Epstein-Barr virus and MS.
Abstract: Epstein-Barr virus (EBV) infection, particularly symptomatic infection, is closely associated with multiple sclerosis (MS). The strength of this association, the interaction of EBV infection with genetic risk factors and emerging immunological data have resulted in some investigators arguing that this association may be causative. Causation, however, is a complex science and more extensive and definitive data is required to convince the wider community that EBV is the pivotal factor in the complex causal pathway that ultimately leads to the development of MS.

KEY WORDS:

MULTIPLE SCLEROSIS, EPSTEIN-BARR VIRUS, CAUSATION, COMPLEX DISEASE
Article Type: Clinical report
Subject: Multiple sclerosis (Risk factors)
Multiple sclerosis (Genetic aspects)
Epstein-Barr virus (Complications and side effects)
Author: Giovannoni, G.
Pub Date: 07/01/2010
Publication: Name: The International MS Journal Publisher: PAREXEL MMS Europe Ltd. Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 PAREXEL MMS Europe Ltd. ISSN: 1352-8963
Issue: Date: July, 2010 Source Volume: 17 Source Issue: 2
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 256365660
Full Text: Background

Multiple sclerosis (MS) is a complex autoimmune disease that cannot be ascribed to a single genetic or environmental factor. (1) There is now overwhelming evidence that MS is determined by strong environmental factors acting at a broad population level. One potential way that the genetic epidemiology can be explained is the action of a ubiquitous infection which almost all the population is exposed to with the disease resulting from host factors. If correct, this hypothesis has important implication for disease prevention; particularly if the infection can be identified; (2) one pivotal factor in a complex web of gene environment interactions may be so critical that if it can be stopped, MS may become a preventable disease. (1) Epstein-Barr virus (EBV) infection is so strongly associated with MS that some investigators are now arguing that this association may be causative. (3-4)

Seroprevalence Studies

Virtually all subjects with MS (>99%) are infected with EBV compared with only ~90% of control subjects. (4-5) The corollary is that MS is very rare in subjects who are not infected with EBV; (4-6) the relative risk of getting MS if you are EBV-negative is close to zero (odds ratio [OR] = 0.06, 95% confidence interval [CI] = 0.03-0.13, P<0.000000001). (4)

Paediatric MS

In a North-American study of 1 37 children with MS and 96 control participants matched by age and geographical region, 108 (86%) of the children with MS were seropositive for remote EBV infection, compared with only 61 (64%) of matched controls (P=0.025); (7) in this study, children with MS did not differ from controls in seroprevalence of the other childhood viruses studied. (7) In a European seroprevalence study of 147 paediatric patients, 99% of children with MS had detectable antibody against EBV virus capsid antigen (VCA) compared with only 72% of age-matched controls (P=0.001). (8) The observation that the EBV prevalence rate in paediatric MS is not 100%, is a strong argument against the association between EBV and MS being causative. However, it is more difficult to diagnose MS in children and there remains some uncertainty about nosology as paediatric cases unexpectedly have a reduced frequency of affected relatives (Canadian Collaborative Study, George Ebers personal communication); usually early onset in complex traits is associated with greater load of risk factors and greater familial risk. Therefore it will be critical to establish with long-term follow-up if the EBV seronegative children with a diagnosis of MS turn out to have 'typical' MS or not.

Temporal Profile of Infection

To prove that EBV causes MS, it is important to show that EBV infection predates the onset of disease. (9) The paediatric data largely supports this temporal criterion with the caveat referred to above. In an elegant study, Levin et al estimated the time of EBV infection by determining antibody titres serially before the onset of MS and compared this with a group of control subjects. (10) They Hd access to medical records of over 8 million US military personnel with stored serum samples. Using a nested, case-control study of 305 people who developed MS and 610 matched controls selected, 10 (3.3%) of cases who developed MS and 32 (5.2%) controls were initially EBV-seronegative. (10) All of the 10 EBV-negative cases had became EBV- positive before the onset MS, in comparison only 35.7% (10) of the 28 controls with follow-up samples seroconverted (P=0.0008). (10)

Lymphocyte Transformation

People with MS have a higher rate of spontaneous ex vivo EBV-associated lymphocyte transformation compared with control subjects. (11) Possible explanations are that people with MS: (i) have higher EBV viral loads, (ii) are infected with a strain of virus with a greater propensity to transform B lymphocytes or (iii) have abnormal immune regulation of EBV-infected lymphocytes.

Infectious Mononucleosis

People with symptomatic EBV infection or infectious mononucleosis (IM) have an increased risk of developing MS compared with people who have not had IM; a systematic review and meta-analysis of 14 case-control and cohort studies reports a combined conservative relative risk of MS after IM of 2.3 (95% CI, 1.7-3.0; P<10-8) (6). This risk has subsequently been confirmed in a large Danish cohort study of over 25 000 Danish patients with suspected IM followed up for the occurrence of MS after the diagnosis of IM or with a negative Paul-Bunnell test; the ratio of observed to expected MS cases was 2.27 (95% CI, 1.87-2.75). (12) The risk of MS was persistently increased for more than 30 years after IM and uniformly distributed across all investigated strata of sex and age. (12) More recently, in the longitudinal Canadian database study, consisting of 14 362 MS index cases and 7671 spousal controls, there was no significant differences between cases and controls for all viral exposures and vaccinations, except for a reported history of IM (OR 2.06, 95% CI, 1.71-2.48; P<0.001). (13) Possible explanations for these observations include infection with a more pathogenic strain of EBV or immune dysregulation; the immune system of people who are destined to develop MS may deal with primary EBV viral infection less efficiently than 'normal' subjects and therefore develop IM.

Anti-EBV Antibody Titres

People with high titres of anti-EBV antibodies have a higher risk of developing MS compared with those with low titres. (4,14-15) This is independent of antibody titres to a cytomegalovirus (CMV) a related herpesvirus, suggesting that it is not a non-specific phenomenon related to immune dysregulation. (14-15) This observation does not necessarily imply causation and may simply represent an association due to abnormal immune responses to EBV infection. People with MS have been shown to have raised antibody titres to a number of other viruses, particularly measles, mumps and rubella. (16) Compared with EBV, primary infection with CMV occurs at a later age and therefore CMV may not necessarily be the best control virus to ascertain whether or not raised anti-EBV antibody titres are specific to EBV or not.

Disease Cluster

An unusual cluster of MS in children attending a school in a small rural Danish community called Fjelso, occurred shortly after an outbreak of glandular fever or IM; all these subjects were infected with the same EBV genotype. (17) It could be argued that in isolation, the outbreak of MS after a local epidemic of IM may have occurred due to chance. (17) This cluster, however, focuses on EBV itself and raises the question of whether or not a specific strain of EBV is more likely to be associated with MS than other strains. Superficially this does not seem to be the case. In a relatively small study, EBV-latent membrane protein-1 (LMP-1) genotyping did not reveal any obvious differences in the distribution of EBV strains between subjects with MS and controls. (18) Another study investigating genetic variability in the genes encoding the EBNA1 and BRRF2 EBV proteins did not show any specific EBV sequence variability unique to 40 subjects with MS compared with controls. (19) Several single nucleotide polymorphisms within the EBNA1 gene, and one within the BRRF2 gene, occurred at marginally different frequencies in EBV strains infecting MS patients versus controls. (19) More detailed analysis of EBV viral strains will be required to see if any viral-specific factors are associated with MS.

Intrathecal Humoral Response

An undefined proportion of antibodies in the spinal fluid of subjects with MS recognize EBV-specific antigens. Four independent studies have extracted peptides homologous with EBV from random peptide libraries using CSF-derived MS IgG and have shown intrathecal IgG synthesis reactive to EBV protein. (20-23) In a recent study of 43 childhood-onset and 50 adult-onset MS patients, and 20 children and 12 adults with other disorders, the intrathecal synthesis of anti-EBV antibodies were detectable in 26% paediatric- and 10% adult-onset MS patients, compared with frequencies ranging from 10-60% for other common neurotropic viruses. (24) Interestingly, the antibody indices (AIs; an index of intrathecal antibody production) for EBV were lower than those for all other viruses, with more than two-fold higher median AIs for measles, rubella and varicella zoster virus (VZV). (24) More recently, Jaquiery et al reported that the anti-VCA and anti-EBNA-1, but not anti-CMV IgG, AIs were increased in early MS compared with controls. (25) This was associated with the intrathecal enrichment in EBV-, but not CMV-specific, CD8+ cytotoxic T-cells in MS, but not controls. (25) If the latter observation is confirmed it would support the argument for central or central nervous system (CNS) role for EBV in the MS causal pathway over a peripheral one. It must be stressed, however, that the intrathecal EBV-targeted humoral immune response in MS is only part of a 'polyspecific' antibody response directed against several neurotropic viruses.

T-cells

Autoimmune MBP-specific T-cells in the circulation of subjects with MS, which are capable of orchestrating an attack on myelin producing cells, also recognize EBV antigens. (26) EBV reactivation induces the expression in peripheral B-cells of [alpha]B-crystallin, a stress protein (27) that has been identified as one of the immunodominant antigens in CNS myelin from subjects with MS. (28) It has been proposed that EBV-induced peripheral [alpha]B-crystallin expression may, therefore, drive a pathogenic autoimmune response in MS. (27) Subjects with MS have a higher number of circulating CD8+ cytotoxic T-cells that recognize EBV than control subjects. (22,29) Whether these are targeting EBV-infected cells or are part of an ongoing autoimmune response, as suggested by Lang et al (26) and Van Noort et al (28) needs to be determined. EBV-encoded nuclear antigen-1 (EBNA1) specific CD4+ T-cell responses from patients with MS preferentially recognize multiple epitopes within the central part of the C-terminal EBNA1 domain, compared with fewer, more restricted, epitopes in healthy EBV carriers. (30) These observations support intra-molecular antigenic spread and suggest that T-cell anti-EBV immune responses in MS are enhanced and may be driving MS disease activity via molecular mimicry.

Lymphoma

Hodgkin's lymphoma, which is aetiologically linked to EBV infection, occurs more frequently in families with MS and in subjects with MS than in the general population. (31) This observation supports the hypothesis that people with MS and their family members handle latent EBV infection differently than the normal population.

MS Disease Activity

There is evidence that during MS relapses EBV is actively replicating compared with periods of remission. (32) Patients with an antibody response to EBV early antigen are more likely to have disease activity as measured using gadolinium-enhanced MRI than patients without an antibody response. (33) In 100 subjects involved in serial imaging studies over a 5-year period (50 clinically isolated syndrome [CIS], 25 relapsing-remitting MS [RRMS], 25 primary progressive MS [PPMS]) anti-EBNA1 IgG titres were highest in patients with RRMS compared with patients with PPMS (P<0.001) and CIS (P<0.001). Gadolinium (Gd)-enhancing lesions on magnetic resonance imaging (MRI) correlated with anti-EBNA1 IgG (r = 0.33, P<0.001) and the anti-EBNA1 :anti-VCA IgG ratio (r = 0.36, P<0.001). (34) Anti-EBNA1 IgG correlated weakly with the change in T2 lesion volume (r = 0.27, P=0.044) and Expanded Disability Status Scale (EDSS) (r = 0.3, P=0.04). (34) In a another study of 50 patients with MS and a mean follow-up of 3.1 years, anti-EBV VCA IgG was negatively correlated with grey matter fraction (r-squared = 0.24, P=0.002) and whole brain parenchymal fraction (BPF) (r-squared = 0.39, P<0.001); (35) the decline in BPF over 3 years was significantly associated with increased baseline anti EBV VCA IgG levels (P<0.001). (35) These results suggest that MS disease activity may be related to latent EBV infection and is associated with MS disease progression. In a recent study, CIS patients had both an increased humoral (P<0.0001) and cellular (P=0.007) immune responses to EBNA1, but not to other EBV-derived proteins. (36) The response to EBNA1 correlated with the number of T2 lesions (P=0.006), the number of Barkhof criteria (P=0.001) at baseline, the number of T2 lesions at 1 and 5 years after presentation (P=0.012), the presence of new T2 lesions at 1 and 5 years after presentation (P [less than or equal to] 0.028), and EDSS score at 1 and 5 years after presentation (P [less than or equal to] 0.015). (36)

MS Pathology

Serafini et al have recently reported evidence of EBV infection in a substantial proportion of brain-infiltrating B-cells and plasma cells in post-mortem MS tissue. (23) In 21 out of 22 cases, examined ectopic B-cell follicles that had formed in the cerebral meninges of cases with secondary progressive MS (SPMS) were identified as sites of expression of viral latent proteins indicative of EBV persistence. (23) Viral reactivation was restricted to ectopic B-cell follicles and acute lesions. Activation of [CD8.sub.+] T-cells with signs of cytotoxicity directed towards EBV-infected plasma cells was noted at sites of EBV-infected cells. (23) These observations were not seen in other inflammatory neurological diseases. (23) The investigators suggest that the EBV persistence and reactivation in the CNS is an important factor in the immunopathogenesis of MS. (23) These findings have not been confirmed. (37-39) Whether or not the disparate results reported in these studies relate to case selection or technical differences needs to be determined.

EBV and HLA

In a single nested case-control study of 148 women with MS and 296 age-matched healthy women the association between anti-EBNA1 antibody titres and MS risk was not affected by adjustment for DR15 and was similar in DR15-positive and DR15-negative women. (40) Interestingly, the relative risk of MS among DR15-positive women with elevated (>1:320) anti-EBNA1 titres was nine times higher than that of DR15-negative women with low (<1:80) anti-EBNA-1 titres. (40) Sundstrom et al have recently reported that subjects with MS have increased antibody reactivity against several EBNA1 domains, of which, antibodies against the EBNA1 domain (amino acid 385-420) in HLA DRB1 * 1501 positive individuals is associated with a 24-fold increased risk of developing MS. (41) If confirmed, these data suggest a specific role for this epitope in the autoimmune pathogenesis of MS.

Other Autoimmune Diseases

The observation that EBV has also been linked to other putative autoimmune diseases, (42-43) in addition to MS, suggests that EBV may be a non-specific trigger in the autoimmune cascade. One hypothesis states that EBV randomly immortalizes B-cells, which as professional antigen presenting cells are capable of presenting auto-antigens to auto-reactive T-cells. (44) As these cells are resistant to apoptosis they are capable of perpetuating the aberrant autoimmune response. (44) Alternative hypotheses to explain the association between EBV and other autoimmune diseases include molecular mimicry eluded to above (26,28) and that EBV operates indirectly by activating the pathogenic expression of endogenous retroviruses such as HERV-W, which has also been postulated to play an aetiological role in MS. (45-47)

Causation

Traditionally, microbiologists used Koch's postulates to establish whether or not a particular microbe caused a specific disease. (48) Koch's postulates are not appropriate for most viral infections; particularly for diseases caused by viruses that are a rare manifestation of a common infection. (49) The latter includes establishing EBV as a possible cause of MS. Because of the difficulties of applying Koch's postulates to viral infections most contemporary investigators have adopted Sir Austin Bradford Hill's criteria for causation. (50) Although these were formulated primarily to establish causation for diseases initiated by environmental exposure to toxins, they have been adapted for causation in relation to infectious disease (49) and more recently for establishing causation in relation to MS (see Giovannoni G et al for review). (9)

Conclusion

There is little doubt that EBV infection, particularly symptomatic infection, is associated with MS. Although the strength of this association is relatively strong it is too premature to claim causation. The interaction of EBV infection with genetic and other environmental risk factors (Vitamin D and smoking) needs to be explained and the emerging immunological data needs further clarification. Causation is a complex science and investigators should respect and appreciate this science. People with MS, their families and the wider community do not deserve premature claims of causation that turn out to be false after more extensive investigation; we are tired of this phenomenon. As a corollary, the sceptics need to acknowledge that EBV may yet turn out to be that pivotal factor in the complex causal pathway that ultimately leads to the development of MS. The challenge is to get the balance right going forward so that appropriate resources are directed to funding fundamental research in this area.

Key Points

* EBV infection, particularly primary symptomatic infection, is an important risk factor for the development of MS.

* A history of infectious mononucleosis and/or raised antibody titre to the EBV protein EBNA1 synergizes with HLA susceptibility locus to greatly increase the risk of developing MS.

* Persistent EBV seronegativity is one of the most powerful predictors of not developing MS.

* An increased adaptive immune response to EBNA1 is associated with MS disease activity.

Conflicts of Interest

No conflicts of interest were declared in relation to this article.

Received: 4 January 2010

Accepted: 15 January 2010

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G Giovannoni

Queen Mary University of London, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London, UK

Address for Correspondence

Gavin Giovannoni Neuroscience and Trauma Centre

Blizard Institute of Cell and Molecular Science

Barts and The London School of Medicine and Dentistry

4 Newark Street

London E1 2AT

UK

Tel: +44 20 7377 7472

Fax: +44 20 7377 7300

E-mail: g.giovannoni@qmul.ac.uk
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