热休克蛋白gp96作为新型疫苗佐剂在细胞免疫与体液免疫应答中的作用机制

Adjuvants are essential components of currently most used vaccines. However, the key mechanisms of adjuvant action are not fully understood. Due to its unique immune activities, heat shock protein gp96 is an ideal model for studying vaccine adjuvants. Heat-shock protein gp96 as a new vaccine adjuvant and immunostimulator, plays important roles in promoting adaptive immune responses against infectious diseases and cancer. Owing to its remarkable immunostimulatory properties, autologous gp96 vacccines have been initiated in clinical trial for the treatment of a range of tumors in the US. We have demonstrated that gp96 could activate hepatitis B virus (HBV) and influenza virus specific cellular and humoral immune responses, which provides a basis fro the development of a novel vaccine adjuvant. However, until now the mechanisms of immunoregulatory and adjuvant founctions of gp96 in antigen presentation and activation of cellular and antibody response are still in hot debate and await further investigation, which hampers further studies of gp96 immune functions and its application as adjuvant. In this study, we will use HBV and influenza virus antigen presentation as study models, and investigate the molecular mechanism of antigen binding by gp96 by crystal structure analysis of gp96-antigen complexes. Moreover, we will explore the molecular basis of gp96-induced cellular and humoral immunity by analyzing the interaction of gp96 with MHC molecules or other antigen presentation molecules, the function of gp96 in antigen relay line, the role of Toll like receptors (TLR)-mediated innate immunity using gene knockout mice models, as well as performing gp96 adjuvant immunization experiments. This study will allow to optimize the gp96 immune function and provide efficient strategies to potentiate gp96-medicated cellular and humoral responses in new influenza, HBV, HIV and TB vaccines. Furthermore, the studies of the project will help to resolve the key mechanisms of adjuvant action and drive the development of novel and specifically directed vaccine adjuvant strategies.

佐剂是目前绝大多数疫苗的基本成分之一，但其关键作用机制并不完全清楚。热休克蛋白gp96作为免疫活性分子具有独特的免疫功能，是研究疫苗佐剂的理想分子模型。我们在研究gp96在乙肝、流感病疫苗引发的细胞免疫和体液免疫功能研究有多年工作积累，在国际上形成了自己的特色。鉴于目前gp96免疫学机制研究存在较大争议,本课题拟以乙肝病毒、流感病毒等抗原呈递做为模型，通过晶体结构分析，查明gp96与病毒抗原结合的分子机制。并通过分析gp96与MHC分子及其它抗原呈递分子的相互作用及抗原传递、应用基因敲除鼠研究Toll样受体（TLR）介导的天然免疫发挥的作用以及佐剂疫苗免疫试验，阐明gp96作为佐剂激活体液免疫与细胞免疫的分子基础，为进一步优化gp96免疫功能和研制新型流感疫苗、乙肝疫苗、HIV疫苗和结核疫苗提供依据。同时，本研究将有助于推动对佐剂增强疫苗免疫应答的本质认识，推动设计新型特异性疫苗佐剂战略。

背景：热休克蛋白gp96作为免疫活性分子具有独特的免疫功能，是研究疫苗佐剂的理想分子模型。.主要研究内容：本项目针对目前疫苗佐剂和gp96免疫学功能研究中存在的关键科学问题，系统查明gp96与抗原结合的结构基础以及与MHC分子相互作用参与抗原呈递的分子机制，进一步明确TLR介导的天然免疫对gp96引发细胞免疫和体液免疫的影响，为gp96佐剂在流感疫苗、乙肝疫苗、HIV疫苗、结核疫苗中的应用提供理论依据和指导。.重要结果和关键数据：首先查明gp96与乙肝病毒HLA-A2限制性表位、流感病毒表位结合，发现一系列新型病毒抗原与表位，包括乙肝病毒HBc60–68 [V60]，HBc123–131 [P130], HBc123–131 [T130], HBc141–149 [S141]和流感病毒NP147–155等，鉴定gp96与抗原结合的结构域。同时发现gp96在肿瘤细胞中由内质网向细胞膜上位移，作为分子伴侣和脚手架蛋白与HER2、uPAR、ER-α36等多种肿瘤抗原相互作用，揭示胞膜gp96作为肝癌、乳腺癌等肿瘤新靶点具有重要科研和药物研发价值。接着验证gp96与MHC I类分子、TAP相互作用，发现gp96-抗原复合物被抗原呈递细胞摄取进入静息早期内体，结合的抗原被蛋白酶体加工，进而进入内质网与MHC I类分子结合并通过高尔基体分泌途径呈递到细胞表面供T细胞识别。gp96与Toll样受体（TLR）相互作用对于T细胞活化至关重要，鉴定了gp96分子伴侣与TLR底物蛋白相互作用的区域，验证突变型gp96不能与TLR2/4结合、失去活化TLR受体通路的能力，野生型gp96对病毒特异性T细胞的活化明显高于突变型。最后，考察gp96作为新型疫苗佐剂，显著提高现有灭活流感疫苗T细胞应答水平，进而提高疫苗的交叉保护功能。gp96明显提高乙肝疫苗、结核疫苗的抗病毒、抗分枝杆菌的活性，为开发新型佐剂疫苗提供依据。.科学意义：（1）查明gp96结合抗原表位、参与抗原呈递和活化特异性T细胞的机制，为开发新型流感、乙肝和结核疫苗提供依据，为设计广普性流感疫苗提供理论基础；（2）发现胞膜gp96作为乙肝感染肝癌、乳腺癌等新靶点，并设计新型靶向药物。