乙型肝炎病毒特异性细胞毒性T淋巴细胞应答与不同临床感染状态的关系

目的 分析人类白细胞抗原(HLA)-A0201限制性的特异性CTL,研究急性肝炎急性期和慢性乙型肝炎活动期患者T淋巴细胞对特异性抗原表位免疫应答的差异.方法 收集HLA-A0201阳性的5例急性肝炎急性期和6例慢性乙型肝炎活动期患者的外周血单个核细胞(PBMC),酶联免疫斑点技术(ELISPOT)测定针对HBV聚合酶区(Pol575-583)、包膜区(Env348-357)和核心区(Core18-27)3个CD8+T淋巴细胞表位肽特异性CTL的数量和功能.数据采用t检验.结果 经Pol575-583、Env348-357和Core18-27三条抗原肽刺激,急性乙型肝炎急性期患者组斑点形成细胞数(SFC)分别为110±13、165±17和185±20;慢性乙型肝炎活动期患者组SFC分别为22±4、23±5和30±5,两组差异有统计学意义(t值分别为10.9、15.2和8.0,均P<0.05).急性乙型肝炎急性期患者各抗原肽特异性CTL的应答能力Pol575-5830.05).非特异性HLA-2402限制性Core117-125刺激也出现SFC增加,但与阴性对照组比较,差异无统计学意义(P>0.05).结论 急性感染者HBV特异性CTL应答水平显著高于慢性HBV感染者,慢性乙型肝炎患者体内的多克隆CTL数量和功能低下.     

丙型肝炎病毒核心区多肽对细胞毒T细胞的作用

目的 探讨丙型肝炎病毒（HCV）感染者体内细胞毒T细胞（CTL）功能缺陷的原因。方法 将HCV核心区多肽皮下注射免疫BALB/c小鼠，用乳酸脱氢酶释放实验检测小鼠脾细胞CTL活性，选择上述多肽中对CTL有抑制作用和增强作用的多肽各2条，交叉组合后共同免疫BALB/c小鼠检测其CTL活性。结果 经单因素方差分析显示，HCV核心区多肽CPA9（39-74位氨基酸），CPB7（67-76位氨基酸），CPB8（71-80位氨基酸）对小鼠CTL有抑制作用，CPA10（5-23位氨基酸），CPB6（63-72位氨基酸），CPB2（131-140位氨基酸），对小鼠CTL有增强作用，CPB2 CPB8，CPB6 CPB8组中效靶比10：1，20：1的CTL活性显著高于对照组，CPB2 CPB7，CPB6 CPB7组与对照组无明显差异。双因素方差分析显示HCV核心区抑制性多肽和增强性多肽有交互作用。结论 HCV核心区多肽CPA9，CPB7，CPB8对小鼠CTL有抑制作用。CPA10，CPB6，CPB2对小鼠CTL有增强作用，HCV核心区抑制性和增强性多肽有交互作用。     

慢性乙型肝炎患者树突状细胞特点及其免疫治疗

树突状细胞（DC）是诱导和维持抗原特异性免疫应答非常重要的抗原提呈细胞（APC），研究表明慢性乙型肝炎（CHB）患者体内DC功能下降，这可能是导致CHB的重要原因之一。DC增强免疫疗法的基本原理是提高CHB患者体内DC功能，诱导强烈的HBV特异性免疫反应清除病毒。     

慢性乙型肝炎患者外周血树突状细胞诱导特异性T淋巴细胞应答

目的 探讨慢性乙型肝炎患者外周血树突状细胞(Dc)是否诱导特异性T细胞应答。方法(1)将研究对象分为慢性乙型肝炎患者组、急性乙型肝炎痊愈组、健康志愿者组,分离各组研究对象的外周血单个核细胞(PBMC),细胞内细胞因子染色方法检测其对细胞毒性T淋巴细胞(CTL)特异表位多肽乙型肝炎病毒核心抗原(HBcAg)18-27的记忆性免疫应答;(2)培养慢性乙型肝炎患者DC,将负载有乙型肝炎抗原表位多肽的DC诱导特异的T细胞应答。采用细胞内细胞因子染色方法检测诱导的T细胞分泌的细胞因子,乳酸脱氢酶释放法测定诱导的T细胞杀伤活性。结果(1)急性乙型肝炎患者PBMC对HBcAg 18-27 CTL特异表位多肽存在记忆的免疫应答,其分泌干扰素-γ的CD8+T细胞占CD8+T细胞总数的(4.3±2.5)％,分泌白细胞介素-2的占总细胞数的(4.8±2.2)％,分泌肿瘤坏死因子-α占总细胞数的(4.6±2.3)％。而慢性乙型肝炎患者和健康志愿者对其记忆应答很低,与急性乙型肝炎患者比较差异有显著性,t值为2.508-3.305,P<0.05。(2)用多肽共孵育过的慢性乙型肝炎患者DC多次诱导的T细胞慢性乙型肝炎患者组,加肽孵育的靶细胞比例为30:1、10:1、3:1时,其杀伤率分别为(57.0±20.3)％、(49.5±20.2)％、(21.8±12.9)％,均高于对照组,表明慢性乙型肝炎患者DC可以诱导特异的T     

尿酸辅助树突细胞诱导乙型肝炎病毒抗原特异性细胞毒性T淋巴细胞效应的观察

近来研究提示,尿酸是免疫系统的一种重要的危险信号,它能刺激DC成熟,促进DC向T淋巴细胞呈递抗原．尿酸可能成为一种有效的免疫佐剂。本研究以尿酸辅助负载HBV表位肽S（28-39）的树突状细胞免疫接种小鼠,对其产生的HBV抗原特异性CTL免疫效应进行了报道。[第一段]     

乙型肝炎病毒核心区基因变异与机体细胞免疫水平的关系

HBV所激发的免疫应答和免疫病理反应是乙型肝炎发病机制的关键。其中细胞毒性T淋巴细胞（CTL）是细胞免疫反应的主要执行者，而针对HBcAg的CTL应答决定病毒是否被清除。当HBV基因发生变异，可改变氨基酸分子的表达，尤其当CTL识别的重要表位发生变异会改变机体对病毒的免疫反应。     

重型肝炎患者乙型肝炎病毒特异性细胞毒性T淋巴细胞应答功能研究

目的 用主要组织相容性复合物（MHC）抗原肽四聚体（Tetramer）流式细胞技术，分析乙型重型肝炎患者外周血中特异性细胞毒性T淋巴细胞（CTL）应答状况，并探讨其临床意义。方法 采用Tetramer流式细胞技术检测乙型重型肝炎患者外周血中受人类白细胞抗原Ⅰ类分子限制的三类特异性CD8^＋CTL细胞数量；采用酶联免疫吸附斑点试验技术，测定经特异性乙型肝炎病毒（HBV）肽段诱导培养的特异性CTL表达膜内细胞因子IFNγ、TNFα、IL-4和IL—10等的水平；采用Promega CytoTox96非放射性细胞毒试验技术，测定经特异性HBV肽段诱导培养的特异性CTL杀伤靶细胞能力。结果 急性乙型重型肝炎组外周血中针对HBVcore18-27表位的特异性CTL数量高于慢性乙型重型肝炎组（P〈0．05），而低于急性乙型肝炎组（P〈0．05）；急性乙型重型肝炎组表达干扰素γ和肿瘤坏死因子α较慢性乙型重型肝炎组高（P值均〈0．05）；急性乙型重型肝炎组HBVcore18—27特异性CTL裂解靶细胞能力高于慢性乙型重型肝炎组（P〈0．05）。结论 急性乙型重型肝炎患者特异性CTL应答作用增强，而慢性乙型重型肝炎患者特异性CTI。应答缺乏。急性乙型重型肝炎患者外周血特异性CTL持续存在，可能与促进病毒清除等相关。     

艾滋病病毒／丙型肝炎病毒双重感染者的艾滋病病毒特异性CTL应答

目的探讨中国艾滋病病毒/丙型肝炎病毒（HIV/HCV）双重感染患者HIV特异性细胞毒性T淋巴细胞（CTL）应答的特征。方法观察对象为HIV/HCV双重患者、单纯HIV感染者。以HIV-1 B亚型构建的全基因组肽库作为抗原,通过酶联免疫斑点（ELISPOT）法检测HIV/AIDS患者HIV特异性CTL应答。并对HIV/HCV双重感染患者的HIV特异性CTL应答进行相关性分析。结果 HIV/HCV双重感染组和单纯HIV感染组的HIV特异性CTL应答强度与频率在肽段水平存在一定差异,与单纯HIV感染组相比,合并HCV感染组反应增强的肽段主要集中在Gag。两组特异性CTL应答强度与频率均呈明显正相关,且合并HCV感染组较单纯感染组正相关性增强。不同肽段累计反应强度的比较,Gag反应强度最高,其次是Nef。CD4计数与特异性CTL应答强度的关系无统计学意义。结论 HIV/HCV双重感染患者HCV对HIV进程的影响可能与对Gag蛋白CTL应答的增强有关。     

rAAV／HCV核心抗原基因转染树突状细胞的免疫功能研究

目的 研究通过rAAV／HCV（hepatitis Cvirus,HCV）核心抗原基因（Coregene）转染树突状细胞（dendritic cell,DC）制备DC疫苗的免疫功能。方法分离外周血单个核细胞（DC前体细胞）,以rAAV／Core／Neo病毒转染DC前体细胞（基因转染组）,同时设293细胞裂解物刺激为对照组,转染12h后,均采用GM-CSF、IL-4、TNF-α诱导成熟。7天后收集细胞,流式细胞仪检测rAAV／Core／Neo病毒转染效率（即HCV核心抗原表达率）及DC表面标志CDl4、CD40、CD80、CD83、CD86的表达情况,混合淋巴细胞实验检测DC刺激自体T细胞增殖的能力,^51Cr释放法检测CTL对抗原阳性靶细胞的杀伤效率及特异性。结果rAAV／Core／Neo转染DC的效率超过90％,成熟DC高表达CD40、CD80、CD83、CD86,转染后的DC具有较强的刺激自体T细胞增殖能力,其CTL对HCV核心抗原阳性靶细胞具有很高的杀伤率及抗原特异性。结论rAAV／Core／Neo能够高效转染DC,转染后的DC能刺激自体T细胞增殖,使CTL对HCV核心抗原阳性靶细胞具有杀伤活性。     

结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。     

Hepatitis C virus eradication associated with hepatitis B virus superinfection and development of a hepatitis B virus specific T cell response

BACKGROUND/AIMS: Specific T cell responses during acute hepatitis B and during chronic hepatitis C have been described in detail. However, the T cell responses during the rare setting of acute hepatitis B virus (HBV) infection in the course of chronic hepatitis C that eventually lead to clearance of both viruses are completely unknown. METHODS: We analyzed the virus specific CD4+ and CD8+ T cell response during an acute HBV superinfection in a patient with chronic hepatitis C. RESULTS: The patient eliminated hepatitis C virus (HCV)-RNA and HBV-DNA from serum soon after the clinical onset of acute hepatitis B. The HBV specific T cell response found in this patient corresponds to the typical response that has been described in acute hepatitis B without chronic HCV infection. In contrast the hepatitis C specific immune response was similar to that generally found in chronic hepatitis C despite the fact that the patient also eliminated HCV-RNA. CONCLUSIONS: We hypothesize that the acute HBV infection induced a HBV specific T cell response which was associated with elimination HBV DNA and HCV-RNA, the latter possibly by bystander mechanisms, e.g. via secretion of cytokines. If such a non-specific bystander mechanism which has proven to be effective in the experimental setting and which is formally described here for a single patient can be shown to be a more general phenomenon, it may support the approach with new antiviral strategies, e.g. the induction of non-specific defense mechanisms against HCV.     

Current therapy for hepatitis C or D or immunodeficiency virus concurrent infection with chronic hepatitis B

Concurrent hepatitis C virus (HCV), hepatitis delta virus (HDV), or human immunodeficiency virus (HIV) infection with chronic hepatitis B virus (HBV) appears to increase the risk of progressive liver disease including liver cirrhosis and hepatocellular carcinoma. There is a 10% prevalence of HCV infection in chronic HBV or HDV infection. Serological evidence of previous exposure to HBV is found in more than 80% of HIV-positive patients in the high risk group. Notably, the most recently acquired virus tends to suppress the pre-existing virus. In chronic HBV infection acquired perinatally or in early childhood, usually HCV is dominant and may suppress or even displace HBV and HDV. Less frequently, HBV or HDV suppresses HCV. It is generally agreed that the dominant virus should be identified in order to make appropriate treatment decisions. Studies with standard interferon (IFN) to treat patients with HCV dominantly dual HBV/HCV infection have showed only limited virological response. But high dose of IFN has been demonstrated with better response rate. Combined ribavirin with standard or pegylated IFN therapy could achieve a sustained HCV clearance rate comparable with those infected with HCV alone. On the contrary, patients with HBV dominantly dual viral infection might indicate more appropriate addition of lamivudine to IFN than ribavirin. Additionally, patients with concurrent infection of HBV and HDV, IFN seems to be the only effective agent. However, the efficacy of IFN is related to the dose. High dose of IFN [9 MU tiw (thrice per week)] and longer treatment duration (at least 2 years) have been shown to achieve adequate virological response. In patients with concurrently infected HBV and HIV, anti-HBV therapy should be considered for all patients with evidence of liver disease, irrespective of the CD4 cell count. In patients not requiring antiretroviral therapy, HBV therapy should be preferentially based on IFN, adefovir, or telbivudine. In contrast, in patients with CD4 cell counts <350 cells/μl or those already on antiretroviral therapy, agents with double anti-HBV and anti-HIV activity are preferred. At present, the evidence of therapeutic efficacy is not sufficient to make a recommendation in treating patients with dual HBV/HCV or HBV/HDV or HBV/HIV infection. Further studies of the well-designed, larger scale are needed to elucidate the role of different regimens or combination in the treatment of dual viral infection.     

腺病毒介导的白细胞介素-12表达对丙型肝炎病毒包膜2基因免疫的调节

目的 观察腺病毒介导表达白细胞介素-12(IL-12)对调节丙型肝炎病毒(HCV)包膜基因2(E2)诱导免疫应答的影响。方法 以NIH 3T3细胞表达HCVE2糖蛋白，纯化后用于酶免疫分析法检测抗E2抗体；以表达HCVE2糖蛋白的SP2／0细胞^3Cr释放法检测细胞毒性T淋巴细胞(CTL)应答；以293细胞繁殖表达重组IL-12亚单位p35和p40的腺病毒ADIL12。6～8周龄BALB／C鼠右后肢股四头肌注射表达HCVE2的质粒，同时经腹腔注射ADIL12。分别于2、3、4周尾静脉采血检测抗E2抗体，4周处死动物分离脾细胞检测CTL应答。结果 表达HCVE2的基因免疫可有效诱导特异性抗E2体液免疫应答。腹腔注射后，外源性IL-12微量表达。微量表达的外源性白细胞介素-12不影响特异性抗E2体液免疫应答。同时，显著增强特异性CTL应答的作用。结论 腺病毒微量表达的IL12对HCV E2基因免疫诱导的特异性CTL应答具有调节作用。     

Chronic ethanol consumption impairs cellular immune responses against HCV NS5 protein due to dendritic cell dysfunction

BACKGROUND & AIMS: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. METHODS: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. RESULTS: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. CONCLUSIONS: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.     

丙型肝炎病毒包膜基因对核心基因DNA疫苗免疫应答强度的影响

目的 研究丙型肝炎病毒(HCV)包膜基因E1E2对核心基因C DNA疫苗诱生的免疫应答作用。方法 将包含HCV C或CE1E2基因片段插入真核表达载体pcDNA3中,构建重组质粒pHCV-C或pHCV-CE1E2,分别免疫Balb/c小鼠,每间隔2wk加强免疫1次,同时剪尾取血。ELISA法检测免疫小鼠血清中HCV C特异性抗体的水平。以pHCV-C转染并表达HCcAg的BLAB/c小鼠骨髓瘤Sp4/0细胞为靶细胞,采用~(51)Cr释放试验检测特异性CTL的杀伤作用。结果 两个实验组20只小鼠均产生抗HCV C特异性抗体,当效/靶细胞比例为100:1时,CTL的杀伤率均明显高于对照组(p<0.01);而pHCV-CE1E2与pHCV-C组之间,无论是抗HCV C抗体的滴度还是CTL的杀伤率均无显著性差异(p>0.05)。结论 E1E2基因的加入,并没有增加HCV C基因DNA疫苗诱导的抗HCcAg特异性抗体的滴度和CTL的杀伤作用。     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMS: The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. METHODS: HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B. RESULTS: An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively. CONCLUSIONS: We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.     

Dendritic cells: The warriors upfront-turned defunct in chronic hepatitis C infection

Hepatitis C virus (HCV) infection causes tremendous morbidity and mortality with over 170 million people infected worldwide. HCV gives rise to a sustained, chronic disease in the majority of infected individuals owing to a failure of the host immune system to clear the virus. In general, an adequate immune response is elicited by an efficient antigen presentation by dendritic cells (DCs), the cells that connect innate and adaptive immune system to generate a specific immune response against a pathogen. However, HCV seems to dysregulate the activity of DCs, making them less proficient antigen presenting cells for the optimal stimulation of virus-specific T cells, hence interfering with an optimal anti-viral immune response. There are discordant reports on the functional status of DCs in chronic HCV infection (CHC), from no phenotypic or functional defects to abnormal functions of DCs. Furthermore, the molecular mechanisms behind the impairment of DC function are even so not completely elucidated during CHC. Understanding the mechanisms of immune dysfunction would help in devising strategies for better management of the disease at the immunological level and help to predict the prognosis of the disease in the patients receiving antiviral therapy. In this review, we have discussed the outcomes of the interaction of DCs with HCV and the mechanisms of DC impairment during HCV infection with its adverse effects on the immune response in the infected host.     

Viral hepatitis: virus/host interaction

Abstract   Hepatitis A virus is considered directly cytopathic to the liver cell. Severity of the liver damage is dictated by viral load. Acute infection is followed by sustained immunity to the virus. Hepatitis B (HBV) and C (HCV) viruses are noncytopathic, hepatotropic viruses that cause acute and chronic hepatitis and hepatoma. Cellular and humoral immune responses are responsible not only for viral clearance but also for hepatocyte damage. T-cell response to HBV is vigorous, polyclonal, and multispecific in acutely infected patients who clear the virus while it is weak and narrowly focused in chronically infected patients. It is mainly executed by cytotoxic T lymphocytes (CTL), which destroy infected hepatocytes and secrete antiviral cytokines that interrupt the HBV life cycle. T-cell response to HCV is strong and multispecific in both acutely and chronically infected patients. Whether HCV is susceptible to a cytokine-mediated type of control is unknown. The ability of HCV to persist despite a strong CTL response suggests that HCV is either less visible to the CTL or less responsive to cytokine-mediated antiviral signals than HBV. Both viruses, but especially HCV, have a high mutation rate, leading to the occurrence of variant viral genomes with growth advantage and the ability of escaping immune recognition.     

Molecular mechanisms of viral hepatitis induced hepatocellular carcinoma

Chronic infection with viral hepatitis affects half a billion individuals worldwide and can lead to cirrhosis, cancer, and liver failure. Liver cancer is the third leading cause of cancer-associated mortality, of which hepatocellular carcinoma (HCC)represents 90%of all primary liver cancers. Solid tumors like HCC are complex and have heterogeneous tumor genomic profiles contributing to complexity in diagnosis and management. Chronic infection with hepatitis B virus (HBV),hepatitis delta virus (HDV), and hepatitis C virus (HCV) are the greatest etiological risk factors for HCC. Due to the significant role of chronic viral infection in HCC development, it is important to investigate direct (viral associated) and indirect (immune-associated) mechanisms involved in the pathogenesis of HCC. Common mechanisms used by HBV, HCV, and HDV that drive hepatocarcinogenesis include persistent liver inflammation with an impaired antiviral immune response, immune and viral protein-mediated oxidative stress, and deregulation of cellular signaling pathways by viral proteins.DNA integration to promote genome instability is a feature of HBV infection, and metabolic reprogramming leading to steatosis is driven by HCV infection. The current review aims to provide a brief overview of HBV, HCV and HDV molecular biology, and highlight specific viral-associated oncogenic mechanisms and common molecular pathways deregulated in HCC, and current as well as emerging treatments for HCC.