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Epstein–Barr virus and multiple sclerosis: Updating Pender's hypothesis

  • Martin Laurence
    Affiliations
    Shipshaw Labs, Montreal, Quebec, Canada
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  • Julián Benito-León
    Correspondence
    Correspondence to: Avda. de la Constitución 73, portal 3, 7° Izquierda, E-28821 Coslada, Madrid, Spain.
    Affiliations
    Department of Neurology, University Hospital “12 de Octubre”Octubre”, Madrid, Spain

    Department of Medicine, Faculty of Medicine, Complutense University, Madrid, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain
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      Highlights

      • Epstein–Barr virus (EBV) is involved in the pathogenesis of MS.
      • EBV DNA generally cannot be detected in central nervous system.
      • EBV may enable the recognition of “forbidden” antigens by memory B cells (Pender's hypothesis).
      • Inefficient EBV episome replication during B cell division results in some descendant B cells becoming EBV-free.
      • Memory B cells in the CNS are mostly EBV-free and can recognize “forbidden” MS-causing antigens in the CNS.

      Abstract

      Substantial epidemiological evidence supports the involvement of the Epstein–Barr virus (EBV) in multiple sclerosis (MS). Mechanisms through which EBV may increase MS risk are reviewed here. Most individuals contract EBV in early childhood yet only develop MS in early adulthood, by which time EBV has been latent for decades. When latent, EBV is confined to a minute subset of memory B cells: about 1000 cells in peripheral blood and 500,000 cells in the lymphoid system, mainly in the mouth. Reactivation of EBV in the central nervous system (CNS) has been proposed as a cause of MS. Alternatively, EBV may enable the recognition of “forbidden” antigens by memory B cells through its presence in this leukocyte type, as first proposed by Pender. Though the requirement for B cells in MS supports both hypotheses, EBV has not been consistently found in MS lesions, as would be expected. EBV episome replication during B cell division is now known to be inefficient, resulting in some descendant B cells becoming EBV-free after a few dozen divisions. EBV-free memory B cells in the CNS may thus have descended from a memory B cell which matured while containing EBV episomes, enabling its B cell receptor to recognize “forbidden” MS-causing antigens in the CNS, even if EBV is absent from this site.

      Keywords

      1. Introduction

      The majority of patients with multiple sclerosis (MS) experience their first symptom between 20 and 40 years of age, making this disease the most common cause of non-traumatic neurological disability in young adults (
      • Benito-León J.
      • Martin E.
      • Vela L.
      • Villar M.
      • Felgueroso B.
      • Marrero C.
      • Guerrero A.
      • Ruiz-Galiana J.
      Multiple sclerosis in Mostoles, central Spain.
      ). MS is a complex heterogeneous, inflammatory disorder characterized by the loss of the myelin sheath surrounding nerve axons in the central nervous system (CNS) (
      • Stadelmann C.
      • Wegner C.
      • Brück W.
      Inflammation, demyelination, and degeneration—recent insights from MS pathology.
      ). This damage can cause a wide range of symptoms that may eventually lead to disability (
      • Benito-León J.
      • Morales J.M.
      • Rivera-Navarro J.
      • Mitchell A.
      A review about the impact of multiple sclerosis on health-related quality of life.
      ). In all forms of MS, inflammation is present when active demyelination and neurodegeneration occur (
      • Stadelmann C.
      • Wegner C.
      • Brück W.
      Inflammation, demyelination, and degeneration—recent insights from MS pathology.
      ). Understanding the mechanisms behind the inflammatory process associated with MS appears to be the largest obstacle in primary prevention and in developing effective therapies.
      The two strongest established risk factors for MS are Epstein–Barr virus (EBV) seropositivity (
      • Almohmeed Y.H.
      • Avenell A.
      • Aucott L.
      • Vickers M.A.
      Systematic review and meta-analysis of the sero-epidemiological association between Epstein Barr virus and multiple sclerosis.
      ,
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ,
      • Pakpoor J.
      • Disanto G.
      • Gerber J.E.
      • Dobson R.
      • Meier U.C.
      • Giovannoni G.
      • Ramagopalan S.V.
      The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
      ) and major histocompatibility complex (MHC) class II gene HLA-DRB1*1501 (
      • Hollenbach J.A.
      • Oksenberg J.R.
      The immunogenetics of multiple sclerosis: a comprehensive review.
      ), each increasing MS risk more than four fold. Possible mechanisms linking EBV and MS have been widely studied and reviewed (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ). The three main hypothetical mechanisms linking EBV and MS through B cells involve either the reactivation of EBV within memory B cells in the CNS (
      • Serafini B.
      • Rosicarelli B.
      • Franciotta D.
      • Magliozzi R.
      • Reynolds R.
      • Cinque P.
      • Andreoni L.
      • Trivedi P.
      • Salvetti M.
      • Faggioni A.
      Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain.
      ), cross-reactivity of anti-EBV antibodies to human proteins in the CNS (molecular mimicry) (
      • Vaughan J.
      • Riise T.
      • Rhodes G.
      • Nguyen M.-D.
      • Barrett-Connor E.
      • Nyland H.
      An Epstein Barr virus-related cross reactive autoimmune response in multiple sclerosis in Norway.
      ), or the facilitation of “forbidden” memory B cells recognizing an antigen in the CNS (
      • Lünemann J.D.
      • Münz C.
      EBV in MS: guilty by association?.
      ,
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ). Other hypothetical mechanisms include a hygiene hypothesis where EBV would be acting as a surrogate for a highly hygienic upbringing (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ), and the inadvertent recognition of human antigens in the CNS by CD8+ (cytotoxic) T cells targeting EBV antigens (molecular mimicry) (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ). No proposed mechanism is fully consistent with observations. In this review, the biology of EBV's lifecycle is examined in relation to MS. Minor modifications to the EBV facilitated “forbidden” memory B cell hypothesis first proposed by Pender (
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ) can bring this hypothesis back in line with most observations, making it standout as the most plausible mechanism reviewed here.

      2. Search strategy and selection criteria

      References for this review were identified by searches of PubMed between 1969 and March 2017, and references from relevant articles. The search terms “multiple sclerosis”, “Epstein–Barr virus”, “antibodies”, and “B cells” were used. There were no language restrictions. The final reference list was generated on the basis of relevance to the topics covered in this review.

      3. Results

      3.1 Antibodies against viruses

      Antibodies against viruses have been widely studied as risk factors for MS (reviewed by
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ). According to three recent meta-analyses, EBV seropositivity substantially increases the risk of MS (4.5-fold, 5.5-fold, or 16-fold, depending on the study) (
      • Almohmeed Y.H.
      • Avenell A.
      • Aucott L.
      • Vickers M.A.
      Systematic review and meta-analysis of the sero-epidemiological association between Epstein Barr virus and multiple sclerosis.
      ,
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ,
      • Pakpoor J.
      • Disanto G.
      • Gerber J.E.
      • Dobson R.
      • Meier U.C.
      • Giovannoni G.
      • Ramagopalan S.V.
      The risk of developing multiple sclerosis in individuals seronegative for Epstein-Barr virus: a meta-analysis.
      ). Among EBV positive individuals, the risk of MS is further increased by high anti-EBV-EBNA antibody titers (
      • Munger K.
      • Levin L.
      • O’Reilly E.
      • Falk K.
      • Ascherio A.
      Anti-Epstein–Barr virus antibodies as serological markers of multiple sclerosis: a prospective study among United States military personnel.
      ), by EBV infections acquired beyond childhood which caused infectious mononucleosis (2.2-fold increase according to the most recent meta-analysis) (
      • 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.
      ), and by co-infections with both EBV types (
      • Santón A.
      • Cristóbal E.
      • Aparicio M.
      • Royuela A.
      • Villar L.M.
      • Álvarez-Cermeño J.C.
      High frequency of co-infection by Epstein–Barr virus types 1 and 2 in patients with multiple sclerosis.
      ). Finally, a very large prospective study of EBV as a risk factor for MS in the US military demonstrated that EBV seroconversion in young adults predisposes them to develop MS in the following decade (mean delay of 3.8 years, range 1.7–7 years) (
      • 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.
      ). The high relative risk of MS in EBV carriers ( > 4.5-fold) is difficult to explain by EBV acting as a surrogate for other factors, since such factors would have be to extremely correlated with EBV exposure (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ). The variety and strength of these studies indicate that EBV is directly involved in most MS cases. However, the vast majority of EBV infections occur in childhood (
      • Balfour H.H.
      • Sifakis F.
      • Sliman J.A.
      • Knight J.A.
      • Schmeling D.O.
      • Thomas W.
      Age-specific prevalence of Epstein–Barr virus infection among individuals aged 6–19 years in the United States and factors affecting its acquisition.
      ), yet the onset of MS is much later, usually in early adulthood (
      • Confavreux C.
      • Vukusic S.
      Natural history of multiple sclerosis: a unifying concept.
      ,
      • Ligouri M.
      • Marrosu M.
      • Pugliatti M.
      • Giuliani F.
      • De Robertis F.
      • Cocco E.
      • Zimatore G.
      • Livrea P.
      • Trojano M.
      Age at onset in multiple sclerosis.
      ), suggesting other environmental factors occurring later in life may be required for MS (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ).
      A second virus consistently associated with MS through serology is herpes simplex virus type 2 (HSV-2) (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ). The increase in risk observed with HSV-2 seropositivity is much lower than for EBV (1.5–2.0-fold increase in risk) (
      • Catalano L.W.
      Herpesvirus hominis antibody in multiple sclerosis and amyotrophic lateral sclerosis.
      ,
      • Ferrante P.
      • Castellani P.
      • Barbi M.
      • Bergamini F.
      The Italian cooperative multiple sclerosis case-control study: preliminary results on viral antibodies.
      ,
      • Hawkes C.H.
      • Giovannoni G.
      • Keir G.
      • Cunnington M.
      • Thompson E.J.
      Seroprevalence of herpes simplex virus type 2 in multiple sclerosis.
      ,
      • Wandinger K.
      • Jabs W.
      • Siekhaus A.
      • Bubel S.
      • Trillenberg P.
      • Wagner H.
      • Wessel K.
      • Kirchner H.
      • Hennig H.
      Association between clinical disease activity and Epstein-Barr virus reactivation in MS.
      ), and could well be due to HSV-2 acting as a surrogate for other environmental factors, as occurred in cervical cancer before the discovery of oncogenic HPV subtypes (
      • Aurelian L.
      • Manak M.M.
      • McKinlay M.
      • Smith C.C.
      • Klacsmann K.T.
      • Gupta P.K.
      “The herpesvirus hypothesis”—Are Koch's postulates satisfied?.
      ,
      • Dahlström L.A.
      • Andersson K.
      • Luostarinen T.
      • Thoresen S.
      • Ögmundsdottír H.
      • Tryggvadottír L.
      • Wiklund F.
      • Skare G.B.
      • Eklund C.
      • Sjölin K.
      Prospective seroepidemiologic study of human papillomavirus and other risk factors in cervical cancer.
      ). In contrast with EBV, the distribution of the age at onset of HSV-2 and MS (
      • Confavreux C.
      • Vukusic S.
      Natural history of multiple sclerosis: a unifying concept.
      ,
      • England P.H.
      Infection report.
      ,
      • Ligouri M.
      • Marrosu M.
      • Pugliatti M.
      • Giuliani F.
      • De Robertis F.
      • Cocco E.
      • Zimatore G.
      • Livrea P.
      • Trojano M.
      Age at onset in multiple sclerosis.
      ) match quite well, and both HSV-2 and MS prevalence is skewed about 2:1 toward females (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ,
      • Hawkes C.H.
      • Giovannoni G.
      • Keir G.
      • Cunnington M.
      • Thompson E.J.
      Seroprevalence of herpes simplex virus type 2 in multiple sclerosis.
      ;

      Xu, F.S., Gottlieb, M.R., Bermen, S.L., Markowitz, S.M., Forhan, L.E., Taylor, S.E., LD, 2010. Seroprevalence of Herpes Simplex Virus Type 2 among persons aged 14–49 years — United States, 2005–2008. MMWR Morbidity and Mortality Weekly Report. 59(15), 4.

      ), which prompted the hypothesis that MS could be directly caused by this virus (
      • Martin J.
      Herpes simplex virus types 1 and 2 and multiple sclerosis.
      ). While EBV infects >99% of MS cases (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ) and thus could be etiologically involved in nearly all cases, HSV-2 infects less than 25% of MS cases in most studies (
      • Catalano L.W.
      Herpesvirus hominis antibody in multiple sclerosis and amyotrophic lateral sclerosis.
      ,
      • Hawkes C.H.
      • Giovannoni G.
      • Keir G.
      • Cunnington M.
      • Thompson E.J.
      Seroprevalence of herpes simplex virus type 2 in multiple sclerosis.
      ,
      • Wandinger K.
      • Jabs W.
      • Siekhaus A.
      • Bubel S.
      • Trillenberg P.
      • Wagner H.
      • Wessel K.
      • Kirchner H.
      • Hennig H.
      Association between clinical disease activity and Epstein-Barr virus reactivation in MS.
      ). This means HSV-2 cannot be involved in most MS cases, and if it does play a direct etiological role, the modest increase in risk suggests a minor contribution.
      Other viruses such as herpes simplex virus type 1 (HSV-1), cytomegalovirus, human herpesvirus 6 (HHV-6), varicella zoster, measles, mumps and rubella viruses have been either inconsistently or not associated with MS through serology (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ). Thus EBV appears to be the only virus strongly associated with MS at this time. A small fraction of pediatric (
      • Alotaibi S.
      • Kennedy J.
      • Tellier R.
      • Stephens D.
      • Banwell B.
      Epstein-Barr virus in pediatric multiple sclerosis.
      ) and adult (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection.
      ) MS cases appear to be EBV seronegative, suggesting either EBV is not a necessary factor in MS, or multiple distinct mechanisms can cause MS, the most common of which would require EBV.

      3.2 B cells

      According to the standard model of memory B cell activation (Fig. 1), when a memory B cell encounters an antigen cognate with its antibody, this antigen is endocytosed and broken into peptides which are presented to CD4+ (helper) T cells through HLA-D molecules and the T cell receptor (TCR) (
      • Kurosaki T.
      • Kometani K.
      • Ise W.
      Memory B cells.
      ). If the CD4+ T cell recognizes this peptide as foreign, a secondary immune response is initiated: the memory B cell differentiates into a plasma cell and synthesizes large quantities of this antibody (
      • Kurosaki T.
      • Kometani K.
      • Ise W.
      Memory B cells.
      ).
      Fig. 1
      Fig. 1Standard model of memory B cell activation. If the CD4+ T cell's TCR has a high affinity to the peptide presented by the HLA-D molecule complex (MHC class II receptor), it will signal this through CD154 and CD40 (not shown). Thereafter the memory B cell will differentiate into a plasma cell, which will secrete large amounts of antibodies (not shown). When this occurs to an EBV infected memory B cell, EBV virions are synthesized by the plasma cell, eliciting a strong CD8+ (cytotoxic) T cell response which rapidly clears the infected plasma cell. In this example, the memory B cell was infected with EBV, shown as three circular DNA episomes in the nucleus. EBV is not believed to affect memory B cell activation, as it is in a latent state in memory B cells, expressing only a single protein coding gene (EBNA1). The antigen shown in this example is a mannoprotein which binds to the BCR through terminal mannose residues, though many other antigens are recognized by human B cells.
      A number of recent studies have shown that B cells play an important role in MS (reviewed by
      • Disanto G.
      • Morahan J.
      • Barnett M.
      • Giovannoni G.
      • Ramagopalan S.
      The evidence for a role of B cells in multiple sclerosis.
      ), especially since their decimation using monoclonal antibodies targeting CD20 has proven to be a very effective intervention (
      • Disanto G.
      • Morahan J.
      • Barnett M.
      • Giovannoni G.
      • Ramagopalan S.
      The evidence for a role of B cells in multiple sclerosis.
      ). Counterintuitively, secreted antibodies in the CNS do not seem to be directly involved in MS because CD20 depletion only targets B cells, and does not immediately affect plasma cells or secreted antibody levels despite improvements in MS lesions (
      • Disanto G.
      • Morahan J.
      • Barnett M.
      • Giovannoni G.
      • Ramagopalan S.
      The evidence for a role of B cells in multiple sclerosis.
      ). This suggests memory B cells in the CNS are directly involved in MS, possibly through antigen presentation to CD4+ T cells, direct cytokine production or trafficking of EBV into the CNS, but not through differentiation into plasma cells (
      • Disanto G.
      • Morahan J.
      • Barnett M.
      • Giovannoni G.
      • Ramagopalan S.
      The evidence for a role of B cells in multiple sclerosis.
      ).
      It is unclear which antigens B cells in the CNS are targeting in MS: antibodies against the most likely candidates, human myelin oligodendrocyte glycoprotein and myelin basic protein, are not associated with MS (
      • Kuhle J.
      • Pohl C.
      • Mehling M.
      • Edan G.
      • Freedman M.S.
      • Hartung H.-P.
      • Polman C.H.
      • Miller D.H.
      • Montalban X.
      • Barkhof F.
      Lack of association between antimyelin antibodies and progression to multiple sclerosis.
      ,
      • Owens G.P.
      • Bennett J.L.
      • Lassmann H.
      • O'Connor K.C.
      • Ritchie A.M.
      • Shearer A.
      • Lam C.
      • Yu X.
      • Birlea M.
      • DuPree C.
      Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid.
      ). A very recent study suggests oligoclonal antibodies in MS target intracellular human proteins which are not specific to the brain, and may be a consequence of tissue destruction rather than a cause (

      Brändle, S.M., Obermeier, B., Senel, M., Bruder, J., Mentele, R., Khademi, M., Olsson, T., Tumani, H., Kristoferitsch, W., Lottspeich, F., 2016. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins. Proceedings of the National Academy of Sciences. 113(28), 7864–7869.

      ).
      B cells could also be targeting foreign antigens present in the CNS due to a chronic infection, with the three most obvious candidates being EBV (which is typically latent and would need to become reactivated in the CNS), HHV-6 (a ubiquitous virus which permanently infects leukocytes in the CNS and elsewhere), and Toxoplasma gondii (an endemic obligate intracellular protist which permanently infects the CNS). The associations between EBV and HHV-6 infections within the CNS and MS have been variable and inconclusive (

      Lassmann, H., Niedobitek, G., Aloisi, F., Middeldorp, J.M., Group, N.E.W., 2011. Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain. A journal of neurology. 134(9), 2772–2786.

      ,

      Venkatesan A., Johnson R.T., 2014. Infections and multiple sclerosis.Handb. Clin. Neurol., vol. 122, pp. 151–171.

      ). Seropositivity to Toxoplasma gondii seems to be protective (
      • Stascheit F.
      • Paul F.
      • Harms L.
      • Rosche B.
      Toxoplasma gondii seropositivity is negatively associated with multiple sclerosis.
      ).
      Alternatively, anti-EBV antibodies present in the CNS (
      • Cepok S.
      • Zhou D.
      • Srivastava R.
      • Nessler S.
      • Stei S.
      • Büssow K.
      • Sommer N.
      • Hemmer B.
      Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis.
      ) could inadvertently have a high affinity to human proteins through molecular mimicry, triggering MS-causing inflammation in this site (
      • Lindsey J.W.
      • Pate K.A.
      • Zhao X.
      Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L.
      ,
      • Vaughan J.
      • Riise T.
      • Rhodes G.
      • Nguyen M.-D.
      • Barrett-Connor E.
      • Nyland H.
      An Epstein Barr virus-related cross reactive autoimmune response in multiple sclerosis in Norway.
      ). However, this simple mechanism fails to explain the multi-year delay between EBV seroconversion and MS onset in young adults, as anti-EBV antibodies precede MS onset by many years (
      • 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.
      ).

      3.3 EBV infected memory B cells

      EBV is mainly found in memory B cells, in the form of DNA episomes within the nucleus of these cells (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). During infectious mononucleosis, EBV episomes can be found in up to half of all memory B cells, gradually declining to about one in 100,000 memory B cells in healthy carriers (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). Though less studied, the fraction of EBV infected memory B cells following an asymptomatic primary infection is similar to that observed in infectious mononucleosis (
      • Silins S.L.
      • Sherritt M.A.
      • Silleri J.M.
      • Cross S.M.
      • Elliott S.L.
      • Bharadwaj M.
      • Le T.T.
      • Morrison L.E.
      • Khanna R.
      • Moss D.J.
      Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.
      ). Thereafter, EBV persists at a very low but stable viral load: about half a million memory B cells are infected in the entire human body, approximately one percent of which circulate in the peripheral blood at a concentration of one infected B cell per 5 ml, with remaining cells located mainly in the lymphoid system, especially near the tonsils (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ).
      EBV remains mostly latent during the rest of an infected individual's lifetime, expressing a single gene (EBNA1) within memory B cells which allows its episome to be copied when the cells divide (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). The EBNA1 protein is not well recognized by CD8+ (cytotoxic) T cells, allowing infected memory B cells to mostly escape detection (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). EBV infected memory B cells periodically reactivate in the mouth and genital tract (
      • Israele V.
      • Shirley P.
      • Sixbey J.W.
      Excretion of the Epstein-Barr virus from the genital tract of men.
      ,
      • Sixbey J.
      • Lemon S.
      • Pagano J.
      A second site for Epstein-Barr virus shedding: the uterine cervix.
      ), differentiating into plasma cells and shedding virions to infect other hosts and to maintain a stable infected memory B cell pool in the same host (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). Plasma cells synthesizing EBV virions are rapidly cleared by CD8+ (cytotoxic) T cells (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ).

      3.4 EBV infected memory B cells in the CNS

      B cells and EBV both seem to be necessary factors in MS, suggesting their interaction may be important (
      • Márquez A.C.
      • Horwitz M.S.
      The role of latently infected B cells in CNS autoimmunity.
      ). The presence of EBV infected memory B cells within the CNS is controversial (

      Lassmann, H., Niedobitek, G., Aloisi, F., Middeldorp, J.M., Group, N.E.W., 2011. Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain. A journal of neurology. 134(9), 2772–2786.

      ,
      • Owens G.P.
      • Bennett J.L.
      Trigger, pathogen, or bystander: the complex nexus linking Epstein–Barr virus and multiple sclerosis.
      ). If the EBV infected memory B cell concentration in the CNS is similar to that found in peripheral blood (0.001% of memory B cells infected), such a low load should not elicit an immune response since EBV is very effective at evading CD8+ (cytotoxic) T cells while latent (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). Were EBV infected memory B cells in the CNS to reactivate, expand and differentiate into plasma cells, viral synthesis would occur, eliciting a strong CD8+ (cytotoxic) T cell immune response (
      • Hislop A.D.
      • Taylor G.S.
      T-Cell responses to EBV, Epstein Barr Virus.
      ). In principle, even a single acute EBV reactivation event in the CNS could lead to a permanent loss of immune tolerance to CNS antigens, though such a hit-and-run mechanism would be difficult to prove (
      • Opsahl M.L.
      • Kennedy P.G.
      An attempt to investigate the presence of Epstein Barr virus in multiple sclerosis and normal control brain tissue.
      ).
      The EBV load in the CNS of MS patients is currently unclear. The first positive study of EBV in the CNS found EBV infected B cells in 21 of 22 cases analyzed using in situ hybridization of EBV-encoded small RNA (EBER) transcripts, and reactivation markers EBV nuclear antigen 2 (EBNA2) and EBV latent membrane protein 1 (LMP1) in respectively 13 and 16 of 18 cases analyzed using immunohistochemistry (
      • Serafini B.
      • Rosicarelli B.
      • Franciotta D.
      • Magliozzi R.
      • Reynolds R.
      • Cinque P.
      • Andreoni L.
      • Trivedi P.
      • Salvetti M.
      • Faggioni A.
      Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain.
      ). However, these findings could not be replicated by several groups (
      • Opsahl M.L.
      • Kennedy P.G.
      An attempt to investigate the presence of Epstein Barr virus in multiple sclerosis and normal control brain tissue.
      ,
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • Giovannoni G.
      • Meier U.
      • Hintzen R.Q.
      • Verjans G.M.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ,
      • Sargsyan S.
      • Shearer A.
      • Ritchie A.
      • Burgoon M.
      • Anderson S.
      • Hemmer B.
      • Stadelmann C.
      • Gattenlöhner S.
      • Owens G.
      • Gilden D.
      Absence of Epstein-Barr virus in the brain and CSF of patients with multiple sclerosis.
      ,
      • Torkildsen Ø.
      • Stansberg C.
      • Angelskår S.M.
      • Kooi E.J.
      • Geurts J.J.
      • Van Der Valk P.
      • Myhr K.M.
      • Steen V.M.
      • Bø L.
      Upregulation of immunoglobulin‐related genes in cortical sections from multiple sclerosis patients.
      ,
      • Willis S.N.
      • Stadelmann C.
      • Rodig S.J.
      • Caron T.
      • Gattenloehner S.
      • Mallozzi S.S.
      • Roughan J.E.
      • Almendinger S.E.
      • Blewett M.M.
      • Brück W.
      Epstein–Barr virus infection is not a characteristic feature of multiple sclerosis brain.
      ), suggesting either important methodological differences occurred between studies or no more than a trace amount of EBV is typically present in the CNS of MS patients (

      Lassmann, H., Niedobitek, G., Aloisi, F., Middeldorp, J.M., Group, N.E.W., 2011. Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain. A journal of neurology. 134(9), 2772–2786.

      ,
      • Owens G.P.
      • Bennett J.L.
      Trigger, pathogen, or bystander: the complex nexus linking Epstein–Barr virus and multiple sclerosis.
      ). In addition to in situ techniques, many of these replication studies used highly sensitive and specific reverse transcription PCR (RT-PCR) assays to detect both EBER transcripts (which are abundantly present in all EBV infected B cells including those where EBV is latent) and messenger RNA transcripts (which indicate EBV reactivation). These replication studies analyzed more than 100 cases, including many cases from the first positive study. Thereafter three studies, including one from an independent group, were published with methods and results similar to the first positive study (
      • Magliozzi R.
      • Serafini B.
      • Rosicarelli B.
      • Chiappetta G.
      • Veroni C.
      • Reynolds R.
      • Aloisi F.
      B-cell enrichment and Epstein-Barr virus infection in inflammatory cortical lesions in secondary progressive multiple sclerosis.
      ,
      • Serafini B.
      • Muzio L.
      • Rosicarelli B.
      • Aloisi F.
      Radioactive in situ hybridization for Epstein–Barr virus–encoded small RNA supports presence of Epstein–Barr virus in the multiple sclerosis brain.
      ,
      • Tzartos J.S.
      • Khan G.
      • Vossenkamper A.
      • Cruz-Sadaba M.
      • Lonardi S.
      • Sefia E.
      • Meager A.
      • Elia A.
      • Middeldorp J.M.
      • Clemens M.
      Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.
      ). Finally two more studies with mixed result were published using PCR and RT-PCR based assays, which found EBV DNA in 9 of 26 MS cases’ CNS (
      • Hassani A.
      • Khan G.
      A simple procedure for the extraction of DNA from long-term formalin-preserved brain tissues for the detection of EBV by PCR.
      ) and EBV RNA in 5 of 31 MS cases’ cerebrospinal fluid (CSF) (
      • Veroni C.
      • Marnetto F.
      • Granieri L.
      • Bertolotto A.
      • Ballerini C.
      • Repice A.M.
      • Schirru L.
      • Coghe G.
      • Cocco E.
      • Anastasiadou E.
      Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.
      ).
      EBV reactivation in the CNS is consistent both with Pender's hypothesis, where reactivation occurs through the recognition of a “forbidden” antigen cognate with the BCR of an EBV infected memory B cell, and with memory B cell reactivation independent of the BCR. However, currently available evidence supporting EBV reactivation in the CNS of MS patients is weak, and the EBV reactivation hypothesis cannot easily account for the multi-year delay observed between EBV acquisition in adults and the onset of 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.
      ). This delay would be expected to be short because the EBV load is ten thousand times higher during primary EBV infections as compared to established EBV infections, transitioning from a high to low viral load over approximately one year (
      • Silins S.L.
      • Sherritt M.A.
      • Silleri J.M.
      • Cross S.M.
      • Elliott S.L.
      • Bharadwaj M.
      • Le T.T.
      • Morrison L.E.
      • Khanna R.
      • Moss D.J.
      Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.
      ,
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ).
      Though EBV reactivation in the CNS is the simplest hypothetical mechanism liking EBV to MS, difficulties in replicating supporting studies and the multi-year lag between EBV acquisition and MS onset suggest other mechanisms should be considered. In particular, EBV may be directly affecting memory B cell populations, which in turn affect MS risk (
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ,
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ).

      3.5 EBV infected memory B cell receptor

      A consensus view as to how EBV affects memory B cell populations is lacking (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). The germinal center model (
      • Thorley-Lawson D.A.
      Epstein-Barr virus: exploiting the immune system.
      ), a leading hypothesis, suggests EBV infects naive B cells, then uses its LMP1 gene to increase the probability of producing long-lived memory B cells by emulating the CD4+ (follicular helper) T cell conjugation phase within B cell follicles of lymph nodes (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). Under normal circumstances, a naive B cell can only mature into a memory B cell after binding to an epitope approximately matching its BCR, endocytosing the entire antigen, breaking it into peptides, presenting some peptides on HLA-D receptors, and finally having some peptides recognized by the TCR of CD4+ T cells (Fig. 2, reviewed by
      • Kurosaki T.
      • Kometani K.
      • Ise W.
      Memory B cells.
      ). When EBV infected, naive B cells can mature into memory B cells without going through all these steps (
      • Caldwell R.G.
      • Wilson J.B.
      • Anderson S.J.
      • Longnecker R.
      Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals.
      ).
      Fig. 2
      Fig. 2Standard model of naive B cell maturation into memory B cell (for a more complete description, see in
      • Kurosaki T.
      • Kometani K.
      • Ise W.
      Memory B cells.
      ). An antigen's epitope binds to a maturing B cell's BCR, followed by endocytosis of the antigen, reduction into short peptides, and presentation of these peptides on HLA-D molecule complexes (MHC class II receptors) to the TCR of CD4+ (follicular helper) T cell. If maturing B cell antigen uptake is efficient (indicating high BCR-antigen affinity) and the T cell's TCR has a high affinity to the peptide presented by the HLA-D molecule complex (shown in red), the T cell recognizes the peptide as foreign and signals to the maturing B cell that it can continue on its path to becoming memory B cell. This signaling is done in large part by CD154 on the T cell binding to CD40 on the B cell (not shown). EBV's LMP1 gene can emulate CD154/CD40 signaling, allowing B cells to mature into the memory compartment while skipping the CD4+ (follicular helper) T cell checkpoint. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      In peripheral blood, memory B cells harboring EBV have undergone somatic hypermutation and express a normal BCR, suggesting they have gone through most or all maturation steps (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). However, some of these memory B cells are suspected of having escaped the germinal center of lymph nodes through the expression of EBV's LMP1 gene (rather than through follicular helper T cells conjugation), resulting in memory B cell's BCR recognizing normally “forbidden” epitopes (Fig. 3 – panels 3 and 4) (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). Based on this hypothesis, EBV could influence the epitopes recognized by memory B cells of various isotypes, for example enabling IgG memory B cells to recognize either self epitopes or commensal microbe epitopes, thus causing undesirable chronic inflammation where these epitopes are found in the body (
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ,
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). Note that EBV may not be strictly necessary to produce memory B cells recognizing “forbidden” epitopes, which could explain EBV seronegative MS cases.
      Fig. 3
      Fig. 3Proposed sequence of events leading to memory B cell recognition of epitopes in the CNS. (1) EBV infects a resting naive B cell with a “random” BCR in near mucosal surfaces (mouth / genitals). (2) EBV activates naive B cell, which divides and forms a blast. (3) EBV infected B cell blast improves BCR affinity through somatic hypermutation in germinal center, skewing the BCR toward antigens present in the lymph node at that time. (4) “Forbidden” B cell escapes germinal center and enters memory compartment. (5) EBV replication and segregation during memory B cell division is not efficient, allowing some memory B cell descendants to become EBV-free after a few dozen generations; since memory B cells divide infrequently, this process is expected to be long (years). (6) Recognition of an epitope present in the CNS by a previously EBV infected memory B cell. The epitope must be similar to that present in the germinal center during affinity maturation, though the antigen itself may be completely different: for example, the epitope can be a ubiquitous terminal glycan of a glycoprotein, with the protein portion being completely different between the mouth and CNS.
      If clonally expanded memory B cells in the CNS matured while EBV infected, one would expect them to contain EBV episomal DNA and EBV EBER transcripts readily detectable using PCR and RT-PCR, yet the detection of EBV in the CNS has been inconsistent, and its presence remains controversial (

      Lassmann, H., Niedobitek, G., Aloisi, F., Middeldorp, J.M., Group, N.E.W., 2011. Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain. A journal of neurology. 134(9), 2772–2786.

      ,
      • Owens G.P.
      • Bennett J.L.
      Trigger, pathogen, or bystander: the complex nexus linking Epstein–Barr virus and multiple sclerosis.
      ). Since EBV transmission to daughter cells during memory B cell division is not very reliable (Fig. 3 – panel 5) (
      • Nanbo A.
      • Sugden A.
      • Sugden B.
      The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.
      ), some EBV infected memory B cells will eventually become EBV free: clonally expanded memory B cells in the CNS could thus have matured from an EBV infected naive B cell, and target a “forbidden” MS-causing epitope. In light of this, Pender's hypothesis can be extended to include memory B cells which were previously infected with EBV, eliminating the requirement of finding EBV in the CNS.

      3.6 EBV reactivation in the mouth

      An EBV-positive memory B cell which never reactivates into a plasma cell in the mouth is a dead end for the virus, because shedding virions in this site is required for transmission. In the absence of EBV, memory B cells’ BCR must bind to a cognate antigen which is also recognized by a CD4+ (helper) T cell to reactivate into a plasma cell (Fig. 1). The mechanism through which EBV-positive memory B cells reactivate has not been established, though it is suspected of being identical to that of uninfected memory B cells: within memory B cells, EBV only expresses a single protein (EBNA1) which is not known to be involved in reactivation (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). Alternative reactivation mechanisms which are strongly skewed toward the mouth appear implausible.
      If EBV infected memory B cell reactivation occurs using the cognate antigen recognition mechanism described in the previous paragraph, then the targeted antigens must be from commensal microbes in the mouth. If the targeted antigens were human, reactivation would not be possible due to a lack of CD4+ (helper) T cell recognition of human proteins. This is consistent with the recent finding that the vast majority of EBV-positive memory B cells do not recognize human proteins (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ).
      The main objection to cognate antigen recognition as a homing method to the mouth is that EBV is thought to infect resting naive B cells which have a “random” BCR, rather than a BCR which recognizes an antigen present in the mouth (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). However, the combined presence of a functional BCR and somatic hypermutations in EBV-positive memory B cells suggest their BCR is not “random”, but rather has been honed within a germinal center to recognize an epitope (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). To reconcile these observations, either EBV must infect antigen experienced proliferating B cell blasts (though this seems unlikely because activated B cells in proliferating B cell blasts don’t express CD21, which is required for virion attachment), or initially “random” BCRs of antigen naive B cells must converge toward an antigen present in the lymph node during affinity maturation (Fig. 3 – panel 3).

      3.7 EBV and host fitness

      Since EBV infects a host for life, it is expected to have evolved mechanisms which limit host morbidity and mortality (
      • Thorley-Lawson D.
      EBV persistence and latent infection in vivo.
      ). The increase in risk of MS caused by EBV seems at odds with its own fitness: why hasn’t EBV evolved to avoid causing MS? For example, if inadvertent molecular mimicry between EBV antigens and human antigens were causing MS, EBV would be expected to evolve non-cross-reactive antigens (if possible).
      To resolve this apparent contradiction, we speculate that EBV requires B cells in the memory compartment to have a high affinity to commensal microbes in the mouth, in order to reactivate in this site and complete its lifecycle. In contrast, optimal host fitness requires filling the memory compartment with B cells specific to pathogens, while avoiding B cells with a high affinity to commensal microbes, as this could result in undesirable chronic inflammation. Thus, EBV would be using its LMP genes not to bypass normal B cell maturation altogether, but rather to allow B cells with a high affinity to mouth commensal microbes to enter the memory compartment. MS would be an unfortunate side-effect of this mechanism which is essential for EBV fitness, but harmful for host fitness.
      This mechanism would require “random” BCR B cells to proliferate in the germinal center despite initially poor antigen affinity, until somatic hypermutations have had time to increase BCR affinity toward any microbial antigen abundantly present in the lymph node. Thereafter, the normal B cell maturation process would produce high affinity memory B cells which recognize an abundant microbial antigen present the mouth, ideal for EBV's lifecycle.
      While most EBV-positive memory B cells are not autoreactive (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ), it is difficult to exclude the presence of a minute subset of autoreactive EBV-positive memory B cells which could inadvertently recognize a human epitope in the CNS—though these cells would be of no use to complete EBV's lifecycle, as they would not reactivate in the mouth.

      4. Conclusion

      Epidemiological evidence strongly supports a role for EBV in MS, yet no proposed mechanism has been able to convincingly explain this link so far (
      • Ascherio A.
      • Munger K.L.
      Epstein-barr virus infection and multiple sclerosis: a review.
      ). EBV is present in >90% of adults, most often as a result of childhood exposure (
      • Balfour H.H.
      • Sifakis F.
      • Sliman J.A.
      • Knight J.A.
      • Schmeling D.O.
      • Thomas W.
      Age-specific prevalence of Epstein–Barr virus infection among individuals aged 6–19 years in the United States and factors affecting its acquisition.
      ), and is restricted to a minute subset of memory B cells (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). The requirement for B cells in MS suggest these cells play a key role, possibly due to being EBV infected. The many links between EBV and MS described here suggest peripheral memory B cells must reach the CNS: deep sequencing studies of the IgG heavy chain of B cells in the CSF and peripheral blood confirm this (
      • Michel L.
      • Touil H.
      • Pikor N.B.
      • Gommerman J.L.
      • Prat A.
      • Bar-Or A.
      B cells in the multiple sclerosis central nervous system: trafficking and contribution to CNS-compartmentalized inflammation.
      ,
      • von Büdingen H.-C.
      • Kuo T.C.
      • Sirota M.
      • van Belle C.J.
      • Apeltsin L.
      • Glanville J.
      • Cree B.A.
      • Gourraud P.-A.
      • Schwartzburg A.
      • Huerta G.
      B cell exchange across the blood-brain barrier in multiple sclerosis.
      ).
      Two important aspects linking EBV and MS are puzzling. First, EBV is present at extremely low levels in the body (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ), meaning a direct immune response against EBV appears to be an unlikely cause of MS. Second, MS is rare in children (
      • Confavreux C.
      • Vukusic S.
      Natural history of multiple sclerosis: a unifying concept.
      ,
      • Ligouri M.
      • Marrosu M.
      • Pugliatti M.
      • Giuliani F.
      • De Robertis F.
      • Cocco E.
      • Zimatore G.
      • Livrea P.
      • Trojano M.
      Age at onset in multiple sclerosis.
      ), suggesting EBV infections are insufficient to cause MS on their own. A partial explanation for the first puzzling aspect has been proposed: EBV may rescue “forbidden” memory B cells which recognize an epitope in the CNS (
      • Pender M.P.
      Infection of autoreactive B lymphocytes with EBV, causing chronic autoimmune diseases.
      ,
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). However, inconsistent detection of EBV in the CNS is somewhat at odds with this hypothesis (

      Lassmann, H., Niedobitek, G., Aloisi, F., Middeldorp, J.M., Group, N.E.W., 2011. Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria. Brain. A journal of neurology. 134(9), 2772–2786.

      ). The unreliable transmission of EBV during memory B cell division, first reported by Nanbo and colleagues in 2007, can explain the absence of EBV in the CNS while still allowing “forbidden” memory B cells to reach this site (
      • Nanbo A.
      • Sugden A.
      • Sugden B.
      The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.
      ). This can also explain the delay of a few years between EBV seroconversion in young adults and MS onset (
      • 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.
      ), which other mechanisms reviewed here do not account for. However, no mechanism reviewed here can explain the multi-decade delay between EBV infections in early childhood and MS in early adulthood. This second puzzling aspect may require additional environmental factors which mainly affect teens and adults.
      Both EBV infected memory B cells and memory B cells present in the CNS in MS have a functional BCR and have undergone somatic hypermutation (
      • Qin Y.
      • Duquette P.
      • Zhang Y.
      • Talbot P.
      • Poole R.
      • Antel J.
      Clonal expansion and somatic hypermutation of V (H) genes of B cells from cerebrospinal fluid in multiple sclerosis.
      ,
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ), though antigens cognate to this BCR are not known in either case. EBV's lifecycle suggests this antigen should be present where EBV infects naive B cells (e.g. in the mouth), allowing EBV infected memory B cells to preferentially reactivate and produce virions in this site, completing its lifecycle (
      • Thorley-Lawson D.A.
      EBV Persistence—Introducing the Virus, Epstein Barr Virus.
      ). It appears these cognate antigens are generally not human proteins (
      • Tracy S.I.
      • Kakalacheva K.
      • Lünemann J.D.
      • Luzuriaga K.
      • Middeldorp J.
      • Thorley-Lawson D.A.
      Persistence of Epstein-Barr virus in self-reactive memory B cells.
      ). To our knowledge, no study has investigated the possibility that EBV infected memory B cells recognize commensal microbe antigens present in the mouth: this seems to be the simplest scenario.
      Based on the evidence reviewed here, we slightly modify Pender's hypothesis as follows. EBV infected memory B cells recognizing a commensal microbe epitope eventually become EBV-free through memory B cell division. Once EBV free, these memory B cells can proliferate and recognize a cognate epitope in the CNS, causing inflammation and MS. The nature of the antigen in the CNS on which this cognate epitope is found remains unclear: it could either be a human antigen or an antigen belonging to a microbe present in the CNS (such as Toxoplasma gondii). Under EBV-free circumstances, B cells recognizing this “forbidden” epitope are generally not allowed to enter the memory compartment, greatly reducing the risk of MS in EBV negative individuals. When EBV infected, the LMP1 gene simulates CD4+ (follicular helper) T cell B cell conjugation in the germinal center, allowing a B cell recognizing a “forbidden” epitope to enter the memory compartment.

      Disclosures

      ML reports no disclosures.
      JBL reports no disclosures.

      Acknowledgments and funding

      JBL is supported by the National Institutes of Health , Bethesda, MD, USA ( NINDS #R01 NS39422 ), the Commission of the European Union (grant ICT-2011–287739 , NeuroTREMOR), and the Spanish Health Research Agency (grant FIS PI12/01602 and grant FIS PI16/00451 ).

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