Results included in the current report indicated that the tPA leader sequence and the C-terminal transmembrane domain/ cytoplasmic region of H5 HA both contribute to better functional antibody responses in H5.tPA DNA vaccines when compared to H5.wt and H5.dTM DNA vaccines. The above findings were different from our previous results on protective antibody responses induced by differently designed flu H1 and H3 HA DNA vaccines [11] . In this previous study, only the full length H1.wt but not transmembrane truncated H1.dTM induced high level HI and MN responses against H1 virus while both the full length H3.wt and truncated H3.dTM induced similar HI and MN responses against H3 virus [11] . These results provide a strong indication that the HA antigens from different influenza A subtypes (H1, H3 and H5) may have different preferences for antigen structure designs in order to generate optimal protective antibody responses.