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

Abstract

Seasonal influenza vaccines have been developed for more than half a century and are considered the most cost-effective approach to prevent influenza and associated severe outcomes. Nevertheless, the vaccine effectiveness ranges from 40% to 60% when the vaccine virus matches the circulating viruses well, and it varies between seasons. The lowest vaccine effectiveness can often be seen in protection against the human H3N2 virus. This can be partially attributed to the presence of egg-adaptive mutations in egg-based vaccines, which remain the predominant type of vaccine formulation compared with cell-based vaccines and recombinant hemagglutinin, the major antigen of the viral envelope. Egg-adaptive mutations facilitate binding to α2,3-linked sialylated glycan receptors in eggs via the receptor-binding site of hemagglutinin, which overlaps with several antigenic sites under higher selection pressure, for higher viral yield. Thus, the occurrence of egg-adaptive mutations can potentially alter immunogenicity and result in the reduction of vaccine effectiveness. Identifying factors that influence the preference of egg-adaptive mutations can help optimize the effectiveness of egg-based influenza vaccine. During the past evolution of human H3N2 viruses, antigenic drift was found to be accompanied by changes in 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-adaptive mutations in egg-based seasonal H3N2 vaccines, this study aims to investigate the mechanism behind egg adaptation based on the natural evolution of hemagglutinin in recent human H3N2 viruses. In this study, it was found that recently emerging egg-adaptive mutations contributed to 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-associated and constrained by natural variants on hemagglutinin of human H3N2 virus, making it predictable when selecting the 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 in receptor binding mode in more recent human H3N2 virus. The coevolution was mediated by the epistatic interaction between them, which coordinates to maintain functional receptor binding, restrict the preference of egg-adaptive mutations, and possibly contribute to major antigenic changes. Neutralization assays performed on plasma from humans indicated potential antigenic mismatch, as evidenced by the overall low capacity in neutralizing recent human H3N2 viruses. Such mismatches also involved the two coevolving natural variants identified to cause major structural changes. Altogether, this study reveals that the preference of egg-adaptive 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 in receptor binding mode, to determine the preference of egg-adaptive mutations, and possibly contribute to immune escape. These findings highlight the importance of intensive surveillance of natural mutations for selecting better vaccine strains.