National Engineering Research Center for Protein Drugs, Beijing, 102206, China.
Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK.
J Neuroimmune Pharmacol. 2021 Sep;16(3):567-580. doi: 10.1007/s11481-020-09948-1. Epub 2020 Aug 18.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), the etiology of which is poorly understood. The most common laboratory abnormality associated with MS is increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands (OCBs) in the brain and cerebrospinal fluid (CSF). However, the major antigenic targets of these antibody responses are unknown. The risk of MS is increased after infectious mononucleosis (IM) due to EBV infection, and MS patients have higher serum titers of anti-EBV antibodies than control populations. Our goal was to identify disease-relevant epitopes of IgG antibodies in MS; to do so, we screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. Using a highly sensitive phage-mediated immuno-PCR assay, we determined specific bindings of the two EBV epitopes to IgG from CSF from 46 MS and 5 inflammatory control (IC) patients. MS CSF IgG have significantly higher bindings to EBNA1 epitope than to EBNA2 epitope, whereas EBNA1 and EBNA2 did not significantly differ in binding to IC CSF IgG. Further, the EBNA1 epitope was recognized by OCBs from multiple MS CSF as shown in blotting assays with samples separated by isoelectric focusing. The EBNA1 epitope is reactive to MS intrathecal antibodies corresponding to oligoclonal bands. This reinforces the potential role of EBV in the etiology of MS. Graphical abstract Antibodies purified from an MS brain plaque were panned by phage display peptide libraries to discern potential antigens. Phage displaying peptide sequences resembling Epstein-Barr Virus Nuclear Antigens 1 & 2 (EBNA1 & 2) epitopes were identified. Antibodies from sera and CSF from other MS patients also reacted to those epitopes.
多发性硬化症（MS）是一种中枢神经系统（CNS）的慢性炎症性脱髓鞘疾病，其病因尚未完全了解。与 MS 最常见的实验室异常是鞘内免疫球蛋白 G（IgG）合成增加，以及脑和脑脊液（CSF）中寡克隆带（OCB）的存在。然而，这些抗体反应的主要抗原靶标尚不清楚。由于 EBV 感染，传染性单核细胞增多症（IM）后 MS 的风险增加，MS 患者的血清抗 EBV 抗体滴度高于对照人群。我们的目标是鉴定 MS 中 IgG 抗体的相关表位；为此，我们使用从急性 MS 患者脑内纯化的总 IgG 抗体筛选噬菌体展示的随机肽文库（12 -mer）。我们通过 ELISA、噬菌体介导的免疫-PCR 和等电聚焦鉴定和表征噬菌体肽与 MS 和对照患者鞘内 IgG 的结合特异性。我们鉴定了两个噬菌体肽，它们分别与 EBV 核抗原 1 和 2（EBNA1 和 EBNA2）具有序列同源性。通过用 MS 脑 IgG 进行淘选发现的 EBV 表位的特异性通过 ELISA 和竞争性抑制试验得到证实。使用高度敏感的噬菌体介导的免疫-PCR 检测法，我们测定了 46 例 MS 和 5 例炎症对照（IC）患者 CSF 中两种 EBV 表位与 IgG 的特异性结合。MS CSF IgG 对 EBNA1 表位的结合明显高于 EBNA2 表位，而 EBNA1 和 EBNA2 在与 IC CSF IgG 的结合上没有显著差异。此外，在印迹分析中，用等电聚焦分离的样本进行检测，发现两种 EBV 表位均被来自多个 MS CSF 的 OCB 识别。EBNA1 表位与多发性硬化症鞘内抗体反应，对应于寡克隆带。这强化了 EBV 在 MS 病因学中的潜在作用。图表摘要从 MS 脑斑块中纯化的抗体通过噬菌体展示肽文库进行淘选，以辨别潜在的抗原。鉴定出与 Epstein-Barr 病毒核抗原 1 & 2（EBNA1 & 2）表位相似的噬菌体展示肽序列。来自其他 MS 患者的血清和 CSF 中的抗体也与这些表位反应。
