Glycosylation profiling of Influenza A Virus H3 HA

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. Despite significant progress, challenges persist in identifying conserved epitopes capable of inducing broadly protective immune responses, understanding the correlates of protection against influenza infection, and ensuring vaccine safety and efficacy across diverse populations. In this study, we explore the influence of glycan evolution on H3 HA from 1968 to the present. Our in-depth analysis unveiled how glycan addition significantly and differentially impacted HA antigenicity and immunogenicity over time. We observe that adding glycans influenced the polyclonal immune response elicited post-immunization. We propose that the appearance of PNGS at N158 potentially impacted the binding of head-specific broadly neutralizing antibodies (bnAbs). We further observe that, while enzymatic de-glycosylation barely impacted HK/68 HA stability, it destabilized and unfolded in the hyperglycosylated head domain and the membrane-proximal region of the stem domain in Sing/16. These insights expand our understanding of glycans beyond their conventional role in protein folding and as immune evasion tactics and highlight the intricate interplay among glycan integration, viral fitness, and immune recognition. Our findings have implications for vaccine design and manufacturing processes, offering valuable insights for developing more effective influenza vaccines. Moreover, they contribute to ongoing efforts to refine influenza research strategies, emphasizing the potential of stem-directed antibodies in creating more effective and universally applicable influenza vaccines.