Epstein-Barr virus and multiple sclerosis: interaction with HLA

Background: Epstein-Barr virus (EBV), a common human lymphotropic herpes virus infecting over 95% of the overall individuals. Strong evidence from recent research that EBV is a contributing factor in multiple sclerosis (MS). The exact mechanism by which EBV might exacerbate MS in risk groups is unknown. Objectives: To determine plasma levels of Autophagy-related proteins 5 (ATG5) and Epstein-Barr virus nuclear antigen1 (EBNA1) IgG. Additionally, assessing EBV DNA load in MS patients in relation to disease activity. Also, studying the relation between EBV DNA burden and the plasma levels of ATG5 and EBNA1 IgG in MS patients. Methodology: The study was conducted on 23 relapsing remitting MS (RRMS) patients in active attack, 23 RRMS patients' in-between attacks and 23 healthy controls. Enzyme-linked immunosorbent assay (ELISA) method was used to measure the amount of ATG5 and EBNA1 IgG in plasma. Real-time polymerase chain reaction (real-time PCR) was used to assess the EBV DNA load in peripheral blood mononuclear cells (PBMCs). Results: No statistically significant difference in ATG5 or EBNA1 IgG level between MS patients and healthy controls. However, all MS patients (100%) were EBNA1 IgG positive while 91.3% of the control group were EBNA1 IgG positive. EBV DNA load did not show statistically significant difference between MS patients and healthy controls. Significant positive correlation between EBV DNA load and ATG5 plasma level in MS patients in between attacks, P value =0.019. Conclusion: ATG5 levels and EBV DNA load could be utilized to predict disease-related activities in predisposed individuals or MS patients.

It is unclear whether smoking interacts with different aspects of Epstein–Barr virus (EBV) infection with regard to multiple sclerosis (MS) risk. We aimed to investigate whether smoking acts synergistically with elevated EBNA-1 antibody levels or infectious mononucleosis (IM) history regarding MS risk. Two Swedish population-based case–control studies were used (6,340 cases and 6,219 matched controls). Subjects with different smoking, EBNA-1 and IM status were compared regarding MS risk, by calculating odds ratios (OR) with 95% confidence intervals (CI) employing logistic regression. Potential interaction on the additive scale was evaluated by calculating the attributable proportion due to interaction (AP). Current and past smokers had higher EBNA-1 antibody levels than never smokers (p < 0.0001). There was an additive interaction between current smoking and high EBNA-1 antibody levels (AP 0.3, 95% CI 0.2–0.4), but not between past smoking and high EBNA-1 antibody levels (AP 0.01, 95% CI − 0.1 to 0.1), with regard to MS risk. An interaction also occurred between current smoking and IM history (AP 0.2, 95% CI 0.004–0.4), but not between past smoking and IM history (AP − 0.06, 95% CI − 0.4 to 0.3). Current smoking increases EBNA-1 antibody levels and acts synergistically with both aspects of EBV infection to increase MS risk, indicating that there is at least one pathway to disease in which both risk factors are involved.

Among multiple sclerosis (MS) patients, an association has been observed between low levels of vitamin D and high Epstein-Barr nuclear antigen 1 (EBNA-1) antibody levels. However, whether sun exposure/vitamin D moderates the role of Epstein-Barr virus (EBV) infection in MS etiology is unclear. We aimed to investigate potential synergistic effects between low sun exposure and elevated EBNA-1 antibody levels regarding MS risk. We used a population-based case-control study involving 2017 incident cases of MS and 2443 matched controls. We used logistic regression models to calculate the odds ratios of MS with 95% confidence intervals (CIs) in subjects with different sun exposure habits and EBNA-1 status. Potential interaction on the additive scale was evaluated by calculating the attributable proportion due to interaction (AP). Low sun exposure acted synergistically with high EBNA-1 antibody levels (AP 0.2, 95% CI 0.03-0.3) in its association to increased MS risk. The interaction was present regardless of HLA-DRB1*15:01status. Low sun exposure may either directly, or indirectly by affecting vitamin D levels, synergistically reinforce pathogenic mechanisms, such as aspects of the adaptive immune response, related to MS risk conveyed by EBV infection.

Background: Little is known about risk factors for neuromyelitis optica (NMO) or transverse myelitis (TM). Objective: The objective of this paper is to evaluate whether established multiple sclerosis (MS) risk factors, including smoking history, a history of infectious mononucleosis (IM), anti-EBNA1 Ab titers and HLA-DR15 are associated with NMO or TM. Methods: We conducted a case-control study among participants in the Accelerated Cure Project for Multiple Sclerosis (ACP) Repository, which includes patients with MS, NMO and TM. Controls include related and unrelated individuals without evidence of demyelinating disease. Analyses included 1237 cases of MS, 98 cases of NMO, 133 cases of TM and 488 healthy controls. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to assess the association between smoking, HLA-DR15, anti-EBNA1 Ab titers and a history of IM adjusting for gender, study site and ethnicity. Results: Overall, the association between smoking, IM, HLA-DR15 and anti-EBNA1 Ab titers and odds of MS were as expected and no significant interactions were observed. However, there was little evidence of association between these MS risk factors and odds of NMO or TM. Conclusions: Established MS risk factors do not appear to be associated with susceptibility to TM or NMO and, among MS patients, these risk factors appear to act independently.

Epstein-Barr virus and multiple sclerosis: supporting causality

Evaluation of Epstein-Barr virus-specific antibodies in Cypriot multiple sclerosis patients

ObjectiveIn multiplex MS families, we determined the humoral immune response to Epstein-Barr virus nuclear antigen 1 (EBNA-1)-specific immunoglobulin γ (IgG) titers in patients with MS, their healthy siblings, and biologically unrelated healthy spouses and investigated the role of specific genetic loci on the antiviral IgG titers.MethodsIgG levels against EBNA-1 and varicella zoster virus (VZV) as control were measured. HLA-DRB1*1501 and HLA-A*02 tagging single-nucleotide polymorphisms (SNPs) were genotyped. We assessed the associations between these SNPs and antiviral IgG titers.ResultsOR for abundant EBNA-1 IgG was the highest in patients with MS and intermediate in their siblings compared with spouses. We confirmed that HLA-DRB1*1501 is associated with abundant EBNA-1 IgG. After stratification for HLA-DRB1*1501, the EBNA-1 IgG gradient was still significant in patients with MS and young siblings compared with spouses. HLA-A*02 was not explanatory for EBNA-1 IgG titer gradient. No associations for VZV IgG were found.ConclusionsIn families with MS, the EBNA-1 IgG gradient being the highest in patients with MS, intermediate in their siblings, and lowest in biologically unrelated spouses indicates a genetic contribution to EBNA-1 IgG levels that is only partially explained by HLA-DRB1*1501 carriership.

Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P < 0.0001) for VCA IgM, 0.889 (P < 0.0001) for VCA IgG, and 0.961 (P < 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.

In a recent review evaluating the evidence concerning the association between smoking and multiple sclerosis (MS), the author specifically calls for: (1) studies investigating this association using objective markers for smoking in prospective datasets; and (2) studies addressing the timing of smoke exposure [Wingerchuk, 2012]. We have recently reported the findings of a nested case-control study where levels of cotinine, a recognized biomarker for tobacco use, were measured in biobank blood samples collected median 9 years prior to disease onset [Salzer et al. 2012a]. Elevated levels of cotinine suggestive of smoking (≥10 ng/ml) [Vineis et al. 2005] were associated with a 50% increased risk of MS when analyzing the entire cohort. When we stratified the cohort according to age at blood sampling, it was evident that it was only among young subjects (below median age at blood sampling, <26.4 years) that this effect was seen. In addition, we used samples collected in the first trimester from 37 pregnant women, whose offspring had later developed MS, and 185 pregnant women whose offspring had not (further described by Salzer and colleagues [Salzer et al. 2012b]) to study the effects of gestational levels of cotinine on the risk of MS in the offspring (previously unpublished data). In this dataset n = 13 (35%) of case mothers and n = 71 (39%) of control mothers had cotinine levels ≥10 ng/ml. There was no association between elevated cotinine levels during gestation and the risk of MS in the offspring (odds ratio [OR] 0.87, 95% confidence interval [CI] 0.41–1.8, matched logistic regression). In conclusion, our data indicate that smoke exposure during young adulthood is associated with an increased MS risk, but exposure during early gestation according to our limited gestational material, or exposure after the age of 26.4 years is not. This age-of-exposure-dependent effect of smoking on MS risk converges with similar findings for other risk factors of MS (vitamin D levels, elevated body mass index [BMI], Epstein–Barr virus [EBV] seroconversion, infectious mononucleosis) suggesting that young adulthood is a crucial time for establishing an individual’s risk of the disease [Hedstrom et al. 2012; Munger et al. 2006; Thacker et al. 2006].

Objective: Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) and the replication origin, oriP, are essential for the replication and maintenance of latent EBV DNA in cells, but no enzymatic activity has been associated with EBNA-1 protein alone. In this study, we have searched for host cellular proteins that interact with EBNA-1 protein in various B cell lines latently infected with EBV, including a recently EBV growth-transformed cell line. Methods: By using gel shift analysis, we investigated the interactions of an oligonucleotide containing a single EBNA-1 recognition site, derived from the family of repeats (FR) element of oriP, with protein from cell extracts. Results: The FR oligonucleotide bound a (72-kD) cellular protein in the absence of EBNA-1 and without induction of the previously reported ‘anti-EBNA-1 proteins’. The FR oligonucleotide formed complexes with additional proteins from EBNA-1-synthesizing cell lines; these complexes were abolished or supershifted by anti-EBNA-1 monoclonal antibodies. SDS-PAGE analyses of ³⁵S-Met-labeled proteins that bound to a biotin- conjugated FR oligonucleotide, fractionated by a glycerol gradient centrifugation and affinity-purified with streptavidin, showed three major bands, a 72-kD protein, the FR binding of which seemed to be independent of EBNA-1, a 64-kD protein in both EBNA-1-transfected and latently EBV-infected cell lines, and a 45-kD protein in EBV-infected cell lines, which was most prominent in a recently EBV growth-transformed cell line. Conclusions: The FR element forms complexes with cellular proteins in the absence and presence of EBNA-1. These 72-, 64- and 45-kD cellular proteins might be involved in the function of the oriP and EBNA-1 system.

The interaction of Epstein-Barr virus encoded transcription factor EBNA2 with multiple sclerosis risk loci is dependent on the risk genotype.

Hepatitis A virus (HAV) infection is usually self-limited and does not progress to chronic liver disease. However, atypical courses such as prolonged cholestatic hepatitis may occur in adults, posing diagnostic and therapeutic challenges.We report the case of a 55-year-old man with hypertension, obesity, and known hepatic steatosis who presented with jaundice, choluria, acholic stools, fatigue, epigastric pain, and nausea. Laboratory evaluation revealed a mixed hepatocellular-cholestatic pattern with predominantly direct hyperbilirubinemia. Acute HAV infection was confirmed (anti-HAV IgM positive), and alternative causes were excluded. Imaging showed no biliary obstruction. Epstein-Barr virus (EBV) serology obtained during the first admission was consistent with past infection (viral capsid antigen (VCA) IgG positive, VCA IgM negative, and EBV nuclear antigen-1 (EBNA-1) IgG positive). The patient improved with supportive care and was discharged after one week.He was readmitted one week later with clinical relapse and severe hyperbilirubinemia (total bilirubin 25.3 mg/dL). Given a household contact with a mononucleosis-like illness, repeat EBV serology showed weak/low-level VCA IgM reactivity (12.1 UA/mL) with persistent VCA IgG positivity. However, subsequent reassessment results returned to VCA IgM negativity with persistent VCA IgG and EBNA-1 IgG positivity, supporting remote EBV infection and suggesting non-specific IgM reactivity (or, less likely, reactivation) rather than primary acute EBV infection. EBV DNA PCR was not pursued due to low clinical and serologic suspicion of active infection and the subsequent resolving course. A genetic study requested during the first admission revealed HFE H63D heterozygosity, with a ferritin level >11,000 ng/mL and transferrin saturation of >80%. The patient improved with ursodeoxycholic acid (UDCA) and individualized iron management. Corticosteroids were not used, given progressive improvement with supportive care and UDCA. Follow-up quantitative liver MRI showed only mild iron overload (43 µmol/g), supporting an acute-phase/inflammatory iron contribution during severe hepatitis, and FibroScan® revealed mild fibrosis (5.3 kPa). This case highlights the importance of stepwise reassessment in prolonged cholestatic HAV, the pitfalls of interpreting transient EBV VCA IgM reactivity in a patient with serology consistent with prior EBV infection, and careful interpretation of marked hyperferritinemia in HFE H63D heterozygotes.

The performance of an immunofiltration assay (IMFA) that detects immunoglobulin M (IgM) antibodies to the Epstein-Barr virus (EBV) ZEBRA (BamHI Z EBV replication activator) protein was evaluated for the diagnosis of EBV infectious mononucleosis (IM) in immunocompetent patients. The test panel consisted of 47 sera displaying an EBV-specific antibody profile compatible with an acute primary EBV infection from patients with clinical and biological features of EBV IM, 20 sera from healthy individuals either with a past EBV infection or who were EBV seronegative, 20 sera displaying an equivocal EBV antibody pattern (viral capsid antigen IgG positive [VCA IgG+], VCA IgM+, and EBV nuclear antigen-1 IgG+), and 15 sera obtained from patients with a mononucleosis-like syndrome owing to cytomegalovirus, human herpesvirus 6, or parvovirus B19. Overall, the sensitivity and the specificity of the assay were found to be 92.5%, and 97.3%, respectively. The sensitivity of the assay for the diagnosis of heterophile antibody-negative EBV IM was 86.2%. The IMFA is rapid, easy to perform, and, thus, suitable for point-of-care testing, and it may be used as a first-line test for the diagnosis of acute EBV IM in immunocompetent patients.

BackgroundMultiple sclerosis (MS) is a neurological disease that is caused by an autoimmune response that results in the neuron’s demyelination in the central nervous system. The exact etiology of MS is not clear; however, several environmental and genetic factors are believed to participate in its initiation and development, including exposure to viruses. This study aims to investigate the association between the seropositivity and antibody titer of selected herpesviruses and MS in Jordanian MS patients.MethodIn this study, 55 MS patients and 40 age- and gender-matching apparently healthy volunteers were recruited from two main hospitals in the north of Jordan. MS patients were grouped into three types of MS based on the clinical presentation of the disease. Blood samples were collected from the participants and the IgG antibodies for human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV) nuclear antigen (EBNA), EBV viral capsid antigen (VCA) and varicella-zoster virus (VZV) were assayed by ELISA. The prevalence of seropositivity and the antibody level for each of the antibodies were compared between MS patients and controls and between the three types of MS.ResultsThere was no significant difference in the prevalence of seropositivity and in the levels of antibodies for HHV-6, EBNA and VCA between MS patients and controls and between the three types of MS. In contrast, the number of seropositive patients and the level of IgG antibodies for VZV were significantly higher in MS patients compared to the control.ConclusionThis study showed that patients with MS in the north of Jordan were more likely to be seropositive for VZV than the general population. Based on this finding, we recommend further studies to evaluate the seropositivity to VZV to be carried out in other parts of Jordan and the greater middle east to find out if there is a correlation between MS and previous infection with VZV.

Objective:Because dysregulated Epstein-Barr virus (EBV)-infected B cells may induce meningeal inflammation, which contributes to cortical pathology in multiple sclerosis (MS), we investigated associations between antibody responses to EBV and development of cortical pathology in MS.Methods:We included 539 patients with MS (369 with relapsing-remitting MS, 135 with secondary progressive MS, and 35 with primary progressive MS), 66 patients with clinically isolated syndrome (CIS), 63 patients with other neurologic diseases (OND), and 178 age- and sex-matched healthy controls (HC). All participants were scanned on 3T MRI. Serum samples were analyzed for IgG antibodies against EBV viral capsid antigen (VCA) and EBV nuclear antigen-1 (EBNA-1), and their quartiles were determined on the whole study sample. Differences between the study groups were assessed using analysis of covariance adjusted for multiple comparisons.Results:More than 30% of patients with MS and CIS presented with the highest quartile of anti-EBV-VCA and -EBNA-1 status compared to ≤10% of HC (p < 0.001). The figures were 9 (14.3%) and 7 (12.3%) for patients with OND. Patients with MS with the highest quartile of anti-EBV-VCA showed significantly increased T2 lesion volume (p = 0.001), T1 lesion number (p = 0.002), and T1 lesion volume (p = 0.04) and decreased gray matter (p = 0.041) and cortical (p = 0.043) volumes compared to patients with MS with lower quartiles. No significant differences of MRI outcomes in patients with CIS, patients with OND, and HC with lower or highest quartiles of anti-EBV-VCA and -EBNA-1 were detected.Conclusions:Humoral response to anti-EBV-VCA and -EBNA-1 is associated with more advanced cortical atrophy, accumulation of chronic T1 black holes, and focal white matter lesions in patients with MS.