In order to rule out that the above finding was not only unique to this H5N1 virus isolate from Hong Kong in 1997, similar designs of HA inserts were produced by using a codon optimized HA DNA gene from the H5N1 strain A/VietNam/1203/04, a well-studied representative isolate for the H5N1 viruses [20, 21] . The pattern of H5-VN HA expression was similar to that of H5-HK HA antigens. Only cell-associated HA antigens were detected with H5-VN.wt and H5-VN.tPA constructs in contrast to that identified with the H5-VN.dTM, which had HA antigen expression in both cell lysate and supernatant fractions (Fig. 3A) . Rabbits were immunized with the electroporation method as previously reported [22] . Similar to H5-HK DNA plasmids, binding antibody responses, as measured by ELISA, showed similar levels among sera elicited by the three H5-VN DNA vaccines with different HA gene insert designs (Fig. 3B) . However, functional antibodies, as measured by HI and MN antibody analyses, revealed again that the H5-VN.tPA design induced the highest levels of functional antibody responses ( Fig. 3C and 3D ). In the case of functional antibody responses against the wild type virus A/VietNam/1203/04, the differences between H5-VN.tPA and the other two forms were statistically significant by both HI and MN assays (p,0.05 or p,0.01).