B7-1、B7-2分子在靶向防龋DNA疫苗增强免疫反应过程中的作用

B7-1 and B7-2 play different roles in the induction of immune response. This project is designed to disclose the different effects of B7-1 and B7-2 in the enhanced immune response induced by a targeted fusion anti-caries DNA vaccine. In vitro studies will be conducted to evaluate the ability of antigen-presenting cells (APC) to capture proteins, the expression of costimulatory molecules on APC, the production of cytokines by APC, the activation of signaling pathways in APC, and the differentiation potential of Th0 cells to Th1, Th2 or Th17 cells in the context of the stimulation of CTLA4 fusion protein and non-fusion protein. In vivo studies will be performed to compare the expression of antigen in draining lymph nodes, the levels of specific antibodies in serum and saliva, the levels of IgG subtypes in serum, and the Th1/Th2/Th17 cytokines produced by splenocytes following immunization of mice with targeted anti-caries DNA vaccine versus with non-targeted anti-caries DNA vaccine. For the purpose of delineating how B7-1 and B7-2 are differently involved in the induction of immune response induced by CTLA4 targeted DNA vaccine, four types of mice including wild-type, B7-1 knockout, B7-2 knockout and B7-1/2 double knockout mice will be used in the above studies. This project would help to deepen our understanding on the differential aspects between B7-1 and B7-2 in the induction of immune response and provide new supportive evidences for the designing of B7-targeted vaccines.

B7-1、B7-2分子在免疫诱导过程中发挥不同的作用，本项目拟通过体外、体内两部分研究探寻B7-1、B7-2在靶向防龋DNA疫苗增强免疫反应过程中的作用机制。体外研究评价在CTLA4融合蛋白和非融合蛋白刺激下，抗原提呈细胞（APC）对两种蛋白的摄取能力，APC的共刺激分子表达、细胞因子产生和信号通路活化，以及T细胞向Th1、Th2和Th17分化能力。体内研究采用靶向和非靶向防龋DNA疫苗免疫小鼠，比较抗原在免疫局部引流淋巴结的表达水平，血清和唾液中的特异性抗体水平，血清IgG亚型，以及脾细胞产生的Th1/Th2/Th17细胞因子水平。研究中拟使用野生型小鼠及B7-1、B7-2、B7-1/2基因敲除鼠做比较，明确B7-1、B7-2如何参与CTLA4靶向疫苗的免疫诱导过程。该项目有助于深入了解B7-1、B7-2分子在免疫调控中的不同作用，可能为靶向B7分子的疫苗的设计提供新的理论依据。

树突状细胞（dendritic cells, DC）是体内最强的抗原提呈细胞，在免疫诱导的过程中发挥重要作用。DC表面表达两种B7共刺激分子，B7-1和B7-2，两者在免疫诱导的过程中发挥不同的作用。研究发现将CTLA4分子与特定的抗原融合表达时，可以增强抗原特异性的抗体反应。抗体反应的增强可能缘于CTLA4与位于DC表面的B7分子的结合，从而将与其融合表达的抗原靶向引导至DC，增加了DC对抗原的摄取。本研究探索了B7-1、B7-2两种分子在CTLA4融合疫苗诱导免疫反应过程中的不同作用，揭示了CTLA4融合疫苗增强免疫反应的分子机制。研究结果发现DC摄取CTLA4融合蛋白的效率明显高于摄取非融合蛋白，DC表面的B7-2分子在DC摄取CTLA-4融合蛋白的过程中发挥关键作用。B7-2分子可以与CTLA4融合蛋白结合，且该结合具有明显的浓度依赖性。与非融合蛋白相比，CTLA4融合蛋白促进DC表面的MHCII分子表达水平提高，该促进作用与B7-2分子相关。与非融合蛋白相比，CTLA4融合蛋白促进DC MAPKs信号通路分子p38和ERK的活化，诱导DC分泌更高水平的IL-10和IL-12p40。不同B7分子对于激活T淋巴细胞有不同影响，B7-1分子可促进T细胞向Th1型分化，抑制Th2型分化，与Th17型分化无明显相关性；而B7-2分子抑制T细胞向Th1型分化，促进Th2型及Th17型分化。B7-1及B7-2分子对T细胞增殖均无明显影响。分别采用CTLA4融合防龋DNA疫苗和非融合防龋DNA疫苗免疫野生型及B7基因敲除小鼠，检测B7-1、B7-2分子对抗体反应强度和免疫类型的影响。结果发现B7-1和B7-2分子均参与调控CTLA4融合防龋DNA疫苗的抗体反应，同时缺失了B7-1和B7-2基因的小鼠接种疫苗后产生特异性抗体的能力显著下降。本项目研究证明了B7-2分子在DC对CTLA4融合蛋白的摄取、DC的活化方面发挥关键作用。B7-1和B7-2分子共同参与调控免疫反应的Th类型和抗体反应的强度。