Anti-viral citrullinated peptides antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) sera contain antibodies specific for deiminated peptide/proteins (ACPA). The deimination (or citrullination) is a post-translational modification catalysed by the calcium dependent enzyme peptidylarginine deiminase: an arginyl residue within protein is converted to a cytrullyl residue, with the consequent loss of a net positive charge. Citrullination is a physiologic event, occurring during cell differentiation, and particularly in inflammation and cell death; on the contrary, antibodies directed towards citrullinated proteins/peptides are highly specific for RA. A number of endogenous proteins have been found to be citrullinated and target of ACPA Recently, a viral peptide corresponding to the sequence 35-58 Epstein Barr Nuclear Antigen-1 containing citrulline in place of arginine (VCP – Viral Citrullinated Peptide) has been shown to be a specific probe for the detection of ACPA. To analyze the immune response to citrullinated Epstein-Barr antigens in RA, we tested peptides derived from different viral proteins and characterised by different rates of citrullination. Studying a large panel of peptides, we found high reactivity against another deiminated viral peptide (DVP). Thus, we measured anti-DVP antibodies of IgG, IgM and IgA isotype in 100 normal sera, 107 RA and 196 disease controls. Setting the threshold value at the 99° percentile of the normal population, IgG anti-DVP were found in 65%; IgM anti-DVP in 44%; and IgA anti-DVP in 48% of RA patients. In some cases IgM or IgA were present in the absence of IgG; moreover, anti-DVP and anti-VCP antibodies were found to be overlapping but distinct populations. Rheumatoid arthritis is a multifactorial disease. Among genes, the major contribution to RA susceptibility is given by HLA-DRB1 alleles encoding the “shared epitope” (SE) sequences. The presence of these alleles is strictly associated with the production of ACPAs. However, the genetic background underlying the production of single ACPA specificities has not been thoroughly investigated. Thus, we analysed the reactivity to two different viral citrullinated peptides (VCP and DVP) and studied their association with genetic variants within HLA-DRB1 SE alleles in a RA Caucasian population. One hundred and seventy-two French RA patients were characterized in terms of HLA-DRB1 genotype, anti VCP-A and anti-DVP antibodies. HLA-DRB1 SE alleles were classified into four SE+ groups (S1, S2, S3P, S3D) and one SE- group (X) according to a new classification of HLA-DRB1 alleles, “reshaping the shared epitope (SE) hypothesis”. Anti-viral citrullinated peptides antibodies were assessed by home made ELISA on VCP and DVP coated plates. We found that anti-VCP and anti-DVP antibodies showed a trend in association to HLA-SE alleles but not significant (odds ratio > 1, P>0.05). However, subgrouping the SE alleles according to the new classification, the presence of anti-VCP or anti-DVP antibodies is associated with S2 allele (OR>1, P<0.05). Analysing the contribution of single SE alleles belonging to the S2 subgroup, anti-VCP and anti-DVP antibodies are associated with HLA-DRB1 *0401 (OR>1, P<0.05). We have tested the peptides for their capacity to bind various HLA-DR molecules. In no instance we found that any of the peptides bound any of the DR molecules at the highest dose tested. Therefore, these results challenge the role of HLA-SE in the pathogenesis of rheumatoid arthritis. More studies are needed to clarify whether HLA-SE may act as a classic immune response gene, thus presenting citrullinated peptide to T cells or whether they may play other roles, directly stimulating immune system cells.