Structural characterization of antibodies binding the conserved central helix and membrane-proximal lower stalk of coronavirus spike glycoprotein 2248624
The ongoing threat of zoonotic coronavirus spillover into humans, exemplified by severe acute respiratory syndrome virus 2 (SARS-CoV-2), underscores the urgent need for pan-coronavirus therapeutics that can be deployed to mitigate future pandemics. Here, we identified monoclonal antibodies from COVID-19 convalescent donors that target two conserved epitopes in the S2 domain of the coronavirus Spike glycoprotein: the central helix (CH) and a membrane-proximal epitope in the heptad repeat 2 (HR2), which we designate the lower stalk (LS). CH-directed antibodies exhibited broad cross-reactivity across betacoronaviruses, whereas LS-directed antibodies demonstrated reactivity primarily within sarbecoviruses. Using cryogenic electron microscopy (cryo-EM), we determined sub-4 Å structures of three cross-reactive CH antibodies–ch.005, ch.007, and ch.010–bound to the prefusion-stabilized SARS-CoV-2 S2 protein, revealing distinct binding poses and contact residues relative to previously described CH antibodies. In parallel, X-ray crystallography studies yielded a sub-2 Å structure of the ls.019 Fab in complex with the LS peptide, providing, to our knowledge, the first structural visualization of a human monoclonal antibody engaging this epitope. Together, these findings advance our knowledge of two conserved and structurally vulnerable sites within the coronavirus S2 subunit—the central helix and the lower stalk—that can guide the development of broad-spectrum antibody therapeutics and vaccines against current and emerging coronaviruses. Howard Hughes Medical Institute Vaccines and Immunotherapy (VAC)