Preclinical Characterization of the Omicron XBB.1.5-Adapted BNT162b2 COVID-19 Vaccine (preprint)

As SARS-CoV-2 continues to evolve, increasing in its potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains the same mRNA backbone as the original BNT162b2 vaccine, modified by the incorporation of XBB.1.5-specific sequence changes in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation that adopts a flexible and predominantly open one-RBD-up state, with high affinity binding to the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and the phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose primary series in naive mice. Strong S-specific Th1 CD4+ and IFN{gamma}+ CD8+ T cell responses were also observed. These findings, together with prior experience with variant-adapted vaccine responses in preclinical and clinical studies, suggest that the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine is anticipated to confer protective immunity against dominant SARS-CoV-2 strains. ONE-SENTENCE SUMMARYThe monovalent Omicron XBB.1.5-adapted BNT162b2 mRNA vaccine encodes a prefusion-stabilized spike immunogen that elicits more potent neutralizing antibody responses against homologous XBB.1.5 and other circulating sublineage pseudoviruses compared to the bivalent Wild Type + Omicron BA.4/5 BNT162b2 vaccine, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.

Community incidence patterns drive the risk of SARS-CoV-2 outbreaks and alter intervention impacts in a high-risk institutional setting (preprint)

Optimization of control measures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in high-risk institutional settings (e.g., prisons, nursing homes, or military bases) depends on how transmission dynamics in the broader community influence outbreak risk locally. We calibrated an individual-based transmission model of a military training camp to the number of RT-PCR positive trainees throughout 2020 and 2021. The predicted number of infected new arrivals closely followed adjusted national incidence and increased early outbreak risk after accounting for vaccination coverage, masking compliance, and virus variants. Outbreak size was strongly correlated with the predicted number of off-base infections among staff during training camp. In addition, off-base infections reduced the impact of arrival screening and masking, while the number of infectious trainees upon arrival reduced the impact of vaccination and staff testing. Our results highlight the importance of outside incidence patterns for modulating risk and the optimal mixture of control measures in institutional settings.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model (preprint)

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery, which improve vaccination compliance and demonstrate efficacy against emerging variants. Here we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprised of stabilized, pre-fusion Spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction of lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Prioritizing interventions for preventing COVID-19 outbreaks in military basic training (preprint)

Like other congregate living settings, military basic training has been subject to outbreaks of COVID-19. We sought to identify improved strategies for preventing outbreaks in this setting using an agent-based model of a hypothetical cohort of trainees on a U.S. Army post. Our analysis revealed unique aspects of basic training that require customized approaches to outbreak prevention, which draws attention to the possibility that customized approaches may be necessary in other settings, too. In particular, we showed that introductions by trainers and support staff may be a major vulnerability, given that those individuals remain at risk of community exposure throughout the training period. We also found that increased testing of trainees upon arrival could actually increase the risk of outbreaks, given the potential for false-positive test results to lead to susceptible individuals becoming infected in group isolation and seeding outbreaks in training units upon release. Until an effective transmission-blocking vaccine is adopted at high coverage by individuals involved with basic training, need will persist for non-pharmaceutical interventions to prevent outbreaks in military basic training. Ongoing uncertainties about virus variants and breakthrough infections necessitate continued vigilance in this setting, even as vaccination coverage increases.

Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery Against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques (preprint)

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Post-pyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that post-pyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. ImportanceSARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here we report that oral administration of live SARS-CoV-2 in non-human primates may offer prophylactic benefits, but that formulation and route of administration will require further optimization.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters (preprint)

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 g) or low (0.2 g) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

SARS-CoV-2 Ferritin Nanoparticle Vaccines Elicit Broad SARS Coronavirus Immunogenicity (preprint)

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of emerging coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion SARS-CoV-2 Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers (ID50 > 10,000), 20-fold greater than convalescent donor serum titers following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations, that also showed potent neutralization against circulating variants of concern. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicited ACE2 blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1 highlighting the broad response elicited by these immunogens.Funding: We acknowledge support from the U.S. Department of Defense, Defense Health Agency (Restoral FY20). This work was also partially executed through a cooperative agreement between the U.S. Department of Defense and the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (W81XWH-18-2- 0040).Declaration of Interest: M.G.J. and K.M. are named as inventors on International Patent Application No. WO/2021/21405 entitled “Vaccines against SARS-CoV-2 and other coronaviruses.” M.G.J. is named as an inventor on International Patent Application No. WO/2018/081318 and U.S. patent 10,960,070 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” Z.B. is named as an inventor on U.S. patent 10,434,167 entitled “Non-toxic adjuvant formulation comprising a monophosphoryl lipid A (MPLA)-containing liposome composition and a saponin.” Z.B. and G.R.M are named inventors on “Compositions And Methods For Vaccine Delivery”, US Patent Application: 16/607,917. M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech and Carnival Corporation and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, Kaleido, and Emergent BioSolutions. S.R., P.M.M., and M.T.E. are employees of AstraZeneca and currently hold AstraZeneca stock or stock options. Zoltan Beck is currently employed at Pfizer.Ethical Approval: All research in this study involving animals was conducted in compliance with the Animal Welfare Act, and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. The research protocol was approved by the Institutional Animal Care and Use Committee of WRAIR. BALB/c and C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).

A spike-ferritin nanoparticle vaccine induces robust innate immune activity and drives polyfunctional SARS-CoV-2-specific T cells (preprint)

Potent cellular responses to viral infections are pivotal for long-lived protection. Evidence is growing that these responses are critical in SARS-CoV-2 immunity. Assessment of a SARS-CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non-classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike-specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike (539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques (preprint)

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 {micro}g RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only [~]2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. Significance StatementThe emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates (preprint)

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 mcg) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response on high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. The potent and broad immunogenicity profile of this vaccine and its resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses.

A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic (preprint)

The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.

A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains (preprint)

The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Here we analyzed SARS-CoV-2 sequence diversity across 5,700 sequences sampled since December 2019. The Spike protein, which is the target immunogen of most vaccine candidates, showed 93 sites with shared polymorphisms; only one of these mutations was found in more than 1% of currently circulating sequences. The minimal diversity found among SARS-CoV-2 sequences can be explained by drift and bottleneck events as the virus spread away from its original epicenter in Wuhan, China. Importantly, there is little evidence that the virus has adapted to its human host since December 2019. Our findings suggest that a single vaccine should be efficacious against current global strains. One Sentence SummaryThe limited diversification of SARS-CoV-2 reflects drift and bottleneck events rather than adaptation to humans as the virus spread.

A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein (preprint)

SARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease--COVID-19-- within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronaviruss surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 [A]. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the "closed" prefusion S structure, but is accessible in "open" conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses. HIGHLIGHTSHigh resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD). Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies. Structure of the SARS-COV-2 RBD in complex with antibody CR3022. Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.