Broadly neutralizing antibodies target the coronavirus fusion peptide (preprint)

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development.

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants (preprint)

The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.

A combination of cross-neutralizing antibodies synergizes to prevent SARS-CoV-2 and SARS-CoV pseudovirus infection (preprint)

Coronaviruses have caused several epidemics and pandemics including the ongoing coronavirus disease 2019 (COVID-19). Some prophylactic vaccines and therapeutic antibodies have already showed striking effectiveness against COVID-19. Nevertheless, concerns remain about antigenic drift in SARS-CoV-2 as well as threats from other sarbecoviruses. Cross-neutralizing antibodies to SARS-related viruses provide opportunities to address such concerns. Here, we report on crystal structures of a cross-neutralizing antibody CV38-142 in complex with the receptor binding domains from SARS-CoV-2 and SARS-CoV. Our structural findings provide mechanistic insights into how this antibody can accommodate antigenic variation in these viruses. CV38-142 synergizes with other cross-neutralizing antibodies, in particular COVA1-16, to enhance neutralization of SARS-CoV-2 and SARS-CoV. Overall, this study provides valuable information for vaccine and therapeutic design to address current and future antigenic drift in SARS-CoV-2 and to protect against zoonotic coronaviruses.

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago (preprint)

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.

Evidence of antigenic imprinting in sequential Sarbecovirus immunization (preprint)

Antigenic imprinting, which describes the bias of antibody response due to previous immune history, can influence vaccine effectiveness and has been reported in different viruses. Give that COVID-19 vaccine development is currently a major focus of the world, there is a lack of understanding of how background immunity influence antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting in Sarbecovirus, which is the subgenus that SARS-CoV-2 belongs to. Specifically, we sequentially immunized mice with two antigenically distinct Sarbecovirus strains, namely SARS-CoV and SARS-CoV-2. We found that the neutralizing antibodies triggered by the sequentially immunization are dominantly against the one that is used for priming. Given that the impact of the background immunity on COVID-19 is still unclear, our results will provide important insights into the pathogenesis of this disease as well as COVID-19 vaccination strategy.

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody (preprint)

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model (preprint)

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 A revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity (preprint)

Most antibodies isolated from COVID-19 patients are specific to SARS-CoV-2. COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here we determined a crystal structure of COVA1-16 Fab with the SARS-CoV-2 RBD, and a negative-stain EM reconstruction with the spike glycoprotein trimer, to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long CDR H3, and competes with ACE2 binding due to steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.

An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain (preprint)

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.

Structural basis of a public antibody response to SARS-CoV-2 (preprint)

Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3-53 neutralizing antibodies +/- Fab CR3022 ranging from 2.33 to 3.11 [A] resolution. The germline-encoded residues of IGHV3-53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.

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

The outbreak of COVID-19, which is caused by SARS-CoV-2 virus, continues to spread globally, but there is currently very little understanding of the epitopes on the virus. In this study, we have determined the crystal structure of the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein in complex with CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient. CR3022 targets a highly conserved epitope that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding site can only be accessed when at least two RBDs on the trimeric S protein are in the "up" conformation. Overall, this study provides structural and molecular insight into the antigenicity of SARS-CoV-2. ONE SENTENCE SUMMARYStructural study of a cross-reactive SARS antibody reveals a conserved epitope on the SARS-CoV-2 receptor-binding domain.