The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.