Michelina Nascimbeni

Immunology of hepatitis B virus and hepatitis C virus infection

More than 500 million people worldwide are persistently infected with the hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite many common features in the pathogenesis of HBV- and HCV-related liver disease, these viruses markedly differ in their virological properties and in their immune escape and survival strategies. This review assesses recent advances in our understanding of viral hepatitis, contrasts mechanisms of virus–host interaction in acute hepatitis B and hepatitis C, and outlines areas for future studies.

Impaired Effector Function of Hepatitis C Virus-Specific CD8+ T Cells in Chronic Hepatitis C Virus Infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-γ production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.

Kinetics of CD4+ and CD8+ memory T-cell responses during hepatitis C virus rechallenge of previously recovered chimpanzees.

ABSTRACT The immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood. Antibodies to HCV structural proteins do not appear to play a key role in clearance of the virus and do not persist after recovery. Here, we studied the kinetics of the cellular immune responses of three HCV-recovered chimpanzees during rechallenge with increasing doses of homologous HCV. Although HCV envelope antibodies remained undetectable throughout the rechallenge, all animals mounted rapid HCV-specific T-cell responses. The pattern of the cellular immune response in blood and liver correlated with the virological outcome. The animal that most rapidly cleared circulating HCV as determined by nested reverse transcription-PCR (RT-PCR) displayed the most vigorous and sustained response of gamma interferon (IFN-γ)-producing and proliferating CD4+ T cells in the blood. Vigorous CD4+ T-cell proliferation during viremia was followed by an increased frequency and a phenotypic and functional change of the tetramer+ CD8+ T-cell population. The second animal cleared HCV initially with strong peripheral and intrahepatic CD4+ T-cell responses but experienced low-level HCV recrudescence 12 weeks later, when HCV-specific T cells became undetectable. The third animal maintained minute amounts of circulating HCV, detectable only by nested RT-PCR, in the face of a weak IFN-γ+ T-cell response. Collectively, the results suggest protective rather than sterilizing immunity after recovery from hepatitis C. The rate of HCV clearance following reexposure depends on the cellular immune response, the quality and quantity of which may vary among chimpanzees that recovered from HCV infection.

Previously Infected and Recovered Chimpanzees Exhibit Rapid Responses That Control Hepatitis C Virus Replication upon Rechallenge

ABSTRACT Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 106 copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 103 copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-γ) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-γ in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.

Effects of antiviral therapy on the cellular immune response in acute hepatitis C

Spontaneous recovery occurs in a minority of patients with acute hepatitis C but is associated with vigorous and long‐lasting cellular immune responses. Treatment‐induced recovery can be achieved in the majority of patients who are treated in the acute phase, but the kinetics and mechanisms of viral clearance and immune responsiveness are not known. Both direct antiviral effects and indirect immune‐mediated effects, such as immune modulation of Th2 to Th1 responses and prevention of exhaustion of cellular responses by rapid reduction of viral titer, have been proposed. To investigate how early antiviral therapy affects hepatitis C virus (HCV)‐specific T cell responses, we performed detailed prospective clinical, virological, and immunological studies on 7 patients with acute hepatitis C who received antiviral therapy and were followed at 2 to 4 week intervals for 1 to 2 years. The total CD4+ and CD8+ cell response was analyzed with 600 overlapping HCV peptides and 6 proteins by ex vivo enzyme‐linked immunospot (ELISpot), intracellular cytokine staining, and proliferation assays. In contrast to earlier studies with selected HCV epitopes, this extended analysis detected multispecific interferon γ+ (IFN‐γ+) responses in each patient, even in the absence of T‐cell proliferation. After initiation of antiviral therapy (at a mean of 20 weeks after infection), all sustained responders demonstrated gradually decreasing, then nearly absent HCV‐specific T‐cell responses, whereas the sole patient who developed viral breakthrough after initial HCV control maintained cellular immune responses. In conclusion, a sustained response to antiviral therapy was not associated with a lasting enhancement of HCV‐specific T‐cell responsiveness in the blood. (HEPATOLOGY 2004;40:87–97.)

Immunization with Hepatitis C Virus-Like Particles Induces Humoral and Cellular Immune Responses in Nonhuman Primates

ABSTRACT We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon+ (IFN-γ+)CD4+ and IFN-γ+CD8+ T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.

The Clearance of Hepatitis C Virus Infection in Chimpanzees May Not Necessarily Correlate with the Appearance of Acquired Immunity

ABSTRACT 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-γ)-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-γ, IFN-α, 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.

Delayed Induction, Not Impaired Recruitment, of Specific CD8+ T Cells Causes the Late Onset of Acute Hepatitis C

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is characterized by lack of immune-mediated liver injury despite a high level of HCV replication during the incubation phase, which lasts about 8 weeks. We investigated whether this results from delayed recruitment of HCV-specific T cells and whether it facilitates HCV persistence. METHODS Six chimpanzees were infected with HCV; blood and liver samples were collected for 28 weeks and analyzed for immune cells and chemokines. RESULTS Two chimpanzees developed self-limited infections, whereas the remaining 4 developed chronic infections. Levels of the chemokines CXCL10, CXCL11, CCL4, and CCL5 increased in blood and liver samples from all chimpanzees within 1 month of HCV infection. Chemokine induction correlated with intrahepatic type I interferon (IFN) responses in vivo and was blocked by neutralizing antibodies against IFN-β in vitro. Despite the early-stage induction of chemokines, the intrahepatic lymphocytic infiltrate started to increase no earlier than 8 weeks after HCV infection, when HCV-specific, tetramer-positive CD8(+) T cells appeared in the circulation. The HCV-specific CD8(+) T cells expressed chemokine receptors when they were initially detected in blood samples, so they could be recruited to the liver as soon as they entered the circulation. CONCLUSIONS Chemokines are induced during early stages of HCV infection, which requires a type I IFN-mediated response. The delayed onset of acute hepatitis does not result from delayed recruitment of HCV-specific T cells, but could instead be related to a primary delay in the induction of HCV-specific T cells. Divergent outcomes occur without evident differences in chemokine induction and T-cell recruitment.

The Frequency of CD127+ Hepatitis C Virus (HCV)-Specific T Cells but Not the Expression of Exhaustion Markers Predicts the Outcome of Acute HCV Infection

ABSTRACT T cells are exhausted and overexpress inhibitory molecules in chronic hepatitis C virus (HCV) infection. It is unclear whether this is the cause or consequence of HCV persistence. By studying serial blood and liver samples of chimpanzees during acute infection, we demonstrate that the early expression of the memory precursor marker CD127 on HCV-specific T cells, but not the expression of inhibitory molecules on those T cells or their ligands in the liver, predicts the outcome of acute infection.

Chronic HCV infection and the clonality of intrahepatic T cells

A broad repertoire of highly specific effector and memory T cells is required to fight the various pathogens that one encounters during life. How is this diversity achieved given that the number of genes encoding the antigen-specific Tcell receptor (TCR) is limited? The majority of T cells have a TCR composed of two chains, a and b, each chain containing one variable (V) region and one constant (C) region. TCR diversity is created during development of T cells in the thymus, when the multiple genes that encode the two chains of the TCR heterodimer are rearranged by somatic recombination. Receptors of different specificities are generated by different combinations of V, D and J gene segments, and by removal and addition of nucleotides at the VJ (TCRa) or VDJ (TCRb) junctions. Both mechanisms maximize diversity in the so called complementarity determining region 3 (CDR3), that varies in size and amino acid sequence and provides the TCR’s unique specificity [1,2]. It is this region that binds immunogenic peptides from invading pathogens and triggers the adaptive cellular immune response. Thus, analysis of the size and sequence of the CDR3 region provides a molecular footprint of the overall TCR repertoire [1]. Not surprisingly, healthy individuals typically display a Gaussian distribution of their CDR3 length repertoire at birth. With increasing age, the diversity of the CDR3 length repertoire decreases and oligoclonal T-cell expansions are frequently found in the memory T-cell population of healthy, older persons [3,4]. These oligoclonal expansions have been attributed to the increasing number of antigens that the immune system responds to during life. Especially common pathogens such as influenza A virus, cytomegalovirus, and Epstein–Barr virus [5–7], that cause latent or repeated infections, may therefore maintain and expand subpopulations of T cells with a specific TCR receptor. In addition, significant expansions of clonal and oligoclonal Tcell subpopulations have been associated with malignancies [8,9] and with autoimmune and chronic inflammatory conditions [10]. An overview of the factors that may contribute to oligoclonal expansions and need to be considered and, if possible, controlled for in studies on CDR3 length diversity, is given in Table 1. In this issue of the Journal, Vigan et al. evaluate the TCRb chain diversity in patients with hepatitis C virus (HCV) infection [11]. HCV is known for its propensity to establish persistent infection and chronic inflammatory liver injury in most infected subjects [12]. An early and multispecific T-cell response has been associated with viral clearance [13] and immune protection [14,15]. In contrast, a less efficient immune response that continues for decades in its futile attempt to eradicate the virus, has been associated with chronic inflammatory liver injury, fibrogenesis and development of liver cirrhosis [16,17]. Whether this unsuccessful immune response is targeted against fewer antigens or consists of smaller effector cell populations than a successful one, is one of the questions currently being investigated. In general, HCV-specific T cells can readily be isolated from liver biopsies and cultured and tested after in vitro expansion. Based on this technique, many studies clearly demonstrate that the intrahepatic T-cell response is multispecific even in persistently infected individuals [18–21]. However, because these studies require in vitro expansion, it is not possible to assess the size of clonal T-cell subpopulations in situ, in the infected liver. Also, it is not possible to study T-cell subpopulations that do not adapt well and do not grow in vitro. Thus, the size and diversity of HCVspecific T-cell subpopulations in the liver and their relevance for the outcome of HCV infection are still unknown. Vigan et al. address this issue with a molecular technique by determining the size and the sequence of the TCRb chain CDR3 region using RNA from paired peripheral blood mononuclear cells (PBMC) and from liver biopsies from patients with chronic HCV infection [11]. This technique, originally established by Pannetier et al. [22] is probably the most powerful and accurate technique to analyze TCR Journal of Hepatology 38 (2003) 677–680