Rapid characterization of spike variants via mammalian cell surface display

Abstract

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.

Expression and characterization of SARS-CoV-2 spike proteins

“…Here, we describe the expression, purification and biophysical characterization of HexaPro, a second-generation prefusion-stabilized SARS-CoV-2 spike glycoprotein from mammalian cells 36 . In addition to developing HexaPro, we have used this protocol to rapidly screen circulating spike mutants, to develop new spike antigens, and for structure-function studies of spike conformations [37][38][39][40] . We also describe three widely available assays to characterize recombinant spikes: (1) spike thermostability; (2) affinity for ACE2; and (3) binding by patient sera or monoclonal antibodies.…”

The ins and outs of SARS-CoV-2 variants of concern (VOCs)

“…Regarding ACE2 mimics, the B.1.427 and B.1.429 variants showed moderate resistance to peptide-based inhibitors such as LCB1, and since the L452R mutation is located near the ACE2-LCB1 interface, it might affect the binding affinity of LCB1 [87,91]. In addition, due to an S13I mutation in the signal peptide cleavage site that causes a significant structural rearrangement of the NTD antigenic supersite, a complete loss of B.1.427/B.1.429 neutralization was observed with a panel of mAbs targeting the NTD [87].…”

ACE2‐based decoy receptors for SARS coronavirus 2

“…To begin with, the designed miniproteins poorly bind S of SARS‐CoV‐1 and are not pan‐specific inhibitors. A deep mutational scan of the SARS‐CoV‐2 RBD also provided hints of possible escape mutations, even if not common, 130 and at least one of the designed miniproteins with exceptionally tight affinity to S from the original Wuhan SARS‐CoV‐2 isolate has a near total loss of affinity to newer virus variants in circulation 132 . Computational calculations predict that the miniproteins in general will have reduced affinity to highly transmissible SARS‐CoV‐2 variants carrying a N501Y mutation in S; 133 this mutation markedly increases affinity between ACE2 and the RBD 48 .…”