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Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap1-17), which represents, along with Ap17-30, an immunodominant epitope of the protein. Peptide Ap1-17 was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap1-17 antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap17-30. Panning of a phage display peptide library with anti-Ap1-17 antibodies from 2 patients identified two novel, partially overlapping motifs, <5Rx(st)xKP10> and <9KPxxPxR15> as the result of the alignment of specific phage clone insert sequences. Anti-Ap1-17 IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap1-17 antigenic motifs. These data show that anti-CENP-A1-17 antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model.

Introduction: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. Methods: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. Results: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA-SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009). Conclusions: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance.

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SummaryThe safety of four different adjuvants was assessed in lupus-prone New Zealand black/New Zealand white ( Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post-test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post-immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti-RNP/Sm autoantibody, as occurred in the CFA group.

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