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EBV and MS: Major cause, minor contribution or red-herring?

  • Sean Burnard
    Correspondence
    Corresponding author.
    Affiliations
    Medical Genetics, Hunter Medical Research Institute, School of Biomedical Sciences, Faculty of Health and Medicine, University of Newcastle, NSW, Australia
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  • Jeannette Lechner-Scott
    Affiliations
    Department of Neurology, John Hunter Hospital, New Lambton, NSW, Australia/Hunter Medical Research Institute, Newcastle, NSW, Australia/School of Health Sciences, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia
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  • Rodney J. Scott
    Affiliations
    Medical Genetics, Hunter Medical Research Institute, School of Biomedical Sciences, Faculty of Health and Medicine, University of Newcastle, NSW, Australia

    Division of Molecular Medicine, Pathology North, John Hunter Hospital, Newcastle, NSW, Australia
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      Highlights

      • EBV has a strong long-standing association with MS but with conflicting evidence.
      • An estimated 90% of the human population are infected by EBV, yet only a small proportion develop ‘EBV related diseases’.
      • EBV presence within the Central Nervous System has not been conclusively shown.
      • Molecular mimicry causing CD8+ T cells to target myelin basic protein can be achieved by pathogens other than just EBV.
      • MS may be the failure of viral clearance and driven by multiple pathogens.

      Abstract

      Multiple Sclerosis (MS) is a chronic neurological disease with genetic and environmental risk factors. Epstein Barr-Virus (EBV) has been closely associated with MS but with a significant amount of conflicting evidence. Some of the evidence for EBV involvement in MS includes: almost 100% of MS patients showing past EBV infection, an association with Infectious Mononucleosis (acute EBV infection), higher titres of EBV antibodies associated with an increased risk of MS development, and an overall altered immune response to EBV found in peripheral blood and the CNS of MS patients. However, evidence for EBV presence in the CSF and T cell responses to EBV in MS have been particularly conflicting. Several hypotheses have been proposed for direct and indirect EBV involvement in MS such as 1) Molecular Mimicry 2) Mistaken Self 3) Bystander Damage and 4) Autoreactive B cells infected with EBV. More recently, an association between EBV and human endogenous retrovirus in MS has been shown, which may provide an alternative pathogenetic target for MS treatment. However, if EBV is not the major contributor to MS and is instead one of several viral or infectious agents able to elicit a similar altered immune response, MS development may be the result of a failure of viral clearance in general. This review aims to evaluate the evidence for the currently discussed theories of EBV involvement in MS pathogenesis.

      Abbreviations:

      CNS (Central Nervous System), CSF (Cerebral Spinal Fluid), EBV (Epstein-Barr Virus), HC (Healthy Control), EAE (Experimental Autoimmune Encephalomyelitis), HERVS (human endogenous retroviruses), LCMV (lymphocytic choriomeningitis virus), MHC (Major Histocompatibility Complex), MOG (Myelin Oligodendrocyte Glycoprotein), MS (Multiple Sclerosis), MSRV (Multiple Sclerosis Associated Retrovirus), PBMCs (Peripheral Blood Mononuclear Cells)

      Keywords

      1. Introduction

      Multiple Sclerosis (MS) is a chronic neurological disease affecting more than 23,000 individuals in Australia and approximately 2.5 million people worldwide (
      • Hollenbach J.A.
      • Oksenberg J.R.
      The immunogenetics of multiple sclerosis: a comprehensive review.
      ). Three times as many woman as men (
      • Ribbons K.
      • Lea R.
      • Tiedeman C.
      • Mackenzie L.
      • Lechner-Scott J.
      Ongoing increase in incidence and prevalence of multiple sclerosis in Newcastle, Australia: a 50-year study.
      ) are now affected by MS and the incidence of MS is increasing in ethnic groups other than European Caucasians. MS is associated with neurodegeneration and central nervous system (CNS) inflammation mediated by an aberrant immune system and characterised by an altered T Cell response (
      • Carbajal K.S.
      • Mironova Y.
      • Ulrich-Lewis J.T.
      • Kulkarni D.
      • Grifka-Walk H.M.
      • Huber A.K.
      • et al.
      Th cell diversity in experimental autoimmune encephalomyelitis and multiple sclerosis.
      ) to self.
      While there have been significant improvements in the treatments of MS over the last decade, the underlying cause and pathogenesis of MS remains unclear. Advances in technologies such as Next Generation Sequencing and bioinformatics has enabled the identities of approximately one third of the genetic susceptibilities to MS to be localized to the Major Histocompatibility Complex (MHC), and an additional 110 polymorphisms centred on 103 discrete loci outside the MHC (
      • Hollenbach J.A.
      • Oksenberg J.R.
      The immunogenetics of multiple sclerosis: a comprehensive review.
      ). Notwithstanding, these genetic risk factors still only account for ~30% of total disease risk. The recognised contribution of genetic risk in MS is likely to increase as technologies improve, multi-centre focused initiatives increase and genetic pathway/ interaction analysis between gene-gene and gene-environmental links are better understood. Epidemiological risk factors for MS have been increasingly implicated with MS pathogenesis but require further investigation to differentiate simple association with genuine pathogenesis. The question remains, are these observations the result of an altered systemic immune system because of MS, or do they have a genuine bearing on MS pathogenesis or disease progression? The environmental risk factors most closely investigated to date, include: obesity, smoking, reduced sunlight exposure (Vitamin D deficiency) and Epstein-Barr Virus (EBV) infection (
      • Goodin D.S.
      Chapter 11 - The epidemiology of multiple sclerosis: insights to a causal cascade†.
      ).
      Out of these risk factors, EBV has been significantly implicated in MS pathogenesis. The most troubling question for EBV involvement in MS is that since the majority of the population are infected by EBV - why do some people develop MS, while others may develop cancer, and most individuals are asymptomatic with no further health problems? This review evaluates the current evidence for EBV involvement in MS in relation to disease pathogenesis.

      2. Epstein-Barr virus biology and route of infection

      EBV is a double stranded human herpesvirus that has infected more than 90% of the population worldwide (
      • Gao J.
      • Luo X.
      • Tang K.
      • Li X.
      • Li G.
      Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji).
      ) and results in lifelong infection. The majority of EBV infections are asymptomatic and occur in early childhood, but if EBV is contracted later in adolescence then Infectious Mononucleosis (IM) is more likely to develop, with varying degrees of clinical severity (
      • Dunmire S.K.
      • Hogquist K.A.
      • Balfour H.H.
      Infectious mononucleosis.
      ). The prevalence of EBV infection among pre-adolescences is lower and varies significantly depending on age, geographic location and race/ethnicity (
      • Condon L.M.
      • Cederberg L.E.
      • Rabinovitch M.D.
      • Liebo R.V.
      • Go J.C.
      • Delaney A.S.
      • et al.
      Age-specific prevalence of Epstein-Barr virus infection among minnesota children: effects of race/ethnicity and family environment.
      ).
      EBV transmission typically occurs via sharing of infected saliva. Nearly all of those who are EBV seropositive shed virus into their saliva and are capable of infecting EBV naïve individuals. However, primary EBV infection is also possible via hematopoietic cell transplantation, solid organ transplantation and blood transfusion (
      • Dunmire S.K.
      • Hogquist K.A.
      • Balfour H.H.
      Infectious mononucleosis.
      ). EBV is capable of infecting epithelial cells, B cells as well as Natural Killer and T cells (
      • Kang M.-S.
      • Kieff E.
      Epstein-Barr virus latent genes.
      ). The primary route of infection initiates via oropharyngeal epithelium where active viral replication occurs, also known as lytic infection (
      • Ok C.Y.
      • Li L.
      • Young K.H.
      EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management.
      ). Following this lytic cycle, EBV infects nearby naïve B cells via viral enveloping of the protruding glycoprotein on EBV GP350, with complement Receptor 2 (CD-21) on B-Cells (
      • Ok C.Y.
      • Li L.
      • Young K.H.
      EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management.
      ,
      • Pender M.P.
      • Burrows S.R.
      Epstein–Barr virus and multiple sclerosis: potential opportunities for immunotherapy.
      ). Once EBV infects B cells it is able to turn them into active B blasts. EBV then activates its ‘growth programme’ (latency III), turning these B blasts into resting memory B cells (
      • Pender M.P.
      • Burrows S.R.
      Epstein–Barr virus and multiple sclerosis: potential opportunities for immunotherapy.
      ). The virus enters the latent stage after infecting naïve B cells, which is characterised by a drastic reduction in the number of proteins and miRNA expressed and can be divided into 3 stages (latency types I-III), depending on the combination of proteins expressed (
      • Kang M.-S.
      • Kieff E.
      Epstein-Barr virus latent genes.
      ). These proteins include: 6 nuclear antigens (EBNA-1, 2, 3A, 3B, 3C and LP), 3 latent membrane proteins (LMP-1, 2A and 2B) and non-coding RNA (EBER-1 and 2) (
      • Ok C.Y.
      • Li L.
      • Young K.H.
      EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management.
      ). During lytic infection the EBV genome is linear and is capable of expressing all of its encoded proteins (approximately 100 viral proteins) and non-coding RNA, but circularises forming an episome in the nucleus of infected B cells during latent infection (
      • Ok C.Y.
      • Li L.
      • Young K.H.
      EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management.
      ).
      An alternative mechanism by which EBV persists within cells is by integrating into the host cell genome. Several studies have shown the ability of EBV to integrate within chromosomes successfully (
      • Gao J.
      • Luo X.
      • Tang K.
      • Li X.
      • Li G.
      Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji).
      ,
      • Santpere G.
      • Darre F.
      • Blanco S.
      • Alcami A.
      • Villoslada P.
      • Mar Alba M.
      • et al.
      Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1000 genomes project.
      ), and indeed EBV integration of B cells in vitro is often used to establish immortal cell lines. However, studies into EBV DNA integration have been difficult due to methylated EBV DNA and multiple copies of viral episomes that create interference impeding mapping studies (
      • Gao J.
      • Luo X.
      • Tang K.
      • Li X.
      • Li G.
      Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji).
      ,
      • Takakuwa T.
      • Luo W.J.
      • Ham M.F.
      • Sakane-Ishikawa F.
      • Wada N.
      • Aozasa K.
      Integration of Epstein-Barr virus into chromosome 6q15 of Burkitt lymphoma cell line (Raji) induces loss of BACH2 expression.
      ). The integration of EBV has been shown to be random in cell lines (
      • Gao J.
      • Luo X.
      • Tang K.
      • Li X.
      • Li G.
      Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji).
      ,
      • Santpere G.
      • Darre F.
      • Blanco S.
      • Alcami A.
      • Villoslada P.
      • Mar Alba M.
      • et al.
      Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1000 genomes project.
      ,
      • Takakuwa T.
      • Luo W.J.
      • Ham M.F.
      • Sakane-Ishikawa F.
      • Wada N.
      • Aozasa K.
      Integration of Epstein-Barr virus into chromosome 6q15 of Burkitt lymphoma cell line (Raji) induces loss of BACH2 expression.
      ), which may explain why some individuals infected by EBV develop EBV-related diseases compared to others. Furthermore, Hernando et. al 2013 has shown in vitro that EBV B cell infection causes hypomethylation, resulting in overexpression of approximately 250 genes (
      • Hernando H.
      • Shannon-Lowe C.
      • Islam A.B.
      • Al-Shahrour F.
      • Rodriguez-Ubreva J.
      • Rodriguez-Cortez V.C.
      • et al.
      The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion.
      ). If this occurs in vivo, in combination with random integration, this could further alter methylation patterns caused by EBV and potentially drive different EBV related diseases such as lymphomas and MS.

      3. The association of EBV and MS

      3.1 History of IM

      As previously mentioned, IM is more commonly caused by late EBV infection compared to asymptomatic individuals who are infected by EBV earlier in life. There is a large amount of evidence associating IM with MS. In 2010 A meta-analysis of past history for IM and development of MS produced a relative risk of 2.17 (95% CI 1.97–2.39) (
      • Handel A.E.
      • Williamson A.J.
      • Disanto G.
      • Handunnetthi L.
      • Giovannoni G.
      • Ramagopalan S.V.
      An updated meta-analysis of risk of multiple sclerosis following infectious Mononucleosis.
      ). Sundqvist et al. later confirmed this in another meta-analysis with an odds ratio of 1.89 (1.45–2.48 95% CI) (
      • Sundqvist E.
      • Sundstrom P.
      • Linden M.
      • Hedstrom A.K.
      • Aloisi F.
      • Hillert J.
      • et al.
      Epstein-Barr virus and multiple sclerosis: interaction with HLA.
      ). It is important to note that some of these studies rely on self-reported data, which is prone to error, some of which is the result of ‘IM like symptoms’ caused by other viruses, such as cytomegalovirus (
      • Dunmire S.K.
      • Hogquist K.A.
      • Balfour H.H.
      Infectious mononucleosis.
      ). However, Goldacre et al. showed a fourfold increase in MS risk following hospital admission with confirmed IM (
      • Goldacre M.J.
      • Wotton C.J.
      • Seagroatt V.
      • Yeates D.
      Multiple sclerosis after infectious mononucleosis: record linkage study.
      ). The mean onset to MS following IM in this study was 14 years (
      • Goldacre M.J.
      • Wotton C.J.
      • Seagroatt V.
      • Yeates D.
      Multiple sclerosis after infectious mononucleosis: record linkage study.
      ) compared to a large Danish cohort, which showed an increased risk of MS after 10 years and persisted even after 30 years following IM (
      • Nielsen T.R.
      • Rostgaard K.
      • Nielsen N.M.
      • Koch-Henriksen N.
      • Haahr S.
      • Sorensen P.S.
      • et al.
      Multiple sclerosis after infectious mononucleosis.
      ). Sundqvist et al. also showed an association between IM infection and the presence of the risk allele DRB1*15 with an attributable proportion score of 0.34 (0.001–0.68 95% CI), compared to those without a history of IM and with the presence or absence of DRB1*15 (
      • Sundqvist E.
      • Sundstrom P.
      • Linden M.
      • Hedstrom A.K.
      • Aloisi F.
      • Hillert J.
      • et al.
      Epstein-Barr virus and multiple sclerosis: interaction with HLA.
      ). This further implicates the role of acute EBV infections in the development of MS.

      3.2 Seropositivity in MS

      Approximately 95% of the world's population is thought to have been infected by EBV at some point in their lives (
      • Luzuriaga K.
      • Sullivan J.L.
      Infectious mononucleosis.
      ). This has been supported by 80–95% of healthy controls (HCs) consistently showing EBV seropositivity (
      • Lucas R.M.
      • Hughes A.M.
      • Lay M.L.
      • Ponsonby A.L.
      • Dwyer D.E.
      • Taylor B.V.
      • et al.
      Epstein-Barr virus and multiple sclerosis.
      ). Intriguingly, nearly every study reporting EBV seropositivity in MS patients has shown >99% are infected. A recent meta-analysis has argued that the true value for MS patients seropositive for EBV is actually 100% since those studies showing anything less, used only a single EBV detection method (
      • Pakpoor J.
      • Disanto G.
      • Gerber J.E.
      • Dobson R.
      • Meier U.C.
      • Giovannoni G.
      • et al.
      The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
      ). Whereas, studies that used a combination of two different EBV test methodologies confirmed 100% of MS patients as seropositive. However, this restricted the meta-analysis to only 14.6% of cases (n=402/2760) (
      • Pakpoor J.
      • Disanto G.
      • Gerber J.E.
      • Dobson R.
      • Meier U.C.
      • Giovannoni G.
      • et al.
      The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
      ). In a longitudinal study of US army personnel, all individuals who were found to be seronegative for EBV seroconverted prior to MS onset, compared to only 35.7% of controls who were initially seronegative (
      • Levin L.I.
      • Munger K.L.
      • O'Reilly E.J.
      • Falk K.I.
      • Ascherio A.
      Primary infection with the epstein-barr virus and risk of multiple sclerosis.
      ). This evidence has given rise to the notion that infection by EBV is a pre-requisite to the development of MS. However, an EBV serology study on paediatric MS patients using two methods of detection (ELISA and Indirect Immunofluorescence) determined 2 out of 147 (1.4%) to be seronegative. This could be the result of false negative results, but this is less likely due to the utilisation of two methodologies applied simultaneously. Furthermore, pathogenesis of paediatric MS may also be slightly different to that of adult onset disease. Therefore, the inconsistency of the results does not confirm the argument that EBV seroconversion is a prerequisite for MS. To resolve this issue further testing using a consistently accurate assay in a large patient cohort is required.
      EBV titre levels were also shown to modulate disease risk in relation to other known genetic and behavioural MS risk factors such as the presence of DRB1*15, absence of A*02 and smoking. Higher Anti-EBNA-1 levels and the presence of DRB1*15 have been shown to be independent risk factors for MS but interact additively (
      • De Jager P.L.
      • Simon K.C.
      • Munger K.L.
      • Rioux J.D.
      • Hafler D.A.
      • Ascherio A.
      Integrating risk factors: hla-drb1*1501 and Epstein-Barr virus in multiple sclerosis.
      ). Jager et al. showed women with DRB1*15 and higher anti-EBNA-1 levels had a nine-fold increased risk of MS compared to those with lower EBNA-1 levels and DRB1*15 presence. Sundqvist et al. confirmed this in a larger cohort and went on to confirm that anti-EBNA-1: 385-420 IgG was a stronger risk marker than anti-EBNA IgG (
      • Sundqvist E.
      • Sundstrom P.
      • Linden M.
      • Hedstrom A.K.
      • Aloisi F.
      • Hillert J.
      • et al.
      Epstein-Barr virus and multiple sclerosis: interaction with HLA.
      ,
      • Sundstrom P.
      • Nystrom M.
      • Ruuth K.
      • Lundgren E.
      Antibodies to specific EBNA-1 domains and HLA DRB1*1501 interact as risk factors for multiple sclerosis.
      ). Furthermore, a 16-fold increase in risk was identified, in an additive but not multiplicative interaction, between higher anti-EBNA-1: 385-420 IgG, presence of DRB1*15 and absence of A*02. Interestingly, higher levels of anti-EBNA IgG were also shown to interact additively with smoking to increase MS risk (
      • Simon K.C.
      • van der Mei I.A.
      • Munger K.L.
      • Ponsonby A.
      • Dickinson J.
      • Dwyer T.
      • et al.
      Combined effects of smoking, anti-EBNA antibodies, and HLA-DRB1*1501 on multiple sclerosis risk.
      ). The incidence of IM and smoking on MS risk was found to compete with one another, producing a negative multiplicative interaction (

      Bjornevik K., Riise T., Bostrom I., Casetta L., Cortese M., Granieri E.. et al. 2016. Negative interaction between smoking and EBV in the risk of multiple sclerosis: The EnvIMS study. Multiple sclerosis (Houndmills, Basingstoke, England).

      ). This antagonistic relationship is intriguing, since IM would be predicted to result in higher circulating EBNA-1 IgG levels compared to that observed in asymptomatic EBV infection carriers. Thus, a higher anti-EBNA-1 titre should reflect a similar competitive risk factor for MS with smoking. Taken together this suggests that additional factors, rather than just the severity of EBV infection, may determine EBNA-1 IgG levels in MS patients. It is worth noting that this latter study relied solely on self-reported data and smoking was recorded with only a ‘yes’ or ‘no’ response, with no clarification on quantity or duration of smoking. Such studies of risk interaction are crucial in aiding identification of how different factors may interplay and be involved in disease pathogenesis.

      3.3 Altered immune responses to EBV in MS

      Studies into EBV immune responses in MS have taken two different approaches: one focuses on measuring CD8+ responses to specific EBV peptides or multimers; and the other utilises EBV infected B cells. The use of peptides essentially bypasses the antigen processing stage seen in normal physiological conditions, but enables scrutiny of peptides to identify which stages of EBV infection are more highly targeted. Whereas studies using EBV infected B cells in autologous B cell lymphoblastoid cell lines (LCL) provides an in vitro method closer to normal physiological conditions utilising each person's natural antigen processing mechanisms. Furthermore, a small proportion of LCLs are in the lytic phase of infection, which means both latent and lytic antigens will be present (
      • Pudney V.A.
      • Leese A.M.
      • Rickinson A.B.
      • Hislop A.D.
      CD8(+) immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells.
      ). However, testing by both methodologies has produced inconsistent results, potentially due to the time of collection in relation to disease activity or the way the methods were applied.
      Pender et. al 2009 using autologous LCL showed a decreased CD8+ T cell response in MS patients (
      • Pender M.P.
      • Csurhes P.A.
      • Lenarczyk A.
      • Pfluger C.M.
      • Burrows S.R.
      Decreased T cell reactivity to Epstein-Barr virus infected lymphoblastoid cell lines in multiple sclerosis.
      ), whereas another study found no difference between MS and HCs (
      • Lindsey J.W.
      • Hatfield L.M.
      Epstein-Barr virus and multiple sclerosis: cellular immune response and cross-reactivity.
      ). A further study found increased levels of IFNγ production by CD8+ T cells in response to LCLs in MS (
      • Cepok S.
      • Zhou D.
      • Srivastava R.
      • Nessler S.
      • Stei S.
      • Bussow K.
      • et al.
      Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis.
      ). Additional evidence supporting an aberrant CD8+ T cell control of EBV infected cells include a trend towards a decreased CD8+ T cell proliferative response to LCL (
      • Lindsey J.W.
      • Hatfield L.M.
      Epstein-Barr virus and multiple sclerosis: cellular immune response and cross-reactivity.
      ) and decreased T cell control of Ig-G secreting B cells after in vitro infection with EBV (
      • Craig J.C.
      • Haire M.
      • Merrett J.D.
      T-cell-mediated suppression of Epstein-Barr virus-induced B lymphocyte activation in multiple sclerosis.
      ). EBV infected B cells from clinically active MS patients compared to inactive MS and HCs have also been shown to have higher rates of immortalisation in vitro (
      • Fraser K.B.
      • Haire M.
      • Millar J.H.
      • McCrea S.
      ). This evidence for a deficiency in T cell control of EBV infected B cells lead Pender in 2014 to test a treatment to increase EBV reactive CD8+ T cells against viral latent proteins in a secondary Progressive MS patient. The results of which showed some promise (
      • Pender M.P.
      • Csurhes P.A.
      • Smith C.
      • Beagley L.
      • Hooper K.D.
      • Raj M.
      • et al.
      Epstein-Barr Virus-specific Adoptive Immunotherapy for Progressive Multiple Sclerosis.
      ), but the data was based on only a single patient and no further trials have been reported so far (discussed in Autoreactive B cells infected with EBV hypothesis).
      Studies using peptides have been equally inconsistent with some finding no difference (
      • Lünemann J.D.
      • Jelčić I.
      • Roberts S.
      • Lutterotti A.
      • Tackenberg B.
      • Martin R.
      • et al.
      EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-γ and IL-2.
      ,
      • Gronen F.
      • Ruprecht K.
      • Weissbrich B.
      • Klinker E.
      • Kroner A.
      • Hofstetter H.H.
      • et al.
      Frequency analysis of HLA-B7-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy controls.
      ), increased (
      • Angelini D.F.
      • Serafini B.
      • Piras E.
      • Severa M.
      • Coccia E.M.
      • Rosicarelli B.
      • et al.
      Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.
      ,
      • Jilek S.
      • Schluep M.
      • Meylan P.
      • Vingerhoets F.
      • Guignard L.
      • Monney A.
      • et al.
      Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis.
      ) or decreased (
      • Jilek S.
      • Schluep M.
      • Harari A.
      • Canales M.
      • Lysandropoulos A.
      • Zekeridou A.
      • et al.
      HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis.
      ) CD8+ T cell responses to EBV between MS and HCs. Again, these discrepancies could be the result of the timing of collection, particularly since different peptides are expressed at different stages of EBV activity. This is highlighted particularly by two recent studies, one of which identified raised EBV responses in MS patients compared to HCs, but only for specific viral proteins. Only 3 out of the 10 EBV proteins tested were found to be raised, which were EBNA-1, sCP and BRRF2. EBNA-1 is expressed during viral latency, while sCP and BRRF2 are expressed by mature virions (
      • Dooley M.M.
      • de Gannes S.L.
      • Fu K.A.
      • Lindsey J.W.
      The increased antibody response to Epstein-Barr virus in multiple sclerosis is restricted to selected virus proteins.
      ). Meanwhile, Angelina et al. found untreated inactive MS patients had a lower lytic antigen CD8+ T cell response compared to active MS patients and HC (
      • Angelini D.F.
      • Serafini B.
      • Piras E.
      • Severa M.
      • Coccia E.M.
      • Rosicarelli B.
      • et al.
      Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.
      ). However, both active and inactive MS patients had a higher frequency of CD8+ T cells specific for EBV lytic and latent antigens compared to HC. Follow-up results found lytic antigens and interactions between CD8+ T cells and EBV infected plasma cells in white matter lesions and meninges of MS patients (
      • Angelini D.F.
      • Serafini B.
      • Piras E.
      • Severa M.
      • Coccia E.M.
      • Rosicarelli B.
      • et al.
      Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.
      ). Furthermore, increased levels of EBV reactive CD8+ T cells have been identified in the Cerebral Spinal Fluid (CSF) of patients with MS and clinically isolated syndrome compared to other neurological diseases (
      • van Nierop G.P.
      • Mautner J.
      • Mitterreiter J.G.
      • Hintzen R.Q.
      • Verjans G.M.
      Intrathecal CD8 T-cells of multiple sclerosis patients recognize lytic Epstein-Barr virus proteins.
      ,
      • Jaquiery E.
      • Jilek S.
      • Schluep M.
      • Meylan P.
      • Lysandropoulos A.
      • Pantaleo G.
      • et al.
      Intrathecal immune responses to EBV in early MS.
      ). More recently, the increased production of IFNγ producing cells from Peripheral Blood Mononuclear Cells (PBMCs) in response to LCL preceding MRI activity has been reported, with a correlation shown as early as 8 weeks prior (
      • Latham L.B.
      • Lee M.J.
      • Lincoln J.A.
      • Ji N.
      • Forsthuber T.G.
      • Lindsey J.W.
      Antivirus immune activity in multiple sclerosis correlates with MRI activity.
      ).
      This evidence supports the notion that the attempt of the immune system to control EBV reactivation leads to clinical relapse or T2 lesions, also known as the ‘Bystander damage hypothesis’ (Section 4.3). However, as outlined in Section 3.4, consistent evidence supporting EBV presence in the CNS of MS patients has been elusive. Meanwhile, primary EBV infection may be involved in priming the immune system for MS development, since a time-delay between initial EBV infection and MS onset, in excess of a decade has been reported (
      • Goldacre M.J.
      • Wotton C.J.
      • Seagroatt V.
      • Yeates D.
      Multiple sclerosis after infectious mononucleosis: record linkage study.
      ,
      • Nielsen T.R.
      • Rostgaard K.
      • Nielsen N.M.
      • Koch-Henriksen N.
      • Haahr S.
      • Sorensen P.S.
      • et al.
      Multiple sclerosis after infectious mononucleosis.
      ).

      3.4 Conflicting evidence of EBV presence in the CNS

      Evidence for the presence of EBV within the brain has been particularly conflicting. Serafini et al. reported that nearly 100% of post-mortem MS brains showed evidence of EBV infected B cells and plasma cells (
      • Serafini B.
      • Rosicarelli B.
      • Franciotta D.
      • Magliozzi R.
      • Reynolds R.
      • Cinque P.
      • et al.
      Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain.
      ). Serafini also detected EBV latency transcripts in all MS brain samples tested (
      • Serafini B.
      • Severa M.
      • Columba-Cabezas S.
      • Rosicarelli B.
      • Veroni C.
      • Chiappetta G.
      • et al.
      Epstein-Barr virus latent infection and BAFF expression in B cells in the multiple sclerosis brain: implications for viral persistence and intrathecal B-cell activation.
      ). These findings were supported by another group who detected latent EBV expression in active MS lesions coinciding with an overexpression of IFNα (
      • Tzartos J.S.
      • Khan G.
      • Vossenkamper A.
      • Cruz-Sadaba M.
      • Lonardi S.
      • Sefia E.
      • et al.
      Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.
      ). However, several other groups have failed to replicate these findings, with only a very limited number of cells showing EBV presence, if at all (
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • et al.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ,
      • Sargsyan S.A.
      • Shearer A.J.
      • Ritchie A.M.
      • Burgoon M.P.
      • Anderson S.
      • Hemmer B.
      • et al.
      Absence of Epstein-Barr virus in the brain and CSF of patients with multiple sclerosis.
      ,
      • Willis S.N.
      • Stadelmann C.
      • Rodig S.J.
      • Caron T.
      • Gattenloehner S.
      • Mallozzi S.S.
      • et al.
      Epstein-Barr virus infection is not a characteristic feature of multiple sclerosis brain.
      ). The failure to replicate the presence of EBV in the brain may be due to varying methodological issues that include differences in tissue handling and preservation, which can have a bearing on protein preservation and subsequent detection. Peferoen et al. attempted to address these issues by using a variety of EBV sensitive methodologies and even a cohort containing the same samples but failed to verify Serafini's report.
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • et al.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      .
      Evidence for the unequivocal presence of EBV DNA in the CSF of MS patients is also lacking. Mancuso et. al 2010 identified only one MS patient (out of 51) with detectable EBV DNA in cell free CSF and none with cell associated EBV DNA (
      • Mancuso R.
      • Hernis A.
      • Cavarretta R.
      • Caputo D.
      • Calabrese E.
      • Nemni R.
      • et al.
      Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis.
      ). Although Cocuzza et al. later found 10 out of 55 RR MS had cell associated EBV DNA in CSF, and 3 out of 51 with cell free EBV DNA; no difference was observed between MS compared to Non-Inflammatory Neurological conditions (
      • Cocuzza C.E.
      • Piazza F.
      • Musumeci R.
      • Oggioni D.
      • Andreoni S.
      • Gardinetti M.
      • et al.
      Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.
      ). Interestingly, no EBV DNA was detected in the CSF of other inflammatory neurological conditions compared to MS (
      • Cocuzza C.E.
      • Piazza F.
      • Musumeci R.
      • Oggioni D.
      • Andreoni S.
      • Gardinetti M.
      • et al.
      Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.
      ).
      The absence of any clear evidence of EBV in the CNS of MS patients reduces the likelihood that EBV is directly involved in CNS MS pathogenesis. This suggests that notions such as the bystander damage hypothesis (Section 4.3) and the Autoreactive B cells infected by EBV hypothesis (Section 4.4) are unlikely to be substantiated. Peferoen et al. did propose the possibility that EBV infected cells within the brain could be ‘hiding’ within other cells of the CNS away from the samples that were tested and a methodological sensitivity issue could be the cause for the lack of validation (
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • et al.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ). Even if EBV is not present at all in the CNS, it may still play an instrumental role in MS pathogenesis outside of the CNS. This could still be a result of molecular mimicry (see Section 4.1).

      3.5 Experimental autoimmune encephalomyelitis

      Experimental autoimmune encephalomyelitis (EAE) is the most common mouse model used to study MS however, unlike MS, it does not follow the same disease trajectory, with EAE being monophasic compared to MS, having limited demyelination (

      Lovett-Racke AE. Contribution of EAE to understanding and treating multiple sclerosis. Journal of Neuroimmunology.

      ) and minimal brain infiltrates (
      • Casiraghi C.
      • Citlali Márquez A.
      • Shanina I.
      • Steven Horwitz M.
      Latent virus infection upregulates CD40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis.
      ). Interestingly, infection by the murine homologue of EBV (γHV-68) prior to EAE induction, produces a more severe clinical pattern (
      • Peacock J.W.
      • Elsawa S.F.
      • Petty C.C.
      • Hickey W.F.
      • Bost K.L.
      Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68.
      ). A recent study showed this approach more closely paralleled MS-like lesions with demyelination in the brain and spinal cord with an equal number of IFNγ producing CD4+ and CD8+ T cells, which are not seen in standard myelin oligodendrocyte glycoprotein (MOG) induced EAE (
      • Casiraghi C.
      • Shanina I.
      • Cho S.
      • Freeman M.L.
      • Blackman M.A.
      • Horwitz M.S.
      Gammaherpesvirus latency accentuates EAE pathogenesis: relevance to Epstein-Barr virus and multiple sclerosis.
      ). Mirroring current evidence of EBV involvement in the CNS of MS, no viral DNA γHV-68 was detected in the CNS of the γHV-68 infected model of EAE (
      • Peacock J.W.
      • Elsawa S.F.
      • Petty C.C.
      • Hickey W.F.
      • Bost K.L.
      Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68.
      ). Moreover, Casiraghi et al. showed that this was all dependant on the latent life cycle of γHV-68 (
      • Casiraghi C.
      • Citlali Márquez A.
      • Shanina I.
      • Steven Horwitz M.
      Latent virus infection upregulates CD40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis.
      ), which may provide a better model to study how an EBV-like infection could cause or exacerbate MS.

      4. Potential roles of EBV in MS pathogenesis/ Mechanisms

      4.1 EBV cross-reactivity/ Molecular mimicry

      Molecular Mimicry is the description given to the notion that pathogenic antigens that are structurally similar to the hosts proteins, such as myelin basic protein (MBP), elicit an immune response which results in the inability to differentiate between the host and pathogen target. Wucherpfennig and Strominger demonstrated that EBV antigens can react with MBP (85–99) reactive T cell clones taken from MS patients (
      • Wucherpfennig K.W.
      • Strominger J.L.
      Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein.
      ). In support of this finding, Lünemann et al. showed that 3–4% of EBNA-1 specific CD4+ T cells react with MBP peptides from both healthy donors and MS patients (
      • Lünemann J.D.
      • Jelčić I.
      • Roberts S.
      • Lutterotti A.
      • Tackenberg B.
      • Martin R.
      • et al.
      EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-γ and IL-2.
      ). EBV is not the only pathogen capable of eliciting such an immune response. Interestingly, Wucherpfennig and Strominger also revealed the same T cell clones that recognised MBP (85–99) are capable of recognising multiple peptides from different pathogens, such as influenza virus A and Herpes Simplex Virus (
      • Wucherpfennig K.W.
      • Strominger J.L.
      Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein.
      ;
      • Wucherpfennig K.W.
      • Sethi D.
      T cell receptor recognition of self and foreign antigens in the induction of autoimmunity.
      ). Although this implicates more viruses than just EBV, the ability of EBV to cause persistent life-long infection and produce EBNA-1 even in the latent stage of infection, still gives credence to EBV having a leading role in MS pathogenesis. This is further supported by evidence from Kakelacheva et al. who have shown that IM produced IgG autoantibodies against MOG in 20% of paediatric IM cases compared to none in controls (no history of IM) (
      • Kakalacheva K.
      • Regenass S.
      • Wiesmayr S.
      • Azzi T.
      • Berger C.
      • Dale R.C.
      • et al.
      Infectious mononucleosis triggers generation of IgG auto-antibodies against native myelin oligodendrocyte glycoprotein.
      ). This suggests a capacity for symptomatic primary EBV infection to prime autoimmune disease in the future, particularly in those individuals that produce autoimmune IgG following IM and have recurring lytic EBV infections. Therefore, it would be interesting to try and determine which genetic and environmental differences impact on the 20% of patients that developed autoimmune IgG antibodies. It would also be prudent to follow these patients over time to see if any go on to develop autoimmune diseases later in life.
      More recently, Hogeboom has proposed the lymphocytic choriomeningitis virus (LCMV) as a more likely candidate for MS pathogenesis compared to EBV (
      • Hogeboom C.
      Peptide motif analysis predicts lymphocytic choriomeningitis virus as trigger for multiple sclerosis.
      ). This was based on a convincing set of information concerning this virus. First, LCMV has greater structural homology to MBP (85–99) than the EBV peptide of EBNA-1 (86% vs 63%, respectively); Second, LCMV was the only viral protein tested that met all the cross-reactivity criteria (surface accessibility, antigenicity, flexibility, hydrophobicity, and hydrophilicity); Third, Hogeboom also argued that LCMV is more consistent with epidemiological features of MS compared to EBV (
      • Hogeboom C.
      Peptide motif analysis predicts lymphocytic choriomeningitis virus as trigger for multiple sclerosis.
      ). These include, a higher prevalence farther from the equator, increased incidence of infection in regions of peak MS incidence and increased concentration of incidence in temperate regions (such as Europe and Southern Australia). Hogeboom also argues that the low concordance of monozygotic twins for MS who share genetic risk factors would suggest an environmental trigger that has low infectivity, which EBV would not fit, unlike LCMV (
      • Hogeboom C.
      Peptide motif analysis predicts lymphocytic choriomeningitis virus as trigger for multiple sclerosis.
      ).

      4.2 αB-Crystallin/ ‘Mistaken Self’

      This theory is also not necessarily EBV specific but EBV is still a candidate for this potential mechanism. This hypothesis postulates that immune tolerance for the heat shock protein αB-Crystallin does not develop due to its limited expression within tissue. Therefore, when αB-Crystallin is expressed by lymphoid cells due to activation by microbial antigens, it provokes a CD4+ T cell response toward αB-Crystallin derived from oligodendrocytes, resulting in inflammatory demyelination. This theory requires a stimulus to increase αB-Crystallin levels within lymphoid cells that infiltrate the CNS as well as within oligodendrocytes. However, this stimulus does not need to be the same to cause αB-Crystallin levels to increase in both lymphoid cells and oligodendrocytes, therefore EBV is a potential candidate since EBV in B cells promotes expression of αB-Crystallin, which is then presented to CD4+ T cells. Ergo, this theory can account for how oligodendrocytes and myelin is targeted by CD4+ T cells. At present this is only a theory as there is no clear evidence to support it (
      • Márquez A.C.
      • Horwitz M.S.
      The role of latently infected B cells in CNS autoimmunity.
      ).

      4.3 Bystander damage hypothesis

      This hypothesis is based on the notion that neurodegenerative damage is instigated by damage from the immune system attempting to keep EBV infection under control. Collateral damage to the CNS is thought to occur via activation of CD8+ and/or CD4+ T cells against EBV antigens, particularly lytic antigens (
      • Angelini D.F.
      • Serafini B.
      • Piras E.
      • Severa M.
      • Coccia E.M.
      • Rosicarelli B.
      • et al.
      Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.
      ,
      • Fernandez-Menendez S.
      • Fernandez-Moran M.
      • Fernandez-Vega I.
      • Perez-Alvarez A.
      • Villafani-Echazu J.
      Epstein-Barr virus and multiple sclerosis. From evidence to therapeutic strategies.
      ,
      • Bhaduri-McIntosh S.
      • Rotenberg M.J.
      • Gardner B.
      • Robert M.
      • Miller G.
      Repertoire and frequency of immune cells reactive to Epstein-Barr virus-derived autologous lymphoblastoid cell lines.
      ). For this theory to hold true, the presence of EBV infected cells and EBV directed CD8+ cells within the CNS would need to be confirmed. Evidence for this is absent (see Section 3.4), since only Serafani et al. has successfully shown consistent (nearly 100% of observed cases) presence of active EBV infection in B cell infiltrates of secondary progressive and acute MS patient brains (
      • Serafini B.
      • Rosicarelli B.
      • Franciotta D.
      • Magliozzi R.
      • Reynolds R.
      • Cinque P.
      • et al.
      Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain.
      ).

      4.4 Autoreactive B cells infected with EBV hypothesis

      In 2003, Pender proposed another more general theory for the causation of chronic autoimmune disease; whereby in individuals genetically predisposed to autoimmune disease, EBV infects autoreactive B cells that then migrate and ‘lodge’ within a particular target organ (
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ). Within this organ they survive and produce pathogenic autoantibodies, thereby acting as an antigen presenting cell. Then, by cross-reactivity (Section 4.1), infectious agents activate CD4+ cells in lymphoid tissue that recognise the target organ antigen and migrate to this region. These autoreactive CD4+ cells survive due to the infected B cells providing co-stimulatory survival signals, preventing apoptosis. Autoreactive T cells are then able to proliferate, produce cytokines, recruit other cells that ultimately result in target organ damage and chronic autoimmune disease.
      This notion relies on three major predictions, some of which have been shown in current MS research. First, MS patients have a predisposition to EBV infection and fail to clear or regulate EBV in the same manner as HCs. MS patients have been shown to have a reduction in EBV reactive CD8+ T cells compared to HCs (
      • Pender M.P.
      • Csurhes P.A.
      • Lenarczyk A.
      • Pfluger C.M.M.
      • Burrows S.R.
      Decreased T cell reactivity to Epstein–Barr virus infected lymphoblastoid cell lines in multiple sclerosis.
      ), the reason for this is unknown; Second, molecular mimicry occurs; Third, is the presence of EBV infected B cells within the brain (similar to the bystander damage hypothesis).
      This theory was tested in a single patient with secondary progressive MS by increasing EBV reactive CD8+ T cells against viral latent proteins to alleviate MS symptoms (
      • Pender M.P.
      • Csurhes P.A.
      • Smith C.
      • Beagley L.
      • Hooper K.D.
      • Raj M.
      • et al.
      Epstein-Barr Virus-specific Adoptive Immunotherapy for Progressive Multiple Sclerosis.
      ). To achieve this, a recombinant adenovirus vector was used that encodes epitopes for the latent EBV protein EBNA1, LMP1 and LMP2A. No serious or lasting adverse events were observed and a reduction in fatigue and pain was reported. An increase in EBV reactive CD8+ cells was observed in circulating blood; as well as reduction in the size of gadolinium-enhancing MRI brain lesions, and a reduction in the CSF IgG index from 0.79 to 0.63. This study only had a 6-week follow up, so it is not known if these levels were maintained over a prolonged period. As the results of this study came from a single patient and appear to be positive, it does merit further investigation. Drugs such as ocrelizumab (
      • Menge T.
      • Dubey D.
      • Warnke C.
      • Hartung H.-P.
      • Stüve O.
      Ocrelizumab for the treatment of relapsing-remitting multiple sclerosis.
      ), ofatumumab and rituximab have all shown a beneficial response correlated with a reduction in B cells in MS (
      • Milo R.
      Therapeutic strategies targeting B-cells in multiple sclerosis.
      ,
      • Sorensen P.S.
      • Blinkenberg M.
      The potential role for ocrelizumab in the treatment of multiple sclerosis: current evidence and future prospects.
      ). In fact, rituximab has been shown to specifically reduce B cells within both peripheral blood and CSF in Primary Progressive MS (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • Antel J.
      • Fox R.J.
      • et al.
      B-cell depletion with rituximab in relapsing–remitting multiple sclerosis.
      ,
      • Monson N.L.
      • Cravens P.D.
      • Frohman E.M.
      • Hawker K.
      • Racke M.K.
      EFfect of rituximab on the peripheral blood and cerebrospinal fluid b cells in patients with primary progressive multiple sclerosis.
      ). More recently, interferon-β treatment has also been shown to reduce the levels of pathogenic memory B cells in MS (
      • Rizzo F.
      • Giacomini E.
      • Mechelli R.
      • Buscarinu M.C.
      • Salvetti M.
      • Severa M.
      • et al.
      Interferon-[beta] therapy specifically reduces pathogenic memory B cells in multiple sclerosis patients by inducing a FAS-mediated apoptosis.
      ). The increasing number of monoclonal agents targeting B cell population depletion with high efficacy in MS only adds credence to the integral role B cells play in the pathogenesis of MS. This means identifying the reason(s) behind erroneous B cell activity in MS is likely to be a major key in understanding the cause of MS.

      4.5 EBV interaction with human endogenous retroviruses

      A more recent role of EBV pathogenesis of MS has been proposed in combination with human endogenous retroviruses (HERVs). Somewhere between 5% and 8% of the human genome is comprised of HERVs, which are estimated to have entered the human genome between 10 and 50 million years ago during primate evolution (
      • Fernandez-Menendez S.
      • Fernandez-Moran M.
      • Fernandez-Vega I.
      • Perez-Alvarez A.
      • Villafani-Echazu J.
      Epstein-Barr virus and multiple sclerosis. From evidence to therapeutic strategies.
      ). Three HERV families have been implicated in MS; HERV-H, HERV-K and HERV-W (
      • Morandi E.
      • Tarlinton R.E.
      • Gran B.
      Multiple sclerosis between genetics and infections: human endogenous retroviruses in monocytes and macrophages.
      ). The strongest association being from multiple sclerosis associated retrovirus (MSRV) and syncytin-1, which are two members of the HERV-W family (
      • Antony J.M.
      • DesLauriers A.M.
      • Bhat R.K.
      • Ellestad K.K.
      • Power C.
      Human endogenous retroviruses and multiple sclerosis: innocent bystanders or disease determinants?.
      ).
      Recently, HERV-W expression has been reported strongest in microglia and macrophages of active lesions, weakly within MS normal appearing white matter and rarely within HC brain (
      • van Horssen J.
      • van der Pol S.
      • Nijland P.
      • Amor S.
      • Perron H.
      Human endogenous retrovirus W in brain lesions: rationale for targeted therapy in multiple sclerosis.
      ). This is in slight contrast to a previous report, which found some evidence of HERV-W expression within normal brain, however only MS affected brains appeared to show strong and specific HERV-W staining patterns (
      • Perron H.
      • Lazarini F.
      • Ruprecht K.
      • Pechoux-Longin C.
      • Seilhean D.
      • Sazdovitch V.
      • et al.
      Human endogenous retrovirus (HERV)-W ENV and gag proteins: physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions.
      ). MRSV titres have also been found to be higher in MS patients’ blood and CSF compared to other neurological patients and HCs (
      • Arru G.
      • Mameli G.
      • Astone V.
      • Serra C.
      • Huang Y.M.
      • Link H.
      • et al.
      Multiple sclerosis and HERV-W/MSRV: a multicentric study.
      ) and have the ability to induce the production of inflammatory cytokines (
      • Duperray A.
      • Barbe D.
      • Raguenez G.
      • Weksler B.B.
      • Romero I.A.
      • Couraud P.O.
      • et al.
      Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.
      ).
      Mameli et al. have associated EBV and HERV expression both in vitro (
      • Mameli G.
      • Poddighe L.
      • Mei A.
      • Uleri E.
      • Sotgiu S.
      • Serra C.
      • et al.
      Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for multiple sclerosis.
      ) and in vivo (
      • Mameli G.
      • Madeddu G.
      • Mei A.
      • Uleri E.
      • Poddighe L.
      • Delogu L.G.
      • et al.
      Activation of MSRV-type endogenous retroviruses during infectious mononucleosis and Epstein-Barr virus latency: the missing link with multiple sclerosis?.
      ). Untreated MS patients were found to have higher levels of MRSV-env in PBMCs, B cells, NK cells and monocytes but not in T cells, which tended to return to levels seen in HCs with treatment on either azathioprine or glatiramer acetate (
      • Mameli G.
      • Poddighe L.
      • Mei A.
      • Uleri E.
      • Sotgiu S.
      • Serra C.
      • et al.
      Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for multiple sclerosis.
      ). In vitro astrocytes express MRSV and synctin-1 upon stimulation by EBVgp350, thereby supporting the role of HERVs in MS. Similarly, PBMCs treated with EBVgp350 resulted in B cells and monocytes, but not T cells or NK cells, expressing MRSVenv and synctin-1 (
      • Mameli G.
      • Poddighe L.
      • Mei A.
      • Uleri E.
      • Sotgiu S.
      • Serra C.
      • et al.
      Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for multiple sclerosis.
      ). IM patients, intriguingly, also have higher levels of MSRV compared to HC (
      • Mameli G.
      • Madeddu G.
      • Mei A.
      • Uleri E.
      • Poddighe L.
      • Delogu L.G.
      • et al.
      Activation of MSRV-type endogenous retroviruses during infectious mononucleosis and Epstein-Barr virus latency: the missing link with multiple sclerosis?.
      ). When the HCs were subdivided into higher and lower EBNA titres and no past EBV infection, an increasing trend in MRSVenv expression with EBNA titres was found, with those with higher EBNA titres showing similar levels to that of IM. Cell analysis by flow cytometry revealed HERV-W presence varied within specific cell subsets dependant on acute (IM) and past EBV infection (
      • Mameli G.
      • Madeddu G.
      • Mei A.
      • Uleri E.
      • Poddighe L.
      • Delogu L.G.
      • et al.
      Activation of MSRV-type endogenous retroviruses during infectious mononucleosis and Epstein-Barr virus latency: the missing link with multiple sclerosis?.
      ).
      Together, these associations suggest HERV-W as another promising target for MS treatment. To that end, GNbAC1 (a monoclonal antibody targeting MRSV-env) has recently completed a phase IIa safety trial (
      • Derfuss T.
      • Curtin F.
      • Guebelin C.
      • Bridel C.
      • Rasenack M.
      • Matthey A.
      • et al.
      A phase IIa randomized clinical study testing GNbAC1, a humanized monoclonal antibody against the envelope protein of multiple sclerosis associated endogenous retrovirus in multiple sclerosis patients - a twelve month follow-up.
      ) and preliminary results from this study are expected sometime in 2017.

      5. Conclusion

      There is an undeniable association between EBV and MS, with evidence that includes higher EBNA-1 titres (
      • Sundqvist E.
      • Sundstrom P.
      • Linden M.
      • Hedstrom A.K.
      • Aloisi F.
      • Hillert J.
      • et al.
      Epstein-Barr virus and multiple sclerosis: interaction with HLA.
      ,
      • Sundstrom P.
      • Nystrom M.
      • Ruuth K.
      • Lundgren E.
      Antibodies to specific EBNA-1 domains and HLA DRB1*1501 interact as risk factors for multiple sclerosis.
      ) and an increased incidence of IM (
      • Nielsen T.R.
      • Rostgaard K.
      • Nielsen N.M.
      • Koch-Henriksen N.
      • Haahr S.
      • Sorensen P.S.
      • et al.
      Multiple sclerosis after infectious mononucleosis.
      ) in MS patients. Another consistent feature of MS is the finding that nearly 100% of MS patients have been shown to be infected with EBV, with one report indicating 100% seroconversion prior to MS onset, suggesting EBV infection is a pre-requisite to MS (
      • Levin L.I.
      • Munger K.L.
      • O'Reilly E.J.
      • Falk K.I.
      • Ascherio A.
      Primary infection with the epstein-barr virus and risk of multiple sclerosis.
      ). Nonetheless, the presence of EBV DNA in the CNS (
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • et al.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ), and an altered EBV reactive CD8+ T Cell response (
      • Pender M.P.
      • Csurhes P.A.
      • Lenarczyk A.
      • Pfluger C.M.
      • Burrows S.R.
      Decreased T cell reactivity to Epstein-Barr virus infected lymphoblastoid cell lines in multiple sclerosis.
      ,
      • Lindsey J.W.
      • Hatfield L.M.
      Epstein-Barr virus and multiple sclerosis: cellular immune response and cross-reactivity.
      ,
      • Cepok S.
      • Zhou D.
      • Srivastava R.
      • Nessler S.
      • Stei S.
      • Bussow K.
      • et al.
      Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis.
      ,
      • Lünemann J.D.
      • Jelčić I.
      • Roberts S.
      • Lutterotti A.
      • Tackenberg B.
      • Martin R.
      • et al.
      EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-γ and IL-2.
      ,
      • Gronen F.
      • Ruprecht K.
      • Weissbrich B.
      • Klinker E.
      • Kroner A.
      • Hofstetter H.H.
      • et al.
      Frequency analysis of HLA-B7-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy controls.
      ,
      • Angelini D.F.
      • Serafini B.
      • Piras E.
      • Severa M.
      • Coccia E.M.
      • Rosicarelli B.
      • et al.
      Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.
      ,
      • Jilek S.
      • Schluep M.
      • Meylan P.
      • Vingerhoets F.
      • Guignard L.
      • Monney A.
      • et al.
      Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis.
      ,
      • Jilek S.
      • Schluep M.
      • Harari A.
      • Canales M.
      • Lysandropoulos A.
      • Zekeridou A.
      • et al.
      HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis.
      ) in MS patients has been inconsistent. Thus, none of the evidence so far has unequivocally implicated EBV involvement in MS pathogenesis. Therefore, the question whether EBV is a causative factor or simply associated with an altered immune system in MS remains to be answered.
      Given the conflicting evidence about EBV involvement in MS there are two further plausible contributions of EBV involvement into MS. If EBV is truly a pre-requisite and/ or the main contributor of MS pathogenesis, then the fact that most of the world's population are infected by EBV yet only a small proportion go on to develop MS (or other EBV related diseases), needs to be addressed. Genetic risks for MS, such as the presence of the HLA allele DRB15*01 and absence of A*02, have been shown to be increased in combination with EBV infection (
      • Sundqvist E.
      • Sundstrom P.
      • Linden M.
      • Hedstrom A.K.
      • Aloisi F.
      • Hillert J.
      • et al.
      Epstein-Barr virus and multiple sclerosis: interaction with HLA.
      ), but this still does not account for 100% of MS cases. It would be interesting to know if individuals with the major genetic risk factors for MS, in the absence of EBV infection, developed MS.
      One possibility that addresses the issue of why some individuals who are infected with EBV are asymptomatic, while others might develop MS or lymphomas, is the notion that differential random integration of EBV in host cells may drive different disease pathologies. EBV integration may cause particular methylation changes resulting in subtle or more discrete expression level changes in regions yet to be identified as risk factors for MS. EBV has already been shown to integrate into random locations (
      • Santpere G.
      • Darre F.
      • Blanco S.
      • Alcami A.
      • Villoslada P.
      • Mar Alba M.
      • et al.
      Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1000 genomes project.
      ,
      • Takakuwa T.
      • Luo W.J.
      • Ham M.F.
      • Sakane-Ishikawa F.
      • Wada N.
      • Aozasa K.
      Integration of Epstein-Barr virus into chromosome 6q15 of Burkitt lymphoma cell line (Raji) induces loss of BACH2 expression.
      ) and cause methylation changes in vitro in B cell lines (
      • Hernando H.
      • Shannon-Lowe C.
      • Islam A.B.
      • Al-Shahrour F.
      • Rodriguez-Ubreva J.
      • Rodriguez-Cortez V.C.
      • et al.
      The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion.
      ). This could also account for the inconsistent findings of EBV within the CNS, since detection of viral load of EBV DNA relies upon a conserved sequence of the EBV genome (
      • Gulley M.L.
      • Tang W.
      Using Epstein-Barr viral load assays to diagnose, monitor, and prevent posttransplant lymphoproliferative disorder.
      ), which may be disrupted in the process of integration.
      Another possibility, since the presence of EBV has not been unequivocally shown in the CNS, the involvement of EBV mediation outside the CNS, by means of molecular mimicry, is still a plausible contributor to MS development or disease progression. However, if molecular mimicry is a key player in MS pathogenesis, then EBV is not the only pathogen that needs to be considered, since other pathogens such as the influenza virus and LCMV can elicit a similar response against MBP as EBV. Thus, there may be a failure of viral clearance in general in MS patients that could provide new therapeutic targets. Whether this is caused by a combination of pathogens augmenting the hosts’ immune system and/or a genetic pre-disposition is worthy of further investigation.

      Conflicts of interest

      Jeannette Lechner-Scott has accepted travel compensation from Novartis, Biogen and Merck Serono. Her institution receives the honoraria for talks and advisory board commitment and also clinical support, as well as research grants from Bayer Health Care, Biogen, Genzyme Sanofi, Merck, Novartis and TEVA.
      All other authors have nothing else to declare.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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