Effective therapeutic
vaccines against
virus infection must induce sufficient levels of cell-mediated immune responses against the target viral
epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural
protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4(+) and CD8(+)
T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV
infection. On the other hand, NS3 possesses
serine protease and
nucleoside triphosphatase (
NTPase)/
RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of
DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the
serine protease (H57A, D81A and S139A) and the
NTPase/
RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the
DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both
serine protease and
NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by
interferon-γ production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both
serine protease and
NTPase/
RNA helicase activities, would be good candidates for safe and efficient therapeutic
DNA vaccines against HCV
infection.