Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis
Sabine Cepok, Dun Zhou, Rajneesh Srivastava, Stefan Nessler, Susanne Stei, Konrad Büssow, Norbert Sommer, Bernhard Hemmer
Sabine Cepok, Dun Zhou, Rajneesh Srivastava, Stefan Nessler, Susanne Stei, Konrad Büssow, Norbert Sommer, Bernhard Hemmer
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Article Neuroscience

Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis

Abstract

MS is a chronic inflammatory and demyelinating disease of the CNS with as yet unknown etiology. A hallmark of this disease is the occurrence of oligoclonal IgG antibodies in the cerebrospinal fluid (CSF). To assess the specificity of these antibodies, we screened protein expression arrays containing 37,000 tagged proteins. The 2 most frequent MS-specific reactivities were further mapped to identify the underlying high-affinity epitopes. In both cases, we identified peptide sequences derived from EBV proteins expressed in latently infected cells. Immunoreactivities to these EBV proteins, BRRF2 and EBNA-1, were significantly higher in the serum and CSF of MS patients than in those of control donors. Oligoclonal CSF IgG from MS patients specifically bound both EBV proteins. Also, CD8+ T cell responses to latent EBV proteins were higher in MS patients than in controls. In summary, these findings demonstrate an increased immune response to EBV in MS patients, which suggests that the virus plays an important role in the pathogenesis of disease.

Authors

Sabine Cepok, Dun Zhou, Rajneesh Srivastava, Stefan Nessler, Susanne Stei, Konrad Büssow, Norbert Sommer, Bernhard Hemmer

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Identification of the CSF IgG-binding epitope. (A) Peptide scan analysis...
Identification of the CSF IgG-binding epitope. (A) Peptide scan analysis with 13-mer peptides that overlapped 11 AAs, covering the entire sequence of protein B3 of pattern I (upper membrane) and protein H5 of pattern II (lower membrane), was used to define the epitopes. Membranes were incubated with CSF (in 1:100 dilution) from MS patients immunoreactive to B3 or H5. Binding of IgG was visualized by anti-human IgG-HRP and TMB substrate. The minimal peptide epitopes were EPARSRSR for motif 1 and EAGAGGGA for motif 2. Similar results were obtained with CSF from 2 additional patients. (B) Substitution analysis was performed in order to define the optimal binding motif for the 8 AA epitopes defined in A. Binding of IgG was visualized by anti-human IgG-HRP and TMB substrate. A representative example for pattern I is shown. 1–8, the substituted AA-positions of the minimal epitope; 1–20, the 20 naturally occurring acids A–Y; *original peptide sequence. Similar results were obtained with 2 additional CSF samples from MS patients. (C) Definitions of 2 consensus motifs were based on the epitope mapping in 3 MS patients. These motifs were used to search the Swiss-Prot database. Database searching revealed 10 proteins matching with motif 1 and 13 proteins with motif 2. Two identified EBV proteins and the genomic locations according to http://www.ncbi.nlm.nih.gov are displayed. (D) Qualitative comparison of CSF IgG binding to peptides matching motif 1 (left) and motif 2 (right). Antibody binding was quantified by gel densitometry (highest signal and integrated density), which revealed the strongest binding to the 2 EBV epitopes BRRF2 and EBNA-1. The analysis was performed with similar results in 2 additional patients.