An optimized expression cassette containing the codonoptimized H5N1-HA was inserted by homologous recombination into a replication-incompetent PAV3 vector containing deletions in the E1/E3 genes (described in [23] ). An AdHu5-HA vaccine was also developed in parallel as a control to compare levels of protection and immune responses. Expression of the PAV3-HA and AdHu5-HA vaccines was evaluated in VRIBL E1, HEK 293, and mouse AB12 cells (Figure 1a ). BALB/c mice were also vaccinated with 10 10 virus particles (vp) and in vivo expression of PAV3-HA or AdHu5-HA in muscle tissue was detected 4 days post-immunization ( Figure 1b) . A robust antibody response is important for the prevention of influenza virus infection. The ability of the PAV3-HA vaccine to generate humoral immune responses following immunization was assayed through detection of HA-specific antibodies by hemagglutination inhibition (HI) or neutralizing antibody (NAB) titres. BALB/c mice were vaccinated with 10 10 vp/mouse of PAV3-HA or AdHu5-HA and the development of HI and NAB antibody responses was monitored from serum collected at days 8, 10, 14, and 21 post-immunization. All samples were treated with receptor-destroying enzyme (RDE), followed by complement inactivation the next day. An HI assay was performed using serial dilutions of the treated serum combined with H5N1-H05 virus and horse red blood cells to detect inhibition of cell agglutination by serum antibody. The reciprocal of the highest dilution which did not agglutinate red blood cells was scored as the HI antibody titre. HI titres of 2060, 120669, 213692, 5336184 were detected for PAV3-HA and 27611, 67623, 213692, 4266184 for AdHu5-HA, respectively, with no statistical difference between the two vaccines ( Figure 2a ). The presence of neutralizing antibodies to inhibit active H5N1-H05 infection was also evaluated as a measure of the humoral response. Serial dilutions of treated serum were incubated with 100 pfu/well of H5N1-H05 virus and cells were monitored for the presence or absence of cytopathic effects (CPE) using a light microscope. The highest serum dilution which did not exhibit CPE was scored as positive for neutralizing antibody and titres were reported as the reciprocal of the dilution. NAB titres of 060, 7612, 7612 and 90650 or 060, 7612, 10610 and 4060 reciprocal dilutions were observed with the PAV3-HA or AdHu5-HA vaccines at 8, 10, 14, and 21 days, respectively (Figure 2b ).