汉滩病毒糖蛋白HLA-A2限制性CTL表位免疫保护性作用的评价

Hemorrhagic fever with renal syndrome (HFRS) is a serious acute infectious disease distributed globally with great harm. As we known, the cellular immune response is one of the effective mechanisms in the antivirus process. However, so far, the research on the pathogen Hantaan virus (HTNV) specific T-cell response is limited. Only several HLA restricted CTL epitopes on HTNV nucleocapsid protein have been reported. In our previous work, we used the computer prediction to screen the candidate HTNV glycoprotein (Gn/Gc) 9-mer peptides restricted by HLA-A2, which is the most popular allele in Chinese population. The synthesized candidate peptides were then screened by the T2 cell binding assay and ELISPOT to define a group of HLA-A2 restricted HTNV-Gn/Gc 9-mer epitopes. In this project, the frequency of the HLA-A2 restricted HTNV-Gn/Gc CTL response in the peripheral blood of HFRS patients and in the HLA-A2 transgenic mice infected by the HTNV will be detected, and the correlation between the responses and the disease severity will be analyzed. Thus the HLA-A2 restricted HTNV-Gn/Gc 9-mer epitopes which can cause the immune protective responses in the HFRS will be defined. Then the way and the mechanism through which HTNV-Gn/Gc specific CTL responses can play the antivirus effect will be studied. Base on these results, the research can provide the important theoretical basis for illustration on the principle of the protective immune responses caused by HLA-A2 restricted HTNV-Gn/Gc specific CTL epitopes，and also for the design of the novel peptides vaccine for HTNV.

肾综合征出血热(HFRS)是全球广泛分布且危害极大的一种急性传染病。已知细胞免疫应答是机体抗病毒的有效机制之一，但目前对引起HFRS的汉滩病毒(HTNV)特异性T细胞应答研究，仅局限于数个核蛋白特异性CTL表位。我们前期研究工作中预测并合成了我国人群中频率最高的HLA-A2限制性候选HTNV糖蛋白9肽，应用T2细胞结合实验以及酶联免疫斑点实验，系统筛选出一组HLA-A2限制性糖蛋白CTL表位。本课题拟在此基础上，测定HLA-A2限制的HTNV糖蛋白表位特异性CTL在不同病情患者外周血中以及HTNV感染HLA-A2转基因小鼠中的应答水平，分析其与病情的相关性，从而鉴定出能够诱导免疫保护作用的HLA-A2限制性HTNV糖蛋白CTL表位，探讨糖蛋白表位特异性CTL发挥抗病毒作用的途径，阐明HLA-A2限制的糖蛋白特异性CTL保护性免疫应答的规律，为新型HTNV多肽疫苗设计提供重要的理论依据。

汉滩病毒（Hantaan virus，HTNV）感染在人类可引起严重的肾综合征出血热（hemorrhagic fever with renal syndrome，HFRS）疾病。已知病毒特异性T细胞应答在抗病毒免疫中发挥着至关重要的作用，且与病情的发生、发展及预后关系密切，但目前对于HTNV糖蛋白（Gn/Gc）上杀伤性T细胞（cytotoxic T cell，CTL）表位的鉴定及其发挥的作用尚未见研究报道。HLA-A2在中国人群中分布最广、频率最高，因此鉴定出HLA-A2限制的HTNV-Gn/Gc 9肽CTL表位，并研究表位特异性CTL应答在HTNV感染中发挥的作用具有重要的意义。. 在前期筛选得到7个HLA-A2高亲和力HTNV-Gn/Gc 9肽表位基础上，对表位特异性CTL在HFRS患者外周血中的频率及分泌细胞因子、杀伤介质以及增殖能力进行检测，并利用HLA-A2.1/Kb转基因小鼠体内HTNV复制模型进行攻毒实验，检测表位免疫后的小鼠各器官中HTNV抗原及病毒载量。. 四聚体染色发现HFRS患者PBMC中可分别检测到7条HTNV-Gn/Gc表位特异性的CTL应答频率，且轻型/中型患者特异性CTL频率明显高于重型/危重型患者（P<0.05）。HTNV-Gn/Gc表位特异的CTL可分泌IFN-γ和TNF-α细胞因子以及颗粒酶B和穿孔素杀伤介质并表达CD107a，并可诱导特异CTL增殖。其中HTNV-Gn/Gc 9肽表位VV9、SL9和LL9分别免疫HLA-A2.1/Kb转基因小鼠后在HTNV攻毒实验中可诱导强而有效的特异性CD8+ T细胞应答，均可显著降低小鼠肝、肾和脾中HTNV RNA载量和HTNV抗原含量，其中LL9免疫组HTNV RNA载量及HTNV抗原水平均显著低于VV9和SL9免疫组（P<0.05）。. 总之，鉴定得到保护性HLA-A2限制性HTNV-Gn/Gc CTL 9肽表位，其中LL9、SL9和VV9可能是主要的免疫保护性表位，以LL9诱导的免疫保护作用最为显著，在HTNV感染的小鼠体内可通过诱导CD8+T细胞应答发挥抗病毒作用。该研究结果可为设计新型HTNV疫苗提供明确的、有价值的实验资料，为深入阐明HFRS发病过程中HTNV-Gn/Gc特异性CTL应答的免疫保护作用机制提供基础。