Mucosal Immunity to Antigens Expressed by V.cholerae

对霍乱弧菌表达的抗原的粘膜免疫

• 批准号: 6892889
• 负责人: Edward T. Ryan
• 金额: $ 38.64万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6892889
• 关键词: Clostridium difficile; Escherichia coli; Vibrio cholerae; active immunization; antigen presentation; attenuated microorganism; bacterial antigens; bacterial genetics; bacterial proteins; bacterial toxins; bacterial vaccines; bactericidal immunity; clostridial infection; gene delivery system; gene expression; host organism interaction; immunologic substance development /preparation; immunomodulators; laboratory mouse; mucosal immunity; oral administration; protein engineering; recombinant DNA; transfection /expression vector; vaccine development; vector vaccine

DESCRIPTION (provided by applicant): Clostridium difficile is the cause of significant morbidity, mortality and cost. Systemic and mucosal immunity to C. difficile toxin A has been associated with protection from clinical disease, amelioration of clinical symptoms, and prevention of relapse. The previous funding period of this grant resulted in several advances in the use of live, oral, attenuated Vibrio cholerae as a vector for immunizing against heterologous antigens. The long term goal of the next, proposed segment of funding is to develop and test in animals a V. cholerae-based vector vaccine that will stimulate systemic and mucosal humoral immunity against toxin A of C. difficile, and that is appropriate for ultimate human use. The current proposal has FOUR SPECIFIC AIMS to achieve this long term goal. These aims are: 1. To develop and analyze in vitro an anti-C. difficile V. cholerae-based vaccine vector. A glutamine auxotroph of V. cholerae vaccine strain CVD 103-HgR (a safe and immunogenic vaccine strain in North American human volunteer studies) will be engineered to express toxin A-HlyA (a fusion protein between a non-toxic 720 amino acid C. difficile toxin A fragment and the E. coli hemolysin A secretion signal) from plasmids of various copy numbers that complement the glutamine auxotrophy. The vaccine vector strains will also express HlyBD (the pore-forming proteins that mediate extracellular secretion of toxin A-HlyA), and an immunoadjuvant molecule, such as LT(R192G) (a non-toxic derivative of Escherichia coli heat-labile enterotoxin that retains immunoadjuvancy). Expression and cellular localization of toxin A-HlyA and LT(R192G) will be evaluated, as well as viability and stability of the vaccines in vitro. 2. The viability, stability, immunogenicity, and reactogenicity of the various oral vaccine constructs will then be evaluated in mice. 3. A combination oral priming and transcutaneous boosting immunization strategy (the latter with C. difficile toxin A toxoid with or without an immunoadjuvant) will be evaluated in mice for production of both mucosal and systemic immunity to toxin A. 4. The immunogenicity, reactogenicity, and protective efficacy of the most promising vaccine strategy will be assessed in rabbits, the latter will be measured using a challenge assay in which purified C. difficile toxin A is injected into ligated ileal loops of vaccinated and control animals.

描述（由申请人提供）：艰难梭菌是导致显着发病率、死亡率和成本的原因。对艰难梭菌毒素 A 的全身和粘膜免疫与预防临床疾病、改善临床症状和预防复发有关。该赠款的前一个资助期在使用活的、口服的、减毒的霍乱弧菌作为异源抗原免疫的载体方面取得了几项进展。下一个拟议资金部分的长期目标是开发一种基于霍乱弧菌的载体疫苗并在动物身上进行测试，该疫苗将刺激针对艰难梭菌毒素 A 的全身和粘膜体液免疫，并且适合最终人类使用。当前的提案有四个具体目标来实现这一长期目标。这些目标是： 1. 开发并体外分析抗 C。基于艰难梭菌霍乱弧菌的疫苗载体。霍乱弧菌疫苗株 CVD 103-HgR（北美人类志愿者研究中的一种安全且具有免疫原性的疫苗株）的谷氨酰胺营养缺陷型将被改造为表达毒素 A-HlyA（一种无毒的 720 个氨基酸 C 和 C 之间的融合蛋白）。艰难梭菌毒素 A 片段和大肠杆菌溶血素 A 分泌信号）来自补充谷氨酰胺营养缺陷的各种拷贝数的质粒。疫苗载体菌株还将表达 HlyBD（介导毒素 A-HlyA 细胞外分泌的成孔蛋白）和免疫佐剂分子，例如 LT(R192G)（大肠杆菌不耐热肠毒素的无毒衍生物，保留免疫佐剂）。将评估毒素 A-HlyA 和 LT(R192G) 的表达和细胞定位，以及疫苗的体外活力和稳定性。 2. 然后将在小鼠中评估各种口服疫苗构建体的活力、稳定性、免疫原性和反应原性。 3. 将在小鼠中评估口服初免和经皮加强免疫策略（后者使用艰难梭菌毒素 A 类毒素，加或不加免疫佐剂），以产生对毒素 A 的粘膜和全身免疫。 4. 免疫原性、反应原性，并且最有希望的疫苗策略的保护功效将在兔子中进行评估，后者将使用挑战试验进行测量，其中将纯化的艰难梭菌毒素 A 注射到连接的体内接种疫苗的动物和对照动物的回肠环。