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

Objective To identify human leucocyte antigen (HLA)-A* 0201-restricted hepatitis C virus (HCV)-cytotoxic T lymphocyte (CTL) epitopes. Methods Based on the prediction results of RANKpep and SYFPEITHI prediction programs, six candidate CTL epitopes were selected and synthesized. The affinity of candidate CTL epitopes to HLA-A* 0201 molecules of T2 cells was explored. Subsequently, enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining (ICS) were utilized to determine whether candidate CTL epitopes could induce the recall positive response in peripheral blood mononuclear cells (PBMC) of HLA-A* 0201 positive HCV-1b-infected patients. Results Among six candidate CTL epitopes, peptides C_181(LLSCLTTPV) and NS2_172 (VLQAGLIRV) had high affinity to HLA-A* 0201 molecules. Moreover, the affinity was proportional to the concentration of peptide. Furthermore, among ten HLA-A* 0201 positive HCV-1b-infected patients, the frequencies of C_181 and NS2_172-specific interferon (IFN)-γ-producing cells were 0-19 spots forming cells (SFC)/1 × 105 PBMC and 0-20 SFC/1 × 105 PBMC, respectively.The percentages of C_ 181 and NS2_172-specific IFN-γ+ CD8+ T lymphocytes in total CD8+ T lymphocytes were 0.006%-0.065% and 0.005%-0.080%, respectively. Conclusion Peptides C_181 (LLSCLTTPV) and NS2_172 (VLQAGLIRV) are identified as novel HLA-A* 0201-restricted HCV-CTL epitopes.     

结核分枝杆菌Rv0440 CTL表位肽的免疫原性研究

目的 体外鉴定结核分枝杆菌（Mycobacterium tuberculosis,Mtb）Rv0440蛋白序列中表位肽362 370 aa和369-377 aa的HLA A＊0201限制性CD8＋ CTL表位的免疫原性,为基于表位的结核疫苗研究提供实验依据.方法 根据T2细胞HLA-A＊ 0201分子与多肽结合力分析实验结果,选取结核分枝杆菌Rv0440蛋白质氨基酸序列中对HLA-A＊ 0201分子高亲合力的Rv0440 1（362-370 aa,KLQERLAKL）和Rv0440-2（369 377 aa,KLAGGVAVI）作为候选表位肽.用候选表位肽刺激PPD（＋＋＋）健康志愿者外周血单个核细胞（peripheral blood mononuclear cells,PBMC）检测细胞分泌IFN γ的水平.用候选表位肽诱导特异性CTL细胞,检测特异性CTL细胞对负载表位肽的T2细胞的杀伤活性,观察Rv0440-1和Rv0440 2的HLA-A＊ 0201限制性CD8＋ CTL表位的免疫原性.结果 ELISPOT实验结果显示,表位肽Rv0440-1能够明显诱导HLA-A＊ 0201（＋）、PPD（＋＋＋）健康志愿者PBMC分泌IFN γ（P＜0.05）;且表位肽Rv0440-1负载DC诱导的CTL在效靶比为10：1时对负载相应表位肽的T2细胞的特异性杀伤活性高于对照组（P＜0.05）;与对照组相比,表位肽Rv0440 2没有诱导能力.结论 表位肽Rv0440-1（362-370 aa,KLQERLAKL）具有良好的免疫原性,是有效的结核分枝杆菌的HLA-A＊0201限制性CTL表位.     

慢性乙型肝炎病毒感染者HBcAg特异性细胞毒性T细胞克隆的建立

目的建立HLA-A*2402限制的HBcAg特异性CTL细胞克隆。方法取HLA-A*2402阳性的慢性HBV感染者的PBMC,用限制性表位肽(HBV core117-125,EYLVSFGVW)和重组HBcAg刺激,并以有限稀释法对活化的T细胞进行克隆化,用免疫荧光染色、流式细胞术以及乳酸脱氢酶释放实验对所获克隆进行鉴定。结果PBMC经表位肽激活后,特异性细胞毒活性达到43.7%;从活化细胞获得11株T细胞克隆;除2株是CD4+T细胞克隆外,其余9株为CD8+T细胞克隆,其中2株为Tc1,3株为Tc2和4株为Tc0。9株CD8+T克隆在效/靶比例为2.5:1时均具有HBcAg特异性靶细胞溶解活性(33.9%～90.2%)。结论用HBVcore117-125能激活HLA-A*2402阳性的慢性HBV感染者外周血CD8+T细胞,随之建立的9株CD8+T克隆均具有HBcAg表位特异性细胞毒活性。     

ELISPOT检测HLA-A0201转基因小鼠对恶性疟原虫红前期候选抗原的CTL应答

用恶性疟原虫红前期多表位候选抗原PfCP-3^tcl（含有1个HLA A*0201限制的CTL表位YLNKIQNSL）免疫人白细胞抗原复合体（HLA）A*0201（HLA-A*0201）转基因小鼠，再用鼠γ干扰素（IFN-γ）酶联免疫吸附斑点法（ELISPOT）检测该转基因小鼠特异性CTL应答，尝试在转基因实验动物中建立评价恶性疟原虫红前期候选抗原CTL应答的方法。结果显示该候选抗原中含有的CTL表位在转基因小鼠体内激发出了特异性的CTL应答，表明该CTL表位在转基因小鼠体内能够正确地加工和递呈。     

结核杆菌抗原Ag85A HLA-A*0201限制性CD8+ CTL表位的预测和鉴定

目的预测结核杆菌（Mtb）抗原Ag85A的HLA-A＊0201限制性CD8^＋细胞毒性T细胞（CTL）表位,并对其进行鉴定。方法应用数据库SYFPEITHI预测可能存在的HLA—A＊0201的限制性CD8^＋CTL表位,并经流式细胞术分析抗原肽与HLA-A＊0201的亲合力,经时间荧光分辨法检测结核（TB）患者外周血单个核细胞（PB-MC）对抗原肽的增殖反应,再通过细胞毒实验研究抗原肽诱导的T细胞毒杀伤活性,逐步鉴定Ag85A的HLA—A＊0201限制性CD8^＋CTL表位。结果位于Ag85A氨基酸序列7（48-56aa）和5（242-250aa）的表位与HLA—A＊0201分子具有较高的亲和力,并能刺激HLA-A＊0201阳性结核患者PBMC增殖,诱导产生具有特异性杀伤活性的CTL。结论肽7GLPVEYLQV（48—56aa）和肽5KLIANNTRV（242—250aa）是Mtb抗原Ag85A的HLA—A＊0201限制性CD8^＋CTL表位。     

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.     

人端粒酶逆转录酶抗原HLA-A0201限制性CTL表位预测及鉴定

目的应用生物信息学方法对人端粒酶逆转录酶(h TERT)HLA-A2+限制性细胞毒性T细胞(CTL)表位进行预测和鉴定,寻找诱导机体特异性杀伤肺癌肿瘤细胞的抗原表位。方法应用生物信息学软件BIMAS、SYFPEITHI对h TERT蛋白进行HLA-A0201限制性CTL抗原表位预测,筛选优势表位;应用肽亲和力实验、乳酸脱氢酶(LDH)释放实验及人干扰素γ(IFN-γ)ELISPOT实验验证表位,筛选出激发机体产生特异性免疫反应的表位。结果生物信息学软件筛选出优势表位为:ILAKFLHWL、ELLRSFFYV及ILSTLLCSL;肽亲和力实验得到优势表位荧光系数(FI)为:ILAKFLHWL0.67、ELLRSFFYV0.66及ILSTLLCSL0.90;LDH释放实验显示ILAKFLHWL所诱导CTLs的杀伤率明显高于其它各表位,也明显高于阴性表位,差异均具有统计学意义(P0.05);人IFN-γELISPOT实验证明ILAKFLHWL所诱导的CTLs产生的IFN-γ斑点数多于其他表位,差异具有统计学意义(P0.05)。结论ILAKFLHWL的免疫原性强,可用于后续制备肺癌多肽疫苗。     

HLA-A2限制性CTL表位HPV18E7_(7-15)分子修饰的实验研究

目的对人类白细胞抗原A2分子(HLA-A2)限制性细胞毒性T淋巴细胞(CTL)表位HPV18E77-15进行氨基酸置换修饰,并探讨修饰后多肽的免疫原性。方法根据量化模体方案,比较置换后的多肽与HLA-A2分子的结合系数,采用标准Fmoc方案合成并纯化多肽、细胞毒实验(51Cr释放法),观察多肽是否能够诱导特异性CTLs。结果修饰肽TLQDIVLHV符合HLA-A2分子限制性细胞毒性T细胞的表位要求,具有特异性细胞毒性T细胞诱导活性。结论修饰肽TLQDIVLHV具有更好的结合力和较强的抗原性,可以作为高危型人乳头瘤病毒(HPV)感染治疗性肽疫苗分子设计的候选表位。     

丙型肝炎病毒特异性细胞毒性T淋巴细胞活性研究

目的阐明丙型肝炎病毒(HCV)特异性细胞毒性T淋巴细胞(CTL)在慢性丙型肝炎病毒感染中的作用.方法用标准铬释放法(以从患者肝组织或外周血单个核细胞中经选择性克隆扩增后的CD8+细胞为效应细胞,经EB病毒转染的自身B淋巴母细胞为靶细胞,由能表达HCV1型核心区基因的重组痘苗病毒作为转导载体)对62例慢性丙型肝炎患者肝组织及外周血单个核细胞(PBMC)中的HCV特异性CTL活性(HCV CTL)进行检测,8例非HCV感染的肝病患者作为对照.结果62例慢性丙型肝炎患者中,共有28例(46 7%)肝组织中检测出HCV CTL活性,但HCV-CTL在PBMC中未检出.对照组患者肝组织及PBMC中均未检出.5例非HCV1感染的丙型肝炎患者检测出针对HCV1型表位的HCV-CTL.HCV-CTL阳性的丙型肝炎患者血清丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)水平及肝组织活动指数均明显高于HCV-CTL阴性的患者,而其血清HCVRNA水平则显著低于后者(P＜0.01).结论1 HCV-CTL主要存在于肝组织内;2存在型交叉性HCV-CTL;3.HCV-CTL活性阳性的患者较HCV-CTL活性阴性者具有较高的疾病活动度及较低的病毒血症水平;4.HCV特异性CTL在丙型肝炎的发病机制及疾病控制中起重要作用.     

胰岛人类白细胞抗原-A2限制性T淋巴细胞表位体外筛选方法的建立

目的 应用表位多肽与人类白细胞抗原(HLA)Ⅰ类分子结合力和解离率分析建立新型T淋巴细胞表位体外筛选方法.方法 采用基于矩阵算法的SYFPEITHI和BIMAS数据库预测6种胰岛细胞自身抗原[包括谷氨酸脱羧酶65(GAD65)、胰岛素瘤相关抗原2(IA-2)、前胰岛素原(PPI)、胰岛特异性葡萄糖6-磷酸酶催化亚基相关蛋白(IGRP)、胰岛淀粉样多肽(IAPP)、神经胶质纤维酸性蛋白(GFAP)]的表位序列,根据预测的结合力指数和已有数据分析筛选并合成15个HLA-A2限制性候选表位多肽.采用HLA-A2转基因的T2细胞检测候选肽与HLA-A2的分子结合力,通过多肽/HLA复合物解离率实验分析复合物的稳定性.采用单因素方差分析进行数据统计.结果 T2细胞肽结合力分析显示,15个候选表位多肽中,IGRP152～160、IGRP215～223、IGRP228-236、PPI2～10、胰岛素B10～18、IA-2172～180、GFAP143～151与HLA-A2的分子结合力＞80%.肽/HLA复合物解离率分析显示,上述结合力较强的7个表位多肽中,胰岛素B10-18、IGRP228～236、GFAP143～151、IA-2 172～180 4 h解离率低于20%.结论 本实验建立的候选多肽结合力与解离率相结合的T淋巴细胞表位体外筛选方法有助于减少研究中目标肽数量,推进1型糖尿病T淋巴细胞诊断方法的研究.     

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.     

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206

HA-1(H) is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1(H)-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1(H) peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1(H), VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206(+) and HLA-A*0201(+) targets (including leukemic blasts) that express HA-1(H) peptide endogenously, whereas an HLA-A*0201-restricted, HA-1(H)-specific CTL clone failed to lyse HLA-A*0206(+) targets. This finding will expand the patient population who can benefit from HA-1(H)-based immunotherapy.     

Comprehensive mapping of HLA-A0201-restricted CD8 T-cell epitopes on PDC-E2 in primary biliary cirrhosis

Growing evidence has implicated the involvement of autoreactive T lymphocytes in the pathogenesis of primary biliary cirrhosis (PBC). We have recently taken advantage of motif prediction analysis of HLA-A*0201 and identified the first major histocompatibility complex (MHC) class I restricted epitope, amino acids 159 to 167 on E2 components of pyruvate dehydrogenase complexes (PDC-E2), the major mitochondrial antigens in PBC. The mechanisms involved in the selection of epitope peptide(s) that comprise the PDC-E2-specific autoreactive cytotoxic T lymphocytes (CTLs) are unknown and likely involve other epitopes on PDC-E2 restricted by MHC class I molecules. To address this issue, a comprehensive mapping of the CTL epitope repertoire on the PDC-E2 molecule that binds HLA-A*0201 was performed to provide further clues regarding the role of CTLs. We used the T2 cell line to screen 79 overlapping 15mer peptides, spanning the entire PDC-E2 molecule. Six of the 79 peptides exhibited significantly higher binding activity to HLA-A*0201 than the other 15mer peptides. Two of these 6 peptides induced CTL lines from patients with PBC. Fine mapping with N-terminus or C-terminus truncated peptides identified 10mer peptide, PDC-E2 amino acids 165 to 174, which is a novel CD8 epitope restricted by HLA-A*0201. In conclusion, using a combination of the 15mer peptide library screening with the T2 binding assay and also the induction of CTL lines with candidate peptides, we have defined a novel HLA-A*0201-restricted epitope PDC-E2 165 to 174 in patients with PBC. These data will become important in the development of altered peptide ligands to modulate disease activity.     

Identification of HLA-A11-restricted HIV-1-specific cytotoxic T-lymphocyte epitopes in China

To fully define HLA-A11-restricted HIV-1-specific cytotoxic T-lymphocyte epitopes in China, a method combining the enzyme-linked immunospot (ELISPOT) assay with intracellular gamma interferon staining (ICS) of peripheral blood mononuclear cells (PBMC) was used to map the optimal epitopes targeted by ELISPOT and then to define the HLA restriction of epitopes by ICS. A novel HLA-A11-restricted CTL epitope and five other published HLA-A11-restricted epitopes previously identified by reverse immunogenetics or other methods were defined. The approach of integrating ELISPOT with ICS is both convenient and useful for the characterization of CTL responses to HIV-1 infection; this method is practical for defining novel epitopes and facilitates in developing new strategies for future vaccine design in China and other Asian countries.     

ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes

Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201-restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89- and p375-86-induced peptide-specific CTL lines were able to specifically release interferon-gamma (IFN-gamma) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus-transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I-restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.     

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.     

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 and characterization of HLA-A*3303-restricted,HIV type 1 Pol- and Gag-derived cytotoxic T cell epitopes

HLA-A*3303 is one of the common HLA alleles in East and Southeast Asia. Identification of HLA-A*3303-restricted HIV-1 cytotoxic T lymphocyte (CTL) epitopes is therefore required to investigate the immunopathogenesis of AIDS and vaccine development in these areas, where AIDS is rapidly expanding. We attempted to identify HLA-A*3303-restricted CTL epitopes derived from relatively conserved proteins Pol, Gag, and Nef of HIV-1 clade B, using reverse immunogenetics. Ninety-nine 8-mer to 11-mer peptides corresponding to the HLA-A*3303-binding peptide motif were selected from the HIV-1 SF2 sequence. Fifty-two of these 99 peptides bound to HLA-A*3303. Six of these binding peptides induced peptide-specific CTLs in PBMCs from at least one of two HIV-1-seropositive individuals. CTL clones specific for three Pol peptides and one Gag peptide killed HLA-A*3303-restricted target cells infected with HIV-1 recombinant vaccinia, indicating that these peptides were naturally processed HLA-A*3303-restricted CTL epitopes. SF2-Pol 594-602 (FYVDGAANR) and SF2-Gag 144-152 (MVHQAISPR) induced specific CTLs in 5 and 4 of 10 chronically HIV-1-infected individuals, respectively, whereas SF2-Pol 60-70 (TLWQRPLVTIR) and SF2-Pol 934-943 (KIQNFRVYYR) induced specific CTLs in 2 and 1 of 10 chronically HIV-1-infected individuals, respectively. Thus, the former are immunodominant epitopes whereas the latter are not. These epitopes are useful for studies of AIDS immunopathogenesis and vaccine development.     

Characterization of humoral and cell-mediated immune responses directed against hepatitis C virus F protein in subjects co-infected with hepatitis C virus and HIV-1

BACKGROUND: Hepatitis C virus (HCV) F protein is encoded in an alternate reading frame overlapping the core protein region. Its precise sequence, biological function and mode of expression are currently unclear. This study was conducted to examine the prevalence and characteristics of host humoral and cell-mediated immune responses directed against F protein in patients co-infected with HCV and HIV-1. METHODS: Mutations were introduced to allow the expression of HCV-1a F protein in the absence of core. This recombinant and a truncated form lacking the first 11 amino acid residues shared with core were expressed in Escherichia coli, and their amino acid sequences were verified by mass spectrometry. Vaccinia-F protein recombinants were used to test F protein-specific cytotoxic T lymphocyte (CTL) activity. The binding of F protein-derived peptides to HLA-A*0201 was studied to identify putative CTL epitopes. RESULTS: Sera from 23 of 39 patients infected with various HCV genotypes recognized the truncated form, including 13 of 25 subjects co-infected with HIV-1, indicative of antigenic crossreactivity and consistent with the conservation of F protein coding sequences between HCV genotypes. Crossreactive F protein-specific CTL precursors were detected in nine of 11 HCV-infected subjects, including seven of nine patients co-infected with HCV and HIV-1. Finally, three novel putative HLA-A*0201-restricted CTL epitopes were identified. CONCLUSION: These results indicate that patients co-infected with HCV and HIV-1 can mount immunoglobulin and CTL responses directed against HCV F protein that are fully comparable in scope and magnitude with those observed in individuals infected with HCV alone.