Approximately 5% of the world population is infected by the hepatitis B virus (HBV) that causes a necroinflammatory liver disease of variable duration and severity. Chronically infected patients with active liver disease carry a high risk of developing cirrhosis and hepatocellular carcinoma. The immune response to HBV-encoded antigens is responsible both for viral clearance and for disease pathogenesis during this infection. While the humoral antibody response to viral envelope antigens contributes to the clearance of circulating virus particles, the cellular immune response to the envelope, nucleocapsid, and polymerase antigens eliminates infected cells. The class I- and class II-restricted T cell responses to the virus are vigorous, polyclonal, and multispecific in acutely infected patients who successfully clear the virus, and the responses are relatively weak and more narrowly focused in chronically infected patients who do not. The pathogenetic and antiviral potential of the cytotoxic T lymphocyte (CTL) response to HBV has been demonstrated by the induction of a severe necroinflammatory liver disease following the adoptive transfer of HBsAg-specific CTL into HBV transgenic mice, and by the noncytolytic suppression of viral gene expression and replication in the same animals by a posttranscriptional mechanism mediated by interferon gamma, tumor necrosis factor alpha, and interleukin 2. The dominant cause of viral persistence during HBV infection is the development of a weak antiviral immune response to the viral antigens. While neonatal tolerance probably plays an important role in viral persistence in patients infected at birth, the basis for poor responsiveness in adult-onset infection is not well understood and requires further analysis. Viral evasion by epitope inactivation and T cell receptor antagonism may contribute to the worsening of viral persistence in the setting of an ineffective immune response, as can the incomplete downregulation of viral gene expression and the infection of immunologically privileged tissues. Chronic liver cell injury and the attendant inflammatory and regenerative responses create the mutagenic and mitogenic stimuli for the development of DNA damage that can cause hepatocellular carcinoma. Elucidation of the immunological and virological basis for HBV persistence may yield immunotherapeutic and antiviral strategies to terminate chronic HBV infection and reduce the risk of its life-threatening sequellae.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.

It has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy or acting as an antagonist for the TCR. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide.

Overproduction of the hepatitis B virus (HBV) large envelope polypeptide by transgenic mice containing the entire HBV envelope coding region leads to the formation of extremely long (up to 800 nm), occasionally branching, filamen-

The HLA class I-restricted cytotoxic T Iymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in seronegative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO + (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCVspecific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.(J. Clin. Invest. 1995. 95:521-530.)

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 wellestablished 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. ( J.

During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virusassoiated pathology. It is generally thought that HBV-speciflc class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic m nism, however, could be lethal if most of the hepatocytes are Infected. In the current study, we demonstrate that class I-retricted HBV-speclfic CTLs profoundly suppress hepatoceliular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength ofwhich greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon y and tumor necrosis factor a, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatoceflular HBV gene expression without kifling the cell.The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection rem to be determined.The control of hepatitis B virus (HBV) infection is thought to be mediated by the class I-restricted cytotoxic T-lymphocyte (CTL) response. Patients with acute viral hepatitis, who successfully clear the virus, mount a multispecific polyclonal CTL response to several HBV-encoded antigens, whereas persistently infected patients with chronic hepatitis do not (1). Hepatitis B surface antigen (HBsAg)-specific murine CTL clones cause a necroinflammatory liver disease when they are injected into HBsAg-positive transgenic mice, and the cytopathic effect of the CTLs is largely mediated by the inflammatory cytokines that they release when they are activated by antigen recognition (2, 3). While the data indicate that the CTL response to HBV plays a critical role in viral clearance and disease pathogenesis, the extent to which viral clearance depends on the destruction of infected cells is not known at this time.We have shown that hepatocellular HBV gene expression in transgenic mice is negatively regulated, noncytopathically, by the pharmacological administration of recombinant interleukin (IL) 2 and tumor necrosis factor (TNF) a and that the IL-2 effect is mediated by TNF-a, which inhibits HBV gene expression by a posttranscriptional mechanism in these animals (4-6). We now report that hepatocellular HBV gene expression is profoundly inhibited, noncytopathically, by HBsAg-specific class I-restricted CTLs activated physiologically by antigen recognition in vivo, and we show that the inhibitory effect of the CTLs, which is mediated by TNF-a and interferon y (IFN-y), greatly exceeds their cytopathic effect in magnitude and duration. MATERIALS AND METHODSHBV Transgenic Mice. The two lineages studied were selected because they do not develop evidence of spontaneous liver disease and they display lineage-specific differences...