Background We described the conformational PDGFRα epitope of VHPAM–Vκ16F4 agonistic autoantibody1, cloned from memory B cells of a SSc patient, that can induce fibrosis in vivo2. We showed that peptides composing this epitope may be specifically recognized by serum IgG of patients with systemic sclerosis (SSc), but not of controls.
Objectives i. To identify the immunodominant peptide within the discontinuous PDGFRα epitope of VHPAM–Vκ16F4; ii. to identify other immunodominant epitopes recognized by agonistic autoantibodies; iii. to use these immunodominant peptides to develop an epitope-based assay for diagnosis of SSc and classification of SSc clinical subtypes.
Methods i.The large PDGFRα peptide library used for epitope mapping of monoclonal anti-PDGFRα antibodies1 was screened with 25 SSc (12 limited, 13 diffuse) and 25 healthy control (HC) serum samples. ii. A smaller PDGFRα peptide library containing only the top 20 conformational binders plus 20 linear and 20 conformational controls was synthesized. 60 conformational and linear peptides of a cognate protein forming a molecular complex with PDGFRα were included in the array. 20 scrambled peptides were added as negative controls. This library was screened with the same 50 serum samples. iii. A third library was synthesized, retaining the top cognate protein peptide binders, and 15 chimeric PDGFRα/cognate protein peptides, chosen among the best binders, with some nonbinding controls. This library was tested as before. Libraries were synthesized by Pepscan Presto, Netherlands. Statistical analysis was performed by Wilcoxon-Mann-Whitney test. Correlations between serological results and clinical status were made.
Results i. An immunodominant peptide discriminating SSc from HC serum samples was identified in the first library. ii. This was confirmed by the second library, which highlighted also one immunodominant epitope from the cognate protein. Statistical analysis identified two cohorts of SSc samples (reactive vs nonreactive, the latter undistinguishable from HC) each composed by limited and diffuse SSc subtypes. iii. The third peptide library identified the chimeric peptide recognized exclusively by the reactive SSc serum samples, which were taken from patients with active, progressive disease regardless of limited vs diffuse classification, whereas the nonreactive SSc samples were taken from subjects with less active, non progressive disease.
Conclusions We developed a conformational epitope-based assay detecting SSc-specific, agonistic, serum autoantibodies. The preliminary results suggest that this novel array may identify SSc patients with active disease, regardless of the canonical classification criteria. We propose this assay for prospective screening of large cohorts of patients affected by, or suspected for, SSc, to validate it as a tool for disease activity assessment and/or early diagnosis.
Moroncini G et al. Epitope specificity determines pathogenicity and detectability of anti-PDGFRα autoantibodies in systemic sclerosis. Arthritis and Rheumatology. 2015; 67(7):1891–1903.
Luchetti MM et al. Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet-Derived Growth Factor Receptor Agonistic Autoantibodies. Arthritis & Rheumatology. 2016; 68(9):2263–73.
Disclosure of Interest None declared