Trigger, pathogen, or bystander: the complex nexus linking Epstein Barr virus and multiple sclerosis

8109MSJ18910.1177/1352458512448109Owens and BennettMultiple Sclerosis Journal Topical Review MULTIPLE SCLEROSIS JOURNAL MSJ Trigger, pathogen, or bystander: the complex nexus linking Epstein Barr virus and multiple sclerosis Multiple Sclerosis Journal 18(9) 1204 1208 The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: 10.1177/1352458512448109 msj.sagepub.com Gregory P Owens 1 and Jeffrey L Bennett 2 Abstract A causal role for virus infection in the pathophysiology of multiple sclerosis (MS) has been suggested and widely debated since the landmark epidemiologic studies of Kurtzke revealed a strong environmental determinant to MS susceptibility. Despite multiple efforts, no virus has been unequivocally associated with lesion formation in the brain either by direct isolation or by indirect methods of detection. In many infectious diseases of the central nervous system, oligoclonal IgG bands are the product of a robust and specific humoral response against the causative agent; yet in MS, immunoreactivity to a primary target has been elusive. In the absence of any infectious agent fulfilling Koch s Postulates, new concepts that could plausibly explain the epidemiology of MS have been postulated. The initiation or activation of a nascent autoimmune response in genetically susceptible individuals following exposure to one or more common infectious agents is now a leading hypothesis to explain MS pathogenesis. Epstein Barr Virus (EBV), a human herpes virus that infects B cells in ~95% of the human population and persists latently in the memory B cell pool throughout life, has received the most attention as a probable candidate; EBV has been implicated as both an environmental trigger and as a direct causative agent of CNS immunopathology. In this review, we will discuss the most salient features of EBV epidemiology, the immunological response to EBV in MS patients and whether EBV infection of the brain is a necessary prerequisite of MS pathology. Keywords demyelination, multiple sclerosis Date received: 13th April 2012; accepted: 19th April 2012 A causal role for virus infection in the pathophysiology of multiple sclerosis (MS) has been suggested and widely debated since the landmark epidemiologic studies of Kurtzke revealed a strong environmental determinant to MS susceptibility. 1 Despite multiple efforts, no virus has been unequivocally associated with lesion formation in the brain either by direct isolation or by indirect methods of detection. Likewise, attempts to link the intrathecal production of immunoglobulin (Ig) and IgG oligoclonal bands to a specific infection have generated a confounding and contradictory body of literature. In many infectious diseases of the CNS, oligoclonal IgG bands are the product of a robust and specific humoral response against the causative agent; yet in MS, immunoreactivity to a primary target has been elusive. In the absence of any infectious agent fulfilling Koch s Postulates, new concepts that could plausibly explain the epidemiology of MS have been postulated. The initiation or activation of a nascent autoimmune response in genetically susceptible individuals following exposure to one or more common infectious agents is now a leading hypothesis to explain MS pathogenesis. 2 Epstein Barr virus (EBV), a human herpes virus that infects B cells in ~95% of the human population and persists latently in the memory B cell pool throughout life, has received the most attention as a probable candidate; 3 EBV has been implicated as both an environmental trigger and as a direct causative agent of central nervous system (CNS) immunopathology. In this review, we will discuss the most salient features of EBV epidemiology, the immunological response to EBV in MS patients and whether EBV 1 Department of Neurology, University of Colorado Denver, USA. 2 Departments of Neurology and Ophthalmology University of Colorado Denver, USA. Corresponding author: Gregory P Owens, University of Colorado Denver, Department of Neurology, 12700 E. 19th Avenue, Research Complex 2, Box B-182 Room 5004, Aurora, CO 80045, USA. Email: greg.owens@ucdenver.edu

Owens and Bennett 1205 infection of the brain is a necessary prerequisite of MS pathology. Infection with EBV and risk of MS There is a persuasive body of evidence linking EBV infection to MS risk. The 5% of the human population without detectable EBV infection have a remarkably low risk for developing MS. In a review of case-control studies, Ascherio and Munch found the incidence of MS to be significantly higher (odds ratio = 13.5) in EBV seropositive compared with seronegative individuals. 4 A more recent study that analyzed serial serum samples from US military personnel for EBV antibodies (Abs) identified 10 healthy patients who were EBV-negative at their time of enrollment, but subsequently developed clinical MS. All 10 of the MS patients became infected with EBV prior to their diagnosis. In comparison, a significantly lower rate (35.7%) of EBV infection over the same time interval was observed in a cohort of age- and sex-matched controls. 5 An associated risk for MS following EBVinfection, but not cytomegalovirus- or HSV1-infection, was also found in cases of pediatric MS, 6,7 and individuals who acquire EBV during adolescence or later, as manifested by infectious mononucleosis, are at an additional 2 3-fold higher risk to develop MS than children infected early in life. 8,9 Heightened humoral immune responses to EBV are found in individuals with MS. Notable differences in antibody titers to EBV antigens, particularly EBNA-1 and the nuclear EBNA complex, are detected in MS patients up to five years before the diagnosis of disease. Compared with matched controls, increased EBV Ab titers indicate an increased risk for developing MS disease and are suggested to be an early event in the disease process. 10,11 The association between enhanced EBV immunity and the role of EBV as a causative agent is complicated by elevated Ab titers in MS serum and cerebrospinal fluid (CSF) to many common viruses and infections prevalent within the human population, which may be more indicative of an overall immune dysregulation of B cell memory responses. 12 Unlike EBV, however, epidemiologic evidence does not show an increased risk of MS following exposure to many other common viral infections including measles, rubella, mumps, HSV-1 and VZV. 13 Cellular immunity is also somewhat altered in MS patients relative to healthy controls. Generally, there are increased numbers of circulating EBV-specific CD8+ cells but not EBV-specific CD4+ T cells, the exception being EBNA-1. 14 EBNA-1-specific memory CD4+ T cells are elevated in MS blood, show increased proliferative capacity and possess higher avidity and broadened peptide specificity compared with demographically and HLA-DRmatched controls. Although the epidemiologic and immunologic data provide a compelling connection between exposure to EBV and risk of MS, alternative explanations that do not necessitate a direct causal link cannot be excluded, the most likely being a shared susceptibility for MS and EBV infection. Indeed, recent work has shown that the mechanism through which class I and class II HLA genes influence the risk of MS may similarly affect the immune control of EBV infection. 15 EBV infection of the brain Disease mechanisms linking EBV infection to MS pathology are to date largely hypothetical. Autoreactive T cells could be activated by EBV through molecular mimicry, as demonstrated in vitro for MBP-specific T cell clones derived from blood of MS patients. 16 Alternatively, enhanced breakdown and presentation of self-antigens, expression of viral superantigens or bystander activation could be putative mechanisms by which EBV infection promotes CNS inflammation. 17 Perhaps most contentious is a direct role for EBV infection in CNS inflammation as a consequence of potential selective enrichment of EBV-infected B cells in ectopic meningeal follicles and perivascular CNS inflammatory infiltrates in MS brain. In 2007, Serafini and colleagues 18 reported that ~90% of B-lymphocytes in meningeal B cell follicles and in perivascular cuffs of multiple acute and chronic active white matter lesions expressed EBV nuclear transcripts (EBERs) that were identified by in situ hybridization; 50 80% of parenchymal plasma cells were also EBERs positive. EBV proteins, LMP1 and LMP2a, were detected by immunohistochemistry and in very severe MS cases of short disease-duration, evidence for the EBV lytic cycle was suggested by numerous cells immunostained with antibody to the early-lytic cycle-associated protein BFRF1. The robust presence of EBV-infected B cells in close to 100% of MS cases examined in this study stands in stark contrast to several later publications that rarely detected EBV in MS brain specimens. 19-21 Concurring reports by Willis et al. 19 and Peferoen et al. 20 found little evidence for EBV infection in a large sampling of MS brain tissue using similar in situ hybridization methodologies. The Willis study examined 23 blocks from 12 MS specimens that were clearly demonstrated to contain CD20+ B cell infiltrates. Peferoen et al. examined 632 brain sections from 94 MS patients for EBV infection, including 12 of the same tissue blocks previously identified as EBV-positive. 20 Although B cells in EBVassociated control tumors were positive by hybridization to EBV-encoded RNA, only one MS sample in these two studies was EBERs positive. In the absence of detectable EBV RNA by in situ hybridization, Willis et al. developed sensitive real-time PCR assays that could amplify EBV genomic DNA or EBER1 RNA from one or two EBV-infected cells processed in the presence of human brain tissue. 19 Using their real-time PCR assay, Willis et al. identified EBV in 0/17 MS white matter sections and in 2/12 MS meningeal sections including

1206 Multiple Sclerosis Journal 18(9) identical tissue samples evaluated in the Serafini study. 18 Our research group was also intrigued by the possibility of a chronic active EBV infection in MS brain and attempted to detect EBV transcripts from single FACS-sorted MS CSF B and plasma blast cell populations and from MS brain tissue. 21 Because plasma blasts in MS CSF are clonally expanded and are cells contributing to the production of CSF IgG oligoclonal bands, 22 we reasoned that a prominent infection of CNS B cells would be detectable within this population, a view validated by repertoire studies showing a strong clonal continuum between B cells populating the meninges, brain parenchyma and CSF of MS patients. 23 Using real-time PCR with a pre-amplification step to detect EBER1 and a cellular gene (RPLP0) in single cells, we failed to identify EBER1 transcripts in 274 single plasma blast cells and 57 single CD19+ B cells sorted from four different MS CSF donors. EBER1 was detected in 100% of single EBV-infected B cells processed in the same manner and RPLP0 was detected in all of the single B and plasma cells analyzed. Fifteen plaques from US and UK tissue banks were further studied for EBV and control gene transcripts. Four of the lesions were verified to contain perivascular B cell infiltrates and another five were previously shown to carry a high EBV-load in the Serafini study 18. Although we readily detected cellular transcripts for GAPDH, human IgG and RPLP0, EBV-specific transcripts (EBER1, EBNA-2, LMP-1 and BFRF-1) were not detected in any of the MS lesions, but were amplified from control EBV-infected lymphomas. The only MS tissues in which EBV transcripts were detected using real-time PCR had been prescreened for EBV DNA from a larger sample of MS brain sections. When a series of paraffin-embedded MS tissue sections including three EBV DNA-positive sections were assayed blinded, we only detected EBER1 RNA, but not other EBV transcripts, indicating the presence of rare Stage 0 latently EBV-infected B cell in these samples. Together, the above three studies concluded that EBVinfection in MS brain tissue is a rare event and unlikely to be a direct cause of CNS inflammation. Two other studies attempting to amplify EBV DNA from MS brain tissue also found EBV infection to be an infrequent event. 24,25 More recently, Aloisi et al. countered that EBV latency transcripts (LMP2a and EBNA-1) are present in MS tissue by PCR, but require pre-amplification from B cell infiltrates that were obtained from MS brain samples by laser-microdissection. 26 They suggested that the previous PCR assays may have been insensitive and limited EBV detection. They also indicated that EBV-infection of the brain is more limited than previously reported. To address the largely divergent body of literature regarding the presence of EBV and its relationship to MS brain inflammation, a workshop was held in Vienna in 2010 under the umbrella of the European Union NeuroProMiSe project. Although a consensus was not reached, a majority view held that EBV-infection of B cells in the brain, if present at all, is limited to very few cells. Issues regarding the specificity and sensitivity of individual assays used to detect EBV were discussed in detail and have been presented in a summary publication. 27 Without recapitulating the many arguments presented in this review, several additional points bear further emphasis. (1) EBERs transcripts are normally quite abundant (>1,000,000 copies per cell) in EBV-infected B cells. The observation that other B cellspecific transcripts including CD20 and IgG were routinely detected by PCR in the MS tissues studied indicates that EBER1 and other EBV transcripts, if present in a high percentage of B cells, should have been detected unless EBERs expression is somehow down-regulated in the CNS. (2) The sensitivity argument does not explain the absence of EBV transcripts in individually FACS-sorted MS CSF plasma blasts and B cells. The idea that CSF B cells are somehow distinct from those in the meninges and brain parenchyma is not supported experimentally. As described above, detailed sequence analyses show that B cell populations within the different compartments of the CNS share a common lineage and bear the phenotype of antigen-selection. 23 (3) Tissue selection has also been suggested as an explanation for the discrepancy in results, even though many of the sections examined in this series of studies are from the same MS patients, tissue banks and, in the Perforoen study, included sections from the exact same tissue block. A recent paper, which was also discussed at the Vienna meeting, reported abundant EBV-positive B cells, including some lytically-infected B cells, in active MS lesions that were characterized by the presence of MHC class II+ cells expressing interferon α (IFN-α). 28 The specificity of the in situ hybridization protocol and/or specificity of EBVinfection to MS brain was somewhat compromised by the presence of EBV-positive cells (presumably B cells) in two stroke brains but not in normal control brain. Subacute sclerosing panencephalitis (SSPE) brain tissue, which is usually highly-infiltrated with B cells, was EBERs negative as well. However, because SSPE is a rare consequence of measles virus infection that occurs primarily in the young, the unknown EBV status of the patient is an important consideration in interpreting results. Whether EBV-infected cells are driving IFN-α production through stimulation of innate TLR3 receptors via the release of EBERs RNA, as suggested by some additional preliminary evidence in this article, or are coincidental to CNS inflammation and demyelination will likely be addressed in future studies. Lastly (4), the notion of EBV driving CNS inflammation is not uniformly supported by studies of the intrathecal immune response. Although enhanced EBV immunity is a feature of MS serology, it does not necessarily translate to antibody responses in CSF. Although one study did show enhanced CSF immunoreactivity to the EBV-specific proteins BRRF2 and EBNA-1 in MS patients, 29 our analyses of paired CSF and serum samples found little evidence for a disease-specific intrathecal Ab response to EBV antigens. 21

Owens and Bennett 1207 Figure 1. Possible interactions linking Epstein Barr virus (EBV) infection to risk of multiple sclerosis. CNS: central nervous system A determination of the CSF IgG index to EBV antigens revealed indices >0.7 in 85% (17/20) of MS CSFs and 60% of non-ms CNS inflammatory disease controls. Patients with an IgG index (>1.5) indicative of intrathecal synthesis of anti-ebv IgG antibody did not differ significantly between MS (3/20) and non-ms CNS inflammatory disease controls (2/5). We further tested recombinant Abs generated from clonally expanded MS CSF plasma cells for binding to EBV antigens. While EBV-infected B95-8 cells were readily stained with control anti-ebv-gp125 antibody and with MS and control CSF, none of the 32 recombinant Abs derived from four patients with relapsing or progressive MS bound to EBV-infected cells. Other studies of Ab responses in MS CSF, including samples obtained at the time of their first clinical event and from pediatric cases, also found intrathecal anti-ebv antibody synthesis to be rare or comparable in frequency to the poly-specific response observed for other common infectious agents. 30 32 Future directions After years of investigation, the exact relationship between EBV infection and MS remains unclear and may involve totally unexplored areas of cause and effect (Figure 1). Epidemiologic data indicate a significant association between EBV infection and MS risk; however, confusing correlation with causation is a long-standing pitfall in research. Is the link between EBV and MS immunologic? Are there MS risk alleles that incur susceptibility to EBV infection or significantly alter the immune response to EBV? Or, does the pathophysiology of MS foster susceptibility to EBV infection? If so, then genetic and immunologic studies comparing the genotypes and immune responses of EBV-infected individuals with and without MS may be directly informative. Alternatively, is the link between EBV and MS causal? Does MS result from a persistent or dysregulated EBV infection? Or does an altered immune response to EBV infection trigger or perpetuate demyelinating disease? 33 Although implicated in the etiology of multiple immunologic disorders such as MS, systemic lupus erythematosus and rheumatoid arthritis, evidence that EBV causes autoimmunity falls short of the strong evidence supporting the role of the virus in lymphoid malignancy. Future research supporting a causal link between EBV and MS will need to show either a reproducible presence of EBV-infected lymphocytes in affected CNS tissue or demonstrate a mechanistic link between a dysregulated EBV immune response and MS immunopathology. Funding This research was supported by Grant Number R01NS072141 from the National Institute Of Neurological Disorders And Stroke to G.P.O, and by funding from the National Multiple Sclerosis Society (Grant Number RG4320) and Guthy-Jackson Foundation to J.L.B. The content is solely the responsibility of the authors and does not necessarily represent official views of the funding agencies. Conflict of interest statement The authors declare that there is no conflict of interest. References 1. Kurtzke JF. Epidemiologic evidence for multiple sclerosis as an infection. Clin Microbiol Rev 1993; 6: 382 427.

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