重塑沙门氏菌脂多糖和外膜蛋白，开发针对肠道致病性细菌的减毒沙门疫苗

Enteric pathogens including Salmonella, Escherichia coli and Shigella spp belonging to the Biodefence agent II listed by NIH, cause ~2 million deaths over the world each year (WHO). These bacteria are also responsible for significant morbidity causing diarrheal and systemic diseases, with increase in antibiotic resistance. Vaccination may be an efficient strategy for controlling enteric bacterial infection and epidemic caused by these pathogens. We have demonstrate that attenuated Salmonella Typhimurium has ability to induce the cross-reactive antibodies against other enteric bacteria such as E. coli, Salmoenlla Enteriditis, Shigella spp. in BALB/c mice by oral administration, and this cross-reactive immunity could be further enhanced by regulating to shut off the Salmonella O-antigen synthesis by arabinose absence in vivo. It is possible that if the Salmonella lipopolysaccharide (LPS) is truncated further, this may result in more effective presentation of conserved outer membrane proteins (OMPs) to the host immune system, which will lead to an immune response that is cross protective against a number of other Salmonella species and to other Gram-negative enteric pathogens. In addition, exposure of the fairly conserved core oligosaccharide and enhanced production of outer membrane vesicles may also aid in production of a cross-protective immune response. .We will construct a series of mutations to regulate O-antigen or core synthesis by arabinose availability, and evaluate the effects of these single mutations on membrane permeability, LPS synthesis and other relevant characteristics in cells grown with or without arabinose. We will also include mutations that up-regulate expression of genes that specify essential ion uptake proteins include ing the iron regulated outer membrane proteins (IROMPs). 9 mutant strains will be further modified by introduction of other mutations including the pagC and ompX genes that are maximally up-regulated in vivo to promote outer membrane vesicles (OMVs) production and △pagP81::Plpp lpxE deletion and insertion to increase the Salmonella safety and decrease the endotoxic acitivity. We will utilize the mice models to evaluate the Salmonella vaccine safety and efficiency. We will measure the total IgG, IgG1 and IgG2a from serum, and SIgA in fecal samples and vaginal washes to evaluate the production of antibodies against OMPs from S. Typhimurium, S. Enteritidis, E. coli O157:H7 and S. flexneri 2a, and we also will measure other criteria to evaluate the T-cell immunity; lastly we will determine the protective efficacy against Salmonella strains, S. flexneri, and Citrobacter rodentium in mice. These novel mutations not only provide the innovative attenuated strategy to develop the attenuated Salmonella or Salmonella vector to deliver heterologous antigens to protect against other pathogens infection for human being and for livestock, they also provide the clues to comprehend the pathogenesis mechanism of Salmonella.

肠道致病性细菌每年引起全球约200万人的死亡（WHO数据），伴随极高的发病率，这些细菌包括沙门氏菌，致病性大肠杆菌和志贺氏菌等，被NIH列为生物恐怖II类病菌，而且很多细菌产生了多种抗药性。疫苗是一种预防肠道疾病感染和流行的有效方法。我们已经证明减毒沙门氏菌能够诱导宿主产生针对其它肠道致病细菌交叉反应的抗体，而且调控沙门O-抗原合成能提高这种交叉反应抗体的产生。在本项目中，我们将通过遗传改造，构建一系列调控O-抗原/核长度的突变体，筛选能最大化提高保守抗原暴露的突变，组合其它突变：体内提高保守外膜蛋白表达的突变，降低内毒素毒性和提高外膜微囊体的分泌的突变，构建减毒沙门疫苗来预防多种肠道细菌性疾病的感染。利用洁净和含有正常菌群的小鼠模型，验证减毒疫苗的有效性和安全性。这些新的减毒技术，为开发人用和兽用沙门疫苗和沙门减毒疫苗载体提供新颖的减毒策略，进而了解沙门氏菌的致病机理和潜在的应用价值。

肠道致病性细菌每年可以引起全球约200万人的死亡（WHO数据），伴随有极高的发病率，这些细菌包括沙门氏菌，致病性大肠杆菌和志贺氏菌等，被NIH列为生物恐怖II类病菌，主要由食物和水污染引起人类感染，更为严重的是很多肠道细菌产生了多种抗药性。疫苗是一种预防肠道疾病感染和流行的有效方法。我们已经证明沙门氏菌减毒疫苗能够诱导宿主产生针对其它肠道致病细菌交叉反应的抗体，而且体内调控沙门氏菌O-抗原合成能提高这种交叉反应抗体的产生。在本项目中，我们将利用基因工程的方法构建一系列调控O-抗原/核长度的突变体，筛选能最大化提高保守抗原免疫性的突变。我们已经从三个方面筛选可作为广谱疫苗的可行性，1）通过调控O-抗原及ECA的合成来构建减毒沙门氏菌疫苗，以实现广谱性交叉免疫及保护；2）通过从不同长度LPS的沙门氏菌突变体纯化外膜蛋白来开发广谱型疫苗；以及3）通过从不同长度LPS的沙门氏菌突变体纯化外膜囊泡来开发广谱型疫苗。实验结果显示，减毒沙门疫苗通过调控LPS及ECA可以实现广谱型交叉保护，针对其他肠道细菌的感染；而且外膜蛋白及外膜囊泡同样也可以诱导交叉保护，尽管来源于不同LPS长度的突变体表现的结果不同。