Journal of Neuroimmune Pharmacology

, Volume 5, Issue 3, pp 271–277

Epstein–Barr Virus Infection and Multiple Sclerosis: A Review

Authors

    • Department of NutritionHarvard School of Public Health
    • Department of EpidemiologyHarvard School of Public Health
  • Kassandra L. Munger
    • Department of NutritionHarvard School of Public Health
BRIEF REPORT

DOI: 10.1007/s11481-010-9201-3

Cite this article as:
Ascherio, A. & Munger, K.L. J Neuroimmune Pharmacol (2010) 5: 271. doi:10.1007/s11481-010-9201-3

Abstract

Epstein–Barr virus (EBV) infection results in a life-long persistence of the virus in the host’s B-lymphocytes and has been associated with numerous cancers including Burkitt’s lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma. There is considerable evidence that EBV infection is a strong risk factor for the development of multiple sclerosis. Early age at primary EBV infection is typically asymptomatic, but primary infection during adolescence or adulthood often manifests as infectious mononucleosis, which has been associated with a two- to threefold increased risk of MS. Most importantly, MS risk is extremely low in individuals who are EBV negative, but it increases several folds following EBV infection. Additional evidence supporting a role for EBV in MS pathogenesis includes the observations of elevated antibodies to EBV antigens (especially EBV nuclear antigen-1) prior to the onset of MS, and an increased risk of MS among EBV-positive children. The biological mechanism by which EBV may cause MS is not known, but several possibilities are discussed.

Keywords

Epstein–Barr virusmultiple sclerosisinfection

Introduction

In this review, we summarize the evidence linking infection with the Epstein–Barr virus (EBV) and multiple sclerosis (MS), focusing primarily on the results of epidemiological studies. We will start, however, with a brief introduction on the natural history of EBV infection and the recognized role of EBV in neoplastic diseases, as this will provide a useful perspective on the balancing act that results—in most individuals—in a life-long asymptomatic infection.

EBV infection

EBV, a potentially oncogenic virus belonging to the gamma-herpesvirus family, was discovered in the early 1960s in lymphoma cells cultivated from tumor biopsies obtained by Burkitt in African children with jaw tumors (Kieff and Rickinson 2007). Further investigation revealed that EBV is present in all populations and infects over 90% of individuals within the first decades of life. The virus exists in two main strains denoted as types 1 and 2, with several variants described within each strain (Gratama and Ernberg 1995). Primary infection usually occurs through contact with infected saliva, and is asymptomatic in young children, but in up to 40% of adolescents and adults it results in infectious mononucleosis (IM), an acute and usually self-limited lymphoproliferative disease of a few weeks duration. The clinical manifestations of IM are largely the results of a marked increase in circulating EBV-specific cytotoxic T lymphocytes and release of inflammatory cytokines (Kutok and Wang 2006). More intense viral transmission or age-related differences in immune responses because of a larger number of cross-reacting CD8 T lymphocytes in older individuals have been proposed to explain the strong correlation between IM and age (Rickinson and Kieff 2007).

Following primary infection, EBV persists for the life of the host in B-lymphocytes, in which the EBV double-stranded DNA forms an episome, typically present as a single copy at a frequency of 1 to 50 per million B-lymphocytes. The shift of EBV-infected B cells from a lymphoproliferative to a resting state seems to parallel the physiological process of B cell maturation (Thorley-Lawson 2001). In the tonsils, EBV infects naïve B cells and expresses nine latent proteins, EBV nuclear antigen (EBNA) 1 through 6, latent membrane protein (LMP) 1, LMP2a, and LMP2b, which promotes B cell activation to proliferating lymphoblasts (growth program). (Thorley-Lawson 2001) These EBV-infected lymphoblasts, like antigen-activated B cells, enter into follicles, where they differentiate into memory B cells. During this transition EBV expresses EBNA1, LMP1, and LMP2a (default program; Thorley-Lawson 2001); LMP1, which is a functional homologue of CD40, and LMP2a, which provides a signal analogous to that of the tonic survival signal of the B cell receptor, protect the cells from apoptosis, whereas EBNA-1 is needed for EBV replication at each cell division. Viral protein expression is shut down after the B cells have differentiated into resting memory B cells, so that these cells escape cytotoxic T lymphocyte detection. Periodic reactivation in the tonsils leads to shedding of infectious EBV in the saliva and transmission to a new host, thus completing the cycle.

EBV and cancer

In the immunosuppressed host, EBV can cause post-transplantation lymphoproliferative disorders, lymphomas, leiomyosarcomas, and oral hairy leukoplakia (Kutok and Wang 2006). Uncontrolled proliferation of EBV-infected B cells is observed in X-linked lymphoproliferative disorder, which results from mutation in a gene known as SAP (signaling lymphocyte activation molecule-associated protein; Kutok and Wang 2006). In the immunocompetent host, EBV is associated with other neoplastic conditions, which tend to have circumscribed geographical distributions, such as Burkitt’s lymphoma in areas holoendemic for Falciparum malaria in Africa and New Guinea (Kutok and Wang 2006), and nasopharyngeal carcinoma in South East Asia, North Africa, and among Inuits. The reasons for these geographical variations are largely unknown, and may include a combination of host genetics, EBV strain variations, and environmental factors, such as other infections (Moormann et al. 2007) and diet (Chang and Adami 2006; Feng et al. 2007). Furthermore, in western countries, EBV has been associated with Hodgkin lymphoma—the virus is present in clonal form in the malignant Reed–Steinberg cells of about 40% of tumors (Kutok and Wang 2006). EBV-positive Hodgkin lymphoma is more common among individuals with history of IM (Hjalgrim et al. 2003). Reed–Steinberg cells are probably germinal center B cells that have acquired crippling mutations in their immunoglobulin genes, but have escaped apoptosis because of activation of the nuclear factor kB (NFkB) that induces antiapoptotic genes (Kutok and Wang 2006); LMP1 is a potent NFkB activator and may thus contribute to the development of Hodgkin lymphoma. In contrast, Burkitt’s lymphoma is defined by the presence of a c-MYC translocation, resulting in the dysregulated expression of c-MYC (Kutok and Wang 2006). EBV may increase the incidence of Burkitt’s lymphoma by increasing genomic instability and the frequency of this translocation or by promoting the survival and proliferation of B-lymphocytes carrying the translocation (Rowe et al. 2009).

EBV and multiple sclerosis

A link between EBV and multiple sclerosis was first proposed to explain the striking similarity between the epidemiology of IM and that of MS in terms of age, geographical distribution, socioeconomic status, and ethnicity (Table 1; Warner and Carp 1981; Ascherio and Munger 2007a). IM, like MS, is rare in developing countries and, more generally, in conditions of poor hygiene, where virtually all children are infected with EBV in the first years of life. A young age at EBV infection and thus few cases of IM are also observed among Inuits (Melbye et al. 1984) and in Japan, (Takeuchi et al. 2006) the latter possibly because of sharing food from the same bowl. In contrast, IM is common in western countries, where about 50% of individuals escape infection in early childhood and acquire EBV during adolescence and young adulthood, most commonly by kissing; in these countries, MS risk is two- to threefold higher among individuals with history of IM (Ahlgren et al. 2009; Nielsen et al. 2007; Ramagopalan et al. 2009; Thacker et al. 2006; Zaadstra et al. 2008).
Table 1

Similarities between multiple sclerosis and infectious mononucleosis epidemiology

Parameter

MS

IM

Age at peak incidence (year)

25–34

15–24

Age at onset

F < M

F < M

Geography

  

 Extremely rare in the tropics

+++

+++

 Latitude gradient within temperate regions

+++

+++

 Rare in Japan

+++

+++

 Rare in Eskimos

++

++

Positive association with SES

+

++

Incidence in Blacks < Whites

+

++

Incidence in Asians < Whites

++

++

+++ strong evidence, ++ moderate evidence, + weak evidence, F female, M male, SES socioeconomic status

Reproduced with permission from Ascherio and Munger (2010)

A possible explanation of the higher MS risk following IM is that both diseases are linked to good hygiene in childhood, which results in a delayed age at infection with EBV and other infectious agents (Fig. 1a).
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Fig. 1

The hygiene hypothesis of MS causation. a The formulation of the hygiene hypothesis that states MS and IM arise from the common cause of “high hygiene” in childhood and are associated due to this common cause. b The “EBV variant” of the hygiene hypothesis, which states that high hygiene in childhood increases the likelihood of a late age at infection with EBV (IM), which then leads to an increased risk of MS. Current epidemiological data support this latter formulation of the hypothesis. See text for additional details

The lower load of infections in infancy and childhood would prime the immune system toward a more inflammatory response and an increased risk of autoimmune diseases (Bach 2002). Alternatively, higher hygiene could be related to an increased MS risk because of the late age at EBV infection (Fig. 1b). Discrimination between these two states of nature is important, because of their different public health implications—only under the diagram in Fig. 1b, interventions on EBV, such as prophylactic infection at an early age or a vaccine providing sterile immunity, would be expected to reduce MS risk. These two states of nature also have opposite predictions on the MS risk of individuals who escape EBV infection. Because these individuals have on average a highly hygienic upbringing, if hygiene itself was a risk factor for MS their MS risk should be high; in contrast, if EBV infection itself was causally related to MS, their MS risk would be very low. The MS risk of EBV-negative individuals can be indirectly estimated from the results of multiple cross-sectional or case–control studies—in a recent meta-analysis of these studies we found this risk to be about 15 times lower than that of EBV-positive individuals (Ascherio and Munger 2007a). This result not only strongly supports the conclusion that the association between IM and MS cannot be explained by confounding by hygiene during childhood, but also suggests that EBV infection at any age is an important risk factor for MS. The latter conclusion is further supported by studies of pediatric MS—EBV-positive children, the large majority of whom were infected in the first years of life and have no history of IM, have a markedly higher MS risk than EBV-negative children (Alotaibi et al. 2004; Banwell et al. 2007; Pohl et al. 2006). The extremely low MS risk in EBV-negative individuals and the high risk in those with a history of IM also suggest that there is a sharp increase in MS risk following IM, which only occurs following primary EBV infection (Fig. 2). An alternative explanation, however, is that individuals who are EBV negative are genetically resistant to both EBV infection and MS.
https://static-content.springer.com/image/art%3A10.1007%2Fs11481-010-9201-3/MediaObjects/11481_2010_9201_Fig2_HTML.gif
Fig. 2

Schematic representation of MS incidence according to EBV infection. Reprinted with permission from Thacker et al. (2006)

More recently, we have been able to follow longitudinally a large population of EBV-negative young adults and directly observe the temporal relation between primary EBV infection and MS risk (Levin et al. 2010). In this population, we observed no cases of MS before EBV infection, thus confirming the very low MS risk inferred from cross-sectional studies. EBV infection, however, occurred at a rate of about 11% per year, and the incidence of MS increased sharply following EBV infection (Levin et al. 2010). The results of this study, by showing that MS risk changes in the same subjects following EBV infection, virtually rule out confounding by genetic factors as an explanation for the low MS risk among EBV-negative individuals and establish EBV infection as a robust risk factor for MS.

Among healthy individuals infected with EBV, MS risk increases monotonically by several folds with increasing serum titers of anti-EBNA complex and anti-EBNA-1 antibodies (Ascherio et al. 2001; DeLorenze et al. 2006; Levin et al. 2005; Sundstrom et al. 2004). This association could in theory be due to confounding by genetic factors, but it was not explained by the MS risk haplotype HLA-DRB1*1501 (De Jager et al. 2008), which is the strongest genetic predictor of MS. Results of preliminary studies suggest that the presence of EBV in plasma (Wagner et al. 2004) and antibodies to the lytic antigen BZLF1 (Massa et al. 2007) may also predict an increased MS risk, but these associations are weaker than those observed for antibodies to EBNA-1. An interesting question is whether anti-EBV antibodies or other markers of EBV immunity are also related to MS severity and progression. Some studies suggest that EBV reactivation may play a role in MS relapses (Wandinger et al. 2000) or disease activity (Buljevac et al. 2005), but overall results are unconvincing. Only recently, attempts have been made to relate the serum titers of anti-EBV antibodies to conversion from a clinically isolated syndrome (CIS), an isolated (in time) event of central nervous system demyelination, to MS and to MS severity and progression. Positive correlations between anti-EBV antibodies and progression among individuals with MS were reported in two independent studies. Zivadinov et al., in a study including 50 patients, reported a positive correlation between anti-VCA antibodies and increased loss of brain volume on MRI over a 3-year period (Zivadinov et al. 2009), whereas Farrell et al. have reported that serum titers of anti-EBNA-1 IgG antibodies, but not anti-VCA antibodies, were correlated with number of gadolinium-enhancing lesions, T2 volume and EDSS score over a period of 5 years in a study including 50 patients with CIS, 25 patients with relapsing remitting MS and 25 patients with primary progressive MS (Farrell et al. 2009). In contrast, no association was found between antibodies to EBV and progression from CIS to MS in a cohort of Austrian patients with positive oligoclonal bands (Abstract presented at the 2009 Austrian Neuroscience Wintermeeting, Kitzbuhel, Austria). Further investigations are clearly needed to clarify these rather inconsistent findings. The results of three placebo-controlled trials of the antiviral alacyclovir or its precursor valacyclovir suggested overall a non-significant benefit in the treated patients, but these studies were too small to be conclusive, and alacyclovir does not decrease the number of latently infected B cells (Bech et al. 2002; Friedman et al. 2005; Lycke et al. 1996). The marked effects of rituximab, a monoclonal antibody that selectively targets and depletes CD20+ B cells, on relapse rates in MS patients is consistent with a continuing role of EBV in MS immunopathology (Hauser et al. 2008), but open to multiple alternative interpretations.

Although the epidemiological evidence linking EBV infection to MS risk is rather compelling, the mechanisms underlying this link remain unclear. One possibility is that EBV-infected B cells infiltrate the MS brain and elicit a cytotoxic T lymphocyte response with damage to surrounding tissue. This hypothesis is supported by the results of a study of 22 MS brains by Serafini et al., in which a high proportion of B cells in ectopic meningeal follicles and perivascular infiltrates in MS lesions were found to be positive by in situ hybridization for EBV RNA transcripts (EBER; Serafini et al. 2007) and by immunohistochemistry for EBNA-2 and LMP-1, two EBV latent proteins that are not normally expressed in circulating resting EBV-infected memory B cells. Based on these and other corroborating findings, the authors concluded that brain infiltration with EBV-infected B cells is common in MS and drives the immunopathology (Serafini et al. 2007). However, the results of follow-up investigations were largely null. Willis et al., who studied a total of 24 MS brains, were unable to find evidence of EBV infection in a first set of 12 MS cases by using a combination of in situ hybridization, immunohistochemical techniques, and real-time polymerase chain reaction (PCR) methods for genomic EBV or EBER (Willis et al. 2009), and only detected low levels of EBV by PCR, but not by in situ hybridization, in two meningeal samples in the second set (Willis et al. 2009). Peferoen et al. screened 94 MS patients, including 12 patients included in the Serafini et al. study, using in situ hybridization for EBER and immunohistochemistry for latent (LMP-1) and lytic (BZLF1, BMRF1, BFRF3, and BLLF1) EBV proteins (Peferoen et al. 2009). Only one tissue specimen was found positive for EBER, and one was found to be negative for EBER but positive for multiple lytic proteins. PCR for EBV genome and encoded RNA applied to five tissue samples was negative. Finally, Sargsyan et al. (Sargsyan et al. 2010) searched for evidence of EBV infection in MS brains using real-time PCR for EBER and other EBV RNA transcripts (EBNA-2, LMP-1, and BFRF1) in 15 fresh-frozen MS plaques, five formalin-fixed paraffin-embedded plaques, including three that tested positive for EBV DNA in a previous study, and in single plasma cells (n = 274) and B cells (n = 67) from MS cerebral spinal fluid (CSF). Evidence of EBV transcripts was only found in the three formalin-fixed plaques known to be positive for EBV DNA. Furthermore, intrathecal synthesis of anti-EBV antibodies did not differ from that of non-MS inflammatory patients and recombinant antibody generated from clonally expanded plasma cells from MS CSF did not bind EBV-infected cells.

A possible explanation for the discordant findings of pathological studies is that EBV-infected B cells are present only in ectopic B cell meningeal follicles and in the early phases of MS lesion formation, which are rarely captured in autopsy material. Alternatively, EBV could contribute to MS onset acting outside of the central nervous system. One hypothesis is that of molecular mimicry between EBV and myelin antigens, which could involve antibodies (Rand et al. 2000; Vaughn et al. 1996) or T lymphocytes.(Hollsberg et al. 2003; Holmoy et al. 2004; Lang et al. 2002). Consistent with this hypothesis are the presence of immune responses in the CSF of MS patients to two EBV peptides, including EBNA-1 (Cepok et al. 2005), the increased frequency and broadened specificity in MS of CD 4+ T cells recognizing EBNA 1, (Lunemann et al. 2006) and the report of EBNA-1-pecific cell lines from MS patients cross-reacting with myelin antigens (Lunemann et al. 2008). Results of the investigations of EBV-specific cytotoxic T cells in MS have been somewhat inconsistent. Gronen et al. have reported no difference in the frequency of cytotoxic T cells against HLA-B7 restricted immunogenic EBV peptides between MS patients and healthy controls (Gronen et al. 2006). In contrast, Jilek et al. found a stronger EBV-specific CD8+ T cell response in patients with clinically isolated syndrome than in healthy controls and a decrease in the intensity of the response among MS patients with increasing disease duration (Jilek et al. 2008), whereas Pender et al. reported an overall decrease in the frequency of EBV-specific CD8+ T cells in MS patients in response to autologous EBV-infected B-cell lymphoblastoid cell lines (Pender et al. 2009). It has also been proposed that EBV may act by transactivation of a HERV-K18, an endogenous retrovirus that encodes a superantigen (Sutkowski et al. 2001; Tai et al. 2008), infection of autoreactive B cells (Pender 2003), or induction and antigenic presentation of alpha-beta crystallin (van Sechel et al. 1999). The recent report that EBER-1, a 167-nucleotide-long RNA expressed in EBV latent infection, binds to cellular proteins and is released in serum where it binds to toll-like receptor 3 and induces type 1 interferon and pro-inflammatory cytokines (Iwakiri et al. 2009) provides an additional potential link between EBV and MS—EBV-infected B cells in meningeal or intraparenchymal vessels could elicit an inflammatory response and facilitate brain infiltration by activated macrophages and lymphocytes.

It is important to note that several aspects of MS epidemiology, including the decline in MS risk with migration from high to low prevalence regions (Gale and Martyn 1995) and the putative epidemics in the Faroe islands (Kurtzke and Heltberg 2001) cannot be explained by EBV infection, unless it is postulated that there is an EBV subtype with a higher propensity to cause MS (Ascherio and Munger 2007a). Mutations in LMP-1 and other EBV genes are being investigated in EBV-related neoplasias, but there are not yet conclusive data. In MS, there is an interesting report of infection with same EBV subtype in a small cluster (Munch et al. 1998), but preliminary studies based on LMP-1 and EBNA-1 sequencing have been inconclusive (Lindsey et al. 2008).

Because the large majority of individuals are infected with EBV, but only about one in 500 will ever get MS, other factors must be critical for MS development. Genetic predisposition certainly plays an important role (Ebers et al. 1995), and growing evidence implicates some environmental factors such as cigarette smoking (Ascherio and Munger 2007b) and low vitamin D (Munger et al. 2004, 2006). Infections that affect the immune response to EBV (Clute et al. 2005; Kim et al. 2005) may also be important. Nevertheless, the extremely low risk of MS among EBV-negative individuals suggests that EBV is a critical component cause in most, if not all, cases of MS. More uncertain is the role of EBV in MS progression.

In future studies, it will be important to elucidate the mechanisms underlying the link between EBV and MS and to determine whether EBV only triggers MS or also contributes to the disease progression. To understand the mechanisms, an important approach is the systematic investigation of polymorphisms in human genes related to EBV infection. So far, studies on EBV-related genes are limited to two preliminary reports, one suggesting a possible association between the EBV transactivated HERV-K18 and MS (Tai et al. 2008) and the other the lack of association between the EBV receptor CR2 (CD21) and MS risk (Simon et al. 2007). The investigation on the role of EBV in MS progression, on the other hand, will need large longitudinal observational investigations. Conclusive proof that EBV is causally related to MS may need to await the development of an effective vaccine, which is under development primarily for the prevention of IM (Moutschen et al. 2007; Sokal et al. 2007); paradoxically, however, a vaccine that delays rather than prevents EBV infection could increase the incidence of both IM and MS.

In summary, there is convincing epidemiological evidence that EBV infection is a strong risk factor for MS development, although the mechanisms remains elusive. The epidemiological data suggest that MS risk could be markedly reduced by preventing EBV infection, which could only be possible with a hypothetical vaccine that confers permanent sterile immunity against EBV or, less effectively, by causing an iatrogenic EBV infection in early childhood, when the adverse effect of infection on MS risk seems mitigated. It remains to be determined whether EBV continues to play a role after disease initiation and thus constitutes a valuable target for MS treatment.

Acknowledgments

We thank Ms. Leslie Unger for technical assistance in the preparation of this manuscript.

Disclaimers

The authors have no conflicts of interest to disclose.

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© Springer Science+Business Media, LLC 2010