key: cord-0878181-zn0dr13v authors: Boots, A.M.H.; Benaissa-Trouw, B. J.; Hesselink, W.; Rijke, E.; Schrier, C.; Hensen, E. J. title: Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein date: 1992-12-31 journal: Vaccine DOI: 10.1016/0264-410x(92)90028-i sha: f182d59842dd585d7bfdfbdd55dfd8f828c51e1a doc_id: 878181 cord_uid: zn0dr13v Abstract Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken. Infectious bronchitis virus (IBV) is the prototype of the Coronaviridae. The virus consists of a lipid-containing membrane, a single-stranded RNA genome and three structural proteins. Apart from the internally localized nucleocapsid protein (N), the virus consists of two glycoproteins anchored in the lipid membrane',2. The integral matrix protein (M ) protrudes only slightly from the membrane in contrast to the spike protein (S) exposed at the surface which gives the virus its coronaviral image. The virus can cause an acute respiratory disease in young chickens and a reduction in egg production in laying hens. Attention has been focused primarily on responses to the S protein. This was based on the observation that neutralizing antibody showed specificity for the S protein3. By generating antigenic variants of the S protein the virus is capable of avoiding elimination by virus neutralizing antibody4. These distinct antigenic variations pose a problem in IBV vaccine design5. To circumvent the problem of the observed antigenic variability of the S protein we directed our attention to the IBV N protein which is more conserved among IBV strains'v6. Observations in other Protection is not only induced by generating cytotoxic T cells (36) , but also by generating T helper cell responses that augment the activity of B cells in production of virus-neutralizing antibody . ' l-l 3 Recently, we have shown that two murine CD4-positive T cell hybridomas generated from an IBV-specific T cell line were responsive to N proteins of several IBV strains14. Now the immunogenicity of recombinant-DNA encoded N protein in relation to the cellular immune response to IBV was studied. First it was shown that delayed-type hypersensitivity (DTH) and lymphocyte proliferative responses to IBV were induced upon immunization of mice with the expressed N protein. The purpose of the work described in this paper was secondly, to assess the role of the N protein in induction of cellular immune responses to IBV in the chicken, thirdly to test whether the N protein can accelerate the induction of virus-neutralizing (VN) and haemagglutination-inhibition (HI) antibodies, and finally to ascertain whether priming of chickens with the N protein results in increased tracheal protection against challenge with IBV. Antigens IBV strain M41 was obtained from egg-grown virus and gradient purified as described". The M42 strain, an IBV laboratory strain, was grown in Vero cells The IBV nucleocapsid pEX clone was constructed as described by Kusters 17 . Briefly, the DNA encoding the N protein was isolated from the IBV M41 cDNA library and thereafter cut with restriction enzymes and cloned in the expression vector pEX TM. The recombinant plasmid was expressed in Escherichia coll. In this system heterologous expression leads to the synthesis of a C-terminal extension of the cro-beta-galactosidase protein (CGZ). The sequence of the insert was checked by sequencing using the dideoxy termination method. The expressed fusion protein included amino acids 2-405 of the IBV M41 N protein. The protein expressed from pEXll, the vector without insert, contained only the CGZ protein and was used as control. To analyse cellular immune responses to the IBV N protein we used a lymphocyte proliferative assay and a classical DTH assay. Lymph node cell proliferative assay. Groups of five mice were immunized subcutaneously in the footpad with either 5 pg gradient-purified, inactivated IBV M41, N fusion protein (pXM41-EP) or CGZ (pEXll control) mixed with 100#g dimethyl dioctadecyl ammonium bromide (DDA, Kodak ). Antigens were injected in 50 pl volumes. At day 7 after immunization mice were killed to obtain the popliteal lymph nodes. Lymph node cell suspensions were prepared in Iscove's modification of Dulbecco's medium containing 1% fetal calf serum (FCS; Gibco, Breda, The Netherlands). Cells were washed and distributed into round-bottomed 96-well microtitration plates in 0.2 ml volumes of Iscove's modification of Dulbecco's medium (Gibco) supplemented with 10% FCS, antibiotics and 2-mercaptoethanol (2 x 10 -5 M) containing 105 cells. Antigens were added in 0.01 ml volumes to triplicate wells and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 and 4 days. [ 3H]-thymidine ( 1/~Ci/well, l Ci mmol-i Amersham) was added and the incorporated radioactivity was measured 18 h later. Immunization and assay for D TH. The immunization and DTH assay were performed as described 19. Briefly, groups of five Balb/c mice were immunized intracutaneously, either with purified inactivated IBV strain M41 (0.3 ~g), or with pEX fusion protein (pXM41-EP or pEX11,0.2 or 5.0/~g). Antigens were diluted in PBS, subsequently mixed with DDA (100/~g/animal) and injected in the vicinity of axillae and groins. Control mice received PBS and DDA. The footpad immunization was followed 6 days later by an injection of antigen in PBS in a 50 pl volume in the left hind footpad. To determine IBV-specific DTH responses, antigen preparations of different sources were used. DTH reactions were measured as the increase in footpad thickness of the left hind footpad between 0, 24 and 48 h. The footpad swelling was measured using an electronic footpad meter. Immunization. Groups of 20, four-week-old, female White Leghorn chickens, derived from a specified pathogen-free (SPF) flock from Intervet (Boxmeer, The Netherlands) were immunized intramuscularly in the leg with the IBV M41 preparation (formalin inactivated according to standard procedures), the N fusion protein, the CGZ protein or with PBS. All antigens were mixed 1:1 with Freund's complete adjuvant (FCA; Difco). Each chicken received ~ 100 #g of protein. All birds were boosted with the formalin-inactivated IBV M41 preparation 6 weeks after the first immunization. Blood mononuclear cell proliferative assay. Two weeks after primary and secondary immunization, chickens were bled by cardiac puncture and blood mononuclear cells were isolated to perform proliferative assays. A modified procedur e as described by Timms z° was used. Heparinized blood samples were centrifuged three times at low speed (7.5 min at 509 at 18-20°C) in glass capillaries containing 0.8 ml heparinized blood. The subsequent plasma layer and white cells were then collected. Lymphoid cells (10 6) were cultured in 150/A HEPES buffered RPMI 1640 (Dutch modification) supplemented with 1 g 1 1 NaHCO3 and 200 IU mlpenicillin and 200#gml -t streptomycin in roundbottomed wells of a microtitration plate. Antigens were added in 50 tA volumes to triplicate wells and plates were incubated for 3 days at 41°C in a humidified 5% CO 2 atmosphere. Six hours prior to harvesting, 0.5/~Ci ~-3I-1 ]-thymidine (5 Ci mmol t ) was added to the wells. Humoral immune responses. Serum was collected at 2, 4 and 6 weeks after the first immunization and at 1, 2 and 4 weeks after the booster immunization. Two techniques were applied to analyse anti-S protein antibodies : the haemagglutination inhibition assay (HI)22.22 and the virus neutralization (VN) assay 2~. For the latter assay the constant virus/diluted serum microneutralization technique on primary chicken embryo kidney cells was applied as described 23 except that the presence or absence of virus in the cultures was assayed by antigen capture ELISA on the culture supernatants ~4. The H1 and VN data were analysed using a two-sample t test (STATISTIX). Induction of tracheal protection. From each vaccination group chickens were transferred to separate isolators and challenged by eye-drop administration of 105 egg infectious doses (EIDso) of the IBV M41 challenge strain (originally supplied by CVL, Weybridge, UK) at 4 weeks after primary immunization and at 4 weeks after secondary immunization. Four days postchallenge the chickens were killed and the tracheae removed. To assess protection of the trachea following challenge with live M41 virus, two methods were used, the ciliostasis assay 25"a6 and a more sensitive method to detect viral antigen in tracheal sections by an indirect immunofluorescence test 17 using an IBV M proteinspecific monoclonal antibody (obtained from G. Koch, Central Veterinary Institute, Lelystad, The Netherlands ). The data were analysed using a linear model of logistic regression (STATISTIX). The immunogenicity of the recombinant-derived N protein in the mouse system was evaluated in the following assays. Lymph node cell proliferative assay. The induction of proliferative cellular immune responses to IBV by immunization with the N fusion protein was studied. Immunization of mice in the footpad resulted in popliteal lymph node cell proliferative responses to IBV ( Table I ) . IBV-specific proliferation was not observed following immunization with the control protein CGZ. Assay for DTH. IBV-specific DTH responses peaked at 24-30 h after the second injection ( Subsequently, the immunogenicity of the expressed N protein was analysed in the chicken. Blood mononuclear cell proliferative assay. After single immunization with IBV only modest proliferative responses to IBV were observed. Out of the group of eight chickens only one, 5083, showed a distinct proliferative response to IBV with a stimulation index (SI) value (antigen-specific counts min-t/control counts min-1) of 5 ( Table 3) . One out of eight chickens immunized with the N fusion protein showed a moderate response to IBV. In contrast, none of the CGZ immunized control chickens responded to IBV. Following the booster immunization with IBV, two out of eight chickens of the IBV primed group showed an IBV-specific proliferative response with SI values of 6 and 17. The data indicate that priming with inactivated IBV followed by an IBV booster induces cellular responses in only 25% of chickens tested. Priming with recombinant-derived N protein followed by a booster immunization with IBV resulted in a proliferative response to IBV in four out of eight chickens with SI values ranging from 5 to 17, suggesting that the N protein can efficiently prime cellular immune responses to IBV. These results should be compared to priming with CGZ, which did not provoke proliferative responses to IBV. To assess an effect of the priming antigen on the kinetics of antibody induction to the IBV S protein, which is the main target of both VN antibody and HI antibody 28, we monitored the humoral immune response to the S protein in chickens before and after the booster injection with IBV. We hypothesized that activation of N protein-specific T-helper cells would accelerate antibody synthesis to the IBV S protein. As expected, the effect of priming with whole IB virus was most prominent. Mean HI and VN titres (VN titres not shown) rose within 2 weeks after primary immunization and rose to maximum levels upon secondary immunization. Chickens primed with N fusion protein, CGZ or PBS (data not shown) showed, as anticipated, no anti-S responses before the booster with IBV (Figure 1) . A significant priming effect of immunization with the N protein was detected within 2 weeks after administration of the IBV booster using the HI assay (p = 0.0009). An anti-S protein antibody response was mounted more rapidly than in control groups, suggesting a role for activated T-helper cells in the anti-S antibody response. These results were confirmed using the VN assay. VN titres rose in weeks 8-10 only in N primed chickens ( p = 0.0025 ). primary IBV immunization one out of eight chickens showed a protection to challenge with live IBV based on ciliary activity ( Table 4 ). In the other groups none of the chickens resisted challenge. After revaccination a minimal percentage (20-40%) of protection was expected in all groups due to single vaccination with inactivated IBV 29'3°. In the IBV primed group all chickens challenged showed protection on the basis of ciliary activity. However, using a more sensitive immunofluorescence assay, virus was detected in one out of eight chickens primed with IBV. In the N protein primed group of chickens eight out of ten showed protection on the basis of ciliary activity. Virus was detected by immunofluorescence in only three out of ten chickens, suggesting that 70% of N-primed chickens showed protection in this group. In the control group several animals showed ciliostasis after challenge and in eight out of 12 chickens virus was detected in tracheal tissue. This indicated that, at the most, 33% demonstrated signs of resistance to live IBV in this group, which equals the expected percentage of protection due to one vaccination with inactivated IBV 29. To test the hypothesis that priming of chickens with IBV or IBV N protein contributes to protection we analysed the data obtained by immunofluorescence microscopy using a linear model (33) ND, not done of logistic regression. We compared the induction of protection following priming with IBV or IBV N protein to priming with the control CGZ protein. A significant effect of IBV priming on tracheal protection was shown (p = 0.013). Analysis of the data in these small groups suggested also an increased protection to tracheal challenge in chickens primed with recombinant-derived N protein (p = 0.083). We have found that the N protein of IBV produced in a bacterial expression system is capable of priming an immune response to intact virus, both in mice and in chickens. Upon immunization of mice with the N protein, lymph node cell proliferative responses and DTH reactions specific for IBV were demonstrated. Thus the data indicated a role for the N protein in activation of T-cells in the response to intact IBV. Subsequently, the role of the N protein in induction of protective immunity to IBV in chickens was explored. Three main findings emerged from this study. First, a priming effect of the N protein on proliferative responses of blood mononuclear cells was seen 2 weeks after secondary immunization. Primary N protein vaccination did not result in a detectable proliferative response to IBV. Primary immunization with inactivated virus showed responses to IBV in 25% of chickens, similar to previous reports 31. After secondary immunization of chickens with IBV, the priming effect of the N protein became manifest and exceeded the outcome of priming with virus twofold. In 50% of chickens a proliferative response to IBV was shown. No responses were observed in chickens vaccinated with CGZ which demonstrates the specificity of the IBV sensitization by N protein priming. Secondly, we have shown that immunization with N protein resulted in accelerated antibody induction to the IBV S surface protein as measured in the HI and VN assays. The observed variations of individual log2 HI and VN titres agree with those reported by Darbyshire 32. Both assays showed increased mean titres specific for the S protein within 2 weeks after secondary immunization. Since it is expected that in the chicken T and B cells follow the rules of cognate interaction as described for mammalian species 33 we explain the present observation by the action of an expanded population of N proteinspecific T cells which could accelerate the expansion and differentiation of primary virus-specific B cells. In individual chickens, however, proliferative responses did not always coincide with increased HI and VN titres, an observation made earlier by Timms and Bracewel131. This finding can be explained by the notion that not all proliferative antigen-specific cells are T-helper cells 34. Finally, a role for the N protein in protection to tracheal challenge was implicated. Single vaccination with inactivated IBV results in little or no protection in the trachea against challenge infection, whereas two vaccinations may result in up to 80 100% protection 29,3°. We hypothesized that a priming effect of immunization with the N protein should be visible within these ranges. Indeed, the data obtained by immunofluorescence microscopy indicated that 70% of N-primed and IBV-boosted chickens had resisted tracheal challenge. Of the chickens primed with CGZ, 33% had resisted challenge. The ciliostasis assay indicated a higher percentage of protected chickens compared to the data obtained by immunofluorescence microscopy. This discrepancy is explained by the observation that in older chickens tracheal symptoms due to virus replication are less severe than in young chickens 3°. It has been suggested that local immunity of the respiratory tract, the primary target organ of IBV infection, is of fundamental importance in IBV resistance 35. Our data confirm earlier data 36 that systemically induced responses to IBV can support protection at a local level. Recombinant-derived proteins have been successful in the induction of cellular immune responses to viral antigens 19'37'3s. In our study we demonstrated the immunogenicity of the IBV N protein fused to CGZ. The CGZ control protein, although known for its capacity to induce T helper and T suppressor responses in the mouse 34 did not influence the response to IBV in chickens. It should be considered, however, that this complex protein could influence the efficacy of a given recombinant vaccine. Little is known about the role of coronaviral N proteins in the response to coronaviral infection. Until now, cell-mediated immune responses to coronaviral N proteins in the target species have not been reported. Our data have shown a role for the IBV N protein in the activation ofT-helper cell responses in the chicken. Our findings add to the pivotal role of internal viral antigens in the induction of protective immunity by activation of cytotoxic or helper T-cell responses 1°'12,13. These internal antigens, usually less subject to antigenic variation than surface proteins, have been shown to generate crossreactive protective immunity 8'9'38. The N proteins oflBV strains also show highly conserved regions 2,6. In a previous paper we reported on T-cell hybridoma responses to N proteins of several IBV strains, supporting the data that indicate a stable antigenicity of the N protein 14. For future vaccine design it would be useful to gain insight into the determinants recognized by T-helper cells. We hypothesize that the N protein on the basis of its immunogenicity and relatively constant antigenicity is relevant for further study. Further evidence that the surface projections are associated with two glycoproteins Sequences of the nucleocapsid genes from two strains of Avian Infectious Bronchitis virus Coronavirus IBV: virus retaining spike glycoprotein $2 but not $1 is unable to induce virus neutralizing or haemagglutination inhibiting antibody, or induce chicken tracheal protection Sequence evidence for in vivo RNA recombination in avian coronavirus IBV Molecular epidemiology of infectious bronchitis virus in The Netherlands Rapid detection and identification of avian infectious bronchitis virus Protection against hepatitis B virus infection by immunization with hepatitis B core antigen Immunization with recombinant-DNA encoded infectious bronchitis nucleocapsid protein Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein Purified influenza virus nucleoprotein protects mice from lethal infection Cytotoxic T-cell recognition of the influenza nucleoprotein and Haemagglutinin expressed in transfected mouse L cells In vitro influenza virus-specific antibody production in man:antigen-specific and HLA restricted induction of helper activity mediated by cloned human T lymphocytes Antibody production to the nucleocapsid and envelope of the hepatitis B virus primed by a single synthetic T-cell site Differential ability of B cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus specific T-cell clones in vivo MHC Class II restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison to Beaudette strains Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome Analysis of an immunodominant region of avian coronavirus IBV Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins Identification of a DTH inducing T-cell epitope on the E2 membrane protein of Semliki Forest virus Cell mediated and humoral immune response in chickens infected with infectious bronchitis Procedures for the haemagglutination and the haemagglutination inhibition tests for avian infectious bronchitis virus A standard technique for haemagglutination inhibition tests for antibodies to avian infectious bronchitis virus Serological classification of recent infectious bronchitis virus isolates by the neutralisation of immunofluorescent loci Detection of IBV antigen by enzyme immunoassay application for diagnostic purposes Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures Evaluation of ciliary movement in tracheal rings to assess immunity against infectious bronchitis virus Possibilities and limitations of combined vaccines Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions Immunity to avian infectious bronchitis virus Diseases of Poultry Cell mediated and humoral immune response of chickens to inactivated oil emulsion infectious bronchitis vaccine Sequential development of humoral immunity and assessment of protection in chickens following vaccination and challenge with avian infectious bronchitis virus Can B-cells turn on virgin T-cells? The relationships between Ts-inducing and Th and Tp-inducing determinants on a large protein antigen Presence of viral antigens and antibody in the trachea of chickens infected with avian infectious bronchitis virus Aspects of local immune response to IBV Mapping of T-cell epitopes using recombinant antigens and synthetic peptides Antiviral immunity induced by recombinant nucleoprotein of influenza A virus I: Characteristics and cross-reactivity of T cell responses The authors are grateful to B.A.M. van der Zeijst, J.G. Kusters (Department of Bacteriology, Institute of Infectious Diseases and Immunology, Utrecht), A. Snijders and C.A. Kraaijveld (Section of Experimental Virology, Eijkman Winkler Laboratory, Utrecht) for helpful discussion and for their contribution to the work described above, and to K.A. Zwaagstra (Department of Bacteriology, Institute of Infectious Diseases and Immunology, Utrecht) and T. Loeffen (Intervet, Boxmeer) for technical assistance. The authors are indebted to I. Joosten for critical reading of the manuscript.