
==== Front
Eur J Neurol
Eur J Neurol
10.1111/(ISSN)1468-1331
ENE
European Journal of Neurology
1351-5101
1468-1331
John Wiley and Sons Inc. Hoboken

39023088
10.1111/ene.16387
ENE16387
EJoN-24-0375.R1
Original Article
Multiple Sclerosis
Rubella virus seropositivity after infection or vaccination as a risk factor for multiple sclerosis
Rubella virus seropositivity and MS
Ingvarsson et al.
Ingvarsson Jens https://orcid.org/0009-0003-4261-9434
1 jens.ingvarsson@umu.se

Grut Viktor https://orcid.org/0000-0002-5415-6567
1
Biström Martin https://orcid.org/0000-0003-3994-2305
1
Berg Linn Persson 2 3
Stridh Pernilla 4 5
Huang Jesse 4
Hillert Jan 4
Alfredsson Lars 4 6
Kockum Ingrid 4
Olsson Tomas 4 5
Waterboer Tim 7
Nilsson Staffan 8
Sundström Peter 1
1 Department of Clinical Sciences, Neurosciences Umeå University Umeå Sweden
2 Department of Infectious Diseases, Institute of Biomedicine University of Gothenburg Gothenburg Sweden
3 Department of Clinical Microbiology Sahlgrenska University Hospital Gothenburg Sweden
4 Department of Clinical Neuroscience Karolinska Institutet Stockholm Sweden
5 Center for Molecular Medicine Karolinska University Hospital Stockholm Sweden
6 Institute of Environmental Medicine Karolinska Institutet Stockholm Sweden
7 Division of Infections and Cancer Epidemiology German Cancer Research Center Heidelberg Germany
8 Department of Laboratory Medicine, Institute of Biomedicine University of Gothenburg Gothenburg Sweden
* Correspondence
Jens Ingvarsson, Department of Clinical Sciences, Neurosciences, Umeå University, Umeå, Sweden.
Email: jens.ingvarsson@umu.se

18 7 2024
10 2024
31 10 10.1111/ene.v31.10 e1638727 5 2024
23 2 2024
31 5 2024
© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

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.

the Neuro foundationForskningsrådet om Hälsa, Arbetsliv och Välfärd 10.13039/501100006636 2015‐00195 2017‐00687 Hjärnfonden 10.13039/501100003792 FO2020‐0077 Margaretha af Ugglas foundationHorizon 2020 MultipleMS733161 OskarfondenNEURO SwedenVetenskapsrådet 10.13039/501100004359 2015‐02419 2016‐02349 2020‐01998 521‐2012‐2917 Research and Development unit Region Jämtland Härjedalen source-schema-version-number2.0
cover-dateOctober 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:20.09.2024
Ingvarsson J , Grut V , Biström M , et al. Rubella virus seropositivity after infection or vaccination as a risk factor for multiple sclerosis. Eur J Neurol. 2024;31 :e16387. doi:10.1111/ene.16387
==== Body
pmcBACKGROUND

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS), characterised by multifocal inflammation and progressive neurodegeneration [1]. The symptomatic burden is often mild to moderate at presentation, although severe impairment of physical, cognitive and socioemotional function may eventually develop if effective disease‐modifying treatment is not implemented [2, 3]. A comprehensive description of the aetiopathogenesis remains to be presented, but it likely involves a dysregulated immune response towards intrinsic structures, including myelin antigens, engaging both adaptive and innate immune cells [1, 4, 5]. In addition, most people with MS (pwMS) have intrathecal production of heterogeneous antibodies, targeting patient‐specific peptide antigens, further strengthening the view of MS as an immune‐mediated disease [6]. Hereditary predisposition increases the risk of MS by 19%, and risk alleles and single nucleotide polymorphisms (SNPs) revealed through genome‐wide association studies (GWAS) explain up to 39% of heritability [5]. Among the most important genetic factors are human leukocyte antigen (HLA) genotypes HLA‐DRB1*15:01, HLA‐B*07 and the absence of HLA‐A*02, which have been associated with increased risk of MS. [7, 8] Environmental factors such as smoking, vitamin D and certain viral infections are also involved in disease development [9, 10]. Such viruses include Epstein–Barr virus (EBV), now firmly established as a risk factor for MS, and human herpesvirus 6A (HHV‐6A), which has been associated with MS risk in some studies [11, 12, 13]. Previous studies have also reported that cytomegalovirus (CMV) infection is associated with a reduced risk of developing MS. [12, 14] In addition, up to 78% of pwMS have intrathecal antibodies towards at least two of measles virus, rubella virus (RV) or varicella‐zoster virus (VZV) [15].

RV typically causes mild disease. However, infection during pregnancy may spread via the placenta, causing congenital infection with risk of miscarriage or congenital rubella syndrome (CRS), with findings such as congenital heart disease, sensorineural hearing impairment and microcephaly in newborns [16]. In addition, CRS has been associated with an increased risk of autoimmune thyroiditis and diabetes [17, 18]. Rubella complications include acute post‐infectious encephalitis and the rare condition progressive rubella panencephalitis (PRPE), which can develop months to years after infection [16]. The pathophysiology of PRPE is unknown, but the absence of RV in the CNS combined with high intrathecal anti‐RV‐antibody titres has led to the hypothesis that the condition is immune‐mediated [16, 19].

The RV has two envelope proteins: E1 and E2. Neutralising antibodies are mainly developed against E1, the viral attachment protein seemingly responsible for entry into the host cell. The biological function of E2 is largely unknown, and the protein remains shielded in the virion in vivo, but has one confirmed region that may elicit the production of neutralising antibodies [20, 21]. Myelin oligodendrocyte glycoprotein (MOG), primarily expressed on the surface of oligodendrocytes, is the only known host receptor for RV, suggesting the ability to infect these cells [22]. Moreover, RV and humans share at least 19 peptide epitopes, several of which are highly immunogenic [23]. One such epitope is present on MOG, containing homologous sequences to RV E2, which has been hypothesised to explain the mechanism behind demyelination seen in rat brain cell cultures treated with anti‐E2‐antibodies in vitro [24].

Because of CRS, vaccination programs against RV have been introduced [25]. The Swedish programme was started in 1974, when grade 6 (approximately 12 years of age) schoolgirls were offered monovalent vaccination with the live attenuated RV strain RA 27/3, reaching a coverage of approximately 84% in 1980 [26, 27, 28]. Simultaneously, pregnant women with low antibody titres of anti‐RV‐immunoglobulin G (IgG) were also offered monovalent vaccination after their pregnancy [29]. In 1982, the combined measles‐mumps‐rubella (MMR) vaccine replaced the monovalent version, consisting of the RA 27/3 strain, as well as the live attenuated Edmonston measles and Jeryl‐Lynn mumps strains. All children were offered vaccination at 18 months of age and during sixth grade of school. Of the children born in 1980, 57.1% received a dose of MMR vaccine at 18 months of age, whereas this number increased to 89.7% in children born in 1981, with coverage rates remaining between 88.5% and 97.4% up to 2015 [28]. Coverage for MMR vaccination in sixth grade was initially 88%, climbing to 92% in 1985. In the same year, seroconversion towards measles, mumps and RVs in MMR‐vaccinated cohorts were 100%, 98% and 100%, respectively [27].

The scope of this exploratory nested case–control study was to examine whether seropositivity towards RV E1 affects the risk of later developing MS and to study the effect of rubella vaccination upon said risk in a Swedish cohort.

METHODS

Study material

The material consisted of biobanked serum or plasma samples collected before the clinical onset of relapsing–remitting MS for 670 cases and 670 individually matched controls. The controls were matched with respect to sex, biobank and as closely as possible regarding both sampling date and age. A more detailed description of the case ascertainment process as well as information on matching accuracy is presented in previous studies [10, 11].

Participants were divided into two cohorts—vaccinated or unvaccinated against RV—based on the Swedish vaccination programme and dates of birth and sampling. Women born between 1962 and 1969 were considered vaccinated if their sample was collected after August in the year the participant turned 12 years of age, that is, if participants had started sixth grade of school, as the Swedish school year begins in the second half of August. Participants born between 1970 and 1979 were considered vaccinated if their sample was collected after they had started sixth grade. Participants born in 1980 onwards were considered vaccinated if their sample was collected after 18 months of age. All other participants were considered unvaccinated. If a case and its matched control were not sorted into the same cohort, both were excluded when calculating odds ratios (ORs), removing four discordant case–control pairs.

Epidemiological survey data

Data on the smoking habits of 455 cases was available, 421 of which originated from data collected in the following Swedish studies: Epidemiological Investigation of MS (EIMS), Genes and Environment in MS (GEMS) and Immunomodulation and Multiple Sclerosis Epidemiology (IMSE) [30, 31]. Additional data for 34 cases and 206 controls not included in these studies was collected through a complementary survey assessing smoking habits.

Laboratory analyses and HLA data

Samples were analysed for antibodies towards RV, EBV, CMV, HHV‐6A, as well as herpes simplex virus type 1 (HSV1), herpes simplex type 2 (HSV2) and VZV at the German Cancer Research Center, Heidelberg, Germany using previously described assays [32, 33]. Dilution of serum was 1:1000. Antigen E1 was used for RV, glycoprotein G (gG) for HSV1 and membrane glycoprotein G unique (mgG unique) for HSV2. Antigens for VZV were glycoproteins E (gE) and I (gI). Previously defined cut‐off values in median fluorescence intensity (MFI) units were used to determine serostatus towards these antigens [32, 33]. Antigens and definitions of serostatus for EBV, HHV‐6A and CMV, as well as the analysis of vitamin D levels in the sample material, have been described elsewhere [10, 11, 14]. The previously determined cut‐off for vitamin D3 (1st–4th quintile vs. 5th quintile) was used when adjusting for vitamin D in the present study [10].

Imputed genotypes for HLA‐DRB1*15:01, HLA‐A*02 and HLA‐B*07 were available for 493 cases, retrieved from EIMS, GEMS or IMSE. The imputation methods have been described previously [5]. In short, participants were genotyped on a custom array designed to densely cover the HLA, and 1967 SNPs were used for HLA imputation using the HLA*IMP software [34]. Saliva samples from 37 additional cases and 207 controls were retrieved using Oragene DNA OG‐500 (DNA Genotek, Ottawa, Ontario, Canada). DNA was extracted from 1–4 mL saliva, using the standard procedure of prepIT®‐L2P and solubilised in Tris‐EDTA buffer. Concentration and purity were determined using NanoDrop. Alleles were determined for HLA‐A*02 and HLA‐B*07 using tag SNPs with the TaqMan method (Applied Biosystems Inc., Foster City, CA, USA) but could not be determined for HLA‐DRB1*15:01 due to low resolution [35]. TaqMan Real‐Time polymerase chain reaction (PCR) probes are detailed in Table S1. This resulted in successful genotyping for an additional 24 cases and 160 controls concerning HLA‐A*02 and 28 cases and 185 controls for HLA‐B*07.

Statistics

Odds ratios (ORs) for developing MS dependent on RV E1, HSV1, HSV2 or VZV serostatus with 95% confidence intervals (CIs) were calculated through conditional logistic regression analysis, stratified by vaccination in univariate and multivariate analysis. In the latter, EBV, CMV and HHV‐6A serostatus and level of vitamin D3 were used as covariates. Fisher's test was used to compare the prevalence of HLA‐DRB1*15:01, HLA‐A*02 and HLA‐B*07 carriers (homozygote or heterozygote) and smoking (ever smoker) between RV E1 seropositive and seronegative cases. A comparison of sample age and age at MS onset between cohorts was performed using the Mann–Whitney U test. Linear mixed models were used to compare antibody reactivity towards RV E1, HSV1, HSV2 and VZV seropositive cases compared with seropositive controls for each virus, respectively. All statistical analyses were performed with IBM SPSS statistics version 28.0.

Ethics statement

This project was approved by the Regional Ethical Review Board in Umeå (2011–198‐31 M) with subsequent amendments and conducted in accordance with the Declaration of Helsinki. All participants were informed via letter and had the possibility of opting out. Study participants who underwent genetic testing signed a written consent.

RESULTS

The prevalence of RV E1 seropositive individuals was higher in cases (93.7%) compared with controls (88.5%, p < 0.001; Table 1). Seropositivity towards RV E1 was associated with a higher risk of MS in the unvaccinated cohort (OR = 3.0, 95% CI 1.4 to 6.4, p = 0.004) and in the vaccinated cohort (OR = 1.7, 95% CI 1.0 to 2.8, p = 0.037; Table 2). When adjusting for EBV, CMV and HHV‐6A serostatus, and levels of vitamin D, the association was statistically significant in the unvaccinated cohort (OR = 4.0, 95% CI 1.8 to 8.8, p < 0.001) but not in the vaccinated cohort (OR = 1.7, 95% CI 1.0 to 2.9, p = 0.056). No significant difference between the effect sizes in the two cohorts was found however. When comparing RV E1 seropositive and seronegative cases, no significant differences in the frequency of HLA‐DRB1*15:01, HLA‐A*02, HLA‐B*07 or smoking were found.

TABLE 1 Characteristics of cases and controls.

Cohort	Statistic	Cases	Controls	
All	n	670	670	
Female/male sex, %	84/16	84/16	
Age at sampling, years	25 (21–29)	25 (21–29)	
Age at disease onset, years	33 (28–40)	n/a	
Time from sampling until disease onset, years	8 (4–13)	n/a	
Rubella virus seropositivity, %	93.7	88.5	
Anti‐RV E1 reactivity for seropositive, MFI	172	148	
Unvaccinated	n	224	224	
Female/male sex, %	75/25	75/25	
Age at sampling, years	28 (22–33)	28 (22–33)	
Age at disease onset, years	40 (33–44)	n/a	
Time from sampling until disease onset, years	11 (5–16)	n/a	
Rubella virus seropositivity, %	93.8	85.7	
Anti‐RV E1 reactivity for seropositive, MFI	220	181	
Vaccinated	n	446	446	
Female/male sex, %	88.6/11.4	88.3/11.7	
Age at sampling, years	24 (20–28)	24 (20–28)	
Age at disease onset, years	31 (26–36)	n/a	
Time from sampling until disease onset, years	6 (3–11)	n/a	
Rubella virus seropositivity, %	93.7	89.9	
Anti‐RV E1 reactivity for seropositive, MFI	151	135	
Note: Unvaccinated = presumed not to have received rubella vaccination. Vaccinated = presumed to have received rubella vaccination. Proportions expressed as percentage value; age and time variables expressed as median (interquartile range); antibody reactivity expressed as geometric mean; n expressed as count.

Abbreviations: E1, envelope protein 1; NA, not applicable; MFI, median fluorescence intensity; RV, rubella virus.

TABLE 2 Rubella virus E1 seropositivity associations with risk of MS.

	n, RV E1 seropositive/total	Univariate	Multivariate	
Cohort	Cases	Controls	OR	95% CI	p	OR*	95% CI	p	
All	628/670	593/670	2.1	1.4–3.1	<0.001	2.2	1.4–3.3	<0.001	
Unvaccinated	208/222	190/222	3.0	1.4–6.4	0.004	4.0	1.8–8.8	<0.001	
Vaccinated	416/444	399/444	1.7	1.0–2.8	0.037	1.7	1.0–2.9	0.056	
Note: Unvaccinated = presumed to not have received rubella vaccination. Vaccinated = presumed to have received rubella vaccination. *OR adjusted for serostatus towards Epstein–Barr virus, cytomegalovirus, human herpesvirus 6A and vitamin D levels.

Abbreviations: CI, confidence interval; E1, envelope protein 1; n, number of participants, expressed as count; OR, odds ratio; RV, rubella virus; total, total number of analysed participants per cohort.

Median ages at sampling and MS onset were significantly higher, and time from sampling to onset was significantly longer in the unvaccinated compared with the vaccinated cohort (p < 0.001 for each test; Table 1). Geometric mean reactivity towards RV E1 was significantly higher for rubella seropositive cases compared with seropositive controls within the unvaccinated cohort (220 vs. 181 MFI, p = 0.039), the vaccinated cohort (151 vs. 135 MFI, p = 0.026) and in seropositive individuals in the unvaccinated compared with the vaccinated cohort (201 vs. 143 [range 31–3877] MFI, p < 0.001). There was no significant difference in antibody reactivity towards HSV1, HSV2 or VZV antigens when comparing cases and controls seropositive towards the respective virus (Table S2). No significant association between seropositivity for HSV1, HSV2 or VZV and MS risk was found in multivariate analysis adjusted as described earlier (Table S3).

DISCUSSION

In this exploratory nested case–control study in a Swedish cohort of biobank samples collected before the clinical onset of MS, cases had higher RV E1 seroreactivity than controls. No such differences were observed for HSV1, HSV2 or VZV. Additionally, seropositivity towards RV E1 was associated with a higher risk of developing MS. This finding was driven by, and in the multivariate analysis only significant in, the unvaccinated cohort, suggesting that rubella infection may be a risk factor for MS. In the vaccinated cohort, the findings were only borderline significant in the multivariate analysis adjusted for vitamin D status and serostatus towards EBV, HHV‐6A and CMV. We further set out to adjust for HLA genotype and smoking. However, due to insufficient quantity of data for controls, these factors could not be included in the multivariate analysis. As a next‐best approach, we compared HLA genotypes and smoking exposure in RV E1 seropositive and seronegative cases. We found similar prevalences with no significant differences in this regard, arguing against any major influence from these risk factors.

With samples from both the pre‐vaccination era and through the different stages of rubella vaccination in Sweden, we explored a unique opportunity to study RV in the field of MS. To the best of our knowledge, this is the first report of an association between RV serostatus and MS in pre‐MS samples. Our findings corroborate a previous retrospective case–control study where questionnaires and serology testing were used to compare the frequency of RV infection in persons with demyelinating disease—including optical neuritis, isolated demyelinating lesions and MS—compared with controls [36]. In that study, concerning subjects unvaccinated towards RV, cases had significantly higher titres of anti‐RV antibodies than controls, and an association was found between RV infection at age >6 years and demyelinating disease; however, no difference in infection frequency was reported [36]. Two previous questionnaire‐based studies showed that RV infection was more common in MS cases than in controls, and one of these studies found more convincing associations when RV infection occurred at a later age [37, 38]. From our data, the age at rubella infection cannot be derived, but the sampling age was higher in the unvaccinated cohort, and the median age at infection could possibly be higher in this cohort compared with vaccinated subjects. Still, age at sampling did not differ between matched cases and controls within cohorts, arguing against such a confounding effect on the result. On the contrary, two other retrospective, population‐based studies found no significant associations between MS and previous rubella, measles, mumps or varicella infection, based on questionnaires or school records [39, 40].

Viruses may elicit autoimmune reactions e.g. through molecular mimicry with host epitopes, or cause local cell destruction, risking the presentation of self‐antigens from destroyed host cells and initialising an autoimmune response through bystander activation [41, 42]. Both these mechanisms are hypothetically plausible for RV. Infection could lead to an autoimmune CNS response via molecular mimicry due to epitope sharing (e.g., homology between MOG and RV E2). Interestingly, around 50% of pwMS display a potent T‐cell reactivity against MOG [43]. Due to the virus' presumed ability to infect oligodendrocytes, bystander activation is another plausible mechanism for autoimmunity after RV infection.

With a sensitivity of 57.6%, the assay used may lead to an underestimation of the seroprevalence. However, a study with sample material from several European countries reported that the prevalence of pre‐vaccination immunisation towards rubella in 20–30‐year‐olds ranged between approximately 80% and 90% [44]. This is well in line with the seroprevalence for RV E1 in the controls of the unvaccinated cohort, suggesting that the number of false‐negatives in our study may be lower than implied by the sensitivity reported in the assay's validation study. Furthermore, the assay's largest strength lies in its high specificity (91.7%), minimising risk of overestimation of the seroprevalence. Our results should, however, still be interpreted with care due to the sensitivity reported in the validation study.

An argument against the specificity of findings in serological MS case–control studies is that pwMS may have inherently increased immune activity in general. This might create a stronger immunisation response in cases compared with controls, explaining findings like the significantly higher geometric mean anti‐RV E1 antibody reactivity in RV seropositive cases compared with seropositive controls in both the vaccinated and the unvaccinated cohort in this study. The recognition of the role of EBV in MS may have been delayed because of concerns such as these, together with the ubiquitousness of EBV infections, the high rate of positive serology in controls, and the possibility that latent EBV may be reactivated due to immune alterations naturally occurring in MS. Such arguments can also be applied for RV. For EBV, both the prevalence of seropositivity and the seroreactivity are increased in MS, but other pieces of evidence, such as the epidemiological association between MS and infectious mononucleosis and the association between EBV antibodies and neurofilament elevation during the MS prodrome, strongly suggest that EBV does indeed play a central part in MS aetiopathogenesis [12, 45]. Regarding the present study, the RV assay has been validated, and the findings in the unvaccinated cohort are clearly significant. The epidemiological support for the RV association is, however, two‐sided, arguing both for and against our findings [36, 37, 38, 39, 40]. In addition, if pwMS tend to develop more forceful antibody responses than healthy controls, this should arguably also affect antibody reactivity towards antigens other than RV E1. The lack of differences between MS cases and controls in the geometric mean reactivity towards HSV1, HSV2 and VZV argues against such an inherent predisposition to explain our results. Still, cases may both have been exposed to RV to a larger extent and exhibit a different response to immunisation towards RV compared with controls, and such a response may be dependent on age at immunisation, as suggested in the case of EBV [11]. One explanation for higher antibody reactivity in unvaccinated cases compared with vaccinated counterparts might be that RV infection has been shown to cause a stronger immunisation response than RV vaccination [46].

Interestingly, in a previous study on antibody decay after vaccination, seropositivity towards EBV was associated with faster decay of serum IgG antibodies towards RV, whereas CMV seropositivity was associated with slower such decay [47]. Since EBV infection is a risk factor for MS, and conversely CMV infection may be a protective factor, this argues against RV seropositivity being only an epiphenomenon secondary to EBV seroreactivity.

We also report a borderline significant, potentially controversial, association between RV E1 seropositivity and MS in participants presumed to have received rubella vaccination. These results contradict most previous studies on rubella and MMR vaccination in MS, though we note that one study found associations between early, but not late, MMR vaccination and MS. [39, 48] Although no significant association was found in multivariate analysis in our study, a tendency was implied, and we found no significant difference in effect size between the cohorts. Thus, we can neither support nor forcefully refute a possible association between RV vaccination and MS based solely on our results.

With the possible exception of some women who may have been offered vaccination after pregnancy, as this information was unavailable, participants in the unvaccinated cohort were not included in any vaccination programmes. Thus, this cohort most likely exclusively consists of truly unvaccinated subjects. In contrast, the vaccination status of participants in the vaccinated cohort is presumed since no information on the vaccination status of individual participants was available. Moreover, the school grade 6 cut‐off is not specific since, to the best of our knowledge, no available records specify when the vaccinations were performed during the school year. Since the same method for dividing participants into vaccination cohorts was used on the whole sample, the potential errors should, however, not be biased towards cases or controls. One may hypothesise that cases or controls were either more or less prone to accept vaccination or to a higher or lower extent were pregnant at some point, possibly affecting the extent of vaccination. Such tendencies would, however, have to be extraordinarily widespread to explain the results in their entirety.

Furthermore, the vaccinated cohort might contain subjects seropositive towards RV due to pre‐vaccination rubella infection. In this cohort, children could have contracted rubella infection earlier in life, since vaccination was performed at approximately 12 years of age for many vaccinated subjects, and infection typically occurs in early childhood [16]. We do not know if participants are seropositive due to vaccination or a possible combination of early infection and later vaccination, which could possibly explain these tendencies. Finally, most rubella‐vaccinated subjects received the combined MMR vaccination, introducing further variables that we at this stage cannot adjust for, in part since this sample is too small to divide into vaccination subcohorts without introducing too great an error due to lack of power.

To conclude, seroreactivity towards RV, but not HSV1, HSV2 or VZV, was higher in cases than in healthy controls, and seropositivity towards the RV E1 antigen in a cohort very likely consisting of unvaccinated subjects (i.e., representing rubella infection) was associated with a higher risk of developing MS. This suggests a broadening of the virus hypothesis in MS aetiology. Although this study may imply rubella infection as a risk factor for MS, our results should be interpreted with caution due to the low sensitivity of our method for defining seropositivity. Further serological and epidemiological studies are needed to support our findings. Molecular mimicry is an attractive hypothesis for a possible mechanism behind these findings, given the homology of an E2 peptide with a MOG sequence [49]. As for vaccinated subjects, although a similar tendency as in unvaccinated counterparts was implied, no significant association between RV E1 seropositivity and MS risk was shown. Rubella and MMR vaccination, as well as the respective viral infections, need to be further studied to elucidate or further refute any such associations.

FUNDING INFORMATION

This project was funded by the Swedish Research Council (grant number 2015‐02419). V.G. was supported by the Research and Development Unit, Region Jämtland Härjedalen, by Oskarfonden, and by NEURO Sweden. P.St. was supported in part by Margaretha af Ugglas foundation, the Neuro Foundation, and Horizon 2020 MultipleMS (grant number 733161). L.A. received grants from the Swedish Research Council (grant numbers 521‐2012‐2917, 2016‐02349 and 2020‐01998), grants from the Swedish Research Council for Health Working Life and Welfare (grant numbers 2015‐00195 and 2017‐00687) and grants from the Swedish Brain Foundation (FO2020‐0077) during the conduct of the study. J.Hu. and I.K. were supported by Horizon 2020 MultipleMS (grant number 733161).

CONFLICT OF INTEREST STATEMENT

J.I., V.G., S.N., P.St., L.P.B., M.B., T.W., L.A. and J.Hu. report no disclosures. P.Su. serves as an unpaid consultant for Moderna and has received lecture honoraria from Merck. T.O. has received advisory board or lecture honoraria from Biogen, Novartis, Merck and Sanofi. The same companies have provided unrestricted multiple sclerosis research grants. J.Hi. declares research grants outside of this study from Biogen, Bristol‐Myers‐Squibb, Janssen, Merck KGaA, Novartis, Roche and Sanofi‐Genzyme, and speaker's fees or fees for serving on advisory boards from Biogen, Bristol‐Myers‐Squibb, Janssen, Merck KGaA, Novartis, Sandoz, Sanofi‐Genzyme and Teva. I.K. has received lecture honoraria from Merck and has a collaborative research grant from Neurogene Inc.

Supporting information

Appendix S1.

ACKNOWLEDGEMENTS

We thank the Swedish multiple sclerosis registry, the study participants, and the biobank staff who collected the serum samples. The staff at Biobanken Norr is gratefully acknowledged for DNA extraction and the handling of saliva samples. We further extend our thanks to Maria Nyström and Niklas Sörlén for the handling of biobank samples locally.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.
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