RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

细菌外毒素的重组类毒素

• 批准号: 2062009
• 负责人: ROBERT JOHN COLLIER
• 金额: $ 21.31万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2062009
• 关键词: Escherichia coli; Salmonella; affinity chromatography; bacterial vaccines; biological products; biotechnology; diphtheria toxin; exotoxins; gene deletion mutation; genetic manipulation; mutant; protein structure function; receptor binding; recombinant DNA; site directed mutagenesis; synthetic vaccines; toxoids

DESCRIPTION (Adapted from applicant's abstract): The major goal of the proposed studies is to apply the current wealth of knowledge about the molecular structure and function of diphtheria toxin (DT) to create genetically inactivated cross- reactive mutant forms of the toxin (CRMs) that will serve as ideal components of future vaccines. The crystallographic structure of DT was solved recently, revealing the topography of the 3 functionally distant domains-the C (catalytic), T (transmembrane), and R (receptor-binding) domains. Using known substitution and deletion mutations that selectively abrogate individual functions, we will seek to define a limited set of CRMs that are appropriate for developing various types of vaccines. First, as an alternative to standard formal intoxoid, the investigators will seek to develop a holotoxin CRM with negligible biologic activity and with minimal alteration in antigenicity and immunogenicity. Second, toxin fragments corresponding to the 3 domains and selected inter- and intra-domain peptide sequences will be investigated as potential immunogens. Third, the investigators will investigate the hypothesis that disruption of the receptor-binding or membrane-translocation functions may alter the immunogenicity of the molecule. Fourth, to create a new vehicle for polysaccharides in conjugate vaccines, the investigators will introduce specific functional residues within CRMs to generate coupling sites for polysaccharides. Fifth, to identify mutant forms of DT appropriate for incorporation into live-vectored vaccines, the investigators will prepare and test multiply mutated CRMs that have suitably low probabilities of reversion. These studies will enhance understanding of the role of each of the critical vaccine antigens which will be: biologically inactive and stable without chemical toxoiding, highly consistent from batch to batch, inexpensively purified by simple affinity chromatography methods, suitable for chemical coupling to bacterial polysaccharide antigens in defined locations, and suitable for insertion into live vectors or for creation of chimeric proteins. The approaches developed in these studies may be applicable to other important vaccine antigens, such as tetanus toxin, pertussis toxin, and other pertussis antigens.

描述（改编自申请人的摘要）：主要目标 拟议的研究的目的是应用当前的财富 有关分子结构和功能的知识 白喉毒素（DT）可产生基因灭活的交叉作用 毒素的反应性突变形式（CRM）将作为理想的选择 未来疫苗的成分。 晶体结构 最近解决了 DT，揭示了 3 个区域的地形 功能上相距较远的结构域——C（催化）、T（跨膜）、 和 R（受体结合）结构域。使用已知的替代和 选择性消除个体功能的缺失突变，我们 将寻求定义一组有限的 CRM，适用于 开发各种类型的疫苗。 首先，作为标准正式毒素的替代品， 研究人员将寻求开发一种可忽略不计的全毒素 CRM 生物活性和抗原性变化最小 免疫原性。 二、3个结构域对应的毒素片段 并且选定的域间和域内肽序列将是 作为潜在的免疫原进行研究。 第三，调查人员将 研究受体结合破坏的假设 或膜易位功能可能会改变免疫原性 分子的。 四、打造新车型 结合疫苗中的多糖，研究人员将 在 CRM 中引入特定的功能残基以生成 多糖的偶联位点。五、识别突变形式 适合纳入活载体疫苗的 DT， 研究人员将准备并测试多重突变的 CRM 适当低的回复概率。 这些研究将增强 了解每种关键疫苗抗原的作用 这将是： 不具有生物活性且稳定，无需化学物质 毒化，批次间高度一致，价格低廉 通过简单的亲和层析方法纯化，适用于 与定义的细菌多糖抗原的化学偶联 位置，并且适合插入到实时向量中或用于创建 的嵌合蛋白。 这些研究中开发的方法可能是 适用于其他重要的疫苗抗原，例如破伤风毒素， 百日咳毒素和其他百日咳抗原。