Structural determinants of human immunity to anthrax

描述(申请人提供)：最近故意传播的炭疽芽孢杆菌导致了严重的发病率和死亡率，以及对基本公共服务的广泛和代价高昂的破坏。由此产生的对当前炭疽病预防和治疗方法的重新评估揭示了我们对这种疾病的基本免疫生物学知识的重大差距，并强调了在没有临床试验的情况下制定疫苗效力的方法的必要性。这项建议中描述的研究将确定目前获得许可的人炭疽疫苗所引发的保护性抗体反应的分子和结构特征。本实验室最近发展的全谱克隆方法将用于在分子水平上建立针对炭疽杆菌保护性抗原(PA)的人抗体全谱的结构多样性、可变的基因使用和体细胞成熟历史。不同的个体利用相同的免疫球蛋白可变基因产物来结合抗原上的特定表位的程度将被确定。通过序列分析，将确定应答抗体克隆经历了体细胞成熟的程度。克隆的PA特异性结合域将在体外表达，能够阻断PA功能活性的子集被确定，并建立了功能的亲和力和价态要求。克隆的抗体结合域将被用来定义人类免疫反应识别的PA相关抗原表位，并精确定位那些引发中和抗体的PA相关表位。我们的发现将对PA亚单位疫苗的合理设计至关重要，并将有助于在体外建立炭疽杆菌感染保护性免疫的相关性。此外，分离的抗体将构成一组完全人类的单抗结合域，具有作为被动免疫原用于治疗的潜力。

DESCRIPTION (provided by applicant): The recent, intentional dissemination of Bacillus anthracis caused significant morbidity and mortality, as well as widespread and costly disruption of essential public services. The resulting re-evaluation of current methods for the prevention and treatment of anthrax has revealed significant gaps in our knowledge of the basic immunobiology of this disease, and highlighted the need to develop methodologies for establishing vaccine efficacy in the absence of clinical trials. The research described in this proposal will define the molecular and structural characteristics of the protective antibody response elicited by the currently licensed human anthrax vaccine. Methods of repertoire cloning recently developed in our laboratory will be used to establish at the molecular level the structural diversity, variable gene usage, and somatic maturational history of the human antibody repertoire specific for the protective antigen (PA) of B. anthracis. The extent to which different individuals utilize the same immunoglobulin variable gene products to bind specific epitopes on the antigen will be determined. Through sequence analysis, the degree to which responding antibody clones have undergone somatic maturation will be ascertained. Clonally derived PA-specific binding domains will be expressed in vitro, the subset capable of blocking PA functional activity identified, and affinity and valence requirements for functionality established. Cloned antibody binding domains will be used to define the PA-associated antigenic epitopes recognized by the human immune response, and to pinpoint those PA-associated epitopes that elicit neutralizing antibodies. Our findings will be crucial for the rational design of PA subunit vaccines, and will aid in establishing in vitro correlates of protective immunity to B. anthracis infection. In addition, the antibodies isolated will constitute a panel of fully human monoclonal binding domains with potential for therapeutic use as passive immunogens.