An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies (preprint)

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed 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) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-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 (RBM). 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. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants.

Structure of a germline-like human antibody defines a neutralizing epitope on the SARS-CoV-2 spike NTD (preprint)

Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germline-like monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against SARS-CoV-2 and determined its structure in complex with the spike glycoprotein by cryo-EM. We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its HCDR3 loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that - because of the conserved nature of the epitope - the mAb maintains binding to viral variants B.1.1.7 and B.1.351. Our study illustrates the feasibility of targeting the NTD to achieve broad neutralization against SARS-CoV-2 variants.