Broadly neutralizing nanobodies target a defined structural pivot site on the RSV fusion protein

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections in infants and elderly individuals. Although nirsevimab represents a recent breakthrough against RSV infection, the emergence of resistant variants highlight the need for additional antiviral strategies. Here, we report two nanobodies (Nbs), 1G9 and 1D8, that target the RSV fusion (F) protein and exhibit high neutralizing activity against RSV subtypes. In vivo, the Nbs-Fc demonstrated robust prophylactic and therapeutic efficacy. Cryo-electron microscopy revealed that 1G9 and 1D8 specifically engage a conformational pivot site within antigenic site IV, crosslinking the metastable heptad repeats B (HRB) and the conformationally stable domain II of the F protein. This interaction stabilizes the prefusion conformation and prevents the structural rearrangement required for membrane fusion. Notably, the binding residues are highly conserved across RSV subtypes, accounting for the broad-spectrum neutralization observed. Together, our findings identify a structurally conserved and functionally critical epitope on RSV F and highlight 1G9 and 1D8 as promising candidates for next-generation prophylactic and therapeutic interventions against RSV.