Barbara Rehermann

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.

Pathogenesis, Natural History, Treatment, and Prevention of Hepatitis C

Dr. T. Jake Liang (Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], National Institutes of Health [NIH], Bethesda, Maryland): The identification of hepatitis C virus (HCV) as the cause of non-A, non-B hepatitis represents a technical tour de force of modern molecular medicine (1). Characterization of the viral genome and the structures and functions of viral gene products has led to a better understanding of the viral life cycle and the pathogenesis of HCV-associated disease. This knowledge will contribute to the development of an effective vaccine and better therapies. Hepatitis C virus is a member of the Flaviviridae family, which includes the flaviviruses and pestiviruses (2). There are at least 6 HCV genotypes and more than 50 subtypes. The virion contains a positive single-stranded RNA genome of 9.5 kilobases (Figure 1). The genome consists of 5 and 3 untranslated regions (5UTR and 3XR) that have little sequence variation among all genotypes and are important for translation of viral proteins and replication of the virus. The viral genome encodes a large single polyprotein of about 3000 amino acids; the N-terminal one third harbors the structural proteins, and the C-terminal two thirds contains the nonstructural proteins. The HCV structural proteins comprise the core protein and the two envelope glycoproteins E1 and E2. The nonstructural proteins, including proteases (NS2/3 and NS3), helicase (an enzyme that unwinds double-stranded nucleic acid) (NS3), and RNA-dependent RNA polymerase (NS5B), perform various functions essential for the viral life cycle (Table 1). Cleavage of structural proteins from the polyprotein is catalyzed by a host signal peptidase, whereas polyprotein cleavage in the nonstructural region requires HCV-encoded proteases. Figure 1. Schematic diagram of the hepatitis C virus genome. 5UTR 3XR Table 1. Functions of Genetic Elements of Hepatitis C Virus Hepatitis C virus enters a susceptible host either directly, through needle inoculation or transfusion of contaminated blood products, or inadvertently, through breakage of a percutaneous barrier (as exemplified by sexual or perinatal transmission) (3). The virus then enters hepatocytes or other susceptible cells, probably through a unique surface molecule or molecules, as the viral receptor (4). After uptake, the virus uncoats and releases the genome to begin replication. The viral genome first serves as the template for translation of the polyprotein. The processed nonstructural proteins then form a complex with the genome and initiate synthesis of the negative strand, which in turn functions as the template for positive strand synthesis. The replication complex probably resides in a membranous compartment in the cytoplasm, presumably derived from the endoplasmic reticulum. The RNA replicative intermediate matures and interacts with the core and envelope proteins to assemble into a virion. Although most of the replicative processes have not been defined, some nonstructural proteins clearly play critical roles in viral replication and productive infection; these proteins are therefore the focus of antiviral development. Immunopathogenesis of Hepatitis C Dr. Barbara Rehermann (Liver Diseases Section, NIDDK, NIH): Unlike other hepatitis viruses, the hepatitis C virus is more likely to cause clinically inapparent, chronic infection in persons who are otherwise considered immunocompetent. Thus, the virus is capable of circumventing an efficient immune response of the host. Components of Antiviral Immune Response The mechanisms whereby HCV circumvents immune response and establishes persistent infection are currently undefined. It is well known that the specific immune response to any viral infection is primed by macrophages and dendritic cells that present viral proteins to B cells, helper T cells, and cytotoxic T cells (Figure 2). In many viral infections, B cells produce antibodies that can clear circulating virus and protect from reinfection. For example, antibodies against the hepatitis B virus surface antigen are critical for viral clearance. Through specific T-cell receptors on the cell surface, helper T cells recognize viral peptides that are derived from phagocytosed and proteolytically cleaved HCV proteins and are presented in the context of class II MHC molecules. Figure 2. Components of the antiviral immune response. HCV On activation of their specific T-cell receptors, HCV-specific helper T cells assist with activation and differentiation of B cells as well as induction (5) and stimulation of virus-specific cytotoxic T cells. Most of these effects are mediated by different sets of immunoregulatory Th1 (interferon- and interleukin-2) or Th2 (interleukin-4, interleukin-5, and interleukin-10) cytokines. In the context of class I MHC molecules, CD8-positive cytotoxic T cells recognize HCV peptides that are synthesized and processed in infected cells (Figure 2). This encounter can lead to lysis of virus-infected cells. Together with helper T cells, cytotoxic T lymphocytes may also secrete cytokines, such as interferon- and tumor necrosis factor-, that inhibit replication and gene expression of several viruses, such as hepatitis B virus, cytomegalovirus, and rotavirus (6-8). Humoral Immune Response Hepatitis C virus can establish persistent infection despite an active humoral and cellular immune response that is generally targeted against all viral proteins. The virus may escape from the humoral immune response if the kinetics of infection and viral replication do not allow complete neutralization of the virus by HCV-specific antibodies after primary infection. Although virus-specific antibodies may interfere with viral entry into host cells and opsonize the virus for elimination by macrophages, they cannot eliminate HCV from infected cells. In addition, HCV has a high mutation rate, especially in the hypervariable region of the envelope proteins that can be recognized by neutralizing antibodies (antibodies that can bind and eliminate virus) (9, 10). Several studies have demonstrated that the humoral immune response can select HCV variants with sequence changes that allow escape from antibody recognition (11-14). However, recent studies in chimpanzees have suggested that HCV can cause persistent infection in the absence of mutations in the hypervariable region (15, 16). Thus, progression to persistent HCV infection is most likely a multifactorial process that depends on multiple aspects of virus-host interaction. Cellular Immune Response The cellular immune response probably plays an important role in the outcome of HCV infection because of its ability to recognize and eliminate virus from infected cells. Most studies have concentrated on the antigen-specific immune response that is mediated by CD4-positive helper T cells and CD8-positive cytotoxic T cells. Because chronic rather than acute infection is diagnosed in most patients, immunologic studies have been performed on patients with persistent infection who could not clear HCV. Only a few studies have analyzed the cellular immune response during the acute phase of infection. These studies suggest that the strength and quality of both helper T-cell (17, 18) and cytotoxic T-cell responses (19) differ between patients who recover and those who develop chronic infection. More important, the viral sequences that are recognized most frequently and vigorously by HCV-specific T cells vary little among all the HCV genotypes. Furthermore, several of these frequently recognized viral peptides bind with high affinity to many different class II MHC molecules, suggesting that they can be efficiently presented and recognized by patients with different MHC haplotypes (17, 20). Thus, these viral sequences could be explored for development of preventive or therapeutic vaccines against HCV. The cellular response against HCV could be interfered with in several ways. First, HCV elicits only a weak T-cell response in patients who develop chronic infection (17, 21). In the blood of patients with chronic hepatitis C, the frequency of cytotoxic T-cell precursors that are specific for individual HCV peptides is much lower than the frequency of T cells that recognize an influenza virus peptide as a recall antigen (22) or peptides of other viruses that can be cleared, such as cytomegalovirus (23). The reasons for this relative weakness of the cellular immune response are not known. Certainly, general immune tolerance or immunosuppression is not the cause of persistent HCV infection, because most chronically infected patients display normal immune responses against other viral agents (22). The emergence of viral mutants or quasi-species with sequence variations in T-cell epitopes may contribute to the apparent ineffectiveness of cell-mediated immune response (24-26). There is also increasing evidence that several HCV proteins, such as core (27), E2 (28), and NS5A (29), interfere with the immune response. Furthermore, infected hepatocytes, which lack co-stimulatory molecules, may be relatively inefficient in priming the immune system, and the liver has been proposed as the major site where activated T cells are destroyed (30). Finally, the cellular immune response is a double-edged sword. An immune response that is ineffective in clearing HCV infection may be more harmful to the liver, causing chronic inflammation, hepatocellular injury, and, over several decades, liver fibrosis and cirrhosis. Progression to persistent infection and the immunologic mechanisms of liver injury are the consequence of complicated interactions between the virus and host. Identification of immunologic correlates of viral clearance may contribute to the development of an effective vaccine and better therapy for HCV infection. Natural History of Hepatitis C Dr. Leonard B. Seeff (Division of Digestive Diseases and Nutrition, NIDDK, NIH): The natural history of hepatitis C continues to be

Cellular immune responses persist and humoral responses decrease twodecades after recovery from a single-source outbreak of hepatitis C

As acute hepatitis C virus (HCV) infection is clinically inapparent in most cases, the immunologic correlates of recovery are not well defined. The cellular immune response is thought to contribute to the elimination of HCV-infected cells and a strong HCV-specific T-helper-cell (Th) response is associated with recovery from acute hepatitis C (ref. 2). However, diagnosis of resolved hepatitis C is based at present on the detection of HCV-specific antibodies and the absence of detectable HCV RNA, and detailed comparison of the humoral and cellular immune response has been hampered by the fact that patient cohorts as well as HCV strains are usually heterogeneous and that clinical data from acute-phase and long-term follow-up after infection generally are not available. We studied a cohort of women accidentally exposed to the same HCV strain of known sequence and found that circulating HCV-specific antibodies were undetectable in many patients 18–20 years after recovery, whereas HCV-specific helper and cytotoxic T-cell responses with an interferon (IFN)-γ-producing (Tc1) phenotype persisted. The data indicate these HCV-specific CD4+ and CD8+ T cells are biomarkers for a prior HCV exposure and recovery. Because of undetectable antibodies against HCV, the incidence of self-limited HCV infections and recovery may be underestimated in the general population.

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.

A variant upstream of IFNL3 ( IL28B ) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus

Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.

Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence

Since the identification of the hepatitis C virus (HCV) 20 years ago, much progress has been made in our understanding of its life cycle and interaction with the host immune system. Much has been learned from HCV itself, which, via decades of coevolution, gained an intricate knowledge of host innate and adaptive immune responses and developed sophisticated ways to preempt, subvert, and antagonize them. This review discusses the clinical, virological, and immunological features of acute and chronic hepatitis C and the role of the immune response in spontaneous and treatment-induced HCV clearance.

Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus.

This study was performed to test the hypothesis that cytotoxic T lymphocyte (CTL) selection of hepatitis C virus (HCV) escape variants plays a role in HCV persistence. The peripheral blood CTL responsiveness of patients with well-established chronic hepatitis C to a panel of 10 prototype HCV peptides (genotype 1a) was compared with the corresponding sequences encoded by the infecting viruses in each patient. Variant viral peptide sequences were threefold more frequent in the presence of a CTL response than in its absence, and CTL responses were detected nearly twice as often in association with variant rather than with prototype viral peptide sequences. Furthermore, over half of the patients were infected with potential CTL escape variants that contained nonimmunogenic and noncross-reactive variant peptides many of which displayed reduced HLA-binding affinity. Surprisingly, follow up analysis over a period of up to 46 mo revealed that, in contrast to the relatively high frequency of escape variants initially observed, the subsequent emergence rate of CTL escape variants was very low. Interestingly, the one escape variant that was detected proved to be a CTL antagonist. Collectively, these observations suggest that CTL selection of epitope variants may have occurred in these patients before their entrance into the study and that it may have played a role in HCV persistence. The low apparent rate of ongoing CTL selection in chronically infected patients, however, suggests that if CTL escape occurs during HCV infection it is probably an early event.

Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are present in the peripheral blood and liver of chronically infected patients. The current study was performed to study the relationship between the strength of the CTL response, liver disease severity, and viral load. The results may be summarized as follows: first, using CTL precursor frequency (CTLpf) analysis to quantitate the peripheral blood CTL response, chronically infected patients were less strongly sensitized to a panel of well-defined HCV epitopes than they were to an epitope within the influenza matrix protein. Second, HCV-specific CTLpf did not correlate with disease activity or viral load in the majority of patients on a cross-sectional basis, although it did increase in three patients concomitant with sharp increases in liver disease. Finally, interferon therapy did not enhance the CTLpf against the HCV epitopes studied in these patients, indicating that its antiviral effect is independent of the CTL response. Since the HCV-specific CTLpf in the blood is actually quite low, the CTL may contribute to ongoing liver disease in these patients while being quantitatively inadequate to destroy all of the infected hepatocytes, thereby facilitating HCV persistence and contributing to chronic liver disease.

Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection.

Clearance of the hepatitis B virus (HBV) during acute hepatitis is associated with a strong, polyclonal, multispecific cytotoxic T lymphocyte (CTL) response to the viral envelope, nucleocapsid and polymerase proteins that persists for decades after clinical recovery. In contrast, chronically infected patients usually fail to mount a strong CTL response to this virus. In this study we demonstrate that chronically infected patients who experience a spontaneous or interferon-induced remission develop a CTL response to HBV that is similar in strength and specificity to patients who have recovered from acute hepatitis. The results suggest that specific immunotherapeutic enhancement of the CTL response to HBV should be possible in chronically infected patients, and that it could lead to viral clearance in these individuals with resolution of chronic liver disease.

Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK cells.

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections account for 57% of cases of liver cirrhosis and 78% of cases of primary liver cancer worldwide and cause a million deaths per year. Although HBV and HCV differ in their genome structures, replication strategies and life cycles, they have common features, including their noncytopathic nature and their capacity to induce chronic liver disease, which is thought to be immune mediated. However, the rate of disease progression from chronic hepatitis to cirrhosis varies greatly among infected individuals, and the factors that regulate it are largely unknown. This review summarizes our current understanding of the roles of antigen-specific and nonspecific immune cells in the pathogenesis of chronic hepatitis B and C and discusses recent findings that identify natural killer cells as regulators of T cell function and liver inflammation.