Rapid characterization of spike variants via mammalian cell surface display (preprint)

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 the N-terminal domain (NTD) highlights a public class of epitopes in the N3 and N5 loops that are recognized by most of the NTD-binding nAbs assayed in this study. 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.1.1351 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 be useful for rapid antigen design, deep scanning mutagenesis, and epitope mapping of antibody interactions for SARS-CoV-2 and other emerging viral threats.

Identification of a conserved neutralizing epitope present on spike proteins from highly pathogenic coronaviruses (preprint)

Three pathogenic human coronaviruses have emerged, with SARS-2 causing a global pandemic. While therapeutic antibodies targeting the SARS-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report three antibodies binding epitopes conserved on the pre-fusion MERS, SARS-1 and SARS-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes in in vitro SARS-2 cell fusion and pseudovirus assays. Hydrogen-Deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, consistent with binding to natural and engineered spike variants. This location at the spike trimer interface suggests 3A3 prevents the S2 conformational rearrangements required for virus-host cell fusion. This work defines a highly conserved vulnerable site on the SARS-2 S2 domain and may help guide design of pan-protective spike immunogens.