| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
1. Novel animal models of HCV infection in mice
We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically inoculated with an expression plasmid DNA encoding HCV structural proteins. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. The intrahepaticaly DNA-inoculated mice developed CD8+, MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells.
2. HCV vaccine
(1) Peptide vaccine : We assessed the capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTL) against hepatitis C virus (HCV). Mice immunized with CTL, CTL+ Th and CTL-Th peptides, but not Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic
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activity induced by immunization with CTL-Th conjugated peptide was much higher than that of CTL+Th and CTL peptide immunization alone. However, effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides.
(2) DNA vaccine : we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-α promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFcE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization.
(3) Recombinant vaccine : We assessed the capability of a novel recombinant vaccine the use of the attenuated tuberculosis bacillus, Bacillus Calmette-Guerin (BCG), as a vaccine vehicle to elicit HCV-specific CTL.BCG was engineered to express CTL epitope peptide of HCV-non structure protein 5a (NS5a) as a chimeric protein with alpha antigen of mycobacteria. Immunization with this recombinant BCG-HCV organism elicited major histocompatibility complex class I-restricted CD8+ HCV-NS5a-specific CTL in mice. These immunized mice showed a substantial reduction in vaccinia virus titer compared with mock-immunized control, when these immunized mice were challenged with recombinant vaccinia virus carrying the HCV-NS5a gene. Less
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