Functional Antibody Repertoire Against S. aureus Leukocidins after Invasive Human Infection

项目摘要/摘要 金黄色葡萄球菌现在是美国儿童最常见的侵袭性细菌病原体， 抗生素耐药率继续上升。需要新的目标和方法，如果安全和 有效的金黄色葡萄球菌疫苗将成为现实。金黄色葡萄球菌的最佳干预靶点 人类特有的病原体，可以通过定义有助于入侵的抗原来最有效地识别 人类感染。LukAB是金黄色葡萄球菌在侵袭性人体环境中产生的一种分泌性白毒素。 它是一种疾病，是病原体在各种模型中免疫逃逸的关键。我们已经提纯了一系列 针对具有广泛中和作用的LukAB的有效中和人类单抗(MAbs) 脓毒症小鼠模型的容量、不同的结合部位和活性机制以及疗效。这个 这一提议的总体目标有两个：第一，检验人类关键方面的假设 抗白血球蛋白适应性反应包括毒素中和机制和白血球蛋白的功能。 以这些毒素为靶标的非中和抗体；第二，测试选定的 具有不同功能的单抗组合将最有效地抑制毒素介导的葡萄球菌 免疫逃避。在严格的模型中仔细验证抗杀白素抗体反应将允许 人源单抗作为干预金黄色葡萄球菌候选试剂的评价。建议进行的研究 将定义自然产生的金黄色葡萄球菌杀白素抗体谱系的多样性 人类感染，鉴于它们在毒力和作为干预目标的前景中的重要性。这将是 通过：1)表征自然产生的人类的多样性和功能能力 儿童侵袭性感染后针对葡萄球菌杀白素的抗体；2)阐明其作用 金黄色葡萄球菌杀白素的非中和抗体，包括抗体依赖补体 沉积和吞噬；3)抗白血球蛋白适应性关键成分的测定 对人血中金黄色葡萄球菌的反应。我们将利用我们现有的工作流程进行净化和 来自侵袭性和非侵袭性S。 一家大型三级保健儿童医院的金黄色葡萄球菌感染。将创建一组人类单抗，以允许 研究中和、交叉毒素活性和其他重要功能的不同机制。 拟议的目标将第一次确定人类宿主作出反应的机制的广度 在侵袭性人类疾病中靶向和中和金黄色葡萄球菌杀白素。这些研究共同解决了 迫切需要新的抗S抗体。金星战略。这部作品旨在制作经过合理选择的作品 根据我们对表达的重要抗原的了解，抗葡萄球菌干预的候选对象 在人类疾病期间，从感染金黄色葡萄球菌的儿童身上获得，在人性化的情况下进行疗效测试 在随后的工作中立即建立动物模型。

PROJECT SUMMARY / ABSTRACT Staphylococcus aureus is now the most common invasive bacterial pathogen in children in the US, and antibiotic resistance rates continue to increase. Novel targets and approaches are needed if a safe and effective S. aureus vaccine is to become a reality. Optimal targets of intervention against S. aureus, a highly human-specific pathogen, may be identified most effectively by defining the antigens that contribute to invasive human infections. LukAB, a secreted leukotoxin, is produced by S. aureus in the setting of invasive human disease and is essential for immune evasion by the pathogen in a variety of models. We have purified a series of potently neutralizing human monoclonal antibodies (mAbs) targeting LukAB, which have broad neutralizing capacity, distinct binding sites and mechanisms of activity, and efficacy in a murine model of sepsis. The overall goals of this proposal are twofold: first, to test the hypothesis that key aspects of the human anti-leukocidin adaptive response include both mechanisms of toxin neutralization and the function of non-neutralizing antibodies targeting these toxins; second, to test the hypothesis that a selected combination of mAbs with distinct functions will most potently inhibit toxin-mediated staphylococcal immune evasion. Careful validation of the anti-leukocidin antibody response in rigorous models will allow for the evaluation of human mAbs as candidate agents of intervention against S. aureus. The proposed studies will define the diversity of the naturally occurring antibody repertoire to S. aureus leukocidins following natural human infection, given their importance in virulence and promise as targets of intervention. This will be accomplished by: 1) Characterizing the diversity and functional capacity of naturally occurring human antibodies targeting the staphylococcal leukocidins following invasive pediatric infection; 2) Elucidating the role of non-neutralizing antibodies specific to S. aureus leukocidins, including antibody-dependent complement deposition and phagocytosis; and 3) Determining the critical components of the anti-leukocidin adaptive response against S. aureus in human blood. We will leverage our existing workflow for the purification and characterization of neutralizing mAbs from prospectively enrolled children with invasive and non-invasive S. aureus infections at a major tertiary care children’s hospital. A panel of human mAbs will be created to allow the investigation of diverse mechanisms of neutralization, cross-toxin activity, and other important functions. The proposed aims will define, for the first time, the breadth of mechanisms by which the human host response targets and neutralizes S. aureus leukocidins during invasive human disease. Together these studies address a critical need for novel anti-S. aureus strategies. This work is designed to produce rationally selected candidates for anti-staphylococcal interventions based on our knowledge of important antigens expressed during human disease, obtained from children with S. aureus infections, to be tested for efficacy in humanized animal models in immediately subsequent work.