Hepatic transcriptome analysis of hepatitis C virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance

Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV) infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3) and cytotoxic granule-associated RNA binding protein (TIA1), associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a "danger signal" leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection.     

Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus

Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14(+) mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4(+) T cells, and HCV-specific CD8(+)-T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection.     

Kinetics of miR-122 Expression in the Liver during Acute HCV Infection

The relationships among micro RNA-122 (miR-122) expression in the liver, hepatitis C virus (HCV) replication and hepatic damage were analyzed in three chimpanzees observed for 180 days after inoculation with HCV genotype 1a. Levels of miR-122 in the liver and serum were measured by real-time RT PCR in serial liver biopsies and serum samples. Hepatic miR-122 levels were normalized separately for each of three chimpanzees with small RNAs and microRNAs that are endogenous to the liver and are stably expressed. Two- to 4-fold rise in hepatic miR-122 levels was observed at the onset of HCV infection (the first 4 weeks) when HCV titers in the liver and serum increased rapidly in all three chimpanzees in concordance with in vitro data indicating the miR-122 significance for HCV replication. Between 10 to 14 weeks after inoculation, when hepatic and serum HCV RNA titers exceeded 3 logs and alanine aminotransferase (ALT) activity was elevated, hepatic miR-122 levels were in decline. Cumulative data derived from all three chimpanzees from 180 days of observation documented an inverse (negative) correlation between hepatic miR-122 and HCV RNA in the liver and serum and positive correlation between level of serum miR-122 and HCV replication. Subsequent rise of miR-122 level during HCV clearance and ALT normalization suggested a tri-phasic occurrence of the relationship among hepatic miR-122 expression, HCV replication and hepatic destruction, which was the most apparent in one chimpanzee but less evident in two other animals. In vivo kinetics of hepatic and serum miR-122, HCV replication and hepatic destruction reflects complexities of the virus-host interaction during the acute phase of HCV infection.     

The clearance of hepatitis C virus infection in chimpanzees may not necessarily correlate with the appearance of acquired immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-gamma)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.     

Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1⁺ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4⁺FoxP3⁺ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.     

CTLA-4 exon 1 +49 polymorphism alone and in a haplotype with ?318 promoter polymorphism may confer susceptibility to chronic HBV infection in Chinese Han patients

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) plays a pivotal role in regulating T cell activation, which is believably critical for the outcome of hepatitis B virus (HBV) infection. The expression and function of CTLA-4 may be affected by gene polymorphisms. This study investigated the influence of CTLA-4 polymorphisms on disease susceptibility in Chinese Han patients with chronic HBV infection. CTLA-4 +49A/G and -318C/T polymorphisms were evaluated by DNA amplification with polymerase chain reaction followed by the restriction fragment length polymorphism analysis. The patients with chronic HBV infection had higher frequencies of genotype AA and allele A of CTLA-4 +49A/G polymorphism. The haplotype +49A-318C was significantly over-represented (P < 0.001) and haplotype +49G-318C under-represented (P = 0.006) in the patients. The +49GG genotype was more frequent (P = 0.009) and +49A allele was less frequent in patients with lower ALT levels (P = 0.012) in HBeAg positive chronic hepatitis B. It is indicated that CTLA-4 +49A/G polymorphism alone and in a haplotype with -318C allele may confer susceptibility to chronic HBV infection in Chinese Han patients.     

Parenchymal expression of CD86/B7.2 contributes to hepatitis C virus-related liver injury

Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single- and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4+ T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4+ T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection.     

Immune-based novel therapies for chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a great public health problem, with an estimated 200 million chronically infected patients worldwide. No vaccines are currently available for HCV, and only a subset of HCV patients responds to interferon-alpha (IFN-alpha) and Ribavirin treatment. Substantial evidence has emerged recently to support the role of the host immune response in the outcome and pathogenesis of HCV infection. Our aims of this article are to present the immune-based novel therapeutic options for HCV infection and the evidence supporting their use in patients with chronic hepatitis C. There is a growing consensus that acute control of HCV infection is associated with a vigorous intrahepatic antiviral CD4+ and CD8+ T cell response. IFN-gamma was detectable in the livers of the chimpanzees that cleared or controlled the virus, raising the possibility that IFN-gamma might perform antiviral effector functions during HCV infection. Based on these observations, therapeutic induction of intrahepatic IFN-gamma by adoptive immunotherapy might be able to control chronic HCV infection. Immune-based novel therapies appear to hold great promise in treating chronic HCV infection.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.     

Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIV(mac251) macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIV(mac251) infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 Ab of macaques chronically infected with SIV(mac251) decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4(+) T cell proliferation.     

Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.     

Prokaryotic expression of woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation of polyclonal antibody to wCTLA-4

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an inhibitory T cell receptor predominately expressed on activated T cells and plays an important role in regulation of specific T cell responses to viral infection. The woodchuck model is an informative animal model for hepatitis B virus (HBV) infection. In this study, the extracellular region of woodchuck CTLA-4 (wCTLA-4) was cloned and the fusion protein of GST-wCTLA-4 was expressed and purified. Polyclonal antibody against GST-wCTLA-4 (anti-GST-wCTLA-4) was prepared. The full length wCTLA-4 protein expressed in transfected baby hamster kidney cells was detected by anti-GST-wCTLA-4 in western blot analysis and immunofluorescence staining. Anti-GST-wCTLA-4 provides a useful tool to study the role of CTLA-4 in T-cell response in the woodchuck model. Further, the blocking of CTLA-4 with anti-GST-wCTLA-4, as a novel therapy approach for chronic hepatitis B virus infection, could be studied in woodchuck model now.     

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

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.     

Galectin-9 and IL-21 Mediate Cross-regulation between Th17 and Treg Cells during Acute Hepatitis C

Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3^high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.     

High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8⁺ and CD4⁺ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.