ACELLULAR VACCINES AGAINST BACTERIAL PATHOGENS

针对细菌病原体的无细胞疫苗

• 批准号: 2065485
• 负责人: Joseph T Barbieri
• 金额: $ 17.03万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2065485
• 关键词: ADP ribosylation; Bordetella pertussis; CHO cells; G protein; Golgi apparatus; Pseudomonas aeruginosa; adenine phosphoribosyltransferase; cell membrane; chemical association; chimeric proteins; diphtheria toxin; enzyme activity; enzyme structure; exoenzyme; exotoxins; guinea pigs; immunological substance; laboratory mouse; laboratory rabbit; pertussis; pertussis toxin; pertussis vaccine; protein sequence; proteolysis; tetanus toxin

DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to contribute to the generation of effective acellular vaccines to prevent pertussis and Pseudomonas aeruginosa infections. The focus of this research is to define functional residues and domains of pertussis toxin and Pseudomonas exoenzyme S which will lead to the generation of noncatalytic toxoids that can be tested as vaccine candidates. These studies will also determine the mechanism by which pertussis toxin intoxicates eukaryotic cells and presents strategies for engineering a trivalent a cellular vaccine to prevent diphtheria, pertussis and tetanus. The specific aims of this proposal are to: (i) define the kinetic constants of mutants of the S1 subunit of PT that express reduced enzymatic activity. These studies will define residues that contribute to NAD binding, G protein binding, and catalysis; (ii) determine how pertussistoxin ADP-ribosylates both plasma membrane- and Golgi- associated G(i) proteins. Experiments will define the rate of ADP- ribosylation of plasma membrane- and Golgi-associated G(i) proteins and correlate this data with the physical localization of S1 during the entry of PT into sensitive cells. These studies will also determine the contribution of the ADP-ribosylation of plasma membrane- and Golgi-associated G proteins in the pathogenesis of pertussis toxin; (iii) identify the minimal amino acid sequence of S1 required for association with B oligomer; (iv) engineer a recombinant trivalent diphtheria-pertussis-tetanus vaccine composed of non-toxic deletion peptides of each respective toxin. These fusion proteins will be analyzed for the elicitation of a neutralizing immune response against their respective toxin; and (v) define the kinetic constants of recombinant Pseudomonas exoenzyme S, which ADP-ribosylates the Ras protooncogene product, and identify residues that contribute to the catalysis of exoenzyme S. Mapping functional residues and domains of pertussis toxin and exoenzyme S will define rational approaches for the production of acellular pertussis and Pseudomonas vaccines. Parallel studies on pertussis toxin and exoenzyme S will define relationships between these two members of the family of bacterial ADP-ribosylating exotoxins and may identify new strategies for engineering effective toxoids against other bacterial pathogens.

描述（改编自申请人的摘要）：此目标 提案旨在促进有效非细胞的产生 预防百日咳和铜绿假单胞菌感染的疫苗。 本研究的重点是定义功能残基和 百日咳毒素和假单胞菌外切酶 S 的结构域 导致可测试的非催化类毒素的产生 作为候选疫苗。 这些研究还将确定其机制 百日咳毒素通过它使真核细胞中毒并呈现 设计三价细胞疫苗以预防的策略 白喉、百日咳和破伤风。 该提案的具体目标是：（i）定义动力学 表达减少的 PT S1 亚基突变体的常数 酶活性。 这些研究将定义有助于 NAD 结合、G 蛋白结合和催化； (ii) 确定如何 百日咳毒素 ADP-核糖基化质膜和高尔基体 相关的 G(i) 蛋白。 实验将确定 ADP- 的速率 质膜和高尔基相关 G(i) 蛋白的核糖基化和 将此数据与 S1 期间的物理定位相关联 PT进入敏感细胞。 这些研究也将 确定质膜 ADP-核糖基化的贡献- 和高尔基相关 G 蛋白在百日咳发病机制中的作用 毒素; (iii)确定S1所需的最小氨基酸序列 用于与B寡聚物缔合； (iv) 设计重组三价 无毒缺失的白喉、百日咳、破伤风疫苗 每种毒素的肽。 这些融合蛋白将 分析引发中和免疫反应 它们各自的毒素； (v) 定义动力学常数 重组假单胞菌外切酶 S，ADP 核糖基化 Ras 原癌基因产物，并鉴定有助于 外酶S的催化作用。 绘制百日咳毒素的功能残基和结构域 外切酶 S 将定义合理的生产方法 无细胞百日咳和假单胞菌疫苗。 平行研究 百日咳毒素和外切酶 S 将定义两者之间的关系 这两个细菌 ADP 核糖基化外毒素家族的成员 并可能确定工程有效类毒素的新策略 对抗其他细菌病原体。