Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, United Kingdom.
Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, United Kingdom.
PLoS Pathog. 2024 Jun 6;20(6):e1012177. doi: 10.1371/journal.ppat.1012177. eCollection 2024 Jun.
Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated.
Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens.
EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP.
Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.
EB 病毒（EBV）是多发性硬化症（MS）发生的必要前提，但具体的发病机制尚不清楚。我们对 MS 患者（pwMS）、健康 EBV 血清阳性对照（HC）和传染性单核细胞增多症（POST-IM）患者的 EBV 抗体和 T 细胞反应进行了研究，检测时间为疾病缓解后 6 个月。此外，我们还对 EBV 特异性 T 细胞对中枢神经系统（CNS）靶抗原的反应能力进行了研究。
我们尽可能地按照性别、年龄和 HLA-DRB1*15:01 对处于缓解-复发期的 MS 患者、POST-IM 患者和 HC 进行匹配。通过 qPCR 测定 EBV 载量，通过 ELISA、免疫荧光和 Western blot 测定关键 EBV 抗原的 IgG 反应，通过多重微珠阵列测定破伤风类毒素抗体反应。通过细胞内细胞因子染色（ICS）体外测定 EBV 特异性 T 细胞反应，并通过针对 9 种新的改良安卡拉痘苗病毒（MVA）的体外扩增反应探测候选 CNS 自身抗原，检测交叉反应性。
与 HC 相比，pwMS 患者外周血单核细胞（PBMC）中的 EBV 载量无变化。在血清学方面，尽管各组之间破伤风类毒素反应无变化，但 EBNA1 和病毒衣壳抗原（VCA）的 IgG 反应明显升高（EBNA1 p = 0.0079，VCA p = 0.0298），但重要的是，pwMS 患者中也更频繁地检测到 EBNA2 和 EBNA3 家族抗原的 IgG 反应（EBNA2 p = 0.042 和 EBNA3 p = 0.005）。在体外试验中，针对自身 EBV 转化的 B 细胞和 EBNA1 的 T 细胞反应在数值上基本不变，但 pwMS 对某些刺激物的 IL-2 产生显著增加。来自 MS 和 HC 的 EBV 特异性多克隆 T 细胞系通过 ICS 显示出对自身抗原的高识别水平，并且包括 MOG、MBP、PLP 和 MOBP 在内的几种神经元蛋白被鉴定为共同靶标。
MS 组中发现 EBV 特异性抗体反应升高超出了 EBNA1，这表明 EBV 特异性抗体反应的失调程度比以前认识到的更大。然而，T 细胞对 EBV 的反应更难辨别，但是用 9 种候选 CNS 自身抗原刺激 EBV 扩增的多克隆 T 细胞系显示出高度的自身反应性，并表明病毒诱导的 T 细胞区室对 CNS 造成损害的能力深远。
