Understanding conformational changes of the coronavirus spike protein is critical for developing broad-spectrum therapies. The pan-coronavirus epitope spike residues 815–825 (centred on the S2′ site) are buried in the prefusion spike but are transiently exposed upon ACE2 binding1,2. Here, using integrated functional and structural analyses, we demonstrate that 76E1, an antibody targeting spike residues 815–825, specifically recognizes an open early fusion intermediate conformation in which this epitope adopts a helical conformation, designated the S2′-helix. SARS-CoV-2 Omicron variants evade such antibodies via steric hindrance resulting from S2′-helix shifts and restricted S1–ACE2 distancing in the early fusion intermediate conformation, together with increased reliance on cathepsin-mediated entry that impairs 76E1 inhibition of S2′ cleavage. The H655Y mutation is central to this evasion. Antibody size directly affects its access to the S2′-helix. Crucially, antibody size minimization reversed the evasion mechanisms and significantly enhanced neutralizing activity against authentic Omicron variants and other human coronaviruses, including SARS-CoV-1 and HCoV-229E. These findings establish small-molecule targeting of the S2′-helix as a strategy for pan-coronavirus therapies.
