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Epstein–Barr virus is now regarded as the critical risk factor for multiple sclerosis. However, Cytomegalovirus and human herpesvirus 6A have also been associated with altered multiple sclerosis risk, suggesting a multifactorial aetiology. Here, we present the first large-scale study of the association between human herpesvirus 7 and the risk of developing multiple sclerosis. A nested case-control study was performed by crosslinking Swedish registries and biobanks, identifying blood samples from 981 cases who later developed multiple sclerosis and 1278 matched controls. Serological testing was performed with a multiplex immunoassay. The association between viral serostatus and the risk of developing multiple sclerosis was analysed with conditional logistic regression, calculating an odds ratio with 95% confidence interval. Interactions between antibodies against human herpesvirus 7 and the Epstein–Barr virus nuclear antigen 1 regarding multiple sclerosis risk were analysed on the additive scale. Serological evidence of human herpesvirus 7 infection was associated with a higher risk of developing multiple sclerosis: odds ratio = 2.2 (95% confidence interval = 1.8–2.7), P < 0.001. The results remained similar when adjusting for cytomegalovirus, Epstein–Barr virus and human herpesvirus 6A serostatus. Synergistic interactions between human herpesvirus 7 and Epstein–Barr virus nuclear antigen 1 seroreactivity were observed: attributable proportion due to interaction = 0.51 (95% confidence interval = 0.34–0.68). These results suggest that human herpesvirus 7 could be a contributing factor in multiple sclerosis aetiology.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A.

A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI).

Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis).

In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Synergistic interactions between human herpesvirus 6A (HHV-6A) and Epstein–Barr virus (EBV) are hypothesized in the etiopathogenesis of multiple sclerosis (MS). This study investigated if HHV-6A and EBV seroreactivities interact regarding the risk of developing MS. Antibodies against viral antigens were analyzed in biobank samples from 670 individuals who later developed MS and matched controls. Additive interactions were analyzed. A significant interaction between HHV-6A and EBNA-1 seroreactivities was observed in study participants above the median age of 24.9 years (attributable proportion due to interaction = 0.45). This finding supports the hypothesis that HHV-6A and EBV infections interact in MS development. ANN NEUROL 2024.

Background: Multiple sclerosis (MS) is a demyelinating disease affecting millions of people worldwide. Hereditary susceptibility and environmental factors contribute to disease risk. Infection with Epstein–Barr virus (EBV) and human herpesvirus 6A (HHV-6A) have previously been associated with MS risk. Other neurotropic viruses, such as rubella virus (RV), are possible candidates in MS aetiopathogenesis, but previous results are limited and conflicting.

Methods: In this nested case–control study of biobank samples in a Swedish cohort, we analysed the serological response towards RV before the clinical onset of MS with a bead-based multiplex assay in subjects vaccinated and unvaccinated towards RV. The association between RV seropositivity and MS risk was analysed with conditional logistic regression.

Results: Seropositivity towards RV was associated with an increased risk of MS for unvaccinated subjects, even when adjusting for plausible confounders including EBV, HHV-6A, cytomegalovirus and vitamin D (adjusted odds ratio [AOR] = 4.0, 95% confidence interval [CI] 1.8–8.8). Cases also had stronger antibody reactivity towards rubella than controls, which was not seen for other neurotropic viruses such as herpes simplex or varicella zoster. Furthermore, we observed an association between RV seropositivity and MS in vaccinated subjects. However, this association was not significant when adjusting for the aforementioned confounders (AOR = 1.7, 95% CI 1.0–2.9).

Conclusions: To our knowledge, these are the first reported associations between early RV seropositivity and later MS development. This suggests a broadening of the virus hypothesis in MS aetiology, where molecular mimicry between rubella epitopes and human central nervous system molecules could be an attractive possible mechanism.

Background: Multiple sclerosis (MS) and presymptomatic axonal injury appear to develop only after an Epstein-Barr virus (EBV) infection. This association remains to be confirmed across a broad preclinical time range, for lytic and latent EBV seroreactivity, and for potential cross-reacting antigens.

Methods: We performed a case-control study with 669 individual serum samples obtained before clinical MS onset, identified through cross-linkage with the Swedish MS register. We assayed antibodies against EBV nuclear antigen 1 (EBNA1), viral capsid antigen p18, glycoprotein 350 (gp350), the potential cross-reacting protein anoctamin 2 (ANO2) and the level of sNfL, a marker of axonal injury.

Results: EBNA1 (latency) seroreactivity increased in the pre-MS group, at 15-20 years before clinical MS onset, followed by gp350 (lytic) seroreactivity (p=0.001-0.009), ANO2 seropositivity appeared shortly after EBNA1-seropositivity in 16.7% of pre-MS cases and 10.0% of controls (p=0.001). With an average lag of almost a decade after EBV, sNfL gradually increased, mainly in the increasing subgroup of seropositive pre-MS cases (p=8.10^-5 compared with non-MS controls). Seropositive pre-MS cases reached higher sNfL levels than seronegative pre-MS (p=0.038). In the EBNA1-seropositive pre-MS group, ANO2 seropositive cases had 26% higher sNfL level (p=0.0026).

Conclusions: Seroreactivity against latent and lytic EBV antigens, and in a subset ANO2, was detectable on average a decade before the appearance of a gradually increasing axonal injury occurring in the last decade before the onset of clinical MS. These findings strengthen the hypothesis of latent EBV involvement in the pathogenesis of MS.

Axonal loss is the main cause of irreversible disability in multiple sclerosis (MS). Serum neurofilament light (sNfL) is a biomarker of axonal disintegration. In this nested case–control study, blood samples from 519 presymptomatic persons (age range 4–39 years) who later received an MS diagnosis showed higher sNfL concentrations than 519 matched controls (p < 0.0001), noticeable at least 10 years before clinical MS onset. Mean values for pre-MS and control groups were 9.6 pg/mL versus 7.4 pg/mL 0–5 years before onset, and 6.4 pg/mL versus 5.8 pg/mL 5–10 years before onset. These results support that axonal injury occurs early in MS pathogenesis.

BACKGROUND AND PURPOSE: High levels of 25-hydroxyvitamin D₃ (25[OH]D₃ ) are associated with a lower risk for multiple sclerosis (MS). The bioavailability of 25(OH)D₃ is regulated by its main plasma carrier, vitamin D-binding protein (DBP). Free 25(OH)D₃ can be estimated by also measuring DBP concentration. In addition, DBP has immunomodulatory functions that may independently affect MS pathogenesis. No previous studies have assessed free 25(OH)D₃ or DBP in presymptomatically collected samples. This study was undertaken to assess free 25(OH)D₃ and DBP as risk factors for MS.

METHODS: A nested case-control study was performed with presymptomatic serum samples identified through cross-linkage of MS registries and Swedish biobanks. Concentration of 25(OH)D₃ was measured with liquid chromatography and DBP levels with sandwich immunoassay. Free 25(OH)D₃ was approximated as free vitamin D₃ index: (25[OH]D₃ /DBP) × 10³ . MS risk was analyzed by conditional logistic regression, calculating odds ratios (ORs) with 95% confidence intervals (CIs).

RESULTS: Serum samples from 660 pairs of matched cases and controls were included. At <20 years of age, high levels of free vitamin D₃ index were associated with a lower risk of MS (highest vs. lowest quintile: OR = 0.37, 95% CI = 0.15-0.91, p for trend across quintiles = 0.04). At age 30-39 years, high levels of DBP were associated with a lower MS risk (highest vs. lowest quintile: OR = 0.36, 95% CI = 0.15-0.85, p for trend = 0.02).

CONCLUSIONS: These findings support the hypothesis that high levels of free 25(OH)D₃ at a young age reduce the risk of MS later in life. They also implicate a role for DBP in MS etiology.

Background: C-reactive protein (CRP) is a marker of systemic inflammation. Increased levels of CRP in young persons have been suggested to decrease the risk of multiple sclerosis (MS).

Objectives: To assess CRP as a risk factor for MS.

Methods: Levels of CRP were measured with a high-sensitive immunoassay in biobank samples from 837 individuals who later developed MS and 984 matched controls. The risk of developing MS was analysed by conditional logistic regression on z-scored CRP values.

Results: Levels of CRP were not associated with MS risk.

Conclusions: We found no association between CRP levels and risk of MS development.

Background and purpose: Epstein–Barr virus (EBV) and human herpesvirus 6A (HHV-6A) are associated with increased risk of multiple sclerosis (MS). Conversely, infection with cytomegalovirus (CMV) has been suggested to reduce the risk of MS but supporting data from presymptomatic studies are lacking. Here, it was sought to increase the understanding of CMV in MS aetiology.

Methods: A nested case–control study was performed with presymptomatically collected blood samples identified through crosslinkage of MS registries and Swedish biobanks. Serological antibody response against CMV, EBV and HHV-6A was determined using a bead-based multiplex assay. Odds ratio (OR) with 95% confidence interval (CI) for CMV seropositivity as a risk factor for MS was calculated by conditional logistic regression and adjusted for EBV and HHV-6A seropositivity. Potential interactions on the additive scale were analysed by calculating the attributable proportion due to interaction (AP).

Results: Serum samples from 670 pairs of matched cases and controls were included. CMV seropositivity was associated with a reduced risk for MS (OR = 0.70, 95% CI 0.56–0.88, p = 0.003). Statistical interactions on the additive scale were observed between seronegativity for CMV and seropositivity against HHV-6A (AP 0.34, 95% CI 0.06–0.61) and EBV antigen EBNA-1 (amino acid 385–420) at age 20–39 years (AP 0.37, 95% CI 0.09–0.65).

Conclusions: Cytomegalovirus seropositivity is associated with a decreased risk for MS. The protective role for CMV infection in MS aetiology is further supported by the interactions between CMV seronegativity and EBV and HHV-6A seropositivity.

Background and purpose: Infections with human herpesvirus 6A (HHV‐6A) and Epstein–Barr virus (EBV) have been linked to multiple sclerosis (MS) development. For EBV, late infection has been proposed as a risk factor, but serological support is lacking. The objective of this study was to investigate how age affects the EBV and HHV‐6A associated risks of developing MS.

Methods:  In this nested case–control study, Swedish biobanks were accessed to find pre‐symptomatically collected blood samples from 670 individuals who later developed relapsing MS and 670 matched controls. A bead‐based multiplex assay was used to determine serological response against EBV and HHV‐6A. Conditional logistic regression was used to calculate odds ratios and 95% confidence intervals.

Results: Seropositivity against EBV exhibited a pattern where associations switched from a decreased risk of developing MS in the group below 20 years of age to an increased risk amongst individuals aged 20–29 and 30–39 years (p for trend 0.020). The age of transition was estimated to be 18.8 years. In contrast, HHV‐6A was associated with increased MS risk in all age groups (total cohort odds ratio 2.1, 95% confidence interval 1.6–2.7).

Conclusions: This study suggests EBV infection after adolescence and age independent HHV‐6A infection as risk factors for MS.