一种新颖的Aβ多表位重组疫苗的建立及其治疗阿尔茨海默病的效应机制研究

The increase and deposition of β-Amyloid peptide(Aβ) are the key pathogenic factors of Alzheimer's Disease, and it is an effective means to make a vaccine which targets the Aβ as the antigen. The B cell epitope of Aβ antigen is able to induce curative humoral immune response, but its T cell epitope can also activates the cytotoxic T cell mediated central nervous system toxicity. Therefore, to improve the immunogenicity of the endogenous small molecule-B cell epitope of Aβ antigen and induce the immune response to develop towards benefiting humoral immunity and deviate from the cellular immunity Th2, are the difficult part and breakthrough point of improving the efficacy and safety of AD vaccine. The applicant and the research team have set up an innovative peptide surface displaying vector based on a clinical applicable polysubunits L-asparaginase B in the previous work, and have proved that the vector is able to enhance the immunogenicity of the displayed epitope through various ways. This project will use the self-established vector as the frame, design and make a novel Aβ multi-epitope recombinant vaccine with high immunogenicity, and conduct the research work on its pharmacological effect and mechanism of immune interrupting of Aβ and AD. This study provides not only an antigen combination strategy of intensifying endogenous small molecule epitope immunogenicity, but also a novel vaccine design and development approach of immune treatment of AD.

β-淀粉样蛋白(Aβ)的增多和沉积是阿尔茨海默病(AD)的关键致病因素，制备抗Aβ的疫苗是治疗AD的有效手段。Aβ的B细胞表位能够诱导具有治疗效应的体液免疫，但其T细胞表位却会激活毒性T细胞介导的中枢神经毒性反应。因此，提高Aβ B细胞表位这一内源性小分子抗原的免疫原性，并诱导免疫反应向利于体液免疫而背离细胞免疫的Th2方向发展，是提高AD疫苗有效性和安全性的难点和突破点。申请人研究团队在前期工作中已基于一种有临床应用的多亚基自聚酶- - L-门冬酰胺酶II建立了创新的多肽表面呈现载体，并证明了该载体能够通过多种途径有效提高所呈现表位的免疫原性。本项目将以该原创性载体为骨架，设计并制备一种新颖的高免疫原性Aβ多表位重组疫苗，并阐明其免疫阻断Aβ进而治疗AD的药理效应与作用机制。该研究成果将提供一种强化内源性小分子表位免疫原性的抗原组合策略，并为AD的免疫治疗提供新的疫苗设计和开发思路。

β-淀粉样肽（β-amyloid peptide, Aβ）的过度表达、聚集和沉积是导致阿尔茨海默病（Alzheimer’s disease, AD）的主要因素。临床前数据显示：针对Aβ的主动免疫能有效抑制Aβ聚集，清除Aβ沉积，改善AD小鼠的认知障碍。临床试验则提示：安全有效的Aβ疫苗应当在激发高水平体液免疫的同时避免T细胞介导的炎性自身免疫。本研究旨在基于剔除Aβ42毒性T表位的功能B表位（Aβ1-15）建立新型的AD表位疫苗。课题组以临床药用的大肠杆菌L-门冬酰胺酶II（E. Coli L-asparaginase B, EcA, EC 3.5.1.1）为骨架，分别融合两种辅助T表位建立了能够作为疫苗载体的新型融合蛋白，进而通过基因重组和蛋白操作将Aβ1-15表位装载其中，制备了两种候选的Aβ表位疫苗，并从中甄选出目标疫苗EPA。在此基础上，又以野生型小鼠为生理模型，以AD转基因小鼠为病理模型，以NGF诱导的PC-12细胞为体外模型对新颖疫苗的免疫应答和AD防治效应机制进行了评价和研究。结果表明，本项目基于EcA建立的新型疫苗载体能够通过简单的基因融合和重组表达完成Aβ1-15表位多肽的装载，并在EcA分子的自聚作用下实现表位的四价呈现；由此制备的Aβ多表位疫苗能够有效提高小分子表位的免疫原性，诱导高水平抗Aβ42抗体的产生，并成功调控免疫应答背离致炎的Th1途径向利于AD防治的Th2方向（以IgG1类抗体生成为主）偏移，有效避免了Aβ42中T表位诱导的炎性自身免疫；此外，新颖疫苗诱导的特异抗体也被证实能够靶向识别病理模型脑内的Aβ斑块并中和Aβ42的神经毒性，体现出有效防治AD的潜力。总之，本项目基于自聚化药用酶建立了一种更加安全、有效的新颖Aβ多表位疫苗候选，同时提供了一种能够多价呈现并强化小分子表位免疫原性的蛋白载体和实施方案，也为其它以Th2免疫为主导的表位疫苗和单克隆抗体开发用抗原的制备提供了新组合、新载体和新方法。

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