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Immunology 1980 40 97

Specificity of antibodies induced by Streptococcus mutans during immunization against dental caries

M. W. RUSSELL, S. J. CHALLACOMBE & T. LEHNER Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London

Acceptedfor publication 27 October 1977*

Summary. Protection against smooth surface dental caries was investigated in fifteen young rhesus monkeys which were immunized subcutaneously with Streptococcus mutans serotype c in Freund's incomplete adjuvant. Monkeys immunized with killed whole organisms developed significantly less caries than control animals. Monkeys immunized with pronasetreated cell walls developed significantly more caries than control animals while monkeys immunized with untreated cell walls showed no such enhancement of caries. Haemagglutinating and complement-fixing antibodies to cell walls and culture supernatant antigens (SN Ag) of S. mutans developed in the sera of all immunized animals to a similar degree. Antibodies to lipoteichoic acid and to an insoluble dextran preparation were found in all immunized animals and showed no relationship to the prevalence of caries. Antibodies to the serotype c polysaccharide were also found in animals immunized with whole cells and pronase-treated cell walls. However, precipitating antibody levels to partially purified antigens I/II and II, derived from SN Ag, but present also in cells, were related to the development of caries. Animals immunized with whole cells and with untreated cell walls
Correspondence: Dr T. Lehner, Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London SEI 9RT. * Publication delayed because Patent Applicaton was pending. 0019-2805/80/0500-0097S02.00 (0 1980 Blackwell Scientific Publications

developed a brisk antibody response to antigen I/II, while those immunized with pronase-treated cell walls responded more slowly. The results suggest that immunization may induce both caries reduction and enhancement, depending on the antibody response which is developed.
INTRODUCTION The induction of immunity to dental caries has been studied in rhesus monkeys vaccinated with cells and extracellular products of Streptococcus mutans serotype c (Lehner, Challacombe & Caldwell, 1975b; Russell, Challacombe & Lehner, 1976). It appears that subcutaneous immunization with killed organisms in Freund's incomplete adjuvant (FIA) induces both systemic antibody and cellular responses (Lehner, Challacombe, Wilton & Caldwell, 1976b) and reduces the development of dental caries. It has been postulated that serum antibodies could reach the site of carious attack by passage through the gingival crevice (Lehner, Challacombe & Caldwell, 1976a; Challacombe, Russell & Hawkes, 1978). Serum antibody levels have been assayed by means of passive haemagglutination and complement fixation tests, using trypsin- or pronase-treated cell walls and an extracellular antigen preparation obtained from culture supernatants (supernatant antigen; SNAg), which has glucosyltransferase (GTF) activity. The results indicate that protection is associated with the early development of complement-fixing anti-

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M. W. Russell, S. J. Challacombe & T. Lehner

bodies, possibly of the IgG class, directed against antigens to be found in both cell walls and culture supernatants (Lehner et al., 1976a). Tests for the presence of antibodies capable of inhibiting GTF activity, suggest that such antibodies are not responsible for the observed protection (Russell et al., 1976). There have been no investigations into the specificity of the antibody response to different antigenic components of S. mutans. Several different antigens of S. mutans serotype c have recently been described, and some have been purified and characterized. They include the serotype c polysaccharide (c PS) (Wetherell & Bleiweis, 1975; Linzer, Gill & Slade, 1976), lipoteichoic acid (LTA) (Markham, Knox, Wicken & Hewett, 1975), dextran-like glucans (the products of GTF activity) (Cisar & Kabat, 1976) and three antigens designated I, II, and III (Russell & Lehner, 1978). All of these can be detected in, or extracted from, both the cells and culture supernatant fluids. It is therefore appropriate to use these antigens to study the antibody response further. It is now clear that antigen I as originally defined, also contains antigen II determinant within the one molecule (Russell, Bergmeier, Zanders & Lehner, submitted for publication) and is now correctly described as antigen 1/11. Immunity to caries has been successfully induced by vaccinating with whole organisms and with broken cell preparations (Bowen, Cohen, Cole & Colman, 1975; Lehner et al., 1975b). Immunization with extracellular products such as crude GTF preparations, has not been successful (Guggenheim, Miihleman, Regolati & Schmid, 1970; Bowen et al., 1975; Russell et al., 1976), although success with purer GTF preparations has been reported in rats and hamsters (Taubman & Smith, 1977). This paper reports the results of the immunization of rhesus monkeys with whole cells of S. mutans and with cell wall preparations. The serum antibody responses were assayed by haemagglutination and complement-fixation tests using pronase-treated cell walls and SNAg. The responses were further analysed in terms of six defined antigens: cPS, LTA, insoluble dextran, and antigens 1/11, II and III. These antibody responses were then related to protection against smooth surface dental caries.
METHODS Animals Fifteen young rhesus monkeys were housed and maintained as described previously (Lehner, Challacombe

& Caldwell, 1975a). The cariogenic human type ofdiet was instituted at the start of the experiment. All monkeys had a fully erupted deciduous dentition. The monkeys were divided into four groups; those in Group A (3) were immunized with formalin-killed whole organisms; those in group B (5) were immunized with pronase-treated cell walls; those in group C (2) were immunized with washed untreated cell walls; and those in group D (5), the controls, were injected with saline. The first injections at week 0 were given with FIA to the monkeys in groups A, B and C. Further injections of antigen (or saline) without adjuvant were given at week 30 to the monkeys in groups A and three monkeys of group D and at 8 or 9 weeks for the monkeys in groups B and C and two monkeys of group D. The monkeys were examined for caries, and blood samples were taken at 3-4 week intervals as described previously (Lehner et al., 1975b), for a period of 35 weeks. One monkey in group B died at week 9, from unknown causes.
Vaccines Whole cells. An organism isolated from dental plaque in a previous series of monkeys and identified at S. mutans serotype c was grown in Todd-Hewitt broth for 24 h at 37. The cells were killed with 0-6% formalin, washed and suspended to give a concentration of 2 x 109 organism per ml, and emulsified with an equal volume of FIA. Volumes of 0-5 ml were injected subcutaneously into one arm and the opposite leg.

Cell walls. The organisms were broken in a Mickle disintegrator as described previously (Lehner et al., 1976a), washed three times in water and freeze-dried. A suspension ofthe crude cell walls in 0 1 M phosphate buffer pH 8 was treated with pronase (0-5 mg/ml) at 370 for 16 h, washed three times in water and freezedried. The untreated and pronase-treated cell walls were suspended in saline 10 mg/ml, emulsified with FIA and injected as described for whole cells.
Test antigens Pronase-treated cell walls were prepared as described above. SNAg was prepared either as HACS (Russell et al., 1976) or as a freeze-dried concentrate ofculture supernatants of the organism grown in a defined medium (see below). Both preparations contained cPS, LTA, antigens I/II, II and III, and possessed GTF activity.

S. mutans and immunization against dental caries


cPS was prepared from cells of S. mutans (Ingbritt) by extraction with hot 5% trichloracetic acid and purified by DEAE-cellulose chromatography (Linzer et al., 1976). The neutral polysaccharide fraction was used after esterification with palmitoyl chloride (Hammerling & Westphal, 1967). LTA was prepared from cells of S. mutans (Ingbritt) by extraction with hot 45% phenol, and purified by digestion with RNase and DNase, followed by chromatography on Sepharose 6B (Wicken, Gibbens & Knox, 1973). The high molecular weight fraction was used. Insoluble dextran was prepared from the culture supernatant of S. mutans (Ingbritt) grown in a glucose supplemented dialysate medium (Russell & Lehner, 1978). The supernatant was adjusted to pH 6-5, sucrose 100 g/l was added and the mixture incubated for 20 h at 37. A small amount of insoluble material and any remaining cells were removed by centrifugation, and the bulk of the polysaccharide was precipitated with 3 volumes of alcohol in the presence of 5% sodium acetate. The gelatinous polysaccharide was collected by centrifugation, washed twice by homogenizing in water, in which it failed to redissolve, dialysed against water and freeze-dried. Antigens I/IT, IT and III were prepared from culture supernatants of S. mutans (Ingbritt) grown in 10 1 of semi-defined medium based on casein hydrolysate (Acidicase 2, low salt; BBL) (Bowden, Hardie & Fillery, 1976). After 48 h, the supernatant was concentrated to about 200 ml by dialysis against solid polyethylene glycol, dialysed exhaustively against distilled water and freeze-dried. Antigens I/IT and III were separated and partially purified by chromatography on DEAE-cellulose (Russell & Lehner, 1978). Antigen II was prepared from 150 mg of freeze-dried antigen I/IT by treatment with 1 mg of pronase dissolved in 5 ml of 0- 1 M tris-HCl pH 7-4 at 37 for 16 h, and purified by chromatography on Bio-Gel P150.

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performed on microscope slides using 1% agarose as previously described (Russell & Lehner, 1978). Precipitin titres were obtained in quantitative immunodiffusion by making serial two-fold dilutions of the sera, and allowing them to diffuse against standard solutions of antigens I/II, II and III, each at 1 mg/ml for 40 h. The highest dilution of serum giving a visible precipitin line after staining was taken as the titre.
RESULTS
Haemagglutinating and complement-fixing antibodies to cell walls and SNAg The mean antibody responses of the four groups of monkeys to cell walls and SNAg are shown in Tables 1 and 2. In the haemagglutination but not complement fixation tests with both antigens, the group A animals showed higher pre-injection titres than those in the other groups. Response to immunization is therefore measured as the increase in titre above the initial level. Haemagglutination titres were higher, both at the start of and throughout the experimental period, than complement fixation titres. This has been observed previously, and may be a reflection of the differing sensitivities of the methods. The controls (group D) showed relatively constant low titres in both tests with both antigens throughout the course of the experiment. Mean log2 haemagglutination titres fluctuated between 2-0 and 3 9 against cell walls, and between 2-9 and 4-2 against SNAg. Similarly, mean log2 complement fixation titres varied between 0-7 and 3-7 for cell walls and 0 0 and 1 9 for SNAg. The difference in titre from week 0 of each subsequent sample-interval was calculated for each individual control monkey in each test, and the standard deviation of the differences calculated for the whole group. These values were: in haemagglutination tests, against cell walls, 2-33 and against SNAg, 1-73; in complement-fixation tests, against cell walls, 2-30 and against SNAg, 1-92. These values gave a measure of the range of increase of titres from week 0 in the control group. Assuming that the titres in animals of the other groups would show a similar tendency to vary irrespective of treatment, any individual titre which showed an increase above its corresponding week 0 value, greater than twice the standard deviation of the corresponding increase in the control titre, may be taken as significant with > 95% confidence. In group A, haemagglutination titres to both antigens increased significantly from week 4 in all

Antibody titrations Complement fixation and passive haemagglutination tests were performed in microtitre trays as previously described (Lehner et al., 1976a). The optimum concentrations established for each antigen preparation were as follows. In complement fixation tests: cell walls, 0 01 or 0 02 mg/ml; SNAg, 0-1 or 0-2 mg/ml; insoluble dextran, 5 mg/ml. In passive haemagglutination tests: cell walls, I mg/ml; SNAg, 1 mg/ml; cPS (5% palmitate ester), 5-10 Mg/ml; LTA, 1 Mg/ml. Immunodiffusion and immunoelectrophoresis were

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M. W. Russell, S. J. Challacombe & T. Lehner


Table 1. Haemagglutinating antibody titres to pronase-treated cell walls and SNAg Mean ( SEM) 10g2 titre at weeks

Test No. of Monkeys immunized with animals antigen

12

16

20

28

35

Whole cells Pronase-treated cell walls Untreated cell walls Controls

3 5

2 5

Cell wall SNAg Cell wall SNAg Cellwall SNAg Cell wall SNAg

6-7(1-76) 6-3(1-45) .3-8(1-25) 43(075) 5-0(0) 4-5(0-5) 2 0(0 49) 3 3(0 42)

13-3(1-45) 12-3(0-67) 8-6(1-17) 8-4(1-25) 12 (1-0) 9-5(0-5) 2 8(0 55) 3 9(0 59)

13-3(1-45) 13-0(1-73) 12-3(1-45) 12-0(1-73) 12-7(2-73) 13-3(2-33) 11-3(0-88) 12-7(2-67 11-3(0-88) 11-0(1-53) 11-7(1-76) 12-0(1-53) 9-8(1-39) 10-3(1-49) 10-8(0-58) 9-5(1-66) 9 3(0 63) 8 8(0 48) 9-6(1-12) 95(1 19) 98(073) 83(085) 83(075) 85(065) 8-0(1-0) 80(0) 9-5(1.5) - - 8-5(1-5) 8-0(0) 7-5(0 5) 6.5(0.5) 8-0(1-0) 8 5(0.5) - - 7-5(0-5) 3 3(0 73) 3-4(0 38) 3 9(0 53) 2 7(0-58) 3 3(0 70) 3 5(0 69) 4-1(0-66) 3.7(0 44) 3 7(0 37) 2 9(C-38) 3-1(0-67) 4 2(0-68)

Table 2. Complement-fixing antibody titres to pronase-treated cell walls and SNAg

Mean ( SEM) log2 titre at weeks

Monkeys immunized with Whole cells Pronase-treated cell walls Untreated cell walls Controls

Test No. of animals antigen 3 5 2

12

16

20

28

35

Cell wall SNAg Cell wall SNAg Cell wall SNAg Cell wall

0-3(0 33) 3-7(1-86) 6 0(0) 1-3(0-33) 5-3(0 33) 5 7(0 88) [-0(1-0) 7 4(0 5) 7-3(0 25) 0-3(0-25) 4-2(1-35) 28(0-85) 1-0(1-0) 4.0* - 7.0* 0 (0) 3-0* - 3-0* 1-2(0-53) 1-0(0-6) 2 9(0 79)

6 7(0 33) 7 3(0.33) 7 7(0 67) 6-7(0.67) 8 3(0 33)

5-3(1-2) 6-3(0 33) 6-0(1-0) 8-3(1 33) 9 0(0 54) 7 0(1-78) 4-0(1 52) 4-2(1-15) 5-0(1-08) - 65(0 49) 5-0* - 4-0(1-0) 1-0* 3 7(0 55) 1-4(0-58) 0-7(0 4)

SNAg

1-0(0-42) 1-0(0-54) 0-9(0 3)


*

1-9(0-31) 1-0(0-33) 0 (0)

5-0(1-0) 6.8(2 25) 4-8(1-54) 60(0) 3-5(0-49) 0-9(0-41) 0 7(0 42)

6-7(1-20) 6-8(2 06) 4-0(1-68)

7-0(1-0) 3-5(0-49) 2-6(0 62)

1-5(0-52)

One sample only.

three animals. Complement-fixation titres to both antigens likewise increased significantly in two animals at week 4, and in all three animals from week 8 onwards. In group B animals, responses were variable; the haemagglutination titres to cell walls showed significant increases in all five animals only after 12 weeks. The haemagglutination titres to SNAg showed significant increases by week 8 in four out of five animals. The complement-fixation titres in group B animals showed some fluctuation; a high mean titre against cell walls was shown at week 4 and all animals showed significant increases in titre by week 12. The mean titres against SNAg were somewhat lower. In group C, both animals responded promptly with haemagglutinating antibodies to both antigens and significant titres were reached from week 4 onwards. Complement-fixing antibodies developed more slowly, reaching significant titres (to cell walls) in both

animals from 16 weeks, while only one animal showed significant titres to SNAg, again from 16 weeks. Previous experience has indicated that the antibody response to an injection of whole organisms in FIA may last for up to 30 weeks or more (Lehner et al., 1976b). Antibody titres were assayed at monthly intervals and booster injections without adjuvant were given as soon as a drop in titre was seen, in order to maintain antibody levels throughout the experiment. These prevented any further drop in titre, but since a significant rise in titre was not apparent, it is not certain that they were strictly necessary.

Immunoelectrophoretic analysis of sera Immunoelectrophoresis of SNAg against serum samples from immunized monkeys revealed precipitating antibodies to a number of distinct antigenic com-

S. mutans and immunization against dental caries


ponents of SNAg (Fig. 1). Qualitatively, the presence or absence of precipitin lines was not consistently related to the immunization method or caries im-

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munity. However, close scrutiny of the lines suggested the possibility of quantitative differences which might be significant. Consequently, the defined antigens known to be present in SNAg were used to quantify the precipitating antibody responses.
Precipitating antibodies A preliminary survey of serum samples was made for precipitating antibodies to LTA and cPS. Most sera with high haemagglutinating titres against LTA (see below) also precipitated with LTA in gel diffusion, but none of the sera precipitated with cPS. A number of sera, however, showed a variety of precipitating antibodies to antigens present in SNAg (Fig. 1) and the precipitin arcs formed resembled those shown by rabbit antisera, by means of which the antigens I/II, II and III have been defined (Russell & Lehner, 1978). Antigen I/II is a high molecular weight (approximately 190,000) material containing 80-85% protein and havI/n

ing two antigenic determinants (Russell et al., 1980). One of these is destroyed by pronase and the other is serologically identical with antigen II. Antigen II is of lower molecular weight (48,000) and contains about 12% carbohydrate (Russell et al., 1980). Antigen III is a protein of molecular weight of 39,000 (Russell, 1979). The separated and partially purified antigens I/II, II and III were therefore used to assay antibody responses by precipitation in gel. Precipitating antibodies to antigen I/I1 were never observed in the controls but all animals immunized with whole cells or untreated cell walls formed precipitating antibodies at titres of up to log2 6 from week 8 onwards (Fig. 2). Animals immunized with pronasetreated cell walls generally responded more slowly; only two animals had detectable antibodies at week 8 and these showed low titres; it was not until 16 weeks that precipitating antibodies were found in all five animals, but from then on, titres were comparable with those in the other immunized animals. Antibody titres to antigen II were different (Fig. 2). Animals immunized with whole cells or untreated cell walls had precipitating antibodies from week 8
m

rH

lz

LTA

Figure 1. Immunoelectrophoresis of SNAg against sera from four immunized monkeys. Anode to the right. Arrows indicate precipitin arcs due to antigens I/II, II, III and IV, and lipoteichoic acid.

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Antigen 1/11
h-

M. W. Russell, S. J. Challacombe & T. Lehner


7
10 -

5 .$ 4 ,/I ~:

I
0

2 3

N
0 4 8 12 16 20

5I
",

I-"

-.?

28

35
oJ

Antigen II
6 5._ 4"e 3 %-

12 16

20

28

35

A/~~~0
4
8

Weeks

12 16 20

28

35

Figure 3. Haemagglutinating antibodies to lipoteichoic acid (mean and standard error). Monkeys immunized with: *, whole cells; o, pronase-treated cell walls; *, untreated cell walls; o, controls.

Antigen II I
o"

5 4

2 00

12 16 20 Weeks

28

35

There was a slight tendency for the LTA-haemagglutinating antibody titre in the control animals to increase during the experimental period. It is possible that this was due to sensitization of these animals with glycerol teichoic acids from S. mutans or other Grampositive organisms of their own dental plaque, or indeed other commensal organisms.
Antibodies to cPS As cPS is a neutral polysaccharide, it is itself incapable of sensitizing red cells, but this property can be conferred without loss of antigenicity by esterification with a long chain fatty acid (Hammerling & Westphal, 1967). Haemagglutinating antibody titres to cPS, as a 5% palmitate ester, showed some differences in response between the groups (Fig. 4). Monkeys immunized with whole cells responded promptly, giving significantly increased titres from week 4 onwards, while animals immunized with untreated cell walls appeared to give no response. In contrast, immunization with pronase-treated cell walls resulted in an antibody response comparable with that shown by animals immunized with whole cells.

Figure 2. Precipitating antibodies to antigens I/II, II and III (mean and standard error). Monkeys immunized with: *, whole cells; o, pronase-treated cell walls; *, untreated cell walls; o, controls.

onwards, with relatively low titres of log2 1-3. Animals immunized with pronase-treated cell walls however, gave higher titres, reaching log2 6 or higher from 12 weeks onwards. Again, controls did not show precipitating antibodies. Precipitating antibodies to antigen III (Fig. 2) appeared only intermittently, at low titre, and not in all animals of any group
Antibodies to LTA LTA readily sensitizes sheep red cells at low concentration and this makes it possible to estimate antibodies by means of haemagglutination. All immunized animals in groups A, B and C responded promptly (Fig. 3), and by week 4 the titres usually reached significant levels, by the same criterion as that discussed above. Consequently, no difference in response could be ascribed to the different immunizing preparations. The haemagglutinating antibody responses to LTA ran in parallel with the haemagglutinating antibodies against cell walls and SNAg. It therefore appears likely that LTA, which is present in cell walls and SNAg, was the principal sensitizing agent for haemagglutination in these preparations.

1---. ,11
x

le

4---*-

.z
----i

"..

'T.-

-?--

00 4
8

12

16 20 Weeks

28

35

Figure 4. Haemagglutinating antibodies to c polysaccharide (mean and standard error). Symbols as in Fig. 3.

S. mutans and immunization against dental caries


Antibodies to dextran Insoluble dextran from S. mutans could not be used to sensitize red cells, nor was it possible to carry out a simple esterification procedure as used for cPS, but it was possible to use it in the complement-fixation test. Most animals in all three immunization groups showed responses to about the same level (Fig. 5). However, there was anti-complementary activity in many serum samples and in consequence the data are incomplete. It has been suggested that the dextrans of S. mutans can form complexes with LTA (Melvaer, Helgeland & Rolla, 1974); indeed the dextran preparation used here contained 0 12% phosphorus which could be accounted for as contamination with approximately I% LTA. This would imply the presence of LTA in the complement-fixation test of about 50 yg/ml, and an antiserum to Lactobacillusfermentii LTA registered a titre of log2 6 in this test. It is therefore possible that complement-fixing antibodies to LTA were measured inadvertently using insoluble dextran. Antibodies against the dextran preparation were measurable in all immunized animals, and paralleled the LTA titres. It appeared, nevertheless, that there was no substantial difference in complement-fixing antibodies between the various immunized groups.

103

20r
15
w
0

LfLl 5l w
cr

C D scores (mean and standard Figure 6. Smooth surface caries error) at 35 weeks (open columns) and 54 weeks (shaded

columns). Monkeys immunized with: A, whole cells; B, pronase-treated cell walls; C, untreated cell walls; D, controls.

Caries scores The incidence of smooth surface caries (approximal and cervical) was scored in the deciduous teeth at 35 weeks in all animals, and again at 54 weeks in the nine animals which were kept until that time. Analysis of the caries incidence in the groups by the x2 test showed it to be significantly different from a random distribution. At 35 weeks, 75 lesions were found between 15

animals in 4 groups; x2 = 21-29, P < 0-001. At 54 weeks, 75 lesions were distributed between 9 animals in 3 groups; X2 = 21-84, P < 0-001. Animals immunized with whole cells showed a much lower caries score than the controls, both at 35 and 54 weeks (Fig. 6). Animals immunized with pronase-treated cell walls, had a distinctly higher incidence of caries at both times. However, both animals immunized with untreated cell walls had a considerably lower caries score at 35 weeks than animals immunized with pronase treated cell walls, although by comparison with the controls at this time, no measure of protection can be demonstrated. DISCUSSION
In previous experiments using the rhesus monkey caries model, immunity has been induced by subcutaneous immunization with killed whole cells of S. mutans serotype c in FIA (Lehner et al., 1975b, 1976b). As the antibody response of successfully immunized animals was monitored using pronase treated cell walls and SNAg as test antigens (Lehner et al., 1976a), these preparations were used for immunization. Three monkeys were vaccinated previously with a supernatant antigen preparation (HACS) containing GTF activity, but this did not induce protection (Russell et al., 1976). In the present experiments, monkeys immunized with pronase-treated cell walls far from showing resistance to caries, developed substantially more caries than the control animals. In contrast, monkeys immunized with untreated cell walls showed

10 I

1-

-F----,
0

12

16

20

28

35

Weeks

Figure 5. Complement-fixing antibodies to inssoluble dextran (mean and standard error). Symbols as in Figy. 3.

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M. W. Russell, S. J. Challacombe & T. Lehner

fewer carious lesions, though not significantly different from the controls. However, there were no consistent differences in antibody responses against cell walls or SNAg which could account for the difference in the incidence of caries between the three immunized groups. The complement-fixing antibodies to cell walls or SNAg were the best indicators of protection against caries in the previous experiments, since a brisk antibody response was found in protected animals, while high titres of haemagglutinating antibodies were developed in most immunized monkeys, whether protected or not (Lehner et al., 1975b; Russell et al., 1976). In this system, haemagglutinating antibodies are predominantly of the IgM class, while complement-fixing antibodies belong mainly to the IgG class (Lehner et al., 1976a). Recent evidence obtained by immunofluorescence using Ig class-specific antisera has demonstrated the prompt response with IgG antibodies to S. mutans cells in protected monkeys, although IgM antibodies were also found at a lower level (Lehner et al., 1976b). It might be possible to consider that the present results obtained with monkeys immunized with whole cells were consistent with the hypothesis that complement-fixing IgG antibodies are important in immunity to caries. However, the finding that monkeys immunized with pronase-treated cell walls showed enhanced caries despite having high titres of complement-fixing antibodies to cell walls and SNAg, needs further explanation. One possible factor, previously neglected, is that pronase-treated cell walls and SNAg are complex antigens with several distinct antigenic components. Analysis of antibody responses to individual antigens was therefore attempted. LTA is probably a membrane rather than a cell wall component, but it has been suggested that it extends into and even through the cell wall lattice (Knox & Wicken, 1973). All immunized animals developed LTA haemagglutinating antibodies promptly, irrespective of the subsequent development of caries. It therefore appeared unlikely that such antibodies were related to caries immunity. The use of the haemagglutination test possibly meant that IgM antibodies made a relatively greater contribution than IgG to the titres obtained. However, precipitating antibodies to LTA were sometimes observed in serum samples from animals immunized with whole cells or pronase-treated cell walls, suggesting that IgG antibodies may also be formed. LTA is widely distributed amongst Gram-positive

organisms, and it possesses a common antigenic determinant in the polyglycerophosphate backbone. Specific side-chain determinants occur in some streptococci and lactobacilli (Knox & Wicken, 1973), but have not yet been found in S. mutans serotype c. The haemagglutinating antibodies to LTA could not be definitely ascribed to either polyglycerophosphate or side-chain determinants. However, some sera from monkeys precipitated with LTA from both S. mutans and Lactobacillusfermentii forming a reaction of identity, which suggested that specificity for side-chain determinants was not involved in these antibody responses. If the LTA antibody response in monkeys immunized with S. mutans is mainly directed against the common polyglycerophosphate backbone, it would not be expected to contribute to specific immunity. Serotype polysaccharides probably constitute the major part of the non-peptidoglycan moiety of S. mutans cell walls (Hardie & Bowden, 1974; Bleiweis, Taylor, Deepak, Brown & Wetherell, 1976). However, it appears that cPS in particular, is not highly immunogenic, and the sensitive haemagglutination test was necessary to detect antibody responses. This showed that cPS antibodies were not related to caries immunity, as similar titres were present in both animals immunized with whole cells, which were protected, and in animals immunized with pronase-treated cell walls which had enhanced caries. Furthermore, the monkeys immunized with untreated cell walls did not develop antibodies to cPS, although in comparison with the monkeys immunized with pronase-treated cell walls they had lower caries scores. Although the haemagglutination technique may have favoured the detection of IgM antibodies, cPS antibody titres were at variance with the subsequent caries score. It has been suggested that protection against caries might involve antibody-mediated inhibition of adherence of S. mutans to hard surfaces, or of adherence of GTF to S. mutans cells, and that antibodies to serotype antigens can achieve this (Genco, Evans & Taubman, 1974; Mukasa & Slade, 1974). Our results suggest that caries immunity in these experiments does not involve such a mechanism. The high concentration of insoluble dextran required for optimal titration of antibody was disturbing. Reasons have already been advanced for believing that the dextran preparation contained small quantities of lipoteichoic acid which could have been the relevant antigenic determinant at the concentration used. In preliminatry trials to establish the optimum concentration of dextran for this test, titres fell sharply

S. mutans and immunization against dental caries


with concentrations less than 1-2 mg/ml. The materials used for immunizing the monkeys were prepared from organisms grown in broth supplemented with glucose but not sucrose, so that the dextran content of the cells and cell walls would have been minimal, and as a result, a marked dextran antibody response was not to be expected. There appeared to be no association of significant levels of dextran antibodies with caries immunity. A role for dextran antibodies may be postulated on the basis that they can inhibit the adherence of GTF to S. mutans cells (Mukasa & Slade, 1974). Our results again suggest that this does not apply in the present experiments. Precipitating antibodies to a supernatant antigen preparation (HACS) have been observed previously (Lehner et al., 1976a), especially in monkeys immunized with whole cells and showing resistance to caries. However, it has been found that HACS contains at least four new antigens (in addition to LTA and cPS) and of these, antigens I/II, II and III are probably also located in or on S. mutans cells (Russell & Lehner, 1978). In a quantitative immunodiffusion test, precipitin titres against antigens I/II and IT of log2 6 or more were recorded in sera from monkeys immunized with whole cells and cell walls while antibodies against antigen III were intermittent and seldom above a titre of log2 1. Most animals immunized with whole cells or untreated cell walls gave high titres (log2> 5) against antigen I/IT by 8 weeks, while animals immunized with pronase-treated cell walls responded with low titres (<2), reaching comparable high titres only after 16-20 weeks. The apparent importance of a brisk antibody response at the crucial initial stages of colonization by S. mutans has been noted previously (Lehner et al., 1975b). As monkeys immunized with whole cells showed resistance to caries, and so also did those immunized with untreated cell walls by comparison with those immunized with pronase-treated cell walls; it appeared that a brisk antibody response to antigen I/TI was associated with protection against caries. However, this did not explain the increased level of caries found in animals immunized with pronase-treated cell walls. These animals showed a higher antibody response to antigen II, than the others, though this developed relatively slowly. Enhanced caries in response to immunization has been recorded previously (Guggenheim et al., 1970; Bowen et al., 1975; Russell et al., 1976). It is therefore possible that enhancement of caries might be related to an increase in antibodies to antigen II, at the expense of true antigen I antibodies.

105

In addition however, there are other factors, especially the ratio of IgG to IgA antibodies against S. mutans. This ratio is significantly higher throughout the experimental period in the.whole cell immunized animals as compared with the animals immunized with pronase-treated cell walls (Lehner, Russell, Scully, Challacombe & Caldwell, 1979). Furthermore, the cell-mediated immune responses are also increased in the former group. Antigen I is destroyed by pronase (Russell & Lehner 1978) and this treatment was used to prepare antigen II from antigen I/II. Cell walls treated with pronase would not be expected to retain the pronase-sensitive antigen I, and animals immunized with such a preparation would therefore not form antibodies to antigen I. It seemed probable that any precipitating antibodies to antigen I/II preparations in monkeys immunized with pronase treated cell walls were directed against the antigen II component. In contrast, monkeys immunized with whole cells or untreated cell walls gave only low titres against antigen II, and therefore the precipitating antibodies against antigen I/TI preparations were probably directed mainly against the pronase-sensitive antigen I. If antibodies to this component are important in caries immunity, this would explain the difference in caries susceptibility between animals immunized with pronase-treated and untreated cell walls. Untreated cell walls possibly contained cellular materials not strictly belonging to the wall structure. It appeared, however, that whole cells and untreated cell walls contained a protective immunogen which was not present in pronase-treated cell walls. It is possible that antibodies to antigen I were the effective component of complement fixing antibodies to SNAg, or of IgG-fluorescent antibodies to whole cells, while the presence of antibodies to antigen II tended to obscure the association of the former with caries immunity in tests which did not discriminate between them. It seems that immunization can induce both reduction and enhancement of caries, depending on the immunizing agents and the balance of antibody responses elicited.

ACKNOWLEDGMENTS
We should like to thank Miss Lesley Bergmeier and Mrs Jill Caldwell for their excellent technical assistance. The support of a project grant from the Medical Research Council is gratefully acknowledged.

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M. W. Russell, S. J. Challacombe & T. Lehner

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