Development of a vaccine against Helicobacter pylori to prevent gastric cancer

描述（由申请人提供）： 胃腺癌在美国每年有19，000例病例和12，000例死亡。这种癌症风险的主要因素是幽门螺杆菌的感染。目的：我们的长期目标是开发一种预防H。幽门螺杆菌感染及其相关并发症，包括胃癌。本申请的具体目标是：a）基于H.幽门螺杆菌尿素酶B：纯化的重组蛋白（done）、DNA质粒（done）和AgAg-尿素酶B（本申请）;和B）在小鼠中测试这三种产物以确定它们的免疫原性和保护效力。健康相关性：该项目属于该机构预防癌症的使命。研究设计：将重组人Ag-S基因克隆到酵母表达载体pYES 2中，构建pYES 2/HBS，制备Ag-Urease B。螺杆然后扩增幽门螺杆菌尿素酶B优势表位（DKSIKEDVQFAD）并插入到HBsAg-S的四个位置之一中：HBsAg基因之前（N-末端）、HBsAg基因之后（C-末端）、第一亲水结构域（aa 32-74）或第二亲水结构域（aa 110- 156）。然后将该构建体用于转化酿酒酵母（INVSc 1）酵母细胞，并通过在含半乳糖培养基中生长细胞诱导蛋白质表达。然后分析上清液和裂解细胞沉淀物中22 nm颗粒的形成以及HBsAg和尿素酶B表位的抗原反应。然后将具有最佳抗原性的构建体与我们先前制备的纯化的尿素酶B蛋白和DNA尿素B疫苗一起沿着用于在一系列实验中免疫BALB B/c小鼠（评价各种产品、各种途径和各种组合），以比较它们的体内免疫原性和它们保护免受H.猫感染创新点：在本申请中提出了两种特别新颖的方法，用于H。幽门疫苗接种：用纯化蛋白和DNA疫苗进行初免-加强免疫，并通过载体蛋白（HBsAg）呈递尿素酶B抗原。重要性：这些实验的结果应当鉴定出一种或多种候选产物和适于进一步开发成有效的H.幽门螺杆菌疫苗。预防H.幽门螺杆菌感染最终应导致消除其相关并发症，包括胃癌。

DESCRIPTION (provided by applicant): Gastric adenocarcinoma accounts for 19,000 cases and 12,000 deaths annually in the USA. The main factor in the risk of this cancer is infection by the bacterium Helicobacter pylori. Aims: Our long-term objective is to develop a vaccine to prevent H. pylori infection and its associated complications, including gastric cancer. The specific aims of this application are: a) to construct 3 candidate products based on H. pylori urease B: purified recombinant protein (done), DNA plasmid (done) and HBsAg-urease B (this application); and b) to test these three products in mice to determine their immunogenicity and protective efficacy. Health relatedness: this project is within the agency's mission to prevent cancer. Research Design: To prepare HBsAg-urease B, the HBsAg-S gene will be cloned into the yeast vector pYES2 to generate the construct pYES2/HBS. The H. pylori urease B dominant epitope (DKSIKEDVQFAD) will then be amplified and inserted into HBsAg-S in one of four locations: before the HBsAg gene (N-terminus), after the HBsAg gene (C-terminus), in the 1st hydrophilic domain (aa 32-74), or in the 2nd hydrophilic domain (aa 110- 156). The constructs will then be used to transform Saccharomyces cerevisiae (INVSc1) yeast cells and protein expression induced by growing cells in galactose-containing medium. The supernatant and lyzed cell pellets will then be analyzed for formation of 22-nm particles and antigenic reaction of the HBsAg and urease B epitopes. The constructs with the best antigenicity will then be used - along with our previously prepared purified urease B protein and DNA ureB vaccine - to immunize BALB/c mice in a series of experiments (evaluating various products, various routes and various combinations) to compare their in vivo immunogenicity and their efficacy to protect against H. felis infection. Innovation: two particularly novel approaches are proposed in this application for H. pylori vaccination: prime-boost immunization with purified protein and DNA vaccine, and presentation of the urease B antigen by a carrier protein (HBsAg). Significance: the results of these experiments should identify one or more candidate products and vaccination strategies suitable for further development into an effective H. pylori vaccine. And, prevention of H. pylori infection should eventually lead to elimination of its associated complications, including gastric cancer.