Ferrets were immunized with HA alone, NP alone, M2 alone, NP+M2, HA+NP+M2, and control using gene-based vaccines delivered in DNA/rAd5 vectors. Since there are no established There was no statistical difference between the control group and groups immunized with NP, NP+M2, or M2. Both the HA and HA+NP+M2 groups showed 100% survival (right panel), whereas the vector control group showed 0% survival after the viral challenge. There was no statistically significant difference between the HA and HA+NP+M2 groups (p = 1.00), but there was a significant difference between these groups and the control (p = 0.008), by a log-rank test. (B) Body weights of the ferrets were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weight (left panel). Ferrets immunized with HA and HA+NP+M2 groups showed no weight loss, while the control group ferrets showed rapid weight loss (right panel). The survival and initial animal numbers in each group on the last day of body weight data collection are indicated next to the curve labels. The survival percentage for each group was analyzed statistically by a log-rank test. doi:10.1371/journal.pone.0009812.g005 M2(KAN-1) antigens. Each bar represents the group mean for the end-point titers of total IgG and IgM, determined in duplicate by series dilution of ELISA assay with the error bars indicating the standard deviation. ANOVA tests were significant for only the responses against HA at the first time point, and for all three antigens after the rAd5 boost. For HA and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.05 are indicated. * represents a p-value between 0.05 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. As expected, the HA alone group elicited significant anti-HA immunity that increased after rAd5 HA boost (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 (middle and right panels). (B) Sera from the NP, M2, NP+M2 or vector controls were collected 14 days after the third DNA immunization (white bars), and the sera from the same animals were also collected 14 days after the recombinant adenovirus boost (solid bars). ANOVA tests were not significant for any of the antigens at the first time point, and for NP and M2 after the rAd5 boost. For NP and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.008 are indicated. * represents a p-value between 0.008 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (p,0.0001) (middle panel). 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 (right panel). doi:10.1371/journal.pone.0009812.g004 assays available to measure cellular responses against HA, NP and M2 in ferrets, the efficacy of these immunogens was evaluated based on the measurement of neutralizing antibody titers through microneutralization and HA-inhibition assays. When HA and NP were present, significant humoral responses were stimulated against these proteins. However, M2 antibodies were only stimulated when M2 was in combination with NP, suggesting possible immune synergy of the two gene products. Similar adjuvant effects of NP in the anti-M2 response have recently been reported in mice [21] . Each group was challenged with a lethal strain of HPAI H5N1, and only vaccines containing HA conferred protection while NP, M2, and NP+M2 groups did not survive.