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ABSTRACTThe coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 770 million cases and >6.9 million deaths worldwide. We identified a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor-binding domain (RBD) (J. A. Duty, T. Kraus, H. Zhou, Y. Zhang, et al., Med 3:705–721, 2022, https://doi.org/10.1016/j.medj.2022.08.002). Representative antibodies from genetically distinct families were evaluated for the inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in the place of VSV-G. One mAb clone denoted FG-10A3 and its therapeutically modified version STI-9167 effectively inhibited infection and in vivo proliferation of early variants of SARS-CoV-2 including Omicron BA.1 and BA.2 and corresponding pseudoviruses and rcVSV-S variants (Duty et al.). To define the epitope of the broadly reactive FG-10A3 mAb, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-electron microscopy (EM). FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif. Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470–490 loop at the Spike RBD tip. Furthermore, neutralization studies using rcVSV-S F486 point mutants and currently-circulating variants Omicron BA.5, XBB.1.5, and BQ.1.1 that contain a V or P at position 486 further supported the model in which residue 486 is an important residue for FG-10A3 inhibition. This work provides an experimental strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants.IMPORTANCEThe COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy.