HBV CTL表位-O糖基化修饰的分子模建研究

研究Ｏ 糖基化 ( ｇｌｙｃｏｓｙｌａｔｉｏｎ)修饰对ＨＢＶ前 Ｓ2上ＣＴＬ表位结构影响。选择ＨＢＶ前 Ｓ2上公认的ＨＬＡ Ａ2限制性ＣＴＬ表位 4 4 53(ＳＩＬＳＫＴＧＤＰＶ) ,以Ｓｅｒ 4 4为糖基化位点 ,进行计算机分子模建。结果表明 ,α ＧａｌＮＡｃ 糖基化可改变表位形态使之更适宜与ＨＬＡ Ａ2类分子结合 ,糖基部分基团可从ＨＬＡ Ａ2结合沟中向外伸出。Ｓｅｒ 4 4α Ｄ ＧａｌＮＡｃ 糖基化修饰能改变前 Ｓ2上ＣＴＬ表位 4 4 53的结构 ,并促进与ＨＬＡ Ａ2分子结合的稳定性 ,表明表位糖基化修饰有可能调节ＣＴＬ应答。HBV CT…     

合成多肽体外诱导乙肝病毒抗原特异性T杀伤细胞实验研究

目的及方法 为探讨ＨＢＶ抗原特异性细胞毒Ｔ细胞（ＨＢＶ－ＣＴＬ），根据ＨＢｃＡｇ的ＨＬＡ分子结合关键序列合成抗原多肽２段，分别与转铁蛋白（Ｔｆ），抗ＣＤ单克隆抗体（ＣＤ３ｍＡｂ）和ＩＬ－２联合体外诱导ＨＢＶ－ＣＴＬ，应用＾３Ｈ－ＴｄＲ释放法分别测定ＨＢＶ－ＣＴＬ对ＨＢＶＤＮＡ转染的ＨｅｐＧ２．２．１５细胞特异性活性及无ＨＢＶＤＮＡ转染的ＨｅＰＧ２细胞的非特异性杀伤率。结果 ＨＢＶ－ＣＴＬ对ＨＢＶＤ     

乙型肝炎病毒变异对其特异性CTL反应的影响

特异性细胞毒Ｔ淋巴细胞（ＣＴＬ）反应是人体清除ＨＢＶ的主要机制，与临床结局密切相关，了解病毒变异对ＣＴＬ反应的影响尤为重要。 １．ＣＴＬ表位区内的变异：ＣＴＬ通过识别ＨＢＶ抗原特异性表位而清除病毒，ＨＢＶ编码的蛋白产物上都有这种表位。表位区内的变异可能引起（１）与ＨＬＡ分子结合的病毒抗原肽“错位变异”，抗原提呈失败；（２）ＣＴＬ不能识别变异的表位；（３）变异性肽配体与ＣＴＬ受体结合，使ＣＴＬ分化信号发生改变而失去对靶抗原的杀伤作用。但ＨＢＶ可表达多种抗原性、且变异绝大多数不在表位区内，个别ＣＴＬ…     

口服丙型肝炎复合多表位DNA减毒鼠伤寒沙门活菌苗的实验研究

目的　利用减毒鼠伤寒沙门菌作为载体,探讨丙型肝炎病毒（ＨＣＶ） 口服ＤＮＡ 疫苗的可行性。方法　把ＨＣＶ 复合多表位抗原基因ＰＣＸ 克隆到真核表达载体ｐｃＤＮＡ３（ＣＭＶ 启动子） ,构建ＨＣＶ 真核表达载体ｐｃＤＮＡ３／ＰＣＸ,转化减毒鼠伤寒沙门菌ＳＬ３２６１ ,获得ＨＣＶ 重组口服ＤＮＡ 活菌苗ＳＬ３２６１ （ｐｃＤＮＡ３／ＰＣＸ） ,免疫小鼠及家兔后,检测特异性免疫应答及安全性。结果　ＳＬ３２６１（ｐｃＤＮＡ３／ＰＣＸ） 在小鼠及家兔中均可诱发低水平的特异性体液免疫及细胞免疫应答,免疫动物未见明显的毒性反应。结论　ＨＣＶ 口服减毒鼠伤寒沙门菌ＤＮＡ 疫苗可诱发特异性的免疫应答,为ＨＣＶ 疫苗的研究提供新的理论及实验依据     

HLA部分不相合异基因外周血干细胞移植造血重建

目的：了解ＨＬＡ部分不相合异基因外周血干细胞移植（ＡＬＬｏ－ＰＢＳＣＴ）后造血功能重建情况,及在发生严重移植物抗宿主病（ＧＶＨＤ）时对造血功能的影响。方法：对患者移植后的血象、骨髓象,生化指标进行动态观察并分析结果。结果：移植后,＋ １３ ｄ 白细胞达２．２×１０９／Ｌ;＋ ２０ ｄ 骨髓增生活跃;＋ ４６ ｄ ＡＢＯ 血型、ＭＮ 血型、ＨＬＡ表型、ＨＬＡ ＤＮＡ分型均转为供型者,显示移植物植入成功。＋ １０ ｄ 开始出现ＧＶＨＤ征象,＋ ２９ ｄ 达ＧＶＨＤⅢ度,经用马抗人胸腺细胞球蛋白（ＡＴＧ）、ＣＤ３ 单抗、ＣＤ２５单抗后,症状消失。同时造血功能明显受抑。结论：①ＡＬＬｏ－ＰＢＳＣＴ造血重建过程中,应用Ｇ－ＣＳＦ可促使移植干细胞加快分化、增殖。②发生严重ＧＶＨＤ后,用ＡＴＧ等药物抑制Ｔ细胞,同时减少Ｔ细胞分泌ＧＭ－ＣＳＦ等相关细胞因子,致造血功能受抑制。     

蛛网膜下腔出血后血清C3、C4、CH50动态检测

目的探讨蛛网膜下腔出血后血清补体变化。方法对２１例蛛网膜下腔出血（ＳＡＨ）患者及１４例高血压脑出血（ＣＨ）患者进行血清总补体溶血活性（ＣＨ５０）及补体成分Ｃ３、Ｃ４的动态检测,观察其值在ＳＡＨ病程中的变化情况。结果（１）ＳＡＨ的出血量与Ｃ３、Ｃ４值呈负相关（Ｐ＜０．０１）,与ＣＨ５０值无相关;（２）Ｃ３、Ｃ４值与脑血管痉挛（ＣＶＳ）有对应性变化,即有ＣＶＳ的患者Ｃ３值在０．８ｇ／Ｌ以下,Ｃ４值在０．４ｇ／Ｌ以下;无ＣＶＳ的患者病后Ｃ３值在０．９ｇ／Ｌ以上,Ｃ４值在０．４ｇ／Ｌ以上,且在ＣＶＳ发生以前Ｃ３、Ｃ４值明显下降;（３）Ｃ４值呈急剧下降且最低值在０．２ｇ／Ｌ以下的ＣＶＳ患者预后极差。结论在ＳＡＨ后的病程中补体成分Ｃ３、Ｃ４值出现有意义的变化,ＣＨ５０值无明显变化;ＳＡＨ后慢性脑血管痉挛（ＣＣＶＳ）发生前Ｃ３、Ｃ４值明显下降,Ｃ３、Ｃ４值可做为预测ＣＣＶＳ发生的指标;在ＣＣＶＳ发生之前Ｃ４值呈急剧下降,在０．２ｇ／Ｌ以下者提示预后不良。     

慢性丙肝RIBA与RIA-2检测抗HCV的价值

目的探讨重组免疫印迹试验（ＲＩＢＡ）在慢性ＨＣＶ感染中的应用价值．方法１９９３年１月～１９９５年３月采用含ＨＣＶ不同编码区三种重组抗原（Ｃ２２，Ｃ３３ｃ，Ｃ１００３）的ＲＩＢＡ，对８５例慢性非甲非乙型肝炎（ＮＡＮＢＨ）患者血清进行检测并将其结果与第二代放免法（ＲＩＡ２）检测抗ＨＣＶ及逆转录多聚酶链反应（ＲＴＰＣＲ）检测ＨＣＶＲＮＡ结果进行比较分析．结果慢性ＮＡＮＢＨ患者血清ＲＩＢＡ阳性（７８８％，６７／８５）结果与ＨＣＶＲＮＡ阳性（７５３％，６４／８５）结果之间有良好相关性和高的符合率；ＲＩＢＡ可检出ＲＩＡ２阴性的ＨＣＶ感染者；单项抗体阳性时仅见一定比例的单项抗Ｃ２２或抗Ｃ３３ｃ阳性血清可检出ＨＣＶＲＮＡ，而单一抗Ｃ１００３阳性者ＨＣＶＲＮＡ均阴性．结论ＲＩＢＡ可提高慢性ＨＣＶ感染的诊断率，ＲＩＢＡ阳性为ＨＣＶ感染及病毒血症存在的标志，可作为ＲＩＡ２检测抗ＨＣＶ的确证实验．     

庚型肝炎病毒同丙型肝炎病毒的联合感染和单独感染

为了建立ＨＧＶ感染的酶免疫诊断方法，用计算机技术分析了ＨＧＶ序列，预测核心区，ＮＳ３区的抗原表位，合成了多段的ＨＧＶ核心和ＮＳ３区合成肽，通过免疫筛选试验，证实Ｃ２９，Ｃ４０和Ｎ３１０三段肽有免疫原性。用这三面肽包被酶标板，用间接免疫试验原理，建立了ＨＧＶ的ＥＩＡ检测方法。用中和抑制试验检测了该方法的特异性。使用ＨＧＶ ＥＩＡ方法检测卫生部临床检测中心提供的ＨＣＶ ＥＩＡ参比血清，发现３／９２抗－     

丙型肝炎病毒核心基因免疫研究

目的研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫用于预防和治疗ＨＣＶ感染的可行性和有效性．方法将ＨＣＶＣ基因片段插入真核表达载体ｐｃＤＮＡ３质粒ＣＭＶ启动子的下游，在证实其可以在小鼠骨髓瘤细胞ＳＰ２／０（Ｈ２ｄ）中表达之后，将重组质粒注射ＢＡＬＢ／ｃ（Ｈ２ｄ）小鼠股四头肌，ＥＬＩＳＡ检测血清中抗体产生水平；３ＨＴｄＲ掺入法测定免疫小鼠淋巴细胞ＨＣＶＣ抗原特异性增殖能力，４ｈ５１Ｃｒ释放法检测免疫小鼠细胞毒Ｔ细胞（ＣＴＬｓ）体外杀伤功能．结果免疫小鼠２０只，初次免疫２ｗｋ后，血清中均出现了ＨＣＶＣ抗体，且增加免疫剂量可提高抗体滴度；淋巴细胞增殖指数为６１０，明显高于对照组（Ｐ＜００１），ＣＴＬｓ体外特异性杀伤率为６３１％，也高于对照组（Ｐ＜００１）．结论ＨＣＶＣ基因免疫不仅可以诱导机体产生特异性的体液免疫，而且产生特异性的细胞免疫，它可能是防治ＨＣＶ的有效方法．     

66例慢性非甲非乙型肝炎患者的丙型肝炎病毒感染研究

以ＨＣＶ－Ｔ３序列为引物，结合ＲＴ－ＰＣＲ和寡聚核甘酸探针Ｓｏｕｔｈｅｒｎ杂交，检测６６例慢性非甲非乙型肝炎（ＮＡＮＢＨ）患者的血浆ＨＣＶ－ＲＮＡ，阳性４２例（６３．６％）。同样病例以相当于ＨＣＶＣ区基因编码和ＮＳ３区编码的人工合成肽抗原检测抗ＨＣＶ，阳性４９例（７４．２％）。这６６例慢性ＮＡＮＢＨ病例，抗ＨＣＶ和ＨＣＶ－ＲＮＡ双阳性者３８例（５７．６％）；抗ＨＣＶ阴性而ＨＣＶ－ＲＮＡ阳性者４例（６．１％）；抗ＨＣＶ阳性而ＨＣＶ－ＲＮＡ阴性者１１例（１６．７％）。其中诊断为散发型ＮＡＮＢＨ者３５例，检出ＨＣＶ－ＲＮＡ者１７例（４８．６％），为输血后ＮＡＮＢＨ者３１例，检出ＨＣＶ－ＲＮＡ者２５例（８０．７％）。     

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus

AIM: Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS: Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs. RESULTS: No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes. CONCLUSION: Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.     

Relation between viral fitness and immune escape within the hepatitis C virus protease

BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.     

Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.     

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.     

Identification of novel hepatitis C virus-specific cytotoxic T lymphocyte epiotpe in NS3 region

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are thought to be effective in limiting viral spread and in clearing virus during infection. Therefore, we attempted to establish HCV-specific CTL and identify novel HCV-specific CTL epitopes in a patient with acute hepatitis C by a novel screening method using recombinant vaccinia viruses (rVV) and synthetic peptides. CD8(+)CD45RA(-) T cells (memory T cells) were isolated from peripheral blood mononuclear cells (PBMC) of a patient with acute hepatitis C. HCV-specific CTL were cloned at limited dilutions and tested for HCV-specific CTL activity using a standard (51)Cr release assay. CTL assay was performed using rVV expressing regions of HCV-J, and overlapping and truncated synthetic peptides from HCV-J. CTL recognizing the NS3 region were isolated by (51)Cr release assay with rVV-HCV. Isolated CTL were restricted by HLA class I molecules B(*)5603. We confirmed that isolated CTL recognized 8-mer amino acids in the NS3 region of HCV-J by (51)Cr release assay with overlapping and truncated synthetic peptides. In conclusion, we isolated HCV-specific CTL restricted by HLA-B(*)5603 and identified a novel HCV-specific CTL epitope (IPFYGKAI, amino acids 1373-1380) in the NS3 region. The identified HCV-specific CTL epitope might be useful for HCV therapy.     

人白细胞抗原-A*0201限制性丙型肝炎病毒细胞毒性T淋巴细胞表位的鉴定

目的 鉴定人白细胞抗原(HLA)-A*0201限制性HCV-CTL表位.方法 基于RANKpep和SYFPEITHI细胞表位预测软件预测结果,选择合成6条候选CTL表位.研究候选CTL表位肽与T2细胞表达的HLA-A*0201分子的亲和力,进一步采用酶联免疫斑点实验(ELISPOT)和细胞内细胞因子染色(ICS)实验研究HLA-A*0201高亲和力肽在HLA-A*0201阳性HCV感染者的外周血单个核细胞(PBMC)中刺激CTL反应情况.结果 在6条候选CTL表位肽中,肽C_181(LLSCLTTPV)和NS2_172(VLQAGLIRV)与HLA-A*0201分子有高亲和力,其亲和力随肽浓度增加而升高.在10例HLA-A*0201阳性HCV-1b感染者每1×105PBMC中,肽C_181和NS2_172刺激后,特异性分泌IFN-γ细胞的斑点形成细胞数(SFC)分别为0～19和0～20.肽C_181和NS2_172特异性IFN-γ+CD8+T淋巴细胞占CD8+T淋巴细胞的比例分别为0.006%～0.065%和0.005%～0.080%.结论 肽C_181(LLSCLTTPV)和NS2_172(VLQAGLIRV)为HLA-A*0201 限制性HCV-CTL表位.     

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection

BACKGROUND/AIMS: It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS: Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS: An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS: Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.     

Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA-expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope.

The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.10(5) cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope.     

Conserved hepatitis C virus sequences are highly immunogenic for CD4(+) T cells: implications for vaccine development.

The HLA class II-restricted T-cell response to hepatitis C virus (HCV) antigens is believed to influence the final outcome of hepatitis C, because it is vigorous in patients who recover from acute hepatitis C, but it is weak in those who develop a chronic infection. For this reason, exogenous stimulation of T-cell responses in chronic HCV infection may represent a strategy to cure patients with chronic hepatitis C by approximating the vigor of their T-cell reactivity to that of patients who succeed in recovering from hepatitis. It may also be a preventive approach to avoid spread of the virus by facilitating the development of a vigorous protective response at the very early stages of infection. T-cell-based vaccines composed of immunodominant, promiscuous, and conserved T-cell epitopes may represent a powerful tool to achieve optimal stimulation of the T-cell reactivity. To identify HLA class II-restricted T-cell epitopes useful for this purpose, 22 subjects with acute HCV infection were studied and followed for an average time of 29 months. Eight of them recovered from hepatitis, and 14 developed a chronic infection. Overlapping 20-mer peptides covering the entire core and NS4 antigens and a panel of peptides representing highly conserved regions of core, NS3, NS4, and NS5 were used. By direct peripheral blood T-cell stimulation and by fine-specificity analysis of HCV-specific T-cell lines and clones, highly immunogenic T-cell epitopes were identified within core, NS3, and NS4. All these epitopes are immunodominant and highly conserved among the known HCV isolates. Moreover, they are promiscuous, because they can be presented to T cells by different HLA class II molecules. Immunodominance, sequence conservation, and promiscuity make these epitopes ideal components of preventive or therapeutic T-cell-based vaccines against HCV.