This program is extended from our continuing efforts to investigate the immune response to the hepatitis C virus (HCV) in both humans and experimental animals. This long-term project can be globally subdivided into four phases. The initial phase is design and construction of immunogenic plasmids. This includes monitoring the development and course of HCV cellular and humoral immunity and selecting the most effective gene delivery systems. The second phase is to determine the immunogenicty of these constructs. During this step, we are evaluating a variety of gene delivery strategies and the use of adjuvants and other immune modifiers. The third phase is to evaluate the immune responses and the final phase is to conduct protectivity studies in chimpanzees. [unreadable] [unreadable] Great efforts have been devoted to evaluate HCV immune response including assay development for measuring cell mediated immunity in mice, chimpanzees and humans. The protectivity study would have to be conducted in an appropriate animal model. Each of these phases has many different components and the development, implementation, evaluation and improvement steps could be over-lapping. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocyte responses, may not only help in preventing initial HCV infection, but may serve as a mechanism for immune modulation to overcome existing infection. Using the mouse model, we were able to demonstrate the induction of both humoral and cell-mediated immune responses, including CTL activities, to several different plasmid constructs containing both HCV structural and non-structural genes. In the past years, we have tested the genetic sequences of many HCV-related immunogens to establish the best candidate DNA vaccine. We have also studied methods of vaccine delivery and immunity augmentation procedures; accumulated extensive experience in measuring humoral and cell-mediated immunity; and developed effective immunization strategies in small experimental animals. During last period, we have conducted studies combining several immunogens to evaluate their interaction or interference. We also developed a protein boost strategy. After recognizing the induction of potent cellular and humoral responses in small animals using our DNA prime-protein boost procedures and formulations, we initiated a prototype multicomponent (core, envelope, NS3, and NS5b) HCV vaccine trial in chimpanzee animal model in FY 2004. [unreadable] [unreadable] The chimpanzee experiment was just completed on August, 2007. The available results indicate that our immunization strategy is capable of inducing vigorous HCV specific CD4+ and CD8+ T-cell immunity, as well as antibody activities in chimpanzee. After challenging two immunized chimpanzees with homologous HCV virus, both animals became infected. However, one animal cleared the infection nine weeks later and remained negative for 18 months. This chimpanzee was subsequently challenged again twice with HCV virus, the animal appears to have sterilizing immunity against homologous virus. The chimpanzee was eventually challenged with heterologous HCV genotype 2b virus, this animal developed only low viremia (< 400 copies/ml) for three weeks then cleared the virus. The other chimpanzee is shown to be chronically infected for more than a year with consistently positive viral load. This chronic chimpanzee has been undertaken immune therapy through various immunization regimens for augmenting the immunity to overcome the infection. [unreadable] [unreadable] To better characterize the immune responses and assess the potential neutralizing antibody activity from two immunized chimpanzees, we started to set up HCV genotype 1a in vitro culture system with the help from Dr. Stanly Lemon. We have established a stable cell line that can continuously produce infectious virus for in vitro assay use. The determination of neutralizing antibody activity for the immunized animals is still in progress. Using HCV in vitro culture system, we are also looking into the possibility of augmenting genes involving in innate immunity signaling pathways, which may be crucial in host defense against HCV infection. Several genes are currently lined up to be tested to see whether enhancement of gene expression can eradicate the virus from a HCV persistently infected cell line.[unreadable] .