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

et al. (2014) 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. PLoS ONE 9(6): e98877. doi:10.1371/journal.pone.0098877 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 Hak Hotta 0 Suratno Lulut Ratnoglik 0 Da-Peng Jiang 0 Chie Aoki 0 Pratiwi Sudarmono 0 Ikuo Shoji 0 Lin Deng 0 Golo Ahlenstiel, University of Sydney, Australia 0 1 Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan, 2 JST/JICA SATREPS Laboratory of Kobe University, Faculty of Medicine, University of Indonesia , Jakarta , Indonesia , 3 Faculty of Medicine, University of Indonesia , Jakarta , Indonesia 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-c 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. - Funding: This study was supported in part by a SATREPS Grant from Japan Science and Technology Agency (JST) and Japan International Cooperation Agency (JICA), a grant from the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare, Japan. This study was also carried out as part of the Global Center of Excellence (G-COE) Program at Kobe University Graduate School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Hepatitis C virus (HCV) is an enveloped RNA virus that belongs to the genus Hepacivirus of the family Flaviviridae. The viral genome encodes a single polyprotein of about 3,000 amino acids, which is cleaved by host and viral proteases to generate at least 10 viral proteins, i.e., envelope 1 (E1) and E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A and NS5B. NS3 is a multi-functional protein with a serine protease domain located in the N-terminal one-third and a nucleoside triphosphatase (NTPase)/RNA helicase domain located in the C terminal twothirds, which are involved in the proteolytic processing of the viral polyprotein and viral RNA replication, respectively [1,2,3]. HCV is a major cause of chronic liver disease, such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. It is estimated that 180 million people are currently infected with HCV worldwide, and that ca. 70% of them become chronically infected [4,5]. The recent approval of NS3 serine protease inhibitors for treatment of HCV genotype 1 infection was a great progress in HCV antiviral development, and combination of a protease inhibitor with interferon (IFN) and ribavirin has increased sustained virological response (SVR) in patients [6]. On the contrary, great success has not been achieved in HCV vaccine development; no effective HCV vaccine is available so far, either for a prophylactic or a therapeutic purpose. While prophylactic HCV vaccines must have capacity to induce protective levels of neutralizing antibodies directed principally to the viral protein E2, effective therapeutic HCV vaccines must elicit strong cell-mediated immune responses against a wide variety of CD4+ and CD8+ epitopes of the viral origin. NS3 is known to carry a variety of CD4+ and CD8+ T cell epitopes to induce strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection [7,8,9,10,11]. Also, the HCV core protein is known to carry a variety of CD4+ and CD8+ epitopes [7,8,9,12,13,14]. From the antigenic point of view, therefore, NS3 and the core protein would be attractive candidates to be used for therapeutic vaccines that elicit T cellmediated immune responses against HCV. Another important aspect to be assessed carefully in vaccine development is a potential risk(s) of the vaccine-derived peptides/ proteins of the viral origin, which might impair or deregulate the normal functions of the host cells. For example, the HCV core protein is known to exhibit oncogenic properties in cell culture systems and transgenic mouse models [15,16,17]. The NS3 serine protease cleaves the mitochondrial antiviral signaling protein MAVS (also referred to as IPS-1, VISA and Cardif) to blockade the RIG-I- and TLR3/TRIF-mediated signaling for the induction of IFN-b production [3,18,19,20,21]. Also, NS3 inactivates T cell protein tyrosine phosphatase and modulates epithelial growth factor (EGF) signaling [22]. Moreover, the NS3 NTPase/RNA helicase, which is principally required for HCV RNA replication [1,2], may concomitantly deregulate cellular RNA helicasemediated functions, such as DNA replication, RNA transcription, splicing, RNA transport, ribosome biogenesis, mRNA translation, RNA storage and decay [3,23,24,25]. These observations imply the possible involvement of NS3 in the development of hepatocellular carcinoma. Therefore, a vaccine expressing the functionally active core protein or NS3 may be disadvantageous to the vaccinees. To avoid those potential (...truncated)