Epidemiological studies suggest an association between infection with Epstein-Barr virus (EBV) and risk of multiple sclerosis (MS).
Objective
To determine whether elevation in
serum antibody titers to EBV viral capsid antigen (VCA), nuclear antigens
(EBNA, EBNA-1, and EBNA-2), and diffuse and restricted early antigen (EA-D
and EA-R) as well as to cytomegalovirus (CMV) precede the occurrence of
MS.
Design, Setting, and Subjects
Prospective, nested case-control
study. Of 62 439 women participating in the Nurses' Health Study (aged
30-55 years in 1976) and Nurses' Health Study II (aged 25-42 years in 1989)
who gave blood samples in 1989-1990 and 1996-1999, respectively, and were
followed up through 1999, 144 women with definite or probable MS and 288
healthy age-matched controls were included in the analysis.
Main Outcome Measure
Serum antibody titers to the specific
EBV and CMV antigens, compared between cases and controls.
Results
We documented 18 cases of MS with
blood collected before disease onset. Compared with their matched controls,
these women had higher serum geometric mean titers (GMTs) of antibodies
to EBV but not CMV. Elevations were significant for antibodies to EBNA-1
(GMT, 515 vs 203; P = .03), EBNA-2 (GMT, 91 vs 40; P = .01), and EA-D (15.9
vs 5.9; P = .04). The strongest association was found for antibodies to
EBNA-2; a 4-fold difference in titers was associated with a relative risk
(RR) of MS of 3.9 (95% confidence interval [CI], 1.1-13.7). The corresponding
RRs were 1.6 (95% CI, 0.7-3.7) for VCA, 2.5 (95% CI, 1.0-6.3) for EBNA,
1.8 (95% CI, 1.0-3.1) for EA-D, and 1.0 (95% CI, 0.6-1.7) for CMV. Significant
but generally weaker elevations in anti-EBV antibodies were also found
in analyses of 126 cases of MS with blood collected after disease onset
and their matched controls.
Conclusions
Our results support a role of EBV
in the etiology of MS.
Full Text
The etiology of multiple sclerosis
(MS) is largely unknown, but evidence supports an autoimmune process triggered
by infection or other environmental factors.(1-3) Epstein-Barr virus (EBV),
a herpesvirus, infects more than 90% of the human population, establishing
a persistent and highly immunogenic infection of B lymphocytes.(4) Antigen-specific
cytotoxic T cells are massively expanded in response to primary infection
and persist at high levels for several years.(5, 6) Autoimmunity could
result if some of these cells carry T-cell receptors that recognize self-peptides.
Epstein-Barr virus has been related to nasopharyngeal carcinoma, Burkitt
lymphoma, and Hodgkin disease,(7) and a relation to autoimmune diseases
has been proposed(8) but remains unproven.
Infection with EBV is usually asymptomatic
in childhood but frequently causes infectious mononucleosis in adolescents
and adults. The similarity in the epidemiology of MS and infectious mononucleosis
led to the proposition that MS could be caused by infection with EBV during
or after adolescence in genetically susceptible individuals.(9) This hypothesis
is supported by observations suggesting an increased risk of MS following
infectious mononucleosis,(10-15) the rarity of MS among individuals without
serum anti-EBV antibodies,(16, 17) and the higher serum titers of anti-EBV
antibodies in MS patients than in controls. Elevated titers have been reported
against both the EBV viral capsid antigen (VCA), which is expressed in
the viral replicative cycle, and the Epstein-Barr nuclear antigen (EBNA),
which is expressed in latently infected B lymphocytes.(18, 19) These elevations
in antibody titers are consistent with an association between infection
with EBV and risk of MS16 but could also simply reflect the immune dysregulation
in MS.(20) To clarify the temporal relation between antibody titers and
MS, antibody status should be assessed before clinical evidence of disease
exists.
To address the possible role of EBV
in the etiology of MS, we prospectively examined the association between
serum anti-EBV antibody titers and risk of developing MS in 2 large cohorts
of US women.
METHODS
Study Population
The study base for this investigation
comprised the subsets of participants who provided a blood sample in 2
large ongoing US cohorts, the Nurses' Health Study (NHS) and the Nurses'
Health Study II (NHS II). The NHS was established in 1976, when 121,700
female registered nurses aged 30 to 55 years from 11 states responded to
a mailed questionnaire about disease history and lifestyle items. The NHS
II was established in 1989, when 116,671 female registered nurses aged
25 to 42 years from 14 states responded to a similar questionnaire. All
participants in both cohorts were invited to provide a blood sample to
investigate several biomarkers of chronic disease. Blood was collected
from 32,826 participants in the NHS between 1989 and 1990 and from 29,613
women in the NHS II between 1996 and 1999. Follow-up questionnaires are
mailed to the participants of both studies every 2 years to update information
on potential risk factors for chronic diseases and to ascertain whether
major medical events have occurred.
Case Ascertainment and Control
Selection
Details on the documentation of MS
cases in these cohorts have been reported previously.(21) Briefly, participants
who reported a new diagnosis of MS in 1 of the follow-up questionnaires
were asked permission to contact their neurologists and review their medical
records. After obtaining permission, we sent the neurologists a questionnaire
addressing the certainty of the diagnosis (definite, probable, possible,
or not MS), the date of onset of neurological symptoms related to MS, other
aspects of the clinical history, and laboratory test results. Since 93%
of all definite and probable diagnoses appeared to conform to the Poser
criteria(22) for diagnosis of MS when applied to the clinical and laboratory
data provided in the questionnaire,(23) we classified as cases women who
had a diagnosis of definite or probable MS according to their neurologists.
The date of onset of the disease
was determined by asking both women with MS and their neurologists for
the date of first neurological symptoms. When the 2 dates were discordant,
the earliest date was considered valid. A total of 149 incident cases of
definite and probable MS were documented between baseline and December
1999 among women with available blood samples. For each case, we randomly
selected 2 women without MS, matched by year of birth and study cohort.
One case was excluded because no serological results were obtained and
4 because of missing dates of onset; thus, 144 women with MS and 288 controls
were included in the analysis.
In 18 of the women with MS, the onset
of neurological symptoms occurred after blood collection (median, 1.9 years;
range, 2 months–6.5 years). The diagnoses of MS in these women were all
made by neurologists and supported by magnetic resonance imaging; the diagnosis
was reported as definite MS in 12 women and probable MS in 6. Because of
the age distribution of the cohorts at the time of blood collection, most
of these women had a late onset of MS (median age, 52 years; range, 39-66
years).
Blood Collection and Laboratory
Analyses
Blood was obtained using collection
kits that were returned to our laboratory via overnight courier. Approximately
97% of the samples arrived within 26 hours of being drawn. On arrival in
our laboratory, the blood samples were centrifuged and the blood components
aliquotted into cryotubes and stored in liquid nitrogen freezers until
laboratory analysis. Serum samples from MS cases and controls were sent
to the laboratory (Virolab Inc, Berkeley, Calif) in triplets containing
the case and the 2 matched controls in random order. The laboratory was
blind to case-control status and unidentified triplets were included for
quality control.
Immunoglobulin G antibodies to EBV
VCA and anti–early antigen complex (diffuse [EA-D] and restricted [EA-R])
were determined by indirect immunofluorescence according to methods of
Henle et al.(24) Immunoglobulin G antibodies against the EBNA family and
2 of its individual components, EBNA-1 and EBNA-2, were determined using
anticomplement immunofluorescence, as previously described.(25) Antibodies
to the VCA and the EA-D components emerge during the late incubation period
or in the course of the acute phase of infectious mononucleosis, whereas
antibodies to EBNA and EA-R arise only weeks or months after onset of the
disease.(24) Antibodies to EBNA-2 arise before those to EBNA-1 and usually
decline over several months, whereas antibodies to EBNA-1 persist indefinitely.
Persistently high anti–EBNA-2 titers and low anti–EBNA-1 titers have been
associated with chronic infectious mononucleosis.(24) Antibody titers against
cytomegalovirus (CMV) were also determined to assess the specificity of
any association that may be found between MS and EBV serology. The methods
used for CMV serology have been previously described.(26) The intra-assay
coefficients of variation were VCA, 8.6%; EBNA, 13.9%; EBNA-1, 9.8%; EBNA-2,
9.1%; EA-D, 13.0%; EA-R, 9.2%; and CMV, 7.3%.
Assessment of Covariates
We considered in the analyses age,
latitude of birthplace (northern, middle, or southern), ancestry (Scandinavian,
Southern European, other white, or nonwhite), and smoking (never, past,
or current), data on which were collected from all participants as part
of the cohort follow-up. The associations of birthplace latitude, ancestry,
and smoking history with risk of MS in these cohorts have been previously
reported.(23, 27)
Statistical Analyses
Antibody geometric mean titers (GMTs)
were compared between cases and controls using generalized linear models
that take into account the matched design of the study and the use of clustered
measurements.(28) We used conditional logistic regression to estimate the
relative risk (RR) of MS associated with a 4-fold difference in specific
antibody titers. In these regression models, we included the base 2 logarithm
of the reciprocal of the dilution of the antibody titers as a continuous
variable. On this scale, the regression coefficient estimates the logarithm
of the RR associated with a 2-fold difference in titers; we doubled and
exponentiated this value to estimate the RR associated with a 4-fold difference.
The main analyses included only women
with MS who provided blood samples before onset of the disease compared
with their matched controls or with all controls combined. However we also
presented results for women with blood collected after onset of MS to assess
whether there is any effect of the disease on antibody titers. Finally,
we classified women according to whether they had evidence of past infection
with EBV or CMV, to estimate the risk of MS associated with prior infection.
For this analysis, women were considered EBV-positive if the antibody titer
to VCA was at least 1:20 or that to EBNA was at least 1:5; women were considered
CMV-positive if the antibody titer to CMV was at least 1:10. The association
between seropositivity and MS was estimated using the Mantel-Haenszel method
for matched data(29) or conditional logistic regression for multivariate
analyses. All P values are 2-tailed. The statistical software SAS (SAS
Institute Inc, Cary, NC) was used for all analyses.
RESULTS
The GMTs of serum antibodies to EBV
were consistently higher among women with MS compared with their matched
controls. In analyses including only the 18 cases with blood collected
before disease onset and their matched controls, these differences were
significant for antibodies to EBNA-1 (GMT for cases, 515 vs for controls,
203; P = .03), EBNA-2 (GMT, 91 vs 40; P = .01), and EA-D (GMT, 15.9 vs
5.9; P = .04). The GMTs to VCA and EBNA were also higher among cases than
controls (GMT for VCA, 1613 vs 1036; GMT for EBNA, 667 vs 333), but these
differences only achieved statistical significance in analyses of the 126
cases with blood collected after onset of MS. There was no difference between
cases and controls in the GMTs of antibodies to EA-R or CMV. The case-control
differences in antibody titers to EBV were of similar magnitude whether
the blood was collected before or after disease onset, although the larger
number of cases with blood collected after disease onset conferred a stronger
degree of statistical significance. A direct comparison of these associations
stratified by time of blood collection is shown in Table 1.
In matched analyses, the RRs of MS
associated with a 4-fold difference in antibody titers before onset of
MS ranged from 1.6 (95% confidence interval [CI], 0.7-3.7) for antibodies
to VCA to 3.9 (95% CI, 1.1-13.7) for antibodies to EBNA-2. Significant
positive associations with antibodies to EBNA-1, EBNA-2, and EA-D were
also observed in age-adjusted analyses including all controls (Table 1).
A plot of this association for EBNA-1 is shown in Figure 1; results for
EBNA-2 were similar. The corresponding RR for antibody titers collected
after onset of MS were smaller, except for VCA (1.7 vs 1.6). The RRs associated
with antibodies to CMV were close to 1.0 for all comparisons.
To address the possibility that the
increased anti-EBV antibody titers among the cases with blood collected
before onset were due to preclinical disease, we calculated the mean antibody
titers among 12 women with MS who provided a blood sample at least 1 year
before the onset of MS and plotted the antibody titers of women with MS
according to the temporal relation between blood collection and disease
onset (Figure 2). Serum antibody titers to EBV antigens were already elevated
among women who gave blood at least 1 year before onset of MS and were
unrelated to the time of blood collection, except for a decline in anti–EA-D
after MS onset (Figure 2).
The 18 women with MS who gave blood
before onset of disease were all positive for both VCA and EBNA, whereas
2 of their matched controls were negative for VCA and 1 was negative for
EBNA (P = .30 and P = .50, respectively). Using the combined sample of
MS subjects, only 1 of 144 women with MS had negative anti-VCA titers vs
18 of 287 controls (OR, 9.0; 95% CI, 1.8-45.2; P = .008) (Table 2). Results
for anti-EBNA titers were similar. In contrast, the proportions of women
with serum anti-CMV antibodies were similar in the case and control groups
(Table 2). These associations were not materially changed in multivariate
analyses.
COMMENT
In this prospective study, we found
that significant elevations in serum anti-EBV antibody titers were present
before onset of MS. Elevations were observed for antibodies against antigens
expressed during replication of the virus (EA-D) as well as in latency
(EBNAs). The strongest association was found with antibodies to EBNA-2;
a 4-fold difference in titers of antibodies to this antigen was associated
with a 4-fold increase in risk of MS.
The nested case-control design of
our investigation makes a biased selection of controls unlikely. Some degradation
of the IgG antibodies or desiccation of blood samples may have occurred
during shipping and storage, but these are probably modest because most
samples reached our serum bank within 26 hours and were kept in closely
monitored liquid nitrogen freezers. Most importantly, blood samples from
cases and controls were handled in the same manner throughout the study,
and triplets composed of a case and 2 matched control samples were assayed
in the same run and in random order by technicians who were blind to disease
status. Under these conditions, any laboratory error should be unrelated
to disease status and would attenuate the difference in antibody titers
between cases and controls.
A potential limitation of our study
is the relatively short period between blood collection and onset of MS
(median, 1.9 years). The onset of the autoimmune process that leads to
demyelination may precede the recognition of neurological symptoms by several
months and so may the immune dysregulation that accompanies the disease.
Thus, the elevation in anti-EBV titers could be an early manifestation
of the preclinical phase of the disease. The fact that antibody titers
in serum samples collected 1 or more years before onset of symptoms were
similar to those in serum samples collected in women with fully clinical
disease provides some evidence against this explanation, but confirmation
of these results in a larger number of serum samples collected years before
MS onset will be important. The late age of onset of MS among women in
our study is atypical, but it is consistent with the age distribution of
our cohorts at the time of blood collection.
A causal association between EBV
and MS was originally suggested 20 years ago,(9) primarily because of the
similarities in the epidemiology of infectious mononucleosis and MS and
of the increased risk of MS among individuals with a history of infectious
mononucleosis.(10-15) If EBV had a critical role in the etiology of MS,
then it would be expected that few or no MS cases would occur among individuals
who are not infected with EBV. This expectation is indirectly supported
by the results of several case-control studies. In a meta-analysis of published
investigations, we estimated that the odds of disease are more than 10
times higher among EBV-positive than EBV-negative persons.16 Since primary
EBV infection is rare among patients with MS,(17, 30) this finding suggest
that either EBV itself increases the risk of MS or that they both are related
to some common factor. A microorganism transmitted in a manner similar
to EBV could be involved, but the EBV-MS association in so strong that
it is unlikely to be fully explained by confounding. A common genetic predisposition
may be more plausible, because of the known association between HLA class
II polymorphism and risk of MS and the recent observation that the HLA
class II protein acts as a cofactor in EBV infection of B lymphocytes.(31)
A study of long-term EBV-negative adults, however, revealed a distribution
of the DR2 alleles commonly associated with MS similar to that of EBV-seropositive
adults,(32) and higher titers of anti-EBV antibodies have been reported
in individuals with MS than in HLA-DR2–matched controls.(33) These observations
suggest that genes are unlikely to explain the strong associations reported
herein. Further evidence that EBV has an active role in MS recently has
been provided by the observation that active viral replication occurs more
commonly in MS patients with exacerbations than in patients with stable
disease.(17)
The elevation of anti-EBV antibody
titers before onset of MS that we found in this study provides further
support for an important role of EBV in the etiopathogenesis of MS. The
observation of elevated anti-EBV serum antibody titers before diagnosis
has contributed to establishment of the causal link between EBV and Burkitt
lymphoma(34) and has been reported in other EBV-related diseases, including
nasopharyngeal carcinoma(35) and Hodgkin disease.(36) The pattern of antibody
response that we observed among women with MS is different from that observed
in immunocompromised hosts or in chronic infectious mononucleosis, which
is characterized by elevated anti-EBNA-2 and reduced anti-EBNA-1 titers.(25)
The simultaneous elevation of titers to VCA, EBNA-1, EBNA-2, and EA-D rather
suggests a more severe or more recent primary infection among women who
developed MS than in women who remained healthy.
Evidence on potential mechanisms
by which EBV could be causally related to MS is limited. The failure to
demonstrate EBV in MS plaques by in situ hybridization(37) or polymerase
chain reaction(38) suggests that direct central nervous system infection
is not involved. Rather, the T-cell response to EBV infection could include
clones that are potentially cross-reactive with self-antigens. In acute
infectious mononucleosis, there is a massive expansion of activated circulating
T cells that are specific for EBV latent and lytic antigens.(6) A high
proportion of these cells is committed to a single viral epitope,5 and
populations of cells that are specific for both latent and lytic antigens
are still present at frequencies of more than 1% up to 3 years after recovery.6
Other potential mechanisms proposed to explain a potential role of EBV
in MS include cross-reactions of anti-EBNA antibodies with epitopes of
a neuroglial antigen(39) and the EBV-induced expression in B lymphocytes
of a-b-crystallin,
a small stress protein that has been reported to be an immunodominant myelin
antigen in MS patients.(40) It has recently been reported that variant
A of human herpesvirus 6 is more commonly reported in MS patients than
in controls.(41-43) Unlike the more common human herpesvirus 6 variant
B, variant A may infect EBV-positive B-cell lines and activate the latent
EBV genome.(44, 45) These observations suggest that interactions between
herpesviruses may have a role in the pathogenesis of MS.
In conclusion, our results, in conjunction
with those of case-control studies, offer evidence that EBV infection may
increase the risk of MS. Because few individuals infected with EBV develop
MS, other cofactors are required. These may include genetic predisposition
and, perhaps, age at primary infection or infection with other microbes.
Final proof of causality could come from the demonstration that a suitable
vaccine prevents MS. Available antiviral drugs have little effect on the
number of infected B lymphocytes and may thus be ineffective in MS treatment.(7)
However, a better understanding of the mechanism that relate EBV to MS
may also lead to novel therapeutic approaches.
Author/Article Information
Author Affiliations: Departments
of Nutrition (Dr Ascherio and Ms Munger), Epidemiology (Drs Ascherio, Spiegelman,
Hernán, Hankinson, and Hunter), and Biostatistics (Dr Spiegelman),
Harvard School of Public Health, Center for Neurological Diseases–Multiple
Sclerosis Unit (Dr Olek), and Channing Laboratory, Department of Medicine
(Drs Hankinson and Hunter), Harvard Medical School and Brigham and Women's
Hospital, Boston, Mass; and Virolab Inc (Dr Lennette), Berkeley, Calif.
Corresponding Author and Reprints:
Alberto Ascherio, MD, DrPH, Harvard School of Public Health, Nutrition
Department, 665 Huntington Ave, Boston, MA 02115.
Author Contributions:
Study concept and design:
Ascherio.
Acquisition of data:
Ascherio, Lennette, Hernán,
Olek, Hankinson, Hunter.
Analysis and interpretation of
data:
Ascherio, Munger, Spiegelman, Olek,
Hankinson, Hunter.
Drafting of the Manuscript:
Ascherio.
Critical revision of the manuscript
for important intellectual content:
Ascherio, Munger, Lennette, Spiegelman,
Hernán, Olek, Hankinson, Hunter.
Statistical expertise:
Ascherio, Spiegelman, Hunter.
Obtained funding:
Ascherio, Hankinson.
Administrative, technical, or
material support:
Ascherio, Munger, Lennette, Hernán,
Hankinson, Hunter.
Study supervision:
Ascherio, Olek.
Funding/Support:
This study was supported by grant
NS35624 from the National Institute of Neurological Disorders and Stroke
and grants CA49449 and CA67262 from the National Cancer Institute.
Acknowledgment:
We are indebted to the participants
of the Nurses' Health Study and the Nurses' Health Study II and to Frank
Speizer, MD, Graham Colditz, MD, DrPH, and Walter Willett, MD, DrPH, principal
investigators of these 2 studies. We thank Karen Corsano, David Coppola,
Lisa Li, Mary Louie, Lisa Dunn, Gary Chase, Barbara Egan, Lori Ward, Jeanne
Sparrow, Rachel Meyer, Todd Reid, and Erin Boyd for their technical assistance
and Nancy Mueller, ScD, for her expert advice.
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