构建新颖的减毒沙门疫苗载体递送来源于革兰氏阴性和阳性细菌的异源多糖抗原

Bacterial pathogens including Gram-negative and Gram-positive bacteria cause millions of deaths in the world each year. Moreover, pathogenic bacteria are developing resistance to numerous antibiotics. Most of bacteria are able to generate the diverse polysaccharide as the pathogenic agents, also as immunogenic agents to stimulate immune response against bacterial infection. The development of a vaccine against pathogenic bacteria is the best choice to decrease or eliminate these diseases. Several polysaccharide-protein conjugated vaccines have been developed based on the polysaccharides. However, clinically used polysaccharide-based vaccines suffer from low yields, non-specific chemical conjugation, high costs, and difficult delivery by needle injection. It is urgent to develop a novel polysaccharide vaccine to decrease the costs and increase productivity and serotypes coverage. .Recombinant attenuated Salmonella vaccines (RASVs) can deliver antigens from a variety of different pathogens, generating a range of immune responses including serum antibodies, mucosal IgA, and a panoply of cell-mediated immune responses at local and distal sites. Currently, RASVs are mainly targeting for synthesizing protein antigens; the specific Salmonella vaccines targeting heterologous polysaccharide synthesis remain to be constructed. The objective of this application is to design and develop innovative RASV platforms which not only can synthesize O-specific polysaccharide from G- bacteria (using Shigella flexneri 2a as a model) but also can synthesize capsular polysaccharide from G+ bacteria (using Streptoccocus pneumoniae 19A as a model) to attach to the adjuvant Monophosphoryl lipid A (MPL) on the surface of vaccine strains, but these RASVs possess a specific feature of in vivo-programmed down-regulation of its own O-antigen expression, occurring after colonization of host lymphoid tissues, which would decrease the immune response against the RASV carrier while triggering a strong immune response against heterologous polysaccharides. The gene operons synthesizing for the enterobacterial common antigen, the β-1,4-glucan polymer bacterial cellulose, colanic acid and O-antigen-like capsule will be deleted from Salmonella chromosome to eliminate their interferences to heterologous polysaccharide synthesis..In addition, a serial of Asd+ T-vector will be constructed to facilicate cloning of large gene cluster such O-antigen gene clusters from Shigella spp and CPS gene clusters from Streptoccocus pneumoniae. .These innovative attenuated Salmonella vaccine carriers together with non-antibiotic marker T-vector systems not only can be as live vaccine to deliver the polysaccharide antigen by oral administration route, also can be served as bio-reactors to produce heterologous polysaccharides coupled with the low endotoxic MPL as adjuvant for multi-valent vaccine development.

致病性细菌引起人类的多种疾病，而且多数产生了抗药性。致病性细菌包括革兰氏阳性和阴性两大类，大多数会产生毒性因子多糖，其也是很好的免疫分子，开发接种多糖疫苗是有效预防细菌感染的方法。减毒沙门作为疫苗载体有诸多优点，已经成为世界上研究热点，但目前的研究都集中在其作为蛋白抗原递送的平台上。在本项目中，我们将构建一种专门用于表达递送异源多糖的减毒沙门疫苗载体，该载体不仅能表达合成G-细菌的O-抗原多糖，而且可能能表达合成G+细菌的胞外多糖，然后连接到佐剂形式的单磷酸lipid A core 上，该疫苗载体还含有其它特性如调控沙门自身的O-抗原合成。另外我们还将构建一系列新无抗生素marker的T-vector系统，方便克隆大片段基因序列。该减毒沙门载体系统不仅可以作为口服疫苗免疫宿主，而且还可以作为生物反应器来产生连接于可作为疫苗佐剂MPL上的异源多糖，进行复合多糖亚单位疫苗的研究。

致病性细菌包括革兰氏阳性和阴性两大类，大多数会产生毒性因子多糖，其也是很好的免疫分子，开发接种多糖疫苗是有效预防细菌感染的方法。减毒沙门作为疫苗载体有诸多优点，已经成为世界上研究热点，但目前的研究都集中在其作为蛋白抗原递送的平台上。该课题成功构建了一种新型的穿梭质粒，用于克隆表达长片段基因序列，同时也构建了一种减毒沙门氏菌疫苗载体用于递送多糖疫苗，我们成功将大肠杆菌O1的多糖基因序列克隆到质粒中，并成功表达；用鸡作为动物模型来验证这种疫苗的有效性，但我们没有成功表达克隆来源革兰氏阳性细菌的多糖。该项目为国家自然基金青年基金，属于前期探索性质的工作，共资助经费为20万元整，研究时间跨度为2013年1月到2015年12月，共3年时间，2个研究生参与实验工作，申请与项目相关的中国专利4项，发表文章4篇，另2篇文章在投，1篇文章在撰写。