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.