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

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

Chronic fatigue syndrome (CFS) is a heterogeneous disorder characterized by severe prolonged unexplained fatigue and a variety of associated symptoms such as arthralgias, myalgias, cognitive dysfunction, and severe sleep disturbances. 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. Unfortunately there is no official test for CFS and no known cure. Moreover, HHV-6 has been associated with many of the neurological and immunological findings in patients with CFS. But the researchers say the study’s power rests in their access to 16 months of blood samples for each patient – a collection allowing for an unprecedented longitudinal look at CFS. Mady Hornig, these samples allowed the researchers to get as close to the brain – long thought to be a key area in chronic fatigue syndrome – as they could. 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.

Most of the postulated etiologies of CFS (e.g., infection, immune and hormone perturbations) affect sleep; and, conversely, primary sleep disorders, sleep deprivation and experimental disruption of sleep produce many of the features of CFS (e.g., fatigue, impaired cognition, joint pain and stiffness) [7–10]. Common physical and laboratory findings for FM include chronic aching, stiffness, sleep disturbances, pain, headaches, anxiety, depression, fatigue, low levels of adenosine triphosphate (ATP) (the energy currency of the cell), and intestinal disturbances among others. With Dr. He most recently discovered a highly dangerous virus that recently jumped into humans called MERS (Middle Eastern Respiratory Syndrome Coronavirus). Most patients are in excellent condition until they experience a sudden attack of “flu-like” symptoms including low-grade fever, sore throat, and muscle and joint pains. It has also been observed that many infectious pathogens implicated in ME/CFS can affect the central nervous system. In his presentation he stated almost 30% of his patients test positive for HHV-6 or human cytomegalovirus (HCMV) (PCR, rapid culture, antigenemia), and a whopping 50% test positive for Epstein-Barr virus (EBNA).

The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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]. Now we have access to 23andme for genetic data we can see specific gene polymorphisms that may affect the person with CFS. In about 10-20% of cases, follow-up shows neurological damage. 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].

pylori modulating the conversation of EBV to it’s lytic phase. showed reduced proliferative responses of lymphocytes and reduced frequencies of CD4+ T cells [14]. The patients produced antibodies specifically designed to identify and neutralize those proteins. Powerful enough for scientists to tell people to not take it with drugs like tamoxifen that block estrogen (even though I’m not sure they understand how phytoestrogens work and the role of estrogen in breast cancer anyway!). 1998;158:908–14. 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]. We propose that the human cardiac myofiber, like the B lymphocyte for EBV and the mononuclear progenitor cell for HCMV, is a site of noninfectious, episome-mediated persistent infection.

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), 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]. Meet Our Team, Volunteer, or Donate. 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.
Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue Syndrome

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 Charité, 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].

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. As at his last public talk, he urged patients to get active and enlist their congressman in  their cause. 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.

Peterson called for double-blinded, placebo-controlled studies to further study Vistide’s efficacy and mechanism of effect. Rats- Lipkin’s  study of New York City’s second most common resident – rats – revealed they carried an amazing array of pathogens including Escherichia coli, Clostridium difficile, and Salmonella enterica, Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. 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. Detection of EBV-DNA in PBMCs was done by nested PCR for EBER-1 with the following primers forward 5′-TCC CGG GTA CAA GTC CCG-3′ and reverse 5′-TGA CCG AAG ACG GCA GAA AG-3′ at 900 nM. Detection has been performed with probe FAM-5′-TGG TGA GGA CGG TGT CTG TGG TTG TGT T-3′-TAMRA (Eurofins MWG Operon, Ebersberg Germany) at 5 µM.

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 5′-CCA GAG CGC AGC TAT CGG T-3′ at 900 nM and reverse 5′-CAC GTT TGG CAT GGA TAG CAC -3′ at 50 nM and the probe FAM – 5′-GCA AGT GAG GCC TAT CTG GTT GGC CTT T-3- TAMRA (Eurofins MWG Operon, Ebersberg Germany) at 5 µM. For BZLF-1 the following primers forward 5′-AAATTTAAGAGATCCTCGTGTAAAACATC-3′ and reverse 5′-CGCCTCCTGTTGAAGCAGAT-3′ at 30 pM were used. Detection has been performed with probe FAM-5′-ATAATGGAGTCAACATCCAGGCTTGGGC-3′-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/µg DNA and BZLF-1 copies/µg cDNA were calculated in accordance to standard EBV-copies [60]. Results ≥35 copies/µg DNA/cDNA were regarded as positive.

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. [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 µg/ml (InvivoGen, CA, USA) in RPMI 1640 (PAA Laboratories, Cölbe, Germany) supplemented with Penicillin/Streptomycin 100× and L-Glutamine at 2 mM and 10% FCS (both Biochrom, Berlin, Germany) and β-Mercaptoethanol at 50 µM (Merck, Darmstadt, Germany) for 7 days at 37°C in 5% CO2. 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). 2.5×106 B cells per well were kept in 1 ml IMDM (PAA Laboratories, Cölbe, Germany) with 10% heat-inactivated FCS (Valley Biomedical, Winchester, VA, USA), 5 µg/ml insulin/transferrin and 5 ng/ml selenium (all Sigma Aldrich, Schnelldorf, Germany), 1.25 µg/ml CpG (Invivogen, CA, USA), 300 U/ml IL-2 (Chiron-Behring, Liederbach, Germany), 12.5 ng/ml IL-10 (ImmunoTools, Friesoythe) and 500 ng/ml IL-21 (ImmunoTools, Friesoythe, Germany) and 0.5 µg/ml anti-CD40 monoclonal antibody (R&D Systems, MN, USA.

Cells were cultured for 7 days at 37°C in 5% CO2. After stimulation, the cells were transferred at a concentration of 1×106/100 µl into a 96-well multiscreen HTS-IP filter plate (Merck Millipore, MA, USA) pre-coated with purified, recombinant EBV-VCA at 0.1 µg/well (tebu-bio, Le-Perray-en-Yvelines, France) and EBV-EBNA-1 at 1 µg/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 µg/well and cells were seeded at a concentration of 1.25×104/100 µl, 6250/100 µl and 3125/100 µl for 6 h. Secreted IgGs were detected using an anti-human IgG, F(ab′)2 fragment coupled to Biotin at 1 µg/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 µg/ml SEB (Sigma-Aldrich, Schnelldorf, Germany), 1 µg/ml EBV total lysate or 1 µg/ml of the EBV peptide EBNA-1 (JPT, Berlin, Germany) for 48 h. 2×106 PBMCs were kept in 1 ml serumfree RPMI (PAA Laboratories, Cölbe, Germany) with 2% Hepes buffer, 1% L-glutamin (Biochrom, Berlin, Deutschland) and 0.5% gentamycin (Merck, Darmstadt, Germany). IFN-γ, IL-10, IL-2 and TNF-α were measured in cell culture supernatants with a MPXHCYTO-60K Multiplex-Immunoassay (Merck Millipore, MA, USA) on a Luminex® 200™ (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 [62]. After overnight incubation of PBMCs in IMDM (PAA Laboratories, Cölbe, Germany) containing 10% AB serum (Valley Biomedical, Winchester, VA, USA) and supplemented with Penicillin/Streptomycin 100× and L-glutamine at 2 mM (both Biochrom, Berlin, Germany) at 37°C in 5% CO2, in 96-well round bottom plates at a concentration of 2×105 cells per well with 50 IU/mL rhIL-2 (Chiron-Behring, Liederbach, Germany) and 10 ng/mL IL-7 (ImmunoTools, Friesoythe, Germany). On day 3, 5 and 7 media and IL-2 at 50 ng/µl were renewed. IL-7 at 5 ng/µl was added on day 7 of culture, and cells were harvested, washed and stained for cytokines.

Intracellular and extracellular staining was applied for T-cell analysis after 10 days of expansion. 2×106 PBMCs were restimulated with an EBNA-1 or CMV-pp65 peptide pool (JPT, Berlin, Germany) at (1 µg/mL) or DMSO (Sigma Aldrich, Schnelldorf, Germany) as negative control for 5 h. Brefeldin A (7.5 µg/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-α and 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.

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