Antigenic landscapes on Staphylococcus aureus pore-forming toxins reveal insights into specificity and cross-neutralization.

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.

金黄色葡萄球菌携带一系列特殊的毒力因子，这些因子有助于免疫逃逸。先前针对金黄色葡萄球菌的特异性疫苗和单克隆抗体（mAbs）的单一靶点方法在临床试验中失败，原因是感染期间释放出多种毒力因子。抗生素耐药菌株的出现需要一种多靶点方法，包括中和不同的、非重叠的致病因子。在几种导致金黄色葡萄球菌发病机制的成孔毒素中，研究工作主要集中在中和α -溶血素（Hla）并靶向受体结合位点的单克隆抗体上。在此，我们分离出两种抗Hla和三种抗潘顿 - 瓦伦丁杀白细胞素（LukSF - PV）的单克隆抗体，并结合氢氘交换质谱（HDX - MS）和丙氨酸扫描诱变来描绘和验证毒素的表位图谱。我们的研究确定了Hla上被2B6和CAN6靶向的两个新的中和表位，它们在体外能抵御溶血活性，并在啮齿动物肺炎模型中对致死性攻击显示出协同作用。在抗LukF单克隆抗体中，SA02和SA131对LukSF - PV显示出特异性中和活性，而SA185对LukSF - PV、γ -溶血素HlgAB和白细胞毒素ED显示出交叉中和活性。我们进一步将这些抗原特异性单克隆抗体与两种广泛中和的单克隆抗体H5（靶向Hla、LukSF - PV、HlgAB、HlgCB和LukED）和SA185（靶向LukSF - PV、HlgAB和LukED）进行比较，并通过HDX - MS确定了成孔毒素中广谱反应性的分子水平标记。为了进一步强调针对杀白细胞素上的交叉反应性表位对于开发广谱疗法的必要性，我们对从多个国家的患者中分离出的Hla序列进行注释，以了解在我们所定义的表位范围内的基因组变异情况。