甲型副伤寒生物偶联多糖蛋白结合疫苗的研究

Conjugate vaccines are largely used to prevent important invasive bacterial diseases such as pneumonia, meningitis and bacteraemia. The polysaccharide antigens are extracted and purified from the bacterial organism and are activated chemically for conjugation. The unconjugated protein carrier is also manufactured by growing the natural bacterial source or a recombinant host system followed by the appropriate purification process. Due to the multiple activation points within the polysaccharide and multiple linkages on the carrier protein, the resulting conjugate is called a cross-linked network, which is highly heterogenous, unsafe and high cost. The N-glycosylation and O-glycosylation pathways of bacteria were found recently, which provide the basic theory to develop a technology of novel bio-conjugate vaccines. This study will be performed to (i) elimination O-antigen ligase gene and (ii) to reconstruct the Neisseria neningitides O-linked protein glycosylation system in Salmonella paratyphi A. The relationships between carrier proteins, O-antigen lengths, sites of glycosylation and the immunogenicity of polysaccharide -conjugated vaccines were researched and explained. For example, the polysaccharide acceptor will be replaced from the model protein PilE to the extensively used carrier proteins— tetanus toxin C fragment (TTc), dipheria toxoid mutant CRM197, cholera toxin B subunit (CTB), exoprotein A of Pseudomonas aeruginosa (EPA) in which the PglL glycosylation sites will be introduced. This new strategy of producing polysaccharide -conjugated vaccines “One-step bioconjugation technology” can develop and produce immunogenic glycoproteins in a simplified biological process that circumvents many of the challenges and uncertainties involved in currently used methods. The combination of glycoengineered bacteria expression system with established quality control methods maybe provide a mechanism for rapid production of vaccines against common severe bacterial infections produced with its unique, proprietary in-vivo glycosylation platform.

多糖蛋白结合疫苗在预防传染病方面发挥着重要作用，如肺炎、脑膜炎和菌血症，但是现有化学交联法制备多糖蛋白结合疫苗存在安全性、产物均一性差、成本高等问题。原核生物糖基化修饰系统的发现为利用生物法合成多糖蛋白结合疫苗提供了理论基础。本研究以甲型副伤寒沙门氏菌为模型，将脑膜炎奈瑟球菌的O-糖基化修饰系统引入其中，通过对底物蛋白的修饰效率、对糖基化位点的筛选、对糖链长度的控制，以及获得的多糖蛋白结合物的免疫学特性等问题的研究，阐明O-糖基化修饰系统与载体蛋白、糖链长度、糖基化位点之间的联系，为获得病原菌多糖直接修饰载体蛋白的多糖蛋白结合物奠定理论基础，初步建立利用生物一步法制备多糖蛋白结合物的生物交联技术，同时为新型多糖蛋白结合疫苗表达系统的建立提供技术基础。

多糖蛋白结合疫苗在预防传染病方面发挥着重要作用，目前在售的多糖蛋白结合疫苗均由化学交联法制备，其生产周期长、生产过程存在一定的生物安全隐患，多糖蛋白交联产物均一性差、结构解析和质控困难。本研究通过遗传改造细菌O-糖基化修饰途径与多糖合成途径，将两个代谢途径偶联，构建了可一步获得细菌多糖修饰的载体蛋白的生物偶联技术平台，通过多肽截断实验、丙氨酸扫描实验确定了最优O-糖基化位点氨基酸保守序列；以甲型副伤寒沙门氏菌为研究模型，通过筛选不同来源的O抗原糖链控制酶，延长了甲型副伤寒沙门氏菌的O抗原链长，并利用生物偶联技术获得了两种多糖蛋白结合物（CTB4573-OPS50973和rEPA4573-OPS50973），免疫学评价表明CTB4573-OPS50973的免疫原性和保护效果更优；以CTB载体蛋白为基础，引入三聚化蛋白结构域，开发了基于CTB的纳米多糖结合疫苗技术平台，免疫学评价发现纳米多糖蛋白结合疫苗的抗原提呈效率更高；以CTB为载体蛋白，优化发酵和纯化工艺，获得了高纯度的CTB4573-OPS50973多糖蛋白结合物，通过质谱和NMR分析多糖蛋白结合物的精确分子量和O抗原的结构，结果表明，CTB4573-OPS50973的多糖蛋白结合物中O抗原链长为14-32重复单位，O抗原糖链结构与文献报道一致。.本研究建立了基于细菌O-糖基化修饰系统的生物偶联技术，并确定了载体蛋白O-糖基化修饰位点的最佳氨基酸保守序列；以甲型副伤寒沙门氏菌为研究模型，通过延长O抗原糖链长度及选择多聚体的载体蛋白，提高了疫苗的免疫原性和保护效果，进而开发稳定的生产工艺，获得了高纯度的多糖蛋白结合物CTB4573-OPS50973，并对其结构进行详细分析；通过自组装载体蛋白将抗原纳米颗粒化，从而进一步提升抗原的淋巴结引流速度和抗原利用度，提升了抗原的提呈能力，大大提高了疫苗的免疫原性及保护效果。综上所述，本研究开发的生物偶联技术及自组装纳米颗粒技术为多糖蛋白结合疫苗的研发开辟了新的路径，在抗原制备、结构解析、质量控制、免疫机制研究等方面提供了坚实的基础。