Unmasking stemspecific neutralizing epitopes by abolishing N-linked glycosylation sites of influenza virus hemagglutinin proteins for vaccine design

Influenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.