Alterations in immunodominance of Streptococcus mutans AgI/II: lessons learned from immunomodulatory antibodies

Abstract

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus.

Keywords: Immune complex; Immunodominance; Monoclonal antibody; Streptococcus; Vaccine design.

Figure 1

Schematic representations of S. mutans Antigen I/II illustrating location of putative T cell epitopes and approximate antibody binding sites. (A) A representation of the primary structure of AgI/II and the recombinant polypeptides used in this study. (B) A three-dimensional model of Ag I/II. Approximate binding sites of monoclonal antibodies are indicated.

Figure 1

Schematic representations of S. mutans Antigen I/II illustrating location of putative T cell epitopes and approximate antibody binding sites. (A) A representation of the primary structure of AgI/II and the recombinant polypeptides used in this study. (B) A three-dimensional model of Ag I/II. Approximate binding sites of monoclonal antibodies are indicated.

Figure 2

Evaluation of S. mutans adherence to SAG and detection of anti-S. mutans IgG. (A) BIAcore SPR analysis was used to evaluate bacterial adherence following incubation of S. mutans with sera collected from mice immunized with full-length P1 (CG14) and truncated derivatives of P1 (NA1, P3C). Values were normalized to the change in resonance units (ΔRU) for S. mutans NG8, in the absence of added serum, detected following the 60-second injection cycle. Immunogens are indicated on the X-axis. Tukey’s Multiple Comparison Test determined differences among the groups; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 (B) The presence of anti-S. mutans IgG in the sera of mice immunized with P1 versus truncated P1 polypeptides was evaluated by serial dilution and standard ELISA. Data are representative of at least three independent experiments. Statistical significance between groups was evaluated by two-way ANOVA and no significant differences were observed.

Figure 3

Evaluation of the specificity and isotype of serum antibody responses by ELISA. The level of anti-NR21-specific IgG1, IgG2a, and IgG2b subclass antibody in the sera collected from sham-immunized mice, mice immunized with full-length P1, or the truncated protein lacking 2 putative T cell epitopes was evaluated by ELISA. All results are expressed as mean ± SEM and data are representative of at least three independent experiments. Statistically significant differences of the T-delete compared to CG14 were determined by two-way ANOVA. p≤0.05 (IgG1) , p≤0.0001 (IgG2a), p≤0.01 (IgG2b)

Figure 4

Functional C-terminal epitopes are masked by intra-molecular interactions. (A) Sera from mice immunized with full-length P1 (CG14), NA1, P3C, or a mixture of NA1 and P3C were used in a competition ELISA to evaluate inhibition of binding of biotin-labeled MAb 3–10E to S. mutans. Serum dilution is indicated on the x-axis. (B) BIAcore SPR analysis was used to evaluate bacterial adherence following incubation of S. mutans with sera collected from immunized mice. Values were normalized to the change in resonance units (ΔRU) for S. mutans NG8, in the absence of added serum, detected following the 60-second injection cycle. Immunogens are indicated on the X-axis. Tukey’s Multiple Comparison Test determined differences among the groups; * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (C) The level of anti-S. mutans IgG in the sera from P1 versus truncated P1 -immunized mice was evaluated by serial dilution and standard ELISA. Data are representative of at least three independent experiments. Statistically significant differences compared to the CG14 group were determined by two-way ANOVA. p≤0.01 (P3C, NA1+P3C), p≤0.0001 (NA1).