Highly potent C-type nanoantibodies neutralize Nipah and Hendra viruses by cavity filling on fusion glycoprotein

Nipah (NiV) and Hendra (HeV) viruses pose a serious threat, causing global outbreaks with high fatality rates. The fusion (F) glycoprotein, located on the viral surface, presents a conserved and promising neutralizing target. Here, we identify two C-type nanoantibodies (C-Nabs), 1A1-3-22 and 2A1-3-8, targeting F protein. 2A1-3-8 exhibits cross-reactivity against both viruses, whereas 1A1-3-22 shows specificity to NiV. The half-life-extended 2A1-3-8 provides 100% protection against NiV lethal challenge in female hamsters. Cryo-EM analysis reveals that 1A1-3-22 and 2A1-3-8 target similar funnel-like epitopes. Based on the unique “cavity-filling” mechanism, we engineered 2A1-3-8 to exhibit enhanced neutralizing activity and converted 1A1-3-22 from a NiV-specific antibody into a broad-spectrum one. In addition, the affinity-matured 2A1-3-8 clones exhibit broad binding activity against F proteins spanning both the Henipavirus and Parahenipavirus genera. Our study not only demonstrates the potential of C-Nabs for clinical application against henipaviruses, but also provides critical epitope information for the future design and engineering of antibodies and the development of other antivirals. Currently, no approved vaccines or antiviral drugs are available against Nipah virus. Here, the authors identify two nanobodies targeting a conserved cavity on the Nipah virus F protein, show that 2A1-3-8-HLE provides complete protection in vivo, and further enhance nanobody activity and breadth through rational design.