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Epstein-Barr virus (EBV) infection is associated with MS; up to 3.3% people with MS are EBV nuclear antigen-1 (EBNA1)-seronegative compared with 6.0% controls.1 EBV serology is complex, and multiple antigens are required to assess seropositive status.2,3 We examined a cohort of seemingly EBV-negative patients with clinically isolated syndrome (CIS). The size of the population enrolled in the International CIS study allowed us to examine the largest population of seemingly EBV-negative patients with CIS gathered to date.

Methods.

The International CIS study is a collaborative study across 33 centers. Inclusion and exclusion criteria and methods for sample and data collection have previously been described.4 Immunoglobulin G (IgG) reactivity against EBNA1 was initially evaluated using commercially available ELISA (ETI-EBNA-G, Diasorin, Italy) according to the manufacturer's instructions.
Samples with anti–EBNA1-IgG reactivity less than the manufacturer's cutoff value (CoV) (<20 AU/mL) for the ETI-EBNA-G ELISA (screen-negative) were tested using well-validated in-house ELISAs based on multiepitope peptides of EBNA1 and virus capsid antigen (VCA) using previously described methods.5,6 The CoV for the in-house ELISA was the mean OD450 value of 4 truly EBNA1- and VCA-negative sera plus twice the SD (mean + 2 SD). OD450 values were normalized against the CoV, and values >1.0 were considered positive. Samples demonstrating negative or borderline (0.8–1.2) results against both EBV antigens were investigated using an EBV-specific immunoblot.5

Results.

Patient details are provided in the table. The CIS cohort has previously been described in detail.4 Forty-one (3.9%) of 1,047 patients were screen-negative. Age, sex, time to serum sampling and clinically definite MS (CDMS) and follow-up duration were not different to the whole cohort. Screen-negative patients were less likely to be CSF oligoclonal band (OCB)-positive (48.8% vs 74.3%; p = 0.0009, Fisher exact test) and less likely to be smokers (p = 0.01). Anti–cytomegalovirus (CMV)-IgG4–negative patients were not less likely to be OCB-negative (52% vs 48%).
Table Details of the 41 patients with CIS who had undetectable anti–EBNA1-IgG levels on initial screening
Of the screen-negative samples, 5/41 had reactivity <1.0 for anti–EBNA1-IgG and 2/41 <1.0 for anti–VCA-IgG and 2/41 had reactivity <1.0 against both EBV antigens. When values obtained from the 2 EBNA1 assays were compared, they demonstrated a correlation coefficient (rSpearman) of 0.57 after 3 outliers with reactivity <CoV of the ETI-EBNA-G ELISA, but high reactivity on the in-house ELISA were excluded (figure, A). A Bland-Altman plot demonstrated no evidence of systemic bias and reasonable agreement between the 2 assays (figure, B). Of the 2 samples with low reactivity to both EBV antigens, 1 showed no reactivity on immunoblot. This patient was OCB-positive and developed CDMS during follow-up.
Figure Scatter graph and Bland-Altman plot demonstrating correlation and agreement between the 2 EBNA1 assays
(A) Scatter graph demonstrating correlation between the 2 Epstein-Barr virus nuclear antigen-1 (EBNA1) assays. (B) Bland-Altman plot demonstrating reasonable agreement between the 2 assays. Given the different arbitrary scales used to report assay results, the in-house ELISA values have been multiplied by the ratio of the mean values of each ELISA to allow them to be reported on the same scale.

Discussion.

Only 1 of 1,047 patients (<0.01%) was truly EBV-negative. Previous literature demonstrates a strong association between MS and EBV infection; in keeping with this, 41 patients (3.9%) showed no EBNA1-reactivity on initial screen. More detailed testing revealed a much higher rate of EBV seropositivity in CIS and MS patients than previously described.
There is significant antigenic diversity of anti–EBV-IgG responses in EBV carriers and patients with EBV-related disease.2,3 Serologic testing against multiple antigens is recommended to accurately define EBV status when examining the link between EBV infection and other diseases. In studies examining MS risk and EBV infection, the rate of EBV positivity in the population is highly dependent on the method used to determine EBV serostatus.7
ELISA against 2 different EBV antigens provides a screening method; immunoblot on apparently seronegative samples increases sensitivity. The ETI-EBNA-G ELISA was validated for Food and Drug Administration's approval against a cell-based EBNA1-anticomplement immunofluorescence. The CoV for this ELISA is likely set relatively high, giving borderline values a negative interpretation. The in-house ELISA that we used utilizes the same peptide antigen, but was validated against purified EBNA1, increasing sensitivity and explaining the correlation between EBNA1 titers in the 2 ELISAs. Some EBNA1-IgG–negative sera are positive against other EBV antigens.
The fact that patients who were EBNA1-screen–negative were less likely to have OCBs provides an avenue for future research; there is likely a biological link between lower EBV immunoreactivity and OCB production. However, in our earlier study, while OCB predicted a second event, lower EBV reactivity did not4—the relationship therefore is more complex than a simple linear or threshold effect. EBNA1-seropositivity occurs later than VCA seroconversion; low titers of anti–EBNA1-IgG may reflect recent EBV infection which has not yet triggered downstream biological events. It may be that patients who are EBNA1-screen–negative have a lower overall level of immune response, resulting in negative OCBs. However, the same relationship is not seen in the CMV-seronegative population, hinting at an EBV-specific link.
With only 1 of 1,047 patients in our large international cohort testing negative for EBV across multiple antigens and 2 platforms, it seems that while it is possible to be truly EBV seronegative and develop MS, this is extremely rare. It seems likely that this indicates a role for EBV in MS development, and further research is needed to examine this further.

Coinvestigators

Files in this Data Supplement:
Coinvestigators - Microsoft Word file

Footnote

Author contributions: R.D. analyzed the data, performed the statistical analysis, drafted the manuscript, and produced the final version. J.K. led the international CIS study, arranged sample analysis, and collated all data prior to analysis. J.M. performed the EBV ELISA and immunoblot. G.G. conceived the study and provided intellectual guidance. All authors revised the draft manuscript and approved the final version.

Supplementary Material

File (supplementary_data_collaborators_v2.docx)

References

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Munger KL, Levin LI, O'Reilly EJ, Falk KI, Ascherio A. Anti-Epstein-Barr virus antibodies as serological markers of multiple sclerosis: a prospective study among United States military personnel. Mult Scler 2011;17:1185–1193.
2.
De Paschale M, Clerici P. Serological diagnosis of Epstein-Barr virus infection: problems and solutions. World J Virol 2012;1:31–43.
3.
Middeldorp J. Epstein-barr virus-specific humoral immune responses in health and disease. Curr Top Microbiol Immunol 2015;391:289–323.
4.
Kuhle J, Disanto G, Dobson R, et al. Conversion from clinically isolated syndrome to multiple sclerosis: a large multicentre study. Mult Scler 2015;21:1013–1024.
5.
de Sanjose S, Bosch R, Schouten T, et al. Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls. Int J Cancer 2007;121:1806–1812.
6.
Jafari N, van Nieropa GP, Verjans GMGM, Osterhaus ADME, Middeldorp JM, Hintzen RQ. No evidence for intrathecal IgG synthesis to Epstein Barr virus nuclear antigen-1 in multiple sclerosis. J Clin Virol 2010;49:26–31.
7.
Pakpoor J, Disanto G, Gerber JE, et al. The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis. Mult Scler 2013;19:162–166.

Information & Authors

Information

Published In

Neurology® Neuroimmunology & Neuroinflammation
Volume 4Number 2March 2017
PubMed: 28203615

Publication History

Received: October 4, 2016
Accepted: November 8, 2016
Published online: February 3, 2017
Published in print: March 2017

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Funding Information

Study funding: This work was supported by institutional funding and in part by the BMBF grant KKNMS (Competence Net Multiple Sclerosis) to H Tumani. The funder had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.Disclosure: R. Dobson received research support from Multiple Sclerosis Society of Great Britain and Northern Ireland. J. Kuhle's institution received research support from Swiss MS Society, Biogen, Novartis, Roche, Genzyme, and Merck Serono; he received research support from Bayer AG, Genzyme, Novartis, Roche Pharma (Schweiz) AG, Swiss National Research Foundation, ECTRIMS, University of Basel, and Swiss MS Society. J.M. Middeldorp serves on the editorial board of the Journal of Clinical Virology and Journal of Virological Methods; receives publishing royalties from Springer; is employed by Cyto-Barr BV; and consulted for Roche Diagnositcs and Argenx. G. Giovannoni served on the scientific advisory board of Biogen-Idec, FivePrime, Genzyme, GW Pharma, Ironwood, Merck Serono, Novartis, Roche, Sanofi-Aventis, Synthon BV, Teva, Vertex Pharmaceuticals, AbbVie, and Canbex; received speaker honoraria from Biogen-Idec, Genzyme, GW Pharma, Merck Serono, Novartis, Roche, and Teva; is an editor for Multiple Sclerosis and Related Disorders; consulted for Biogen-Idec, FivePrime, Genzyme, GW Pharma, Ironwood, Merck Serono, Novartis, Roche, Sanofi-Aventis, Synthon BV, Teva, Vertex Pharmaceuticals, Abbvie, and Canbex; is on the speakers' bureau for Novartis, and Teva; and received research support from Genzyme and Merck. Go to Neurology.org/nn for full disclosure forms. The Article Processing Charge was paid by Queen Mary University.

Authors

Affiliations & Disclosures

Ruth Dobson, PhD, MRCP
From the Blizard Institute (R.D., J.K., G.G.), Queen Mary University London; Department of Neurology (R.D.), St Georges Hospital, London, UK; Neurology, Departments of Medicine (J.K.), Biomedicine and Clinical Research, University Hospital Basel, Switzerland; and Department of Pathology (J.M.), VU University Medical Centre, Amsterdam.
Disclosure
Scientific Advisory Boards:
1.
NONE
Gifts:
1.
NONE
Funding for Travel or Speaker Honoraria:
1.
NONE
Editorial Boards:
1.
NONE
Patents:
1.
NONE
Publishing Royalties:
1.
NONE
Employment, Commercial Entity:
1.
(1) Multiple sclerosis society of Great Britain and Northern Ireland/Association of British Neurologists funded my previous position as a Clinical Research Fellow.
Consultancies:
1.
NONE
Speakers' Bureaus:
1.
NONE
Other Activities:
1.
NONE
Clinical Procedures or Imaging Studies:
1.
NONE
Research Support, Commercial Entities:
1.
NONE
Research Support, Government Entities:
1.
NONE
Research Support, Academic Entities:
1.
NONE
Research Support, Foundations and Societies:
1.
(1) Multiple sclerosis society of Great Britain and Northern Ireland/Association of British Neurologists funded my previous position as a Clinical Research Fellow.
Stock/stock Options/board of Directors Compensation:
1.
NONE
License Fee Payments, Technology or Inventions:
1.
NONE
Royalty Payments, Technology or Inventions:
1.
NONE
Stock/stock Options, Research Sponsor:
1.
NONE
Stock/stock Options, Medical Equipment & Materials:
1.
NONE
Legal Proceedings:
1.
NONE
Jens Kuhle, PhD
From the Blizard Institute (R.D., J.K., G.G.), Queen Mary University London; Department of Neurology (R.D.), St Georges Hospital, London, UK; Neurology, Departments of Medicine (J.K.), Biomedicine and Clinical Research, University Hospital Basel, Switzerland; and Department of Pathology (J.M.), VU University Medical Centre, Amsterdam.
Disclosure
Scientific Advisory Boards:
1.
NONE
Gifts:
1.
NONE
Funding for Travel or Speaker Honoraria:
1.
JK?s institution (University Hospital Basel) received in the last 3 years and used exclusively for research support: Speaker honoraria: (1) Swiss MS Society (2) Biogen (3) Novartis (4) Roche (5) Genzyme Travel expenses: (1) Merck Serono (2) Novartis (3) Roche
Editorial Boards:
1.
NONE
Patents:
1.
NONE
Publishing Royalties:
1.
NONE
Employment, Commercial Entity:
1.
NONE
Consultancies:
1.
NONE
Speakers' Bureaus:
1.
NONE
Other Activities:
1.
NONE
Clinical Procedures or Imaging Studies:
1.
NONE
Research Support, Commercial Entities:
1.
(1) Bayer AG (2) Genzyme (3) Novartis (4) Roche Pharma (Schweiz) AG
Research Support, Government Entities:
1.
(1) Swiss National Research Foundation
Research Support, Academic Entities:
1.
NONE
Research Support, Foundations and Societies:
1.
(1) ECTRIMS Research Fellowship Programme (2) University of Basel (3) Swiss MS Society
Stock/stock Options/board of Directors Compensation:
1.
NONE
License Fee Payments, Technology or Inventions:
1.
NONE
Royalty Payments, Technology or Inventions:
1.
NONE
Stock/stock Options, Research Sponsor:
1.
NONE
Stock/stock Options, Medical Equipment & Materials:
1.
NONE
Legal Proceedings:
1.
NONE
Jaap Middeldorp, PhD
From the Blizard Institute (R.D., J.K., G.G.), Queen Mary University London; Department of Neurology (R.D.), St Georges Hospital, London, UK; Neurology, Departments of Medicine (J.K.), Biomedicine and Clinical Research, University Hospital Basel, Switzerland; and Department of Pathology (J.M.), VU University Medical Centre, Amsterdam.
Disclosure
Scientific Advisory Boards:
1.
NONE
Gifts:
1.
NONE
Funding for Travel or Speaker Honoraria:
1.
NONE
Editorial Boards:
1.
editorial board member: J. Clinical Virology J. Virological Methods
Patents:
1.
NONE
Publishing Royalties:
1.
Current Topics in Microbiology and Immunology; Epstein- Barr virus, Volume 2,(2015). Publ. Springer
Employment, Commercial Entity:
1.
1. VU University medical center, Amsterdam, The Netherlands (Professor; 0.5 fte) 2. Cyto-Barr BV, Zuidhorn, The Netherlands (CEO)
Consultancies:
1.
Roche Diagnostics, Germany. Argen-x, Belgium
Speakers' Bureaus:
1.
NONE
Other Activities:
1.
NONE
Clinical Procedures or Imaging Studies:
1.
NONE
Research Support, Commercial Entities:
1.
NONE
Research Support, Government Entities:
1.
NONE
Research Support, Academic Entities:
1.
NONE
Research Support, Foundations and Societies:
1.
NONE
Stock/stock Options/board of Directors Compensation:
1.
NONE
License Fee Payments, Technology or Inventions:
1.
NONE
Royalty Payments, Technology or Inventions:
1.
NONE
Stock/stock Options, Research Sponsor:
1.
NONE
Stock/stock Options, Medical Equipment & Materials:
1.
NONE
Legal Proceedings:
1.
NONE
Gavin Giovannoni, PhD, FRCP
From the Blizard Institute (R.D., J.K., G.G.), Queen Mary University London; Department of Neurology (R.D.), St Georges Hospital, London, UK; Neurology, Departments of Medicine (J.K.), Biomedicine and Clinical Research, University Hospital Basel, Switzerland; and Department of Pathology (J.M.), VU University Medical Centre, Amsterdam.
Disclosure
Scientific Advisory Boards:
1.
(1)Biogen-Idec (2) Fiveprime (3) Genzyme (4) GW Pharma (5) Ironwood (6) Merck-Serono (7) Novartis (8) Roche (9) Sanofi-Aventis (10) Synthon BV (11) Teva (12) Vertex Pharmaceuticals (13) Abbvie (14) Canbex
Gifts:
1.
NONE
Funding for Travel or Speaker Honoraria:
1.
Speaker honoraria: (1)Biogen-Idec (2) Genzyme (3) GW Pharma (4) Merck-Serono (5) Novartis (6) Roche (7) Teva
Editorial Boards:
1.
(1) Multiple Sclerosis and Related Disorders, Editor since 2011
Patents:
1.
NONE
Publishing Royalties:
1.
NONE
Employment, Commercial Entity:
1.
NONE
Consultancies:
1.
(1)Biogen-Idec (2) Fiveprime (3) Genzyme (4) GW Pharma (5) Ironwood (6) Merck-Serono (7) Novartis (8) Roche (9) Sanofi-Aventis (10) Synthon BV (11) Teva (12) Vertex Pharmaceuticals (13) Abbvie (14) Canbex
Speakers' Bureaus:
1.
(1) Novartis (2) Teva
Other Activities:
1.
NONE
Clinical Procedures or Imaging Studies:
1.
NONE
Research Support, Commercial Entities:
1.
Genzyme 2012-2014 Merck 2012-2014
Research Support, Government Entities:
1.
NONE
Research Support, Academic Entities:
1.
NONE
Research Support, Foundations and Societies:
1.
NONE
Stock/stock Options/board of Directors Compensation:
1.
NONE
License Fee Payments, Technology or Inventions:
1.
NONE
Royalty Payments, Technology or Inventions:
1.
NONE
Stock/stock Options, Research Sponsor:
1.
NONE
Stock/stock Options, Medical Equipment & Materials:
1.
NONE
Legal Proceedings:
1.
NONE
On behalf of the International CIS study investigators

Notes

Correspondence to Dr. Dobson: [email protected]
Full list of investigators and centers can be found in the coinvestigators list at Neurology.org/nn.

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