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Chronically HBV infected subjects are more than 260 million worldwide; cirrhosis and liver cancer represent possible outcomes which affect around 700,000 patients per year. Both innate and adaptive immune responses are necessary for viral control and both have been shown to be defective in chronic patients. Metabolic remodeling is an essential process in T cell biology, particularly for T cell activation, differentiation and survival. Cellular metabolism relies on the conversion of nutrients into energy to support intracellular processes, and to generate fundamental intermediate components for cell proliferation and growth. Adaptive immune responses are the central mechanisms for the resolution of primary human infections leading to the activation of pathogen-specific B and T cell functions. In chronic HBV infection the anti-viral immune response fails to contain the virus and leads to persistent hepatic tissue damage which may finally result in liver cirrhosis and cancer. This T cell failure is associated with metabolic alterations suggesting that control of nutrient uptake and intracellular utilization as well as correct regulation of intracellular metabolic pathways are strategic for T cell differentiation during persistent chronic infections. This review will discuss some of the main features of the T cell metabolic processes which are relevant to the generation of an efficient antiviral response, with specific focus on their clinical relevance in chronic HBV infection in the perspective of possible strategies to correct deregulated metabolic pathways underlying T cell dysfunction of chronic HBV patients., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

A great effort of research has been devoted in the last few years to developing new anti-HBV therapies of finite duration that also provide effective sustained control of virus replication and antigen production. Among the potential therapeutic strategies, immune-modulation represents a promising option to cure HBV infection and the adaptive immune response is a rational target for novel therapeutic interventions, in consideration of the key role played by T cells in the control of virus infections. HBV-specific T cells are severely dysfunctional in chronic HBV infection as a result of several inhibitory mechanisms which are simultaneously active within the chronically inflamed liver. Indeed, the liver is a tolerogenic organ harboring different non-parenchymal cell populations which can serve as antigen presenting cells (APC) but are poorly efficient in effector T cell priming, with propensity to induce T cell tolerance rather than T cell activation, because of a poor expression of co-stimulatory molecules, up-regulation of the co-inhibitory ligands PD-L1 and PD-L2 upon IFN stimulation, and production of immune regulatory cytokines, such as IL10 and TGF-β. They include resident dendritic cells (DCs), comprising myeloid and plasmacytoid DCs, liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), hepatic stellate cells (HSCs) as well as the hepatocytes themselves. Additional regulatory mechanisms which contribute to T cell attrition in the chronically infected liver are the high levels of soluble mediators, such as arginase, indoleamine 2,3-dioxygenase (IDO) and suppressive cytokines, the up-regulation of inhibitory checkpoint receptor/ligand pairs, the expansion of regulatory cells, such as CD4+FOXp3+ Treg cells, myeloid-derived suppressor cells and NK cells. This review will deal with the interactions between immune cells and liver environment discussing the different mechanisms which contribute to T cell dysfunction in chronic hepatitis B, some of which are specifically activated in HBV infection and others which are instead common to chronic inflammatory liver diseases in general. Therapeutic interventions targeting dysregulated pathways and cellular functions will be also delineated., (Copyright © 2020 Fisicaro, Barili, Rossi, Montali, Vecchi, Acerbi, Laccabue, Zecca, Penna, Missale, Ferrari and Boni.)

Objective: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability., Methods: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry., Results: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity)., Conclusions: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy., (Copyright © 2018. Published by Elsevier Ltd.)

Chronic hepatitis B virus (HBV) infection represents a worldwide public health concern with approximately 250 million people chronically infected and at risk of developing liver cirrhosis and hepatocellular carcinoma. Nucleos(t)ide analogues (NUC) are the most widely used therapies for HBV infection, but they often require long-lasting administration to avoid the risk of HBV reactivation at withdrawal. Therefore, there is an urgent need to develop novel treatments to shorten the duration of NUC therapy by accelerating virus control, and to complement the effect of available anti-viral therapies. In chronic HBV infection, virus-specific T cells are functionally defective, and this exhaustion state is a key determinant of virus persistence. Reconstitution of an efficient anti-viral T cell response may thus represent a rational strategy to treat chronic HBV patients. In this perspective, the enhancement of adaptive immune responses by a checkpoint inhibitor blockade, specific T cell vaccines, lymphocyte metabolism targeting, and autologous T cell engineering, including chimeric antigen receptor (CAR) and TCR-redirected T cells, constitutes a promising immune modulatory approach for a therapeutic restoration of protective immunity. The advances of the emerging immune-based therapies in the setting of the HBV research field will be outlined.

Background: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation., Methods: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry., Results: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001)., Conclusions: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Background & Aims: In patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, antiviral functions of T cells are impaired; these might be increased by blocking T-cell co-inhibitory pathways, such as preventing interaction between the receptor programmed death (PD)-1 and its ligand, PD-L1. We attempted to optimize the restoration of T-cell functions in patients with chronic HBV or HCV infection with a combination of reagents that block PD-1 interaction with PD-L1 and stimulate T-cell signaling via CD137, a member of the tumor necrosis factor-receptor family., Methods: We assessed the effects of CD137 stimulation (via CD137L), alone or in combination with antibodies that block PD-1 interaction with PD-L1 (anti-PD-L1), on proliferation and production of interferon-γ and interleukin-2 by intrahepatic and peripheral T cells from patients with chronic HBV or HCV infection. We also analyzed expression of different co-stimulatory molecules on virus-specific CD8+ and forkhead box P3+CD4+ cells by flow cytometry., Results: Incubation of intrahepatic T cells with CD137L and anti-PD-L1 increased their responses to HBV, but not HCV. However, HCV-specific T cells isolated from peripheral blood were sensitive to these reagents. Virus-specific T cells from some, but not all patients, had increased responses to anti-PD-L1 when CD137L was added because in some cases the combination of anti-PD-L1 and CD137L overstimulated T cells, leading to their inhibition. Intrahepatic HBV- and HCV-specific CD8+ T cells had different costimulatory profiles; liver cells from patients with chronic HBV infection had a higher proportion of forkhead box P3+ regulatory T cells, with higher levels of PD-1, compared with liver cells from patients with chronic HCV infection., Conclusions: A combination of reagents that prevent interaction between PD-1 and its ligand and activate CD137 signaling increase responses of intrahepatic HBV-specific T cells and circulating HCV-specific T cells. This strategy might be developed to increase T-cell responses to these viruses in patients with chronic hepatitis B or C, and tailoring the dose of CD137L administered will help optimize results., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: In patients with chronic hepatitis B virus (HBV) infection, persistent exposure to high concentrations of antigen can disrupt T-cell functions. It is not clear to what extent long-term suppression of HBV by nucleos(t)ide analogues can restore antiviral T-cell functions. We compared HBV-specific T-cell responses of patients treated with nucleos(t)ide analogues with those detected in other conditions of HBV control., Methods: We analyzed intracellular levels of interferon gamma, interleukin-2, and tumor necrosis factor α in HBV-specific T cells after 10 days of stimulation with peptides covering the overall HBV genotype D sequence and ex vivo with selected CD8 epitopes and the corresponding HLA-A2 dextramers. Findings from patients treated with nucleos(t)ide analogues who had complete (HBV DNA negative/antibody to hepatitis B surface antigen positive) or partial (HBV DNA negative/hepatitis B surface antigen positive) control of their infections were compared with those of patients with spontaneous or interferon alfa-induced resolution of acute or chronic infections, inactive HBV carriers, or untreated hepatitis B e antigen-negative patients with chronic infections., Results: Although HBV-specific T cells from nucleos(t)ide analogue-treated patients with complete control of infection were dysfunctional ex vivo, they had efficient responses after in vitro expansion. These responses were comparable to those of patients who spontaneously resolved acute HBV infection. Nucleos(t)ide analogue-treated patients who were HBV DNA negative but hepatitis B surface antigen positive had lower levels of T-cell responses but responses greater than those of untreated patients with chronic infection., Conclusions: In vitro reactivity can be restored to T cells from patients with suppressed HBV infection following long-term treatment with nucleos(t)ide analogues, despite prolonged exposure to large loads of antigen. Immune therapies that increase the antiviral T-cell response might increase the likelihood of complete HBV control in patients undergoing long-term nucleos(t)ide analogue treatment., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The effect of IFN-α therapy on HBV-specific T-cell responses in HBeAg-negative, genotype D, chronic hepatitis B is largely undefined. Understanding to what extent IFN-α can modulate HBV-specific T-cells is important to define strategies to optimize IFN efficacy and to identify immunological parameters to predict response to therapy., Methods: HBV-specific T-cell responses were analyzed longitudinally ex vivo and after expansion in vitro in 15 patients with genotype D, HBeAg-negative chronic hepatitis B treated with peginterferon-α-2a. HBV proteins and synthetic peptides were used to stimulate T-cell responses. Analysis of the CD4 and CD8 T-cell functions was performed by ELISPOT, intracellular cytokine and tetramer staining. The effect of anti-PD-L1 on T-cell functions was also analyzed., Results: Ex vivo IFN-γ production by total HBV-specific T-cells was significantly greater before therapy in patients who showed HBV DNA <50 IU/ml at weeks 24 and/or 48 of therapy. No significant improvement of T-cell proliferation, Th1 cytokine production and cytotoxicity was observed during IFN therapy by both ex vivo and in vitro analysis. PD-1/PD-L1 blockade showed a modest improvement of cytokine production in a total of 15% of T-cell lines., Conclusions: IFN-α did not improve peripheral blood HBV-specific T-cell responses in the first 24 weeks of treatment, consistent either with a predominant antiviral/antiproliferative effect or with an immunomodulatory activity on other arms of the immune system which were not analyzed in our study. A better pre-treatment ex vivo IFN-γ production was associated with better chances to control HBV replication during therapy and represents a promising predictor of IFN efficacy., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The antiviral function of peripheral hepatitis B virus (HBV)-specific T cells can be increased in patients with chronic hepatitis B by blocking the interaction of programmed death (PD)-1 with its ligand PD-L1. However, no information is available about the effects of this blockade on intrahepatic lymphocytes. We studied T-cell exhaustion and the effects of PD-1/PD-L1 blockade on intrahepatic and circulating HBV-specific T cells in patients with chronic hepatitis B., Methods: A total of 42 patients with chronic HBV infection who underwent liver biopsy were studied. The ex vivo phenotype of peripheral and intrahepatic HBV-specific CD8(+) T cells was assessed by flow cytometry with class I tetramers and antibodies to T-cell differentiation molecules. Functional recovery was evaluated by analyzing expansion and production of interferon (IFN)-gamma and interleukin (IL)-2 after short-term incubation of T cells with HBV peptides in the presence of anti-PD-L1 or control antibodies., Results: Intrahepatic HBV-specific CD8(+) cells expressed higher levels of PD-1 and lower levels of CD127 than their peripheral counterparts. Blockade of PD-1/PD-L1 interaction increased CD8(+) cell proliferation and IFN-gamma and IL-2 production by circulating intrahepatic lymphocytes, even though anti-PD-L1 had a stronger effect on intrahepatic compared with peripheral T cells., Conclusions: T-cell exhaustion by high antigen concentrations promotes HBV-specific T-cell dysfunction by affecting phenotype and function of peripheral and intrahepatic T cells. By restoring antiviral T-cell functions, not only in peripheral but also in intrahepatic lymphocytes, anti-PD-L1 might be a good therapeutic candidate for chronic HBV infection.

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA in serum and/or in the liver of patients negative for hepatitis B surface antigen (HBsAg). Occult infection may impact in several different clinical contexts including the risk of HBV transmission with transfusion or transplantation, and endogenous viral reactivation. The gold standard test for detection of occult infection is the amplification of HBV DNA. However, the serological assay for the long-lasting antibody response to the highly immunogenic HBV core antigen (anti-HBc) represents a qualified candidate as a surrogate for DNA amplification, or for increasing overall sensitivity when assessing the risk of occult hepatitis in peripheral blood. The risk of occult hepatitis associated with anti-HBc seropositivity has been demonstrated extensively, and the presence of antibody response to HBc can be considered a sentinel marker of occult HBV infection.

Background & Aims: Hepatitis B virus (HBV) DNA detection in serum and/or in the liver of hepatitis B surface antigen (HBsAg)-negative patients with or without serologic markers of previous viral exposure is defined as occult HBV infection. Because the role of the adaptive response in keeping HBV replication under control in occult infection still is undefined, this study was performed to characterize the features of the HBV-specific T-cell response in this condition., Methods: HBV-specific T-cell frequency and function were tested ex vivo and after in vitro expansion in 32 HBsAg-negative patients undergoing diagnostic liver biopsy for chronic hepatitis C: 18 with occult HBV infection (11 anti-HBc-negative and 7 anti-HBc-positive patients) defined by the detection of intrahepatic HBV DNA by polymerase chain reaction; 14 without detectable intrahepatic HBV DNA (5 anti-HBc-positive and 9 anti-HBc-negative patients). Six patients with chronic hepatitis B and 7 HBsAg-inactive carriers were studied for comparison., Results: The presence or absence of serologic HBV markers defined 2 profiles of HBV-specific T-cell responses in occult infection. Anti-HBc-positive patients showed a T-cell response typical of protective memory, suggesting that this condition represents a resolved infection with immune-mediated virus control. In contrast, HBV-specific T cells in anti-HBc-negative patients did not readily expand and produce interferon-gamma in vitro, suggesting the possibility of a low-dose infection insufficient to allow maturation of protective memory., Conclusions: Our results suggest different mechanisms of control of viral replication in seropositive and seronegative occult infections. Additional studies aimed at understanding possible different clinical implications are needed.

Dysfunctional CD8+ T cells present in chronic virus infections can express programmed death 1 (PD-1) molecules, and the inhibition of the engagement of PD-1 with its ligand (PD-L1) has been reported to enhance the antiviral function of these T cells. We took advantage of the wide fluctuations in levels of viremia which are typical of chronic hepatitis B virus (HBV) infection to comprehensively analyze the impact of prolonged exposure to different virus quantities on virus-specific T-cell dysfunction and on its reversibility through the blocking of the PD-1/PD-L1 pathway. We confirm that chronic HBV infection has a profound effect on the HBV-specific T-cell repertoire. Despite the use of a comprehensive panel of peptides covering all HBV proteins, HBV-specific T cells were rarely observed directly ex vivo in samples from patients with chronic infection, in contrast to those from patients with acute HBV infection. In chronic HBV infection, virus-specific T cells were detected mainly in patients with lower levels of viremia. These HBV-specific CD8+ T cells expressed PD-1, and their function was improved by the blocking of PD-1/PD-L1 engagement. Thus, a broad spectrum of anti-HBV immunity is expressed by patients with chronic HBV infection and this spectrum is proportional to HBV replication levels and can be improved by blocking the PD-1/PD-L1 pathway. This information may be useful for the design of immunotherapeutic strategies to complement and optimize available antiviral therapies.

To characterize acute-phase hepatitis B virus (HBV)-specific T cell responses associated with self-limited and persistent HBV infections, we compared a patient with acute HBV/HCV coinfection, who was able to control HCV but developed chronic hepatitis B, with patients who resolved acute HBV infection spontaneously. Acute-phase CD4 responses were efficient in self-limited infections but undetectable in the coinfected patient with HBV persistence. CD8 responses were multispecific irrespective of the outcome of infection, but the CD8 repertoire associated with HBV persistence lacked the most dominant specificities detectable in self-limited infections. In conclusion, insufficient CD4 help and defective CD8 repertoire may play a role at the early stages of infection in influencing HBV persistence.

Previous studies on CTL responses in HIV-exposed uninfected individuals assumed that the patients were exposed to replicating HIV, but the possibility that the immune responses detected were primed by exposure to a defective virus or viral antigen could not be excluded. Epidemiological and laboratory analysis of a nosocomial outbreak of acute hepatitis B unequivocally allowed the identification of an HIV-1- and HBV-co-infected patient with high plasma levels of both viruses, as the source case of the epidemics. This clinical setting provided a natural model for testing the HIV-specific T cell response in patients exposed to blood from a patient with highly replicating HIV. Parenteral exposure to both viruses led to acute hepatitis B in five subjects without evidence of HIV-1 infection. Cryopreserved lymphocytes derived from three exposed patients were tested ex vivo in an ELISPOT assay for IFN-gamma release upon stimulation with peptides from structural and non-structural HIV proteins; one of the patients was also tested with four HLA/class I tetramers. Circulating HIV-specific CD8 cells were detected by tetramer staining and a high frequency of T cells were able to release IFN-gamma upon stimulation with HIV peptides, showing in vivo T cell priming by HIV. These results unequivocally demonstrate a HIV-specific cell-mediated immune response in the absence of infection after exposure to highly replicating HIV.

Background and Aims: Occult hepatitis B virus (HBV) infection is characterized by persistence of HBV DNA into the tissue of hepatitis B surface antigen-negative individuals. The clinical relevance of this peculiar infection is still under debate. In particular, the impact of occult HBV infection in cases of hepatocellular carcinoma (HCC) is uncertain. We investigated the prevalence and molecular status of occult HBV in patients with HCC., Methods: We tested tumor tissues from 107 patients with HCC and the corresponding nontumor liver tissue from 72 of these patients for HBV DNA. We also examined liver specimens from 192 patients with chronic hepatitis. All cases were hepatitis B surface antigen negative. Covalently closed circular HBV genomes, HBV transcripts, and viral integrated forms were investigated in cases of HCC found positive for occult HBV., Results: Viral DNA was detected in 68 of 107 cases of HCC (63.5%) and in 63 of 192 cases of chronic hepatitis (32.8%) (P < 0.0001; odds ratio, 3.6; 95% confidence interval, 2.2-5.9). The significant association of occult HBV with HCC was irrespective of age, sex, and contemporary hepatitis C virus infection. Both integrated viral DNA and covalently closed circular HBV genomes were detected in patients with occult HBV. Moreover, the presence of free HBV genomes was associated with persistence of viral transcription and replication., Conclusions: Our findings provide clear evidence that occult HBV is a risk factor for development of HCC and show that the potential mechanisms whereby overt HBV might induce tumor formation are mostly maintained in cases of occult infection.

Background/aims: Lamivudine therapy in patients with chronic hepatitis B can induce the recovery of antiviral T cell responses. It is unknown whether the recovery of T cell responsiveness is long-lasting and persists throughout the treatment and whether the elevation of viremia which follows therapy withdrawal can restore a condition of T cell unresponsiveness., Methods: Frequency and function of circulating hepatitis B virus (HBV)-specific CD4 and CD8 cells from 12 hepatitis e surface antigen + patients with chronic hepatitis B were studied longitudinally before, during and after lamivudine therapy by intracellular cytokine staining, proliferation and cytotoxicity assays against HBV proteins and peptides. CD4-mediated responses were analyzed in all patients, whereas CD8 cells were studied in 6 HLA-A2+ patients., Results: HBV-specific CD4 and CD8 reactivity showed a bi-phasic behavior under lamivudine therapy with an early enhancement of T cell frequency and intensity of responses followed by a persistent decline starting from the 5th to 6th month of treatment., Conclusions: Since restoration of HBV-specific T cell reactivity is only transient, our study indicates that therapeutic stimulation of HBV-specific T cell responses to complement lamivudine treatment should be done early after the initiation of lamivudine. Moreover, the transient nature of the immune reconstitution may represent a favorable condition for virus reactivation once lamivudine therapy is withdrawn.

Hepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7(-) CD45RA(-)). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-gamma) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-gamma production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a 10-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic peptide-based immunotherapeutic strategies to terminate persistent viral infection.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.