基于TLR4识别的FimH结构优化及其增强防龋疫苗粘膜免疫效应和机制研究

FimH, a component of type I pili on the bacterial outer membrane, which is uptake through glycoprotein 2 by M cell. It is also a novel TLR4 ligand. FimH can induce a signal cascade mediated by binding to the TLR4, and rapidly culminates the production of proinflammatory mediator such as cytokines, chemokines and costimulatory molecules which mediate both innate inflammatory response as well as adaptive immuoresponse. Based on these immunological characters, FimH has been recognized as a new candidate of adjuvant. However, its possible toxic side effect limit its application used as adjuvant. In order to optimize flagellin for use as mucosal adjuvant, internal deletion FimH molecular mimicry (designated as FimH△72-80 ) with point mutations were constructed. Thus, nFimH△72-80 were used as adjuvant to programming the magnitude and persistence of antibody responses to anti-caries vaccine . Firstly, nFimH△72-80 were selected according to the level of cytokine. Next, nanoparticles were constructed through wrapping nFimH△72-80 and anti-caries vaccine with chitosan, immunogenic characterization and protection against Streptococcus mutans infection of the nanoparticles were determined by oral or nasal immunization. Ultimately the influence of gene expression on DC and its cellular regulation of T cells were analyzed, cellular components and microenvironment of local mucosa-associated lymphoid tissue were also discussed to explore the possible mechanism of nFimH△72-80 in enhancing the effect of mucosal immunity by anti-caries Vaccine.

本课题组前期研发的防龋疫苗佐剂FimH能特异性识别M细胞表面的GP2，也是TLR4的新配体，发挥M细胞特异性GP2受体和TLR4受体双重靶向效应,其中FimH与TLR4结合所诱导的生物学功能是增强疫苗免疫活性的关键，然而，其潜在的毒副效应不容忽视。因此，本课题以FimH对TLR4的识别诱导的免疫活性为切入点，在明确FimH中TLR4识别结构域，FimH结构和免疫活性、毒副反应相关性的基础上，通过毒性相关区缺失和点突变优化FimH结构，构建粘附力和免疫活性增强而毒副反应降低的新型粘膜免疫佐剂分子。以壳聚糖包被新型FimH和防龋疫苗制备纳米粒子，经粘膜途径接种，检测其诱导防龋的特异性抗体水平，免疫反应类型和强度以及对龋齿的预防保护能力，评价FiHH增强防龋疫苗粘膜免疫效能，分析TLR4介导的FimH对粘膜上皮细胞、树突状细胞，B细胞活化的功能调控，以及其对局部免疫微环境的影响，系统深入探索Fi

本项目成功构建 FimH突变体，证明其TLR4 依赖树突状细胞活化。研发新型 FimH佐剂评价其免疫活性功能效应和毒性，经粘膜途径接种能够有效地加强防龋疫苗粘膜免疫效果。有效探索CS-TLR2激动剂，CS-TLR4激动剂（包括FimH）的PRRS联合佐剂效应及相关机制，联合佐剂可有效地加强防龋疫苗粘膜免疫效果。 LKB1分子调控DC可能与FimH活化DC的免疫佐剂效应相关，深入研究提示LAKB1调控口腔黏膜朗格汉斯细胞发育及稳态作用，明确LKB1不依赖经典LKB1-AMPK信号通路调控口腔黏膜LC，其可能通过正向调控β-catenin来调控口腔黏膜LC。