INFECTION AND IMMUNITY, Sept. 1978, p. 843-851 0019-9567/78/0021-0843$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 21, No. 3 Printed in U.S.A.

Effects of Local Immunization with Glucosyltransferase Fractions from Streptococcus mutans on Dental Caries in Hamsters Caused by Homologous and Heterologous Serotypes of Streptococcus mutans DANIEL J. SMITH,* MARTIN A. TAUBMAN, AND JEFFREY L. EBERSOLE Department of Immunology, Forsyth Dental Center, Boston, Massachusetts 02115 Received for publication 28 June 1978

Seven serotypes of Streptococcus mutans have been identified. The biochemical, genetic, and serological characteristics of these serotypes have indicated that certain serotypes are quite similar, whereas others are quite distinct. The effect of local immunization with glucosyltransferase (GTF) enzymes from serotypes a, c, or g on infection and disease caused by homologous or heterologous cariogenic S. mutans is reported. Organisms with either similar (a and g) or different (c and g) biochemical and serological characteristics were selected for heterologous challenge. NIH white hamsters were injected four times at weekly intervals with GTF prepared by 6 M guanidine-hydrochloride elution from water-insoluble glucan of serotypes a, c, or g, which resulted in enzyme (homologous) inhibitory activity in sera and salivas. After infection of GTF-immunized and sham-immunized groups of hamsters with cariogenic S. mutans of the same serotype as the injected antigen (homologous infection) or with S. mutans of a different serotype from the injected antigen (heterologous infection), the numbers of streptomycinlabeled S. mutans, caries, and lesions were determined. Immunization with GTF preparations from each of the three serotypes resulted in statistically significant reductions in the extent of infection and disease and number of lesions caused by infections with homologous cariogenic S. mutans. Statistically significant reductions in these three parameters were also observed in groups immunized with enzyme from serotype a (strain E49) and challenged with cariogenic serotype g (strain 6715) organisms; or immunized with enzyme from serotype c (strain Ingbritt) and challenged with cariogenic serotype g (strain 6715) organisms; or immunized with enzyme from serotype g (strain 6715) and challenged with cariogenic serotype c (strain Ingbritt) organisms. These studies suggest that soluble antigen preparations containing GTF from one serotype may elicit a protective immune response against infection with cariogenic S. mutans from many or possibly all serotypes.

Glucosyltransferase (GTF) enzymes play an At least seven serotypes of S. mutans have been

important role in the ultimate pathogenic effects of cariogenic Streptococcus mutans on the tooth surface. This role is thought to be accomplished by the synthesis of water-soluble and -insoluble glucans from sucrose, which then participate in the formation of dental plaques (11). Because of this, GTF preparations have received attention as potential antigens in immunization experiments designed to interfere immunologically with dental caries caused by S. mutans (1, 9, 12, 17, 31). In rodent models, antigenic preparations containing GTF have elicited a protective (caries-reducing) immune response against a bacterial challenge of the same serotype as that from which the enzyme antigen was derived (12, 31).

843

identified after isolation from human dental plaque (2, 24). GTF antigens from most of these serotypes, including the most frequent human isolate (serotype c) (3), have yet to be evaluated for their protective effect in the rodent caries model. Furthermore, the ability of GTF antigen preparations to elicit an effective immune response against cariogenic S. mutans of serotypes different from the source of antigen has not been reported. Whereas in vitro inhibition assays suggest that two or three antigenic subsets of GTF exist among the seven serotypes of S. mutans (7, 16, 26), antigen-binding analyses suggest a closer antigenic relationship (32). The purpose of the present investigation was to determine

844

SMITH, TAUBMAN, AND EBERSOLE

the effect of immunization with preparations containing GTF from serotypes a, c, and g on infection with cariogenic S. mutans of the same serotype or on infection with S. mutans serotypes that were either closely (a and g) or more distantly (c and g) related to the serotype used as the source of antigen for injection. MATERLS AND METHODS Bacteria. S. mutans strains E49 (serotype a), Ingbritt (serotype c), and 6715 (serotype g) used in these experiments are cariogenic in hamsters and are mutants resistant to streptomycin at concentrations of 2,000 Ig/ml. GTF antigen preparations. S. mutans organisms were grown anaerobically (10% CC2, 90% N2) for 24 h at 37°C in 6 to 10 liters of a chemically defined medium (28). A cell-free supernatant, obtained by centrifugation (13,700 x g), was brought to pH 6.5. Waterinsoluble polysaccharide was then synthesized by the addition of sucrose to 10% at 37°C for 48 h. Bacterial growth was inhibited by addition of 0.02% sodium azide. The water-insoluble polysaccharide that formed was collected by centrifugation at 13,700 x g (4°C), then washed extensively with cold distilled water and 0.01 M sodium phosphate (pH 6.8) to which 0.02% sodium azide had been added. GTF enzymes were then eluted from the washed, water-insoluble polysaccharide by 1 h of incubation (4°C) with a volume of 6 M guanidine-hydrochloride that was twice the weight of the polysaccharide, as reported previously (D. J. Smith, M. A. Taubman, and J. L. Ebersole, J. Dent. Res. 66:A132, 1977). Following elution, guanidine was removed by dialysis, and, after concentration, the eluates were separated by gel filtration on columns of 8% agarose in 0.01 M sodium phosphate (pH 6.8). Column fractions were monitored for protein spectrophotometrically (280 nm) and for enzymatic activity by the Somogyi (29) and Glucostat (Worthington Biochemical) assays. GTF activity from eluates of all three serotypes (a, c, g) was found to elute at the column void volume. Fructose was the principal sugar released from sucrose with GTF from serotype a (79%), serotype c (75%), or serotype g (78%). In none of the GTF preparations could teichoic acid or serotype-specific antigen be detected when tested in gel diffusion using an antiserum directed to the PGP backbone (which showed a visible precipitin band with approximately 0.3 nM lipoteichoic acid) or antisera to purified serotype antigens (13). In GTF preparations from serotypes g and a, 10.4 and 4.7 jg of carbohydrate (5) per jig of protein (20) were detected. Although the amount of carbohydrate (presumably glucan) was not measured in the serotype c GTF void volume peak, antisera raised to this preparation gave a positive reaction against glucan from serotype g in immunodiffusion analysis. Guanidine-eluted GTF preparations from each serotype formed water-insoluble and watersoluble polysaccharide when incubated with 0.125 M sucrose for 4 h at 37°C. Immunization experiments. Three immunization experiments (H1, H2, H3) were performed in NIH white hamsters. The general protocol for these experiments is shown in Fig. 1. In each experiment hamsters

INFECT. IMMUN. were divided into five groups: (I) nonimmunized and noninfected; (II) sham immunized with 0.1 ml of phosphate-buffered saline incorporated into 0.1 ml of complete Freund adjuvant and infected with a cariogenic strain of S. mutans from which the GTF antigen was derived (homologous infection); (III) sham immunized as group II but infected with a cariogenic strain of S. mutans of a different serotype from that used as the antigen source (heterologous infection); (IV) immunized with 0.1 ml of GTF enzyme incorporated into 0.1 ml of complete Freund adjuvant and homologously infected; and (V) immunized as group IV and heterologously infected. The source of GTF antigen and the infecting strains used in each experiment are listed in Table 1. Immunization was initiated in H1, H2, and H3 when hamsters were 26, 20 to 23, and 22 to 23 days of age, respectively. The animals in groups II and V were injected subcutaneously in two to four sites at 7to 12-day intervals in the vicinity of the parotid and submandibular glands four times prior to infection. Hamsters in groups IV and V of experiment H1 were injected with GTF from serotype a strain E49 (GTFE:4), which contained 1.2 units of activity (unit the amount of enzyme converting 1 mg of sucrose to glucan in 1 h, releasing 0.52 mg of fructose [15]) per injection. In experiment H2, each injection of strain Ingbritt GTF (GTFlngbritt) contained 0.8 unit of activity, while in experiment H3 each injection of strain 6715 GTF (GTF6715) contained 0.7 unit of activity. All hamsters were maintained on pelleted Purina Mouse Chow from weaning to 3 days prior to infection, when diet 2000 (14) was initiated. Salivas and sera. Salivas and sera were collected and treated prior to antibody assay as previously described (30). In addition, saliva to be used in the inhibition of ['4C]glucose incorporation assays was dialyzed, first against phosphate-buffered saline containing 0.001 M ethylenediaminetetraacetic acid, then against 0.01 M sodium phosphate (pH 6.8). Assay for inhibition of GTF activity. The procedure for determining inhibition of GTF activity by serum and saliva, a modification of the method of Evans and Genco (7), has been described previously (26). GTF activity was measured by determining ['4C]glucose incorporation from glucosyl-labeled sucrose into ethanol-insoluble polysaccharide, which contains both water-insoluble and water-soluble polysaccharide. Inhibition of GTF activity was expressed as the percentage of reduction in counts incorporated into precipitated polysaccharide by enzyme in the presence of immune sera (salivas) compared with incorporation by enzyme in the presence of control (group I) sera or salivas. Culture supernatants of strains E49, Ingbritt, and 6715, precipitated at 60% (NH4)2SO4, were used as sources of GTF activity for inhibition experiments. Inhibition in these assays reflects specific antibody activity as demonstrated by the marked inhibitory effect of purified immunoglobulin G containing anti-GTF antibody activity (25, 26). In addition, specific precipitation and removal of immunoglobulin A from immune rat saliva leaves insignificant inhibitory activity (8). Infection. The hamsters in groups II, III, IV, and V were orally infected with 0.4 ml of 20-h cultures of the appropriate strain of S. mutans (Table 1) (approx-

CROSS-PROTECTIVE EFFECTS OF GTF IMMUNIZATION

VOL. 21, 1978

845

PROTOCOL - Pre"Wecion

GENERAL

P-sinwecxim i, kijectiion in SGV

GROUP I

II IV III

i

i

Infection

i- ; -

e

I

I

kifetion

V .

20

40

M-

60

80

100

AGE (Days) FIG. 1. Typical protocol for hamster experiments 1, 2, and 3 (HI, H2, and H3). The horizontal bars opposite the group designation represent experimental duration with respect to age. Approximate times of injection in the salivary gland vicinity (SGV) are indicated by solid vertical lines for GTF-injected groups IV and V and by broken vertical lines for sham-injected groups II and III. Actual injection days for HI were 26, 33, 40, 47, and 68; for H2 they were 21, 32, 39, 47, and 63; for H3 they were 23, 30, 37, 44, 66, and 96. Initial infection times for the experiments are indicated by the transition from solid to broken bar and were on day 55 (HI), day 59 (H2), and day 51 (H3). The hamsters were infected with approximately I0 colony-forming units ofstreptomycinresistant S. mutans of the same strain used for antigen preparation (homologous infection) or a strain of a different serotype from the GTF used for injection (heterologous infection). Infection continued for 46 days in H1, 38 days in H2, and 54 days in H3.

Expt

Hi

TABLE 1. Summary of hamster experiments GTF antigen source Homologous infecting strain Heterologous infecting strain S. mutans E49 (a)a E49 (a) 6715 (g) S. mutans Ingbritt (c) 6715 (g) Ingbritt (c) S. mutans 6715 (g) 6715 (g) Ingbritt (c)

H2 H3 ° Serotypes indicated in parentheses. imately 10' colony-forming units) 7 to 12 days after completion of the initial immunization regimen (Fig. 2). Hamsters were infected for 2 consecutive days starting on day 51, 55, or 59. Prior to this time, salivary (and serum) GTF-inhibiting activity could be demonstrated in all the immunized animals. The flora of all

animals were periodically monitored after infection as described previously (30, 31). At the termination of the experiment (38 to 54 days after infection), saliva was collected and the animals were exsanguinated. All bacterial plaque was removed from the buccal and lingual surfaces and, after sonic disruption for 10 s, was appropriately diluted and plated on mitis-salivarius (MS) agar and MS agar containing 200 pg of streptomycin per ml (MSS) as previously described (30). The whole jaws were then defleshed, and all caries and lesions were scored by a modified Keyes method (30) without knowledge of the group designation of the animal. Individual means were compared by analysis of variance. In all experiments comparisons were made between the immunized group IV and sham-immunized group I or between immunized group V and sham-immunized group III.

RESUL TS

Functional inhibition of GTF enzymes. Both sera and salivas of hamsters taken prior to infection and at the termination of the experiment were analyzed (Fig. 2) for functional inhibition of GTF-mediated glucose incorporation into total polysaccharide. Before infection, the sera of hamsters immunized four times showed mean inhibition of from 52 to 59% of the enzyme from the same serotype as that used for injection. Sera from GTFE49-immunized hamsters (experiment H1) also strongly inhibited enzyme from serotype g strain 6715 to be used for heterologous infection (78.1 ± 3.2% inhibition). On the other hand, sera from GTFIgbritt (experiment H2)- or GTF6715 (experiment H3)-injected hamsters showed low but significant inhibition of enzyme from strains to be used in the heterologous phases of those experiments (16.5 ± 5.0% and 11.7 ± 1.1% inhibition, respectively). At termination, after the animals had been infected

846

SMITH, TAUBMAN, AND EBERSOLE

INFECT. IMMUN.

SERUM

SALIVA Preinfection

70 60

50

40

30

1|

20

L

4'~1111rt

10

114411

ENZYME

IV .V

E49

11HII

IV .V

ING

11 V

FIII

114411

6715

E49

ING

11 IV III v

IV III, v

6715

Termination 80 70

.50

znm 40 301

30

sot

20

20

ENZYME

E49

ING

67,15

E49

ING

,, fv III v

6715

H1 H2 H3 H2 H3 H1 FIG. 2. Inhibition of GTF-mediated [14C]glucose incorporation into ethanol-insoluble polysaccharide by sera and salivas in hamster experiments 1, 2, and 3 (Hi, H2, and H3). Inhibition is expressed as the percentage of reduction of counts per minute incorporated into total ethanol-insoluble polysaccharide by GTF enzyme contained in ammonium sulfate precipitates of culture supernatants from S. mutans strains E49, Ingbritt (Ingj, or 6715. Source of enzyme used for inhibition is shown beneath the bars. Approximately 100 pg of precipitate was used per assay, which incorporated between 900 and 4,000 cpm of [/4Clglucose into ethanolprecipitable polysaccharide. Sera and salivas were tested against only the strain of enzyme used for injection. Each bar represents the mean percentage of inhibition of 4 to 22 salivas or sera. Brackets enclose two standard errors. Data are shown for each experiment from sera or salivas taken either before infection or at the termination of the experiment: experiment HI (days 52, 101); H2 (days 56, 97); and H3 (days 49, 105). The group designations (II, III, IV, and V) below the bars are as shown in Fig. 1. and immunized one additional time, the level of serum inhibition of the homologous enzyme activity was at least as great as preinfection levels.

the homologous serotype before infection (12 to 19%) and at experiment termination (7 to 43%). Although reductions in [14C]glucose incorporaSera from GTF-infected hamsters also formed tion into ethanol-insoluble polysaccharide were at least one precipitin band with homologous low, immune salivary inhibition was always guanidine-eluted enzyme preparations in gel dif- greater than that occurring in the salivas of fusion analyses. The sera of sham-immunized sham-immunized animals in each experiment. hamsters showed no significant effect on enzy- Insufficient amounts of saliva were available for matic activity, nor did they precipitate with testing of heterologous GTF inhibitory activity. enzyme from any of the three serotypes. However, results of previous experiments indiSalivas of hamsters immunized with serotype cated that salivary cross-inhibitory patterns rea (experiment Hi), serotype c (experiment H2), flect those observed in serum (26). or serotypeg (experiment H3) GTF preparations Bacterial studies. Since inhibitory activity inhibited polysaccharide formation by GTF of in serum and saliva could be elicited by local

CROSS-PROTECTIVE EFFECTS OF GTF IMMUNIZATION

VOL. 21, 1978

injection with GTF-enzyme antigen, it was of interest to evaluate the effects of infection of immunized hamsters with cariogenic streptococci, the GTF of which was either closely (serotypes a and g) or more distantly (serotypes c and g) related to the injected GTF. Therefore, on the days indicated in Fig. 1, all groups but the group I control animals in each experiment were infected with the homologous or heterologous cariogenic and streptomycin-resistant strains of S. mutans indicated in Table 1. The extent of S. mutans infection was qualitatively estimated by systematic swabbing of molar surfaces twice during the infection period and by smooth-surface plaque removal at experiment termination. A comparison of the identically challenged sham- and GTF-immunized groups in the homologous phases of the three experiments reveals significantly lower S. mutans recoveries in GTF-immunized groups on seven of eight swabbing occasions (Table 2). Likewise, a similar comparison in the heterologous aspect of these experiments also reveals the recovery of significantly fewer S. mutans on seven of eight swabbing occasions. At experiment termination, the molar surfaces of the GTF-immunized groups also showed reduction in the number of

847

S. mutans when compared to the sham-imnunized groups. These reductions were statistically significant in both homologous and heterologous infection aspects of experiment 2. Comparison of total streptococci recovered at the termination of the experiment did not demonstrate significant differences between sham- and GTF-immunized groups in any experiment. Although these types of data are of a qualitative nature, the consistency of the observation strongly suggests that reductions in the recovery of S. mutans from immunized groups occurred whether the bacterial challenge and source of antigen were of the same or different serotype. Caries scores and lesions. To assess the effects of injection of GTF-containing preparations derived from serotype a, c, andg organisms on the effects of such infection with organisms of the same strain, the mean caries scores and lesions of the groups representing the homologous aspect of the hamster experiment (groups I, II, and IV) were determined (Table 3). The GTF-immunized group IV had significantly lower mean caries scores and lower mean numbers of lesions than the respective sham-immunized group II in experiment H1 (serotype a) and experiment H2 (serotype c), indicating that

TABLE 2. Bacterial recoveries from molar surfaces of hamsters during infection and at experiment termination Swabbing occaExpt termination sion'

Expt

Group

Treatment

Infecting strain

1st S.

2nd S.

tans (x

Total colonies

104)

(X 10')

0 445 103 91 11

3 127 56 45 20

0

0

238d

46d 16 57d 25d

3 115 32 125 51

mutans mutans

Hi

H2

H3

I II IV III V

Noninjected Sham-injected GTF,:9-injected Sham-injected

I II IV III V

Noninjected Sham-injected

None

GTFIngorittinjected GTF,.ghitt-injected

6715 6715

Noninjected Sham-injected GTF6715-injected Sham-injected

None 6715 6715

I

GTFgA9-injected

Sham-injected

None E49 E49 6715 6715

Ingbritt Ingbritt

(x 10') 0 310c 30c 6' le

0 340 90

502" 46" 0

(X

10:) 0

447w 26" 110 6

17"d

773' 43' 0

S.mu-

0 ND 736 225 123 605 187 Ingbritt 416' 516 150 Ingbritt 33c 66e GTF671s-injected 372 95 a Procedure was performed 4 and 18 days (H1), 4 and 21 days (H2), and 14 and 33 days (H3) postinfection. b Procedure was performed 46 days (H1), 38 days (H2), and 54 days (H3) postinfection. ND, Not done. e Statistically significant, P < 0.001. dStatistically significant, P < 0.05. ' Statistically significant, P < 0.005. f Statistically significant, P < 0.01.

II IV III V

504f 87f 556c

816"

848

SMITH, TAUBMAN, AND EBERSOLE

INFECT. IMMUN.

TABLE 3. Effect of immunization with GTF on pathogenesis of homologous cariogenic S. mutans Expt Group Expt Group

H1

H2

H3

Treatment

Mean caries SEa score

6

4.8 ± 0.6 51.3 ± 9.1" 27.0 ± 4.4b

16.4 ± 1.2 42.7 ± 2.0 30.9 ± 2.4d

2.5 ± 0.6 14.0 ± 2.6c 5.0 ± 0.9c

9.8 ± 2.0 38.5 ± 2.3c 15.5 ± 1.7c

4.4 ± 1.1 59.0 ± 7.3d 26.6 ± 5.7d

13.0 ± 2.6 46.8 ± 3.4c 20.8 ± 2.3c

I II IV

Noninfected; nonimmunized Sham-immunized; E49-infected GTFE49-immunized; E49-infected

11 11

I II IV

Noninfected; nonimmunized Sham-immunized; Ingbritt-infected GTFIngbrtt-IMMUniZed; Ingbritt-infected

11

I

Noninfected; nonimmunized

II Sham-immunized; 6715-infected IV GTF6715-immunized; 6715-infected a SE, Standard error. b Statistically significant, P < 0.05. c Statistically significant, P < 0.001. d Statistically significant, P < 0.005.

soluble GTF preparations from these two strains can also elicit a caries-protective immune response. Disease in the immune group IV of experiment H3 (serotype g) was also significantly lower than the sham group, confirming observations made previously with antigen of this serotype, prepared in a different fashion

(31).

N

The mean caries scores and lesion counts of control group I, sham group III, and GTF-injected group V in the heterologous phases of each experiment are presented in Table 4. Marked reductions in disease occurred in immunized compared to sham-injected groups when serotypeg strain 6715 organisms were used to infect hamsters injected with serotype a strain E49 GTF (experiment H1). These reductions were statistically significant when mean numbers of lesions were evaluated. When serotype g organisms were used to infect hamsters injected with serotype c strain Ingbritt GTF (experiment H2), similar reductions occurred and were statistically significant for both disease parameters. The third experiment represented the converse of experiment 2. In experiment H3, hamsters injected with serotype g strain 6715 GTF and infected with serotype c strain Ingbritt (group V) had significantly lower caries scores and numbers of lesions than the identically challenged sham-immunized group III. Weights of animals in experiments H1, H2, and H3 were monitored throughout each experiment and were not significantly different among all groups on any occasion. The caries scores and lesions of GTF-immunized and control animals were determined with respect to surface. The scores and lesions of the sham-immunized groups of animals were then compared with those of the corresponding immune animals by calculating the percentages of reduction of immune versus sham (Table 5).

6 12

7 11 11

~~~~±

Mean SE lesions a

Reductions in numbers of lesions and in caries scores were always seen in immunized animals on both occlusal and smooth (buccal and lingual) surfaces. Disease on smooth surfaces was somewhat more reduced than on occlusal surfaces in both homologous and heterologous aspects of experiments H1, H2, and H3. A comparison of homologous with heterologous percentage reductions revealed no significant differences, suggesting a similarity in effectiveness of the GTF antigen within each experiment. This pattern supports the concept that an interference with the production of lesions, primarily on smooth surfaces, has occurred in immunized animals, whether the GTF antigen is derived from the same or a different serotype.

DISCUSSION Immunization with GTF preparations from serotypes a, c, and g resulted in significant reductions in the extent of infection and disease and the number of lesions on hamster molar surfaces caused by infection with homologous cariogenic S. mutans. These findings extend the observation of the effectiveness of GTF antigens from serotype g (26) and suggest that GTF antigen preparations from each serotype of S. mutans might be effective in eliciting a protective immune response against homologous infection. Antigen preparations containing GTF from serotype c have not been effective in primate models (1). This may result from differences in the model (e.g., oral flora, immune system) or may be related to other experimental factors such as the immunization protocol or bacterial challenge (27). Although differences in antigen preparation may be a factor, it is worth noting that our use of guanidine-hydrochloride-eluted enzyme represents a third different purification technique used to obtain an effective antigen from strain 6715 of the g serotype.

849

CROSS-PROTECTIVE EFFECTS OF GTF IMMUNIZATION

VOL. 21, 1978

TABLE 4. Effect of immunization with GTF on pathogenesis of heterologous cariogenic S. mutans Expt Group Group Expt

Treatment

N

Mean caries SE score

Mean SEa lesions ±

4.8 ± 0.6 29.8 ± 6.7 14.4 ± 4.1

16.4 ± 1.3 34.3 ± 3.6b 23.2 ± 2.7b

~~~~±

a

I III V

Noninfected; nonimmunized Sham-immunized; 6715-infected

GTFEs-immunized; 6715-infected

6 11 11

H2

I III V

Noninfected; nonimmunized Sham-immunized; 6715-infected GTFjgbrtt-immunized; 6715-infected

6 12 12

2.5 ± 0.6 30.4 ± 5.2c 13.6 ± 3.2c

9.8 ± 2.0 53.3 ± 3.8d 24.1 ± 2.8d

H3

I III V

Noninfected; nonimmunized

7 12 10

4.4 ± 1.1 45.1 ± 9.7b 18.6 ± 5.1b

13.0 ± 2.6 43.8 ± 4.2d 20.4 ± 3.5d

Hi

Sham-immunized; Ingbritt-infected GTF6715-immunized; Ingbritt-infected

aSE, Standard error.

b Statistically significant, P < 0.05. ¢ Statistically significant, P < 0.025. dStatistically significant, P < 0.001.

TABLE 5. Reduction of caries scores or lesions on occlusal or smooth surfaces of immunized and shamimmunized hamster groups Percentage of reduction i n

Type of infection

Expt

Caries'

Serotype of i-

fecting strain

Occlusal

H1 H2 H3

Homologous

a

Heterologous

g

Homologous

c

Heterologous

g

Homologous Heterologous

g

'100 [mean caries -

100]. l 100

group)

-

x

Lesionsh

Buccal and

surfaces

lingual sur-

39 36 67 50

Occiusal

Buccal and

faces

surfaces

lingual sur-

53 53

18 15

51 57

69 60

50 61

76 63

faces

52 51 58 58 52 66 45 62 score of immune group/mean caries score of identically challenged sham group) x c

[(mean number of lesions of immune group/mean number of lesions of identically challenged sham 100].

The potential use of soluble antigen preparations containing GTF in a vaccine is extended by the observations of cross-protection between serotypes a and g and between serotypes c and g. Cross-protection between the former two strains might be expected, given the close antigenic relationship (Fig. 2) of their GTF in vitro (26) as well as the similarities in base ratios (4, 6) and the cross-reactions between serotypic antigens of the parent organisms (13, 18). On the other hand, these characteristics are relatively dissimilar for serotype c and g strains of S. mutans (3), and antisera (Table 2) or salivas (26) directed to GTF from either strain show relatively low levels of inhibition. Despite this, immunization with either serotype c (H2) org (H3) GTF preparations significantly reduced disease caused by infection with the heterologous strain. The similarities in percentage reductions in disease in the heterologous phases of either exper-

iment would not be totally anticipated from the inhibition data (Fig. 2). Although some in vitro inhibition of total glucan synthesis does occur between these strains with immune sera (Fig. 2) or salivas (26), it is less than that seen with homologous enzyme. On the other hand, comparison of the inhibition of formation of waterinsoluble glucan from several serotypes with antisera directed to serotype a GTF showed similar and significant reductions in glucan formed among serotypes a, b, c, d, and e (19). Antigenbinding experiments performed either with radiolabeled [3H]GTF or with GTF affixed to polystyrene plates (enzyme-linked immunosorbent assay [ELISA] technique) also give evidence for antigenic similarty between GTF of serotypes c and g (32). The caries-protective immune response in vivo is still poorly understood and probably functions in ways not totally measured by individual in vitro assays (32). Thus, while

850

SMITH, TAUBMAN, AND EBERSOLE

sufficient in vitro evidence exists to predict that some degree of cross-protection might occur among serotypes of S. mutans when using GTF preparations as antigen, the mechanisms leading to immune reduction in disease remain to be elucidated. Another related issue concerns the antigenic basis of the protection and cross-protection. To limit the number of non-GTF antigens in the preparations used in the present experiment, chemically defined medium was used for bacterial culture. No teichoic acid or serotype antigen was detected in the enzyme preparations, nor was an antibody response to these antigens observed by immunodiffusion techniques. Measurements of glucan synthesis or release of "C from radiolabeled dextran failed to reveal dextranase activity, although it may have been present in an inactive form. Radioactive incorporation techniques and chemical assays indicated little or no fructosyltransferase. The antigen preparations used in the present study contain glucan, GTF, and an additional unidentified component. However, of particular importance is the finding that disc gel (5%) electrophoresis of strain 6715 GTF antigen preparations, followed by reaction of gel slices with antisera from experiment H3, showed the principal region of immunoprecipitation to be associated with bands of enzymatic activity. Although hamsters in experiment 3 responded with serum antibody to glucan (strain 6715), hamsters immunized with GTF prepared in the absence of substrate (30), demonstrating no response to glucan, had very similar levels of protection after identical immunization (31). This comparison might suggest that the presence of glucan may not enhance the protective or cross-protective features of the antigen preparation. However, since antibody to GTF or glucan or other components in the GTF complex (such as dextran-binding protein [21]) may in theory be protective (31), each needs to be evaluated individually in caries immunization experiments. These studies emphasize the effectiveness of soluble vaccines containing GTF in diminishing the pathogenic effects of S. mutans on the tooth surface. Serotypes c and dig represent by far the most frequently isolated group of S. mutans obtained individually, or coinfecting human dental surfaces (3). Since GIF antigen preparations from either c or g S. mutans serotypes were effective against experimental disease caused by either serotype, vaccines containing GTF from either serotype might be expected to interfere with most human S. mutans dental infections. In fact, the extensive cross-protection seen experimentally among the three serotypes studied suggests that enzyme antigens from one, or pos-

INFECT. IMMUN.

sibly any, serotype may elicit a protective response to all S. mutans serotypes. ACKNOWLEDGMENTS This research was performed pursuant to Public Health Service contract n6. DE-42438 and grant no. DE-04733 with the National Institute of Dental Research and was also supported by Public Health Service Research Career Development Awards DE-70122 (to M.A.T.) and DE-00024 (to D.J.S.) from the National Institute of Dental Research. We thank M. Dhond and W. King for expert technical assistance, C. Raymond for secretarial assistance, and T. Myoda (Dupont Research Institute) for generously supplying us with an antiserum directed toward teichoic acid.

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