Abstract：
Staphylococcus aureus carries an exceptional repertoire of virulence factors that aid in immune evasion. Previous single-target approaches for S. aureus-specific vaccines and monoclonal antibodies (mAbs) have failed in clinical trials due to the multitude of virulence factors released during infection. Emergence of antibiotic-resistant strains demands a multi-target approach involving neutralization of different, non-overlapping pathogenic factors. Of the several pore-forming toxins that contribute to S. aureus pathogenesis, efforts have largely focused on mAbs that neutralize α-hemolysin (Hla) and target the receptor-binding site. Here, we isolated two anti-Hla and three anti-Panton-Valentine Leukocidin (LukSF-PV) mAbs, and used a combination of hydrogen deuterium exchange mass spectrometry (HDX-MS) and alanine scanning mutagenesis to delineate and validate the toxins\' epitope landscape. Our studies identified two novel, neutralizing epitopes targeted by 2B6 and CAN6 on Hla that provided protection from hemolytic activity in vitro and showed synergy in rodent pneumonia model against lethal challenge. Of the anti-LukF mAbs, SA02 and SA131 showed specific neutralization activity to LukSF-PV while SA185 showed cross-neutralization activity to LukSF-PV, γ-hemolysin HlgAB, and leukotoxin ED. We further compared these antigen-specific mAbs to two broadly neutralizing mAbs, H5 (targets Hla, LukSF-PV, HlgAB, HlgCB, and LukED) and SA185 (targeting LukSF-PV, HlgAB, and LukED), and identified molecular level markers for broad-spectrum reactivity among the pore-forming toxins by HDX-MS. To further underscore the need to target the cross-reactive epitopes on leukocidins for the development of broad-spectrum therapies, we annotated Hla sequences isolated from patients in multiple countries for genomic variations within the perspective of our defined epitopes.
摘要：
金黄色葡萄球菌携带有助于免疫逃避的特殊毒力因子库。先前用于金黄色葡萄球菌特异性疫苗和单克隆抗体(mAb)的单靶标方法由于感染期间释放的多种毒力因子而在临床试验中失败。抗生素抗性菌株的出现需要多目标方法，涉及中和不同的，非重叠致病因素。在几种促进金黄色葡萄球菌发病机制的成孔毒素中，努力主要集中在中和α-溶血素(Hla)并靶向受体结合位点的mAb上。这里,我们分离了两种抗Hla和三种抗Panton-ValentineLeukocidin（LukSF-PV）单克隆抗体，并结合使用氢氘交换质谱（HDX-MS）和丙氨酸扫描诱变来描绘和验证毒素的表位景观。我们的研究发现了两部小说，由Hla上的2B6和CAN6靶向的中和表位提供体外溶血活性的保护，并在啮齿动物肺炎模型中显示出针对致命攻击的协同作用。在反LukF的MAB中，SA02和SA131对LukSF-PV显示出特定的中和活性，而SA185对LukSF-PV显示出交叉中和活性，γ-溶血素HlgAB,和白细胞毒素ED。我们进一步将这些抗原特异性mAb与两种广泛中和的mAb进行了比较，H5（目标Hla，LukSF-PV，HlgAB,HlgCB,和LukED)和SA185(针对LukSF-PV，HlgAB,和LukED），并通过HDX-MS鉴定了成孔毒素之间广谱反应性的分子水平标记。为了进一步强调需要靶向杀白细胞素上的交叉反应表位，以开发广谱疗法，我们注释了从多个国家的患者中分离出的Hla序列，以了解我们定义的表位。
