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

项目总结/摘要 金黄色葡萄球菌现在是美国儿童中最常见的侵入性细菌病原体， 抗生素耐药率继续上升。如果一个安全和有效的方法能够被应用， 有效S。金黄色葡萄球菌疫苗即将成为现实。针对S.的最佳干预目标。金黄色葡萄球菌，一种高度 人类特异性病原体，可以最有效地确定通过定义抗原，有助于入侵 人类感染。LukAB是一种分泌型白细胞毒素，由S.金黄色葡萄球菌在侵袭性人类环境中的作用 疾病，并在各种模型中对病原体的免疫逃避至关重要。我们净化了一系列 靶向LukAB的有效中和人单克隆抗体（mAb），其具有广泛的中和作用， 能力、不同的结合位点和活性机制以及在败血症鼠模型中的功效。的 这项建议的总体目标是双重的：首先，检验假设，即人类的关键方面， 抗杀白细胞素适应性反应包括毒素中和机制和 针对这些毒素的非中和抗体;第二，为了测试假设， 具有不同功能的单克隆抗体的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细胞素抗体应答将允许 人源单克隆抗体作为抗沙门氏菌的候选药物的评价。金黄色。拟议的研究 将定义天然存在的抗S抗体库的多样性。金黄色葡萄球菌杀白细胞素 考虑到它们在毒力方面的重要性和作为干预目标的前景，这将是 完成：1）表征自然发生的人类的多样性和功能能力 针对侵袭性儿科感染后葡萄球菌杀白细胞素的抗体; 2）阐明 非中和抗体的特异性S.金黄色葡萄球菌杀白细胞素，包括抗体依赖性补体 沉积和吞噬作用;和3）确定抗杀白细胞素适应性的关键成分 针对S.金黄色葡萄球菌我们将利用现有的工作流程进行纯化， 来自前瞻性入组的侵袭性和非侵袭性S. 金黄色葡萄球菌感染在一个主要的三级保健儿童医院。将创建一组人类mAb， 研究不同的中和机制、交叉毒素活性和其他重要功能。 拟议的目标将首次界定人类宿主反应机制的广度， 靶向并中和S.金黄色葡萄球菌杀白细胞素在侵袭性人类疾病。这些研究共同探讨了 急需新型抗S抗体。aureus战略。这项工作的目的是产生合理选择 根据我们对重要抗原表达的了解， 在人类疾病期间，从患有S.金黄色葡萄球菌感染，以测试在人源化 动物模型在随后的工作。

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.