Deficient EBV-Specific B- and T-Cell Response in Patients
with Chronic Fatigue Syndrome
, Kristin Strohschein
, Carolin Giannini
, Uwe Koelsch
, Sandra Bauer
, Sybill Thomas
, Nadine Unterwalder
, Volker von Baehr
, Petra Reinke
, Leif G. Hanitsch
, Christian Meisel
, Hans-Dieter Volk
, Carmen Scheibenbogen
1Institute for Medical Immunology, Charite
´University Medicine Berlin, Campus Virchow, Berlin, Germany, 2Julius Wolff Institute, Charite
´University Medicine Berlin,
Campus Virchow, Berlin, Germany, 3Labor Berlin GmbH, Immunology Department, Charite
´University Medicine Berlin, Campus Virchow, Berlin, Germany, 4Institute for
Medical Diagnostics, Berlin, Germany, 5Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charite
´University Medicine Berlin, Germany, 6Department
´University Medicine Berlin, Germany
Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a
subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific
antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell
response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear
antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory
B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76%
of patients. Moreover, the ex vivo EBV-induced secretion of TNF-aand IFN-cwas significantly lower in patients. Multicolor
flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-a/IFN-c/IL-2 producing CD4
subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When
comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients
compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a
deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps
of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.
Citation: Loebel M, Strohschein K, Giannini C, Koelsch U, Bauer S, et al. (2014) Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue
Syndrome. PLoS ONE 9(1): e85387. doi:10.1371/journal.pone.0085387
Editor: Marc S. Horwitz, University of British Columbia, Canada
Received September 17, 2013; Accepted November 26, 2013; Published January 15, 2014
Copyright: ß2014 Loebel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the ‘‘Fatigatio e.V. Bundesverband Chronisches Erscho
¨pfungssyndrom’’ and the ‘‘Europa
¨ischen Fonds fu
regionale Entwicklung (EFRE)’’ of the European Union grant ‘‘Investition in Ihre Zukunft’’. The funder had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Madlen.email@example.com
.These authors contributed equally to this work.
Chronic Fatigue Syndrome (CFS) is characterized by severe
fatigue with typical post-exertional delay to recover from
exhaustion, cognitive dysfunctions and flu-like symptoms ,
. CFS is diagnosed based on clinical Center of Disease Control
criteria scores known as Fukuda criteria  or on the Canadian
Consensus Definition from 2004 . Diagnosis of CFS is often
restrained as many symptoms are not disease-specific and no
diagnostic test could be established for CFS so far , , , .
Hallmarks of CFS are immune dysregulation and immune
activation , , . Diminished natural killer (NK)-cell
cytotoxicity and reduced NK-cell derived perforin have been
repeatedly reported for CFS patients , , . Furthermore,
increased frequencies of activated HLA-DR class II-positive CD8
T cells were proposed as immunological activation markers in CFS
, , . Straus et al. showed reduced proliferative
responses of lymphocytes and reduced frequencies of CD4
cells . Similarly Curriu et al. reported diminished proliferation
of T cells but enhanced frequencies of regulatory T cells .
Broderick and colleagues described a dysregulation of Th-17
priming by enhanced levels of IL-13, IL-2 and IL-8 but decreased
levels of IL-5 and IL-23 in post-infectious CFS patients , .
Furthermore, the group of Skowera et al. reported an effector
memory cell responsiveness bias towards type 2 in patients with
CFS onset typically goes along with a viral illness. Various
viruses have been reported to trigger CFS. In 2009, it was
published that the retrovirus XMRV is linked to CFS. Although
this turned out to be a laboratory contamination, it called
attention to this so far neglected disease , , , .
Herpes viruses as cause of CFS have been discussed for decades.
However, stringent evidence for a clear association of enhanced or
altered viral load and disease is still lacking , , , ,
, . Further, in CFS data about altered serological
responses against viruses of the herpes group are not consistent.
Several groups reported more frequent detection of HHV6/7 load
and elevated antibody titers , , , ,  a finding
that was not confirmed by others , . Increased IgG to
human cytomegalovirus (CMV), EBV viral capsid antigen (VCA),
PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e85387
HHV-6, Herpes-Simplex Virus (HSV)-1, HSV-2 and Coxsackie
viruses were reported in CFS in some studies , , , but
not in others , .
Numerous studies have tried to find evidence for an association
of CFS with EBV. In a subset of patients, CFS begins with
infectious mononucleosis and enhanced EBV-specific antibody
titers have been reported. Lerner et al. found serum IgM antibodies
to EBV-VCA in CFS patients but not in controls and recently
reported elevated antibodies against EBV-dUTPase and EBV-
DNA polymerase in a subset of CFS patients , . Consistent
with these data, elevated titers of early antigen (EA)-IgG and
antibodies to ZEBRA, a product of the immediate early EBV gene
BamHI Z fragment leftward open reading frame (BZLF)-1, were
detected in CFS patients , . No differences in IgG titers
against EBV-VCA, EBV nuclear antigen (EBNA)-1 and EA were
reported in other studies , , .
The orally transmitted EBV initially targets the mucosal
epithelium and remains in a life-long latency in memory B cells
, , . In healthy subjects the EBV genome usually
remains latent in the so-called latency phase 0 and EBV
replication is latent and without production of infectious virions
, . , . This latency is controlled by NK- and T-cell
responses. Replication occurs in different cycles, including latency
I characterized by the expression of EBNA-1, latency II
characterized by latent membrane proteins (LMP)-1 and LMP-2,
and latency III when EBNA-2, -3 and -6 are also expressed ,
. During lytic reactivation the EBV immediate-early genes
BZLF-1 and BRLF-1 are expressed. These genes activate viral and
cellular promoters that induce early, lytic and late viral gene
expression and high amplification of the EBV genome . EBER
genes encode for regulatory RNAs. EBER-DNA can be used as a
sensitive tool for the detection of EBV-infected cells, and the
EBER-DNA copy number is related to the copy number of EBV-
DNA molecules [54,55]. No clear differences in EBV-DNA levels
in blood and gastro-intestinal biopsies of CFS patients could be
demonstrated yet , , .
At the Charite´, we take care of patients with CFS in our
outpatient clinic for adult immunodeficiencies as a subset of our
CFS patients have concomitant immunoglobulin deficiency. Our
observation of both elevated VCA-IgM and lack of EBNA-IgG in
a subset of patients with CFS prompted us to perform a
comprehensive analysis of the EBV-specific immune response.
By comparing memory B- and T-cell responses of CFS patients
with healthy EBV-infected subjects, we observed a profound
deficiency in EBV-specific B- and T-cell memory response in the
majority of CFS patients resembling the deficiency of EBV
memory responses described in autoimmune diseases  and
chronic HIV infection , , .
Materials and Methods
The study was approved by the institutional ethics committee –
Charite´ Universita¨tsmedizin Berlin and written informed consent
was provided by all subjects. Only adults were included.
Study population and specimen collection
Patients were diagnosed with CFS according to Fukuda criteria
at our outpatient clinic between 2007 and 2013 . Patients with
other medical or neurological diseases were excluded. Patients
who had a concomitant immunoglobulin deficiency were excluded
when they fulfilled the diagnostic criteria for CVID or required
immunoglobulin substitution due to recurrent bacterial infections.
Two consecutive cohorts of patients (Table 1) were analyzed for
EBV antibodies. We excluded 1/64 seronegative patients (1.6%)
and 4/61 controls (7%) from our analyses in cohort 1 and 28/411
(6.8%) patients in cohort 2. Due to similar numbers of
seronegative patients and controls, the interpretation of our data
is not affected. A subset of patients from cohort 1 was analyzed for
B-cell memory response by ELISpot, and EBV-induced T-cell
cytokines. A subset of patients from cohort 2 was analyzed for
EBV viral load. Patients of both cohorts were analyzed for EBV-
specific T-cell responses by flow cytometry. All subgroups of
patients were randomly selected but were representative for the
respective cohort 1 or 2 in age, disease score and duration.
Blood and serum was obtained from CFS patients and healthy
subjects. Peripheral blood mononuclear cells (PBMCs) were
isolated by density gradient centrifugation using Ficoll Hypaque
and either cryopreserved for T-cell analysis or directly used in cell
culture stimulation assay for memory B-cell analysis.
Quantitative real-time PCR
Detection of EBV-DNA in PBMCs was done by nested PCR for
EBER-1 with the following primers forward 59-TCC CGG GTA
CAA GTC CCG-39and reverse 59-TGA CCG AAG ACG GCA
GAA AG-39at 900 nM. Detection has been performed with probe
FAM-59-TGG TGA GGA CGG TGT CTG TGG TTG TGT T-
39-TAMRA (Eurofins MWG Operon, Ebersberg Germany) at
5mM. Amplification data were analyzed by an ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, California,
USA). Successful DNA isolation was verified by histone replication
with the primers forward 59-CCA GAG CGC AGC TAT CGG
T-39at 900 nM and reverse 59-CAC GTT TGG CAT GGA
TAG CAC -39at 50 nM and the probe FAM - 59-GCA AGT
GAG GCC TAT CTG GTT GGC CTT T-3- TAMRA (Eurofins
MWG Operon, Ebersberg Germany) at 5 mM. For BZLF-1 the
following primers forward 59-AAATTTAAGAGATCCTCGTG-
TAAAACATC-39and reverse 59-CGCCTCCTGTTGAAGCA-
GAT-39at 30 pM were used. Detection has been performed with
39-TAMRA (Eurofins MWG Operon, Ebersberg Germany) at
10 pM. For detection of BZLF-1 RNA, isolated total RNA was
reverse transcribed according to the manufacture’s instructions
(Life Technologies, Darmstadt, Germany). EBER copies/mg DNA
and BZLF-1 copies/mg cDNA were calculated in accordance to
Table 1. Patient characteristics.
Cohort 1 Cohort 2
Age, mean, range 47, 27–63 45, 20–78
Women, n (%) 46 (73) 245 (63)
Bell score, mean, range 30, 10–50 30, 10–70
Disease duration in years, mean, range 7.4,1–30 7.4, ,1–39
History of autoimmune disease (%) 11.1* 9.3
Deficiency of IgG/IgA/IgM (%) 4.4/3.6/3.1 15.2/2.9/0
Deficiency of IgG
(%) 8.18 16.7
*Hashimoto thyreoiditis (n = 6), lichen sclerosus (n = 1);
Hashimoto thyreoi ditis (n = 30), psoriasis (n = 3), colitis ulcerosa (n = 1), Morbus
Bechterew (n = 2).
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e85387
standard EBV-copies . Results $35 copies/mg DNA/cDNA
were regarded as positive.
Enzyme-linked immunosorbent assay (ELISA)/Enzyme
EBNA-IgG, VCA-IgG and VCA-IgM were detected using an
immuno chemiluminescence assay (CLIA, DiaSorin, S.p.A.,
Saluggia, Italy) according to the manufacturer’s instructions. EIA
was used to detect EBV EBNA-1-IgG at the Labor Berlin GmbH.
Analysis of memory B cells was adapted from Crotty et al. .
PBMCs were stimulated unspecifically with Pokeweed mitogen
(PWM) at 10 ng/ml (Sigma Aldrich, Schnelldorf, Germany),
Staphylococcus aureus Cowan at 1:10000 dilution (Merck,
Darmstadt, Germany) and CpG at 6 mg/ml (InvivoGen, CA,
USA) in RPMI 1640 (PAA Laboratories, Co¨lbe, Germany)
supplemented with Penicillin/Streptomycin 1006and L-Gluta-
mine at 2 mM and 10% FCS (both Biochrom, Berlin, Germany)
and b-Mercaptoethanol at 50 mM (Merck, Darmstadt, Germany)
for 7 days at 37uCin5%CO
. For T-cell independent stimulation
B cells from CFS patients were enriched with a RosetteSep CD3
depletion kit according to the manufacturer’s instructions
(Stemcell Technologies, Grenoble, France). 2.5610
B cells per
well were kept in 1 ml IMDM (PAA Laboratories, Co¨lbe,
Germany) with 10% heat-inactivated FCS (Valley Biomedical,
Winchester, VA, USA), 5 mg/ml insulin/transferrin and 5 ng/ml
selenium (all Sigma Aldrich, Schnelldorf, Germany), 1.25 mg/ml
CpG (Invivogen, CA, USA), 300 U/ml IL-2 (Chiron-Behring,
Liederbach, Germany), 12.5 ng/ml IL-10 (ImmunoTools, Frie-
soythe) and 500 ng/ml IL-21 (ImmunoTools, Friesoythe, Ger-
many) and 0.5 mg/ml anti-CD40 monoclonal antibody (R&D
Systems, MN, USA. Cells were cultured for 7 days at 37uCin5%
. After stimulation, the cells were transferred at a concentra-
tion of 1610
/100 ml into a 96-well multiscreen HTS-IP filter
plate (Merck Millipore, MA, USA) pre-coated with purified,
recombinant EBV-VCA at 0.1 mg/well (tebu-bio, Le-Perray-en-
Yvelines, France) and EBV-EBNA-1 at 1 mg/well (tebu-bio, Le-
Perray-en-Yvelines, France) and purified EBV-lysate at 1:20
dilution (tebu-bio, Le-Perray-en-Yvelines, France). For the analysis
of total IgG, anti-human IgG-Fc-fragment antibody (Jackson
Immunoresearch, PA, USA) was coated at a concentration of
1.2 mg/well and cells were seeded at a concentration of 1.25610
100 ml, 6250/100 ml and 3125/100 ml for 6 h. Secreted IgGs were
detected using an anti-human IgG, F(ab9)2 fragment coupled to
Biotin at 1 mg/ml (Biosource, Life Technologies, Darmstadt,
Germany) and Horseradish Peroxidase Avidin D at 5 ng/ml
(Vector Laboratories, MI, USA). IgG spots were visualized by
adding 3-Amino-9-ethylcarbazole (Sigma-Aldrich, Schnelldorf,
Germany). Plates were scanned and spots enumerated on a CTL
Immunoplate reader using Immunospot Academic software
(Cellular Technology Ltd, OH, USA). Frequencies were expressed
as the ratio of the mean number of antigen-specific spots and mean
number of total IgG spots.
Antigen-specific T-cell response was measured by cytokine
production in cell culture supernatants of PBMCs stimulated with
either 1 mg/ml SEB (Sigma-Aldrich, Schnelldorf, Germany),
1mg/ml EBV total lysate or 1 mg/ml of the EBV peptide
EBNA-1 (JPT, Berlin, Germany) for 48 h. 2610
kept in 1 ml serumfree RPMI (PAA Laboratories, Co¨lbe,
Germany) with 2% Hepes buffer, 1% L-glutamin (Biochrom,
Berlin, Deutschland) and 0.5% gentamycin (Merck, Darmstadt,
Germany). IFN-c, IL-10, IL-2 and TNF-awere measured in cell
culture supernatants with a MPXHCYTO-60K Multiplex-Immu-
noassay (Merck Millipore, MA, USA) on a LuminexH200
(Luminex, TX, USA) according to manufacturer’s instructions.
EBV-specific memory T cells were analyzed after stimulation
with EBNA-1 or CMV-pp65 peptides and expansion in vitro as
recently described . After overnight incubation of PBMCs in
IMDM (PAA Laboratories, Co¨lbe, Germany) containing 10% AB
serum (Valley Biomedical, Winchester, VA, USA) and supple-
mented with Penicillin/Streptomycin 1006and L-glutamine at
2 mM (both Biochrom, Berlin, Germany) at 37uCin5%CO
96-well round bottom plates at a concentration of 2610
well with 50 IU/mL rhIL-2 (Chiron-Behring, Liederbach, Ger-
many) and 10 ng/mL IL-7 (ImmunoTools, Friesoythe, Germany).
On day 3, 5 and 7 media and IL-2 at 50 ng/ml were renewed. IL-7
at 5 ng/ml was added on day 7 of culture, and cells were harvested,
washed and stained for cytokines.
Multiparameter flow cytometry
Intracellular and extracellular staining was applied for T-cell
analysis after 10 days of expansion. 2610
restimulated with an EBNA-1 or CMV-pp65 peptide pool (JPT,
Berlin, Germany) at (1 mg/mL) or DMSO (Sigma Aldrich,
Schnelldorf, Germany) as negative control for 5 h. Brefeldin A
(7.5 mg/mL) (Sigma Aldrich, Schnelldorf, Germany) was added
after 1 h of stimulation. Live/dead cells were discriminated using
an amine reactive dye (Invitrogen, Life Technologies, Darmstadt,
Germany) and stained with fluorescence conjugated monoclonal
antibodies against CD3, CD4, CD8, PD-1, IFN-y, TNF-aand IL-
2 (BD Biosciences, NJ, USA). Background events in DMSO
controls were subtracted from events counted in response to
EBNA-1 or CMV-pp65 stimulation. Data acquisition was
performed on BD LSR II (Becton Dickinson, NJ, USA) and
analysis was done using FlowJo software.
Statistical data analysis was done using the software SPSS
Statistics 19 and GraphPad Prism 5. Nonparametric statistical
methods were used. Continuous variables were expressed as
median and interquartile range (IQR), if not indicated otherwise.
Univariate comparisons of two independent groups were done
using the Mann-Whitney-U test. For association analysis Fisher’s
exact test was used. A p-value of ,0.05 was considered statistically
A significant subset of CFS patients shows abnormal EBV
First, we analyzed the EBV-specific antibody response from
CFS patients. We compared serum EBV-VCA-IgG, -IgM, and
EBNA -IgG from patients of cohort 1 (n = 63, Table 1) and
healthy controls (n = 57) (Figure 1A). While we did not observe a
difference in levels of VCA-IgG, IgG antibodies against EBNA
were undetectable (#20 U/ml) in 12.7% of CFS patients in
contrast to 3.5% of healthy controls (p =0.06, Figure 1B). When
excluding the values which were out of range, we obtained similar
results with comparable levels for VCA-IgG and a significant
difference for EBNA-IgG (p = 0.05, data not shown).
Further, elevated VCA-IgM was found more frequently in
patients compared to healthy controls (17.5 vs. 3.5% p = 0.013,
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e85387
Figure 1C). In contrast, CMV-specific IgM was elevated in only 2
patients and levels of IgG revealed no difference between CFS
patients and healthy controls (Figure 1D and Figure 1E). Within
this cohort of patients only one patient with positive EBV-VCA
IgM showed lack of EBNA-IgG. Thus, an abnormal EBV serology
(positive VCA-IgM or negative EBNA-1-IgG) was detected in 30%
of CFS patients.
In a second consecutive cohort with 387 CFS patients (Table 1),
EBV-specific antibodies were measured by ELISAs that determine
IgG against a mixture of various EBV proteins or EBNA-1,
respectively. Similarly, we observed a lack of EBNA-1-IgG in
9.8% of EBV-IgG positive patients (Figure 2A). In a randomly
selected subset of 8 EBNA-1-IgG positive and 7 negative patients
we further comparatively analyzed total IgG levels, frequencies of
B cells, and B-cell subsets. No difference in total IgG (Figure 2B)
was found in EBNA-1-IgG negative compared to EBNA-1-IgG
positive CFS patients. Moreover, the absolute B-cell numbers
(Figure 2C) and frequencies of memory B-cell subsets were not
different among EBNA-1 negative and positive patients (Figure 2D
and Figure 2E) and were within the normal range compared to the
reference values of our immune diagnostic laboratory.
EBV-specific memory B cells are low or absent in most
Serum IgG antibodies are mostly derived from long-living
plasma cells, which have settled in the bone marrow, often many
years before. Thus, disturbance of memory B-cells can be
overlooked for long time if only IgG serum levels are analyzed.
Therefore, we studied the frequencies of specific antibody-
secreting cells (ASCs) derived from EBV-specific memory B cells
after in vitro restimulation by activating PBMCs with a mixture of
CpG, SAC, and PWM for 7 days. ASCs were analyzed in the
ELISpot assay. No difference between CFS patients and healthy
controls was detected for total IgG secreting memory B cells
(Figure 3A). However, the CFS patients had significantly reduced
frequencies of B cells producing antibodies against EBV antigen
mixture (EBV-lysate). In addition, by using overlapping peptide
pools, VCA-specific and EBNA-1-IgG secreting B cells were
analyzed. A diminished B-cell memory response was defined by
frequencies below the interquartile range of the control group. As
shown in Figure 3B a diminished B-cell memory response against
EBV-VCA was observed in 59% of CFS patients, and against
EBV-EBNA in 76% of patients, respectively. We found no
correlation between frequencies of VCA- or EBNA-specific
memory B cells and levels of IgG antibodies (VCA r = 20.1242,
p = 0.67 and EBNA r = 0.07913, p = 0.8). Patients analyzed for
memory B cells were derived from cohort 1 and their IgG titers
were comparable to the IgG titers of the whole cohort 1 (median
VCA-IgG 350 vs. 450 U/ml, EBNA-IgG 100 vs. 180 U/ml,
shown in Figure 1B). We determined HSV- and CMV lysate-
specific ASCs in a subgroup of these patients detecting no
difference between patients and controls further excluding a
general B-cell defect (Figure 3C and Figure 3D). To assess whether
the reduction of EBV-specific memory B cells is possibly due to the
presence of suppressive T cells in culture, enriched B cells were
cultivated without T cells in the presence of feeder LL8 cells.
Soluble CD40 ligand together with a cytokine mix and CpG were
used to induce memory B-cell differentiation. Similar numbers of
EBV-specific antibody secreting B cells were detected in patients in
the T-cell dependent and independent analysis (Figure 3E). Taken
together, the lack or reduced level of memory B cells shows a more
profound deficiency of the EBV-specific B-cell response as evident
CFS patients show diminished T-cell cytokine response to
In a next series of experiments, we analyzed the EBV-specific T-
cell response in patients and healthy controls. First, EBV-lysate
induced production of several cytokines that were tested in whole
blood, revealed a significantly reduced number of IFN-c
Figure 1. EBNA antibody response is reduced in CFS patients. (A) Serum IgG titers were assessed for healthy controls and CFS patients by
ELISA for EBV VCA-IgG (control n = 57, CFS n = 63), (B) EBNA-IgG (control n = 57, CFS n = 63), (C) EBV VCA-IgM (control n = 57, CFS n = 63), (D) CMV-IgG
(control n = 32, CFS n = 41) and (E) CMV-IgM (control n = 32, CFS n = 41). Statistical analysis was performed using the two-tailed Mann-Whitney-U test
and for EBNA-IgG and EBV VCA-IgM Fisher’s exact one-tailed test for association analysis with * p,0.05.
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e85387
responders in the patient group with 50% (n = 11/22) vs. 69%
(n = 20/29) in the control group (Figure 4A). Using whole protein-
spanning overlapping 15-mer peptides from EBNA-1 protein for
stimulation, no patient showed a detectable IFN-cresponse
(Figure 4A). Similar IFN-clevels were observed in response to the
T-cell superantigen SEB in patients and controls. In addition, a
significant reduction of TNF-aand a lower number of patients
producing IL-2 was observed in response to EBV-lysate, while the
IL-10 response was not diminished (Figure 4B).
CFS patients show reduced EBV-specific multifunctional
memory T cells
To analyze the EBV-specific memory T-cell response in more
detail, we stimulated PBMCs with EBNA-1 peptides and
expanded them in vitro in the presence of IL-2 and IL-7 for 10
days as recently described . IFN-c
T cells were analyzed by
flow cytometry. Frequencies of EBV-specific T cells were lower in
CFS patients (n = 23) compared to the control group (n = 17,
Figure 5A). The difference was most evident in the IFN-c
triple producing - so called multifunctional - CD4
T cells. In addition, the frequencies of the IFN-c
double producers were significantly lower in the patient group. No
difference was observed for the single producers. A diminished
EBV-specific T-cell response was defined by frequencies below the
interquartile range of the control group in CD4
cytokine producing T cells. Based on this definition a diminished
response was detected in 48% of CFS patients for triple CD4
cells and in 52% of patients for triple CD8
T cells. As control, we
analyzed the T-cell response against CMV peptide pp65 in a
subset of CMV-reactive subjects (Figure 5A, right panel) showing
no difference among triple and double CMV-reactive T cells
between patients (n = 5) and controls (n = 7). Comparing the total
cell numbers of cultures stimulated with EBNA-1, no difference
was observed (patients median 77%, range 44%–106% of initial
cell number, controls median 93%, range 28%–148% of initial cell
number). Next, we analyzed the expression of the exhaustion
marker PD-1 on EBNA-1- and CMV-pp65-specific T cells but
found no difference in the frequencies of PD-1 expression between
patients and controls as shown for the population of the IFN-c
double producing CD4
T cells in Figure 5B
(n = 8).
Evidence of enhanced latent EBV replication in CFS
As a measure of the EBV load, we determined EBER DNA by
real-time PCR in whole blood. EBER-DNA was detectable in 21
of 290 patients (7.2%) in a low copy number (,1000 – 2.930
copies/ml). In a subset of patients and controls we comparatively
analyzed EBV DNA in isolated PBMCs. EBER-DNA was
Figure 2. EBNA-1-IgG is reduced in a subset of patients but total IgG and B-cell subpopulations are not different in EBNA-1-IgG
positive and -negative CFS patients. (A) Serum IgG titers were assessed in CFS patients for EBV-IgG and EBV-EBNA-1-IgG (n = 387), (B–E) EBNA-1
negative (neg, n = 7) and positive (pos, n = 8) CFS patients were compared for (B) total IgG, (C) the absolute numbers of CD19
B cells/nl blood, (D)
frequencies of IgD
marginal zone B cells, and (E) frequencies of IgD
class switched memory B cells. Statistical analysis was
performed using the two-tailed Mann-Whitney-U test and for IgG Fisher’s exact one-tailed test for association analysis with *** p,0.0001.
EBV Response in CFS Patients
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detectable in 55% of patients (n = 11/20) compared to 15%
(n = 3/20) of healthy controls (p,0.01) (Figure 6A). No EBER-
DNA was detected in the plasma (data not shown). Additionally,
we tested 4 EBV seronegative CFS patients and detected no EBER
DNA in PBMCs (data not shown). As a marker for lytic replication
we further analyzed RNA levels of the lytic protein BZLF-1 in
PBMCs (n = 20) but could not detect BZLF-1 cDNA in patients or
controls (Figure 6B). As positive control copies/mg cDNA of EBV
cell line 293T/B95-8 were determined.
For many years, researchers have suspected EBV to be involved
in CFS. A hallmark of CFS is chronic activation of the immune
system, which can be triggered by infections or non-infectious
agents , , . Although altered EBV-specific antibody
titers have been repeatedly demonstrated in CFS, no clear
evidence for chronic EBV replication has been obtained so far.
To contribute to the understanding of CFS, our present study aims
to further elucidate the immune response to EBV in CFS.
First of all we could confirm previous reports providing
serological evidence of EBV reactivation by demonstrating
elevated IgM antibodies against the late VCA antigen in a subset
of patients , , . Remarkably, in line with this finding
we could provide evidence of enhanced viral load of EBV by
detection of EBV DNA in a significantly higher proportion of
patients compared to healthy controls. However, no patient
showed a high viral load as seen in EBV-associated post-transplant
lymphoproliferative disorder or acute infectious mononucleosis.
Further, we had no evidence of lytic replication as we could
neither detect EBER DNA in plasma nor BZLF-1 RNA in
PBMCs. Thus, our findings suggest a higher level of latency-
associated replication in CFS patients.
A key finding in our study was the lack of IgG antibodies to
EBNA-1 antigen, observed in a subset of patients in 2 different
cohorts. EBNA-1 is one of the few gene products expressed in type
Figure 3. EBV-specific antibody secreting cells are reduced in CFS patients. (A–D) Frequencies of ASCs in healthy controls and CFS patients
7 days after polyclonal stimulation of total PBMCs. Secreted total and specific IgG was assessed with the ELISpot assay. IgG-secreting B cells are shown
as frequencies from 1610
seeded cells for (A) total IgG (control n = 12, CFS n = 17), (B) EBV-lysate-specific IgG (control n = 12, CFS n = 17), VCA-specific
IgG (control n = 12, CFS n = 17), IgG against EBNA-1 peptides (control n = 12, CFS n = 16) and (C) HSV- (control n = 8, CFS n = 11) and (D) CMV-lysate-
specific IgG (control n = 6, CFS n = 6). (E) Comparison of frequencies of ASCs in polyclonal stimulation of total PBMCs (T-cell dependent) and
stimulation of isolated B cells with CD40L (T-cell independent) in CFS patients for total IgG, EBV-lysate, (n = 8), VCA or EBNA-1 peptides (n = 6).
Statistical analysis was performed using the two-tailed Mann-Whitney-U test with * p,0.05.
EBV Response in CFS Patients
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I latency. Assessment of EBNA-IgG is used for diagnosis of EBV
infection, as it is mounted rather late during primary infection and
a failure to produce EBNA-IgG had been observed both in severe
infectious mononucleosis and chronic active EBV disease ,
. However, serum IgG levels do not reflect the number of B-
cell memory cells as serum IgG is derived from long-lived plasma
cells, which often have been acquired many years ago. To
investigate the memory B-cell response in more detail, we
analyzed the in vitro differentiated EBV-specific memory B-cell
pool revealing a much more profound defect in EBV-directed B-
cell response with low or undetectable EBV-specific ASCs in the
majority of patients. Remarkably, memory B-cell responses not
only against EBNA-1, but also against the late lytic antigen VCA
were low to absent in the majority of patients despite normal IgG-
VCA titers indicating a secondary exhaustion of the memory B-
cell pool. Alternatively, an impaired ability to mount a sufficient
number of EBV-specific memory B cells upon primary infection
may be discussed. This deficiency seems to be specific for EBV as
total IgG, CMV-specific antibodies and CMV- and HSV-specific
B-cell memory responses were not different compared to healthy
controls. Further, no differences in the amount of total IgG or B
cells and memory B cells was observed between EBNA-1-IgG
negative and -positive patients. The failure to detect ASCs in vitro
might either be related to a loss of memory B cells or their failure
to differentiate into ASCs. To exclude that T cells in CFS patients
inhibit or kill in vitro differentiating EBV-memory B cells, we
additionally used a T-cell independent ASCs-inducing protocol
showing similar results.
In accordance with the diminished EBV-specific memory B-cell
response, we could demonstrate an impaired EBV-specific T-cell
response, particularly of the triple and double cytokine producers.
Based on our findings we assume frequent EBV reactivation as a
mechanism for the impaired EBV-specific B- and T-cell immune
responses in CFS patients. An impairment of specific T-cell
responses is observed in various chronic infections. In HIV and
HCV infection diminished specific polyfunctional CD8
were described [68,69,70,71]. Further, selective lack of polyfunc-
tional T cells was demonstrated in animal models of chronic SIV
and Leishmania infection accompanied by the absence of
circulating antibodies , . The persistence and continuous
exposure to antigen may drive T cells into exhaustion. This
process is often accompanied by the presence of high levels of IL-
10 and upregulation of immune suppressor molecules .
Our findings in CFS have similarities to recent studies in
systemic lupus erythematosus (SLE) as well, in which EBV
reactivation is thought to play an important role in disease
Figure 4. CFS patients show diminished cytokine response against EBV. Whole blood of healthy controls and CFS patients was analyzed by
Multiplex-Immunoassay for (A) IFN-cproduction after stimulation with either EBV-lysate (control n = 29, CFS n =22), EBNA-1 peptide (control n = 24,
CFS n = 11) or SEB (control n = 21, CFS n = 11) and (B) after EBV-lysate stimulation for TNF-a(control n = 29, CFS n = 22), IL-2 (control n = 29, CFS n = 22)
and IL-10 (control n = 25, CFS n = 13). Statistical analysis was performed using the two-tailed Mann-Whitney-U test with * p,0.05 and *** p,0.001.
EBV Response in CFS Patients
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Figure 5. CFS patients show reduced EBV-specific memory T-cell response. (A) Comparison of cytokine producing CD4
(upper panels) and
T cells (lower panels) of CFS patients and healthy controls after 10 days of stimulation with EBNA-1 (left panel, Control n= 17, CFS n = 23).
Boolean gating strategy was applied to analyze IFN-c/TNF-a/IL-2 triple, IFN-c/TNF-adouble, and IFN-cand TNF-asingle cytokine producing T cells
after intracellular staining of isolated PBMCs incubated with Brefeldin A for 16 h. Stimulation with CMV pp65 (right panel, Control n = 7, CFS n = 5) is
shown for IFN-c/TNF-a/IL-2 triple, and IFN-csingle cytokine producing T cells. (B) Frequencies of PD-1 expression were analyzed for IFN-c/TNF-a
double producing CD4
T cells after 10 days of stimulation with EBNA-1 or pp65 (n= 8). Statistical analysis was performed using the two-
tailed Mann-Whitney-U test with ** p,0.01.
Figure 6. Latent EBV can be detected more frequently in CFS patients. (A) EBV DNA was analyzed via nested real-time PCR in total PBMCs of
20 healthy donors and CFS patients for EBER-1. EBER-copies were calculated in accordance to Namalwa standard. (B) BZLF-1 RNA was analyzed via
nested real-time PCR in total PBMCs of 20 healthy donors and CFS patients but no BZLF-1 cDNA was detected. cDNA of EBV cell line 293T/B95-8 was
used as positive control. Statistical analysis was performed using the one-tailed Mann-Whitney-U test with ** p,0.01.
EBV Response in CFS Patients
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pathogenesis . It is thought that the increased viral load or an
altered presentation of EBV proteins that cross-react with cellular
antigens may trigger pathogenic processes through molecular
mimicry , , , , . Abnormal expression of both
viral lytic genes and impaired EBV-specific T-cell responses were
demonstrated in SLE patients , . However, in contrast to
our findings in CFS, increased levels of EBV/EBNA-1 directed
antibodies were reported for SLE as well as for patients with
multiple sclerosis , . Further EBV-specific polyfunctional
T cells were shown to have higher levels of PD-1 in SLE compared
to healthy controls , a finding we did not observe in our
Finally, we think the altered pattern of the specific immune
response to EBV may be suitable as a diagnostic marker for CFS.
The most prominent finding is the very low or absent B-cell
memory response to EBV in the majority of CFS compared to
healthy controls. Therefore we are currently evaluating the
antibody responses against a broad variety of EBV peptides from
8 different proteins by an EBV seroarray. Further, we are
evaluating fluorescence-labelled EBNA- and VCA peptides for the
ex vivo quantitation of specific memory B cells by flow cytometry.
Taken together, our study provides clear evidence that
deficiency of EBV-specific immune response is present in CFS.
As EBV is known to be controlled by cell-mediated immunity, a
diminished memory T- and B-cell response may result in impaired
control of EBV. EBV replication is risk factor for development of
lymphomas and autoimmune diseases both occurring at enhanced
frequencies in CFS patients [83,84].
This paper is dedicated to Ruediger von Baehr who passed on June 21,
Conceived and designed the experiments: ML KS HDV CS. Performed
the experiments: ML KS SB ST. Analyzed the data: ML KS CG SB CS
MK. Contributed reagents/materials/analysis tools: CG UK NU LH CD
VvB CM PR. Wrote the paper: ML KS CS HDV.
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