Hepatitis C virus (HCV) infection is most often clinically inapparent and rarely associated with symptoms of acute hepatitis. It is therefore difficult to study immune correlates of viral clearance during the very early phase of HCV infection in humans. The chimpanzee is the only infectious animal model to study the role of the HCV specific immune response. Transfection of a chimpanzee with RNA transcribed from a full length HCV cDNA clone has now provided the opportunity to prospectively study the peripheral blood as well as the vigor, kinetics and specificity of the intrahepatic T cell response with antigens of a single, defined sequence and to rechallenge recovered animals with virus of precisely the same sequence. Because virus-specific T lymphocytes recognize immunogenic viral peptides in the context of MHC molecules expressed on the surface of infected cells, it is important to identify and characterize chimpanzee MHC, also called Patr-alleles and to identify the panel of HCV peptides that are generated and the repertoire of T cell specificities that is induced during HCV infection. Chimpanzee MHC alleles are closely related to, but distinct from human alleles. In order to identify chimpanzee Patr alleles that correspond to the most frequent human ones, we cloned and sequenced Patr alleles of 13 chimpanzees and identified a total of 21 different alleles, two of them previously unknown and three only partially reported. Individual Patr alleles were expressed in MHC class I negative cell lines, highly positive transfectants were sorted and cloned, and binding of biotinylated peptides characteristic for the HLA-A1, A2, A3, B7 supertype was evaluated by FACS analysis. In addition, tetrameric Patr-HCV peptide complexes were generated and demonstrated to bind to CD8+ T cells from HCV infected animals. We then used a panel of 27 HCV peptides to study cytokine production and lytic function of chimpanzee CD8+T cells prior to and after HCV inoculation. Direct ex vivo Elispot analysis confirmed that IFNg producing T cells were induced in vivo in response to HCV infection and that these T cells were targeted against the peptides that we had predicted based on the Patr-type of the individual animals. Importantly, these included HCV epitopes that we had previously shown to be recognized by memory T cells isolated from the blood of the rare patients who recover from HCV infection (Takaki et al. Nature Medicine, 6:578, 2000). HCV peptide specific T cell lines were then expanded in vitro and tested for cytotoxicity against a panel of transfectant cell lines that expressed individual Patr-alleles and presented the peptide of interest. In each case, Patr-restriction of the peptide specific T cell line could be demonstrated. In conclusion, these and previous studies on HLA pocket structures and binding motifs confirm a close homology between individual Patr- and HLA-alleles. In addition, we demonstrate that HCV infection induces a common panel of HCV peptides that is recognized by chimpanzees and humans. This allows selection of animals according to the most frequent human MHC types for infection and vaccine studies and to select HCV peptides and peptide-tetramers to characterize the kinetics and function of the HCV-specific CD8+ T cell response during the early stages of infection in this animal model.
Showing the most recent 10 out of 50 publications
