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.     

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。     

HLA B44-restricted cytotoxic T lymphocyte responses to the peptides of HCV nucleoprotein residues 81–100 in patients with chronic hepatitis C

Human leukocyte antigen B44-restricted cytotoxic T lymphocytes (CTLs) recognize an epitope in hepatitis C virus (HCV) nucleoprotein residues 81–100. CTLs that recognize two wild-type peptides 81–100 of HCV genotypes 1b/II and 2a/III were generated from peripheral blood lymphocytes of each of three patients studied. Although CTLs that recognize a wild-type peptide 81–100 of HCV genotypes 1a/I and 2b/IV were not generated from any patient, CTLs that recognize peptide 81–100 of a rate HCV isolate of type 1a/I were generated from two patients. The results suggest that HLA B44-restricted CTLs recognize most, if not all, HCV isolates of types 1b/II and 2a/III and rare variants of type 1a/I and that the wild-type HCV isolates of genotypes 1a/I and 2b/IV may be less immunogenic for HLA B44-restricted CTLs.     

Impaired cytotoxic T lymphocyte inductivity by dendritic cells derived from patients with hepatitis C virus-positive hepatocellular carcinoma

Aim: Peptide-based therapeutic vaccines are being developed. The aim of this study was to determine the feasibility of immunotherapy to hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) by assessing the inductivity of peptide-specific cytotoxic T lymphocyte (CTL) by dendritic cells. Methods: The inductivity of CTL was characterized in six patients with HCV-positive HCC, and compared to seven healthy volunteers and six patients with chronic HCV hepatitis (control). Results: Peptide-specific CTL was comparably induced in controls, but not induced in any patients with HCC. To characterize this, the cytokine profile and the expression of surface molecules interacting between dendritic and T cells were evaluated. Among the cytokines, production of interferon (IFN)-gamma was found to be impaired and closely related to the results of CTL assays, while the expression of surface molecules showed no significant changes. Conclusions: In HCV-positive HCC patients, CTL inductivity by dendritic cells is impaired. This may be related to the impaired production of IFN-gamma.     

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.     

Generation of hepatitis C virus-specific cytotoxic T lymphocytes from healthy individuals with peptide-pulsed dendritic cells

BACKGROUND AND AIMS: In hepatitis C virus (HCV) infection, cytotoxic T lymphocytes (CTL) are involved in liver inflammation and contribute to the reduction of viral load. Antibodies for HCV-CTL precursor frequencies (CTLpf) are relatively low in chronic hepatitis C, and this may be related to the poor CTL response in vivo. The aim of this study was to assess the efficacy of dendritic cells (DC) as antigen-presenting cells in CTL generation from low CTLpf. METHODS: To confirm the rationale of using DC to prime naive T cells, five HCV-uninfected individuals were enrolled in the study. We obtained DC by maturation from peripheral progenitors under stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and IL-1alpha. Autologous T cells were cultured with DC or concanavalin-A-induced blasts loaded with four HCV-derived peptides bearing human leukocyte antigen (HLA)-A*0201 or -A24 motifs for 28 days under IL-7 and IL-2 stimulation. The lytic activity against peptide-pulsed targets was assessed by using a [51Cr]-releasing assay. RESULTS: The DC strongly expressed HLA class I, II, B7-1 and B7-2, but not phenotypic markers of T-, B-, natural killer (NK)-cells or monocytes. The CD8-positive, HLA-class I-restricted and HCV peptide-specific CTL were generated with DC from HLA-A antigen-matched subjects, whereas no CTL activity was detected with concavalin (Con-A) blasts. We were thus able to generate HCV specific CTL from naive precursors with peptide-pulsed DC. CONCLUSIONS: This DC-based system can be used to generate CTL of desired antigen specificity, even from a source with low CTLpf.     

人类免疫缺陷病毒和丙型肝炎病毒重叠感染者的临床及免疫特征

目的 观察人类免疫缺陷病毒(HIV)和HCV重叠感染者与慢性丙型肝炎患者临床特征及HCV特异性细胞毒性T淋巴细胞(CTL)的数量及功能,探讨两组患者免疫功能的差异及其可能的影响因素.方法 以HIV和HCV重叠感染患者59例、慢性丙型肝炎患者36例为研究对象,取治疗前外周血检测肝脏生物化学指标、血常规、外周血T淋巴细胞亚群(CD4+T、CD8+T淋巴细胞计数)及HIV、HCV病毒载量,以酶联免疫斑点法检测HCV特异性CTL的数量和功能,统计学分析两组问免疫功能的差异及与上述检测指标的相关性. 结果 中国河南省有偿献血、单采血浆人群HIV感染者中HIV和HCV重叠感染率达60.8%.ALT、AST值在重叠感染组与HCV组间差异无统计学意义;球蛋白在重叠感染组为(40.3±5.8)g/L,HCV组为(32.8±6.3)g/L,差异有统计学意义(P＜0.01).重叠感染组外周血CD4+T淋巴细胞数明显低于HCV组(P＜0.01),而CD8+T淋巴细胞数高于HCV组(P＜0.01).重叠感染组HCV RNA定量高于HCV组(P＜0.01).重叠感染组对HCV-NS3区肽段的反应强度(每106个外周血单个核淋巴细胞中斑点形成细胞的个数)较HCV组弱,649.34±685.90对比1233.70±1085.16,差异有统计学意义(P＜0.05).重叠感染组白蛋白与HCV病毒载量呈现负相关(r=0.540);重叠感染组对HCV-NS3区肽段反应强度与HIV病毒载量负相关(r=0.356);重叠感染患者CD4+T淋巴细胞数与血小板正相关(P＜0.05).但未见重叠感染组HCV RNA与CD4+T淋巴细胞数量及HIVRNA水平有相关关系.结论 重叠HIV感染有利于HCV的复制,而HIV载量可影响针对HCV的特异性免疫反应,HIV载量高则不利于HCV的清除.慢性丙型肝炎患者重叠HIV感染时,病情易慢性化,预后更差.     

Identification of human leucocyte antigen (HLA)‐A*0201‐restricted cytotoxic T lymphocyte epitopes derived from HLA‐DOβ as a novel target for multiple myeloma

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)‐A*0201 (HLA‐A2)‐restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA‐DOβ (DOB) as a potential target for MM. By DNA microarray analysis, the HLA‐DOB expression in MM cells was significantly higher than that in normal plasma cells. Twenty‐five peptides were predicted to bind to HLA‐A2 from the amino acid sequence of HLA‐DOB. When screened for the immunogenicity in HLA‐A2‐transgenic mice immunized with HLA‐DOB cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA‐A2‐immunoprecipitates of MM cell lines, only two epitopes, HLA‐DOB_232–240 (FLLGLIFLL) and HLA‐DOB_185–193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated in vitro with these two peptides and assessed by antigen‐specific γ‐interferon secretion, HLA‐DOB_232–240 was more immunogenic than HLA‐DOB_185–193. Additionally, the HLA‐DOB_232–240‐specific CTLs, but not the HLA‐DOB_185–193‐specific CTLs, displayed an major histocompatibility complex class I‐restricted reactivity against MM cell lines expressing both HLA‐A2 and HLA‐DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA‐DOB_232–240 might be useful for developing a novel immunotherapy against MM.     

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。     

HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new Plasmodium falciparum epitopes by IFN-gamma ELISPOT

The role of antigen specific CD8+ T-lymphocytes in mediating protection against sporozoite-induced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-specific CD8+ T-lymphocytes. Nevertheless, the low frequency of circulating specific cells together with the lack of sensitive methods to quantify them has hampered the direct assessment of their function. Using a combination of short-term cell culture and IFN-gamma ELISPOT, we studied CD8+ T-lymphocyte responses to a panel of HLA-A*0201 binding peptides. In addition to confirming the response to already described epitopes, we also identified five new CD8+ T-lymphocyte epitopes. These epitopes are presented in pre-erythrocytic stages gene products of Plasmodium falciparum 7G8 strain and correspond to the following protein segments: circumsporozoite (CS) 64-72, 104-113, 299-308 and 403-411; liver stage antigen (LSA-1) repeat region; sporozoite surface protein 2 or thrombospondin related anonymous protein (SSP2/TRAP) 78-88 and 504-513. Four of these peptides are conserved amongst all published sequences of P. falciparum strains. We conclude that the modified IFN-gamma ELISPOT assay is a sensitive technique to monitor antigen-specific CD8+ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the efficacy of malaria subunit vaccines.     

Induction of HIV-1 Nef-specific cytotoxic T lymphocytes by Nef-expressing DNA vaccine

Recently, some individuals who have remained seronegative despite definite exposure to HIV-1 have been reported. Among these individuals, an unusually high frequency of HIV-1 Nef-specific cytotoxic T lymphocytes was observed. Direct injection of plasmid DNA encoding foreign antigen can elicit both cell-mediated immunity and antibody responses (DNA vaccine). We constructed an HIV-1 Nef-expressing plasmid, and we induced HIV-1 Nef-specific cytotoxic T lymphocytes. This is the first report of inducing HIV-1 Nef-specific cytotoxic T lymphocytes by DNA vaccine. © 1996 Wiley-Liss, Inc.     

Identification of hepatitis C virus 1b-derived peptides recognized by both cellular and humoral immune systems in HCV1b(+) HLA-A24(+) patients.

Despite scientific advances, the therapeutic options for hepatitis C virus (HCV) are limited by poor response rates. HCV1b is particularly resistant to standard interferon therapy. The inhibition of the progression of chronic hepatitis and liver cirrhosis and the prevention of the occurrence and recurrence of hepatocellular carcinoma (HCC) are important and thus, there is a need for new therapeutic modalities for HCV1b infection. We, therefore, investigated highly immunogenic peptides and report in this study three novel candidate peptides (at positions 711-720 in envelope 2 protein, 885-893 in non-structural protein 2 and 1716-1724 in non-structural protein 4B) among 35 peptides of conserved regions of HCV1b proteins containing HLA-A24 binding motifs tested. Namely, HCV(711-720), HCV(885-893) and HCV(1716-1724) induced HLA-A24-restricted and peptide-specific cytotoxic T lymphocytes (CTLs) activity in peripheral blood mononuclear cells (PBMCs) from 7, 6 and 5 of 12 patients and also were recognized by plasma of 8, 5 and 7 of 12 HCV1b(+) patients, respectively. These results may provide new insight into the development of a peptide-based specific immunotherapy for HCV1b(+) HLA-A24(+) patients.     

PD-1分子对慢性丙型肝炎患者T细胞免疫功能的影响

目的 探讨慢性丙型肝炎患者外周血T细胞表面程序性死亡因子-1（PD-1）分子在T细胞免疫应答中的作用。方法受试对象包括40例慢性丙型肝炎患者,10例非病毒性肝炎的肝病患者,以及20名健康对照。取外周血,采用流式细胞术检测CD4^＋及CD8^＋T细胞上PD-1的表达水平;ELIsA法测定混合淋巴细胞培养上清中IFN—γ及IL-2的浓度。结果慢性丙型肝炎患者外周血CD4^＋及CD8^＋T细胞上PD-1阳性表达率[（38．61±4．35）％、（48．17±5．16）％]明显高于其他肝病患者及健康对照（P〈0．01）。慢性丙型肝炎患者产生Ⅰ型细胞因子的能力明显降低,阻断PD-1共刺激信号途径能够增强患者T细胞分泌I型细胞因子的能力。结论慢性丙型肝炎患者外周血T细胞上PD-1表达水平的升高,可能是导致T淋巴细胞应答能力下降的重要原因。     

Characterization of an immunologically conserved epitope from hepatitis C virus E2 glycoprotein recognized by HLA-A2 restricted cytotoxic T lymphocytes

BACKGROUND/AIMS: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins. METHODS: Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays. RESULTS: Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL. CONCLUSIONS: These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.     

Identification of new immunogenic peptides in conserved regions of hepatitis C virus (HCV) 1b with the potentiality to generate cytotoxic T lymphocytes in HCV1b+ HLA-A24+ patients

Aim: Hepatitis C virus (HCV) 1b is resistant to standard interferon therapy and has a high risk of developing into hepatocellular carcinoma at the late stage of infection. Therefore, new therapeutic modalities for HCV1b infection must be developed. One approach would be active specific immunotherapy with highly immunogenic HCV1b peptides. Methods: HCV1b-derived 44 synthetic peptides were selected based on their binding scores to HLA-A24. Peptide-specific IgG were measured by ELISA. Peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in vitro by repeated peptide-stimulation. Results: We identified three novel candidate peptides of HCV1b proteins containing HLA-A24 binding motifs. Each of them had the ability to induce HLA-A24-restricted and peptide-specific CTL activity, and IgGs specific to each of them were detected in the plasma of HCV1b patients. Among these three peptides, a peptide NS5A 2132-2142 was recognized by both cellular and humoral immunities in the majority of blood samples of patients tested. More importantly, the peptide-stimulated peripheral blood mononuclear cells (PBMCs) showed cytotoxicity against cells cotransfected with NS5A and HLA-A2402 genes in an HLA-restricted manner. This is an additional report to our previous study. Conclusion: These findings may provide a new insight into the development of a peptide-based specific immunotherapy for HCV1b-infected patients.     

乙型肝炎患者HBV X蛋白特异性CTL表位变异分析

目的 比较急性乙型肝炎、慢性乙型肝炎与慢性重型乙型肝炎HBV X 蛋白特异性CTL表位变异差异,探讨乙型肝炎重症化和慢性化的可能机理.方法 对393例乙型肝炎患者的血清样本进行HLA-A2分型;用巢式PCR扩增血清HBV前S/S基因与X基因并对PCR产物进行序列测定;根据HBV前S/S基因序列,用VirusBlast软件鉴定患者感染的HBV基因型;用Vector NTI软件对目前已知的6个HLA-A2限制性X蛋白特异性CTL表位序列分析,对患者各个表位变异的差异进行卡方检验.结果 190例(48.35%)患者HLA-A2阳性,其中急性乙型肝炎(AHB)67例,慢性乙型肝炎(CHB)52例,慢性重型乙型肝炎(CSHB)71例.CTL表位变异分析结果如下:对三组所有患者进行比较时,X92-100表位变异发生频数三组患者比较有非常显著性差异(P＜0.01);对三组中HBV C基因型患者进行比较时,X92-100和X115-123表位变异发生频数三组患者比较有显著性差异(P＜0.05);对三组中HBV B基因型患者进行比较时,各表位变异发生频数三组患者比较无显著差异.结论 某些HBV X蛋白特异性CTL表位在AHB、CHB和CSHB患者间变异有明显差异且受病毒基因型影响,CTL表位变异可能与乙型肝炎的重症化和慢性化机制相关.