Multiple sclerosis (MS) is an autoimmune disease caused by the infiltration of autoreactive lymphocytes into the central nervous system (CNS). How these lymphocytes become activated against the CNS remains unclear. Several infectious agents have been postulated to trigger the development of autoimmune diseases. The role of Epstein-Barr virus (EBV) in the pathogenesis of MS has been investigated in several clinical trials [1
]. EBV, a B-lymphotropic herpes virus, is present in all populations and infects over 90% of individuals before adulthood [5
Once infected, the virus is able to persist in the host in a dormant state for an entire lifetime. Periodically, the virus can be reactivated, at which time the viral replication cycle is initiated and lymphocytes are exposed to viral antigens [7
]. Activation and expansion of EBV-specific lymphocytes will then force the virus back into its latent state. If primary infection is delayed beyond the first decade of life, it often results in infectious mononucleosis (IM), a self-limiting disease with flu-like symptoms. Thacker et al.
postulated that EBV infection, which manifests itself as IM in adolescents and young adults, constitutes a risk factor for MS [8
]. Furthermore, one study showed evidence of EBV infection in a substantial proportion of B cells and plasma cells found in post mortem
MS brain tissue [3
]. Moreover, there seems to be an increased risk of developing MS when high titers of anti-EBV antibodies are present in the serum [9
Thus far, the analyses of a correlation between brain reactivity and a positive EBV response were limited due to the fact that there were no reliable parameters reflecting cellular autoimmunity to CNS antigens in MS. In several trials the EBV serum antibody titer has been correlated with clinical and magnetic resonance imaging (MRI) evidence of disease activity [10
]. The major drawback of these studies was that neither MRI lesions nor the Expanded Disability Status Scale (EDSS) were reflective of the cellular immunity to brain antigens. We have recently introduced an enzyme-linked immunospot (ELISPOT) assay for the detection of brain-specific B cells in the blood of patients with MS. These B cells only occurred in patients with clinically isolated syndrome or definite MS and were absent in healthy donors and in patients with other inflammatory and non-inflammatory neurological diseases as well as other autoimmune disorders [12
]. In addition, the presence of directly ex vivo
detectable brain antigen-specific B cells during relapse was associated with a significantly increased risk of the development of a subsequent relapse within the next few months [13
In the following, we used this assay to study the correlation between the EBV-, Cytomegalovirus (CMV)- and brain-specific B cell response as detected in the blood of patients with MS. The data show that there was no difference in the EBV-specific B cell response in the blood or the previous viral reactivation status comparing healthy donors and MS patients. Along these lines, the B cell response status to EBV did not have a direct clinical impact on the course and severity of established MS. Interestingly, however, there was an association between the frequencies of CMV- and brain-reactive B cells in the blood and disease activity in MS.
The foundations of an efficient and functional immune system are both high diversity and specificity of the lymphocyte pool. A side effect of the high diversity is that besides pathogen-specific cells, also autoreactive lymphocytes are released from the bone marrow and thymus. Circulating autoreactive B cells were found in healthy individuals without any history of an autoimmune disease [20
]. The question, which has remained unclear, is which mechanisms trigger the activation of these naïve autoreactive lymphocytes and initiate the development of an autoimmune disease only in some individuals compared to those individuals whose autoreactive cells remain in a dormant state and will never cause tissue damage. Various studies have focused on EBV and CMV as potential triggers of MS [1
]. There are several possible mechanisms including molecular mimicry, epitope spreading or bystander activation explaining the link between an anti-viral immune response and MS [24
]. These mechanisms linking viral infections to MS pathology are largely hypothetical [25
]. Molecular mimicry occurs when peptides from pathogens share structural similarities with self-antigens of the CNS, which leads to the activation of autoreactive lymphocytes. Infection with various pathogens, each with its individual molecular resemblance to a CNS antigen, may explain the inability of investigators to link one specific virus to MS [26
]. Wucherpfennig and Strominger showed that EBV peptides could activate myelin basic protein (MBP)-specific T cell clones isolated from the blood of MS patients [27
]. Bystander activation is based on the fact that viral infections lead to inflammation and activation of antigen-presenting cells (APC) such as dendritic cells. These activated APC could potentially activate autoreactive lymphocytes, which may then initiate autoimmune diseases [28
]. However, at the same time, the hypothesis of epitope spreading and bystander activation highlights the notion that the immunopathology may diverge in disease evolution and manifested MS.
The consensus of these hypotheses is that there is a correlation and/or cross-reactivity between brain- and virus-specific lymphocytes. In this study we used the ELISPOT approach to detect brain- and virus-specific B cells in the blood of MS patients and healthy controls. To our knowledge this is the first study comparing the B cell response to EBV, CMV and brain antigens using a cell-based assay. In this assay we used an EBV lysate as antigen mix to coat the plates. There are studies pointing out that EBV lysates are more immunogenic than EBV-encoded nuclear antigen 1 (EBNA-1). Loebel et al.
could show that EBV lysate induced production of several cytokines in particular interferon (IFN)-γ in whole blood in 50% of chronic fatigue syndrome (CFS) patients. Using EBNA-1 protein for stimulation, no patient showed a detectable IFN-γ response [29
]. We suggest that the use of EBV lysate has the advantage that it covers various immunogenic EBV proteins, which minimizes the risk of false negative results.
Our data demonstrate a correlation between EBV-specific and brain-reactive B cells in the blood of MS patients in remission. We also could detect a significant association between the frequencies of EBV-specific B cells and the disease activity in MS patients.
These results go in line with earlier studies. Latham et al.
postulated a correlation between the frequencies of EBV-specific T cells in the blood and the number of active lesions on MRI scans [30
] and Levin et al.
found high serum levels of IgG antibodies to EBV to be a strong predictor of MS [31
]. Serafini et al.
observed the presence of EBV-infected B cells in the brain in almost 100% of MS cases in addition to EBV reactivation in plasma cells in acute MS lesions and ectopic B cell follicles [3
]. These findings support a role for EBV infection in B cell activation in the MS brain, which may contribute to the disruption of B cell tolerance [32
]. Still, EBV-triggered B cell activation may be rather a consequence and not the cause of B cell activation paralleling polyclonal activation of serum antibodies against several viral agents in MS that is referred to as MRZ (measles-rubella-zoster) reaction. The increased viral load in the brain as compared to the blood suggested that a locally dysregulated viral infection could support the autoimmune response and tissue damage in the brain [3
]. These earlier results may explain why we could not detect increased numbers of EBV-specific B cells and antibody titers in MS patients experiencing an acute relapse due to the fact that EBV-specific cells might accumulate in the brain during acute inflammation. Under these conditions EBV-specific B cells circulating as memory B cells could become readily detectable in the blood during remission, which was the case in our study.
The findings by Serafini et al.
are in strong contrast to later studies that rarely detected EBV-infected B cells in MS brains. Willis et al.
studied a large cohort of MS specimens containing white matter lesions with parenchymal and meningeal B cell infiltrates and they could not detect EBV in any of the specimens using multiple techniques including in situ
hybridization, immunohistochemistry and two independent real-time PCR approaches [33
]. Moreover, Sargsyan et al.
failed to identify EBV infection in cerebrospinal fluid (CSF) B and plasma blast cell populations and EBV-specific transcripts were not detected in MS lesions. In addition, the extent of intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease patients [34
]. Overall, there is a largely divergent body of literature regarding the relationship between EBV and MS brain inflammation.
The influence of CMV on MS is also disputed. There are studies supporting a detrimental role of CMV as a trigger of MS, whereas most of the studies describe CMV infection as disease limiting. CMV has been found in demyelinating lesions and a T cell response against CMV epitopes has been observed within CD8+ cells derived from chronic inflammatory lesions [35
]. Other researchers found that the time to relapse decreased and the number of relapses increased with anti-CMV IgG positivity [36
]. In this study we were able to show that there was a significant correlation between the CMV- and brain antigen-specific B cell response in MS patients experiencing an acute relapse. Furthermore, an elevated B cell response to CMV correlated with a higher disease activity. In earlier studies we demonstrated that treatment-related effects had no impact on the ELISPOT results since the number of brain antigen-specific B cell positive MS patients was independent of the treatment status [12
]. However, to further analyze the impact of the immune modulatory treatment on our results, we correlated the frequencies of CMV-specific B cells with disease activity in patients in remission who were untreated (n
= 11). The Spearman’s rank correlation was 0.69 and the p
-value 0.035. This shows that the treatment does not significantly impact the results of the correlation analysis.
Earlier studies detected a correlation between CMV serum antibody titers and an increased MS disease risk. Sundqvist et al.
could show that CMV seropositivity was associated with a decreased risk of developing MS [37
]. Additionally, Zivadinov et al.
investigated an association between clinical and MRI measures of disease activity and the presence and titer of IgG antibodies against CMV in n
= 140 patients with definite MS and n
= 131 healthy controls [38
]. In their study there was an association between antibody positivity against CMV, a higher titer and better clinical and MRI outcomes [38
]. Limitations and a reason for the discrepancy in the results of the earlier studies could be the low sensitivity, which is due to the measurement of serum antibody titers rather than detecting virus-specific cells, and the lack of the technique reflecting cellular autoimmunity to brain antigens. Along these lines, in an independent currently ongoing study we were able to show that measurements of serum antibodies frequently failed to reveal CMV exposure in humans, which may be better identified by direct detection of CMV-specific memory lymphocytes [39
]. In the current study the tendency for a correlation between the CMV- and brain antigen-specific B cell response in MS patients experiencing a relapse remained similar when excluding CMV seronegative subjects. However, the correlation was not significant most likely due to the low number of CMV seropositive MS patients.