基于广谱中和单抗的通用型流感疫苗设计及其结构基础研究

Influenza is a long-term challenge to human health. There is a great need to develop a universal influenza vaccine to provide broad-based, universal protection against different types of influenza virus infection. Several studies have demonstrated that some broadly neutralizing antibody epitopes exist in the hemagglutinin (HA), the major surface glycoprotein of the influenza virus and they can induce broadly neutralizing antibodies against different types of influenza virus. This finding provide scientific basis for the development of universal influenza vaccine. However, almost all these non-immunodominant epitopes are conformational and extremely difficult to express in current expression systems. In our previous studies, we has explored an effective anti-idiotype antibody vaccine strategies which can display broadly neutralizing antibody epitopes in hemagglutinin. However, it is still unclear why and how the anti-idiotype antibody can display such conformational epitopes. This proposal aims to continue to raise more anti-idiotype antibodies using hemagglutinin specific broadly neutralizing monoclonal antibody as immunogen, and select three kinds of anti-idiotype antibody (broad spectrum, medium broad spectrum, and narrow spectrum) using cell-based micro-well neutralization assay and lethal-challenge animal model based prophylactic evaluation assay. The potential key residues in the epitopes of these three types of anti-idiotype antibody will be determined using X-ray crystallographic analysis and further verified using chimeric antibody technique, point mutation analysis and immune response analysis. The expected results of this study will be helpful to illustrate the structural basis of conformational epitopes of hemagglutinin reconstructed by anti-idiotype antibody and provide better understanding of the molecular mechanism of the expression of conformational epitopes, as well as facilitate the development of universal influenza vaccine and structure vaccinology.

流感长期危害人类健康，研制通用型流感疫苗是流感病毒学和疫苗学领域的重要研究方向。研究发现流感病毒表面糖蛋白血凝素中存在可诱导产生广谱抗体的中和表位，为研制通用型流感疫苗奠定了科学基础，然而这些广谱表位多是非免疫优势的不易重建表达的构象性表位，因此，如何重建表达血凝素构象性表位成为重要的科学问题。前期研究我们已探索出一种有效的能展示血凝素中和单抗识别表位的抗独特型抗体疫苗策略，本项目拟在此基础上，继续应用流感血凝素广谱中和单抗去免疫构建出能展示血凝素构象表位的抗独特型抗体库，通过细胞中和试验、病毒感染小鼠保护性实验等功能实验筛选出可诱导广谱、中等广谱和窄谱保护性抗体的三类抗独特型抗体，然后应用蛋白质晶体结构解析和嵌合抗体突变验证等方法阐明抗独特型抗体重建的血凝素构象性广谱中和表位的结构基础和抗独特型抗体模拟构象性表位的重建表达规律，为通用型流感疫苗的研发设计和结构疫苗学的发展提供科学依据。

研发通用流感疫苗将是人类战胜流感病毒的希望。近年来流感病毒血凝素广谱中和表位的发现，为通用流感疫苗研究带来新希望，但这些广谱表位多为构象性表位，如何体外重塑构象性表位并表达出有天然活性的重组蛋白疫苗已成为通用流感疫苗研究中的关键难题。为此，本项目研究策略之一是尝试用抗独特型抗体展示血凝素构象性广谱中和表位，首先我们通过小鼠单克隆抗体技术筛选出一批流感血凝素广谱中和单抗，然后首选乙型流感广谱中和单抗12G6为免疫原结合大鼠单克隆抗体技术制备出抗12G6独特性抗体1C12，竞争ELISA显示1C12能竞争12G6与乙型流感病毒血凝素的结合活性，且能在小鼠中诱导出乙型流感病毒广谱活性，能广谱保护两种变异亚系乙型流感病毒对小鼠的致死性感染，说明利用抗独特型抗体展示血凝素构象性中和表位是通用流感疫苗研究的可行策略。随着结构疫苗学技术的发展，本项目也尝试基于结构生物学的疫苗设计研究，首先利用晶体结构学方法解析了12G6抗体与血凝素的复合物三维结构，通过关键表位氨基酸位点分析，设计了多个模拟12G6识别表位的肽段，然后利用体外重组表达和蛋白偶联技术还原重塑12G6表位，结果显示12G6-P1段多肽模拟展示的12G6表位效果最佳，其免疫血清的广谱结合活性和在小鼠体内的抗病毒广谱保护活性最佳，证明了基于结构的表位疫苗设计也是通用流感疫苗研究中的可行策略。另外，本项目还研究了糖基化位点对血凝素隐匿性关键表位展示表达的影响，初步证明血凝素糖基化位点对血凝素广谱抗体应答具有显著影响，提示未来的通用流感疫苗设计研究应重视血凝素糖基化位点的改造。另外，本项目实施期间初步建立的基于Rosetta的表位架展示技术、基于反向遗传学技术的荧光报告流感病毒、基于ELISPOT的流感快速滴定技术都为后续通用流感疫苗研究奠定重要工作基础。