Structural and functional characterization of a conserved cryptic epitope on SARS-CoV-2 spike S2 subunit

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone extensive evolution since its emergence in 2019, underscoring the continuous need for vaccines and therapeutics effective against multiple variants of concern (VOCs). The S2 subunit of the viral spike (S) glycoprotein is highly conserved among sarbecoviruses, making it an attractive target for broadly protective countermeasures. To elucidate the S2 antigenic landscape, we employed yeast surface display to isolate S2-targeted antibodies from COVID-19 convalescent donors. Biophysical characterization revealed that these S2 apex-directed antibodies preferentially bind to open spike conformations and a stabilized S2 construct but not to the closed, trimeric prefusion spike. Cryo-electron microscopy structures defined a cryptic epitope encompassing the upper helix and fusion peptide proximal region on S2. This epitope is conserved among sarbecoviruses but remains largely occluded in the closed prefusion conformation of the spikes. As a result, the antibodies exhibited weak neutralization activity against SARS-CoV-2 pseudoviruses, failed to neutralize authentic viruses, and did not provide protection in a lethal mouse challenge model using a mouse-adapted SARS-CoV-2 strain. These findings highlight a non-neutralizing epitope on S2 capable of eliciting antibodies during SARS-CoV-2 infection in humans and provide valuable reagents for probing S2 conformational dynamics and optimizing S2-based vaccine antigens.Author summary: The rapidly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge vaccines and antibody therapeutics that primarily target the variable S1 subunit of the spike (S) glycoprotein. In contrast, the fusion machinery, the S2 subunit, is more conserved among sarbecoviruses and represents a potential target for broadly reactive antibodies. Here, we used yeast surface display to isolate S2-directed antibodies from COVID-19 convalescent donors and characterized their structural and functional properties. Biophysical analyses showed preferential binding to open spike conformations and a stabilized S2 construct, but not to the closed prefusion trimer. Cryo-electron microscopy structures revealed a conserved, cryptic epitope spanning the upper helix and fusion peptide–proximal region (FPPR) that is largely occluded in the closed spike. Despite cross-reactivity, these antibodies weakly neutralized SARS-CoV-2 pseudoviruses, failed to neutralize authentic virus, and did not protect mice in a lethal challenge model. Collectively, our findings define a conserved but non-neutralizing cryptic S2 epitope targeted by infection-elicited antibodies and provide reagents to probe spike conformational dynamics and inform the rational design of S2-based immunogens that minimize exposure of non-protective epitopes.