PD-1表达对急性乙型肝炎患者HBV特异性CD8＋T细胞功能的影响

目的 观察急性乙型肝炎发病过程中患者体内病毒特异性CD8^＋细胞（CTL）上免疫抑制性分子程序性细胞死亡受体1（PD-1）的表达及临床意义。方法合成4种识别HBV不同抗原表位的五聚体，同时采集16例HLA-A2阳性急性乙型肝炎患者外周血，用五聚体染色后，通过流式细胞仪分析病毒特异性CTL细胞的频率及其表面PD-1分子的表达特点。同时进行肝功能、乙型肝炎病毒（HBV）血清标志物和血清HBVDNA检测。结果所有急性乙型肝炎患者发病早期均表现出多特异性的CTL反应，且PD-1在这些细胞上表达明显上调，显著高于来自同一患者的巨细胞病毒（CMV）和Flu特异性CTL上PD-1分子的表达水平。体外阻断PD-1／PD-L1间的相互作用能够恢复HBV特异性CTL分泌细胞因子的能力。相关性分析显示PD-1分子的上调表达与转氨酶水平正相关。结论在HBV急性感染过程中，尤其是在HBV基本被清除后，HBV特异性CTL细胞PD-1分子表达明显升高，有效抑制病毒特异性CTL分泌IFN-γ的能力，后者可能有利于避免急性乙型肝炎患者肝脏遭受更为严重的免疫损伤。     

乙型肝炎病毒感染临床转归与宿主细胞免疫的相关性研究

目的 比较慢性乙型肝炎患者、HBV携带者、HBV既往无症状感染者之间T细胞亚群和HBV特异性CD4+ T细胞应答强度的差异,分析宿主的细胞免疫状态对HBV感染后临床转归的影响,探讨乙型肝炎的发病机制,为慢性乙型肝炎的治疗提供新的线索.方法 选取2004年2-10月在北京协和医院肝炎门诊就诊的慢性乙型肝炎患者30例、HBV携带者22例、HBV既往无症状感染者9例以及正常对照11例,使用流式细胞仪检测其T细胞亚群,使用酶联免疫斑点法检测病毒特异性CD4+T细胞应答强度,分析其差异及临床意义.结果 慢性乙型肝炎组CD4+T细胞计数显著低于HBV既往无症状感染组和正常对照组;慢性乙型肝炎组、HBV携带者组、HBV既往无症状感染组病毒特异性CD4+ T细胞应答强度分别为(156±105)、(56±68)、(229±114)SFC/106PBMC(每106个外周血单个核细胞中斑点形成细胞的个数);慢性乙型肝炎组明显高于HBV携带者组(P＜0.01),而低于HBV既往无症状感染组(P＜0.05).结论 慢性乙型肝炎患者、HBV携带者、HBV既往无症状感染者病毒特异性T细胞应答强度存在差异,这种差异可能是造成HBV感染后不同临床转归的主要因素之一.     

Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection

BACKGROUND & AIMS: Cytotoxic T cells have been suggested to be responsible for lysis of hepatitis B virus (HBV)-infected hepatocytes and control of virus infection. The frequency, kinetics, phenotype, and capacity for clonal expansion of circulating HBV-specific CD8 cells were analyzed directly in patients with acute HBV infection to clarify their pathogenetic role. METHODS: Three HLA-A2 peptide tetramers able to visualize HBV core, envelope, and polymerase epitope-specific cytotoxic T lymphocytes were synthesized and used for flow cytometric analysis of antigen-specific populations. RESULTS: Tetramer-positive cells specific for the core 18-27 epitope were found at a higher frequency than those specific for polymerase 575-583 and envelope 335-343 epitopes in most patients with acute HBV. The number of HBV-specific CD8 cells was highest during the clinically acute stage of infection and decreased after recovery. These cells expressed an activated phenotype and had an impaired capacity to expand in vitro and to display cytolytic activity in response to peptide stimulation. Recovery of these functions was observed when the frequency of specific CD8 cells decreased, coincident with a progressive decrease in their expression of activation markers. CONCLUSIONS: This study provides the first ex vivo evidence that the highest frequency of circulating HBV-specific CD8 cells coincides with the clinically acute phase of hepatitis B. These cells exhibit an activated phenotype with limited further proliferative capacity that is restored during recovery.     

Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen

BACKGROUND & AIMS: Impaired T-cell reactivity is believed to be the dominant cause of chronic hepatitis B virus (HBV) infection. We characterized HBV-specific T-cell responses in chronic hepatitis B surface antigen carriers who received bone marrow from HLA-identical donors with natural immunity to HBV and seroconverted to antibody to hepatitis B surface antigen. METHODS: T-cell reactivity to HBV antigens and peptides was assessed in a proliferation assay, the frequency of HBV core- and surface-specific T cells was quantified directly by ELISPOT assays, and T-cell subsets were analyzed by flow cytometry. RESULTS: CD4+ T-cell reactivity to HBV core was common in bone marrow donors and the corresponding recipients after hepatitis B surface antigen clearance, whereas none reacted to surface, pre-S1, or pre-S2 antigens. Furthermore, CD4+ T cells from donor/recipient pairs recognized similar epitopes on hepatitis B core antigen; using polymerase chain reaction for the Y chromosome, the recipients' CD4+ T lymphocytes were confirmed to be of donor origin. The frequency of core-specific CD4+ and CD8+ T cells was several-fold higher than those specific for surface antigen. CONCLUSIONS: This study provides the first evidence in humans that transfer of hepatitis B core antigen-reactive T cells is associated with resolution of chronic HBV infection. Therapeutic immunization with HBV core gene or protein deserves further investigation in patients with chronic hepatitis B.     

急性乙型肝炎患者程序性细胞死亡受体1表达与记忆性T淋巴细胞形成的关系

目的 观察急性自限性乙型肝炎发病过程中患者体内病毒抗原特异性细胞毒性T淋巴细胞(CTL)上程序性细胞死亡受体1(PD-1)表达的动态变化特点及其与记忆性抗原特异性CD8+T淋巴细胞形成的关系. 方法 针对不同表位合成4种五聚体,长期随访收集11例人类白细胞抗原(HLA)-A2阳性的急性乙型肝炎患者的外周血,流式细胞仪检测病毒特异性CTL上免疫抑制性分子PD-1、记忆性分子(CCR7、CD45RA、CD127)和活化标志物CD38的表达情况,并分析其相关性.同时进行肝功能,HBsAg、抗-HBs和血清HBV DNA载量检测.结果所有急性自限性乙型肝炎患者发病早期均表现出高频度、病毒抗原多表位的特异性CTL反应,而晚期各表位CTL频率均明显下降.CTL上PD-1表达在早期明显上调;与早期比较,晚期PD-1分子的表达明显降低(t=4.314,P＜0.01).同时CTL高表达记忆性分子CCR7,CD45RA和CD127,而低表达活化标志物CD38.提示病毒清除后记忆性CD8+T淋巴细胞形成. 结论 在急性自限性乙型肝炎发病过程中,HBV特异性CTL上PD-1分子表达的动态变化与记忆性T淋巴细胞的形成密切相关.     

Programmed death-1 expression is associated with the disease status in hepatitis B virus infection

AIM： TO define the potential role of programmed death-i/programmed death-ligand （PD-1/PD-L） pathway in different hepatitis B virus （HBV） infection disease status; we examined the expression of PD-1 on antigen specific CD8＋T cells in peripheral blood of patients with chronic hepatitis B （CriB） and acute exacerbation of hepatitis B （AEHB） infection.
METHODS： The PD-1 level on CD8＋ T lymphocytes and the number of HBV specific CD8＋ T lymphocytes in patients and healthy controls （HCs） were analyzed by staining with pentameric peptide-human leukocyte antigen2 （HLA2） complexes combined with flow cytometry. Real-time quantitative polymerase chain reaction （PCR） was used to measure the serum HBV- DNA levels.
RESULTS： The level of PD-1 expression on total CD8＋ T cells in CHB patients （13.86% ± 3.38%） was significantly higher than that in AEHB patients （6.80%± 2.19%, P 〈 0.01） and healthy individuals （4.63% ± 1.23%, P 〈 0.01）. Compared to AEHB patients （0.81% ± 0.73%）, lower frequency of HBV-specific CD8＋ T cells was detected in chronic hepatitis B patients （0.37% ± 0.43%, P 〈 0.05）. There was an inverse correlation between the strength of HBV-specific CD8＋ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8＋ T cells in CriB and AEHB subjects （R = 0.541, P 〈 0.01）. However, PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase （ALT） levels （R = 0.066, P 〉 0.05）.
CONCLUSION： Our results confirm previous reports that HBV specific CD8＋T-cell response in the peripheral blood is more intense in patients with AEHB than in chronic hepatitis B wlth persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8＋ T cell response.     

Depletion of CD25^＋CD4^＋T cells （Tregs） enhances the H BV-specific CD8^＋ T cell response primed by DNA immunization

AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.     

Analysis of intrahepatic HBV-specific cytotoxic T-cells during and after acute HBV infection in humans

BACKGROUND/AIMS: Characteristics of the intrahepatic virus-specific T-cell response in patients with acute hepatitis B virus (HBV) infection have not been studied due to the risk of complications associated with standard liver biopsies. In this study we aimed to characterize the virus-specific CD8 + T-cell response in the liver of patients with acute HBV infection using fine-needle aspiration-biopsy (FNAB). METHODS: In HLA-A2 positive patients with acute HBV infection a FNAB was performed at first presentation, at the time of HBsAg-seroconversion and 3 months after HBsAg-seroconversion. HLA-A2 tetramers were used to identify HBV-specific CD8 + T-cells in FNAB-cytology and peripheral blood (PB). RESULTS: At first presentation there was a correlation between the frequency of intrahepatic CD8 + T-cells and the degree of liver damage. At all time points there was sequestering of HBV-specific CD8 + T-cells in the liver, and the percentage of intrahepatic HLA-DR expressing HBV-specific CD8 + T-cells was higher than in PB. Three months after HBsAg-seroconversion the frequency of intrahepatic HBV-specific CD8 + T-cells remained high. CONCLUSIONS: HBV-specific CD8 + T-cells are compartmentalized in the liver during acute HBV infection. Their presence in the liver may suggest a role in the resolution of the infection. Intrahepatic HBV-specific CD8 + T cells remain detectable at high frequencies after HBsAg-seroconversion.     

Antiviral CD8-mediated responses in chronic HCV carriers with HBV superinfection

Hepatitis B virus (HBV) superinfection in chronic hepatitis C represents a natural model to investigate whether or not hepatitis C virus (HCV) can influence priming and maturation of antiviral T cells; whether or not HBV superinfection, which is known to determine control of HCV replication, can restore HCV-specific T cell responsiveness; and whether or not cytokines stimulated by HBV infection can contribute to HCV control. To address these issues, the function of CD8 cells specific for HBV and HCV was studied longitudinally in two chronic HCV patients superinfected with HBV. Patients with acute hepatitis B were also examined. Frequency and function of HBV tetramer+ CD8 cells were comparable in patients acutely infected with HBV with or without chronic HCV infection. HBV-specific CD8 cell function was efficiently expressed irrespective of serum HCV-RNA levels. Moreover, fluctuations of HCV viremia at the time of HBV superinfection were not associated with evident changes of CD8 responsiveness to HCV. Finally, no correlation was found between serum levels of interferon alpha, interleukin (IL)-12, IL-10, or IL-18 and control of HCV replication. In conclusion, HCV did not affect the induction of primary and memory HBV-specific CD8 responses. HCV-specific CD8 responses were undetectable when HCV-RNA was negative, showing that inhibition of HCV replication in the setting of a HBV superinfection was not sufficient to induce a restoration of CD8 reactivity against HCV.     

Liver injury is associated with enhanced regulatory T-cell activity in patients with chronic hepatitis B

Chronic hepatitis B virus (HBV) infection is associated with impairment of HBV-specific immune responses. Recently, it has been shown that regulatory T (Treg) cells downregulate HBV-specific immune responses but their role in chronic hepatitis B is still controversial. We hypothesized that liver injury enhances the influence of Treg cells on HBV-specific immune responses. The frequency of Treg cell and the in vitro expansion of HBV-specific CD8+ T cell detected by the tetramer method were investigated in 79 patients with chronic hepatitis B. Thirty-three healthy volunteers were enrolled to measure the frequency of Treg cell as controls. The results showed that in chronic hepatitis B cases, the frequency of Treg cells in peripheral blood was significantly higher than that in normal volunteers. The higher level of serum transaminase was associated with higher frequency of Treg cells, which both had a linear correlation relationship. HBV-DNA level, HBe status, age and sex had no statistical association with Treg cell frequency. Furthermore, in patients with higher serum transaminase levels, the expansion of HBV-specific CD8+ T cells was higher after removal of Treg cells when compared with patients with lower serum transaminase levels. In conclusion, our data indicate a significant association between serum transaminase level and frequency/activity of Treg cells. Based on this observation, we propose that liver-injury enhances Treg cell frequency/activity in chronic hepatitis B patients.     

Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice

The strength of antiviral T cell responses correlates with clearance of hepatitis B virus (HBV) infection, but the immunological mechanisms mitigating or suppressing HBV-specific T cells are still poorly understood. In this study, we examined the role of CD4(+) Foxp3(+) regulatory T cells (Tregs) in a mouse model of acute HBV infection. We initiated HBV infection via an adenoviral vector transferring a 1.3-fold overlength HBV genome (AdHBV) into transgenic DEREG mice, where Tregs can be transiently but selectively depleted by injection of diphtheria toxin. The effect of Treg depletion on the outcome of HBV infection was characterized by detailed virological, immunological, and histopathological analysis. Numbers of Tregs increase in the liver rapidly after initiation of HBV replication. Initial depletion of Tregs revealed their complex regulatory function during acute infection. Tregs mitigated immunomediated liver damage by down-regulating the antiviral activity of effector T cells by limiting cytokine production and cytotoxicity, but did not influence development of HBV-specific CD8 T cells or development of memory T cells. Furthermore, Tregs controlled the recruitment of innate immune cells such as macrophages and dendritic cells to the infected liver. As a consequence, Tregs significantly delayed clearance of HBV from blood and infected hepatocytes. Conclusion: Tregs limit immunomediated liver damage early after an acute infection of the liver, thereby contributing to conservation of tissue integrity and organ function at the cost of prolonging virus clearance. (HEPATOLOGY 2012;56:873-883).     

The impact of HBV-DNA fluctuations on virus-specific CD8+ T cells in HBeAg+ chronic hepatitis B patients treated with a steroid and lamivudine

Restoration of anti-viral immune response may be a requisite for sustained virological response to treatment in chronic hepatitis B patients. Over a 13-month period, we examined the dynamics of hepatitis B virus (HBV)-specific CD8+ cells in six human leucocyte antigen (HLA)-A2+ hepatitis B e antigen (HBeAg)+ 'immunotolerant' chronic hepatitis B patients treated sequentially with corticosteroid and lamivudine. Our results show that the combination treatment did not result in a sustained restoration of anti-viral specific CD8+ cells in five of the six patients studied. However, HBV-specific CD8+ cells, despite being severely compromised, were not totally deleted. Paradoxically, steroid treatment was not associated with inhibition but with a minimal increase of the HBV-specific CD8 response, and we observed that nucleocapsid-specific CD8 responses were not rescued by stable and prolonged inhibition but became detectable after rapid rebounds of HBV replication. In most patients, the transient and minimal restoration of HBV-specific immunity was not associated with clinical benefits. Our results describe a dynamic relationship between HBV-specific CD8+ cells and HBV-DNA values, that could potentially be used for a better design of HBV treatment in HBeAg+ 'immunotolerant' chronic hepatitis B patients.     

Kinetics of peripheral hepatitis B virus-specific CD8+ T cells in patients with onset of viral reactivation

Background

Patients with resolved hepatitis B virus (HBV) infection undergoing chemotherapy or immunosuppressive therapy are potentially at risk of HBV reactivation. However, it remains unclear how liver disease develops after HBV reactivation. To compare the host immune response against HBV, we performed immunological analyses of six HBV reactivation patients.

Methods

The numbers of peripheral HBV-specific CD8⁺ T cells were investigated longitudinally in six HLA-A2- and/or A24-positive patients with HBV reactivation. In addition, 34 patients with resolved HBV, 17 patients with inactive chronic hepatitis B (ICHB), 17 patients with chronic hepatitis B (CHB) and 12 healthy controls were analyzed. The number and function of HBV-specific CD8⁺ T cells were assessed by flow cytometry using tetramer staining and intracellular IFN-γ production. Furthermore, the numbers of CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells and serum inflammatory cytokine levels were analyzed.

Results

The frequency of HBV-specific CD8⁺ T cells was significantly increased in HBV reactivation patients compared with ICHB and CHB patients. In addition, the number of HBV-specific CD8⁺ T cells was increased in resolved HBV patients compared with ICHB patients. PD-1 expression was decreased in HBV reactivation patients compared with ICHB and CHB patients. The numbers of HBV-specific CD8⁺ T cells and CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T cells were negatively correlated following onset of HBV reactivation.

Conclusions

During HBV reactivation, the frequency of HBV-specific CD8⁺ T cells increased even though the administration of immunosuppressive drugs and interactions with CD4⁺ regulatory T cells may be important for the onset of liver disease.     

DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.     

慢性乙型肝炎患者外周血CD8~+T细胞CD95表达的研究

目的检测慢性乙型肝炎患者外周血CD8+T细胞CD95分子的表达水平,了解CD95分子表达与炎症进展的关系。方法采集37例慢性乙型肝炎患者及健康对照者外周血,分离外周血单个核细胞(PBMCs),用流式细胞术检测外周血CD8+T细胞CD95分子的表达情况。结果高病毒载量组和低病毒载量组CD8+T细胞CD95的表达均显著高于健康对照组(P0.05);CD95的表达及CD8+T细胞频率之间存在负相关关系(r=-0.3486)。结论慢性乙型肝炎患者CD95的高表达可能促进了CD8+T细胞的凋亡,使CD8+T细胞的抗病毒作用减弱,可能与HBV持续感染、慢性乙型肝炎的形成机制有关。     

Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.     

Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection

An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4(hi) Bim(hi) tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-γ)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-HBV responses by either CTLA-4 or PD-L1 blockade is nonredundant. CONCLUSION: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease.     

Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.     

Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.