Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis

Abstract 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.

with Single molecule array technology.The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for EBV 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).

Introduction
Multiple sclerosis is an immune-mediated chronic disease affecting the CNS. 1 According to the prevailing hypothesis, the disease is triggered by an interplay of environmental risk factors in individuals with genetic susceptibility to multiple sclerosis. 2Virtually all patients with multiple sclerosis are seropositive for Epstein-Barr virus (EBV), which has thus been suggested as a prerequisite for the disease in adults. 3,4However, the high seroprevalence of EBV in the healthy population shows that EBV is not a sufficient cause of multiple sclerosis.Additional environmental factors are likely contributing to its aetiology.One such candidate is human herpesvirus 6 (HHV-6), which has been repeatedly associated with multiple sclerosis risk. 57][8][9] As a neurotrophic virus, HHV-6A can infect oligodendrocytes. 10These cells produce myelin, often regarded as the target for the inflammatory processes in multiple sclerosis. 13] Higher levels of antibodies against HHV-6 have also been detected in serum and cerebrospinal fluid from patients with multiple sclerosis. 7,14The level of these antibodies is associated with the risk of relapse in multiple sclerosis. 15In addition, higher levels of HHV-6 DNA have been observed in blood plasma from patients with multiple sclerosis. 16Interestingly, these signs of reactivated HHV-6 infection were only observed during relapses or exacerbations of multiple sclerosis. 16,17ious research suggests that HHV-6 may be involved in the early stages of multiple sclerosis pathogenesis. 9,18The initiation and early phase of multiple sclerosis need to be better studied, but such investigations are complicated by the recently recognised prodrome. 19For example, two recent studies reported higher levels of serum neurofilament light chain (sNfL)a marker of axonal injuryin biobank samples from individuals who several years later developed clinical signs of multiple sclerosis. 20,21These ultra-sensitive analyses of sNfL have also been utilized to investigate the order of events in EBV seroconversion and the onset of multiple sclerosis, strengthening their association: a nested case-control study identified samples from 801 individuals who later developed multiple sclerosis.Only 35 of these individuals were EBV - seronegative and all but one seroconverted before the onset of multiple sclerosis.Furthermore, EBV seroconversion preceded the sNfL increase. 22imed to investigate the possible causative role of HHV-6A in multiple sclerosis by analysing HHV-6A serology and levels of sNfL in the prodromal phase of multiple sclerosis.The first objective of this study was to determine if HHV-6A infection before the clinical onset of multiple sclerosis was associated with higher levels of sNfL.The second objective was to determine if increasing HHV-6A seroreactivity was detectable before the increase of sNfL in the multiple sclerosis prodrome.

Materials and methods
A nested case-control study was performed by cross-linking multiple sclerosis registries and six Swedish microbiological biobanks, as previously described in detail. 23Through this process, we identified and retrieved plasma or serum samples from individuals who later developed relapsingremitting multiple sclerosis.These samples are remnants from serological testing.All included samples were collected before the clinical onset of multiple sclerosis and before the age of 40 (Supplementary Fig. 1).For each case, one control without multiple sclerosis was randomly selected and matched for biobank, sex, blood sampling date and birth date (in order of priority).
The samples were categorized both according to sampling age and time to clinical onset of multiple sclerosis.
The study was performed in accordance with the Declaration of Helsinki and approved by the
The data on sNfL, CRP, HHV-6A, EBV and CMV have been published previously. 8,9,21,26,28In this study, we combined these data in expanded analyses to assess a different hypothesis.

Serostatus of HHV-6A and levels of sNfL
The proportions of HHV-6A-seropositive samples in matched cases and controls were compared with McNemar test.Serum NfL was log10-transformed and analysed with a paired-samples t-test, comparing each case with its matched control.The log10 sNfL in HHV-6A-seropositive andnegative samples were compared with an independent samples t-test, stratified by case-control status.To adjust for the natural age-dependent increase of sNfL, 29 a within-pair ratio of sNfL was calculated for each case and its age-matched control.The ratios were log10-transformed and compared between HHV-6A-seropositive and -negative cases with an independent-samples t-test.

A C C E P T E D
To further investigate the levels of sNfL in HHV-6A-seropositive and -negative samples, we plotted the within-pair ratios of sNfL against the time from serum sampling to the clinical onset of multiple sclerosis.These plots were stratified for HHV-6A serostatus and analysed with locally estimated scatterplot smoothing (loess) regression with 95% confidence intervals (CI).
As an additional approach to adjust for sampling age, we used age-adjusted z-scores of sNfL.These z-scores were calculated for each case and control using the sNfL Reference Application based on measurements from 4532 controls. 30Data regarding body mass index (BMI) at the time of serum sampling was not available and a standard BMI value of 25 was thus used in these calculations.
Age-adjusted sNfL z-scores in HHV-6A-seropositive and -negative cases and controls were compared with a t-test.
The association between HHV-6A seropositivity and log10 sNfL was analysed with multiple linear regression, stratified by case-control status.The regression model included sampling age and serostatus for EBV.Since EBV is an age-dependent risk factor for multiple sclerosis, 9 these analyses were stratified by median sampling age.The model was also adjusted for sex, indirectly associated with sNfL through the risk of trauma.
To further assess the association between HHV-6A serostatus and elevated sNfL, a logistic regression was performed, calculating the odds ratio (OR) with 95% CI for HHV-6A as a risk factor for elevated sNfL.Elevated sNfL was defined as age-adjusted sNfL z-score >2.The model also included serostatus of EBV and sex.These analyses were stratified by case-control status and median sampling age.
To determine if combined HHV-6A and EBV seropositivity affected sNfL levels, we performed a t-test of log10 sNfL in cases with singular versus combined seropositivity for HHV-6A and EBV.
Sampling age was accounted for by analysing age-adjusted sNfL z-scores.

Time relation of HHV-6A antibodies and sNfL
The included samples were donated up to 33 years (median 10.4 years) before the clinical onset of multiple sclerosis.Together with the matched samples, this material reflects a wide time span, enabling temporal analysis of the order of events on group level.
The natural variation of sNfL is too large to decide if an individual sample was drawn during the multiple sclerosis prodrome.However, by analysing many samples from a wide time range, the transient shift towards increasing levels of sNfL is revealed. 21Events occurring before the increasing sNfL levels are likely occurring before the subclinical onset of multiple sclerosis.
To assess if increasing HHV-6A seroreactivity was detectable before the rise of sNfL, we calculated the within-pair ratio of HHV-6A seroreactivity (measured as MFI) for each matched case-control pair.To reduce the effect of the random technical noise in the seronegative samples, and avoid division by zero, these plots were calculated with a limit of detection (LOD) of 50 MFI.These ratios were plotted against the time from the serum sampling to the clinical onset of multiple sclerosis and analysed using loess regression with 95% CI.This plot was then compared with the previously reported loess regression of within-pair ratio of sNfL against the time to the clinical onset of multiple sclerosis. 21For comparison, we also calculated loess regressions of within-pair MFI ratios for herpesviruses HSV-1, HSV-2, VZV, CMV, HHV-6B, and HHV-7, plotted against the time to the clinical onset of multiple sclerosis.These plots were also calculated with a LOD of 50 MFI.

Sensitivity analyses
Elevated levels of NfL have been reported in many other conditions, such as traumatic brain injury and CNS infections. 31,32To exclude individuals with biochemical signs of acute disease or trauma, sensitivity analyses were performed in samples with CRP <5 mg/L.
The method to calculate age-adjusted sNfL z-scores is currently not applicable to children below the age of 17, for whom (n = 91 of 1038) the z-scores were calculated as if these individuals were 17 years old.Sensitivity analyses were performed to account for the uncertainty of these z-scores, excluding samples drawn before the age of 17.A few samples were collected closely before the clinical onset of multiple sclerosis (Supplementary Fig. 1), conferring uncertainty about whether the samples were truly presymptomatically collected.A set of sensitivity analyses were thus performed limited to samples collected more than 1-4 years before the clinical onset.
Since seropositivity against EBV appears to be necessary for developing multiple sclerosis, the prodromal phase may also be dependent on EBV.Additional sensitivity analyses were thus limited to EBV seropositive samples.
Statistical tests were two-tailed.The significance level was 0.05.Statistical analyses were performed in R, version 4.2.1, and SPSS, version 28.All graphs were constructed in R.

Results
We identified and retrieved samples from a total of 670 matched case-control pairs.Due to insufficient sample volume, 151 sets were excluded, leaving 519 matched case-control pairs for the final analyses.The median age at sampling was 24.7 years and the median time to clinical onset of multiple sclerosis was 9.5 years.Demographic data has been described previously. 21

Serostatus of HHV-6A and levels of sNfL
The proportion of HHV-6A-seropositive samples was significantly higher in cases than in controls (table 1).A significantly higher level of sNfL was observed in cases compared to age-matched controls.Comparing HHV-6A-seropositive and -negative cases, the levels of sNfL were significantly higher in the seropositive cases.Similar results were observed for age-adjusted values, although not statistically significant (table 1).
The level of sNfL was significantly higher in HHV-6A-seropositive cases than in their matched controls from more than 6 years prior to the clinical onset of multiple sclerosis (Fig. 1A).However, a similar increase of sNfL was also observed in HHV-6A-seronegative cases, where sNfL was significantly higher than in their matched controls from more than 4 years before the clinical onset (Fig. 1B).The confidence intervals were overlapping, and the loess regressions of these two groups were not significantly different.
We observed a significant association between HHV-6A seropositivity and higher sNfL among cases, but not in controls (table 2).The association between HHV-6A and sNfL was driven by findings in samples drawn before the median age of 24.7 years.
The level of sNfL was significantly higher in cases with combined seropositivity for HHV-6A and EBV, compared to those only seropositive for EBV (geometric mean 7.67 vs 6.76, P = 0.03).Similar findings were observed when accounting for age, although not statistically significant; mean age-adjusted sNfL z-score 1.10 vs 0.89, P = 0.06.
Only eight cases were seropositive against HHV-6A and simultaneously EBV seronegative, limiting further interaction analyses.Levels of sNfL in categories with different combinations of EBV and HHV-6A serostatus are presented in Supplementary Table 2.

Time relation of HHV-6A antibodies and sNfL
The within-pair ratios of HHV-6A seroreactivity and sNfL were plotted against the time to the clinical onset and analysed with loess regression.As visualized by the 95% CI of the loess regression, a statistically significant ratio of HHV-6A seroreactivity was detectable from more than 13.5 years before the clinical onset of multiple sclerosis (Fig. 2A).As previously reported, the sNfL ratio increased from 10 years before the clinical onset. 21A significant sNfL ratio was observed from 6.6 years before the clinical onset (Fig. 2B).The increasing HHV-6A seroreactivity was thus detectable before the rise of sNfL in samples from individuals who later developed multiple sclerosis.For comparison, the ratios of seroreactivities for human herpesviruses HSV-1, HSV-2, VZV, CMV, HHV-6B and HHV-7 were also plotted.No significant increases in MFI ratios were observed for these viruses (Supplementary Fig. 2).

Sensitivity analyses
To identify and exclude samples drawn at acute disease, we used available CRP estimates. 28We selected those with CRP <5 mg/L (300 cases and 310 controls) for these sensitivity analyses.The level of sNfL remained significantly higher in HHV-6A-seropositive cases than in -negative cases (geometric mean 7.91 vs 6.68, P = 0.01).The within-pair ratio of sNfL in HHV-6A-seropositive and -negative cases (geometric mean ratio 1.29 vs 1.10, P = 0.12) were similar to ratios for all samples.This was calculated for pairs where both the case and the control had CRP <5 mg/mL (n = 191).The association between HHV-6A and higher levels of sNfL remained similar in all sampling ages (+14%, 95% CI 0%-29%, P = 0.06) and still significant in the younger group (+26%, 95% CI +4%-52%, P = 0.02).Cases with combined seropositivity for HHV-6A and EBV still had significantly higher levels of sNfL compared to those only seropositive for EBV (geometric mean 7.99 vs 6.69, P = 0.01).
In the sensitivity analyses of age-adjusted sNfL z-scores, we also excluded samples drawn before the age of 17.Still, the results remained similar.The sNfL z-scores were significantly higher in HHV-6A-seropositive than in -negative cases (mean z-score 1.20 vs 0.91, P = 0.045).Cases with combined seropositivity for HHV-6A and EBV had significantly higher sNfL z-scores than those only seropositive for EBV (mean z-score 1.19 vs 0.90, P = 0.04).The logistic regression, assessing HHV-6A as a risk factor for elevated sNfL, also remained similar in cases below median age (OR = 2.19, 95% CI 0.92-5.23,P = 0.08).
Sensitivity analyses of samples drawn >1-4 years before the clinical onset of multiple sclerosis yielded similar results (Supplementary Table 3).
Finally, highly similar results were also observed when excluding EBV seronegative samples.As in the primary analysis, HHV-6A seropositivity was significantly associated with the level of sNfL in the younger half of the cases, but not among the older cases (Supplementary Table 4).

Discussion
In this study, we investigated the relationship between HHV-6A serostatus and sNfL levels in samples collected many years prior to the clinical onset of multiple sclerosis.
In the linear regression analysis, HHV-6A seropositivity was significantly associated with higher levels of sNfL, also when adjusted for sampling age, EBV serostatus, and sex.The association was only present in samples collected from cases at a young (below median, 24.7 years) age, where HHV-6A seropositivity was associated with a doubled risk of elevated sNfL.This finding may reflect that this is the age when HHV-6A may contribute to the multiple sclerosis prodrome.The level of sNfL was also higher in cases with combined seropositivity for HHV-6A and EBV compared to those only seropositive for EBV.Similar, although not statistically significant, results were observed when analysing mean age-adjusted sNfL z-scores and within-pair sNfL ratios.The results also remained similar in sensitivity analyses.No corresponding associations between HHV-6A and sNfL were observed in the control group.
Using presymptomatically collected serum samples, the current study design minimizes the risk of reverse causation.Spanning from early childhood to adulthood, the collection of samples provides a unique opportunity to assess the order of events in the pathogenesis of multiple sclerosis.Increasing seroreactivity against HHV-6A was detectable in cases many years before the increase of sNfL, suggesting a time relation where HHV-6A antibodies precede demyelination and axonal injury.This was not part of a non-specific increase of antibodies belonging to a subclinical multiple sclerosis pathological process, as similar findings were not found for six other human herpesviruses (Supplementary Fig. 2).
This study also entails limitations that need to be addressed.The conversion to age-adjusted sNfL z-scores resulted in a mean sNfL z-score of 0.74 in the control samples, which is higher than expected.One possible reason would be that many samples were initially collected to diagnose acute disease, which may affect the levels of sNfL.Unfortunately, no clinical data at the time of serum sampling was available for cases or controls.However, since sensitivity analyses, excluding samples with CRP ≥5 mg/L, yielded highly similar results, this may not be the sole explanation.Other factors such as evaporation may also affect the levels of sNfL.Still, this would affect samples from cases and controls equally, and would not affect the validity of our findings.Furthermore, we did not have access to height or weight data at the time of serum sampling.The level of sNfL decreases with higher BMI, likely by dilution in the larger circulating blood volume. 33Adolescent overweight is a recognized risk factor for multiple sclerosis. 34,35It can thus be assumed that the cases had a higher mean BMI than controls, which confers the risk of introducing a systematic bias.However, such a bias would lead to an underestimation of the sNfL z-scores in cases, resulting in a false negative result.This limitation should thus not question the validity of the results.In addition, the levels of sNfL might also be affected by differences between the cohorts and the laboratory methods in the current study and the sNfL reference database.
In the plots and loess-regressions, the age-dependent variation of sNfL is accounted for by the use of the within-pair ratio, providing age-matched comparisons.Using the same approach for seroreactivity, which is not necessarily age-dependant, might seem unjustified.However, this approach enables plotting seroreactivity against the time to multiple sclerosis onset, simultaneously providing comparisons with controls matched for biobank, sex, and age.Furthermore, this method enables comparisons of the time points for increasing seroreactivity and sNfL levels.
The antigens and MFI cut-offs for determining serostatus of HSV-1, HSV-2, VZV, EBV, and CMV have all been validated against established reference assays. 27However, this is not the case for HHV-6A, for which no reliable reference assay is yet available.The assay has only been partly validated for specificity in sera from children with exanthema subitum (also known as roseola infantum or sixth disease), which is caused by HHV-6B. 36The validation indicated negligent cross-reactivity for IE1A regarding HHV-6A and -6B. 8Since no differences between cases and controls were observed for HHV-6B seroreactivity, 9 the association between HHV-6B and sNfL was not assessed here.Furthermore, no validated cut-off has been established for IE1A.In consistence with our previous studies, we defined HHV-6A seropositivity as a seroresponse against IE1A >50 MFI. 9,26Other studies have used the 75 th percentile of IE1A MFI in controls as the cut-off. 8,37In the current sample, that value corresponds to an MFI of 50.7.The different definitions of serostatus would thus yield highly similar or identical results.Furthermore, the cut-off used in the current study has previously been tested in sensitivity analyses and shown to be robust. 9Still, the method to differentiate HHV-6A serostatus is less certain than the approach for EBV serostatus.This is a limitation, as in all current studies of HHV-6A serology.
Finally, it remains possible that the association between HHV-6A and multiple sclerosis represents an epiphenomenon due to viral reactivation in injured oligodendrocytes.However, the observation that increasing HHV-6A antibodies were detectable before increasing sNfL argues against such a mechanism, which would likely result in the reversed time-order.
Several hypotheses for HHV-6A in the pathogenesis of multiple sclerosis have been suggested. 5,38,39A direct injury from lytic HHV-6A infection in oligodendrocytes could cause cell death, demyelination, and subsequent inflammation.Similar processes have been observed for JC Polyomavirus in progressive multifocal leukoencephalopathy and measles virus in subacute sclerosing panencephalitis. 38It has also been suggested that the pathogenic effect could be caused by molecular mimicry. 38This concept implies that viral proteins share amino acid sequences or structures with host cell proteins.These viral antigens are thereby suggested to activate autoreactive immune-competent cells, causing aberrant inflammation and cell damage.For EBV, two epitopes of EBNA-1 display amino acid homology with CNS host antigens, namely Anoctamin 2 (ANO2) 40 and GlialCAM. 41Carriage of ANO2 antibodies associate with an increased risk of multiple sclerosis, highly suggestive of a pathogenic role. 40A homologous amino acid sequence with potential for molecular mimicry has also been described for an HHV-6 peptide and myelin basic protein, a putative autoantigen for multiple sclerosis. 42An alternative mechanism of virus-induced autoimmunity have also been suggested: during viral replication, the envelope is formed from the host cell membrane and can thus incorporate lipids and proteins from the host.As a neurotrophic virus, the envelope of HHV-6A may contain proteins and lipids from oligodendrocytes, which could trigger autoimmune reactions directed against these cells. 44ver, the previous observations of total EBV seroprevalence in adults with multiple sclerosis suggest that EBV is a prerequisite, but insufficient cause of the disease. 3,4Neither do the results of the current study indicate that HHV-6A is the sole cause of multiple sclerosis, instead providing an increased risk.The level of sNfL increased significantly before the clinical onset of multiple sclerosis in both HHV-6A-seropositive and -negative cases (Figure 1A-B), indicating that HHV-6A is no prerequisite for multiple sclerosis but rather a contributing factor in a proportion of cases.
Only eight of 519 samples from individuals who later developed multiple sclerosis were seropositive against HHV-6A and simultaneously seronegative against EBV.All these samples were drawn more than ten years before the clinical onset of multiple sclerosis, leaving ample time for late EBV infection.Isolated HHV-6A infection is thus rare during the presymptomatic phase of multiple sclerosis and virtually non-existent when the disease is fully developed. 3,4Instead, the observations in the current study support the hypothesis that both EBV and HHV-6A contribute to the development of multiple sclerosis. 396][47] Furthermore, additive interactions have been observed between EBV and HHV-6A seropositivity regarding the risk of multiple sclerosis. 8Infection from EBV during early childhood is typically mild or asymptomatic, while infection at later age often causes infectious mononucleosis.The latter is highly associated with the risk of multiple sclerosis.Late EBV infection, causing infectious mononucleosis, might thus be sufficient to cause multiple sclerosis in individuals with genetic susceptibility and/or other risk factors.In contrast, early EBV infection could require a second hit by HHV-6A infection to reactivate latent EBV and initiate the inflammatory cascade leading to multiple sclerosis.This hypothesis is consistent with the observations in the present study, where increasing seroreactivity against HHV-6A was detectable years before the rising levels of sNfL.This indicates that elevated HHV-6A antibodies precede the immune-mediated axonal injury in a subset of individuals that will develop multiple sclerosis.
However, the current study can merely indicate the order of events at the group level.While the large number of samples and the many years observed between increasing HHV-6A seroreactivity and sNfL support the relevance of these observations, the lack of serial samples limits the possibility to suggest causality.Testing the suggested hypothesis requires an extensive longitudinal study of presymptomatically collected serial samples, identifying the time points for both EBV and HHV-6A seroconversion.Furthermore, there is a need for PCR studies of HHV-6A and -6B excretion in several clinical materials, including saliva and/or white blood cells, combined with type-specific HHV-6 serology.
In conclusion, we report a significant association between HHV-6A seropositivity and the level of sNfL in samples from individuals who later developed multiple sclerosis, likely reflecting the prodromal phase of the disease.In addition, increasing seroreactivity against HHV-6A was detectable before the rise of sNfL.These findings support the hypothesis that HHV -6A may contribute to the pathogenesis of multiple sclerosis in a proportion of cases, but do not show that HHV-6A infection is essential for disease development.

Figure 1
Figure 1 Locally estimated scatterplot smoothing (loess) regression with 95% confidence intervals for within-pair sNfL ratio against time to onset of multiple sclerosis.(A) HHV-6Aseropositive cases.(B) HHV-6A-seronegative cases.Within-pair sNfL ratio was calculated for each matched case-control pair.Matched controls were included regardless of their HHV-6A serostatus.The time to onset of multiple sclerosis was calculated as the interval from the sampling date to the date of the first symptom indicative of multiple sclerosis.The time-points for significant within-pair ratios of MFI (95% CI > 1.0) are marked with vertical red lines.Log<sub>2</sub> scaled y-axis, limited to a ratio of 2 in either direction.HHV-6A seropositivity was defined as a seroresponse >50 MFI against HHV-6A antigen IE1A.HHV-6A, Human herpesvirus 6A; MFI, median fluorescence intensity; sNfL, serum neurofilament light chain; IE1A, truncated immediateearly protein 1 from HHV-6A.

Figure 2
Figure 2Time relation of HHV-6A antibodies and sNfL.(A) Loess regression with 95% confidence intervals for within-pair ratio of seroreactivity (MFI) for HHV-6A antigen IE1A against time to clinical onset of MS. <b>(B)</b> Loess regression of within-pair sNfL ratio against time to onset of multiple sclerosis, as previously reported.21Within-pair ratios were calculated for each case and matched control and a cut-off of 50 MFI.The time to onset of multiple sclerosis was calculated as the interval from the sampling date to the date of the first symptom indicative of multiple sclerosis.The time-points for significant within-pair ratios (95% CI > 1.0) are marked with vertical lines.HHV-6A, human herpesvirus 6A; sNfL, serum neurofilament light chain; MFI, median fluorescence intensity; loess, locally estimated scatterplot smoothing.A C C E P T E D M A N U S C R I P T Figure 1 159x58 mm ( x DPI) Figure 2 159x106 mm ( x DPI) R I P T