Objective Epstein-Barr virus (EBV) infection has been thought to be a key environmental factor in the development of multiple sclerosis (MS). The aim of this study is to investigate the association of EBV infection with neuromyelitis optica (NMO).
Methods We measured levels of serum antibodies against EBV antigens, including anti-viral capsid antigen (VCA) IgM, anti-VCA IgG, anti-early antigen (EA) IgM, anti-EA IgG and anti-EBV nuclear antigen-1 IgG, in 50 patients with NMO (including 12 partial form with antiaquaporin 4 antibodies), 51 patients with MS, and 52 healthy controls, and cerebrospinal fluid (CSF) antibodies in 37 patients with NMO and 33 patients with MS with ELISA.
Result Compared with patients with MS and normal participants, patients with NMO more frequently had serum anti-EA IgG antibodies (52%), indicating more active viral replication than patients with MS (26%) and controls (25%). The antibody titres were significantly higher in the NMO group than in the MS (p=0.005) and control (p=0.005) groups. The CSF antibody titres were also higher in patients with NMO than in those with MS (p=0.03).
Conclusions Our results raise the hypothesis that persistent, active EBV replication is present in NMO, and may contribute to the immunological alterations that play a pathogenetic role in the disorder.
- MULTIPLE SCLEROSIS
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Multiple sclerosis (MS) is presently regarded as a disease with multifactorial aetiologies, including genetic and environmental factors. Epstein-Barr virus (EBV) infection has long been considered to be a key environmental factor in MS.1–3 However, various infectious agents have been reported to be associated with neuromyelitis optica (NMO). Single case reports have suggested that viral infections such as those caused by cytomegalovirus (CMV), human simplex virus (HSV), varicella zoster virus (VZV), HIV and human T lymphotropic virus type 1, and bacterial infections such as those caused by Mycoplasma pneumoniae, Mycobacterium tuberculosis and Treponema pallidum are related to the development of NMO.4 ,5 It has recently been reported that Helicobacter pylori and Chlamydia pneumoniae infections are risk factors for antiaquaporin-4 (AQP4) antibody-positive NMO6 and that amplified AQP4-specific T cells recognise the Clostridium ABC transporter exhibiting Th17 bias in NMO.7 However, the evidence for the associations of NMO with infectious agents has not been completely accumulated. The present study aimed to investigate the association of EBV infection and NMO comparing MS.
Fifty patients with NMO, including 38 patients with definite NMO who met the 2006 Wingerchuk criteria8 and 12 patients with partial NMO defined as anti-AQP4 antibody-positive optic neuritis and/or transverse myelitis,9 and 51 patients with MS who met the 2005 revised McDonald criteria10 were included in the present study. Fifty-two volunteers (11 males and 41 females; median age=32.0 years) were recruited as healthy controls (HCs).
We reviewed the gender, age at sample collection, disease duration, expanded disability status scale (EDSS) scores, cell counts of cerebrospinal fluid (CSF) and proportion of patients who exhibited oligoclonal band positivity and were receiving any immunotherapy.
The ethics committee of the Chiba University School of Medicine, Chiba, Japan approved the study, and written informed consent was obtained from all the study participants.
Serum and CSF samples of the patients with NMO and MS were collected at the time of clinical relapse; additional serum samples were collected during the remission phase. All the samples were immediately stored at −80°C until analysis.
Measurements of levels of antibodies against EBV-specific antigens
The following serum antibodies against five EBV-specific antigens were evaluated: anti-viral capsid antigen (VCA) IgM, anti-VCA IgG, anti-early antigen (EA) IgM, anti-EA IgG and anti-EBV nuclear antigen (EBNA)-1 IgG. These antibody titres were measured using ELISA kits (IBL, Hamburg, Germany) according to the manufacturer's instructions. The levels of CSF anti-EA IgG antibody were also measured. The serum was tested at 1 μL/well, and CSF was measured at 100 μL/well. The optical density (OD) was measured at 450 nm (reference wavelength: 630 nm), and the concentration (U/mL) was calculated by referring to a standard curve. As recommended by the manufacturer, serum anti-VCA IgM, VCA IgG, EA IgG and EBNA-1 IgG antibodies were considered positive if >22 U/mL, and the anti-EA IgM antibody was positive if >1.2 U/mL.
Detection of IgM antibodies against CMV, HSV and VZV
The serum IgM antibodies against CMV, HSV and VZV in the 45 patients with NMO and the 51 patients with MS were analysed by SRL Inc, a commercial laboratory in Tokyo, Japan; these measurements were performed using enzyme immunoassay kits (DENKA SEIKEN, Tokyo, Japan). The serum was tested at 0.5 μL/well, and OD was measured at 450 nm (reference wavelength: 630 nm). The antibody index was defined as the OD ratio of the target sample to the cut-off control sample and was considered positive if >1.2.
Antibody index for EA IgG
To determine whether the increased CSF anti-EA IgG antibody levels in patients with NMO were explained simply by higher total IgG concentrations in the CSF, we compared the measured CSF IgG concentrations between patients with NMO and MS. In addition, we measured the antibody index, which is a method to rival or surpass other indexes for sensitive detection and quantification of intrathecal synthesis of virus-specific antibodies, for EA IgG in patients with NMO, as previously described.11 ,12 At first, the Qspec and QIgG was calculated as the ratio of EA IgG antibody titre in CSF to EA IgG antibody titre in serum and the ratio of total IgG in CSF to total IgG in serum, respectively. The antibody index was determined as Qspec/QIgG when QIgG<Qlim or as Qspec/Qlim when QIgG>Qlim, and was considered positive if ≥1.4.11 ,12
Correlation of demographic features with patients with NMO according to the anti-EA IgG antibody status
We analysed the correlation between clinical and laboratory findings such as the male/female ratio, age at sample collection, disease duration, EDSS scores, positivity rate and OD value at 450 nm of the serum anti-AQP4 antibody using ELISA or frequency of receiving any immunotherapy among serum anti-EA IgG antibody-positive and antibody-negative patients with NMO.
For baseline variables, the groups were compared using Fisher's exact test for categorical outcomes and the Mann-Whitney U test for continuous variables, as appropriate. Spearman's rank correlation coefficient was used to test the associations. All comparisons were planned, and tests were two-sided; a p value <0.05 was considered statistically significant. In addition, to consider multiple testing problems, we applied the Bonferroni correction on the computed p values to reduce type I errors. All statistical analyses were performed using SPSS V.16.0J (SPSS Japan Inc, Tokyo, Japan).
Clinical and laboratory profiles of patients
The clinical and laboratory characteristics of the patients with NMO and MS are summarised in table 1. The male/female ratio, age at sample collection, EDSS scores as well as the proportion of patients who exhibited oligoclonal band positivity and were receiving any immunotherapy and low-dose oral prednisolone therapy (5–20 mg/day) were significantly higher in patients with NMO than in patients with MS.
Determination of levels of serum antibodies against EBV-specific antigens
The seropositivity rates of the antibodies against the EBV-specific antigens in patients with NMO and patients with MS and HCs are summarised in table 2.
Elevated serum anti-VCA IgG antibodies were found in 48 of 51 patients with MS (94.1%), in 38 of 50 patients with NMO (76.0%) and in 42 of 52 HCs (80.8%). The seropositivity rates for anti-VCA IgG antibodies were significantly higher in the patients with MS than in those with NMO (p=0.01). Elevated serum anti-EA IgG antibodies were found in 26 of 50 patients with NMO (52.0%), in 13 of 51 patients with MS (25.5%) and in 13 of 52 HCs (25.0%). The test for the serum anti-EA IgG antibody was thus more frequently positive in the patients with NMO than in those with MS (p=0.005) and HCs (p=0.005). No significant differences were observed in the seropositivity rates of the antibodies against VCA IgM, EA IgM or EBNA-1 IgG among the patients with NMO, patients with MS and HCs.
The titres of the serum antibodies against the EBV-specific antigens in the patients with NMO, patients with MS and HCs are shown in figure 1. The titres of the anti-VCA IgG antibodies were significantly higher in the patients with MS than in those with NMO (p=0.005). The anti-EA IgM antibody titres were significantly higher in the patients with NMO and MS than in HCs (p <0.001 and p<0.001, respectively). The titres of the anti-EA IgG antibodies were significantly higher in the patients with NMO than in those with MS (p=0.005) and HCs (p=0.005) and were similar between prednisolone-treated and prednisolone-untreated patients with NMO (data not shown). The anti-EBNA-1 IgG antibody titres were significantly higher in the patients with MS than in those with NMO (p<0.001) and HCs (p=0.005). No significant differences were observed in the anti-VCA IgM antibody titres among the patients with NMO, patients with MS and HCs.
Thus, only the anti-EA IgG antibody showed a significantly higher seropositivity rate and titres in patients with NMO than in patients with MS and HCs. Therefore, we focused on the anti-EA IgG antibody-positive patients with NMO.
Frequency of IgM antibodies against CMV, HSV and VZV
The seropositivity rates of the IgM antibodies against CMV, HSV and VZV were 1/45 (2.2%), 2/45 (4.4%) and 0/45 (0.0%), respectively, in the patients with NMO and 3/50 (6.0%), 0/51 (0.0%) and 0/50 (0.0%), respectively, in the patients with MS. There were no significant differences in the frequencies of serum IgM antibodies against CMV, HSV and VZV between the patients with NMO and MS. None of the anti-EA IgG antibody-seropositive patients with NMO showed positive results for these antibodies.
Determination of the level of the CSF anti-EA IgG antibody
The CSF anti-EA IgG antibody titres were significantly higher in the 37 patients with NMO than in the 33 patients with MS (p=0.03; figure 2A). There was a significant positive correlation between the serum and CSF anti-EA IgG antibody titres among the patients with NMO and MS (r=0.563; p<0.001; figure 2B).
Longitudinal changes of the serum anti-EA IgG antibody titres
Figure 3 shows the serum anti-EA IgG antibody titres at the time of clinical relapse and during the remission phase. Clinical relapse was defined as an episode of neurological disturbance, as seen in MS, lasting at least 24 h. The remission phase was defined as the period when patients’ neurological condition had been stable for more than 3 months and the next relapse did not occur for a further 3 months at least. Among the 20 anti-EA IgG antibody-seropositive patients (14 with NMO and 6 with MS), the titres remained at high levels and had not changed between the time of clinical relapse and the remission phase.
Antibody index for EA IgG
CSF IgG concentrations were measured in 37 patients with NMO and 33 patients with MS in this study. The mean (SD) IgG concentrations were 7.8 (9.2) mg/dL in patients with NMO and 5.3 (3.4) mg/dL in patients with MS, and the differences were not significant. Thus, we believe that the higher CSF anti-EA IgG antibody levels in patients with NMO than in patients with MS were not due to higher IgG concentrations in patients with NMO. In addition, we calculated the antibody index of the 37 patients with NMO to determine whether the elevated CSF anti-EA IgG antibody levels were produced centrally or peripherally. The results were shown in the online supplementary file. The antibody index for EA IgG was positive in 16/37 (43%) patients with NMO who were measured with a CSF anti-EA IgG antibody.
Correlation of demographic features with patients with NMO according to the anti-EA IgG antibody status
Among the anti-EA IgG antibody-seropositive and antibody-seronegative patients with NMO, there were no significant differences in any demographic features, including the male/female ratio, age at sample collection, disease duration or EDSS scores. In addition, there was no difference in the frequencies of serum anti-AQP4 antibody positivity using ELISA or the receipt of any immunotherapy and low-dose oral prednisolone therapy (data not shown). On the other hand, among 26 anti-EA IgG antibody-seropositive patients with NMO, there was a positive correlation between the titres of the serum anti-AQP4 antibody and the serum anti-EA IgG antibody (r=0.397; p=0.046; figure 4).
In MS, EBV has long been thought to be a key environmental factor in MS. In contrast, although a relationship between NMO and various infectious agents has been reported, this association has not been completely elucidated. However, there have been studies about the association between EBV and some autoimmune diseases such as systemic lupus erythematosus and Sjögren's syndrome, which occasionally complicates NMO.13 ,14 Therefore, we studied the association between NMO and EBV.
EBV is a human herpes virus with a unique ability to infect, activate and persist in a latent form in B lymphocytes throughout the infected individual's lifetime, mostly as asymptomatic. The different stages of EBV infection are characterised by the appearance of different antibodies against the viral antigens. In the present study as well as in previous reports,15–17 compared with patients with NMO and HCs, we found an elevation of the anti-EBNA-1 IgG antibody, which could indicate past infection, in patients with MS. Furthermore, compared with patients with MS and HCs, our study showed a significant elevation of serum anti-EA IgG antibodies, which could indicate active viral replication, in patients with NMO.
The anti-EA IgG antibody levels in the CSF of patients with NMO were also higher than those in the CSF of patients with MS and positively correlated with the serum anti-EA IgG antibody levels. The antibody index, which is a test for antigen-specific intrathecal IgG synthesis,11 ,12 for EA IgG was positive in 43% of patients with NMO with a cut-off value of 1.4, and 38% with a value of 1.5 in our study. To the best of our knowledge, the antibody index for EA IgG has never been studied. However, it was reported that antibody indices for anti-VCA and anti-EBNA-1 antibody were positive in a small proportion of MS (2.5% and 6.3%, respectively) when the indices was considered positive if ≥1.5.18 Therefore, our results suggest that the CSF EA IgG antibody was produced centrally at least in a specific subset of patients with NMO.
The titres of anti-EA IgG antibody-seropositive patients had remained high and did not differ between the time of clinical relapse and the remission phase. Serial measurements of the serum of a patient with partial NMO revealed that the anti-EA IgG antibody titres increased before the onset of partial NMO and remained at a high level after its onset. According to the anti-EA IgG antibody status, our results suggest that persistent active EBV replication may chronically occur in patients with NMO.19
Moreover, anti-EA IgG antibody-seropositive patients with NMO showed a positive correlation with serum anti-AQP4 antibody levels. A limited number of reports on cytokine profiles have revealed that EBV activation induces a wide variety of cytokines, including tumour necrosis factor-α, interferon-γ, interleukin (IL)-6, IL-10 and viral IL-10, which have been shown to share considerable amino acid homology and biological properties with human IL-10.20 ,21 We previously reported that the serum and CSF IL-6 levels are elevated in NMO,22 and recent studies have suggested that IL-6 promotes anti-AQP4 antibody production from plasmablasts in NMO.23 These results may support the possibility that cytokine imbalances induced by EBV activation and anti-AQP4 antibody production promoted by IL-6 may play a role in the pathogenesis of NMO.
At present, the evidence for the presence of EBV-infected cells in the brain of patients with MS remains controversial.24 Recent reports showed that a substantial proportion of the B cells and plasma cells in the brain of patients with MS are infected with EBV by in situ hybridisation and immunohistochemistry and also showed that ectopic B cell follicles are the main intracerebral sites of viral persistence and reactivation.25 However, subsequent studies using similar technologies were unable to detect EBV in the brain tissue of patients with MS.26 ,27 Adequate studies about the presence of EBV in the brain and spinal cord of patients with NMO are lacking; thus, further investigation will be required.
Our results show that elevated serum antibodies against EBV VCA-IgG, VCA-IgM, EBNA-1 IgG, EA IgG or any of them were found in 94%, 7.8%, 98%, 26% and 98% of patients with MS and 81%, 3.9%, 90%, 25% and 92% of HCs, respectively. These findings are comparatively similar to the results of previous reports. In a study in Norway by Myhr et al,28 these antibodies were detected in 97.9%, 1.4%, 99.3%, 68.8% and 100% of patients with MS and 81.2%, 0.7%, 94.1%, 46.5% and 95.3% of HCs, respectively.
In our study, the positive rates of serum anti-EA IgG antibodies were similar between patients with MS (26%) and HCs (25%), and there was no significant difference in between these groups. Thus, we believe that there was minimal association between persistent EBV infection and MS. In the Myhr's report,28 elevated serum anti-EA IgG antibodies were observed in 69% of patients with MS and 47% of HCs, and the positive rates were significantly different between the two groups. These differences between the findings of the present and previous studies may be due to the different assays used to measure anti-EA IgG antibodies or due to the differences in the patients’ environmental and genetic backgrounds.
In conclusion, our results showed that compared with patients with MS and HCs, the anti-EA IgG antibody was significantly elevated in patients with NMO; this antibody remained at a high level at the time of clinical relapse and during the remission phase. According to the anti-EA IgG antibody status, our results support the hypothesis that persistent, active EBV replication is frequent in NMO and may contribute to the immunological alternation that plays a pathogenetic role in the disorder.
Part of this study was presented in a poster session at the 6th PACTRIMS (Pan-Asian Committees on Treatment and Research in Multiple Sclerosis) held in Kyoto, Japan (November 2013).
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online supplement
Contributors SM, MM, KA and SK designed the study and drafted the manuscript. AU, MM, TU and HM contributed the clinical data and samples. SM measured the antibodies in the samples and performed the statistical analysis.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics committee of the Chiba University School of Medicine.
Provenance and peer review Not commissioned; externally peer reviewed.
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