Immunology 1976 30 619

Serum glucosyltransferase-inhibiting antibodies and dental caries in Rhesus monkeys immunized against Streptococcus mutans

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

Received 3 September 1975; acceptedfor publication 30 October 1975

INTRODUCTION

Summary. Serum antibodies to glucosyltransferase (GTF) of Streptococcus mutans serotype c were assayed sequentially by means of an enzyme inhibition radio-assay in twenty-six Rhesus monkeys immunized with S. mutans. Pre-immune and control sera had a GTF-enhancing effect which was shown also by albumin and non-immune immunoglobulin fractions. GTF-inhibitory activity was found in IgG fractions from some immune sera and could be absorbed by S. mutans cells possessing cell-bound GTF. Inhibitory antibodies to GTF developed in the sera of four monkeys immunized with hydroxylapatite extract of culture supernatant (HACS), and in four out of fifteen monkeys immunized with S. mutans cells, but in none of the seven sham-immunized control animals. The monkeys immunized with HACS showed no reduction in caries. A correlation has been demonstrated between protection against caries and the early development of serum IgG antibodies to antigens present in HACS but there was no consistent association between pro-

A model for the immunological study of dental caries has been established in Rhesus monkeys (Lehner, Challacombe and Caldwell, 1975a). These animals develop caries in the deciduous teeth when maintained on a human type of diet containing 15 per cent sucrose and caries is associated with bacterial plaque containing naturally acquired Streptococcus mutans serotype c (Bratthall, 1970). A significant reduction in the incidence of smooth surface caries was achieved in Rhesus monkeys by immunization with S. mutans (serotype c) in Freund's incomplete adjuvant (FIA) (Lehner, Challacombe and Caldwell, 1975b, c). S. mutans produces extracellular glucosyltransferases (GTF) which may be partially purified from the culture supernatant by absorption to hydroxylapatite (Guggenheim and Newbrun, 1969). GTF synthesizes glucans from sucrose, and it is thought to play an important part in the aetiology of caries by mediating the adherence of S. mutans to the tooth and producing plaque polysaccharide (Gibbons and Nygaard, 1968; Mukasa and Slade, 1973; Kuramitsu, 1974). Antibodies against GTF might be expected to play a part in immunity to caries by inhibiting the synthesis of glucans. GTF activity can be inhibited by antisera raised by immunization with whole organisms or crude GTF preparations in rabbits

tection against caries and GTF-inhibitory antibodies. The results also suggest the possibility that other antibodies, possibly present in the IgM or IgA fractions and having an enhancing effect on GTF, may increase the incidence of caries. Correspondence: Dr M. W. Russell, Department of Oral Immunology and Microbiology, Guy's Hospital, London, SEI 9RT.

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

(Evans and Genco, 1973; Challacombe, Guggenheim and Lehner, 1973) or in rats (Hayashi, Shklair and Bahn, 1972; Evans et al., 1974). In man, sera with increased haemagglutinating titres against S. mutans showed greater inhibitory effects on GTF activity than sera with lower titre (Challacombe et al., 1973). However, Guggenheim et al. (1970) failed to protect rats against caries by immunization with S. mutans and crude GTF. The purpose of this investigation was to use an enzyme inhibition radio-assay to evaluate sequentially, over a period of 50 weeks, the role of serum antibodies to GTF in immunity to dental caries. This was carried out in Rhesus monkeys immunized with whole organisms and a crude GTF preparation of S. mutans. MATERIALS AND METHODS Immunization procedure, collection of serum and saliva, examination for caries and titration of antibodies Rhesus monkeys, housed and maintained on a human type of diet containing 15 per cent sucrose, were divided into five groups. Six animals were immunized subcutaneously (SC) with 5 x 108 heat-killed S. mutans (strain Ingbritt) in FIA at weeks 0 and 36 and in saline at weeks 4, 8, 12 and 16. Another group of six animals was immunized by the same schedule submucosally (SM) in the oral cavity. Three animals were immunized SC by the same schedule with 2 5 mg of hydroxylapatite extract of culture supernatant (HACS) of S. mutans and a fourth animal received HACS in Freund's complete adjuvant (FCA) intramuscularly at week 0 followed by HACS in saline SC at weeks 4, 8 and 16. Three animals were immunized SC with monkey-passaged S. mutans in FIA at week 0 and in saline at weeks 4, 8 and 16. Seven controls were sham-immunized either SC with saline at weeks 0, 4, 8, 12 and 36 (three animals) or with FIA at weeks 0 and 36 and saline at the intervening times (four animals). The full details of the immunizations together with the methods of collection of serum and whole saliva, examinations for caries and titration of antibodies have been described previously (Lehner et al., 1975a, b, 1976).

Glucosyltransferase (GTF) Crude GTF was prepared as a hydroxylapatite ex-

tract of culture supernatant (HACS) of S. mutans strain Ingbritt (serotype c). Ammonium sulphateprecipitated material from a bulk culture of S. mutans in a medium similar to that of Cybulska and Pakula (1963) was blended in 500 ml water and centrifuged at 10,000 g for 30 min. This supernatant was stirred into 1 litre of a 25 per cent (v/v) suspension of hydroxylapatite (Bio-Gel HT; Bio-Rad Laboratories Ltd) for 1 h at room temperature (Guggenheim and Newbrun, 1969). The hydroxylapatite was collected by centrifuging at 1000 r.p.m. for 1 min, washed twice with 500 ml of 0 01 M phosphate, pH 6-0, and eluted successively with 0-2 M, then 0-5 M potassium phosphate, pH 6-0, using 2 x 250 ml of each. The 0 5 M extract was concentrated to 20 ml by ultrafiltration, desalted by passage through a column (30 x 2 cm) of Sephadex G-25 in distilled water and freeze-dried. This material contained 9 3 units/mg of GTF activity, 1 unit being defined as the amount of enzyme which synthesizes 100 ,pg of 70 per cent ethanol precipitable polysaccharide, from 0 125 M sucrose in 1 h at 370, in 0 5 ml of 0 05 M phosphate buffer, pH 6 8. Protein content was 23 per cent as measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard; carbohydrate content was 4 per cent by the method of Dubois et al. (1956) with glucose as a standard, and total phosphorus amounted to 5-7 per cent (Chen, Toribara and Warner, 1956). The material was heterogeneous on polyacrylamide gel electrophoresis which revealed about twenty protein bands of varying intensities. GTF activity, revealed by incubating duplicate gels in 10 per cent sucrose in phosphate buffer, pH 6-8, was located in one major broad zone, not readily identifiable with a particular protein band. GTF inhibition assay An aliquot of 5 pg of crude GTF was incubated in 0.4 ml of 0 05 M phosphate, pH 6 8, with 20 ,1 of serum for 1 h at 370, to allow antigen-antibody complexes to form. Then 0-1 ml of 0 625 M sucrose, containing 0 2 uCi of [U-14C]sucrose (Radiochemical Centre, Amersham) was added and incubation continued for 16 h. The reaction was stopped by the addition of 1 ml of 95 per cent ethanol and the insoluble polysaccharide was collected on a glass fibre filter disc (Whatman GF/C). Washings from the reaction tube were transferred to the filter which was washed twice with 10 ml of 70 per cent ethanol. The filter was placed in 4 ml of Instagel (Packard) scintillation fluid and counted with an efficiency of about

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GTF-inhibiting antibodies and dental caries 93 per cent. Controls consisted of substrate incubated without GTF, and serum incubated with substrate but without GTF to determine the level of residual [14C]sucrose retained by the filter. This value was subtracted from those resulting with GTF. The specific activity of the sucrose substrate was determined to calibrate the results in terms of the amount of polysaccharide synthesized. Each individual serum sample was tested in quadruplicate giving a mean coefficient of variation of 3 75 per cent for each of the 235 samples. All the sequential serum samples over 50 weeks from each animal were tested simultaneously. The activity of GTF in the presence of each serum sample was expressed as a percentage of activity in the presence of the pre-immune sample which therefore assumed a value of 100 per cent.

Fractionation of serum Serum samples of 5 ml were fractionated to separate the three main immunoglobulin components, by a modification of the method of Heide and Schwick (1973). This involved euglobulin precipitation followed by chromatography on Sephadex G-200 to yield IgM, DEAE-cellulose chromatography of the pseudoglobulin fraction to yield pure IgG and also IgA which was further purified by zinc sulphate precipitation and Sephadex G-200 chromatography. The immunoglobulin concentrations of the fractions were estimated by radial immunodiffusion (Mancini, Carbonara and Heremans, 1965) using anti-human IgG, IgM and IgA sera (Wellcome Reagents Ltd) and thereby related to the concentrations estimated in the original sera. Crossreactions of monkey with human Ig are adequate for this purpose (Monte-Wicher, Wicher and Arbesman, 1970) and this was confirmed in double-diffusion precipitation tests with these antisera. However, the absolute figures obtained should be treated with caution. The preparations were also analysed by immunoelectrophoresis. No other components were detectable in the IgG fractions. The IgM and IgA fractions contained small but measurable amounts of contaminating IgG together with traces of another component of fl-mobility. Albumin was partially purified from 5 ml of shamimmunized monkey serum, by means of precipitation with 50-75 per cent saturated ammonium sulphate and with 40 per cent ethanol at pH 5 2. This material contained no immunoglobulin on gel immunodiffusion against IgG, IgM or IgA antisera. Immuno-

electrophoresis revealed two components, albumin and a component of fl-mobility. The protein concentration was estimated by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Absorption of sera Aliquots of 0-2 ml of serum were absorbed twice with an equal volume of packed S. mutans (Ingbritt) cells for 1 h at 370 followed by 16 h at 4°. The organisms were grown in tryptone-soy broth (Oxoid) and killed with 01 per cent formaldehyde. RESULTS

Serum and salivary antibody responses, and caries incidence in monkeys immunized with HACS Of the three animals immunized with whole cells and HACS in FIA, only one showed a positive complement-fixing titre (log2 = 1) against HACS before immunization. Following immunization, the mean (±s.e.m.) titre rose to 3-7 (±09) at 16 weeks but then declined (Fig. 1). The salivary haemagglutinating antibody titres remained relatively constant at log2 1-2. All three animals developed smooth surface caries from week 20, reaching a mean (± s.e.m.) score of 14-3 ( ±2.2) at 100 weeks (Fig. 1). There was no reduction of caries in comparison with the control monkeys (13-25 ± 3 6) (Lehner et al., 1976). Effect of non-immune serum on GTF activity All samples of serum taken from each animal before immunization showed an enhancing effect upon the GTF activity of HACS. The mean (± s.e.m.) quantity of polysaccharide synthesized by 5 ug of HACS in 16 h without serum was 63± 3-24 4ug (N = 12), while the corresponding figure in the presence of 20 ul of pre-immune serum was 136 ±4-66 ,ug of polysaccharide (N = 23). This represents a mean enhancement of 2-2 ± 0 3 times.

Sequential studies of GFT-inhibitory activity in sera Serum from control monkeys, which had been shamimmunized with saline or FIA, continued to show GTF-enhancing activity at about the same level as the pre-immune sera (Fig. 2a). Serum from the three monkeys which received HACS in FIA developed a GTF-inhibitory activity from week 12, reaching maximum values of 20-40 per cent inhibition at

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Figure 2. Sequential GTF inhibitory activities of monkey sera. (a) Controls, sham-immunized with saline (-), FIA --(b) Immunized with HACS in FIA (-), or HACS in FCA ( - -). (c) Immunized SC with S. mulans in FIA. (d) Immunized SM with S. mutans in FIA. (e) Immunized SC with passaged S. mutans in FIA.

GTF-inhibiting antibodies and dental caries

effect on GTF activity. Approximately two-fold enhancement was achieved with 50 pug of protein (Fig. 3a). A similar result was obtained with pure crystalline bovine serum albumin. The antagonistic effects of immune IgG and albumin are illustrated in Fig. 3b, from which it may be seen that the inhibitory effect of 0 35 mg of immune IgG was reversed by 1 mg of albumin.

weeks 16 and 20 after which inhibitory activity declined (Figs 1 and 2b). The booster immunization with HACS in FIA at week 36 was followed by an increased GTF-inhibitory activity of up to 60 per cent inhibition. One monkey which was immunized with HACS in FCA developed serum GTF-inhibitory activity from 4 weeks, reaching a maximum of about 50 per cent inhibition at 12 weeks (Fig 2b). The six monkeys immunized SC with S. mutans in FIA showed varying responses (Fig. 2c). Three animals failed to develop significant GTF-inhibitory activity over 50 weeks, while three others slowly developed GTF-inhibitory activity from about week 20, and reached maximum values of 30-50 per cent inhibition after the booster immunization at 36 weeks. Only one monkey out of six immunized SM with whole cells developed GTF-inhibitory activity, commencing at week 12 and reaching a maximum of 40 per cent inhibition at week 24 (Fig. 2d). Of the three monkeys immunized with passaged S. mutans, two showed no GTF-inhibitory activity and the third showed a fairly constant low inhibitory activity from 4 weeks (Fig. 2e).

Distribution of GTF-inhibitory and enhancing activity in serum fractions GTF inhibition tests were carried out with 20 ,ul of four selected sera and their immunoglobulin fractions in amounts equivalent to those found in the corresponding unfractionated sera. In addition the immunoglobulin fractions were tested in the presence of 100 pg of crude monkey albumin (Table 1). Serum from sham-immunized monkeys and their immunoglobulin fractions showed GTF-enhancing activity. In the presence of albumin, the IgG fraction revealed a small but significant inhibitory effect (P 0-05 E

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Serum glucosyltransferase-inhibiting antibodies and dental caries in rhesus monkeys immunized against Streptococcus mutans.

Immunology 1976 30 619 Serum glucosyltransferase-inhibiting antibodies and dental caries in Rhesus monkeys immunized against Streptococcus mutans M...
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