HBV相关抗原HLA-A3超型CTL表位的预测与鉴定

发布时间：2018-07-04 07:50

本文选题：HLA + 超型　；参考：《第三军医大学》2011年硕士论文

【摘要】：研究背景:由乙型肝炎病毒(HBV)引起的慢性乙型肝炎是我国常见的慢性传染病之一,严重危害人民健康。以往研究表明,抗原特异性的CD8+细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)对体内HBV的清除起关键作用。在乙肝患者体内,来源于HBV相关抗原的、MHC-I类分子限制性表位肽被认为是特异性CD8+CTL识别病毒感染细胞的靶标。虽然慢性HBV感染患者外周血中存在抗原肽特异性CTL,但这种病毒特异性CD8+CTL的频率及功能减低,是HBV得以持续存在的一个重要原因。因此,通过主动免疫方式增强HBV相关抗原肽特异性CTL反应,是治疗慢性HBV感染的理想策略。既往对于病毒抗原表位肽的研究多针对单一主要组织相容性复合体(Major histocompatibility complex, MHC)限制性,缺乏覆盖更多人白细胞抗原(human leucocyte antigen ,HLA)背景人群的治疗性表位肽疫苗。HLA分子的高度多态性与T细胞识别的MHC限制性是研制广谱高效表位肽疫苗的最大障碍。HLA超型是指具有相同的肽结合特异性的不同HLA分子的总合。这一概念的提出,为基于HBV相关抗原超型表位肽疫苗的设计提供了新的思路。研究目的:本研究初步分析了慢性HBV感染与HLA-A超型的相关性,预测并初步鉴定了HLA-A3超型限制性HBV相关抗原特异性CTL表位肽,为基于超型表位的慢性HBV感染的治疗提供实验基础及新的候选靶标。研究方法:分别从西南医院肝炎门诊和献血中心收集慢性HBV感染者及健康志愿者外周血,通过聚合酶链反应/序列特异性引物技术(PCR-SSP)对其HLA型别进行检测,分析慢性HBV感染与HLA-A超型之间的关系。基于SYFPEITHI、BIMAS、SVMHV、IEDB、EPIJEN、NETMHC 6个表位预测网站,分别预测出HLA-A3超型各等位基因(HLA-A*0301、HLA-A*1101、HLA-A*3101、HLA-A*3301、HLA-A*6801)限制性表位,并对预测结果进行整合,预测出与HLA-A3超型各等位基因具有高亲和力的乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)候选表位肽。鉴于HLA超型分子具有结合相同或相似表位肽的特点,并且在HLA-A3超型中,HLA-A*1101分子表型频率最高,因此,我们选择首先鉴定候选超型表位肽HLA-A*1101限制性。在体外细胞功能试验中,通过Elispot Assay检测候选表位肽诱导外周血单核细胞(PBMC)IFN-γ的分泌;胞内因子染色流式细胞术分析表位肽特异性CD8+CTL IFN-γ及GranB的分泌;CFSE标记检测表位肽诱导的特异性CD8+CTL的增殖;Pentamer染色流式细胞术分析法检测HLA-A*1101健康志愿者PBMC经肽诱导后所产生的特异性CD8+CTL频率,以及慢性HBV感染患者外周血表位肽特异性CD8+CTL的频率。最后通过Elispot assay初步鉴定候选表位肽对其他HLA-A3超型等位基因的健康人PBMC所产生的诱导效应。研究结果:在我们收集的44例来自西南医院献血中心的健康志愿者中,HLA-A3超型为主要HLA-I类分子超型基因,其中以HLA-A*1101频率最高,在49例慢性HBV感染者中,HLA-A2超型为最高频超型等位基因。结合多个在线表位预测网站,采用整合法预测的超型表位兼顾了与HLA-A3超型全部等位基因具有高亲和力的表位(P5, P6, P7, P8, P9)。根据应答指数以及胞内因子染色结果证实五条候选表位肽均能在体外诱导HLA-A*1101健康人外周血表位肽特异性CD8+CTL增殖并分泌IFN-γ和GranB。在HLA-A*1101阳性的慢性HBV感染患者中,P7肽诱导产生的特异性CD8+CTL频率明显高于已知的阳性表位P6肽。五条候选超型表位肽均可以诱导带有HLA-A3超型等位基因个体的PBMC分泌IFN-γ,但是对同属于HLA-A3超型的不同HLA等位基因,其限制性CTL分泌IFN-γ的能力存在差异。研究结论:结合本研究中各部分的实验结果,HLA-A3超型在人群中分布频率最高,同时,HLA-A*1101是HLA-A3超型中最高频等位基因。利用整合法预测的5条HBV相关抗原候选表位,经初步鉴定为HLA-A3超型限制性CTL表位。而且,从表位肽特异性CTL的频率与功能水平上分析,较目前已知的HLA-A*1101限制性阳性表位肽(P6),P7肽是一个明确的免疫优势性的HLA-A*1101限制性表位肽。
[Abstract]:Background: chronic hepatitis B caused by hepatitis B virus (HBV) is one of the common chronic infectious diseases in China, which seriously endangers the people's health. Previous studies have shown that the antigen specific CD8+ cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) plays a key role in the clearance of HBV in the body. In hepatitis B patients, it comes from HBV phase. Antigenic, MHC-I class molecular restrictive epitopes are considered to be specific CD8+CTL targets for identifying virus infected cells. Although antigenic peptide specific CTL exists in peripheral blood of patients with chronic HBV infection, the frequency and function of this virus specific CD8+CTL is an important reason for the persistence of HBV. Pestilence enhanced HBV related antigen peptide specific CTL response, which is an ideal strategy for the treatment of chronic HBV infection. Previous studies of viral antigen epitopes are mostly limited to a single major histocompatibility complex (Major histocompatibility complex, MHC), and lack of more human leukocyte antigen (human leucocyte antigen, HLA) back. The high polymorphism of the therapeutic epitope peptide vaccine.HLA molecule and the MHC restriction of T cell recognition are the biggest obstacle to the development of the broad spectrum epitope peptide vaccine.HLA super type is the combination of the different HLA molecules with the same peptide binding specificity. This concept is designed for the design of the super epitope peptide vaccine based on the HBV related antigen. A new way of thinking is provided.
Objective: This study preliminarily analyzed the correlation between chronic HBV infection and HLA-A super type, and predicted and preliminarily identified the HLA-A3 super restrictive HBV related antigen specific CTL epitope, providing experimental basis for the treatment of chronic HBV infection based on super epitopes and a new candidate target.
Methods: the peripheral blood of chronic HBV infected persons and healthy volunteers were collected from the Southwest Hospital hepatitis clinic and blood donation center respectively. The HLA types were detected by polymerase chain reaction / sequence specific primer technique (PCR-SSP), and the relationship between the chronic HBV infection and the HLA-A type was analyzed. Based on SYFPEITHI, BIMAS, SVMHV, IEDB, EPIJEN, NETMHC. 6 epitope prediction sites were used to predict the restriction epitopes of HLA-A3 super type alleles (HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*6801), and the prediction results were integrated to predict the high affinity hepatitis B surface antigen (HBsAg) and the hepatitis B core antigen (HBcAg) candidate epitope with the HLA-A3 super type alleles. In view of the characteristics of HLA super type molecules having the same or similar epitopes and the highest phenotypic frequency of HLA-A*1101 molecules in the HLA-A3 super type, we chose to identify the HLA-A*1101 restriction of the candidate super epitopes first. In vitro cell function test, the candidate epitope was detected by Elispot Assay to induce peripheral blood mononuclear cells (P). BMC) secretion of IFN- gamma; intracellular factor dyed flow cytometry analysis of the secretion of epitope specific CD8+CTL IFN- gamma and GranB; CFSE markers to detect the proliferation of specific CD8+CTL induced by epitope peptide; Pentamer staining flow cytometry analysis of the specific CD8+CTL frequency of PBMC in HLA-A*1101 healthy volunteers after peptide induction. The frequency of peripheral blood epitoped peptide specific CD8+CTL in patients with chronic HBV infection. Finally, the inducement effect of candidate epitopes on the healthy human PBMC of other HLA-A3 super type alleles was preliminarily identified by Elispot assay.
Results: in 44 healthy volunteers from the blood donation center of Southwest Hospital, the HLA-A3 super type was the main HLA-I molecular super type gene, of which the frequency of HLA-A*1101 was the highest. Among the 49 cases of chronic HBV infection, the HLA-A2 super type was the most high-frequency super type allele. The super epitopes have high affinity to all alleles of HLA-A3 super type (P5, P6, P7, P8, P9). According to the response index and intracellular factor staining results, it is confirmed that all five candidate epitopes can induce the proliferation of the peripheral blood epitoped peptide specific CD8+CTL in HLA-A*1101 healthy human and secrete IFN- gamma and GranB. in HLA-A*1101 in vitro. In the patients with positive chronic HBV infection, the specific CD8+CTL frequency induced by P7 peptide is significantly higher than the known positive epitopes P6 peptide. Five candidate super epitopes can induce PBMC secreting IFN- gamma with HLA-A3 super type allele, but the restrictive CTL secretes IFN- gamma for the same HLA alleles belonging to HLA-A3 super type. There is a difference in ability.
Conclusion: combined with the experimental results of various parts of this study, the HLA-A3 super type is the highest frequency in the population, while HLA-A*1101 is the most high frequency allele in the HLA-A3 super type. The 5 HBV associated antigen candidate epitopes predicted by the integration method are preliminarily identified as HLA-A3 super limited CTL epitopes. Moreover, the frequency of the epitopes specific CTL Compared with the current known HLA-A*1101 restrictive positive epitopes (P6), the P7 peptide is a definite immune dominant HLA-A*1101 restrictive epitope.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】