Identification and characterization of a broadly neutralizing and protective nanobody against the HA1 domain of H5 avian influenza virus hemagglutinin

The highly pathogenic avian influenza viruses (HPAIVs) of subtype H5, particularly those of the currently circulating clades 2.3.2.1 and 2.3.4.4, are largely responsible for the sporadic human infections that frequently present with a high case fatality rate. Consequently, there is an urgent necessity for the development of advanced antiviral therapeutic options against the H5 HPAIVs. Herein, the yeast two-hybrid system was employed for identifying seven nanobodies that bind the HA1 domain of hemagglutinin (HA). Among these nanobodies, Nb10 was found to exhibit high-affinity and broad-spectrum neutralization capacity against viruses of clades 2.3.2.1 and 2.3.4.4 under both <i>in vitro</i> and <i>in vivo</i> conditions. Surprisingly, Nb10 exhibited excellent efficacy against the recombinant viruses Re6/PR8, Re8/PR8, Re10/PR8, Re11/PR8, and Re14/PR8 of the subtype H5, with average half-maximal inhibitory concentrations ranging from 0.01 to 0.42 µg/mL in a microneutralization assay. Furthermore, the intratracheal administration of Nb10 resulted in remarkable prophylactic and therapeutic efficacy in mice. The findings herein reveal that the virus-neutralizing effect of Nb10 is achieved by obstructing viral entrance into host cells. Moreover, Western blot analysis and enzyme-linked immunosorbent assay revealed that Nb10 recognizes a conformational epitope located in the region spanning amino acid residues 50-271 of the protein HA1 displayed on the surface of yeast cells. The predicted structure of the binding pocket indicates that Nb10 recognizes the highly conserved receptor-binding site of HA1. Taken together, the current study offers valuable insights for the development of protective therapeutics with broad-spectrum activity and the design of broadly protective influenza vaccines.IMPORTANCEHPAIVs of subtype H5 have raised substantial public health concerns regarding the potential for viral adaptation and sustained human-to-human transmission. The prevention and treatment of the disease are replete with numerous challenges due to frequent antigenic alterations in the virus. Nanobodies have significant potential for clinical applications and therapies owing to their small size and robust tissue-penetrating capabilities. Herein, we describe the identification of Nb10, a broad-spectrum virus-neutralizing and protective nanobody that is effective against the currently circulating H5 HPAIVs of clades 2.3.2.1 and 2.3.4.4. The intratracheal administration of Nb10 afforded significant protection in mice infected with the H5 virus. This result provides novel insights for the rational design of antiviral pharmaceuticals. Furthermore, an analysis of the binding site of the target protein HA1 may be useful for the development of more effective vaccinations against influenza viruses of the subtype H5.