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EBNA-1 MS Biomarker

Epstein-Barr Virus Antibodies to Differentiate Multiple Sclerosis From Other Neuroinflammatory Diseases

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Year of Publication: 2026

Authors: Vietzen H, et al.

Journal: JAMA Neurology

Citation: JAMA Neurol. 2026;83(5):461-470. DOI: 10.1001/jamaneurol.2026.0240

Link: https://doi.org/10.1001/jamaneurol.2026.0240

Bottom Line

Persistent high-level EBNA-1 peptide antibodies across serial samples are a highly specific biomarker for MS versus MOGAD, NMOSD (including AQP4-seronegative cases), and other neuroinflammatory diseases, and may serve as a useful diagnostic adjunct.

Major Points

  • In the test cohort, 96.2% of MS patients had high-level EBNA-1 peptide titers in >=2 of 4 samples vs only 7.7% MOGAD (OR 303.4) and 18.0% NMOSD (OR 114.9).
  • In AQP4-seronegative NMOSD, only 11.1% had persistent high-level EBNA-1 peptide antibodies vs 96.7% of matched MS patients (OR 236.0, 95% CI 18.6-2588.0).
  • Findings were replicated in an independent validation cohort (MS 95.1% vs MOGAD 16.7% vs NMOSD 17.6%).
  • High-level titers in >=2 plasma samples outperformed CSF oligoclonal bands for indicating an MS diagnosis.
  • Kinetics analysis showed MS titers remain elevated over time while controls decline, and 4.6 months is sufficient interval between measurements.
  • MOGAD and NMOSD both showed transient declines in EBNA-1 peptide titers over follow-up, suggesting distinct immunopathogenesis from MS.

Design

Study Type: Retrospective multicenter case-control diagnostic biomarker study with independent validation cohort

Randomization:

Blinding: Not applicable (retrospective serological assay)

Enrollment Period: 2001 - 2023

Follow-up Duration: 2 years (mean sampling interval 13.2-13.9 months across 4 time points)

Centers: 3

Countries: Austria, Germany, USA

Sample Size: 4067

Analysis: Fisher exact test, chi-square, linear mixed models with random intercept, Sidak-adjusted pairwise comparisons

Inclusion Criteria

  • Diagnosis of relapsing-remitting MS per 2017 McDonald criteria
  • OR MOGAD diagnosis by live cell-based assay with MOG-IgG >=1:160; clear positive titers (>=1:640) required fulfillment of core clinical criteria alone, while low-positive titers (1:160-1:320) required both core and supporting clinical/imaging features
  • OR NMOSD diagnosis (AQP4-IgG positive by CBA, or double-seronegative for AQP4-IgG and MOG-IgG)
  • OR other inflammatory neurological disease (autoimmune/viral/bacterial encephalitis) as inflammatory controls
  • OR other noninflammatory neurological disease (headache, migraine, seizure, stroke) as controls
  • OR healthy blood donors without autoimmune/neuroinfectious/neuroinflammatory disease as healthy controls
  • EBV seropositive with longitudinal plasma samples available (for longitudinal analysis)
  • Recruited 2001-2023 at Medical University of Vienna, US Network of Pediatric MS Centers, or BIOMARKER study (Children's Hospital Datteln)

Exclusion Criteria

  • Development of autoimmune, neuroinfectious, or neuroinflammatory disease during follow-up (for healthy controls)
  • Insufficient longitudinal plasma samples for kinetics analysis
  • MOG-IgG below 1:160 (does not meet MOGAD positivity threshold), or low-positive MOG-IgG (1:160-1:320) without both core and supporting clinical/imaging features (for MOGAD group)
  • Missing baseline serology or clinical diagnostic data
  • Not meeting diagnostic criteria for assigned group

Baseline Characteristics

CharacteristicMS (n=1039)MOGAD (n=205)NMOSD (n=94)OIND (n=318)OND (n=715)Healthy Controls (n=1548)
Sex - Female50.0%36.1%86.1%55.7%53.8%38.4%
Sex - Male50.0%63.9%13.9%
Age, mean (range), y24.1 (4-72)32.7 (1-86)49.2 (14-89)30.1 (7-74)39.9 (6-90)42.2 (5-70)
Pediatric patients52.3%32.2%5.3%
EBV VCA IgG+92.1%61.0%80.9% (76/94)
EBV EBNA-1 IgG+92.1%83.6%77.9%78%
MOG IgG+100%
MOG IgG >=1:640100%
AQP4 IgG+87.2%

Arms

FieldMultiple Sclerosis (MS)ControlControlControlControlControl
InterventionSerial EBNA-1 peptide IgG measurementSerial EBNA-1 peptide IgG measurementSerial EBNA-1 peptide IgG measurementSerial EBNA-1 peptide IgG measurementSerial EBNA-1 peptide IgG measurementEBNA-1 peptide IgG measurement
DurationBaseline + 3 follow-up samples over ~2 yearsBaseline + 3 follow-up samplesBaseline + 3 follow-up samplesBaseline + 3 follow-up samplesBaseline + 3 follow-up samples1-2 samples

Outcomes

OutcomeTypeControlInterventionHR / OR / RRP-value
Diagnostic utility of persistent high-level EBNA-1 peptide antibody titers (>=2 of 4 samples) for MS vs MOGAD/NMOSD in test cohortPrimary<.001
MS vs AQP4-seronegative NMOSD (persistent high-level titers)Secondary236<.001
MS vs OIND (persistent high-level titers, test cohort)Secondary717.9<.001
MS vs OND (persistent high-level titers, test cohort)Secondary271.8<.001
Validation cohort: MS vs MOGADSecondary96.4<.001
Validation cohort: MS vs NMOSDSecondary90<.001
Validation cohort: MS vs MOGAD MOG-IgG 160-320 (low-positive)Secondary242.5<.001
Diagnostic accuracy MS vs MOGAD (test)SecondaryMOGADMSaccuracy 95.2%
Diagnostic accuracy MS vs NMOSD (test)SecondaryNMOSDMSaccuracy 92.7%
EBNA-1 titers exceed controls by 4.6 months post-baseline (kinetics)SecondaryHealthyMS<.05 (Sidak)

Criticisms

  • Retrospective case-control design; prospective validation cohorts needed to confirm diagnostic performance
  • A few MS patients (6 total) had consistently nondetectable EBNA-1 peptide antibodies, with no identifiable clinical or treatment features distinguishing them
  • Discovery cohort had relatively low female proportion driven by heavy pediatric enrollment, though the validation cohort was more representative of adult-onset RRMS
  • Race and ethnicity data not collected due to European legal/privacy restrictions, limiting generalizability across populations
  • AQP4-seronegative NMOSD subgroup was small (n=9-12), limiting precision of the key MS vs AQP4-negative NMOSD comparison despite highly significant results
  • Assay is not standardized/commercially available; requires the specific EBNA-1 381-452 peptide ELISA with established optical density cutoff (>=1.7)
  • Cannot replace current standard diagnostic techniques (McDonald criteria, MOG-IgG, AQP4-IgG); positioned as adjunct only

Subgroup Analysis

Findings consistent across adult and pediatric patients, and across both sexes; also robust in MS-RIS, MS-CIS, and MS with low MOG-IgG (<1:160) subgroups in validation cohort; Fabry disease patients with MS-mimicking white-matter lesions did not show persistent high-level EBNA-1 titers

Funding

Center for Virology and Department of Neurology, Medical University of Vienna; Austrian Science Fund SYNABS I6565-B; Austrian Society of Neurology; Austrian MS Society; National MS Society (RG150705285, HC0165); NIH (HC150606233, R01NS071463)

Based on: EBNA-1 MS Biomarker (JAMA Neurology, 2026)

Authors: Vietzen H, et al.

Citation: JAMA Neurol. 2026;83(5):461-470. DOI: 10.1001/jamaneurol.2026.0240

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