An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody

The 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.