A modular three in one mucosal vaccine against three antigenic clusters of ACE2 using sarbecoviruses

The recurrent emergence of ACE2‑using sarbecovirus underscores the need for a broadly protective vaccine. Here, we mapped the antigenic landscape of sarbecovirus receptor-binding domains (RBDs) and identified three distinct clusters. We then engineered a single "three‑in‑one" immunogen, 3Rs-NC, incorporating representative RBDs from each cluster into a single scaffold. Intranasal administration of 3Rs-NC with a flagellin-derived mucosal adjuvant (KFD), which possess excellent safety profile potential for clinical usage, elicited high titers of RBD-specific serum IgG and mucosal IgA, as well as potent neutralizing antibody responses in mice. Furthermore, KFD-adjuvanted 3Rs-NC conferred sustained protection in both the upper and lower respiratory tracts against SARS-CoV-2 Omicron BA.1 and SARS-like coronavirus WIV1. Additionally, 3Rs-NC immunization protected mice from lethal challenge of SARS-like coronavirus rRsSHC014S, with more efficient protection observed in female mice than male mice. This needle-free formulation offers a potent, broad-spectrum vaccine candidate against current and emerging ACE2-using sarbecoviruses, functioning as a modular "three-in-one" vaccine platform ready for rapid deployment in future coronavirus outbreaks.