Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

Peptides 76 (2016) 87–95

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Application of synthetic peptides for detection of anti-citrullinated peptide antibodies Nicole Hartwig Trier a , Bettina Eide Holm a , Ole Slot b , Henning Locht c , Hanne Lindegaard d , Anders Svendsen e , Christoffer Tandrup Nielsen f , Søren Jacobsen f , Elke Theander g , Gunnar Houen a,∗ a

Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark Department of Rheumatology, Glostrup Hospital, Nordre Ringvej 57, 2600 Glostrup, Denmark c Department of Rheumatology, Frederiksberg Hospital, Nordre Fasanvej 57, 2000 Frederiksberg, Denmark d Department of Rheumatology, Odense University Hospital, Søndre Boulevard 29, 5000 Odense C, Denmark e Epidemiology, Biostatistics and Bio-demography, Institute of Public Health, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark f Department of Rheumatology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark g Department of Rheumatology, Skåne University Hospital, Lund University, S-20502 Malmø, Sweden b

a r t i c l e

i n f o

Article history: Received 1 October 2015 Received in revised form 4 January 2016 Accepted 8 January 2016 Available online 12 January 2016 Keywords: Citrullinated epitopes EBNA-1 Streptavidin capture ELISA Peptides

a b s t r a c t Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n = 60), healthy controls (n = 40), systemic lupus erythematosus (n = 20), Sjögren’s syndrome (n = 40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRGCit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the peripheral joints with inflammation of the synovium and subsequent pannus formation, ultimately leading to joint destruction and loss of function. Therefore, early diagnosis and intervention is important to prevent rapid progression of the disease [1,2].

∗ Corresponding author. Fax: +45 32583876. E-mail address: [email protected] (G. Houen). http://dx.doi.org/10.1016/j.peptides.2016.01.005 0196-9781/© 2016 Elsevier Inc. All rights reserved.

Being an autoimmune disease, RA is associated with the presence of autoantibodies such as rheumatoid factors (RFs) [3]. RFs were originally used as the sole autoantibody for detection of RA. However, RFs have a modest RA disease specificity and are found in other autoimmune rheumatic diseases [4,5], hence other serological markers of RA have been requested. The search for novel and more disease-specific autoantibodies to RA has among others led to identification of anti-citrullinated protein antibodies (ACPAs) and anti-carbamylated protein antibodies (anti-CarP) [6–9]. Especially the detection of ACPA has become a key point for the diagnosis of RA and ACPA was recently added to the new RA classification criteria, in addition to RF [10]. ACPAs are detected in 70–80% of patients with RA and render a specificity of

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98% [6,7,11] and they may precede the clinical appearance of RA by more than 10 years and predict more severe clinical outcomes [12–17]. The two major genetic risk factors for RA, the shared epitope (SE) and PTPN22, are both independently associated with smoking and the generation of ACPAs [18,19]. Another proposed environmental risk factor is the bacterium Porphyromonas gingivalis, a periodontal pathogen that has been shown to be associated with RA, smoking and the SE [20]. ACPAs that target human citrullinated ␣-enolase cross-react with highly conserved sequences of P. gingivalis enolase, suggesting a possible mechanism of autoimmunity in RA [21,22]. In general, several studies of ACPA reactivity to viral citrullinated proteins and peptides in RA have been described [23–26]. Several specificities of ACPAs directed to citrullinated antigens such as vimentin, fibrinogen, collagen, ␣-enolase have been described [27–30]. Apart from the seemingly very important CitGly motif, no sequence similarity seems to prevail between the citrullinated epitopes. Comparative studies of protein sequences confirm that the main feature of citrullinated peptides recognized is the presence of Cit flanked by neutral amino acids such as Gly, Ser or Thr [6,31,32]. Similar repeats are commonly found in viral proteins [25,26]. One of the nuclear proteins encoded by Epstein-Barr virus (EBV), EBNA-1, contains in its N-terminal region a sequence (aa 35–58, viral citrullinated peptide (VCP) 1) with several ArgGly repeats, which, modified by the peptidyl arginine deiminase enzyme, are converted into Cit-Gly motifs [23]. This posttranslational modification of arginine residues is necessary to generate epitopes recognized by ACPAs [6,7]. The VCP1 peptide has been widely studied and has been found to detect RA-associated ACPAs. Similar studies have been described using a viral peptide corresponding to amino acids 338–358 (VCP2) of EBNA-2, which also contains several Cit-Gly motifs [26]. Antibodies to this peptide were found to be specific for RA and to be associated with erosive arthritis. Besides studies describing ACPA reactivity to the two citrullinated EBNA peptides VCP1 and VCP2, only limited knowledge is present in relation to EBV-related ACPA reactivity. In a recent study by Westergaard et al. describing EBV-induced immune responses in patients with RA, it was found that RA patients were prone to have increased IgA, IgG and IgM antibodies towards EBNA-1, however, when comparing the ACPA reactivity to individual sera that were positive to EBNA-1 IgA and IgG antibodies, no differences in ACPA levels were found, indicating that although RA patients have high titers of IgA and IgG to EBNA-1, are these responses not exclusively related to citrullinated epitopes [33]. These findings have been confirmed by studies describing immune responses to EBNA-1 in RA patients, where antibody reactivity specifically to the C-terminal amino acids 451–641 of EBNA-1 was found, where only three ArgGly motives are present [34]. In this study, we analyzed RA-associated antibody reactivity to overlapping citrullinated peptides covering the EBNA-1 sequence to determine their potential as a substrate for detection of ACPA. By studying the antibodies reactive with overlapping peptides in terms of their frequency, isotypes and correlations with anti-CCP2 and RF levels, we confirmed that antibodies to citrullinated EBNA-1 peptides have a high specificity and sensitivity for RA.

2. Materials and methods 2.1. Patient sera 10 CCP2-positive sera and 10 CCP2-negative sera were obtained from the biobank at Statens Serum Institut. The authors did not have direct contact with any of the patients and were neither involved in drawing or collection of samples. The sera were used anonymously.

60 RA sera diagnosed according to the American College of Rheumatology (ACR) classification criteria were enrolled in this study [35]. The sera were evaluated with respect to RF levels and anti-CCP2 concentrations. Sera from individuals with RA were obtained from the Department of Rheumatology, Glostrup Hospital, Department of Rheumatology, Frederiksberg Hospital, Department of Rheumatology, Odense University Hospital and Epidemiology, Biostatistics and Bio-demography, Institute of Public Health, University of Southern Denmark. 100 controls were studied, comprising following patient groups: healthy controls (n = 40), systemic lupus erythematosus (SLE) (n = 20) and Sjögren’s syndrome (SjS) (n = 40). SLE was diagnosed according to ACR criteria [36]. SjS was diagnosed according to the criteria of the American–European Consensus Group [37]. Sera from healthy donors were obtained anonymously from Rigshospitalet, Copenhagen, sera from individuals with pSS were obtained from the Department of Rheumatology at the Skåne University Hospital in Malmö. Sera from individuals with SLE were provided by the Department of Rheumatology, Rigshospitalet. 2.2. Peptides and peptide synthesis The viral EBNA-1 sequence (Uniprot KB ID: P03211.1), comprising 641 aa, was used as template to synthesize 68 overlapping peptides, which covered the aa sequence 1–111 and aa 320–641. The aa sequence 111–320 was left out, as this sequence only contains repetitive GA sequences and no Arg residues. Each peptide was 20 aa long, except from peptide 68, containing 21 aa. For every Arg-Gly motif, the Arg residue was replaced with a Cit residue. To ensure specific reactivity to a single citrullinated peptide, only one Cit residue was present in each peptide. Thus 20mer peptides containing several Arg-Gly motifs were synthesized several times. E.g., peptides 4–9, 10–14, 20–23, 24–28, 29–32, 33–35, 36–39 and 40–42 contained the same template sequence with the only difference being the localization of the Cit residue. Peptides 55 and 61 were left out as peptides of high purity could not be obtained. However, neither of these peptides contained ArgGly motifs. At the N-terminus, a biotin moiety was added unless otherwise stated. All peptides used are listed in Supplementary materials S1. Moreover, two synthetic peptides without relation of EBNA1 were applied in these studies as well, the cyclic citrullinated peptide 1 (CCP) 1 (HQCHQEST(Cit)GRSRGRCGRSGSK) [7,38], which originally was used for detection of ACPAs and a truncated version (SHQEST(Cit)GRSRGRS) of this peptide as well, which has been found to be very efficient for ACPA detection as well [32]. Moreover, a fusion peptide, containing the reactive regions of peptides 32-36-39 was added (RGGSGGRRG-Cit-GRERGERARACit-GGSREGRGRG-Cit-GEKRGRGRG). The peptides used for this study were obtained from Schäfer-N (Lyngby, Denmark) using standard Fmoc solid-phase peptide synthesis as previously described [39]. 2.3. Detection of antibodies by streptavidin capture enzyme-linked immunosorbent assay Citrullinated EBNA-1 peptide antibodies were detected in sera by streptavidin capture enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well maxisorp microtiter plates (Nunc, Roskilde, Denmark) were precoated with streptavidin (Sigma–Aldrich, St. Louis, Mo, USA) diluted in carbonate buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , 0.001% phenolred, pH 9.6) (SSI diagnostic, Hillerød, Denmark) to 1 ␮g/mL for 2 h at room temperature (RT) followed by coating with overlapping biotinylated EBNA-1 peptides diluted in PBS (10 mM Na2 HPO4 /NaH2 PO4 , 0.15 M NaCl, pH 7.2) (Statens Serum Institut) to 1 ␮g/mL for 2 h at RT. Sera diluted 1:200 in Tris-Tween-NaCl (TTN) buffer (0.05 M Tris, 0.3 M NaCl, 1% Tween

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Fig. 1. Antibody reactivity to overlapping EBNA-1 peptides analysed by streptavidin capture ELISA. (A) Reactivity of IgG anti-CCP2-positive sera (n = 10). (B) Reactivity of IgG anti-CCP2-negative sera (n = 10). (C) Reactivity of IgG healthy control sera (n = 10).

20, pH 7.4) (SSI diagnostic, Hillerød, Denmark) were then incubated in duplicates for 1 h at RT. After washing with TTN buffer, alkaline phosphatase (AP)-conjugated goat-anti human IgG, IgA or IgM (Sigma–Aldrich, St. Louis, Mo, USA), diluted in TTN to 1 ␮g/mL, was added to the wells and the plates incubated for 1 h at RT. For quantification of bound Abs, AP activity was determined with p-nitrophenylphosphate (Sigma–Aldrich, St. Louis, Mo, USA) (1 mg/mL) diluted in AP substrate buffer (1 M diethanolamine, 0.5 mM MgCl2 , pH 9.8) (SSI diagnostica, Hillerød, Denmark). The absorbance was measured at 405 nm, with background subtraction at 650 nm, using a Thermomax microtitre plate reader (Molecular Devices, Menlo Park, CA, USA). Samples were corrected for nonspecific reactivity in non-coated wells.

peptides screening using healthy donor sera, a non-specific reactivity of maximum 7% to the citrullinated peptides was tolerated. Based on these estimations, a cutoff of 0.5 was introduced. In order to ensure comparable reactivity between experiments, a positive control, containing a given specific citrullinated peptide and an ACPA positive donor pool, was obtained on each plate to ensure least possible intra-assay variations. Intra-assay variations within a −/+ range of 0.5 absorbance units were accepted. Absorbances of 2.8 were generally obtained for the positive controls.

3. Results 3.1. Antigenic regions within EBNA-1

2.4. Data interpretation Non-coated peptide wells were used to determine background levels and subtracted prior to data analysis. Based on individual

Citrullinated EBNA-1 peptides were synthesized as free Nterminally biotinylated peptides and used in a streptavidin capture ELISA to analyse antibody reactivity from 10 CCP2-positive sera, 10

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Fig. 2. Antibody reactivity to selected EBNA-1 peptides analysed by streptavidin capture ELISA. (A) Reactivity of IgG RA sera (n = 20). (B) Reactivity of IgG SLE sera (n = 20). (C) Reactivity of IgG healthy control sera (n = 40).

CCP2-negative sera (suspected to have RA) and 10 healthy donor sera. In total, 66 peptides were screened for ACPA reactivity (Fig. 1). The EBNA-1 peptides reacted primarily with IgG antibodies (Fig. 1), although occasionally sporadic reactivity was found with IgM antibodies as well (results not shown). Healthy donor sera (IgG) reacted with several of the peptides, as reactivity in clusters of peptides 4–19, 40–50, 53 and 66–67 was found. CCP2-negative IgG antibody reactivity was mainly found to peptides 17–19 and 39–63, although sporadic reactivity was found to the remaining peptides. Specific CCP2-positive IgG antibody reactivity was found to peptides 20–39 and peptides 57–58. Based on these findings the last-mentioned peptides were selected for further analysis. We then tested the reactivity of RA sera (n = 20), SLE sera (n = 20) and healthy control sera (n = 20) to the selected EBNA peptides

(20–39, 57, 58) by streptavidin capture ELISA (Fig. 2). No significant reactivity of ACPAs of the IgM and IgA isotype was observed (results not shown). Only the reactivity of IgG isotype from RA patient sera differentiated clearly from healthy controls and SLE disease controls. Collectively, the 22 selected EBNA-1 peptides reacted with all 10 of the RA sera positive for ACPA antibodies of the IgG isotype, whereas peptide 39 alone reacted with 8 out of 10 of the RA sera and one SLE serum. Moreover, antibody reactivity to the peptides differentiated between the location of the Cit residue within the peptide sequence, e.g., between the peptide template series analyzed (20–23, 24–28, 29–32, 33–35, 36–39). Here, a correlation between the number of reactive sera and the location of the Cit residue was found, as peptides containing Cit in the central region

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Fig. 3. Reactivity of RA sera (n = 60) to N- and C-terminal biotinylated EBNA-1 peptides analysed by streptavidin capture ELISA. N and C represent the actual location of the biotin moiety relative to the selected peptides 32, 36 and 39 to be analysed.

ranging from position 9–16 were notably better recognized than peptides containing Cit close to the N- or C-terminal end (aa 1–3 and 17–20). In general, the peptide template spanning peptides 36–39 (ARGGSRERARGRGRGRGEKR) with several Arg-Gly motifs was found to be the peptide template recognized the most, with the peptide, ARGGSRERARGRGRG-Cit-GEKR, containing the Cit residue in the central region 9–16, being the most reactive. 3.2. Citrulline presentation within the peptide epitopes As systematic overlapping peptides were used to determine ACPA reactivity, the actual location of the Cit residue within the peptides varied notably. This may cause sterical hindrance of the antibody access to the epitope, if Cit is positioned next to the biotin moiety. To determine the possible effect of sterical hindrance on antibody reactivity further, selected citrullinated EBNA-1 peptides 32 (GSGGRGRGGSGGRRG-Cit-GRER), 36 (A-CitGGSRERARGRGRGRGEKR) and 39 (ARGGSRERARGRGRG-Cit-GEKR) where equipped with either a N- or C-terminal biotin moiety and tested for reactivity in streptavidin capture ELISA (Fig. 3) using RA sera (n = 20). Among the sera tested, significant difference in reactivity was found. Most pronounced difference was observed with peptide 39, where 25% of the RA sera reacted with this peptide presented with a C-terminal biotin, while 70% of the RA sera reacted with the N-terminal biotinylated peptide. In all three examples, antibody reactivity was reduced as the Cit residue moved closer to the biotin moiety, as a reduction in reactive sera to peptides 32, 36 and 39 by 50, 32 and 67% was observed, respectively. These findings indicate that the location of Cit within the peptide and possibly relative to the location of biotin is essential for antibody reactivity. To avoid a possible sterical hindrance from the presence of biotin-streptavidin coating, we then tested the reactivity of RA sera (n = 20) to extended peptides (31-32-33, 35-36-37, 38-3940) with centered Cit residues by streptavidin capture ELISA. Moreover, a fusion peptide, containing the reactive regions of peptides 32-36-39 was added (RGGSGGRRG-Cit-GRERGERARACit-GGSREGRGRG-Cit-GEKRGRGRG), which, as the only peptide examined, contained three Cit residues. Peptide versions containing either a C- or a N-terminal biotin moiety were then screened for reactivity (Fig. 4). No significant difference in the number of reactive sera was found between the extended peptides and the 20mer peptides. In fact, one of the 20mer peptides reacted with more RA sera than the extended peptides, as peptide 32 with a N-terminal biotin reacted with 40% of the RA sera, while the extended peptide versions 31-

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Fig. 4. Reactivity of RA sera (n = 20) to selected 20mer and 40mer N- and C-terminal biotinylated peptides analysed by streptavidin capture ELISA. N and C represent the actual location of the biotin moiety relative to the selected peptides. The extended peptides (31-32-33, 36-37-38, 38-39-40) contained the full-length center peptide, which was extended with 20 amino acids in each terminal ends relative to citrulline, generating 41mer peptides, which covered the three specific peptides in question. Moreover, a fusion peptide, containing the reactive regions of peptides 32-36-39 was added (RGGSGGRRG-Cit-GRERGERARACit-GGSREGRGRG-Cit-GEKRGRGRG).

32-33 with N- and C-terminal biotinylation reacted with 15%. 70% of the RA sera reacted with peptide 39 with a N-terminal biotin, while 70% and 75% reacted with the extended versions 38-3940, containing either a C-terminal or a N-terminal biotinylation. A slight increase in the number of reactive RA sera was found to the extended peptide 35-36-37 with a N-terminal biotin compared to peptide 36, as the extended peptide was recognized by 50% of the RA sera, while peptide 36 with a C-terminal biotin was recognized by 40% of the sera. Thus, no general effect on antibody reactivity could be ascribed to the use of extended peptides. The mutual difference in antibody reactivity to N- and Cterminal biotinylated peptides was not restricted to the 20mer peptides containing Cit in the terminal ends, as the same difference in antibody reactivity was found to the extended peptides as well, although the 41mer peptides contained Cit in a central position. Interestingly, the fusion peptide 38-39-40 with a N-terminal biotin moiety reacted with 75% of the RA sera, however further studies, screening the reactivity of 40 healthy controls, revealed that 75% of the healthy controls reacted with the fusion peptide as well (results not shown). 3.3. Antibody binding to citrullinated filaggrin-derived peptides and selected citrullinated EBNA-1 peptides Next, we investigated the reactivity of RA sera (n = 60), healthy controls (n = 40), SjS sera (n = 40) and SLE sera (n = 20) to a selected EBNA-1 peptide panel, composed of peptides 28, 35, 36, 39 and 57, by streptavidin capture ELISA. The peptides were selected for further analysis, as some of them showed the highest reactivity with the first 20 RA sera screened for reactivity and as they complemented each other the most. CCP1 (HQCHQEST(Cit)GRSRGRCGRSGSK), originally used for detection of ACPA [38], was screened for reactivity, and a truncated version of this peptide, which previously has been found to be effectively recognized by a human monoclonal antibody [32] (Table 1). Peptides 39, 57 and 35 reacted with 53, 42 and 32% of the RA sera, respectively. In total, the five selected EBNA-1 peptides reacted with 67% of the RA sera. Moreover, disease specificity was determined in relation to SLE and SjS groups. Among the groups tested, two SLE patient sera were positive for reactivity to the peptide panel; 7 SjS patient sera were positive for reactivity to the peptide panel, while 6 healthy control sera were positive for reactivity to the peptide panel, yielding specificities of 90, 83 and 85%, respectively.

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Table 1 Reactivity of RA sera (n = 60), SLE sera (n = 20), SSA/SSB sera (n = 40) and healthy donor controls (n = 40) to citrullinated peptides analysed by streptavidin capture ELISA. Sera # Samples

RA 60

Sensitivity

SLE 20

Specificity SLE

SjS 40

Specificity SjS

HD 40

Specificity HD

28 35 36 39 57

5 19 8 32 25

8 32 13 53 42

0 0 2 1 0

100 100 90 95 100

1 1 4 3 4

98 98 90 93 90

1 3 3 2 0

98 93 93 95 100

CCP1/C-term Fil. C-term

43 30

72 50

2 0

90 100

6 3

85 93

4 0

90 100

EBNA-1 peptides Anti-CCP2 assay EBNA-1 pep/anti-CCP2a

40 46 52

67 77 87

2 2 –

90 90 –

7 – –

83 – –

6 0 –

85 100 –

EBNA-1 pos + anti-CCP2 pos EBNA-1 pep neg + anti-CCP2 pos EBNA-1 pep pos + anti-CCP2 neg EBNA-1 pep neg + anti-CCP2 neg

34 12 6 8

57 20 10 13

– – – –

– – – –

– – – –

– – – –

– – – –

– – – –

a

Either positive for antibodies to CCP2 or EBNA-1 peptides.

Table 2 Antibody specificities in reactive RA sera (n = 60).

Table 3 Antibody reactivity to citrullinated EBNA-1 peptides.

Antibody specificity

Reactive sera

Reactivity in%

No. of EBNA-1 peptides recognized

Reactive sera

Reactivity in%

Anti-CCP2, anti-EBNA-1, RF Anti-CCP2, anti-EBNA-1 Anti-CCP2, RF Anti-EBNA-1, RF RF Anti-CCP2 Anti-EBNA-1 No antibodies

34 0 11 3 3 1 3 5

57 0 18 5 5 2 5 8

0 1 2 3 4 5

19 9 16 11 5 0

32 15 27 18 8 0

Based on the presence of anti-CCP2, anti-EBNA-1 and RFs, reactive RA sera were divided into groups. As seen, more than 50% of the RA sera screened for antibody reactivity were positive for antibodies to all three antibody specificities.

When comparing the antibody reactivity of the selected EBNA1 peptides to the linear pro-filaggrin peptide and CCP1, 72% of the RA sera reacted with CCP1. However, 15% of the SjS sera reacted with this peptide as well (Table 1). 50% of the RA sera reacted with the linear pro-filaggrin peptide, which yielded disease specificity scores of 97%, 100% and 97% for SLE sera, SjS sera and healthy donor sera, respectively. Among the 60 RA sera screened for reactivity, 45% were positive for antibodies to citrullinated EBNA-1 peptides, CCP1 and the linear pro-filaggrin peptide. 10% were positive for antibodies to EBNA-1 peptides and CCP1, while 8% were positive for antibodies to CCP1 or EBNA-1 peptides, respectively. 18% of the sera tested were negative for all three groups of antibodies. We also investigated whether the antibody reactivity levels to the EBNA-1 panel varied with the presence/absence of CCP2 antibodies and RFs, however no correlation was found (results not shown). Moreover, the presence of antibody specificities in the RA sera was determined (Table 2). Among the RA sera analysed, 57% were positive for anti-CCP2, anti-EBNA-1 and RFs. In addition, 18% were only positive for antibodies to EBNA-1 peptides and RFs, while the remaining sera primarily contained single antibody specificities, e.g., antibodies to CCP2, RFs or EBNA-1. Finally, 8% of the RA sera did not contain any of the analysed antibodies. Finally, we analysed the individual reactivity of the RA sera to the selected EBNA-1 peptides (Table 3). As seen, 47% of the RA sera were found react with two or three citrullinated EBNA-1 peptides, whereas only 8% of the RA sere reacted with four peptides. None of the 60 sera analysed reacted with all of the EBNA-1 peptides. 4. Discussion The present study describes the reactivity of RA sera to citrullinated EBNA-1 peptides. As, seen antibody reactivity was found

Number of EBNA-1 peptides recognized by RA sera (n = 60).

to several citrullinated peptides (Fig. 2). Antibodies from RA sera bound with highest reactivity to peptides 35, 39 and 57, with 53% of sera analyzed recognizing peptide 39. In total, the five peptides that complemented each other the most, peptides 28, 35, 36, 39 and 57, reacted with 67% of the RA sera, which is slightly reduced compared to the CCP2 assay, which yielded a sensitivity of 77%. Preliminary screenings of the EBNA-1 peptides, 1–68 (Fig. 1), revealed that antibodies of IgG isotypes were detected almost exclusively in RA sera, whereas no significant reactivity was found with IgA or IgM isotypes, as previously described [40]. These findings are in accordance with clinical associations and that the predictive value of ACPA so far only has been established by detecting IgG ACPA. Nevertheless, a high proportion of RA sera have also been suggested to contain IgA and IgM ACPA [33], although we could not confirm these results. In some of the most reactive peptides, 32, 36 and 39, the Cit residue was located close to the terminal ends, which may cause sterical hindrance, e.g., if the Cit residue is positioned close to biotin. In these studies, primarily N-terminal biotinylated peptides were used in order to generate a stable point of origin for screening of antibody reactivity, as peptides may coat differently when coated directly to the solid surface [41–43]. Only in a few cases is biotin attached to the C-terminus of the peptides [44]. As seen, the distance between the Cit residue and biotin influenced the interaction between antibodies and peptides (Fig. 3). In all cases, the antibody reactivity was reduced as the Cit was positioned closer to biotin. These findings are in accordance with studies previously described, illustrating that the position of biotin within a peptide sequence markedly influences antibody reactivity [45]. By increasing the length of the peptides and hence the distance from the Cit residue to biotin, one would expect an increase in the number of reactive sera. However, this was not observed when analyzing antibody reactivity to the extended 41mer peptides compared to the 20mer peptides (Fig. 4), indicating that the effect of the biotin label is not sufficient to account for the observed reactivity pattern.

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Moreover, notable differences in antibody reactivity was found for the fusion peptide with different biotin labels, as antibody reactivity to the C-terminal biotin-labelled peptide was almost 100% higher compared to the N-terminal biotin-labelled peptide. This effect may be ascribed to differences in the peptide folding, however the exact reason remains to be determined. Previous findings do, however, not support this hypothesis, as studies using 20-mer peptides indicated that the secondary structure of the peptides was not altered when replacing a N-terminal biotin label with a C-terminal biotin label [45]. When a panel of sera was analyzed on the selected EBNA-1 peptides, 28, 35, 36, 39 and 57, pro-filaggrin peptides and the CCP2 assay, a high correlation overall in the reactivity of the antibodies was found. E.g. a correlation between the reactivity of antibodies to the EBNA-1 peptide panel and the CCP2 assay of 67% was found. Moreover, 50% of sera reacted with two or three of the EBNA-1 peptides. In a recent study analyzing ACPA reactivity, cross-reactivity between a citrullinated peptide derived from the EBNA-1 protein (amino acids 35–58) and human citrullinated fibrin was described [46], hence it is possible that the sera which recognized three of the EBNA-1 peptides were in fact cross-reactive and not just related to simultaneous reactivity in two populations of ACPA co-existing in the sample. IgG antibodies that bound to peptide 39 were detected in 53% of RA sera. Previous studies describing ACPA reactivity to a citrullinated EBNA-1 peptide, constituting amino acids 35–58 (GGDNHGRGRGRGRGRGGGPRGAPG), have previously been detected by 45–50% of RA sera [23–25]. However, it appears that these studies were actually based on application of the reverse amino acid sequence (GPAGPRGGGRGRGRGRGRGHNDGG), thus at this moment peptide 39 can be considered as the lead substrate candidate of EBNA-1 for detection ACPA. Furthermore, findings in this study could not determine specific antibody reactivity to single citrullinated peptides covering amino acids 35–58, as healthy controls were found to react with peptides within this region. The combination of these citrullinated EBNA-1 peptides could be used as a supplementary tool for the diagnosis of RA. The reactivity of RA sera to new EBNA-1 sequences encoded by EBV that could be used as a substrate for ACPA detection once again raises the issue of the role of EBV in inducing ACPA and possible mechanisms leading to the production of ACPAs and RA [26,47]. EBV is considered to be one of the environmental agents that contribute to the onset of RA. EBV infection is widespread and 95% of the population display serologic signs of an EBV infection. Moreover, it is known that patients with RA have elevated antibody levels to latent and replicative EBV proteins [33,48] and in particular to EBNA-1 [49,50]. In addition, it has been demonstrated that patients with RA are less efficient in killing autologous EBV-infected cells [51,52] and that RA patients possess increased amounts of EBV DNA [53–55]. Alternatively, EBV-derived citrullinated peptides could be targeted by ACPA simply because of a similarity in sequence or conformation with other ACPA targets. The question whether EBNA1 is a true autoantigen of ACPA remains to be determined. However, in these studies we only found IgG antibody reactivity towards the citrullinated epitopes, which are antibodies with high affinity to their targets. Moreover for these studies, TTN buffer was applied in the immunoassays. This buffer contains high levels of salt and detergent, which limit the binding of low affinity antibodies [56]. Hence, based on the current findings, the EBNA1 peptides are believed to be high affinity antigens for detection of ACPA. Previous adsorption studies analyzing reactivity to the noncitrullinated EBNA-1 peptide, VCP1, showed that antibodies to this peptide were detected in healthy individuals and in patients with other EBV-related diseases [25,57]. In fact, antibodies to VCP1 was

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found in 30% of healthy controls, 25% of sera from patients with RA and 38% of sera from patients with SLE. However, when this peptide was citrullinated and screened for antibody reactivity, no reactivity with healthy controls and SLE was found, instead 54% of the RA patient sera reacted with the VCP1 peptide, which was found to be specific towards citrullinated VCP1 [25,57]. These findings were confirmed in similar studies, where ACPA reactivity to different citrullinated epitopes were determined [7,58]. Moreover, it previously has been described that immune responses towards the EBNA-1 in patients with RA, primarily is located to the proline-rich C-terminal of the proteins, more specific to amino acids 451–641 [34]. These findings are in accordance with studies conducted using a EBNA1 mosaic, only covering amino acids 1–90 and 408–498 which obtained an EBNA1 immune response similar to full-length EBNA-1 (unpublished results). In the current study, results depicted in Fig. 1, illustrated that specific antibody reactivity primary was found to peptides 20–39, which cover amino acids 321–389. Based on these findings, we believe that the antibody responses described within this study is directly related to the ACPA reactivity to citrullinated epitopes and not the presence of antibodies to EBNA-1. Collectively, these observations indicate that EBNA-1 peptides may be considered as a substrate for the detection of ACPA and that deiminated viral proteins encoded by EBV may contribute to the production of ACPA as suggested by Pratesi et al. [25].

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