MOLECULAR DETERMINANTS OF HUMAN PNEUMOCOCCAL IMMUNITY

针对肺炎链球菌的衣壳多糖的抗体可以保护人类免受感染，并通过接种多糖或连接到蛋白质载体的多糖来激发抗体。在拟议的研究中，将通过全套克隆和序列分析来确定针对肺炎链球菌衣壳6B、14和23F型的人类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-蛋白质结合疫苗配方对表达谱的影响，并确定这些胸腺非依赖和胸腺依赖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、定点突变和分子模拟来确定。序列定义的Fab片段将在体外表达，确定它们的亲和力和良好的特异性，并使用体外吞噬细胞实验建立抗体亲和力和保护效果之间的关系。我们的总体假设是，与多克隆反应相比，寡克隆抗体反应的质量，如在人类中看到的对细菌荚膜多糖的反应，受到单个抗体结合域亲和力的影响更大。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突变体可能会导致整体抗体质量的降低。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产生的反应的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出现在无关的个体中，以及单一克隆型是否主导了对给定特异性的反应。明确结合部位亲和力和抗体功能质量之间的关系将提供更好的保护性免疫替代标志物。了解形成人类肺炎链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。

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.