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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 &amp; 2 (EBNA1 &amp; 2) epitopes were identified. Antibodies from sera and CSF from other MS patients also reacted to those epitopes.

Antibodies from Multiple Sclerosis Brain Identified Epstein-Barr Virus Nuclear Antigen 1 & 2 Epitopes which Are Recognized by Oligoclonal Bands