We evaluated the immunogenicity and protective activity of plasmid DNA vaccines encoding the influenza virus NP gene (pNP) alone or in combination with the herpes simplex virus type 1 protein 22 gene (pVP22). Optimal immune responses were observed in BALB/c mice immunized with the combination of pVP22 plus pNP, as assessed by enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICCS). These mice also showed maximal resistance following challenge with the A/PR/8/34 (H1N1) and A/Udron/72 (H3N2) strains of influenza virus. The susceptibility of immunized mice to virus infection was significantly increased following depletion of either CD4(+) or CD8(+) T cells. These results indicate that a plasmid DNA vaccine encoding pVP22 plus NP induces a high level of cross-protective immunity against influenza virus subtypes.