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（根据申请人的摘要改编）：校长 研究人员建议使用纤维霍利拉（Vibrio Cholerae） 霍乱，作为活口量衰减疫苗载体，以提供免疫原性 肠道大肠杆菌（EHEC）的抗原 刺激常见的粘膜免疫反应。 EHEC，一种引起的细菌 出血性结肠炎和溶血性尿毒症综合征，是一个重要的 人类的病原体，像V一样，霍乱，通过坚持 肠的粘膜表面并产生毒素。调查人员 假设口服EHEC可能导致对EHEC的保护性免疫 胃肠道感染。检查免疫 对载体菌株感染的反应可能有助于阐明 对EHEC的保护性免疫机制，可能会进一步 了解粘膜表面保护性免疫。 来自EHEC的RDEC-1和STXB1编码EAEA和ESPB的克隆基因 将放置在大肠杆菌溶血分泌系统中以出口 来自弧菌霍乱疫苗菌株。这些基因将被放大，并且 克隆成Hlya的内部缺失，使该基因融合在 大肠杆菌溶血素基因的羧基末端的框架。结果 构造将通过限制摘要和序列分析来确认， 并通过电穿孔放入0395-NT的霍乱弧菌菌株中。 EAEA，ESPB和STXB1在细胞室中的定位将是 通过ELISA或使用多克隆抗EAEA进行的ELISA或Western印迹分析， ESPB和STXB1抗体。 将使用蛇形霍乱定殖的兔模型来检查 载体菌株表达的EAEA，ESPB和STXB1的免疫反应。 血清和粘膜分泌物将从兔子那里收集 口服接种载体菌株，并检查是否存在 响应免疫产生的特异性抗体。 RDEC-H19A 口头挑战模型将用于评估疾病的保护 通过疫苗菌株免疫诱导。免疫兔子 对矢量表示的EAEA，ESPB或STXB1产生免疫反应 兔病原体RDEC-H19A的菌株将是口服挑战，并且 通过检查保护性免疫的诱导将通过检查 临床疾病和组织学病变的动物 肠。 该提案分为三个具体目的：1） EAEA，ESPB和STXB1到大肠杆菌溶血素分泌信号；和 分析杂交生产的表达和定位。 2）使用 兔模型检查对EAEA，ESPB和STXB1的免疫反应 由向量菌株表示。 3）使用RDEC-H19A兔子挑战 评估免疫赋予保护的模型。