Abstract Background Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. Methods We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. Results EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Conclusion Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

Citation: Loebel M, Eckey M, Sotzny F, Hahn E, Bauer S, Grabowski P, et al. (2017) Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray. PLoS ONE 12(6): e0179124. https://doi.org/10.1371/journal.pone.0179124 Editor: Joseph S. Pagano, University of North Carolina at Chapel Hill, UNITED STATES Received: June 22, 2016; Accepted: May 24, 2017; Published: June 12, 2017 Copyright: © 2017 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. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant 10158447 from the "Europäischen Fonds für regionale Entwicklung (EFRE)" of the European Union grant "Investition in Ihre Zukunft". The funder provided support in the form of salaries for authors PH, EM, UR, JZ and ML, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. The work was further supported by the Lost Voices Foundation e.V., and by a flex fund grant (07-905) from the German Center for Infection Research. The authors ME, JZ, PH and UR are employed by a commercial company, JPT Peptide Technologies GmbH, and received support in the form of salaries. JPT Peptide Technologies is not the funder of this study, but the recipient of the funding. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Maren Eckey, Johannes Zerweck, Pavlo Holenya and Ulf Reimer are employed by JPT Peptide Technologies GmbH. The commercial affiliation (JPT Peptide Technologies GmbH) does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Introduction Epstein-Barr Virus (EBV) infection plays a critical role in various autoimmune diseases such as Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) or Sjögren's syndrome [1–6]. There is a link between infectious mononucleosis and an increased risk for MS and seroprevalence of EBV is close to 100% in MS [7–9]. Several studies show homologies of EBV sequences with human autoantigens such as myelin basic protein for MS [10, 11] and smith antigen for SLE [12]. With an estimated prevalence of 0.3% CFS affects more people than MS and SLE [13]. CFS is a multisystem disorder characterized by severe fatigue with the hallmark symptom of exertion intolerance and post-exertional delay to recover, and cognitive dysfunctions [14, 15]. CFS is probably a heterogeneous disease, but currently no diagnostic biomarkers are available [16–20]. Some studies describe immune dysregulation and activation in CFS patients [21–23]. 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 evidence for a potential role of EBV in CFS comes from many studies. In 1984 DuBois first described patients with the mononucleosis syndrome suffering from long-lasting fatigue and serological evidence of EBV reactivation [24] followed by a number of studies describing patients with CFS with serological evidence of chronic active EBV infection [25]. A first placebo-controlled trial with acyclovir in CFS patients with serological active EBV infection performed by Straus et al. showed no efficacy [26] while trials with valacyclovir and valganciclovir showed moderate improvement in some patients [27, 28]. Enhanced EBV-specific antibodies against VCA, early antigen and EBV DNAse as well as persistent IgM antibodies were described in numerous studies [29–37] but findings were not consistent [38–40]. In a previous study, we found an elevated IgM response against the late VCA antigen but a lack of antibodies and memory B cells against EBNA-1 in another subset of EBV-positive CFS patients [41]. A diminished EBNA-1 IgG production was also reported in severe infectious mononucleosis and chronic active EBV infection [42–44]. In the present study, we therefore wanted to comprehensively analyse the antibody response against all major EBV proteins in a large cohort of CFS patients. Further, we wanted to answer the question if reactivation of EBV occurs more frequently in CFS patients. The EBV DNA genome is large and contains over 100 protein-coding genes [45, 46]. In primary infection, EBV undergoes a short period of lytic replication in oral and nasal epithelium [47–49]. The orally transmitted EBV initially targets the mucosal epithelium and remains in a life-long latency in memory B cells [50–52]. In healthy subjects the EBV genome in B cells usually remains latent in the so-called latency phase 0 which maintains the genome in a quiescent state [53–56]. This latency is controlled by NK- and T-cell responses. Frequent replication occurs in epithelial cells of the pharynx. Latency I is characterized by the expression of EBNA-1, latency II by latent membrane proteins (LMP)-1 and LMP-2, and latency III by EBNAs 2–6 [57, 58]. 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 [59]. There are EBV type-specific sequence variations in the EBNA genes that are used to characterize the two EBV major subtypes I or II [44, 60]. The EBV types differ in their capacity to immortalize human B cells with type II EBV as the less efficient strain [61]. In this study, we performed a mapping of the IgG response against 14 EBV proteins by seroarray using overlapping peptide pools in CFS patients and healthy controls. The HHV4 proteome consists of close to 90 proteins of which 8 have been functionally classified as capsid, 14 as membrane protein, 6 as nucleotide metabolism, 9 as latency, 5 as packaging, 7 as replication, 11 as transcription factors, transactivators or involved in signaling and 11 as tegument. We selected one protein from each of these classes except for the latency class where we selected 5 and replication where three were selected. Reactivity against these EBV proteins observed in CFS and healthy controls in our study was rather similar with an enhanced reactivity in CFS patients against a repeat region in EBNA-6.

Materials and methods Study population CFS patients were diagnosed at the Charité outpatient clinic for immunodeficiencies at the Institute of Medical Immunology at the Charité Universitätsmedizin Berlin between 2011 and 2015. Patients with MS were recruited at the Department of Neurology and patients with SLE at the Department of Rheumatology at the Charité Universitätsmedizin Berlin [8]. Samples of 50 patients with cancer-related fatigue and 50 without fatigue following chemotherapy for Hodgkin’s lymphoma were provided by the University Hospital of Cologne and the German Hodgkin Study Group (GHSG). Diagnosis of CFS was based on Canadian Criteria [62] and exclusion of other medical or neurological diseases which may cause fatigue. Patients with systemic steroid or immunosuppressant therapy or a diagnosis of primary immunodeficiency were excluded from this study. Around 80% of our patients had an onset of disease after acute infection. Baseline demographic characteristics of the patients are shown in Table 1. Controls were recruited from staff and did not suffer from fatigue. However, neither clinical nor laboratory assessment was performed for controls. The study was approved by the Ethics Committee of Charité Universitätsmedizin Berlin in accordance with the 1964 Declaration of Helsinki and its later amendments and patients gave written informed consent. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Demographic characteristics. https://doi.org/10.1371/journal.pone.0179124.t001 Blood samples Serum obtained from patients and healthy persons was stored in aliquots at -80°C. Peptide seroarray IgG antibody responses to EBV peptides were determined by peptide microarrays as described before [8]. In brief, peptides were synthesized using SPOT synthesis and immobilized onto glass slides (JPT Peptide Technologies, Berlin). Serum samples of healthy donor controls and patients were incubated for 1h and specific antibodies were detected by fluorescently labelled anti-IgG antibodies. Signals were further processed for data analysis and statistical evaluation. Data are reported as arbitrary fluorescence units. Based on our previous study [8] in which we had included two EBV seronegative healthy controls (EBV-VCA- and EBNA-IgG negative) revealing no or only little background reactivity, responder samples were defined as a signal intensity of > 5,000 Units to exclude background signals. The peptide library contained 3054 peptides covering the full-length EBV proteins BALF-2, BALF-5, BFRF-3 (VP26), BLLF-1, BLLF-3, BLRF-2, BMRF-1, BZLF-1, EBNA-1, EBNA-3, EBNA-4, EBNA-6, LMP-1 and LMP-2 of different EBV strains. Additionally, a relatively conserved region from the N-terminus of VP1 (AA 42–75) of different Coxsackieviruses from the genus Enteroviridae which is described as highly reactive with patient sera was covered by 164 overlapping peptides [63]. The library was printed onto functionalized glass slides in triplicates. The proteins were represented as peptides consisting of 15 amino acids (15-mer) which exhibited optimal overlap of 11 amino acids in most cases to cover the EBV sequence diversity including but not limited to strains of type I (B95.8) and type II (AG876) at minimum peptide numbers (S1 and S2 Files). Multiwell assay IgG antibody responses against a selection of 128 EBV peptides were determined as described elsewhere [64]. In brief, sera were incubated in parallel on multiple identical mini-arrays containing identical copies of triplicates of the peptide library combined on one microarray slide where four slides yielded an incubation frame possessing the dimensions of a 96-well microtiter plate and enabling downstream procedure identical to ELISA (S1 File). The selection of the peptides was based firstly on the p-value for the comparison of the healthy and the CFS patients, respectively. Secondly, peptides with high variances in the signals and negative controls were added. ELISA Selected peptides were synthesized containing an additional C-terminal glycine, a spacer molecule and biotin (JPT Peptide Technologies). 100 nM of biotinylated peptide was coated on a 96 well Streptavidin-plate (Nunc Thermo Scientific) for 1 h at RT. After blocking for 1 h at 30°C serum samples were diluted 1:1,000 and 1:10,000 and incubated for 1 h at 30°C. Secondary antibody (anti-human IgG-HRP (goat), Invitrogen) was diluted 1:4,000 and incubated for 1 h at 30°C, then TMB substrate was added (Sigma Aldrich). Plates were measured at Tecan GENios at 620 nm (S1 File). Detection of EBNA-6 protein EBNA-6 was expressed in HEK293T cells as C-terminally hexahistidine-tagged full-length protein and purified from cell lysates using Nickel-NTA beads (Qiagen). Approximately 50 ng of purified EBNA-6 were spotted onto nitrocellulose membranes (Millipore) and allowed to dry. Subsequently, membranes were blocked with 5% skim milk powder in PBS for 1 h before overnight incubation with patient sera diluted 1:1,000 in 3% skim milk/PBS. Next day, the membranes were incubated for 1 h with a 1:10,000 dilution of IRDye 800CW goat anti-human IgG (Rockland Immunochemicals) in TBS + 0.05% Tween-20 (TBST) and then washed 4 x 5 min in TBST. IR detection was performed with an Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified with the analysis software provided. EBV load and calculation of EBER copies Detection of EBV viral load was done by a quantitative real-time PCR analysis (qPCR) for EBV EBER-1 (Table 2). The DNA samples from throat washing samples were obtained by gargling with 10 ml of water and washed twice with PBS. The pellet was resuspended in 200 μl of AL-Lysis buffer (QIAamp DNA Blood mini Kit from QIAGEN) for DNA preparation according to manufacturer’s instructions. 100 ng DNA template per reaction was added to a final volume of 25 μl. A dilution series of Namalwa DNA, a positive EBV cell line with known EBER copies per specific amount of Namalwa DNA, was used as positive control and to calculate the EBER copies per μg DNA/cDNA of each analysed sample. Results over 35 EBER copies per template DNA or cDNA were regarded as positive. Duplicate measurements of each sample were performed on a 7500 Real Time PCR System (Applied Biosystems Life Technologies). The amplification was performed at 42 cycles with 2 min 50°C, 10 min 95°C and 15 sec 95°C and finally 1 min at 60°C. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Primer and probe for EBV load and type PCR. https://doi.org/10.1371/journal.pone.0179124.t002 Detection of EBV type I and II PCR was performed using primer spanning the EBNA-6 (EBNA-3C) gene (Table 2) as previously described [44]. The primer flank regions in the EBNA-6 gene of type-specific variations resulting in different product fragments with a size of 153 bp for EBV type I and 246 bp for EBV type II. 100 ng DNA template of the throat washing samples were used per reaction. The amplified products were investigated by gel electrophoreses analysis in a 2% agarose gel and visualized by ethidium bromide. Statistical analysis Statistical data analyses were done using R (www.r-project.org) and the software GraphPad Prism 6.0. Nonparametric statistical methods were used. Continuous variables were expressed as median and interquartile range (IQR). Univariate comparisons of two independent groups were done using the Mann-Whitney-U test. Contingency analysis was done by Fisher’s exact test. Correlation analysis was performed by nonparametric Spearman coefficient r. A two-tailed p-value of <0.05 was considered statistically significant.

Discussion In this study we performed a comprehensive analysis of IgG responses against a peptide library of the major EBV proteins. In healthy controls we observed the strongest and broadest antibody responses against the latency proteins EBNA-1, -3, -4, and -6, and the lytic proteins BALF2, BALF5, and BZLF1. This finding is in accordance with studies analysing the IgG response against EBV proteins in which strong IgG responses against both EBNA proteins and BZLF1 are found and used for serodiagnosis [67–73]. The strong enhanced IgG response against various EBV proteins in MS and SLE shown in previous studies was confirmed in this and for MS in our prior study [8]. In contrast, although total IgG response against EBV peptides was higher in CFS the pattern of IgG responses was more similar to healthy controls. Further, we had performed a subgroup analysis in patients with Hodgkin lymphoma showing no difference in IgG EBV response pattern in patients with and without fatigue. Interestingly, we observed a significantly enhanced IgG response against EBNA-6 peptides spanning a repeat region in CFS patients compared to healthy controls. A 21 aa long peptide comprising the repeat region of EBNA-6 was already described by Rajnavölgyi et al. as a HLA-DR restricted T cell epitope [74]. Further we could show a close correlation between IgG responses against EBNA-6 peptide and a recombinant EBNA-6 protein suggesting that this peptide is recognized in the protein and is an immunodominant epitope. If the enhanced IgG response against EBNA-6 may be of diagnostic relevance in a subgroup of patients needs to be analysed in a longitudinal study. Cross-reactivity of EBV-specific IgG with human antigens due to antigenic mimicry is known to trigger pathogenic immune responses in SLE and MS [75–77]. We thus performed a sequence comparison of the EBNA-6 repeat region with human proteins and identified a 7 amino acids homologous sequence in the LPO protein, an enzyme producing oxidants and secreted by mammary, salivary and mucous glands of the bronchia. LPO is involved in immune defence with broad activity against bacteria and viruses [78–80]. Interestingly, we detected a correlation of EBNA-6 peptide and protein IgG with LPO peptide IgG levels. However, we observed no elevated levels of LPO protein IgG in patients with CFS suggesting that the peptide is not recognized in the recombinant protein. Interestingly, human TPO shows a sequence homology with 5 (comprising 4 identical aa) of the 7 amino acids of the EBNA-6 repeat region as well. Autoantibodies against TPO and hashimoto's thyroiditis are detected in 10–20% of patients with CFS and lead to thyroid destruction through antibody dependent cellular cytotoxicity and complement activation [81]. We could, however, not observe elevated IgG responses against the TPO peptide in our study. Two other proteins showing homology to the EBNA-6 repeat region, the enzymes OTC and PFK arouse our interest because of their metabolic function (Table 3). Data from Yamano et al. revealed higher ornithine/citrulline ratio in CFS patient which could be explained by reduced OTC activity [66]. The PFK catalyses the rate limiting step of glycolysis and inhibition of the enzyme could play a role in metabolic alterations in CFS [65, 66]. However, as for LPO protein, we observed no reactivity of CFS patients against these two proteins. There exist two major EBV subtypes type I and type II EBV with sequence variations in several EBV proteins. The prevalence of EBV serotypes varies in different geographic regions [51]. While healthy subjects usually are resistant to coinfection with another subtype, in immunodeficiency infection with two EBV types is frequently found [82, 83]. In our study, there was a significantly lower IgG response against peptides from type II specific regions compared to type I specific regions in both CFS patients and controls. EBV frequently reactivates in the oropharynx and can be detected in throat washing samples in 20–70% in healthy donors [84, 85]. Our data show a similar prevalence of EBV reactivation in 30–40% of samples and the infection with type I EBV in both healthy controls and CFS patients providing no evidence for EBV coinfection or prevalence of another EBV serotype in CFS patients. The prevalence of EBV type I is in accordance with another study showing that it is more frequent in northern countries [51]. The observed antibody responses against peptides derived from sequences of EBV type II strains might be due to cross reactivity. In conclusion, we could show the suitability of the EBV peptide microarray to analyse the seroresponse against EBV. Further, we could identify multiple peptide epitopes of EBV proteins recognized in healthy controls and patients. Our seroarray data and the similar prevalence of EBV in throat washings in CFS compared to healthy controls, argue against a pathogenic role of EBV reactivation in CFS. The enhanced IgG response against an EBNA-6 repeat sequence may point to a potential antigenic mimicry and requires further studies.

Author Contributions Conceptualization: CS. Data curation: UR ML. Formal analysis: ML ME PG UR UB CS. Funding acquisition: UR CS. Investigation: ML ME EH SB PG JZ PH LGH KW CM FS. Methodology: ML ME EH SB PG JZ PH LGH KW CM UR CS. Project administration: UR CS. Resources: PB JUR FH KR HDV UB UR CS. Supervision: UR CS. Visualization: ML EH SB FS. Writing – original draft: ML CS. Writing – review & editing: ME UR LGH KW PG JZ PH UB KR FS.