Immunology 1980 40 97

Specificity of antibodies induced by Streptococcus mutans during immunizationagainst dental caries

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

Acceptedforpublication 27 October 1977*

Summary. Protection against smooth surface dentalcaries was investigated in fifteen young rhesus mon-keys which were immunized subcutaneously withStreptococcus mutans serotype c in Freund's incom-plete adjuvant. Monkeys immunized with killed wholeorganisms developed significantly less caries than con-trol animals. Monkeys immunized with pronase-treated cell walls developed significantly more cariesthan control animals while monkeys immunized withuntreated cell walls showed no such enhancement ofcaries. Haemagglutinating and complement-fixingantibodies to cell walls and culture supernatantantigens (SN Ag) of S. mutans developed in the sera ofall immunized animals to a similar degree. Antibodiesto lipoteichoic acid and to an insoluble dextran prepa-ration were found in all immunized animals andshowed no relationship to the prevalence of caries.Antibodies to the serotype c polysaccharide were alsofound in animals immunized with whole cells andpronase-treated cell walls. However, precipitatingantibody levels to partially purified antigens I/II andII, derived from SN Ag, but present also in cells, wererelated to the development of caries. Animals im-munized with whole cells and with untreated cell walls

Correspondence: Dr T. Lehner, Department of Oral Im-munology and Microbiology, Guy's Hospital Medical andDental Schools, London SEI 9RT.

* Publication delayed because Patent Applicaton waspending.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 wallsresponded more slowly. The results suggest that im-munization may induce both caries reduction and en-hancement, depending on the antibody responsewhich is developed.

INTRODUCTION

The induction of immunity to dental caries has beenstudied in rhesus monkeys vaccinated with cells andextracellular products of Streptococcus mutans sero-type c (Lehner, Challacombe & Caldwell, 1975b; Rus-sell, Challacombe & Lehner, 1976). It appears thatsubcutaneous immunization with killed organisms inFreund's incomplete adjuvant (FIA) induces both sys-temic antibody and cellular responses (Lehner, Chal-lacombe, Wilton & Caldwell, 1976b) and reduces thedevelopment of dental caries. It has been postulatedthat serum antibodies could reach the site of cariousattack by passage through the gingival crevice(Lehner, Challacombe & Caldwell, 1976a; Challa-combe, Russell & Hawkes, 1978).Serum antibody levels have been assayed by means

of passive haemagglutination and complement fixa-tion tests, using trypsin- or pronase-treated cell wallsand an extracellular antigen preparation obtainedfrom culture supernatants (supernatant antigen;SNAg), which has glucosyltransferase (GTF) activity.The results indicate that protection is associated withthe 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 againstantigens to be found in both cell walls and culturesupernatants (Lehner et al., 1976a). Tests for the pre-sence ofantibodies capable of inhibitingGTF activity,suggest that such antibodies are not responsible for theobserved protection (Russell et al., 1976).There have been no investigations into the speci-

ficity of the antibody response to different antigeniccomponents of S. mutans. Several different antigens ofS. mutans serotype c have recently been described, andsome have been purified and characterized. They in-clude the serotype c polysaccharide (c PS) (Wetherell& Bleiweis, 1975; Linzer, Gill & Slade, 1976), lipotei-choic acid (LTA) (Markham, Knox, Wicken & Hew-ett, 1975), dextran-like glucans (the products ofGTFactivity) (Cisar & Kabat, 1976) and three antigensdesignated I, II, and III (Russell & Lehner, 1978). Allof these can be detected in, or extracted from, both thecells and culture supernatant fluids. It is thereforeappropriate to use these antigens to study the antibodyresponse further. It is now clear that antigen I asoriginally defined, also contains antigen II deter-minant within the one molecule (Russell, Bergmeier,Zanders & Lehner, submitted for publication) and isnow correctly described as antigen 1/11.Immunity to caries has been successfully induced by

vaccinating with whole organisms and with brokencell preparations (Bowen, Cohen, Cole & Colman,1975; Lehner et al., 1975b). Immunization with extra-cellular products such as crudeGTF preparations, hasnot been successful (Guggenheim, Miihleman, Rego-lati & Schmid, 1970; Bowen et al., 1975; Russell et al.,1976), although success with purer GTF preparationshas been reported in rats and hamsters (Taubman &Smith, 1977).

This paper reports the results of the immunizationof rhesus monkeys with whole cells of S. mutans andwith cell wall preparations. The serum antibody re-sponses were assayed by haemagglutination and com-plement-fixation tests using pronase-treated cell wallsand SNAg. The responses were further analysed interms of six defined antigens: cPS, LTA, insolubledextran, and antigens 1/11, II and III. These antibodyresponses were then related to protection againstsmooth surface dental caries.

METHODSAnimalsFifteen young rhesus monkeys were housed and main-tained as described previously (Lehner, Challacombe

& Caldwell, 1975a). The cariogenic human type ofdietwas instituted at the start of the experiment. All mon-keys had a fully erupted deciduous dentition.The monkeys were divided into four groups; those

in Group A (3) were immunized with formalin-killedwhole organisms; those in group B (5) were immunizedwith pronase-treated cell walls; those in group C (2)were immunized with washed untreated cell walls; andthose in group D (5), the controls, were injected withsaline. The first injections at week 0 were given withFIA to the monkeys in groups A, B and C. Furtherinjections of antigen (or saline) without adjuvant weregiven at week 30 to the monkeys in groupsA and threemonkeys of group D and at 8 or 9 weeks for themonkeys in groups B and C and two monkeys ofgroupD.The monkeys were examined for caries, and blood

samples were taken at 3-4 week intervals as describedpreviously (Lehner et al., 1975b), for a period of 35weeks. One monkey in group B died at week 9, fromunknown causes.

Vaccines

Whole cells. An organism isolated from dental pla-que in a previous series ofmonkeys and identified at S.mutans serotype c was grown in Todd-Hewitt brothfor 24 h at 37°. The cells were killed with 0-6% forma-lin, washed and suspended to give a concentration of2 x 109 organism per ml, and emulsified with an equalvolume of FIA. Volumes of 0-5 ml were injected sub-cutaneously into one arm and the opposite leg.

Cell walls. The organisms were broken in a Mickledisintegrator 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 phosphatebuffer pH 8 was treated with pronase (0-5 mg/ml) at370 for 16 h, washed three times in water and freeze-dried. The untreated and pronase-treated cell wallswere suspended in saline 10 mg/ml, emulsified withFIA and injected as described for whole cells.

Test antigensPronase-treated cell walls were prepared as describedabove.SNAg was prepared either as HACS (Russell et al.,

1976) or as a freeze-dried concentrate ofculture super-natants 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.

98

S. mutans and immunization against dental caries

cPS was prepared from cells of S. mutans (Ingbritt)by extraction with hot 5% trichloracetic acid and puri-fied by DEAE-cellulose chromatography (Linzer etal., 1976). The neutral polysaccharide fraction was

used after esterification with palmitoyl chloride (Ham-merling & Westphal, 1967).LTA was prepared from cells of S. mutans (Ingbritt)

by extraction with hot 45% phenol, and purified bydigestion with RNase and DNase, followed by chro-matography 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 glucosesupplemented dialysate medium (Russell & Lehner,1978). The supernatant was adjusted to pH 6-5, suc-

rose 100 g/l was added and the mixture incubated for20 h at 37°. A small amount of insoluble material andany remaining cells were removed by centrifugation,and the bulk of the polysaccharide was precipitatedwith 3 volumes of alcohol in the presence of 5%sodium acetate. The gelatinous polysaccharide was

collected by centrifugation, washed twice by homo-genizing in water, in which it failed to redissolve,dialysed against water and freeze-dried.

Antigens I/IT, IT and III were prepared from culturesupernatants of S. mutans (Ingbritt) grown in 10 1 ofsemi-defined medium based on casein hydrolysate(Acidicase 2, low salt; BBL) (Bowden, Hardie & Fil-lery, 1976). After 48 h, the supernatant was concen-

trated to about 200 ml by dialysis against solidpolyethylene glycol, dialysed exhaustively against dis-tilled water and freeze-dried. Antigens I/IT and IIIwere separated and partially purified by chromatogra-phy on DEAE-cellulose (Russell & Lehner, 1978).Antigen II was prepared from 150 mg of freeze-driedantigen I/IT by treatment with 1 mg of pronase dis-solved 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.

Antibody titrationsComplement fixation and passive haemagglutinationtests were performed in microtitre trays as previouslydescribed (Lehner et al., 1976a). The optimum concen-

trations 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; insolubledextran, 5 mg/ml. In passive haemagglutination tests:cell walls, I mg/ml; SNAg, 1 mg/ml; cPS (5% palmitateester), 5-10 Mg/ml; LTA, 1 Mg/ml.

Immunodiffusion and immunoelectrophoresis were

performed on microscope slides using 1% agarose aspreviously described (Russell & Lehner, 1978). Preci-pitin titres were obtained in quantitative immunodif-fusion by making serial two-fold dilutions of the sera,and allowing them to diffuse against standard solu-tions ofantigens I/II, II and III, each at 1 mg/ml for 40h. The highest dilution of serum giving a visible preci-pitin line after staining was taken as the titre.

RESULTS

Haemagglutinating and complement-fixing antibodiesto cell walls and SNAgThe mean antibody responses of the four groups ofmonkeys to cell walls and SNAg are shown in Tables 1and 2. In the haemagglutination but not complementfixation tests with both antigens, the group A animalsshowed higher pre-injection titres than those in theother groups. Response to immunization is thereforemeasured as the increase in titre above the initial level.Haemagglutination titres were higher, both at the startofand throughout the experimental period, than com-plement fixation titres. This has been observed pre-viously, and may be a reflection ofthe differing sensiti-vities of the methods.The controls (group D) showed relatively constant

low titres in both tests with both antigens throughoutthe course of the experiment. Mean log2 haemagglu-tination titres fluctuated between 2-0 and 3 9 againstcell walls, and between 2-9 and 4-2 against SNAg.Similarly, mean log2 complement fixation titres variedbetween 0-7 and 3-7 for cell walls and 0 0 and 1 9 forSNAg. The difference in titre from week 0 of eachsubsequent sample-interval was calculated for eachindividual control monkey in each test, and the stan-dard deviation of the differences calculated for thewhole group. These values were: in haemagglutinationtests, against cell walls, 2-33 and against SNAg, 1-73;in complement-fixation tests, against cell walls, 2-30and against SNAg, 1-92. These values gave a measureof the range of increase of titres from week 0 in thecontrol group. Assuming that the titres in animals ofthe other groups would show a similar tendency tovary irrespective of treatment, any individual titrewhich showed an increase above its correspondingweek 0 value, greater than twice the standard devia-tion of the corresponding increase in the control titre,may be taken as significant with > 95% confidence.

In group A, haemagglutination titres to bothantigens increased significantly from week 4 in all

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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 weeksMonkeys No. of Testimmunized with animals antigen 0 4 8 12 16 20 28 35

Whole 3 Cell wall 6-7(1-76) 13-3(1-45) 13-3(1-45) 13-0(1-73) 12-3(1-45) 12-0(1-73) 12-7(2-73) 13-3(2-33)cells SNAg 6-3(1-45) 12-3(0-67) 11-3(0-88) 12-7(2-67 11-3(0-88) 11-0(1-53) 11-7(1-76) 12-0(1-53)Pronase-treated 5 Cell wall .3-8(1-25) 8-6(1-17) 9-8(1-39) 10-3(1-49) 10-8(0-58) 9-5(1-66) 9 3(0 63) 8 8(0 48)cell walls SNAg 43(075) 8-4(1-25) 9-6(1-12) 95(1 19) 98(073) 83(085) 83(075) 85(065)Untreated 2 Cellwall 5-0(0) 12 (1-0) 9-5(1.5) - - 8-5(1-5) 8-0(0) 80(0) 8-0(1-0)cell walls SNAg 4-5(0-5) 9-5(0-5) 8 5(0.5) - - 7-5(0-5) 7-5(0 5) 6.5(0.5) 8-0(1-0)Controls 5 Cell wall 2 0(0 49) 2 8(0 55) 3 3(0 73) 3-4(0 38) 3 9(0 53) 2 7(0-58) 3 3(0 70) 3 5(0 69)

SNAg 3 3(0 42) 3 9(0 59) 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 weeksMonkeys No. of Testimmunized with animals antigen 0 4 8 12 16 20 28 35

Whole 3 Cell wall 0-3(0 33) 3-7(1-86) 6 0(0) 6 7(0 33) 7 3(0.33) 7 7(0 67) 6-7(0.67) 8 3(0 33)cells SNAg 1-3(0-33) 5-3(0 33) 5 7(0 88) 5-3(1-2) 6-3(0 33) 6-0(1-0) 5-0(1-0) 6-7(1-20)Pronase-treated 5 Cell wall [-0(1-0) 7 4(0 5) 7-3(0 25) 8-3(1 33) 9 0(0 54) 7 0(1-78) 6.8(2 25) 6-8(2 06)cell walls SNAg 0-3(0-25) 4-2(1-35) 28(0-85) 4-0(1 52) 4-2(1-15) 5-0(1-08) 4-8(1-54) 4-0(1-68)Untreated 2 Cell wall 1-0(1-0) 4.0* - 7.0* - - - 65(0 49) 5-0* - 60(0) 7-0(1-0)cell walls SNAg 0 (0) 3-0* - 3-0* - - - 4-0(1-0) 1-0* - 3-5(0-49) 3-5(0-49)Controls S Cell wall 1-2(0-53) 1-0(0-6) 2 9(0 79) 3 7(0 55) 1-4(0-58) 0-7(0 4) 0-9(0-41) 2-6(0 62)

SNAg 1-0(0-42) 1-0(0-54) 0-9(0 3) 1-9(0-31) 1-0(0-33) 0 (0) 0 7(0 42) 1-5(0-52)

* One sample only.

three animals. Complement-fixation titres to bothantigens likewise increased significantly in two ani-mals at week 4, and in all three animals from week 8onwards. In group B animals, responses were variable;the haemagglutination titres to cell walls showed sig-nificant increases in all five animals only after 12weeks. The haemagglutination titres to SNAg showedsignificant increases by week 8 in four out of fiveanimals. The complement-fixation titres in group Banimals showed some fluctuation; a high mean titreagainst cell walls was shown at week 4 and all animalsshowed significant increases in titre by week 12. Themean titres against SNAg were somewhat lower. Ingroup C, both animals responded promptly with hae-magglutinating antibodies to both antigens and sig-nificant 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 showedsignificant titres to SNAg, again from 16 weeks.

Previous experience has indicated that the antibodyresponse to an injection of whole organisms in FIAmay last for up to 30 weeks or more (Lehner et al.,1976b). Antibody titres were assayed at monthly inter-vals and booster injections without adjuvant weregiven as soon as a drop in titre was seen, in order tomaintain antibody levels throughout the experiment.These prevented any further drop in titre, but since asignificant rise in titre was not apparent, it is notcertain that they were strictly necessary.

Immunoelectrophoretic analysis of sera

Immunoelectrophoresis of SNAg against serum sam-ples from immunized monkeys revealed precipitatingantibodies to a number of distinct antigenic com-

100

S. mutans and immunization against dental caries

ponents of SNAg (Fig. 1). Qualitatively, the presence

or absence of precipitin lines was not consistentlyrelated to the immunization method or caries im-munity. However, close scrutiny of the lines suggestedthe possibility of quantitative differences which mightbe significant. Consequently, the defined antigensknown to be present in SNAg were used to quantifythe precipitating antibody responses.

Precipitating antibodiesA preliminary survey of serum samples was made forprecipitating antibodies to LTA and cPS. Most sera

with high haemagglutinating titres against LTA (seebelow) also precipitated with LTA in gel diffusion, butnone of the sera precipitated with cPS. A number ofsera, however, showed a variety of precipitating anti-bodies to antigens present in SNAg (Fig. 1) and theprecipitin arcs formed resembled those shown by rab-bit antisera, by means ofwhich the antigens I/II, II andIII have been defined (Russell & Lehner, 1978).Antigen I/II is a high molecular weight (approximately190,000) material containing 80-85% protein and hav-

I/n rH m lz

ing two antigenic determinants (Russell et al., 1980).One of these is destroyed by pronase and the other isserologically identical with antigen II. Antigen II is oflower molecular weight (48,000) and contains about12% carbohydrate (Russell et al., 1980). Antigen III isa protein of molecular weight of 39,000 (Russell,1979). The separated and partially purified antigensI/II, II and III were therefore used to assay antibodyresponses by precipitation in gel.

Precipitating antibodies to antigen I/I1 were never

observed in the controls but all animals immunizedwith whole cells or untreated cell walls formed precipi-tating antibodies at titres of up to log2 6 from week 8onwards (Fig. 2). Animals immunized with pronase-

treated cell walls generally responded more slowly;only two animals had detectable antibodies at week 8and these showed low titres; it was not until 16 weeksthat precipitating antibodies were found in all fiveanimals, but from then on, titres were comparablewith those in the other immunized animals.Antibody titres to antigen II were different (Fig. 2).

Animals immunized with whole cells or untreated cellwalls had precipitating antibodies from week 8

LTA

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

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

Antigen 1/117

h- 5 -.$ 4 -

I -0

Antigen II

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

Antigen II I5 -

o" 4 -

02-

/I ~:

0 4 8 12 16 20

10 -

,-

N 5-

2 3

28 35

A/~~~-0 4 8 12 16 20 28 35

0 4 8 12 16 20 28 35

Weeks

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 cellwalls; o, controls.

onwards, with relatively low titres of log2 1-3. Animalsimmunized with pronase-treated cell walls however,gave higher titres, reaching log2 6 or higher from 12weeks onwards. Again, controls did not show precipi-tating antibodies.

Precipitating antibodies to antigen III (Fig. 2)appeared only intermittently, at low titre, and not inall animals of any group

Antibodies to LTALTA readily sensitizes sheep red cells at low concen-tration and this makes it possible to estimate anti-bodies by means of haemagglutination. All im-munized animals in groups A, B and C respondedpromptly (Fig. 3), and by week 4 the titres usuallyreached significant levels, by the same criterion as thatdiscussed above. Consequently, no difference in re-sponse could be ascribed to the different immunizingpreparations. The haemagglutinating antibody re-sponses to LTA ran in parallel with the haemagglu-tinating antibodies against cell walls and SNAg. Ittherefore appears likely that LTA, which is present incell walls and SNAg, was the principal sensitizingagent for haemagglutination in these preparations.

0 4 8 12 16 20Weeks

28 35

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

There was a slight tendency for the LTA-haemagg-lutinating antibody titre in the control animals toincrease during the experimental period. It is possiblethat this was due to sensitization ofthese animals withglycerol teichoic acids from S. mutans or other Gram-positive organisms of their own dental plaque, or in-deed other commensal organisms.

Antibodies to cPSAs cPS is a neutral polysaccharide, it is itself incapableof sensitizing red cells, but this property can be con-ferred without loss of antigenicity by esterificationwith a long chain fatty acid (Hammerling& Westphal,1967). Haemagglutinating antibody titres to cPS, as a5% palmitate ester, showed some differences in re-sponse between the groups (Fig. 4). Monkeys im-munized with whole cells responded promptly, givingsignificantly increased titres from week 4 onwards,while animals immunized with untreated cell wallsappeared to give no response. In contrast, immuniza-tion with pronase-treated cell walls resulted in an anti-body response comparable with that shown by ani-mals immunized with whole cells.

0-0 4 8 12 16 20

Weeks28 35

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

102

I I-" -.?

f",

o J

1---.,11 .z

x 4-le

"..

'T.- --?-- ---*- ----i

S. mutans and immunization against dental caries

Antibodies to dextranInsoluble dextran from S. mutans could not be used tosensitize red cells, nor was it possible to carry out asimple esterification procedure as used for cPS, but itwas possible to use it in the complement-fixation test.Most animals in all three immunization groupsshowed responses to about the same level (Fig. 5).However, there was anti-complementary activity inmany serum samples and in consequence the data areincomplete.

It has been suggested that the dextrans of S. mutanscan form complexes with LTA (Melvaer, Helgeland &Rolla, 1974); indeed the dextran preparation used herecontained 0 12% phosphorus which could beaccounted for as contamination with approximatelyI% LTA. This would imply the presence ofLTA in thecomplement-fixation test of about 50 yg/ml, and anantiserum to Lactobacillusfermentii LTA registered atitre of log2 6 in this test. It is therefore possible thatcomplement-fixing antibodies to LTA were measuredinadvertently using insoluble dextran. Antibodiesagainst the dextran preparation were measurable in allimmunized animals, and paralleled the LTA titres. Itappeared, nevertheless, that there was no substantialdifference in complement-fixing antibodies betweenthe various immunized groups.

Caries scoresThe incidence of smooth surface caries (approximaland cervical) was scored in the deciduous teeth at 35weeks in all animals, and again at 54 weeks in the nineanimals which were kept until that time. Analysis ofthe caries incidence in the groups by the x2 test showedit to be significantly different from a random distribu-tion. At 35 weeks, 75 lesions were found between 15

10 I

0

0 4 8 12 16 20Weeks

Figure 5. Complement-fixing antibodies to ins(mean and standard error). Symbols as in Fig

-1-

-F----,

28 35

soluble dextrany. 3.

20r

15w0

LfLl5lw

cr

A B C D

Figure 6. Smooth surface caries scores (mean and standarderror) at 35 weeks (open columns) and 54 weeks (shadedcolumns). Monkeys immunized with: A, whole cells; B, pro-nase-treated cell walls; C, untreated cell walls; D, controls.

animals in 4groups; x2=21-29, P< 0-001. At 54weeks,75 lesions were distributed between 9 animals in 3groups; X2= 21-84, P < 0-001. Animals immunizedwith whole cells showed a much lower caries scorethan 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 bothtimes. However, both animals immunized with un-treated cell walls had a considerably lower caries scoreat 35 weeks than animals immunized with pronasetreated cell walls, although by comparison with thecontrols at this time, no measure of protection can bedemonstrated.

DISCUSSION

In previous experiments using the rhesus monkeycaries model, immunity has been induced by subcu-taneous immunization with killed whole cells of S.mutans serotype c in FIA (Lehner et al., 1975b, 1976b).As the antibody response of successfully immunizedanimals was monitored using pronase treated cellwalls and SNAg as test antigens (Lehner et al., 1976a),these preparations were used for immunization. Threemonkeys were vaccinated previously with a superna-tant antigen preparation (HACS) containing GTFactivity, but this did not induce protection (Russell etal., 1976). In the present experiments, monkeys im-munized with pronase-treated cell walls far fromshowing resistance to caries, developed substantiallymore caries than the control animals. In contrast,monkeys immunized with untreated cell walls showed

103

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

fewer carious lesions, though not significantly differ-ent from the controls. However, there were no consis-tent differences in antibody responses against cell wallsor SNAg which could account for the difference in theincidence of caries between the three immunizedgroups.The complement-fixing antibodies to cell walls or

SNAg were the best indicators of protection againstcaries in the previous experiments, since a brisk anti-body response was found in protected animals, whilehigh titres ofhaemagglutinating antibodies were deve-loped in most immunized monkeys, whether protectedor not (Lehner et al., 1975b; Russell et al., 1976). Inthis system, haemagglutinating antibodies are predo-minantly of the IgM class, while complement-fixingantibodies belong mainly to the IgG class (Lehner etal., 1976a). Recent evidence obtained by immuno-fluorescence using Ig class-specific antisera has demon-strated the prompt response with IgG antibodies to S.mutans cells in protected monkeys, although IgM anti-bodies were also found at a lower level (Lehner et al.,1976b). It might be possible to consider that the pre-sent results obtained with monkeys immunized withwhole cells were consistent with the hypothesis thatcomplement-fixing IgG antibodies are important inimmunity to caries. However, the finding that mon-keys immunized with pronase-treated cell wallsshowed enhanced caries despite having high titres ofcomplement-fixing antibodies to cell walls and SNAg,needs further explanation.One possible factor, previously neglected, is that

pronase-treated cell walls and SNAg are complexantigens with several distinct antigenic components.Analysis of antibody responses to individual antigenswas therefore attempted.LTA is probably a membrane rather than a cell wall

component, but it has been suggested that it extendsinto and even through the cell wall lattice (Knox &Wicken, 1973). All immunized animals developedLTA haemagglutinating antibodies promptly, irres-pective of the subsequent development of caries. Ittherefore appeared unlikely that such antibodies wererelated to caries immunity. The use of the haemagglu-tination test possibly meant that IgM antibodies madea relatively greater contribution than IgG to the titresobtained. However, precipitating antibodies to LTAwere sometimes observed in serum samples from ani-mals immunized with whole cells or pronase-treatedcell walls, suggesting that IgG antibodies may also beformed.LTA is widely distributed amongst Gram-positive

organisms, and it possesses a common antigenic deter-minant in the polyglycerophosphate backbone. Speci-fic side-chain determinants occur in some streptococciand lactobacilli (Knox & Wicken, 1973), but have notyet been found in S. mutans serotype c. The haemagg-lutinating antibodies to LTA could not be definitelyascribed to either polyglycerophosphate or side-chaindeterminants. However, some sera from monkeys pre-cipitated with LTA from both S. mutans and Lactoba-cillusfermentii forming a reaction of identity, whichsuggested that specificity for side-chain determinantswas not involved in these antibody responses. If theLTA antibody response in monkeys immunized withS. mutans is mainly directed against the commonpolyglycerophosphate backbone, it would not beexpected to contribute to specific immunity.

Serotype polysaccharides probably constitute themajor 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 immuno-genic, and the sensitive haemagglutination test wasnecessary to detect antibody responses. This showedthat cPS antibodies were not related to caries im-munity, as similar titres were present in both animalsimmunized with whole cells, which were protected,and in animals immunized with pronase-treated cellwalls which had enhanced caries. Furthermore, themonkeys immunized with untreated cell walls did notdevelop antibodies to cPS, although in comparisonwith the monkeys immunized with pronase-treatedcell walls they had lower caries scores. Although thehaemagglutination technique may have favoured thedetection of IgM antibodies, cPS antibody titres wereat variance with the subsequent caries score.

It has been suggested that protection against cariesmight involve antibody-mediated inhibition of adher-ence of S. mutans to hard surfaces, or of adherence ofGTF to S. mutans cells, and that antibodies to sero-type antigens can achieve this (Genco, Evans& Taub-man, 1974; Mukasa & Slade, 1974). Our results sug-gest that caries immunity in these experiments doesnot involve such a mechanism.The high concentration of insoluble dextran

required for optimal titration ofantibody was disturb-ing. Reasons have already been advanced for believingthat the dextran preparation contained small quanti-ties of lipoteichoic acid which could have been therelevant antigenic determinant at the concentrationused. In preliminatry trials to establish the optimumconcentration ofdextran for this test, titres fell sharply

104

S. mutans and immunization against dental caries

with concentrations less than 1-2 mg/ml. The mater-ials used for immunizing the monkeys were preparedfrom organisms grown in broth supplemented withglucose but not sucrose, so that the dextran content ofthe cells and cell walls would have been minimal, andas a result, a marked dextran antibody response was

not to be expected. There appeared to be no associ-ation of significant levels of dextran antibodies withcaries immunity. A role for dextran antibodies may bepostulated on the basis that they can inhibit the adher-ence of GTF to S. mutans cells (Mukasa & Slade,1974). Our results again suggest that this does notapply in the present experiments.

Precipitating antibodies to a supernatant antigenpreparation (HACS) have been observed previously(Lehner et al., 1976a), especially in monkeys im-munized with whole cells and showing resistance tocaries. However, it has been found that HACS con-

tains at least four new antigens (in addition to LTAand 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 immunodiffusiontest, precipitin titres against antigens I/II and IT of log26 or more were recorded in sera from monkeys im-munized with whole cells and cell walls while anti-bodies against antigen III were intermittent and sel-dom above a titre of log2 1. Most animals immunizedwith whole cells or untreated cell walls gave high titres(log2> 5) against antigen I/IT by 8 weeks, while ani-mals immunized with pronase-treated cell walls res-

ponded with low titres (<2), reaching comparablehigh titres only after 16-20 weeks. The apparent im-portance of a brisk antibody response at the crucialinitial stages of colonization by S. mutans has beennoted previously (Lehner et al., 1975b). As monkeysimmunized with whole cells showed resistance tocaries, and so also did those immunized with untreatedcell walls by comparison with those immunized withpronase-treated cell walls; it appeared that a briskantibody response to antigen I/TI was associated withprotection against caries. However, this did notexplain the increased level of caries found in animalsimmunized with pronase-treated cell walls. These ani-mals showed a higher antibody response to antigen II,than the others, though this developed relativelyslowly. Enhanced caries in response to immunizationhas been recorded previously (Guggenheim et al.,1970; Bowen et al., 1975; Russell et al., 1976). It istherefore possible that enhancement ofcaries might berelated to an increase in antibodies to antigen II, at theexpense of true antigen I antibodies.

In addition however, there are other factors, espe-cially the ratio of IgG to IgA antibodies against S.mutans. This ratio is significantly higher throughoutthe experimental period in the.whole cell immunizedanimals as compared with the animals immunizedwith pronase-treated cell walls (Lehner, Russell,Scully, Challacombe & Caldwell, 1979). Furthermore,the cell-mediated immune responses are also increasedin the former group.

Antigen I is destroyed by pronase (Russell& Lehner1978) and this treatment was used to prepare antigenII from antigen I/II. Cell walls treated with pronasewould not be expected to retain the pronase-sensitiveantigen I, and animals immunized with such a prepa-ration would therefore not form antibodies to antigenI. It seemed probable that any precipitating antibodiesto antigen I/II preparations in monkeys immunizedwith pronase treated cell walls were directed againstthe antigen II component. In contrast, monkeys im-munized with whole cells or untreated cell walls gaveonly low titres against antigen II, and therefore theprecipitating antibodies against antigen I/TI prepa-rations were probably directed mainly against the pro-nase-sensitive antigen I. If antibodies to this com-ponent are important in caries immunity, this wouldexplain the difference in caries susceptibility betweenanimals immunized with pronase-treated and un-treated cell walls. Untreated cell walls possibly con-tained cellular materials not strictly belonging to thewall structure. It appeared, however, that whole cellsand untreated cell walls contained a protective im-munogen which was not present in pronase-treatedcell walls.

It is possible that antibodies to antigen I were theeffective component of complement fixing antibodiesto SNAg, or of IgG-fluorescent antibodies to wholecells, while the presence of antibodies to antigen IItended to obscure the association of the former withcaries immunity in tests which did not discriminatebetween them. It seems that immunization can induceboth reduction and enhancement of caries, dependingon the immunizing agents and the balance ofantibodyresponses elicited.

ACKNOWLEDGMENTS

We should like to thank Miss Lesley Bergmeier andMrs Jill Caldwell for their excellent technical assist-ance. The support of a project grant from the MedicalResearch Council is gratefully acknowledged.

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

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