The first set of H5 HA DNA vaccines was produced by cloning codon optimized HA genes based on the amino acid sequences of HA antigen from an H5N1 influenza isolate A/HongKong/156/ 97, which was responsible for the first outbreak of H5N1 avian influenza in humans in 1997, into a DNA vaccine vector [19] . The expression of HA antigens from three different H5-HK DNA vaccines was examined in transiently transfected 293T cells ( Fig. 2A) . Regardless of whether the natural HA leader or tPA leader was used, HA proteins expressed from two full length H5-HK DNA vaccine constructs (HA-HK.wt and HA-HK.tPA) were detected in cell lysate but not in the supernatant, suggesting they are mainly cell-associated. In contrast, truncation of the Cterminal segment, including the removal of the TM domain in the H5-HK.dTM DNA vaccine construct, was able to significantly increase the secretion of HA protein (detected in supernatant) ( Fig. 2A) . H5-HK HA proteins expressed by all of three HA DNA vaccine designs were able to be cleaved into HA1 and HA2 subunits ( Fig. 2A) .