Rapid characterization of spike variants via mammalian cell surface display

Abstract

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with 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 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 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.

Analysis of Immune Escape Variants from Antibody-Based Therapeutics against COVID-19: A Systematic Review

“…Deep mutational scanning (DMS) predicts protein expression, ACE2 binding, and mAb binding [ 5 ]. The method was first deployed with yeast display libraries [ 6 ], then evolved to phage display libraries ( ) [ 7 ] and finally mammalian cell surface display [ 8 ]. nAb binding is common within the fusion peptide and in the linker region before heptad repeat (HR) region 2.…”

Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant

“…The emergence of variants of concern (VOCs) with the enhanced transmissibility and infectivity profile including the D614G variant [54][55][56][57], B.1.1.7 (alpha) [58][59][60][61], B.1.351 (beta) [62,63], B.1.1.28/P.1 (gamma) [64], and B.1.1.427/B.1.429 (epsilon) variants [65,66] have attracted enormous attention in the scientific community and a considerable variety of the proposed mechanisms explaining functional observations from structural and biochemical perspectives. The detection of common mutational changes such as D614G, E484K, N501Y, and K417N that are shared among major circulating variants B.1.1.7, B.1.351, and B.1.1.28/P.1 indicated that these positions could be particularly critical for modulation of the SARS-CoV-2 S protein responses.…”

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering

“…The emergence of variants of concern (VOC's) with the enhanced transmissibility and infectivity profile including D614G variant [54][55][56][57], B.1.1.7 (alpha) [58][59][60][61], B. 1.351 (beta) [62,63], B.1.1.28/P.1 (gamma) [64], and B.1.1.427/B.1.429 (epsilon) variants [65,66] revealed complex mechanisms underlying function and dynamics of the S proteins in different biological environments. Structural and biophysical studies characterized a diversity of S functional states for B.1.1.7 (B.1.1.7), B.1.351 (beta), P1 (gamma), and B.1.1.427/B.1.429 (epsilon) variants and showed that conformational plasticity of S proteins is modulated by mutations and determines the ability to evade host immunity and incur resistance to antibodies [67][68][69].…”

“…SARS-CoV-2 S mutants with the enhanced infectivity profile have attracted an enormous attention in the scientific community following the evidence of the mutation enrichment via epidemiological surveillance, resulting in proliferation of experimental data and a considerable variety of the proposed mechanisms explaining functional observations. The emergence of variants of concern (VOC’s) with the enhanced transmissibility and infectivity profile including D614G variant [ 54 , 55 , 56 , 57 ], B.1.1.7 (alpha) [ 58 , 59 , 60 , 61 ], B.1.351 (beta) [ 62 , 63 ], B.1.1.28/P.1 (gamma) [ 64 ], and B.1.1.427/B.1.429 (epsilon) variants [ 65 , 66 ] revealed complex mechanisms underlying function and dynamics of the S proteins in different biological environments. Structural and biophysical studies characterized a diversity of S functional states for B.1.1.7 (B.1.1.7), B.1.351 (beta), P1 (gamma), and B.1.1.427/B.1.429 (epsilon) variants and showed that conformational plasticity of S proteins is modulated by mutations and determines the ability to evade host immunity and incur resistance to antibodies [ 67 , 68 , 69 ].…”