High-Level EBNA-1 Antibodies: A Precise Biomarker for Differentiating Multiple Sclerosis From MOGAD and NMOSD

High-Level EBNA-1 Antibodies: A Precise Biomarker for Differentiating Multiple Sclerosis From MOGAD and NMOSD

Highlight

High-level titers of antibodies against the Epstein-Barr nuclear antigen 1 (EBNA-1) peptide are found in over 95% of patients with Multiple Sclerosis (MS), but are significantly less common in patients with Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease (MOGAD) and Neuromyelitis Optica Spectrum Disorder (NMOSD).

In longitudinal assessments, persistent high-level EBNA-1 titers demonstrated an odds ratio of 303.4 for differentiating MS from MOGAD and 114.9 for differentiating MS from NMOSD.

The biomarker is particularly valuable for diagnosing AQP4-seronegative NMOSD, where diagnostic ambiguity is highest; only 11.1% of these patients showed high-level EBNA-1 titers compared to 96.7% of matched MS patients.

Background

The landscape of neuroinflammatory diseases has shifted dramatically over the last two decades. Once grouped under the broad umbrella of Multiple Sclerosis (MS), conditions such as Neuromyelitis Optica Spectrum Disorder (NMOSD) and Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease (MOGAD) are now recognized as distinct clinical and pathological entities. However, the clinical and radiological presentation of these diseases—optic neuritis, transverse myelitis, and brainstem lesions—often overlaps significantly, especially in the early stages of disease progression.

Accurate early diagnosis is paramount because the therapeutic strategies for these conditions are fundamentally different. For instance, several disease-modifying therapies (DMTs) used to treat MS, such as interferon-beta or natalizumab, can actually exacerbate NMOSD. While the discovery of aquaporin-4 (AQP4) and MOG antibodies has improved diagnostic specificity, a significant subset of patients remains seronegative for these markers, leaving clinicians in a diagnostic gray area.

Recent epidemiological and mechanistic research has solidified the link between Epstein-Barr Virus (EBV) infection and the development of MS. Specifically, antibodies targeting the EBNA-1 peptide have emerged as a potential marker of the dysregulated immune response characteristic of MS. This study, published in JAMA Neurology, investigates whether longitudinal measurements of EBNA-1 peptide antibodies can reliably distinguish MS from its clinical mimics.

Study Design

This investigation was a retrospective, multicenter, longitudinal, case-control study involving patients from Austria, Germany, and the United States. The researchers analyzed samples from two independent cohorts: a test cohort and a validation cohort. The primary objective was to assess the diagnostic utility of persistent EBNA-1 peptide antibody levels across multiple time points.

Participants and Methodology

The study included plasma samples from 2,091 patients with neuroinflammatory diseases (mean age 31.0 years; 54.4% female) and 1,976 healthy controls. Patients were recruited between 2001 and 2023 and were followed for a period of two years.

The test cohort consisted of 310 patients (184 with MS, 65 with MOGAD, and 61 with NMOSD). Notably, 12 of the NMOSD patients were seronegative for AQP4-IgG. The validation cohort included 183 patients (142 with MS, 24 with MOGAD, and 17 with NMOSD). Plasma EBNA-1 peptide immunoglobulin G (IgG) titers were measured using enzyme-linked immunosorbent assay (ELISA) at baseline (after diagnosis) and at three subsequent follow-up intervals.

Key Findings

The results of the study underscore the remarkable specificity of high-level EBNA-1 titers for Multiple Sclerosis compared to other neuroinflammatory conditions.

Distinguishing MS from MOGAD and NMOSD

In the test cohort, 177 of the 184 patients with MS (96.2%) maintained high-level EBNA-1 titers in at least two out of four follow-up samples. In contrast, only 5 patients (7.7%) with MOGAD and 11 patients (18.0%) with NMOSD exhibited these persistent high titers. The statistical strength of this differentiation was profound, with an odds ratio (OR) of 303.4 (95% CI, 94.4-908.6) for MS versus MOGAD and 114.9 (95% CI, 43.0-280.0) for MS versus NMOSD.

Addressing the Seronegative Challenge

One of the most clinically relevant findings involved the AQP4-seronegative NMOSD subgroup. Among these patients, who are often the most difficult to distinguish from MS based on imaging and clinical symptoms alone, only one individual (11.1%) had persistent high-level EBNA-1 titers. When compared to 61 matched patients with MS, the OR for differentiation was 236.0 (95% CI, 18.6-2588.0). This suggests that EBNA-1 testing could serve as a vital rule-out or rule-in tool when AQP4 and MOG antibody tests are negative.

Validation Results

The validation cohort confirmed these findings. In this group, 95.1% of patients with MS had persistent high-level titers, compared to 16.7% of patients with MOGAD (OR, 96.4) and 17.6% of patients with NMOSD (OR, 90.0). These consistent results across independent cohorts from different geographic regions enhance the generalizability of the findings.

Expert Commentary

The clinical utility of EBNA-1 peptide antibody titers lies in their ability to reflect the unique host-pathogen interaction that precedes or accompanies MS. While EBV infection is nearly ubiquitous in the general adult population, the specific high-titer response to the EBNA-1 peptide appears to be a hallmark of the MS disease process, likely linked to molecular mimicry or a specific failure in immune regulation.

From a clinical perspective, the persistent nature of these titers is a major advantage. Unlike some biomarkers that fluctuate wildly with disease activity or treatment, EBNA-1 titers remained stable over the two-year follow-up period in this study. This stability makes it a robust diagnostic marker regardless of when the patient is tested during the early course of their disease.

However, some limitations must be considered. While the specificity is high, the presence of high EBNA-1 titers is not 100% exclusive to MS, as a small percentage of NMOSD and MOGAD patients also exhibited elevated levels. Furthermore, as a retrospective study, prospective validation in real-world clinical settings—where clinicians are faced with undifferentiated first demyelinating events—is still necessary. The biological mechanism through which EBNA-1 contributes to MS pathology (whether as a driver or a bystander of immune dysregulation) also remains a subject of intense ongoing research.

Conclusion

This large-scale, longitudinal study provides compelling evidence that persistent high-level EBNA-1 peptide antibody titers are a reliable biomarker for differentiating Multiple Sclerosis from MOGAD and NMOSD. In an era where precision medicine is becoming the standard of care in neurology, such a biomarker could significantly reduce the time to accurate diagnosis, prevent the administration of potentially harmful treatments, and ensure that patients receive the most appropriate disease-modifying therapies as early as possible. For clinicians managing diagnostic uncertainty in neuroinflammatory diseases, the inclusion of EBNA-1 peptide antibody testing may soon become a critical component of the diagnostic workup.

References

Vietzen H, Kühner LM, Berger SM, et al. Epstein-Barr Virus Antibodies to Differentiate Multiple Sclerosis From Other Neuroinflammatory Diseases. JAMA Neurology. 2026; doi:10.1001/jamaneurol.2025.XXXX (Published March 9, 2026). PMID: 41801194.

Bittel AJ, et al. Longitudinal analysis of EBV antibodies in multiple sclerosis. Science. 2022;375(6578):296-301.

Wingerchuk DM, et al. International consensus diagnostic criteria for neuromyelitis optica spectrum disorder. Neurology. 2015;85(2):177-189.