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Logo of neurologyNeurologyAmerican Academy of Neurology
Neurology. 2008 September 23; 71(13): 1033–1035.
PMCID: PMC2676958


J D. Lünemann, MD, P Huppke, MD, S Roberts, BSc, W Brück, MD, J Gärtner, MD, and C Münz, PhD

Epidemiologic studies suggest that childhood viral exposures are important determinants of multiple sclerosis (MS) risk in both adults and children1 and, so far, Epstein-Barr virus (EBV) stands out as the infectious agent for which there is the most compelling evidence for an association with the disease.1–3

Patients, healthy donors, and methods.

Twenty-three children with a diagnosis of definite MS and 17 demographically matched healthy peers were enrolled at the Department of Pediatrics and Pediatric Neurology, University of Göttingen, Germany (table e-1 on the Neurology® Web site at Virus-specific antibody responses were assessed using standardized ELISA kits (Bio-Quant, San Diego, CA) for IgG antibodies directed against the EBV- encoded viral capsid antigen (EBV-VCA), the HCMV-derived early antigens (HCMV-EA), the immunogenic C-terminus of the latent EBV nuclear antigen-1 (EBNA1, aa 458-641), and toward EBV-infected B cell lysates.4 In order to assess the specificity of EBNA1-targeting IgG, 211 dodecamer peptides, overlapping by nine amino acids and covering the entire sequence of EBNA1 (aa 1-641), were covalently linked to cellulose membranes as described previously.5 Positive spots were identified as blue or purple. Individual membranes were processed and evaluated blinded to the clinical diagnosis.


A total of 91% and 87% of MS cases compared to 64% and 58% of controls showed IgG responses toward EBV-VCA and EBNA1, respectively. The frequency of HCMV-EA seropositivity did not differ between both cohorts. IgG titers to EBV-VCA, EBV-infected B cell lysates which predominantly contain lytic EBV antigens,6 and to HCMV-EA did not significantly differ between the cohorts (figure, A). By contrast, children with MS showed a moderate but significant increase of EBNA1-specific IgG1 antibody titers (p = 0.02) (figure, B). The difference in EBNA1 recognition between patients and controls was preserved after eliminating the samples derived from treated patients (8/23) from our analysis (p = 0.03). These data suggest a primary deregulation of EBNA1-, but not generally of EBV-specific immune responses in pediatric MS.

figure znl0370858350001
Figure Broadened and elevated humoral immune response to EBNA1 in pediatric MS

We next determined target epitopes of EBNA1-specific IgG in 10 patients and 10 controls, in whom IgG responses to EBNA1 were detectable by ELISA (figure, C). The overall titer of EBNA1-specific IgG antibodies, including all IgG isotypes, did not statistically differ between these subgroups (mean IgG titer ± SEM in MS vs HD: 1,024 ± 152 vs 711 ± 106, p = 0.15). Antibody specificities were primarily directed toward the glycine-alanine repeat domain of EBNA1 (aa 88-323). In addition, pediatric MS sera bound to distinct epitopes which were not recognized by any of the control sera. However, the broadened recognition of EBNA1-specific IgG was not restricted to a distinct part of the antigen. As shown in the figure, D, patients recognized a higher number of epitopes within all three domains of the protein (p < 0.0001). Although we cannot completely exclude that the broadening of the antibody response resulted from the elevated IgG1 titers, we consider this explanation unlikely, because the overall immunoglobulin levels for EBNA1 were not increased in this subgroup of patients. Instead, our data point toward an upregulated and qualitatively distinct immune recognition of EBNA1 in children with MS.


EBNA1 is the only EBV antigen consistently expressed in proliferating EBV-infected B cells of healthy virus carriers. Increased titers of EBNA1-specifc antibodies were found to be associated with various autoimmune diseases including SLE and MS1 as well as more recently with pediatric SLE and pediatric MS.2 Cepok et al.7 reported that the two most frequent MS-specific and high affinity epitopes, recognized by CSF-derived oligoclonal IgG in MS, are both derived from EBV, i.e., EBNA1 and BRRF2, suggesting that EBNA1-specific antibodies are not only systemically elevated in MS, but also enriched in the CSF.

EBNA1 represents a key target antigen for CD4+ T cell-mediated immune control of latent EBV infection and we suggest that the observed quantitatively and qualitatively altered IgG1 responses to EBNA1 in pediatric patients with MS reflect the finding that adult patients show increased frequencies and broadened specificities of EBNA1-specifc CD4+ T helper 1 cells.3 Increased availability of EBNA1 protein due to higher frequencies of EBV-infected and potentially autoreactive B cells in MS or continuous cross-stimulation of EBNA1-specific T cells by a disease-relevant autoantigen could trigger a selective increase of EBNA1-targeting immune responses in pediatric and adult MS.

We conclude that the increase and broadened recognition of EBNA1-specific IgG suggests an early dysregulation of EBV specific immune control in pediatric MS development. Further studies are necessary to elucidate the mechanisms by which altered EBV and in particular EBNA1-specific immune responses could potentially contribute to the early pathobiology of MS.

Supplementary Material

[Data Supplement]


Supplemental data at

J.D.L. is a recipient of the Dana Foundation and Irvington Institute’s Human Immunology Fellowship provided by the Cancer Research Institute and is supported by a Pilot Grant from the National Multiple Sclerosis Society (PP1145) as well as by an Institutional Clinical and Translational Science Pilot and Collaborative Project Grant (to the Rockefeller University Hospital). C.M. is supported by the Dana Foundation’s Neuroimmunology program, the Arnold and Mabel Beckman Foundation, the Alexandrine and Alexander Sinsheimer Foundation, the Burroughs Wellcome Fund, the National Cancer Institute (R01CA108609 and R01CA101741), the National Institute of Allergy and Infectious diseases (RFP-NIH-NIAID-DAIDS-BAA-06-19), the Foundation for the NIH (Grand Challenges in Global Health), and an Institutional Clinical and Translational Science Award (to the Rockefeller University Hospital).

Disclosure: The authors report no disclosures.

Received February 19, 2008. Accepted in final form May 5, 2008.

Address correspondence and reprint requests to Dr. Christian Münz, Laboratory of Viral Immunobiology, Christopher H. Browne Center for Immunology and Immune Diseases, Rockefeller University, Box 390, 1230 York Avenue, New York, NY 10021-6399; ude.rellefekcor@cznum


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3. Lünemann JD, Edwards N, Muraro PA, et al. Increased frequency and broadened specificity of latent EBV nuclear antigen-1-specific T cells in multiple sclerosis. Brain 2006;129:1493–1506. [PubMed]
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