MOLECULAR DETERMINANTS OF HUMAN PNEUMOCOCCAL IMMUNITY

针对病原体肺炎链球菌荚膜多糖的抗体保护人类免受感染，并通过接种多糖或多糖结合蛋白载体引起。在本研究中，将通过全库克隆和序列分析来确定6B、14和23F荚膜肺炎链球菌血清型特异性人抗体的可变区基因使用和连接多样性。将研究普通多糖和多糖蛋白结合疫苗制剂对表达库的影响，并确定这些胸腺独立和胸腺依赖形式的疫苗诱导体细胞突变和亲和成熟的程度。抗多糖抗体亲和力的结构决定因素将通过序列比较、位点定向诱变和分子模型来确定。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，并通过体外调理噬细胞实验建立抗体亲和力与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，如人类对细菌荚膜多糖的反应，在更大程度上受到单个抗体结合域的亲和力的影响，而不是多克隆反应。这些抗体亲和力的差异是可变区域基因使用、连接多样性和体细胞突变的结果。因此，通过体细胞突变产生亲和损失变异可能导致整体抗体质量的降低。这些研究将确定针对结构上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了反应的总体亲和力。这些研究还将确定相同的克隆型是否发生在不相关的个体中，以及单一克隆型是否对给定特异性的反应占主导地位。明确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物。了解形成针对肺炎球菌多糖的人抗体库的分子机制也可能提出促进开发更有效疫苗的策略。

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.