At least nine weeks after the rAd5 boost, ferrets were challenged with a high dose of A/Vietnam/1203/2004 (H5N1) virus. All groups that lacked HA, including NP alone, NP+M2, and M2 alone, developed severe disease, manifested by significant weight loss and neurological dysfunction less than seven days after the viral challenge, similar to the clinical symptoms observed in the control group ( Figure 5A, left panel) . The animals were euthanized due to severity of symptoms. The body weight loss among these groups was very similar ( Figure 5B, left panel) . In contrast, the HA and HA+NP+M2 groups were completely protected from lethality after a high dose of influenza virus challenge compared to no survival in empty vector immunized controls ( Figure 5A, right panel) . Three animals showed very mild clinical signs such as slight temperature elevation or weight loss three days after the viral challenge, but these symptoms disappeared within two days. Body weight of the two groups remained steady post-viral challenge in HA-immunized ferrets ( Figure 5B , right panel), in contrast to the controls. Viral titers from the nasal washes confirmed the antiviral effects of HAbut not NP-containing vaccines (data not shown). Control animals showed peak viral titers at day 3, and subsequently died by day 6. Both HA and HA+NP+M2 groups showed moderate viral titers at day 3, followed by quick clearance with no viral titers detectable at day 5 and day 7 (data not shown). Survival, body weight, and viral titers are indistinguishable between the HA and HA+NP+M2 groups after viral challenge, suggesting that the NP and M2 did not contribute to immune protection in ferrets. HA immune responses alone were necessary and sufficient to protect ferrets from the lethal effects of infection under these challenge conditions.