The full complementary DNA (cDNA) sequence from the A/ Hanoi/30408/2005 H5N1 hemagglutinin (H05-HA) gene was obtained, codon optimized, and synthesized from overlapping oligonucleotide primers, as previously described [36] . The H05-HA gene was first cloned into the pCAGa plasmid, containing a chicken-b-actin (CAG) promoter, to generate the pCAGa-HA construct. The pCAGa-HA expression cassette was then excised and inserted into two shuttle vector systems: pShuttle2 (Clontech) and pPAV227 (VIDO, University of Saskatchewan). Insertion of the expression cassette replaced the existing pShuttle2 cytomegalovirus (CMV) promoter with the CAG promoter, resulting in the pShuttle2-HA construct. Transfer plasmid pPAV227 was also modified to include the SV40 polyadenylation signal from pShuttle2, generating pPAV227-HA-PolyA.