To determine the comparative efficacy of alternative gene-based vaccines in ferrets, we immunized ferrets with these gene products in different combinations in a triple DNA inmmunization. In this experiment, animals received a recombinant adenovirus serotype 5 (rAd5) boost in order to increase the immunogenicity of DNA priming. Ferrets were immunized with HA alone (n = 4); HA+NP+M2 (n = 4); NP alone (n = 4); NP+M2 (n = 5); M2 alone (n = 4); or empty vector controls (n = 4+5). ELISA titers to HA, NP and M2 were determined after DNA immunization (Figure 4 , white bars) and after the rAd5 boost (Figure 4 , black bars). As expected, after DNA immunization, the HA alone group elicited Each immunized group was compared to controls as well as other groups containing relevant immunogens. ANOVA tests were significant for the responses against HA and M2, but not against NP. For HA and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.05/6 = 0.0083 (Bonferroni Correction) were considered significant for these pairwise comparisons. A single asterisk (*) represents a p-value between 0.008 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. All HA-containing groups showed significant antibody responses against HA compared to controls (p,0.0013), but did not differ significantly among themselves. Differences between NP-immunized groups were at the border of statistical significance by ANOVA (p = 0.0504), as was the comparison between HA+NP and HA groups (p = 0.008) when adjusted for multiple comparisons. The only M2-containing group, HA+NP+M2, elicited a significant humoral response against M2 protein compared to control (p,0.0001). doi:10.1371/journal.pone.0009812.g002 significant anti-HA immunity that increased after rAd5 HA boost ( Figure 4A , left panel) compared to controls (p,0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (p = 0.0006) and anti-M2 responses (p,0.0001) after the rAd boost ( Figure 4A , middle and right panels) when compared to controls. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (p,0.0001) ( Figure 4B , middle panel) compared to controls. Significant anti-M2 humoral responses were detected in animals immunized with NP+M2 post-rAd boost (p = 0.0005), but not in the M2 alone group ( Figure 4B , right panel) relative to controls. Anti-M2 humoral responses were not detectable in most cases, except after DNA/rAd5 immunization with M2 in combination with NP.