Single amino acid substitution in Hendra virus attachment glycoprotein induces cross-neutralizing antibodies against Nipah virus

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic henipaviruses within the Paramyxoviridae family, causing severe respiratory and neurological diseases in humans and animals with fatality rates up to 75%, and no licensed human vaccines or therapeutics. In this study, we identified a unique vulnerable epitope on the NiV attachment glycoprotein (G) recognized by the potent neutralizing antibody 14F8, which targets a receptor-binding site and neutralizes NiV effectively. Using the 2.8 Å crystal structure of the 14F8 Fab-NiV-G complex as a guide, we reconstructed this epitope on HeV-G via a single amino acid substitution (S586N), creating the HeV-G_S586N mutant. Immunization with HeV-G_S586N in BALB/c mice and cynomolgus monkeys elicited robust, broadly neutralizing antibody responses against both NiV and HeV, achieving higher NiV-neutralizing titers post-prime compared to wild-type HeV-G, as confirmed by pseudovirus and live-virus assays. Crystal structures of HeV-G_S586N (3.3 Å) and its 14F8 complex (3.2 Å) showed the S586N substitution induced a 9 Å conformational rearrangement in β-propeller blade 6, reshaping the molecular skeleton and solvent-accessible surface without direct N586-14F8 interaction, thus mimicking the NiV epitope. These findings position HeV-G_S586N as a promising broad-spectrum antigen for henipavirus prevention and demonstrate the value of structure-guided epitope reconstruction in universal vaccine design for emerging viral threats.