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

Kristina E. Atanasoff, Luca Brambilla, Daniel C. Adelsberg, Shreyas Kowdle, Christian S. Stevens, Stefan Slamanig, Chuan Tien Hung, Yanwen Fu, Reyna Lim, Linh Tran, Robert Allen, Weina Sun, J. Andrew Duty, Goran Bajic, Benhur Lee, Domenico Tortorella

Research output: Contribution to journalArticlepeer-review


The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 770 millioncases and >6.9 million deaths worldwide. We identifieda 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, 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 modifiedversion STI-9167 effectivelyinhibited 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 definethe 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 identifiedF486 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-stabilized470 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 definethe neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. IMPORTANCE The COVID-19 pandemic remains a significantpublic 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 effectivetherapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificityof 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 workflowcan serve to predict the efficacyof antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Original languageEnglish
Issue number1
StatePublished - Jan 2024


  • Cryo-EM
  • Epitope mapping
  • Neutralizing antibody
  • SARS-CoV-2
  • Structural analysis
  • Viral resistance


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