The highly conserved viral genes NP and M2 have become a focus for the development of broad, cross-protective or ''universal'' influenza vaccines. In mice, several studies have shown that genebased immunization with NP and M2 induce strong humoral and cellular responses, and protect against lethal H5N1 challenges [1, 4, 6, 9, 22, 35, [46] [47] [48] . In this study, mice were immunized with DNA vaccines encoding HA alone, NP alone, HA+NP, and HA+NP+M2. All HA-containing groups and the M2-containing group generated significant antibody responses against HA and M2 proteins, respectively. However, only the HA+NP group elicited a response against NP protein, and that was marginally significant at best. Moreover, while all HA-containing groups were protected against lethal H5N1 challenge with a survival rate of at least 70%, immunization with NP alone did not protect mice from lethal H5N1 challenge (Figure 3) . Neutralization was determined by lentiviral inhibition assay, hemagglutinin inhibition assay, and microneutralization assay. Sera from the indicated mouse and ferret immunizations with the indicated viral antigens by DNA alone or DNA/rAd5 before the viral challenge were evaluated by pseudotyped lentiviral inhibition, hemagglutinin inhibition (HAI), and microneutralization assays (MN). UD represents serum samples with undetectable neutralization activities even at the lowest dilutions, while NA represents samples that were not available, and therefore not assessed. In both mice and ferrets, only HA-containing groups stimulated strong humoral responses. doi:10.1371/journal.pone.0009812.t001