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We report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement during the coronavirus disease 2019 (COVID-19) pandemic. Delta SARS-CoV-2 efficiently outcompetes the Alpha variant in human lung epithelial cells and primary human airway tissues. The Delta spike mutation P681R is located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduces the replication of the Delta variant to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhances the cleavage of the full-length spike to S1 and S2, which could improve cell-surface-mediated virus entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the cleavage of the Alpha spike is reduced compared with the Delta spike. Our results suggest P681R as a key mutation in enhancing Delta-variant replication via increased S1/S2 cleavage., Competing Interests: Declaration of interests X.X., V.D.M., and P.-Y.S. have filed a patent on the reverse genetic system and reporter SARS-CoV-2., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962)., Competing Interests: Declaration of interests G.M., C.P.M., J.W., T.C., G.L., K.F., L.Q., J.T.S., J.M., A.K., K.A., K-F.W., I.M., R.T., A.R., M.A.R., A-M.S., R.Jo., S.N., R.J., K.L., S.B., J.B.U., A.H.S., and D.Y. are current or former employees of the GSK group of companies and may own GSK shares and/or restricted GSK shares. G.M., J.W., L.Q., K.L., J.B.U., and D.Y. are inventors on a patent application claiming subject matter related to the SARS-CoV-2 SAM vaccine candidates described herein. P.Y.S. is a member of the Scientific Advisory Boards of AbImmune and is Founder of FlaviTech. X.X. and P.-Y.S. have filed a patent on the reverse genetic system of SARS-CoV-2. M.A.A., M.G., and C.S. received compensation from GSK to perform the rat toxicity and biodistribution assays. The other authors declare no other competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Two doses of the BNT162b2 mRNA vaccine are highly effective against SARS-CoV-2. Here, we tested the antibody neutralization against Omicron SARS-CoV-2 after 2 and 3 doses of BNT162b2. Serum from vaccinated individuals was serially tested for its ability to neutralize wild-type SARS-CoV-2 (USA-WA1/2020) and an engineered USA-WA1/2020 bearing the Omicron spike glycoprotein. At 2 or 4 weeks post dose 2, the neutralization geometric mean titers (GMTs) against the wild-type and Omicron-spike viruses were 511 and 20, respectively; at 1 month post dose 3, the neutralization GMTs increased to 1,342 and 336; and at 4 months post dose 3, the neutralization GMTs decreased to 820 and 171. The data support a 3-dose vaccination strategy and provide a glimpse into the durability of the neutralization response against Omicron., Competing Interests: Declaration of interests X.X. and P.-Y.S. have filed a patent on the reverse genetic system of SARS-CoV-2. H.X., J.Z., C.K., X.X., and P.-Y.S. received compensation from Pfizer to perform the project. H.C., Q.Y., M.C., D.C., K.U.J., and K.A.S. are employees of Pfizer and may hold stock options. A.M. is an employee of BioNTech and may hold stock options., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs., Competing Interests: Declaration of interests D.H.F. is a founder of Courier Therapeutics. A.H.E. is a consultant for Inbios and Fimbrion Therapeutics. M.S.D. is a consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals and is on the Scientific Advisory Board of Moderna and Immunome. D.H.F., M.S.D., and A.H.E. have received unrelated funding support from Emergent BioSolutions. M.S.D. has sponsored research agreements from Moderna and Vir Biotechnology, A.H.E. has a sponsored research agreement from Abbvie, and D.H.F. has a sponsored research agreement from Mallinckrodt Pharmaceuticals., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

With the emergence of severe acute respiratory syndrome coronavirus 2 (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 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants., Competing Interests: Declaration of interests 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, and Emergent BioSolutions. Some of the mAbs described in this study have been licensed by Washington University to Bio X Cell. D.H.F is a founder of Courier Therapeutics and has received funding support in a sponsored research agreement from Emergent BioSolutions. J.E.C. has served as a consultant for Eli Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from AstraZeneca and IDBiologics. The Boon laboratory has received funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences, AbbVie, and Nano Targeting & Therapy Biopharma., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern., Competing Interests: Declaration of interests The Ellebedy laboratory received funding under sponsored research agreements that are unrelated to the data presented in the current study from Emergent BioSolutions and from AbbVie. A.H.E. is a consultant for Mubadala Investment Company and the founder of ImmuneBio Consulting LLC. M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, Carnival Corporation and on the Scientific Advisory Board of Moderna and Immunome. The Diamond laboratory has received unrelated sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., and Nano targeting & Therapy Biopharma Inc. The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of a SARS-CoV-2 mAb. A.J.S., J.S.T., W.B.A., J.B.C., S.P.J.W., M.S.D., A.C.M.B., and A.H.E. are recipients of a licensing agreement with Abbvie Inc., for commercial development of a SARS-CoV-2 mAb. A patent application related to this work has been filed by Washington University School of Medicine. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list Florian Krammer as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. Florian Krammer has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. The Shi laboratory has received sponsored research agreements from Pfizer, Gilead, Merck, and IGM Sciences Inc. The Whelan laboratory has received unrelated funding support in sponsored research agreements with Vir Biotechnology, AbbVie, and sAB therapeutics. All other authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, Fotress Biotech, and Carnival Corporation and on the Scientific Advisory Board of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. M.S.D. is on the Advisory Board of Moderna. M.S.D., I.P.D., D.T.C., and A.O.H. have filed a disclosure with Washington University for possible commercial development of ChAd-SARS-CoV-2-S. D.T.C. is an equity holder in Precision Virologics, Inc., which has optioned the ChAd-SARS-CoV-2-S vaccine., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to have a global impact. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 spike receptor-binding domain. Here, we investigated the capacity of modified versions of one lead miniprotein, LCB1, to protect against SARS-CoV-2-mediated lung disease in mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung when given as many as 5 days before or 2 days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation, and is on the scientific advisory boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. L. Cao, I.G., L.S., J.B.C., M.S.D., and D.B. are coinventors on a provisional patent application that incorporates discoveries described in this manuscript. D.B. is a cofounder of Neoleukin Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development., Competing Interests: Declaration of interests X.Z., X.X., and P.-Y.S. have filed a patent on the trans-complementation system of SARS-CoV-2. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, 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 Moderna, Vir Biotechnology, and Emergent BioSolutions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8 + T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8 + T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2., Competing Interests: Declaration of interests R.R.A., Sailaja Gangadhara, and N.K.R. are co-inventors of the MVA/S vaccine technology. Emory University filed a patent on this technology. R.R.A. serves as a SAB member for Heat Biologics Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 106 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures. • A reverse genetic system has been established for SARS-CoV-2 • Recombinant SARS-CoV-2 replicates as efficiently as the original clinical isolate • A stable mNeonGreen reporter SARS-CoV-2 has been developed • The mNeonGreen SARS-CoV-2 can be used to screen antiviral inhibitors Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic. Xie et al. generated an infectious cDNA clone of SARS-CoV-2 and a mNeonGreen reporter virus. Recombinant SARS-CoV-2 and reporter virus replicate as efficiently as the original clinical isolate. [ABSTRACT FROM AUTHOR]

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes. [Display omitted] • Natural SARS-2 infection induces a subset of potent N-terminal domain-specific mAbs • N-terminal domain reactive human monoclonal antibodies can neutralize live virus • COV2-2676 and COV2-2489 offer protection in a hACE2-transgenic mouse model • COV2-2676 and COV2-2489 Fc-effector functions are essential for optimal protection Suryadevara et al. find human neutralizing antibodies to the spike protein N-terminal domain that arise from natural infection with SARS-CoV-2. These antibodies inhibit post-attachment steps of the viral cycle and initiate protective immune responses via the antibody Fc domain. [ABSTRACT FROM AUTHOR]