MUCOSAL IMMUNITY TO CHOLERA VECTORED EHEC ANTIGENS

对霍乱载体大肠杆菌抗原的粘膜免疫

• 批准号: 2867933
• 负责人: JOAN R BUTTERTON
• 金额: $ 9.89万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2867933
• 关键词: Escherichia coli; Escherichia coli infections; Vibrio cholerae; attenuated microorganism; bacterial antigens; bacterial genetics; bacterial vaccines; fusion gene; gene expression; hemolysin; histology; laboratory rabbit; live vaccine; molecular cloning; mucosal immunity; oral administration; vaccine development; vector vaccine

DESCRIPTION (Adapted from the applicant's abstract): The principal investigator proposes to use Vibrio cholerae, the causative agent of cholera, as a live oral attenuated vaccine vector to deliver immunogenic antigens of enterohemmorrhagic Escherichia coli (EHEC) to the intestine to stimulate a common mucosal immune response. EHEC, a bacterium that causes hemorrhagic colitis and hemolytic uremic syndrome, is an important pathogen of humans which, like V, cholerae, acts by adhering to the mucosal surface of the intestine and producing toxin. The investigators hypothesize that oral EHEC may lead to protective immunity against EHEC infection in the gastrointestinal tract. Examination of the immune response to infection with vector strains may aid in elucidating the mechanisms of protective immunity to EHEC and may further the understanding of protective immunity at the mucosal surface. The cloned genes encoding EaeA and EspB from RDEC-1 and StxB1 from EHEC will be placed within the E. coli hemolysis secretion system for export from Vibrio cholerae vaccine strains. The genes will be amplified and cloned into an internal deletion of hlyA such that the gene is fused in frame to the carboxy-terminus of the E. coli hemolysin gene. The resulting constructs will be confirmed by restriction digests and sequence analysis, and placed into V. cholerae strain 0395-NT by electroporation. Localization of EaeA, EspB, and StxB1 in cellular compartments will be performed by ELISA or Western blot analysis using polyclonal anti-EaeA, EspB, and StxB1 antibodies. A rabbit model of Vibrio cholerae colonization will be used to examine the immune response to EaeA, EspB, and StxB1 expressed by the vector strains. Serum and mucosal secretions will be collected from rabbits that have been orally inoculated with the vector strains, and examined for the presence of specific antibody produced in response to immunization. The RDEC-H19A oral challenge model will be used to evaluate protection from the disease induced by immunization with the vaccine strains. Immunized rabbits that develop an immune response to EaeA, EspB, or StxB1 expressed by the vector strains will be orally challenge with the rabbit pathogen RDEC-H19A, and the induction of protective immunity will be evaluated by examining the animals for clinical disease and for histological lesions in the intestine. The proposal is divided into three specific aims: 1) Construct fusions of eaeA, espB, and stxB1 to the E. coli hemolysin secretion signal; and analyze the expression and localization of the hybrid productions. 2) Use the rabbit model to examine the immune responses to EaeA, EspB, and StxB1 expressed by the vector strains. 3) Use the RDEC-H19A rabbit challenge model to assess protection conferred by the immunization.

描述（改编自申请人的摘要）： 校长 研究人员建议使用霍乱弧菌，它是霍乱的病原体 霍乱，作为活口服减毒疫苗载体来提供免疫原性 肠出血性大肠杆菌 (EHEC) 抗原到达肠道 刺激共同的粘膜免疫反应。 EHEC，一种导致 出血性结肠炎和溶血性尿毒症综合征是重要的 人类病原体，与霍乱弧菌一样，通过粘附在 肠道粘膜表面并产生毒素。调查人员 假设口服肠出血性大肠杆菌可能会产生针对肠出血性大肠杆菌的保护性免疫力 胃肠道感染。免疫检查 对载体菌株感染的反应可能有助于阐明 EHEC 的保护性免疫机制，并可能进一步 了解粘膜表面的保护性免疫。 编码来自 RDEC-1 的 EaeA 和 EspB 以及来自 EHEC 的 StxB1 的克隆基因 将置于大肠杆菌溶血分泌系统内用于出口 来自霍乱弧菌疫苗株。基因将被放大并 克隆到 hlyA 的内部缺失中，使得该基因融合在 框架连接到大肠杆菌溶血素基因的羧基末端。由此产生的 构建体将通过限制性消化和序列分析来确认， 并通过电穿孔置于霍乱弧菌菌株0395-NT中。 EaeA、EspB 和 StxB1 在细胞区室中的定位将是 使用多克隆抗 EaeA 通过 ELISA 或蛋白质印迹分析进行， EspB 和 StxB1 抗体。 霍乱弧菌定植的兔子模型将用于检查 对载体菌株表达的 EaeA、EspB 和 StxB1 的免疫反应。 采集兔的血清和粘膜分泌物 口服接种载体菌株，并检查是否存在 因免疫接种而产生的特异性抗体。 RDEC-H19A 口服激发模型将用于评估对疾病的保护作用 通过疫苗株免疫诱导。免疫兔子 对载体表达的 EaeA、EspB 或 StxB1 产生免疫反应 菌株将接受兔病原体 RDEC-H19A 的口服攻击，并且 保护性免疫的诱导将通过检查来评估 用于临床疾病和组织学病变的动物 肠。 该提案分为三个具体目标： 1）构建融合 eaeA、espB 和 stxB1 为大肠杆菌溶血素分泌信号；和 分析混合产物的表达和本地化。 2）使用 兔子模型检查对 EaeA、EspB 和 StxB1 的免疫反应 由载体菌株表达。 3) 使用RDEC-H19A兔子挑战 评估免疫接种所提供的保护的模型。