Critical haemagglutinin residues shape antigenic diversity in H5 avian influenza viruses
Research led by The Pirbright Institute has identified specific mutations in the haemagglutinin (HA) protein of H5 avian influenza viruses that significantly influence how well poultry vaccines work, providing insights that could improve global vaccine design and disease control strategies.
Since emerging in 1996, H5 avian influenza virus lineage A/Goose/Guangdong/1/1996 (Gs/GD) has evolved into more than 30 genetically and antigenically distinct clades. Among these, clade 2.3.4.4b has become one of the most widespread and concerning, responsible for recent global outbreaks affecting domestic poultry, wild birds and an expanding range of mammalian hosts.
Whilst vaccination remains a key strategy for controlling avian influenza in poultry and reducing the risk of zoonotic transmission to humans, the virus evolves rapidly, often leading to mismatches between vaccine strains and circulating field viruses. These differences can weaken vaccine protection and enable the virus to escape immune responses.
Pirbright researchers, investigated the molecular drivers of these antigenic differences. Using reverse genetics, the team generated recombinant viruses representing multiple H5 clades and analysed their antigenic relationships using haemagglutination inhibition (HI) assays and antigenic cartography. These methods allowed the scientists to visualise how different viral strains relate to one another immunologically.
Published today in the Journal of Virology, their findings show viruses belonging to clade 2.3.4.4 are clearly distinct from other H5 lineages and exhibit substantial diversity even within the clade itself. By combining antigenic data with genetic comparisons, the researchers identified 48 candidate amino acid positions in the HA protein that may influence antigenic variation.
Lead author Professor Munir Iqbal, Head of the Avian Influenza and Newcastle Disease group at The Pirbright Institute, said: “Our analysis offers practical implications for improving vaccine seed strain selection and developing broader, more durable vaccines for poultry. Such advances could help reduce the economic impact of avian influenza outbreaks while lowering the risk of virus spillover into humans and other mammals.”
To test predictions, individual mutations were introduced into a candidate HA protein through site-directed mutagenesis. Subsequent antigenic testing showed that four mutation (R82K, A83T, T204I and F229Y) produced significant changes in antigenicity. Three of these mutations (R82K, T204I and F229Y) represent newly identified antigenic determinants.
Notably, several of these mutations have already appeared sporadically in recent outbreaks, including the ongoing H5N1 cattle epizootic in North America, and their emergence suggests they may play a role in shaping future viral evolution and vaccine effectiveness.
The study also shows clade 2.3.4.4 viruses are not only genetically distinct but also antigenically separated from other H5 clades, with significant variation even among closely related subclades. This diversity complicates vaccine strain selection and highlights the need for more precise antigenic matching when developing poultry vaccines.
In addition, the researchers identified numerous variations in potential glycosylation sites on the HA protein. Modifications can shield viral epitopes from antibody recognition and further contribute to immune escape.
By integrating phylogenetic analysis, antigenic cartography, structural modelling, and experimental validation, the study provides a framework for identifying the molecular determinants that drive antigenic drift in avian influenza viruses.
Researchers say the approach could also assist in monitoring emerging viral variants and predicting which mutations are most likely to affect vaccine performance.
Read the paper: Mapping Haemagglutinin Residues Driving Antigenic Diversity in H5Nx Avian Influenza Viruses.