人锌转运蛋白8的HLA-A~*0201限制性CTL表位的预测及初步验证

目的预测和初步鉴定1型糖尿病(T1DM)主要自身抗原锌转运蛋白8(ZnT8)的HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)表位,为基于ZnT8抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS预测工具预测该抗原HLA-A*0201限制性结合肽,人工合成待测表位肽,利用T2细胞株测定各肽与HLA-A*0201分子的结合力。利用酶联免疫斑点检测(enzyme-linked immunospotassay,ELISPOT)方法检测候选肽刺激T1DM患者外周血单个核细胞分泌IFN-γ和IL-2的能力,利用标准51Cr释放试验检测特异性CTL诱导活性。结果在所筛选的5个候选CTL表位中,ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)与HLA-A*0201分子具有较高的结合荧光强度,可在体外有效诱导抗原特异性CTL的产生,刺激T1DM患者PBMC分泌IFN-γ和IL-2,并对抗原肽负载的T2细胞具有明显的杀伤效应。结论 ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)可能是HLA-A*0201限制性CTL表位,为基于人ZnT8抗原表位的特异性免疫治疗奠定理论基础。     

负载HCMV pp65抗原肽的HLA-A*1101-GPI四聚体的制备和鉴定

为体外复性制备负载人巨细胞病毒(HCMV)pp65抗原肽的HLA-A*1101-GPI四聚体,研究优化HLA-A*1101重链与生物素化酶底物肽融合蛋白(HLA-A*1101-BSP)在大肠杆菌中的诱导表达的温度、时间和诱导剂IPTG浓度,并以抗HLA-A*0201抗血清进行免疫印迹鉴定HLA-A*1101-BSP的表达水平。将初步纯化的HLA-A*1101-BSP与β2-微球蛋白(β2m)和HCMV抗原肽pp6516-24(GPISGHVLK,简称GPI)一起利用稀释法进行重折叠复性,获得可溶性HLA-A*1101-GPI单体,经生物素化和纯化后,与Streptavidin-PE结合成四聚体。流式细胞仪分析显示HLA-A*1101-GPI四聚体具有与特异性细胞毒T细胞(CTL)的结合活性,表明成功获得可溶性HLA-A*1101-GPI四聚体,为研究HLA-A*1101限制性CTL的免疫应答打下基础。     

HIV-1 pol抗原HLA-A＊0201限制性低亲和性CTL表位预测及改造分析

目的:初步筛选HIV-1 pol抗原的HLA-A*0201 限制性低亲和性CTL表位,预测并初步鉴定修饰后的表位与HLA-A*0201之间亲和性的变化.方法:采用超基序、蛋白酶解、HLA结合力等预测相结合的办法筛选HLA-A*0201限制性低亲和性CTL表位,通过氨基酸置换适当修饰,并以T2细胞检测HLA-A*0201分子与肽的亲和力和稳定性来评价修饰后表位.结果:筛选出的低亲和性CTL候选表位,经修饰后与HLA-A*0201 之间的亲和性均有不同程度的提高.其中,YVSLSFPQI (pol52-60Y1),YVSQIIEQL(pol673-681Y1),YIQKETWEA(pol548-556Y1)HLA-A*0201呈高亲和力结合,同时肽-HLA-A*0201复合物半数解离时间(Dissociation Complex50,DC50)均大于8 h.结论:预测的pol抗原表位经过修饰可能会成为潜在的HLA-A*0201限制性表位.     

HIV-1 Gag抗原HLA-A*0201限制性低亲和性CTL表位预测及改造分析

初步筛选HIV-1 Gag抗原的HLA-A*0201限制性低亲和性CTL表位,预测并初步鉴定修饰后的表位与HLA-A*0201之间亲和性的变化。采用超基序、蛋白酶解预测等相结合的办法筛选HLA-A*0201限制性低亲和性CTL表位,通过氨基酸置换适当修饰,并以T2细胞株测定肽与HLA-A*0201分子的亲和力和稳定性试验来评价修饰后表位与HLA-A*0201之间亲和性。结果:筛选出3个低亲和性CTL候选表位,经修饰后的表位与HLA-A*0201之间的亲和性均有不同程度的提高。YIYKRWIIL(259-267Y1),YQANFLGKI(429-437Y1)和YTNNPPIPV(249-257Y1)与HLA-A*0201呈高亲和力结合,荧光系数(flurorescene index,FI)分别为2.68、2.54和2.35,同时肽-HLA-A*0201复合物半数解离时间(dissociation complex50,DC50)均大于8h。预测的低亲和力表位经过修饰可能会成为潜在的HLA-A*0201限制性表位。     

肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

目的 预测并鉴定新的人乙酰肝素酶(Hpa)抗原的HLA-A2限制性CTL表位,为恶性肿瘤多肽疫苗的免疫治疗提供依据. 方法 采用SYFPEITHI和BIMAS软件预测方法,对肝素酶HLA-A2限制性CTL表位进行预测,合成候选表位肽;利用T2细胞特点,对合成的候选肽与HLA-A2分子进行亲和力分析;利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性;利用ELISPOT检测T细胞活性. 结果 在所筛选的6条候选CTL表位肽中,Hpa(310～318)FLNPDVLDI与HLA-A2分子具有高亲和力,在体外可有效诱导肝素酶特异性CTL的产生,对肝素酶阳性且HLA-A2限制性的HCC-LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应,且能有效诱导IFN-γ分泌,增强免疫活性. 结论 首次发现Hpa(310～318)FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位.     

三种常见HBV抗原肽-HLA-A*0201复合物四聚体的构建及初步检测应用

目的 体外构建三种常见HBV抗原肽-HLA-A*0201复合物四聚体,并初步用该三种四聚体对抗原肽特异性的细胞毒性T细胞(CTL)进行了初步检测.方法 原核高效表达的HLA-A*0201-BSP和β2m蛋白,分别和三种HBV抗原肽(HBVCore18-27,Env335-343,Po1575-583)体外复性折叠成可溶性的HLA-A*0201抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体,分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成相应的三种抗原肽四聚体.最后进行流式细胞仪检测.结果 Dot-ELISA和ELISA检测显示获得了三种具有天然构象的生物素化的HBV抗原肽-HLA-A*0201复合物单体.构建的三种四聚体均可以检测到相应特异性的CTL,在自限性感染患者体内针对乙肝核心抗原(core18-27)的CTL细胞频数(0.18%)高于针对聚合酶抗原(po1575-583)(0.08%)和包膜抗原(env335-343)(0.06%).结论 成功构建了三种具有完整构象的生物素化HBV抗原肽-HLA-A*0201复合物单体,所构建的三种四聚体都可以检测到抗原特异性的CTL.     

人Ⅱ型胶原的HLA-A~*0201限制性CTL表位的预测及初步鉴定

目的预测和初步鉴定类风湿关节炎(rheumatoid arthritis,RA)主要自身抗原Ⅱ型胶原(CollagenⅡ,CⅡ)的HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)表位,为基于CⅡ抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen预测工具预测该抗原HLA-A*0201限制性结合肽;人工合成待测表位肽,利用T2细胞株通过直接免疫荧光法测定各肽与HLA-A*0201分子的结合力。利用酶联免疫斑点检测(enzyme-linked immunospotassay,ELISPOT)方法检测候选肽刺激关节滑液单个核细胞(synovial fluid mononuclear cell,SFMC)分泌IFN-γ的能力。结果综合BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen预测结果筛选出来可能与HLA-A*0201结合的5条肽。MHC亲和力实验表明,在候选的5条肽中,P1261、P1365及P1399具有与HLA-A*0201分子结合的能力,平均荧光强度分别为:1.35、2.53、1.78。ELISPOT试验结果表明,P1365具有刺激SFMC分泌IFN-γ的能力。结论表位预测结果与初步鉴定结果具有一致性,两者联合应用初步认为P1365是HLA-A*0201限制性CTL表位的可能性最大,为下一步表位鉴定及基于人CⅡ抗原表位的特异性免疫治疗奠定理论基础。     

原发性胆汁性肝硬化HLA-A*0201限制性PDC-E2特异性CTL功能分析

目的：探讨原发性胆汁性肝硬化（PBC）中HLA-A＊ 0201限制的PDC-E2159-167特异性CTL与PDC-E2165-174特异性CTL功能。方法：分析15例HLA-A＊0201阳性（A2＋）PBC患者抗原肽PDC-E2159-167、PDC-E2165-174诱导的CTL对以表达HLA-A＊ 0201分子的T2细胞作为靶细胞的细胞毒性作用，并利用四聚体技术结合细胞内因子染色研究PDC-E2159-167特异性CTL与PDC-E2165-174特异性CTL分泌细胞因子（IFN-γ、IL-4和TNF-α）情况。结果：15例A2＋PBC患者中，12例PDC-E2159-167诱导的CTL和10例PDC-E2165-174诱导的CTL对负载相应抗原肽的靶细胞特异杀伤活性大于10%（E∶T=40∶1），而对照组则均小于10%；15例A2＋PBC患者从PBMC诱导的PDC-E2159-167特异性CTL和PDC-E2165-174特异性CTL中分泌IFN-γ细胞比值分别为0.33%±0.24%（0%～0.86%）、0.27%±0.24%（0%～0.75%），未检测到分泌TNF-α或IL-4的PDC-E2159-167或PDC-E2165-174特异性CTL。结论：结果表明，大部分PBC患者中HLA-A＊ 0201限制性PDC-E2159-167特异性CTL和PDC-E2165-174特异性CTL具有特异杀伤活性，HLA-A＊ 0201限制性PDC-E2特异性CTL主要分泌IFN-γ并非IL-4、TNF-α。     

肿瘤抗原PIWIL2的HLA-A2限制性CTL表位鉴定

目的:鉴定肿瘤抗原PIWIL2的人类白细胞抗原A2(HLA-A2)限制性细胞毒性T淋巴细胞(CTL)表位。方法:首先运用RT-PCR、Western blot方法检测PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中的表达情况,然后通过BIMAS、Rank Pep、Net MHC、Net CTL1.2及IEDB软件预测打分来选取PIWIL2的HLA-A2限制性的表位。候选表位肽通过标准的Fmoc化学合成法合成,结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力,ELISPOT实验检测候选表位肽诱导CTL分泌IFN-γ的能力,体外细胞毒性实验检测候选肽诱导CTL的能力。结果:PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中均有表达。候选肽P485、P493、P965具有中等结合力。ELISPOT实验结果显示表位肽P485和P965诱导的CTL具有分泌IFN-γ的能力。细胞毒性实验结果显示表位肽P485和P965对MCF-7细胞均有一定的杀伤作用。结论:表位肽P485和P965是优秀的PIWIL2抗原HLAA2限制性CTL候选表位,可能成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

癌-睾丸抗原MAGEC2 HLA-A3限制性细胞毒性T淋巴细胞表位的鉴定

目的:预测并初步鉴定HLA-A3超型限制性MAGEC2抗原特异性细胞毒性T细胞(CTL)表位肽,为基于超型表位的MAGEC2治疗提供实验基础及新的候选靶标。方法:通过BIMAS、SYFPEITHI和IEDB软件预测打分来选取MAGEC2的HLA-A3限制性表位;结合力实验用于检测候选表位与T2A3细胞表面HLA-A3分子的结合能力,ELISPOT实验检测候选表位肽诱导的CTL分泌IFN-γ的能力,体外细胞毒实验检测侯选表位肽诱导的CTL杀伤靶细胞的能力。结果 :表位肽P147、P167、P196、P229和P251具有较好的HLA-A3结合力。ELISPOT实验结果显示表位肽P167、P196和P251诱导的CTL具有分泌IFN-γ的能力。细胞毒实验结果显示表位肽P196和P251诱导的CTL对靶细胞有一定的杀伤作用(P0.05或P0.01)。结论 :P196和P251有更高的HLA-A3分子亲和力,保留了原有的免疫原性,是优秀的MAGEC2抗原的HLA-A3限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.     

Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Identification of an HLA-A*0201-restrictive CTL epitope from MUC4 for applicable vaccine therapy

Recent research has indicated that MUC4 plays an important role in the development of many tumors and may prove useful as a novel cancer immunotherapy target. We aimed to identify HLA-A*0201-restrictive cytotoxic T lymphocyte (CTL) epitopes of the cancer-associated antigen MUC4. The MUC4 sequence was scanned for immunogenic peptides using HLA-binding prediction software. Dendritic cells (DCs) from peripheral blood mononuclear cells (PBMCs) were induced by cytokines. Five possible CTL epitopes were selected by software analysis, synthesized, and used to pulse mature DCs. The CD8⁺ T cells from PBMCs from an HLA-A*0201 healthy donor were stimulated with autologous MUC4-peptide-loaded DCs and expanded in vitro. T cell activation was assessed by ELISPOT, and cytotoxicity was determined by ⁵¹chromium (⁵¹Cr)-release assays. Our results show that CTLs induced by peptide P01204 could lyse T2 cells pulsed with peptide P01204 and HCT-116 cells (MUC4⁺, HLA-A2⁺). Compared with a control peptide, P01204 increased the number of IFN-γ producing T cells. Overall, these results suggest that P01204 is a novel HLA-A*0201-restrictive CTL epitope of the cancer-associated antigen MUC4. This will provide a foundation for the development of tumor-specific peptide vaccines.     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

Identification of HLA-A*3101-restricted cytotoxic T-lymphocyte response to human immunodeficiency virus type 1 (HIV-1) in patients with chronic HIV-1 infection

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infections. As several HIV-1 CTL epitopes restricted to many HLA types are already known, we aimed at identifying the CTL epitopes restricted by HLA-A*3101 in an effort to expand the epitope repertoire available for the development of potential T cell-mediated therapeutic measures and protective vaccines. Scanning of HIV-1 clade B SF2 strain proteins for the presence of peptides containing HLA-A*3101-binding motifs revealed 88 nine- to 11-mer peptides that had been synthesized and assayed for binding to HLA-A*3101 molecules. Peptides with medium to high HLA-binding affinity were tested for their ability to stimulate a CTL response in the peripheral blood mononuclear cells (PBMCs) from selected HIV-1-infected patients. Two of these binding peptides, Env769-779 (RLRDLLLIAAR) and Nef192-200 (KLAFHHMAR), induced peptide-specific CTLs in PBMCs from at least two of five HIV-1-seropositive individuals. CTL clones specific for the two peptides killed HLA-A*3101-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed HLA-A*3101-restricted CTL epitopes. Identification of T-cell epitopes on HIV-1 proteins will increase our understanding of the role of CD8+ T cells in HIV-1 infections and assist in the design of new protective strategies.     

十肽表位HPV-16E711-20氨基酸置换修饰的研究

目的 采用氨基酸置换对人乳头状瘤病毒16型口抗原的人白细胞抗原A2分子限制性细胞毒性T细胞表位HPV-16E7 11-20进行修饰和鉴定。方法 运用量化模体方案对置换后的多肽与人白细胞抗原A2分子的结合系数进行比较，以分子模拟方法确定合成序列，采用标准Fmoc方案进行合成与纯化多肽以及标准^51 Cr释放试验检测特异性细胞毒性T细胞诱导活性。结果 修饰多肽符合人白细胞抗原A2分子限制性细胞毒性T细胞的表位要求，十肽YLLDLQPEVT具有特异性细胞毒性T细胞诱导活性。结论 修饰表位YLLDLQPEVT具有更好的结合力和较强的抗原性，可以代替原有序列E7 11-20（YMLDLQPEIT）作为人乳头状瘤病毒感染治疗性肽疫苗分子设计的新表位。