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.     

Hepatitis C virus-derived lipopeptides differentially induce epitope-specific immune responses in vitro

We studied the induction of T cell responses against hepatitis C virus (HCV)-related cytotoxic T cell (CTL) epitopes in lymphocytes from human leukocyte antigen (HLA)-A2-positive, HCV-naive individuals. Peptide and lipopeptides corresponding to the epitope at amino acids (aa) 1073-1081 induced equivalent numbers of tetramer-positive CD8+ T cells and detectable interferon-gamma and granzyme B spot-forming cells (sfcs). Epitope aa 35-44 induction of CTLs was not achieved, despite detectable tetramer-positive CD8+ T cells. IFN-gamma sfcs could only be induced with lipopeptide aa 20-44 but not by conventional antigens, which indicates that lipopeptides may alter T cell functions, depending on the epitope.     

Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV-specific CTL in a transgenic mouse model.

BACKGROUND & AIMS: The ability to induce cytotoxic T cells is considered an important feature of a candidate hepatitis C virus (HCV) vaccine. We used an oral immunization strategy with attenuated HCV-NS3-transformed Salmonella typhimurium to deliver DNA directly to the gut-associated lymphoid tissue. METHODS: HLA-A2.1 transgenic mice were immunized once with transformed attenuated Salmonella. HCV-specific CD8+ T cells were analyzed in vitro as well as in vivo by challenge of mice with recombinant HCV-NS3 vaccinia virus. RESULTS: Salmonella (10(8) colony-forming units; 20 microg plasmid DNA) induced cytotoxic and IFN-gamma-producing CD8+ T cells specific for the immunodominant epitope NS3-1073 in 26 of 30 mice (86%) that persisted for at least 10 months. A second epitope (NS3-1169) was also recognized by cytotoxic and IFN-gamma-producing T cells, whereas a third one (NS3-1406) stimulated IFN-gamma production without cytotoxicity. The minimal amount of plasmid DNA required to induce CTLs was 2 ng. Upon challenge with recombinant HCV-NS3-expressing vaccinia virus, vaccinia titers were significantly lower in mice immunized with Salmonella-NS3 than in mice immunized with control Salmonella, demonstrating the in vivo function of CTLs. CONCLUSIONS: Oral immunization with attenuated Salmonella typhimurium as a carrier for HCV DNA induces long-lasting T-cell responses.     

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.     

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.     

Immunogenicity of Multi-Epitope DNA and Peptide Vaccine Candidates Based on Core,E2, NS3 and NS5B HCV Epitopes in BALB/c Mice

Background:

Hypervariability of HCV proteins is an important obstacle to design an efficient vaccine for HCV infection. Multi-epitope vaccines containing conserved epitopes of the virus could be a promising approach for protection against HCV.

Objectives:

Cellular and humoral immune responses against multi-epitope DNA and peptide vaccines were evaluated in BALB/c mice.

Materials and Methods:

In this experimental study, multi-epitope DNA- and peptide-based vaccines for HCV infection harboring immunodominant CD8+ T cell epitopes (HLA-A2 and H2-Dd) from Core (132-142), NS3 (1073-1081) and NS5B (2727-2735), a Th CD4+ epitope from NS3 (1248-1262) and a B-cell epitope from E2 (412-426) were designed. Multi-epitope DNA and peptide vaccines were tested in two regimens as heterologous DNA/peptide (group 1) and homologous peptide/peptide (group 2) prime/boost vaccine in BALB/c mice model. Electroporation was used for delivery of the DNA vaccine. Peptide vaccine was formulated with Montanide ISA 720 (M720) as adjuvant. Cytokine assay and antibody detection were performed to analyze the immune responses.

Results:

Mice immunized with multi-epitope peptide formulated with M720 developed higher HCV-specific levels of total IgG, IgG1 and IgG2a than those immunized with multi-epitope DNA vaccine. IFN-γ levels in group 2 were significantly higher than group 1 (i.e. 3 weeks after the last immunization; 37.61 ± 2.39 vs. 14.43 ± 0.43, P < 0.05). Moreover, group 2 had a higher IFN-γ/IL-4 ratio compared to group 1, suggesting a shift toward Th1 response. In addition, in the present study, induced immune responses were long lasting and stable after 9 weeks of the last immunization.

Conclusions:

Evaluation of multi-epitope DNA and peptide-vaccines confirmed their specific immunogenicity in BALB/c mice. However, lower Th1 immune responses in mice immunized with DNA vaccine suggests further investigations to improve the immunogenicity of the multi-epitope DNA vaccine through immune enhancers.     

Expression and immunoreactivity of HCV/HBV epitopes

AIM: To develop the epitope-based vaccines to prevent Hepatitis C virus (HCV)/Hepatitis B virus (HBV) infections. METHODS: The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD (residuals aa2-21) and C2 VKFPGGGQIVGGVYLLPRR (residuals aa22-40), envelope epitope E GHRMAWDMMMNWSP (residuals aa315-328) and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC (residuals aa124-147) were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E.coli cells (pET-3 system). Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined. RESULTS: The expressed chimeric protein carrying the HCV epitopes C1, C2, E (two times), L3C1-I2E-L1C2-L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with anti-HCV and anti-HBs antibodies. CONCLUSION: These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.     

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.     

Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8(+) CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5-12 log(10)) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8(+) cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1(+) humans.     

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.     

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.     

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 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.     

Quantitative analysis of hepatitis C virus-specific CD8+ T cells in peripheral blood and liver using peptide-MHC tetramers

It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.     

DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.     

丙型肝炎病毒NS3蛋白C末端细胞毒T细胞表位的动力学分析

目的研究丙型肝炎病毒（ＨＣＶ）ＮＳ３蛋白Ｃ末端细胞毒Ｔ细胞（ｃｙｔｏｔｏｘｉｃＴｌｙｍｐｈｏｃｙｔｅ,ＣＴＬ）表位的长期演变规律。方法血清学法检测了２例慢性ＨＣＶ感染者的ＨＬＡ分型,并以ＨＬＡ结合基序为依据,预测了ＨＬＡ限制的ＣＴＬ表位。血清样本中的ＨＣＶ基因片段用ＰＣＲ扩增,Ｓａｎｇｒｒ法测定核苷酸序列,并翻译成蛋白质。用计算机软件分析了ＣＴＬ表位的改变。结果５年和３年内预测的ＨＬＡ—Ａ２和ＨＬＡ—Ａ１１限制的ＣＴＬ表位在２例携带者中都未发生改变。结论ＨＣＶＮＳ３蛋白Ｃ末端的ＣＴＬ表位可能与ＨＣＶ慢性化无关。     

Significant effect of hepatitis C virus specific CTLs on viral clearance in patients with type C chronic hepatitis treated with antiviral agents.

Aim: To evaluate the correlation between hepatitis C virus (HCV) specific cytotoxic T lymphocytes (CTLs) and viral clearance in antiviral treated patients, we examined the number and function of HCV epitope-specific CTLs and the viral load in 12 HLA-A2-positive patients with chronic hepatitis C, after undergoing interferon therapy. Methods: Peripheral blood mononuclear cells (PBMC) were analyzed on days 0, 3, 7, 14 and 28 of undergoing antiviral therapies. To investigate the quantity of the antigen specific CTLs, CD8-positive T cells were isolated using microbeads and were stained for HLA-A*0201 tetramers. To investigate the function of CTLs, PBMC were stimulated with the same synthetic epitope peptides and analyzed to determine their interferon (IFN)-gamma expression. Results: In seven patients, HCV-RNA became undetectable 4 weeks after antiviral therapies (EVR), but five patients were non-responders (NR). In peptide NS3 1406 on day 3 and day 7 of therapy and in NS3 1073 on day 3 of therapy, the level of IFN-gamma expression on CD8+ T cells was significantly higher in the EVR group than in the NR group. In other peptides, the number of and cytokine production from the CTLs in the EVR group were also higher than in the NR group, but not significantly. Conclusion: After antiviral therapy, analysis of the number and function of antigen-specific CTLs in the early phase was thus found to be useful for predicting viral clearance in chronic hepatitis C patients.     

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.     

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.