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.

Figure 1

Analysis of CSF IgG immunoreactivity in MS patients by protein expression arrays. (A) Incubation of the protein expression array with CSF from a representative MS patient (left) and a control donor (right). A 3 cm × 3 cm section of the 24 cm × 24 cm array is shown. IgG immunoreacitivity of the MS CSF to the expression clone B3 (spotted in duplicate) is marked by a circle. IgG concentration was adjusted to 1 mg/l IgG in MS and control CSF. (B) Western blot with purified protein B3. Immunoractivity was observed in the CSF of a representative MS patient (left) but not the NIND (middle) or OIND (right) control donors. All CSF samples were adjusted to 10 mg/l IgG. IgG binding was developed with ECL. M, molecular weight marker. (C) Analysis of immunoreactivity to B3 protein (left) and control protein GAPDH (right) with CSF (1:5 diluted) of 132 MS patients and 125 NIND patients by ELISA. Antibody titers were significantly higher in MS patients. Dot points represent the OD of a single CSF sample. Cut-off point, OD > 0.3 (mean ± 6 SEM). (D) CSF from a patient with high immunoreacitivty to patterns I and II proteins was separated by IEF and blotted against proteins B3, C6, C5, F4, and G4 as well as the control protein GAPDH. Binding of proteins from both patterns was similar with little overlap between the patterns. Similar results were obtained with CSF from another patient (data not shown). (E) IEF immunoblot with CSF (C) and serum (S) from 3 MS patients (adjusted to 10 mg IgG/l) was performed to compare IgG binding patterns to proteins B3, C5, and G4. A stronger and more focused immune response to the proteins was observed in the CSF of 10 patients analyzed in total. *P < 0.001, Fisher’s exact test.

Figure 2

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.

Figure 3

Expression and immunoreactivity to EBNA-1 and BRRF2. (A) BRRF2 and EBNA-1 RNA transcripts were detected in the B95 cell line (B95) and the EBV-transformed B cell line (BC) by BRRF2- (61 bp) and EBNA-1–specific (107 bp) RT-PCR. To exclude contamination with residual DNA, a control sample without reverse transcription (no RT) was included in the experiment. (B). Expression of full-length and partial BRRF2 RNA in the B95 cell line verified by RT-PCR. (C) BRRF2 expression in E. coli. The BRRF2 protein was cloned as partial (16 kDa) and full-length (58 kDa) protein with a 30-kDa GST tag resulting in 46-kDa partial and 88-kDa full-length band on SDS-PAGE (SDS) after Coomassie staining (left). Western blot (WB) and immunostaining with CSF of a control donor (Ctr; middle) and of a representative MS patient (right) confirmed the specific binding of CSF IgG to the BRRF2 proteins. (D) Immunoreactivity to recombinant BRRF2 (upper panels) and EBNA-1 (lower panels) was investigated by ELISA in CSF (left, 1:5 dilution) and serum (right, 1:100 dilution) of 130 MS patients compared with 115 NIND and 85 OIND patients. The immunoreactivities for each sample are given as OD values. Mean ODs and P values comparing the extent of immunoreactivity by Student’s t test are displayed above each group. Fisher’s exact test was applied to compare the frequencies of reactive patients: *Significant compared with NIND patients; ^#significant compared with OIND patients. RT, reverse transcription.

Figure 4

Specific intrathecal IgG response to EBV proteins. (A) Intrathecal IgG response to BRRF2 proteins in MS patients and control donors detected by ELISA. CSF and serum were adjusted to 10 mg/l IgG and the ratio of OD CSF/OD serum determined. Ratios above 1.2 indicate intrathecal synthesis. MS patients showed intrathecal BRRF2-specific IgG synthesis more frequently than controls. (B) IEF-immunoblot demonstrating specific binding of CSF oligoclonal IgG bands from 2 MS patients to the EBV proteins. The membranes were coated with BRRF2, EBNA-1, or a solution containing 10% milk alone as indicated. Affinity-blotted IgG was detected with anti-human IgG-HRP and visualized by TMB substrate. (C) IEF-immunoblot for BRRF2-specific OCBs (right) and total OCB pattern (left). Detection of bound IgG was performed as described in B. BRRF2-specific OCBs correspond to some of the major bands in the OCB pattern of the MS patient. (D) Loss of OCBs by preabsorption with EBNA-1 but not GAPDH. 2D-electrophoresis and IgG immunoblot of CSF from a patient with EBNA-1 immunoreactivity. No, no preabsorption of CSF-IgG was performed with EBNA-1 or GAPDH. (E) Solution phase assays demonstrate high affinity and specificity of CSF antibodies to EBNA-1 (left) and BRRF2 (right). Soluble EBV proteins at different dilutions were incubated with CSF and, subsequently, the remaining immunoreactivity measured by ELISA. Competition by soluble antigens is displayed. No competition was measured with GAPDH protein. For each assay, 4 MS patients were analyzed. *Fisher's exact probability test.

Figure 5

Frequency and phenotype of EBV-specific T cells in MS patients and healthy donors. (A) Strategy for detection of EBV-specific T cells in PBMC samples by intracellular IFN-γ staining and flow cytometry. PBMCs and autologous EBV-transformed B cell lines were either cultured separately (nonspecific activation, left graph) or in short-term coculture (EBV-specific activation, right graph) before staining. Shown are analyses of CD8⁺ T cells in the CD28⁺ and CD28^– compartments of an MS patient. One percent of CD8⁺CD28⁺ T cells were EBV-specific in this patient. (B) Frequency of EBV-specific CD4⁺ and CD8⁺ T cells in 11 MS patients and 14 healthy donors (HD). No significant differences were observed in the CD4⁺ T cell compartments, whereas a higher frequency of EBV-specific CD8⁺ T cells was observed in MS patients. Further characterization revealed a significantly higher frequency of EBV-specific CD8⁺CD28⁺ in MS patients compared with healthy controls. Student’s t test was applied.