Development of a Preclinical Assay to Predict Efficacy of Ricin Toxin Subunit Vaccines

项目摘要 美国国家过敏和传染病研究所(NIAID)将蓖麻毒素(RT)归类为 新发传染病优先病原体。NIAID和国防部各自 支持开发有效的RT亚单位疫苗的努力。发展RT的主要障碍 疫苗缺乏一种功能性的临床前试验来预测对人类的疗效。趁它好的时候 证实了对RT的免疫是由抗体介导的，对蓖麻毒素A的免疫反应 基于亚单位(RTA)的抗原主要由(&gt；90%)非中和抗体组成。的确， RTA特异性小鼠和骆驼单抗的详细功能和结构分析 (单抗)揭示毒素中和活性(TNA)与数量非常有限的B RTA表面的表位“团簇”。传统的基于细胞的细胞毒性检测不是 足够灵敏，可以检测到一小部分毒素中和抗体 保护豁免权。从疫苗发展的角度来看，开发一种更多的 灵敏而直接的检测针对关键保护性表位的血清抗体。 因此，该计划的目标是开发一种基于临床前酶联免疫吸附试验的竞争分析方法，将 预测RT亚单位疫苗在人类中的效果。目标1将建立一个原型竞赛 基于酶联免疫吸附试验确定与保护性免疫相关的血清抗体图谱 反对RT。Aim 2将在NHP模型中鉴定基于竞争ELISA法的检测以确定抗体 与雾化RT防护相关的配置文件。最后，Aim 3将验证 使用来自第一和第二阶段研究的人血清的竞争酶联免疫吸附试验。作为该计划的一部分，我们将 建立单抗竞争和保护性免疫之间的联系，期望 测量表位特异性中和抗体反应的能力将构成对 评估候选RT亚单位疫苗在人类中的效力。

Project Summary Ricin toxin (RT) is classified by the National Institute of Allergy and Infectious Diseases (NIAID) as an Emerging Infectious Disease Priority Pathogen. NIAID and the Department of Defense are each supporting efforts to develop an effective RT subunit vaccine. A major roadblock to developing RT vaccines is the absence of a functional preclinical assay to predict efficacy in humans. While it is well established that immunity to RT is mediated by antibodies, the immune response against ricin toxin A subunit (RTA)-based antigens consists predominantly (>90%) of non-neutralizing antibodies. Indeed, detailed functional and structural analyses of RTA-specific murine and camelid monoclonal antibodies (MAbs) has revealed that toxin-neutralizing activity (TNA) is associated a very limited number of B epitope “clusters” on the surface of RTA. Conventional cell-based cytotoxicity assays are not sufficiently sensitive to detect the small fraction of toxin-neutralizing antibodies responsible for protective immunity. From the standpoint of vaccine development, it is critical to develop a more sensitive and direct assay to measure serum antibodies against the key protective epitopes. Therefore, the goal of this program is to develop a preclinical ELISA-based competition assay that will predict the efficacy of RT subunit vaccines in humans. Aim 1 will e stablish a prototype competition ELISA-based assay that identifies a serum antibody profile associated with protective immunity against RT. Aim 2 will qualify the competition ELISA-based assay in a NHP model to define antibody profile that is associated with protection against aerosolized RT. Finally, Aim 3 will validate the competition ELISA using human sera from Phase I and II studies. As part of this program we will establish the link between MAb competition and protective immunity, with the expectation that the ability to measure epitope-specific neutralizing antibody responses will constitute a major advance in assessing the efficacy of candidate RT subunit vaccines in humans.