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.

Delta variants of SARS-CoV-2 cause significantly increased vaccine breakthrough COVID-19 cases in Houston, Texas

Genetic variants of SARS-CoV-2 have repeatedly altered the course of the COVID-19 pandemic. Delta variants of concern are now the focus of intense international attention because they are causing widespread COVID-19 disease globally and are associated with vaccine breakthrough cases. We sequenced the genomes of 16,965 SARS-CoV-2 from samples acquired March 15, 2021 through September 20, 2021 in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared to all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared to 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Individuals with vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold (Ct) value (a proxy for high virus load). This value was closely similar to the median Ct value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. Our integrated analysis emphasizes that vaccines used in the United States are highly effective in decreasing severe COVID-19 disease, hospitalizations, and deaths.

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.

Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer [≥]160 was 80% or greater with anti-RBD or anti-ECD titers of [≥]1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers [≥]160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of [≥]1:1350, and evidence of VN [≥]1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of [≥]1:1350 may provide critical information about protection against COVID-19 disease.

Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas

BackgroundCOVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. MethodsPatients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 - April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. ResultsAt baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. ConclusionsThe data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.