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 gene-based immunization with NP and M2 induce strong humoral and cellular responses, and protect against lethal H5N1 challenges 
. 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 ().
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 
, 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 
. 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 
. 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 
. 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 
Despite showing no neutralizing antibody responses, mice immunized with HA+NP+M2 were fully protected against lethal challenge. Lalor et al. showed a similar result in which a Vaxfectin-formulated DNA vaccine encoding H5+NP+M2 consensus genes protected mice against H5N1 challenge, despite low HAI responses. This is suggestive of other useful mechanisms of protection, such as cytotoxic T-lymphocytes, although this was not assessed in our study. A previous study with DNA/rAd5 immunization has also suggested that cellular immunity may contribute to protection in this model 
While the mouse model has been used for immunogenicity studies, the murine disease does not have the same pathogenicity as human infection and is not ideal for H5N1 infection studies. These differences may be due to species changes in HA receptor specificity and distribution, as well as differential immunopathogenicity 
. In contrast, it is generally accepted that ferrets exhibit pathology more similar to humans after H5N1 infection, including severe lethargy, fever, weight loss, transient lymphopenia, and viral replication in the upper/lower respiratory tracts and multiple systemic organs 
. Furthermore, human isolates of influenza virus have been shown to attach and infect ferret airways 
, indicating that humans and ferrets share similar HA receptor specificity 
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 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 
. 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.
Lalor et al. showed NP+M2 to be protective in mice, but only when formulated with Vaxfectin and in a vaccine dose 6.6 times greater than that used in this study. However, in ferrets, in the absence of HA, this combination only resulted in delayed illness and death 
. Price et al. showed that NP+M2 protects ferrets against H5N1 in a triple-prime rAd boost regimen similar to the one used here, but this difference in results may be due to the higher dose of DNA in the primary immunization (ten-fold greater than the present study) as well as a much lower viral challenge dose (5×LD50 
compared to 3×105
in this study). However, due to differences in experimental parameters such as the immunization regimen and assay standardization, direct comparisons of NP and M2 immune responses between studies are difficult. In addition, different vaccinations may alter various antibody and cellular immune responses which may affect the protective immunity in various animal models. Nonetheless, the evidence suggests while NP or NP+M2 may provide moderate levels of protection against low dose viral challenges in ferrets, they are insufficient against high challenge doses of HPAI. On the other hand, HA elicits effective immune protection even against very high HPAI viral challenge doses. Although vaccines encoding NP or M2 alone are not required for protection against H5N1, they could potentially augment HA-encoded vaccines, particularly when there is a mismatch between the vaccine and viral HA proteins. Studies in mice have shown that M2 antibodies may help to reduce viral replication 
, while studies in ferrets have shown M2 to be associated with reductions in viral recovery 
. However, based on our H5N1 challenge results in ferrets, HA DNA immunization is superior to NP and M2 DNA immunization in terms of protection. These highly conserved viral genes may require combinatorial vaccination with HA to be suitable candidates for universal influenza vaccines.