Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements

Abstract

Associations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-S-transferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD(450) readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins. High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.

Fig. 1

Interference by rheumatoid factor in the ELISA and concentrations of IgG, IgA and IgM proteins in sera from patients with systemic lupus erythematosus (SLE), primary Sjögrens’ syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc), polymyositis/dermatomyositis (PM/DM) or mixed connective tissue disease (MCTD). (a, b) Rheumatoid factor was added to nine rheumatoid factor-negative sera in the concentration range of 125–2000 U/ml. Each of the nine sera are represented by individual symbols. (a) OD₄₅₀ readings in ELISA for IgG antibodies to EBNA-2(N). (b) OD₄₅₀ readings in ELISA for IgG antibodies to EBNA-1(C).

Fig. 2

OD₄₅₀ readings of IgG antibodies to EBNA-2(N) and EBNA-1(C) in sera from patients with SLE, SS, RA, SSc or MCTD. (a) OD₄₅₀ readings of IgG antibodies to EBNA-2(N) in sera from patients with SLE, SS, RA, SSc, PM/DM or MCTD. (b) OD₄₅₀ readings of IgG antibodies to EBNA-2(N) in sera from patients with secondary SS. (c) OD₄₅₀ readings of IgG antibodies to EBNA-1(C) in sera from patients with SLE, SS, RA, SSc, PM/DM or MCTD. (d) OD₄₅₀ readings of IgG antibodies to EBNA-1(C) in sera from patients with secondary SS.

Fig. 3

Immunoglobulin concentrations in sera from patients with SLE, SS, RA, SSc or MCTD. (a) IgG concentration. (b) IgA concentration. (c) IgM concentration. Median values are indicated by the horizontal bars in the scattergrams. Median values of immunoglobulin concentrations are indicated by the horizontal bars in the scattergrams.

Fig. 4

Specific levels of IgG antibodies to EBNA-2(N) and to EBNA-1(C) as defined by ELISA OD₄₅₀ normalized to whole IgG concentration. (a) Specific OD₄₅₀ readings of IgG antibodies to EBNA-2(N) in sera from patients with SLE, SS, RA, SSc or MCTD. (b) Specific OD₄₅₀ readings of IgG antibodies to EBNA-2(N) in sera from patients with secondary SS. (c) Specific OD₄₅₀ readings of IgG antibodies to EBNA-1(C) in sera from patients with SLE, SS, RA, SSc or MCTD. Median values are indicated by the horizontal bars in the scattergrams.