RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

细菌外毒素的重组类毒素

• 批准号: 2062010
• 负责人: ROBERT JOHN COLLIER
• 金额: $ 26.04万
• 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/2062010
• 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（改编自申请人摘要）：主要目标 建议的研究是应用现有的财富， 分子结构的知识， 功能 白喉毒素 (DT)来创造基因失活的交叉 毒素的反应性突变形式（CRM），将作为理想的 未来的疫苗。 的结晶结构 DT最近得到解决，揭示了3 功能上遥远的结构域-C（催化），T（跨膜）， 和R（受体结合）结构域。使用已知的替换和 选择性地消除个体功能的缺失突变，我们 将寻求定义一组有限的CRM，适合于 研制各种疫苗。 第一，作为替代方案， 标准的正式类醉药， 研究人员将寻求开发一种全毒素CRM， 生物活性，抗原性变化极小， 免疫原性 第二，对应于3个结构域的毒素片段 并且选择的结构域间和结构域内肽序列将被 作为潜在的免疫原。 第三，调查人员 将 调查的假设，中断的 受体结合 或膜转位功能可能改变免疫原性 的分子。 第四，创造新的交通工具， 结合疫苗中的多糖，研究人员将 在CRM内引入特定功能残基以产生 多糖的偶联位点。第五，识别突变形式 对于适于掺入活载体疫苗的DT， 研究人员将准备和测试多重突变的标准物质， 适当降低逆转的可能性。 这些研究将提高 了解每种关键疫苗抗原的作用 这将是： 无生物活性且稳定，不含化学物质 类毒素化，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 与细菌多糖抗原的化学偶联 位置，并适合插入到活载体或创建 嵌合蛋白质。 这些研究中开发的方法可能是 适用于其它重要的疫苗抗原，如破伤风毒素， 百日咳毒素和其它百日咳抗原。