Division of Infectious Disease, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
Department of Computer Science, Emory University, Atlanta, Georgia, USA.
mBio. 2021 Apr 20;12(2):e03679-20. doi: 10.1128/mBio.03679-20.
causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition. Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.
霍乱弧菌引起严重的腹泻病霍乱。临床疾病和当前的口服霍乱疫苗会产生与保护相关的抗体反应。人们认为免疫主要由脂多糖 (LPS) 特异性抗体介导，主要针对 O 抗原。然而，功能相关抗体的特性和保护机制尚未得到很好的定义。我们之前曾在孟加拉国患者的队列中报告过霍乱的早期 B 细胞反应，并从中鉴定了一组从急性诱导的浆母细胞中分离出的人单克隆抗体 (Mab)。在之前的研究中，所有抗体均以 IgG1 为骨架表达，与其原始同种型无关。为了清楚地确定亲和力、免疫球蛋白同种型和亚类对这些 Mab 功能特性的影响，我们重新设计了一组低亲和力和高亲和力抗体，使其具有不同的同种型和亚类免疫球蛋白骨架，并研究了这些变化对结合、杀弧菌、凝集和运动抑制活性的影响。虽然高亲和力抗体与 O 抗原的结合相似，与同种型无关，但低亲和力抗体显示出明显的亲和力差异。有趣的是，尽管低亲和力 Mab 衍生的变体表现出较低的结合特性，但它们在凝集和运动抑制特性方面与强效结合抗体相当，这表明 Mab 与 O 抗原的结合方式可能对功能至关重要。此外，不仅五聚体 IgM 和二聚体 IgA，而且单体 IgA，在抑制运动方面也比其 IgG 对应物强得多。最后，分析这些抗体的高度纯化 F(ab) 版本，我们表明 LPS 交联对于运动抑制至关重要。针对严重腹泻病霍乱的免疫主要由脂多糖 (LPS)-特异性抗体介导。然而，功能相关抗体的特性和保护机制尚未得到很好的定义。在这里，我们在不同的免疫球蛋白骨架中设计了低亲和力和高亲和力 LPS 特异性抗体，以评估亲和力、免疫球蛋白同种型和亚类对结合、杀弧菌、凝集和运动抑制功能特性的影响。重要的是，我们发现亲和力并没有直接决定功能效力，因为低亲和力 Mab 衍生的变体具有与强效结合抗体相当的凝集和运动抑制特性。这表明抗体的结合方式可能对功能至关重要。此外，不仅五聚体 IgM 和二聚体 IgA，而且单体 IgA，在抑制运动方面也比其 IgG 对应物强得多。最后，分析这些抗体的高度纯化 F(ab) 版本，我们表明 LPS 交联对于运动抑制至关重要。
