FliC中TLR-5与Ipaf识别位点在其黏膜佐剂活性中的作用研究

Most infectious agents spread through the mucosal surfaces and therefore mucosal immune responses function as a first line of defense. Flagellin (FliC) derived from bacteria is composed of hypervariable and conserved domain, and previously we confermed that the deletion of hypervarialbe region of FliC retained its mucosal adjuvancy for antigens PAc (S. mutans) and HIV p24, thus the conserved domain of FliC is the most important region for its potential of adjuvancy. TLR-5 and Ipaf signaling pathways inducing innative and antigen-specific adaptive immunity are uniquely activated by separated determinants in the conserved region of FliC respectively, we accordingly speculated that TLR-5/Ipaf binding epitopes of flagellin play central role in its mucosal adjuvancy. To verify this hypothesis, we attempt to systemaically analysis the cellular stimulation activities and infammatory responses of the diverse FliC mutants (integrety FliC, TLR-5-recognition-domain deleted FliC, Ipaf-recognition-domain deleted FliC, and double deleteted FliC), and effort to illucidate the differences of mucosal adjuvancy for the distinct recombinant proteins. The focus of this project is to clarify the function of TLR-5/Ipaf binding epitopes in mucosal adjuvancy of FliC, and to lay the theoretical basis for trying to clear mechanism of safe and effective recombinant FliC-based mucosal adjuvants.

绝大部分传染性疾病通过黏膜途径传播，严重威胁人类自身的健康。单一免疫原难以诱导有效的黏膜免疫应答。我们前期工作业已证实：细菌来源的鞭毛素蛋白（FliC）能显著增强变型链球菌PAc、HIV p24黏膜IgA应答，且免疫增强作用主要来自FliC的保守区，但更进一步的分子机制尚不明确。TLR-5与Ipaf信号通路由FliC保守区上不同位点激活，因此我们推测:FliC上TLR-5与Ipaf识别位点是其实现黏膜免疫佐剂效应的核心区域。为了验证此假说，本项目将以TLR-5与Ipaf信号通路为切入点，系统研究TLR-5或Ipaf识别位点缺失FliC突变体蛋白的细胞刺激活性、及其对实验动物的炎症应答效应，并重点分析突变蛋白在黏膜免疫佐剂效应上的特征，旨在阐明细菌鞭毛素蛋白中TLR-5和Ipaf激活区域在其黏膜免疫佐剂效应中的具体作用，力图为研发机制明确、安全有效的重组鞭毛素为基础的黏膜佐剂奠定理论依据。

黏膜组织是众多病原体入侵的主要场所，诱导黏膜免疫应答对抵御病毒入侵有重要意义。然而单一免疫原难以诱导有效的黏膜免疫应答。我们前期工作业已证实：细菌来源的鞭毛素蛋白（FliC）能显著增强变型链球菌PAc、HIV p24黏膜IgA应答，且免疫增强作用主要来自FliC的保守区，但更进一步的分子机制尚不明确。TLR-5与Ipaf信号通路由FliC保守区上不同位点激活，因此我们推测:FliC上TLR-5与Ipaf识别位点是其实现黏膜免疫佐剂效应的核心区域。.为了探究FliC上两个关键识别位点（TLR-5, Ipaf）在其黏膜应答中的作用，我们构建了各重组鞭毛素与HIV核心蛋白p24的融合蛋白，即FliC-p24、FliC-L3A-p24、FliCΔ90-97-p24和FliCΔ90-97:L3A-p24。首先通过生物学活性检测显示重组鞭毛素蛋白C端融合抗原并不降低其激活TLR5通路的能力，但是影响激活NLRC4通路的能力。然后我们比较了滴鼻免疫途径诱导的系统和黏膜抗体应答，发现只融合激活TLR5通路的鞭毛素蛋白突变体FliC-L3A诱导血清中的抗原特异性的IgG和IgG1抗体的能力要强于融合FliC蛋白，而在诱导黏膜部位产生的IgA抗体水平上二者无显著差异。融合激活NLRC4通路的重组鞭毛素蛋白FliCΔ90-97表现的佐剂效果最弱。总体而言，我们通过研究发现，TLR-5识别位点（90-97位氨基酸）是鞭毛素蛋白黏膜佐剂活性的主要位点，而Ipaf位点缺失(ΔL3A,C端3氨基酸缺失)的FliC抑制鞭毛素蛋白的粘膜免疫佐剂活性。.本项目将以TLR-5与Ipaf信号通路为切入点，系统研究TLR-5或Ipaf识别位点缺失FliC突变体蛋白的细胞刺激活性、及其对实验动物的炎症应答效应，并重点分析突变蛋白在黏膜免疫佐剂效应上的特征，阐明了细菌鞭毛素蛋白中TLR-5和Ipaf激活区域在其黏膜免疫佐剂效应中的具体作用，力图为研发机制明确、安全有效的重组鞭毛素为基础的黏膜佐剂奠定理论依据。