title: Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities.
abstract: 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.
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan; Faculty of Medicine, University of Indonesia, Jakarta, Indonesia.
authors.forename: Suratno Lulut
authors.initials: SL
authors.lastname: Ratnoglik
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Da-Peng
authors.initials: DP
authors.lastname: Jiang
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan; JST/JICA SATREPS Laboratory of Kobe University, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia.
authors.forename: Chie
authors.initials: C
authors.lastname: Aoki
authors.affiliation: Faculty of Medicine, University of Indonesia, Jakarta, Indonesia.
authors.forename: Pratiwi
authors.initials: P
authors.lastname: Sudarmono
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Shoji
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Lin
authors.initials: L
authors.lastname: Deng
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Hak
authors.initials: H
authors.lastname: Hotta
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D016679:Genome, Viral; D006526:Hepatitis C / Q000276:immunology / Q000517:prevention & control*; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis; D008297:Male; D051379:Mice; D009154:Mutation; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D019444:Vaccines, DNA / Q000276:immunology*; D014761:Viral Hepatitis Vaccines / Q000276:immunology*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D007371:Interferon-gamma; D019444:Vaccines, DNA; D014761:Viral Hepatitis Vaccines; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases