Screening and Characterization of Nanobody Against the Emerging Dairy Cattle H5N1 Influenza Virus

Background: The ongoing spillover of highly pathogenic avian influenza H5N1 virus into mammalian species, including recent outbreaks in dairy cattle, underscores an urgent need for broadly effective therapeutics. Nanobody, the variable domain of heavy chain-only antibody, offers unique advantages for viral neutralization due to its small size, stability, and accessibility to cryptic epitopes. Methods: In this study, we isolated a human nanobody, nAb35, targeting the hemagglutinin of the dairy cattle H5N1 virus A/Texas/37/2024 (clade 2.3.4.4b) via phage display of a human nanobody library. After three rounds of panning against the recombinant H5 ectodomain, a dominant clone (nAb35) was highly enriched. Then nAb35 was fused with a human IgG1 Fc fragment and expressed as a bivalent dimer. Results: Biochemical characterization demonstrated that nAb35 specifically binds to H5 under both denaturing and native conditions and recognizes a linear epitope accessible on H5 expressed on the cell surface. In vitro neutralization assays using H5 pseudovirus showed that nAb35 inhibited viral entry with a half-maximal effective concentration of approximately 10 μg/mL, as determined by both luciferase reporter and immunofluorescence assays. Conclusion: These findings identify nAb35 as a promising candidate for further therapeutic development against emerging H5N1 virus.