慢性乙型肝炎患者外周血及肝组织中CD4+CD25+调节性T细胞和乙型肝炎病毒特异性细胞毒性T淋巴细胞的表达及意义

目的 研究CD4+CD25+调节性T细胞和HBV特异性CTL在慢性乙型肝炎患者外周血和肝组织中的表达和临床意义.方法 流式细胞分析技术和流式细胞术细胞因子测定法(CFC)检测157例HBV感染者(包括急性乙型肝炎20例、慢性乙型肝炎115例、乙型肝炎肝硬化22例)和20例健康对照组外周血和部分肝组织中CD4+CD25+调节性T细胞和HBV特异性CTL的表达.组间分析采用t检验.结果急性乙型肝炎,慢性乙型肝炎轻、中、重度患者外周血中CD4+CD25+调节性T细胞分别为(2.87±0.94)%、(3.53±1.56)%、(4.59±2.98)%和(3.65±1.73)%,明显高于对照组的(2.36±0.60)%(t值分别为2.04、5.97、3.30和3.17,P<0.01);慢性乙型肝炎轻、中、重度和乙型肝炎肝硬化患者外周血HBV特异性CTL为(0.189土0.152)%、(0.103±0.110)%、(0.118±0.120)%和(0.098±0.101)%,明显低于急性乙型肝炎患者的(0.815±0.360)%(t值分别为10.09、11.87、9.17和8.96,P<0.01).肝组织中CD4+CD25+调节性T细胞和HBV特异性CTL的表达高于外周血.结论 CD4+CD25+调节性T细胞可能通过抑制CD8+T淋巴细胞在机体抗病毒过程中发挥重要作用.     

慢性HBV感染患者外周血CD4~+CD25~+调节性T细胞表达及其临床意义

目的探讨CD4+CD25+调节性T细胞与慢性HBV感染后不同临床转归和临床特点的相关性。方法在26例慢性乙型肝炎(CHB)患者、15例无症状HBsAg携带者(ASC)和11例肝炎肝硬化(LC)患者和16例正常对照者,分离外周血单个核细胞(PBMC),采用流式细胞仪检测CD4+CD25+调节性T细胞的表达水平。结果CHB组和ASC组的CD4+CD25+调节性T细胞占CD4+T细胞的百分率分别为4.40±2.76%和4.43±2.10%,均高于正常对照组(2.70±0.97%),差异显著(P0.01);CD4+CD25+调节性T细胞的表达水平与HBVDNA水平无相关性(r=0.018,P0.05);在HBeAg阳性与阴性组患者CD4+CD25+调节性T细胞的表达也无明显的差异(P0.05)。结论慢性HBV感染者外周血CD4+CD25+调节性T细胞水平升高,可能与HBV感染的慢性化有关。     

CD4+ CD25+调节性T细胞在重型乙型肝炎发病中的作用

目的探讨CD4 CD25 调节性T细胞在重型乙型肝炎病人发病机制中的作用。方法采用流式细胞仪检测30例重型乙型肝炎患者、20例慢性乙型肝炎患者、20例无症状乙肝病毒携带者和10例健康的外周血CD4 CD25 调节性T细胞(CD4 CD25 Treg)的水平,并应用荧光定量PCR方法测定上述研究对象血中HBVDNA滴度。结果(1)重型乙型肝炎组外周血CD4 CD25 调节性T细胞的平均百分率为(2.63±0.83)%,较慢性乙型肝炎组的(4.15±1.17)%有显著差异(P<0.05),较无症状乙肝病毒携带者组及健康对照组则有极其显著差异(P均<0.01);(2)慢性乙型肝炎组外周血CD4 CD25 调节性T细胞的百分率与无症状乙肝病毒携带者及健康对照组,有显著的差异(P<0.05);无症状乙型肝炎携带者组外周血调节CD4 CD25 T细胞的百分率为(8.32±2.72)%,与健康对照组(8.10±2.65)%比较无差异;(3)重型乙型肝炎组外周血HBVDNA滴度为1.2×104拷贝/ml,与慢性乙型肝炎组(2.3×106拷贝/ml)和无症状乙肝病毒携带者(7.8×105拷贝/ml)相比较,有极其显著差异(P均<0.01);慢性乙型肝炎组外周血HBVDNA滴度与无症状乙肝病毒携带者相比,无差异;(4)无症状乙型肝炎携带者;慢性乙型肝炎和重型乙型肝炎,患者外周血CD4 CD25 调节性T细胞的百分率与HBVDNA滴度呈正相关。结论(1)CD4 CD25 调节性T细胞能抑制T细胞对乙型肝炎病毒的免疫反应,从而抑制乙型肝炎病毒诱导的对肝细胞的免疫攻击的发生;(2)不同乙型肝炎病人外周血CD4 CD25 调节性T细胞的百分率与HBVDNA滴度呈正相关,表明CD4 CD25 调节性T细胞数量对体内HBV载量具有较大的影响;(3)CD4 CD25 调节性T细胞在重型乙型肝炎的病情进展及病毒抑制清除等方面起着重要的作用。     

乙型肝炎病毒感染不同阶段患者外周血CD4+CD25+调节性T细胞的测定

目的 测定HBV感染不同阶段患者外周血CD4+CD25+调节性T细胞(Treg)的频率及标记分子,并分析其与临床指标的相关性.方法 采集79例慢性乙型肝炎(CHB)、12例急性乙型肝炎(AHB)患者、26例无症状HBV携带者(ASC)和20例健康对照的外周血,流式细胞仪分析Treg频率、Treg细胞表面和胞内特征性分子的表达.普通RT-PCR和相对荧光定量PCR测定叉头/翼状转录因子3(Foxp3)在CD25+Treg细胞的表达水平.所有患者及健康对照均经ELISA检测HBV血清标记物水平,实时荧光定量PCR测定血清HBV DNA载量,并进行肝功能检测.结果 总CD4+CD25+T细胞占外周血CD4+T细胞的比率,在各患者组和健康对照组之间差异均无统计学意义(P＞0.05).HBeAg阳性CHB组CD4+CD25高表达T细胞频率(3.42%±0.81%)与HBeAg阴性CHB组(3.19%±0.67%)、ASC组(3.05%±0.64%)比较,差异无统计学意义(均P＞0.05),但明显高于健康对照组(2.72%±0.71%,P=0.034)和AHB组(2.25%±0.54%,P=0.013).CD4+CD25高表达T细胞表面高表达CD45RO、CD25分子,低表达CD45RA,细胞内高表达细胞毒性T淋巴细胞相关抗原-4(CTLA-4)和Foxp3.各患者组及健康对照组Treg中Foxp3表达水平的差异无统计学意义(P＞0.05).CHB组患者的Treg频率与血清病毒载量呈正相关(r=0.48,P=0.018).结论 Treg可能通过抑制T细胞免疫应答反应而影响病毒清除,并与CHB患者的持续感染密切相关.     

CD4+CD25+Foxp3+调节性T细胞在慢性乙型肝炎患者中的作用

目的 探讨CD4+CD25+Foxp3+调节性T细胞与乙型肝炎慢性化和病毒清除之间的关系.方法 收集慢性活动性乙型肝炎(CAH)患者19例、HBV携带者(AsC)21例、HBV感染恢复者12例和健康对照者15例.通过流式细胞术分析外周血CD4+CD25+Foxp3+T细胞的表型和频率,磁珠分选(MACS)CD4+CD25+T细胞,实时荧光定量PCR方法 分析Foxp3 mRNA基因在CD4+CD25+T细胞的表达水平.统计学处理采用单因素方差分析或非参数检验.结果CAH或AsC组外周血CD4+CD25+Foxp3+T细胞频率以及CD4+CD25+T细胞中Foxp3 mRNA的表达水平显著高于健康对照组或HBV感染恢复者(F=6.8,F=3.72,均P<0.05).免疫组织化学染色发现,CAH患者肝组织Foxp3'T细胞浸润累积较对照组明显增高,但AsC较CAH减少.HBeAg阳性患者(包括CAH和AsC)CD4'CD25'T细胞频率显著高于HBeAg阴性患者(t=2.3,P<0.05),抗-HBe阴性患者显著高于抗-HBe阳性患者(t=2.4,P<0.05).CD4+CD25+Foxp3+T细胞频率与慢性乙型肝炎患者血清中HBV病毒载量存在正相关(r=0.56,P<0.01).结论 慢性乙型肝炎患者CD4+CD25+Foxp3+调节性T细胞异常与乙型肝炎慢性化和病毒清除有关.     

慢性乙型肝炎患者CD3+CD56+淋巴细胞及其表型与病情的关系

目的 探讨CD3+CD56+淋巴细胞与慢性乙型肝炎(CHB)患者病情变化和转归的关系.方法 CHB患者53例,HBV携带者17例,19名健康体检者为对照组.研究对象均抽取外周血2～3ml,采用流式细胞技术测定CD3+CD56+淋巴细胞,并进一步分析CD3+CD56+淋巴细胞表面CD4,CD8、T细胞抗原受体(TCR)V α 24,TCR α/β以及TCR γ/δ的表达.结果CHB组CD3+CD56+淋巴细胞为7.4%±4.6%,慢性HBV携带者组为4.5%±3.5%,对照组为4.4%±3.7%,CHB组CD3+CD56+淋巴细胞明显升高.3组人群CD3+CD56+淋巴细胞TCR V α 24的表达,差异无统计学意义.慢性HBV携带者组CD3 CD56+细胞表达的TCR V α 24为2.8%±1.4%,明显高于对照组1.7%±1.0%.CHB组CD3+CD56+细胞CD8和TCRα/β的表达分别为61.9%±16.8%和68.1%±16.9%,对照组为49.2%±15.6%和56.4%±17.9%,CHB组均明显高于对照组.CHB组和HBV携带者组TCR γ/δ的表达,分别为29.6%±15.4%和30.5%±14.8%,CHB组和HBV携带者明显低于对照组41.4%±19.4%.CHB重度患者CD3+CD56 1细胞CD8和TCR α/β的表达分别为69.0%±14.0%和76.1%±12.9%,CHB中度患者CD8的表达为66.4%±14.9%,均明显高于CHB轻度患者51.4%±16.2%和62.1%±14.6%. 结论 慢性乙型肝炎的活动可能与CD3+CD56+淋巴细胞的CD8高表达有关.     

慢性乙型肝炎患者外周血中T淋巴细胞亚群和CD4~+CD25~+调节性T淋巴细胞的表达水平与HBV DNA定量的相关分析

目的探讨慢性乙型肝炎(CHB)患者外周血中T淋巴细胞亚群(CD3+T、CD4+T、CD8+T、CD4+/CD8+T)和CD4+CD25+调节性T淋巴细胞(Treg)表达水平与HBV DNA定量的相关性。方法选取2012年7月-12月在兰州市第二人民医院就诊的175例HBV感染者作为研究对象,分为CHB组(63例)、HBV携带者(ASC组)(112例),以同期50例健康体检者作为对照组。采用流式细胞术检测3组患者血清中的CD3+T、CD4+T、CD8+T、CD4+T/CD8+T和CD4+CD25+Treg表达水平,实时荧光定量PCR法检测HBV DNA载量。多组间均数比较采用方差分析,进一步两两比较采用LSD-t检验,计数资料组间比较采用χ2检验;采用Pearson进行相关性分析。结果 CHB患者外周血CD4+CD25+Treg表达水平(30.97±18.78)%显著高于对照组(27.30±17.59)%,差异具有统计学意义(P0.05);与对照组相比,CHB组、ASC组的CD3+T、CD4+T淋巴细胞百分数和CD4+与CD8+T淋巴细胞百分数比值显著降低,差异均有统计学意义(P值均0.05),CD8+T淋巴细胞百分数显著升高,差异均有统计学意义(P值均0.05);ASC组和CHB组CD4+CD25+Treg表达水平与HBV DNA呈正相关(r值分别为0.501、0.682,P值均0.01)。结论 HBV感染者存在T淋巴细胞亚群比例异常,HBV DNA可以促进CD4+CD25+Treg表达水平升高,说明CD4+CD25+Treg和CD4+与CD8+T淋巴细胞比例失衡在乙型肝炎慢性化过程中发挥重要作用。     

慢性HBV感染及其相关肝病患者外周血T细胞亚群的变化

目的观察不同临床类型HBV感染者外周血T淋巴细胞亚群的差异,探讨HBV对人体T细胞免疫的影响及其可能机制,不同类型慢性HBV感染者免疫失衡的规律。方法用流式细胞仪技术检测患者外周血T细胞亚群。慢性HBV感染者318例,其中HBV携带者8例,慢性乙型肝炎231例,肝炎后肝硬化61例,原发性肝癌18例,观察患者的T淋巴细胞亚群、HBV DNA等。同时收集22名健康志愿者的新鲜血检测T淋巴细胞亚群。结果不同临床类型HBV感染者外周血CD3＋T、CD4＋T、CD8＋T细胞百分数低于正常对照组,差别均有统计学意义（P〈0.05或P〈0.01）,CD4＋/CD8＋比正常对照组低,但差异均无统计学意义（P〉0.05）。结论慢性HBV感染随着病情进展,由慢性肝炎→肝硬化→肝癌,直至细胞免疫功能逐渐衰退。慢性HBV感染的不同阶段的细胞免疫紊乱各具特点,就不同类型患者应采用不同的免疫调节治疗手段。     

乙肝病毒感染者外周血CD4^＋CD25^＋调节性T细胞的表达及临床意义

目的 探讨乙肝病毒感染者外周血CD4 CD25 调节性T细胞(CD4 CD25 Tr)表达水平及其临床意义.方法 选择乙肝病毒携带者25例、急性乙肝患者26例、慢性乙肝患者38例,26例正常对照,用流式细胞仪检测外周血中CD4 CD25 Tr表达水平;用PCR法检测患者HBV DNA载量.结果 乙肝病毒携带者组与慢性乙肝患者组CD4 CD25 Tr水平(9.93±3.51,11.21±3.29)明显高于对照组(8.12±2.80)(P＜0.01或0.05),急性肝炎组与对照组相比,差异没有显著性.结论 持续性HBV感染者CD4 CD25 Tr表达增加,提示CD4 CD25 Tr在慢性乙型肝炎中担负着重要的免疫调节作用,可能抑制HBV感染者特异性细胞免疫反应,与乙肝病毒感染的慢性化及肝病的临床发生、发展有关.     

慢性乙型肝炎患者干扰素治疗前后血清IL-7及调节性T细胞的变化

目的研究慢性乙型肝炎患者干扰素治疗前后血清IL-7及调节性T细胞的变化。方法本研究共有慢性乙型肝炎(CHB)患者67例,慢性乙型肝炎病毒(HBV)携带者18例,健康对照者14例。CHB组患者予以派罗欣(聚乙二醇干扰素α-2a注射液180μg,1次/周)抗病毒治疗24周,治疗结束后,按HBV-DNA定量不同再分为完全应答组(16例)、部分应答组(15例)和无应答组(36例);HBV携带组和健康组不做任何治疗。分别于0周、24周检测外周血IL-7、CD4+CD25+调节性T细胞百分比。结果在0周时,外周血IL-7含量和CD4+CD25+调节性T细胞在完全应答组、部分应答组及无应答组比较,差异均无统计学意义(P0.05);24周时,完全应答组与部分应答组、无应答组间比较,差异均有统计学意义(P0.05);完全应答组治疗前后比较,差异也有统计学意义(P0.05)。结论慢性乙型肝炎患者干扰素治疗后血清IL-7升高,调节性T细胞降低,可能与病毒应答有关。     

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.     

Parallel decline of CD8＋CD38＋ lymphocytes and viremia in treated hepatitis B patients

AIM:To assess the peripheral T lymphocyte subsets in chronic hepatitis B virus(HBV) infection,and their dynamics in response to adefovir dipivoxil monotherapy.METHODS:Proportions and absolute counts of peripheral natural killer cells,B cells,CD8+,CD4+,CD8+ CD38+,CD8+CD28+ and CD4+CD28+ T cells were determined using three-color flow cytometry in chronic hepatitis B patients(n = 35),HBV carriers(n = 25) and healthy controls(n = 35).Adefovir dipivoxil was initiated in 17 chronic hepatitis B patients who were r...     

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.     

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.     

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.     

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.     

In Vivo Bioluminescence Imaging of HBV Replicating Hepatocytes Allows for the Monitoring of Anti-Viral Immunity

Immunity against hepatitis B virus (HBV) infection is complex and not entirely understood so far, including the decisive factors leading to the development of chronic hepatitis B. This lack of a mechanistic understanding of HBV-specific immunity is also caused by a limited number of suitable animal models. Here, we describe the generation of a recombinant adenovirus expressing an HBV 1.3-overlength genome linked to luciferase (Ad-HBV-Luc) allowing for precise analysis of the quantity of infected hepatocytes. This enables sensitive and close-meshed monitoring of HBV-specific CD8 T cells and the onset of anti-viral immunity in mice. A high dose of Ad-HBV-Luc developed into chronic hepatitis B accompanied by dysfunctional CD8 T cells characterized by high expression of PD1 and TOX and low expression of KLRG1 and GzmB. In contrast, a low dose of Ad-HBV-Luc infection resulted in acute hepatitis with CD8 T cell-mediated elimination of HBV-replicating hepatocytes associated with elevated sALT levels and increased numbers of cytotoxic HBV-specific CD8 T cells. Thus, the infectious dose was a critical factor to induce either acute self-limited or chronic HBV infection in mice. Taken together, the new Ad-HBV-Luc vector will allow for highly sensitive and time-resolved analysis of HBV-specific immune responses during acute and chronic infection.     

Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B

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.     

Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers

Despite the availability of effective hepatitis B vaccines for many years, over 370 million people remain persistently infected with hepatitis B virus (HBV). Viral persistence is thought to be related to poor HBV-specific T-cell responses. A phase I clinical trial was performed in chronic HBV carriers to investigate whether HBV DNA vaccination could restore T-cell responsiveness. Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV-specific T-cell responses were assessed by proliferation, ELISpot assays, and tetramer staining. Secondary end points included safety and the monitoring of HBV viraemia and serological markers. Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV-specific interferon gamma-secreting T cells were detectable before immunization, but the frequency of such responses was significantly increased by 3 DNA injections. Immunization was well tolerated. Serum HBV DNA levels decreased in 5 patients after 3 vaccine injections, and complete clearance was observed in 1 patient. In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective. We demonstrate that DNA vaccination can specifically but transiently activate T-cell responses in some chronic HBV carriers who do not respond to current antiviral therapies.