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Background and Objectives Myasthenia gravis (MG), an autoimmune disease characterized by fluctuating muscle weakness, is believed to result from complex gene-environment interactions, yet few risk factors have been identified. The objective of this study was to determine the effect of nicotine and alcohol on MG disease risk.

Methods The Genes and Environment in Myasthenia Gravis study is a Swedish, nationwide cross-sectional case-control study where prevalent patients with MG were invited to submit an extensive questionnaire on lifestyle and environment. Data collection took place between November 2018 and August 2019, and cases were matched by sex and year of birth to population controls. Year of disease onset was used as index year. Associations between use of alcohol, tobacco smoke, Swedish snuff, and MG risk were investigated using multivariable logistic regression.

Results A total of 1,067 patients with MG (mean age at onset 48 (SD 21) years, 53% female) were matched to 2,087 controls. Any alcohol consumption was associated with a lower MG risk compared with not drinking at all (odds ratio [OR] 0.48, 95% CI 0.39-0.59, p < 0.001, exposed cases n = 616). Effects were observed in a similar direction across disease subtypes, with the strongest association in the late-onset MG group (onset ≥50 years). Although neither cigarette smoke nor use of Swedish snuff affected the disease risk of the whole group, subset specific effects were observed. Smoking at onset was associated with an increased risk of early-onset MG (EOMG, onset 18-49 years; OR 1.60, 95% CI 1.17-2.20, p = 0.003, n = 133), which was accentuated in acetylcholine receptor antibody-positive EOMG (OR 2.08, 95% CI 1.34-3.25, p = 0.001, n = 74). Use of Swedish snuff, which contains high levels of nicotine, at disease onset was also associated with an increased risk of EOMG (OR 1.61, 95% CI 1.02-2.54, p = 0.039, n = 43).

Discussion We observed an inve rse correlation of MG risk and alcohol consumption. Furthermore, smoking and the use of Swedish snuff at disease onset were positively associated with EOMG. We recognize limitations related to retrospective data and limited number of available controls. However, multiple sensitivity analyses were performed supporting the robustness of our results.

Background: The placebo-controlled RINOMAX trial (NCT02950155) demonstrated superiority up to 12 months of rituximab over standard-of-care in new-onset generalized myasthenia gravis (MG), but benefit–risk over longer time frames remains unknown.

Methods: RINOMAX included 47 participants with a Quantitative Myasthenia Gravis (QMG) score ≥ 6. Twenty-five patients were randomized to a single intravenous infusion of 500 mg rituximab, and 22 to placebo of which 16 received rituximab after the double-blinded phase (7 ± 2.9 months). Data were extracted from the Swedish MG registry to track hospitalizations, treatments including rescue, and disease activity scores.

Results: Compared to the placebo arm, lower mean time-weighted QMG scores at 12 months (mean difference [MD]: 2.9, 95% CI: 0.9, 4.9; p = 0.005) and 24 months (MD: 2.6, 95% CI: 0.3, 4.9; p = 0.027) were observed in the RTX arm. The incidence rate of rescue from 48 weeks up to 5 years was numerically higher in the placebo arm than RTX (0.16 vs. 0.09/person-year; p = 0.121). Compared to delayed RTX, early exposure displayed lower QMG, risk of hospitalization (HR 0.24, 95% CI 0.07, 0.83), and rescue (HR 0.46, 95% CI 0.14, 1.57), but also the six patients never receiving RTX showed lower hospitalization risk (HR 0.08, 95% CI 0.01, 0.96). Corticosteroid doses were low globally throughout. Overall, 12.5% and 18.8% and of patients with early and delayed RTX, respectively, suffered a severe infection.

Conclusion: Disease activity and treatment burden, including hospitalization and rescue treatments, remained low, indicating a potential benefit of rituximab on the long-term disease trajectory. Infection risk with B cell depletion, however, remains a concern.

Epstein-Barr virus (EBV) infection has been advocated as a prerequisite for developing multiple sclerosis (MS) and possibly the propagation of the disease. However, the precise mechanisms for such influences are still unclear. A large-scale study investigating the host genetics of EBV serology and related clinical manifestations, such as infectious mononucleosis (IM), may help us better understand the role of EBV in MS pathogenesis. This study evaluates the host genetic factors that influence serological response against EBV and history of IM and cross-evaluates them with MS risk and genetic susceptibility in the Swedish population. Plasma IgG antibody levels against EBV nuclear antigen-1 [EBNA-1, truncated = amino acids (aa) (325-641), peptide = aa(385-420)] and viral capsid antigen p18 (VCAp18) were measured using bead-based multiplex serology for 8744 MS cases and 7229 population-matched control subjects. The MS risk association for high/low EBV antibody levels and history of IM was compared to relevant clinical measures along with sex, age at sampling, and associated HLA allele variants. Genome-wide and HLA allele association analyses were also performed to identify genetic risk factors for EBV antibody response and IM history. Higher antibody levels against VCAp18 [odds ratio (OR) = 1.74, 95% confidence interval (CI) = 1.60-1.88] and EBNA-1, particularly the peptide (OR = 3.13, 95% CI = 2.93-3.35), were associated with an increased risk for MS. The risk increased with higher anti-EBNA-1 IgG levels up to 12× the reference risk. We also identified several independent HLA haplotypes associated with EBV serology overlapping with known MS risk alleles (e.g. DRB1*15:01). Although there were several candidates, no variants outside the HLA region reached genome-wide significance. Cumulative HLA risk for anti-EBNA-1 IgG levels, particularly the peptide fragment, was strongly associated with MS. In contrast, the genetic risk for high anti-VCAp18 IgG levels was not as strongly associated with MS risk. IM history was not associated with class II HLA genes but negatively associated with A*02:01, which is protective against MS. Our findings emphasize that the risk association between anti-EBNA-1 IgG levels and MS may be partly due to overlapping HLA associations. Additionally, the increasing MS risk with increasing anti-EBNA-1 levels would be consistent with a pathogenic role of the EBNA-1 immune response, perhaps through molecular mimicry. Given that high anti-EBNA-1 antibodies may reflect a poorly controlled T-cell defence against the virus, our findings would be consistent with DRB1*15:01 being a poor class II antigen in the immune defence against EBV. Last, the difference in genetic control of IM supports the independent roles of EBNA-1 and IM in MS susceptibility.

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 the most common chronic demyelinating disease of the central nervous system and the major non-traumatic cause of permanent disability in young adults. Several migration studies have been performed over the years suggesting a pattern of higher disease disability in certain ethnic groups. To our knowledge, differences in disease progression in immigrants have not been studied in Sweden before. Thus, the aims of our study were to estimate the prevalence of multiple sclerosis among first-generation immigrants in the City of Malmö and to compare differences in disease severity with the native population.

Methods: All persons with multiple sclerosis living in Malmö on prevalence day 31 Dec 2010 were included. Cases were classified according to the country of birth into Scandinavians, Western and non-Western.

Results: The crude prevalence was 100/100,000 (95% CI, 80–124) among first-generation immigrants, 154/100,000 (95% CI, 137–173) among individuals with Scandinavian background, 123/100,000 (95% CI, 94–162) in the Western group and 76/100,000 (95% CI, 53–108) in the non-Western group. The mean Multiple Sclerosis Severity Score (MSSS) value among Scandinavians was 4.2 (SD 3.5), whereas the figures in the immigrant group were 4.6 (SD 3.3) and 5.2 (SD 3.7) among Westerns respectively non-Westerns, which differences were not statistically significant. When adjusting for gender, age at onset and initial disease course, the mean MSSS difference between the non-Western and the Scandinavian individuals was 1.7 (95% CI 0.18–3.3, p = 0.030). There were no differences on time to diagnosis or the time from diagnosis to treatment initiation between the three groups.

Conclusions: We found a lower prevalence among Western and non-Western first-generation immigrants compared to the Scandinavian population and a more severe disease in non-Western immigrants than in Scandinavians.

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.

Peripheral B cell depletion via anti-CD20 treatment is a highly effective disease-modifying treatment for reducing new relapses in multiple sclerosis (MS) patients. A drawback of rituximab (RTX) and other anti-CD20 antibodies is a poor immune response to vaccination. While this can be mitigated by treatment interruption of at least six months prior to vaccination, the timing to resume treatment while maintaining subsequent vaccine responses remains undetermined. Here, we characterized SARS-CoV-2 S-directed antibody and B cell responses throughout three BNT162b2 mRNA vaccine doses in RTX-treated MS patients, with the first two doses given during treatment interruption. We examined B-cell mediated immune responses in blood samples from patients with RTX-treated MS throughout three BNT162b2 vaccine doses, compared to an age- and sex-matched healthy control group. The first vaccine dose was given 1.3 years (median) after the last RTX infusion, the second dose one month after the first, and the third dose four weeks after treatment re-initiation. We analyzed SARS-CoV-2 S-directed antibody levels using enzyme-linked immunosorbent assay (ELISA), and the neutralization capacity of patient serum against SARS-CoV-2 S-pseudotyped lentivirus using luciferase reporter assay. In addition, we assessed switched memory (CD19⁺CD20⁺CD27⁺IgD^-), unswitched memory (CD19⁺CD20⁺CD27⁺IgD⁺), naïve (CD19⁺CD20⁺CD27^-IgD⁺), and double negative (DN, CD19⁺CD20⁺CD27^-IgD^-) B cell frequencies, as well as their SARS-CoV-2 S-specific (CoV⁺) and Decay Accelerating Factor-negative (DAF^-) subpopulations, using flow cytometry. After two vaccine doses, S-binding antibody levels and neutralization capacity in SARS-CoV-2-naïve MS patients were comparable to vaccinated healthy controls, albeit with greater variation. Higher antibody response levels and CoV⁺-DN B cell frequencies after the second vaccine dose were predictive of a boost effect after the third dose, even after re-initiation of rituximab treatment. MS patients also exhibited lower frequencies of DAF^- memory B cells, a suggested proxy for germinal centre activity, than control individuals. S-binding antibody levels in RTX-treated MS patients after two vaccine doses could help determine which individuals would need to move up their next vaccine booster dose or postpone their next RTX infusion. Our findings also offer first indications on the potential importance of antigenic stimulation of DN B cells and long-term impairment of germinal centre activity in rituximab-treated MS patients.

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.