Download Epstein–Barr virus early antigen diffuse (EBV-EA/D)

280
Scand J Rheumatol 2012;41:280–289
Epstein–Barr virus early antigen diffuse (EBV-EA/D)-directed immunoglobulin A
antibodies in systemic lupus erythematosus patients
AH Draborg1, JM Jørgensen1*, H Müller1*, CT Nielsen1, S Jacobsen2, LV Iversen3, E Theander4, LP Nielsen5, G Houen1,
K Duus1
Department of Clinical Biochemistry and Immunology, Statens Serum Institut, 2Department of Rheumatology, Copenhagen University
Hospital, Rigshospitalet, 3Department of Dermatology, Copenhagen University Hospital, Bispebjerg, Copenhagen, Denmark,
4
Department of Rheumatology, Lund University, Skåne University Hospital, Malmö, Sweden, and 5Department of Virology, Statens
Serum Institut, Copenhagen, Denmark
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1
Objective: We sought to determine whether the serological response towards lytic cycle antigens of Epstein–Barr virus
(EBV) is altered in systemic lupus erythematosus (SLE) patients.
Method: We used enzyme-linked immunosorbent assay (ELISA) to investigate the prevalence of EBV early antigen
diffuse (EBV-EA/D) antibodies in sera from 60 patients with SLE, 40 with scleroderma (SSc), 20 with primary Sjögren’s
syndrome (pSS), 20 with rheumatoid arthritis (RA), 20 healthy controls, and also subjects with various circulating
autoantibodies. Samples from patients were obtained from clinics specialized within the diseases in Denmark and Sweden
and samples from healthy controls were obtained from volunteers.
Results: A significant elevated titre of immunoglobulin (Ig)A, IgG, and IgM EBV-EA/D antibodies was found in SLE
patients compared to healthy controls, a finding not explained by immunosuppressive treatment or disease activity. The
largest difference was observed for IgA EBV-EA/D antibodies (p ¼ 0.0013) with a seropositive rate of 58% in SLE
patients and 0% in healthy controls. RA and SSc patients and individuals seropositive for anti-Scl-70 were additionally
found to have elevated titres of IgA EBV-EA/D antibodies (40%, p ¼ 0.014; 60%, p ¼ 0.015; and 38.5%, p ¼ 0.045,
respectively). However, the titres were generally lower than in SLE patients.
Conclusion: Our findings support an association between EBV and SLE. The elevated titre of EBV-EA/D-directed IgA
antibodies found in SLE patients could suggest reactivation of EBV in epithelial cells or reinfection of epithelial cells after
reactivation in B cells, indicating lack of control of the latent infection.
Systemic lupus erythematosus (SLE) is an autoimmune
connective tissue disease with an incidence of 6–35 new
cases per 10 0000 per year. SLE typically presents in
women (90%) of reproductive age (1–3). It is characterized
by the production of autoantibodies directed against conserved cellular components, including autoantibodies
directed against double-stranded DNA (dsDNA), histones,
Ro/SSA, La/SSB, Sm, and ribonucleoproteins (3–5).
A connection between SLE and Epstein–Barr virus
(EBV) infection has been suggested. Nearly all SLE
patients are infected with EBV (99.5%); however, a
large proportion of healthy adults are also infected
(94.5%) (6). Studies have shown an increased number
of latently EBV-infected cells (7) and an abnormally high
Karen Duus, Department of Clinical Biochemistry and Immunology,
Statens Serum Institut, Ørestads Boulevard 5, DK-2300 Copenhagen,
Denmark.
E-mail: [email protected]
*Both authors contributed equally to this work.
Accepted 8 February 2012
viral load in the peripheral blood mononuclear cells of
SLE patients (8–10). An impaired EBV-specific T-cell
response is also observed in SLE patients (8, 11, 12).
EBV infection during childhood is asymptomatic but
infection in adolescence causes infectious mononucleosis
in 30–70% of cases (13, 14). EBV is transmitted by saliva
and replicates initially in nasopharyngeal epithelial cells
and B cells. After the primary infection, EBV persists in a
latent form within immortalized memory B cells and
expresses a limited number of genes including EB nuclear
antigen 1 (EBNA-1) (15–17).
Occasionally, EBV reactivates and switches to the lytic
cycle activating viral and cellular promoters, resulting in
viral gene expression and viral genome replication (18).
This gives rise to the release of virus that can infect other
B cells and epithelial cells (17). The virus is thus a constant antigenic challenge to the host.
The EBV BMRF-1 gene encoding EBV early antigen
diffuse (EBV-EA/D) (also known as the DNA polymerase accessory protein) is required in the initiation of lytic
replication and is one of the first genes transcribed when
switching to the lytic cycle (19–22). EBV-EA/D is
© 2012 Taylor & Francis on license from Scandinavian Rheumatology Research Foundation
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DOI: 10.3109/03009742.2012.665944
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EBV-EA/D IgA antibodies in SLE patients
localized in both the nucleus and the cytoplasm of
infected cells (23, 24) and is a dsDNA-binding protein
(25–29) acting as a cofactor of the viral DNA polymerase
(26–28).
The humoral immune response is initiated during EBV
infection and individuals infected with EBV have distinct
serological profiles. In early stages of the infection, antibodies towards EBV-EA/D and EBV-viral capsid antigen
(VCA) are produced, whereas EBNA-1 antibodies
develop later. EBV-VCA IgM antibodies are used diagnostically for recent active infection (30). Antibodies of
the immunoglobulin G (IgG) subclass towards EBVVCA and EBNA-1 persist throughout life (31, 32) and
antibodies towards EBV-EA/D are known as a strong
indication of lytic replication (32, 33).
Previous studies on EBV-directed antibodies in sera
from SLE patients showed no difference in titres of IgG
and IgM antibodies towards EBNA-1 or EBV-VCA
(34–37). However, an elevated amount of both EBNA-1
and EBV-VCA IgA antibodies has been observed in SLE
patients (38–40) and the titre of EBV-EA IgG antibodies
has been shown to be elevated in SLE patients (35–37, 41,
42). One study demonstrated, by immunofluorescence,
that five out of 34 SLE patients were seropositive for
EBV-EA IgA antibodies (43).
In this study, these results were substantiated and
extended by analysing sera from SLE patients for the
presence of EBV-EA/D IgA antibodies by enzyme-linked
immunosorbent assay (ELISA) and showed a seropositive rate of 58% compared to 0% in healthy controls.
Method
Patients and controls
Serum samples were obtained from 60 unrelated Danish
SLE patients attending the Department of Rheumatology,
Rigshospitalet, Copenhagen University Hospital,
Denmark. Serum samples from 20 apparently healthy,
ANA and anti-dsDNA negative, controls were obtained
from volunteers at Statens Serum Institute, Copenhagen,
Denmark. 40 patients with SSc were recruited from the
Department of Dermatology, Bispebjerg, Copenhagen
University Hospital, Denmark and serum samples from 20
RA patients attending the Department of Rheumatology,
Rigshospitalet, Copenhagen University Hospital, Denmark
were also included. Sera from 20 randomly chosen outpatient clinic pSS patients were recruited from Department
of Rheumatology, Lund University, Skåne University
Hospital, Malmö, Sweden. 75% of the pSS patients were
positive for SSA/B, those lacking these autoantibodies
had lower lip salivary gland biopsy with focal sialadenitis
and a focus score of one or more. All patients included
fulfilled internationally accepted classification criteria
for the autoimmune diseases investigated (44–47).
Furthermore, Serum samples were also obtained from
17 individuals seropositive for dsDNA antibodies, 13
individuals seropositive for Scl-70 antibodies, 20 indivi-
281
duals seropositive for SSA/B antibodies and 20 individuals seropositive for CCP antibodies. Access to the latter
samples was gained from the routine diagnostic laboratory at the Department of Clinical Biochemistry and
Immunology at Statens Serum Institut in Copenhagen,
Denmark. Informed consent for the studies was obtained
from all patients in accordance with the protocol as approved
by the Scientific-Ethical Committee of the Capital Region of
Denmark.
ELISA
All incubations and washings were performed at room
temperature on a shaking table using 100 μl in each well
for incubations and 250 μl in each well for washing and
blocking. TTN (0.025 M Tris, 0.5% Tween 20, 0.15 M
NaCl, pH 7.4) was used for blocking, incubation and
washing. Recombinant EBV-EA/D (Prospec, NessZiona, Israel; Produced in E. Coli, C terminus regions
amino acids 306-390, >95% pure) was coated at 1 μg/ml
onto the NUNC MaxiSorp microtitre plates (Thermo
Fisher Scientific, Denmark) using carbonate buffer (50
mM sodium carbonate, pH 9.6) as the coating buffer.
After coating overnight at 4 C, plates were washed three
times, for 1 minute, followed by blocking for 30 minutes.
Subsequently, sera diluted 1:20 for IgA and IgM detection
and 1:100 for IgG detection were incubated in the wells for
1 hour. Each serum sample was tested in duplicate in both
coated and non-coated wells. After another three washes
the plates were incubated for 1 hour with alkaline phosphatase (AP)-conjugated goat anti-human IgG/IgA/IgM
(1:2000) (Sigma-Aldrich, St. Louis, Missouri, USA).
Following another three washes, immunoglobulins were
quantified using p-nitrophenylphosphate (p-NPP) (SigmaAldrich, St. Louis, Missouri, USA) (1 mg/ml) diluted in
AP substrate buffer (1M diethanolamine, 0.5 mM MgCl2,
pH 9.8). The absorbance was read at 405 nm, with background subtraction at 650 nm, on a Versamax microplate
reader (Molecular Devices, Sunnyvale, CA, USA). For
each serum sample the blank values from the non-coated
wells were subtracted. A standard curve was created for
each assay using a high titre serum and all absorbance
values were normalized relative to this standard. A serum
sample was considered seropositive for EBV-EA/D antibodies if the antibody binding was higher than the cut-off
value (0.2, 0.035 and 0.1, regarding IgG-, IgA- and IgMEBV-EA/D antibodies, respectively). Cut-offs was estimated by the use of ROC-curves.
Epitope mapping was performed using 20 amino acids
long peptides with 10 amino acids overlaps covering the
C-terminal region of EBV-EA/D (amino acids 306-390)
equalling the recombinant EBV-EA/D coated in ELISA
wells for detection of EBV-EA/D directed antibodies.
Peptides were coated at 10 μg/ml onto a NUNC
MaxiSorp microtitre plate. Subsequently, sera were incubated in wells and immunoglobulins were quantified as
described above.
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AH Draborg et al
Antibodies against EBNA-1 and EBV-VCA were
detected using ELISA test kits (Demeditec Diagnostics,
Kiel-Wellsee, Germany) according to the manufacturer’s
instructions.
Immunofluorescence
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IgG antibodies against EBV-EA were determined by immunofluorescence using EBV-EA IFA Kit (Diagnostic
Automation/Cortez Diagnostics Inc, Calabasas, CA, USA)
according to the manufacturer’s instructions. Approximately
5-15% of the Raji cells on the 10-well substrate slides
express EBV-EA.
Statistical analysis
Analysis of data was carried out using GraphPad Prism
software 5 (GraphPad Software, Inc, La Jolla, CA, USA).
Comparison of antibody binding between patients and
controls was performed using the nonparametric unpaired
two-tailed t test and considered significant at p-values less
than 0.05. Data are presented with mean SEM (standard
error of the mean).
Table 2. Clinical characteristics of SSc, RA, and pSS patients.
SSc patients
No. of individuals
Age (years), mean (range)
Females (%)
ANA positive (%)
Limited/diffuse SSc (%)
anti-Scl-70 positive (%)
Skin score, mean (range)
RA patients
No. of individuals
Age (years), mean (range)
Females (%)
Time since diagnosis (years), mean (range)
C-reactive protein (mg/L), mean (range)
anti-CCP positive (%)
Rheumafactor positive (%)
IgA
IgM
Total SHARP score, mean (range)
pSS patients
No. of individuals
Average age (years), mean (range)
Females (%)
ANA positive (%)
anti-SSA/B positive (%)
With low C3 (%)
With low C4 (%)
40
57.3 (33–76)
85
95
70/30
77.5
10.0 (3–30)
20
52.4 (27–72)
70
15.7 (1–40)
6.7 (1–28)
70
80
50
102.2 (2–321)
20
53.8 (30–78)
90
95
75
20
15
Results
Clinical characteristics of SLE patients and controls
The average age was 39.4 years for the SLE patients with the
youngest being 21 and the oldest 76 years. For the healthy
controls the average age was 32.1 years with a range of 25 to
63 years. The proportion of females was 92% among the
patients and 60% among the healthy controls. The average
Systemic Lupus Erythematosus Disease Activity Index
(SLEDAI) score for the SLE patients was 5 ranging from
0 to 21 and the average time since diagnosis was 10.8 years
ranging from 0 to 37 years. Table 1 shows more details of
SLE patients and Table 2 and 3 shows clinical characteristics of disease control groups and healthy controls.
Table 1. Clinical characteristics of SLE patients.
No. of individuals
Age (years), mean (range)
Females (%)
Time since diagnosis (years), mean (range)
SLEDAI score, mean (range)
ANA positive (%)
anti-dsDNA positive (%)
On immunosuppressive medication (%)
Average amount of immunoglobulins (g/L)
IgG
IgA
IgM
C-reactive protein (mg/L), mean (range)
With low C3 (%)
With low C4 (%)
60
39.4 (21–76)
92
10.8 (0–37)
5 (0–21)
80
46.7
61.7
12.4
2.56
1.19
4.3 (0–21)
51.7
66.7
SLEDAI, Systemic Lupus Erythematosus Disease Activity Index;
ANA, antinuclear antibodies.
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High prevalence of IgA, IgG and IgM antibodies against
EBV-EA/D in sera of SLE patients
Sera from the 60 SLE patients and 20 healthy controls
were examined by the use of ELISA for IgA, IgG and IgM
antibodies against EBV-EA/D. As illustrated in
Figure 1A there was a significantly elevated antibody
binding of IgA, IgG and IgM antibodies to EBV-EA/D
in SLE patients compared to the healthy controls (with
p-values of 0.0013, <0.0001, and 0.012, respectively).
The use of ELISA showing reactivity of antibodies directed against EBV-EA/D was confirmed by immunofluorescence on serum samples from nine of the SLE patients
and nine of the healthy controls. Analyses of serum from
the SLE patients showed a positive signal illustrated by
the whole cell/cytoplasmic fluorescence pattern characteristic of EBV-EA expression, whereas the healthy controls revealed no fluorescent cells. Figure 1B shows a
representative image of the outcome of analyses on Raji
cells expressing EBV-EA of two of the serum samples
from SLE patients and healthy controls, respectively.
To compile the results from Figure 1A, the EBV-EA/D
reacting antibody isotypes were grouped for each individual. A cut-off value of the antibody binding between
positive and negative serum samples was used and sera
were assigned one point value for each of the three antibody isotypes reacting with EBV-EA/D. The value 1
indicates a serum positive for EBV-EA/D antibodies of
either the IgG, IgM or IgA isotype or the value 3 indicates
a serum positive for all three antibody classes (Figure 1C).
A total of 65% of the SLE patients were positive for two
EBV-EA/D IgA antibodies in SLE patients
283
Table 3. Characteristics of heatlhy controls and groups of individuals seropositive for various autoantibodies.
Control group
No. of individuals
Age (years), mean (range)
% females
Anti-dsDNA positive
Anti-Scl-70 positive
Anti-SSA/B positive
17
13
20
41.6 (27–81)
47.2 (24–65)
58.3 (25–85)
95
62
85
Anti-CCP positive
Healthy controls
20
20
54.8 (24–78)
32.1 (25–63)
85
60
A
(i)
(iii)
1.0
0.8
0.6
0.4
0.2
0.8
EBV-EA/D lgM antibody binding
EBV-EA/D lgA antibody binding
EBV-EA/D lgG antibody binding
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70.5 (47–200)
All positive
Anti-Ro52 (n ¼ 19): 78.5 (10–100)
Anti-Ro60 (n ¼ 15): 75.3 (12–100)
Anti-La (n ¼ 3): 65.3 (12–100)
708.5 (28.2–2401.7)
–
(ii)
1.0
0.8
0.6
0.4
0.2
0.0
0.0
Healthy
controls
SLE
patients
0.6
0.4
0.2
0.0
SLE
patients
SLE
patients
Healthy
controls
B
Healthy
controls
C
(i)
(ii)
100
SLE patients
Healthy controls
80
50 µm
Titre of autoantibodies (U/mL), mean (range)
10 µm
(iii)
50 µm
60
10 µm
(iv)
%
40
20
0
0
50 µm
10 µm
50 µm
10 µm
1
2
3
0
1
No. of antibody isotypes
2
3
Figure 1. The titre of EBV-EA/D antibodies in serum from SLE patients is elevated compared to healthy controls. (A) EBV-EA/D (i) IgG, (ii) IgA, and
(iii) IgM antibody binding of SLE patients (n ¼ 60) and healthy controls (n ¼ 20) analysed on EBV-EA/D-coated microtitre plates using AP-conjugated
anti-human IgG, IgA, or IgM and p-NPP for detection of EBV-EA/D-binding antibodies. Each serum sample was applied in duplicate on both coated and
non-coated wells and the blank values were subtracted. The absorbance values were normalized to the standard. Middle horizontal bar indicates mean SEM. p-values for comparison of SLE patients and healthy controls are < 0.0001, 0.0013, and 0.012, for IgG, IgA, and IgM EBV-EA/D antibodies,
respectively. (B) EBV-EA IgG antibodies in sera from ANA-negative SLE patients ((i), (ii)) and healthy controls ((iii), (iv)) analysed by confocal laser
scanning microscopy on EBV-EA expressing Raji cells. (C) Percentage of SLE patients (n ¼ 60) and healthy controls (n ¼ 20) seropositive for none, one,
two, or three EBV-EA/D antibody isotypes (IgG, IgA, and IgM). A serum sample was considered seropositive if the antibody binding was higher than
cut-off (0.2, 0.035, and 0.1 for IgG, IgA, and IgM EBV-EA/D antibodies, respectively).
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284
(i)
(ii)
EBV-EA/D IgA antibody binding
EBV-EA/D IgG antibody binding
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
no IS
IS
(i)
0.6
0.4
0.2
0.0
0.0
no IS
IS
no IS
(ii)
1.0
EBV-EA/D IgA antibody binding
EBV-EA/D IgG antibody binding
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0.0
EBV-EA/D IgM antibody binding
1.0
1.0
B
(iii)
0.8
0.8
0.6
0.4
0.2
0.0
(iii)
0.8
0.6
0.4
0.2
0.0
anti-dsDNA
positive
anti-dsDNA
negative
IS
0.8
1.0
EBV-EA/D IgM antibody binding
A
AH Draborg et al
anti-dsDNA
positive
anti-dsDNA
negative
0.6
0.4
0.2
0.0
anti-dsDNA
positive
anti-dsDNA
negative
Figure 2. No observed association between EBV-EA/D antibodie in SLE Serum Samples and immunosuppressive treatment or the presence of antidsDNA. The EBV-EA/D (i) IgG, (ii) IgA, and (iii) IgM antibody binding in SLE patients and healthy controls observed in Figure 1A was plotted against
(A) immunosuppressive (IS) treatment and (B) the presence of anti-dsDNA. Middle horizontal bar indicates mean SEM.
or more antibody isotypes whereas only 10% of the
healthy controls were positive for two isotypes and 65%
for none. The greatest difference was observed with IgA
antibodies directed against EBV-EA/D, where none of the
sera from healthy controls showed IgA antibody binding
towards EBV-EA/D whereas 35 of the 60 (58%) SLE
patients did.
To investigate whether the larger production of EBV-EA/
D antibodies in SLE patients is an effect of
immunosuppressive treatment and thereby an altered
immune control of the latent virus, a possible correlation
between immunosuppressive medication and serum EBVEA/D antibodies was examined. As demonstrated in
Figure 2A, no association was observed, indicating that the
antibodies directed against EBV-EA/D do not occur upon
reactivation of EBV after medicated immunosuppression.
In addition, the presence of anti-dsDNA in the SLE patients
and the disease activity (SLEDAI score) was platted against
the anti-EBV-EA/D load and showed no association
(Figure 2B) (results not shown).
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Specificity of EBV-EA/D antibodies in serum of SLE patients
To ensure the specificity of the serum antibodies towards
EBV-EA/D, the antigen–antibody binding was investigated. ELISA plates coated with recombinant EBV-EA/D
were incubated with serum from three SLE patients for
different periods of time (Figure 3A) and at different concentrations (Figure 3B). The results show an increase in
binding between EBV-EA/D and the serum antibodies
when the incubation time and serum concentration were
increased, revealing a saturable, time and concentration
dependency of the interaction. The inhibition of EBV-EA/D
IgG antibody binding to immobilized EBV-EA/D was also
conducted by adding EBV-EA/D in the solution phase,
showing a strong and concentration-dependent inhibition
of binding, ensuring the specificity and recognition of the
soluble antigen (Figure 3C). The epitopes on EBV-EA/D
were also examined using 20-amino-acid- long peptides
with 10 amino acids overlaps covering the C-terminal
region of EBV-EA/D (amino acids 306–390). The results
0.5
0.0
0
50
100
150
Sera incubation time (min)
Absorbance (405–650 nm)
IgG
3
2
Patient 1
Patient 2
Patient 3
1
0
0
1.5
1.0
Patient 1
Patient 2
Patient 3
0.5
0.0
0
50
100
150
Sera incubation time (min)
Absorbance (405–650 nm)
1.0
IgA
IgM
2.0
1.5
1.0
Patient 1
Patient 2
Patient 3
0.5
0.0
0
IgA
2.0
Patient 1
Patient 2
Patient 3
1.5
1.0
0.5
0
5
10
Sera concentration (%)
0
50
100
150
Sera incubation time (min)
IgM
5
10
Sera concentration (%)
Absorbance (405–650 nm)
Patient 1
Patient 2
Patient 3
1.5
B
3
2
Patient 1
Patient 2
Patient 3
1
0
0
5
10
Sera concentration (%)
C
Absorbance (405–650 nm)
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IgG
Absorbance (405–650 nm)
Absorbance (405–650 nm)
A
2.0
285
Absorbance (405–650 nm)
EBV-EA/D IgA antibodies in SLE patients
3
2
1
0
0
20
40
60
80
[EA/D]inhibition (µg/ml)
100
Figure 3. EBV-EA/D antibodies in SLE sera are antigen specific. Absorbance measurements (405–650 nm) of microtitre plates coated with recombinant
EBV-EA/D and incubated with serum from three SLE patients at (A) variable periods of time and (B) variable concentrations and detected with APconjugated anti-human IgG, IgA, or IgM and p-NPP. (C) Inhibition assay using serum from an SLE patient incubated with different concentrations of
recombinant EBV-EA/D in the solution phase for 2 h and subsequently transferred to a microtitre plate coated with recombinant EBV-EA/D (1 μg/mL). The
quantity of bound antibody was detected by absorbance measurements (405–650 nm) using AP-conjugated anti-human IgG and p-NPP.
for three of the six SLE patients examined showed that
antibodies reacted with peptides covering different parts of
EBV-EA/D, showing that the antibody epitopes are different and for a large part three-dimensional (data not shown).
SLE patients and healthy controls (Figure 4B), making
EBV-EA/D antibodies the best observed EBV marker to
distinguish SLE patients from healthy controls.
Prevalence of antibodies against other EBV antigens in
serum of SLE patients
EBV-EA/D antibodies in individuals seropositive for different
autoantibodies
The presence of EBNA-1 IgG antibodies is a known
marker for previous EBV infection. As demonstrated in
Figure 4A, the majority of the healthy controls and SLE
patients were seropositive for EBNA-1 IgG antibodies,
which is consistent with the background population. To
test whether SLE patient sera contained antibodies
towards other lytic cycle antigens in addition to EBVEA/D, the seropositivity of EBV-VCA antibodies was
examined. Only a minor difference was observed between
To investigate whether the elevated level of EBV-EA/D
antibodies was specific to SLE patients, serum samples
from individuals seropositive for different autoantibodies
including anti-dsDNA positive, anti-Scl-70 positive, antiSSA/B positive, and anti-CCP positive were tested by
ELISA for antibodies against EBV-EA/D (Figure 5A).
Sera positive for anti-dsDNA showed a significant elevated antibody binding of IgG and IgA antibodies to
EBV-EA/D compared to controls (p-value of 0.026 and
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286
AH Draborg et al
anti-EBNA-1 seropositivity (%)
A
100
SLE patients
Healthy controls
80
Discussion
60
40
20
0
IgG
IgA
IgM
anti-EBV-VCA seropositivity (%)
B
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patients (p ¼ 0.0013). In addition, sera from SSc, pSS,
and RA patients had an elevated EBV-EA/D IgM antibody binding compared to sera from healthy controls
(Figure 5B).
100
SLE patients
Healthy controls
80
60
40
20
0
IgG
IgA
IgM
Figure 4. Antibodies towards EBNA-1 and EBV-VCA in serum from
SLE patients and healthy controls. Percentage of SLE patients (n ¼ 60)
and healthy controls (n ¼ 20) with seropositivity of (A) EBNA-1- and
(B) EBV-VCA-directed IgG, IgA, and IgM antibodies in serum. Data are
depicted as percentage of positive samples. Cut-off was set as determined by the manufacturer.
0.0066, respectively), consistent with the results in
Figure 1A. In addition, anti-Scl-70 positive individuals
showed a significant elevated IgA antibody binding compared to healthy controls (p-value ¼ 0.045). All classes of
sera positive for different autoantibodies had a significant
high prevalence of EBV-EA/D IgM antibodies in sera
compared to healthy controls (Figure 5A).
EBV-EA/D antibodies in systemic autoimmune diseases
Individuals seropositive for Scl-70 antibodies and those
positive for dsDNA antibodies were found to have an
increased frequency of EBV-EA/D IgA antibodies,
whereas no increased titres of EBV-EA/D IgA antibodies
were found in anti-SSA/B- and anti-CCP-positive sera.
Patients with SLE, pSS, SSc, and RA were included to
confirm the results obtained from the seropositive sera.
As demonstrated in Figure 5B, a significant higher prevalence of IgA antibodies against EBV-EA/D was
observed in sera from SSc and RA patients compared to
healthy controls (p-values of 0.015 and 0.014, respectively), although not to the same extent as for the SLE
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The results of this study show a significantly elevated titre of
IgA EBV-EA/D-directed antibodies in SLE patients compared to healthy controls analysed by ELISA. The seropositive rate of IgA EBV-EA/D antibodies was 58% for SLE
patients, whereas none of the serum samples from the
healthy controls showed IgA antibody binding towards
EBV-EA/D. Significantly elevated titres of IgG and IgM
EBV-EA/D antibodies were also found, although with less
specificity. None of the results could be explained by intake
of immunosuppressive medication. Analysis of the antigen–
antibody binding ensured that the serum antibodies towards
EBV-EA/D were specific by showing that the interaction
was time and concentration dependent and saturable and
could be inhibited by the soluble antigen.
To examine whether the humoral EBV immune
response observed in SLE patients was directed against
all EBV lytic cycle proteins, antibodies towards another
lytic antigen, EBV-VCA, was examined. No significant
difference was detected, indicating that EBV-EA/D is a
special lytic cycle EBV antigen in SLE patients. EBVEA/D is one of the first antigens expressed by EBV in the
initiation of the lytic cycle and EBV-EA/D is therefore an
obvious target of the immune system. Therefore, EBVEA/D-directed antibodies could be anticipated as an
immune response to control reactivation of the virus.
Previous serological studies on EBV-VCA-directed
IgA antibodies showed elevated titres in SLE patients
compared to healthy controls (38, 39). These results
were obtained with SLE patients from Taiwan and the
differences between the outcomes of the studies might be
explained by geoepidemiological or ethnic differences in
the populations used in the respective studies.
The majority of the SLE patients (80%) and healthy
controls (75%) were shown to be seropositive for antiEBNA-1 IgG antibodies revealing previous EBV infection. This ensures that the lack of antibody activity
towards EBV-EA/D observed in the healthy controls is
not a result of uninfected individuals.
EBV-EA/D is a lytic cycle antigen of EBV and is
known to play an essential role in the initiation of lytic
replication when the virus switches from latent to lytic
cycle. SLE is a disease with symptoms in the skin and
mucosa. As IgA antibodies are fundamental in mucosal
immunity and EBV is known to infect epithelial cells, the
connection between EBV infection and production of IgA
antibodies towards EBV-EA/D is perhaps not surprising.
The fact that more than half of the SLE patients examined
in this study produced IgA antibodies towards this lytic
cycle antigen suggests a reactivation of the virus in
epithelial cells in SLE patients. The IgA EBV-EA/D
antibodies may reflect the host’s attempt to control an
EBV-EA/D IgA antibodies in SLE patients
(iii)
(i)
(ii)
0.6
0.4
0.2
0.0
1.0
EBV-EA/D IgM antibody binding
EBV-EA/D IgA antibody binding
0.8
0.8
0.6
0.4
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0.0
1.0
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ls
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EBV-EA/D IgM antibody binding
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EBV-EA/D IgA antibody binding
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EBV-EA/D IgG antibody binding
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EBV-EA/D IgG antibody binding
A
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287
Figure 5. EBV-EA/D antibodies in serum from patients with other autoimmune diseases. (A) EBV-EA/D (i) IgG, (ii) IgA, and (iii) IgM antibody binding
of anti-dsDNA-positive individuals (n ¼ 17), anti-Scl-70-positive individuals (n ¼ 13), anti-SSA/B-positive individuals (n ¼ 20), anti-CCP-positive
individuals (n ¼ 20), and healthy controls (n ¼ 20). All were analysed in EBV-EA/D-coated microtitre wells and detected with AP-conjugated antihuman IgG, IgA, or IgM and p-NPP. All serum samples were applied in duplicates in both coated and non-coated wells and the blank absorbances were
subtracted and the values normalized to the standard. Middle horizontal bar indicates mean SEM. p-values for comparison of anti-dsDNA antibody
positive individuals and healthy controls are 0.026, 0.0066, and 0.001, for IgG, IgA, and IgM EBV-EA/D antibodies, respectively. p-values of
comparison of anti-Scl-70 positive individuals with healthy controls are 0.045 and 0.0018 for IgA and IgM antibodies, respectively. The p-values for
comparison of individuals positive for anti-SSA/B and anti-CCP and healthy controls are 0.027 and 0.0003 for IgM EBV-EA/D antibodies, respectively.
(B) EBV-EA/D (i) IgG, (ii) IgA, and (iii) IgM antibody binding of SLE (n ¼ 60), SSc (n ¼ 40), pSS (n ¼ 20), RA (n ¼ 20), and healthy controls (n ¼ 20)
analysed on EBV-EA/D-coated microtitre plates with detection by AP-conjugated anti-human IgG, IgA, or IgM and p-NPP. The serum samples were
analysed in duplicate in both coated and non-coated wells and the blank values were subtracted. Each absorbance value was then normalized to the
standard. Middle horizontal bar indicates mean SEM. p-values for comparison of SSc patients and healthy controls are 0.015 and < 0.0001 for IgA and
IgM EBV-EA/D antibodies, respectively, and the p-values for comparison of RA patients and healthy controls are 0.014 and < 0.0001 for IgA and IgM
EBV-EA/D antibodies, respectively. The p-value for comparison of SS patients and healthy controls is 0.0015 for IgM EBV-EA/D antibodies.
EBV reactivation. It could be hypothesized that lack of
control of the EBV infection could lead to an increased
amount of EBV infected cells and thereby more apoptosis
and amplified wasteload. This could be recognized by the
immune system as ‘non-self’, which gives rise to the
development of autoantibodies. The amplified wasteload
also gives rise to release of EBV antigens including EBVEA/D, which results in the production of EBV-EA/D-
directed antibodies. Lack of control of EBV infection
might therefore be a contributing agent to the development of SLE. Furthermore, the observed lack of
any significant association between serum EBV-EA/D
antibodies and immunosuppressive medication emphasizes that EBV-EA/D IgA antibodies are not caused by
EBV reactivation as a result of an iatrogenically suppressed immune system. However, it cannot be ruled
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288
out that the main findings of this study relate to more
specific intrinsic defects of the immune system. Previous
research has shown that the defective control of the virus
could involve potential T-cell defects in SLE patients comprising a defect in EBV-specific CD8þ T -cell cytotoxicity
and a compensatory increased frequency of EBV-specific
CD4þ T cells (8, 11, 12). Furthermore, specific genetic
variations in the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene promoter has also been shown to be
associated with the seroprevalence of EBV-directed IgA
antibodies (40).
In addition to our findings for SLE patients, a statistically significant higher prevalence of EBV-EA/D IgA
antibodies was found in the disease control groups containing SSc and RA patients and in individuals seropositive for anti-Scl-70 compared to healthy controls. This
could indicate a common immune deficiency connecting
EBV infection with the development of these diseases,
where the lack of control of the latent EBV infection in
these individuals could result in different autoimmune
manifestations according to where the reactivation or
reinfection takes place in the body. No antibody binding
towards EBV-EA/D was observed in the other disease
control groups.
The presence of EBV-EA-directed IgA antibodies in
SLE patients has also been investigated by another
research group using indirect immunofluorescence and
they reported only a slightly elevated prevalence compared to healthy controls (15% compared to 0%).
However, only a small sample size was used (n ¼ 34)
(43). Our results elaborate on these findings by the use of
a larger cohort of SLE patients confirmed by different
methods. Our results led to a higher prevalence of antiEBV-EA/D IgA-responding SLE patients (58% compared to 0%) possibly because of the use of ELISA,
which is a more sensitive method than immunofluorescence. In addition, the occurrence of IgG antibodies
towards EBV-EA has been intensively studied and
observed to be elevated in approximately half of SLE
patients compared to 8–17% of healthy controls
(36, 37, 41). Our results support these previous findings
for EBV-EA IgG antibodies in SLE patients.
In the SLE cohort used in this study, 32 out of the 60
SLE patients were anti-dsDNA negative and in 53% of
these anti-dsDNA-negative SLE patients, high titres of
EBV-EA/D IgA antibodies were detected. As no association was observed between serum EBV-EA/D antibodies
and the presence of anti-dsDNA, high titres of IgA EBVEA/D antibodies could in the future be useful as a supplementary serological tool to identify SLE patients
without dsDNA antibodies. However, more validating
research with more SLE patients, disease groups, and
healthy controls needs to be examined for EBV-EA/D
-directed antibodies to confirm these findings. The specificity of this test should be further investigated because
elevated titres of EBV-EA/D IgA antibodies were also
observed in some of our disease control groups. In addition, several studies have shown that the presence of
www.scandjrheumatol.dk
AH Draborg et al
EBV-EA IgA antibodies is common in patients with
nasopharyngeal carcinoma (48–50).
In conclusion, our results support previously suggested
associations between EBV infection and the development
of SLE. Numerous research groups have demonstrated
that SLE patients have an abnormally high viral load
(7–10) indicating difficulties with suppressing the latent
infection. The defective control of the virus has been
shown to involve an impaired EBV-specific T-cell
response in SLE patients (8, 11, 12). Various research
groups have investigated EBV-directed antibodies in SLE
patients, but the prevalence of IgA antibodies towards
EBV-EA/D has not been thoroughly examined until
now. The clear differentiation between SLE patients and
healthy controls demonstrated in this study by the presence of EBV-EA/D-directed IgA antibodies (58% compared to 0%) has not previously been established. The
present results corroborate the notion of EBV infections
having either a primary or a secondary pathogenetic role
in the development of SLE.
Acknowledgements
We thank Esin Djebir Güven, Department of Clinical Biochemistry and
Immunology, Statens Serum Institut, Copenhagen, Denmark for collecting blood samples and for technical assistance, and Lone Troelsen from
the Department of Rheumatology, Copenhagen University Hospital,
Rigshospitalet, Copenhagen, Denmark for collection of RA serum
samples.
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