For either H5-HK or H5-VN HA DNA vaccines, three versions of codon optimized HA gene inserts were cloned into DNA vaccine vector pSW3891 [43] . For the first version, the full length H5-HK or H5-VN HA gene insert (568 aa,) with their natural HA leader sequences subcloned, individually, into the pSW3891 vector at the PstI and BamHI sites, designated as H5-HK.wt or H5-VN.wt DNA vaccine constructs. For the second version, the HA natural leader sequence (the first 15 aa at N-terminus for both H5-HK and H5-VN) was replaced by a human tissue plasminogen activator (tPA) leader sequence. The H5-HK and H5-VN HA gene inserts coding for aa 16-568 were PCR amplified from the full length codon optimized H5 HA genes using the following primers: H5-HA-opt-1 (gtcgctccgctagc GACCAGATCTGCATC-GGCTAC) and H5-HA-opt-2 (agtcacggatcc TCAGATGCA-GATCCGGCACTG). The individual H5-HK or H5-VN HA gene was cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.tPA or H5-VN.tPA DNA vaccine constructs. For the third version, the HA natural leader sequence was replaced by a tPA leader sequence and the transmembrane (TM) and cytoplasmic region (37 aa at the C-terminus) of H5 and HA was deleted for both H5-HK and H5-VN. The truncated H5-HK or H5-VN HA genes were PCR amplified from the full length codon optimized H5-HK or H5-VN HA gene using primer pairs: H5-HA-opt-1 and H5-HA-opt-4 (agtcac ggatccTCACTGGTAG-GTGCCCATGCTCTC), or H5-HA-opt-1 and H5-HA-opt-8 (agtcacggatccTCACTGGTAGATGCCGATGCTTTC), respectively. The truncated H5-HK or H5-VN gene insert was individually cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.dTM or H5-VN.dTM.