肺炎链球菌疫苗D39△CPS-TA引起的Th17型免疫应答机制研究

Streptococcus pneumoniae strain D39△CPS-TA, a mutant strain we found previously was shown to be an effective live attenuated vaccine candidate which could significantly reduce the colonization of wild Streptococcus pneumoniae strain on the upper respiratory tract in mice. At the same time, there was dramastic increase of IL-17A concentration in vaccinated mice. It is reported that IL-17A,a key factor involved in host aquired musocal immune response could assist the host against colonization of pathogenic bacteria. Thus it is indicated that the immune protection effect induced by this vaccine strain could be related to the activation of Th17 immune response. However, the mechanism involved in the the activation of Th17 immune response is still not clear.. In this study the mechanisms of Th17 immune response induced by D39△CPS-TA will be systematically explored from several aspects including the TLRs combined with D39△CPS-TA, the activation and maturation of dendritic cells stimulated with D39△CPS-TA,the activation of T lymphocytes and the function of effective factors. Gene knockout mice mode and cell mode will be established and techniques such as real-time PCR, expression spectrum genechips, flow cytometry and laser scanning confocal microscope will be used. As a result, the mechanism figure of "recognition by receptors- activation of signal path- activation of lyphocytes- immune response of host" will be described.. This research will not only provide theoretical basis and experimental basis for further development of this candidate vaccine but also afford some novel ideas for exploring the immune protection mechanisms of other kinds of Streptococcus pneumoniae vaccines as well as vaccines of other bacteria strains.

肺炎链球菌D39△CPS-TA是本课题组前期发现的一株高效的疫苗候选菌株，粘膜免疫小鼠后，可以显著降低肺炎链球菌在小鼠鼻咽部的定植，同时免疫小鼠体内IL-17A水平也明显升高。文献报道IL-17A在宿主获得性粘膜免疫应答中起关键作用，可抵抗病原菌在宿主体内定植。因此提示疫苗D39△CPS-TA的免疫保护效应与Th17型免疫应答有关，但具体机制不详。. 本研究拟借助缺陷小鼠模型、体外细胞模型，采用荧光定量PCR、功能分类表达谱芯片、流式细胞术、激光共聚焦等技术，从与抗原结合的受体、树突状细胞的成熟、淋巴细胞的活化以及效应因子的作用等方面系统研究D39△CPS-TA引起Th17型免疫应答机制，绘制其"受体识别-信号通路激活-淋巴细胞活化-宿主免疫效应"的机制图谱。. 本研究可为该疫苗的进一步研发提供理论依据，并为其它类型的肺炎链球菌疫苗乃至其它菌种疫苗的免疫保护机制研究提供新思路。

肺炎链球菌D39△CPS-TA（SPY1）是本课题组前期发现的一株高效的疫苗候选菌株，粘膜免疫小鼠后，可以显著降低肺炎链球菌在小鼠鼻咽部的定植，同时免疫小鼠体内IL-17A水平也明显升高。因此提示疫苗D39△CPS-TA的免疫保护效应与Th17型免疫应答有关，但具体机制不详。. 研究内容包括：采用IL-17缺陷小鼠观察细菌定植量及炎症细胞浸润的情况，以阐明IL-17A在小鼠鼻咽部细菌定植中的作用及机制；采用定量PCR筛选参与介导SPY1引起Th17型免疫保护作用的TLR种类； Western Blot检测MAPKs和NF-κB的磷酸化情况，以研究SPY1参与DC成熟活化的信号通路；DC与CD4+ T细胞共培养检测T细胞的极化情况；采用过继转移研究SPY1活化的DC在小鼠体内引起的免疫应答类型。. 体内实验结果显示：SPY1免疫组IL-17缺陷小鼠的鼻腔灌洗液及肺匀浆中的细菌载量与对照组小鼠无明显差别，表明Th17细胞免疫反应参与了SPY1对细菌定植的保护。组织切片和流式细胞术结果表明SPY1诱导的Th17免疫反应是通过招募炎症细胞如中性粒细胞和巨噬细胞等至感染发生部位，参与SPY1对细菌定植的保护。. 体外实验结果显示：参与SPY1保护作用的模式识别受体主要有TLR1、TLR2和MARCO，通过这些受体SPY1能够引起抗原递呈细胞（树突状细胞和巨噬细胞）的p-38, JNK, ERK和NF-κB p65发生磷酸化，产生TNF-α、IL-6、IL-12及IL-10等多种细胞因子，而这些因子共同作用于初始T细胞，使其向Th1和Th17极化，引起Th1和Th17免疫应答。过继转移结果显示SPY1致敏的DC过继转移到小鼠后，可产生明显的Th1和Th17应答。而野生株D39几乎不能活化DC和巨噬细胞，不能引起有效的免疫应答。. 通过以上研究，阐明了SPY1“受体识别-信号通路激活-淋巴细胞极化-宿主免疫效应”的机制，为该疫苗的进一步研发提供了理论依据，并为其他类型的肺炎链球菌疫苗乃至其他菌种疫苗的免疫保护机制研究提供思路。