INFECTION AND IMMUNITY, Dec. 2004, p. 6951–6960 0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.12.6951–6960.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 12
Redirecting the Humoral Immune Response against Streptococcus mutans Antigen P1 with Monoclonal Antibodies Monika W. Oli, Nikki Rhodin, William P. McArthur, and L. Jeannine Brady* Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida Received 10 June 2004/Returned for modification 23 July 2004/Accepted 12 August 2004
The adhesin P1 of Streptococcus mutans has been studied as an anticaries vaccine antigen. An anti-P1 monoclonal antibody (MAb) bound to S. mutans prior to mucosal immunization of mice was shown previously to alter the amount, specificity, isotype, and biological activity of anti-P1 antibodies. The present study was undertaken to screen this and four additional anti-P1 MAbs for immunomodulatory activity when complexed with S. mutans and administered by a systemic route and to evaluate sera from immunized mice for the ability to inhibit adherence of S. mutans to immobilized human salivary agglutinin. All five MAbs tested influenced murine anti-P1 serum antibody responses in terms of subclass distribution and/or specificity. The effects varied depending on which MAb was used and its coating concentration. Two MAbs promoted a more effective, and two others a less effective, adherence inhibition response. An inverse relationship was observed between the ability of the MAbs themselves to inhibit adherence and the ability of antibodies elicited following immunization with immune complexes to inhibit adherence. Statistically significant correlations were demonstrated between the levels of anti-P1 serum immunoglobulin G2a (IgG2a) and IgG2b, but not of IgG1 or IgG3, and the ability of sera from immunized animals to inhibit bacterial adherence. These results indicate that multiple anti-P1 MAbs can mediate changes in the immune response and that certain alterations are potentially more biologically relevant than others. Immunomodulation by anti-P1 MAbs represents a useful strategy to improve the beneficial immune response against S. mutans. Numerous reports over many decades indicate that both salivary secretory immunoglobulin A (sIgA) and serum IgG against these and other S. mutans antigens can be associated with decreases in S. mutans colonization and/or caries formation in vaccinated as well as naturally sensitized hosts. However, a definitive correlate of protection has not been entirely elucidated. In some cases, salivary and serum antibodies against S. mutans antigens, including P1, have been reported to be nonprotective (32). The importance of the specificity of the immune response against S. mutans has been pointed out in studies whereby reactivity against different determinants can be documented between caries-susceptible and -resistant individuals (15, 37). In addition, a passively applied monoclonal antibody (MAb) directed against antigen I/II has been reported to confer long-term protection against S. mutans colonization in human clinical trials; however, this property is not shared by all anti-antigen I/II MAbs (45). Disruption of hostpathogen interactions can be accomplished with molecules that bind to or compete with relevant regions of ligands or receptors. Adherence of S. mutans can be inhibited in vitro by addition of fluid-phase P1 and by some but not all anti-P1 antibodies or defined P1 peptides (13, 38). Taken together, these results suggest that qualitative rather than quantitative aspects of the antibody response may be more important for immune protection against S. mutans colonization and cariogenicity. It is becoming increasingly evident that some past assessments of antibody-mediated immunity against pathogenic microorganisms may have been overly simplistic and that protection by certain antibodies may in fact result from indirect mechanisms (65, 71, 81). A delicate balance of antibodies can modulate inflammatory responses and alter outcome, with
Oral microorganisms colonize, survive, and multiply in a dynamic host environment that is continually altered by dietary components as well as the host immune system. Streptococcus mutans is the predominant etiologic agent of dental caries (29). This and related members of the oral microflora have also been associated with bacteremias and endocarditis (7, 18, 26, 30). S. mutans possesses a number of virulence factors that allow it to colonize and eventually dominate its niche in the oral cavity (9). The wall-associated Mr ⬃185,000 multidomain fibrillar lectin, known variously as P1, antigen I/II, or PAc, is widely presumed to allow attachment to the acquired pellicle of teeth via calcium-dependent binding to a high-molecularweight glycoprotein, salivary agglutinin (22), now also known as gp340 (59). Structurally analogous antigen I/II family surface antigens also contribute to salivary pellicle attachment and microbial interactions (40). An isogenic mutant of S. mutans was constructed by eliminating spaP encoding P1 (19). This enabled characterization of the cell surface-localized protein as an adhesive component of S. mutans and demonstrated its contribution to caries formation and invasion of dentinal tubules (19, 44). P1 has been studied as an anticaries vaccine antigen. Parenteral and mucosal administration, as well as genetic immunization with plasmid DNA, have been evaluated, all with reported success (25, 27, 39, 51, 68). Data supporting a role for humoral immunity in protection against dental caries have been documented, with P1, glucosyltransferases, and glucan binding proteins showing the most promise as potential therapeutic immunogens (39, 45, 51). * Corresponding author. Mailing address: Department of Oral Biology, P.O. Box 100424, Health Science Center, University of Florida, Gainesville, FL 32610-0424. Phone: (352) 846-0785. Fax: (352) 8460786. E-mail:
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seemingly subtle changes in antibody specificity and/or concentration of given antibody isotypes significantly influencing the host-pathogen interaction (50). Our investigators showed previously that anti-P1 MAb 6-11A that was bound to the surface of S. mutans prior to immunization of mice by gastric intubation or intranasal administration altered the amount, specificity, and subclass composition of anti-P1 serum IgG antibodies and depended on the coating concentration of the MAb as well as on the route of immunization (14). Changes in the specificity of the sIgA response were similar to those observed for serum IgG, and changes in serum IgG antibodies were associated with differences in biological activities (61). Sera from mice immunized by gastric intubation with whole bacteria coated with a subsaturating concentration of MAb 6-11A demonstrated significantly greater inhibition of S. mutans adherence to human salivary agglutinin-coated hydroxyapatite beads than sera from mice immunized with bacteria alone. The present study was undertaken to screen five anti-P1 MAbs for immunomodulatory activity when complexed with S. mutans and administered intraperitoneally. All five MAbs tested influenced murine anti-P1 serum responses in terms of IgG subclass distribution and/or specificity of elicited antibodies in various ways and to various extents. As a reflection of potentially protective immunity, sera from immunized mice were also evaluated for the ability to inhibit adherence of S. mutans to immobilized human salivary agglutinin. Two MAbs, including 6-11A, promoted a more effective adherence inhibition response. Two others promoted a less effective response, and one did not influence adherence inhibition. Statistically significant positive correlations were identified between the measured levels of anti-P1 antibodies of the IgG2a and IgG2b isotypes and the ability of murine sera to inhibit S. mutans adherence. MATERIALS AND METHODS Growth conditions of bacteria. Serotype c S. mutans NG8 (kindly provided by K. W. Knox, Institute for Dental Research, Sydney, Australia) was grown aerobically to stationary phase for 16 h in Todd-Hewitt broth supplemented with 0.3% yeast extract (BBL, Cockeysville, Md.). Bacteria were centrifuged and washed twice with phosphate-buffered saline (PBS), pH 7.2, and resuspended to ⬃1.5 ⫻ 1010 CFU/ml. Anti-P1 MAbs. Five murine IgG1 MAbs, 3-10E, 4-9D, 4-10A, 1-6F, and 6-11A (8), were tested for immunomodulatory activity. MAbs 3-3B and 3-8D were used to identify amino- and carboxy-terminal fragments of P1 generated by cleavage with N-chlorosuccinimide (NCS). All MAbs were affinity purified from murine ascites fluid by using a protein A cartridge on a BioLogic HR workstation (Bio-Rad, Hercules, Calif.), dialyzed against PBS (pH 7.2) containing 0.3% sodium azide, aliquoted, and stored at ⫺20°C. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Sigma) with bovine serum albumin as the standard. Immunizations and sample collections. Groups of six 8-week-old female BALB/c mice (Charles River Laboratories, Wilmington, Mass.) were immunized intraperitoneally with ⬃1.5 ⫻ 109 CFU of S. mutans in 150 l of PBS or the same amount of S. mutans coated with either a saturating or 0.1⫻ subsaturating concentration of each MAb. The dilution of MAb necessary to saturate the bacterial cells was predetermined by serial titration and dot blot analysis as previously described (14). In each experiment, a negative control group received PBS only. To exclude the possibility of anti-idiotype effects and to control for other potential effects of MAbs occurring independently of antigen, additional control groups received MAb alone at the concentration required to saturate the immunizing dose of bacteria. Mice were prebled 1 week before the first inoculation, immunized on days 0 and 14, and exsanguinated on day 45. Measurement of anti-P1 immune response by ELISA. Serum samples were assayed for anti-P1 total immunoglobulin and IgG1, IgG2a, IgG2b, and IgG3 isotype antibodies by enzyme-linked immunosorbent assay (ELISA) as described elsewhere (14). ELISA plate wells were coated with approximately 3 ng of native
INFECT. IMMUN. P1, purified from a phosphate extract of S. mutans as described previously (12), and diluted in carbonate-bicarbonate buffer, pH 9.6. Mouse immune sera were serially diluted twofold beginning at 1:1,000. Antibody reactivity was detected with affinity-purified peroxidase-labeled goat anti-mouse (H⫹L chain) IgG (Southern Biotech, Birmingham, Ala.) at a 1:1,000 dilution. Peroxidase-conjugated IgG subclass-specific antibodies (Southern Biotech) were used at the following dilutions: anti-IgG1, 1:2,000; anti-IgG2a, 1:1,000; anti-IgG2b, 1:2,000; anti-IgG3, 1:2,000. Plates were developed with 0.1 M o-phenylenediamine dihydrochloride and 0.012% hydrogen peroxide in 0.01 M phosphate citrate buffer. Plates were incubated for 30 min at room temperature in the dark, and the absorbance at 450 nm was recorded by using an MPM Titertek model 550 ELISA plate reader (Bio-Rad). Concentrations of anti-P1 antibodies were calculated by interpolation on standard curves generated using purified mouse subclass reagents (Southern Biotech). All control sera were nonreactive with P1. NCS digestion of P1 and anti-P1 specificity analysis. To assess potential changes in antibody specificity, full-length P1 was partially cleaved at tryptophan residues with NCS as previously described (14). This method yields two predominant amino- and carboxy-terminal clusters of P1 polypeptide fragments at 85 and 120 kDa, respectively. The products of NCS digestion were electrophoresed on four replicate sodium dodecyl sulfate–10% polyacrylamide preparatory slab gels and electroblotted onto nitrocellulose filters. Each nitrocellulose filter was cut into strips, and replicate strips were reacted with serum samples from individual mice in each of the three treatment groups. Blot strips were washed and reacted with horseradish peroxidase–goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 secondary antibodies followed by washing and development with 4-chloro1-naphthol solution (7 ml of PBS, 1 ml of 4-chloro-1-naphthol [Sigma; 3 mg/ml in ice-cold methanol], and 8 l of 30% hydrogen peroxide). Appropriate digestion was confirmed on control blot strips reacted with either MAb 3-8D or 3-3B. These MAbs recognize amino- and carboxy-terminal determinants within P1, respectively (12, 20). Following development, blot strips were scanned at ⱖ1,200 dots per in., and densitometry analysis was performed using the FluorChem IS-8800 software (Alpha Innotech, San Leandro, Calif.). Mean densitometry profiles for the six animals in each of the three experimental groups were generated for each IgG subclass and compared. This procedure was performed independently for each of the five individual immunomodulation experiments. Inhibition of S. mutans adherence. Salivary agglutinin was prepared by a modification of the technique of Rundegren and Arnold (13, 63). Inhibition of adherence of S. mutans whole cells to salivary agglutinin immobilized on an F1 sensor chip was assayed using the BIAcore 3000 machine (BIAcore AB, Uppsala, Sweden). Salivary agglutinin was immobilized on the BIAcore F1 sensor chip surface in flow cell 2 by amine coupling, and the dextran matrix was activated with 35 l of an equal mixture of N-hydrosuccinimide (11.5 mg/ml) and N-ethylN⬘-(dimethylaminopropyl)carbodiimide (75 mg/ml) as suggested by the manufacturer. Agglutinin (100 ng/ml) was diluted 1:5 in acetate buffer (pH 5), and 2 to 20 l was injected manually until the change in refractive units was ⬎1,000. The remaining activated dextran was inactivated by injection of two aliquots of 35 l of 1 M ethanolamine. Flow cell 1 was treated in the same way but the addition of agglutinin was omitted, and it served as a reference surface. The flow rate was 10 l/min throughout the experiment. Adherence buffer (AB; 0.78 mM KH2PO4, 1.22 mM K2HPO4, 50 mM KCl, 1 mM CaCl2 䡠 6H2O; pH 7.2) was used as running buffer. Whole S. mutans NG8 cells (109 CFU/ml) in AB were sonicated to dechain the cells and injected at a flow rate of 10 l/min for 60 s, totaling ⬃1 ⫻ 107 cells per injection. The surface was regenerated with 5 to 25 l of PBS containing 0.03% Tween, 10 mM EDTA, 100 mM NaCl, and 100 mM NaOH. A spaP-negative isogenic mutant of S. mutans devoid of P1 (19) that does not adhere above background levels to the control surface is used in this assay (M. W. Oli, W. P. McArthur, and L. J. Brady, submitted for publication). This indicates that the detected change in resonance signal is entirely P1 mediated and is not due to an interaction of non-P1 cell surface components with low levels of potential contaminants, such as sIgA in the agglutinin preparation. Adherence inhibition was performed with murine sera pooled from each treatment group or with a protein A-purified anti-P1 MAb. One hundred microliters of a suspension with a 1010CFU/ml density was incubated with a 1:100 dilution of pooled sera or with 20 g of each anti-P1 MAb/ml, and the mixtures were rotated end over end at room temperature for 1 h. The cells were harvested by centrifugation, washed twice with AB, and then used for BIAcore analysis. Percent inhibition of adherence was determined for immune sera, with percent inhibition of control sera subtracted as background. Percent inhibition of adherence was calculated as follows: {[(percent adherence without antibody) ⫺ (percent adherence with antibody)]/(percent adherence without antibody)} ⫻ 100. Assays were performed in triplicate. Statistical analysis. Linear regression analysis was used to test the correlation of anti-P1 IgG subclass antibodies, measured by ELISA in the pooled sera of mice from each of the 15 treatment groups in the five separate experiments, with
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FIG. 1. Measurement of anti-P1 serum IgG by ELISA. The anti-P1 MAb tested is indicated in the upper left-hand corner of each panel. The experimental groups in each experiment included mice immunized with S. mutans alone (S. mutans), with S. mutans coated with a saturating concentration of the test MAb (High), or with S. mutans coated with a 0.1⫻ subsaturating concentration of the test MAb (Low). The measured amount of anti-P1 serum IgG for each of the six mice in each treatment group is indicated. The solid line represents the mean Ig level for each treatment group. ⴱ, statistically significant decrease compared to the S. mutans control group (P ⬍ 0.01). the percent inhibition of S. mutans binding to salivary agglutinin. The nonparametric Kruskal-Wallis test at ␣ ⫽ 0.05 was used to detect whether differences in the treatment groups existed. If a significant difference was detected, the data were reanalyzed to determine which treatment groups were significantly different from each other. A pair-wise comparison procedure was applied, after rank transformation. Correlation analysis was used to examine whether a global relationship between concentration of a given IgG isotype and percent inhibition of adherence existed. Statistical analysis was performed using the SAS statistical analysis system (Cary, N.C.). Data processing and graphing were performed using MS Excel (Microsoft) and Sigma Plot (Rockware Inc.).
RESULTS Evaluation of MAb-mediated effects on elicited anti-P1 serum antibodies. To determine if coating S. mutans with any of the anti-P1 MAbs prior to immunization affected the magnitude of the antibody response, total serum Ig against P1 was measured by ELISA. Anti-P1 antibodies were detected in the sera of all mice that received S. mutans as well as those that received S. mutans coated with saturating (high) or subsaturating (low) concentrations of MAb (Fig. 1). There was no measurable anti-P1 response in control mice that received buffer only or MAb only (data not shown). All experiments were performed using the some protocol; however, they were performed at different times with different batches of mice.
Hence, differences in the mean anti-P1 response among the groups of mice immunized only with S. mutans were noted and not unexpected. Statistical comparisons between treatment groups were made in comparison with the S. mutans-only control group for that particular experiment. When the effect of MAb on total anti-P1 serum Ig was analyzed, the high coating concentration of MAb 4-9D was found to result in a significant decrease in anti-P1 antibodies (P ⬍ 0.01). None of the other MAbs had a significant effect on the total level of anti-P1 serum Ig produced compared to the level in the respective S. mutans-only control group for that experiment. The subclass(es) of pathogen-specific antibodies can substantially influence the protective efficacy of an immune response (33, 43, 82). To determine if any of the MAbs tested in this study affected the IgG subclass distribution of elicited antibodies, levels of anti-P1 serum IgG1, IgG2a, IgG2b, and IgG3 also were measured by ELISA. Anti-P1 antibodies of all four subclasses were detectable, with IgG3 representing the least prevalent subclass. The influence of each of the five test MAbs on the IgG subclass response to P1 is graphically illustrated as the percent increase or decrease in the mean concentration of each subclass in the high and low MAb treatment groups compared to the response in the S. mutans-only control
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FIG. 2. Comparison of anti-P1 serum IgG subclass responses. The levels of anti-P1-specific serum IgG1, IgG2a, IgG2b, and IgG3 were measured by ELISA for the six animals in the three treatment groups for each of the five experiments. The percent increase or decrease in the mean values of the mice that received S. mutans coated with the high concentration of MAb (light bars) or S. mutans coated with the low concentration of MAb (dark bars) compared to the S. mutans-only control group are illustrated in the histogram. The MAb used in each experiment is indicated on the x axis.
group for that experiment (Fig. 2). Each MAb influenced the anti-P1 subclass response, albeit to various extents and in various ways depending on the MAb and its coating concentration. In general, MAbs 4-9D and 1-6F both appeared to have predominantly negative effects on the production of anti-P1 antibodies, while more positive effects were observed for MAbs 3-10E, 6-11A, and 4-10A. Our group showed previously that coating S. mutans with MAb 6-11A prior to administration by gastric intubation altered the specificity of anti-P1 serum IgG and mucosal sIgA responses as assessed using NCS digestion fragments of P1 as antigens on Western blotting (14, 61). This procedure enables discrimination of antibodies that recognize different epitopes within P1 that are not apparent when intact protein is used as antigen. A lack of observable differences by this method, however, does not mean that there are not additional unrecognized changes. To determine whether coating S. mutans with any of the five MAbs prior to intraperitoneal immunization influenced the specificity of the elicited anti-P1 antibodies of any of the four serum IgG subclasses, the same approach was used the present study. Sera from individual mice were reacted with replicate blot strips, and the mean densitometry profiles of the six animals in each of the three treatment groups were compared for each MAb. All five of the MAbs tested influenced the specificity of the anti-P1 response, and the results for MAb 3-10E are shown as an example (Fig. 3). Little if any change in the reactivity of the IgG1 response against NCS-digested P1 was observed. Sera from mice that received S. mutans coated
with the low concentration of 3-10E demonstrated more IgG2a against the 85-kDa P1 fragment. Sera from mice that received S. mutans coated with a high concentration of 3-10E demonstrated less IgG2b against the 120-kDa fragment and more reactivity against the 85-kDa fragment. Those mice that received S. mutans coated with the low concentration of 3-10E demonstrated less IgG2b against the 85-kDa fragment. Sera from mice in both the high- and low-MAb treatment groups had higher IgG3 reactivity against both P1 fragments compared to the S. mutans-only group. Other MAbs also demonstrated changes in serum IgG subclass reactivity against P1 fragments, predominantly within the IgG2a and IgG2b subclasses (data not shown). However, there were no obvious patterns with regard to reactivity of these antibodies with the 85- and 120-kDa P1 fragments and the ability of the sera to inhibit adherence of S. mutans to immobilized agglutinin. The only change demonstrated by MAb 4-10A was an increase in IgG3 reactivity against the 120-kDa fragment in the high-MAb treatment group, and the only MAb to affect IgG1 reactivity against NCS-digested P1 was MAb 1-6F, with an increase in the high-MAb group against the 85-kDa fragment. In summary, all of the anti-P1 MAbs affected the specificity of the anti-P1 response, and the changes were most evident within the IgG2a and IgG2b subclasses. As stated above, while this approach is useful to identify that changes in the specificity of elicited antibodies have occurred, it is not fully definitive of what all those changes are.
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FIG. 3. Binding of serum IgG subclass antibodies 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 from the experiment with anti-P1 MAb 3-10E. The ordinate corresponds to the intensity of the bands detected by the serum antibodies, and the abscissa indicates the apparent molecular masses (in kilodaltons) of the corresponding P1 fragments calculated from the electrophoretic migration. Solid line, S. mutans-only group; dotted-dashed line, S. mutans coated with the high concentration of MAb; dashed line, S. mutans coated with the low concentration of MAb.
Inhibition of S. mutans binding to human salivary agglutinin. S. mutans coated with a subsaturating concentration of MAb 6-11A and administered by gastric intubation triggered a serum anti-P1 response that was more inhibitory of S. mutans binding to salivary agglutinin than that triggered by immunization with bacteria alone (61). To determine whether intraperitoneal immunization with S. mutans complexed with any of the five MAbs tested in this study, including 6-11A, resulted in the formation of antibodies more inhibitory of bacterial adherence, a whole-cell BIAcore surface plasmon resonance assay was employed. The percent inhibition of S. mutans adherence by the pooled sera from the three treatment groups for each experiment is summarized in Fig. 4. Compared to sera from mice that received S. mutans alone, sera from mice that received S. mutans coated with the high concentration of MAb 3-10E and sera from mice that received S. mutans coated with the low concentration of MAb 6-11A were significantly more inhibitory of adherence (P ⬍ 0.05). Sera from mice that received S. mutans coated with both high and low concentrations of MAb 4-9D or with a high concentration of MAb 1-6F were significantly less inhibitory of adherence (P ⬍ 0.05). Coating S. mutans with MAb 4-10A at either concentration had no effect on the ability of elicited antibodies to inhibit bacterial adherence. To determine if the inhibitory behavior of the anti-P1 MAb itself was related to promotion of a more effective or a less effective immune response, the ability of each MAb to directly inhibit S. mutans adherence to immobilized agglutinin was also tested (Fig. 5). MAbs 3-10E and 6-11A did not inhibit adherence to a greater extent than an isotype-matched IgG1 irrelevant control MAb (anti-Actinobacillus actinomycetemcomitans). In contrast, MAbs 4-10A, 4-9D, and 1-6F all demonstrated inhibitory
activities that were significantly greater than that of the control antibody (P ⬍ 0.0001). The degree of inhibition of adherence by MAbs observed using the BIAcore assay paralleled that seen previously using an agglutinin-coated hydroxyapatite bead assay (13). When the immunomodulatory effect of coating S. mutans with each MAb on the ability of elicited anti-P1 serum antibodies to inhibit adherence was compared, it was observed that the two MAbs that themselves did not inhibit adherence promoted a more effective response, while the three MAbs that significantly inhibited adherence themselves promoted a less effective response or did not influence the efficacy of the response (Table 1). Correlation of adherence inhibition with IgG subclass of anti-P1 antibodies. As shown above, coating S. mutans with anti-P1 MAbs prior to intraperitoneal immunization resulted in changes in the IgG subclass composition and specificity of elicited antibodies and in the ability of those elicited antibodies to inhibit adherence of S. mutans to immobilized salivary agglutinin. To determine whether the ability of the anti-P1 antibodies contained in the sera of animals in the different treatment groups to inhibit S. mutans adherence was related to their isotype, linear regression analysis was performed with data compiled from all five experiments. The mean serum concentrations of anti-P1 IgG1, IgG2a, IgG2b, and IgG3 in the pooled sera from the six mice in each of the 15 experimental groups (S. mutans alone and S. mutans coated with the high and low concentrations of each MAb for five experiments) were plotted against the percent inhibition of adherence (Fig. 6). There were significant correlations (P ⬍ 0.005) between adherence inhibition and anti-P1 IgG2a (r2 ⫽ 0.47) and IgG2b (r2 ⫽ 0.68), but not for IgG1 (r2 ⫽ 0.006) or IgG3 (r2 ⫽ 0.12) antibodies.
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FIG. 4. Percent inhibition by serum antibodies of S. mutans adherence to immobilized human salivary agglutinin as measured by BIAcore surface plasmon resonance. The MAb tested for immunomodulatory activity is indicated in the upper left-hand corner of each panel. Pooled sera from the six mice in each treatment group were tested. Results are presented as the mean percent inhibition of S. mutans adherence ⫾ the standard deviation. Dark gray bars, S. mutans-only groups; light gray bars, S. mutans coated with the high concentration of MAb; medium gray bars, S. mutans coated with the low concentration of MAb. ⴱ, statistically significant difference from the S. mutans-only group (P ⬍ 0.05). ⴱⴱ, statistically significant difference from the other MAb treatment group (P ⬍ 0.05).
DISCUSSION MAb 6-11A was shown previously to alter the response against P1 when mice were mucosally immunized (14, 61). The results presented in the present report provide evidence that multiple anti-P1 MAbs, including 6-11A, demonstrate immunomodulatory activity when bound to the surface of S. mutans and administered intraperitoneally as part of an immune complex. The effects varied depending on the MAb and its coating concentration. Certain alterations in the elicited responses were apparently more biologically effective than others, as supported by relative differences in inhibition of bacterial adherence to P1’s known physiologic ligand, human salivary agglutinin. In the present study two MAbs, 6-11A and 3-10E, promoted the formation of antibodies more inhibitory of the S. mutans-agglutinin interaction. Two other MAbs, 4-9D and 1-6F, promoted the formation of less inhibitory antibodies, and MAb 4-10A did not influence the ability of elicited antibodies to inhibit adherence. An inverse correlation was observed between the ability of the MAbs to promote a more effective adherence inhibition response and the ability of the MAbs to inhibit adherence directly. As discussed below, this relation-
ship appears to relate to the specificity of the MAbs. Changes in the binding of elicited antibodies to NCS-generated P1 polypeptide fragments indicate that all of the MAbs influenced the composite specificity of the anti-P1 polyclonal antibodies in the sera of immunized animals, particularly those of the IgG2a and IgG2b subclasses. These isotypes appear to be involved in changes in biological activity that resulted from coating S. mutans with the five different anti-P1 MAbs. Statistically significant correlations were found between levels of anti-P1 IgG2a and IgG2b and the degree to which immune sera inhibited binding of S. mutans to immobilized agglutinin. The effects of actively induced and passively transferred antibodies can have measurable and complex effects on initial inflammatory and subsequent immune responses that would be critical during host-microbe interactions (17, 31, 65, 66). The ability to modulate an immune response by parenteral or mucosal immunization with an antibody bound to an antigen has been demonstrated (6, 14, 56, 58, 61, 71, 73, 83). Although factors which dictate whether an antibody complexed with an antigen will change an immune response to that antigen are not fully understood, a number of mechanisms have been suggested and include
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FIG. 5. Percent inhibition by anti-P1 MAbs of S. mutans adherence to immobilized human salivary agglutinin as measured by BIAcore surface plasmon resonance. Results are presented as the mean percent inhibition of S. mutans adherence ⫾ the standard deviation. The control was an IgG1 isotype-matched MAb against A. actinomycetemcomitans. ⴱ, statistically significant decrease compared to the control MAb (P ⬍ 0.0001).
the following: increased uptake of antigen via Fc receptors on antigen-presenting cells (46); changes in cytokine expression resulting from immune complex ligation of macrophage Fc receptors (4); masking of epitopes by antibody (10, 47); exposure of cryptic epitopes induced by antibody binding (67, 78); enhanced germinal center formation and induction of somatic hypermutation (41, 56); and/or alterations in proteolysis and antigen processing (46, 48, 67). The mechanism(s) by which anti-P1 MAbs exert their effects is not yet known, but speculations can begin to be made. All the MAbs tested are of the IgG1 subclass. Therefore, if an Fc␥R-mediated mechanism is primarily operational, the results might be expected to be similar for each MAb. This was not the case. However, considering the influence of all five MAbs on antibody specificity and isotype, changes in antigen presentation and/or cytokine expression via engagement of Fc␥Rs cannot be entirely ruled out (2, 3, 6). A change in susceptibility of a protein to proteolytic digestion is an indication of an alteration in its conformation (34, 36, 77, 80). We have demonstrated that anti-P1 MAb 6-11A bound to S. mutans substantially increases the rate and degree of digestion of cell-associated P1 by numerous proteases, as well as altering the products of digestion, without influencing breakdown of non-P1 proteins (61). In general, destabilization of structure is associated with exposure of cryptic target sites
and faster and more extensive digestion. Native and destabilized proteins vary with regard to their immunogenicity (55, 62, 69). Increases in structural flexibility and susceptibility to proteolysis are associated with a stronger and broader helper T-cell response (16, 21, 70), and change of a single cleavage site can dramatically influence the presentation of multiple T-cell epitopes (5). Investigators have shown that presentation of particular antigen-specific T-cell determinants can be enhanced or suppressed as a direct consequence of antibody modulation of antigen processing (6, 48, 67, 78), and changes in the specificity of T-cell epitopes modulate the fine specificity of an antibody response (42, 49). In addition, Milch et al. (52) and Akdis et al. (1) have reported that conformational variants of antigens can result in differences in cytokine expression by helper T cells, a factor in determination of murine Ig isotype (1, 72, 79). We speculate that changes in the susceptibility of P1 to proteolysis as a result of binding of anti-P1 MAbs would result in a different spectrum of peptides that interact with the antigen-specific T-cell repertoire and influence the spectrum of antibodies elicited during a polyclonal response. The influence of T-cell specificity on the ultimate antibody response may provide an explanation for the observation of Kelly et al. (37) that individuals with low caries susceptibility preferentially rec-
TABLE 1. Comparison of inhibition of S. mutans adherence Adherence inhibition activity of MAb compared to controla Inhibitory reagent
MAb alone MAb-modulated anti-P1 antiserum
3–10E
6–11A
4–9D
1–6F
4–10A
No effect Increased
No effect Increased
Inhibitory Decreased
Inhibitory Decreased
Inhibitory No difference
a The control for the anti-P1 MAbs was an irrelevant isotype-matched IgG1 MAb. The control for each MAb-modulated anti-P1 polyclonal antiserum was antiserum raised against S. mutans alone.
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FIG. 6. Linear regression analysis to assess the relationship between anti-P1 serum IgG subclass and inhibition of S. mutans adherence. The mean level of anti-P1 serum antibodies in each of the 15 groups from all five immunomodulation experiments was plotted against the percent inhibition of S. mutans adherence determined for each group. The IgG subclass measured is indicated in each panel. Correlation coefficients (r2) are indicated on the graphs. Statistically significant correlations (P ⬍ 0.005) were observed between adherence inhibition and levels of anti-P1 IgG2a and IgG2b.
ognized a particular T-cell epitope of P1 compared to individuals with high caries susceptibility. The modulatory effect of each anti-P1 MAb on the elicited immune response appears to be related to its specificity. MAb 4-9D was the only reagent shown to decrease the overall magnitude of the anti-P1 response. Both 4-9D and 1-6F promoted the formation of anti-P1 antibodies less inhibitory of bacterial adherence and demonstrated predominantly negative effects on IgG2a and IgG2b responses. MAbs 4-9D and 1-6F share the common property of recognizing epitopes contributed to by the intervening segment of P1 between the alanine-rich and proline-rich repeat regions (12). They are themselves strong inhibitors of bacterial adherence and therefore may mask an agglutinin-binding site or block recognition of epitopes that are important targets of a beneficial response (10, 47). On the other hand, the MAbs that are not inhibitory of P1’s interaction with immobilized agglutinin, but that promote the formation of antibodies that are, may do so by an influence on protein structure and exposure of cryptic or partially masked epitopes (67, 78). MAbs 6-11A and 3-10E both promoted the formation of inhibitory antibodies but are not themselves inhibitory of adherence. MAb 6-11A’s epitope has been shown to depend on an interaction between the alanine- and prolinerich repeat domains of P1 and is also contributed to by sequence immediately upstream of the alanine-rich region (11, 60, 64). MAb 3-10E shares both of these properties (unpublished data). Interestingly, deletion of P1 residues upstream of the alanine-rich repeats increases binding of the adherenceinhibiting MAb 1-6F (unpublished data). Therefore, elimination of pre-A-region sequence may have a similar effect on P1 structure and consequent exposure of important targets of protection as contact of MAbs 3-10E and 6-11A with this
segment. The epitope recognized by 4-10A, the MAb that did not affect the adherence inhibition response, is dependent on an interaction between the alanine- and proline-rich domains but does not involve sequence upstream of the alanine-rich domain (unpublished data). Several studies suggest that the interaction of the antigen I/II family of molecules with salivary glycoproteins is complex and involves multiple binding sites (28, 35). The ability of isolated fragments of P1 to interact with salivary components has been reported (20, 23, 37, 53, 54, 57), while other studies indicate that interactions of discontinuous segments of P1 contribute to function and antigenicity (13, 14, 60, 61, 64, 74, 75). Hence, protective antibodies elicited during a polyclonal response would be expected to recognize linear as well as conformational determinants and may act directly or via allosteric effects. Rhodin et al. have demonstrated that the anti-P1 response elicited by immunization with whole S. mutans cells is in large part directed against discontinuous epitopes (61). Immunomodulatory MAbs may also mediate their effects by direct interaction with ligand binding sites or indirectly by influencing protein structure. Detection of MAbmediated changes in antibody specificity against P1 partially digested with NCS likely reflects changes in recognition of complex epitopes, because analyses of sera do not demonstrate differential reactivities with smaller recombinant P1 polypeptides spanning the molecule (61). In addition to sIgA, IgG and IgM are detectable in human saliva, presumably via transudation through the gingival crevice (24). Salivary and serum antibodies have both been reported to contribute to protective immunity against S. mutans (33, 82). The influence of the form of S. mutans antigens on the serum IgG subclass response has been described in a study utilizing a chimeric protein consisting of the saliva-binding
IMMUNOMODULATION BY MAbs TO S. MUTANS P1 ADHESIN
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region of P1 and the glucan-binding region of glucosyltransferase (82). Effective antibody-mediated protection depends on both the isotype and the specificity of the antibodies (14, 76, 82). The results presented in this report indicate that immunomodulatory anti-P1 MAbs can influence both the isotype and specificity of the anti-P1 response and that the more effective antibodies with regard to inhibition of bacterial adherence in vitro appear to be of the IgG2a and IgG2b subclasses. Elucidation of the mechanism(s) of immunomodulation, characterization of the relevant epitopes of P1 recognized by IgG2a and IgG2b antibodies, and the contributions of antibodies of these subclasses to protection against S. mutans colonization and cariogenicity in vivo remain to be seen and will be the focus of future work. Our results indicate that multiple MAbs against S. mutans P1 possess immunomodulatory activity, that changes in the immune response vary depending on the MAb and its coating concentration, that certain alterations of the serum antibody response affect biological function more than others, and that it is possible to utilize MAb-mediated immunomodulation to increase the beneficial response against a pathogen. This approach represents a useful strategy to elicit more effective antibodies against the P1 adhesin molecule and provides a tool to dissect beneficial and nonbeneficial immune responses against this widely studied candidate vaccine antigen. Beyond the impact of these results on the development of a therapeutic approach against dental caries, the well-characterized P1 antigen and MAbs against it represent a model system to begin to understand the consequences and underlying molecular mechanisms of immunomodulation by antibody that are important in the consideration of active and passive immunization approaches against microbial pathogens. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant DE13882 and training grant T32-DE07200. REFERENCES 1. Akdis, C. A., T. Blesken, D. Wymann, M. Akdis, and K. Blaser. 1998. Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants. Eur. J. Immunol. 28:914–925. 2. Anderson, C. F., J. S. Gerber, and D. M. Mosser. 2002. Modulating macrophage function with IgG immune complexes. J. Endotoxin Res. 8:477–481. 3. Anderson, C. F., and D. M. Mosser. 2002. Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J. Immunol. 168:3697–3701. 4. Anderson, C. F., and D. M. Mosser. 2002. A novel phenotype for an activated macrophage: the type 2 activated macrophage. J. Leukoc. Biol. 72:101–106. 5. Antoniou, A. N., S. L. Blackwood, D. Mazzeo, and C. Watts. 2000. Control of antigen presentation by a single protease cleavage site. Immunity 12:391–398. 6. Antoniou, A. N., and C. Watts. 2002. Antibody modulation of antigen presentation: positive and negative effects on presentation of the tetanus toxin antigen via the murine B cell isoform of Fc␥RII. Eur. J. Immunol. 32:530–540. 7. Ayakawa, G. Y., A. S. Bleiweis, P. J. Crowley, and M. W. Cunningham. 1988. Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin. J. Immunol. 140:253–257. 8. Ayakawa, G. Y., L. W. Boushell, P. J. Crowley, G. W. Erdos, W. P. McArthur, and A. S. Bleiweis. 1987. Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci. Infect. Immun. 55:2759–2767. 9. Banas, J. A. 2004. Virulence properties of Streptococcus mutans. Front. Biosci. 9:1267–1277. 10. Berzofsky, J. A. 1983. T-B reciprocity. An Ia-restricted epitope-specific circuit regulating T cell-B cell interaction and antibody specificity. Surv. Immunol. Res. 2:223–229. 11. Brady, L. J., D. G. Cvitkovitch, C. M. Geric, M. N. Addison, J. C. Joyce, P. J. Crowley, and A. S. Bleiweis. 1998. Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule. Infect. Immun. 66:4274–4282.
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