Lytic Epstein-Barr virus infection and emerging autoantibodies precede multiple sclerosis prodrome

Epstein-Barr virus (EBV) is a critical risk factor for multiple sclerosis (MS), but the pathogenic mechanisms remain elusive. To clarify its role in MS, we examined the sequence of EBV-related antibody responses over a broad period before the clinical onset of MS, from early childhood to middle age. A nested case-control study was performed by linking Swedish MS registries with biobanks to identify pre-symptomatic samples from individuals who later developed MS and matched controls. Leveraging these samples, we analysed the immune responses to EBV in and before the prodromal phase of MS. Antibodies against EBV (viral capsid antigen, VCA; EBV nuclear antigen 1, EBNA1; early antigen-diffuse, EA-D) and the putative autoantibody target Anoctamin 2 (ANO2) were quantified by immunoassay. Serum neurofilament light chain (S-NfL), a marker of axonal injury, was quantified by single-molecule array. Ratios of these markers were calculated within each matched case-control set, plotted against time to the clinical onset of MS, and analysed with Loess regression to visualise the temporal order of events on group level. Samples from 981 cases and 1278 controls were included. Median age at blood sampling was 22 years and 32% of the participants were children or adolescents. The median time from the blood sample to the clinical onset of MS was 9 years. On the group level, we observed the following sequence of events: Seroreactivity against lytic EBV antigens (VCA-IgG, VCA-IgM, EA-D) - characterizing primary EBV infection and reactivation - were significantly higher in cases than in controls more than 20 years before MS onset and onwards. Increased seroreactivity against the latent antigen EBNA1 was observed 15 years before onset, followed by increased ANO2 seroreactivity from 9 years and onwards. Finally, a significant elevation of S-NfL was observed from 7 years prior to MS onset. These findings demonstrate a more extensive lytic EBV infection in the decades before MS onset, consistent with the hypothesis that EBV is a driver of MS development. The sequence of increasing seroreactivity against EBNA1 and ANO2 was closely followed by biochemical signs of neuroaxonal injury, suggesting epitope spreading from EBNA1 before the subclinical onset of neuroaxonal damage. This sequence of events supports the hypothesis that EBV infection promotes autoreactive immune cells contributing to MS pathogenesis.