manufacturing sciences, especially into process development. infrastructure gaps in gmp manufacturing and fill-finish were also identified and limitations in gmo regulation and regulatory and ethical approvals, especially for outbreak pathogens required new policy initiatives. the uk vaccine network has since begun work to correct for these limitations with a series of funding calls and development programmes. this paper seeks to summarise the vaccine development tool and its key findings. timely response to epidemics requires the development, manufacture and distribution of vaccine. activities by the uk vaccine network (ukvn) and the who have seen the identification of several pathogens that have epidemic potential [ ] . however, the development of effective vaccines against these pathogens requires improved coordination between policy makers, funders, researchers, vaccine manufacturers, and regulators, if we are to develop and deploy vaccines to mitigate the spread of epi-or pandemics [ ] . set in the context of the ebola outbreak that began in , multiple stakeholders were mobilised to respond to the humanitarian crisis of this escalating outbreak. although there was no licenced ebola vaccine available, approximately fifteen different vaccines were in preclinical development, including dna vaccines, virus-like particles and viral vector [ ] . levine et al. [ ] described how these candidates had largely been developed as part of the bioterrorism preparedness programme, bioshield, in the united states. due to the relatively low geographic prevalence of infection there was a limited market and thus a poor case for industry to develop a vaccine for commercial purposes. however, concerns over the potential use of ebolavirus as a bioterrorism agent resulted in a number of stockpiled sources that had undergone testing in animal models [ ] . as the ebola epidemic worsened, the availability of small vaccine stockpiles, taken together with the threat of further spread, catalysed stakeholder engagement to test the vaccine in humans to determine safety for potential deployment. researchers, industrialists, regulators, and funders worked together to expedite the process of carrying out first-in-man trials of the ebola vaccine e-mail addresses: siobhan.jolliffe@homeoffice.gov.uk (s. jolliffe), ucbetkm@ucl. ac.uk (t.k. mukhopadhyay). unprecedented international consortium assembled to accelerate collaborative multi-site trials of candidate ebola vaccine, wellcome trust, th august (https://wellcome.ac.uk/press-release/unprecedented-international-consortiumassembled-accelerate-collaborative-multi-site). last accessed august . vaccine ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] contents lists available at sciencedirect vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e vaccines were deployed as part of the international response [ ] although this outbreak also served to highlight the necessity of effective public health systems and infrastructure, and community engagement in order to mitigate disease spread through cultural or social practices [ ] . in the case of ebola, it was fortuitous that several candidate vaccines were already in development and immense global efforts made the vaccine available. however, this also conspicuously highlighted that nations around the world need to be better prepared for outbreaks of all infectious diseases, particularly those with the potential for high levels of morbidity and mortality, and that other measures to control infection spread should also be implemented [ ] . the united kingdom (uk) has a strong international reputation for scientific research and innovation, and a long history in vaccinology. along with other countries, the uk government mobilised resources to manage the ebola outbreak, as well as played a role in testing ebola vaccines in the uk, and west africa. alongside these efforts, the uk chief medical officer for the department of health and social care (dhsc), dame sally davies, raised the question to government and related stakeholders -what could the uk do to be better prepared for future outbreaks of infectious diseases? a partnership was instigated across the dhsc, the uk medical research council (mrc) and the biotechnology and biological sciences research council (bbsrc), who then worked together to establish a 'uk vaccine network' (ukvn, www.gov.uk/government/groups/ uk-vaccines-network) bringing together a small number of specialists from industry, academia and relevant funding bodies to make targeted investments in specific vaccines and vaccine technology for infectious diseases with the potential to cause an epidemic. at its inaugural meeting in july the ukvn discussed potential priority areas for investment. members agreed that four working groups (wgs) with the following foci: wg : to prioritise - pathogens most likely to cause an epidemic or pandemic in the short to medium term. wg : to rationalise where and when vaccine development resources should be prioritised when intervention is possible. wg : to understand the challenges in vaccine development and the key rate limiting steps for any given vaccine in development. wg : to address manufacturing capacity for vaccines. this paper describes the activities undertaken by working group on vaccine development. the group created a visual process mapping tool to better understand the potentially ratelimiting bottlenecks associated in moving a vaccine candidate from discovery through to development and early phase clinical trials; then into clinical manufacture and phase trials. the primary purpose of the tool was to act as a visual aid for early discovery and development scientists, highlighting the major steps required to fully develop any experimental vaccine. the secondary, and crucial purpose of the tool, was to identify any generic bottlenecks that may slow down development. identifying such strategic limitations may identify corrective actions, that may in turn help to overcome potential delays or setbacks when expediting vaccine development during epidemics. actions could be taken by policy makers to correct for limitations in regulation, funders to identify limits in scientific knowledge that could be corrected with appropriate investment, and for government and trans-national organisations to consider local and global response. though the genesis of this tool was considered in the context of epidemic preparedness, the tool itself is sufficiently far-reaching that it can be utilized by the wider vaccine community. considering the global nature of vaccines, it is important to understand the interdependencies that exist between discovery, development and manufacture. this tool gives scientists vision of the considerations that will need to be made along the development process. in taking a much more holistic approach to vaccine development it is hoped that this can sup-port other international such as the global vaccine action plan [ ] , which relies on the continued supply of vaccines to meet immunization targets. thus, the tool serves multiple users and purposes, and we report on its main findings in this paper. after initial literature consultations, a basic schematic overview of the major steps was created, which then fell into four main areas: (i) pre-clinical discovery; (ii) pre-clinical development; (iii) manufacturing; and (iv) clinical trials. several subgroups were convened to discuss these areas; membership was drawn from the ukvn, a core group of specialists that made up wg and further expertise consulted according to need. process schematics were made, and members undertook interactive discussions to add in, remove, and reorganise steps. the ukvn secretariat led on organising a series of iterative workshops that repeated this process several times; each time a skills gap analysis was carried out to ensure appropriate expertise was representing different disease/vaccine interests, and from across the vaccine sector from academic and industry discovery, development, regulation, manufacturing and clinical trials. the repeated process of interactive mapping, refinement of core documents, and presentation of progress back to the subgroups stimulated dialogue on the map as a whole. this was a concerted effort to break down silos between disciplines and sectors, and address how choices made in early discovery may affect the later development pathway. once the iterative, interactive working subgroups had met to agree the final nodes and bottlenecks on the map, the final figures were transcribed into powerpoint. these images were then used as the basis for interactive webpages, which were constructed by the mrc regulatory support centre (mrc rsc, https://mrc.ukri.org/research/facilities-and-resources-for-researchers/regulatory-support-centre/). the mrc rsc offered designs for the main menu, layout and colour scheme and once agreed with the secretariat, all graphics for the tool were drawn using adobe illustrator then saved in a web-friendly format. the website was built in dreamweaver using html coding. efforts were made to make the site responsive to different devices, and while this was possible for the text it was not possible to do so for the maps due to the complexity of the subject. the final version can be seen at www.vaccinedevelopment.org.uk. the process mapping workshops culminated in production of a website, with standalone pages for maps that distinguished the following major subdivisions: ( ) target product profile (tpp); ( ) pre-clinical discovery; ( ) pre-clinical development; ( ) clinical development; and ( ) regulatory affairs and ethical approvals (fig. ). due to the mandate and remit of the tool to be used by the ukvn for the development of vaccines for outbreaks, wg agreed that the tool did not need to address steps for market access and authorisation, as outbreak vaccines would not normally undergo this process -instead being deployed in small populations according to need once necessary evidence on safety and immunogenicity had been achieved. the target product profile (tpp) is a descriptive document that sets out clear parameters for the characteristics of the desired end product, its intended use, and market. it is the designated entry point into the process map. this came about through repetitive discussions that prevailed on the importance of the tpp at all stages in development; in simple terms, to know what it is you are making, and for whom. wg agreed that the significance of the tpp and the relevance to even the earliest exploratory basic science work, should not be underestimated, therefore prominently placing the tpp, aimed to engage early discovery scientists to consider what the end product is, and how the choices made during pre-clinical discovery and development influence them. the consensus opinion within the working group was that the tpp should be seen as a strategic planning document that should be utilised as a benchmark as data emerges. during normal development, this is a tool for novel vaccine creation. however, in an outbreak situation, the tpp acts as an essential checklist under accelerated timescales. it is worth noting that that the importance of tpps is widely shared and recent work by the who and cepi includes creating standard tpps for priority pathogens. iterative workshops hosted ongoing discussion on what was involved in each step, and where might rate limiting steps slow down development. there was agreement that almost anything could be described as a bottleneck in that each step is necessary, and by the nature of discovery research and development, such endeavours take time. however, in order to scrutinize the bottlenecks that could potentially be ameliorated by corrective action or investments by governments, research funders and policy makers, wg agreed that the tool should only identify steps where corrective action could be taken that could potentially accelerate vaccine development. the main bottlenecks and the corrective action required are summarised in table . an additional important consideration, was that the identification of these bottleneck 'nodes' should be sufficiently generic so as to be applied to a range of vaccine types, and pathogens -thus wg refined the key steps to common denominators that were shared across approaches. overall, many of the bottlenecks focused around gaps in funding for scientific knowledge, training, and infrastructure. an additional item was harmonisation of regulation, especially with regards to the release of genetically modified organisms (gmos). the bottlenecks can readily be seen on the process map by clicking 'show bottlenecks' on each map page; below we describe the main bottlenecks highlighted at each stage. within pre-clinical discovery, understanding pathogen biology and developing the appropriate animal challenge models are seen as significant limiting factors, especially with emerging infectious disease. the ability to screen, test, and verify potential new antigens is of critical importance. increased research funding into standardised methodologies for in vitro and in vivo testing and work into human-livestock host immunology would be critical to increase capacity. as lead candidates are identified, ensuring that the work has the appropriate freedom to operate becomes increasingly important. a review of the intellectual property landscape including searches on the antigen, cell line, and biological methods employed to create them are required and time to completion could be as little as three months, but increases with complexity. conducting a freedom to operate review is an important step towards commercialisation. yet, in an outbreak scenario, special regulations such as compulsory licensure and step-in rights may allow this issue to be side-stepped, but would require further clarification in policy. pre-clinical development is a complex, multi-step, and timeconsuming process (fig. ) . it requires that vaccine candidates undergo strain development and confirmation of antigen presentation, verified by animal studies. this is followed by process development to ensure that a scalable, robust, and gmp compliant manufacturing process is created. material generated at the end of pre-clinical development can be used for animal toxicology studies and forms the basis of a clinical trial application. the first bottleneck encountered is in strain development. some development work can occur using a research cell bank, however research cell banks are generally not to gmp standards therefore delays at later stages can be avoided by ensuring banks are gmp compliant. this will often require re-cloning into traceable cell lines that are gmp certified and free of any adventitious agent. major bottlenecks are around process development to create a scalable, gmp compliant, manufacturing process and the determination of the critical process parameters. at this stage manufacturers must address if the vaccine can in fact be manufactured in a scalable process. there is no generic manufacturing process for vaccines, thus almost all processes are designed from the bottom up, which adds costs and delays to any future intervention plan. platform manufacturing technologies are a method to overcome this issue by streamlining manufacturing with generic operations into which your selected antigen(s) can be introduced. viral vectors are one such platform technology that was effectively employed during the ebola outbreak [ , ] . looking more broadly, recombinant sub-unit vaccine technologies are another platform technology that could be used in other epidemics and has been successfully demonstrated in context of influenza vaccines [ ] . other advances in nucleic acid based vaccines, such as dna and rna, offer a future, though as yet unrealised potential, for generic manufacturing and release testing of vaccines. the determination of the range of physical, chemical, biological, or microbiological properties that ensure the quality of the desired product, is described as 'critical quality attributes' (cqa) and requires welldefined analytics which in the long term can reduce quality control (qc) costs. finding suitable analytics that can resolve complex antigen structure, increase understanding of degradation pathways and formulation needs is a continuing bottleneck. funders must ensure that process development, manufacturing, and analytics are not overlooked in future research calls. further bottlenecks are encountered during animal in vivo studies; characterising immunogenicity and reactogenecity in relevant animal models is imperative for taking forward vaccine candidates into phase . with this model there are further potential bottlenecks associated with home office approvals, and containment level and facilities depending on the pathogen of study, and supply of animals. gmp manufacturing is an infrastructure bottleneck that requires funders and policy makers to examine the pressures upon the current manufacturing model. almost all vaccine manufacturers operate at capacity and are dedicated to production of a single product. ' step-in' rights in an emergency scenario will disrupt supply of an essential vaccine, but also cause delay. recruiting a cmo to the task may help to alleviate the situation, but essential training and know-how is required. furthermore, some sites may only be suitable for certain vaccines classes, which can range from protein sub-unit vaccines to live viral and bacterial vaccines. based on vaccine class, facilities may not have the appropriate containment level for bulk manufacture of the drug substance, or the fill-finish for drug product. indeed, many facilities do not have fill-finish operations co-located and fill-finish is also a bottleneck in clinical development due to the limited number of appropriate sites and the volume of vials required for phase and trials. the uk government, with the ukvn providing evidence and advice, has identified manufacturing infrastructure as a significant bottleneck, and it has sought to invest into a new vaccine development and manufacturing centre (https://www.gov. uk/government/news/medicine-and-vaccine-manufacturing-centres-apply-for-funding). other organisations, such as cepi [ ] , have also issued calls for proposals into new platform manufacturing technologies (cepi.net). the situation exists under the status quo that we may have a vaccine that can be utilised to fight a future epidemic, but not have the manufacturing capacity to enable production and distribution. these latest calls seek to reverse current limitations. many of the priority pathogens are prone to sporadic outbreaks, and due to potential lethality, it will not be possible to conduct full phase trials, and even phase studies may prove challenging in a human population due to the unpredictable nature of these outbreaks. nonetheless, the sequential clinical trial steps that would comprise the 'normal' steps in development of a vaccine being taken forward for licensure have been incorporated into the tool. clinical trials require great investment and resource to execute, and the lengthy nature of the process could itself be described as a bottleneck; for the purposes of the tool however, we highlight fives steps within clinical development. current gmo regulations do not distinguish vaccines based on viral vectors, or live, replication incompetent vectors based on bacteria and viruses. current regulation stipulates that use of gmo vaccines is done so under 'contained use' or 'deliberate release' protocols to prevent release into the environment. consequently, this may involve obtaining separate approvals, modifications to the trial site, modified procedures for sterilising, administering and disposing, separate storage facilities and special training of staff to handle gmos. overall, this whole area would benefit a revision of regulations and derogating the use of common vaccine vectors in separate regulations from the currently applicable gmo guidance would have a substantial beneficial impact in speeding up approvals. obtaining regulatory approval may be especially difficult in the context of an international trial, where local rules may differ from country to country, or the national regulatory authority (nra) itself may have insufficient experience and seek expert opinions, which can further delay the process. engagement with the nra at an early stage is essential, as seeking opinion on the proposed trial design can capture any 'in country' requirements. the availability of trial sites may also impede progress; particularly if sites are not approved to work with gmo vaccines, or there are other limitations such as lack of infrastructure, skilled personnel, and capacity. working with credible and experienced partners will help to minimise delay and several clinical trial networks already exists to facilitate clinical development, some of which are pathogen specific that can further minimise this bottleneck. to overcome these bottlenecks, finding clinical trial sites and experienced nras that can act as credible authorities is essential, and early discussions need to occur in tandem to development and production to avoid delays. the process mapping tool emphasises the importance of ethical and regulatory considerations throughout the entire process, as the iterative workshops continually highlighted the importance of early engagement so the appropriate advice and input shape the r&d. the tool provides an overview and signposting to other organisations and information sources, as many outbreak vaccines may never go through market authorisation due to there being no commercial incentive for the manufacturers. nevertheless, there will be several stages where necessary approvals must be obtained in order to proceed with developing and testing a candidate. for regular marketing authorization in the eu, vaccine developers must provide a full development dataset of pre-clinical and clinical trial results to evidence clinical safety and efficacy, as well as evaluation of a positive benefit-risk ratio. the mapping tool highlights that: to discuss the tpp the uk medicines and healthcare products regulatory agency (mhra) can be approached at any time. animal work is regulated under the animals (scientific procedures) act (aspa). a uk government home office licensure must be provided at an institutional, personal and project level. the use of gmo vaccines is regulated by separate legislation, and approval must be given by the health and safety executive (hse). to carry out experimental medicine studies of immune responses in humans the health research authority (hra) provides ethical approvals through the research ethics service (res) for projects in the nhs (led from england). clinical trial authorisation (cta) is made through a formal application to the mhra. trialists must provide information on the mode of action, nature of the target, animal model studies and an imp dossier with preclinical toxicology data, and any human mechanistic/proof of concept studies. though these regulations are specific for the uk, many other countries require a similar level of scrutiny. in the case of life-threatening infections, it is conceivable that many of the priority vaccines under development will ultimately never undergo the standard clinical trials process and be used in an outbreak situation under eual [ ] . in these circumstances, it is imperative to test novel vaccine candidates in well characterised animal models. previously applied to defence vaccines due to the limitations of clinical testing, the fda animal rule [ ] may provide a pathway to test novel vaccine candidates. in outbreak settings, as was the case with ebola, it is possible to undertake an emergency use protocol and expedite authorisation to ensure a promising vaccine candidate can be deployed for human use, if it is justified as major interest for public health and/or therapeutic innovation. once a generic framework that could be applied to vaccine r&d broadly was established, the working group undertook a series of meetings applying the process map to three case studies of different vaccine candidates under development for three of the ukvn's priority pathogens: mers viral vector vaccine, zika protein subunit/vlp vaccine (fig. ) , and the prokarium vaxonella Ò plague vaccine (fig. ) . the case studies can be accessed through the main landing page of the process map . mers was selected as there are several candidates in later stages of development, including the viral vectored vaccine candidate chadox mers. the viral vector approach overcomes a number of bottlenecks to speed up development, mainly impacting on the preclinical development stages to overcome delays that would be associated with masterseed bank production, pre-formulation work/in vitro and in vivo, and lead identification. antigen identification bottlenecks are also overcome, through straightforward selection of the spike protein (the major external antigen of coronaviruses) which elicits neutralising antibodies to the receptor binding domain [ ] . the bottlenecks that remains relevant from the generic tool are: basic understanding of human/livestock host immunology; using an appropriate animal challenge model (camels would be suitable, but few places have facilities for such a large model); and the three main bottlenecks in clinical development of obtaining ethical and regulatory approval, working with vaccines classified as gmos, as the viral vector platform has been altered genetically from its original form, and the availability of overseas trial sites. more bottlenecks were identified when applying the vaccine development process map to a novel zika vaccine. at the time of the ukvn formation, zika was little heard of, and had only modest investment as a potential emerging virus in uk research funding portfolios. as the work of the ukvn progressed, the zika outbreak unfolded, and funders in the uk and internationally were mobilised to increase investments. the ukvn immediately added zika to the priority pathogen list, and contributed to a number of funding streams to support vaccine r&d. applying the process mapping tool to such an emerging pathogen, allowed the visualisation of the significant number of unknowns and challenges when dealing with a little-known emerging disease without platform approaches (fig. ) . in the earliest pre-clinical discovery stage, pathogen biology and host immunology were both rate limiting, particularly in identifying regions of the virus that could be used as a possible antigen. in vitro studies and deploying an in vivo challenge model were also rate limiting as the generation of reagents and the appropriate assays need to be established. without a robust set of assays and generation of some antigen in the early discovery phase, the bottleneck would be overcome however identification of the surrogate/correlate of protection in the appropriate challenge models will be important. it should be noted that many vaccines have launched without the establishment of correlates of protection and will depend on the licensure sought and the severity of the disease and its outbreak. in pre-clinical development, due to the novelty of the vaccine, process development, assays, adjuvantisation and formulations were identified as bottlenecks. time could be saved if a platform technology was used for this vaccine, circumventing many steps in process development and formulation. in clinical development, deploying a human challenge model to study the response to a vaccine studies were considered rate-limiting, and it must be noted that in the case of emergency or experimental licensure, a strategy might be to establish some animal challenge data and seek to establish safety data in a phase trial before expansion into a phase a/b trial. however, demonstrating efficacy can only be one in endemic or outbreak situations and will require early engagement of the nra, and locating suitable overseas trial sites has the potential to be rate-limiting. a plague vaccine potentially has dual application: the development path could be in collaboration with government agencies in producing an anti-bioterror vaccine but could be expanded and developed as a vaccine against endemic disease in regions such as madagascar. this dual nature affects the way we think about the tpp and development path. as with the mers case study the plague vaccine exploited a platform technology -prokarium's vaxonella platform -thus many bottlenecks, particularly in preclinical development, were circumvented (fig. ) . the main bottlenecks are in the clinical phase and animal rule regulations necessary to follow for licensure and are particular to this pathogen and vaccine approach, and included: quality control; phase ii regulatory requirements for two animal models; fill finish; and in phase ii safety studies, meeting requirements for licensure regulations. the case studies have demonstrated how useful the process map can be in vaccine development, and how using platform technologies enables many development steps to be circumvented and thus overcome rate-limiting steps, which could be vital in an outbreak situation where speed of development must be minimised where possible. as an additional feature, the tool also presents some of the work completed by wg , which was undertaken to better understand the feasibility and challenges of developing vaccines for the major ukvn prioritised pathogens. the ukvn members agreed that to develop a new vaccine might not always be the most appropriate response in an outbreak setting, particularly if a candidate vaccine is early stage and presents significant r&d or manufacturing challenges. thus, it may be more appropriate to deploy other healthcare interventions to manage the disease, such a therapeutic antibodies or containment procedures. the prioritisation guide addresses the 'technical feasibility' of creating a vaccine together with 'public health value' based on pathogen severity and alternatives to vaccination. a final consideration was the time scale and cost of development, with the single criterion to address the available vaccine candidate(s), considering the stage in development of any existing candidates. this included manufacturing considerations, and whether any vaccine may already be stockpiled. each criteria has a red, amber or green status, and taken together will enable decision-making on the suitability of pursuing vaccine r&d in an outbreak setting. this tool represents a first step towards understanding the limiting factors in vaccine development and epidemic preparedness. however, there are still several key outstanding challenges it does not fully capture. the first is sustained funding for this problem, which is partly address through the creation of the coalition for epidemic preparedness innovation (cepi, www.cepi.net), however improved co-ordination is still required between industry, academia, government and international organizations, such as the who. this will be a key priority for policy makers in the future. infrastructure and manufacturing capacity needs to be accurately assessed, especially fill-finish capacity for the different vaccine types/classes. additionally, continued research is required to better understand the correlates of protection, appropriate animal models and, of course, the discovery of new antigens that can protect against these emerging pathogens. an extraordinary array of technologies are under development that have the potential to change our ability to respond to emerging infectious diseases. amongst these, viral vector and subunit vaccines are at the forefront, however future innovations with nucleic acid based vaccines are on the horizon. currently, there are no dna or rna vaccines licensed for human use. yet should this milestone be reached, many of the bottlenecks described currently will be eliminated. in the meantime, funders, governments and policy makers must all focus to overcome the existing bottlenecks identified and employ better co-ordination to overcome future epidemic threats. the author declares that there is no conflict of interest. . plague case study summary. the total number of bottlenecks is reduced due to the use of a live bacterial vector system being exploited as a platform technology. plague antigens are already known and well characterised, thus, many of the bottlenecks shift to clinical development and testing of the vaccine candidate. who's blueprint list of priority diseases the need for global r&d coordination for infectious diseases with epidemic potential clinical development of ebola vaccines how the current west african ebola virus disease epidemic is altering views on the need for vaccines and is galvanizing a global effort to field-test leading candidate vaccines a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines lessons learnt from the ebola outbreak in west-africa cdc's evolving approach to emergency response collaborating to achieve global vaccine action plan goals ebola ça suffit ring vaccination trial consortium. the ring vaccination trial: a novel cluster randomised controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to ebola efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination clusterrandomised trial a fast track influenza virus vaccine produced in insect cells cepi-a new global r&d organisation for epidemic preparedness and response world health organization. emergency use assessment and listing procedure (eual) for candidate medicines for use in the context of a public health establishing efficacy of human products using animals: the us food and drug administration's ''animal rule chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice the authors would like to thank the members of the uk vaccine network, mrc, bbsrc, the mrc regulatory support centre, and the many subject matter experts who have helped put this tool together. key: cord- -mwnnmwnw authors: herst, c.v.; burkholz, s.; sidney, j.; sette, a.; harris, p.e.; massey, s.; brasel, t.; cunha-neto, e.; rosa, d.s.; chao, w.c.h.; carback, r.; hodge, t.; wang, l.; ciotlos, s.; lloyd, p.; rubsamen, r. title: an effective ctl peptide vaccine for ebola zaire based on survivors’ cd + targeting of a particular nucleocapsid protein epitope with potential implications for covid- vaccine design date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: mwnnmwnw the - west africa ebov epidemic was the biggest ebov outbreak to date. an analysis of virus-specific cd + t-cell immunity in survivors showed that of those individuals had a cd + response to at least one ebov protein. the dominant response ( / subjects) was specific to the ebov nucleocapsid protein (np). it has been suggested that epitopes on the ebov np could form an important part of an effective t-cell vaccine for ebola zaire. we show that a -amino-acid peptide np - (yqvnnleei) located in a conserved region of ebov np provides protection against morbidity and mortality after mouse adapted ebov challenge. a single vaccination in a c bl/ mouse using an adjuvanted microsphere peptide vaccine formulation containing np - is enough to confer immunity in mice. our work suggests that a peptide vaccine based on cd + t-cell immunity in ebov survivors is conceptually sound and feasible. nucleocapsid proteins within sars-cov- contain multiple class i epitopes with predicted hla restrictions consistent with broad population coverage. a similar approach to a ctl vaccine design may be possible for that virus. mouse using an adjuvanted microsphere peptide vaccine formulation containing np - is enough to confer immunity in mice. our work suggests that a peptide vaccine based on cd + t-cell immunity in ebov survivors is conceptually sound and feasible. nucleocapsid proteins within sars-cov- contain multiple class i epitopes with predicted hla restrictions consistent with broad population coverage. a similar approach to a ctl vaccine design may be possible for that virus. keywords: ebola zaire vaccine, ctl vaccine, controller, yqvnnleei, covid- , sars-cov- , flow focusing development of safe and effective vaccines for some viruses such as hiv and ebov has been challenging [ ] . although vaccine development has been almost exclusively focused on eliciting a humoral immune response in the host through inoculation with whole protein antigen [ ] [ ] [ ] [ ], ctl peptide vaccines producing a t-cell response may offer an important alternative approach [ ] . for hiv and ebov and influenza in particular, the potential of ctl vaccines has been discussed [ ] [ ] [ ] . although computational prediction alone has been used for t-cell vaccine design [ ] [ ], we saw a unique opportunity to see if a preventative ebov t-cell vaccine could be successfully designed based on the specific epitopes targeted by survivors of documented ebov infection. the notion of hla restricted hiv control has been described [ ] . pereyra- heckerman conducted an analysis of virus-specific cd + t-cell immunity in individuals living with hiv [ ] . they reported that hiv controllers, individuals living with hiv not undergoing treatment who do not progress to aids, have cd + cells targeting different hla restricted class i epitopes on hiv compared with progressors, individuals with hiv who progress to aids in the absence of therapy. pereyra-heckerman suggested that this observation could guide the in-silico development of a ctl vaccine for hiv and other diseases. acquired immunity has been documented after ebov infection [ ] . antibody as well as t-cell responses have been described [ ] . sakebe et al. have shown that of subjects surviving the - ebov outbreak in west africa, cd + t-cells from of those survivors responded to at least one ebov antigen, with of the responders targeting epitopes on ebov np [ ] . one of the most commonly targeted ebov eptitopes on ebov np in the survivor group (targeted by cd + cells from four survivors) was np - (ipvyqvnnleeicqliiqaf). they also suggested that a ctl vaccine could be designed using epitopes targeted by cd + t-cells identified in these ebov controllers. human pathogen-derived peptide antigens that are also recognized by c bl/ t-cells have been previously described. these include peptides from vesicular stomatitis virus (vsv) rgyvyqgl [ ] , and human immunodeficiency virus (hiv) rgpgrafvti [ ] . the existence of such epitopes makes a range of pre- clinical vaccine experiments possible without having to rely on non-human primates and expensive and complex-to-manage humanized mouse models. wilson et al. showed that the ebov nucleoprotein (np) is an immunogen that provides protective, ctl-mediated immunity against ebov in a c bl/ mouse model and that this protection was conferred by a peptide sequence within ebola zaire: np - (vyqvnnleeic) [ ] . wilson we set out to see if we could drive ctl expansion directed against np - to occur after vaccinating c bl/ mice with ebola zaire np - (vyqvnnleeic), and to subsequently conduct an in-vivo ebov challenge study to see if this peptide was protective. we fabricated adjuvanted microspheres for this study as a room temperature stable dry powder using the flow focusing process to be µm in diameter so as to prevent more than one microsphere from being phagocytosed by any given antigen presenting cell (apc) at the same time [ ] . by loading only one peptide sequence per microsphere, we maximized the peptide payload and mitigated the possibility of multiple, different peptide sequences being delivered to the apc simultaneously, which could possibly result in competitive inhibition at the motif which could interfere with antigen presentation and subsequent t-cell expansion (supplementary material section ). we also set out to see if a similar approach to a ctl vaccine design for sars-cov- would be feasible based on an analysis of the hla binding characteristics of peptide sequences on sars-cov- nucleocapsid. we used a previously described biodegradable dry powder, plga microsphere, synthetic vaccine platform adjuvanted with tlr- and tlr- agonists for this study [ ] . in that article, we showed that the tlr- and tlr- agonists given together with a peptide in a mouse model did not produce t-cell expansion by elispot and that microencapsulation of the peptide and the tlr- ligand, with the tlr- ligand in the injectate solution, was required to elicit an immune response to the delivered peptide antigen as determined by elispot. that study also demonstrated that the microencapsulated peptides alone were insufficient to induce an adequate immune response without the presence of the tlr- and tlr- agonists administered as described. the tlr agonists used for this vaccine formulation are used in fda approved vaccines and can be sourced as non-gmp or gmp material for pre-clinical and clinical studies. we show here that the h -d b restricted epitopes vsv (rgyvyqgl) and ova (siinfekl), when administered to c bl/ mice, each produce a cd + we used this adjuvanted microsphere peptide vaccine platform to immunize c bl/ mice with np - , the ctl+ class i peptide antigen from the ebola ziare np protein identified as protective by wilson et al. [ ] . microspheres containing np - and cpg were prepared as a dry powder formulation and suspended before use in a pbs injectate solution containing mpla, and administered intradermally via injection at the base of the tail into mice as described in a previous publication [ ] . as illustrated in figure c , there was no statistically significant difference between the elispot data for the vaccinated mice versus the response seen in the negative elispot controls. wilson reported that protection seen in her experiment was due to a peptide sequence within np- - . we hypothesized that the np - epitope was inefficiently processed into mhc binding sub-sequences during antigen presentation. in order to explore possible h -d b matches for peptide sequences contained within ebola zaire np - (vyqvnnleeic), we prepared three peptide vaccine formulations, each containing one of the three possible mer sub-sequences within np - . these sequences are shown in table . we then vaccinated, via intradermal (tail) injection, three groups of mice with microspheres containing one of the three mer sub-sequences of np - ( per group). elispot analysis was performed, stimulating harvested splenocytes with the three possible mer sub-sequences. splenocytes from mice receiving the np - sub-sequence had a statistically higher elispot response than mice vaccinated with the other two possible sub-sequence mers (p < . ) as shown in figure a . this is consistent with the predicted h -d b binding affinity of yqvnnleei as shown in supplementary material table . we then loaded one population of adjuvanted microspheres with np - . this peptide has a predicted favorable h -i b binding affinity as shown in supplementary material table . we showed that vaccination of mice with the adjuvanted microsphere vaccine loaded with vg and np - showed an elispot response to np - whereas mice vaccinated with adjuvanted microspheres not loaded with peptide did not ( figure d ). we also showed that mice vaccinated with vg alone did not show an elispot response to np - ( figure a ) and, conversely, mice vaccinated with np - did not show a response to vg (figure b ). we conducted a pilot study demonstrating that intraperitoneal injection of the adjuvanted microsphere vaccine produced a statistically superior immune response by elispot compared with the same dose delivered by intradermal tail or intramuscular injection in c bl/ mice (supplementary material section ). based on the data from that study, and the fact that the volume of the intraperitoneal space would allow larger amounts of microsphere suspension to be delivered, we chose to proceed with intraperitoneal administration for the challenge portion of this study delivering mg of microspheres per dose. we dosed three groups of mice, ten mice per group, with the adjuvanted mi- table . peak mortality across all groups tested was seen in mice challenged with , pfu maebov versus pbs buffer control as shown in the survival curve in we saw what appears to be an innate immune response at the , pfu ebov exposure level. it has been suggested that ebov can mediate an innate immunity response through stimulation of tlr- [ ] . because the adjuvanted this provides some evidence that the protective effect of vaccination using this adjuvanted microsphere vaccine is reproducible. serum samples from sacrificed animals exposed to ebov who did not receive vaccine were quantitatively assayed for various cytokines using bioplex plates. animals having unwitnessed demise did not have serum samples collected. a pearson correlation analysis was performed to assess relationships between specific cytokine levels and survival. the results are shown in table . we observed low levels of il- in surviving mice. nhps infected with ebov have been determined by other researchers to have elevated levels of il- in plasma and serum [ ] [ ] . ebov infected humans have also shown elevated il- levels and these elevated levels have been associated with increased mortality [ ] . similarly, we observed low levels of mcp- , il- and gm-csf in survivors. [ ] [ ] and elevated levels of mcp- were associated with fatalities in ebov infected human subjects [ ] . human survivors of ebov have been found to have very low levels of circulating cytokines il- and elevated levels of gm-csf have been associated with fatality in humans exposed to ebov [ ] . we lating t-cells targeting sars-cov- nucleocapsid two years after initial infection. [ ] we decided to investigate the feasibility of designing a sars-cov- peptide vaccine targeting sars-cov- nucleocapsid. all available sars-cov- protein sequences were obtained from the ncbi viral genomes resource within genbank, an nih genetic sequence database [ ] . retrieved sequences were processed using multiple sequence alignment (msa) via clustal for the nucleocapsid phosphoprotein [ ] . the nucleocapsid phosphoprotein sequences were trimmed down to every possible peptide sequence dosing [ ] . predicted values of these peptides were cross referenced with actual in-vitro binding measurements from identical mer peptides when that data was available. most preventative vaccines are designed to elicit a humoral immune response, typically via the administration of whole protein from a pathogen. antibody antigens and the hla restricted nature of ctl vaccines have limited their utility to protect individuals from infectious disease [ ] . however, observations derived from individuals able to control hiv infection [ ] and ebov infection [ ] demonstrating that control may be associated with specific ctl targeting behavior, suggest that there may be an important role for hla-restricted pep- ebov can cause severe pulmonary problems in exposed subjects [ ] . these problems can be especially severe when the virus is delivered by aerosol [ ] [ ]. interaction of ebov specific antibody, nhp lung tissue and ebov delivered to nhps via aerosol can produce a more lethal effect than in nhps without circulating anti-ebov antibody exposed to aerosolized ebov (unpublished conference presentation). this suggests that a ctl vaccine may be more effective for prophylaxis against filovirus protection than an antibody vaccine if the anticipated route of ebov exposure is via aerosol. coproteins present on the surface, show a high degree of conservation. epitopes within these internal proteins often stimulate t-cell-mediated immune responses [ ] . as a result, vaccines stimulating influenza specific t-cell immunity have been considered as candidates for a universal influenza vaccine [ ] . response to sars-cov- nucleocapsid two years after infection. [ ] this suggests that the same approach could be applied to sars-cov- which has con- table and supplementary material table . the remaining sars-cov- peptides listed in could also be further qualified as potential vaccine candidates by confirming mhc binding predictions by in-vitro binding affinity and/or binding stability studies [ ] [ ] [ ] . another approach to evaluating the sars-cov- candidate vaccine peptides though in-vitro testing is also possible. as we have shown in this paper, a peptide targeted by ebov controllers could form the basis of a preventative vaccine for ebov. elispot analysis of pbmcs taken from the peripheral blood of covid- controllers and progressors to assess the presence of a differential response to the peptides could lead to a broadly applicable protective ctl vaccine against sars-cov- by incorporating peptides into the vaccine that are more commonly targeted for cd + attack by the controllers versus the progressors. a peptide vaccine for sars-cov- , unlike a typical antibody vaccine, is not limited to virus surface antigen targets. this provides opportunities to attack other targets on sars-cov- besides spike which may be prone to mutation [ ] . in addition, a peptide vaccine mitigates the risk of antibody disease enhancement (ade) seen in the context of a non-neutralizing antibody response to a whole protein vaccine [ ] [ ] . also, neutralizing antibodies directed against spike protein in sars-cov- patients have been associated with an increased risk of acute lung injury (ali) [ ] . specifically, patients succumbing to sars-cov- were found to develop a neutralizing antibody (nab) response to spike protein faster than survivors after the onset of symptoms and the nab titers were higher in the patients who died compared with those who recovered [ ] . to the extent to which antibody vaccines producing an antibody response against the spike protein in sars-cov- could increase the risk of ali, this risk could also be mitigated by a using peptide vaccine as an alternative approach. the extent of the covid- outbreak should allow many more controllers to be identified than the thirty individuals studied by sakabe and the seven individuals identified in the peng study [ ] [ ] . furthermore, sakebe and peng did not report progressor data perhaps because of the difficulty in obtaining blood samples from those patients. if researchers act now during the covid- outbreak, perhaps controller and progressor blood samples could be collected and prospectively analyzed, quickly creating a database of optimal candidate class i peptides for inclusion into a ctl vaccine with potentially broad hla coverage for subsequent rapid manufacture and deployment. it would be interesting to see the extent to which the peptides favored by controllers appear on sars-cov- nucleocapsid, making sars-cov- a second example, across two different viruses, of controllers exhibiting ctl attack preferentially on the nucleocapsid protein. all animal handling was done in accordance with nih and institutional an- gapped sequence alignment using artificial neural networks: application to the mhc class i system aerosol transmission of filoviruses high-resolution structure of hla-a* in complex with sars nucleocapsid peptide vaccine-elicited human t cells recognizing conserved protein regions inhibit hiv- aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques. veterinary pathology insight into the ebola virus nucleocapsid assembly mechanism: crystal structure of ebola virus nucleoprotein core domain at . å resolution host response dynamics following lethal infection of rhesus macaques with zaire ebolavirus identification and characterisation of peptide binding motifs of six autoimmune disease-associated human leukocyte antigen-class i molecules including hla-b* : ebola virus: from discovery to vaccine barriers to antigenic escape by pathogens: trade-off between reproductive rate and antigenic mutability a vaccine against ebola: problems and opportunities t-cell-inducing vaccines -what's the future? cd -mediated protection against ebola virus infection is perforin dependent profound early control of highly pathogenic siv by an effector memory t-cell vaccine large scale analysis of peptide-hla class i interactions. iedb ebola virus glycoprotein induces an innate immune response in vivo via tlr clustal w and clustal x version . . bioinformatics anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection t-cell immunity of sars-cov: implications for vaccine development against mers-cov flow focusing: a versatile technology to produce size-controlled and specific-morphology microparticles human ebola virus infection results in substantial immune activation immunopathogenesis of severe acute respiratory disease in zaire ebolavirus-infected pigs reliable prediction of t-cell epitopes using neural networks with novel sequence representations synthetic peptides coupled to the surface of liposomes effectively induce sars coronavirus-specific cytotoxic t lymphocytes and viral clearance in hla-a* transgenic mice long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sarsrecovered patients hiv control is mediated in part by cd + t-cell targeting of specific epitopes ebola: an analysis of immunity at the molecular level large scale analysis of peptide -hla-i stability large scale analysis of peptide -hla-i stability t cell responses to viral infectionsopportunities for peptide vaccination eliciting cytotoxic t-lymphocyte responses from synthetic vectors containing one or two epitopes in a c bl/ mouse model using peptide-containing biodegradable microspheres and adjuvants conserved peptide vaccine candidates containing multiple ebola nucleoprotein epitopes display interactions with diverse hla molecules analysis of cd + t cell response during the - ebola epidemic in west africa phase trials of rvsv ebola vaccine in africa and europe peptide binding to the most frequent hla-a class i alleles measured by quantitative molecular binding assays measurement of mhc/peptide interactions by gel filtration or monoclonal antibody capture viral-induced enhanced disease illness heterosubtypic t-cell immunity to influenza in humans: challenges for universal t-cell influenza vaccines cellular immune correlates of protection against symptomatic pandemic influenza the international hiv controllers study et al. the major genetic determinants of hiv- control affect hla class i peptide presentation development of a preventive vaccine for ebola virus infection in primates sars ctl vaccine candidateshla supertype, genome-wide scanning and biochemical validation ebola virus proteins np, vp , and vp are essential and sufficient to mediate nucleocapsid transport on the origin and continuing evolution of sars-cov- the length distribution of class i-restricted t cell epitopes is determined by both peptide supply and mhc allele-specific binding preference hla-a t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins the immune epitope database (iedb) . broadly protective strategies against influenza viruses: universal vaccines and therapeutics structure and assembly of the ebola virus nucleocapsid on the defing rules for interactions between the t cell receptor and its ligand: a critical role for a specific amino acid residue of the t cell receptor b chain homologous and heterologous protection of nonhuman primates by ebola and sudan virus-like particles ebola virus-like particles protect from lethal ebola virus infection human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis plotkin sa, orenstein wa protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein reasoning of spike glycoproteins being more vulnerable to mutations among coronavirus proteins from different species recent advances in the vaccine development against middle east respiratory syndrome-coronavirus antibody responses against sars coronavirus are correlated with disease outcome of infected individuals advances in the study of hla-restricted epitope vaccines screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes sars-cov- nucleocapsid top candidate peptides with associated predicted hla restricted binding affinities peptide start key: cord- -uq fsqh authors: dwivedi, varun; manickam, cordelia; patterson, ruthi; dodson, katie; weeman, matthew; renukaradhya, gourapura j. title: intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: uq fsqh porcine reproductive and respiratory syndrome (prrs) is an economically important disease to pork producers worldwide. commercially, both live and killed prrsv vaccines are available to control prrs, but they are not always successful. based on the results of mucosal immunization studies in other viral models, a good mucosal vaccine may be an effective way to elicit protective immunity to control prrs outbreaks. in the present study, mucosal adjuvanticity of mycobacterium tuberculosis whole cell lysate (mtb wcl) was evaluated in pigs administered a modified live prrs virus vaccine (prrs-mlv) intranasally. a mtb wcl mediated increase in the frequency of nk cells, cd (+)and cd (+) t cells, and γδ t cells in pig lungs were detected. importantly, an increased and early generation of prrsv specific neutralizing antibodies were detected in prrs-mlv+ mtb wcl compared to pigs inoculated with vaccine alone. in addition, there was an increased secretion of th cytokines (ifnγ and il- ) that correlated with a reciprocal reduction in the production of immunosuppressive cytokines (il- and tgfβ) as well as t-regulatory cells in pigs vaccinated with prrs-mlv+ mtb wcl. further, a complete rescue in arginase levels in the lungs mediated through mtb wcl was observed in pigs inoculated with prrs-mlv. in conclusion, mtb wcl may be a potent mucosal adjuvant for prrs-mlv in order to potentiate the anti-prrsv specific immune responses to control prrs effectively. porcine reproductive and respiratory syndrome (prrs) is one of the most economically important chronic viral diseases of pigs [ ] . the causal organism of this disease is prrs virus (prrsv), which belongs to the family arteviridae in the order nidovirales [ ] . the clinical signs of prrs are reproductive failure, abortion, and high pre-weaning mortality [ ] . prrsv causes immunosuppression resulting in susceptibility of pigs to other poly-microbial infections [ , ] . the virus induces weak, innate immune responses as a result of reduced ifn-␣ production and dampened natural killer (nk) cell mediated cytotoxicity [ , ] , which lead to a weak/delayed adaptive immune response. although increased prrsv specific antibodies are generated early post-infection, virus neutralizing (vn) antibodies appear quite late and remain at low levels [ ] . a killed prrsv vaccine is available but it has failed to provide adequate protection. a modified-live prrs virus vaccine (prrs-mlv) has been in use to control clinical prrs in young pigs. unfortunately, like the prrsv infection, prrs-mlv also induces delayed neutralizing antibody and dampened cell-mediated immune (cmi) responses [ ] [ ] [ ] . therefore, it is important to improve the efficacy of prrsv live vaccines to control prrs effectively. induction of the ifn-␥ response by vaccination is important for viral clearance. the pro-inflammatory cytokine il- produced by myeloid cells initiates the virus specific adaptive immune response [ ] . additionally, the cmi response is manipulated by the metabolism of an essential amino acid, l-arginine, whose level in the body is modulated by the enzymes arginase ( and ) and nitric oxide synthase [ ] . the role of arginase in viral infections of the respiratory tract or in vaccination is limited, however, arginase deficient mice have an increased susceptibility to viral infections [ ] . the requirement of arginase to abort the multiplication of herpes simplex virus (hsv) has been reported [ ] . in contrast, uncontrolled replication of leishmania was correlated with enhanced arginase activity [ ] . mucosal surfaces cover the largest surface area in the body and almost % of the total immune cell population is present at mucosal sites. nasopharyngeal lymphoid tissues contain the entire repertoire of immune cells and are strategically located to orchestrate regional immune functions against airborne infections [ ] . therefore, effective mucosal vaccination is an appropriate strategy to provide protection against various infectious agents. protective mucosal immunity is mediated by cd + t helper (th) cells, cd + cytotoxic lymphocytes (ctls), and nk cells in hsv genital infections [ ] . similarly, intranasal delivery of an influenza vaccine flumist (medimmune, gaithersburg, md) provided immunity comparable to that induced by a natural infection [ , ] . an added advantage of mucosal vaccination is that it can induce both mucosal and systemic immune responses [ ] . intranasal immunization of hivliposome resulted in an effective virus specific immune response at both mucosal and systemic sites [ ] . to increase the efficacy of mucosal immunization, a potent adjuvant or delivery system is needed to overcome the immune tolerant mechanisms at mucosal sites [ , ] . mucosal administration of a live attenuated vaccine with a suitable adjuvant induces long lasting protection in various studies performed in bovine herpes virus- , influenza, and parainfluenza- virus [ ] [ ] [ ] . killed mycobacterium tuberculosis is an excellent candidate adjuvant used in the preparation of freund's complete adjuvant [ ] , but its use in humans and in food animals is contraindicated due to a severe granulomatous inflammatory reaction induced at the injection site. this adverse effect results from toxic cell wall components of mtb (such as mycolic acids, arabinogalactan, wax d) [ , ] . however, adjuvanticity of various purified components of mtb have been evaluated individually with satisfactory results [ , ] . in particular, certain individual components and a total fraction of whole cell lysate (wcl) of mtb free from its toxic cell wall constituents have been demonstrated to possess superior adjuvanticity in rodents, guinea pigs, and rabbits [ ] [ ] [ ] [ ] . however, the knowledge related to mucosal adjuvanticity of mtb wcl to protect against viral infections of the respiratory tract is limited. the purpose of this study was to enhance the efficacy of prrs-mlv with the help of mtb wcl by inducing protective mucosal immunity. initially, we performed studies in pigs to choose a suitable bacterial candidate to use as a mucosal adjuvant by inoculating nine different bacterial preparations belonging to mycobacterium, vibrio, and streptococcus species intranasally; the detailed results of which will be presented elsewhere. from that study, we selected mtb wcl for its potent adjuvant properties such as its ability to augment prrsv specific th cytokines and immune cells, and suppress immunosuppressive responses. in this study, immune responses of pigs administered prrs-mlv+/− mtb wcl intranasally were evaluated at both mucosal and systemic sites. stable mycoplasma-free marc- cells which support the growth of prrsv [ ] were used to prepare prrsv antigens and to perform immunological assays. marc- cells were maintained in dmem (lonza, riverside, ca) with % fetal bovine serum (atlanta biologicals, lawrenceville, ga) at • c with % co . prrs-mlv (ingelvac ® prrs) was provided by dr. mike roof (bio-r&d, boehringer ingelheim vetmedica inc., st. joseph, mo). wild-type prrsv vr was provided by eric nelson (south dakota state university). m. tuberculosis whole cell lysate (mtb wcl) was provided by drs. dobos and belisle (nih/niaid funded contract "tb vaccine testing and research materials"; colorado state university, fort collins, co). purified and biotin labeled cytokine specific anti-porcine antibodies, il- , il- , and il- (r&d systems, minneapolis, mn), ifn-␥ (bd pharmingen, san diego, ca), tgf-␤ (invitrogen, camarillo, ca) were purchased from commercial sources and used to perform the sandwich elisa. conventional large white-duroc crossbred weaned specificpathogen-free piglets were transported to the animal facilities of the food animal health research program at the ohio agricultural research and development center, wooster, oh. the swine herd was confirmed seronegative for antibodies by elisa to prrsv, porcine respiratory corona virus, transmissible gastroenteritis virus, and porcine circo virus . piglets were bled on arrival and tested to confirm the absence of prrsv antibodies. pigs were allowed to acclimate for one week before initiation of our study. for the duration of our study, all animals received food and water ad libitum. all inoculations such as adjuvant (mtb wcl, mg/pig) and vaccine ( × tcid per pig) were administered intranasally. all pigs were maintained, samples collected, and were euthanized as per the protocol approved by the institutional animal care and use committee (iacuc) and institutional biosafety committee (ibc), the ohio state university, ohio. twenty-two pigs were randomly allocated to one of three groups: group , mock pigs (n = ) received normal saline ( ml); groups and (each group n = ) were vaccinated with prrs-mlv+/− mtb wcl. both vaccine and adjuvant each in ml volume were administered at the same time into each nostril. three pigs each from groups and were euthanized on , , and post-immunization day (pid). mock inoculated pigs (n = ) were euthanized separately prior to euthanasia of any vaccinated pigs. in another study (data shown in fig. ), nine pigs were allocated into three groups (n = per group), and inoculated intranasally with prrs-mlv+/− mtb wcl as described above, and pigs were euthanized at pid . mock inoculated pigs (n = ) were euthanized separately prior to euthanasia of any vaccinated pigs. three to ml of blood was collected on pid , , , , , , , , , , , and . serum was separated from the blood, aliquoted, and stored at − • c. lung homogenates were prepared as described previously [ ] . for isolation of peripheral blood mononuclear cells (pbmc), blood was collected in acid citrate dextrose solution. for isolation of bronchoalveolar lavage fluid (bal) cells, lung mononuclear cells (lung mnc) and mnc from tracheobronchial lymph nodes (tbln) draining the lungs, the procedure was followed as described previously [ , , ] . to analyze prrsv isotype specific iga and igg antibodies in serum, elisa plates were coated with pre-titrated crude killed prrsv (vr ) antigens ( g/ml) in carbonate-bicarbonate buffer (ph . ) and incubated overnight at • c. plates were washed in pbs-tween- and then treated with blocking buffer ( % bsa in pbs) for h at rt. test serum was added and incubated at rt for h. plates were washed and the bound prrsv isotype specific antibody was detected using anti-pig iga and anti-pig igg secondary antibodies conjugated with hrp (kpl, gaithersburg, md). plates were developed using a chromogen abts and the od was read at nm. to eliminate the background activity, a control plate with bicarbonate buffer (ph . ) and without prrsv antigens was blocked, treated with the test serum, and developed separately. the od values obtained from control wells were subtracted from the respective experimental wells to obtain the corrected values. a standard indirect immunofluorescence assay (ifa) was followed for determining the virus neutralizing antibody titers [ , ] . briefly, serum was heat treated for complement inactivation, diluted two fold in dmem, and incubated with an equal volume of prrsv vr containing tcid per well for h at • c. one hundred microliters of that suspension was transferred into a -well microtiter plate containing a confluent monolayer of marc- cells, and the plate was incubated for h at • c in a co incubator. cytopathic effects were examined following fixation with acetone water and the addition of anti-prrsv nucleocapsid mab (clone sdow ) and alexa- conjugated antimouse igg(h+l) secondary antibody. these cytopathic effects were observed under a fluorescent microscope after mounting with glycerol-pbs ( : ratio). nk assay to determine the pig nk-cell mediated cytotoxicity was performed as described previously [ ] . briefly, pbmc were used as the source of nk cells (effectors) against k- (human myeloblastoid cell line) or yac- (mouse t lymphoma cell line) target cells. effectors and targets were incubated at different e:t ratios and the amount of released lactate dehydrogenase (ldh) was measured by a colorimetric assay. released ldh is directly proportional to the nk specific lysis of target cells. five million lung mnc were subjected to in vitro restimulation in a -well tissue culture plate in the presence of killed crude prrsv vr antigens ( g/ml) in enriched rpmi- [ ] for h at • c. harvested culture supernatant was analyzed for cytokines by elisa. cells cultured in the absence of any antigens were included as a control, and the amount of cytokines secreted by these cells was subtracted from the respective restimulated experimental well values. lung homogenates and cell culture supernatants were analyzed for cytokines ifn-␥, il- , il- ,il- , and tgf␤ by elisa as described previously [ , ] . flow cytometric analysis was performed to determine the phenotype and the frequency of different immune cells by a multicolor immunoassay as described previously [ , , ] . immunostained cells were acquired using a facs ariaii flow cytometer (bd biosciences). the analysis was performed using a flowjo software (tree star, inc., or, usa) to enumerate different immune cell populations based on the cell surface marker expression as follows: nk cells (cd − cd − cd ␣ + ) [ ] ; t-helper cells (cd + cd + cd − ); cytotoxic t lymphocytes (ctls) (cd + cd − cd + ); ␥␦ t cells (cd ␣ + tcr n + ); t-regulatory cells (cd + cd + foxp + ); myeloid cells (cd + ). frequencies of individual lymphoid and myeloid cell subsets were analyzed from a total , to , events. a colorimetric assay was used to measure arginase levels in the pig lungs, and the protocol was standardized based on previously described methods [ ] . bal cells were lysed using lysis buffer containing . % tritonx- and protease inhibitor cocktail for min on ice. cell debris and nuclei were clarified by centrifugation and the clear supernatant was aliquoted. each aliquot was treated with mm mncl and heated for min at • c. another identical aliquot not treated with mncl was kept at rt. l-arginine . m (ph . ) was added to all the tubes and incubated for min at • c. the reaction was stopped using a mixture of % h so , % h po , and h o mixed in the ratio of : : . the enzyme activity was measured after adding % ␣-isonitrosopropiophenone and heat treating for min at - • c. standard concentration of urea ( . m) was diluted ten fold and subjected to similar treatment as described above. optical density was measured at nm and the values were converted to units based on the od values of the urea standards. one unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of mol of urea per min. all of the data were expressed as the mean of three or six pigs ± sem. statistical analyses were performed using nonparametric wilcoxon t-test when functionality was compared between two study groups, and paired t-test with repeated measures when functionality was compared among different pid (sas software, sas institute inc., cary, nc). statistical significance was assessed as p < . . initially, at pid , prrsv specific mtb wcl-mediated adjuvant effect was analyzed in pigs. lung homogenates from prrs-mlv inoculated pigs had significantly higher amounts of the immunosuppressive cytokine il- and lower levels of the pro-inflammatory cytokine il- ( fig. a) . also, lung-mnc of prrs-mlv inoculated pigs secreted significantly higher amounts of il- and lower amounts of il- compared to pigs that received prrs-mlv+ mtb wcl (fig. b) . as an indicator of a systemic immune response, pbmc from pigs receiving prrs-mlv secreted more of an another immunosuppressive cytokine tgf-␤ and less of th response inducing cytokine il- compared to pigs received prrs-mlv+ mtb wcl (fig. c ). an increased immunosuppressive cytokine response was associated with a significant increase in the frequency of t-regulatory cells (tregs) in the lungs of pigs receiving prrs-mlv compared to pigs inoculated with prrs-mlv+ mtb wcl (fig. d) . in addition, the frequency of myeloid cells (cd + ) in tbln of prrs-mlv inoculated pigs was significantly less (fig. d) ; particularly the dendritic cells (dcs) rich fraction (cd + cd c + sla class ii + ) (data not shown) when compared to pigs vaccinated with prrs-mlv+ mtb wcl. to note, mtb wcl did not induce any adverse effects in the pig respiratory tract. we also performed an in vivo dose kinetics study of mtb wcl in pigs vaccinated intranasally with prrs-mlv and detected an enhanced anti-prrsv specific immune response when mtb wcl was used at mg per pig (data not shown). serum samples were analyzed for prrsv specific total igg and iga antibodies; and surprisingly, a significantly reduced secre- a standard colorimetric nk cell cytotoxicity assay was used to determine the nk cell function. pbmc were used as effectors against k targets and the released ldh was measured using a ldh substrate. each bar or the data point on the line graph represents the average % nk cells or percent nk specific lysis, respectively, from three pigs ± sem. asterisk denotes statistically significant difference between pigs inoculated with prrs-mlv+/− mtb wcl. tion of igg at pid was detected from pigs that received prrs-mlv+ mtb wcl (fig. aii) . the virus specific total iga secretion was also less (but not statistically significant) at pid , and it was significantly reduced at pid in pigs vaccinated with prrs-mlv+ mtb wcl compared to vaccine alone inoculated pigs ( fig. bii and biii). in contrast, prrsv specific vn titers at pid , and were significantly higher in prrs-mlv+ mtb wcl vaccinated pigs compared to prrs-mlv alone inoculated animals (fig. c) . therefore, our results suggested that mtb wcl has induced the increased prrsv specific vn titers in prrs-mlv inoculated pigs, and interestingly it was independent of total prrsv specific antibodies. lung immune cells were immunostained to identify the frequency of nk cells. a significant increase in the frequency of nk cells at pid , and a moderate increase at pid and in the lungs of pigs vaccinated with prrs-mlv+ mtb wcl, compared to mock and vaccine alone received pigs was detected (fig. a-c) . further, a colorimetric nk cell cytotoxic assay was performed and detected a moderate rescue in the nk cell cytotoxicity only at e:t ratio : in prrs-mlv+ mtb wcl received pigs at pid ( fig. d ). increased frequency of nk cells at pid and did not result in the rescue of nk cell killing function (data not shown). lung mnc of prrs-mlv+ mtb wcl vaccinated pigs secreted significantly higher levels of the th cytokines (ifn␥ and il- ) and significantly reduced levels of the immunosuppressive cytokine il- at pid compared to pigs that received prrs-mlv alone and mock-inoculated pigs ( fig. a-c) . also at pid and , a significant increase in secretion of the cytokine ifn-␥ and a moderately reduced secretion of the cytokine il- by lung mnc were detected (fig. a-d) . however, a considerable difference in the secretion of the cytokine il- was not observed between pig groups at pid and (data not shown). the phenotype and frequency of various lymphoid immune cells in pigs were analyzed by flow cytometry. the frequency of mock-infected pig immune cells is shown separately ( table ). the lungs from prrs-mlv+ mtb wcl vaccinated pigs had a higher frequency (but not significant) of total lymphocytes (cd + t cells) and cd + t cells at all pid (table a and b) . at pid , a significant increase in the frequency of cd + t cells was detected in the lungs of prrs-mlv+ mtb wcl vaccinated pigs compared to lungs from pigs inoculated with prrs-mlv alone (table c ). an increased (but not significant) frequency of cd + t cells was also detected in the (table c ). in addition, increased (but not significant) frequency of ␥␦ t cells in the lungs of prrs-mlv+ mtb wcl vaccinated pigs was detected at all the pid (table d ). in contrast, the frequency of tregs in the lungs of prrs-mlv+ mtb wcl vaccinated pigs was significantly less compared to the frequency of tregs in the lungs of prrs-mlv inoculated pigs at pid (table e) . in blood, a significant increase in the frequency of cd + t cells was detected at pid and in prrs-mlv+ mtb wcl inoculated pigs compared to pigs inoculated with prrs-mlv alone (table a ). significant modulation of treg populations was not observed in the blood as prrs is predominantly a disease of the pig lung (tables e and b ). information related to inhibition of arginase in the setting of respiratory viral infection and/or vaccination is limited. here we report that pigs vaccinated intranasally with prrs-mlv had signif- icantly reduced intracellular arginase levels in their lungs at pid compared to physiological levels from the mock pigs. in contrast, arginase levels in pigs administered prrs-mlv+ mtb wcl were completely rescued. interestingly, the rescue was significantly higher than in pigs inoculated with prrs-mlv at pid and (fig. ). repeated analysis of arginase from pigs receiving prrs-mlv+/− mtb wcl at all the pid was also statistically significant with reference to increase or decrease in lung arginase levels, respectively (fig. ) . three mock-inoculated and nine pigs each inoculated with prrs-mlv or prrs-mlv+ mtb wcl intranasally were euthanized on pid , or (n = pigs at each pid), and lymphoid cell subsets were enumerated by flow cytometry. a cd + and cd − cells were gated to enumerate cd and cd ␣ expression. b cd + cells were gated to enumerate cd and foxp expression and the percent of total cd + cd + foxp + cells are shown. each number is an average percent of immune cells from three pigs ± sem. * statistically significant difference (p < . ) between prrs-mlv+/− mtb wcl inoculated pig groups. a marked immunosuppressive response was observed in pigs inoculated intranasally with prrs-mlv alone which was likely attributed to induction of tregs, increased levels of il- , delayed and reduced secretion of ifn-␥, as well as low vn titers. similarly, earlier studies also reported comparable immunosuppressive responses in pigs administered prrs-mlv, also by parenteral route [ ] [ ] [ ] . apart from delaying the ifn-␥ response, prrsv preferentially promotes the synthesis of non-neutralizing antibodies [ ] . currently, development of mucosal vaccines against a variety of viral pathogens is gaining momentum to protect against predominant mucosal infections, such as influenza, parainfluenza, respiratory syncytial virus, rotavirus, and hiv/siv [ , , , ] . consistent with a high degree of compartmentalization, the mucosal immune system is populated by functionally distinct b cells, t cells, and accessory cell subpopulations comparable to populations present in systemic lymphoid tissues. the mucosal immune system is modulated by a variety of mechanisms involving innate immune cells such as dcs, nk cells, and mast cells which contribute significantly to host defense against pathogens [ , ] . studies have also demonstrated the mechanisms of induction of mucosal immune responses to live and inactivated, viral and bacterial vaccines with the help of potent adjuvants [ , , ] . the innate nk cell is one of the major players in antiviral defense. we detected an increased frequency of nk cells in prrs-mlv+ mtb wcl inoculated pigs; however, the nk cell frequency was comparable to mock pigs in prrs-mlv alone received pigs. these findings suggest that prrsv modulates nk cell function rather than its frequency in pig lungs. nk cells play two major roles in antiviral defense such as secretion of cytokines (predominantly ifn-␥), and lysis of infected and transformed cells. prrsv infection suppresses the nk cell cytotoxic function in pigs [ ] . in a recent study, pigs experimentally infected with a prrsv showed approximately a % reduction in their nk cell killing function just two days post-infection (dwivedi and renukaradhya , unpublished data) . now we report that prrs-mlv delivered intranasally also suppresses the nk cell cytotoxic function; however, when prrs-mlv was administered with mtb wcl, an increased frequency with moderate rescue in their function was observed. enhanced production of theth cytokine il- in the lungs of prrs-mlv+ mtb wcl inoculated pigs was detected early (pid and ). il- is critical for augmenting the innate nk cell function and is responsible for the induction of protective mucosal immunity against intracellular pathogens [ , ] . direct nk-dcs crosstalk in mucosally vaccinated animals with adjuvant resulted in enhanced innate and adaptive immune responses [ ] . neutralizing antibody response is a major component of the protective immunity to prrsv infection [ , ] . it has been demonstrated that prrsv infection induces a polyclonal activation of b cells in pigs [ , ] , leading to generation of non-neutralizing as well as auto-antibodies [ ] . also in prrsv-mlv administered pigs, enhanced production of non-neutralizing antibodies directed mainly to prrsv-nucleocapsid protein (internal viral protein), and also antibodies directed to anti-decoy neutralizing epitopes of the virus was detected [ , ] . in contrast, our study shows a reduction in the total prrsv specific antibodies with increased vn titers mediated by mtb wcl to prrs-mlv. generation of antibodies against mucosal pathogens and soluble protein antigens is dependent on cd + th cells [ , ] . consistent with that information, a polarized th -based response mediated via cd + and cd + t cells, and enhanced secretion of th and pro-inflammatory cytokines were detected in prrs-mlv+ mtb wcl inoculated pigs. generally, the th and th immune responses mutually suppress each other when either of one is significantly upregulated. therefore, our results clearly suggest that the mucosal adjuvant mtb wcl suppressed the secretion of prrsv specific non-neutralizing antibodies and at the same time promoted the secretion of neutralizing antibodies critical for prrsv clearance. apart from the effective humoral response, robust cmi response is critical for protective prrsv immunity [ , , , , ] . pathogen specific mucosal cd + t cells are required for the clearance of intracellular pathogens at both enteric and respiratory mucosal sites [ ] [ ] [ ] . consistent with those details, in prrs-mlv+ mtb wcl received pigs there was an increased frequency of cd + t cells and secretion of th cytokines in pig lungs. studies have reported enhanced immunosuppressive responses in prrsv infected pigs mediated through increased il- and reduced ifn-␥ production [ , ] . in pigs, il- reportedly inhibits ifn-␥ production by t cells [ ] . infiltration of tregs in the infected pig lung microenvironment contributes to secretion of high levels of il- and tgf␤ [ ] . in prrsv infected pigs, an increased frequency of tregs was detected indicating their involvement in disease progression [ ] [ ] [ ] . the immunosuppressive nature of prrsv-mlv was also reported to be mediated by increased tregs and il- which results in delayed and reduced ifn-␥ secretion [ ] [ ] [ ] . to elicit a protective cmi response to prrs-mlv, it is important to counteract the virus-induced immunosuppressive response. in our study, mtb wcl mediated an increased frequency of cd + and cd + t cells with enhanced secretion of th cytokines (ifn␥ and il- ), counteracted the virus induced immunosuppressive response by reciprocally downregulating the frequency of tregs and secretion of immunosuppressive cytokines. therefore, it is possible to protect pigs from both prrsv outbreaks and poly-microbial infections by adapting mucosal vaccination strategies with the use of a potent adjuvant. the pro-inflammatory cytokine, il- , plays an important role in initiating the adaptive immune response by influencing the proliferation of professional antigen presenting cells such as macrophages [ , ] . in our study, at an early time point (pid ), significantly increased secretion of il- mediated through mtb wcl was detected in pig lungs. associated with that finding, there was a significant increase in the myeloid cell population (cd + ) in tbln of pigs vaccinated with prrs-mlv+ mtb wcl. as a lymphoid organ, tbln is a site where sensitization and activation of immune cells take place; therefore, increased frequency of myeloid cells in tbln might indicate the mtb wcl-mediated initiation of the cmi response. arginase helps in the metabolism of l-arginine into l-ornithine and subsequently in the generation of l-proline, l-glutamate, and l-glutamine [ ] . a physiological level of arginase is essential in the body as arginase knockout mice die by weeks after birth [ ] . glutamine is an important fuel for lymphocytes and macrophages, and plasma glutamine is decreased in hiv infected individuals, where a decrease in the levels of glutamine impairs lymphocyte function [ ] . the amino acid proline promotes the growth and differentiation of b-cells and also nk cell activity [ , ] . therefore, one of the immune dysregulation mechanisms of prrsv in pig lungs appears to be mediated through downregulation of arginase, because reduced intracellular arginase was detected in pigs inoculated intranasally with prrs-mlv. therefore, mucosal immunization with mtb wcl has the potential to rescue an important molecular mechanism mediated by the enzyme arginase in pigs vaccinated with prrs-mlv. considering the modulation of function of arginase by prrs-mlv, the direct role of arginase on anti-prrsv specific mucosal immunity needs to be investigated in detail. in conclusion, mtb wcl is considered as a potent mucosal adjuvant for prrs-mlv in the pig respiratory tract. this adjuvant has the ability to potentiate innate, humoral, and cmi responses by modulating both cellular and molecular mechanisms required for generation of anti-prrsv specific protective immunity. further, in continuation of this study, adjuvanticity of mtb wcl was also confirmed in post-challenge studies which detected a cross-protective immunity to a genetically variant prrsv mn [ ] . considering the fact 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supplementation: two randomized trials a colostral protein that induces the growth and differentiation of resting b lymphocytes the role of natural killer cells in viral infections cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant this work was supported by national pork board award (npb # - ) and usda-nifa prrs cap award ( - - ) to rjg. salaries and research support were provided by the state and federal funds appropriated to oardc, the ohio state university. drs. juliette hanson, mahesh khatri, and hadi yassine and todd root helped us in animal studies. drs. dobos, belisle, eric nelson, and mike roof provided reagents. we also thank bert bishop for statistics, and dr. michele williams for editing the manuscript.role of the funding source: sponsors have no role in study design, in the writing of the report, and in the decision to submit the paper for publication. key: cord- -jnmogy s authors: yang, lin; ma, stefan; chen, ping yan; he, jian feng; chan, king pan; chow, angela; ou, chun quan; deng, ai ping; hedley, anthony j.; wong, chit ming; peiris, j.s. malik title: influenza associated mortality in the subtropics and tropics: results from three asian cities date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jnmogy s influenza has been well documented to significantly contribute to winter increase of mortality in the temperate countries, but its severity in the subtropics and tropics was not recognized until recently and geographical variations of disease burden in these regions remain poorly understood. in this study, we applied a standardized modeling strategy to the mortality and virology data from three asian cities: subtropical guangzhou and hong kong, and tropical singapore, to estimate the disease burden of influenza in these cities. we found that influenza was associated with . , . and . deaths per , population in guangzhou, hong kong and singapore, respectively. the annual rates of excess deaths in the elders were estimated highest in guangzhou and lowest in singapore. the excess death rate attributable to a/h n subtype was found slightly higher than the rates attributable to a/h n during the study period of – based on the data from hong kong and guangzhou. our study revealed a geographical variation in the disease burden of influenza in these subtropical and tropical cities. these results highlight a need to explore the determinants for severity of seasonal influenza. influenza is one of the respiratory pathogens that can cause serious health problems in both mortality and morbidity worldwide. however, unspecific presenting symptoms of influenza, absence of timely laboratory tests in clinical practice and frequent secondary bacterial pneumonia make it difficult to estimate the disease burden of influenza directly from the clinical diagnosed cases [ , ] . it is therefore necessary to assess influenza associated excess mortality or hospitalization by statistical modeling and use the estimate as a measurement for severity of influenza epidemics [ ] . some studies reported that the disease burden of influenza was nearly equivalent across some temperate countries [ ] , but others noticed that abbreviations: ili, influenza-like illness; icd, international classification of diseases; crd, cardiorespiratory diseases; p&i, pneumonia and influenza; copd, chronic obstructive pulmonary diseases; ihd, ischemic heart diseases. effects of influenza occasionally exhibited disparities between geographical areas even in the same influenza season [ ] . it has been proposed that socioeconomic factors and various circulating virus strains could play an important role in determining the severity of influenza epidemics [ ] [ ] [ ] . some environmental factors that are critical for survival and transmission of viruses, such as temperature and humidity, could also be involved as effect modifiers [ ] . however, geographical variations in influenza associated disease burden in the tropics and subtropics have not been explored. a recent large-scale phylogenetic study postulated that novel influenza viruses first emerge in the tropics and subsequently spread into the temperate when environmental factors favor virus transmissions [ ] . particularly, east and southeast asian countries are proposed as the potential reservoirs for dormant influenza viruses, although the mechanism remains unclear [ ] . unfortunately, there are few disease burden studies in this region to provide the evidence for or against the postulation, largely due to a lack of data from well-designed long-standing surveillance. this situation was further complicated by the unpredictable influenza seasonality in these regions. in this study we applied a standardized modeling strategy to three metropolitan cities in east and southeast asia: guangzhou, hong kong and singapore, all of which have well-organized surveillance networks for influenza for several years. guangzhou and hong kong, both located at southern china, share a similar subtropical climate with a mean temperature around • c, but the latter is more economically developed with a higher gdp per capita of $ , in hong kong as compared with that of $ in guangzhou in [ , ] . singapore is a tropical city with a higher mean temperature of . • c than guangzhou and hong kong. the socioeconomic level of singapore is comparable to hong kong, with a gdp per capita of $ , in [ ] . the difference and similarity between these cities, in terms of socioeconomic status and environmental factors, provide us a good opportunity to explore the factors that may affect the disparity or similarity across geographical areas. the influenza virology data in guangzhou, hong kong and singapore during - were obtained from the guang dong provincial center for disease control and prevention (gdcdc), the hong kong department of health (dh) and the singapore ministry of health (moh), respectively. the nasopharyngeal aspirate specimens were collected from the outpatients or inpatients presenting influenza-like illness (ili) symptoms of fever (≥ • c), cough or sore throat and then were tested for influenza by direct immunofluorescence or cell culture. positive samples were later typed or subtyped into a/h n , a/h n and b by haemagglutination inhibition (hi) test with strain-specific antiserum provided by the world health organization (who). mortality data for each city were obtained from hong kong census and statistics department (coded according to the international classification of diseases tenth revision, icd- ), guangzhou department of health (coded in icd- ) and singapore registry of births and deaths (coded in icd- ), respectively. we aggregated the weekly numbers of deaths with underlying cause of cardiorespiratory diseases (crd, icd- - ; icd- i -i , j -j ), pneumonia and influenza (p&i, icd- - ; icd- j -j ), chronic obstructive pulmonary diseases (copd, icd- - ; icd- j -j ), ischemic heart diseases (ihd, icd- - ; icd- i -i ) and all causes (icd- - ; icd- a -t ). two age groups were considered: all-ages and the elders aged years or over. we obtained the weekly mean temperature and humidity for three cities, from the national meteorological information center in china, hong kong observatory and singapore national environment agency, respectively. we fitted a poisson regression model to weekly numbers of mortality with the aim to assess the percentage of excess mortality associated with increased influenza activity in the population, as described in our previous studies [ , ] . briefly, long-term trends and seasonal patterns of mortality counts, as well as weekly average temperature and relative humidity, were added as confounders into the core models with their natural cubic spline smoothing functions. both temperature and humidity have been found to affect influenza virus transmission [ ] and also been associated with mortality in previous studies [ , ] . a typical form of core model is where yt denotes the death numbers at week t; ns(t), ns(temp t ) and ns(humd t ) denote the natural spline smoothing functions of time, temperature and relative humidity, respectively. auto-regressive terms in the residuals may be added to remove significant autocorrelations of residuals in first four weeks, so that time varying confounding could be adequately controlled. the weekly proportions of specimens positive for specific influenza types/subtypes (named as influenza proportions, flu t ) were simultaneously entered into the core model, to estimate influenza effects: the annual effects of influenza on mortality for each year were measured by excess mortality associated with influenza, which is defined as the sum of differences between the observed and expected death numbers when influenza proportions were assumed to be zero during that entire year [ ] . the % confidence intervals (ci) for estimates were derived by bootstrapping the scaled pearson residuals for times. we further derived the excess death rates associated with influenza per , population to render influenza effects comparable between cities. the percentage of excess deaths was calculated as the excess deaths divided by the total number of observed deaths. all the analyses were performed using the mgcv package of r software (version . . .) [ ] . two subtropical cities guangzhou and hong kong shared similar meteorological conditions, while the tropical city singapore tended to be hotter and more humid with much less variations in both temperature and humidity (table ) . among the three cities, hong kong had the largest population, while singapore had the youngest ( table ). the crude mortality rate of guangzhou was slightly higher than that of hong kong and singapore, respectively. more than % of deaths were in the or older age group in guangzhou and hong kong, whereas this number was only % in singapore. crd was the major cause of death in all three cities, but p&i mortality in guangzhou and copd in singapore was the lowest among the three cities ( table ) . the proportion of specimens positive for influenza a or b in singapore had an average of . %, which was lower than that in guangzhou and hong kong ( . % and . %, respectively) ( table ). this could be due to less stringent diagnosis criteria adopted by singapore for recruitment of patients into its surveillance network, using the case definition of acute respiratory illness instead of ili. influenza seasonality was similar between hong kong and guangzhou, but the winter peak was less pronounced in the latter. there were no clear epidemic periods in singapore, despite one sharp spike around june of (fig. ) influenza was significantly (p < . ) associated with all-cause mortality as well as with underlying cause of crd, p&i, copd and ihd for the all-ages group in all three cities, with the exceptions of copd in guangzhou and singapore, and ihd in singapore (table ) . during the study period of - , hong kong had the highest percentages of excess deaths associated with influenza among the three cities, with the only exception of ihd deaths. for the all-ages group, hong kong had the highest excess deaths for all causes and for underlying causes of p&i and copd, whereas guangzhou had the highest excess deaths due to crd and ihd. the excess death rates for the old population were highest in guangzhou for all-cause and crd death. note: ci, confidence interval; crd, cardiorespiratory diseases; p&i, pneumonia and influenza; copd, chronic obstructive pulmonary diseases; ihd, ischemic heart diseases. the annual death rates attributable to influenza revealed the greatest disease burden of influenza in the year for guangzhou and singapore, but in for hong kong. the year with the lowest disease burden was for hong kong and singapore, and for guangzhou (fig. ) . in guangzhou, an annual rate of . between the two types of influenza virus, influenza a accounted for the majority of excess deaths in all three cities (data not shown). as the denominator of subtype proportion of singapore was not comparable with that of the other two cities, we only estimated deaths attributable to influenza a subtypes, a/h n and a/h n , in guangzhou and hong kong. comparison between the two subtypes showed that a/h n accounted for slightly more excess deaths in the all-ages group for all the disease categories under study in both guangzhou and hong kong (fig. ) . this pattern was also observed for the + age group. to our best knowledge, this is the first comparative study between asian subtropical and tropical cities for influenza disease burden. the surveillance networks for influenza in guangzhou, hong kong and singapore were established under the who global influenza surveillance network [ ] , which allowed us to estimate the disease burdens in a standardized procedure and to compare across cities. here we adopted the poisson regression models to assess the age-disease specific excess mortality rates associated with influenza. allowing for flexible adjustment of seasonal factors, this poisson model has been widely used in many other disease burdens studies and is recommended for the subtropical and tropical regions with unpredictable influenza seasonality in a recently published who guideline [ ] [ ] [ ] [ ] [ ] . our recent validation study using an empirical dataset of laboratory confirmed influenza cases demonstrated that this model was able to provide reliable estimates for disease burden of influenza [ ] . although this approach requires sufficient long-standing year-round surveillance data, such data have become available in many subtropical and tropical regions in recent years. our study may serve as a good example for the disease burden studies across these regions. two previous studies found that the influenza associated mortality rates were nearly equivalent between hong kong and singapore, and singapore even had a higher excess rate for the older population [ , ] . however, these results need careful interpretation as these two studies differed in terms of statistical models, data aggregation and study periods covered. the present study adopted a standardized modeling approach to show that the overall influenza burden was comparable between the two subtropical cities guangzhou and hong kong, but lower in the tropical singapore. this geographical disparity was particularly evident in the elders, with the highest excess mortality rates associated with influenza found in guangzhou, which has a smaller proportion of old population than hong kong but is the least developed among the three cities. cohen et al. also found the mortality impact of seasonal influenza was higher in the elders of south africa than in those of the united states and this difference did not diminish after adjustment for age structures [ ] . together with our findings, it seems that the socioeconomic factors may associate with the mortality burden of influenza, particularly for the elders. another fig. . annual excess all-cause mortality rates associated with influenza (per , population) between guangzhou, hong kong and singapore, all-ages group. important factor that needs to be considered in interpreting our results is the different underlying susceptibility of population to severe outcomes of influenza infection across three cities. for example, the lower overall impact of influenza in singapore may not be due to its tropical climate, but partially due to its younger age structure. this heterogeneity of susceptibility could possibly been adjusted for by calculating age standardized excess mortality rates for each city, as the elders tend to be more fragile and have a higher mortality risk attributable to influenza. however, it is difficult to obtain reliable estimates for younger age groups due to small numbers of deaths. a future study with a longer study period shall be able to provide estimates with proper adjustment for age structures. singapore had the lowest disease burden from influenza among three cities, although fewer people in singapore had received influenza vaccination annually. in , doses of influenza vaccine were distributed for every total population in singapore [ ] , but a corresponding vaccination rate doses/ total population was reported in hong kong [ ] and doses/ total population in guangzhou (he jf, unpublished data). during - the annual distribution doses of seasonal influenza vaccines in singapore were around doses/ total population, less than half of those in hong kong [ ] . however, the effectiveness of influenza vaccines is largely complicated by age structure of population and also by various levels of herd immunity. moreover, the severity of infections could be aggravated by the environmental factors favoring virus transmission, such as air pollution [ ] . it has been widely cited that the disease burden of influenza, in terms of excess p&i mortality and hospitalization, tended to be higher in the a/h n dominant seasons than those with a/h n and b as the dominant virus strains [ ] [ ] [ ] . a/h n is believed to transmit more efficiently in human population than a/h n and antigenic drifts occurred more frequently in this subtype [ , ] . surprisingly, we found that the disease burden attributable to a/h n was slightly lower than the burden attributable to a/h n in guangzhou and hong kong. we noticed that after the outbreak of severe acute respiratory syndrome (sars) viruses in , all the three cities observed dramatically increased vaccination rates for influenza [ ] . we compared the excess mortality attributable to each subtype during the pre-sars period of - in hong kong, and the results showed that h n were associated with significantly more deaths than h n (data not shown). although a new strain a/california/ / -like gradually became prevalent in the late year of , it did not dramatically increase the mortality risks associated with influenza according to our results. some studies have shown that this novel strain mainly attacked the young adults and the vaccine recommended for the northern hemisphere during the - season was still able to provide crossprotection against it [ , ] . as a result, those susceptible people (the elders and chronic patients) might have still been benefited from increased vaccination and exempted from deaths. however, the disease burden of a/h n may have long been neglected, probably owing to its less frequent antigenic drifts compared to a/h n in the human community. recent experimental studies have found that seasonal h n strains can replicate as efficiently as pandemic h n and avian h n strains in ex vivo-infected lung tissues [ , ] . further experimental studies on virulence of different subtypes circulating in - may help to solve this mystery. more epidemiology studies with longer time series data from different climates are also needed to compare the relative severity of a/h n and a/h n strains. our study has potential caveats. firstly, these three cities vary in the organization of their influenza surveillance networks. most specimens in hong kong were mainly from the inpatients and therefore more severe cases of influenza infections were recruited into the surveillance. the sentinel doctors of guangzhou were all from the outpatient sectors, and the virology data exhibited greater variation than the other two cities due to limited numbers of samples taken each week. singapore had the lowest positive rates of influenza specimens, probably due to the fact this city included a mixture of samples from both inpatient and outpatient settings. nonetheless, we standardized the comparison procedure by using the age-specific excess mortality rate as measurement for influenza effects, which could at least partially exclude the bias introduced by different surveillance systems in these cities. secondly, although we carefully controlled for the confounding seasonal factors by building core models a priori, it is possible that there are still many unmeasured confounding factors. moreover, to what extent such confounding factors should be adjusted for was subject to personal judgement and over-fitting of core models cannot be completely avoided, as revealed by the negative estimates in some disease categories. thirdly, all three cities in this study might be more socioeconomically developed than the rest of asian cities; therefore, extra caution is needed to extrapolate our findings to the other cities/countries. last but not least, we did not quantify the statistical significance of differences between the city specific estimates, because they were derived from different models. but their overlapping confidence intervals suggest that the differences might not be statistically significant. in conclusion, by using a standardized modeling approach, we found a geographical disparity in mortality rates attributable to influenza, which could be partially explained by socioeconomic factors and age structure. in future, a multinational study comprising regions across temperate, subtropical and tropical countries like our present study could provide more information about mechanisms and severity of seasonal influenza and thereby help the policy makers to refine their regional specific control measures against influenza. clinical signs and 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be found, in the online version, at doi: . /j.vaccine. . . . key: cord- -xtwqv fk authors: hussain, althaf i.; cordeiro, melissa; sevilla, elizabeth; liu, jonathan title: comparison of egg and high yielding mdck cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xtwqv fk currently medimmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (laiv) from specific-pathogen free (spf) chicken eggs. difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. as part of medimmune's effort to develop the live attenuated influenza vaccine (laiv) using cell culture production technologies we have investigated the use of high yielding, cloned mdck cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both spf egg and mdck cell production technologies. in addition to cloned mdck cells the indicator cell lines used to evaluate the impact of producing laiv in cells on host range and replication included two human cell lines: human lung carcinoma (a ) cells and human muco-epidermoid bronchiolar carcinoma (nci h ) cells. the influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the – influenza seasonal trivalent vaccine (a/new caledonia/ / (h n ), a/wisconsin/ / (h n ) and b/malaysia/ / ). results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental mdck cell produced vaccine production platforms. medimmune's high yielding cloned mdck cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. both the a and nci h cells regardless of production system were less permissive to influenza a and b viruses than the mdck cells. irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. based on these study results we conclude that the mdck cell produced and egg produced vaccine strains are highly comparable. the currently licensed egg-derived influenza vaccines are efficacious, protecting up to percent of vaccine recipients [ ] [ ] [ ] [ ] . the recent threat of pandemic influenza outbreaks and anticipated increase in demand for seasonal influenza vaccine owing to expanded vaccination recommendations require evaluation of alternative non-egg based technologies for manufacturing influenza vaccines [ ] . the production of the egg-derived vaccine is not easily scalable and is limited by the availability of chicken eggs [ ] . the egg-based influenza vaccine manufacturing process is labor intensive requiring long term planning and long annual production cycles [ ] . these difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses have prompted the evaluation of cell culture vaccine production platforms as alternatives to egg dependent production [ ] . medimmune currently manufactures, a live attenuated virus (laiv) vaccine indicated for the active immunization of individuals - years of age against influenza disease caused by influenza virus subtypes a and b [ ] [ ] [ ] . as part of medimmune's effort to develop laiv using cell culture production technologies, we have investigated a biologically cloned madin-darby canine kid-ney (mdck) cell line that produces high titer of influenza virus as a substrate for production [ ] . to evaluate the impact of producing laiv in high yielding cloned mdck cells, we have assessed in vitro susceptibility and virus replication kinetics of influenza virus produced from these cells or using specific-pathogen free (spf) eggs. in addition to med-immune's cloned mdck cells we used two human cell lines to evaluate and compare infectivity and replication to assess whether the production system had an impact. the human cell lines used for evaluation are human lung carcinoma (a ) cells and human muco-epidermoid bronchiolar carcinoma (nci h ) cells. these cells previously have shown to have different susceptibilities to influenza viruses. a cells were used in this study because they support productive viral replication to levels and kinetics similar to the human primary lung cells [ ] . nci h cells have been previously shown to be good for isolation and replication of many human viruses including vaccinia virus, hsv, adenovirus, polyoma bk virus and paramyxoviruses [ ] . nci h cells have also been suggested as an alternate to primary rhesus monkey kidney cells for isolation and replication of human paramyxoviruses [ ] . viruses that do not replicate in nci h cells include cmv, vzv, sv- , respiratory coronaviruses and some influenza a and type b viruses. additionally, a and nci h cells have been studied to evaluate influenza virus replication, proinflammatory cytokine responses and influenza virus induced apoptosis [ , ] . the viruses used for these studies were the virus strains that were components of the northern hemisphere - influenza seasonal trivalent vaccine: ca a/new caledonia/ / (h n ), ca a/wisconsin/ / (h n ) and ca b/malaysia/ / ; each strain was produced in both mdck cultures as well as eggs [ ] . each vaccine strain was used to infect mdck, a and nci h cell lines at a specific moi. cell culture supernatants, cell lysates and formalin-fixed cell monolayers were collected through h post-infection. the respective test samples were analyzed by fluorescent focus assay (ffa), real-time qrt-pcr, immunofluorescence (if), western blot analysis and hemagglutination assay (ha). here we report the evaluation of the influenza virus produced in a high yielding mdck cell clone and compared it to the virus produced in spf chicken eggs. the high yielding mdck cells were very permissive to both egg produced (ep) and cell produced (cp) attenuated vaccine strains. both the a and nci h cells were less permissive to influenza a strains compared to mdck cells regardless of the production system. based on these study results we conclude that the cloned mdck cell produced and egg produced influenza vaccine strains are highly comparable. the three - seasonal vaccine components were produced using the egg and mdck cell production platforms. the test virus used represented egg and cell produced h n , h n and influenza b virus represented by a/new caledonia/ / , a/wisconsin/ / and b/malaysia/ / virus, respectively. the three seasonal strains were produced by a classical reassortment process according to the currently licensed laiv master virus seed (mvs) process. the cell produced virus for the study was generated from the same virus seeds used to manufacture the eggderived - viruses. briefly, the egg produced virus strains were produced in -to -day-old spf eggs inoculated with . ml of . log tcid /ml each. the inoculated eggs were incubated at ± in the presence of % humidity for h for h n and h n virus and h for influenza b virus. the virus containing allantoic fluid was harvested, stabilized with sucrose phosphate buffer and further processed for downstream purification. the final purified bulk was tested for virus concentration and stored at or below • c until further use. the egg produced viruses were subjected to four passages in eggs during the mvs manufacturing process. the mdck cell produced viruses were propagated in mdck cells using sufficient quantity of cells for bioreactor inoculation. the cells were allowed to attach to the microcarriers with controlled ph in the presence of co . viral infection of the mdck cell culture was achieved by allowing the microcarriers to settle and by addition of infection medium and appropriate volume of master virus seed stock. upon addition of infection medium and virus inoculum the cell culture was maintained at ± • c for h. at the end of infection the cell culture supernatant was collected and further processed through downstream purification. the final purified bulk was tested for virus concentration and stored at or below • c until further use. the human lung carcinoma (a ) cells atcc/catalogue # cc- lot no. were cultured in ham's f- k media supplemented with % fetal bovine serum (fbs) at ± • c in the presence of % co . the human muco-epidermoid pulmonary carcinoma (nci h ) cells atcc/catalogue # crl- lot were propagated in rpmi medium supplemented with % fbs. the mdck cloned cells a proprietary cell line developed in medimmune were first grown in roller bottles and then seeded in -well tissue culture (tc- ) plates prior to infection. a and nci h cells were all expanded in t- tissue culture flasks prior to infection in tc- well plates. the cells were seeded into tc- well plates at a seeding density of . × cells/well with ml complete growth media. the cells were incubated - h at ± • c and % co until confluent. the tc- well plates with confluent wells were washed once with ml hanks balanced salt solution (hbss) prior to infection. based on the cell count per well and virus titers, the egg produced (ep) and cell produced (cp) viruses were diluted in complete growth medium and then used to infect cells at an moi of . except for a which was also infected at an moi of . . the tc- well plates were inoculated with . ml inoculum and incubated at ± • c, % co for min. following virus adsorption the cell monolayer was washed with hbss and wells replaced with ml complete growth media. cell culture supernatants and cell lysates were collected at , , , , , , and h post-infection (pi) and stored at ≤− • c until testing. the test samples included clarified and stabilized cell culture supernatant, cell lysates for both rna extraction and western blot analysis and formalin-fixed cell monolayers. ffa, an antibody binding and staining method was used for the potency determination of influenza virus titers. cell supernatants collected through h pi were each appropriately diluted in virus growth medium. in the case of samples with low virus concentrations the dilution scheme was altered. briefly, -day-old atcc madin-darby canine kidney (mdck) cells in -well tissue culture plates were infected by inoculating l of appropriate virus dilutions and incubating the plates at ± • c in the presence of % co for h. polyclonal chicken antisera against the virus were used as the primary antibody and rabbit antichicken igg conjugated with fitc was used as the secondary antibody. they were used as to detect the infected cells prior to titer determination. total rna was isolated from each cell line using the rneasy ® mini kit (qiagen) according to the manufacturer's instructions, with minor modifications. briefly, l of rlt buffer was added to each tc- well and allowed to incubate at room temperature for min. the cells were then scraped and total rna was extracted with an rneasy ® spin column. purified rna was eluted into l of nuclease-free water and stored at ≤− • c until use. uninfected total cell rna was also extracted and used as control. rna was isolated from × sucrose phosphate buffer stabilized cell supernatants using the rneasy ® kit (qiagen) according to the manufacturer's spin technology instructions, with minor modifications. rna was extracted with an rneasy ® spin column plate. purified rna was eluted into l of nuclease-free water and stored at ≤− • c until use. real-time one-step rt-pcr reactions were carried out using the abi taqman ® one-step rt-pcr master mix according to the manufacturer's instructions with a total volume of l. no template control (ntc) reactions contained l of nucleasefree water. each sample was tested in duplicate. a -fold dilution series of purified influenza a or b rna with known virus titers (log ffu/ml) was performed and l of each standard dilution was run in triplicate to construct a -point standard curve. thermal cycling was performed in a fast real-time pcr system ht (abi). the rt step involved incubation at • c for min. the pcr cycling conditions included • c for min, cycles of • c for s and • c for min. the quantity, or copy number, for the test samples was calculated based on the standard curve and reported as rt-pcr log copies/ml. reverse transcription of rna with random hexamers was performed in a final volume of l with unit rnase inhibitor and l of sample or standard rna using the high capacity cdna reverse transcription kit (abi). multiplex real-time pcr reactions were carried out using the taqman ® universal pcr master mix according to the manufacturer's instructions with a total volume of l. primers and probes for m, pb , and np segments for both influenza a and influenza b were obtained from abi. in each reaction, the custom primers and probe were used at a final concentration of nm and nm, respectively, along with a × final concentration of eukaryotic s rrna taqman ® endogenous control, vic ® /mgb (abi). each sample was tested in triplicate. thermal cycling was performed in a fast real-time pcr system ht (abi) in standard mode. the pcr cycling conditions included a ung incubation of • c for min, denaturation of • c for min, followed by cycles of • c for s and • c for min. the quantity, or copy number, for the test samples is calculated and reported as log copies/ml. following harvesting the supernatants at each of the time points the cells in the tc plates were washed with × in pbs and fixed with % formalin for min at • c. the plates were washed twice with ml/well of pbs. sheep anti-a/new caledonia/ / , a/hiroshima/ / (cross-reactive with a/wisconsin/ / ) and b/malaysia/ / were used as primary antibodies at : dilutions. primary antibody treated plates were incubated at ± • c for h and washed with ml of tpbs (pbs with . % tween- ) with min interval between washes. : diluted fitc-conjugated secondary antibodies was added and plates incubated at ± • c for h and washed × with ml/well of tpbs. all of the wash fluid was removed and plates were air-dried at room temperature. the plates were screened for fluorescence using a fluorescent microscope and images were captured for analysis. proteins in the virus infected cell lysates were separated by electrophoresis using the biorad pre-cast gels under denaturing and reducing conditions. ten-well - % sds-polyacrylamide gels were used to size separate proteins in the samples. samples were denatured using sample buffer with ␤-mercaptoethanol and heated at • c for min. samples were normalized by protein concentration. biorad kaleidoscope and rainbow marker (amersham biosciences) molecular weight marker was used on each gel. electrophoresis was performed in running buffer at - v for . - h. following electrophoresis the size-separated proteins were transferred to pre-cut pvdf membranes in a biorad blotting module using × transfer buffer. proteins were transferred for h at v in an ice bath. pvdf membranes with transferred proteins were blocked overnight in a % solution of nonfat dry milk solution containing . % tween- . the blocked pvdf membranes were washed × in wash buffer and reacted for h on a rocker plate at room temperature with appropriate dilutions of sheep antisera to a/new caledonia/ / (h n ), a/hiroshima/ / (h n ), and b/malaysia/ / , respectively. after washing the pvdf membrane were reacted with goat anti-sheep igg-hrp conjugate for h on the rocker plate at room temperature. the viral proteins were detected using pierce, supersignal west pico chemiluminescent substrate on film. the hemagglutination (ha) assay was performed using freshly prepared . % chicken, turkey and guinea pig rbcs prepared in pbs. the culture supernatants harvested from through h postinfection were tested for hemagglutination activity. briefly, l of undiluted cell supernatant was added to the first well of a vbottom -well plates containing l/well dpbs. serial two-fold dilutions were performed and l of . % appropriate red blood cell suspension was added to each well and the -well plate was incubated undisturbed at room temperature (rt) for - min. ha results were recorded and results reported as log ha units. rna from supernatants from virus infected wells was extracted using qiaamp ® viral rna mini kit (qiagen) per manufacturer's instructions and l of it was used for generation of ha and na rt-pcr products for sequencing. following rt-pcr amplification single-tube microspin s- spin columns were used to clean up the amplicons. analysis and pcr product concentration were determined by agarose gel electrophoresis. sequencing was performed using prism bigdye ® terminator cycle sequencing ready reaction kit v. . (abi) using standard cycle sequencing conditions. unincorporated dye terminators were removed using a performa tm dtr gel filtration block and purified products were collected in a microamp ® pcr plate and electrophoresis for sequencing was performed using dna analyzer (abi) and the data files were processed using sequencher tm software fluorescent focus assay (ffa) was performed to quantitate infectious virus and compare replication kinetics of ep and cp influenza viruses in mdck, a and nci h cells. the virus titer measured using ffa was found to be comparable for both the ep and cp influenza viruses in all the indicator cells. in mdck cells infected at . moi, a difference of no more than . log ffu/ml in virus peak titer was observed between ep and cp a/new caledonia/ / , a/wisconsin/ / and b/malaysia/ / virus (fig. ) . results from the replication kinetics suggest mdck cells to be highly permissive to both cp and ep influenza a and b viruses. the peak virus titers for ep and cp h n , h n and influenza b virus were determined to be . ± . and . ± . , . ± . and . ± . and . ± . and . ± . , respectively. for both a and nci h cells the virus titer was below the limit of detection of the ffa (< . log ffu/ml) when infected with either ep or cp a/new caledonia/ / and a/wisconsin/ / at moi of . . detectable virus titers for ep and cp b/malaysia/ / virus infected a and nci h cells were observed for infection at moi of . . however, the peak virus titers were < log ffu/ml and suggest restricted permissiveness of both a and nci h cells to influenza virus infection using the study protocol (data not shown). results demonstrate medimmune's cloned mdck cells are highly permissive to influenza virus infection and irrespective of the production platform generate high titers of ca attenuated influenza viruses. to evaluate the effect of production substrate on replication kinetics of the influenza viruses total viral rna (log copies/ml) was measured in ep and cp infected mdck, a and nci h cells culture supernatants using qrt-pcr. comparison of ep and cp virus replication kinetics showed both were capable of efficient replication in mdck cells. the peak viral rna copy numbers for ep and cp a/new caledonia/ / , a/wisconsin/ / and b/malaysia/ / in mdck cell supernatant were determined to be . ± . and . ± . , . ± . and . ± . and . ± . and . ± . , respectively. similar to mdck cells, replication of both ep and cp influenza viruses in a and nci h cells was found to be comparable. in a cells infected at moi of . for egg and cp a/new caledonia/ / and b/malaysia/ / virus the peak viral copy number was determined to be . ± . and . ± . and . ± . and . ± . , respectively. the ep and cp a/wisconsin/ / virus replicated poorly in a cells. in nci h cells virus replication was severely restricted with h n and h n viral rna not detectable in culture supernatants for both the ep and cp viruses. similar to replication in a cells, replication of b/malaysia/ / was restricted in nci h cells for both ep and cp viruses (fig. ) . replication kinetics measured using viral rna copies like the ffa assay confirm that the virus production platform has minimal impact on replication of cold-adapted influenza viruses. the results also suggest cloned mdck cell to be highly permissive for both ep and cp influenza viruses. efficiency of intracellular viral replication was also evaluated by measuring pb , m and np segment rna expression kinetics using viral rna accumulation in infected whole cell lysates was comparable. accumulation of pb , m and np viral rna in infected mdck was higher for all three viruses compared to a and nci h cells and confirmed the highly permissive nature of mdck cells to influenza virus infection (fig. ) . results demonstrate intracellular influenza segment rna expression to be efficient in mdck cells compared to restricted replication kinetics in a and nci h cells. the level of pb , m and np segment rna in mdck cell lysates was found to be similar for both ep and cp influenza viruses. these results confirm minimal impact of the egg and cell production platforms on intracellular viral expression kinetics. to assess the impact of production platform on influenza virus protein expression western blotting was performed on cell lysates from cp and ep infected mdck, a and nci h cells. in mdck cells infected with a/new caledonia/ / , a/wisconsin/ / and b/malaysia/ / virus protein expression detected by western blot showed three major protein bands corresponding to ha , ha and ha as early as - h post-infection (fig. ) . the profile of ha protein expression was similar for both ep and cp h n , h n and b influenza virus, respectively. similarly viral proteins were also detected in a cells infected with a/new caledonia/ / and b/malaysia/ / virus and in nci h cells infected with both and egg and cell-based b/malsysia/ / virus (data not shown). the viral protein expression of ep and cp viruses was found to be comparable and confirm minimal impact of production platform on virus replication. to assess the effect of production platform on virus receptor binding properties hemagglutination activity in cell culture supernatants from cp and ep virus infected mdck, a and nci h cells was measured. the ha titers using chicken, turkey and guinea pig red blood cells for the ep and cp influenza viruses from infected mdck, a and nci h cells found to be comparable. [ ] ) when compared to wild-type ha sequence. the hemagglutinationinhibition (hai) analysis from this previous study comparing virus with and without the a n substitution revealed no differences between viruses. a broad range of cell substrates are available for development of human biologicals. however, only a few including wi- , vero, mrc- , cef and cho cells have earned acceptability among the regulatory agencies. different cell substrates for manufacturing biologicals are being considered for the development of new biologicals. this is especially true in the case of both pandemic and seasonal inactivated influenza vaccines. the cell substrates being evaluated and in use include vero, suspension and adherent mdck, table comparison of hemagglutination activity in cell and egg produced influenza b virus infected a cells. perc and insect cells [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . transgenic plants have also been shown useful to generate subunit influenza vaccines [ ] . these new substrates while useful for vaccine development also raise concerns of safety. assessing the safety and risks of using these novel substrates takes into account the advances made in manufacturing processes, purification technologies and methodologies to evaluate risks. the high yielding mdck clone used by medimmune is well characterized and has been shown to be safe for manufacturing laiv (data not discussed) [ ] . the cloned mdck cell line was developed from atcc cell line and a bank was established and later adapted to grow in serum-free growth medium. briefly, the high yielding mdck cells were initially cloned in serum containing medium and screened for virus productivity. these clones were later adapted to grow in serum-free conditions for generation of the accession and master cell bank. the cloned mdck cells have been well characterized and shown to be free of adventitious agents and have been demonstrated to be non-tumorigenic in animal models. the egg and cell produced influenza viruses showed remarkable similarity in their ability to infect cloned mdck, a and nci h cells. cloned mdck cells were highly permissive to infection by all three egg and cell produced influenza virus. a and nci h cells in comparison to cloned mdck cells were not permissive to a/new caledonia/ / and a/wisconsin/ / virus but allowed for limited but restricted replication of b/malaysis/ / virus representing both egg and cell production processes. based on in vitro substrate susceptibility, infection progression, receptor binding ability, replication kinetics, viral rna and protein expression and ha and na sequence comparisons, cold-adapted influenza virus generated using the cloned mdck cell-based production technology were found to be very similar to the laiv generated using the egg based manufacturing process. safety, efficacy and effectiveness of cold-adapted, live, attenuated, trivalent intranasal influenza vaccine in adults and children safety, efficacy and effectiveness 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vaccine influenza virus infection increased p activity: role of p in cell death and viral replication tnfand ifn-enhance influenza a virus induced chemokine gene expression in human a lung epithelial cells sensitivity of nci-h human lung mucoepidermoid cells for respiratory and other human viruses influenza a virus induced apoptosis in bronchiolar epithelial (nci-h ) cells limits pro-inflammatory cytokine release recommended composition of influenza virus vaccines for use in - influenza season. who genetic stability of live, cold-adapted influenza virus components of the flumist ® /caiv-t vaccine throughout the manufacturing process african green monkey (vero) cells provide and alternate host cell system for influenza a and b viruses cloning and assessment of tumorigenecity and oncogenicity of a madin-darby canine kidney (mdck) cell line for influenza vaccine production mdck-siat cell show improved isolation rates for recent human influenza viruses compared to conventional mdck cells the growth of attenuated influenza vaccine donor strains in continuous cell lines distinct host range of influenza h n virus isolates in vero and mdck cells is determined by cell specific glycosylation pattern safety and immunogenicity of a trivalent, inactivated, mammalian cell culturederived influenza vaccine in health adults, seniors and children the human cell line per.c provides a new manufacturing system for the production of influenza vaccines suitability of per.c cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics development of a novel recombinant influenza vaccine in insect cells plant expressed ha as a season influenza vaccine candidate egg egg mdck abbreviations used: crbc-chicken red blood cells; trbc-turkey red blood cells; gprbc-guinea pig red blood cells. key: cord- - xsbpid authors: condit, richard c; kim, denny; robertson, james s.; excler, jean-louis; gurwith, marc; monath, thomas p.; pavlakis, george; fast, patricia e.; smith, jonathan; smith, emily r.; chen, robert t.; kochhar, sonali title: the brighton collaboration standardized template for collection of key information for benefit-risk assessment of viral vector vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xsbpid many of the vaccines under development for covid- involve the use of viral vectors. the brighton collaboration benefit-risk assessment of vaccines by technology (bravato, formerly the viral vector vaccine safety working group, v swg) working group has prepared a standardized template to describe the key considerations for the benefit-risk assessment of viral vector vaccines. this will facilitate key stakeholders to anticipate potential safety issues and interpret or assess safety data. this would also help improve communication and public acceptance of licensed viral vector vaccines. in , the speed of vaccine development for covid- is unprecedented [ ] . keeping in mind the volume and pace of vaccine development, a systematic and deliberate approach to vaccine safety that is understandable and accessible to diverse stakeholders is of considerable importance. several viral vectored vaccines are among the covid- vaccines in development. the brighton collaboration (www.brightoncollaboration.us) was launched in to improve the science of vaccine safety [ ] . the brighton collaboration formed the viral vector vaccines safety working group (v swg) in october to improve the ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when a viral vector vaccine is licensed [ ] . pursuant to this goal, the v swg developed a standardized template that the coalition for epidemic preparedness innovations (cepi) and other key stakeholders could use to evaluate and communicate key considerations for the benefit-risk assessment of viral vectors and viral vector vaccines. the information in the template will help in the communication of technical and complex data among key stakeholders, and increase the comprehension, transparency and comparability of essential information (see table ). the viral vector vaccine template and the mission of the v swg has evolved over time. the first version of the template (v . ) was used for the standardized benefit-risk assessment of several new viral vectors or viral vector vaccines [ ] [ ] [ ] , including a vaccine targeting ebola. the who global advisory committee on vaccine safety (gacvs) endorsed the use of the viral vector template for other new candidate ebola vaccines ''as it is a structured approach to vaccine safety" [ ] . a second version of the template (v . ) was used to describe viral vectors based on adenovirus and modified vaccinia virus ankara (in preparation). experience with earlier versions of the viral vector template and with other vaccine platform templates under development by the v swg inspired improvements included with the template presented here. a detailed history of the development of the viral vector vaccine template is archived on the brighton collaboration website (https://brightoncollaboration.us/bravato/). the v wsg has recently been renamed to the benefit-risk assessment of vaccines by technology (bra-vato) working group to reflect its expanded role in development of templates for additional vaccine platforms, namely nucleic acid based, live attenuated, inactivated, and protein based vaccines [ ] . viral vector vaccines are laboratory-generated, chimeric viruses that are based upon replicating or non-replicating virus vectors into which have been spliced genes encoding antigenic proteins for a target pathogen. consideration of safety issues associated with viral vector vaccines requires a clear understanding of the agents used for construction of the vaccine. these include ( ) the wild type virus from which the vector is derived, referred to in the template as ''wild type virus"; ( ) the vector itself before incorporation of the foreign antigen, referred to in the template as ''viral vector"; and ( ) the final recombinant viral vector vaccine, referred to in the template as ''vaccine". wild type viruses used as vectors may originate from human or animal hosts and may have low or high pathogenic potential in humans regardless of species of origin. understanding the characteristics of the wild type virus as directed in the template is critical in anticipating the potential behavior of any vector adapted from the wild type virus. viral vectors can originate from attenuated viral vaccines used in humans (e.g. yellow fever, modified vaccinia virus ankara); from attenuated human or animal viruses (e.g. human adenovirus, vesicular stomatitis virus); or from human or animal viruses with low pathogenic potential (e.g. adeno associated virus, chimp adenovirus). viral vectors can be replicating (e.g. vesicular stomatitis virus) or non-replicating (e.g. modified vaccinia virus ankara). viral vectors usually, but not always, have properties in a human host that differ from the wild type virus from which they were derived. incorporation of a target antigen into a viral vector to create a vaccine may alter the properties of the vector such that the vaccine may have properties that differ from the vector. this updated version of the brighton collaboration vaccine vector template is designed for dual use. it may be used to describe exclusively viral vectors into which transgenes may be incorporated to create vaccines, or it may be used to describe viral vector vaccines for specific pathogens. thus, the template has two main parts. part i is used to describe a viral vector and part ii is used additionally to describe a specific vaccine, where this is the intent. pursuant to understanding completely the characteristics of a given vector, part i considers the wild type virus from which the vector is derived (section ) in addition to characteristics of the vector itself (sections and ). pursuant to understanding completely the characteristics of a vaccine, part ii additionally considers the target pathogen (section ) and the potential impact of transgene insertion to create a vaccine (section ). each part contains its own sections evaluating the toxicology, adverse effects and overall assessment of either the vector alone or a vaccine. when the template is being used to characterize a viral vector vaccine, it is understood that there may be limited information concerning the vector itself, especially concerning toxicology and potency of the vector (section ), and section on adverse events may not be relevant. vaccine developers should nevertheless complete section i to what extent this is feasible. bravato intends that this template focuses on key questions related to the essential safety and benefit-risk issues relevant for the intrinsic properties of the vaccine components. we recognize that there are many other aspects of manufacturing, quality, and implementation that can play an important role in the safety of a vaccine, but we have chosen to keep some of those issues out of scope for the template in order to summarize information that is the most useful to the most stakeholders. the latest version of the template can be accessed on https:// brightoncollaboration.us/bravato/. vaccine developers are encouraged to complete the relevant templates for their vaccine candidate platform or vaccine candidate and collaborate with bravato. the draft templates would be shared for review by bra-vato and submitted for publication. similarly, updates to the templates by the vaccine developers should be submitted to the brighton collaboration website for bravato review. see supplementary material for definitions and additional guidance for completing this template. please read these instructions before you complete the thirteen sections. send questions to:brightoncollaborationv swg@gmail.com the first section entitled ''authorship and affiliation" should include your name, your affiliation and the latest date completing the form. if you are working with someone else to complete this form, their name and affiliation should be provided as well. if you are updating the form, please provide the updated date. these co-authors will be included in the final published template in vaccine once reviewed and approved by bravato and in subsequent wiki updates on the bravato website. part i collects information regarding a viral vector alone, while part ii collects information regarding a vaccine based on the viral vector. if the template is being used to describe a vector only, then complete part i only. if the template is being used to describe a vector vaccine, then complete both parts i and ii. within part i, sections - collect information regarding the wild type virus (section ) and the vector (sections and - ). within part ii, section collects information regarding the target pathogen and population while sections - collect information regarding the vaccine based on the vector. depending on the circumstances, some sections may be redundant, for example if a vector is in fact identical to the wild type virus. in cases of redundancies, an answer may simply refer to the answer in another section. furthermore, some sections may not be applicable, for example if safety evaluations have been conducted only in the context of a vaccine and not with an empty viral vector alone. in such cases the answer should include ''not applicable" or ''not tested", whichever is relevant. whether competing only part i or both parts i and ii, any supplementary information should be added in section . answer questions by responding in the column entitled 'information.' if you have any comments or concerns regarding the question or your answer to the question, note these in the 'comments/concerns' column. finally, please provide references wherever possible in both the ''information" and ''comments/concerns" columns. referencing should use the vaccine journal format, with references numbered sequentially in the text and full citations listed in sequence at the end of the form. more than one reference can be used per question. sections , , and have column titles that differ from preceding sections intended to provide a summary assessment of adverse effects and toxicity of the vector. please summarize adverse effect and toxicities as requested and rate the risk in the following fashion: none, minimal, low, moderate, high, or unknown. if there is insufficient data for use of the vector in humans to accurately make these assessments, please state so in response to the questions. when completing information on adverse effects in sections and , please provide as many details as possible based on the brighton collaboration guidelines for collection, analysis and presentation of vaccine safety data in pre-and postlicensure clinical studies [ ] . in the references, unpublished data and non-peer reviewed published data are acceptable, though we do wish that you include the source and contact information. if a literature search was conducted to complete any of the sections (strongly encouraged), please provide the following information in the reference section: ( ) the findings, opinions, conclusions, and assertions contained in this consensus document are those of the individual members of the working group. they do not necessarily represent the official positions of any participant's organization (e.g., government, university, or corporations) and should not be construed to represent any agency determination or policy. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper the covid- vaccine development landscape the brighton collaboration: addressing the need for standardized case definitions of adverse table (continued) events following immunization (aefi) the brighton collaboration viral vector vaccines safety working group (v swg) live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment rvsvdg-zebov-gp (also designated v ) recombinant vesicular stomatitis virus pseudotyped with ebola zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment global advisory committee on vaccine safety the brighton collaboration standardized templates for collection of key information for benefit-risk assessment of vaccines by technology (bravato; formerly v swg). vaccine guidelines for collection, analysis and presentation of vaccine safety data in pre-and post-licensure clinical studies we thank the following colleagues for their helpful advice: ( ) margaret liu, brighton collaboration members; and ( ) other members of bravato during the preparation of this document: david wood, karin bok, najwa khuri-bulos, bettina klug, and merita kucuku. we acknowledge the financial support provided by the coalition for epidemic preparedness innovations (cepi) for our work under a service order entitled safety platform for emergency vaccines (speac) project with the brighton collaboration, a program of the task force for global health, decatur, ga. key: cord- -pho dksc authors: huang, jun; ma, rui; wu, chang-you title: immunization with sars-cov s dna vaccine generates memory cd (+) and cd (+) t cell immune responses date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: pho dksc an effective vaccine for severe acute respiratory syndrome (sars) will probably require the generation and maintenance of both humoral and cellular immune responses. it has been reported that after natural infection in humans and immunization in animals with sars-cov vaccine, antibody is produced and persistent for a long period of time. in the present study, mice were immunized i.m. with sars-cov s dna vaccine, and three different methods (elisa, elispot and facs) were used to evaluate the immune responses when the cells were stimulated in vitro with a pool of peptides overlapping entire sars spike protein. the results show that prime-immunization with sars-cov s dna vaccine can induce both cd (+) and cd (+) t cell responses. boosting with the same vaccine enhances cd (+) and cd (+) t cell responses in both lymphoid and nonlymphoid organs and were persistent over two months. the sars-cov s-specific cd (+) and cd (+) t cells were cd l(−), a marker for memory cells, and − to % of the cells expressed il- rα (cd ), a marker for the capacity of effector cells to develop into memory cells. in addition, immunization with the dna vaccine elicited high levels of antibody production. taken together, these data demonstrate that immunization with sars-cov s dna vaccine can generate antigen-specific humoral and cellular immune responses that may contribute to long-term protection. severe acute respiratory syndrome (sars) is a new emerging infectious disease that is caused by a novel coronavirus named as sars-cov [ , ] . the sars-cov genome consists of approximately , bp and encodes four main structure proteins including spike protein (s), membrane protein (m), envelope protein (e) and nucleocapsid protein (n). among them spike protein is responsible for binding to specific receptor on the susceptible cells. additionally, the gene sequence of the spike protein is comparably constant [ , ] . it has been reported that patients infected with sars-cov had high levels of antibodies in sera. these antibodies could inhibit the infection of cells with pseudotyped lentiviral particles bearing sars-cov s protein and be used to treat sars-cov-infected patients [ ] [ ] [ ] [ ] . recently, several reports including ours have demonstrated that sars-cov s specific memory cd + t cells were generated and maintained in vivo in the patients with fully recovered from sars-cov infection [ ] [ ] [ ] . these data indicate that the spike protein is a good target for the vaccine to generate immune responses against the sars-cov infection. in animal studies, a number of increasing evidence demonstrated that immunization of animals with sars-cov s dna vaccine lead to the generation of long-term neutralizing antibodies. these antibodies are effective in reducing sars-cov replication in lungs when mice immunized with sars-cov s dna vaccine were then challenged with sars-cov [ , ] . although it has been reported that cd + t cell-mediated immune responses against sars-cov s protein in animals are generated after sars-cov s dna vaccination, it remains unclear whether cd + and cd + t cells are able to become memory cells. in this regard, our study was mainly focused on determining sars-cov s specific effector/memory t cell responses after mice are immunized i.m. with sars-cov s dna vaccine. our results demonstrate that immunization of mice with sars-cov s dna vaccine results in the generation of both cd + and cd + t cell responses. these cd + and cd + t cells are persisted for over a long period of time with a phenotype of memory cells. these data provide important information in animal models for analyzing antigenspecific memory cd + and cd + t cell responses and rational design effective vaccine against sars-cov infection based on sars s protein since it is not known whether sars-cov reoccurs and infects humans through animal reservoirs. female balb/c mice, - weeks old, were purchased from zhongshan university animal center (guangzhou, china) and maintained in our animal care facility under pathogen-free conditions. complete rpmi- media was purchased from gibco, and supplemented with % heat-inactivated fcs, . % -me, u/ml penicillin and ug/ml streptomycin. purified anti-cd and anti-cd /cd , anti-cd -percp, anti-cd l-fitc, anti-cd -apc-cy , anti-ifn-␥-pe, anti-il- -apc and isotype-matched control abs were purchased from bd/pharmingen (san diego, ca). anti-il- r␣-fitc was obtained from ebioscience (san diego, ca). plasmids encoding sars-cov spike (s) protein was constructed as described and kindly provided by dr. gary j. nabel from vaccine research center, niaid, national institutes of health, bethesda, md, usa. plasmid dna was purified by plasmid purified kit (qiagen, usa). the / ratios ranged from . to . . the endotoxin content from purified plasmid dna was found below units/ml. the endotoxin level within this range had no effect on the immune response. a pool of peptides ( - -mer) overlapping by spanning the entire sars-cov s protein were synthesized and kindly provided by dr. richard karp from vaccine research center, niaid, national institutes of health, bethesda, md, usa. female balb/c mice were injected either i.m. (thigh) or s.c. (foot-pad) with ug/mouse of sars-cov s plasmid dna in ul of sterile pbs. mice were boosted at - week intervals. mice were sacrificed. inguinal and popliteal lymph nodes (ln), spleen and lungs from individual mice were harvested one week before and after boost vaccinations. single-cell suspensions were prepared and plated in triplicate in a -well microtiter plate at × cells/ ul. a pool of sars-cov s peptides at ug/ml of the final concentration of each peptide and anti-cd mab ( ug/ml) were added to cultures. in each experiment, as a negative control, ug/ml anti-cd was added at the absence of sars-cov s peptides. seventy-two hours after incubation, cell-free culture supernatants were collected, and levels of ifn-␥ and il- were assessed by specific elisa kit (bd pharmingen) according to the manufacturer's protocol. the lower limits of detection for ifn-␥ and il- were both . pg/ml. the titers of mouse anti-sars s protein antibody were measured as described previously [ ] . briefly, -well plates were pre-coated overnight with transmembrane-domaintruncated sars-cov s protein-cell supernatants. serum was obtained from mice before and after vaccination. the plates were washed, blocked for h in % fbs and then washed again. the diluted serum samples were added to the wells in triplicate for h at room temperature, the plates were washed, and horseradish peroxidase-conjugated anti-mouse igg (jakson, usa) were added at / dilution for h at room temperature. after washing, the plates were developed with tetramethylbenzidine (tmb) and hydrogen peroxide (bd pharmingen) and read it using elx universal microplate reader (bio-tek, usa). ifn-␥-producing cells were assessed by specific elispot kit (diaclone, france) according to the manufacturer's protocol. in brief, single-cell suspensions were prepared from lymph nodes and spleens of mice after vaccination, and plated in -well microplates pre-coated with anti-ifn-␥ antibody specific for elispot. cells were incubated overnight in the presence or absence of pooled sars-cov s peptides ( ug/ml) and anti-cd ( ug/ml). the plates were then washed and alkaline phosphatase-conjugated anti-mouse ifn-␥ antibody was added, developed with ready-to-use bcip/nbt, and read by champspotii elispot reader (sage creation, china). single-cell suspensions from the lymph nodes, spleens and lungs of mice after vaccination were stimulated with or without pooled sars-cov s peptides at ug/ml of the final concentration of each peptide and anti-cd ( ug/ml) for a total of h. brefeldin a (bfa, ug/ml) was added into the culture at the end of first hour during the incubation. the cells were washed, fixed with % paraformaldehyde, and permeabilized with pbs containing . % saponin (fluka, sigma, usa) plus . % bsa buffer overnight at • c. the cells were then blocked for min with ug/ml of anti-cd /cd mabs in pbs containing . % bsa, and stained with anti-cd , anti-cd , anti-ifn-␥, anti-il- , anti-il- r␣ and cd l for min at • c. cells ( , - , ) were acquired on a facscalibu flow cytometer (bd biosciences) and facs data were analyzed using cellquest software (bd biosciences). isotype-matched controls for cytokines were included in each staining. statistical evaluation of differences between means of experimental groups was done by analysis of variance and a non-parametric two-tailed t-test. a value of < . was considered to be significant. to assess the production of cytokines following the priming injections, balb/c mice were immunized i.m. with sars-cov s dna vaccine. seven days after vaccination, mice were sacrificed and cells were prepared from spleen and lymph nodes. the cells were stimulated with or without a pool of sars-cov s individual peptides overlapping entire sars s antigen and anti-cd mab to assess sars-cov s-specific immune responses. the levels of ifn-␥ and il- in the cell-free culture supernatants were determined by elisa. as a negative control, cells stimulated with anti-cd mab alone did not produce any cytokines, indicating that cytokine production was specific for peptide antigen (data not shown). the frequency of ifn-␥-producing cells at the single cell level was determined by elispot assay before and after boost immunization. as shown in fig. , seven days after fig. . frequency of sars-cov s-specific ifn-␥ producing cells are increased following prime and boost dna immunization. mice were vaccinated in a similar manner as described. one week after boost, cells from spleen and lymph node were cultured at a density of × cells/well with ug/ml of pooled peptide and anti-cd in the well pre-coated elispot plate. after incubation for h, elispot assay was performed as described in section . the empty circles represent the results in the absence of peptides and solid circles represent the results in the presence of peptides. * p < . ; ** p < . ; n.s. p > . . prime immunization with sars s dna vaccine, cells from spleens but not from lymph nodes produced ifn-␥ at a range of - spots in × cells following stimulation with a pool of sars s peptides plus anti-cd . moreover, mice boosted with sars s dna vaccine exhibited a - -fold increase in the frequency of ifn-␥-producing cells in spleens (p < . ) and lymph nodes (p < . ), respectively (fig. ) , compared with the prime immunization. these data demonstrate the striking enhancement of cellular immune responses after boost immunization with sars s dna vaccine. we next analyzed the immune responses of t cell subsets generated after prime and boost immunization using flow cytometry. to determine t cell responses after prime vaccination, mice were immunized i.m. with sars-cov s dna vaccine. seven days after immunization, cells from lymph nodes, spleens and lungs were prepared and stimulated with a pool of sars-cov s peptides. intracellular cytokines and cell surface markers were stained. initially, a lymphocyte-enriched population was selected from total cells by forward/side scatter gating, cd + and cd + t cells ( fig. a) were then gated and analyzed for their expression of ifn-␥ and il- . as shown in fig. b , sars-cov s-specific cd + and cd + ifn-␥-producing t cells were detected in fig. . sars-cov s-specific cd + and cd + t cells are generated following prime and boost vaccination. mice were vaccinated as described in fig. . cells were prepared from lymph nodes (ln), spleen and lungs, and incubated with a pool of sars-cov s peptides and anti-cd for h. cd + and cd + t cells were first gated (a), ifn-␥ and il- -producing cd + and cd + t cells were determined by intracellular cytokines staining as described in section following prime (b) and boost (c) vaccination. the frequency of ifn-␥ + and il- + cells was indicated as percentage of cd + and cd + t cells. lymph nodes, spleens and lungs after prime immunization. the frequency of ifn-␥ + cells in the cd + t cell population was markedly higher than that in the cd + t cells in each of the organs tested. in the same experiment, the frequency of il- -producing cells in the cd + and cd + t cell populations were analyzed in a similar manner. as shown in fig. b , il- + cells were detected in cd + t cells but very few il- -producing cells were detected in cd + t cells. to further ascertain whether the frequency of sars-cov s specific cd + and cd + t cell responses was increased after boost vaccination, mice were boosted i.m. with sars-cov s dna vaccine, seven days after injection, ifn-␥-and il- -producing cd + and cd + t cells were determined in lymph nodes, spleen and lungs. as shown in fig. c , the vaccine boost dramatically increased the frequency of cd + and cd + ifn-␥-producing t cells in spleens and lungs but not in lymph nodes. in the same experiment, the frequencies of cd + and cd + il- -producing t cells did not change in any of the tissues tested. moreover, we compared the route of immunization with sars-cov s dna vaccine side by side. in this regard, mice were immunized and boosted either s.c. or i.m. and the frequency of antigen-specific ifn-␥-producing cd + and cd + t cells were determined by facs. the results showed that s.c. immunization induced the highest immune responses of cd + t cells in lymph nodes than in spleen and lungs (fig. a) , whereas there was no markedly different responses of cd + t cells among all organs (fig. b ). immunization of mice by i.m. injection elicited the highest frequency of antigen-specific ifn-␥ expression of cd + t cells in lung than in lymph nodes and spleen. similar results were obtained for cd + t cell responses. in general, vaccination via i.m. induced better immune responses than s.c. injection. thus, we choose i.m. immunization for further study. to explore cell subsets of ifn-␥ and il- -producing cd + and cd + t cells at the single cell level, the intracellular cytokine staining were performed and analyzed. as shown in fig. , based on the expression of ifn-␥ and il- , the cd + t cells could be divided into three subpopulations: ifn-␥ + , il- + and ifn-␥ + il- + . however, in the cd + t cells majority of the cells were ifn-␥ + , only a small population of the ifn-␥ + il- + cd + t cells could be detected in the spleen and very few single il- + cd + t cells were observed. these data demonstrate that the ifn-␥-and il- -producing cells constitute distinct, but over-lapping subsets of cd + t and cd + effector/memory populations. in addition, it is noted that in figs. and , before dna vaccine boost immunization, the frequency of ifn-␥ + cells in cd + and cd + t cell population was no different in lymphoid organs (nodes and spleen) and non-lymphoid organ (lungs). however, after boost vaccination, the frequency of ifn-␥ + t cell population was dramatically increased in the fig. . immunization via i.m. results in higher frequency of sars-cov s-specific ifn-␥ producing cells than that of s.c. mice were immunized via either i.m. or s.c. with sars-cov s dna vaccine. one week after boost, cells from lymph nodes, spleen and lungs were cultured and stained as described in fig. . the results were expressed as a mean percentage (three to five mice) of ifn-␥-producing cells in cd + t cells (a) and cd + t cells (b). spleens and lungs, especially in the lungs for the cd + t cells population. in the same experiment, sars-cov s antigenspecific cd + t cells expressed higher frequency of il- producing cells than cd + t cells in spleens and lung, consist with enrichment of effector/memory t cells from spleen to non-lymphoid organs. in previous experiment, we assessed the cellular immune responses seven days after prime or boost vaccination. here, we extended our study to further characterize sars-cov s specific long-persistent memory t cell response weeks after the final immunization. cd + and cd + t cells from lymph nodes, spleens and lungs were divided into il- r␣ (cd ) + and cd l + subpopulations which have been shown in mice to distinguish naïve and memory t cells [ , ] . as shown in fig. a , ifn-␥-producing cd + t cells from lymph nodes, spleen and lungs were il- r␣ + . in contrast, ifn-␥-producing cd + t cells were cd l − . similar results were also obtained for il- -producing cd + t cells (fig. b ). in addition, ifn-␥-producing cd + t cells could fig. . distinct populations of sars-cov s specific ifn-␥ and il- -producing cd + and cd + t cells are persistent in lymphoid and non-lymphoid organs following prime and boost dna vaccination. mice were vaccinated as indicated in fig. following prime (a) and boost (b) dna vaccination. cells were prepared from lymph nodes (ln), spleens and lungs, and incubated with a pool of sars-cov s peptides and anti-cd for h. cd + and cd + t cells were first gated, the frequency of ifn-␥ and/or il- -producing cells were analyzed within the population of cd + and cd + t cells. results are representative of three separate experiments with similar results. the numbers at the corner in each sample represent the percentage of cytokine-producing cells. be separated into il- r␣ + and il- r␣ − subsets (fig. a) . however, consistent with ifn-␥-producing cd + t cells, ifn-␥-producing cd + t cells were cd l − . interestingly, the expression of il- r␣ and cd l in il- -producing cd + t cells were similar to il- -producing cd + t cells (fig. b) . these data demonstrate that long-lasting cd + and cd + t cells specific for sars-cov s antigen in vivo were memory phenotype. we finally assessed the production of antigen specific antibody induced by the sars-cov s dna vaccination. as shown in fig. , the sera obtained from the immunized mice but not from non-immunized mice had significantly higher amount of sars-cov s specific antibody, indicating that sars-cov s dna vaccine could induce strong humoral immune response that is consistent with the previous report. it has been reported that dna vaccination is a practical and effective way to induce humoral and cellular immune responses and has shown great promise for protective immune responses against several diseases in experimental animal models including hiv, tuberculosis and malaria [ ] [ ] [ ] . in the present study, we have demonstrated that prime-boost immunization of mice with sars-cov spike dna vaccine induces antigen-specific cellular and humoral immune responses. the cellular immune responses are mediated by both cd + and cd + t cell. functional study indicates that antigen-specific cells produced cytokines ifn-␥ and il- from cd + and cd + t cells after short stimulation in vitro with a pool of sars-s peptides. ifn-␥ is an effector cytokine that is able to activate macrophage cells and inhibit viral replication [ ] , and il- is a growth factor that probably maintains memory cells and mediates expansion of both cd + and cd + t cells [ ] . based on ifn-␥ and il- expression, memory t cells are able to divide into three subpopulations including ifn-␥ + , ifn-␥ and il- + , il- + cells, indicating that memory t cells are composed of distinct functional subsets. phenotype analysis indicates that sars-cov s specific cd + and cd + t cells are cd l − . some of the memory cd + and cd + t cells express il- r␣. these populations of the cells may have the potential differentiate into long-term memory cells [ ] . it has been reported that il- is mainly secreted by the stromal cells and plays a key role in sustaining the homeostasis of memory cd + t-cell in vivo [ , ] . in line with other studies, we also find most of the memory cd + and cd + t cells were identified in non-lymphoid tissues (lungs) in which these memory t cells are called effector memory cells [ , ] . thus, by their location, effector mem-ory t cells can respond rapidly when re-exposed to a pathogen and may play the most important role in the defence when re-encountering a pathogen. furthermore, the function of non-lymphoid effector memory cells has been demonstrated in a study in which sendai virus specific memory cd + t cells persist in the lungs, and transfer of these cells into ␤ -microglobulin-dificient mice confers a significant level of protection against re-infection [ ] . consistent with our observations, several reports demonstrate that natural infection in humans induces memory cd + t cells responses [ ] . moreover, immunization of mice with adno-vector encoding codon-optimized sars-cov s protein generates two h- brestricted epitopes and one h- d -restricted epitopes of cd + t cells [ ] . in addition, yang et al., reported that vaccination of mice with sars-cov s dna generated cellular and humoral immunity to sars-cov s glycoprotein. the neutralizing antibody can inhibit sars-cov replication and results in protective immunity in a murine challenge model. in adoptive transfer study, they found that antibodies but not fig. . sars-cov s protein-specific antibody responses following dna vaccination. balb/c mice were immunized i.m. as described in section . sera from normal and immunized mice were collected at day after boost vaccination. antibody titers in sera were determined at different dilutions by elisa specific for sars-s protein. data are expressed as the mean value ± s.d. for each group. a non-parametric two-tailed t-test was used for statistical analysis with similar result; * p < . . t cells play a critical role in protection against sars-cov challenge [ ] . thus, it is still unclear whether t cells may play a role in the generation of protective immune responses. we assume that antigen-specific cd + t cells reveal a direct killing of virus-infected cells and cd + t cells may provide help for antibody isotype switching and generation of memory b cells and memory cd + t cells [ , ] . in support of our expectation, other studies of animal coronavirus have suggested that both cellular and humoral immunity contribute to protection during persistent infection [ ] [ ] [ ] [ ] [ ] . taken together, our results show that immunized mice with sars-cov s dna vaccine can generate both cellular and humoral immunity that may be useful for preventive and therapeutic vaccine for sars-cov infection. coronavirus as a 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t cells to severe acute respiratory syndrome (sars) coronavirus in recovered sars patients and healthy individuals long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine detection of specific antibodies to severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein for serodiagnosis of sars coronavirus pneumonia functional properties and lineage relationship of cd + t cell subsets identified by expression of il- receptor alpha and cd l selective expression of the interleukin- receptor identifies effector cd t cells that give rise to long-lived memory cells dna vaccines against human immunodeficiency virus type tuberculosis vaccines: current progress progress in dna-based heterologous prime-boost immunization strategies for malaria distinct lineages of t h cells have differential capacities for memory cell generation in vivo il- and related cytokines can promote t cell survival by activating akt cytokines control of memory t-cells development and survival interleukin regulates the survival and generation of memory cd cells lineage relationship and protective immunity of memory cd t cell subsets similarities and differences in cd + and cd + effector and memory t cell generation differential contributions of central and effector memory t cells to recall responses identification of murine cd t cell epitopes in codonoptimized sars-associated coronavirus spike protein cd + t cells are required for secondary expansion and memory in cd + t lymphocytes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice mucosal immunisation of african green monkeys (cercopithecusaethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity induce of th type response by dna vaccinations with n, m and e genes against sars-cov in mice augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines we are grateful to drs. gary j. nabel, zhiyong yang, key: cord- -wyy rvqb authors: ashwell, douglas; murray, niki title: when being positive might be negative: an analysis of australian and new zealand newspaper framing of vaccination post australia's no jab no pay legislation date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: wyy rvqb vaccination rates are an ongoing global concern. many developing and developed countries have rates of vaccination below rates required for herd immunity, for differing reasons. one way in which to communicate information about vaccination to the wider public is via the use of the news media. communication agenda-setting and framing theory generally hold that the news media sets the issues of importance to society and also tells us how we should think about those issues. emphasis framing theory however, would suggest that positively-framed statements in the media may actually be viewed as persuasive in a coercing way, leading to resistance to the messages. further, this theory claims that negative news media is viewed as more credible and therefore, more easily accepted. we were interested to explore the framing of news reports about vaccination and the potential effects this framing may have had on the wider public over the years – in both australia and new zealand (when changes in vaccination policy and publicity respectively were on the agenda). we undertook a content analysis of articles and emphasis frame, type of message, and other variables recorded. in both australia and new zealand, the news media messages were predominately positively framed and yet the vaccination rates of new zealand particularly (where no policy changes mandating vaccination took place) have been decreasing. we suggest the media emphasis on positive vaccination reporting may be having the opposite effect of engendering resistance to vaccination within those who are vaccine-hesitant. vaccination is a contentious issue worldwide (see [ ] for a review). the world health organisation (who) sets immunisation rates of at least % vaccination coverage by [ ] to enhance herd immunity and protect populations from potentially serious diseases. however, rates of immunisation for different antigens vary significantly. global coverage rates in ranged from approximately % for rotaviruses, % for pneumococcal diseases, % for hepatitis b, % for measles (first dose) % for measles (second dose), and % for diphtheria-tetanus-pertussis [ ] . more people now question the necessity of vaccinations in a largely disease-free first-world population [ , ] , and with recent developments in terms of covid- it would be reasonable to assume that this questioning would be minimised. unfortunately, however, recent research has modelled that in fact the opposite might occur with anti-vaccination views being held by the majority in ten years' time if interventions are not put in place [ ] . concerns exist in many countries about falling vaccination rates. for example, the who has expressed concerns about the rise of the anti-vaccination movement in italy, with the incoming government during their electoral campaign ''promising to oppose the pre-existing law that made vaccinations mandatory" [ ] . in both australia and new zealand controversy emerged around the screening of the anti-vaccination film 'vaxxed'. the film attempts to link the measles, mumps, and rubella (mmr) vaccine to autism and is directed by ''discredited gastroenterologist andrew wakefield" [ ] . at the time of writing this article a billboard produced by the new zealand anti-vaccination group warnings about vaccination expectations (waves) was placed on auckland's southern motorway posing the question ''if you knew the ingredients in a vaccine would you risk it?" the billboard was removed the day it was erected after complaints were received by the advertising standards authority [ ] . these examples illustrate how prevalent and current the issue of vaccination is in many countries. the news media play a vital role in communicating vaccination messages to the public [ , ] . while increasing numbers of individuals find their news through social media, over % of stories they are exposed to come from news websites [ ] . therefore, information supplied by the media about vaccines can influence the public's attitudes and beliefs towards them (see [ , , ] ). although the news media can promote pro-vaccination messages, they have also been held responsible for exacerbating the efforts of anti-vaccination groups because of an ''inadequate scientific knowledge base within the media, and an irresponsible tendency towards the emotional" [ ] . speir [ ] goes further arguing the media are tempted to turn single, alleged incidents of adverse vaccine effects into ''major disasters" whereas ''by contrast the successful prevention of diseases in terms of millions of individuals is virtually ignored" (p. s ). given the influence of the media on public attitudes and behaviours toward vaccination, it is important to understand how the media frame vaccination and how these frames impact the wider public. a small number of studies have examined the news media's reporting of vaccination in new zealand and australia separately. in a study of newspaper articles occurring between and , leask and chapman [ ] found the australian newsprint media reported vaccination with an emphasis frame on vaccine-preventable diseases and the issue of low immunisation rates. the coverage also emphasised that the responsibility for vaccination resides with the individual. the threat of vaccine-preventable diseases was conveyed in a number of ways including personification, panic language, stories of personal tragedy, and tales from the pre-vaccination past. these messages were often delivered by representatives of professional medical bodies [ ] . using a larger sample of stories ( ) leask and chapman [ ] found . % of the stories had anti-vaccination statements. these statements contained one or more of the following subtexts: ( ) cover-up -arguments that the real facts about the safety of vaccines was being suppressed. ( ) excavation of the 'facts' -real 'truth' seekers had to find the true facts of vaccination. ( ) unholy alliance for profit -accusations of collusion between government and big pharmaceutical companies, so the latter could increase profits. ( ) towards totalitarianism -vaccination was 'forced' on the community by the government. ( ) us and them -those opposed to vaccination were portrayed as caring parents against the impersonal medical profession. ( ) vaccines as poisonous chemical cocktails -argues vaccines contain dangerous chemicals. ( ) vaccines as cause of idiopathic ills -vaccination blamed for a range of conditions including autism. ( ) back to nature -the body was perfect and able to defend itself without the need to turn to antibiotics and vaccines. a number of stories also portrayed the vaccine debate as a religious crusade between those for and opposed to vaccines [ ] . a more recent study examined the reporting of the hpv vaccine by the australian press finding a number of themes including these: australian pride in vaccine development; details and progress of the national vaccination program; vaccine safety; hpv vaccination's future; whether or not males could and/or should get the vaccine; issues related to sexual activity and the vaccine; and issues about decision-making for acceptance of the hpv vaccine [ ] . a study of new zealand print media between and found the coverage was predominantly neutral towards vaccination ( %) with % of stories framed as positive for vaccination and % opposed [ ] . another study examining headlines used in new zealand newspaper stories reporting the meningococcal b immunisation campaign found that % of the headlines were scientifically inaccurate and another % were misleading [ ] . since the previous australian and new zealand studies cited, a number of events have exacerbated the vaccine debate, including the lancet retraction of the now infamous wakefield study linking the mmr vaccine to autism and the vaxxed film mentioned prior. more importantly for this study is the introduction by australia of the no jab -no pay law in january , a policy not adopted by new zealand. the law meant parents who had children under the age of who were fully immunised or under a government recognised catch-up scheme could receive ''the child care benefit, the child care rebate and the family tax benefit part a end-of-year supplement" [ ] . families who did not vaccinate their children would lose the equivalent of $ . per fortnight, $ per annum, in family tax credits. parents could no longer use conscientious objection or objections on non-medical grounds as reasons to be exempted from losing their tax credits. some states in australia go further with unvaccinated children not being allowed to enrol in pre-schools or childcare centres unless they are vaccinated or have a medical exemption to vaccination. given these more recent events and the policy difference between the two countries regarding vaccination the current study compares newspapers from both countries (a first cross-cultural comparison) to understand any differences in how the reporting is framed, who are the main news sources, the arguments both for and against vaccination that are contained in the stories, and how those who are unwilling to vaccinate their children are labelled. we specifically focus in on emphasis framing theory in making sense of our results. emphasis framing theory argues that when stories or statements are framed in a negative way, they are deemed more credible to readers [ ] . further, positive frames of stories or statements are viewed by readers in a negative light also as attempting to persuade them in some way; for example, advertisements that positively review a product are rightly viewed as attempting to persuade people to purchase that product. these understandings will, it is hoped, lead to implications for the media on best practice reporting of vaccination information. four australian and four new zealand newspapers were selected for the study. newspapers were chosen to replicate previous studies and also for ease of access to archives in contrast to broadcast news. according to roy morgan [ , ] over million new zealanders and . million australians read print newspapers in . most of new zealand's relatively small population (approximately million), is served by four large metropolitan dailies; two in the north island, the dominion post and the new zealand herald, and two in the south island, the press and the otago daily times. there is no national daily newspaper as new zealand's unique and rugged geography has historically hindered the development of one or two national daily newspapers. in contrast australia has two national dailies with the australian, chosen for this sample, having the largest circulation. in order to have an equal number of newspapers from both countries a further three, large, well established metropolitan dailies, two from new south wales, the daily telegraph and sydney morning herald and one from victoria, the the age were selected on advice from an australian colleague (see table ). limiting the sample to these four australian newspapers did mean newspapers from queensland, western australia, south australia, and tasmania were not included. however, newspapers in these states have smaller circulations in comparison to the newspapers chosen. all items, including opinion pieces and letters to the editor (ltes) relating to vaccination or immunisation, were considered for inclusion for the period / / through / / . items were found via a search on the australian/new zealand reference centre database via ebsco host using the following key words: vaccine, vaccinate, vaccination, immunise, and immunization. the first date coincided with the introduction of australia's no jab-no pay law and the cut-off date was decided to be the end of the month in which the study commenced. sourcing articles from the database meant the researchers did not have access to the original newspaper articles and therefore accurate measurements of column inches could not be conducted. in addition, database stories did not always contain photographs and so these were unable to be examined as part of the analysis. a total of articles were found and all were carefully read to ascertain what type of article they were. for example, columns and opinion pieces were identified as different to news articles as the author's name and occupation were often quoted at the beginning of an article and there were often no sources quoted. for example, a story entitled ''attacks on science a call to arms for academics" by the new zealand herald [ ] began with ''dr jarrod gilbert is a sociologist at the university of canterbury. . ." illustrating it was an opinion piece rather than a news story. likewise, ltes were also easily identified as they were short, contained within a group of likeminded items, and the authors were identified by name and the town they came from. once all the items were read, were discarded as they were not about vaccination. for example, one dealt with immunity in terms of legal prosecution. the remaining articles were subjected to a content analysis coding for the following: type of article sources used emphasis of the frame of the story e.g., anti (negative) or pro (positive) towards vaccination labels to describe people unwilling to vaccinate and those who do vaccinate emotive or loaded terms appearing type of anti-vaccination argument leask and chapman's [ ] subtexts were used to identify the anti-vaccination arguments included in articles. we used emphasis framing to determine if the argument of the media story was largely positive in nature (pro-vaccination) or negative in nature (anti-vaccination). a sample of % of the coding of one australian and one new zealand newspaper was subjected to a test of intercoder reliability. agreement was % on framing of the article, % on the type of article, % on number of sources seen, and % on the type of sources. although we found some discrepancies in the type of sources seen, these were minor e.g., a school official versus a state education spokesperson or a medical professional versus a health spokesperson. from the / / through / / the selected newspapers published stories concerning vaccination. a slightly higher number of stories appeared in new zealand newspapers ( versus in the australian newspapers). the majority of the stories were news stories % ( ), % were opinion pieces, and the remaining % were letters to the editor. the number of sources appearing in all the stories were . these included: medical sources -gps, nurses, and representatives from government health bodies politicians -australian prime minister, and mps from both government and opposition parties in both countries, education officials -school principals, school board members university researchers parents of children affected by vaccine preventable diseases anti vax spokespeople government officials -officials from government departments other than health bodies adult sufferer -adults who are suffering from vaccine preventable diseases non-vax parent -parents who do not vaccinate their children parent -sources identified as simply parents and the vaccination status of their children is unknown other -sources who were not lte authors and whose affiliation could not be ascertained fig. shows the differences between sources in the australian and new zealand stories. more medical or health sources were used in the new zealand stories ( vs. ) whereas more political sources appeared in australian newspapers ( vs. ). in all the stories only anti-vaccination activists or spokespersons were used as sources. overall, stories of the entire sample were found to contain a 'negative' emphasis using some of the anti-vaccination arguments identified by leask and chapman [ ] . the most common of these was 'vaccination causes idiopathic ills" appearing times in the stories. the next largest argument mentioned was 'towards totalitarianism' appearing times in the stories. 'unholy alliance' appeared four times, 'cover-up' three, 'poisons' two, and 'back to nature' and 'us and them' once each. 'excavation of facts' did not appear at all. in a number of instances these arguments were not raised by anti-vaccination campaigners, instead those in favour of vaccination raised them to refute these arguments. in addition to analysing the stories for anti-vaccination arguments, the research also identified stories with a positive emphasis, and noted the types of pro-vaccination arguments. these types of arguments appeared in of the stories with stories containing more than one pro-vaccination argument. table illustrates these arguments and their distribution between the australian and new zealand newspapers. as shown in table , the australian newspapers appeared to talk of vaccination more in terms of protecting the community or society than new zealand newspapers which strongly argued that vaccines prevented and protected against disease. another aspect of the stories analysed was the type of emotive terms used by the newspapers analysed. these are shown in table . the new zealand newspapers were more likely to describe increased cases of particular diseases as 'outbreaks' or 'epidemics' unlike their australian counterparts. australian newspapers were more likely to speak about 'vaccine-preventable diseases' and also refer to 'deadly diseases'. twelve stories contained more than one emotive term. both australian and new zealand newspapers were inclined to suggest those opposed to vaccinations were putting their own and other children at risk. the final analysis examined the labels used to describe those persons opposed to vaccination and those persons who supported vaccination. these terms are listed below: two or three terms were used to describe persons who were pro-vaccination. doctors were ''pushing vaccines" and one was called a ''pharma whore". finally, vaccines were labelled ''a victim of their own success". in cross-country comparisons, australia and new zealand do not differ largely from each other in the number of stories published in the / / through / / period. differences were found in the sources most prevalent in vaccination stories between the two countries. the medical/health profession dominated new zealand stories, but political sources dominated the australian media, largely due, it is suggested, to the january arrival of the no jab -no pay campaign, making vaccinations mandatory for families that receive certain government benefits. given the change in the political environment surrounding vaccination in australia, it may be that medical arguments for vaccination become of less import to readers than the potential financial impacts of non-vaccination choices. rates of explicit opting-out of the vaccination schedule for children (known as vaccinerefusal or conscientious objection) are often reported as quite low ( . %) in australia [ ] , ranging from . % to . % in new zealand [ ] . considering that a vaccine refuser's medical concerns regarding vaccination may not be superseded by financial concerns, this suggests that the change in the australian political landscape may have instead impacted on the decisions of those who are vaccine-hesitant (or face other barriers to vaccination such as costs and transport [ ] or antipathy [ ] , but do not identify as against vaccination. emphasis framing theory has been applied in several situations, including psychology, political communication, and public opinion [ ] , and the implications are vast for a number of fields including health. koch and peter [ ] posit that people learn socially to expect credible news from traditional mass news media and to expect this news media to be negative; therefore, the association between negative news media and credibility is built. when news media messages are positive, the credibility relationship is weakened. as positive news media messages about vaccination could also be viewed as advertising, a sense of coercion results from positively framed messages, suggesting that readers may find them less credible in terms of factual information. it could be that readers do not wish to see persuasive information (perceived as akin to advertising) in a perceived 'factual' or 'neutral/balanced' space, particularly with contentious topics such as vaccination. wallack and dorfman [ ] note that the traditional view of mass media with regard to public health is to view it as an ''educational strategy primarily to provide individuals with more information to make better health choices" (p. , italics in original). this view of the media with regard to vaccination positions the media as a 'teacher' or 'authority' on the topic, which may in turn lead to resistance to the message particularly when a reader's own or known other's experiences of vaccination differ [ ] . happer and philo [ ] also argue that journalism is not only about balanced reporting but also sensationalising topics to sell papers claiming that ''news reporting is increasingly shaped by this construction of polarisation and conflict. . ." (p. ). this polarisation in reporting on climate change in happer and philo's [ ] study led to opposing opinions to those emphasised in the media. when looking at sources in the news stories, happer and philo [ ] also found that although readers/viewers trusted the scientist or expert on their topic of climate change, they did not trust the science itself. public trust in politician statements about climate change was also very low. this led happer and philo [ ] to conclude that ''[i]n spite of general sympathy towards the issue and a recognition of its importance, the overall picture of current audience reception was therefore one of confusion, cynicism and distrust about public communications." (p. ). further, even when met with compelling evidence about climate change, the majority of participants who emphasised its importance and changed their attitude toward it, did not show any behavioural changes six months on [ ] . this framing effect has implications for vaccination messages in the media. indeed, studies have found a link between negative information about vaccines in news reports and negative messages about vaccination spreading via social media [ ] , and negative hpv vaccine news reports and subsequent low vaccination rates [ ] . in a systematic review, catalan-matamoros and penafiel-saiz [ ] , noted that of the studies from a variety of countries, % (n = ) were framed around negative information about vaccines. only two studies showed positive framing and messaging about vaccines in news articles [ ] . these findings illustrate the difficulty faced by those trying to communicate to vaccine-hesitant parents. as shown above, negative information is linked to negative messages being spread in social media and lowered vaccination rates. whilst positive information can be seen as a form of persuasion and advertising and thus can cause those who read such information to distrust or resist the messages. in the current study we found that the majority of stories in new zealand and australian media were presented in a positive frame. if the framing effect implied above is correct, positive framing would suggest that readers are less likely to view these messages as credible and view the stories as an attempt to persuade them. when people consciously feel an attempt at persuasion, they tend to resist those messages (known as reactance) [ , , ] , and react negatively to its points (reactance lowers the perceived truth of a message) [ , , ] . therefore, news media that provide a positive frame on vaccination as an activity that people should undertake, may actually be contributing to reactance in the form of lower vaccination uptake and/or lower belief in the efficacy/ need for vaccines. as our study showed, emotively loaded terms and pejorative labels were used in the popular media to argue against anti-vaccination. use of these terms could cause readers to react negatively to the pro-vaccination message. as found by comrie et al., [ ] , immunisation decision-makers reported being disconcerted by vaccination promotional material that denigrated non-vaccinators. in particular, the nz slogan 'be wise, immunise' was touted as suggesting that anyone who did not vaccinate was stupid, which led to defensiveness and reactance. indeed, in new zealand since december , immunisation coverage rates have been decreasing [ ] . this is due to a variety of factors (see [ ] , but the possibility exists that media portrayal of vaccination may be contributing to this decline. it must be noted here however, that vaccine hesitancy and/or uptake is a complex decision-making area, and we do not wish to imply that news articles alone are responsible for declining vaccination rates. social media and how these sites influence vaccination attitudes and actions or inactions is another space of study which needs further research. indeed, it would be useful to determine the relationships (if any) between social media messages and traditional print/online messages, and how those messages are portrayed and taken up by readers/viewers in similar or different ways. decades of framing theory research suggest that the traditional media such as newspapers influence people's views on topics (although it was/is often thought that the framing implies the view e.g., a positive frame should imply a positive view). in this sense however, the positive framing in the media does not appear to be having a positive effect. as a comparison, australian vaccination rates are increasing (for one-and five-year olds since ) [ ] and the majority of the australian stories were also found to be positively framed. however, the political environment of australia is different from that of new zealand, where vaccinations are mandatory to receive specific financial benefits. this would suggest that any media effect is perhaps moderated by the impact of pressing financial concerns for persons receiving such benefits. the emphasis framing interpretation does seem to be a conundrum given that negative reports about vaccination, particularly if viewed as more credible, may have similar negative flow-on effects on vaccination uptake and belief in efficacy or the need for vaccines. the answer may lie in the presentation of neutral, balanced information only in media reports with the goal of informing the general news media public. however, this is easier said than done. balancing positive and negative vaccination information is a contentious field with scientific studies supporting vaccination often 'balanced' in media stories at the same level of credibility as vaccine deniers whom are without a basis in scientific studies. balance as defined by end-users in a previous study [ ] , noted that caregivers making decisions about vaccination for pre-school children wanted clear factual information only, e.g., the number of deaths attributed to the vaccine versus the number of deaths attributed to the disease, and then be left to make their own decisions. this information also needed to come from a credible source. sources that are obviously positive towards vaccination (e.g., government health departments) could be viewed as attempting to persuade, so where this information is to come from and who is to deliver it is a question. comrie et al. [ ] found end-users were particularly convinced by health information from health professionals they trusted. trusting relationships between health professionals and vaccination decision-makers are key but must be combined with the presentation of factual information and perhaps the viewing of that information as neutral and factual, rather than positive/persuading or negatively framed. if information presented in a negative frame leads to perceived heightened credibility and trustworthiness of the source and the message, the implication is that information could be presented in terms of general negative 'worst case scenario's' or the use of 'negative' pictures of children inflicted with the disease. critics of this approach note that picturing the diseases themselves can be perceived as 'fearmongering' by the public and even by health professionals [ , ] . therefore, use of such pictures could be viewed as 'persuasive' but in a negative light, which may have the same negative affect on behaviour as positively framed persuasion. comrie and colleagues [ ] also found that immunisation decisionmakers in general welcomed the pictures of diseases (partial pictures rather than whole child pictures though) as 'factual' information so they could make an informed choice about vaccination. subtilities in presenting such information should be explored in future research. it is interesting to note that some of the negatively framed statements made in media stories were attributed to vaccination supporters who raised these statements only to refute them. therefore, these negative frames were present in a positivelyemphasised article. future research would benefit from focusing on the impact of negatively-framed or positively-framed statements in the context of opposite-emphasised media stories. overall, . % of the stories in the entire sample contained a 'negative' emphasis, whereas leask and chapman [ ] found in their sample of articles in australia that only . % contained a negative emphasis of some type. a partial possibility for this difference could be that a new zealand sample was included in the current study, where a focus seemed to be more on medical information presented (regarding the vaccines), in contrast with the australian media's focus more on political information and community/social benefits. the type of information reported in forms of medical vs political (and individual vs. social), could mean that more negative emphases on vaccination could be found in articles with a more individualistic medical focus. anti-vaccination arguments are often made in terms of the impact on the individual [ ] . this argument also suggests future research to investigate the impact of individualistic vs. community-focused communications on subsequent intention to vaccinate. interestingly, the type of anti-vaccination arguments found by leask and chapman [ ] , were found also in the current study (bar one), albeit at differing frequencies. the findings suggest that the same types of anti-vaccination messages are continuing to be revisited over time, further suggesting that communications in the media and elsewhere have had little effect on addressing concerns behind these messages in the last two decades. leask and chapman [ ] note that the majority of their articles showed a positive or promotional message regarding vaccination, which was also replicated in this study. indeed, this study adds nine types of pro-vaccination messages as developed from the content analysis to leask and chapman's [ ] eight types of antivaccination messages. these types of anti-and pro-vaccination messages provide a framework from which to determine changes or continual patterns in communications over time. in terms of patterns of types of anti-vaccination messages, the top two arguments mentioned in our data were 'vaccination causes idiopathic ills' and 'towards totalitarianism'. leask and chapman's [ ] top two anti-vaccination messages were 'excavation of the facts' (which did not appear in our sample at all) and 'us and them' (which appeared only once). it may be that although similar antivaccination arguments occur over time, the prevalence of these arguments is changing. further, using the new nine types of provaccination messages, as noted above, we can see distinct types of arguments being used in australia and new zealand, who have different political approaches to vaccination. the one similarity is the 'vaccines prevent disease' message which topped both countries' pro-vaccination arguments, but from there, the countries diverge with new zealand focused on 'vaccines protect against disease' and australia focused on 'vaccines protect community/society'. this again could be due to the different individualistic vs. community ideologies of the two nations proposed by this study with regard to vaccination. leask has extended this work in , identifying five parental vaccination group types including the 'unquestioning acceptors', the 'cautious acceptors', the 'hesitant', the 'late or selective vaccinator', and the 'refuser' [ ] . these authors found that different communication strategies were needed in health professional and parent interactions for each of these groups to advocate for quality decisions on vaccination. therefore, it is not only the content of the anti-vaccination argument that must be addressed, but such content must be approached in line with the communication strategies of the parents. this study is a content analysis of articles from two countries. the focus of analysis is on the media reports, and links to wider society's views and actions are suggested. however, this study did not investigate the direct and/or indirect links between media consumption and vaccine uptake and/or views. therefore, this link is an assumption of the research, which may or may not be accurate. some information does suggest that a link exists between media reports on vaccination and subsequent uptake rates or adverse vaccine event reporting (for example, see [ ] and [ ] . although the majority of news media articles studied were positively framed, suggesting that vaccination is portrayed in the media as a useful health activity to engage in, the subsequent expected influence on positive uptake of vaccination and views towards vaccination were not evidenced in wider society. many factors impact a caregiver's vaccination decision, only one of which is the influence of the media. however, it is also possible that the positive emphasis of media stories has an unexpected reverse effect when looked at via emphasis framing theory and its view that negative media stories are viewed as credible, while positive news media stories on topics such as vaccination may lead to unwanted feelings of coercion and therefore reactance/resistance. further research is needed to confirm the relationship indicated by these findings. journalists and media commentators must reflect on the impact of their positioning of health articles. neutral, factual reporting to help construct an informed public is needed, rather than positively or negatively slanted articles. credit authorship contribution statement douglas ashwell: conceptualization, methodology, investigation, writing -original draft, writing -review & editing. improving new zealand's childhood immunisation rates vaccinology: past achievements, present roadblocks and future promises australian department of health. no jab, no pay new requirements fact sheet. immunisation. immunise australia programme australian department of health. immunisation coverage rates for all children australian immunisation register. national vaccine objection (conscientious objection) data making sense of perceptions of risk of diseases and vaccinations: a qualitative study combing models of health beliefs, decisionmaking and risk perception anti-vaccination movements and their interpretations a content analysis of news coverage of the hpv vaccine by how is communication of vaccines in traditional media: a systematic review mass media coverage and influenza vaccine uptake australian newspaper coverage of human papillomavirus vaccination communicating infant immunisation information: resource development and evaluation how the anti-vaxxers are winning in italy. the independent improving childhood vaccination rates bad news: the influence of news coverage and google searches on gardasil adverse event reporting attacks on science a call to arms for academics immunization in the print media -perspectives presented by the press the role of the media in the construction of public belief and social change the online competition between pro-and anti-vaccination views the hpv vaccine and the media: how has the topic been covered and what are the effects on knowledge about the virus and cervical cancer? effects of equivalence framing on the perceived truth of political messages and the trustworthiness of politicians helpful or harmful? how frequent repetition affects perceived statement credibility an attempt to swindle nature': press anti-immunisation reportage - the cold hard facts' immunisation and vaccinepreventable diseases in australia's newsprint media - communicating with parents about vaccination: a framework for health professionals from social media to mainstream news: the information flow of the vaccine-autism controversy in the us, canada, and the uk vaccination communication strategies: what have we learned, and lost, in years i approve this message: effects of sponsorship, ad tone and reactance in presidential advertising national and dhb immunisation data reinforcement or reactance? examining the effect of an explicit persuasive appeal following an entertainment-education narrative emphasis framing and political decision making commenting and tagging: effects of sharing news stories on facebook giving boys a shot: the hpv's vaccine portrayal in canadian newspapers misinformation lingers in memory: failure of three pro-vaccination strategies the use of gain-or loss-frame messages and efficacy appeals to dissuade excessive alcohol consumption among college students: a test of psychological reactance theory australian newspaper readership, months to over million new zealanders read newspapers in perception of risk of vaccine adverse events: a historical perspective vaxxed: from cover-up to catastrophe ( ) information for parents anti-vaccine billboard highlights lack of trust in authorities petousis-harris h. the use and misuse of media headlines: lessons from the menzb tm immunisation campaign media advocacy: a strategy for advancing policy and promoting health world health organisation global and regional immunization profile niki murray: conceptualization, investigation, writing -original draft, validation, writing -review & editing. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- - gnf j authors: cheung, ying-kit; cheng, samuel chak-sum; sin, fion wan-yee; chan, kin-tak; xie, yong title: induction of t-cell response by a dna vaccine encoding a novel hla-a* severe acute respiratory syndrome coronavirus epitope date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gnf j the severe acute respiratory syndrome coronavirus nucleocapsid protein (sars-cov n) is one of the major targets for sars vaccine due to its high potency in triggering immune responses. in this study, we have identified a novel hla-a* restricted epitope, n (lalllldrl), of the sars-cov n-protein through bioinformatics analysis. the n-protein peptide n shows a high binding affinity towards human mhc class i in t -cells, and is capable of activating cytotoxic t-cells in human peripheral blood mononuclear cells (pbmcs). the application of using the n peptide sequence with a single-chain-trimer (sct) approach to produce a potential dna vaccine candidate was investigated in hla-a . k(b) transgenic mice. cytotoxicity assay clearly showed that the t-cells obtained from the vaccinated animals were able to kill the n-protein expressing cells with a cytotoxicity level of % in an effector cells/target cells ratio of : one week after the last vaccination, which is significantly higher than other n-protein peptides previously described. the novel immunogenic n-protein peptide revealed in the present study provides valuable information for therapeutic sars vaccine design. severe acute respiratory syndrome (sars) is caused by a novel coronavirus known as sars-associated coronavirus (sars-cov) and the investigation of the sars-cov immunity has garnered significant attention since the worldwide outbreak spreaded to countries in [ ] [ ] [ ] . the sars-cov is an enveloped positive-stranded rna virus encoding four major structural proteins, known as a spike glycoprotein (s), a small envelope protein (e), an integral membrane glycoprotein (m), and a nucleocapsid rna-binding protein (n) [ ] . upon viral infection, the viral proteins expressed within the infected cells are degraded in the cytosol by proteasomes. peptides generated by the proteasomes are transported by the transporter associated with antigen processing (tap) into the lumen of endoplasmic reticulum (er), where an er aminopeptidase produces the mature mhc class i-peptide complex by trimming the peptide to eight or nine amino acids [ ] . the resulting mhc class i-peptide complex expressed on the surface of professional antigen presenting cells, such as dendritic cells, plays important roles in the activation of specific cytotoxic t-cells for infected cell killing. on the other hand, the mhc class i-peptide complexes expressed on the virus infected cells is a "marker" for being killed. therefore, formation of stable mhc class i-peptide complexes is critical to elicit cytotoxic t-cell response to eliminate the infected cells. immunogenicity of a peptide sequence for the cytotoxic t-cell is determined by its binding affinity towards the human mhc class i molecule and its availability upon proteasome digestion of the parental protein. subunit vaccines containing hla-a* restricted peptides is highly effective for the induction of strong cytotoxic t-cell response against infectious virus [ ] . among the four structure proteins of the sars-cov, the s and n proteins are the major targets for vaccine research studies due to their potency in triggering immune responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the s protein contains amino acids and is involved in viral entry through - x/$ -see front matter © elsevier ltd. all angiotensin-converting enzyme receptor on host cell surface [ , ] and the n protein contains amino acids and is involved in the viral rna packaging [ , ] . previous studies suggested that hla-a* restricted sars s and n protein peptides can trigger specific human cytotoxic tcell response against sars in vitro [ , ] . in the present study, we have identified a noval hla-a* restricted nprotein peptide (n : lalllldrl). the binding affinity of the n peptide towards human mhc class i molecule is comparable to the n peptide and is higher than the n and n peptides previously described [ ] . moreover, in order to stablize the mhc class i-peptide complex for antigen presentation, the n sequence was genetically linked to the cdna of human ␤ -microglobulin and the alpha- and alpha- domains of the human mhc class i heavy chain to form a single-chain-trimer (sct) in our dna vaccine design. the sct approach makes the mhc class i molecules less dependent on chaperone assistance for peptide loading and hence a stable mhc class i-peptide complex for antigen presentation is produced [ ] [ ] [ ] . the potency of the n dna vaccine to trigger cytotoxic t-cell response against n-protein expressing cells was demonstrated in a hla-a . k b transgenic mouse model and our results indicated that the sars protein n peptide is a good vaccine candidate for sars vaccine development. the potential sars n-protein peptide sequence for human mhc class i binding was searched by a hla peptide binding prediction program, syfpeithi (http://www.syfpeithi.de) [ ] . seven nine-amino acid peptides with high scores for human mhc class i binding (n - alntpkdhi, n - lqlpqgttl, n - lalllldrl, n - llldrlnql, n - rlnqlesky, n - gmsrigmev, n - illnkhid) were synthesized by a solid-phase strategy using the fmoc-based protocol on an automated peptide synthesizer. the crude products were purified on a reverse-phase preparative high performance liquid chromatography (hplc) column and the homogeneity of the final products was analyzed by reverse-phase hplc of purity of %. the peptides were stored in dimethyl sulfoxide (dmso, sigma) at − • c until use. the dna sequence encoding the n-protein was cloned into a expression vector pet b (novagen) and the recombinant n-protein is expressed in a escherichia coli system bl -condonplus (de )-ril (stratagene) followed by purification with ni-nta his-bind resin (novagen). after protein purification, the purity and the quantity of the recombinant protein was analyzed by sds-page analysis. to establish a n-protein expressing cell-line for the cytotoxicity assay, lung tissues from the hla-a . k b transgenic mice were isolated, minced and treated with collagenase (gibco) at u/ml for min. tissue samples were seeded on a plate and cultured for week, fibroblasts were then collected and immortalized by transduction of a recombinant retrovirus containing the sars n gene and the hpv e -e genes [ ] . expression of the nprotein in the lung fibroblast cell-line was confirmed by rt-pcr (with primers: n: sense: -atgtctgataatgg- , antisense: -ttatgcctgagttg- ; e e : sense: -atgtataaaactaaggg cgtaacc- , antisense: -ttatggtttctgagaacagatgg- ), and the cell-line is named as n/e e /a . k b . the t -cell ( × cem.t ), which are hla-a positive, but deficient for the tap protein for endogenous antigen presentation, were cultured according to the procedure stated in the atcc manual. to determine whether the selected n-protein peptides could bind to the human mhc class i molecule, the t -cells ( × ) were pulsed with each of the n-protein peptides ( g/ml) in the presence of g/ml human ␤ -microglobulin (sigma) for h. after peptide pulsing, the t -cells were washed twice with cold pbs containing % fbs and then stained with a mouse anti-human hla-a antibody (bb . ) followed by a goat anti-mouse igg fitc antibody (zymed). the peptide bound t -cells were fixed with % paraformaldehyde (sigma) and subjected to flow cytometry to measure the relative amount of mhc class i/peptide complexes formed. the results were presented as the fluorescent index (fi) calculated by the mean fluorescence intensity (mfi) of t cells using the formula: fresh human hla-a* positive peripheral blood mononuclear cells (pbmcs) were isolated by ficoll-hypaque density gradients (amersham), followed by enrichment of cd + t-cells by human cd microbeads (macs). the purified human cd + t cells were then subjected to autologous dendritic cells mediated t-cell activation in vitro. in brief, the pbmcs were seeded on dishes for h and the non-adherent cells were removed. the adherent monocytes were then cultured in aim-v medium (invitrogen) supplemented with % human ab serum, u/ml recombinant granulocyte-macrophage colony-stimulating factor (gm-cff) and u/ml recombinant human interleukin (il- ) to obtain dendritic cells. on day , the dendritic cells were matured by addition of u/ml recombinant human interleukin beta (il- ␤), u/ml recombinant human interleukin (il- ), ng/ml recombinant human tumor necrosis factor-alpha (tnf-␣) and g/ml prostaglandin e (pge ). on day , the mature dendritic cells were loaded with the selected n-protein peptides in serum free medium for h. the peptide-loaded dendritic cells were then cocultured with the purified cd + t cells in the presence of u/ml recombinant human interleukin (il- ) and ng/ml recombinant human interleukin (il- ) for t-cell stimulation. the stimulation procedure was repeated once a week and totally three times of t-cell stimulation by the peptide-loaded dendritic cells was conducted. activation of t-cell was investigated by an ifn-␥ elispot assay as previously described with minor modifications [ ] . briefly, a -well elispot plate (multiscreen-ha, millipore) coated with anti-human ifn-␥ antibodies ( g/ml, ebioscience) was blocked with aim-v medium containing % human ab serum. the stimulated cd + t cells ( × ) were then added into the wells together with autologous n-protein loaded b-cells ( × ) and incubated for h. subsequently, the plate was washed and followed by incubation with . g/ml biotinylated ifn-␥ antibody (ebioscience) at • c for another h. after washing, g/ml streptavidin-ap (zymed) was added and incubated at room temperature for min. spots were developed by adding a bcip/nbt solution (invitrogen) and digitally recorded with an elispot plate reader (service provided by beijing swan tech co., china). the dna sequence containing the human ␤ -microglobulin and the chimeric mhc heavy chain, was synthesized by connecting the mouse ␤ -microglobulin cdna, human hla-a* ␣- cdna, hla-a* ␣- cdna and mouse h -k b ␣- domain cdna together by overlapping pcr to construct an mhk gene (m = mouse ␤ -microglobulin, h = human hla-a* ␣- and ␣- , k = mouse h -k b ␣- ) using two templates. the ova ␤ mk b sct gene which was a generous gift from prof. hansen was the template for mouse ␤ -microglobulin and mouse h -k b ␣- domain [ ] . the hhd gene, which was provided by prof. lemonnier, was another template for the human hla-a* ␣- and hla-a* ␣- [ ] . the dna fragments encoding the selected n-protein peptides were then linked to the n-terminal of the mhk gene by pcr using the primers with the corresponding dna sequence at the region to generate single-chain-trimers. the constructed single-chain-trimer dna fragments were cloned into agei and xhoi sites of pvax (invitrogen) to construct n-protein peptide-expressing plasmids, n mhkpvax , n mhkpvax , n mhkpvax , and n mhkpvax for vaccination purpose. ovamhkpvax was also constructed as a control plasmid expressing a siinfekl peptide of the ovalbumin (ova ). [ ] were bred and maintained under pathogen-free conditions (animal care center, hkust, hong kong). the dna vaccine was delivered into - weeks old transgenic mice with a gene gun device as previously described with some minor modifications [ ] . briefly, cartridges were prepared by precipitating the plasmid dna on m gold particles dissolved in . m spermidine and m cacl . the microcarrier/dna suspension was coated on plastic tubing in the presence of . mg/ml polyvinylpyrrolidone (sigma). the coated tubing was cut into . inch cartridges so that each cartridge contained g of dna. dna-coated gold particles were delivered to the shaved abdominal region of mice using a helios gene gun (bio-rad) with a discharge pressure of psi. each mouse was administrated with g dna per injection for a total of three vaccinations within a -week interval. to investigate the cell mediated cytotoxic response triggered by the sct-dna vaccine, mice were sacrificed week after the last vaccination. splenocytes were obtained and cultured in imdm medium (gibco) containing g/ml of the corresponding target peptides and units of interleukin- (peprotech) in a -well plate (nunc) at • c for days. t-cell cells from spleen were harvested by ficoll-hypaque (amersham) density gradient centrifugation and used as effector cells in a cytotoxicity assay. the n-protein transduced n/e e / . k b cells were used as target cells. in the cytotoxicity assay, the effector cells and the target cells were co-cultured in the ratios of : , : , : , : and : . after h incubation, the culture plates were centrifuged and the medium was collected for further analysis using a lactate dehydrogenase (ldh) cytotoxicity detection kit (roche) according to the procedures stated by the manufacturer. the absorbance of the samples was measured by elisa reader at nm with nm as reference wavelength. the spontaneous release of ldh by target cells or effector cells the full-length amino acid sequence of the sars n-protein was subjected to bioinformatic analysis to search for hla-a* restricted nine-amino acid peptides. the scores of the peptides are within the range from − to , and the amino acid sequence of the seven peptides selected that have the high scores are listed in the middle column. a flu m peptide (gilgfvftl) (which is used as a positive control) shows a score of is listed at the bottom. was assessed by incubation of target cells in the absence of effector cells and vice versa. the maximum release of ldh was determined by incubation of the target cells in % triton x- in assay medium. the percentage of specific cell mediated cytotoxicity was determined by the following equation: specific cytotoxicity (%) = experimental value − effector cell spontaneous release − target cell spontaneous release maximum target cell release × in order to search for the hla-a* restricted peptide sequence of the sars n-protein, the syfpetithi program was employed to compare the binding affinity of the nprotein peptides towards the human mhc class i molecule through a bioinformatic analysis. seven high score peptides were selected (table ) and the binding activity of these peptides towards the human mhc class i molecules was investigated by comparison of their ability to stabilize the empty mhc class i molecules expressed on the cell surface in the presence of ␤ -microglobulin. the presence of the stable mhc class i-peptide complex was then detected by the antibody (bb . ) through facs. the results clearly show that among the seven n-protein peptides, n and n display the highest binding affinity towards the human mhc class i and the resulting fluorescence intensities are higher than the positive control flu m peptide ( - , gilgfvftl) [ ] ( fig. ) , suggesting a high binding affinity of the n and n peptides towards the human mhc class i. the t-cell immunogenicity of the seven selected peptides was further tested by their ability to stimulate human cd + t cells isolated from healthy donor pbmcs. the purified cd + t-cells were primed three times with autologous mature peptide-loaded dendritic cells and the level of tcell activation was then investigated by the release of ifn-␥ through ifn-␥-elispot in the presence of autologous bcells loaded with the recombinant n-protein. if the target peptide of the n-protein can be generated and presented by the target cells, the primed cd + t-cells could specifically recognize the peptide-mhc complex and release ifn-␥. the elispot results show that t-cells primed with n (lll-drlnql) and n (lalllldrl) produce the highest number of spots that are six to seven times higher than that found in the irrelevant flu peptide primed t-cells and is significantly higher than that of the other selected n-protein peptides (fig. ) . the results of the t-cell stimulation assay demonstrated that the novel n-protein peptide revealed in the present study is able to trigger specific cytotoxic t-cell response in human pbmcs. the four most immunogenic peptides (n , n , n and n ) selected in the t -cell binding assay and the human t-cell stimulation assay were further tested for their potency in triggering immune response against the sars n-protein expressing cells in an animal model. to facilitate antigen presentation, the dna sequence of the target peptides was genetically linked to the cdna sequence of the mouse ␤ -microglobulin and the chimeric mhc class i heavy chain domains (fig. ) that the peptide can be translated as a peptide-spacer-␤ -microglobulin-space-h-chain complex for t-cell activation. under this covalently linked mhc class i-peptide construction, the linked mhc molecule is at least -fold less accessible to exogenous peptides than normal mhc loaded with endogenous peptide and is more potent in the stimulation of cytotoxic t-cells [ ] . the cytotoxic tcell response triggered by the dna vaccine was investigated by the activity of the spleen t-cells to kill the target cells n/e e /a . k b , which are immortalized primary a . k b fibroblasts expressing the sars n-gene (fig. ), week after the last vaccination. the cytotoxicity level was measured by the amount of lactate dehydrogenase (ldh) released from the target cells. the results demonstrated that the dna vaccines encoding the n-protein peptides n and n trigger the highest t-cell cytotoxicity towards the n-protein expressing cells with and % cytotoxicity level, respectively, in effector cells/target cells ratio of : compared to the cytotoxicity level of the previously described peptide n , which shows only % cytotoxicity, and n , which is similar to the negative control using an irrelevant ova peptide for vaccination (fig. ). the worldwide outbreak of sars in caused a severe economical loss and the development of sars vaccine is one of the most effective ways to prevent the outbreak in the future. an ideal vaccine against infectious virus should be able to trigger both the neutralizing antibody production and the cytotoxic t-cell responses that play a critical role in the elimination of virus infected cells in controlling viral pathogensis. during sars-cov infection, the n-protein is reported to be highly immunogenic [ ] and it has been the y-axis indicates the percentage of cytotoxicity. four groups of mice were vaccinated with the n-protein peptide plasmids, n mhkpvax ( ); n mhkpvax ( ); n mhkpvax ( ); and n mhkpvax ( ). mice vaccinated with an irrelevant plasmid, ovamhkpvax ( ), was used as a negative control. the cytotoxicity level was calculated as previously described. the differences in cytotoxicity level between all peptides with various effector cells: target cells ratios were calculated with t-test (p < . ). shown that n vaccine can trigger specific t-cell response in mice [ ] . however, only six t-cell specific epitopes of the n-protein have been reported up-to-date [ , , ] . the objective of this study is to identify immunogenic n-protein peptides that can serve as cytotoxic t-cell epitope in sars vaccine. a peptide sequence useful for inducing the cytotoxic t-cell response should be presented as endogenous peptide epitope through proteasome digestion and have a high binding affinity towards the human mhc class i molecules. in contrast to the conventional method of screening numerous amino acid sequences manually from synthetic peptide library that is costly and time consuming, bioinformatics analysis was employed to search for the most immunogenic peptide sequences of the sars n-protein. although the efficiency of the bioinformatics analysis in searching the immunogenic peptide is high, discrepant results could also be obtained from different programs online. for instance, although the novel immunogenic n peptide revealed in the present study is the third potent peptide found in the syfpetithi program, it is ranked according to the hla peptide motif search provided by the national institutes of health and was totally ignored in the previous study about searching the t-cell epitopes in the n-protein [ ] . to investigate whether the peptide predicted from the computer program has a high binding affinity to human mhc, a t -cell binding assay was conduced that is based on the binding affinity of the peptide to the empty mhc class i molecules on the cell surface and to stabilize the mhc class i-peptide complex formed. the results clearly show that the novel peptide, n , identified in the present study has a high binding affinity towards the human mhc class i molecule hla-a . that is comparable to the n peptide and is significantly higher than that of the n and n peptides previously described [ ] . previous studies suggested that a peptide sequence with a high binding affinity to hla-a . is not able to trigger t-cell response if the peptide sequence is not generated from endogenous process through the proteasome system. for instance, although the ny-eso- peptide - has a high affinity towards the hla-a molecules, it is not able to elicit cytotoxic t-cell response since the peptide is not processed naturally from the parental ny-eso- protein by the proteasome [ , , ] . to address the question concerning with intracellular protein processing, a t-cell activation assay with n-protein loaded b-cells, which is able to cross-present protein antigen through the proteasome system was preformed [ , ] . the t-cell activation assay demonstrated that when the n-protein loaded b-cells were used as target cells, the n primed t-cells were induced for ifn-␥ production and the level of ifn-␥ produced is comparable to the n peptide and is significantly higher than that of the n and n peptides previously described [ ] , that is consistent to the results obtained in the t -cell binding assay. although a study has previously mapped the three hla-a* restricted n-protein peptides (n , n and n ) in vaccinated transgenic mice [ ] , and the vaccine potency has not been addressed. in the present study, the potency of the novel n peptide together with the three previously described peptides [ ] , vaccine was investigated in a transgenic mouse model expressing the human hla-a . . in contrast to the conventional method using expensive synthetic peptides for injection [ ] that is infeasible for practical used in a large vaccination program, a sct display dna vaccine mechanism was employed. a dna vaccine is much less costly compared to the synthetic peptides and when couple with the sct display system, the immunogenic peptide is translated together with the ␤ -microglobulin and the mhc class i heavy chain molecule as a complex. after translation, the whole covalently linked mhc class i-peptide complex can be transferred from the er to the cell surface for antigen presentation. the high stability of the covalently linked mhc class i-peptide complex produced the sct system excludes competing peptides and is a potent stimulator for t-cells [ ] . thus, it eliminates the uncertainty of the antigen processing in the professional antigen presenting cells and the cytotoxic t-cells can be primed more directly to ensure that the peptide encoded in the vaccine can be presented for vaccination. in our study, the dna vaccine was injected into the transgenic mice for vaccination and the cytotoxicity assay demonstrated that vaccination of the n peptide is able to trigger the cytotoxic t-cell response against the n-protein expressing cells as early as week after the last vaccination even in the absence of any adjuvants. comparative results obtained from cytotoxicity assay among the tested peptides indicated that the n peptide represents as one of the most potent amino acid sequences of the n-protein that is able to trigger the cytotoxic t-cell response. interestingly, all the reported immunogenic t-cell epitopes of n-protein, including the n revealed in the present study, are mostly clustered between the amino acid residues and . therefore it will be interested to investigate whether a vaccine composed of this amino acid peptide (n to n ) coupled with the known sars b-cell epitopes previously described [ , ] is sufficient to trigger a protective immune response against the sars-cov in human. in summary, we have identified a novel hla-a . specific sars-cov n protein epitope (n -n lalllldrl) which can activate cytotoxic t-cells in vitro and when used with the sct system, it is sufficient to elicit cytotoxic tcell response against n-protein expressing cells in the hla-a . k b transgenic mouse model. the findings of the novel cytotoxic t-cell epitope presented in this study provide worth information in sars vaccine design that may contribute to the sars controlling program in the future. sars: clinical presentation, transmission, pathogenesis and treatment options the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review the aetiology, origins, and diagnosis of severe acute respiratory syndrome characterization of viral proteins encoded by the sars-coronavirus genome cellular mechanisms governing 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established tumors with a novel vaccine that enhances major histocompatibility class ii presentation of tumor antigen transduction of dendritic cells with recombinant adenovirus encoding hca activates autologous cytotoxic t lymphocytes to target hepatoma cells cutting edge: single-chain trimers of mhc class i molecules form stable structures that potently stimulate antigen-specific t cells and b cells perarnau b. hla-a . -restricted education and cytolytic activity of cd (+) t lymphocytes from ␤ -microglobulin (␤ m) hla-a . monochain transgenic h- db ␤ m double knockout mice analysis of the hla-restricted influenza-specific cytotoxic t lymphocyte response in transgenic mice carrying a chimeric human-mouse class i major histocompatibility complex the minimum peptide epitope from the influenza virus matrix protein. extra and intracellular loading of hla-a induction of th type response by dna vaccinations with n, m, and e genes against sars-cov in mice long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients multiepitope cd (+) t cell response to a ny-eso- peptide vaccine results in imprecise tumor targeting cross-presentation of hla class iu epitopes from exogenous ny-eso- polypeptides by nonprofessional apc cpg-dna aided cross-priming by cross-presenting b cells b lymphocytes participate in crosspresentation of antigen following gene gun vaccination antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein we thank the hong kong research fund for the control of infectious diseases for funding this project. key: cord- -nabxpyw authors: bell, sadie; clarke, richard; mounier-jack, sandra; walker, jemma l; paterson, pauline title: parents’ and guardians’ views on the acceptability of a future covid- vaccine: a multi-methods study in england date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nabxpyw background the availability of a covid- vaccine has been heralded as key to controlling the covid- pandemic. covid- vaccination programme success will rely on public willingness to be vaccinated. methods we used a multi-methods approach - involving an online cross-sectional survey and semi-structured interviews - to investigate parents’ and guardians’ views on the acceptability of a future covid- vaccine. parents and guardians (aged + years) who reported living in england with a child aged months or under completed the survey. nineteen survey participants were interviewed. findings most survey participants reported they would likely accept a covid- vaccine for themselves (definitely . %; unsure but leaning towards yes . %) and their child/children (definitely . %; unsure but leaning towards yes . %). less than % of survey participants reported that they would definitely not accept a covid- vaccine. survey participants were more likely to accept a covid- vaccine for themselves than their child/children. participants that self-reported as black, asian, chinese, mixed or other ethnicity were almost times more likely to reject a covid- vaccine for themselves and their children than white british, white irish and white other participants. survey participants from lower-income households were also more likely to reject a covid- vaccine. in open-text survey responses and interviews, self-protection from covid- was reported as the main reason for vaccine acceptance. common concerns identified in open-text responses and interviews were around covid- vaccine safety and effectiveness, mostly prompted by the newness and rapid development of the vaccine. conclusion information on how covid- vaccines are developed and tested, including their safety and efficacy, must be communicated clearly to the public. to prevent inequalities in uptake, it is crucial to understand and address factors that may affect covid- vaccine acceptability in ethnic minority and lower-income groups who are disproportionately affected by covid- . parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england and "likely to reject" (those that answered no, definitely not or unsure but leaning towards no). of interest such as underrepresented populations in the survey (e.g. participants from ethnic minority groups or reporting a lower household income) and/or indicated they would likely refuse a covid- vaccine, for their child or themselves. we did not solely interview participants who were likely to refuse a covid- vaccine as we were keen to explore the nuances of reasons participants had for accepting or refusing a covid- vaccine. participants were emailed an information sheet, fully detailing the study objectives and explaining all aspects of participation, including the right to withdraw from the research. written informed consent was obtained from each participant. interviews lasted parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england insert table here . % of survey participants (n= ) provided their details to be contacted for a follow-on interview. in total, parents were contacted to participate. of these took part in interviews ( women and one man), did not respond to recruitment emails, two responded initially but did not follow through with an interview. the characteristics of interviewees are outlined in table ). interestingly, one interviewee (# ) that was leaning towards accepting the vaccine at the time of completing the survey discussed that she was leaning towards refusing the vaccination (for herself and her child) at the time of interview. the following reasons were given for covid- vaccine acceptance for self and for child/children, in order of how often they were mentioned by survey participants and importance to interviewees. to protect self and others of survey participants expressing positive intentions to vaccinate and leaving an open-text response, the most prevalent reason was to provide protection from covid- to the parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england protecting other people (for self: . %, n= ; for child: . %, n= ), including family members (for self: . %, n= ; for child: . %, n= ). participants also reported that they would vaccinate to protect someone known to them in a risk group for covid- (for self: . %, n= ; for child: . %, n= ). . % (n= ) of survey participants specifically mentioned that they wanted to receive the vaccine to stay healthy to look after their child/children. . this is of concern given the evidence that black, covid- ) vaccine became available would you accept the vaccine for your child/children?". a -point likert scale was used to encourage participants to take a stance on the vaccine (rather than selecting a 'do not know' answer). the likert scale options were "yes, definitely a paired samples t-test was used to compare acceptance of a covid- vaccine for self and for the participant's child. two subsequent logistic regressions were then conducted to determine the demographic factors associated with rejection of the covid- vaccine for both self-vaccination and that of the participant's child age, household income, ethnicity, location, and employment were included as predictive variables in the logistic regression models. in the logistic regression model for child vaccination to perform the logistic regressions ethnicity was dichotomised into 'white' (i -£ , ) and "high income" (>£ , ), and the vaccine acceptance variables were dichotomised into parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england around the content of the questionnaire, were used to assist the interviews. the interviews included two questions related to a covid- vaccination, the first focused on parents' and guardians' views on receiving a new covid- vaccine for themselves, should one become publicly available, and the second on their views on their child of accepting a new covid- vaccine for their child. interview participants received a £ gift voucher as a thank you for their time and contribution. the interviews took place between th april and th may and clarke [ ]: data familiarisation, coding and theme identification and refinement. the transcribed interviews were read and coded by sb. to enhance the rigour of the analysis, coding approaches and subsequent theme generation and guardians completed the survey (see participant characteristics in table ) of the survey participants parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england quantitative findings -factors associated with covid- vaccine rejection for self a forward stepwise logistic regression analysis was performed with a dichotomised version of the self-vaccination against covid- variable as the discrete variable. age, household income, ethnicity, location and employment were included as predictive variables. the final model included three predictor variables (household income, employment and ethnicity) and significantly predicted 'likely to reject' (omnibus chi-square = . , df = , p < . ) . ) as likely to reject a covid- vaccine than participants with a medium household income (£ , -£ , ) participants that self-reported as black, asian, chinese, mixed or other ethnicity were . times ( %ci: . - . ) more likely to reject a covid- vaccine than white british white irish and white other participants. there was also some indication that those that parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england a forward stepwise logistic regression analysis was performed with a dichotomised version of the child vaccination against covid- variable as the discrete variable. age, household income, ethnicity, location, employment and number of children were included as predictive variables. the final model included three predictor variables (income, ethnicity and number of children) and significantly predicted 'likely to reject age, location and employment did not significantly predict 'likely to reject', as such they were excluded from the model. the hosmer-lemeshow test demonstrates that the model adequately fits the data chi-square = . , df = , p = . mixed or other ethnicity were . times ( %ci: . - . ) more likely to reject a covid- vaccine for their child than white british, white irish and white other participants. this finding was also found for income, with participants in the lower household income bracket (<£ , ) being . times ( %ci: . - . ) as likely to reject a covid- vaccine for their child than participants with a medium household income (£ , -£ , ). participants with more than four children were also found to be around four times /or publication of this article: the research was funded by the national institute for health research health protection research unit immunisation at the london school of hygiene and tropical medicine (lshtm) in partnership with public health england (phe). the views expressed are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health or european centre for disease prevention and control (ecdc) coronavirus (covid- ) in the uk. the rt hon boris johnson mp. prime minister's statement on coronavirus (covid- staying alert and safe (social distancing) government launches vaccine taskforce to combat coronavirus world health organisation. the push for a covid- vaccine millions could be vaccinated against covid- as uk secures strong portfolio of promising vaccines ensuring global access to covid- vaccines jcvi: updated interim advice on priority groups for covid- vaccination using thematic analysis in psychology parents' and guardians' views on the acceptability of a future covid- vaccine: a multi sabat, i, once we have it, will we use it? a european survey on willingness to be vaccinated against covid- determinants of covid- vaccine acceptance in the willingness to vaccinate against covid- in australia more vaccines for children? parents' views. vaccine parents with doubts about vaccines: which vaccines and reasons why politics and public trust shape vaccine risk perceptions tracking the global spread of vaccine sentiments: the global response to japan's suspension of its hpv vaccine recommendation acceptance of a covid- vaccine in southeast asia: a cross-sectional study in indonesia susceptibility to and transmission of covid- amongst children and adolescents compared with adults: a systematic review and meta-analysis. medrxiv disparities in the risk and outcomes of covid- reasons for non-vaccination: parental vaccine hesitancy and the childhood influenza vaccination school pilot programme in england childhood vaccination coverage by ethnicity within london between ethnic differences in human papillomavirus awareness and vaccine acceptability inequalities in childhood vaccination timing and completion in london patient and practice level factors associated with seasonal influenza vaccine uptake among at-risk adults in england, to : an age-stratified retrospective cohort study effect of socioeconomic deprivation on uptake of measles, mumps and rubella vaccination in liverpool, uk over years: a longitudinal ecological study. epidemiol infect managing the covid- pandemic: new results from the european survey on policy support, confidence and vaccination attitudes office for national statistics. ks uk ethnic group, local authorities in the united kingdom (excel sheet kb) average household income, uk: financial year ending parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england parents' and guardians' views on the acceptability of a future covid- key: cord- -u vgycib authors: volkmann, ariane; williamson, anna-lise; weidenthaler, heinz; meyer, thomas p.h.; robertson, james s.; excler, jean-louis; condit, richard c.; evans, eric; smith, emily r.; kim, denny; chen, robert t. title: the brighton collaboration standardized template for collection of key information for risk/benefit assessment of a modified vaccinia ankara (mva) vaccine platform date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: u vgycib the brighton collaboration viral vector vaccines safety working group (v swg) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. the modified vaccinia ankara (mva) vector system is being explored as a platform for development of multiple vaccines. this paper reviews the molecular and biological features specifically of the mva-bn vector system, followed by a template with details on the safety and characteristics of an mva-bn based vaccine against zaire ebolavirus and other filovirus strains. the mva-bn-filo vaccine is based on a live, highly attenuated poxviral vector incapable of replicating in human cells and encodes glycoproteins of ebola virus zaire, sudan virus and marburg virus and the nucleoprotein of the thai forest virus. this vaccine has been approved in the european union in july as part of a heterologous ebola vaccination regimen. the mva-bn vector is attenuated following over serial passages in eggs, showing restricted host tropism and incompetence to replicate in human cells. mva has six major deletions and other mutations of genes outside these deletions, which all contribute to the replication deficiency in human and other mammalian cells. attenuation of mva-bn was demonstrated by safe administration in immunocompromised mice and non-human primates. in multiple clinical trials with the mva-bn backbone, more than participants have been vaccinated, demonstrating a safety profile consistent with other licensed, modern vaccines. mva-bn has been approved as smallpox vaccine in europe and canada in , and as smallpox and monkeypox vaccine in the us in . no signal for inflammatory cardiac disorders was identified throughout the mva-bn development program. this is in sharp contrast to the older, replicating vaccinia smallpox vaccines, which have a known risk for myocarditis and/or pericarditis in up to in vaccinees. mva-bn-filo as part of a heterologous ebola vaccination regimen (ad .zebov/mva-bn-filo) has undergone clinical testing including phase iii in west africa and is currently in use in large scale vaccination studies in central african countries. this paper provides a comprehensive picture of the mva-bn vector, which has reached regulatory approvals, both as mva-bn backbone for smallpox/monkeypox, as well as for the mva-bn-filo construct as part of an ebola vaccination regimen, and therefore aims to provide solutions to prevent disease from high-consequence human pathogens. the brighton collaboration viral vector vaccines safety working group (v swg) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. the modified vaccinia ankara (mva) vector system is being explored as a platform for development of multiple vaccines. this paper reviews the molecular and biological features specifically of the mva-bn vector system, followed by a template with details on the safety and characteristics of an mva-bn based vaccine against zaire ebolavirus and other filovirus strains. the mva-bn-filo vaccine is based on a live, highly attenuated poxviral vector incapable of replicating in human cells and encodes glycoproteins of ebola virus zaire, sudan virus and marburg virus and the nucleoprotein of the thai forest virus. this vaccine has been approved in the european union in july as part of a heterologous ebola vaccination regimen. the mva-bn vector is attenuated following over serial passages in eggs, showing restricted host tropism and incompetence to replicate in human cells. mva has six major deletions and other mutations of genes outside these deletions, which all contribute to the replication deficiency in human and other mammalian cells. attenuation of mva-bn was demonstrated by safe administration in immunocompromised mice and non-human primates. in multiple clinical trials with the mva-bn backbone, more than participants have been vaccinated, demonstrating a safety profile consistent with other licensed, modern vaccines. mva-bn has been approved as smallpox vaccine in europe and canada in , and as smallpox and monkeypox vaccine in the us in . no signal for inflammatory cardiac disorders was identified throughout the mva-bn development program. this is in sharp contrast to the older, replicating vaccinia smallpox vaccines, which have a known risk for myocarditis and/or pericarditis in up to in vaccinees. mva-bn-filo as part of a heterologous ebola vaccination regimen (ad .zebov/mva-bn-filo) has undergone clinical testing including phase iii in west africa and is currently in use in large scale vaccination studies in central african countries. this paper provides a comprehensive picture of the mva-bn vector, which has reached regulatory approvals, both as mva-bn backbone for smallpox/monkeypox, as well as for the mva-bn-filo construct as part of an ebola vaccination regimen, and therefore aims to provide solutions to prevent disease from high-consequence human pathogens. Ó the authors. published by elsevier ltd. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). the brighton collaboration (www.brightoncollaboration.org) was launched in to improve the science of vaccine safety [ ] . the brighton collaboration formed the viral vector vaccines safety working group (v swg) in october to improve our ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when a viral vector vaccine is licensed [ ] . the v swg has developed a standardized template describing the key characteristics of a novel viral vaccine vector to facilitate the scientific discourse among key stakeholders and increase the transparency and comparability of information. this introduction and the ''specific instructions" provide definitions and additional guidance for completing the template (v . ) that follows. viral vector vaccines are laboratory-generated, chimeric viruses that are based upon replicating or non-replicating virus vectors into which have been spliced genes encoding antigenic proteins for a target pathogen. consideration of safety issues associated with viral vector vaccines requires a clear understanding of the agents used for construction of the vaccine. these include ( ) the wild type virus from which the vector is derived, referred to in the template as ''wild type virus"; ( ) the vector itself before incorporation of the foreign antigen, referred to in the template as ''viral vector"; and ( ) the final recombinant viral vector vaccine, referred to in the template as ''vaccine". wild type viruses used as vectors may originate from human or non-human hosts and may have low or high pathogenic potential in humans regardless of species of origin. viral vectors can originate from attenuated human vaccines, from attenuated human viruses, from human viruses with low pathogenic potential, from animal viruses with low human pathogenic potential, and from vectors (for the expression of proteins) which are then adapted as a viral vector (such as dna plasmids or baculovirus vector vaccines) to be used as a vaccine in humans or animals. thus, viral vectors usually, but not always, have properties in a human host that differ from wild type virus from which they were derived. incorporation of a target antigen into a viral vector to create a vaccine may alter the properties of the vector such that the vaccine may have properties that differ from the vector. the brighton collaboration vaccine vector template is designed to describe vectors into which transgenes may be incorporated to create vaccines. however, pursuant to understanding completely the safety aspects of a given vector, consideration is given to the wild type virus from which the vector is derived ( the world was declared to be free of smallpox in may and this was because of global vaccination with vaccinia virus (vacv) based vaccines [ ] . although these vaccines were very successful at preventing smallpox there were serious adverse events indicating the need for a less virulent vaccine [ , ] . due to these often-severe post-vaccination complications associated with vaccinia viruses, there were several attempts to generate a more attenuated, safe smallpox vaccine. modified vaccinia ankara (mva) originates from the dermal vaccinia virus ankara strain (chorioallantois vaccinia virus ankara, cva) that was maintained in the vaccination institute ankara for many years and used as the basis for vaccination of humans. during the period of to , prof. anton mayr and his colleagues (university of munich, germany, institute for microbiology and infectious diseases of animals) succeeded in attenuating cva by over continuous passages in primary cef (chicken embryo fibroblast) cells. a reduced virulence of cva was reported from passage on cef cells [ ] . from passage , the attenuated cva virus was renamed mva to discriminate it from other attenuated vaccinia virus strains [ ] [ ] [ ] . in clinical trials with mva, the pock lesions associated with vaccinia virus vaccination are not seen [ ] . this attenuated mva vaccine was used in more than , vaccinees for priming prior to administration of a conventional smallpox vaccine in a two-step protocol used in the s in europe [ , , ] . in the last decades, multiple recombinant mva vectors have been tested as vaccine candidates against various pathogens, such as human immunodeficiency viruses, mycobacterium tuberculosis, plasmodium falciparum or middle east respiratory syndrome virus [ , ] . mva-bn, that is derived from the mva strain developed in prof. anton mayr's laboratory, is a further attenuated mva strain, which has lost its ability to replicate in most mammalian cell types, including human cell lines and is safe in severely immune compromised animals [ , ] . the hallmark of mva-bn is the fact that it does not productively replicate in the human keratinocyte cell line hacat, the human cervix adenocarcinoma cell line hela, the human embryo kidney cell line (hek ), and the human bone osteosarcoma cell line b [ , ] . however, like other mva strains, mva-bn effectively infects mammalian cells. infection of mammalian cells results in transcription of the viral genes, but no mva-bn virus is released from the cells due to a genetic block in the viral assembly and egress. the infected cells eventually undergo apoptosis (programmed cell death) [ ] [ ] [ ] . there are several deletions and other mutations in mva that account for the change in host-range of the virus. six major deletions mainly account for a reduction in the size of the original vaccinia genome from . kb to kb for the mva strain [ , ] . sequencing of the genome revealed that these deletions included immune evasion genes, host interactive protein genes and some structural proteins [ ] . due to the lack of replication competence in many mammalian cells including human cells, mva-bn can be safely administered to immunocompromised humans. this safety feature has also been confirmed in severely immunocompromised animals [ , ] . mva-bn is now a licensed smallpox vaccine (since in eu and canada and since in the us, where it is also licensed as mva-bn was shown to be more attenuated compared to two other mva isolates, namely mva- and mva-i , and even fails to replicate in immune compromised animals [ , ] . despite its high attenuation and reduced virulence, mva-bn has been shown to elicit both humoral and cellular immune responses to vaccinia virus and foreign genes cloned into the mva-bn genome [ ] [ ] [ ] [ ] [ ] [ ] . mva is a potent inducer of type i interferon (ifn) in human cells. mva expresses a soluble interleukin- receptor, which has been implicated as an antivirulence factor for certain poxviruses. mva does not express soluble receptors for ifn-c, ifn-a/-b, tumor necrosis factor and cc chemokines [ ] . neurovirulence assessment of vaccinia virus based smallpox vaccines demonstrated the inability of mva to replicate in suckling mouse brains following intracranial inoculation of - plaque forming units (pfu) of virus, while all other vaccinia virus strains tested (dryvax Ò , lister, copenhagen, ihd-j, wr) replicated to peak titers of to pfu per gram tissue. moreover, none of the doses of mva tested, i.e. up to pfu administered intracranially, induced death in the suckling mice. in contrast, mortality induced by dryvax Ò started at a dose of pfu; lister, copenhagen, ihd-j and wr induced death in some mice at pfu and injection of pfu was % lethal confirming neurovirulence reported for these strains [ ] . mva-bn replicates extensively and rapidly in cef cells and also in certain other avian cell lines. (replicating or non-replicating) mva-bn is a non-replicating vector in humans. vacv is the virus used for the replicating smallpox vaccine that was utilized during eradication and now acam . [ ] . what is the natural host for the wild type virus? the original host is unknown, but vacv can replicate in a range of animals including primates, rodents, lagomorphs and ungulates as well as humans. the origin of the vacv is debated and there is some evidence that it originated from a horse poxvirus which was able to infect cows. in brazil and in india vacv is endemic in animals, with occasional transmission to humans, and is thought to originate from smallpox vaccine campaigns. [ [ ] [ ] [ ] [ ] [ ] . . how is the wild type virus normally transmitted? the typical manifestation of the wildtype virus infection are vesiculopustular lesions or dermal vesicles (pox lesions). these lesions contain infectious virus particles. transmission can occur by close contact with infected area. there is no evidence that vacv is transmitted via airborne infection. there is evidence that shedding of vacv from the vaccination lesion of healthy primary vaccinees occurs from about the third day to the end of the third week after vaccination. there are rare reports of transmission of vacv. [ ] . . does the wild type virus establish a latent or persistent infection? no, the infections are acute [ ] . . does the wild type virus replicate in the nucleus? no. poxviruses replicate in the cytoplasm. [ ] (continued on next page) [ , ] reference for myocarditis and/or pericarditis frequency: [ ] in immunocompromised humans can be fatal and so vaccination with vacv is contraindicated. applicable for the replicating vacciniabased smallpox vaccines [ , ] in human neonates, infants, children children < months of age have an increased rate of the complications listed above for healthy human host [ , ] during pregnancy and in the unborn in humans poxviruses make excellent vaccine delivery vehicles since their genomes allow large insertions of foreign dna [ , ] . conventionally, foreign genes are inserted into poxviruses by homologous recombination into non-essential genes or into intergenic regions [ ] . the genes are under the control of a poxvirus promoter and may have a reporter gene or selection marker to aid selection of recombinants [ ] [ ] [ ] [ ] . the foreign genes are usually modified to remove the poxvirus early transcription termination signals (tttttnt) [ ] and must be devoid of introns. recently a horsepox virus genome has been made by chemical synthesis and rescued by coinfection with shope fibroma virus [ ] demonstrating that this strategy can potentially be used in the future to synthesize other poxviruses. one of the most successful poxvirus vectored vaccines is the vacv vectored rabies vaccine distributed in oral baits for foxes, which has almost completely eradicated terrestrial rabies in parts of europe [ , ] . host restricted poxviruses, such as the canary poxvirus, alvac, have been registered as commercial vaccine vectors for a number of veterinary diseases including equine influenza, canine distemper, rabies, feline leukemia and west-nile fever [ ] . this publication presents the properties of mva-bn as a vaccine vector and specifically focuses on mva-bn-filo as a component of an ebola vaccine two-dose regimen (ad .zebov/mva-bn-filo) which was granted marketing authorization by the european commission on july , [ ] (table ). the findings, 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live, nonreplicating) prescribing information vaccinating against monkeypox in the democratic republic of the congo smallpox vaccination and its consequences: first experiences with the highly attenuated smallpox vaccine ''mva" key: cord- -yf lvbs authors: von linstow, marie-louise; nordmann winther, thilde; eltvedt, anna; bybeck nielsen, allan; yde nielsen, alex; poulsen, anja title: self-reported immunity and opinions on vaccination of hospital personnel among paediatric healthcare workers in denmark date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: yf lvbs background: denmark has no general recommendations for vaccination of healthcare workers (hcws). we explored the self-reported immunity to varicella, measles, mumps, and rubella, reasons for receiving the influenza vaccine or not, and opinions on vaccination of hcws against varicella, mmr, pertussis, diphtheria, and influenza among staff from departments with a high risk of exposure to infectious agents. methods: from may to august , a structured questionnaire was distributed to clinical and non-clinical hcws at a tertiary and a general paediatric department in denmark. self-reported immunity was defined as either previous infection or vaccination against the disease. results: of employed hcws, ( %) were included. a large proportion were unsure of or denied previous vaccination or infection with measles ( . %), mumps ( . %), rubella ( . %), varicella ( . %), pertussis ( . %), and diphtheria ( . %). non-clinical personnel and employees born in – had the lowest level of self-reported immunity. mandatory vaccination of non-immune hcws was approved by – . % of participants, and any kind of vaccination (mandatory or as an offer at hospitals) was approved of up to . % of all participants depending on the disease. during the season / , ( . %) hcws received the influenza vaccine, including . % of non-clinical staff, . % of nurses and . % of doctors (p < . ). reasons for lack of vaccine uptake were mainly employees considering themselves rarely sick, the vaccine was not regarded as necessary, forgetfulness or lack of time. only . % was in favour of mandatory influenza vaccination. conclusions: a large proportion of paediatric hcws were not aware of their immune status against important vaccine-preventable diseases. > % supported vaccination of hcws, with two out of three supporting mandatory mmr, pertussis and diphtheria vaccination. better information and an official immunisation policy of non-immune hcws in denmark is warranted. healthcare workers (hcws) in paediatric settings are often exposed to communicable diseases and represent a source of infection for susceptible patients, parents, and colleagues. measles, mumps, rubella, varicella, influenza and pertussis are highly conta-gious infectious diseases that can cause serious complications, particularly in immunocompromised patients, infants and pregnant women. the child vaccination programme in denmark includes two doses of measles, mumps, rubella (mmr) vaccine and a + regimen of diphtheria, tetanus, acellular pertussis, polio, and haemophilus influenzae type b (dtap-ipv-hib) vaccine with a booster dose of dtap-ipv at five years. varicella vaccination and further booster doses of dtap are not implemented in the program and influenza vaccination is only given to certain risk groups. despite high vaccination coverage (> % for most vaccines), and a measles elimination status since , cases of measles occurred in denmark in , six of them as imported cases [ ] . https://doi.org/ . /j.vaccine. . . - x/ published by elsevier ltd. many vaccine-preventable diseases (vpds) spread rapidly in closed settings even before onset of symptoms or when symptoms are mild and unspecific. vaccination is the best way to protect susceptible individuals and prevent transmission of infections, but vaccination status and immunity of danish hcws have only attracted little attention. it is estimated that hcws have a - fold increased risk of acquiring measles compared to the general population [ ] and numerous outbreaks of measles, rubella, pertussis, varicella, and influenza have been traced to hcws [ ] [ ] [ ] [ ] [ ] [ ] . in a recent italian study, five of hcws involved in a nosocomial measles outbreak had no direct contact to patients [ ] . denmark experienced a national pertussis epidemic from june until lockdown of the country due to covid- in mid-march with up to cases/ . per year in some areas [ ] . to date, no outbreaks in hospital settings were recorded. a national initiative was taken to vaccinate pregnant women for pertussis until the end of . no booster vaccination programme of front-line hcws was undertaken and no booster vaccinations in teenagers and adults were implemented. during each season, % of hcws are estimated to contract influenza [ ] , and a large group of hcws continue to go to work despite of symptoms [ , ] . a recent study showed that nearly half of hcws with laboratory-confirmed influenza were afebrile, posing a risk of influenza transmission to patients and coworkers [ ] . influenza vaccination of hcws is an inexpensive and safe way to reduce transmission [ ] . as opposed to many other european countries [ ] , denmark has no national recommendations for vaccination of hcws except for hepatitis b to specific groups, and there is no requirement of up-to-date vaccinations for new employees. vaccination of hcws is highly debated globally and mandatory vaccination against e.g. influenza, pertussis, mmr and diphtheria has been implemented as a requirement to be employed at some institutions in the united states and europe [ , ] . refusal of mandatory vaccination may result in transfer to a post with no patient contact, a fine or employment termination. however, not all countries have a regulatory policy or penalty in case of mandatory vaccine refusal [ ] . before considering implementing recommendations for vaccination of hcws it is important to know the magnitude of the need and understand staff attitudes and perceptions of such an initiative. we explored the self-reported immunity to varicella and the mmr diseases, reasons for receiving the influenza vaccine or not, and opinions on vaccination of hcws against varicella, mmr, pertussis, diphtheria, and influenza among staff from two departments with a high risk of exposure to infectious agents. denmark has general paediatric departments and tertiary referral paediatric departments employing approximately hcws. in this cross-sectional survey, all hcws employed at the department of paediatrics and adolescent medicine, rigshospitalet, a tertiary care centre including oncology, cardiology and bone marrow transplant units, and the department of paediatrics and adolescent medicine, hillerød hospital, a general hospital including a neonatal ward were invited to participate in the study in the period from may to august . both centres have a paediatric emergency ward and outpatient clinics. hcws included nurses, physicians, medical and nursing students, secretaries, dieticians, cleaning staff, clowns and others with direct or indirect con-tact to patients or access to patient rooms. hcws were full-time employed or only temporarily associated to the departments. initially, hcws were informed about the project by flyers distributed to all units followed by short information meetings. during the study period, all hcws were approached personally and offered the opportunity to participate in the study. interested staff received oral and written information from one of four physicians involved in the project. after written informed consent, participants filled-in a hard copy of a structured questionnaire including ) sociodemographic and professional characteristics (sex, age, number of children at home, profession, year of graduation, years in present job, work place (ward, outpatient clinic or both)), ) self-reported immunity status and vaccine uptake (history of infection or vaccination against the following vpds: varicella, measles, mumps, rubella, pertussis and diphtheria, and history of influenza vaccination), ) knowledge of side-effects to vaccines against the above mentioned diseases marked as ''great knowledge", ''little knowledge" or ''no knowledge"), and ) attitudes towards vaccination of hcws in denmark (registered as the answer ''yes", ''no" or ''don't know" to the question ''do you approve mandatory vaccination of hcws" and ''do you approve vaccination as an offer to hcws" for each of the investigated diseases) . reasons for rejecting or accepting influenza vaccination were written in free text promoting open-minded answers and later arranged into appropriate categories. the questionnaire was developed for the purpose of the study and was pilot-tested in a subset of nurses and doctors. it was reviewed and approved by the ethics committee of the capital region of denmark and the data protection agency before project start. data was registered anonymously. we defined self-reported immunity against varicella and the mmr diseases as either previous infection or vaccination against the disease. susceptibility was defined as either no history or no knowledge of previous infection or vaccination. pertussis and diphtheria were not included in these definitions due to waning immunity following disease or vaccination. chi-square tests were applied to compare the associations between self-reported history of disease and vaccination with age and occupation. further, chi-square tests were used to investigate the association between reasons for influenza vaccine refusal and occupation, and to compare opinion on mandatory vaccination with age and own influenza vaccine uptake. logistic regression analysis was applied to investigate factors significantly associated with self-reported immunity to vpds. self-reported immunity as the outcome variable was coded as a binary variable (yes/no) with yes defined as answering yes to either vaccination or previous infection and no defined as answering no or uncertain to both vaccination and previous infection. the following independent variables were assessed in the univariate analysis: sex, age, profession, children in household, workplace, years in present job, knowledge of sideeffects. variables with a significance level of p < . in the univariate analysis were entered in the multiple regression model (forward selection). a p-value < . was considered significant. data were analysed using the spss software, version for windows. we included ( %) of all hcws from the two departments. seventeen ( . %) declined participation, and ( . %) were never approached mainly due to variable work schedules. characteristics of participants are shown in table . almost % were females, and more than half were nurses. of note, at both centres almost one third of participants had been less than two years in their present job, including . % of physicians, which is mainly due to planned rotation among young doctors, and . % of nurses, indicating a great deal of replacement on these wards. participants' self-reported vaccination status and disease history are shown in table . a high proportion of hcws was not aware of their vaccination status. the lack of knowledge regarding previous vaccination to the mmr diseases was highest in the age group - (born in - ), with % of respondents in doubt. similarly, a high proportion of hcws did not know if they had previously been infected, ranging from . % for varicella to . % for pertussis. as for vaccination, the age group - years was the group with least knowledge regarding previous infection. self-reported immunity was seen in . % for varicella, . % for measles, . % for mumps and . % for rubella. . % of female hcws below the age of years, and therefore potentially fertile, reported to be non-immune to rubella. of those with no or uncertain previous infection with measles, mumps or rubella, between % and % had a positive vaccination status (fig. ) . the relation between self-reported immunity and age group is illustrated in fig. . for mumps, immunity declined by age, and for all diseases, immunity was more prevalent in the youngest age group compared to the - year-olds. non-clinical personnel had the lowest level of self-reported immunity for all four diseases (fig. ) , while dieticians and physiotherapists had the highest level of immunity to measles and varicella, followed by physicians. age and profession were the only variables associated with immunity in the multiple logistic regression analysis: compared to the oldest age group, self-reported immunity to mumps was higher in the youngest age group (adjusted odds ratio (aor) . ), and immunity to measles was lower in the - year-olds (aor . ). compared to nurses, physicians had an aor of . of reported immunity to measles, while non-clinical staff had an aor of . for immunity to all three mmr diseases (suppl . table) . there was no difference in immunity according to sex, workplace, or years in present job. during the season / , ( . %) hcws from the two departments received the influenza vaccine, including . % of non-clinical staff, . % of nurses and . % of doctors (p < . ). staff > years were vaccinated in . % of cases compared to . % of those below years (p < . ). only . % of those with no knowledge of side-effects received the influenza vaccine compared to . % of those with great knowledge and . % of those with little knowledge (p < . ). all but one recipient stated self-protection as the reason to get the vaccine, and all recipients mentioned the protection of patients and others as the reason. three nurses felt a pressure from colleagues to receive the vaccine. reasons for vaccine refusal are illustrated in table . decliners most frequently regarded themselves as healthy individuals who rarely got sick and therefore considered the influenza vaccine redundant. only . % did not want the vaccine and . % were concerned of side-effects. physicians most often did not have time or forgot to get vaccinated. most participants were in favour of mandatory vaccination of non-immune hcws ranging from % for varicella to . % for measles. as seen from table , significantly more personnel from the younger age group < years favoured mandatory vaccination. an exception was the influenza vaccine, where only . % of all participants were positive to mandatory vaccination. only . % of hcws in favour of mandatory influenza vaccine received the vaccine themselves in the season / . a large group ( - % depending on the disease) was in doubt regarding their opinion for or against mandatory vaccinations. however, among those not in favour of mandatory vaccinations, between . % and . % thought vaccinations should be offered hcws at hospitals. in total, any kind of vaccination (mandatory or as an offer at hospitals) was approved by . % (varicella) to . % (rubella) of all participants depending on the disease. there was no difference in attitudes between occupational groups (data not shown). a quarter of participants anticipated to have great knowledge of side effects to the varicella, mmr, pertussis and diphtheria vaccines. however, half mentioned they had limited knowledge. a quarter did not know anything about the side effects, and this group was more often in doubt about their opinion on mandatory vaccination or not. a great difference in knowledge was seen according to profession (fig. ) . physicians was the only occupational group with> % having great knowledge of side-effects. despite a significant reduction and control of several infectious diseases after universal implementation of vaccination programs, outbreaks of vpds continue to occur. in our study, a high proportion of hcws was not aware of their immunity status to the vpds investigated. another large proportion claimed not to be immune, so totally - % of hcws were potentially at risk of transmitting diseases in the health-care setting. in paediatrics, diseases like measles and pertussis and their complications are well-known, and hcws regularly provide advice and remind parents to vaccinate their children. therefore, it is of concern that such a large proportion of the staff had not thought of their own immunity and risk of getting sick. for both measles and rubella, the lack of self-reported immunity was highest in employees born in - . in denmark, the mmr vaccine was introduced in with a catch-up program for those born in - . a proportion from this generation may not have received the vaccine, and due to herd immunity, they have not had the diseases either. this age group constitutes a large part of the workforce in most centres including ours, making it of outmost importance to act upon. non-clinical personnel have previously been involved in outbreaks of vpds [ , ] . in our study, this occupational group had the lowest level of self-reported immunity, emphasizing the importance of including all groups of hcws in immunisation programs. likewise, healthcare students can go unnoticed because of their shared time between hospital wards and universities. implementing (mandatory) immunisation programs at medical and nursing schools could contribute to the promotion of a sense of care and increase immunity of future employed hcws [ ] . the uptake of influenza vaccination in our study was . %. other european countries have reported influenza vaccine cover-age ranging from % to . % in / [ ] , and uptakes of . - . % are reality in places with a mandatory vaccination policy [ , ] . like in our setting, most studies report lower vaccination uptake by nurses compared to physicians, which is of concern as nurses commonly spend more time with the patients than physicians do. the finding reflects different levels of knowledge and attitudes about influenza and its prevention, suggesting a need for specific and profession-tailored programs [ ] [ ] [ ] [ ] . self-protection and protection of patients were the main motivators for getting the influenza vaccine, which is in line with findings from others that self-protection is the strongest and most consistent driver of hcw's decisions to accept vaccination followed by prevention of illness in patients [ , ] . vaccine refusal was mainly explained by the assumption that healthy individuals were not in need of the vaccine. inattention and a lack of time as a barrier to vaccination was more than tripled in physicians compared to nurses and increased six-fold compared to other occupational groups, emphasizing a need for easier vaccine access for certain groups. in our study, - % of hcws supported a mandatory vaccination policy for measles, mumps, rubella, varicella, pertussis and diphtheria, most pronounced in those under years of age. on the other hand, the majority of hcws did not support the idea of mandatory occupational influenza vaccination. reasons for this can be many such as suboptimal efficacy, which varies by influenza season, and the fact that the vaccination is needed every year. the finding is different from attitudes in paediatric hcws in greece with . % supporting mandatory vaccinations, less for mumps ( . %) and most for influenza ( . %) [ ] . in a paediatric tertiary care hospital in philadelphia, . % of employees supported a mandatory influenza vaccination policy, but a majority also felt that the policy was coercive [ ] . a recent study involving european countries found an overall of . % hcws favouring mandatory immunisation for hcws involved in clinical work. however, rates differed significantly among countries [ ] . interestingly, when asking the public, > % agree that hcws have an obligation to be vaccinated against influenza and pertussis and just as many support vaccination for childcare workers [ ] . several ethical issues are associated with hcw immunisation and mandatory vaccination is far from danish principles. a voluntary vaccination program seems the best approach to foster greater employee cooperation and trust in the system [ ] . education regarding the potential health consequences for patients, diagnosis, treatment, modes of transmission and easy access to free vaccines including worksite vaccination during all shifts will undoubtedly increase vaccination uptake and must be prioritised before considering mandatory programs. however, in countries experiencing increasing vaccination hesitancy and refusal, mandatory vaccination policies might increasingly be implemented in the next years [ ] . a strength of this study is the high response rate of %, which was possible due to in-person contact with each employee and distribution of hard-copy questionnaires, which would not have been possible in a larger setting. we are not aware of similar studies involving all hcw categories including non-clinical personnel, students and volunteers thus considering the voice of groups that are often not perceived as hcws. the opportunity to provide anonymous responses increases the probability of honest responses and comments. however, the findings that just over half of those supporting the idea of mandatory influenza vaccination were vaccinated themselves, could lead to the assumption that some workers stating they would accept vaccination, would in reality not. however, the main reason for abstaining was a lack of time and not an active choice against vaccination. this study has some limitations. information about previous disease and vaccination was self-reported based on memory and knowledge and not on vaccination cards or health files. a larger part than registered in this study may have been protected from vpds. a mandatory electronic vaccination registry was implemented in , but vaccinations given prior to are not all found electronically explaining why some respondents could not find proof of previous vaccinations and had to rely on information from their parents if possible. this could, however, lead to both an under-and overestimate of immunity. as this is a cross-sectional study the results are limited in time. in conclusion, large immunity gaps might exist in danish paediatric hcws. there is a great interest in improving protection from vpds with up to . % of all hcws supporting vaccination depending on the disease and two out of three hcws supporting mandatory mmr, pertussis and diphtheria vaccination of unprotected employees. however, the support for mandatory influenza vaccination was markedly lower. educational campaigns about the benefit of vaccinations and implementation of national recommendations for vaccination of hcws in denmark is warranted. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. epi-nyt week / - measles among healthcare workers: a potential for nosocomial outbreaks variation in risk for nosocomial chickenpox after inadvertent exposure a nosocomial outbreak of pandemic influenza a(h n ) 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implication of health care personnel in measles transmission: the need for updated immunization status in the move towards eradication of measles in catalonia vaccinations among medical and nursing students: coverage and opportunities seasonal influenza vaccination in europe -vaccination recommendations and coverage rates for - and - . european centre for disease prevention and control employee designation and health care worker support of an influenza vaccine mandate at a large pediatric tertiary care hospital influenza vaccination of health care workers in hospitals-a review of studies on attitudes and predictors influenza vaccination: opinions of health care professionals working in pediatric emergency departments selfreported influenza vaccination rates and attitudes towards vaccination among health care workers: results of a survey in a german university hospital knowledge and attitudes towards influenza vaccination of health care workers in emergency services factors associated with healthcare worker acceptance of vaccination: a systematic review and meta-analysis attitudes regarding occupational vaccines and vaccination coverage against vaccine-preventable diseases among healthcare workers working in pediatric departments in greece immunization related behaviour among healthcare workers in europe: results of the hproimmune survey should professionals caring for children be vaccinated? community perspectives on health care and child care worker immunisation the authors want to thank the healthcare workers who replied to the questionnaire for their great interest in participating in the study. we are grateful to the danish health foundation and aase and ejnar danielsens foundation for supporting the study. author's contributions mll, ap and ayn initiated and designed the study. mll, tnw, ae and abn coordinated the study, organized recruitment, and collected data. mll performed statistical analyses and prepared the first draft of the manuscript. all authors participated in manuscript preparation and approved the final manuscript. the study was funded by the danish health foundation grant no -b- and aase and ejnar danilelsens foundation, denmark grant no - - . the sponsors were not involved in study design, in the collection, analysis and interpretation of data, or in the writing of the report. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- - qfe kok authors: xia, yufei; fan, qingze; hao, dongxia; wu, jie; ma, guanghui; su, zhiguo title: chitosan-based mucosal adjuvants: sunrise on the ocean date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: qfe kok mucosal vaccination, which is shown to elicit systemic and mucosal immune responses, serves as a non-invasive and convenient alternative to parenteral administration, with stronger capability in combatting diseases at the site of entry. the exploration of potent mucosal adjuvants is emerging as a significant area, based on the continued necessity to amplify the immune responses to a wide array of antigens that are poorly immunogenic at the mucosal sites. as one of the inspirations from the ocean, chitosan-based mucosal adjuvants have been developed with unique advantages, such as, ability of mucosal adhesion, distinct trait of opening the junctions to allow the paracellular transport of antigen, good tolerability and biocompatibility, which guaranteed the great potential in capitalizing on their application in human clinical trials. in this review, the state of art of chitosan and its derivatives as mucosal adjuvants, including thermo-sensitive chitosan system as mucosal adjuvant that were newly developed by author's group, was described, as well as the clinical application perspective. after a brief introduction of mucosal adjuvants, chitosan and its derivatives as robust immune potentiator were discussed in detail and depth, in regard to the metabolism, safety profile, mode of actions and preclinical and clinical applications, which may shed light on the massive clinical application of chitosan as mucosal adjuvant. vaccination has arguably been the most successful and effective medical intervention for preventing infectious diseases, resulting in decreased mortality, extended life expectancy, and improved quality of life [ ] [ ] [ ] . more than million vaccine formulations are now administered by subcutaneous or intramuscular injections, according to who records [ ] . however, some shortcomings have persisted throughout the development of the vaccine industry, such as needle-stick injuries, the potential for contamination, and the need for highly trained personnel [ ] . furthermore, most pathogens invade the body at mucosal sites. the mucosal immune system, comprising epithelial cells, mucosa-associated lymphoid tissues (malts), and lamina propria, contains almost % of the immunocytes in the human body [ ] and constitutes the first line of defense against infection [ ] . unfortunately, vaccination via injection fails to achieve acceptable immune protection at mucosal sites [ ] . needle-free vaccination offers immense potential in terms of patient compliance and ease of administration, and has attracted great attention recently [ , [ ] [ ] [ ] . the significance of mucosal immunity and its potential for improving global health has inspired research demonstrating that mucosal vaccines could be an attractive alternative to parenteral administration [ , ] . nonetheless, the administration of antigens via mucosal routes normally leads to poor immunogenic stimulation. to overcome this, live pathogens have been employed in commercial vaccines such as oral polio [ , ] , oral cholera [ ] [ ] [ ] [ ] , oral rotavirus [ ] [ ] [ ] and nasal influenza (flumist tm [ ] [ ] [ ] , nasovac tm [ ] ), but these still retain some potential hazards for large-scale application. hence, rational design of an appropriate adjuvant system with a potent capacity to stimulate immunity is essential [ ] . modern manufacturing technology is obliged to meet the following requirements: ( ) stability of the antigen in an acidic, alkaline, or enzymatic environment [ ] [ ] [ ] ; ( ) retention of the antigen despite constant clearance at mucosal sites [ , ] ; ( ) potent induction of both systemic and mucosal immunity [ ] ; and ( ) a balance between enhanced mucosal immunity and the possibility of immune tolerance when using high doses of antigen [ ] . as a promising solution, chitosan possesses well-defined properties including biocompatibility, low cost [ ] , high levels of tolerance, a broad antibody response, and the capacity to stimulate robust th responses [ , ] . chitosan has been demonstrated to have distinct mucoadhesivity, which prolongs the antigen http://dx.doi.org/ . /j.vaccine. . . - x/© elsevier ltd. all rights reserved. retention time at mucosal sites [ ] , as well as the ability to open the tight junctions between epithelial cells to intensify the transport of antigen to sites of immune induction and effector responses [ , , , ] . moreover, the versatility of chitosan enables a diversity of formulations for chitosan-based delivery systems, such as solutions, powders, micro/nanoparticles, hydrogels, and microneedle patches, to meet the various needs of novel vaccination methods [ , , , ] . recent research has proposed several promising chitosan-based formulations that are highly suitable as mucosal adjuvants with outstanding safety profiles. this article aims to provide an up-to-date review of pre-clinical and clinical applications for these novel adjuvant systems. although chitosan has been investigated for many years as a basic material for mucosal adjuvants, it is not yet present in any marketed vaccine formulation. the main obstacles may be a lack of understanding of its metabolism and concerns regarding safety issues. hence, a more comprehensive evaluation of its metabolism, safety profile, modes of action, and clinical applications is also presented in the following sections. chitosan, a natural cationic polysaccharide, is made up of copolymers of glucosamine (␤( - )-linked -amino- -deoxyd-glucose) and n-acetylglucosamine ( -acetamido- -deoxy-dglucose), and is derived by the partial deacetylation of chitin obtained from the shells of crustaceans or the mycelium of fungi [ ] . a wide range of chitosan polymers is available with diverse molecular weights (mw), degrees of de-acetylation (dd), and modifications, all of which may impact on their solubility, biocompatibility, mucoadhesive behavior, and degradation, and thus influence their immunogenicity [ ] . scherließ et al. successfully showed that three commercialized chitosans with different mw and dd possessed varied levels of adjuvant activity, and the impurities (protein contaminants) in the chitosan were found to play only a minor role. they also pointed out that these diverse adjuvant effects cannot be attributed to a single factor but rather an interplay between molecular weight, degree of deacetylation, particle size, and solubility [ ] . to overcome the poor solubility of chitosan at neutral ph [ ] , various chitosan derivatives have been developed by chemical modification of hydrophilic groups or by simply grafting on water-soluble polymers (e.g., polyethylene glycol) [ ] [ ] [ ] . the derivatization of chitosan not only enhances its water solubility, but also improves the cationic nature of the material, which enhances its interactions with antigens or cell membranes, boosting antigen adsorption/encapsulation and cellular internalization [ ] , as well as facilitating the residence time at mucosal sites [ ] . among the water-soluble chitosan derivatives, n-trimethyl chitosan (tmc) and n-( -hydroxy) propyl- -trimethyl ammonium chitosan chloride (htcc) are the most commonly used mucosal adjuvants. the chemical structures and main properties are summarized in table [ ]. a comprehensive understanding of the metabolism of a polymer is a prerequisite for its development as a novel adjuvant [ ] . as a natural polysaccharide, chitosan is usually regarded as a nontoxic, biologically compatible polymer [ , ] . in vitro studies have proven that chitosan can be degraded efficiently by lysozyme, resulting in approximately a % decrease in the weight of the chitosan hydrogel within days [ ] . three enzymatically active forms of human chitinases have been identified in investigations of the degradation of chitosan and its derivatives [ ] , namely acidic mammalian chitinase (amcase), di-n-acetylchitobiase, and chitotriosidase, which have been found in the gastrointestinal tract, liver, and plasma, respectively [ ] [ ] [ ] [ ] . recent research has revealed that the degradation kinetics depend strongly on both the molecular weight and the degree of deacetylation [ , ] . typically, a higher enzyme-reaction rate was found for the degradation of high-mw chitosan. low-dd chitosan was degraded more rapidly, and the remaining to acetyl groups may serve as essential contacts in the active sites of chitosanases [ ] . tmc was degraded by lysozyme at a similar rate to native chitosan, but the degradation rate was independent of ph [ ] . in a study of in vivo degradation, chitosan underwent partial digestion and uptake by the gastrointestinal tract, and was then deposited in tissues, or circulated in the plasma to eventually be eliminated from the body [ ] . limited data on the degradation and distribution of chitosan have been presented, because of difficulties tracking trace amounts of chitosan in the living body [ ] . however, some conclusions can be deduced. first of all, the in vivo degradation and distribution of chitosan is largely dependent on the molecular weight. fitc-labeled chitosan with a small molecular weight ( . kda) was absorbed by the gastrointestinal tract to give a significant plasma concentration. however, chitosan with a mw higher than kda (with similar dd) was excreted in the feces without any uptake [ ] , which indicated that it was not chitosan polymers but chitosan oligosaccharides that could be adsorbed in the gut. in addition, the optimal mw of chitosan for renal clearance was estimated to be kda. if the mw of the adsorbed chitosan oligomers was larger than that, they would undergo further biodegradation [ ] . the failure of intranasal formulations in clinical trials suggests that toxicity may be caused by the uptake of a degradation product by the brain and irritation of the nasal mucosa during degradation. this may lead to serious local symptoms or neurological disorders such as bell's palsy, which is a facial nerve paralysis caused by the intranasal administration of a heat-labile enterotoxin used as an adjuvant. in the case of chitosan-based mucosal adjuvants, understanding the distribution and degradation of chitosan is vitally important for dispelling safety concerns. recently, smith et al. pointed out that high-dd chitosan was degraded much more slowly under the local ph ( . - . ) of the nasal cavity and might guarantee that the administered chitosan would be cleared by mucociliary movement and swallowed into the alimentary tract, which might protect the body from severe adverse effects and toxicological responses [ ] . chitosan with a high dd can probably serve as an ideal raw material for developing intranasal vaccines. most of the above results have concentrated on the metabolic behavior of chitosan polymers. on the other hand, the metabolism of chitosan powder and smaller particles may be a very different story. although trimethyl-chitosan oligomers/dna nanoparticles were shown to be absorbed in the gastric tract [ ] , there is still insufficient evidence to support the deduction that other chitosan formulations would be degraded in a similar manner. with regard to the safety profile of chitosan, cytotoxicity is dependent on the concentration, molecular weight, and degree of de-acetylation, with a half-maximal inhibitory concentration (ic ) of . - . mg/ml in most cell models [ , , ] . in addition, different forms of chitosan have shown similar cytotoxic effects [ , ] . a number of studies have shown the good tolerance and safety performance of chitosan administered via oral routes. in table currently employed chitosan derivatives as mucosal permeation enhancer. vivo, very few adverse effects have occurred in both mouse and rat models after oral intake of chitosan in doses of - g/kg/day for months [ ] . in addition, no relevant clinical signs were found in healthy human volunteers after taking oral doses of up to . g/day [ ] . moreover, people with shrimp allergies have shown no allergic reactions after oral administration of shellfishderived glucosamine [ ] . archimedes and other groups have conducted a large number of preclinical or clinical studies on chitosan administered via the intranasal route. they concluded that chitosan could be well tolerated with no adverse effects or only mild symptoms such as erythema, itchy nose, rhinorrhea, and sore throat [ ] . in view of the above facts, chitosan is generally thought to be a biodegradable and biocompatible biomaterial with no or minor toxicity and offers the potential to be a safe pharmaceutical material for clinical vaccine formulations [ , ] . consequently, the raw material should be selected carefully with regard to the molecular weight, degree of deacetylation, and purity. moreover, it is worth noticing that the physiochemical and biological properties of chitosan are also influenced by the original raw material, the manufacturing procedure, and even by the season and weather. batch-to-batch consistency and stability of the raw material are other challenges when selecting chitosanbased adjuvants for clinical trials. the greatest difference between vaccine adjuvants and drug delivery systems is that vaccines are administered to healthy people, especially populations at high risk of contracting infectious diseases such as infants and the elderly, which means any side effects must be avoided [ ] [ ] . nevertheless, the truth about adjuvants is that "the nearer they get to killing you, the more effective they are". there is an appropriate level of inflammatory response that is undesirable in a drug-delivery system but obligatory for immune stimulation. future research must manage the balance between toxicity and adjuvanticity, in order to advance the clinical application of chitosan-based mucosal adjuvants. chitosan-based formulations have been found to orchestrate both cellular and humoral responses, and represent the most promising candidates for adjuvant and delivery systems for mucosal vaccines [ ] . therefore, it is essential to understand the mechanisms of mucosal immunity and the mode of action of chitosan-based adjuvants, to further the rational design of a chitosan-based adjuvant system with a profound ability to stimulate immunity and a distinct safety performance [ ] . numerous studies have demonstrated the potent induction of cellular responses, activation of the innate immune system, and positive effects on mucosal adjuvanticity [ , , ] . in this section, general strategies for developing chitosan into a promising mucosal adjuvant are addressed in detail with reference to recent reports. the innate immune system not only initiates immediate and consistent responses towards infection, but also directly triggers the adaptive immune system. recognition by the innate immune system requires the activation of pattern recognition receptors (prrs), such as toll-like receptors (tlrs, one of the membranebound prrs) and nucleotide-binding domain leucine-rich repeat containing receptors (nlrs, one of the cytoplasmic prrs), which are specific for conserved ligands associated with cellular damage (danger associated molecular patterns, damps) or invading pathogens (pathogen associated molecular patterns, pamps) [ ] . upon stimulation by danger signals, dcs undergo activation/maturation to an active antigen-presenting phenotype [ ] , with up-regulation of co-stimulatory membrane proteins such as cd and cd , and secretion of proinflammatory cytokines such as tnf (tumor necrosis factor), interleukin (il)- , or il- . recent evidence has showed that all three signals are key factors for tlymphocyte activation [ ] . villiers et al. have demonstrated that chitosan induces the activation of dcs at the level of membranereceptor recognition. chitosan promoted the activation of dcs via a tlr -dependent mechanism, with up-regulation of membrane proteins such as cd and cd [ ] . however, it failed to elicit the robust cytokine production that activates t cells, which indicates that chitosan alone was unable to evoke a t-cell response. nevertheless, when combined with antigen, chitosan and its derivatives stimulated the expression of major histocompatibility complex (mhc) class i molecules, which profoundly up-regulate the expression of co-stimulator molecules, namely cd , cd , and cd , as well as the secretion of the cytokines il- , il- , mip- , and tnf-␣. [ ] this led to stronger cd + cells proliferation and cytotoxic t lymphocyte (ctl) responses [ ] . as one of the best characterized nlr family members, the nlr family pyrin domain containing (nlrp ) was shown to be associated with potent inflammatory responses, which were activated by a multi-protein complex composed of nlrp , the adaptor apoptosis-associated speck-like protein (asc), and the protease caspase- (fig. ) . currently, chitosan particles are proven triggers of a potent adaptive inflammatory response through the nlrp pathway, which facilitates the recruitment of neutrophils [ ] [ ] [ ] the protonation of the amino groups ("proton sponge effect") leads to an extensive inflow of ions and water into the lysosome, which caused the osmotic swelling and deconstruction of the lysosome. the entrapped components (csp and ag) were released and finally presented onto mhc i, by cytoplasm degradation (with proteosome and er involved); after the rupture, lysosome enzymes, cathepsin b, was also leaked into the cytoplasm, which induced the assembly and activation of nlrp complex. the capacity of tlr stimulation of csp also played an important role in the intracellular synthesis of pro il- ␤ and triggered the secretion of il- ␤ and inflammatory responses, together with nlrp activation. (ag: antigen; csp: chitosan particle.) and maturation of apcs, and creates a pro-inflammatory environment characterized by the secretion of cytokines such as il- ␤ and il- [ , ] . as shown in fig. , the disruption of lysosomes is thought to trigger the chitosan-particle-induced nlrp response. after rupture, the leakage of lysosomal enzymes such as cathepsin b, which can prompt the assembly and activation of nlrp complex, stimulates the secretion of il- ␤ and subsequent inflammatory responses [ ] [ ] [ ] . recent research has suggested that the activation of the inflammasome by chitosan particles (csps) is dependent upon the physiochemical properties of csps, such as particle size, charge [ , ] , and surface texture [ ] . neumann et al. found that only positively charged chitosan nanoparticles and alum induced significant il- ␤ secretion after pre-incubation with lps to activate tlr in vitro [ ] . cellular immunity, which is caused by cd + t-cell activation, is vital for the success of prophylactic and therapeutic vaccines [ ] . typically, exogenous antigens are endocytosed by professional antigen presenting cells (apcs), and are then degraded into short peptides within their endosomes. these degraded peptides are loaded onto mhc class ii molecules to present antigen to cd + helper t cells, which leads to humoral immunity. only some of the intracellular antigens would escape from the endosome and be presented to cd + t lymphocytes through the mhc class i pathway, to stimulate cellular immune responses. traditional adjuvant systems using alum, mf , and as , failed to elicit potent cellular immunity [ ] . on the other hand, chitosan particles possess a robust capacity to elicit cross-presentation of exogenous antigens and induce potent cellular immune responses. cross-presentation can be induced by several means such as tlr activation [ , ] , mannose receptor-mediated uptake [ ] , and up-regulation of the expression of heat shock proteins [ , ] . in the case of csps, tuning the cellular trafficking of antigen is triggered by the "lysosome escape" pathway, which indicates that the internalized chitosan particles can disrupt the lysosome [ , ] and escape into the cytoplasm through a "proton sponge effect" [ ] . as shown in fig. , the protonation of amino groups present on the chitosan chains leads to an extensive inflow of ions and water into the lysosome. when the "proton sponge effect" becomes strong enough, the entrapped components (csp and antigen) are released by rupture of the lysosomes [ , ] . the escaped exogenous antigen can be cross-presented through the mhc class i pathway, like endogenous antigens, to prime cd + t cells and evoke robust ctl responses [ , ] . briefly, mucosal surfaces are constructed of a single layer of epithelial cells joined by tight junctions [ ] , which acts as a natural barrier to protect the body from the outside world. when pathogens frequently breach the epithelial barrier, epithelial tissues are activated and serve as sensors for detecting danger signals through pattern-recognition receptors such as tlrs [ , ] . they respond by releasing chemokines and cytokines to recruit malt-resident immunocytes and trigger innate and adaptive immune responses [ ] . recently, chitosan-based adjuvant systems have been proven to have mucoadhesive qualities and the unique ability to open the tight junctions between epithelial cells at natural portals, which stimulates a robust mucosal immune response by facilitating the transport of antigens via an alternative yet effective route for antigen delivery [ , , , , ] . numerous studies have confirmed the mucoadhesive properties of chitosan that prolong the residence time of formulations at mucosal sites, including the nasal mucosa [ , , , ] , gastrointestinal surfaces [ ] , and rectal and genital tracts [ ] . our group employed an in vivo imaging system to evaluate the ability of a thermo-sensitive htcc hydrogel to prolong the residence time of cy labeled h n antigen in the nasal cavity. as shown in fig. a , no fluorescent signal was detected in the pbs/h n group h post administration. however, strong fluorescence intensity could be observed in the htcc/h n group. more than % of the administered antigens persisted in the nasal cavity after h (fig. b) , which shows the ability of chitosan to offer a sustained antigen-mucosal interaction without being cleared away [ ] . the distinguishing trait of chitosan to mimic pathogen entry by opening tight junctions between epithelial cells has also been investigated in recent research [ , ] . artursson's group suggested that the ability to open junctions was due to the positively charged amino group on the c- position of chitosan, which could interact with the negatively charged mucosal surface [ ] . in another study, they compared differences in the transport of [ c]mannitol, among three chitosan-derivative solutions, including chitosan hydrochloride, chitosan glutamate, and tmc, all with a similar degree of de-acetylation. in the intestinal epithelial cell line caco- , the transport of [ c]-mannitol was increased -fold by chitosan hydrochloride, -fold by chitosan glutamate, and -fold by tmc at . % w/v concentrations in vitro, compared with the administration of mannitol alone [ ] . by determining the change in transepithelial electrical resistance to indicate the tightness of the junctions and using confocal laser scanning microscopy in vitro, they further demonstrated the interactions between chitosan and zona occludens (zo)- protein, which is regarded as the target of chitosan and its derivatives [ ] [ ] [ ] . hsu et al. elucidated the signaling mechanism underlying the opening of tight junctions by chitosan in caco- cell monolayers. as shown in fig. , the cascade of tight-junction opening is accompanied by the clustering of integrins (␣ v ␤ ) along the cell periphery, which led to the phosphorylation of focal adhesion kinase (fak, also known as protein tyrosine kinase , ptk ) and src (a family of non-specific tyrosine kinases). this also resulted in the regulation of tight-junction permeability via the redistribution of cldn (a tight-junction protein) from the cell membrane to the cytosol and its eventual degradation in lysosomes [ , ] . in pre-clinical and clinical research, chitosan and its derivatives have demonstrated low-toxicity, mucoadhesive capabilities, the ability to enhance the internalization of antigens by apcs, and immune-stimulatory properties, which makes them promising candidates for effective mucosal adjuvants [ ] . the current evidence supporting chitosan as a potent mucosal adjuvant is summarized and recapitulated in table , in a multitudinous array of vaccine formulations, different chitosan types, dosage forms, model animals, and routes of administration. in the following section, applications for adjuvant systems based on chitosan . the signaling mechanism of cs-mediated tj opening [ ] . and its derivatives are reviewed with regard to their routes of administration. since the adoption and widespread administration of the sabin oral polio vaccine (opv) [ ] , oral delivery has been considered the preferred route of administration for vaccines, especially for children [ ] . recently, chitosan has been extensively studied as a promising candidate for oral administration to promote the internalization of antigen and activate both systemic and mucosal immunity [ ] . several studies have demonstrated that associating the antigens with csps can enhance their uptake by m cells and reduce the risk of vaccine degradation. van der lubben et al. reported that the size of microparticles (mps) should be m for efficient uptake by m cells [ ] . they also found that fluorescently labeled chitosan mps can be taken up by m cells in peyer's patches at intestinal surfaces [ ] . in later research, van der lubben et al. further showed that antigen was released from mps after its uptake by m cells [ ] . moreover, a dose-dependent immune reaction was revealed for mice vaccinated with different doses of antigen associated with chitosan mps [ ] , which proved the ability of chitosan to promote the internalization of vaccines. with the aid of an enteric coating, chitosan delivery systems can be protected from stomach acid and released from within the enteric coating after reaching the intestine [ ] . hori et al. developed ovalbumin (ova)-containing chitosan mps (chi-ova), and coated them with eudragit l (er-chi-ova). compared to an uncoated chi-ova delivery system, significantly higher intestinal mucosal iga and systemic igg responses were detected after oral administration of coated chi-ova. [ ] . in another study, cho et al. presented a mucoadhesive and ph-sensitive thiolated eudragit-coated chitosan microparticle, with an enhanced capacity for retaining the structural integrity of the encapsulated protein [ ] . recently, hbsag-loaded chitosan microparticles were formulated. to overcome the limitations imposed by enzymatic degradation and permeation barriers, bacitracin was introduced as a protease inhibitor, which resulted in better protective levels of immunity after oral administration compared with aprotinin as a protease inhibitor. moreover, the mps remained stable at room temperature for up to four months [ ] . the nasal route is an excellent alternative to conventional forms of vaccination, and is effective at inducing systemic and mucosal immunity in not only the upper and lower respiratory tracts, but also the gastric and genital tracts via cross-protection [ , ] . as mentioned above, the chitosan-based adjuvant system can provide effective immune stimulation through potent enhancement of antigen retention, mucosal permeability, cellular uptake, and protection of the antigen [ ] , as well as a good safety profile [ ] . the past few years have witnessed the wide application of chitosan-based adjuvants in pre-clinical research on intranasal vaccines. vila et al. prepared tetanus toxoid-encapsulated chitosan nanoparticles as an intranasal antigen delivery system, which resulted in an increasing and long-lasting humoral immune response (igg and iga levels) [ ] . the controlled release of antigen by chitosan particles is another important factor contributing to adjuvanticity. mokarram et al. has demonstrated the effect of peg (polyethylene glycol) modifications on the immune-stimulating performance of chitosan nanoparticles, using diphtheria toxoidloaded chitosan nanoparticles with or without peg modifications (cs-peg and cs). in the study, cs-peg and cs nanoparticles both elicited profoundly higher iga and igg levels compared with fluid antigen. in addition, the performance of cs-peg as an adjuvant was significantly better, which may be due to the controlled releasing of antigen [ ] . scherließ et al. proposed a novel spray-dried and ␤-irradiated sterilized vaccine with outstanding stability for intranasal administration against influenza a(h n )pdm in nonhuman primates, and employed chitosan and a stabilizing and protecting solution (sps). the chitosan in the formulation not only served as an inducer of immunity, but also stabilized the antigen, especially in combination with sps. the seroconversion results revealed that the effectiveness of the pilot formulation was comparable to pandemrix ® . moreover, the hemagglutination-inhibition titer of the group administered sterilized vaccine showed no difference from the group administered unsterilized vaccine [ ] . chitosan derivatives, such as tmc [ ] , cg [ ] , and chitosan hydrochloride salt [ ] , have also been employed in the intranasal delivery of antigen. our group has developed a thermosensitive hydrogel formulated from the chitosan derivative htcc and ␣,␤-glycerophosphate (␣,␤-gp) as an intranasal vaccine delivery system for the h n split antigen [ ] . this hydrogel was designed to gelate in a short time at body temperature on the surface of the nasal mucosa, which significantly reduced mucociliary clearance and prolonged the antigen residence time in the nasal cavity. using confocal laser scanning microscopy and scanning electron microscopy, we observed the stained nasal epithelia after intranasal administration of chitosan hydrogel, and have provided significant evidence for the disorganization of zo- protein in nasal epithelial tissue. the thermo-sensitive hydrogel facilitated the transportation of antigen into the sub-mucosa via the paracellular pathway rather than the endocytosis pathway (fig. ) . when employed in the intranasal delivery of h n split antigen [ ] and adenovirus antigen [ ] , the hydrogel system enhanced robust serum and mucosal antibody levels, the secretion of cytokines, and a potent response by antigen-specific memory cd + t cells. when chitosan and its derivatives were added to a hybrid system, an increase in the adjuvanticity of other biodegradable polymers was observed. jaganathan et al. reported that intranasal administration of chitosan hydrochloride-modified plga mps led to significantly higher anti-hbsag igg and iga titers and levels of cytokines (il- and ifn-␥) in spleen homogenates than unmodified plga mps [ ] . in another study, pawar et al. prepared uncoated plga nanoparticles, chitosan-modified plga np (cs-plga), and glycol chitosan-modified plga np (gc-plga) for encapsulation of the antigen, and administered these via the nasal tract. the results showed that gc-plga exhibited the highest level of mucus adhesion and elicited the highest igg and iga titers, as well as the secretion of il- , il- , and inf-␥ [ ] . vicente et al. developed a polymer/oil-based core-corona nanocapsule, formulated with an oily core of miglyol ® , a neutral oil composed of triglycerides of medium chain fatty acids, and a positively charged shell of chitosan [ ] . through co-delivery of rhbsag antigen and the tlr agonist imiquimod, the vaccine orchestrated greater secretion of pro-inflammatory cytokines (il- ␣, il- , and tnf-␣), increasing igg levels over time and boosting both cellular and humoral immunity [ ] . chitosan-based adjuvant systems have also been employed in mucosal vaccination via other routes. chen et al. developed a novel transdermal delivery system consisting of embeddable chitosan microneedles with a supporting array composed of a polylactic acid (pla) polymer, for the sustained delivery of encapsulated antigen, which in this case was ova. the chitosan microneedle remained in the epidermis and dermis (at a depth of approximately m) for days as the chitosan degraded (fig. ) . the antibody levels were significantly higher in the microneedle group than the intramuscular-administration group [ ] . for pulmonary administration, microparticles containing diphtheria toxoid (dt) and chitosan were prepared using a spray-drying technique with a supercritical mixture of carbon dioxide (co ) and ethanol. after immunization, a strong immune response was observed in the group receiving chitosan-dt, with higher igm, igg, igg , and igg titers than the group receiving alum-absorbed dt, which supports the potential of chitosan as a potent delivery system for pulmonary vaccines [ ] . in the food industry, chitosan has been designated generallyrecognized-as-safe (gras) in the us, japan, and italy [ ] . however, chitosan has still not been utilized in any marketed vaccine formulations. only a few studies have been published on the clinical applications of chitosan as an adjuvant. mills et al. have reported the safety and immune-stimulatory capacity of a chitosan-glutamate intranasal delivery system for the diphtheria toxoid antigen crm , among healthy volunteers. in phase a of the study, three groups of subjects were administered intranasal diphtheria vaccine ( g of crm with . mg of chitosan glutamate , and . mg of mannitol), antigen alone (on day and ), or alum-adsorbed diphtheria toxoid vaccine via intramuscular injection (single administration on day ). serum igg and iga from nasal wash were collected on days and . the results suggested that the chitosan intranasal formulation was well tolerated with only transient mild-to-moderate adverse effects (aes), such as nasal discharge, blockage, and discomfort. there were no significant differences in the circulating antibody titers of the groups after first administration, but the antibody levels of the group receiving crm and chitosan intranasally were boosted after a second vaccination [ ] . norovirus infection is the most common cause of viral gastroenteritis in humans [ , ] . parra et al. developed a norovirus virus-like particle (vlp) vaccine, which orchestrated distinct immune responses in the rabbit after intranasal vaccination [ ] . the vaccine (nv-vlp) was formulated as spray-dried powder, which was composed of chitosan (chisys tm ) and norovirus vlp antigen, with mpl (monophosphoryl lipid) as an immune enhancer. chisys tm is a drug delivery technology based on chitosan, which is provided by archimedes development limited. in phase i clinical studies, two randomized, double-blinded, controlled studies of nv-vlp vaccine have been performed. healthy volunteers ( - years old) were randomized to receive two intranasal administrations of nv-vlp or act as controls (with a interval of days). in study , g, g, and g dosages of norwalk antigen and chitosan alone were evaluated. in study , four groups of healthy subjects were administered mpl/chitosan ( or g vlp per dose, as a powder), chitosan only, or placebo (puff of air). the symptoms were recorded for days after vaccination, and the safety evaluation was followed up for days. samples of serum and peripheral blood mononuclear cells were also collected. the results indicated that the most common symptoms were nasal stuffiness and discharge, and sneezing. no vaccine-related serious adverse effects (saes) were observed. the most significant immune response was found for the -g dose, with igg and iga titers increased by . and . -fold, respectively. all subjects who received the vaccine dose of or g produced iga antibodysecreting cells (ascs), with surface molecules that are associated with homing to mucosal and peripheral lymphoid tissues (cd +, cd +, cd l−, integrin ␣ /␤ +, cd +, cd +, cd l+, and integrin ␣ /␤ +). the results indicated that intranasal administration of nv-vlp may evoke iga secretion by intestinal mucosal tissues (the entry site of norovirus) [ ] . in other studies, healthy adults ( - years of age) were randomized and received primary and booster injections of either vaccine ( g vlp per dose) or placebo, administered weeks apart. three weeks after the second injection, the subjects were challenged with norovirus. serology data were collected and the clinical efficacy was assessed by the occurrence of infection, the onset severity, and the duration of the illness caused by norovirus. aes of nasal stuffiness, nasal discharge, and sneezing were also reported in this trial, and occurred with similar frequency among vaccine and placebo recipients. a significant iga response (a -fold increase) was detected in % of vaccine recipients. moreover, the occurrence of norovirus-induced gastroenteritis ( % of placebo recipients vs. % of vaccine recipients, p = . ) and virus infection ( % of placebo recipients vs. % of vaccine recipients, p = . ) was significantly reduced by nv-vlp vaccination [ , ] . chitosan-based adjuvant systems have been developed with versatility in formulation, excellent tolerance, distinct mucoadhesivity, and a robust capacity to stimulate cellular and humoral immunity in both clinical and pre-clinical studies. application of these adjuvant systems would significantly improve vaccine development to meet the needs of reduced cost, increased stability, and easier administration. however, only a few chitosan adjuvant systems have been approved for clinical trials ( on-going clinical experiments), and none of them have yet been included in a licensed vaccine product. numerous challenges are still hindering the development of chitosan-based mucosal adjuvant systems in humans. diverse types of chitosan at different concentrations with various dd and mw are now being studied by separate research groups using a diverse range of antigens (table ) , which could also lead to controversy and confusion regarding its potential application in human medicine. future research is needed to investigate the mode of action of chitosan, based on its different aforementioned properties, and establish criteria for advancing chitosan development. we envisage a booming future for chitosan as an adjuvant in novel mucosal vaccine formulations. 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results of an in vitro and in vivo study effect of salt forms and molecular weight of chitosans on in vitro permeability enhancement in intestinal epithelial cells (caco- ) chitosans as absorption enhancers for poorly absorbable drugs: influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (caco- ) cells transmucosal macromolecular drug delivery a comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery the safety of chitosan as a pharmaceutical excipient chitosan-based drug nanocarriers: where do we stand? do shrimpallergic individuals tolerate shrimp-derived glucosamine chitosan a promising safe and immuneenhancing adjuvant for intranasal vaccines evaluation of the effectiveness and safety of chitosan derivatives as adjuvants for intranasal vaccines challenges in evaluation of chitosan and trimethylated chitosan (tmc) as mucosal permeation enhancers: from synthesis to in vitro application novel vaccine delivery system induces robust humoral and cellular immune responses based on multiple mechanisms a polymer/oil based nanovaccine as a single-dose immunization approach the effects of chitosan oligosaccharide on the activation of murine spleen cd c+ dendritic cells via toll-like receptor toll-like receptors: key mediators of microbe detection cd t cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal cytokine from secretome analysis to immunology: chitosan induces major alterations in the activation of dendritic cells via a tlr -dependent mechanism the effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells contribution of il- to resistance to streptococcus pneumoniae infection role of interleukin- and myd -dependent signaling in rhinovirus infection airway epithelial myd restores control of pseudomonas aeruginosa murine infection via an il- -dependent pathway cutting edge inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp interleukin- promotes coagulation, which is necessary for protective immunity in the lung against streptococcus pneumoniae infection the nlrp inflammasome: a sensor of immune danger signals uptake of particulate vaccine adjuvants by dendritic cells activates the nalp inflammasome chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis surface charge and cellular processing of covalently functionalized multiwall carbon nanotubes determine pulmonary toxicity tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation activation of the nlrp inflammasome is not a feature of all particulate vaccine adjuvants key roles of adjuvants in modern vaccines vaccine adjuvant formulations: a pharmaceutical perspective systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity intracellular mechanisms of antigen cross presentation in dendritic cells distinct pathways of antigen uptake and intracellular routing in cd and cd t cell activation heat shock protein (hsp ) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells heat shock proteins as regulators of the immune response engineering synthetic vaccines using cues from natural immunity steady-state antigen scavenging, cross-presentation, and cd (+) t cell priming: a new role for lymphatic endothelial cells effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection designing polymeric particles for antigen delivery improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone endosomal escape pathways for delivery of biologicals epithelial cells as sensors for microbial infection immunity, inflammation, and allergy in the gut a thermosensitive hydrogel based on quaternized chitosan and poly(ethylene glycol) for nasal drug delivery system local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration chitosan-coated liposomes for topical vaginal therapy: assuring localized drug effect oral transmucosal drug delivery for pediatric use effect of chitosan on the permeability of monolayers of intestinal epithelial cells (caco- ) comparison of the effect of different chitosan salts and n-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (caco- ) in vitro permeability across caco- cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth transport properties are not altered across caco- cells with heightened teer despite underlying physiological and ultrastructural changes oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid) and a chitosan derivative functionalized by trimethylation mechanism and consequence of chitosan-mediated reversible epithelial tight junction opening elucidating the signaling mechanism of an epithelial tight-junction opening induced by chitosan low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice preparation and evaluation of chitosan nanoparticles containing diphtheria toxoid as new carriers for nasal vaccine delivery in mice n-trimethyl chitosan (tmc) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model the potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization diphtheria toxoid-containing microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles development and characterization of chitosan coated poly-(varepsilon-caprolactone) nanoparticulate system for effective immunization against influenza in vitro and in vivo study of n-trimethyl chitosan nanoparticles for oral protein delivery covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination co-delivery of viral proteins and a tlr agonist from polysaccharide nanocapsules: a needle-free vaccination strategy development and characterization of surface modified plga nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity evaluation of the immune response following a short oral vaccination schedule with hepatitis b antigen encapsulated into alginate-coated chitosan nanoparticles a single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge fully embeddable chitosan microneedles as a sustained release depot for intradermal vaccination first in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice novel thermal-sensitive hydrogel enhances both humoral and cell-mediated immune responses by intranasal vaccine delivery chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine peyer's patches transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal m-cell model chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice chitosan chemistry and pharmaceutical perspectives evaluation of eudragit-coated chitosan microparticles as an oral immune delivery system ph-sensitive and mucoadhesive thiolated eudragit-coated chitosan microspheres formulation, characterization and optimization of hepatitis b surface antigen (hbsag)-loaded chitosan microspheres for oral delivery bioadhesive-based dosage forms: the next generation chitosan as a novel nasal delivery system for vaccines induction of protective immunity against h n influenza a(h n )pdm with spray-dried and electron-beam sterilised vaccines in non-human primates biodistribution and lymph node retention of polysaccharide-based immunostimulating nanocapsules chitosan gras notice protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a human susceptibility and resistance to norwalk virus infection characterisation of a gii- norovirus variant-specific surface-exposed site involved in antibody binding immunogenicity and specificity of norovirus consensus gii. virus-like particles in monovalent and bivalent vaccine formulations adjuvanted intranasal norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues norovirus vaccine against experimental human norwalk virus illness intranasal vaccination with an adjuvanted norwalk virus-like particle vaccine elicits antigen-specific b memory responses in human adult volunteers we thank the financial support provided by national key