Cross-reaction between antibodies to the major epitope of Ro60 kD autoantigen and a homologous peptide of Coxsackie virus 2B protein

Abstract

Coxsackie virus RNA has recently been detected in biopsy specimens of minor salivary glands from patients with primary Sjögren's Syndrome (pSS). A peptide derived from Coxsackie virus 2B protein (pepCoxs) presents 87% sequence homology with the 222-229 region of the major linear B-cell epitope of Ro60 kD autoantigen (pep216-232). Synthetic peptides corresponding to pep216-232: (216)KALSVETEKLLKYLEAV(232) and pepCoxs: (31)MVTSTITEKL LKNLVKI(47), were prepared. Sera from 42 patients with pSS and 43 patients with systemic lupus erythematosus (SLE) as well as sera from 27 healthy individuals (normal controls) and sera from 30 patients with rheumatoid arthritis (disease controls) were tested against the two homologous peptides. Twenty-five percent of SLE sera and 33.3% of pSS sera reacted against pep216-232, whereas 28% of SLE sera and 37% of pSS sera recognized the pepCoxs. The sera reacting with pep216-232 were apparently the same as those reacting with pepCoxs. Normal sera and disease control sera presented only a limited reactivity against both peptides (ranging from 3.7% to 10%). Both peptides reacted more prominently with anti-Ro/La (+) sera from pSS patients. Thus, pep216-232 was recognized by 17% of the anti-Ro (+) sera and by 42% of the anti-Ro/La (+) sera, whereas pepCoxs was recognized by 28.5% and 38% of the a-Ro(+) and a-Ro/La(+) sera, respectively. Purified anti-pep216-232 antibodies readily reacted with both peptides while inhibition experiments revealed the specificity of this reaction. These results suggest a possible cross-reaction between antibodies to the major linear B-cell epitope of Ro60 kD autoantigen and the homologous pepCoxs in pSS patients. This cross-reaction might potentially play a role in autoantibody formation and the perpetuation of the autoimmune response against Ro/SSA and La/SSB.

Fig. 3

Anti-peptide reactivity of purified anti-pep216–232 antibodies. (a) Chromatogram representing the purification procedure of specific anti-pep216–232 antibodies from a representative serum by affinity chromatography A: flowthrough, B: low affinity antibodies eluted with phosphate buffer pH:7·4 containing 0·6 M NaCl C: high affinity antibodies eluted with HCl-glycine pH:2·7 (b) Purified anti-pep216–232 antibodies, from 3 patient sera, exhibited high reactivity in anti-pep216–232 (▪), and anti-pepCoxs (□) ELISAs. In contrast, low affinity antibodies (eluted with 0·6 M NaCl) and normal human serum, gave low OD₄₁₀ values.

Fig. 1

Anti-peptide reactivity of autoimmune and normal sera. Twenty-five percent of SLE sera, 33·3% of pSS sera and 10% of RA sera reacted with pep216–232 (a), whereas 28% of SLE sera, 37% of pSS sera and 10% of RA sera reacted with pepCoxs (b) (dotted lines represent the cut off values). The reactivity of pSS (c) and SLE (d) sera against pep216–232 and pepCoxs varied in parallel in contrast to the reactivity against control peptide, which was remained in low levels. Sera with OD values greater than the cut-off value for each peptide are marked with the ‘+’ symbol.

Fig. 2

Anti-peptide reactivity of autoimmune sera according to their autoantibody profile. A stronger recognition of the two homologous peptides by anti-Ro/La positive sera versus anti-Ro positive sera was observed: 17% of anti-Ro positive sera and 42% of anti-Ro/La positive sera gave a positive reaction against pep216–232 (a), whereas the reactivity of anti-Ro positive sera and anti-Ro/La positive sera against pepCoxs was 28·5% and 38%, respectively (b). Amongst pSS patients anti-Ro/La positive sera displayed greater reactivity (median OD₄₁₀ values) against the two homologous peptides comparing to anti-Ro positive sera (c), whilst SLE patients’ sera did not display statistically different reactivity according to their autoantibody profile (d).

Fig. 4

Homologous and heterologous inhibition of a pSS patient serum. Using pep216–232 (•) and pepCoxs (▪) as inhibitors, the reactivity of a serum sample from a patient with pSS was inhibited by 60% and 46%, respectively, in an anti-pep216–232 ELISA (a) and by 46% and 40%, respectively, in an anti-pepCoxs ELISA (b). Using an irrelevant peptide (▴) as inhibitor, no inhibition was observed.

Fig. 5

Homologous and heterologous inhibition of purified anti-pep216–232 antibodies. (a) Using pep216–232 (•) and pepCoxs (▴) as inhibitors, the binding of purified anti-pep216–232 antibodies was inhibited by 86% and 60%, respectively, in an anti-pep216–232 ELISA. (b) When pepCoxs was used as coating peptide, the inhibition rate was 81% for the pep216–232 (•) and 52% for pepCoxs (▴), respectively. Using an irrelevant peptide (pepCtrl, ♦) as inhibitor, no inhibition was observed.