key: cord-0941501-oxkdcto0
authors: Lai, Chih-Yun; To, Albert; Ann S. Wong, Teri; Lieberman, Michael M.; Clements, David E.; Senda, James T.; Ball, Aquena H.; Pessaint, Laurent; Andersen, Hanne; Furuyama, Wakako; Marzi, Andrea; Donini, Oreola; Lehrer, Axel T.
title: Recombinant protein subunit SARS-CoV-2 vaccines formulated with CoVaccine HT adjuvant induce broad, Th1 biased, humoral and cellular immune responses in mice
date: 2021-11-05
journal: Vaccine X
DOI: 10.1016/j.jvacx.2021.100126
sha: 43c5dc23e994775b532c87027707e6e01af417df
doc_id: 941501
cord_uid: oxkdcto0

The speed at which several COVID-19 vaccines went from conception to receiving FDA and EMA approval for emergency use is an achievement unrivaled in the history of vaccine development. Mass vaccination efforts using the highly effective vaccines are currently underway to generate sufficient herd immunity and reduce transmission of the SARS-CoV-2 virus. Despite the most advanced vaccine technology, global recipient coverage, especially in resource-poor areas remains a challenge as genetic drift in naïve population pockets threatens overall vaccine efficacy. In this study, we described the production of insect-cell expressed SARS-CoV-2 spike protein ectodomain constructs and examined their immunogenicity in mice. We demonstrated that, when formulated with CoVaccine HT(TM) adjuvant, an oil-in-water nanoemulsion compatible with lyophilization, our vaccine candidates elicit a broad-spectrum IgG response, high neutralizing antibody (NtAb) titers against SARS-CoV-2 prototype and variants of concern, specifically B.1.351 (Beta) and P.1. (Gamma), and an antigen-specific IFN-γ secreting response in outbred mice. Of note, different ectodomain constructs yielded variations in NtAb titers against the prototype strain and some VOC. Dose response experiments indicated that NtAb titers increased with antigen dose, but not adjuvant dose, and may be higher with a lower adjuvant dose. Our findings lay the immunological foundation for the development of a dry-thermostabilized vaccine that is deployable without refrigeration.

The emergence and rapid spread of a new infectious respiratory disease, Coronavirus Disease 2019 has caused an unprecedented public health emergency worldwide since emerging in December 2019. A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is closely related to SARS-CoV, was identified as the etiologic agent of the new respiratory disease. As of July 2021, there are over 185 million confirmed cases with over 4 million deaths worldwide and over 33 million cases and 606,000 fatalities reported in the U.S. [1] , accentuating the continued need for safe and effective, inexpensive and easily deployable vaccines that would rapidly establish global herd immunity. Facilitated by the broad nature of the outbreak, significant SARS-CoV-2 variants have been identified, some of which appear to be more resistant to first-generation vaccines [2] . More than 200 COVID-19 vaccine candidates using various technology platforms are in development [3] . Three vaccines including two of the mRNA platforms, Pfizer/BioNTech BNT162b2 and Moderna mRNA-1273, and the third, a viral-vector platform, Janssen Ad26.COV2.S, with reported interim efficacy rates of 95%, 94.1% and 85% respectively, have been approved by the U.S. Food and Drug Administration (FDA) for emergency use as of July 2021 [4] [5] [6] . These vaccines are now being administered in the U.S. and have also been approved for use in other jurisdictions. As no single vaccine can be produced rapidly in sufficient quantities to satisfy the global demand, diversification using different vaccine platforms would enable worldwide vaccine coverage as well as address the needs of the most vulnerable populations, particularly elderly and immunocompromised individuals or those with other co-morbidities. With mutations emerging even in the absence of strong selective pressure placed on SARS-CoV-2, variants of concern (VOC) will continue to arise under inequitable vaccine availability [2, 7] . Thus, continued research into adaptable and more easily distributed vaccines, compatible with rapid deployment and significant cost efficiencies, must continue unabated.

The recombinant subunit vaccine platform offers a safety advantage over virally vectored vaccines and a distribution advantage relative to many other vaccine platforms. Purified recombinant protein antigens can be engineered to achieve optimal immunogenicity and protective efficacy. Furthermore, a thermostabilized subunit vaccine can be deployed in the field, eliminating stringent cold-chain requirements.

Formulation of the vaccine immunogen with a potent adjuvant enhances and focuses immunogenicity while lowering the antigen dose requirement, thereby enabling vaccination of more people with a product carrying significantly more clinical and regulatory precedence compared to nucleic acid-based and viral-vectored approaches.

CoVaccine HT™, is an oil-in-water nanoemulsion adjuvant with an excellent safety profile [8] . In combination with properly selected antigens, CoVaccine HT™ can achieve potent immunogenicity and protective efficacy in rodents and non-human primates (NHPs) [9] [10] [11] [12] . In previous work, we have successfully demonstrated the use of the recombinant protein subunit Drosophila S2 expression system in combination with CoVaccine HT™ to produce vaccines to combat global health threats such as Zika virus (ZIKV) and Ebola virus (EBOV). Immunization with recombinant ZIKV E protein induced potent neutralizing titers in mice [10] and non-human primates [9] and protection against viremia after viral challenge. Similarly, immunization with recombinant subunit formulations consisting of the EBOV glycoprotein and matrix proteins VP40 and VP24 was able to induce potent antibody titers and protection in both mouse [11] and guinea pig models of EBOV disease [12] . In healthy adults, CoVaccine HT TM was shown to be safe and well-tolerated with no risk of severe adverse effects in phase 1 clinical trials (NCT01015703) [8] .

The spike (S) glycoprotein, comprised of a receptor binding subunit (S1) and a membrane-fusing subunit (S2) [13] is the main surface protein and present as homotrimers on the viral envelope of SARS-CoV-2. Based on previous preclinical studies of vaccines against the highly pathogenic SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) [14] [15] [16] as well as recent studies of patients with SARS-CoV-2 infections [17] [18] [19] [20] , the S protein appears to be the antigenic target of both neutralizing antibody and T cell responses. The majority of current COVID-19 vaccines under preclinical and clinical development use full-length S proteins as antigen targets with further modifications such as removal of the polybasic sites [21] [22] [23] , introduction of proline mutations [21, 24, 25] , or addition of trimerization domains to preserve the native-like trimeric prefusion structure of S proteins. These antigens have been shown to mimic the native S protein presented on viral particles and preserve neutralization-sensitive epitopes [16, 26] . In a prior study, we evaluated the utility of CoVaccine HT™ adjuvant to induce properly balanced immunity against SARS-CoV-2, when formulated with a commercially available SARS-CoV-2 spike S1 protein [27] . In the current study we produced a native-like trimeric S protein ectodomain with and without stabilizing mutations using the Drosophila S2 cell expression system and assessed the immunogenicity of these S ectodoman trimers formulated with CoVaccine HT™ in mice. The scope of this work demonstrates that CoVaccine HT™ is an effective adjuvant that promotes rapid induction of balanced humoral and cellular immune responses and in combination with spike proteins warrants further development as an effective medical intervention against coronavirus disease.

All animal work was conducted in accordance with the Animal Welfare Act and the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals.

All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Hawaii at Manoa (UHM) and carried out in the UHM American Association for Accreditation of Laboratory Animal Care (AAALAC) accredited Laboratory Animal Facility.

Plasmids were generated to express the native-like, trimeric, transmembrane (TM)deleted spike (S) glycoprotein (SdTM) from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank Accession number NC_045512). The SdTM sequence was designed to encode the SARS-CoV-2 S protein sequence spanning Gln14 to Ser1147. The SdTM gene was produced by de novo synthesis (ATUM, Newark, CA). The gene was also codonoptimized for expression in Drosophila S2 cells, with an altered furin cleavage site (RRAR changed to GSAR) between S1 and S2 domains to prevent cleavage, and contains a trimerization domain of T4 bacteriophage fibritin (foldon) at the C-terminus.

Two additional proline substitutions (K986P and V987P) between the heptad repeat 1 and central helix regions and the removal of the S2' protease cleavage site were introduced by site-directed mutagenesis to generate the stabilized prefusion structure of S protein (SdTM2P). The SARS-CoV-2 receptor binding domain with a foldon trimerization domain at the C-terminus (RBD-F) was also prepared. The RBD-F sequence was designed to encode the SARS-CoV-2 S protein sequence spanning coupled with 2 mg of a his-tagged human angiotensin I converting enzyme 2 (hACE2), which was also produced using the Drosophila S2 cell expression system and purified by Ni-affinity chromatography in our laboratory. Purified recombinant S proteins were concentrated using Amicon filtration devices (EMD Millipore, Billerica, MA), buffer-exchanged into PBS and analyzed by SDS-PAGE and Western blotting. Antigens were quantified by UV absorbance at 280 nm and stored at -80°C.

A conventional immunoaffinity chromatography (IAC) method was also established to purify SdTM2P proteins. For this, the monoclonal antibody (mAb) CR3022 (provided by Mapp Biopharmaceutical), was coupled to NHS-activated Sepharose at a concentration of 10 mg/mL and used for IAC in tandem with a HiPrep 26/10 desalting column (Cytiva, Marlborough, MA) equilibrated with PBS allowing quick buffer exchange of the eluted protein from low pH buffer into PBS.

Groups (n=7 or 15 per group) of 7 to 10-week-old Swiss Webster mice of both sexes 

The IgG antibody in mouse sera was measured by a multiplex microsphere-based immunoassay as described previously [10, 27, 28] . Briefly, internally dyed, magnetic MagPlex® microspheres (Luminex Corporation, Austin, TX) were coupled to purified receptor binding domain (RBD-F), spike protein (SdTM2P) or bovine serum albumin (BSA) as control [10, 27] . A mixture of RBD, spike SdTM2P, and BSA-coupled beads (approximately 1,250 beads each) was incubated with diluted sera in black-sided 96well plates for 3 hours at 37C with gentle agitation in the dark. The IgG subclass profile in serum samples was analyzed using IgG subclass-specific secondary antibodies (Southern Biotech, Birmingham, AL), and the ratios of IgG2a/IgG1 and IgG2b/IgG1 were calculated using the MFI readouts at the serum dilution (1:2000) that is within the linear range of the antibody binding standard curve.

Replication-competent rVSV expressing SARS-CoV-2 S protein (Wuhan-Hu-1) with a described previously [29] and the virus stocks were amplified in Vero E6 cells. For the plaque reduction neutralization test (PRNT) using rVSV-SARS-CoV-2-S, pooled or individual mouse serum samples were heat-inactivated at 56C for 30 minutes. Eight 3fold serial dilutions of serum samples starting at final 1:10 dilution were prepared and incubated with 100 plaque-forming units (PFU) of rVSV-SARS-CoV-2-S at 37C for 1 hour. Antibody-virus complexes were added to Vero E6 cell monolayers in 6-well plates and incubated at 37C for another hour followed by addition of overlay media mixed with 1% agarose. Three days later, cells were fixed and stained with a solution containing 1% formaldehyde, 1% methanol, and 0.05% crystal violet overnight for plaque enumeration. The neutralization titers (PRNT 50 ) were defined as the highest serum dilution that resulted in 50% reduction in the number of plaques.

PRNT was also performed in a biosafety level 3 facility at BIOQUAL, Inc. (BEI NR-54982) variants, in an equal volume of culture medium (DMEM with 10% FBS and gentamicin) for 1 hour at 37°C. The serum-virus mixtures were added to a monolayer of confluent Vero E6 cells and incubated for 1 hour at 37°C in 5% CO 2 . Each well was then overlaid with 1 ml of culture medium containing 0.5% methylcellulose and incubated for 3 days at 37°C in 5% CO 2 . The plates were then fixed with methanol at -20°C for 30 minutes and stained with 0.2% crystal violet for 30 min at room temperature. Neutralization titers (PRNT 50 ) were defined as the highest final serum dilution that resulted in 50% reduction in the number of plaques.

Three or five mouse spleens from each group were harvested seven days after the first and second vaccinations, and single cell suspensions were prepared using a 

Statistical analysis was performed using a one-sample t test and Wilcoxon test or Mann-Whitney t test to compare the neutralization titers, and IFN- secreting response between adjuvanted and unadjuvanted groups. The difference in the neutralizing antibody titers and the numbers of IFN- secreting cells between groups receiving different dosages of vaccines, or IgG subclass antibody profile, anti-S and anti-RBD IgG concentrations between unadjuvanted and adjuvanted groups were determined by oneway, or two-way, ANOVA with Tukey's multiple comparison test. The correlation between the measured neutralization titers using rVSV-SARS-CoV-2 and authentic SARS-CoV-2 was analyzed by Pearson correlation analysis.

Using the Drosophila S2 cell expression system, we generated trimeric SARS-CoV-2 S protein devoid of transmembrane domain (SdTM) as well as a stabilized prefusion structure of S protein (SdTM2P). To explore different methods for purification of S protein, we employed two methods of purification using hACE2 protein, a known receptor for SARS-CoV-2, for AC, and the SARS-CoV RBD specific mAb CR3022 for IAC. This mAb, originally constructed from an immune svFc phage display library of lymphocytes of a convalescent SARS-CoV patient, was selected due to its crossreactivity with both SARS-CoV and SARS-CoV 2 receptor binding sites, which contain the majority of neutralizing epitopes [30, 31] . The immunogenicity of recombinant S proteins was evaluated with and without CoVaccine HT™ adjuvant in outbred Swiss Webster mice. Animals were immunized with one or two doses at a 3-week interval by intramuscular injection with 5 g of SARS-CoV-2 SdTM or SdTM2P (purified by hACE2 AC) alone or formulated with 1 mg CoVaccine HT™ (Fig. 1A) . We developed a quantitative IgG assay in which purified anti-S polyclonal antibody was used as a standard and therefore the antigen-specific IgG levels are expressed as concentration (ng/mL) (Fig. 1B) . Analysis of IgG antibody in sera obtained on days 7, 14, 28, and 35

indicated that the co-formulation of CoVaccine HT™ with SdTM or SdTM2P significantly increased anti-S IgG antibody levels over those induced by proteins alone (Fig. 1C ). In addition, two doses of CoVaccine HT™ adjuvanted SdTM or SdTM2P elicited high levels of IgG antibodies to RBD, which is the target of neutralizing antibodies (Fig. 1D ).

To further optimize vaccine formulations, we evaluated the IgG antibody responses in mice that received a lower dosage (2.5 or 1.25 g) of SdTM2P proteins or that received a reduced amount (0.3 mg) of adjuvant. The results demonstrated that vaccination with reduced amounts of antigen or adjuvant did not decrease the levels of antigen-specific IgG antibodies (Fig. 1E ).

We next examined whether our vaccine candidates induced neutralizing antibody responses. Measurement of SARS-CoV-2 neutralizing antibodies requires biosafety level 3 (BSL3) laboratory facilities, and therefore, alternative assays have been developed, such as one utilizing replication-competent rVSV-ΔG expressing SARS-CoV-2 S glycoprotein (rVSV-SARS-CoV-2-S) [32, 33] . We first compared the neutralizing antibody titers of pooled mouse sera using both SARS-CoV-2 and rVSV-SARS-CoV-2-S ( Table 1 ) and found that the titers are significantly correlated between these two assays (Fig. 2C ). To further evaluate the neutralizing antibodies of individual animals, we employed only the PRNT using rVSV-SARS-CoV-2-S. The PRNT 50 titers of sera obtained from mice vaccinated with SdTM or SdTM2P in combination with CoVaccine HT™ were significantly higher than those from animals receiving protein alone ( Fig. 2A) , suggesting that the CoVaccine HT™ adjuvant enhances induction of neutralizing antibody responses. Furthermore, a trend of dose-dependent decreases of neutralization titers was observed when animals were given lower dosages of SdTM2P proteins (Fig. 2B) . Of note, the serum PRNT 50 titers of mice receiving a lower amount (0.3 mg) of CoVaccine HT™ were comparable or even higher than those of mice given 1 mg of adjuvant indicating that the optimal vaccine formulation may be achieved using lower dosages of adjuvant. Similar trends were seen with the pooled sera in PRNT using the SARS-CoV-2 WT, lineage B1.351 Beta, and P.1 Gamma VOC (Table 1) . Although two doses of 5 g SdTM or SdTM2P formulated with CoVaccine HT TM yielded PRNT 50 

Vaccine-associated enhanced respiratory disease (VARED) has been reported in infants and young children immunized with inactivated whole virus vaccine against respiratory syncytial virus (RSV) and measles virus [35] [36] [37] , and associated with Th2biased immune responses [38] . A similar pulmonary immunopathology was also observed in animals with SARS-CoV vaccines [39] [40] [41] . Thus, we evaluated the balance of Th1 and Th2 by comparing the levels of S-specific IgG2a/b and IgG1, which are indicative of Th1 and Th2 responses, respectively. Both SdTM and SdTM2P adjuvanted with CoVaccine HT™ elicited high anti-S IgG2a/2b and IgG1 subclass antibodies whereas protein alone induced high IgG1 with lower IgG2a and IgG2b (Fig. 3A) . To further assess the effect of CoVaccine HT™ adjuvanticity on IgG subclass antibody profiles, the ratios of IgG2a versus IgG1 as well as IgG2b versus IgG1 were calculated.

The results indicate that CoVaccine HT™ enhanced the induction of Th1 responses as evidenced by the significantly higher ratios of IgG2a/IgG1 and IgG2g/IgG1 obtained from groups given either SdTM or SdTM2P with adjuvant compared to those without adjuvant (Fig. 3B, 3C ).

To assess vaccine-induced T cell responses, we analyzed the number of IFN- secreting cells after ex vivo stimulation of splenocytes with SARS-CoV-2 spike peptides by a FluoroSpot assay. Stimulation of cells prepared from mice receiving 2 doses of SdTM or SdTM2P with CoVaccine HT™ resulted in robust production of IFN- secreting cells (Fig. 4A) . Interestingly, one dose of CoVaccine HT™ -adjuvanted S proteins also elicited a rapid IFN- secreting response (Fig. 4A) . Additionally, a slight decrease in the numbers of IFN- secreting cells was observed when mice received a lower amount (2.5 or 1.25 g) of adjuvanted SdTM2P proteins or a lower amount (0.3 mg) of CoVaccine HT™ (Fig. 4B) . However, these differences were not statistically significant. In addition, spike peptide stimulation of immune splenocytes was also analyzed for production of IL-4, but no significant IL-4 production was detected at any timepoint with less than 10 spot forming cells (SFCs) per million cells (data not shown). Altogether, our platform shows great potential towards achieving a rapid, robust, and Th1-focused T cell response even in outbred populations.

An ideal COVID-19 vaccine is expected to induce both humoral and cellular immunity, high titers of neutralizing antibodies and Th1-biased response to reduce potential risk of vaccine-associated enhancement of disease [42] [43] [44] . Using a well-established insect cell expression system, we have generated two versions of the SARS-CoV-2 S protein ectodomain and formulated them with a potent adjuvant, CoVaccine HT™. The vaccine candidates elicit both neutralizing antibody and cellular immunity with a balanced Th1/Th2 response in an outbred mouse model. The results obtained in this model may thus inform future vaccine development in animal models more closely related to humans [45] . These data support further preclinical and clinical development of CoVaccine HT™ adjuvanted SARS-CoV-2 vaccines to mitigate the ongoing COVID-19 pandemic.

Stabilization of S proteins in the prefusion trimeric conformation results in increased expression, conformational homogeneity, and production of potent neutralizing antibody responses [16, 25] . Current SARS-CoV-2 vaccines under development use either RBD or full-length S protein with or without modifications for stabilization of prefusion conformation as the major antigen targets [46] [47] [48] [49] . Although RBD is a primary target for potent neutralizing antibodies, it lacks other neutralizing epitopes present on full-length S. This might suggest that full-length S-based vaccines would broaden the neutralizing repertoire cross-neutralize circulating spike variants of concern. In the current work, we produced trimeric S ectodomains in which the furin cleavage site was mutated (SdTM) and that were further stabilized in the prefusion S form by removing the S2' protease cleavage site and introducing two proline substitutions (SdTM2P). The introduction of two prolines in the S2 subunit resulted in significantly greater production yield (~3-fold increase) in Drosophila S2 cells (unpublished data). Vaccination with adjuvanted SdTM or SdTM2P elicits comparable levels of IgG antibody and IFN- cellular responses; however, the SdTM2P vaccine generated a slightly higher level of neutralizing antibodies than SdTM, which was also reported in studies of other SARS-CoV-2 and MERS-CoV vaccines [16, 22] .

CoVaccine HT™ is a novel adjuvant that consists of a sucrose fatty acid sulfate ester (SFASE) immobilized on the oil droplets of a submicrometer emulsion of squalane in water (oil-in-water emulsion) [50] . It has been used for influenza virus and malaria vaccines and shown to enhance humoral and cellular protective immunity, in particular antibody response [51] [52] [53] [54] [55] . In addition, we have successfully utilized CoVaccine HT™ in our previous recombinant subunit EBOV, ZIKV and preliminary SARS-CoV-2 vaccine studies and have shown this adjuvant to elicit robust antibody responses [9] [10] [11] 27] . Use of CoVaccine HT™ with SdTM and SdTM2P yielded significantly enhanced total IgG and neutralizing antibody responses after both the first and second dose as compared to protein alone which reached similar IgG concentrations after two doses as did a single dose of the adjuvanted formulations. Although not directly comparable, the elicitation of S-and RBD-specific IgG and neutralizing antibody titers, along with IFN-γ secreting cells, after each dose followed similar kinetics as other 2P-S subunit vaccine candidates formulated with saponin or CpG based adjuvants [56, 57] , while similarities in IgG and neutralizing antibody titers were observed compared with alum onlyformulated candidates [58, 59] . Interestingly antibody levels (total IgG or neutralizing) did not decrease with a decreased dose of adjuvant; instead, a pronounced increase in neutralizing antibody titers was observed, indicating further opportunity for formulation optimization.

The decrease in neutralization capacity of the humoral response against the B.1.351 Beta and P.1 Gamma VOC is consistent with clinical data of vaccines using the prefusion stabilized spike [34, 60] that are still protective with only a marginal decrease in efficacy [61] [62] [63] [64] . Specifically, NVX-CoV2373 vaccine, a subunit vaccine in a Phase 1/2 clinical trial where the most common circulating strain is the B.1.351 Beta VOC showed sufficient protection against COVID-19 [65] . It is likely that a similar vaccine formulated with CoVaccine HT TM , based on our sustained neutralization results, may yield similar or better results in an appropriate challenge model.

In addition to improved kinetics, the addition of CoVaccine HT TM modulated the humoral response more towards Th1 type relative to protein alone, as indicated by higher levels of IgG2a and IgG2b. Antigen-specific IFN-γ production after splenocyte restimulation ex vivo was more variable, but also increased with the use of adjuvant, particularly after two doses, and was not strongly dependent on adjuvant concentration. These robust The funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in writing of the report; and in the decision to submit the article for publication. Table 1 . MIA. The ratios of IgG2a to IgG1 (B) or IgG2b to IgG1 (C) from individual animals were calculated using the MFI values at the serum dilution of 1:2,000. Bars represent mean ± SEM of each group. Statistically significant differences in the anti-S IgG1, IgG2a, and IgG2b antibody titers, or ratios of IgG2a/IgG1 and IgG2b/IgG1 between adjuvanted and protein alone groups were determined by a one-way ANOVA with Tukey's multiple comparisons test (*p < 0.05; ***p < 0.001; ****p < 0.0001) (*p < 0.05). 

COVID-19 case tracker

Immune Evasion of SARS-CoV-2 Emerging Variants: What Have We Learnt So Far? Viruses

Draft landscape of COVID-19 candidate vaccines

Moderna COVID-19 Vaccine

The Advisory Committee on Immunization Practices' Interim Recommendation for

Vaccine -United States

The Spike of Concern-The Novel Variants of SARS-CoV-2

Open-label Safety and Tolerability Study of CoVaccine HT™ in Healthy Volunteers. clinicaltrials.gov

A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

Recombinant Zika Virus Subunits Are Immunogenic and Efficacious in Mice. mSphere

Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice

Recombinant subunit vaccines protect guinea pigs from lethal Ebola virus challenge

Structural insights into coronavirus entry

Recent Advances in the Vaccine Development Against Middle East Respiratory Syndrome-Coronavirus

A decade after SARS: strategies for controlling emerging coronaviruses

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients. Emerging infectious diseases

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model. bioRxiv : the preprint server for biology

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

Phase 1-2 Trial of a SARS

Recombinant Spike Protein Nanoparticle Vaccine

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

Structurebased design of prefusion-stabilized SARS-CoV-2 spikes

Structure-Based Vaccine Antigen Design. Annual review of medicine

CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1

Effect of serum heat-inactivation and dilution on detection of anti-WNV antibodies in mice by West Nile virus E-protein microsphere immunoassay

Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine. bioRxiv : the preprint server for biology

Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses. The Journal of experimental medicine

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine

Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population

Altered reactivity to measles virus. Atypical measles in children previously immunized with inactivated measles virus vaccines

Summary of the Vaccines and Related Biological Products Advisory Committee meeting held to consider evaluation of vaccine candidates for the prevention of respiratory syncytial virus disease in RSV-naïve infants

Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets

A double

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Immunological considerations for COVID-19 vaccine strategies

SARS-CoV-2 vaccines in development

Rapid COVID-19 vaccine development

Author Correction: Comparing phenotypic variation between inbred and outbred mice

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity

Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters. bioRxiv : the preprint server for biology

Yeast-expressed SARS-CoV recombinant receptor-binding domain (RBD219-N1) formulated with aluminum hydroxide induces protective immunity and reduces immune enhancement

SARS-CoV-2 S1 is superior to the RBD as a COVID-19 subunit vaccine antigen

Sucrose fatty acid sulphate esters as novel vaccine adjuvants: effect of the chemical composition

A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets

Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits

Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques

Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques

CoVaccine HT™ adjuvant is superior to

Freund's adjuvants in eliciting antibodies against the endogenous alarmin HMGB1

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice

Development of CpGadjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19

SARS-CoV-2 spike produced in insect cells elicits high neutralization titres in non-human primates

Mice Immunized with the Vaccine Candidate HexaPro Spike Produce Neutralizing Antibodies against SARS-CoV-2. Vaccines (Basel)

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv : the preprint server for biology

Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant. The New England journal of medicine

Single-vial filovirus glycoprotein vaccines: Biophysical characteristics and immunogenicity after colyophilization with adjuvant

Thermostable Ebola virus vaccine formulations lyophilized in the presence of aluminum hydroxide. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV

Preservation of Quaternary Structure in Thermostable, Lyophilized Filovirus Glycoprotein Vaccines: A Search for Stability-Indicating Assays

Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine

Albert To: Conceptualization, Methodology, Validation, Investigation, Resources, Data curation, Writing-original draft, Writingreview & editing

Formal analysis, Writing -review & editing. David E. Clements: Methodology, Resources, Writing -review & editing. James T. Senda: Methodology, Resources, Writing -review & editing. Aquena H. Ball: Investigation, Writing-original draft. Laurent Pessaint: Investigation. Hanne Andersen: Investigation

Wakako Furuyama: Resources. Andrea Marzi: Resources. Oreola Donini: Writingoriginal draft, Writing -review & editing. Axel T. Lehrer: Conceptualization, Methodology, Validation, Resources, Writing -review & editing, Visualization, Supervision

Lehrer and Oreola Donini are named inventors on a patent application covering a recombinant subunit vaccine for SARS-CoV-2. David E. Clements and James T. Senda, are current employees of Hawaii Biotech Inc. Laurent Pessaint and Hanne Andersen are current employees of BIOQUAL, Inc. Oreola Donini is a current employee of Soligenix Inc