Human cytomegalovirus pp65 lower matrix protein: a humoral immunogen for systemic lupus erythematosus patients and autoantibody accelerator for NZB/W F1 mice

Abstract

Both the infection of human cytomegalovirus (HCMV) and the immunization of its recombinant glycoprotein (gB) in mice have been known to induce autoimmunity, resulting in symptoms similar to those of human systemic lupus erythematosus (SLE). Research has also found that the murine cytomegalovirus (MCMV)-specific monoclonal antibody (mAb) is able to react with a human U1-70K-like autoantigen. To investigate HCMV involvement in autoimmunity, we analysed the humoral responses to HCMV by autoimmune patients and normal adults. Our studies show unambiguously that sera from SLE patients exhibited an elevated IgG titre to HCMV when compared with those observed in controls and other connective tissue disease (CTD) patients (P < 0.001). The IgM titres to HCMV and IgG to HBV were evaluated, and no significant differences were noted among all testing groups. In addition to initiating T cell activity, as reported by many investigators, we found that the HCMV pp65 antigen (also known as lower matrix protein) was able to induce humoral responses in SLE patients. Immunoblot assays showed that 82.56% of sera from SLE patients reacted with the HCMV pp65 antigen, but only 11.11%, 23.53% and 31.17% of patients from normal control, rheumatoid arthritis (RA) and CTD patients, respectively, reacted to it. Unlike HCMV pp65, HCMV pp150 induced B cell activity in most collected sera (92.22%-98.04%). Finally, female NZB/W F1 mice immunized with plasmids encoding HCMV pp65 open reading frame (pcDNApp65) developed an early onset of autoantibody activity and more severe glomerulonephritis. Thus, we conclude that the HCMV pp65 antigen triggers humoral immunity in SLE patients and autoimmune-prone mice and that it could very well exacerbate the autoimmune responses in susceptible animals.

Fig. 1

Detection of IgG and IgM anti-human cytomegalovirus (HCMV) and anti-hepatitis B virus (HBV) in sera from systemic lupus erythematosus (SLE) (n = 86), connective tissue diseases (CTD) (n = 77), rheumatoid arthritis (RA) (n = 51) patients and normal controls (n = 90). Sera were tested by enzyme-linked immunosorbent assay (ELISA) against purified HCMV (a,b), HBV (c, d) and EBV (e) viral particles. (a,c,e) IgG reactivity to the viruses; (b,d) IgM reactivity to the same virus. *P < 0·001 (Student's t-test, compared to normal controls).

Fig. 2

A representative immunoblot analysis showing IgG reactivity to human cytomegalovirus (HCMV) antigens among systemic lupus erythematosus (SLE) patients (a), connective tissue diseases (CTD) (b), normal controls (c) and rheumatoid arthritis (RA) (d) patients. Markers, the HCMV pp150 and pp65 are labelled on the left.

Fig. 3

Detection of IgG anti-human cytomegalovirus (HCMV) pp65 and pp150 in sera from systemic lupus erythematosus (SLE) (n = 86), connective tissue diseases (CTD) (n = 77), rheumatoid arthritis (RA) (n = 51) patients and normal controls (n = 90). Sera were tested by enzyme-linked immunosorbent assay (ELISA) against blot purified HCMV pp65 (a) and 150 (b). The error bars show the s.e.m. *P < 0·001, **P = 0·0014 (Student's t-test, compared to normal controls).

Fig. 4

Mean IgG development against the human cytomegalovirus (HCMV) pp65 (± s.e.m.) antigen in sera from mice (NZB/W) immunized with plasmids encoding HCMV pp65, murine cytomegalovirus (MCMV) M83, M84 and vector only. Sera were tested by enzyme-linked immunosorbent assay (ELISA) against the blot purified HCMV pp65 antigen. The error bars show s.e.m. P-values are listed at the top (Student's t-test, compared to vector control). Tests were conducted in triplicate.

Fig. 5

Mean IgG development against dsDNA and nuclear components (± s.e.m.) in sera from mice (NZB/W, C57BL/6, BALB/c) immunized with plasmids encoding human cytomegalovirus (HCMV) pp65, murine cytomegalovirus (MCMV) M83, M84 and vector only. Sera were tested by enzyme-linked immunosorbent assay (ELISA) against dsDNA or nuclear extract. (a,c) Anti-dsDNA activities by NZB/W F₁ or C57BL/6 and BALB/c, respectively. (b) Anti-nuclear reactivity by NZB/W F₁ mice. The error bars show s.e.m. P-values are listed at the top (Student's t-test, compared to vector control). Tests were conducted in triplicate.

Fig. 6

The effect of human cytomegalovirus (HCMV) pp65 and murine cytomegalovirus (MCMV) M83 and M84 immunization on glomerular lesion counts. Mice received pp65, M83, M84 and the plasmid had a mean renal severity score of 61, 60, 54 and 50, respectively (a). The error bars show s.e.m. P-values are listed at the top (Student's t-test, compared to vector control).

Fig. 7

Histological staining of renal tissues from mice that received DNA vaccinations.