Human scFv inhibiting broad coronavirus replication: a pandemic preparedness candidate

Abstract

The ongoing emergence of zoonotic coronaviruses (CoVs) and the rapid evolution of viral variants underscore the need for broadly active antiviral agents. The relatively conserved S2 subunit of the coronavirus spike protein is an attractive target for developing pan-coronavirus therapeutics. Fully human single-chain antibody variable fragments (HuscFvs) targeting the S2 subunit were generated by phage display. Candidate antibodies were evaluated against multiple SARS-CoV-2 variants and representative Alpha-, Gamma-, and Delta-coronaviruses, including human coronavirus 229E, porcine epidemic diarrhea virus, infectious bronchitis virus, and porcine deltacoronavirus. Antiviral activity and mechanism of action were investigated in TMPRSS2-negative cells. A SARS-CoV-2 spike-mediated cell–cell fusion assay was used to assess fusion inhibition. Antibody–epitope interactions were characterized using mimotope-based epitope mapping, comparative sequence analysis, AlphaFold-based co-folding, and molecular dynamics simulations. HuscFv39 exhibited potent inhibitory activity against multiple tested SARS-CoV-2 variants and cross-neutralizing activity against representative coronaviruses from all four coronavirus genera. In TMPRSS2-negative cells, HuscFv39 restricted viral infection to late endosomal compartments, thereby limiting subsequent cytoplasmic replication. HuscFv39 also significantly inhibited spike-mediated membrane fusion, supporting a fusion-interference mechanism. Structural and computational analyses identified a previously uncharacterized conformational epitope spanning conserved regions of the S1/S2 junction, S2′ cleavage site, fusion peptide/intermediate fusion peptide, and heptad repeat 1 (HR1). Recognition of this conserved structural epitope may contribute to the broad antiviral activity observed across genetically diverse coronaviruses. HuscFv39 is a fully human, Fc-free antibody fragment with broad anti-coronavirus activity. Targeting a conserved structural epitope within the S2 subunit highlights a promising strategy for developing broadly active coronavirus therapeutics and supports further evaluation against additional human and emerging coronaviruses.