Secretory Hemagglutinin Ectodomains Trimerized by Interprotomer Disulfide Bonds Provide Strong Cross-protection Against Influenza A and B Viruses

Abstract

Abstract The stable secretory expression of HA ectodomain would in principle enhance the yield of influenza vaccine. However, without the transmembrane domain, the folded conformation of the ectodomain polypeptide would most likely deviate from its native form, which poses a risk of antigenicity impairment. Here we comprehensively describe all putative patterns of interprotomer disulfide bridges in HA using computer simulation, among which some pairs of cysteine insets forcefully drive the formation of homotrimers with strong antigenicity to a panel of anti-stem cross-reactive antibodies. Negative-stain 2-D class averaging showed the vast majority of the NC99#2 H1 molecules as homogeneous trimers, while the unmodified H1 protomers were irregularly aggregated. Immunizations of mice with the stabilized HA improved cross-protection by the robust elicitation of serum cross-neutralization instead of hemagglutination inhibition activities. Our study bears significant practical implications for developing secretory HA-based influenza vaccines.