利用 Epivolve 噬菌体展示技术生成针对 BamA（TP0326）细胞外环 4 上一个亚显性表位的调理活性单克隆抗体。
Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of BamA (TP0326).
机构信息
Department of Molecular Sciences, Abbratech, Branford, CT, United States.
Department of Medicine, UConn Health, Farmington, CT, United States.
出版信息
Front Immunol. 2023 Aug 22;14:1222267. doi: 10.3389/fimmu.2023.1222267. eCollection 2023.
INTRODUCTION
Syphilis, a sexually transmitted infection caused by the spirochete (), is resurging globally. 's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of by rabbit peritoneal macrophages.
METHODS
Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a thioredoxin scaffold (Trx). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (, opsonization) and validate the mouse assay. Sera from -infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using Trx and overlapping ECL4 peptides in immunoblotting and ELISA assays.
RESULTS
Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of Trx. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with Trx produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.
DISCUSSION
Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by infection.
简介
梅毒是一种由螺旋体（）引起的性传播感染，在全球范围内再次出现。的外膜蛋白（OMP）谱包括 BamA（β-桶组装机制亚基 A/TP0326），这是一种由 16 个β-桶组成的二部分蛋白，有 9 个细胞外环（ECL）和 5 个周质 POTRA（多肽转运相关）结构域。BamA ECL4 抗血清促进兔腹膜巨噬细胞内化。
方法
使用三种重叠的 BamA ECL4 肽和一种称为“Epivolve”（用于表位进化）的两阶段噬菌体展示策略来产生单链可变片段（scFv）。此外，用 BamA ECL4 展示的硫氧还蛋白支架（Trx）免疫小鼠和兔产生的抗血清。用免疫印迹和 ELISA 评估 MAb 和抗血清的反应性。通过比较鼠和兔的调理吞噬作用试验来评估 Abs（调理作用）的功能能力，并验证鼠试验。使用 Trx 和重叠 ECL4 肽在免疫印迹和 ELISA 试验中评估感染的小鼠（MSS）和兔（IRS）血清中的 ECL4 特异性 Abs。
结果
五种 MAb 中的每一种都通过免疫印迹和 ELISA 对纳克量的 Trx 表现出反应性。一种 MAb，其轻链和重链都具有独特的氨基酸序列，在鼠调理吞噬作用试验中具有活性。用 Trx 高度免疫的小鼠和兔产生的调理抗血清强烈识别在异源支架和重叠 ECL4 肽中呈现的 ECL，包括 S2。相比之下，在小鼠和兔感染期间产生的 Abs 对肽的识别能力较差，表明 S2 包含一个亚显性表位。
讨论
Epivolve 产生的 MAb 靶向 BamA ECL4 中的亚显性调理表位，这是一种顶级梅毒疫苗候选物。调理吞噬作用试验可以作为一种替代模型来研究疫苗接种基因的调理潜力。BamA ECL4 特异性 Abs 的详细特征提供了一种剖析由感染引起的 Abs 反应的方法。
Zotero 插件