Double-stranded RNA Antiviral Signature in Early Multiple Sclerosis

Vasileios Gouzouasis^1,2Ioannis Sarrigeorgiou³Margaritis Tsifintaris¹Nikos Markoglou⁴Urania Georgopoulou⁵Peggy Lymberi³Maria Anagnostouli⁴Lesley Probert²Antonis Giannakakis^1*

• ¹Department of Molecular Biology & Genetics, Democritus University of Thrace, Alexandroupolis, Greece
• ²Laboratory of Molecular Genetics, Department of Immunology, Hellenic Pasteur Institute, Athens, Greece
• ³Laboratory of Immunology, Department of Immunology, Hellenic Pasteur Institute, Athens, Greece
• ⁴Multiple Sclerosis and Demyelinating Diseases Unit, 1st Department of Neurology, School of Medicine, National and Kapodistrian University of Athens, NKUA, Aeginition University Hospital, Athens, Greece
• ⁵Laboratory of Molecular Virology, Department of Microbiology, Hellenic Pasteur Institute, Athens, Greece

Viruses, particularly Epstein–Barr virus (EBV), are strongly implicated in multiple sclerosis (MS) pathogenesis, yet reliable biomarkers of active viral replication remain limited. Here, we developed a sandwich ELISA to quantify the levels of double-stranded RNA (dsRNA), the hallmark of viral replication, in matched plasma and cerebrospinal fluid (CSF) samples from 70 treatment-naïve MS patients at first clinical onset and plasma from 26 sex-and age-matched healthy controls. Plasma dsRNA levels were significantly elevated in MS patients compared to control samples and positively correlated with plasma antiviral cytokines, including GM-CSF, IFN-λ1, IFN-λ2/3, IFN-γ, IFN-α2, and IL-12p70. CSF samples also showed elevated levels of IP-10 and IL-8, and one patient positive for dsRNA in the CSF presented amongst the highest concentrations of both CSF cytokines. Notably, a subset of ten patients (14%) with serological evidence of atypical EBV reactivation (EBNA1 IgG⁺/IgM⁺) exhibited higher plasma dsRNA and antiviral cytokine levels compared to all other patients. We also developed an indirect ELISA to measure levels of anti-dsRNA antibodies, using a poly(I:C) synthetic dsRNA analogue target. Levels of anti-dsRNA IgM antibodies, not IgG nor IgA, correlated positively with dsRNA levels in the plasma of MS patients and controls. However, anti-dsRNA IgM antibody levels were significantly reduced in MS patients compared to healthy controls. Our results identify increased plasma viral dsRNA coupled with reduced anti-dsRNA IgM antibody levels as potential biomarkers for a subpopulation of early MS patients and indicate a dysregulated anti-viral immune response.