MOLECULAR DETERMINANTS OF HUMAN PNEUMOCOCCAL IMMUNITY

人类肺炎球菌免疫的分子决定因素

• 批准号: 6511229
• 负责人: Donald C Reason
• 金额: $ 34.81万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511229
• 关键词: Streptococcus pneumoniae; Streptococcus pneumoniae vaccine; antibacterial antibody; antibody titering; bacterial polysaccharides; chemical conjugate; clinical research; gene mutation; human subject; human therapy evaluation; immunoconjugates; immunoglobulin genes; immunoglobulin structure; molecular cloning; nucleic acid sequence; phagocytosis; site directed mutagenesis; vaccine development; young adult human (21-34)

Antibodies directed against the capsular polysaccharides of the pathogen Streptococcus pneumoniae protect humans against infection, and are elicited by vaccination with polysaccharide or polysaccharide conjugated to protein carriers. In the proposed study the variable region gene usage and junctional diversity of human antibodies specific for S. pneumoniae capsular serotypes 6B, 14, and 23F will be determined by repertoire cloning and sequence analysis. The influence of plain polysaccharide and polysaccharide-protein conjugate vaccine formulations on the expressed repertoire will be investigated, and the degree to which these thymus- independent and thymus-dependent forms of the vaccine induce somatic mutations and affinity maturation will be determined. The structural determinants of anti-polysaccharide antibody affinity will be defined by sequence comparison, site directed mutagenesis, and molecular modeling. Sequence-defined Fab fragments will be expressed in vitro , their affinity and fine specificity determined, and the relationship between antibody affinity and protective efficacy established using an in vitro opsonophagocytosis assay. Our overall hypothesis is that the quality of an oligoclonal antibody response, such as that seen in humans to bacterial capsular polysaccharides, is influenced to a greater degree by the affinities of the individual antibody binding domains than would be a polyclonal response. These differences in antibody affinity arise as a consequence of variable region gene usage, junctional diversity, and somatic mutation. The generation of affinity loss variants by somatic mutation could therefore leads a diminution of overall antibody quality. These studies will determine if antibodies to structurally distinct polysaccharides utilize the same or distinct variable region genes and the degree to which maturation of the response through somatic mutation determines overall affinity of the response. These studies will also determine if the same clonotypes occur in unrelated individuals, and if a single clonotype predominates the response to a given specificity. Defining the relationship between binding site affinity and antibody functional quality will provide better surrogate markers of protective immunity. Understanding the molecular mechanisms that shape the human antibody repertoire to pneumococcal polysaccharides may also suggest strategies that would facilitate the development of more efficacious vaccines.

针对病原体肺炎链球菌荚膜多糖的抗体可以保护人类免受感染，并且通过用多糖或与蛋白质载体缀合的多糖进行疫苗接种来引发。在拟议的研究中，肺炎链球菌荚膜血清型 6B、14 和 23F 特异性人抗体的可变区基因使用和连接多样性将通过库克隆和序列分析来确定。将研究普通多糖和多糖-蛋白质缀合物疫苗制剂对表达库的影响，并将确定这些胸腺非依赖性和胸腺依赖性形式的疫苗诱导体细胞突变和亲和力成熟的程度。抗多糖抗体亲和力的结构决定因素将通过序列比较、定点诱变和分子建模来定义。序列定义的 Fab 片段将在体外表达，确定其亲和力和精细特异性，并使用体外调理吞噬作用测定建立抗体亲和力和保护功效之间的关系。我们的总体假设是，寡克隆抗体反应的质量，例如人类对细菌荚膜多糖的反应，与多克隆反应相比，在更大程度上受到各个抗体结合域的亲和力的影响。抗体亲和力的这些差异是可变区基因使用、连接多样性和体细胞突变的结果。因此，体细胞突变产生的亲和力丧失变体可能导致整体抗体质量下降。这些研究将确定针对结构不同的多糖的抗体是否利用相同或不同的可变区基因，以及通过体细胞突变的反应成熟程度决定反应的总体亲和力。这些研究还将确定相同的克隆型是否出现在不相关的个​​体中，以及单一克隆型是否主导对给定特异性的反应。定义结合位点亲和力与抗体功能质量之间的关系将为保护性免疫提供更好的替代标记。了解形成人类肺炎球菌多糖抗体库的分子机制也可能提出有助于开发更有效疫苗的策略。