Mapping Haemagglutinin Residues Driving Antigenic Diversity in H5Nx Avian Influenza Viruses

Since its emergence in 1996, the H5 avian influenza virus (AIV) A/Goose/Guangdong/1/1996 (Gs/GD) haemagglutinin (HA) has evolved into over 30 genetically and antigenically distinct clades, including the widespread clade 2.3.4.4b. Vaccination is widely used in endemic regions to reduce poultry losses and zoonotic risk. However, the evolving antigenic diversity and global co-circulation of multiple clades challenges protective efficacy of poultry vaccines with poor antigenic matching to field strains, resulting in immune escape and vaccine failure. This study aimed to improve vaccine seed selection by identifying HA epitopes contributing to inter-clade antigenic differences. Recombinant clade-representative viruses were generated using HA genes from circulating H5 AIVs via reverse genetics with A/Puerto Rico/8/1934 (PR8) internal and neuraminidase genes. Antigenic relationships were assessed using haemagglutination inhibition (HI) assays with homologous and heterologous chicken antisera. Antigenic cartography revealed a clear distinction of clade 2.3.4.4 from others and notable intra-clade diversity. Pairwise antigenic and genetic comparisons identified 48 putative antigenic residues. These were individually introduced into a candidate HA by site-directed mutagenesis, and antigenic influence assessed by HI using sera raised against the non-mutated HA. Four residues R82K, A83T, T204I, and F229Y had significant antigenic effects, with three (R82K, T204I, F229Y) being novel. These findings demonstrate that combining serology and in silico residue analysis can identify key antigenic determinants. This work highlights the need for precise antigenic matching in vaccine design and highlights the value of combining molecular and immunological tools to optimise vaccine seed selection against diverse and evolving H5 AIV strains. ImportanceThe continued evolution of H5 avian influenza viruses (AIVs), particularly the Gs/GD lineage, poses major challenges for poultry disease control and zoonotic risk mitigation. Vaccine effectiveness is undermined by antigenic drift and the co-circulation of diverse clades, often leading to mismatches between vaccine and field strains. This study addresses the critical need to improve vaccine strain selection by identifying haemagglutinin (HA) residues driving antigenic variation across H5 clades. Using recombinant viruses, antigenic cartography, haemagglutination inhibition (HI) assays, and mutagenesis, 48 putative antigenic residues were identified, with four R82K, A83T, T204I, and F229Y having major antigenic effects, three of which were novel. These findings advance our understanding of H5 antigenic evolution and provide a framework for predicting vaccine performance. By integrating molecular and serological data, our work informs rational vaccine seed strain selection, contributing to more broadly protective vaccines and improved control of H5 AIV in poultry, while reducing the risk of zoonotic transmission.