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Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilize VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bind both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD – a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions., Graphical Abstract, Rapp et al. determine structures of three human VH1-2-derived SARS-CoV-2 neutralizing antibodies and define a prevalent multi-donor antibody class which recognizes the receptor-binding domain (RBD) with convergent heavy and light chain modules. They further show the structural basis for RBD-up and RBD-down recognition and quaternary recognition-mediated spike conformation change.

Numerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies target a common surface, bordered by glycans N17, N74, N122, and N149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies appear to target a single supersite., Graphical Abstract, Cerutti et al. report structural analysis of seven potent neutralizing antibodies targeting the N-Terminal domain of SARS-CoV-2 spike. All antibodies recognize a common glycan-free, electropositive surface comprised of a mobile β-hairpin and flexible loops. While RBD-directed antibodies recognize non-overlapping epitopes, these findings indicate NTD-directed antibodies predominantly target a single supersite.

SummaryNumerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycansN17,N74,N122, andN149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures—at serological and endosomal pH—delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted “up” conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824–858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody., Graphical Abstract, Highlights • Determine cryo-EM structures of SARS-CoV-2 spike along its endosomal entry pathway • Reveal structural basis by which a pH-dependent switch mediates RBD positioning • Show spike to exclusively adopt an all-RBD-down conformation at low pH • Suggest low-pH all-RBD-down conformation to provide a basis for immune evasion, Zhou et al. determine 12 structures of the SARS-CoV-2 spike, bound by ACE2 receptor and ligand free, that reveal a pH-dependent switch to mediate positioning of spike receptor-binding domains (RBDs). At low pH, the spike adopts an all-RBD-down conformation, which provides a potential means of immune evasion from RBD-up-recognizing antibody.

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2., Graphical abstract, Banach et al. report a SARS-CoV-2 neutralizing antibody along with genetic, structural, and functional features of public antibody responses targeting SARS-CoV-2. These data reveal how structural interactions with the SARS-CoV-2 receptor-binding domain correlate with viral neutralization and demonstrate the importance of native antibody heavy:light pairings in convergent antibody responses.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. But what controls RBD positioning? As SARS-CoV-2 utilizes endosomal pathways as a major mode of entry, we investigated the impact of both low pH and ACE2 binding. Cryo-EM structures –at serological and endosomal pH– delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the ‘up’ conformation required for ACE2 interaction, primarily through RBD movement combined with subtle alterations in position and orientation of neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a single all-down conformation at lower pH, which biochemical studies suggested might provide immune evasion from RBD-up-recognizing antibody. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and appeared to mediate RBD positioning in concert with coordinated movements through the entire trimer apex. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), Federal funds from the Frederick National Laboratory for Cancer Research under Contract HHSN261200800001E (A.S., T.S., Y.T.). Cryo-EM data for the spike-ACE2 complexes were collected at Columbia University Cryo-EM Center at the Zuckerman Institute, and at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539,) and by grants from the Simons Foundation (SF349247) and NY State Assembly. Cryo-EM datasets for individual spike proteins were collected at the National CryoEM Facility (NCEF) of the National Cancer Institute. This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. We are especially grateful to U. Baxa, A. Wier, M. Hutchison, and T. Edwards of NCEF for collecting cryo-EM data and for technical assistance with cryo-EM data processing. Frederick Research Computing Environment (FRCE) high-performance computing cluster was used for processing cryo-EM datasets of individual spike proteins. Conflict of Interest: The authors declare no competing interest.

SummaryUnderstanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2.HighlightsA molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1–57 and 2–7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1–57 and 2–7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1–57 and 2–7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1–57 and 2–7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape., Graphical abstract, Cerutti et al. report the structural characterization of two potent neutralizing antibodies, 1–57 and 2–7, bound to the SARS-CoV-2 spike. The cryo-EM structures elucidate distinct mechanisms to accommodate the RBD mutations observed in the B.1.1.7 and B.1.351 variants. Both antibodies represent low-frequency immune responses and their use as therapeutics is suggested.