Previous studies with NP immunization have yielded mixed results, likely dependent on the mode of gene-based vaccination and on the dose of challenge virus. Though the first studies using NP DNA immunization conferred protection against lethal influenza challenge [5, 48] , in retrospect, this result may be seen with relatively low challenge doses. In a more recent study, a challenge dose that overcame NP DNA vaccine protection was similar to the amount used here [2] . NP immunization with DNA/ rAd derived from H1N1 strain A/PR/8 resulted in protection in mice against challenge with a heterologous virus strain, H5N1 A/ HK/156, but no protection was seen against a more virulent strain, A/HK/483 [1] . It appears that protection afforded by immunization with NP in mice diminishes markedly as the dose and virulence of the challenge virus increase. However, DNA vaccination with NP in combination with M2 has been shown to protect mice in both Vaxfectin formulations and rAd-boost regimens [7, 20, 21] . While we did not investigate M2 alone in mice, a previous study has shown that vaccination with M2 alone is capable of protecting mice against heterologous strains of influenza virus challenge, including H5N1 [9] .