| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
Aim: To speculate a tri-dimensional structure of envelope protein of hepatitis C virus (HCV) and develop a more effective vaccine against HCV.
Method: We have already reported that a minimal region of immunogenic HCV envelope protein was amino acid (aa) 406-644. We constructed 10 kinds of mutant expression vectors that were designed to delete glycosilation possibility in this region, and analyze its antigenicity using sera from patients with chronic hepatitis C. Secondly, We constructed DNA vaccine including this region (1) with cytomegalovirus promoter, or (2) hepatitis B virus surface (HBs) chimeric antigen, and (3) otherwise recombinant HBs protein vaccine. Six mice in each group were immunized by these vaccines and their sera were taken every three weeks after immunization. The antibody titers and their isotype of their sera were analyzed by their specific ELISA system.
Results: Among the ten mutants, the mutations in aa 476, 532, 540 has no influence to the antigenicity. That of aa
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623 modified a little, and those of aa 417, 556 modified rather strongly the reactivity. The mutations at aa 423, 430, 448, 576 completely block the immune reactivity. These data suggested that the aa positions of 406, 423, 430 ,448, 576, 644 were localized at outer site of this protein, and others were not. This is helpful to image a tri-dimensional structure of envelope protein of HCV. In the second experiment, the antibody titers of both DNA vaccines increased until 12 weeks after immunization, and maintain high titers for a long time. Their isotype was IgG2a, suggesting induction of cytotoxic immune reaction. The first vaccine induced a stronger reaction than the second one, indicating that HBs could not to act as an adjuvant in this protocol. The third vaccine induced relatively low titer of antibody with peak of the titer at 9 weeks after injection. The titer decreased after then quickly, suggesting poor immunogenicity of protein vaccine.
Conclusions: (1) We could imagine a tri-dimensional structure of envelope protein of HCV through the immune reactivity between mutant HCV proteins and patients antibody. (2) DNA vaccine is a superior immunogenic vaccine to recombinant protein vaccine. We could not find an adjuvant eftect of HBs antigen in DNA vaccine system. Less
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