Mapping hemagglutinin residues driving antigenic diversity in H5Nx avian influenza viruses

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 optimize vaccine seed selection against diverse and evolving H5 strains. IMPORTANCE: The 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 hemagglutinin (HA) residues driving antigenic variation across H5 clades. Using recombinant viruses, antigenic cartography, hemagglutination inhibition assays, and mutagenesis, we pinpointed 48 key residues, with four R82K, A83T, T204I, and F229Y having major antigenic effects, including three novel markers. 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.