Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA vaccines

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of RA-expressing DNA vaccines by using codon-optimized HA sequences for either an III serotype (A/NewCaV20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal RA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated RA, were produced. Both forms of RA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of RA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 RA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher III and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of III and NAb responses. These data indicate that III and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of RA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.