Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic region of mutans streptococcal glucosyltransferase protects

against dental caries.

M A Taubman, C J Holmberg and D J Smith Infect. Immun. 1995, 63(8):3088.

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INFECTION AND IMMUNITY, Aug. 1995, p. 30883093 0019-9567/95/$04.000 Copyright 1995, American Society for Microbiology

Vol. 63, No. 8

Immunization of Rats with Synthetic Peptide Constructs from the Glucan-Binding or Catalytic Region of Mutans Streptococcal Glucosyltransferase Protects against Dental Caries
MARTIN A. TAUBMAN,* CYNTHIA J. HOLMBERG,
AND

DANIEL J. SMITH

Department of Immunology, Forsyth Dental Center, Boston, Massachusetts 02115

Received 3 February 1995/Returned for modication 24 March 1995/Accepted 8 May 1995

Previously, we have described peptide constructs from two regions of glucosyltransferase (GTF) of mutans streptococci. A putative catalytic site in the amino-terminal half of the molecule and a repeated glucan-binding site in the carboxyl-terminal half of GTF were the regions upon which sequences were based. The present study explored the effects of immunization with these peptide constructs (called CAT or GLU) and with streptococcal GTFs from Streptococcus sobrinus and S. mutans on immunological, microbiological, and disease parameters. Groups of immunized Sprague-Dawley rats were infected with either 108 S. sobrinus 6715 or 108 S. mutans SJ32 organisms. Serum immunoglobulin G antibody levels, determined by enzyme-linked immunosorbent assay, to the respective peptide constructs and to the appropriate streptococcal GTF were signicantly increased (after immunization) prior to infection and at the end of the experiment. Also, serum antibody from CAT-, GLU-, and S. sobrinus GTF-immunized rats inhibited S. sobrinus GTF-mediated insoluble glucan synthesis (all) and S. mutans GTF-mediated soluble glucan synthesis (all except anti-GLU) from sucrose. Immunization with the CAT or GLU peptide construct resulted in signicantly reduced smooth surface and sulcal caries after infection with S. sobrinus. Sulcal dental caries after infection with S. mutans SJ32 were also signicantly reduced in CAT- and GLU-immunized rats. Thus, immunization with peptides whose sequences are based on putative functional domains of mutans streptococcal GTF are protective toward a cariogenic S. sobrinus or S. mutans infection. The etiology of dental caries has been associated with the acid by-products of bacterial metabolism. The production of these by-products has been related to a group of aciduric oral microorganisms collectively called the mutans streptococci (4). Important microorganisms in this group which are found in humans include Streptococcus mutans and S. sobrinus (12). An additional feature of the molecular pathogenesis of dental caries appears to be the role of accumulation of these, or like microorganisms, in dental plaque whose formation is dependent on synthesis of glucan from sucrose catalyzed by glucosyltransferase (GTF) enzymes. Previous investigations have indicated that the use of this enzyme as an antigen may result in protection from experimental dental caries in rodents (22, 32) and in the induction of salivary immunoglobulin A (IgA) antibody in humans accompanied by interference with reaccumulation of indigenous mutans streptococci after dental prophylaxis (21, 25). Although the exact basis for experimental protection with such GTF-type vaccines is presently unknown, it appears likely that such protection can involve functional inhibition of the catalytic and/or the glucan-binding activity of GTF. Antibody-mediated inhibition of these functional activities has been demonstrated (33, 34). The glucan-binding and catalytic sites appear to reside in different GTF domains. Thus, the GLU region(s) is found in the C-terminal third of the GTF (1, 19, 36), whereas the catalytic domain appears to be located in the N-terminal third of GTF (1, 3, 19). We have synthesized peptide constructs representative of the glucan-binding domain and the catalytic domain of mutans streptococcal GTF (24, 26). These peptides were found to be immunogenic in a variety of rodent species
* Corresponding author. 3088

(24, 26). Also, the antibody so elicited could interfere with GTF function (24, 26). Therefore, this study evaluated each of these peptide constructs for the ability to elicit antibody which might affect experimental dental caries. MATERIALS AND METHODS
Synthetic peptides and antigens. CAT peptide. A nonapeptide, DSIRV DAVD, located between residues 448 and 457 of the published sequence of the GTF-I of S. downei contains an aspartic acid which has been shown to be involved in the catalytic reaction of GTF with sucrose (17). The sequence of the CAT peptide used in the present study (DANFDSIRVDAVDNVDADLLQ) contained this nonapeptide. An identical sequence is found in a similar region of S. mutans GTF-I (20). The residues within the DSIRVDAVD peptide are either identical to those of or conserved in S. sobrinus GTFs that produce waterinsoluble glucan (IG) (1). The peptide was synthesized (Applied Diagnostics, Foster City, Calif.) by using the stepwise solid-phase method of Merrield (15) on a core matrix of three lysines to yield a multiple antigenic peptide macromolecule with four identical 21-mer peptides per molecule, after the method of Tam (28). Purity (90%) was assessed by high-pressure liquid chromatography, amino acid analysis, and molecular mass determination by mass spectrometry. This peptide-multiple antigenic peptide construct, referred to as CAT, was used for immunization and antibody analyses. GLU peptide. Repeating sequences within the C-terminal third of the GTF molecule have been associated with binding of glucan by these enzymes (6, 19, 36). The composition of a 22-mer GLU peptide, sequence TGAQTIKGQKLY FKANGQQVKG, was based on the derived sequence of one of the repeating regions of S. downei GTF-I (residues 1303 to 1324) which was 86% homologous with an S. sobrinus GTF-I sequence (1) and 77% homologous with S. mutans GTF-I (20) in the same region. Residues 1303 to 1319 are also substantially the same as a consensus sequence for putative carbohydrate-binding regions of other proteins that bind glucan polymers (35). The GLU peptide was synthesized (Applied Diagnostics) on a core matrix of three lysines to yield a macromolecule with four identical 22-mer peptides per molecule, as described above. GTF. GTFs from S. sobrinus 6715 and S. mutans SJ32 were obtained as previously described (24). Briey, after bacterial growth in glucose-containing dened media, enzymes were isolated by chromatography on Sephadex G-100 (Pharmacia Biotech Inc., Piscataway, N.J.), using 3 M guanidine HCl as the eluting solvent. These GTF-rich pools were then subjected to fast protein liquid

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chromatography on Superose 6 (Pharmacia), using 6 M guanidine for elution. The gel ltration step removes non-GTF and other glucan-binding proteins from GTF preparations of S. mutans and S. sobrinus, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which only components with enzyme activity were observed. The S. mutans GTF preparation taken to this level of enrichment synthesized 51 to 81% water-soluble glucan (SG) by lter assay (32) and thus was analogous to a mixture of the gtfB, gtfC, and gtfD gene products of S. mutans GS5 (9, 11). This preparation was designated GTF-Sm and was used for injection and enzyme-linked immunosorbent assay (ELISA). S. sobrinus GTF preparations obtained after gel ltration on Superose 6 contained a mixture of GTF-I (IG product), GTF-Sd (primer-dependent SG product), and GTF-Si (primer-independent SG product) (13, 14). This preparation was designated GTF-Ss and was used for injection and ELISA. Animals. Gnotobiotic Sprague-Dawley rats of strain CD (SD), originally reared in the isolator facility of Charles River Laboratories, Wilmington, Mass., were infected with a dened ora and then screened and found to be free of indigenous mutans streptococci. These rats were bred in our facility, and the female progeny, weaned at approximately 20 days and raised on high-sucrose Diet 2000 (27), were used in the experiments described below. Protocol for animal experiments. Groups of 20-day-old rats were subcutaneously injected in the salivary gland vicinity (31) with CAT or GLU peptide constructs or with streptococcal GTFs or phosphate-buffered saline (PBS) (control animals), all incorporating complete Freund adjuvant. One week later, rats were reinjected with PBS or with the same antigen and dose in incomplete Freund adjuvant. One week after the second injection, some groups (described below) were orally infected with approximately 108 S. sobrinus or S. mutans organisms for 3 consecutive days (27). Rats placed into six experimental groups of six to eight animals each were treated as follows: group 1, sham immunized, noninfected; group 2A, sham immunized, infected with S. sobrinus 6715; group 2B, sham immunized, infected with S. mutans SJ32; group 3A, immunized with 50 g of CAT construct, infected with S. sobrinus; group 3B, immunized with 50 g of CAT, infected with S. mutans; group 4A, immunized with 50 g of GLU construct, infected with S. sobrinus; group 4B, immunized with 50 g of GLU, infected with S. mutans; group 5A, immunized with 50 g of GTF-Ss, infected with S. sobrinus; group 5B, immunized with 50 g of GTF-Ss, infected with S. mutans; group 6A, immunized with 25 g of GTF-Sm, infected with S. sobrinus; group 6B, immunized with 25 g of GTF-Sm, infected with S. mutans. One week after the second injection, animals were bled from the retroorbital plexus and saliva was collected after injection of pilocarpine (1.0 mg/100 g of body weight; Sigma Chemical Co., St. Louis, Mo.). Animals infected with S. mutans were terminated 60 days following initial infection; those infected with S. sobrinus were terminated 83 days after infection. Antibody analyses. Serum and saliva were tested for the presence of antibody by a previously described ELISA performed in microtiter plates (27). The antigens used on plates were as follows: 0.5 g of CAT per well, 0.5 g of GLU per well, 0.15 g of GTF-Ss per well, and 0.05 g of GTF-Sm per well. Isotypespecic rabbit anti-rat IgA or IgG (32) was used with goat anti-rabbit IgGalkaline phosphatase (TAGO, Inc., Burlingame, Calif.). The plates were developed with p-nitrophenyl-phosphate (Sigma) and read on a photometric scanner (Dynatech, Winooski, Vt.) at 405 nm. Antibody of each isotype (IgG and IgA) was expressed separately as ELISA units of a particular isotype, which were calculated relative to the titration of appropriate reference sera from SpragueDawley rats hyperimmunized with each of the antigens mentioned above (24, 26). The following dilutions for serum IgG were considered 100 ELISA units: for anti-CAT, 1/1,600; anti-GLU, 1/6,400; anti-GTF-Ss, 1/25,600; and anti-GTF-Sm, 1/6,400. For IgA (100 ELISA units), all sera were diluted 1/100. GTF inhibition assay. Rat sera (preinfection and termination) were evaluated for the ability to inhibit glucan synthesis by GTF-Ss or GTF-Sm, using a modied lter assay previously described (26). Briey, serum samples (1 l each) were combined with the respective GTF preparation in a nal volume of 100 l in 0.02 M sodium PBS0.02% sodium azide (pH 6.5) and were incubated for 2 h at 37C. To this was added 100 l of PBS-sodium azide containing 0.85 mg of sucrose and 22 nCi of [14C-glucose]sucrose (approximately 50,000 cpm), and the mixture was incubated for 2 h at 37C. IG was collected on Whatman GF/F glass ber lters and washed with PBS-sodium azide, and the radioactivity was determined as previously described (34). SG in the ltrate was precipitated with 70% ethanol after the addition of 4 mg of carrier dextran T10 and centrifuged, and the radioactivity was determined. Bacterial recoveries. The mutans streptococcal ora was assessed at termination. Systematic swabbing of teeth, sonication, and plating of appropriate dilutions on mitis salivarius agar (total streptococci; Difco Laboratories, Detroit, Mich.) were all performed as previously described (27). The numbers of mutans streptococci on these plates, as identied by colonial morphology, are presented as a percentage of the total streptococci. Caries assessment. The extent and depth of carious lesions in all rat molar teeth (caries score) were microscopically evaluated by a modied Keyes method, as previously described (31). These combined caries scores were determined separately on smooth and on sulcal dental surfaces.

TABLE 1. Serum IgG antibody levels to CAT or GLU constructs in rats after two injections and at the experiment termination
ELISA units (mean SE)b Group Injected antigena Test antigen Preinfection S. sobrinus infection termination S. mutans infection termination

1 2 3 1 2 4
a b

Sham Sham CAT Sham Sham GLU

CAT CAT CAT

5 1c (16) NT 33 9e (13)

20 3d (7) 22 4d (7) 53 19d (6)

20 3 (7) 17 2 (6) 17 1 (5) 4 0.5 (6) 2 0.6c (5) 635 98f (6)

GLU 1 0.3c (18) 4 0.5 (6) GLU NT 6 1c (7) GLU 232 48f (11) 464 20f (5)

Sham, sham immunized, uninfected; sham, sham immunized, infected. Determined prior to infection and 7 days after two immunizations and at termination. Values in parentheses are numbers of animals tested. NT, not tested. c The value for this sham group is statistically signicantly different from the values for the groups indicated by footnotes e and f by one-way analysis of variance followed by unpaired t test. d P 0.07 (one-way analysis of variance). e P 0.01. f P 0.001.

RESULTS Serum IgG antibody. The CAT- or GLU-immunized groups formed signicant preinfection antibody to their homologous peptide constructs in ELISAs (Table 1). Thus, rats immunized with CAT showed signicant serum antibody levels to CAT (P 0.01) compared with sham immunized rats, as did sera from GLU-immunized rats when tested against GLU (P 0.001). At termination after infection with S. sobrinus, the level of antibody to CAT was elevated (P 0.07; analysis of variance) and the level of antibody to GLU was signicantly elevated (at least P 0.01) as it was after S. mutans infection (P 0.001) compared with the sham positive group. After two immunizations, statistically signicant antibody levels to GTF-Ss (Fig. 1A) were detected in the groups of animals immunized with CAT (P 0.05) and GLU (P 0.01) and also with GTF-Ss and GTF-Sm (P 0.001) compared with sera from sham-injected rats. Also, these immunized animals demonstrated signicant increases in antibody to GTF-Sm (Fig. 1B) after immunization with GLU (P 0.01) or GTF-Ss or GTF-Sm (P 0.001). In each case, the serum antibody levels at the termination of the experiments are shown to the GTF preparation corresponding to the infecting bacterial strain (Fig. 1A and B). Signicant serum IgG antibody to GTF-Ss (P 0.001) or GTF-Sm (at least P 0.01) was found in groups of animals immunized with GTF-Ss or GTF-Sm when tested against GTF-Ss (Fig. 1A) and similarly when tested against GTF-Sm (Fig. 1B). At termination, after infection with S. sobrinus, there was a signicant elevation of antibody to GTF-Ss (at least P 0.03; t test) in animals injected with either peptide antigen or GTFs after infection with either mutans streptococcus compared with preinfection antibody levels. Therefore, all immunized animals demonstrated serum antibody to GTFs before infection, and for the most part, antibody levels increased (compared with preinfection) after infection with S. sobrinus or S. mutans. Salivary IgA antibody. Since only small amounts of saliva were obtained at 34 days of age after immunization, only antibody to the GTF preparation of the subsequent infecting mutans streptococcal strain was tested (Tables 2 and 3). At the termination of the experiments, animals injected with CAT

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INFECT. IMMUN. TABLE 3. Salivary IgA antibody levels to S. mutans SJ32 GTFs in rats after two injections and at the experiment termination
ELISA units (mean SE)b Group Injected antigena Preinfection S. sobrinus infection termination S. mutans infection termination

1 2 3 4 5 6
a b

Sham Sham CAT GLU GTF-Ss GTF-Sm

NT NT NT NT NT 156 9 (14)

120 33c (7) 560 111d (7) NT NT NT 486 99d (7)

120 33c (7) 486 144e (5) 612 101f (6) 1,070 303d (6) 683 268e (5) 757 242e (6)

Sham, sham immunized, uninfected; sham, sham immunized, infected. Determined prior to infection and 7 days after two immunizations and at termination. Values in parentheses are numbers of animals tested. NT, not tested. c The value for this sham group is statistically signicantly different from the values for the groups indicated by footnotes d to f. d P 0.01. e P 0.05. f P 0.001.

FIG. 1. Serum antibody (IgG) in rats to GTF antigens from S. sobrinus 6715 (A) or S. mutans SJ32 (B). Sera were taken prior to infection (preinfection) and at the termination of the experiment (S. sobrinus after 83 to 87 days; S. mutans after 60 to 62 days). Bars indicate the mean antibody level of serum from 5 to 17 rats in each of the designated groups, expressed in ELISA units (EU). Error bars indicate the standard error of the mean. Differences are statistically signicant at the following levels compared with the sham immunized, uninfected (preinfection) or sham immunized, infected (termination) sham group by one-way analysis of variance followed by unpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001. , sham, uninfected; s, sham, infected; o, CAT, infected; d, GLU, infected; p, GTF-ss, infected; s, GTF-sm, infected.

and infected with S. sobrinus (Table 2) or S. mutans (Table 3) had signicantly elevated salivary IgA antibody to GTF-Ss and GTF-Sm, respectively, compared with preinfection sham immunized rats and uninfected sham immunized animals at ter-

mination. Antibody to GTF-Ss in the GLU-immunized group was elevated compared with the uninfected sham-minus group after infection with S. sobrinus (Table 2). Antibody also increased in the GTF-Sm-immunized group after infection with S. mutans (Table 3). The salivary IgA levels in both peptideimmunized groups, when tested with the homologous peptide construct (i.e., CAT or GLU [Table 4]), were signicantly elevated in animals infected with S. sobrinus or S. mutans compared with levels in sham immunized, noninfected animals. Serum antibody-mediated inhibition of GTF function. Sera taken prior to and after infection were tested for inhibition of the function of GTF enzymes from S. sobrinus or S. mutans. Both IG (Fig. 2) and SG syntheses by the GTF enzymes from S. sobrinus 6715 were signicantly inhibited by serum from animals immunized with CAT, GLU, and GTF-Ss. Immunization with GTF-Ss gave the greatest IG inhibition. Also, SG, but not IG, synthesis by GTF-Sm was signicantly inhibited by sera from animals immunized with CAT, GLU, and GTF-Ss (Fig. 3). The combination of immunization and infection also resulted in inhibitory antibody.

TABLE 2. Salivary IgA antibody levels to S. sobrinus 6715 GTFs in rats after two injections and at the experiment termination
ELISA units (mean SE)b Group Injected antigena Preinfection S. sobrinus infection termination S. mutans infection termination

TABLE 4. Salivary IgA antibody levels to CAT or GLU constructs in rats after two injections and at the experiment termination
ELISA units (mean SE)b Group Injected antigena Test antigen Preinfection S. sobrinus infection termination S. mutans infection termination

1 2 3 4 5 6
a b

Sham Sham CAT GLU GTF-Ss GTF-Sm

24 9 (16) NT NT NT 47 15 (16) NT

10 1c (6) 45 7d (7) 56 4d (6) 57 12e (7) 43 8e (6) 128 35e (7)

10 1 (6) 197 108 (4) NT NT 76 42 (5) NT

1 2 3 1 2 4
a b

Sham Sham CAT Sham Sham GLU

CAT CAT CAT GLU GLU GLU

NT NT 4 1.0 (15) NT NT 37 5 (15)

11 2c (6) 11 3 (6) 26 6d (7) 14 6c (7) 18 3 (7) 28 3e (7)

11 2 (6) 7 2 (5) 17 3 (6) 14 6c (7) 26 7 (5) 20 1d (6)

Sham, sham immunized, uninfected; sham, sham immunized, infected. Determined prior to infection and 7 days after two immunizations and at termination. Values in parentheses are numbers of animals tested. NT, not tested. c The value for this sham group is statistically signicantly different from the values for the groups indicated by footnotes d and e. d P 0.001. e P 0.01.

Sham, sham immunized uninfected; sham, sham immunized, infected. Determined prior to infection and 7 days after two immunizations and at termination. Values in parentheses are numbers of animals tested. NT, not tested. c The value for this sham group is statistically signicantly different from the values for the groups indicated by footnotes d and e. d P 0.05. e P 0.01.

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FIG. 2. Inhibition of IG or SG synthesis by S. sobrinus GTF by serum antibody. Inhibition is tested by incorporation of glucose from 14C-glucose-labelled sucrose into IG or SG and is shown for sera taken prior to infection (n 6 to 8) and at the termination of the experiment (n 4 to 9) after infection with S. sobrinus. All groups are indicated by antigen used for injection, with S indicating sham immunized, uninfected, and S indicating sham immunized, infected mice. Bars indicate the mean percent inhibition ( standard error). Counts incorporated into IG and SG in the presence of sham immunized, uninfected serum taken prior to infection (n 8) were 1,928 92 and 857 22, respectively. The counts incorporated into IG and SG in the presence of sham immunized, uninfected sera (n 3) taken at termination were 2,006 31 and 839 19, respectively. Differences are statistically signicant at the following levels compared with the sera from the sham immunized, uninfected group (preinfection and at termination) by one-way analysis of variance followed by unpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001.

FIG. 4. Dental caries scores of animals immunized and infected with S. sobrinus (n 6 to 7) or with S. mutans (n 6 to 7). Bars show the mean caries scores on smooth surfaces and on sulcal surfaces and the standard errors. Differences are statistically signicant at the following levels compared with the sham immunized, infected group by one-way analysis of variance followed by unpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001.

Relative recovery of mutans streptococci and dental caries assessment at experiment termination. At the termination of the experiment, relative recovery of mutans streptococci, expressed as a percentage of the total streptococci from the immunized groups, was compared with recovery from the sham immunized, infected animals. After infection with S. sobrinus, CAT- (mean mutans streptococci as percentage of total streptococci standard error 54.7% 8.9%; n 7), GLU(74.4% 4.9%; n 6), GTF-Ss- (46.7% 9.1%; n 6), and GTF-Sm-immunized (55.8 10.9; n 7) groups showed statistically signicant reductions (at least P 0.05; except GLU) compared with the sham-plus group (88.5% 3.1%; n 6). However, no differences were found after S. mutans infection. Comparison of each immunized S. sobrinus-infected group with the sham immunized S. sobrinus-infected group indicated statistically signicant reductions (P 0.001; Student-Newman-Keul test) in total caries in the CAT-, GLU-, GTF-Ss-, and GTF-Sm-immunized groups (not shown). Comparison of each immunized S. mutans-infected group with the sham immunized S. mutans-infected group also indicated statistically signicant reductions in total caries in the CAT- (P 0.001), GLU- (P 0.05), GTF-Ss- (P 0.05), and GTF-Sm-immunized (P 0.05) groups (not shown). Highly signicant reductions in dental caries in sulci (at least P 0.01) occurred after immunization of rats with CAT, GLU, GTF-Ss, or GTF-Sm and infection with S. sobrinus or S. mutans (Fig. 4). Reductions in smooth surface caries, relative to the sham immunized, infected group, were statistically signicant after immunization with CAT, GLU, GTF-Ss, and GTF-Sm following infection with S. sobrinus; after S. mutans infection they were only signicant after immunization with the CAT construct (Fig. 4). DISCUSSION There has been abundant evidence to support the involvement of mutans streptococcal GTF in the pathogenesis of dental caries. Mutants defective in GTF activity were shown to be decient in cariogenic ability compared with wild-type organisms. Conversely, mutants which were superproducers of GTF demonstrated more caries than the wild type (5, 16, 30). Recently, Yamashita and his colleagues (39) have used inser-

FIG. 3. Inhibition of IG or SG synthesis by S. mutans GTF by serum taken prior to infection (n 6) and at termination (n 4 to 5) after infection with S. mutans. Bars indicate the mean percent inhibition ( standard error) of incorporation of glucose from 14C-glucose-labelled sucrose into IG or SG. The counts incorporated into IG and SG, in the presence of sham immunized, uninfected sera (n 8) taken prior to infection, were 477 53 and 989 42, respectively. The counts incorporated into IG and SG in the presence of sham immunized, uninfected sera (n 3) taken at termination were 369 31 and 819 40, respectively. Differences are statistically signicant at the following levels compared with sera from the sham immunized, uninfected group (preinfection or termination) by one-way analysis of variance followed by unpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001.

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INFECT. IMMUN.

tionally inactivated S. mutans GTF genes to replace the functional wild-type copy of the gene with the defective counterpart in organisms used to infect rodents (39). Caries was markedly diminished when the gtfB and gtfC genes required for IG synthesis were inactivated. The gftD gene coding synthesis of SGs also appeared to be signicant in the pathogenesis of caries, although its role may be less signicant in the presence of lower amounts of sucrose (39). In this paper, we explored the suggestion that antibody to functionally signicant domains of GTF from mutans streptococci, which blocked the expression of function by this enzyme, could interfere with enzyme-mediated aspects of the molecular pathogenesis of dental caries. The data presented herein conrm this notion. Immunization of rats with peptide constructs of CAT or GLU resulted in serum IgG and salivary IgA antibodies to the respective peptide and to S. sobrinus and S. mutans GTFs. Functional inhibition of S. sobrinus GTF IG and SG syntheses was demonstrated by serum antibody to CAT, GLU, and S. sobrinus GTFs. Inhibition of S. mutans GTF SG synthesis by serum antibody to CAT, GLU, and S. sobrinus GTFs was also shown. Previously, we had demonstrated inhibition of GTF-Ss IG synthesis by rat anti-GLU antibody (24) and mouse IgM antiCAT monoclonal antibody (26) and of SG synthesized by GTF-Sm and by mouse monoclonal and polyclonal rat sera to CAT (26). Importantly, the current experiments indicated that serum antibody to CAT or GLU peptides could inhibit synthesis of IG and SG mediated by mixtures of GTF enzyme from S. sobrinus and SG mediated by GTF enzymes from S. mutans. The ndings support the notion that the sequences chosen can achieve a broad protective effect. In the enzyme inhibition systems employed, there is never 100% inhibition because of the excess of substrate. Inhibition by sera from rats immunized with S. sobrinus GTF was in the order of 50 to 70%. Therefore, 15 to 20% inhibition of IG represents signicant but not maximal inhibition. The inhibition may not be maximal for several reasons: (i) the catalytic site may not be directly or completely accessible; (ii) the potential limited specicity of the anti-CAT may restrict inhibition; and (iii) the conformation of the CAT peptide construct may be different than in the intact GTF and conformationally active determinants may not be recognized. Thus, the extent of conformational integrity of the multiple antigenic peptides is essentially unknown. Presumably, the antibody elicited in the current experiments effectively inhibited the function of the GTFD protein enzyme of S. mutans (Fig. 3; preinfection) and that of the GTF-I, -Si, and -Sd enzymes of S. sobrinus (Fig. 2, preinfection and termination), and this led to the protection seen. Anti-CAT and anti-GLU signicantly inhibited SG synthesis by S. mutans GTFs. Since GTFD can be signicant in smooth caries in the rat model (39), the results of S. mutans infection leading to caries (Fig. 4) may be explained by an effect of SG inhibition on sulcal caries. Interestingly, the sequence of the GLU peptide as synthesized was shown to have 50% homology with the deduced sequence of an S. mutans glucan-binding protein (24). Also, polyclonal rodent antibody to GLU reacted in Western blot (immunoblot) with a separate discrete glucan-binding protein from S. sobrinus (24), and it has been demonstrated that such antibody can inhibit glucan-binding function (38). Thus, a combination of antibody-mediated inhibition of glucan binding by both GTF and glucan-binding protein could conceivably interfere with both S. mutans and S. sobrinus glucan-mediated accumulation. Also, after infection with S. sobrinus highly signif-

icant reductions in dental caries (P 0.001) were observed. Reductions were seen on smooth surfaces and also in sulcal caries after S. sobrinus infection. However, smooth surface reductions were less signicant after S. mutans infection, suggesting that there may be a differential protective effect possibly based on inhibition of IG synthesis which was more significant when tested against GTF-Ss (Fig. 2) than against GTF-Sm (Fig. 3). This may reinforce the notion that IG may be most signicant in smooth surface caries (39). It has recently been suggested that intra- and interspecies variation between streptococcal enzymes may be an ongoing process that could circumvent the idea of using conserved GLU epitopes as immunogens (7). Others (37) have demonstrated that these types of repeats are highly immunogenic, as we have shown (24), and also that protein binding to glucan can be inhibited by antibody to sequences based on these repeats (24, 38). It may be that there are sufcient shared or homologous epitopic repeats on GTFs and glucan-binding proteins to render GLU a highly effective immunogen. We also found that the percentage of total streptococci which were recoverable as mutans streptococci were reduced in the CAT-, GTF-Ss-, and GTF-Sm-immunized animals compared with sham immunized groups similarly infected. Since absolute numbers of mutans streptococci were not monitored, some caution should be taken in drawing the conclusion that antibodies were responsible for this nding. Most importantly, caries was signicantly reduced after infection with S. sobrinus 6715 following immunization with CAT, GLU, or S. sobrinus GTF. Caries was also signicantly reduced after infection with S. mutans SJ32 after immunization with CAT, GLU, S. sobrinus 6715 GTF, or S. mutans SJ32 GTF. Therefore, immunization with CAT or GLU peptide constructs can result in reductions in caries caused by S. sobrinus or S. mutans. These data would appear to conrm the notion that important functional activities of the GTF molecule are encompassed by the regions described as CAT or GLU. Interestingly, Chia and colleagues (2) found that monoclonal antibody to a 19-mer sequence in the N-terminal third of S. mutans GTF inhibited GTFC enzymatic activity and attachment of S. mutans to glass surfaces but not similar activities of GTFD despite the fact that this sequence was completely conserved in both GTF species. The 19-amino-acid sequence was identical among GTFs from several mutans streptococci and was near the reported active site for sucrose binding (residues 435 to 453 of GTFC and residues 487 to 507 of GTFB). Mouse IgM anti-CAT monoclonal antibody to the CAT construct also inhibited S. sobrinus GTF-I activity, and polyclonal rodent antibody to CAT or GLU each inhibited a mixture of S. sobrinus GTF enzymes (26). Although an active site responsible for sucrose binding was proposed by identication of a peptide from the glucosyl-enzyme complex (17, 18) and was conrmed by site-directed mutagenesis (10), others (3) reported that rabbit IgG antibody to two 22-mer peptides in this region reacted weakly with native GTF and did not inhibit GTF-I or GTF-S from S. sobrinus. This lack of reactivity may be partially attributed to the monovalent nature of the peptide used for immunization, possibly giving rise to low-afnity antibody, to an inability of the antibody to access the active site, or to an inability to recognize the native conformation (3). Despite these ndings, it would appear that protection with respect to dental caries can be conferred by administration of either the CAT or the GLU construct as described herein. Signicant advances have been attained by synthesizing multicomponent vaccines which incorporate epitopes from separate regions of the same molecule (29). The two peptides used in the current experiments, which we have described previously (24, 26),

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would be excellent candidates for use in multicomponent or hybrid vaccines.

ACKNOWLEDGMENTS

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21.

This study was supported by grant DE-04733 from the National Institute of Dental Research. We thank William King for enzyme preparation and Jan Schafer for expert secretarial assistance.
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