In the past year, we have conducted the following studies in the chimpanzee model of hepatitis C virus infection: (i) Identification and characterization of novel chimpanzee MHC class I and class II alleles T cells recognize viral peptides in the context of MHC class I and II molecules on the surface of cells that are either infected by the virus or take up and process exogenous viral antigens. Chimpanzee (Pan troglodytes) MHC alleles have been designated Patr alleles. Here, we have continued our efforts to identify and characterize (i) the Patr class I and II alleles of chimpanzees and (ii) the HCV peptides that they present. In a collaboration with extramural investigators, we identified a new Patr-B*01 variant, Par-B*0102, by sequence-based typing. The sequence has been submitted to the IPD/MHC-NHP database (Robinson et al., Nucleic Acids Research 2003: 31:311-14) and to GenBank. Further, we have identified a panel of specific HCV epitopes that are presented by this Patr allele. In in vitro studies using T cells from chimpanzees with acute HCV infection we identified the minimal optimal sequence and Patr-restriction of these epitopes. The identification and characterization of Patr alleles and Patr-binding viral peptides is important for the generation of immunological tools such as Patr-peptide tetramers which can then be used to characterize effective cellular immune responses ex vivo in liver biopsies and blood samples throughout the course of HCV infection in this animal model. (ii) Characterization of HCV-specific memory T cells during vaccine-induced or spontaneous recovery from hepatitis C T-cell responses are considered an important component of a successful vaccine against hepatitis C virus (HCV). However, immunological markers that differentiate successful, vaccine-induced memory T-cells from regular memory T-cells induced during natural HCV infection have not been described. In this project, we mapped minimal T-cell epitopes, generated 6 MHC/peptide tetramers and performed a detailed multi-color flow cytometry analysis of HCV-specific memory T-cells of 5 chimpanzees, which were part of a previously published adenovirus/DNA-based HCV-NS3-5 vaccine trial (Nature Medicine 12:190, 2006). HCV-specific memory T-cells were detectable not earlier than week 11 after HCV-challenge in 2 non-vaccinated chimpanzees, whereas they were detectable immediately after vaccination in 3 vaccinated chimpanzees and their numbers increased as early as week 4 after HCV-challenge. However, irrespective of the chimpanzees vaccination status, HCV-specific memory T-cells were about 44% central memory (CCR7+CD45RA-) and 47% effector memory (CCR7-CD45RA-) cells. At the time of their peak frequency, HCV-specific memory cells were maximally activated in non-vaccinated chimpanzees with high levels of CD38 and PD-1 expression (mean fluorescence intensity (MFI) for CD38=39,061;PD-1(MFI)=25,202). In contrast, HCV-specific memory cells were only modestly activated in vaccinated chimpanzees (MFI for CD38=2,949;PD-1(MFI)=2,549;p=0.007 and p=0.004, respectively), and their activation status remained controlled even during HCV challenge. Consistent with these findings in the peripheral blood, the PD-1/CD8 mRNA ratio in the liver was higher for non-vaccinated chimpanzees than for vaccinated chimpanzees (2.47 versus 0.75;p=0.029). The limited activation of vaccine-induced T-cells was unexpected as these cells showed excellent functional responses (proliferation, IFN-g production) that were superior to those of memory T-cells induced in non-vaccinated chimpanzees during the natural course of HCV infection. Thus, vaccine-induced T-cells appeared to achieve more (better effector function) with less effort (less activation). Taken together, a successful T-cell vaccine induces effective T-cell immunity by optimal rather than maximal activation of HCV-specific CD8 T-cells.
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