Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera (preprint)

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination (preprint)

Increasing evidence points to the microbial exposome as a critical factor in maturing and shaping the host immune system, thereby influencing responses to immune challenges such as infections or vaccines. To investigate the effect of early-life viral exposures on immune development and vaccine responses, we inoculated mice with six distinct viral pathogens in sequence beginning in the neonatal period, and then evaluated their immune signatures before and after intramuscular or intranasal vaccination against SARS-CoV-2. Sequential viral infection drove profound changes in all aspects of the immune system, including increasing circulating leukocytes, altering innate and adaptive immune cell lineages in tissues, and markedly influencing serum cytokine and total antibody levels. Beyond these immune responses changes, these exposures also modulated the composition of the endogenous intestinal microbiota. Although sequentially-infected mice exhibited increased systemic immune activation and T cell responses after intramuscular and intranasal SARS-CoV-2 immunization, we observed decreased vaccine-induced antibody responses in these animals. These results suggest that early-life viral exposures are sufficient to diminish antibody responses to vaccination in mice, and highlight their potential importance of considering prior microbial exposures when investigating vaccine responses.

NVX-CoV2373 ancestral and NVX-CoV2540 BA.5 protein nanoparticle vaccines protect against Omicron BA.5 variant in Syrian hamsters (preprint)

The emergence of SARS-CoV-2 variants with greater transmissibility or immune evasion properties has jeopardized the existing vaccine and antibody-based countermeasures. Here, we evaluated the efficacy of boosting with the protein nanoparticle NVX-CoV2373 or NVX-CoV2540 vaccines containing ancestral or BA.5 S proteins, respectively, in mRNA-immunized pre-immune hamsters, against challenge with the Omicron BA.5 variant of SARS-CoV-2. Serum antibody binding and neutralization titers were quantified before challenge, and viral loads were measured 3 days after challenge. Compared to an mRNA vaccine boost, NVX-CoV2373 or NVX-CoV2540 induced higher serum antibody binding responses against ancestral Wuhan-1 or BA.5 spike proteins, and greater neutralization of Omicron BA.1 and BA.5 variants. One and three months after vaccine boosting, hamsters were challenged with the Omicron BA.5 variant. NVX-CoV2373 and NVX-CoV2540 boosted hamsters showed reduced viral infection in the nasal washes, nasal turbinates, and lungs compared to unvaccinated animals. Also, NVX-CoV2540 BA.5 boosted animals had fewer breakthrough infections than NVX-CoV2373 or mRNA-vaccinated hamsters. Thus, immunity induced by NVX-CoV2373 or NVX-CoV2540 boosting can protect against the Omicron BA.5 variant in the Syrian hamster model.

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants (preprint)

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

An Immunostimulatory Glycolipid That Blocks SARS-CoV-2, RSV, and Influenza Infections In Vivo (preprint)

The fear and devastation caused by the COVID-19 pandemic has been mitigated by the successful development and deployment of prophylactic vaccines that substantially lowered the incidences of symptomatic infection, hospitalization, and death. However, as the causative agent SARS-CoV-2 continues to spread and evolve worldwide, vaccine-breakthrough infections have become frequent, especially after the emergence of viral variants that are antigenically distant from the ancestral strain used in the current vaccines. Additional approaches are therefore needed in our prevention tool kit. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon- gamma. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics that include currently ongoing influenza, SARS-CoV-2 and respiratory syncytia virus- associated tripledemic long before vaccines or drugs are developed.

Potent and pan-neutralization of SARS-CoV-2 variants of concern by DARPins (preprint)

We report the engineering and selection of two synthetic proteins -- FSR16m and FSR22 -- for possible treatment of SARS-CoV-2 infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon and exhibit broad spectrum neutralization of SARS-Cov-2 strains. The IC50 values of FSR16m against authentic B.1.351, B.1.617.2 and BA.1.1 variants are 3.4 ng/mL, 2.2 ng/mL and 7.4 ng/mL, respectively, comparable to currently used therapeutic antibodies. Despite the use of the spike protein from a now historical wild-type virus for design, FSR16m and FSR22 both exhibit increased neutralization against newly-emerged variants of concern (39- to 296-fold) in pseudovirus assays. Cryo-EM structures revealed that these DARPins recognize a region of the receptor binding domain (RBD, residues 455-456, 486-489) overlapping a critical portion of the ACE2-binding surface. K18-hACE2 transgenic mice inoculated with a B.1.617.2 variant and receiving intranasally-administered FSR16m were protected as judged by less weight loss and 10-100-fold reductions in viral burden in the upper and lower respiratory tracts. The strong and broad neutralization potency make FSR16m and FSR22 promising candidates for prevention and treatment of infection by current and potential future strains of SARS-CoV-2.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron/BA.2 (preprint)

The recent emergence of SARS-CoV-2 Omicron variants possessing large numbers of mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies, and antiviral drugs for COVID-19 against these variants1,2. While the original Omicron lineage, BA.1, has become dominant in many countries, BA.2 has been detected in at least 67 countries and has become dominant in the Philippines, India, and Denmark. Here, we evaluated the replicative ability and pathogenicity of an authentic infectious BA.2 isolate in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone3, we observed similar infectivity and pathogenicity in mice and hamsters between BA.2 and BA.1, and less pathogenicity compared to early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from COVID-19 convalescent individuals and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987/REGN10933, COV2-2196/COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir, and S-217622) can restrict viral infection in the respiratory organs of hamsters infected with BA.2. These findings suggest that the replication and pathogenicity of BA.2 is comparable to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron/BA.2 variants.

Nasally-delivered interferon-λ protects mice against upper and lower respiratory tract infection of SARS-CoV-2 variants including Omicron (preprint)

SUMMARY Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with SARS-CoV-2 infection, variants with constellations of mutations in the spike gene threaten their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine IFN-λ has been proposed as a possible treatment based on correlative studies in human COVID-19 patients. Here, we show IFN-λ protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron)variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally-delivered IFN-λ2 limited infection of historical or variant (B.1.351 and B.1.1.529) SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-λ was produced preferentially in epithelial cells and acted on radio-resistant cells to protect against of SARS-CoV-2 infection. Thus, inhaled IFN-λ may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.

The SARS-CoV-2 B.1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters (preprint)

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2 (preprint)

ABSTRACT Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune evasion properties have arisen, which could jeopardize recently deployed vaccine and antibody-based countermeasures. Here, we evaluated in mice and hamsters the efficacy of preclinical non-GMP Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) South American variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. Immunization of 129S2 and K18-human ACE2 transgenic mice with mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. Thus, immunity induced by mRNA-1273 or Ad26.COV2.S vaccines can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.

An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by therapeutic monoclonal antibodies (preprint)

The emergence of the highly-transmissible B.1.1.529 Omicron variant of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is concerning for antibody countermeasure efficacy because of the number of mutations in the spike protein. Here, we tested a panel of anti-receptor binding domain monoclonal antibodies (mAbs) corresponding to those in clinical use by Vir Biotechnology (S309, the parent mAb of VIR-7831 [Sotrovimab]), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59) for their ability to neutralize an infectious B.1.1.529 Omicron isolate. Several mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost neutralizing activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (COV2-2196 and COV2-2130 combination, ~12-fold decrease) or minimally affected (S309). Our results suggest that several, but not all, of the antibodies in clinical use may lose efficacy against the B.1.1.529 Omicron variant.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617 (preprint)

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at ~3 Angstrom resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic resulting in millions of deaths worldwide. Despite the development and deployment of highly effective antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. Indeed, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant is especially concerning because of the number of mutations, deletions, and insertions in the spike protein. Here, using a panel of anti-receptor binding domain (RBD) monoclonal antibodies (mAbs) corresponding to those with emergency use authorization (EUA) or in advanced clinical development by Vir Biotechnology (S309, the parent mAbs of VIR-7381), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59), we report the impact on neutralization of a prevailing, infectious B.1.1.529 Omicron isolate compared to a historical WA1/2020 D614G strain. Several highly neutralizing mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost inhibitory activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (~12-fold decrease, COV2-2196 and COV2-2130 combination) or minimally affected (S309). Our results suggest that several, but not all, of the antibody products in clinical use will lose efficacy against the B.1.1.529 Omicron variant and related strains.

Rationally Designed Immunogens Enable Immune Focusing Following SARS-CoV-2 Spike Imprinting (preprint)

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit spread of related viruses with pandemic potential. Here, we leveraged rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding, we directed murine serum antibody responses to conserved receptor binding motif (RBM) and domain (RBD) epitopes in the context of SARS-CoV-2 spike imprinting. Whereas all engineered immunogens elicited a robust SARS-CoV-2-neutralizing serum response, the RBM-focusing immunogens exhibited increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defined a conserved epitope. Furthermore, the RBM-focused sera conferred protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. Broadly, these engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes.Funding: We acknowledge funding from NIH R01s AI146779 (AGS), AI124378, AI137057 and AI153098 (DL), R01 AI157155 (MSD) and a Massachusetts Consortium on Pathogenesis Readiness (MassCPR) grant (AGS); training grants: NIGMS T32 GM007753 (BMH, TMC); T32 AI007245 (JF); F31 Al138368 (MS); F30 AI160908 (BMH). ABB is supported by the National Institutes for Drug Abuse (NIDA) Avenir New Innovator Award DP2DA040254, the MGH Transformative Scholars Program as well as funding from the Charles H. Hood Foundation (ABB). This independent research was supported by the Gilead Sciences Research Scholars Program in HIV (ABB). JBC is supported by a Helen Hay Whitney Foundation postdoctoral fellowship.Declaration of Interests: BMH, TMC, and AGS have filed a provisional patent for the described immunogens. MSD is a consultant for Inbios, Vir Biotechnology, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, and Emergent BioSolutions.Ethics Approval Statement: All experiments were conducted with institutional IACUC approval (MGH protocol 2014N000252). Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01).

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains (preprint)

SUMMARY Although mRNA vaccines prevent COVID-19, variants jeopardize their efficacy as immunity wanes. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike) or modified (mRNA-1273.351, designed for B.1.351 spike) preclinical Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Immunization with high or low dose formulations of mRNA vaccines induced neutralizing antibodies in serum against ancestral SARS-CoV-2 and several variants, although levels were lower particularly against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. Nonetheless, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, and serve as a possible model for waning immunity, showed breakthrough lung infection and pneumonia with B.1.617.2. Thus, as levels of immunity induced by mRNA vaccines decline, breakthrough infection and disease likely will occur with some SARS-CoV-2 variants, suggesting a need for additional booster regimens.

Mesalamine Reduces Intestinal ACE2 Expression Without Modifying SARS-CoV-2 Infection or Disease Severity in Mice. (preprint)

Introduction: Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study (SECURE-IBD) reported that the standardized mortality ratio trends higher in IBD patients (1.5-1.8) and that mesalamine/sulfasalazine therapy correlates with poor outcome. The goal of our study was to experimentally address the relationship between mesalamine and SARS-CoV-2 entry, replication, and/or pathogenesis. Methods: Viral infection was performed with a chimeric vesicular stomatitis virus expressing SARS-CoV-2 spike protein and EGFP (VSV-SARS-CoV-2) and SARS-CoV-2 virus derived from an infectious cDNA clone of 2019n-CoV/USA_WA1/2020. Primary human ileal spheroids derived from healthy donors were grown as 3D spheroids or on 2D transwells. We assessed the effect of 10 mM mesalamine (Millipore Sigma) on viral RNA levels, as well as the expression of the SARS-CoV-2 receptor angiotensin II-converting enzyme 2 (ACE2), Transmembrane Serine Protease 2 (TMPRSS2), TMPRSS4, Cathepsin B (CTSB) and CTSL by qRT-PCR. 8-12 week old K18-ACE2 were treated orally with PBS or mesalamine at 200 mg/kg daily. Mice were inoculated intranasally with 1x10^3 FFU of SARS-CoV-2. Mice were weighed daily and viral titers were determined 7 days post infection (dpi) by qRT-PCR. For the intestinal viral entry model, VSV-SARS-CoV-2 was injected into a ligated intestinal loop of anesthetized K18-ACE2 mice and tissues were harvested 6 hours post-infection. Results: We found no change in viral RNA levels in human intestinal epithelial cells in response to mesalamine. Expression of ACE2 was reduced following mesalamine treatment in enteroids, while CTSL expression was increased. Mice receiving mesalamine lost weight at similar rates compared to mice receiving vehicle control. Mesalamine treatment did not change viral load in the lung, heart, or intestinal tissues harvested at 7 dpi. Pretreatment with mesalamine did not modulate intestinal entry of the chimeric VSV-SARS-CoV-2 in K18-ACE2 mice. Conclusions: Mesalamine did not alter viral entry, replication, or pathogenesis in vitro or in mouse models. Mesalamine treatment reduced expression of the viral receptor ACE2 while concurrently increasing CTSL expression in human ileum organoids.

Detection of antibodies neutralizing historical and emerging SARS-CoV-2 strains using a thermodynamically coupled de novo biosensor system (preprint)

With global vaccination efforts against SARS-CoV-2 underway, there is a need for rapid quantification methods for neutralizing antibodies elicited by vaccination and characterization of their strain dependence. Here, we describe a designed protein biosensor that enables sensitive and rapid detection of neutralizing antibodies against wild type and variant SARS-CoV-2 in serum samples. More generally, our thermodynamic coupling approach can better distinguish sample to sample differences in analyte binding affinity and abundance than traditional competition based assays.

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 potently neutralizing anti-SARS-CoV-2 antibody inhibits variants of concern by binding a highly conserved epitope (preprint)

With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 mAbs that bind the receptor binding domain of the spike protein at distinct epitopes and block virus attachment to cells and its receptor, human angiotensin converting enzyme-2 (hACE2). While several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by historical SARS-CoV-2 strains, others induced escape variants in vivo and lost activity against emerging strains. We identified one mAb, SARS2-38, that potently neutralizes all SARS-CoV-2 variants of concern tested and protects mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engages a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of inhibitory antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains (preprint)

Rapidly-emerging variants jeopardize antibody-based countermeasures against SARS-CoV-2. While recent cell culture experiments have demonstrated loss of potency of several anti-spike neutralizing antibodies against SARS-CoV-2 variant strains1-3, the in vivo significance of these results remains uncertain. Here, using a panel of monoclonal antibodies (mAbs) corresponding to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron, and Lilly we report the impact on protection in animals against authentic SARS-CoV-2 variants including WA1/2020 strains, a B.1.1.7 isolate, and chimeric strains with South African (B.1.351) or Brazilian (B.1.1.28) spike genes. Although some individual mAbs showed reduced or abrogated neutralizing activity against B.1.351 and B.1.1.28 viruses with E484K spike protein mutations in cell culture, low prophylactic doses of mAb combinations protected against infection in K18-hACE2 transgenic mice, 129S2 immunocompetent mice, and hamsters without emergence of resistance. Two exceptions were mAb LY-CoV555 monotherapy which lost all protective activity in vivo, and AbbVie 2B04/47D11, which showed partial loss of activity. When administered after infection as therapy, higher doses of mAb cocktails protected in vivo against viruses displaying a B.1.351 spike gene. Thus, many, but not all, of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing SARS-CoV-2 variant strains.