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Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue Syndrome

Authors:
  • Labor Berlin - Charité Vivantes

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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-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4(+) and CD8(+) T-cell 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.
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Deficient EBV-Specific B- and T-Cell Response in Patients
with Chronic Fatigue Syndrome
Madlen Loebel
1
*
.
, Kristin Strohschein
1,2.
, Carolin Giannini
1
, Uwe Koelsch
3
, Sandra Bauer
1
,
Cornelia Doebis
4
, Sybill Thomas
1
, Nadine Unterwalder
3
, Volker von Baehr
4
, Petra Reinke
5,6
,
Michael Knops
1
, Leif G. Hanitsch
1
, Christian Meisel
1,3
, Hans-Dieter Volk
1,5
, Carmen Scheibenbogen
1,5
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
Nephrology, Charite
´University Medicine Berlin, Germany
Abstract
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
+
and CD8
+
T-cell
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
¨r
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.loebel@charite.de
.These authors contributed equally to this work.
Introduction
Chronic Fatigue Syndrome (CFS) is characterized by severe
fatigue with typical post-exertional delay to recover from
exhaustion, cognitive dysfunctions and flu-like symptoms [1],
[2]. CFS is diagnosed based on clinical Center of Disease Control
criteria scores known as Fukuda criteria [3] or on the Canadian
Consensus Definition from 2004 [1]. Diagnosis of CFS is often
restrained as many symptoms are not disease-specific and no
diagnostic test could be established for CFS so far [4], [5], [6], [7].
Hallmarks of CFS are immune dysregulation and immune
activation [8], [9], [10]. Diminished natural killer (NK)-cell
cytotoxicity and reduced NK-cell derived perforin have been
repeatedly reported for CFS patients [8], [10], [11]. Furthermore,
increased frequencies of activated HLA-DR class II-positive CD8
+
T cells were proposed as immunological activation markers in CFS
[10], [12], [13]. Straus et al. showed reduced proliferative
responses of lymphocytes and reduced frequencies of CD4
+
T
cells [14]. Similarly Curriu et al. reported diminished proliferation
of T cells but enhanced frequencies of regulatory T cells [15].
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 [16], [17].
Furthermore, the group of Skowera et al. reported an effector
memory cell responsiveness bias towards type 2 in patients with
CFS [12].
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 [18], [19], [20], [21].
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 [22], [23], [24], [25],
[26], [27]. 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 [27], [28], [29], [30], [31] a finding
that was not confirmed by others [32], [33]. 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 [34], [35], [36], but
not in others [37], [38].
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 [39], [40]. 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 [31], [41]. No differences in IgG titers
against EBV-VCA, EBV nuclear antigen (EBNA)-1 and EA were
reported in other studies [37], [42], [43].
The orally transmitted EBV initially targets the mucosal
epithelium and remains in a life-long latency in memory B cells
[44], [45], [46]. 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
[47], [48]. [49], [50]. 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 [51],
[52]. 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 [53]. 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 [33], [37], [42].
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 [56] and
chronic HIV infection [57], [58], [59].
Materials and Methods
Ethics statement
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 [3]. 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 samples
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
probe FAM-59-ATAATGGAGTCAACATCCAGGCTTGGGC-
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
n63387
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
3
(%) 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).
doi:10.1371/journal.pone.0085387.t001
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e85387
standard EBV-copies [60]. Results $35 copies/mg DNA/cDNA
were regarded as positive.
Enzyme-linked immunosorbent assay (ELISA)/Enzyme
immunoassay (EIA)
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.
ELISpot assay
Analysis of memory B cells was adapted from Crotty et al. [61].
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
2
. 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
6
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%
CO
2
. After stimulation, the cells were transferred at a concentra-
tion of 1610
6
/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
4
/
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.
Cytokine analysis
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
6
PBMCs were
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
TM
(Luminex, TX, USA) according to manufacturer’s instructions.
T-cell expansion
EBV-specific memory T cells were analyzed after stimulation
with EBNA-1 or CMV-pp65 peptides and expansion in vitro as
recently described [62]. 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
2
,in
96-well round bottom plates at a concentration of 2610
5
cells per
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
6
PBMCs were
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 analysis
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
significant.
Results
A significant subset of CFS patients shows abnormal EBV
serology
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
CFS patients
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
from serology.
CFS patients show diminished T-cell cytokine response to
EBV
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.
doi:10.1371/journal.pone.0085387.g001
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 [60]. IFN-c
+
TNF-a
+
and IL-2
+
producing CD3
+
CD4
+
and CD3
+
CD8
+
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
+
TNF-
a
+
IL-2
+
triple producing - so called multifunctional - CD4
+
and
CD8
+
T cells. In addition, the frequencies of the IFN-c
+
TNF-a
+
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
+
and CD8
+
triple
cytokine producing T cells. Based on this definition a diminished
response was detected in 48% of CFS patients for triple CD4
+
T
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
+
TNF-a
+
double producing CD4
+
and CD8
+
T cells in Figure 5B
(n = 8).
Evidence of enhanced latent EBV replication in CFS
patients
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
+
IgM
+
CD27
+
marginal zone B cells, and (E) frequencies of IgD
2
CD27
+
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.
doi:10.1371/journal.pone.0085387.g002
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e85387
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.
Discussion
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 [63], [64], [65]. 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 [39], [31], [41]. 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
6
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.
doi:10.1371/journal.pone.0085387.g003
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 [66],
[67]. 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
+
T cells
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 [72], [73]. 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 [74].
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.
doi:10.1371/journal.pone.0085387.g004
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e85387
Figure 5. CFS patients show reduced EBV-specific memory T-cell response. (A) Comparison of cytokine producing CD4
+
(upper panels) and
CD8
+
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
+
and CD8
+
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.
doi:10.1371/journal.pone.0085387.g005
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.
doi:10.1371/journal.pone.0085387.g006
EBV Response in CFS Patients
PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e85387
pathogenesis [75]. 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 [56], [76], [77], [78], [79]. Abnormal expression of both
viral lytic genes and impaired EBV-specific T-cell responses were
demonstrated in SLE patients [80], [81]. 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 [80], [82]. Further EBV-specific polyfunctional
T cells were shown to have higher levels of PD-1 in SLE compared
to healthy controls [81], a finding we did not observe in our
patients.
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].
Acknowledgments
This paper is dedicated to Ruediger von Baehr who passed on June 21,
2012.
Author Contributions
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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EBV Response in CFS Patients
PLOS ONE | www.plosone.org 10 January 2014 | Volume 9 | Issue 1 | e85387
... 114 Several lines of research suggest that viral reactivation or viral persistence underlies ME/CFS, 98,112 including evidence of HHV-6 antigen in peripheral blood mononuclear cells in patients with CFS, 115 evidence of active and latent HHV-6/HHV-7 infection in plasma samples of CFS patients, 116 and deficient EBV-specific B-and T-cell James and Georgopoulos 5 memory response indicative of impaired ability to control early steps involved in EBV reactivation in CFS patients. 117 ME/ CFS is also associated with autoimmunity, 118,119 and recent evidence suggest that molecular mimicry between viral and human proteins may contribute to the condition. 120 Finally, both Class I and Class II HLA have been implicated in ME/CFS, linking immune-mediated pathways targeting foreign pathogens in the pathogenesis of the chronic, debilitating condition. ...
Article
Full-text available
Several foreign antigens such as those derived from viruses and bacteria have been linked to long-term deleterious effects on the brain and other organs; yet, health outcomes subsequent to foreign antigen exposure vary depending in large part on the host’s immune system, in general, and on human leukocyte antigen (HLA) composition, in particular. Here we first provide a brief description of 3 conditions characterized by persistent long-term symptoms, namely long-COVID-19, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and Gulf War Illness (GWI), followed by a brief overview of the role of HLA in the immune response to foreign antigens. We then discuss our Persistent Antigen (PA) hypothesis and highlight associations between antigen persistence due to HLA-antigen incongruence and chronic health conditions in general and the 3 “long” diseases above in particular. This review is not intended to cover the breadth and depth of symptomatology of those diseases but is specifically focused on the hypothesis that the presence of persistent antigens underlies their pathogenesis.
... However, current evidence remains inconclusive on whether the prevalence of these reactivations is either higher or lower in patients than in healthy controls (12). This conflicting evidence notwithstanding, ME/CFS patients show deficient B-and T-cell responses against EBV and altered antibody profiles when compared with healthy controls (10,(13)(14)(15). Finally, CD4+ T cells recognizing self-peptides on HLA-DR15, the strongest genetic risk factor for multiple sclerosis, have been shown to cross-react with peptides derived from EBV (16). ...
Article
Full-text available
Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.
... Furthermore, our results support a role for EBV dUTPase protein in this process by stimulating an extrafollicular Ab response as evidenced by the observed increase in splenic iNKT FH and MZ B cells, which could result in the formation of autoreactive B cells and, subsequently, the production of autoreactive Abs. We are proposing that in a subset of patients with ME/CFS there is increased abortive lytic replication of EBV (8) and possibly HHV-6, especially in those patients exhibiting a diminished EBV-specific B and T cell response (48), resulting in the increased release of EBV dUTPase possibly in exosomes (13). The release of virus dUTPase protein in the cellular microenvironment could result in chronic stimulation of the host immune system, potentially impairing the extrafollicular and GC Ab responses. ...
Article
Full-text available
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, debilitating multisystem illness of unknown etiology for which there is no cure and no diagnostic tests available. Despite increasing evidence implicating EBV and human herpesvirus-6A (HHV-6A) as potential causative infectious agents in a subset of ME/CFS patients, there are few mechanistic studies to address a causal relationship. In this study we examined a large ME/CFS cohort (n=351) and 77 controls and demonstrate a significant increase in activin A and IL-21serum levels, which correlated with seropositivity for antibodies to the EBV and HHV-6 protein deoxyuridine-triphosphate nucleotidohydrolase (dUTPase), but not CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation, generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH-function in ME/CFS. In vitro studies revealed that virus-dUTPases strongly induced activin A secretion while in vivo, EBV-dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Altogether, our data indicate abnormal germinal center (GC) activity in ME/CFS subjects and highlight a mechanism by which EBV and HHV6-dUTPases may alter GC and extrafollicular Ab responses.
... Altered EBVspecific antibodies and a lack of B-cell memory response to EBV have been detected and provide serological evidence of a deficiency of EBV-specific immune response in a subset of ME/CFS patients. These findings might also have potential for a future diagnostic marker (105). One hypothesis of an infectious related pathogenesis is that a virus as a trigger infection interacts with a dysfunctional cellular immunity including T and B cells as well as altered activity of NK cells leading to viral reactivation (10,94). ...
Article
Full-text available
Myalgic encephalomyelitis (ME) or Chronic Fatigue Syndrome (CFS) is a neglected, debilitating multi-systemic disease without diagnostic marker or therapy. Despite evidence for neurological, immunological, infectious, muscular and endocrine pathophysiological abnormalities, the etiology and a clear pathophysiology remains unclear. The gut microbiome gained much attention in the last decade with manifold implications in health and disease. Here we review the current state of knowledge on the interplay between ME/CFS and the microbiome, to identify potential diagnostic or interventional approaches, and propose areas where further research is needed. We iteratively selected and elaborated on key theories about a correlation between microbiome state and ME/CFS pathology, developing further hypotheses. Based on the literature we hypothesize that antibiotic use throughout life favours an intestinal microbiota composition which might be a risk factor for ME/CFS. Main proposed pathomechanisms include gut dysbiosis, altered gut-brain axis activity, increased gut permeability with concomitant bacterial translocation and reduced levels of short-chain-fatty acids, D-lactic acidosis, an abnormal tryptophan metabolism and low activity of the kynurenine pathway. We review options for microbiome manipulation in ME/CFS patients including probiotic and dietary interventions as well as fecal microbiota transplantations. Beyond increasing gut permeability and bacterial translocation, specific dysbiosis may modify fermentation products, affecting peripheral mitochondria. Considering the gut-brain axis we strongly suspect that the microbiome may contribute to neurocognitive impairments of ME/CFS patients. Further larger studies are needed, above all to clarify whether D-lactic acidosis and early-life antibiotic use may be part of ME/CFS etiology and what role changes in the tryptophan metabolism might play. An association between the gut microbiome and the disease ME/CFS is plausible. As causality remains unclear, we recommend longitudinal studies. Activity levels, bedridden hours and disease progression should be compared to antibiotic exposure, drug intakes and alterations in the composition of the microbiota. The therapeutic potential of fecal microbiota transfer and of targeted dietary interventions should be systematically evaluated.
... All these immune evasion m e c h a n i s m s t r i g g e r e d b y l a t e n t c e l l s i n d u c e a n immunodeficiency that allows EBV-transformed B cells, especially EBV latent I cells, to escape from immune surveillance. This could potentially help explaining the reduced EBNA-1-specific CD4 T-cell response, increased EBV latent cells (77), and increased EBV abortive lytic replication (EBV dUTPase in exosomes) (75), observed in a subgroup of patients with ME/ CFS. If the infiltration and proliferation of EBV-transformed B cells in the intestinal mucosa is related to the chronic inflammation that is observed in some ME/CFS patients (15,(92)(93)(94), remains unknown. ...
Article
Full-text available
Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) affects approximately 1% of the general population. It is a chronic, disabling, multi-system disease for which there is no effective treatment. This is probably related to the limited knowledge about its origin. Here, we summarized the current knowledge about the pathogenesis of ME/CFS and revisit the immunopathobiology of Epstein-Barr virus (EBV) infection. Given the similarities between EBV-associated autoimmune diseases and cancer in terms of poor T cell surveillance of cells with EBV latency, expanded EBV-infected cells in peripheral blood and increased antibodies against EBV, we hypothesize that there could be a common etiology generated by cells with EBV latency that escape immune surveillance. Albeit inconclusive, multiple studies in patients with ME/CFS have suggested an altered cellular immunity and augmented Th2 response that could result from mechanisms of evasion to some pathogens such as EBV, which has been identified as a risk factor in a subset of ME/CFS patients. Namely, cells with latency may evade the immune system in individuals with genetic predisposition to develop ME/CFS and in consequence, there could be poor CD4 T cell immunity to mitogens and other specific antigens, as it has been described in some individuals. Ultimately, we hypothesize that within ME/CFS there is a subgroup of patients with DRB1 and DQB1 alleles that could confer greater susceptibility to EBV, where immune evasion mechanisms generated by cells with latency induce immunodeficiency. Accordingly, we propose new endeavors to investigate if anti-EBV therapies could be effective in selected ME/CFS patients.
... Infectious pathogens such as viruses could be the original cause of the inflammatory state by activating antiviral immune responses, which then trigger systemic inflammation [5,26,34,45]. The virus infection most widely reported in relation to CFS is Epstein-Barr virus (EBV) since a considerable number of patients report symptom onset after contracting EBV [47][48][49]. However, it should be noted that an estimate of >90% of the adult population generally test positive for past EBV infection, and most do not develop ME/CFS. ...
Article
Full-text available
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic systemic disease that manifests via various symptoms such as chronic fatigue, post-exertional malaise, and cognitive impairment described as “brain fog”. These symptoms often prevent patients from keeping up their pre-disease onset lifestyle, as extended periods of physical or mental activity become almost impossible. However, the disease presents heterogeneously with varying severity across patients. Therefore, consensus criteria have been designed to provide a diagnosis based on symptoms. To date, no biomarker-based tests or diagnoses are available, since the molecular changes observed also largely differ from patient to patient. In this review, we discuss the infectious, genetic, and hormonal components that may be involved in CFS pathogenesis, we scrutinize the role of gut microbiota in disease progression, we highlight the potential of non-coding RNA (ncRNA) for the development of diagnostic tools and briefly mention the possibility of SARS-CoV-2 infection causing CFS.
Preprint
Full-text available
Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls. In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analysed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could be used to distinguish patients from healthy controls. A similar finding was obtained when comparing patients with noninfectious or unknown disease trigger to healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. When a bioinformatic analysis was performed on these epitopes, it revealed a potential molecular mimicry with several human proteins. To confirm these promising findings, a follow-up study will be conducted in a separate cohort of patients.
Chapter
There is increasingly compelling evidence that microorganisms may play an etiological role in the emergence of mental illness in a subset of the population. Historically, most work has focused on the neurotrophic herpesviruses, herpes simplex virus type 1 (HSV-1), cytomegalovirus (CMV), and Epstein–Barr virus (EBV) as well as the protozoan, Toxoplasma gondii. In this chapter, we provide an umbrella review of this literature and additionally highlight prospective studies that allow more mechanistic conclusions to be drawn. Next, we focus on clinical trials of anti-microbial medications for the treatment of psychiatric disorders. We critically evaluate six trials that tested the impact of anti-herpes medications on inflammatory outcomes in the context of a medical disorder, nine clinical trials utilizing anti-herpetic medications for the treatment of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) or schizophrenia, and four clinical trials utilizing anti-parasitic medications for the treatment of schizophrenia. We then turn our attention to evidence for a gut dysbiosis and altered microbiome in psychiatric disorders, and the potential therapeutic effects of probiotics, including an analysis of more than 10 randomized controlled trials of probiotics in the context of schizophrenia, bipolar disorder (BD), and major depressive disorder (MDD).KeywordsBipolar disorderClinical trialCytomegalovirusEpstein–Barr virusHerpes simplex virusMajor depressive disorderMicrobiomeSchizophrenia Toxoplasma gondii
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A quantitative PCR assay for the detection of human herpesvirus 6 (HHV-6) (variants A and B) and HHV-7 DNAs in clinical samples was developed. The assay uses a nonhomologous internal standard (IS) for each virus that is coamplified with the wild-type target sequence in the same vial and with the same pair of primers. This method allows for a correction of the variability of efficiency of the PCR technique. A standard curve is constructed for each experiment by coamplification of known quantities of the cloned HHV-6 or HHV-7 target templates with the respective IS. Absolute quantitation of the test samples is then achieved by determining the viral target/IS ratio of the hybridization signals of the amplification products and plotting this value against the standard curve. Using this assay, we quantitated the amount of HHV-6 or HHV-7 DNA in infected cell cultures and demonstrated an inhibitory effect of phosphonoformic acid on the replication of HHV-6 and HHV-7 in vitro. As the first clinical application of this procedure, we performed preliminary measurements of the loads of HHV-6 and HHV-7 in lymph nodes from patients with Hodgkin's disease and AIDS. Application of this quantitative PCR method should be helpful for elucidating the pathogenic roles of HHV-6 and HHV-7.
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• The complexities of the chronic fatigue syndrome and the methodologic problems associated with its study indicate the need for a comprehensive, system­ atic, and integrated approach to the evaluation, classi­ fication, and study of persons with this condition and other fatiguing illnesses. We propose a conceptual framework and a set of guidelines that provide such an approach. Our guidelines include recommendations for the clinical evaluation of fatigued persons, a revised case definition of the chronic fatigue syndrome, and a strategy for subgrouping fatigued persons in formal investigations.
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Background Chronic Fatigue Syndrome (CFS) is a debilitating neuro-immune disorder of unknown etiology diagnosed by an array of clinical manifestations. Although several immunological abnormalities have been described in CFS, their heterogeneity has limited diagnostic applicability. Methods Immunological features of CFS were screened in 22 CFS diagnosed individuals fulfilling Fukuda criteria and 30 control healthy individuals. Peripheral blood T, B and NK cell function and phenotype were analyzed by flow cytometry in both groups. Results CFS diagnosed individuals showed similar absolute numbers of T, B and NK cells, with minor differences in the percentage of CD4+ and CD8+ T cells. B cells showed similar subset frequencies and proliferative responses between groups. Conversely, significant differences were observed in T cell subsets. CFS individuals showed increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells, and lower proliferative responses in vitro and in vivo. Moreover, CD8 T cells from the CFS group showed significantly lower activation and frequency of effector memory cells. No clear signs of T-cell immunosenescence were observed. NK cells from CFS individuals displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined. Overall, T cell and NK cell features clearly clustered CFS individuals. Conclusions Our findings suggest that alterations in T-cell phenotype and proliferative response along with the specific signature of NK cell phenotype may be useful to identify CFS individuals. The striking down modulation of T cell mediated immunity may help to understand intercurrent viral infections in CFS.
Article
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A defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar "clinical" conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbach's alpha. Antibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13-16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS. Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls. There is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.
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Human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) are associated with a variety of conditions including rash, fever, and encephalitis and may play a role in several neurological diseases. Here luciferase immunoprecipitation systems (LIPS) was used to develop HHV-6 serologic diagnostic tests using antigens encoded by the U11 gene from HHV-6A (p100) and HHV-6B (p101). Analysis of the antibody responses against Renilla luciferase fusions with different HHV-6B p101 fragments identified an antigenic fragment (amino acids 389 to 858) that demonstrated ~86% seropositivity in serum samples from healthy US blood donors. Additional experiments detected a HHV-6A antigenic fragment (amino acids 751-870) that showed ~48% antibody seropositivity in samples from Mali, Africa, a known HHV-6A endemic region. In contrast to the high levels of HHV-6A immunoreactivity seen in the African samples, testing of US blood donors with the HHV-6A p100 antigenic fragment revealed little immunoreactivity. To potentially explore the role of HHV-6 infection in human disease, a blinded cohort of controls (n=59) and chronic fatigue syndrome (CFS) patients (n=72) from the US was examined for serum antibodies. While only a few of the controls and CFS patients showed high level immunoreactivity with HHV-6A, a majority of both the controls and CFS patients showed significant immunoreactivity with HHV-6B. However, no statistically significant differences in antibody levels or frequency of HHV-6A or HHV-6B infection were detected between the controls and CFS patients. These findings highlight the utility of LIPS for exploring the seroepidemiology of HHV-6A and HHV-6B infection, but suggest that these viruses are unlikely to play a role in the pathogenesis of CFS.
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Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.
Article
Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.
Article
▪A workshop was held 18 to 19 March 1991 at the National Institutes of Health to address critical issues in research concerning the chronic fatigue syndrome (CFS). Case definition, confounding diagnoses, and medical outcome assessment by laboratory and other means were considered from the perspectives of key medical specialties involved in CFS research. It was recommended that published Centers for Disease Control (CDC) case-definition criteria be modified to exclude fewer patients from analysis because of a history of psychiatric disorder. Specific recommendations were made concerning the inclusion or exclusion of other major confounding diagnoses, and a standard panel of laboratory tests was specified for initial patient evaluation. The workshop emphasized the importance of recognizing other conditions that could explain the patient's symptoms and that may be treatable. It was viewed as essential for the investigator to screen for psychiatric disorder using a combination of self-report instruments followed by at least one structured interview to identify patients who should be excluded from studies or considered as a separate subgroup in data analysis. Because CFS is not a homogeneous abnormality and because there is no single pathogenic mechanism, research progress may depend upon delineation of these and other patient subgroups for separate data analysis. Despite preliminary data, no physical finding or laboratory test was deemed confirmatory of the diagnosis of CFS. For assessment of clinical status, investigators must rely on the use of standardized instruments for patient self-reporting of fatigue, mood disturbance, functional status, sleep disorder, global well-being, and pain. Further research is needed to develop better instruments for quantifying these domains in patients with CFS.