Absence of Epstein-Barr virus in the brain and CSF of patients with multiple sclerosis

Abstract

Objective: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that becomes latent in B-lymphocytes and has been implicated in the pathogenesis of multiple sclerosis (MS). We searched for latent and active EBV infection in MS brain and CSF.

Methods: Nested and non-nested real-time PCR were used to detect cell-specific and EBV-specific transcripts in 15 fresh-frozen and 5 formalin-fixed paraffin-embedded MS plaques and in single MS CSF B-lymphocytes and plasma cells. Intrathecal anti-EBV antibody synthesis was measured by ELISA. Immunocytochemistry was used to detect binding of MS CSF and recombinant antibodies (rAbs) generated from clonally expanded plasma cells in MS CSF to EBV-infected cells.

Results: No EBV RNA was found in MS CSF B-lymphocytes or plasma cells. In active MS plaques, EBV-encoded RNA (EBER)-1 was the only and rarely detected transcript. The frequency of detected intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease control patients. Anti-EBV antibodies were detected in the CSF of patients with MS, but MS rAbs did not react with EBV.

Conclusions: Application of real-time PCR to multiple sclerosis brain and single B-lymphocytes in CSF did not reveal any evidence of active Epstein-Barr virus infection.

Figure 1 Histology of active multiple sclerosis (MS) plaques used to screen for Epstein-Barr virus infection Luxol fast blue–stained white matter lesions of MS1987 (A), MS160 (B), MS2180 (C), and MS03-A1.C1 (D). MS03-A1.C1 stained with anti-CD68 antibody (E), anti-CD138 antibody (F), and anti-human-immunoglobulin G (IgG) antibody (G). Normal human brain white matter stained with anti-human IgG (H). Perivascular B-lymphocyte cuffs in chronic-active lesions of MS121 (I) and MS160 (J and K) stained with anti-CD20 antibody. An adjacent section of a lesion from MS160 stained with secondary antibody alone (L). Scale bars: A–D = 1 mm, E–H = 100 μm, I–L = 200 μm.

Figure 2 Anti–Epstein-Barr virus (EBV) antibody detection in multiple sclerosis (MS) CSF (A) Intrathecal anti-EBV antibody production in the CSF of MS and non-MS inflammatory disease control (IC) patients. The anti-EBV immunoglobulin G (IgG) antibody index (AI) was calculated based on 20 MS and 5 IC patients' CSF and plasma-serum: if Q_IgG < Q_Lim, then AI = Q_EBV/Q_IgG; otherwise, AI = Q_EBV/Q_Lim. The distribution of each patient's AI is presented relative to the diagonal line (AI = 1.5). Three of 20 MS and 2 of 5 IC patients demonstrated intrathecal anti-EBV IgG antibody synthesis (AI ≥1.5). The frequency of intrathecal anti-EBV antibody synthesis did not differ between MS and IC patients (p = 0.25, Fisher exact test). (B) EBV-infected B95–8 cells stained with MS CSF and with rAbs generated from clonally expanded plasma cells of MS CSF. B95–8 cells stained with mouse anti-EBV-gp125 antibody (a) and (f) followed by alkaline-phosphatase conjugated anti-mouse IgG; B95–8 cells stained with CSF of MS subjects MS02–19 (b), MS03–1 (c), MS04–2 (d) followed by alkaline phosphatase-conjugated anti-human IgG; B95–8 cells stained with alkaline phosphatase-conjugated anti-human IgG alone (e); B95–8 cells stained with MS recombinant antibodies (rAbs) MS02–19 rAb 11 (g), MS03–1 rAb 15 (h), MS04–2 rAb 13 (i) followed by mouse anti-flag IgG and alkaline phosphatase-conjugated anti-mouse IgG; B95–8 cells stained with mouse anti-flag IgG and alkaline phosphatase-conjugated anti-mouse IgG (j). Immunostainings of B95–8 cells with CSF or rAbs obtained from the same patient with MS are shown in pairs: (b) and (g) represent the CSF and rAb from MS02–19; (c) and (h) represent the CSF and rAb from MS03–1; and (d) and (i) represent the CSF and rAb from MS04–2. Scale = 100 μm.