T cells are considered an important component of a future HCV vaccine but the determinants of successful, vaccine-induced T cells are not known. Here, we compared the phenotype, function and kinetics of vaccine-induced and infection-induced using multicolor flow cytometry and realtime PCR. For this project we used cryopreserved blood lymphocytes and liver biopsies from nonhuman primates that had previously been infected in a study designed to evaluate a candidate vaccine (Nat Med 2006, 12:190-7). This candidate vaccine consisted of a prime with a recombinant adenovirus encoding HCV NS3-NS5 sequences and a DNA boost. To evaluate and characterize successful T cell responses that mediate HCV clearance we compared vaccine-induced CD8 T cells with infection-induced CD8 T cells. We identified minimal CD8 T cell epitopes, determined their major histocompatibility (MHC) restriction and synthesized MHC tetramers loaded with HCV peptides to detect HCV-specific CD8 T cells by multicolor flow cytometry. In addition, intrahepatic mRNA levels of T cell markers and their ligands were studied. We found that HCV-specific T cells appeared earlier, maintained better functionality and persisted at higher frequencies for a longer time after HCV infection than infection-induced T cells. In addition, vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower PD-1 levels than infection-induced T cells. Vaccine-induced, but not infection-induced T cells were multifunctional and their ability to secrete IFN-gamma and TNF-alpha correlated to early CD127 but not PD-1 expression. A side-by-side comparison of vaccine-induced and infection-induced T cells in the same host revealed that the CD127+ memory precursor phenotype was induced by the vaccine itself and not by the low viremia. In contrast, the induction of PD-1 on HCV-specific T cells correlated to viremia, and intrahepatic PD-1, PD-L1 and 2,5-OAS-1 mRNA levels correlated to peak HCV titers. Collectively, these results show that vaccination induced HCV-specific CD127+ T cells with high functionality that persisted at higher levels for a longer time than infection-induced T cells. Control of viremia prevented PD-1 upregulation on T cells, and PD-1, PD-L1 and 2,5-OAS-1 induction in the liver. Thus, early development of a memory T cell phenotype and, via control of viremia, attenuation of the inhibitory PD1/PD-L1 pathway may be regarded as necessary components of successful vaccine-induced protection against HCV. In a second project, we cloned the dominant T cell receptors of vaccine-induced T cells into retroviral vectors and re-expressed them in blood lymphocytes that were not specific for HCV. Using a similar retroviral vector approach we expressed a chimeric, antibody-based receptor (CAR) to HBsAg (Gastroenterology 2008;134:239-244) in blood lymphocytes that were not specific for HBV. Upon transduction these lymphocytes gained a broad spectrum of effector functions (secretion of IFN-gamma, cytotoxicity and proliferation) that were specific to the cognate antigens of the re-expressed TCRs and CAR, respectively. The transduced T cells were expanded to high numbers with anti-CD3 plus IL-21/IL-15 stimulation, which resulted in a preservation of their differentiation status and a lower frequency of immunosuppressive regulatory T cells than conventional stimulation with anti-CD3 plus IL-2. These results demonstrate that the combination of retroviral TCR gene transfer together with IL-21/IL-15 stimulation can efficiently redirect the antigen specificity of resting primary T cells and generate a large number of functional effector T cells. These expanded effector T cells will be used in adoptive immunotherapy protocols to restore and enhance virus-specific immune responses in chronic infection.

Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$605,248
Indirect Cost
City
State
Country
Zip Code
Bolte, Fabian J; O'Keefe, Ashley C; Webb, Lauren M et al. (2017) Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation. Gastroenterology 153:1392-1403.e2
Rehermann, Barbara (2017) Mature peritoneal macrophages take an avascular route into the injured liver and promote tissue repair. Hepatology 65:376-379
Kugler, David G; Flomerfelt, Francis A; Costa, Diego L et al. (2016) Systemic toxoplasma infection triggers a long-term defect in the generation and function of naive T lymphocytes. J Exp Med 213:3041-3056
Didion, John P; Morgan, Andrew P; Yadgary, Liran et al. (2016) R2d2 Drives Selfish Sweeps in the House Mouse. Mol Biol Evol 33:1381-95
Holz, Lauren; Rehermann, Barbara (2015) T cell responses in hepatitis C virus infection: historical overview and goals for future research. Antiviral Res 114:96-105
Rehermann, Barbara; Bertoletti, Antonio (2015) Immunological aspects of antiviral therapy of chronic hepatitis B virus and hepatitis C virus infections. Hepatology 61:712-21
Park, Su-Hyung; Rehermann, Barbara (2014) Immune responses to HCV and other hepatitis viruses. Immunity 40:13-24
Veerapu, Naga Suresh; Park, Su-Hyung; Tully, Damien C et al. (2014) Trace amounts of sporadically reappearing HCV RNA can cause infection. J Clin Invest 124:3469-78
Hara, Koji; Rivera, Maria M; Koh, Christopher et al. (2014) Sequence analysis of hepatitis C virus from patients with relapse after a sustained virological response: relapse or reinfection? J Infect Dis 209:38-45
Park, Su-Hyung; Veerapu, Naga Suresh; Shin, Eui-Cheol et al. (2013) Subinfectious hepatitis C virus exposures suppress T cell responses against subsequent acute infection. Nat Med 19:1638-42

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