Antibodies from multiple sclerosis brain identified Epstein-Barr virus nuclear antigen 1 & 2 epitopes which are recognized by oligoclonal bands

Abstract

Background 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 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. Objectives To identify disease-relevant epitopes of IgG antibodies in MS. Methods 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. Results Two phage peptides were identified 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. Conclusions 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.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM

“…The peptides were gradient eluted into a Lumos Fusion Tribrid (ThermoFisher Scienti c) mass spectrometer using a 120min gradient from 5% to 35% solvent B (90% acetonitrile, 0.5% acetic acid), followed by 10 minutes from 35% to 45% solvent B and 10 min from 45 to 100% B. High resolution full MS spectra were acquired with a resolution of 120,000, an AGC target of 4x10 5 , a maximum ion time of 50 ms, and scan range of 400 to 1800 m/z. Following each full MS scan as many data-dependent HCD MS/MS spectra were acquired in the orbitrap as possible in a 3 second cycle time.…”

“…6). Anti-EBNA1-reactivity has been implicated in MS epidemiology 20 , and the region AA365-425 is the target of stronger antibody responses in MS than in healthy individuals 5,[9][10][11][12] .…”

“…Crystal structure reveals key residues of MS39p2w174-Fab / EBNA1 AA386-405 interaction While the presence of antibodies against the broader EBNA1 region AA365-425 has been described in MS 5,[9][10][11][12] , their relevance remains elusive. We solved the crystal structure of EBNA1 AA386-405 in complex with the MS39p2w174-Fab at a resolution of 2.5 Å (Fig.…”