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); 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的囊囊多糖的抗体可保护人类免受感染的影响，并通过用多糖或多糖与蛋白质载体结合的疫苗接种而引起。在拟议的研究中，人类抗体的可变区域基因使用和对肺炎链球菌囊型血清型6B，14和23F的连接性多样性将通过曲目克隆和序列分析确定。将研究普通的多糖和多糖蛋白结合疫苗制剂对表达曲目的影响，并确定这些胸腺独立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。抗溶酶抗体抗体亲和力的结构决定因素将通过序列比较，定向诱变和分子建模来定义。序列定义的FAB片段将在体外表达，它们的亲和力和精细的特异性，以及使用体外倾向性胞毒性测定法建立的抗体亲和力与保护性疗效之间的关系。我们的总体假设是，寡克隆抗体反应的质量，例如人类对细菌囊囊多糖的质量，受单个抗体结合结构域的亲和力的影响更大，而不是多克隆反应。抗体亲和力的这些差异是由于可变区域基因使用，连接性多样性和体细胞突变而产生的。因此，通过体细胞突变产生的亲和力损失变异可能会导致整体抗体质量的降低。这些研究将确定对结构上不同多糖的抗体是否利用相同或不同的可变区域基因以及通过体细胞突变成熟的响应的程度决定了反应的总体亲和力。这些研究还将确定在不相关的个​​体中是否出现相同的克隆型，以及单一的克隆型是否占对给定特异性的响应。定义结合位点亲和力与抗体功能质量之间的关系将提供更好的保护性免疫标记。了解塑造人类抗体库的分子机制，这也可能提出策略，以促进更有效的疫苗的发展。