Further characterization of immunomodulation by a monoclonal antibody against Streptococcus mutans antigen P1

Abstract

We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion with N-chlorosuccinimide but not in isotype distribution or overall reactivity with S. mutans or intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with either S. mutans alone or S. mutans coated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlapping spaP subclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface of S. mutans alters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.

FIG. 1.

Schematic of P1, spaP subclones, and predicted NCS cleavage products.

FIG. 2.

Binding of sIgA from vaginal wash fluids from immunized mice to NCS-generated P1 polypeptides, as demonstrated by computer-assisted Western blot analysis. Each curve represents the mean densitometry profiles of the six mice in the designated treatment group. The ordinate corresponds to the intensity of bands detected by antibodies, and the abscissa indicates the apparent molecular masses (in kilodaltons) of the corresponding antigens. Group 2, NG8 alone; group 3, NG8[High MAb]; group 4, NG8[Low MAb].

FIG. 3.

Percent inhibition by serum antibodies of adherence of S. mutans to salivary-agglutinin-coated hydroxyapatite beads. The asterisk indicates significantly higher percent inhibition than with group 2 (P < 0.02). Group 1, buffer-only control; group 2, NG8 alone; group 3, NG8[High MAb]; group 4, NG8[Low MAb].

FIG. 4.

Serum IgG antibody reactivity with purified P1 polypeptide fragments measured by ELISA. An asterisk indicates significantly higher levels of serum IgG detected than with group 1 (P < 0.01). Group 1, buffer-only control; group 2, NG8 alone; group 3, NG8[High MAb]; group 4, NG8[Low MAb].

FIG. 5.

Serum IgG antibody reactivity with recombinant full-length P1 and P1ΔP as measured by ELISA. A single asterisk indicates higher serum IgG reactivity than with group 1 (P < 0.01). Two asterisks indicate lower serum IgG reactivity than with full-length P1 in the same treatment group: for group 2, P = 0.0218; for group 3, P = 0.0078; for group 4, P = 0.0005. Group 1, buffer only control; group 2, NG8 alone; group 3, NG8[High MAb]; group 4, NG8[Low MAb].

FIG.6.

Effect of MAb 6-11A binding to S. mutans on susceptibility of cell surface P1 to protease digestion. For colloidal gold stain of products of V8 protease treatment of S. mutans with and without bound MAb 6-11A, polypeptides associated with cell pellets (A) and polypeptides released into the supernatant (B) are shown. For Western blot of products of V8 protease treatment of S. mutans with and without bound MAb 6-11A traced with anti-P1 antiserum 209, polypeptides associated with cell pellets (C) and polypeptides released into the supernatant (D) are shown. For Western blot of polypeptide products released into the supernatant following chymotrypsin treatment of S. mutans with and without bound MAb 6-11A, anti-P1 antiserum 209 (E) and anti-P1 antiserum 218 (F) are shown. For Western blot of polypeptide products released into the supernatant following endoproteinase Arg-C treatment of S. mutans with and without bound MAb 6-11A, anti-P1 antiserum 218 (G) is shown.