骨髓瘤抗原Sp17 HLA-A*0201限制性CTL表位预测及初步鉴定

目的鉴定骨髓瘤抗原Sp17的HLA-A＊0201限制性CTL表位。方法采用超基序和量化基序法预测Sp17的HLA-A＊0201限制性CTL表位；用T2细胞亲和力实验和稳定性实验对该表位进行初步鉴定。结果超基序和量化基序法预测得出Sp17抗原的4个CTL表位（LLEGLTREI（19-27）、ILREQPDNI（27-35）、SLLEKREKT（45-53）、KEKEEVAAV（111-119）），亲和力实验结果表明LLEGLTREI（19-27）、ILREQPDNI（27-35）、SLLEKREKT（45-53）有较高亲和力，而KEKEEVAAV（111-119）肽亲和力较低；稳定性实验表明ILREQPDNI（27-35）、SLLEKREKT（45-53）与HLA-A＊0201分子结合稳定性较好。结论ILREQPDNI（27-35）、SLLEKREKT（45-53）有可能是骨髓瘤抗原Sa17 HLA-A＊0201限制件CTL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。     

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.     

慢性白血病抗原CML28 HLA-A*0201限制性CTL表位预测

目的鉴定慢性白血病肿瘤抗原CML28的HIA-A*0201限制性CTL表位。方法 采用超基序和量化基序结合的方法初步预测CM128的HLA-A*0201限制性CTL表位；利用T2细胞亲合力实验初步验证预测结果。结果 在预测的4个候选CTL表位中，ALVDAGVPM和ALDSDGTLV有较高的亲和力，ALFCGVACA和SLLACCLNA仅有低度亲和力。结论ALVDAGVPM与ALDSDGTLV最有可能是慢性白血病肿瘤抗原CML28的HLA-A*0201限制性CTL表位。     

轮状病毒结构蛋白VP7 HLA-A2.1限制性CTL表位的初步鉴定

目的 预测并初步鉴定轮状病毒Wa株结构蛋白VP7 HLA—A2．1限制性CTL表位。方法 用BIMAS软件预测VP7 HLA—A2．1限制性CTL表位；分子模拟技术对分值最高的4条肽进行分子模建；最后测定候选肽与HLA-A2．1分子的亲和力及结合稳定性。结果 结合BIMAS及分子模拟的预测结果，选择4条肽QLYCDYNLV（132—140）、LLNYILKSV（18—26）、VLMKYDQSL（140—148）及VNWKKWWQV（287—295）作为候选表位肽；4条肽与HLA—A2．1结合的荧光系数（FI）值分别为2．58、3．83、3．19及0．82，肽-HLA-A2．1复合物半数解离时间（DC50）分别为2-4、6-8、8及2h。结论 QLYCDYNLV（132—140）、LLNYILKSV（18—26）及VLMKYDQSL（140—148）为潜在的HLA-A2．1限制性CTL表位。     

加载白血病抗原肽PRl的HLA-A*0201四聚体制备

目的制备加载白血病抗原肽PR1的HLA-A＊0201四聚体。方法构建HLA-A＊0201-BSP和β2-微球蛋白（β2M）原核表达载体，进行目的蛋白表达、纯化。在体外将PR1（VLQELNVIV）与纯化的HLA-A＊0201-BSP、β2M通过稀释复性折叠成HLA-A＊0201-PR1复合物。利用BirA酶进行生物素标记。再经阴离子交换纯化得到生物素化的HLA-A＊0201-PR1复合物单体。此单体与PE标记的链霉亲和素按4：1比例混合，即可形成四聚体。结果成功制备了HLA-A＊0201-PRl四聚体。结论制备的HLA-A＊0201-PR1四聚体为定量检测白血病患者体内PR1抗原特异性的细胞毒性T淋巴细胞提供了有力工具。     

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。     

基于SCORE函数估算HLA-A*0201限制性CTL表位亲和性

目的 建立HLA-A*0201限制性CTL表位亲和性的定量预测方法。方法基于SCORE打分函数,运用定量构效关系的理论和方法研究了HLA-A*0201限制性CTL表位九肽结构与亲和性间的定量关系,并建立了SCORE得分与亲和性的定量关系模型,并用外部样本(5个HLA-A*0201限制性CTL表位九肽)作为预测集用于检验模型的预测能力。结果基于SCORE打分函数建立的定量模型具有较好的相关性(r=0．9165,RMS=0．38)和对外部样本的预测能力(rpred=0.9847,RMS=0.135)。结论基于SCORE打分函数,运用定量构效关系研究的理论和方法建立了HLA-A*0201限制性CTL表位亲和性的定量预测方法,为实验鉴定高亲和性HLA-A*0201限制性CTL表位提供了理论依据。     

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。     

人乳头状瘤病毒11型E7抗原HLA-A*0201限制性细胞毒性T淋巴细胞表位的功能鉴定

目的 筛选和鉴定人乳头状瘤病毒11型E7抗原(HPVllE7)HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位.方法 预测HPVllE7抗原HLA-A*0201限制性CTL表位并合成相对应的表位多肽和四聚体(tetramer),即HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)和82-90(LLLGTLNIV).从健康HLA-A*0201成人外周血单一核细胞诱导树突状细胞(DC)并负载上述表位多肽,流式细胞技术检测DC成熟分化标记及ELISA法检测DC分泌的IL-12;成熟DC负载各组多肽后观察DC激活T淋巴细胞的效应,ELISA法检测T细胞分泌的IFN-γ;四聚体检测抗原特异性CD8+ T细胞及乳酸脱氢酶(LDH)释放法评价DC诱导的CTL对靶细胞的特异性体外杀伤效应.结果 预测的5条HPVllE7表位多肽均能诱导DC的成熟分化;E7 7-15、82-90和15-23多肽负载的DC能激活T淋巴细胞分泌高水平IFN-γ;E7 7-15多肽负载的DC能刺激特异性tetramer+CD8+细胞增殖且其诱导的CTL对HPVllE7/293细胞产生高效率的特异性杀伤作用(P<0.05).结论 筛选并鉴定出1条HPVllE7HLA-A*0201限制性CTL表位E7 7-15(TLKDIVLDL),负载该表位肽的DC体外可诱导高效、特异性的CTL效应,抗原性较强,有可能作为HPV感染治疗用肽疫苗的候选表位.     

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。     

Immunogenic variation between multiple HLA-A*0201-restricted, Hepatitis C Virus-derived epitopes for cytotoxic T lymphocytes

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the immune control of Hepatitis C Virus (HCV) infection. In the current study, a number of HLA-A*0201-restricted CTL epitopes derived from HCV were evaluated by examining the peptide-binding affinity for major histocompatibility complex (MHC) class I molecules, the stability of peptide-MHC complexes, killing activities of peptide-induced CTLs, and frequencies of intracellular interferon (IFN)-gamma-positive CD8+ T cells. Among 24 peptides tested, 15 peptides induced high or medium killing activities of peptide-specific CTLs. Thirteen of the 15 peptides exhibited high or medium binding affinities for HLA-A*0201 molecules, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. In contrast, the stability of peptide-MHC class I complexes was not correlated with killing activities of peptide-induced CTLs. Furthermore, only a limited number of peptides could induce high or medium frequencies of IFN-gamma-producing CD8+ T cells, which were generally considered to play a crucial role for the clearance of HCV. Analyses of the immunogenicity of CTL epitopes such as in the current study should provide important information about the design of an efficient HCV vaccine that induces vigorous, sustained, and broad HCV-specific CTL responses.     

Identification of novel HLA-A*0201-restricted epitopes from anterior gradient-2 as a tumor-associated antigen against colorectal cancer

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration and cellular transformation and its enhanced expression is almost completely restricted to malignant tissues, thus making AGR2 an interesting target for the development of immunotherapeutic strategies. We investigated whether the AGR2 molecule comprises human leukocyte antigen (HLA)-A*0201-binding epitopes recognized by human cytotoxic T lymphocytes (CTLs), which could be targeted in dendritic cell (DC)-based cancer immunotherapy against colorectal cancer (CRC). We reviewed the sequence of AGR2 for peptides that could potentially bind to HLA-A*0201 with the aid of a computer-based program. Five candidate peptides with different binding scores were synthesized and tested. These peptides were then assessed for their immunogenicity to elicit specific immune responses mediated by CTLs in vitro by means of enzyme-linked immunospot assays and CTL assays. AGR2 was highly expressed in several CRC cell lines, including DK01, DLD1, KM12C, HCT-8 and HT-29. DCs pulsed with AGR2-P2 (aa 11-19; LLVALSYTL) or AGR2-P4 (aa 127-135; RIMFVDPSL) generated potent CTLs that could lyse T2 cells pulsed with AGR2-P2 or AGR2-P4 and HLA-A0201(+) AGR2-positive CRC cell lines in a strong dose-dependent and HLA-A*0201-restricted manner. In conclusion, these novel epitopes derived from AGR2 protein may be attractive candidates for DC-based immunotherapy for CRC.     

SARS冠状病毒N蛋白HLA-A*0201限制性CTL表位的预测

目的 预测SARS冠状病毒N蛋白的HLA—A^*0201限制性CTL表位。方法 用人工神经网络和量化矩阵相结合的方法预测出HLA—A^*0201结合肽，再用蛋白酶体矩阵法从中筛选出CTL表位。结果 预测出了6个九肽CTL表位。结论 通过对SARS冠状病毒N蛋白抗原CTL表位进行预测，从而为SARS冠状病毒N蛋白之CTL表位的实验探测和鉴定提供了线索，为认识SARS冠状病毒的免疫保护和免疫病理机制及疫苗的研制提供了基础资料。     

Secondary anchor substitutions in an HLA-A*0201-restricted T-cell epitope derived from Her-2/neu

We investigated analogues of GP2 (IISAVVGIL), an HLA-A*0201-restricted T-cell epitope derived from residues 654-662 in the tumor-associated antigen (TAA) Her-2/neu. One limiting factor of GP2 is its poor affinity for HLA-A*0201. Conformational analysis revealed the P5-P7 region in GP2 appears to be linked to the stability of P9 side chain interaction with the MHC molecule. To identify variants of GP2 with enhanced presentation to HLA-A*0201, we tested V6S, V6T, V6Q, G7P, G7F, T6F7, and Q6F7 for their capacity to stabilize cell surface HLA-A*0201 molecules. Of the mono-substituted variants, V6Q and G7F exhibited superior stabilization as compared to GP2. Molecular dynamics simulations suggest the improved binding can be attributed to concerted motions in the central and C-terminal regions of the peptide. These data support the notion that amino acids in HLA-A*0201 epitopes may be inter-dependent. Priming HLA-A*0201 transgenic mice with G7F-loaded syngeneic dendritic cells stimulated mouse T cells to produce a higher level of INFgamma than mice immunized with GP2.