Antibody escape and cryptic cross-domain stabilization in the SARS CoV-2 Omicron spike protein

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.

Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

Rapid characterization of spike variants via mammalian cell surface display

The SARS-CoV-2 spike (S) protein is a critical component of subunit vaccines and a target for neutralizing antibodies. Spike is also undergoing immunogenic selection with clinical variants that increase infectivity and partially escape convalescent plasma. Here, we describe spike display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed [~]200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by thirteen neutralizing antibodies (nAbs). An alanine scan of all five N-terminal domain (NTD) loops highlights a public class of epitopes in the N1, N3, and N5 loops that are recognized by most of the NTD-binding nAbs. Some clinical NTD substitutions abrogate binding to these epitopes but are circulating at low frequencies around the globe. NTD mutations in variants of concern B.1.1.7 (United Kingdom), B.1.351 (South Africa), B.1.1.248 (Brazil), and B.1.427/B.1.429 (California) impact spike expression and escape most NTD-targeting nAbs. However, two classes of NTD nAbs still bind B.1.1.7 spikes and neutralize in pseudoviral assays. B.1.351 and B.1.1.248 include compensatory mutations that either increase spike expression or increase ACE2 binding affinity. Finally, B.1.351 and B.1.1.248 completely escape a potent ACE2 peptide mimic. We anticipate that spike display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 [A]2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.