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Systemic lupus erythematosus (SLE) is an autoimmune disease, which has been associated with Epstein–Barr virus (EBV) and Cytomegalovirus (CMV) infection. Drug-induced lupus (DIL) is a lupus-like disease caused by the intake of therapeutic drugs, which has been estimated to cause approximately 10–15% of lupus-like cases. Although SLE and DIL share common clinical symptoms, there are some fundamental differences between DIL and SLE onset. Moreover, it remains to be examined whether environmental factors, such as EBV and CMV infections, may contribute to the development of DIL. This study focused on examining the possible association between DIL and EBV and CMV infections, by examining IgG titers to EBV and CMV antigens in serum samples by enzyme-linked immunosorbent assays. Antibody titers to EBV early antigen–diffuse and CMV pp52 were found to be significantly elevated in both SLE and DIL patients compared to healthy controls, although no correlation was found for antibodies to the two virus antigens in the respective disease groups. Moreover, total IgG titers were reduced in SLE and DIL serum samples, which may reflect a general lymphocytopenia, which commonly is associated with SLE. The current findings support that EBV and CMV infections may contribute to the development of DIL and that onset of both diseases are related.

Purpose of the study Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. Patients and methods We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. Results Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. Conclusion Collectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens (EBNA1, EBNA2, and EBNA3) in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2, (EBNA2-A, GQGRGRWRG-Cit-GSKGRGRMH) reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets. Keywords: calreticulin; epitope mapping; frameshift mutations; myeloproliferative neoplasms; peptide antibodies.

Multiple sclerosis (MS) is a neurologic disease affecting myelinated nerves in the central nervous system (CNS). The disease often debuts as a clinically isolated syndrome, e.g., optic neuritis (ON), which later develops into relapsing-remitting (RR) MS, with temporal attacks or primary progressive (PP) MS. Characteristic features of MS are inflammatory foci in the CNS and intrathecal synthesis of immunoglobulins (Igs), measured as an IgG index, oligoclonal bands (OCBs), or specific antibody indexes. Major predisposing factors for MS are certain tissue types (e.g., HLA DRB1*15:01), vitamin D deficiency, smoking, obesity, and infection with Epstein-Barr virus (EBV). Many of the clinical signs of MS described above can be explained by chronic/recurrent EBV infection and current models of EBV involvement suggest that RRMS may be caused by repeated entry of EBV-transformed B cells to the CNS in connection with attacks, while PPMS may be caused by more chronic activity of EBV-transformed B cells in the CNS. In line with the model of EBV’s role in MS, new treatments based on monoclonal antibodies (MAbs) targeting B cells have shown good efficacy in clinical trials both for RRMS and PPMS, while MAbs inhibiting B cell mobilization and entry to the CNS have shown efficacy in RRMS. Thus, these agents, which are now first line therapy in many patients, may be hypothesized to function by counteracting a chronic EBV infection.

Nicole H. Trier, Bettina E. Holm, Paul R. Hansen, Ole Slot, Henning Locht and Gunnar Houen.

Epstein-Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals. Diagnosis of RA and SLE relies on clinical criteria in combination with the presence of characteristic autoantibodies. In addition, antibodies to several EBV antigens have been shown to be elevated in patients with these diseases compared to healthy controls (HC). Here, we elaborated improved enzyme-linked immunosorbent assays for antibodies (IgM, IgA, IgG) to the EBV proteins Epstein-Barr Virus nuclear antigen (EBNA)1 and early antigen diffuse (EAD) in order to determine their potential diagnostic role. We showed that especially EBNA1 IgM distinguished RA from SLE and HCs and also distinguished SLE from HCs. EBNA1 IgA was almost as effective in differentiating RA from SLE and HC, while EAD IgG and IgA were able to discern SLE patients from RA patients and HCs. Collectively, these findings illustrate the potential diagnostic use of antibodies to EBV proteins to diagnose RA and to differentiate SLE from RA.

Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Anti-citrullinated protein antibodies (ACPA) are crucial for the serological diagnosis of RA, where Epstein-Barr virus (EBV) has been suggested to be an environmental agent in triggering the onset of the disease. This study aimed to analyse antibody reactivity to citrullinated EBV nuclear antigen-2 (EBNA-2) peptides from three different EBV strains (B95-8, GD1 and AG876) using streptavidin capture enzyme-linked immunosorbent assay. One peptide, only found in a single strain (AG876), obtained a sensitivity and specificity of 77% and 95%, respectively and showed high sequence similarity to the filaggrin peptide originally used for ACPA detection. Comparison of antibody reactivity to commercial assays found that the citrullinated peptide was as effective in detecting ACPA as highly sensitive and specific commercial assays. The data presented demonstrate that the citrullinated EBNA-2 peptide indeed is recognised specifically by RA sera and that the single peptide is able to compete with assays containing multiple peptides. Furthermore, it could be hypothesized that RA may be caused by (a) specific strain(s) of EBV.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 1–2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs), which have been found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs.

In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures constituting amino acids 130–135 and 136–141, respectively. Moreover, comparison of antibody reactivity to N OVA revealed that in the streptavidin-capture ELISA, antibody reactivity was notably reduced compared to ELISA employing surface-bound OVA. Collectively, immunization with native OVA preferentially generates highly specific antibodies reacting with three-dimensional epitopes, whereas immunization with denatured OVA generates antibodies occasionally reacting with continuous epitopes. Moreover, as differences in monoclonal antibody reactivity was found between the two assays, monoclonal antibodies always should be selected by an assay mimicking the desired use of the final antibodies as closely as possible.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by progressive joint destruction and disability. Classical autoantibodies of RA are rheumatoid factors and citrulline antibodies. Patients positive for these autoantibodies are usually associated with a progressive disease course. A subgroup of RA patients does not express citrulline antibodies, instead are approximately 35% of these anti-citrulline-negative patients reported to express autoantibodies to the heterogeneous nucleoriboprotein A2, a ribonucleoprotein involved in RNA transport and processing also referred to as RA33. In the absence of citrulline antibodies, RA33 antibodies have been suggested to be associated with a milder disease course. In this study we screened the reactivity of a monoclonal antibody to RA33-derived peptides by modified enzyme-linked immunosorbent assays (ELISA). Terminally truncated resin-bound peptides were applied for determination of the functional epitope necessary for antibody recognition. In addition, screening of substituted peptides by modified ELISA identified amino acids necessary for antibody reactivity. A potential epitope was identified in the region 71-79 (PHSIDGRVV), where the amino acids Ser, Ile and Asp were found to be essential for antibody reactivity. These amino acids were found to contribute to the antibody-antigen interface through side-chain interactions, possibly in combination with a positively charged amino acid in position 77. Moreover, the amino acids in the N-terminal end (Pro and His) were found to contribute to the interface through backbone contributions. No notable reactivity was found with RA-positive patient sera, thus screening of RA33 antibodies does not seem to be a supplementary for the diagnosis of RA.