The quality and strength of the HCV-specific cellular immune response contributes tot he outcome of primary infection. In addition, we have now shown in the chimpanzee model that the vigor and quality of the memory T cell response after spontaneous recovery protects upon reexposure to the virus and prevents persistent infection. Specifically, vigorous proliferation of IFN-g-producing, circulating CD4+ T cells was followed by an increased frequency and a phenotypic and functional change of the tetramer+ CD8+ T-cell population from CCR7+ central memory to CCR7-, IFN-g-producing effector memory cells. At the same time, upregulation of IFN-g-mRNA was detectable in the liver suggesting that IFN-g-production by HCV-specific T cells contributed to viral clearance. This is supported by recent publications that show in vitro inhibition of HCV replication in the HuH-7/Replicon system by IFN-g. To test this hypothesis in vivo, we analyzed the immunologic and virologic effects of liver-specific IFN-g expression in chronic hepatitis C. Liver-specific expression of human IFN-g was achieved with a recombinant, replication-deficient HBV vector (rHBV-IFN-g), and with an adenoviral vector (Ad-IFN-g) that expressed IFN-g under the HBV preS2/S-promoter and the liver-specific transthyretin promoter, respectively. A transient peak of IFN-g human and chimpanzee IFN-g RNA were detectable and followed by expression of IP-10, an IFN-g-responsive chemokine. IFN-g expression at the level obtained was not associated with liver injury as measured by ALT levels. Phenotypic and functional characterization of cellular immune responses in the blood and in the liver as well as virological analysis of HCV titer and sequence are currently in progress to evaluate the effects of intrahepatic IFN-g expression. In a second vaccine approach we hypothesized that the poor priming of cellular immune responses in persistently HCV-infected patients may be due to the noncytopathic nature of HCV and to the subsequent absence or scarcity of virus-infected apoptotic or dead cells as source of exogenous antigens for crosspresentation. Because cross-priming contributes to the induction of CD8+ T cells in vivo (Nature 1999; 398:77-80), we hypothesized that cross-priming with dendritic cells (DCs) containing self-replicating RNA might be useful to induce strong, HCV-specific cellular immune responses. For this purpose, we generated a recombinant cytopathic pestivirus self-replicating RNA which was used to transfect murine dendritic cells and to immunize HLA-A2 transgenic mice subcutaneously. Cellular immune responses were evaluated and transfer of cellular material from the vaccine DCs to the endogenous APCs, a phenomenon that underlies crosspriming, was directly visualized in vivo and functionally demonstrated in vitro. Finally, the protective effect and the in vivo function of the induced HCV-specific T cells were directly demonstrated in a surrogate challenge model with recombinant, HCV NS3 encoding vaccinia virus. Whereas 10e4 to 10e7 pfu were detected in nonvaccinated control mice, no vaccinia virus was detected in mice vaccinated with replicon-transfected DCs, indicating complete protection. In summary, these findings support the hypothesis that experimental forcing of exogenous antigens into the MHC class I pathway and subsequent promotion of cross-priming is particularly important to generate T cell responses against noncytopathic and tissue tropic viruses such as HCV.
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