Supporting data for "Egg-adaptive mutations and antigenicity shaped by natural variants on hemagglutinin: improving the design of seasonal influenza (H3N2) vaccine"

Seasonal influenza vaccines have been developed for more than half a century and considered to be the most cost-effective approach to prevent influenza or associated severe outcomes. Nevertheless, the vaccine effectiveness ranges from only 40% to 60% when vaccine virus matches the circulating viruses well and varies between seasons. The lowest vaccine effectiveness can often be seen in protection against human H3N2 virus. This can be partially due to egg-adaptive mutations in egg-based vaccines, which remain the prevalent type of vaccine formulation compared to cell-based and recombinant hemagglutinin, the major antigen of virus envelope. Egg-adaptive mutations facilitate the binding to α2,3-linked sialylated glycan receptors in eggs, via the receptor-bindings site of hemagglutinin that overlaps with several antigenic sites under higher selection pressure, for higher viral yield. Thus, the occurrence of egg-adaptive mutations can potentially alter the immunogenicity and result in the reduction of vaccine effectiveness. Identifying factors that influence the preference of egg-adaptive mutation can help optimize the effectiveness of egg-based influenza vaccine.

During the past evolution of human H3N2 viruses, antigenic drifts were found to be accompanied by changes of hemagglutinin structure and receptor binding mode influenced by several natural mutations adjacent to the receptor-binding site. These changes could also subsequently influence the fitness of adaptive mutations. As little is known about the antigenic effects and rules of selecting egg-adaptative mutations in egg-based human H3N2 vaccines, this study aims to investigate the mechanism behind egg-adaptation based on the natural evolution of hemagglutinin in recent human H3N2 virus.

In this study, I found that recently emerged egg-adaptive mutations contributed to an altered immunogenicity and antigenicity of human H3N2 vaccine in a mouse model. Using deep mutational scanning and mutagenesis experiments, the preference of egg-adaptive mutations was found to be strain or clade-dependent and constrained by natural variants on hemagglutinin of human H3N2 virus, making it predictable when selecting vaccine virus. However, the preference could change with the evolution of hemagglutinin in circulating influenza viruses. Using X-ray crystallography and glycan array, two coevolving natural mutations on hemagglutinin were further identified to cause major changes of receptor binding mode in more recent human H3N2 virus. The coevolution was mediated by the epistatic interaction between them, which coordinates to maintain the functional receptor binding, restrict the preference of egg-adaptive mutations, and possibly contribute to major antigenic changes. By neutralization assay on plasma from human, potential antigenic mismatch was indicated by the overall low capacity in neutralizing recent human H3N2 viruses. Such mismatch also involved the coevolving natural variants identified above.

Altogether, this study reveals that preference of egg-adaptative mutations is constrained by natural variants on hemagglutinin and changes as these natural variants evolve. At least two natural variants coevolve to cause major changes of receptor binding mode, determine the preference of egg-adaptive mutations, and possibly immune escape. These highlight the important of intensive surveillance on natural mutations for selecting better vaccine strains.