Infect. Immun.-1995-Taubman-3088-93.pdf

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1995, 63(8):3088.Infect. Immun.

M A Taubman, C J Holmberg and D J Smithcaries.

glucosyltransferase protects against dentalregion of mutans streptococcalconstructs from the glucan-binding or catalyticImmunization of rats with synthetic peptide

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INFECTION AND IMMUNITY, Aug. 1995, p. 30883093 Vol. 63, No. 80019-9567/95/$04.000Copyright 1995, American Society for Microbiology

Immunization of Rats with Synthetic Peptide Constructsfrom the Glucan-Binding or Catalytic Region of

Mutans Streptococcal Glucosyltransferase

Protects against Dental CariesMARTIN A. TAUBMAN,* CYNTHIA J. HOLMBERG, AND DANIEL J. SMITH

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

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

Previously, we have described peptide constructs from two regions of glucosyltransferase (GTF) of mutansstreptococci. A putative catalytic site in the amino-terminal half of the molecule and a repeated glucan-bindingsite in the carboxyl-terminal half of GTF were the regions upon which sequences were based. The present studyexplored the effects of immunization with these peptide constructs (called CAT or GLU) and with streptococcalGTFs 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 SJ32organisms. Serum immunoglobulin G antibody levels, determined by enzyme-linked immunosorbent assay, tothe respective peptide constructs and to the appropriate streptococcal GTF were significantly increased (after

immunization) prior to infection and at the end of the experiment. Also, serum antibody from CAT-, GLU-, andS. 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 theCAT or GLU peptide construct resulted in significantly reduced smooth surface and sulcal caries afterinfection with S. sobrinus. Sulcal dental caries after infection with S. mutans SJ32 were also significantlyreduced in CAT- and GLU-immunized rats. Thus, immunization with peptides whose sequences are based onputative 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 theacid by-products of bacterial metabolism. The production ofthese by-products has been related to a group of aciduric oralmicroorganisms collectively called the mutans streptococci (4).Important microorganisms in this group which are found in

humans include Streptococcus mutans and S. sobrinus (12). Anadditional feature of the molecular pathogenesis of dentalcaries appears to be the role of accumulation of these, or likemicroorganisms, in dental plaque whose formation is depen-dent on synthesis of glucan from sucrose catalyzed by glucosyl-transferase (GTF) enzymes.

Previous investigations have indicated that the use of thisenzyme as an antigen may result in protection from experi-mental dental caries in rodents (22, 32) and in the induction ofsalivary immunoglobulin A (IgA) antibody in humans accom-panied by interference with reaccumulation of indigenous mu-tans streptococci after dental prophylaxis (21, 25). Althoughthe exact basis for experimental protection with such GTF-type

vaccines is presently unknown, it appears likely that such pro-tection can involve functional inhibition of the catalytic and/orthe glucan-binding activity of GTF. Antibody-mediated inhibi-tion of these functional activities has been demonstrated (33,34). The glucan-binding and catalytic sites appear to reside indifferent GTF domains. Thus, the GLU region(s) is found inthe C-terminal third of the GTF (1, 19, 36), whereas the cat-alytic domain appears to be located in the N-terminal third ofGTF (1, 3, 19). We have synthesized peptide constructs rep-resentative of the glucan-binding domain and the catalytic do-main of mutans streptococcal GTF (24, 26). These peptides

were found to be immunogenic in a variety of rodent species

(24, 26). Also, the antibody so elicited could interfere withGTF function (24, 26). Therefore, this study evaluated each ofthese peptide constructs for the ability to elicit antibody whichmight affect experimental dental caries.

MATERIALS AND METHODS

Synthetic peptides and antigens. CAT peptide. A nonapeptide, DSIRVDAVD, located between residues 448 and 457 of the published sequence of theGTF-I of S. downei contains an aspartic acid which has been shown to beinvolved in the catalytic reaction of GTF with sucrose (17). The sequence of theCAT peptide used in the present study (DANFDSIRVDAVDNVDADLLQ)contained this nonapeptide. An identical sequence is found in a similar region ofS. mutans GTF-I (20). The residues within the DSIRVDAVD peptide are eitheridentical to those of or conserved in S. sobrinus GTFs that produce water-insoluble glucan (IG) (1). The peptide was synthesized (Applied Diagnostics,Foster City, Calif.) by using the stepwise solid-phase method of Merrifield (15)on a core matrix of three lysines to yield a multiple antigenic peptide macro-molecule with four identical 21-mer peptides per molecule, after the method ofTam (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, wasused 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 TGAQTIKGQKLYFKANGQQVKG, was based on the derived sequence of one of the repeatingregions ofS. downei GTF-I (residues 1303 to 1324) which was 86% homologous

with an S. sobrinus GTF-I sequence (1) and 77% homologous with S. mutansGTF-I (20) in the same region. Residues 1303 to 1319 are also substantially thesame as a consensus sequence for putative carbohydrate-binding regions of otherproteins 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 p er molecule, as described above.GTF. GTFs from S. sobrinus 6715 and S. mutans SJ32 were obtained as

previously described (24). Briefly, after bacterial growth in glucose-containingdefined media, enzymes were isolated by chromatography on Sephadex G-100(Pharmacia Biotech Inc., Piscataway, N.J.), using 3 M guanidine HCl as theeluting solvent. These GTF-rich pools were then subjected to fast protein liquid* Corresponding author.

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chromatography on Superose 6 (Pharmacia), using 6 M guanidine for elution.The gel filtration step removes non-GTF and other glucan-binding proteins fromGTF preparations of S. mutans and S. sobrinus, as demonstrated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, after which only components

with enzyme activity were observed. The S. mutans GTF preparation taken tothis level of enrichment synthesized 51 to 81% water-soluble glucan (SG) by filterassay (32) and thus was analogous to a mixture of the gtfB, gtfC, and gtfD geneproducts ofS. 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 filtration on Superose 6contained a mixture of GTF-I (IG product), GTF-Sd (primer-dependent SGproduct), and GTF-Si (primer-independent SG product) (13, 14). This prepara-tion was designated GTF-Ss and was used for injection and ELISA.

Animals. Gnotobiotic Sprague-Dawley rats of strain CD (SD), originallyreared in the isolator facility of Charles River Laboratories, Wilmington, Mass.,

were infected with a defined flora and then screened and found to be free ofindigenous mutans streptococci. These rats were bred in our facility, and thefemale progeny, weaned at approximately 20 days and raised on high-sucroseDiet 2000 (27), were used in the experiments described below.

Protocol for animal experiments. Groups of 20-day-old rats were subcutane-ously injected in the salivary gland vicinity (31) with CAT or GLU peptideconstructs or with streptococcal GTFs or phosphate-buffered saline (PBS) (con-trol animals), all incorporating complete Freund adjuvant. One week later, rats

were reinjected with PBS or with the same antigen and dose in incompleteFreund adjuvant. One week after the second injection, some groups (describedbelow) were orally infected with approximately 108 S. sobrinus or S. mutansorganisms for 3 consecutive days (27).

Rats placed into six experimental groups of six to eight animals each weretreated as follows: group 1, sham immunized, noninfected; group 2A, shamimmunized, infected with S. sobrinus 6715; group 2B, sham immunized, infected

with S. mutans SJ32; group 3A, immunized with 50 g of CAT construct, infectedwith 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 25g of GTF-Sm, infected with S. mutans.

One week after the second injection, animals were bled from the retroorbitalplexus and saliva was collected after injection of pilocarpine (1.0 mg/100 g ofbody 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 antibodyby a previously described ELISA performed in microtiter plates (27). The anti-gens 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. Isotype-specific rabbit anti-rat IgA or IgG (32) was used with goat anti-rabbit IgG-alkaline phosphatase (TAGO, Inc., Burlingame, Calif.). The plates were devel-oped 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 werecalculated relative to the titration of appropriate reference sera from Sprague-Dawley rats hyperimmunized with each of the antigens mentioned above (24,26). The following dilutions for serum IgG were considered 100 ELISA units: foranti-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 evaluatedfor the ability to inhibit glucan synthesis by GTF-Ss or GTF-Sm, using a modifiedfilter assay previously described (26). Briefly, serum samples (1 l each) werecombined with the respective GTF preparation in a final volume of 100 l in 0.02M 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 and22 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 fiber filtersand washed with PBS-sodium azide, and the radioactivity was determined aspreviously described (34). SG in the filtrate was precipitated with 70% ethanolafter the addition of 4 mg of carrier dextran T10 and centrifuged, and theradioactivity was determined.

Bacterial recoveries. The mutans streptococcal flora was assessed at termina-tion. Systematic swabbing of teeth, sonication, and plating of appropriate dilu-tions on mitis salivarius agar (total streptococci; Difco Laboratories, Detroit,Mich.) were all performed as previously described (27). The numbers of mutansstreptococci on these plates, as identified by colonial morphology, are presentedas a percentage of the total streptococci.

Caries assessment. The extent and depth of carious lesions in all rat molarteeth (caries score) were microscopically evaluated by a modified Keyes method,as previously described (31). These combined caries scores were determinedseparately on smooth and on sulcal dental surfaces.

RESULTS

Serum IgG antibody. The CAT- or GLU-immunized groupsformed significant preinfection antibody to their homologouspeptide constructs in ELISAs (Table 1). Thus, rats immunized

with CAT showed significant serum antibody levels to CAT (P 0.01) compared with sham immunized rats, as did sera fromGLU-immunized rats when tested against GLU (P 0.001).

At termination after infection with S. sobrinus, the level ofantibody to CAT was elevated (P 0.07; analysis of variance)and the level of antibody to GLU was significantly elevated (atleast P 0.01) as it was after S. mutans infection (P 0.001)compared with the sham positive group.

After two immunizations, statistically significant antibody

levels to GTF-Ss (Fig. 1A) were detected in the groups ofanimals immunized with CAT (P 0.05) and GLU (P 0.01)and also with GTF-Ss and GTF-Sm (P 0.001) compared withsera from sham-injected rats. Also, these immunized animalsdemonstrated significant increases in antibody to GTF-Sm(Fig. 1B) after immunization with GLU (P 0.01) or GTF-Ssor GTF-Sm (P 0.001). In each case, the serum antibodylevels at the termination of the experiments are shown to theGTF preparation corresponding to the infecting bacterialstrain (Fig. 1A and B). Significant serum IgG antibody toGTF-Ss (P 0.001) or GTF-Sm (at least P 0.01) was foundin groups of animals immunized with GTF-Ss or GTF-Sm

when tested against GTF-Ss (Fig. 1A) and similarly whentested against GTF-Sm (Fig. 1B).

At termination, after infection with S. sobrinus, there was asignificant elevation of antibody to GTF-Ss (at least P 0.03;t test) in animals injected with either peptide antigen or GTFsafter infection with either mutans streptococcus compared

with preinfection antibody levels. Therefore, all immunizedanimals demonstrated serum antibody to GTFs before infec-tion, and for the most part, antibody levels increased (com-pared 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 an-tibody to the GTF preparation of the subsequent infectingmutans streptococcal strain was tested (Tables 2 and 3). At thetermination of the experiments, animals injected with CAT

TABLE 1. Serum IgG antibody levels to CAT or GLU constructsin rats after two injections and at the experiment termination

GroupInjectedantigena

Testantigen

ELISA units (mean SE)b

PreinfectionS. sobrinusinfection

termination

S. mutansinfection

termination

1 Sham CAT 5 1c (16) 20 3d (7) 20 3 (7)

2 Sham CAT NT 22 4d (7) 17 2 (6)3 CAT CAT 33 9e (13) 53 19d (6) 17 1 (5)

1 Sham GLU 1 0.3c (18) 4 0.5 (6) 4 0.5 (6)2 Sham GLU NT 6 1c (7) 2 0.6c (5)4 GLU GLU 232 48f (11) 464 20f (5) 635 98f (6)

a Sham, sham immunized, uninfected; sham, sham immunized, infected.b Determined prior to infection and 7 days after two immunizations and at

termination. Values in parentheses are numbers of animals tested. NT, nottested.

c The value for this sham group is statistically significantly different from thevalues for the groups indicated by footnotes e and f by one-way analysis ofvariance followed by u npaired t test.

dP 0.07 (one-way analysis of variance).eP 0.01.fP 0.001.

VOL. 63, 1995 CARIES PROTECTION AFTER IMMUNIZATION WITH GTF PEPTIDES 3089


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

mination. Antibody to GTF-Ss in the GLU-immunized groupwas elevated compared with the uninfected sham-minus groupafter infection with S. sobrinus (Table 2). Antibody also in-creased in the GTF-Sm-immunized group after infection withS. mutans (Table 3). The salivary IgA levels in both peptide-immunized groups, when tested with the homologous peptideconstruct (i.e., CAT or GLU [Table 4]), were significantly ele-

vated in animals infected with S. sobrinus or S. mutans com-pared with levels in sham immunized, noninfected animals.

Serum antibody-mediated inhibition of GTF function. Serataken prior to and after infection were tested for inhibition ofthe function of GTF enzymes from S. sobrinus or S. mutans.Both IG (Fig. 2) and SG syntheses by the GTF enzymes fromS. sobrinus 6715 were significantly inhibited by serum fromanimals immunized with CAT, GLU, and GTF-Ss. Immuniza-

tion with GTF-Ss gave the greatest IG inhibition. Also, SG, butnot IG, synthesis by GTF-Sm was significantly inhibited by serafrom animals immunized with CAT, GLU, and GTF-Ss (Fig.3). The combination of immunization and infection also re-sulted in inhibitory antibody.

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) andat the termination of the experiment (S. sobrinus after 83 to 87 days; S. mutansafter 60 to 62 days). Bars indicate the mean antibody level of serum from 5 to 17rats in each of the designated groups, expressed in ELISA units (EU). Error barsindicate the standard error of the mean. Differences are statistically significant atthe following levels compared with the sham immunized, uninfected (preinfec-tion) or sham immunized, infected (termination) sham group by one-way analysisof 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.

TABLE 2. Salivary IgA antibody levels to S. sobrinus 6715 GTFs inrats after two injections and at the experiment termination




termination

S. mutansinfection

termination

1 Sham 24 9 (16) 10 1c

(6) 10 1 (6)2 Sham NT 45 7d (7) 197 108 (4)3 CAT NT 56 4d (6) NT4 GLU NT 57 12e (7) NT5 GTF-Ss 47 15 (16) 43 8e (6) 76 42 (5)6 GTF-Sm NT 128 35e (7) NT



c The value for this sham group is statistically significantly different from thevalues for the groups indicated by footnotes d and e.

dP 0.001.eP 0.01.

TABLE 3. Salivary IgA antibody levels to S. mutans SJ32 GTFs inrats after two injections and at the experiment termination




termination

S. mutansinfection

termination

1 Sham NT 120 33c (7) 120 33c (7)

2 Sham NT 560 111d (7) 486 144e (5)3 CAT NT NT 612 101f (6)4 GLU NT NT 1,070 303d (6)5 GTF-Ss NT NT 683 268e (5)6 GTF-Sm 1 56 9 (14) 486 99d (7) 757 242e (6)



c The value for this sham group is statistically significantly different from thevalues for the groups indicated by footnotes d to f.

dP 0.01.eP 0.05.fP 0.001.

TABLE 4. Salivary IgA antibody levels to CAT or GLU constructsin rats after two injections and at the experiment termination


Testantigen



termination

S. mutansinfection

termination

1 Sham CAT NT 11 2c (6) 11 2 (6)

2 Sham CAT NT 11 3 (6) 7 2 (5)3 CAT CAT 4 1.0 (15) 26 6d (7) 17 3 (6)

1 Sham GLU NT 14 6c (7) 14 6c (7)2 Sham GLU NT 18 3 (7) 26 7 (5)4 GLU GLU 37 5 (15) 28 3e (7) 20 1d (6)

a Sham, sham immunized uninfected; sham, sham immunized, infected.b Determined prior to infection and 7 days after two immunizations and at


c The value for this sham group is statistically significantly different from thevalues for the groups indicated by footnotes d and e.

dP 0.05.eP 0.01.

3090 TAUBMAN ET AL. INFECT. IMMUN.


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Relative recovery of mutans streptococci and dental cariesassessment at experiment termination. At the termination ofthe experiment, relative recovery of mutans streptococci, ex-pressed as a percentage of the total streptococci from theimmunized groups, was compared with recovery from the shamimmunized, infected animals. After infection with S. sobrinus,CAT- (mean mutans streptococci as percentage of total strep-tococci standard error 54.7% 8.9%; n 7), GLU-(74.4% 4.9%; n 6), GTF-Ss- (46.7% 9.1%; n 6), andGTF-Sm-immunized (55.8 10.9; n 7) groups showed sta-tistically significant 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 groupwith the sham immunized S. sobrinus-infected group indicatedstatistically significant reductions (P 0.001; Student-New-man-Keul test) in total caries in the CAT-, GLU-, GTF-Ss-,and GTF-Sm-immunized groups (not shown). Comparison ofeach immunized S. mutans-infected group with the sham im-munized S. mutans-infected group also indicated statisticallysignificant reductions in total caries in the CAT- (P 0.001),GLU- (P 0.05), GTF-Ss- (P 0.05), and GTF-Sm-immu-nized (P 0.05) groups (not shown). Highly significant reduc-tions in dental caries in sulci (at least P 0.01) occurred afterimmunization of rats with CAT, GLU, GTF-Ss, or GTF-Smand infection with S. sobrinus or S. mutans (Fig. 4). Reductionsin smooth surface caries, relative to the sham immunized,infected group, were statistically significant after immunization

with CAT, GLU, GTF-Ss, and GTF-Sm following infection

with S. sobrinus; after S. mutans infection they were only sig-nificant after immunization with the CAT construct (Fig. 4).

DISCUSSION

There has been abundant evidence to support the involve-ment of mutans streptococcal GTF in the pathogenesis ofdental caries. Mutants defective in GTF activity were shown tobe deficient in cariogenic ability compared with wild-type or-ganisms. Conversely, mutants which were superproducers ofGTF demonstrated more caries than the wild type (5, 16, 30).Recently, Yamashita and his colleagues (39) have used inser-

FIG. 2. Inhibition of IG or SG synthesis by S. sobrinus GTF by serum anti-body. Inhibition is tested by incorporation of glucose from 14C-glucose-labelledsucrose 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 indi-cating sham immunized, uninfected, and S indicating sham immunized, in-fected 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 shamimmunized, uninfected sera (n 3) taken at termination were 2,006 31 and839 19, respectively. Differences are statistically significant at the followinglevels compared with the sera from the sham immunized, uninfected group(preinfection and at termination) by one-way analysis of variance followed byunpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001.

FIG. 3. Inhibition of IG or SG synthesis by S. mutans GTF by serum takenprior 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 incor-poration of glucose from 14C-glucose-labelled sucrose into IG or SG. The countsincorporated into IG and SG, in the presence of sham immunized, uninfectedsera (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 significant at the following levels com-pared with sera from the sham immunized, uninfected group (preinfection ortermination) by one-way analysis of variance followed by unpaired t test: *, P0.05; **, 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 cariesscores on smooth surfaces and on sulcal surfaces and the standard errors. Dif-ferences are statistically significant at the following levels compared with thesham immunized, infected group by one-way analysis of variance followed byunpaired t test: *, P 0.05; **, P 0.01; ***, P 0.001.



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tionally inactivated S. mutans GTF genes to replace the func-tional wild-type copy of the gene with the defective counterpartin organisms used to infect rodents (39). Caries was markedlydiminished when the gtfB and gtfC genes required for IG syn-thesis were inactivated. The gftD gene coding synthesis of SGsalso appeared to be significant in the pathogenesis of caries,although its role may be less significant in the presence oflower amounts of sucrose (39).

In this paper, we explored the suggestion that antibody tofunctionally significant domains of GTF from mutans strepto-cocci, which blocked the expression of function by this enzyme,could interfere with enzyme-mediated aspects of the molecularpathogenesis of dental caries. The data presented herein con-firm this notion.

Immunization of rats with peptide constructs of CAT orGLU resulted in serum IgG and salivary IgA antibodies to therespective peptide and to S. sobrinus and S. mutans GTFs.Functional inhibition ofS. 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 byserum antibody to CAT, GLU, and S. sobrinus GTFs was alsoshown.

Previously, we had demonstrated inhibition of GTF-Ss IG

synthesis by rat anti-GLU antibody (24) and mouse IgM anti-CAT monoclonal antibody (26) and of SG synthesized byGTF-Sm and by mouse monoclonal and polyclonal rat sera toCAT (26). Importantly, the current experiments indicated thatserum antibody to CAT or GLU peptides could inhibit synthe-sis of IG and SG mediated by mixtures of GTF enzyme from S.

sobrinus and SG mediated by GTF enzymes from S. mutans.The findings support the notion that the sequences chosen canachieve a broad protective effect.

In the enzyme inhibition systems employed, there is never100% inhibition because of the excess of substrate. Inhibitionby sera from rats immunized with S. sobrinus GTF was in theorder of 50 to 70%. Therefore, 15 to 20% inhibition of IGrepresents significant but not maximal inhibition. The inhibi-tion may not be maximal for several reasons: (i) the catalytic

site may not be directly or completely accessible; (ii) the po-tential limited specificity of the anti-CAT may restrict inhibi-tion; and (iii) the conformation of the CAT peptide constructmay be different than in the intact GTF and conformationallyactive determinants may not be recognized. Thus, the extent ofconformational integrity of the multiple antigenic peptides isessentially unknown.

Presumably, the antibody elicited in the current experimentseffectively inhibited the function of the GTFD protein enzymeof S. mutans (Fig. 3; preinfection) and that of the GTF-I, -Si,and -Sd enzymes of S. sobrinus (Fig. 2, preinfection and ter-mination), and this led to the protection seen. Anti-CAT andanti-GLU significantly inhibited SG synthesis by S. mutansGTFs. Since GTFD can be significant in smooth caries in therat model (39), the results of S. mutans infection leading tocaries (Fig. 4) may be explained by an effect of SG inhibitionon sulcal caries.

Interestingly, the sequence of the GLU peptide as synthe-sized was shown to have 50% homology with the deducedsequence 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 proteinfrom S. sobrinus (24), and it has been demonstrated that suchantibody can inhibit glucan-binding function (38). Thus, a com-bination of antibody-mediated inhibition of glucan binding byboth GTF and glucan-binding protein could conceivably inter-fere with both S. mutans and S. sobrinus glucan-mediated ac-cumulation. 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 sulcalcaries after S. sobrinus infection. However, smooth surfacereductions were less significant after S. mutans infection, sug-gesting that there may be a differential protective effect possi-bly based on inhibition of IG synthesis which was more signif-icant when tested against GTF-Ss (Fig. 2) than againstGTF-Sm (Fig. 3). This may reinforce the notion that IG may

be most significant in smooth surface caries (39).It has recently been suggested that intra- and interspecies

variation between streptococcal enzymes may be an ongoingprocess that could circumvent the idea of using conservedGLU epitopes as immunogens (7). Others (37) have demon-strated that these types of repeats are highly immunogenic, as

we have shown (24), and also that protein binding to glucancan be inhibited by antibody to sequences based on theserepeats (24, 38). It may be that there are sufficient shared orhomologous epitopic repeats on GTFs and glucan-binding pro-teins to render GLU a highly effective immunogen.

We also found that the percentage of total streptococciwhich were recoverable as mutans streptococci were reducedin the CAT-, GTF-Ss-, and GTF-Sm-immunized animals com-pared with sham immunized groups similarly infected. Since

absolute numbers of mutans streptococci were not monitored,some caution should be taken in drawing the conclusion thatantibodies were responsible for this finding. Most importantly,caries was significantly reduced after infection with S. sobrinus6715 following immunization with CAT, GLU, or S. sobrinusGTF. Caries was also significantly reduced after infection withS. mutans SJ32 after immunization with CAT, GLU, S. sobri-nus 6715 GTF, or S. mutans SJ32 GTF. Therefore, immuniza-tion with CAT or GLU peptide constructs can result in reduc-tions in caries caused by S. sobrinus or S. mutans. These data

would appear to confirm the notion that important functionalactivities of the GTF molecule are encompassed by the regionsdescribed as CAT or GLU.

Interestingly, Chia and colleagues (2) found that monoclo-nal antibody to a 19-mer sequence in the N-terminal third of S.

mutans GTF inhibited GTFC enzymatic activity and attach-ment ofS. mutans to glass surfaces but not similar activities ofGTFD despite the fact that this sequence was completely con-served in both GTF species. The 19-amino-acid sequence wasidentical among GTFs from several mutans streptococci and

was near the reported active site for sucrose binding (residues435 to 453 of GTFC and residues 487 to 507 of GTFB). MouseIgM anti-CAT monoclonal antibody to the CAT construct alsoinhibited S. sobrinus GTF-I activity, and polyclonal rodentantibody to CAT or GLU each inhibited a mixture of S. sob-

rinus GTF enzymes (26). Although an active site responsiblefor sucrose binding was proposed by identification of a peptidefrom the glucosyl-enzyme complex (17, 18) and was confirmedby site-directed mutagenesis (10), others (3) reported that rab-bit IgG antibody to two 22-mer peptides in this region reacted

weakly with native GTF and did not inhibit GTF-I or GTF-Sfrom S. sobrinus. This lack of reactivity may be partially attrib-uted to the monovalent nature of the peptide used for immu-nization, possibly giving rise to low-affinity antibody, to aninability of the antibody to access the active site, or to aninability to recognize the native conformation (3). Despitethese findings, it would appear that protection with respect todental caries can be conferred by administration of either theCAT or the GLU construct as described herein. Significantadvances have been attained by synthesizing multicomponent

vaccines which incorporate epitopes from separate regions ofthe same molecule (29). The two peptides used in the currentexperiments, which we have described previously (24, 26),

3092 TAUBMAN ET AL. INFECT. IMMUN.


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would be excellent candidates for use in multicomponent orhybrid vaccines.

ACKNOWLEDGMENTS

This study was supported by grant DE-04733 from the NationalInstitute of Dental Research.

We thank William King for enzyme preparation and Jan Schafer for

expert secretarial assistance.

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