Benoit Callendret

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1)

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti–PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti–PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

ABSTRACT Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ∼5.5 log10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness.

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8+ T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS31629-1637, displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.

Selection-Driven Immune Escape Is Not a Significant Factor in the Failure of CD4 T Cell Responses in Persistent Hepatitis C Virus Infection

Immune escape driven by selection pressure from virus‐specific CD8 T cells has been demonstrated in both chimpanzees and humans infected with the hepatitis C virus (HCV). Although escape mutations have also been characterized in major histocompatibility complex (MHC) class II–restricted HCV epitopes, it is unknown whether selection‐driven immune escape by CD4 T cell epitopes is a significant factor in the failure of these responses or contributes to persistent infection. To address this issue, evolution of MHC class I– and class II–restricted HCV epitopes was compared in four chimpanzees persistently infected with the virus for more than 10 years. We identified an amino acid change in a CD4 epitope of the HCV NS3 protein in one of the chimpanzees 3 years after infection. This mutation resulted in diminished activation, cytokine production (interferon‐γ and interleukin‐2), and proliferation by an epitope‐specific CD4 T cell line. We expanded our analysis to determine if mutations were common in multiple CD4 versus CD8 T cell epitopes in the four chronically infected animals. Whereas we observed mutations in over 75% of CD8 T cell epitopes analyzed in this study, only 18% of CD4 T cell epitopes analyzed showed amino acid changes. The frequency of changes in class II epitopes was not different from flanking regions, so CD4 T cells rarely exert selection pressure against the HCV genome. Conclusion: Apparent mutational escape can occur in MHC class II–restricted epitopes, but this is uncommon when compared with class I–restricted epitopes in the same individual. This indicates that other mechanisms for silencing CD4 T cells are dominant in persistent HCV infections. (HEPATOLOGY 2009.)

Dominance of the CD4+ T helper cell response during acute resolving hepatitis A virus infection

CD4+ T cells play a dominant role in control of acute HAV infection in chimpanzees.

T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee.

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T‐cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)‐free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct‐acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T‐cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T‐cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape‐prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. Conclusions: The intrahepatic HCV‐specific CD8+ T‐cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T‐cell responses and reduce the risk of a second persistent infection. (Hepatology 2014;60:1531–1540)

Transmission of Clonal Hepatitis C Virus Genomes Reveals the Dominant but Transitory Role of CD8+ T Cells in Early Viral Evolution

ABSTRACT The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8+ T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8+ T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8+ T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.

Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a direct‐acting antiviral

Exhaustion of antiviral CD8+ T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T‐cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct‐acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3‐NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8+ T‐cell response. However, these antiviral CD8+ T cells did not prevent persistent replication of DAA‐resistant HCV variants that emerged during treatment. Most vaccine‐induced CD8+ T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8+ T‐cell targeting‐conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8+ T‐cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8+ T‐cell expression of the inhibitory receptor programmed cell death 1 increased 25‐fold compared with those in circulation. Conclusion: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T‐cell response, primarily against nonconserved class I epitopes; exhaustion of liver‐infiltrating CD8+ T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants. (Hepatology 2016;63:1442‐1454)

Persistent HCV replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and direct acting antiviral

Exhaustion of antiviral CD8+ T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T‐cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct‐acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3‐NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8+ T‐cell response. However, these antiviral CD8+ T cells did not prevent persistent replication of DAA‐resistant HCV variants that emerged during treatment. Most vaccine‐induced CD8+ T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8+ T‐cell targeting‐conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8+ T‐cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8+ T‐cell expression of the inhibitory receptor programmed cell death 1 increased 25‐fold compared with those in circulation. Conclusion: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T‐cell response, primarily against nonconserved class I epitopes; exhaustion of liver‐infiltrating CD8+ T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants. (Hepatology 2016;63:1442‐1454)

T cell immunity and HCV reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T‐cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)‐free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct‐acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T‐cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T‐cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape‐prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. Conclusions: The intrahepatic HCV‐specific CD8+ T‐cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T‐cell responses and reduce the risk of a second persistent infection. (Hepatology 2014;60:1531–1540)