登革病毒特异性CTL识别相同及相似CTL表位时细胞因子分泌模式及功能研究

The functions of dengue virus (DENV)-specific cytotoxic T lymphocytes (CTL) during recognizing cognate or epitope variants are uncertain. Based on the amino acid sequences of previously identified DENV-1-derived HLA–A*0201-, A*1101-, A*2402-restricted CTL epitopes, the epitope variants in other DENV serotypes would be synthesized；The peripheral blood mononuclear cells are isolated from the venous blood of convalescent patients infected with DENV-1 and are stimulated with cognate epitopes and epitope variants. The cytokine secretion pattern and the effect of secreted cytokines on vascular endothelial permeability are explored. DENV–1-derived epitopes are used to immunize HLA-A*0201, HLA-A*1101, HLA-A*2402 transgenic mice, the cytokine secretion pattern of induced CTL during recognizing cognate epitopes or epitope variants and the effect of secreted cytokines on vascular endothelial permeability are determined. The cytotoxic activity of induced CTL against DENV-infected cells are also explored. A129 mice are backcrossed with HLA transgenic mice and the novel mouse model is produced. After being immunized with DENV–1-derived epitopes, the novel mouse model would receive DENV attack. The viral titers in blood and organs, the serum ALT/AST and cytokine levels, are detected. Organ pathological change detection and in vivo vascular permeability assay are performed. This research would contribute to the identification of protective epitopes and the development of dengue vaccine.

体内预存在登革病毒（DENV）特异性细胞毒性T细胞（CTL）在识别相同、相似表位时功能不清。本项目基于DENV–1特异性HLA–A*0201、A*1101、A*2402限制性CTL表位，合成其它血清型中相似表位；各表位体外刺激DENV–1感染恢复者外周血单个核细胞，探讨CTL识别相同或相似表位时细胞因子分泌模式及所分泌细胞因子对血管内皮通透性影响；DENV–1表位分别免疫HLA–A*0201或A*1101或A*2402转基因小鼠，研究所诱导CTL识别相同或相似表位时细胞因子分泌模式及所分泌细胞因子对血管内皮通透性影响，并研究所诱导CTL杀伤DENV感染的细胞的效应；DENV–1表位免疫由A129小鼠与HLA转基因小鼠杂交产生的新型小鼠，DENV攻击小鼠，检测小鼠体内DENV量、血中转氨酶及细胞因子水平，检测肝、脾病变，进行在体血管通透性研究。本研究有望鉴定出保护性表位、促进登革疫苗研发。

研究已证实登革病毒（DENV）特异性细胞毒性T细胞（CTL）具保护作用，但DENV特异性CTL在识别相同或相似表位时功能尚不清楚。本项目合成了14条登革病毒1型（DENV1）特异性HLA-A*0201，HLA-A*1101，HLA-A*2402限制性CTL表位（NS4A140-148, NS2A144-152, NS4B40-48, NS5183-191，NS4B159-167，NS5561-569，NS5505-513，NS399-107，E39-47，NS4A40-48，NS5880-888，NS3472-480，NS3548-556和NS322-30）及在DENV2, 3, 4中具相似序列多肽；MHC-肽复合物稳定性实验和竞争性肽结合实验证实DENV2, 3, 4中具有相似序列多肽同上述HLA分子具有高结合力；基于DENV1感染者外周血单个核细胞（PBMC）的ELISPOT实验表明:除DENV3-NS3472-480以外，DENV2, 3, 4中具有相似序列多肽均为CTL表位；基于HLA-A*0201，HLA-A*1101，HLA-A*2402转基因小鼠，通过ELISPOT和ELISA检测IFN-gamma和TNF-alpha的分泌证实单一多肽免疫小鼠诱导的CTL可交叉识别具有相似序列多肽；病毒空斑实验证实4条DENV1特异性HLA-A*0201限制性CTL表位（NS4A140-148, NS2A144-152, NS4B40-48, NS5183-191）或5条DENV1特异性HLA-A*1101限制性CTL表位（NS4B159-167，NS5561-569，NS5505-513，NS399-107，E39-47）或5条DENV1特异性HLA-A*2402限制性CTL表位（NS4A40-48，NS5880-888，NS3472-480，NS3548-556和NS322-30）免疫小鼠诱导的CD8+ T细胞可保护小鼠抵抗DENV1，2，3，4攻击。本研究表明DENV1特异性CTL在识别相同或相似CTL表位时细胞因子（IFN-gamma和TNF-alpha）分泌模式和浓度没有发生改变，DENV1特异性CTL表位免疫小鼠诱导的CTL对不同血清型DENV具有保护效应。我们的研究成果将促进研发基于交叉CTL反应的登革病毒新型疫苗。