In order to ensure that the difference in protective antibody responses between sera elicited by H5-VN.wt and H5-VN.tPA was not the result of repeated immunizations, sera collected after one or three immunizations were also measured (Fig. 4) . Pseudotyped viruses expressing VN HA antigen were used to measure the neutralizing antibody activities in rabbit sera with less immunizations. The strength of protective antibodies was measured at two levels: inhibition concentrations that can block either 50% (IC50) or 90% (IC90) of virus infection to target cells. Both measurements showed that H5-VN.tPA-elicited rabbit sera had significantly higher titers of neutralizing antibody activities than the wild type H5 HA design, especially when using the more stringent IC90 as a cut-off (p,0.05 or p,0.01) (Fig. 4) . Sensitivity to deglycosylation treatment for HA antigens expressed by different forms of H5-VN HA DNA vaccines Additional studies were conducted to ask if glycosylation of HA has been affected with the use of a different leader sequence which may influence the immune responses. Our previous study with a hepatitis B surface antigen suggested that post-translational modifications including glycosylation may affect the immunogenicity of DNA vaccine delivered antigens [23] . The Asn (N)-linked glycosylation of influenza HA proteins are essential for virus infectivity and vaccine immunogenicity [24, 25] . Based on sequence analysis, there are 8 to 9 N-linked glycosylation sites (Asn-X-Ser/ Thr) in H5N1 HA proteins. We next investigated whether the HA antigens expressed by different designs of H5 HA inserts in the above DNA vaccinations have similar levels of N-linked glycosylation. The HA antigens expressed in 293T cell lysate transfected with the H5-VN.wt, H5-VN.tPA or H5-VN.dTM DNA vaccines and HA antigen expressed in the 293T cell supernatant transfected with the H5-VN.dTM DNA vaccine were analyzed for their susceptibility to PNGase treatment, which can cleave any type of Nlinked sugar including complex oligosaccharide structures resulting from the maturation of high mannose moieties during transport of the glycoprotein through the Golgi.