Therefore, it is very likely that HA antigen expressed with the tPA leader may be more effective in eliciting conformational antibodies. This hypothesis is supported by two pieces of evidence. First, there was no difference in the levels of binding antibodies as measured by ELISA, which indicated that there was no difference in the general immunogenicity between WT and tPA leader designs; only a difference in the functional antibody was observed. Second, the HA.dTM DNA vaccine design also used the tPA leader but did not have better functional antibodies, proving that the proper folding of the HA antigen in the presence of a tPA leader is important and is dependent on the presence of an intact HA2 domain. It is possible that such an HA antigen conformation is part of a trimer structure of HA since the HA2 domain is involved in the formation of HA trimers. Only antibodies against the trimer structure are more functionally relevant to block the trimer form of HA spikes on viral particles. By using the optimal H5.tPA HA insert design, studies in this report further demonstrated that there are good levels of cross protection by one H5 HA DNA vaccine against multiple H5N1 viruses from different clades. It is well documented that cross protection among H5N1 viruses can be detected [33, 34, 35] . Clade 1 H5N1 vaccines (VN) cross protect against both clade 1 and clade 2 (Indonesia) viruses in ferrets with the use of a strong adjuvant [36] . Using live attenuated cold adapted (ca) viruses expressing HA and NA from 1997, cross protection was observed against the late H5 virus from 1997 to 2005 [37] and ferrets [37] and ferrets [38] and mice [39, 40] and mice [39, 40] .