Background: The pathogenesis of relapsing-remitting multiple sclerosis (RRMS) is linked to autoimmune attacks against myelin proteins, and reactivation of Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6). However, the connection between viral reactivation and autoimmune biomarkers has remained unclear. Objectives: To investigate immunoglobulin (Ig)G/IgA/IgM responses targeting myelin-related proteins in association with EBV and HHV-6 replication markers in RRMS. Methods: We recruited 55 patients with RRMS and 63 healthy controls and assessed IgG/IgA/IgM responses against seven myelin-related components, as well as EBV nuclear antigen 1 (EBNA-1) and deoxyuridine-triphosphate nucleotidohydrolase (dUTPases). Disability was evaluated using the Expanded Disability Status Scale (EDSS) and disease progression using the Multiple Sclerosis Severity Score (MSSS). Results: IgG/IgA/IgM levels targeting seven myelin-related proteins were significantly higher in RRMS than in controls. IgG against myelin basic protein (MBP) (IgG-MBP), IgM-myelin-associated glycoprotein (IgM-MAG)-37-60, IgA-MBP, and IgA-myelin-oligodendrocyte-glycoprotein (IgA-MOG-31-55) distinguished RRMS from controls with a predictive accuracy of 96.6% (sensitivity = 95.7%, specificity = 95.2%) and an area under the ROC curve of 0.991. A large part of the variance in the EDSS (around 75%) and MSSS score (62.8%) was explained by IgG-MBP, IgM-MBP, IgA-MOG-31-55, and IgM-MAG. Part of the variance (47.4%) in the IgG/IgA/IgM responses to myelin-related proteins was explained by immune responses to EBNA and deoxyuridine-triphosphate nucleotidohydrolases of EBV and HHV-6. Conclusions: Autoimmune reactivities targeting myelin-related proteins are valuable biomarkers of RRMS and the severity and progression of RRMS. Reactivation of EBV and HHV-6 may trigger or maintain these autoimmune responses thereby impacting disease progression.

The authors have declared no competing interest.

AFA received funding for the project from the C2F program at Chulalongkorn University in Thailand, with grant number 64.310/436/2565. The Thailand Science Research, and Innovation Fund at Chulalongkorn University (HEA663000016) and the Sompoch Endowment Fund (Faculty of Medicine) MDCU (RA66/016) provided funding to MM. Immunosciences Lab., Inc., Los Angeles, CA, USA, and Cyrex Labs, LLC, Phoenix, AZ, USA, provided funding for the execution of all antibody assays.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Ethics Committee of the College of Medical Technology at the Islamic University of Najaf, Iraq, granted sanction for the investigation (Document No. 11/2021). Written informed consent was obtained from all patients and control participants, and all procedures were conducted in accordance with Iraqi and international ethical standards.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The corresponding author (MM) is available to provide access to the dataset related to this study upon receiving a valid request and following a thorough review of the data.