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. 2024 Mar 18;81(5):515–524. doi: 10.1001/jamaneurol.2024.0272

Serologic Response to the Epstein-Barr Virus Peptidome and the Risk for Multiple Sclerosis

Marianna Cortese 1,✉, Yumei Leng 2,3, Kjetil Bjornevik 1,4, Moriah Mitchell 2,3,5, Brian C Healy 6,7,8, Michael J Mina 9, James D Mancuso 10, David W Niebuhr 10, Kassandra L Munger 1,11, Stephen J Elledge 2,3, Alberto Ascherio 1,4,12
PMCID: PMC10949154  PMID: 38497939

Key Points

Question

Do individuals with multiple sclerosis (MS) have antibody titers against 1 or more Epstein-Barr virus (EBV) peptides that do not commonly generate an antibody response in healthy individuals of the same age, sex, and race and ethnicity?

Findings

In this nested case-control study of 30 individuals with MS and 30 matched controls, the antibody response to the EBV peptidome was stronger overall in those with MS, but individuals with MS and controls recognized the same epitopes. No single EBV peptide appeared to clearly separate individuals with MS from controls after accounting for the overall higher anti–EBV nuclear antigen 1 titers among individuals with MS.

Meaning

The findings suggest that antibodies against a specific EBV epitope may be a risk factor for MS, but alternative mechanisms should be investigated.

This case-control study assesses the serologic response to the Epstein-Barr virus peptidome before first symptoms of multiple sclerosis occur and whether specific epitopes may drive this response.

Abstract

Importance

It remains unclear why only a small proportion of individuals infected with the Epstein-Barr virus (EBV) develop multiple sclerosis (MS) and what the underlying mechanisms are.

Objective

To assess the serologic response to all EBV peptides before the first symptoms of MS occur, determine whether the disease is associated with a distinct immune response to EBV, and evaluate whether specific EBV epitopes drive this response.

Design, Setting, and Participants

In this prospective, nested case-control study, individuals were selected among US military personnel with serum samples stored in the US Department of Defense Serum Repository. Individuals with MS had serum collected at a median 1 year before onset (reported to the military in 2000-2011) and were matched to controls for age, sex, race and ethnicity, blood collection, and military branch. No individuals were excluded. The data were analyzed between September 1, 2022, and August 31, 2023.

Exposure

Antibodies (enrichment z scores) to the human virome measured using VirScan (phage-displayed immunoprecipitation and sequencing).

Main Outcome and Measure

Rate ratios (RRs) for MS for antibodies to 2263 EBV peptides (the EBV peptidome) were estimated using conditional logistic regression, adjusting for total anti–EBV nuclear antigen 1 (EBNA-1) antibodies, which have consistently been associated with a higher MS risk. The role of antibodies against other viral peptides was also explored.

Results

A total of 30 individuals with MS were matched with 30 controls. Mean (SD) age at sample collection was 27.8 (6.5) years; 46 of 60 participants (76.7%) were male. The antibody response to the EBV peptidome was stronger in individuals with MS, but without a discernible pattern. The antibody responses to 66 EBV peptides, the majority mapping to EBNA antigens, were significantly higher in preonset sera from individuals with MS (RR of highest vs lowest tertile of antibody enrichment, 33.4; 95% CI, 2.5-448.4; P for trend = .008). Higher total anti-EBNA-1 antibodies were also associated with an elevated MS risk (top vs bottom tertile: RR, 27.6; 95% CI, 2.3-327.6; P for trend = .008). After adjusting for total anti-EBNA-1 antibodies, risk estimates from most EBV peptides analyses were attenuated, with 4 remaining significantly associated with MS, the strongest within EBNA-6/EBNA-3C, while the association between total anti-EBNA-1 antibodies and MS persisted.

Conclusion and Relevance

These findings suggest that antibody response to EBNA-1 may be the strongest serologic risk factor for MS. No single EBV peptide stood out as being selectively targeted in individuals with MS but not controls. Larger investigations are needed to explore possible heterogeneity of anti-EBV humoral immunity in MS.

Introduction

Infection with the Epstein-Barr virus (EBV) is the leading cause of multiple sclerosis (MS),1 but it remains unclear why only a small proportion of infected individuals develop the disease and what the underlying mechanisms are by which the virus causes MS.2 Furthermore, it is not known whether we can identify individuals who may develop MS based on their immune response to EBV.

Among individuals infected with EBV, a higher IgG antibody titer to EBV nuclear antigens (EBNAs), especially the EBNA complex and its component EBNA-1, is the strongest risk factor for MS.3 This reactivity is consistent with the hypothesis that anti-EBNA-1 antibodies cause MS by cross-reacting with proteins expressed in the central nervous system,4 including anoctamin-2 (ANO2),5 glial cell adhesion molecule GlialCAM,6 myelin basic protein,7 and others.8,9,10 These findings, however, were for the most part based on antibody titers measured in blood samples collected in patients with MS after diagnosis and could therefore be a consequence of longer established disease,5,6,7 or were derived from molecular modeling studies.8 Moreover, the immune response to EBV antigens other than EBNA-1 has been implicated in priming autoreactive T cells and in cross-reactivity, challenging the notion of specific pathologically relevant cross-reactive antibodies targeting the same EBV epitope in all individuals with MS.10,11,12 Overall, the serologic response to only a few selected EBV proteins was assessed, and it is unknown whether there is a unique immune response to EBV in MS.

To address these gaps, we conducted a comprehensive assessment of the preclinical antibody response to the EBV peptidome using VirScan, covering the entire proteome of human pathogenic viruses, including more than 100 000 peptides, of which 2263 belong to EBV.13,14 A previous article1 reported a list of viral peptides with stronger antibody responses in MS cases or controls. Here, our aim was to determine whether there is an anti-EBV antibody signature that discriminates MS cases from matched controls better than the overall anti-EBNA-1 antibody titers.

Methods

Study Population and Design

We conducted a prospective, nested case-control study within the US military cohort comprising more than 10 million active-duty personnel. As of 2021, more than 62 million blood samples had been collected from this cohort for routine HIV screening and deployment tests.1 These samples are stored at −30 °C in the Department of Defense Serum Repository.15

The research protocol was approved by the institutional review boards of the Uniformed Services University of the Health Sciences and Harvard T. H. Chan School of Public Health, who granted a waiver of informed consent because the study was based on archived samples and deidentified data. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.

Case Ascertainment

Procedures for the identification of case patients with MS and controls have been previously described.1 Briefly, we identified all individuals with Veterans Affairs Schedule for Rating Disabilities code 8018 for MS in the electronic databases of the services’ physical disability agencies responsible for evaluating fitness for continued military service among members diagnosed with conditions that may prevent them from performing their duties. The databases are required to contain all available medical evidence related to the medical conditions. We reviewed the medical records and abstracted relevant information, including the neurologist’s diagnosis, first symptom and diagnosis dates, results of CNS magnetic resonance imaging, MS type, and number of relapses.

For this study, we randomly selected 30 individuals with MS with a first report to the military between 2000 and 2011 (further details described elsewhere16) and who had a blood sample collected before MS onset (range, 0-3 years). We matched these individuals to 30 controls based on sex, age (within 1 year), military branch (Army, Marines, Navy, or Air Force), race and ethnicity (Black, Hispanic, White, or other as classified by the US military), and blood collection (within 30 days). Information on race and ethnicity is collected for all military personnel when they join the US military, and the data are stored in the Defense Medical Surveillance System database. Controls were closely matched on the number of covariates, including race and ethnicity, to account for any possible differences according to those covariates. Control individuals had to be on active duty and free of MS on the onset date of the case they were matched to (risk set sampling).

VirScan

VirScan uses phage-displayed immunoprecipitation and sequencing technology14,17 for the comprehensive serologic profiling of antiviral antibodies in humans.14 The performance of VirScan concords strongly with that of traditional methods, such as enzyme-linked immunosorbent assays and Western blots.13,14

The peptide library of VirScan, version 2.0 was designed to cover the entire proteome of all viruses (approximately 200 viruses and approximately 110 000 viral peptides) with known human tropism listed in the UniProt database, collapsed on 90% identity.13 The sequences were parsed into 56–amino acid (aa)-long peptides with 28-aa-long overlap. The library was programmed into Agilent DNA microarrays, amplified, cloned into the DNA of T7 bacteriophages, and packaged into viral particles that display the encoded epitopes on the surface.

The assay was run according to a previously described protocol.13 To ensure reproducibility, every sample was assessed in 2 independent runs (technical replicates). After incubation with serum, antibody-bound phages were separated by immunoprecipitation, then their DNA was amplified and sequenced. The peptide-specific read counts were converted into z scores indicating the relative antibody enrichment against each epitope, a relative measure of the antibody titer, as previously described.1,13 An antibody-epitope hit as an indicator for the presence of antibodies against a particular epitope in individuals’ sera was defined as having an enrichment z score greater than or equal to 3.5 standard units in each of the technical replicates from the same sample run separately.13

Statistical Analysis

We performed the data analysis between September 1, 2022, and August 31, 2023. We started our analyses by focusing on the 30 EBV peptides previously identified as having significantly higher antibody binding in individuals with MS compared with controls, as measured in serum samples collected prior to MS onset.1 First, we descriptively compared the aa sequences of these peptides with the aa sequences of human proteins previously proposed as cross-reactivity targets. Next, we assessed the enrichment z scores (person-specific average from technical replicates) of antibodies to these 30 EBV peptides in the groups using heat maps and plotting mean differences. Subsequently, we expanded the analyses to the entire EBV peptidome. We compared the serologic profile (peptide-specific means) of cases and controls and examined whether it discriminated individuals with MS by conducting partial least squares discriminant analysis (PLS-DA) followed by a permutation test with 1000 permutations to obtain a P value. We also calculated variable importance in the projection (VIP) scores to assess which EBV peptides were the most important ones in the PLS-DA model. A VIP score greater than 1 is considered important and indicative of variables that contribute most in discriminating the comparison groups.

Subsequently, we removed any nonimmunogenic peptides, which we defined as those that had no antibody hit across our study population. We created z score tertiles to compare individuals ranked in the top tertile with those in the bottom tertile of antibody levels. Then, we ran separate conditional logistic regression models for each peptide, which were adjusted for the matching factors by design, and calculated odds ratios, which can be interpreted as rate ratios (RRs) due to the risk set sampling of controls, and 95% CIs. Furthermore, we derived total anti-EBNA-1 antibodies by summing the enrichment z scores for all EBNA-1–related peptides and assessed their association with MS in a conditional logistic regression model as a continuous variable (standardized on a mean of 0 and SD of 1) and categorically in tertiles. We also included the total anti-EBNA-1 antibodies (continuously) as a covariate in separate regression models of the individual EBV peptides to assess potential confounding and whether any EBV peptide stood out despite the adjustment for this risk factor associated with the onset of MS.

Finally, we assessed whether sex, age, or race and ethnicity were associated with the total anti-EBNA-1 antibody response using linear regression. We also examined cytomegalovirus (CMV) seropositivity, another herpesvirus that has been associated with a lower MS risk and believed to be mediated through modifying the immune response to EBV.1,18 Seropositivity for CMV was defined as having an epitope hit (z score ≥3.5) to the previously reported immunogenic envelope glycoprotein M (UniProt ID P16733) peptide aa337-372.14

The statistical analyses were conducted using R, version 4.0 (R Foundation for Statistical Computing). Figures were created using ggplot2.

Results

Selected characteristics of the study population were similarly distributed among cases and controls due to the matched design (Table). Individuals with MS comprised 23 men (76.7%) and 7 women (23.3%) with a mean (SD) age of 27.8 (6.5) years at serum sampling and 28.5 (6.4) years at MS onset, of whom 9 (30.0%) identified as Black, 3 (10.0%) as Hispanic, and 18 (60.0%) as White. Controls comprised the same number of men and women and same distribution of race and ethnicity. Mean (SD) age of serum sampling for controls was 27.9 (6.5) years.

Table. Selected Sample Characteristics .

Characteristic No. (%)
Individuals with MS (n = 30) Controls (n = 30)
Sex
Female 7 (23.3) 7 (23.3)
Male 23 (76.7) 23 (76.7)
Race and ethnicity
Black 9 (30.0) 9 (30.0)
Hispanic 3 (10.0) 3 (10.0)
White 18 (60.0) 18 (60.0)
Other 0 0
Age, y
Serum samplinga
Mean (SD) 27.8 (6.5) 27.9 (6.5)
Range 18.0-42.0 19.0-42.0
MS onset
Mean (SD) 28.5 (6.4) NA
Range 19.0-42.0 NA
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Abbreviations: MS, multiple sclerosis; NA, not applicable.

a

Serum sample collected before MS onset.

As previously reported,1 the antibody response to the human virome was similar in individuals with MS and controls, except for EBV, and 30 of 32 peptides with a higher antibody binding in individuals with MS mapped to EBV. In our descriptive comparison, several previously proposed mimics mapped to these 30 top EBV peptides within EBNA-1 (UniProt ID P03211) (eTable in Supplement 1). The magnitude of antibody response to these EBV peptides is shown in Figure 1A and eFigure 1 in Supplement 1. The ranking of these peptides according to immune response strength was similar between groups (Figure 1B). The EBV antibody response among controls presented with less interindividual variability than among cases (eFigure 2 in Supplement 1).

Figure 1. Magnitude of Antibody Response to Epstein-Barr Virus (EBV) Peptides in Cases With Multiple Sclerosis (MS) Compared With Matched Controls.

Figure 1.

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Antibody response to the 30 EBV peptides in preonset serum samples ranked by statistical significance (r1-r30) and indicating the EBV protein, UniProt ID, and amino acid (aa) sequence. A, Derived as the mean z score in identical replicates, with higher levels indicating a stronger response. A z score greater than or equal to 3.5 is an epitope hit and indicates presence of antibodies to a peptide in VirScan. Matched pairs of cases and controls are shown in the same order. Compare with eFigure 1 in Supplement 1, which shows the peptide grouped by EBV protein. B, Ordered according to strength of the immune response to peptides in the cases. EBNA indicates Epstein-Barr virus nuclear antigen.

We next examined the mean serologic profiles to the entire EBV peptidome. These profiles were remarkably similar in case patients and controls (Figure 2A and B); however, the serologic response was able to discriminate between the groups in PLS-DA (P = .04 by permutation test with 1000 permutations after conducting PLS-DA) (Figure 2C). Among the 2263 EBV peptides, 762 (34%) had a VIP score of 1 or higher and 73 (3%) had a VIP score of 2 or higher, including numerous peptides belonging to EBNA and other EBV proteins (eFigure 3 in Supplement 1).

Figure 2. Serologic Profiles for the Epstein-Barr Virus (EBV) Peptidome in Cases With Multiple Sclerosis (MS) and Matched Controls and Their Discriminatory Potential.

Figure 2.

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A, Antibody response profiles to all 2263 EBV peptides are shown. B, Amino acid (aa) sequences for full-length EBV nuclear antigen 1 (EBNA-1), UniProt ID P03211 are shown. C, Includes the antibody response to all 2263 EBV peptides (permutation test with 1000 permutations, P = .04 for the separation of cases and controls). The EBV peptides contributing most to the separation according to variable importance in the projection scores are shown in eFigure 3 in Supplement 1.

Among the 2263 EBV peptides, there was at least 1 hit (z score ≥3.5 in identical replicates in at least 1 study individual) in 625 peptides, which we analyzed further in separate conditional logistic regression models. The serologic response to 66 EBV peptides was significantly associated with MS (Figure 3; eFigure 4 in Supplement 1), with the strongest estimate for EBNA-1, P03211 aa365-420 (highest vs lowest tertile of antibody enrichment: RR, 33.4; 95% CI, 2.5-448.4; P for trend = .008), which was the top-ranked peptide in previous findings1 (Figure 1A). The 10 peptides most strongly associated with a higher MS risk (top to bottom tertile RRs >14; P for trend = .03) all mapped to EBNA antigens.

Figure 3. Antibody Response to Epstein-Barr Virus (EBV) Peptidome and Risk for Multiple Sclerosis (MS).

Figure 3.

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Rate ratios (RRs) for MS from conditional logistic regression models comparing top vs bottom tertile of antibody response to the 625 EBV peptides with at least 1 epitope hit (z score ≥3.5 in technical replicates) in 1 individual and P value for trend across tertiles. The models in panel B were additionally continuously adjusted for standardized total anti–EBV nuclear antigen 1 (EBNA-1) antibody level (total z score to EBNA-1 peptides). Statistically significant results are plotted above the dotted horizontal line (P for trend <.05). The EBV peptides with RRs less than 0.01 or greater than 100 (all with P >.05) are not shown to facilitate plotting. Of the 10 excluded peptides, the antibody response to 4 of these was associated with MS in unconditional logistic regression models adjusted for sex, age, race and ethnicity, and total anti-EBNA-1 antibodies (comparing top vs bottom tertiles, Q07286 aa57-112: odds ratio [OR], 25.12 [95% CI, 3.23-388.77; P for trend = .006]; Q1EH47 aa18-73: OR, 18.47 [95% CI, 2.50-230.59; P for trend = .01]; P03213 aa85-140: OR, 7.63 [95% CI, 1.41-55.08; P for trend = .03]; Q3KSU8 aa673-728: OR, 0.12 [95% CI, 0.01-0.65; P for trend = .02]).

A higher total anti-EBNA-1 antibody level was associated with a higher MS risk (top vs bottom tertile: RR, 27.6 [95% CI, 2.3-327.6; P for trend = .008]; 1-SD increases: RR, 6.2 [95% CI, 1.4-26.6; P = .02]). After adjusting the separate regression models continuously for the total anti-EBNA-1 antibody enrichment, the associations of the antibody response to individual EBV peptides with MS were attenuated (P03211 aa365-420, top vs bottom tertile: adjusted RR [ARR], 10.07; 95% CI, 0.72-140.80; P for trend = .09). Of the 66 EBV peptides initially associated with a higher MS risk, only 4 remained associated with MS according to the P for trend across tertiles, some with stronger risk estimates, located within EBNA-6/EBNA-3C and latent membrane protein 1 (LMP-1) (top vs bottom tertile of antibodies to EBNA-6/EBNA-3C, Q69140 aa421-476: ARR, 34.63 [95% CI, 1.24-969.20; P for trend = .03]; EBNA-6/EBNA-3C, P03204 aa337-392: ARR, 15.55 [95% CI, 1.22-197.96; P for trend = .03]; LMP-1, G1CS57 aa29-84: ARR, 11.38 [95% CI, 0.98-131.79; P for trend = .04]; EBNA-3/EBNA-3A, P12977 aa813-868: ARR, 12.75 [95% CI, 0.81-201.82; P for trend = .04]) (Figure 4; eFigure 4 in Supplement 1). Among the 625 assessed EBV peptides, 1 within BDLF3.5 showed a stronger association and was statistically significant after adjusting for total EBNA-1 (A0A0C7TUY8 aa1-56: ARR, 15.76; 95% CI, 0.86-289.28; P for trend = .04) (Figure 4).

Figure 4. Antibody Response to Epstein-Barr Virus (EBV) Peptides Associated With Multiple Sclerosis (MS) Before and After Adjusting for Total Anti–EBV Nuclear Antigen 1 (EBNA-1) Antibodies.

Figure 4.

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Rate ratios (RRs) for MS from conditional logistic regression models comparing the top to the bottom tertile of antibody response to EBV peptides with at least 1 epitope hit (z score ≥3.5 in technical replicates) in 1 individual. All peptides that were statistically significantly associated with MS before adjusting for total anti-EBNA-1 are shown (62 of 71 EBV peptides described in the Results with both P <.05 comparing the top vs bottom tertile and P for trend across tertiles <.05).

aRemained statistically significant (P for trend <.05) after adjusting continuously for standardized total anti-EBNA-1 antibody level (total z score to EBNA-1 peptides).

The serologic response to 5 peptides was initially associated with a lower MS risk in P-for-trend analyses across tertiles (BRLF1, Q69126 aa57-112; EBNA2, Q69022 aa337-392; envelope glycoprotein H, Q1HVD2 aa29-84; triplex capsid protein 1, P03187 aa169-224; and tegument protein BKRF4, P0C724 aa57-112) (eFigure 4 in Supplement 1) but were no longer associated after adjusting for total anti-EBNA-1 levels (Figures 3 and 4; eFigure 4 in Supplement 1).

In contrast, the associations between total anti-EBNA-1 antibodies and MS risk remained strong in most instances in the mutually adjusted models. For 96% (n = 600) of all 625 associations and for 75% (n = 62) of the associations including the 71 EBV peptides associated with MS before adjustment, there was a less than 20% change in the estimates for anti-EBNA-1 (RRs remaining >4.96). While the antibody response to 5 of the 625 assessed peptides was significantly associated with a higher MS risk in mutually adjusted models, associations of total anti-EBNA-1 antibody response with MS remained significant in more than 98% (n = 613) of the associations. In the model of the initially strongest associated peptide EBNA-1, P03211 aa365-420, the RR for MS was 3.60 (95% CI, 0.87-14.96, P = .08) per 1-SD increase in total anti-EBNA-1 antibodies. Overall, these results differ from what would be observed if the total anti-EBNA-1 antibodies or the antibody response to a single EBV peptide were the sole risk factor for MS (simulations for these 2 scenarios shown in eFigure 5 in Supplement 1).

In this study population, 14 case patients (46.7%) and 15 controls (50.0%) were CMV positive. Sex, age, race and ethnicity, and CMV seropositivity were not associated with the total anti-EBNA-1 antibody response, both in separate and mutually adjusted linear regression models. The individuals with MS who were positive for CMV had lower standardized anti-EBNA-1 antibody levels than those negative for CMV (−0.36 SDs) in models adjusted for sex, age, and race and ethnicity, but the difference was not significant.

Discussion

In this first in-depth investigation of the entire anti-EBV peptidome in prediagnostic MS sera, the antibody profiles appeared similar in individuals with MS and matched controls, with an overall stronger response in those with MS. No single EBV peptide stood out for being recognized by antibodies among cases with MS but not among controls. Although it is possible that antibodies against a specific EBV peptide may be a risk factor for a subset of individuals with MS, our findings point toward a broader dysregulation of the host-virus balance and suggest that MS prevention and treatment may not be achieved by therapeutic strategies focusing exclusively on the humoral immune responses to a specific EBV epitope.

Previous studies have reported differences in the serologic response to specific EBV peptides in patients with MS and controls, especially to epitopes within EBNA-1,19,20,21,22 but in most of these studies, the authors did not adjust for total anti-EBNA antibodies. In 1 of the few studies, in which the authors adjusted for total anti-EBNA antibodies, the number of EBV epitopes associated with MS was markedly reduced.20 A prospective study suggesting cross-reactivity to ANO2 found that neither ANO2 seropositivity nor anti-ANO2 antibody levels was associated with MS after adjustment for EBNA-1 antibodies in individuals with MS with preonset samples and matched controls.5

This study builds on a previous virome analysis in this study population drawn from a large, prospective US military cohort.2 Antibodies to EBV peptides were consistently higher among individuals with MS than among controls, with little or no signal for antibody responses to other viruses. The serologic response to human herpesvirus 6A, for example, specifically to peptides within immediate-early protein 1, previously suggested to play a role in MS etiology,23,24 were also not significantly different in cases and controls.2

The risk of MS increases strongly and monotonically with higher levels of circulating anti-EBNA antibodies3,25,26,27,28 consistently across sex and race and ethnicity.3 The risk for MS is elevated based on higher anti-EBNA antibodies many years prior to the onset of the disease.26 Few indicators of total EBNA-1 antibody titers have been identified. In a Japanese population, antibody titers to EBNA-1 were found to correlate with the frequency of circulating cytotoxic T cells against autologous EBV-transformed lymphoblastic cell lines.29 Associations with high EBNA-1-IgG have also been reported for HLA-DRB1*1501 carrier status,30,31 the strongest genetic risk factor for MS,32 and other genetic risk variants,33 as well as history of mononucleosis.34,35 Infection with CMV, a herpesvirus also transmitted through saliva and displaying socioeconomic status and racial and ethnic differences for age at first infection,36 similar to EBV,37 has been associated with a lower MS risk1,18 presumably because it may modify the immune response to EBV.18 We did not find an association among CMV positivity, age, sex, or race and ethnicity, and total EBNA-1 antibodies in our study either overall or in cases and controls separately.

It is important to note that risk estimates for some EBV peptides were, although attenuated, still strong (with an RR of approximately 10) after accounting for total anti-EBNA-1 levels, suggesting that the immune response to specific peptides may still contribute to the risk for MS. The finding that antibodies to EBNA-6/EBNA-3C, a gene involved in the immortalization of B lymphocytes, and BDLF3.5, a gene involved in immune evasion in the early lytic phase, are also elevated in preclinical MS sera after accounting for total anti-EBNA-1 antibody response is novel and remains to be confirmed in larger investigations. Antibodies were also increased against EBNA-3/EBNA-3A and LMP-1, both involved in B-cell transformation and establishment of latent infection, and previously implicated in MS.20,38 Overall, although our results do not contradict the hypothesis that cross-reactivity is an important mechanism in MS pathogenesis, they point toward the existence of a broader dysregulation of EBV-host homeostasis. Among the mechanisms to be considered is the transformation of B cells,39,40 for example, through epigenetic modifications.41 Recent evidence has suggested that B cells from newly diagnosed, untreated patients with MS are globally hypomethylated, which leads to B-cell activation and dysfunction.42 More convincingly, the increased frequency of CD8+ T-cell responses against EBV in individuals with MS, and particularly the high proportion of CD8+ T cells against EBV lytic antigens in the cerebrospinal fluid of individuals with MS, may point toward a direct role of EBV reactivation in causing MS.43

Limitations

There are limitations to our study that need to be considered. Since the study sample is relatively small, we could have missed patterns of serologic response to specific EBV peptides that may be present in a subset of patients. Furthermore, VirScan captures antibodies to linear epitopes and cannot identify immune signatures to conformational epitopes or reliably identify different EBV strains. We also did not validate our findings using specific quantitative methods, such as enzyme-linked immunosorbent assay. Finally, we could not assess whether genetic factors modify the associations, as we did not have genotype information for our sample.

Conclusions

The findings suggest that the overall immune response to EBNA-1 is the strongest serologic factor associated with MS. Specific EBV epitopes may contribute to disease risk, but none that we have definitive evidence for in this study.

Supplement 1.

eTable. Descriptive Comparison: Mapping Selected Potential Cross-Reactivity Targets to Epstein-Barr virus (EBV) Peptides

eFigure 1. Magnitude of Antibody Response to EBV Peptides in Cases With MS Compared to Matched Controls

eFigure 2. Variability in Antibody Response to EBV Peptides in Cases With MS and Matched Controls

eFigure 3. EBV Peptides Contributing Most to the Separation of Cases With MS and Controls in PLS-DA

eFigure 4. Rate Ratios and 95% CIs for Multiple Sclerosis According to Tertiles of Antibody Response to the Associated EBV Peptides

eFigure 5. Distribution of Odds Ratios (ORs) and Medians From the Simulation of 2 States of Nature

jamaneurol-e240272-s001.pdf (1,006.4KB, pdf)
Supplement 2.

Data Sharing Statement

jamaneurol-e240272-s002.pdf (15.5KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1.

eTable. Descriptive Comparison: Mapping Selected Potential Cross-Reactivity Targets to Epstein-Barr virus (EBV) Peptides

eFigure 1. Magnitude of Antibody Response to EBV Peptides in Cases With MS Compared to Matched Controls

eFigure 2. Variability in Antibody Response to EBV Peptides in Cases With MS and Matched Controls

eFigure 3. EBV Peptides Contributing Most to the Separation of Cases With MS and Controls in PLS-DA

eFigure 4. Rate Ratios and 95% CIs for Multiple Sclerosis According to Tertiles of Antibody Response to the Associated EBV Peptides

eFigure 5. Distribution of Odds Ratios (ORs) and Medians From the Simulation of 2 States of Nature

jamaneurol-e240272-s001.pdf (1,006.4KB, pdf)
Supplement 2.

Data Sharing Statement

jamaneurol-e240272-s002.pdf (15.5KB, pdf)

Articles from JAMA Neurology are provided here courtesy of American Medical Association

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