B-cell epitope mapping of DNA topoisomerase I defines epitopes strongly associated with pulmonary fibrosis in systemic sclerosis

We hypothesized that B-cell epitope mapping of DNA Topoisomerase I (type-I topoisomerase, or Topo I) may define epitopes strongly associated with pulm onary interstitial fibrosis (PIF) in systemic sclerosis (SSc). B-cell epito pe mapping of Topo I was performed using 63 20-mer peptides overlapping by eight residues and spanning the entire length of the Topo I sequence. These peptides, coupled to polystyrene pins, were tested for antibody binding by enzyme-linked immunosorbent assays (ELISAs) using immunoglobulin G fractio ns from anti-Topo I, anticentromere, anti-U3RNP-positive, and normal sera. Four major epitopes were recognized by anti-Topo I sera, but not from the c ontrol sera: WWEEERYPEGIKWKFLEHKG (205-224, epitope I), RIANFKIEPPGLFRGRGNH P (349-368, epitope II), PGHKWKEVRHDNK-VTWLVSW (397-416, epitope III), and ELDGQEYVVEFDFLGKDSIR (517-536, epitope IV), Peptide-epitopes were then synt hesized in their soluble forms and ELISA systems were developed. Epitopes I I to IV are localized at highly exposed sites of the Topo I tertiary struct ure, whereas epitope I is localized at a less accessible site. In a cohort of 81 patients with SSc with clinical data on the evolution of their diseas e, patients with antibodies in their sera recognizing at least three of the four epitopes had 3.1 times (P = 0.02) the hazard of developing PIF compar ed with patients whose sera recognized no epitopes or only one or two of th e four epitopes. The discrimination was much stronger than that achieved by the simple determination of Topo I antibodies by counterimmunoelectrophore sis and immunoblot (hazard ratio 1.7, P = 0.30) in the same patients. B-cel l epitope mapping of the anti-Topo I response has identified four major epi topes which cumulatively show a strong association with the development of PIF in SSc.