Acute infectious mononucleosis generates persistent, functional EBNA-1 antibodies with high cross-reactivity to alpha-crystalline beta

Krishna Kumar Ganta; Margaret McManus; Ross Blanc; Qixin Wang; Wonyeong Jung; Robin Brody; Mary Carrington; Robert Paris; Sumana Chandramouli; Ryan P McNamara; Katherine Luzuriaga

doi:10.1016/j.celrep.2025.115709

. Author manuscript; available in PMC: 2025 Jul 8.

Published in final edited form as: Cell Rep. 2025 May 13;44(5):115709. doi: 10.1016/j.celrep.2025.115709

Acute infectious mononucleosis generates persistent, functional EBNA-1 antibodies with high cross-reactivity to alpha-crystalline beta

Krishna Kumar Ganta ¹, Margaret McManus ¹, Ross Blanc ^2,³, Qixin Wang ^2,³, Wonyeong Jung ², Robin Brody ¹, Mary Carrington ^2,^4,⁵, Robert Paris ⁶, Sumana Chandramouli ⁶, Ryan P McNamara ^2,^3,^*, Katherine Luzuriaga ^1,^7,^*

PMCID: PMC12235536 NIHMSID: NIHMS2085665 PMID: 40372913

SUMMARY

We investigate the magnitude, specificity, and functional properties of Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1)-specific antibodies in young adults over the course of primary infection. EBNA-1-specific binding antibodies, as well as antibodies capable of antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement deposition (ADCD), are detected. These antibodies primarily target a region of EBNA-1 known to elicit cross-reactive antibodies to several self-peptides. Higher EBNA-1 binding and ADCD antibodies are observed in individuals with at least one HLA-DRB1*15:01 allele. Alpha-crystallin beta (CRYAB) binding and complement-fixing antibodies are detected at 6 months and 1 year following infectious mononucleosis, and CRYAB antibodies are resistant to denaturation, consistent with an affinity-matured response. Blocking experiments show that CRYAB antibodies are cross-reactive with EBNA-1. Altogether, high levels of functional EBNA-1 antibodies are generated in primary EBV infection, some of which are cross-reactive with CRYAB. Further investigation is warranted to determine whether these responses contribute to autoimmunity.

Graphical Abstract

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In brief

Ganta et al. report that durable EBNA-1 antibodies mediating phagocytosis and complement deposition are detected after infectious mononucleosis (IM). Antibodies primarily target a region of EBNA-1 that shares sequence homology with several human proteins. High-avidity, functional antibodies to CRYAB are persistently detected following IM through shared EBNA-1 sequence homology.

INTRODUCTION

Epstein-Barr virus (EBV) establishes persistent infection in >95% of the world’s population by the fourth decade of life.¹ Young children experience only mild or non-specific symptoms with primary EBV infection; however, primary EBV infection in older children, adolescents, and adults commonly causes infectious mononucleosis (IM).¹ EBV has also been implicated in the development of various cancers and autoimmune disorders, most notably multiple sclerosis (MS)^2,3; for example, a history of symptomatic IM increases an individual’s risk of MS 2- to 3-fold.⁴ Evidence supporting a direct causal relationship between EBV and MS was provided by a recent study that compared antibody responses to the entire human virome in a large cohort of young military recruits who developed MS to those who did not develop MS.³ In this study, EBV infection, but not infection with any other virus, conferred an increased risk of developing MS; EBV infection acquired prior to 18 years of age conferred a 26-fold risk, while infection later in life conferred a 32-fold risk of MS.

EBV nuclear antigen 1 (EBNA-1) is a DNA-binding protein necessary for the replication and maintenance of the EBV genome in latently infected cells; it is the only EBV protein expressed in all EBV-associated cancers.^5,6 Multiple studies have linked high levels of EBNA-1 antibodies or T cell responses to a higher risk of development of autoimmune disorders.^3,7 In particular, high levels of antibodies that target a region of EBNA-1 (aa 365–459) with known homology to several human proteins have been linked to an elevated risk of developing MS³; risk is especially high (15-fold) in individuals who have at least one HLA-DRB1*15:01 allele and who are HLA-A*02:01 negative.⁸ A history of IM combined with high EBNA-1 antibody levels in individuals with HLA-DRB1*15:01 and who lack an HLA A*0201 allele further increases the risk (20-fold) of developing MS.⁷

Several recent studies have demonstrated that sequence similarities between EBNA-1 peptides and self-peptides (“molecular mimicry”) result in the generation of autoreactive antibodies or T cell responses, providing a potential mechanism through which EBV may trigger autoimmune disorders. For example, several studies have reported the detection of EBNA-1 binding antibodies that cross-react with epitopes in alpha-crystalline beta (CRYAB),⁹ anoctamin 2 (ANO2),¹⁰ or glial cell adhesion molecule (GlialCAM)¹¹ in individuals with MS.

The present work evaluated EBNA-1-specific antibody magnitude, specificity, and function at presentation with and following acute IM to evaluate how they might contribute to the pathogenesis of EBV-related autoimmune disorders. Through the evaluation of antibody subclass, Fc receptor binding, and functions, including phagocytosis, complement deposition, and cross-reactivity with self-peptides, we provide a deeper understanding of the evolution of EBNA-1-specific and cross-reactive antibodies in primary EBV infection.

RESULTS

High levels of EBNA-1 IgG1 and IgG3 antibodies develop post-IM and target an EBNA-1 region implicated in autoimmunity

Plasma samples were obtained from 97 young adults (median age: 19 years) at IM presentation and again at 6 weeks, 6 months, and 1 year post-IM presentation; 50 EBV-seropositive young adults (median age: 18.8 years); and 10 EBV-seronegative young adults (median age: 18.5 years; Table S1). IM diagnosis was based on clinical symptoms and confirmatory serology, as previously described.^12,13 Age, gender, race, and HLA types of the study cohort are provided in Table S1. When surveyed at the time of specimen collection, 20 EBV-seropositive participants reported a history of IM; 30 EBV-seropositive participants did not recall/did not report a history of IM.

We used a systems serology approach to comprehensively assess antibody responses. To achieve this, we used several peptides spanning the EBNA-1 protein, enabling a comprehensive evaluation of antibody responses to different regions of the antigen (Figure S1). Overall (Figure S2), immunoglobulin (Ig) G1 and IgG3 responses were significantly more pronounced across primary EBV infection compared to IgG2, IgG4, and IgM. IgM was detected early and waned shortly thereafter, consistent with acute infection.^1,14 This was true for all EBNA-1 peptides tested (Figure S2). EBNA-1-specific IgG1 and IgG3 antibodies were infrequently detected at presentation with IM; levels of these antibodies were significantly higher at 6 months and 1 year compared to levels at IM presentation (Figures 1A and 1C). Along with the transient elevation in EBNA-1 IgM levels, the low levels of IgG1 and IgG3 to EBNA-1 during acute-phase and 6-week post-IM diagnosis and their increasing titers over time likely represent a primary antibody response. Antibody class-switching and de novo affinity maturation during this observation period are further supported by IgG1 binding to EBNA-1 in the presence of urea only at later time points (Figure 6). This strongly argues against a recall response where IgG1 and IgG3 can be quickly mobilized from existing memory plasma cells.¹⁵

Figure 1. — (A) Violin and boxplot showing IgG1 responses toward EBNA-1 C-terminal domain peptides at acute presentation (n = 97), 6 weeks (n = 67), 6 months (n = 30), and 1 year (n = 67) post-IM diagnosis. EBV-seropositive (SP-noHx [seropositive without a history of IM], n = 30; and SP-Hx [seropositive with a history of IM], n = 20) and EBV-seronegative (SN; n = 10) controls are also included. The y axis units are IgG1-binding levels quantified through median fluorescence intensity (MFI) as arbitrary units (A.U.).

(B) Overall IgG1-binding heatmap to regions of EBNA-1. Shown on the right-hand side are the peptides used. Influenza hemagglutinin (HA) is used as a positive control, and Ebolavirus glycoprotein (GP) is used as a negative control. At the top are the time points analyzed for the IM cohort, as well as SP and SN individuals as controls. Binding is shown as a fraction of maximum row binding, with the heatmap legend shown on the far right.

(C) Same as (A) but for IgG3 responses to EBNA-1 C-terminal domain peptides.

(D) Same as (B) but for IgG3-binding heatmap to regions of EBNA-1.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Statistical comparisons were conducted across all time points; however, as acute and 6 weeks post-infection did not significantly differ, only acute comparisons and p values were retained to avoid figure crowding; non-significant comparisons are not shown.

Figure 6. — (A) Violin and boxplots showing IgG binding to CRYAB across different EBV infection stages: acute, 6 weeks, 6 months, and 1 year post-IM. EBV-seropositive (SP-noHx, n = 30 and SP-Hx, n = 20) and EBV-seronegative (SN) individuals are included as controls.

(B) Violin and boxplots showing IgG binding to CRYAB in the presence (green) or absence (brown) of 3 M urea across different EBV infection stages: acute, 6 weeks, 6 months, and 1 year post-IM. EBV-SP and EBV-SN individuals are included as controls.

(C) Violin and boxplots showing ADCD to CRYAB across different EBV infection stages: acute, 6 weeks, 6 months, and 1 year post-IM. EBV-SP and EBV-SN individuals are included as controls. See also Figure S6, where we show data for GlialCAM and Ano2.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Comparisons were made for all time points, but as there were no significant differences between acute and 6 weeks post-infection, only acute comparisons and p values are shown to avoid figure crowding; non-significant comparisons are not shown.

EBNA-1 IgG1 and IgG3 antibodies primarily targeted peptides spanning amino acids 365–459, 377–459, and 393–448. At 1 year, EBNA-1-specific IgG1 levels to these peptides did not differ significantly from those measured in EBV-seropositive individuals with or without a history of IM. Of interest, IgG3 antibodies directed at aa 365–420 and 393–448 were similar to those in seropositive individuals with a history of IM but significantly higher at 1 year following IM than levels detected in seropositive individuals without a history of IM (Figures 1B and 1D). Antibodies to these EBNA-1 peptides have previously been reported to be elevated in individuals with MS, with some being cross-reactive to peptides from human proteins.^9–11 Interestingly, binding IgG1 levels to three EBNA-1 peptides (aa 365–420, 377–459, and 393–448) were highly correlated with each other (R > 0.9; Figure S3), suggesting that these peptides target a common immunodominant epitope. Altogether, these data demonstrate that high levels of antibodies to an EBNA-1 region implicated in autoimmunity are commonly detected within 6 months of presentation with IM and persist beyond primary infection.

Higher levels of EBNA-1 binding antibodies are detected in individuals with HLA-DRB1*15:01

Individuals with a history of IM, high titers of an EBNA-1 region implicated in autoimmunity, and certain HLA alleles have a particularly elevated risk of MS.^7,8,16,17 We, therefore, explored the relationship between binding EBNA-1 antibody levels and these reported risk factors. HLA-DRB1*15:01 is the strongest genetic factor associated with elevated MS risk.⁸ Approximately 20% of individuals with IM and seropositive controls in our cohort had at least 1 HLA-DRB1*15:01 allele (Table S1). Individuals with IM with at least one HLA-DRB1*15:01 allele had significantly higher IgG1-binding antibody levels targeting EBNA-1 peptides between aa 365 and 448 at 1 year post-infection compared to those without an HLA- DRB1*15:01 allele (Figure 2A). Individuals with IM and at least one HLA-DRB1*15:01 allele also had significantly higher fold expansion of IgG1 antibodies at 1 year relative to acute levels than those without an HLA-DRB1*15:01 allele (Figure 2B). A mixed-model analysis confirmed a potential interaction between the EBV infection stage and HLA-DRB1*15:01 status. Trend plots (Figure S4) for EBNA-1 IgG1 antibody responses targeting the EBNA-1 region linked to autoimmunity demonstrated significantly higher antibody levels in individuals with HLA-DRB1*15:01 compared to those without the allele; this difference is most pronounced at 1 year post-infection. Additionally, while we observed significant differences in both absolute antibody levels and fold changes from acute infection to 1 year post-IM in individuals with at least one HLA-DRB1*15:01 allele compared to those without the allele, no significant differences in EBNA-1 antibody levels were detected among healthy, EBV-seropositive individuals regardless of HLA-DRB1*15:01 status. These findings highlight the need for further longitudinal studies to determine how EBNA-1 antibody responses evolve over time in relation to HLA genotype and better understand the potential long-term implications of these immune differences. Individuals with at least one HLA-DQB1*06:02 or HLA-DQA1*01:02 allele also showed significantly higher fold changes from acute infection through convalescence, though their absolute antibody levels were not significantly different (Figure S5). Of note, the HLA-DQB1*06:02 and HLA-DQA1*01:02 alleles are in linkage disequilibrium with HLA-DRB1*15:01, and this haplotype confers an elevated risk of MS in European populations.¹⁸ We did not find significant differences in EBNA-1 antibody levels either individually or in combination with other HLA alleles. Altogether, these results link the detection of high titers of antibodies targeting a segment of EBNA-1 that shares sequence homology (Figure S1) with human proteins with an HLA haplotype (HLA-DRB1*15:01, DQB1*06:02, and DQA1*01:02) previously shown to be associated with MS risk.

Figure 2. — (A) Violin and boxplot showing IgG1 responses toward EBNA-1 C-terminal domain peptides (aa 365–420, 377–459, and 393–448) at acute, 6 months, and 1 year post-IM. Groups were distinguished based on being positive (blue) or negative (red) for at least 1 DRB1*15:01 allele. Shown for reference are SP individuals and SP controls in individuals stratified by HLA DRB1*15:01 status.

(B) Fold changes in IgG1 levels at 1 year post-IM relative to acute to EBNA-1 C-terminal domain peptides (aa 365–420, 377–459, and 393–448) were plotted using box and whisker plots for individuals who were positive (blue) or negative (red) for at least one DRB1*15:01 allele. This comparison was made to control potential baseline/acute differences between the groups. The numbers of individuals in the IM cohort with at least one HLA-DRB1*15:01 allele: 19 of 97 participants (20%) at the acute visit, 8 of 30 (27%) participants at 6 months, and 11 of 67 (16%) participants at 1 year. In the SP individuals, there are 11 HLA-DRB1*15:01-positive and 38 HLA-DRB1*15:01-negative participants.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Non-significant comparisons are not shown.

EBNA-1 C-terminal domain-targeting antibodies are polyfunctional through FcγR engagement

A growing body of evidence indicates that antibody engagement of effectors through their Fc domains is critical for mediating immune effector functions, including phagocytosis and complement activation, that are associated with infection outcomes.¹⁹ We, therefore, measured the binding of EBNA-1 antibodies to Fc receptors. EBNA-1 antibodies capable of binding Fc receptors FcγRIIa and FcγRIIIa were detected at 6 months and 1 year and again primarily targeted peptides between aa 365 and 459 (Figure 3). Thus, the timing of the development of antibodies capable of engaging Fc receptors, as well as the antibody specificities, was similar to that observed for binding antibodies. This differs from reports of some vaccine responses, where binding antibodies persisted but FcR-binding antibodies waned,²⁰ suggesting a durable, functionally active antibody response after IM.

Figure 3. — (A) Violin and boxplot showing FcγRIIA-binding antibody responses toward EBNA-1 C-terminal domain peptides at acute presentation (n = 97), 6 weeks (n = 67), 6 months (n = 30), and 1 year (n = 67) post-IM diagnosis. EBV-seropositive (SP-noHx, n = 30 and SP-Hx, n = 20) and EBV-seronegative (SN; n = 10) controls are also included as controls. The y axis units are FcγRIIA-binding antibody levels quantified through MFI as arbitrary units (A.U.).

(B) Overall FcγRIIA-binding antibody heatmap to regions of EBNA-1. Shown on the right-hand side are the peptides used. Influenza HA is used as positive control, and Ebolavirus GP is used as a negative control. On top are the time points analyzed for the IM cohort, as well as SP and SN individuals as controls. Binding is shown as a fraction of maximum row binding, with the heatmap legend shown on the far right.

(C) Same as (A) but for FcγRIIIA-binding antibody responses toward EBNA-1 C-terminal domain peptides.

(D) Same as (C) but for FcγRIIIA-binding antibody heatmap to regions of EBNA-1.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Statistical comparisons were performed across all time points; however, as no significant differences were found between acute and 6 weeks post-infection, only acute comparisons and p values were retained to avoid figure crowding; non-significant results are not shown.

We evaluated the functional capacity of antibodies targeting EBNA-1 peptides using an assay that measured phagocytosis of immune complexes composed of plasma antibodies bound to EBNA-1 peptide (aa 365–420, 377–459, and 393–448)-coated beads. Due to limitations in multiplexing, antibody-dependent cellular phagocytosis (ADCP) assays were performed on only three peptides using a neutravidin-biotin interaction system. Plasma ADCP activity at the 6-month and 1-year visits differed significantly from ADCP activity in plasma samples from the acute visit (Figure 4); plasma ADCP activity was also detected in plasma samples from EBV-seropositive individuals. This is consistent with the persistently high FcγR-binding antibodies in this cohort. We did not observe significant differences in the levels of binding to Fc receptors or ADCP responses between seropositive individuals with or without a history of IM.

Figure 4. — Violin and boxplots showing antibody-dependent cellular phagocytosis by monocytes (ADCP) quantified through phagoscores (see STAR Methods). Phagoscores were quantified against EBNA-1 peptides (aa 377–459, 365–420, and 393–448) across different EBV infection stages: acute, 6 weeks, 6 months, and 1 year post-IM. EBV-seropositive (SP-noHx, n = 30 and SP-Hx, n = 20) and EBV-seronegative (SN) individuals are included as controls.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Statistical comparisons were conducted across all time points; however, as acute and 6 weeks post-infection did not significantly differ, only acute comparisons and p values were retained to avoid figure crowding; non-significant comparisons are not shown.

EBNA-1 antibodies mediate complement, activation, and deposition

Having measured high levels of EBNA-1 binding antibodies and Fc receptor binding, we next evaluated the capacity of these antibodies to activate and fix complement. Antibodies targeting EBNA-1 peptides and capable of fixing complement were detected at significantly higher levels at 6 months and 1 year than at IM presentation (Figures 5A and 5B). A mixed-model analysis (Figure 5C) revealed that by 1 year post-infection, individuals with at least one DRB1*1501 allele had significantly higher antibody-dependent complement deposition (ADCD) activity compared to individuals without a DRB1*1501 allele in response to EBNA-1 C-terminal peptides 365–420 (p < 0.001) and 377–459 (p < 0.001).

Figure 5. — (A) Violin and boxplots showing antibody-dependent complement deposition (ADCD) quantified through C3 deposition units for EBNA-1 peptides (aa 377–459, 365–420, and 393–448) across different EBV infection stages: acute, 6 weeks, 6 months, and 1 year post-IM. EBV-seropositive (SP-noHx, n = 30 and SP-Hx, n = 20) and EBV-seronegative (SN) individuals are included as controls.

For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. Statistical comparisons were conducted across all time points; however, as acute and 6 weeks post-infection did not significantly differ, only acute comparisons and p values were retained to avoid figure crowding; non-significant comparisons are not shown.

(B) Overall ADCD to regions of EBNA-1. Shown on the right-hand side are the peptides used. Influenza HA is used as positive control, and Ebolavirus GP is used as negative control. At the top are the time points analyzed for the IM cohort, as well as SP and SN individuals as controls. The scale is shown as a fraction of the maximum row ADCD, with the heatmap legend shown on the far right.

(C) Line graphs showing ADCD over time to EBNA-1 peptides (aa 377–459, 365–420, and 393–448) for individuals who were positive (blue) or negative (red) for at least one DRB1*15:01 allele. Statistical analysis was performed using a linear mixed model with interaction terms for EBV infection stage and DRB1*15:01 status. Significant increases in ADCD were observed at 1 year compared to acute, 6 weeks, and 6 months for all peptides (p < 0.001). Interaction effects at the 1-year time point revealed that DRB1*15:01-positive individuals showed a more pronounced ADCD response compared to those negative for the peptides aa 377–459 (p = 0.000737) and 365–420 (p = 0.000875). No significant interaction effects were observed at earlier time points.

Complement-fixing EBNA-1 antibodies were also detected in EBV-seropositive individuals. On a questionnaire completed at the first study visit, 30 EBV-seropositive individuals in our cohort did not recall a history of IM, while 20 seropositive individuals reported a prior diagnosis of IM. Significantly higher levels of EBNA-1 antibodies capable of complement fixing were detected in individuals who related a history of IM compared to individuals without a history of IM (Figure 5A).

High levels of binding and ADCD antibodies to CRYAB are detected following IM

We next evaluated whether individuals with IM develop antibody responses to self-peptides (GlialCAM, ANO2, and CRYAB) that share sequence homology with EBNA-1 (Figure S1). Figure 6A shows that binding IgG antibodies targeting CRYAB were detected at 6 months and 1 year. The detection of binding antibodies to self-proteins in our IM cohort is compatible with at least 2 reports of transient detection of antibodies to self-peptides in IM^21,22; in our study, binding IgG antibodies to CRYAB were not transient but persisted through 1 year and were also detected in EBV-seropositive individuals. As mentioned earlier, we observed significantly higher EBNA-1 antibody levels in individuals with HLA-DRB1*15:01 compared to individuals without DRB1*15:01; however, this difference was not observed for CRYAB-specific antibody responses. Additionally, no significant difference was observed between seropositive individuals with and without a history of IM. Of note, we did not detect antibodies to CRYAB in EBV-seronegative individuals, nor did we detect antibodies to ANO2 or GlialCAM in any individuals at or after presentation with IM (Figure S6).

We next evaluated the susceptibility to antibody binding to dissociation on exposure to urea. This approach has been previously used to distinguish avid antibodies from non-specific antibodies.^23–25 Interestingly, antibody binding was resistant to dissociation in the presence of urea (Figure 6B), implying that the CRYAB antibodies detected at 6 months and 1 year following IM, as well as those detected in EBV-seropositive individuals, were of high avidity.

The complement system is increasingly recognized for its role in the pathogenesis of neuroinflammatory and neurodegenerative diseases, including MS.²⁶ We, therefore, went on to evaluate whether the CRYAB IgG antibodies detected in binding assays were capable of mediating ADCD. We observed a significant increase in ADCD activity to CRYAB from the acute phase of IM through 6 months and 1 year; we also detected CRYAB-specific ADCD in seropositive individuals (Figure 6) but not in EBV-seronegative individuals.

CRYAB antibodies are cross-reactive with EBNA-1 antibodies

To further test whether EBNA-1 antibodies could bind to CRYAB, we set up an antigen-specific antibody depletion assay using a peptide-blocking approach.⁹ Serum samples were spiked with EBNA-1 peptides to deplete EBNA-1-specific antibodies, and their cross-reactivity with CRYAB was assessed. If EBNA-1 antibodies were cross-reactive, then their depletion would result in a lack of detectable binding to CRYAB. To validate the specificity of this approach, we performed a control blockade by spiking the same samples with flu peptides to ensure that flu-specific antibodies do not interfere with anti-CRYAB antibody levels. Additionally, viral capsid antigen (VCA) IgG levels were also measured under both blockade conditions to confirm that the depletion is specific to EBNA-1 or flu antibodies without affecting other antibody populations.

Anti-CRYAB IgG levels were significantly reduced (>90%) when plasma samples from convalescent and seropositive individuals were depleted with EBNA-1 peptides (365–420, 377–449, and 393–449), indicating a specific cross-reactivity between CRYAB antibodies and EBNA-1 (Figure 7). By contrast, treatment with a flu-hemagglutinin (HA) peptide did not reduce CRYAB total IgG levels, confirming the specificity of this cross-reactivity. Anti-flu-HA total IgG reactivity was reduced by the flu-HA peptide but not by EBNA-1 peptides; anti-VCA IgG levels remained unchanged under both blocking conditions, demonstrating the specificity of the observed CRYAB and EBNA-1 cross-reactivity. Altogether, these results confirm that CRYAB and EBNA-1 cross-reactive antibodies are commonly generated shortly following primary EBV infection.

Figure 7. — (A) Schematic of the blocking assay: EBNA-1 peptides were used to deplete Igs targeting the viral protein. If these antibodies are cross-reactive with CRYAB, then the EBNA-1 immunodepletion should result in a loss of CRYAB binding. As a control, the same serum was blocked with flu-HA peptides to control non-specific immunodepletion. Lastly, responses to VCA were quantified for both EBNA-1 and flu-HA immunodepletions, as this is not cross-reactive with either or should not result in any binding loss.

(B) Violin and boxplots depicting IgG antibody reactivity to CRYAB, flu-HA, and EBV VCA across different treatments (EBNA-1 peptides or flu-HA peptides). Samples included pooled 6 months–1 year (n = 55), EBV seropositive (SP; n = 25), and EBV seronegative (SN; n = 10). For all comparisons, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a Wilcoxon test followed by Bonferroni correction for multiple comparisons. All time points were statistically compared; however, given the lack of significant differences between acute and 6 weeks post-infection, only acute comparisons and p values were shown to avoid figure crowding; non-significant comparisons are not shown.

DISCUSSION

Multiple epidemiologic studies over the years have linked EBV infection to the risk of developing autoimmune disorders, including MS.^2,27,28 A history of symptomatic IM confers additional risk (2- to 3-fold).^4,27 Molecular mimicry between EBNA-1 and self-peptides has been proposed as a plausible mechanism.^9–11,22 We thus undertook this study, which aimed to characterize the development of EBNA-1-specific antibody responses post-IM and determine whether EBNA-1 antibodies cross-reactive with self-peptides are generated following primary EBV infection. High levels of IgG1-, IgG3-, and FcγR-binding antibodies were detected against the EBNA-1 C-terminal domain at 6 months and 1 year following IM (Figures 1, 2, and 3). EBNA-1-specific antibodies demonstrated strong functional activity, including antibody-mediated phagocytosis and complement deposition (Figures 4 and 5). Levels of binding and complement-fixing EBNA-1 antibodies were higher in individuals with an HLA-DRB1*15:01 allele than in individuals without HLA-DRB1*1501 (Figures 2 and 5). These EBNA-1 C-terminal domain antibodies showed high cross-reactivity and avidity to CRYAB (Figures 6 and 7). Other self-targeting antibodies, such as those to ANO2 and GlialCAM, were not detected.

EBNA-1 plays a key role in EBV persistence within latently infected B cells and elicits antibody responses in most individuals with EBV infection. Prior studies have identified a region of EBNA-1 associated with autoimmune diseases.¹⁷ For example, several studies have documented elevated levels of binding antibodies specifically targeting EBNA-1 aa 365–448 or EBNA-1 aa 377–459 in individuals with MS.^3,29 A recent case-control study of military recruits described an elevated risk of developing MS in individuals with high EBNA-1 antibodies to C-terminal peptides (particularly aa 365–420). In these individuals, elevated levels of serum neurofilament light chain, a marker for neuronal damage, were detected a median of 6 years prior to MS onset.³ In our study, we demonstrate a significant increase in the levels of antibodies targeting these EBNA-1 peptides within 6 months of IM.

Significantly higher levels of IgG1-binding antibodies against C-terminal EBNA-1 peptides were detected in individuals carrying at least one HLA-DRB1*15:01 allele, which has been identified as the strongest identified genetic risk factor for MS.³⁰ Our data are compatible with prior studies demonstrating an association between elevated binding antibody responses to EBNA-1 and HLA-DRB1*15:01.^7,17 Interestingly, we also observed that individuals carrying alleles (DQB1*06:02 and DQA1*01:02) in linkage disequilibrium with HLA DRB1*1501³¹ exhibited significantly higher fold changes in EBNA-1 antibody levels over time (Figure S5). This suggests that this haplotype influences antibody production and function in ways that warrant further investigation.

Many individuals with elevated EBNA-1 IgG-binding antibody titers also had antibodies capable of mediating phagocytosis and complement deposition (Figures 4 and 5). However, we also identified individuals with lower binding antibody levels and high functional activity, implying that antibody efficiency can compensate for lower titers,³² likely driven by the antibody’s Fc region and its ability to engage Fc receptors.^19,33 This finding underscores the importance of measuring both binding and functional activity to fully understand the mechanisms through which antibodies may be active in disease pathogenesis. Our mixed-model analysis revealed that individuals with the HLA-DRB1*15:01 allele showed significantly higher levels of complement deposition activity at 1 year post-infection, particularly in response to EBNA-1 peptides within amino acids 365–420 and 377–459 (Figure 5C). Of interest is that studies of postmortem brain tissue from individuals with MS have consistently shown complement deposition in white matter plaques and gray matter lesions, suggesting that antibody-mediated complement activation may play a role in disease pathogenesis.^26,34

The detection of binding and complement-fixing antibodies to CRYAB in most individuals with IM, as well as their detection at high levels in healthy, EBV-seropositive individuals, was surprising. The detection of antibodies cross-reactive between EBNA-1 and CRYAB, a protein expressed in oligodendrocytes, was also surprising. The urea dissociation assays suggest that affinity-matured IgG responses, rather than IgM, are primarily responsible for this complement-fixing activity. While prior studies have demonstrated the presence of EBNA-1 antibodies cross-reactive with CRYAB in individuals with MS,⁹ it is important to note that our study did not study individuals with IM long enough to determine any association between the detection of cross-reactive antibodies in IM and eventual development of MS. Our findings indicate that the generation of EBNA-1 and CRYAB cross-reactive antibodies occurs frequently in IM and in excess of the number of individuals expected to develop MS following IM. Thus, factors in addition to the development of CRYAB or EBNA-1-CRYAB cross-reactive antibodies likely contribute to the ultimate development of MS.

EBNA-1 is a nuclear antigen, and CRYAB is a cytosolic protein, which raises the question of how antibody responses are generated to these proteins. Viral infection³⁵ or cellular stress can lead to cell surface expression or extracellular release of intracellular proteins, rendering them targets for immune recognition. Antibodies activating the complement system can exacerbate inflammation and tissue damage and have been detected in MS lesions.^34,36 The latter suggests that ADCD antibodies may be active in MS; however, we acknowledge that our data do not provide direct evidence to support this.

Limitations of the study

Our study has several limitations that must be acknowledged. First and foremost, the follow-up period in this study was limited to 1 year post-infection. As a result, we are unable to determine whether the elevated antibody responses and functional activities observed, particularly in individuals with the DRB1*1501 allele, persist beyond this time frame or whether they are associated long term with the development of MS. Additionally, while we identified the frequent development of cross-reactive antibodies to EBNA-1 and CRYAB following IM, our study does not provide evidence of a direct pathological role for these antibodies in vivo. Retrospective analysis of longitudinal samples from individuals with MS, as well as long-term follow-up of individuals experiencing IM, may provide insights into whether these early immune responses are predictive of future disease risk. The history of IM in seropositive individuals was self-reported, and medical records were not available to confirm participant reports. Although gender is known to influence immune responses, we did not observe statistically significant differences in EBNA-1 binding antibodies or functional antibody responses between males and females. However, given the gender imbalance across study groups, future studies may be helpful in evaluating potential sex-specific immune variations. Another limitation of our study is that due to limited plasma availability, we were unable to fractionate IgG1 and IgG3 separately to assess their individual contributions to cross-reactivity with CRYAB. Instead, cross-reactivity was evaluated using total IgG, which does not allow for subclass-specific interpretation. Future studies using purified IgG subclasses will be necessary to determine whether IgG1, IgG3, or both are responsible for CRYAB cross-reactivity. Finally, while we focused on the antibody responses and their functional capacities, other aspects of the immune response, such as T cell responses, were not evaluated. Future research integrating analyses of cell-mediated immunity could provide a more comprehensive, systems-level understanding of the immune response to EBV infection in IM and its role in autoimmunity.

In conclusion, our study documents the generation of high levels of antibodies targeting the C terminus of EBNA-1 following IM. Levels of EBNA-1 binding and complement-fixing antibodies were highest in individuals with the HLA-DRB1*15:01 allele. The sustained antibody responses, their ability to activate complement, and their cross-reactivity with a central nervous system protein, CRYAB, suggest that EBNA-1-specific antibodies generated in early infection may play a role in the initiation of MS. Additional studies defining whether EBNA-1 antibody specificity and function can aid in early identification of individuals at risk of developing MS, as well as their potential role in the development of disease, could inform the development of strategies for early diagnosis and therapeutic intervention.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Katherine Luzuriaga (mailto:katherine.luzuriaga@umassmed.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Luminex and flow cytometry mean fluorescence intensity (MFI) data generated for this study have been deposited to GitHub at https://github.com/HSPHSystemsSerology/ProjID_KG20241203 and will be publicly available upon publication. The repository link is listed in the key resources table.
This paper does not report the original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-guinea pig complement C3 goat IgG fraction, FITC	MP Biomedicals	Cat # 0855385, RRID:AB_2334913
Anti-human IgG1 PE [HP6001]	Southern Biotech	Cat #9054–09, RRID:AB_2796628
Anti-human IgG3 PE [HP6050]	Southern Biotech	Cat #9210–09, RRID:AB_2796701
Biological samples
LowTox Guinea Pig Complement	CedarLane Labs	Cat # CL4051
Chemicals, peptides, and recombinant proteins
Human soluble FcγR2A	Duke University	Custom Order
Human soluble FcyR3A	Duke University	Custom Order
LC-LC-Sulfo-NHS Biotin	ThermoFisher	Cat # A35358
Streptavidin-R-PE	Prozyme	Cat # PJ31S
Recombinant Epstein-Barr virus 1–90	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 377–459	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 460–641	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 29–84	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 365–420 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 393–448 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 393–448 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 421–476 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 589–641 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 589–641 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 1–56 GD1	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 1–56 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 421–476 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 449–504n B95-8	SinoBiological	Custom Synthesis
Influenza AH1N1(A/Ohio/UR06-0091/2007)	SinoBiological	Custom Synthesis
Ebolavirus glycoprotein (GP) (Subtype Bundibugyo, strain Uganda 2007) GP1/Glycoprotein histag(40368-V08B)	SinoBiological	Custom Synthesis
CRYAB (aa 1–27): MDIAIHHPWIRRPFFPFHSPSRLFDQFGGGEQKLISEEDLGGGMDIAIHHPWIRRPFFPFHSPSRLFDQFGGGHHHHHH	Alan Scientific	Custom Synthesis
GlialCAM (aa 370–389): ATGRTHSSPPRAPSSPGRSRGGGEQKLISEEDLGGGATGRTHSSPPRAPSSPGRSRGGGEQKLISEEDLHHHHHH	Alan Scientific	Custom Synthesis
ANO2 (aa 140–149): PGDIELGPLDGGGEQKLISEEDLGGGPGDIELGPLDGGGEQKLISEEDLGG HHHHHH	Alan Scientific	Custom Synthesis
Myc control (spacer): EQKLISEEDL GGG EQKLISEEDL GGG EQKLISEEDL GGG EQKLISEEDLGGG HHHHHH	Alan scientific	Custom Synthesis
Critical commercial assays
NHS-Sulfo-LC-LC Kit	ThermoFisher	21435
Zeba-Spin Desalting and Chromatography Columns	ThermoFisher	89882
EDC	ThermoFisher	22980
Deposited data
Luminex and Flow Cytometry MFI Data	GitHub	HSPHSystemsSerology/ProjID_KG20241203)
Experimental models: Cell lines
THP-1 monocytes	ATCC	ATCC TIB-202/RRID: CVCL_0006
Software and algorithms
iQue Forecyt 9.1	Sartorius	60028
R Studio V 4.0	R Project for Statistical Computing	RRID:SCR_000432
Other
384-well HydroSpeed Plate Washer	Tecan	30190112
IntelliFlex Luminex Dual-Report	Luminex	APX2021
iQue Screener Plus	Intellicyt/Sartorius	11811
MagPlex Microspheres	Diasorin	MC12001-01 (Cataloged by region)
Scarlet Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis
Green Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis
Red Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis

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STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

IM cohort

Ninety-seven individuals who presented with IM were enrolled into this study. IM diagnosis was based on clinical symptoms and confirmatory serology, as previously described.^12,13 Of the 97 initial enrollees, 67 had sufficient volumes of plasma available for testing at IM presentation through 1 year following IM. A smaller number (N = 30) had sufficient plasma sample volumes for testing only at IM presentation and 6 months. Fifty healthy EBV seropositive young adults (median age 18.8 years) and 10 healthy EBV seronegative young adults (Median age 18.5 yrs) were also studied. When surveyed at the time of specimen collection, 20 EBV seropositive participants reported a history of IM; 30 EBV seropositive participants did not recall a history of IM. Age, gender, race, and HLA types of the study cohort are provided in Table S1.

Cell line authentication

THP-1 cells (ATCC TIB-202), a human monocytic leukemia cell line, were purchased from the American Type Culture Collection (ATCC). Cells were used within five passages of thawing and maintained as suspension cultures according to ATCC recommendations. Authentication was performed by ATCC using short tandem repeat (STR) profiling. The cell line was confirmed to express key monocytic markers including HLA (A2, A9, B5, DRw2), complement C3, and Fc receptors, and retains phagocytic activity. All cultures were routinely tested and confirmed to be free of mycoplasma contamination.

Ethical considerations

This project was approved by the Institutional Review Boards (IRBs) at UMass Chan Medical School and Massachusetts General Hospital. Written informed consent was obtained from all participants.

METHOD DETAILS

HLA typing

HLA class I and II genotyping was performed using a targeted next-generation sequencing method as described previously³⁷ with some modifications. Locus-specific primers were used to amplify 25 polymorphic exons of HLA-A and -B (exons 1–4), HLA-C (exons 1–5), HLA-E (exon 3), DPA1 (exon 2), DPB1 (exons 2–4), DQA1 (exons 1–3), DQB1 (exons 2 and 3), DRB1 (exons 2 and 3), and DRB3, 4, 5 (exon 2) genes with Fluidigm Access Array System and Juno LP 48.48 IFCs (Fluidigm). The 25 Fluidigm PCR amplicons were pooled and subjected to sequencing on an Illumina MiSeq sequencer (Illumina). HLA alleles and genotypes were called using the Omixon HLA Explore (version 2.0.0) software (Omixon).³⁷

Antigens

EBNA-1 peptides spanning EBNA-1 minus the repeat sequences (Figure S1) were synthesized by Sino Biologicals (key resources table). Cross-reactive peptides were synthesized by Alan Scientific (key resources table).

Antibody isotypes and Fc-receptor binding activities measurement

The level of antibodies and Fc-receptor binding activities were measured based on multiplexing Luminex microsphere-based method.^15,38 Luminex beads were coupled to EBNA-1 peptides or cross-reactive peptides using a previously described method.²⁰ Briefly, the carboxylate microspheres (Luminex) were conjugated covalently with the antigens (key resources table) via NHS-ester linkages after activation from co-incubation with Sulfo-HNS and EDC (ThermoFisher). Sera were diluted at 1:200 for IgG1 and IgG3, 1:25 for IgG2, IgG4, IgM, and IgA, and 1:400 for Fc gamma receptors IIA and IIIA (FcγRIIA and FcγRIIIA). The samples were incubated with coupled microspheres in 384-well plates for 16 h at 4°C, shaking at 750 rpm, to form immune complexes. Then, the plates were washed with assay buffer (0.1% BSA and 0.02% Tween 20 in PBS) 3 times and incubated with PE-conjugated mouse anti-human antibodies for 1 h at room temperature, at 750 rpm. Then, the plates were washed with the assay buffer 3 times, and all the samples were acquired by iQue Screener Plus (Intellicyt/Sartorius), or IntelliFlex Luminex Dual-Report (Luminex), and the median fluorescence intensities (MFI) were measured. All samples were performed in technical duplicates and averaged for final output.

Antibody-dependent complement deposition

Antibody-dependent complement deposition (ADCD) was conducted using a 384-well plate format to evaluate the functional activity of antibodies in plasma samples.³⁹ A Multiplex bead mixture was prepared by adding 15 μL of individual EBNA-1 peptide antigen-coupled beads to 18 mL of Luminex assay buffer. antigen-coated beads were incubated with individual plasma samples diluted at 1:10 for 2 h at 37°C to facilitate immune complex formation. After incubation, the beads were washed to eliminate unbound material. Guinea pig complement (Cedarlane), reconstituted according to the manufacturer’s instructions, was added in gelatin veronal buffer containing calcium and magnesium (GBV++; Boston BioProducts) and incubated for 30 min. Complement component C3 deposition was then detected using a fluorescein-conjugated goat anti-guinea pig C3 antibody (MP Biomedicals) and results were acquired by flow cytometry with iQue (Intellicyt).³⁹

Antibody-dependent cellular phagocytosis

Antibody-dependent cellular phagocytosis by monocytes (ADCP) was conducted to assess the phagocytic activity of THP-1 cells in response to antigen-coated beads opsonized with antibodies from plasma samples, following a previously published method.⁴⁰ Briefly, biotinylated antigens were conjugated to Neutravidin FTIC beads, incubated with diluted plasma, and then exposed to THP-1 cells. After incubation and washing, cells were fixed and analyzed using the (iQue, Intellicyt). The phagoscore is calculated by taking the mean fluorescence intensity (MFI) of the beads that have been engulfed by the cells and multiplying it by the percentage of cells that have taken up the beads out of the total cell population. This product is then divided by 10,000 to yield the final phagoscore. The phagoscore provides a measure of both the intensity of bead uptake and the proportion of cells involved in phagocytosis, offering a comprehensive assessment of phagocytic activity in the sample.

Peptide blocking assay to evaluate EBNA-1 and alpha crystalline beta cross-reactivity

Plasma samples from individuals with CRYAB antibody levels above the median (MFI = 1979) were selected for the peptide blocking assay⁹ (IM cohort: 6 months (n = 11), 1-year (n = 44), Seropositive (n = 25), Seronegative (n = 10). Plasma samples were diluted at 1:50 and treated overnight with 1 μM each of the EBNA-1 peptides (aa 377–449, 365–420, 393–448). Influenza HA was used as a standardized control. Following incubation, CRYAB-coupled Luminex beads were added to the plasma samples and incubated for 2 h. The samples were then washed three times with Luminex assay buffer. After washing, total IgG anti-human secondary antibody tagged with PE was added and incubated at room temperature for 1 h. This was followed by three additional washes with Luminex assay buffer (PBS 1X, 0.01% BSA, and 0.05% Tween 20). Binding was quantified using the xMAP INTELLIFLEX System.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were performed using R software (version 4.4.1, 2024-06-14, ucrt). All graphical visualizations were done using ggplot2⁴¹ (version 3.5.1) package. Central tendency and dispersion are visualized using box and violin plots, which reflect median values and distribution across the cohort. Specific statistical comparisons are detailed in each figure legend. To control differences in overall antibody levels between samples, MFI values obtained from all Luminex-based assays were normalized by dividing each value by the total IgG concentration measured in the corresponding plasma sample. To compare antibody responses across different EBV infection stages, we used Wilcoxon paired comparisons, with p-values adjusted for multiple comparisons using the False Discovery Rate (FDR) correction. For comparisons between DRB1*1501 positive and DRB1*1501 negative individuals, or other HLA alleles of interest, the two-tailed Mann-Whitney U test (Wilcoxon rank-sum test) was employed, with FDR correction applied to p-values. Fold changes between the acute and 1-year time points were calculated and these fold changes were compared between DRB1*1501 positive and DRB1*1501 negative groups using the Mann-Whitney U test. The lower limit of detection (LLOD) is defined as the mean of EBV seronegative (n = 10) values plus three times the standard deviation. Values below the LLOD were set to the lower limit of detection before analysis. All p-values reported throughout the manuscript are adjusted for multiple comparisons using the Benjamini-Hochberg false FDR correction, unless otherwise noted. Statistical significance was defined as adjusted p values p < 0.05, with significance levels denoted by stars: ***p < 0.001, **p < 0.01, *p < 0.05. Results that did not generate significant comparisons were left blank.

Mixed model analysis

We used linear mixed-effects models (LMMs) to assess EBNA-1-specific antibody responses over time. Separate models were conducted for each peptide and antibody measurement (including IgG1 and complement deposition) with fixed effects for EBV infection timepoint (Acute, 6 weeks, 6 months, 1 year), HLA-DRB1*1501 status (Positive or Negative), and their interaction (EBV infection timepoint × DRB1*1501 status). Participant ID was included as a random intercept to account for repeated measures. Models were fitted using restricted maximum likelihood (REML) via the lmer() function from the lme4 package (version 1.1.36) in R (version 4.4.1, 2024-06-14, ucrt), with significance testing performed using the lmerTest package (version 3.1.3), applying the Satterthwaite approximation for degrees of freedom estimation. Post-hoc contrasts and pairwise comparisons were conducted using the emmeans package (version 1.10.6), with Tukey adjustment for multiple comparisons. Model residuals were assessed to ensure normal distribution and equal variance of residuals. Reported p-values reflect these adjusted post-hoc comparisons unless otherwise specified, with p < 0.05 considered statistically significant, with significance levels denoted by stars: ***p < 0.001, **p < 0.01, *p < 0.05. Results that did not generate significant comparisons were left blank.

Supplementary Material

NIHMS2085665-supplement-1.pdf^{(879.8KB, pdf)}

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.115709.

Highlights.

Infectious mononucleosis induces EBNA-1 antibodies that bind and fix complement
HLA-DRB1*15:01+ individuals have higher EBNA-1 antibodies
EBNA-1 antibodies target peptides with sequence homologies to human proteins
CRYAB antibodies cross-reactive with EBNA-1 are detected in most individuals

ACKNOWLEDGMENTS

We would like to extend our gratitude to the individuals who participated in these studies. We thank Yuko Yuki for HLA typing. Funding for this project was provided by Moderna Therapeutics to R.P.M. and K.L., and clinical research resources were provided by the UMass Center for Clinical and Translational Science (NIH UL1TR001453). This project has been funded in whole or in part with federal funds from the Frederick National Laboratory for Cancer Research under contract no. 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. This research was supported in part by the Intramural Research Program of the NIH, Frederick National Laboratory for Cancer Research.

DECLARATION OF INTERESTS

R.P.M. serves as an advisor/consultant to the International Vaccine Institute (IVI). K.L. has consulted for Gilead Sciences, Inc., and Sanofi; has received research funding from the National Institutes of Health and Moderna, Inc.; and has received funding for clinical research from Gilead Sciences, Inc., Moderna, Inc., and Pfizer, Inc. S.C. and R.P. are employees of Moderna, Inc., and hold stock/stock options in the company.

REFERENCES

1.Luzuriaga K, and Sullivan JL (2010). Infectious mononucleosis. N. Engl. J. Med 362, 1993–2000. 10.1056/NEJMcp1001116. [DOI] [PubMed] [Google Scholar]
2.Ascherio A, Munger KL, Lennette ET, Spiegelman D, Hernán MA, Olek MJ, Hankinson SE, and Hunter DJ (2001). Epstein-Barr virus antibodies and risk of multiple sclerosis: a prospective study. JAMA 286, 3083–3088. 10.1001/jama.286.24.3083. [DOI] [PubMed] [Google Scholar]
3.Bjornevik K, Cortese M, Healy BC, Kuhle J, Mina MJ, Leng Y, Elledge SJ, Niebuhr DW, Scher AI, Munger KL, and Ascherio A (2022). Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296–301. 10.1126/science.abj8222. [DOI] [PubMed] [Google Scholar]
4.Nielsen TR, Rostgaard K, Nielsen NM, Koch-Henriksen N, Haahr S, Sørensen PS, and Hjalgrim H (2007). Multiple sclerosis after infectious mononucleosis. Arch. Neurol 64, 72–75. 10.1001/archneur.64.1.72. [DOI] [PubMed] [Google Scholar]
5.Frappier L (2012). The Epstein-Barr Virus EBNA1 Protein. Scientifica (Cairo) 2012, 438204. 10.6064/2012/438204. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Frappier L (2015). EBNA1. In Epstein Barr Virus Volume 2: One Herpes Virus: Many Diseases, Münz C, ed. (Springer International Publishing; ), pp. 3–34. 10.1007/978-3-319-22834-1_1. [DOI] [Google Scholar]
7.Hedstrom AK, Huang J, Michel A, Butt J, Brenner N, Hillert J, Waterboer T, Kockum I, Olsson T, and Alfredsson L (2019). High Levels of Epstein-Barr Virus Nuclear Antigen-1-Specific Antibodies and Infectious Mononucleosis Act Both Independently and Synergistically to Increase Multiple Sclerosis Risk. Front. Neurol 10, 1368. 10.3389/fneur.2019.01368. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Sundqvist E, Sundström P, Lindén M, Hedström AK, Aloisi F, Hillert J, Kockum I, Alfredsson L, and Olsson T (2012). Epstein-Barr virus and multiple sclerosis: interaction with HLA. Genes Immun. 13, 14–20. 10.1038/gene.2011.42. [DOI] [PubMed] [Google Scholar]
9.Thomas OG, Bronge M, Tengvall K, Akpinar B, Nilsson OB, Holmgren E, Hessa T, Gafvelin G, Khademi M, Alfredsson L, et al. (2023). Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv 9, eadg3032. 10.1126/sciadv.adg3032. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Tengvall K, Huang J, Hellström C, Kammer P, Biström M, Ayoglu B, Lima Bomfim I, Stridh P, Butt J, Brenner N, et al. (2019). Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk. Proc. Natl. Acad. Sci. USA 116, 16955–16960. 10.1073/pnas.1902623116. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Lanz TV, Brewer RC, Ho PP, Moon JS, Jude KM, Fernandez D, Fernandes RA, Gomez AM, Nadj GS, Bartley CM, et al. (2022). Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321–327. 10.1038/s41586-022-04432-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Weiss ER, Alter G, Ogembo JG, Henderson JL, Tabak B, Bakiş Y, Somasundaran M, Garber M, Selin L, and Luzuriaga K (2017). High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis. J. Virol 91, e01562–16–e01516. 10.1128/JVI.01562-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Weiss ER, Lamers SL, Henderson JL, Melnikov A, Somasundaran M, Garber M, Selin L, Nusbaum C, and Luzuriaga K (2018). Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection. J. Virol 92, e01466–17. 10.1128/JVI.01466-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Henie G, Henle W, and Horwitz CA (1974). Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J. Infect. Dis 130, 231–239. 10.1093/infdis/130.3.231. [DOI] [PubMed] [Google Scholar]
15.McNamara RP, Maron JS, Boucau J, Roy V, Webb NE, Bertera HL, Barczak AK, Positives Study Staff T, Franko N, Logue JK, et al. (2023). Anamnestic humoral correlates of immunity across SARS-CoV-2 variants of concern. mBio 14, e0090223. 10.1128/mbio.00902-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.International Multiple Sclerosis Genetics Consortium, Hafler DA, Compston A, Sawcer S, Lander ES, Daly MJ, De Jager PL, de Bakker PIW, Gabriel SB, Mirel DB, et al. (2007). Risk alleles for multiple sclerosis identified by a genomewide study. N. Engl. J. Med 357, 851–862. 10.1056/NEJMoa073493. [DOI] [PubMed] [Google Scholar]
17.Sundstrom P, Nystrom M, Ruuth K, and Lundgren E (2009). Antibodies to specific EBNA-1 domains and HLA DRB1*1501 interact as risk factors for multiple sclerosis. J. Neuroimmunol 215, 102–107. 10.1016/j.jneuroim.2009.08.004. [DOI] [PubMed] [Google Scholar]
18.Martin R, Sospedra M, Eiermann T, and Olsson T (2021). Multiple sclerosis: doubling down on MHC. Trends Genet. 37, 784–797. 10.1016/j.tig.2021.04.012. [DOI] [PubMed] [Google Scholar]
19.Lu LL, Suscovich TJ, Fortune SM, and Alter G (2018). Beyond binding: antibody effector functions in infectious diseases. Nat. Rev. Immunol 18, 46–61. 10.1038/nri.2017.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Tong X, McNamara RP, Avendaño MJ, Serrano EF, García-Salum T, Pardo-Roa C, Bertera HL, Chicz TM, Levican J, Poblete E, et al. (2023). Waning and boosting of antibody Fc-effector functions upon SARS-CoV-2 vaccination. Nat. Commun 14, 4174. 10.1038/s41467-023-39189-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.McClain MT, Rapp EC, Harley JB, and James JA (2003). Infectious mononucleosis patients temporarily recognize a unique, cross-reactive epitope of Epstein-Barr virus nuclear antigen-1. J. Med. Virol 70, 253–257. 10.1002/jmv.10385. [DOI] [PubMed] [Google Scholar]
22.Dempsey LA (2022). Molecular mimicry in MS. Nat. Immunol 23, 343. 10.1038/s41590-022-01156-8. [DOI] [PubMed] [Google Scholar]
23.Singh G, Abbad A, Tcheou J, Mendu DR, Firpo-Betancourt A, Gleason C, Srivastava K, Cordon-Cardo C, Simon V, Krammer F, and Carreño JM (2023). Binding and Avidity Signatures of Polyclonal Sera From Individuals With Different Exposure Histories to Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Vaccination, and Omicron Breakthrough Infections. J. Infect. Dis 228, 564–575. 10.1093/infdis/jiad116. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Hickey TE, Kemp TJ, Bullock J, Bouk A, Metz J, Neish A, Cherry J, Lowy DR, and Pinto LA (2023). SARS-CoV-2 IgG Spike antibody levels and avidity in natural infection or following vaccination with mRNA-1273 or BNT162b2 vaccines. Hum. Vaccin. Immunother 19, 2215677. 10.1080/21645515.2023.2215677. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Piepenbrink MS, Khalil AM, Chang A, Mostafa A, Basu M, Sarkar S, Panjwani S, Ha YH, Ma Y, Ye C, et al. (2024). Potent neutralization by a RBD antibody with broad specificity for SARS-CoV-2 JN.1 and other variants. Npj Viruses 2, 55. 10.1038/s44298-024-00063-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Oechtering J, Stein K, Schaedelin SA, Maceski AM, Orleth A, Meier S, Willemse E, Qureshi F, Heijnen I, Regeniter A, et al. (2024). Complement Activation Is Associated With Disease Severity in Multiple Sclerosis. Neurol. Neuroimmunol. Neuroinflamm. 11, e200212. 10.1212/NXI.0000000000200212. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Goldacre R (2024). Risk of multiple sclerosis in individuals with infectious mononucleosis: a national population-based cohort study using hospital records in England, 2003–2023. Mult. Scler 30, 489–495. 10.1177/13524585241237707. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Loosen SH, Doege C, Meuth SG, Luedde T, Kostev K, and Roderburg C (2022). Infectious mononucleosis is associated with an increased incidence of multiple sclerosis: Results from a cohort study of 32,116 outpatients in Germany. Front. Immunol 13, 937583. 10.3389/fimmu.2022.937583. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Wang Z, Kennedy PG, Dupree C, Wang M, Lee C, Pointon T, Langford TD, Graner MW, and Yu X (2021). Antibodies from Multiple Sclerosis Brain Identified Epstein-Barr Virus Nuclear Antigen 1 & 2 Epitopes which Are Recognized by Oligoclonal Bands. J. Neuroimmune Pharmacol 16, 567–580. 10.1007/s11481-020-09948-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.De Jager PL, Simon KC, Munger KL, Rioux JD, Hafler DA, and Ascherio A (2008). Integrating risk factors: HLA-DRB1*1501 and Epstein-Barr virus in multiple sclerosis. Neurology 70, 1113–1118. 10.1212/01.wnl.0000294325.63006.f8. [DOI] [PubMed] [Google Scholar]
31.Gyllensten UB, and Erlich HA (1993). MHC class II haplotypes and linkage disequilibrium in primates. Hum. Immunol 36, 1–10. 10.1016/0198-8859(93)90002-i. [DOI] [PubMed] [Google Scholar]
32.Daeron M (2024). The function of antibodies. Immunol. Rev 328, 113–125. 10.1111/imr.13387. [DOI] [PubMed] [Google Scholar]
33.Tong X, Deng Y, Cizmeci D, Fontana L, Carlock MA, Hanley HB, McNamara RP, Lingwood D, Ross TM, and Alter G (2024). Distinct Functional Humoral Immune Responses Are Induced after Live Attenuated and Inactivated Seasonal Influenza Vaccination. J. Immunol 212, 24–34. 10.4049/jimmunol.2200956. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Barnett MH, Parratt JDE, Cho ES, and Prineas JW (2009). Immunoglobulins and complement in postmortem multiple sclerosis tissue. Ann. Neurol 65, 32–46. 10.1002/ana.21524. [DOI] [PubMed] [Google Scholar]
35.van Sechel AC, Bajramovic JJ, van Stipdonk MJ, Persoon-Deen C, Geutskens SB, and van Noort JM (1999). EBV-induced expression and HLA-DR-restricted presentation by human B cells of alpha B-crystallin, a candidate autoantigen in multiple sclerosis. J. Immunol 162, 129–135. [PubMed] [Google Scholar]
36.Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, Robertson NP, Neal JW, and Morgan BP (2014). Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol. Commun 2, 53. 10.1186/2051-5960-2-53. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Bashirova AA, Viard M, Naranbhai V, Grifoni A, Garcia-Beltran W, Akdag M, Yuki Y, Gao X, O’HUigin C, Raghavan M, et al. (2020). HLA tapasin independence: broader peptide repertoire and HIV control. Proc. Natl. Acad. Sci. USA 117, 28232–28238. 10.1073/pnas.2013554117. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Brown EP, Dowell KG, Boesch AW, Normandin E, Mahan AE, Chu T, Barouch DH, Bailey-Kellogg C, Alter G, and Ackerman ME (2017). Multiplexed Fc array for evaluation of antigen-specific antibody effector profiles. J. Immunol. Methods 443, 33–44. 10.1016/j.jim.2017.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Fischinger S, Fallon JK, Michell AR, Broge T, Suscovich TJ, Streeck H, and Alter G (2019). A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation. J. Immunol. Methods 473, 112630. 10.1016/j.jim.2019.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Butler AL, Fallon JK, and Alter G (2019). A Sample-Sparing Multiplexed ADCP Assay. Front. Immunol 10, 1851. 10.3389/fimmu.2019.01851. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis, 2 Edition (Springer-Verlag New York; ). 10.1007/978-3-319-24277-4. [DOI] [Google Scholar]

Associated Data

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

Supplementary Materials

NIHMS2085665-supplement-1.pdf^{(879.8KB, pdf)}

Data Availability Statement

Luminex and flow cytometry mean fluorescence intensity (MFI) data generated for this study have been deposited to GitHub at https://github.com/HSPHSystemsSerology/ProjID_KG20241203 and will be publicly available upon publication. The repository link is listed in the key resources table.
This paper does not report the original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-guinea pig complement C3 goat IgG fraction, FITC	MP Biomedicals	Cat # 0855385, RRID:AB_2334913
Anti-human IgG1 PE [HP6001]	Southern Biotech	Cat #9054–09, RRID:AB_2796628
Anti-human IgG3 PE [HP6050]	Southern Biotech	Cat #9210–09, RRID:AB_2796701
Biological samples
LowTox Guinea Pig Complement	CedarLane Labs	Cat # CL4051
Chemicals, peptides, and recombinant proteins
Human soluble FcγR2A	Duke University	Custom Order
Human soluble FcyR3A	Duke University	Custom Order
LC-LC-Sulfo-NHS Biotin	ThermoFisher	Cat # A35358
Streptavidin-R-PE	Prozyme	Cat # PJ31S
Recombinant Epstein-Barr virus 1–90	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 377–459	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 460–641	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 29–84	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 365–420 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 393–448 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus 393–448 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 421–476 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 589–641 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 589–641 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 1–56 GD1	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 1–56 B95-8	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 421–476 AG876	SinoBiological	Custom Synthesis
Recombinant Epstein-Barr virus EBNA-1 449–504n B95-8	SinoBiological	Custom Synthesis
Influenza AH1N1(A/Ohio/UR06-0091/2007)	SinoBiological	Custom Synthesis
Ebolavirus glycoprotein (GP) (Subtype Bundibugyo, strain Uganda 2007) GP1/Glycoprotein histag(40368-V08B)	SinoBiological	Custom Synthesis
CRYAB (aa 1–27): MDIAIHHPWIRRPFFPFHSPSRLFDQFGGGEQKLISEEDLGGGMDIAIHHPWIRRPFFPFHSPSRLFDQFGGGHHHHHH	Alan Scientific	Custom Synthesis
GlialCAM (aa 370–389): ATGRTHSSPPRAPSSPGRSRGGGEQKLISEEDLGGGATGRTHSSPPRAPSSPGRSRGGGEQKLISEEDLHHHHHH	Alan Scientific	Custom Synthesis
ANO2 (aa 140–149): PGDIELGPLDGGGEQKLISEEDLGGGPGDIELGPLDGGGEQKLISEEDLGG HHHHHH	Alan Scientific	Custom Synthesis
Myc control (spacer): EQKLISEEDL GGG EQKLISEEDL GGG EQKLISEEDL GGG EQKLISEEDLGGG HHHHHH	Alan scientific	Custom Synthesis
Critical commercial assays
NHS-Sulfo-LC-LC Kit	ThermoFisher	21435
Zeba-Spin Desalting and Chromatography Columns	ThermoFisher	89882
EDC	ThermoFisher	22980
Deposited data
Luminex and Flow Cytometry MFI Data	GitHub	HSPHSystemsSerology/ProjID_KG20241203)
Experimental models: Cell lines
THP-1 monocytes	ATCC	ATCC TIB-202/RRID: CVCL_0006
Software and algorithms
iQue Forecyt 9.1	Sartorius	60028
R Studio V 4.0	R Project for Statistical Computing	RRID:SCR_000432
Other
384-well HydroSpeed Plate Washer	Tecan	30190112
IntelliFlex Luminex Dual-Report	Luminex	APX2021
iQue Screener Plus	Intellicyt/Sartorius	11811
MagPlex Microspheres	Diasorin	MC12001-01 (Cataloged by region)
Scarlet Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis
Green Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis
Red Fluorescent Neutravidin Microspheres	ThermoFisher	Custom Synthesis

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[R1] 1.Luzuriaga K, and Sullivan JL (2010). Infectious mononucleosis. N. Engl. J. Med 362, 1993–2000. 10.1056/NEJMcp1001116. [DOI] [PubMed] [Google Scholar]

[R2] 2.Ascherio A, Munger KL, Lennette ET, Spiegelman D, Hernán MA, Olek MJ, Hankinson SE, and Hunter DJ (2001). Epstein-Barr virus antibodies and risk of multiple sclerosis: a prospective study. JAMA 286, 3083–3088. 10.1001/jama.286.24.3083. [DOI] [PubMed] [Google Scholar]

[R3] 3.Bjornevik K, Cortese M, Healy BC, Kuhle J, Mina MJ, Leng Y, Elledge SJ, Niebuhr DW, Scher AI, Munger KL, and Ascherio A (2022). Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296–301. 10.1126/science.abj8222. [DOI] [PubMed] [Google Scholar]

[R4] 4.Nielsen TR, Rostgaard K, Nielsen NM, Koch-Henriksen N, Haahr S, Sørensen PS, and Hjalgrim H (2007). Multiple sclerosis after infectious mononucleosis. Arch. Neurol 64, 72–75. 10.1001/archneur.64.1.72. [DOI] [PubMed] [Google Scholar]

[R5] 5.Frappier L (2012). The Epstein-Barr Virus EBNA1 Protein. Scientifica (Cairo) 2012, 438204. 10.6064/2012/438204. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Frappier L (2015). EBNA1. In Epstein Barr Virus Volume 2: One Herpes Virus: Many Diseases, Münz C, ed. (Springer International Publishing; ), pp. 3–34. 10.1007/978-3-319-22834-1_1. [DOI] [Google Scholar]

[R7] 7.Hedstrom AK, Huang J, Michel A, Butt J, Brenner N, Hillert J, Waterboer T, Kockum I, Olsson T, and Alfredsson L (2019). High Levels of Epstein-Barr Virus Nuclear Antigen-1-Specific Antibodies and Infectious Mononucleosis Act Both Independently and Synergistically to Increase Multiple Sclerosis Risk. Front. Neurol 10, 1368. 10.3389/fneur.2019.01368. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Sundqvist E, Sundström P, Lindén M, Hedström AK, Aloisi F, Hillert J, Kockum I, Alfredsson L, and Olsson T (2012). Epstein-Barr virus and multiple sclerosis: interaction with HLA. Genes Immun. 13, 14–20. 10.1038/gene.2011.42. [DOI] [PubMed] [Google Scholar]

[R9] 9.Thomas OG, Bronge M, Tengvall K, Akpinar B, Nilsson OB, Holmgren E, Hessa T, Gafvelin G, Khademi M, Alfredsson L, et al. (2023). Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv 9, eadg3032. 10.1126/sciadv.adg3032. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Tengvall K, Huang J, Hellström C, Kammer P, Biström M, Ayoglu B, Lima Bomfim I, Stridh P, Butt J, Brenner N, et al. (2019). Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk. Proc. Natl. Acad. Sci. USA 116, 16955–16960. 10.1073/pnas.1902623116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Lanz TV, Brewer RC, Ho PP, Moon JS, Jude KM, Fernandez D, Fernandes RA, Gomez AM, Nadj GS, Bartley CM, et al. (2022). Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321–327. 10.1038/s41586-022-04432-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Weiss ER, Alter G, Ogembo JG, Henderson JL, Tabak B, Bakiş Y, Somasundaran M, Garber M, Selin L, and Luzuriaga K (2017). High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis. J. Virol 91, e01562–16–e01516. 10.1128/JVI.01562-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Weiss ER, Lamers SL, Henderson JL, Melnikov A, Somasundaran M, Garber M, Selin L, Nusbaum C, and Luzuriaga K (2018). Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection. J. Virol 92, e01466–17. 10.1128/JVI.01466-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Henie G, Henle W, and Horwitz CA (1974). Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J. Infect. Dis 130, 231–239. 10.1093/infdis/130.3.231. [DOI] [PubMed] [Google Scholar]

[R15] 15.McNamara RP, Maron JS, Boucau J, Roy V, Webb NE, Bertera HL, Barczak AK, Positives Study Staff T, Franko N, Logue JK, et al. (2023). Anamnestic humoral correlates of immunity across SARS-CoV-2 variants of concern. mBio 14, e0090223. 10.1128/mbio.00902-23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.International Multiple Sclerosis Genetics Consortium, Hafler DA, Compston A, Sawcer S, Lander ES, Daly MJ, De Jager PL, de Bakker PIW, Gabriel SB, Mirel DB, et al. (2007). Risk alleles for multiple sclerosis identified by a genomewide study. N. Engl. J. Med 357, 851–862. 10.1056/NEJMoa073493. [DOI] [PubMed] [Google Scholar]

[R17] 17.Sundstrom P, Nystrom M, Ruuth K, and Lundgren E (2009). Antibodies to specific EBNA-1 domains and HLA DRB1*1501 interact as risk factors for multiple sclerosis. J. Neuroimmunol 215, 102–107. 10.1016/j.jneuroim.2009.08.004. [DOI] [PubMed] [Google Scholar]

[R18] 18.Martin R, Sospedra M, Eiermann T, and Olsson T (2021). Multiple sclerosis: doubling down on MHC. Trends Genet. 37, 784–797. 10.1016/j.tig.2021.04.012. [DOI] [PubMed] [Google Scholar]

[R19] 19.Lu LL, Suscovich TJ, Fortune SM, and Alter G (2018). Beyond binding: antibody effector functions in infectious diseases. Nat. Rev. Immunol 18, 46–61. 10.1038/nri.2017.106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Tong X, McNamara RP, Avendaño MJ, Serrano EF, García-Salum T, Pardo-Roa C, Bertera HL, Chicz TM, Levican J, Poblete E, et al. (2023). Waning and boosting of antibody Fc-effector functions upon SARS-CoV-2 vaccination. Nat. Commun 14, 4174. 10.1038/s41467-023-39189-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.McClain MT, Rapp EC, Harley JB, and James JA (2003). Infectious mononucleosis patients temporarily recognize a unique, cross-reactive epitope of Epstein-Barr virus nuclear antigen-1. J. Med. Virol 70, 253–257. 10.1002/jmv.10385. [DOI] [PubMed] [Google Scholar]

[R22] 22.Dempsey LA (2022). Molecular mimicry in MS. Nat. Immunol 23, 343. 10.1038/s41590-022-01156-8. [DOI] [PubMed] [Google Scholar]

[R23] 23.Singh G, Abbad A, Tcheou J, Mendu DR, Firpo-Betancourt A, Gleason C, Srivastava K, Cordon-Cardo C, Simon V, Krammer F, and Carreño JM (2023). Binding and Avidity Signatures of Polyclonal Sera From Individuals With Different Exposure Histories to Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Vaccination, and Omicron Breakthrough Infections. J. Infect. Dis 228, 564–575. 10.1093/infdis/jiad116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Hickey TE, Kemp TJ, Bullock J, Bouk A, Metz J, Neish A, Cherry J, Lowy DR, and Pinto LA (2023). SARS-CoV-2 IgG Spike antibody levels and avidity in natural infection or following vaccination with mRNA-1273 or BNT162b2 vaccines. Hum. Vaccin. Immunother 19, 2215677. 10.1080/21645515.2023.2215677. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Piepenbrink MS, Khalil AM, Chang A, Mostafa A, Basu M, Sarkar S, Panjwani S, Ha YH, Ma Y, Ye C, et al. (2024). Potent neutralization by a RBD antibody with broad specificity for SARS-CoV-2 JN.1 and other variants. Npj Viruses 2, 55. 10.1038/s44298-024-00063-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Oechtering J, Stein K, Schaedelin SA, Maceski AM, Orleth A, Meier S, Willemse E, Qureshi F, Heijnen I, Regeniter A, et al. (2024). Complement Activation Is Associated With Disease Severity in Multiple Sclerosis. Neurol. Neuroimmunol. Neuroinflamm. 11, e200212. 10.1212/NXI.0000000000200212. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Goldacre R (2024). Risk of multiple sclerosis in individuals with infectious mononucleosis: a national population-based cohort study using hospital records in England, 2003–2023. Mult. Scler 30, 489–495. 10.1177/13524585241237707. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Loosen SH, Doege C, Meuth SG, Luedde T, Kostev K, and Roderburg C (2022). Infectious mononucleosis is associated with an increased incidence of multiple sclerosis: Results from a cohort study of 32,116 outpatients in Germany. Front. Immunol 13, 937583. 10.3389/fimmu.2022.937583. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Wang Z, Kennedy PG, Dupree C, Wang M, Lee C, Pointon T, Langford TD, Graner MW, and Yu X (2021). Antibodies from Multiple Sclerosis Brain Identified Epstein-Barr Virus Nuclear Antigen 1 & 2 Epitopes which Are Recognized by Oligoclonal Bands. J. Neuroimmune Pharmacol 16, 567–580. 10.1007/s11481-020-09948-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.De Jager PL, Simon KC, Munger KL, Rioux JD, Hafler DA, and Ascherio A (2008). Integrating risk factors: HLA-DRB1*1501 and Epstein-Barr virus in multiple sclerosis. Neurology 70, 1113–1118. 10.1212/01.wnl.0000294325.63006.f8. [DOI] [PubMed] [Google Scholar]

[R31] 31.Gyllensten UB, and Erlich HA (1993). MHC class II haplotypes and linkage disequilibrium in primates. Hum. Immunol 36, 1–10. 10.1016/0198-8859(93)90002-i. [DOI] [PubMed] [Google Scholar]

[R32] 32.Daeron M (2024). The function of antibodies. Immunol. Rev 328, 113–125. 10.1111/imr.13387. [DOI] [PubMed] [Google Scholar]

[R33] 33.Tong X, Deng Y, Cizmeci D, Fontana L, Carlock MA, Hanley HB, McNamara RP, Lingwood D, Ross TM, and Alter G (2024). Distinct Functional Humoral Immune Responses Are Induced after Live Attenuated and Inactivated Seasonal Influenza Vaccination. J. Immunol 212, 24–34. 10.4049/jimmunol.2200956. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Barnett MH, Parratt JDE, Cho ES, and Prineas JW (2009). Immunoglobulins and complement in postmortem multiple sclerosis tissue. Ann. Neurol 65, 32–46. 10.1002/ana.21524. [DOI] [PubMed] [Google Scholar]

[R35] 35.van Sechel AC, Bajramovic JJ, van Stipdonk MJ, Persoon-Deen C, Geutskens SB, and van Noort JM (1999). EBV-induced expression and HLA-DR-restricted presentation by human B cells of alpha B-crystallin, a candidate autoantigen in multiple sclerosis. J. Immunol 162, 129–135. [PubMed] [Google Scholar]

[R36] 36.Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, Robertson NP, Neal JW, and Morgan BP (2014). Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol. Commun 2, 53. 10.1186/2051-5960-2-53. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R37] 37.Bashirova AA, Viard M, Naranbhai V, Grifoni A, Garcia-Beltran W, Akdag M, Yuki Y, Gao X, O’HUigin C, Raghavan M, et al. (2020). HLA tapasin independence: broader peptide repertoire and HIV control. Proc. Natl. Acad. Sci. USA 117, 28232–28238. 10.1073/pnas.2013554117. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Brown EP, Dowell KG, Boesch AW, Normandin E, Mahan AE, Chu T, Barouch DH, Bailey-Kellogg C, Alter G, and Ackerman ME (2017). Multiplexed Fc array for evaluation of antigen-specific antibody effector profiles. J. Immunol. Methods 443, 33–44. 10.1016/j.jim.2017.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Fischinger S, Fallon JK, Michell AR, Broge T, Suscovich TJ, Streeck H, and Alter G (2019). A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation. J. Immunol. Methods 473, 112630. 10.1016/j.jim.2019.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Butler AL, Fallon JK, and Alter G (2019). A Sample-Sparing Multiplexed ADCP Assay. Front. Immunol 10, 1851. 10.3389/fimmu.2019.01851. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis, 2 Edition (Springer-Verlag New York; ). 10.1007/978-3-319-24277-4. [DOI] [Google Scholar]

PERMALINK

Acute infectious mononucleosis generates persistent, functional EBNA-1 antibodies with high cross-reactivity to alpha-crystalline beta

Krishna Kumar Ganta

Margaret McManus

Ross Blanc

Qixin Wang

Wonyeong Jung

Robin Brody

Mary Carrington

Robert Paris

Sumana Chandramouli

Ryan P McNamara

Katherine Luzuriaga

SUMMARY

Graphical Abstract

In brief

INTRODUCTION

RESULTS

High levels of EBNA-1 IgG1 and IgG3 antibodies develop post-IM and target an EBNA-1 region implicated in autoimmunity

Figure 1. IgG1 and IgG3 binding to EBNA-1 C-terminal peptides.

Figure 6. EBNA-1 antibodies are cross-reactive to CRYAB and retain avidity and ADCD to the self-peptide.

Higher levels of EBNA-1 binding antibodies are detected in individuals with HLA-DRB1*15:01

Figure 2. The presence of the HLA DRB1*15:01 allele enhances EBNA-1 IgG1 responses.

EBNA-1 C-terminal domain-targeting antibodies are polyfunctional through FcγR engagement

Figure 3. FcγRIIA- and FcγRIIIA-binding antibodies targeting EBNA-1 peptides persist up to 1 year post-IM.

Figure 4. Antibody-dependent cellular phagocytosis is activated post-IM to the EBNA-1 C-terminal domain.

EBNA-1 antibodies mediate complement, activation, and deposition

Figure 5. Antibody-dependent complement deposition to EBNA-1 C-terminal peptides increases over time and is enhanced in DRB1*15:01-positive individuals.

High levels of binding and ADCD antibodies to CRYAB are detected following IM

CRYAB antibodies are cross-reactive with EBNA-1 antibodies

Figure 7. EBNA-1 specific antibodies from post-IM individuals cross-react with CRYAB.

DISCUSSION

Limitations of the study

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

IM cohort

Cell line authentication

Ethical considerations

METHOD DETAILS

HLA typing

Antigens

Antibody isotypes and Fc-receptor binding activities measurement

Antibody-dependent complement deposition

Antibody-dependent cellular phagocytosis

Peptide blocking assay to evaluate EBNA-1 and alpha crystalline beta cross-reactivity

QUANTIFICATION AND STATISTICAL ANALYSIS

Mixed model analysis

Supplementary Material

Highlights.

ACKNOWLEDGMENTS

DECLARATION OF INTERESTS

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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