改造沙门菌类脂A结构，开发新型减毒疫苗载体及疫苗佐剂

As recombinant attenuated Salmonella vaccine (RASV) has ability to stimulate mucosal, cell and humoral-mediated immunity after oral administration or other vaccinated routes, RASV is the best choice to develop a vaccine carrier to deliver heterologous antigens from bacterial, viral pathogens or to cure the cancer. How to genetically construct Salmonella vaccine is the key point for its delivering heterologous antigens while causing no-symptoms. Candidate RASVs are attenuated in animal models, when administered to humans they can be reactogenic, producing unwanted side effects, including abdominal pain, gastrointestinal disorder, and fever. Many of these symptoms are consistent with known reactions to lipid A..Previous work has demonstrated that Salmonella was capable of being engineered to alter the number of fatty acid of lipid A to decrease the endotoxic activity. Here, we propose novel strategies including modifying the length of the acyl chains and removing of phosphate group from the specific lipid A structure to remodel Salmonella lipid A to reduce endotoxicity/reactogenicity without compromising overall immunogenicity, and develop innovative vaccine adjuvant based on the Salmonella lipid A structures. The mutants will be characterized in vivo and in vitro including phenotypes identification, lipid A structure determination, animal experiments to check the LD50, colonization ability. The optimal mutations combination will be introduced to the vaccine vector strains to evaluate the effects on the immunogenicity. We will introduce a test antigen, the pneumococcal gene pspA into the strains to test the humoral-mediated immunity，and a flu virus (rWSN) test antigen gene, Hemagglutinin(HA), will also be introduced into the specific vaccine strains to test the cell-mediated immunity; To assess and select the optimal lipid A structure for anti-tumor in Salmonella vaccine, the mutations combinations relating to the lipid A modification will also be introduced to the specific parent vaccine and will be evaluated in B16F10 melanoma animal model.

重组减毒沙门疫苗经口服或其它途径免疫后能产生多种免疫反应，已被世界上许多科学家首选为疫苗载体递送异源抗原 。如何构建沙门菌载体是沙门菌有效发挥其递送抗原功能而又不引起症状的关键。沙门引起发热，腹痛和神经紊乱症状，与内毒素有关。前期工作证明可以通过遗传工程改变lipid A脂肪酸链数量的方法改造内毒素的结构来降低其毒性。本项目拟利用全新的遗传策略通过改变lipid A脂肪酸链的长度或改变特殊形式lipid A的磷酸基团重组lipid A结构来降低其内毒素毒性而保持佐剂活性，和筛选体外用疫苗佐剂。突变体将进行一系列生物学性状鉴定，lipid A结构鉴定等体外实验和动物毒性和定殖能力测定等体内实验。我们将用三种动物模型（一种基于体液免疫为主，一种基于细胞免疫为主和治疗性癌症疫苗为主）来验证筛选含有改造lipid A减毒沙门菌的载体递送异源抗原和诱导免疫效果的能力。

减毒沙门氏菌可以诱导多种免疫反应，而且遗传操作较为容易及生物学性状了解相对清楚，因此重组减毒沙门氏菌被首选为作为疫苗载体进行异源抗原的递送，开发针对其它感染性病原菌、肿瘤、代谢疾病等的预防性疫苗或治疗性疫苗。如何构建沙门菌载体是沙门菌有效发挥其递送抗原功能而又不引起症状的关键。沙门氏菌脂多糖是其主要的致病因子之一，也是引起宿主发热，腹疼等症状的关键因素。脂多糖由类脂A及多糖组成，类脂A是活性部位，而多糖部分为免疫成分，可以用异源多糖替代来开发疫苗。本课题的主要工作是对LPS进行改造，用于减毒沙门氏菌疫苗的构建，包括类脂A和O-抗原多糖的遗传改造。.针对类脂A的改造，我们构建了三种lipid A结构不同的减毒沙门氏菌或大肠杆菌，包括1）通过基因删除改变lipid A链数量的多个基因（pagL, pagP, lpxR, eptA, arnT 和lpxT），沙门氏菌含有有最大毒性的lipid A结构，最大激活内源免疫，可用于肿瘤疫苗等的构建；2）通过删除msbB基因及过量表达pagL和lpxE基因，产生最小毒性lipid A的结构，该工作在大肠杆菌中完成，可用于蛋白的纯化。我们也在沙门氏菌中完成了该构建，但毒性影响较大，无法实现口服活沙门菌免疫；3）通过异源替代lpxA 或lpxD基因，产生改变链长的lipid A结构，可用于粘膜免疫为主的疫苗构建，目前该项工作扔在开展。针对多糖的改造，我们完成了多项工作，1）通过基因替换，使鼠伤寒沙门菌的B-group的O-抗原变为D-group或A-group, C-group为主的O-抗原，来构建针对以上抗原的减毒沙门疫苗，也同时证明改变鼠伤寒沙门氏菌的O-抗原，对毒性有一定影响；2）通过在鼠伤寒沙门菌中调控表达人伤寒沙门菌中的Vi抗原和B，D-group抗原的表达，可以诱导产生针对三种多糖的免疫反应，构建多价疫苗；3）表达大肠杆菌的O-抗原，构建鸡大肠杆菌病多价疫苗；4）基于外膜囊泡的纳米疫苗，用于递送多糖抗原也有一定的进展，来开发多价疫苗。.通过遗传改造及合成生物学的方法，对沙门氏菌减毒疫苗进行构建及基于细菌自身的亚单位纳米疫苗的构建，是未来疫苗开发的主战场，将会替代目前所用的疫苗产品，尤其是兽用疫苗领域。