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J Dent Res 83(4): 354-358, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Therapeutic Vaccine against Streptococcus sobrinus-induced Caries

M. Dinis1,5, D. Tavares1,2,5, A.J.M.M. Fonseca3,4, R. Faria1, A. Ribeiro1,2, A.M. Silvério Cabrita4, and P. Ferreira1,2,*

1 Laboratory of Immunology, ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Lg. Prof. Abel Salazar 2, 4099-003 Porto,
2 Instituto de Biologia Molecular e Celular, Porto,
3 Faculdade de Medicina de Coimbra-Hospitais da Universidade de Coimbra, and
4 Faculdade de Medicina de Coimbra (Instituto de Patologia Experimental), Coimbra, Portugal;
5 authors contributing equally to this work;

* corresponding author, pauferr{at}icbas.up.pt


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus sobrinus produces a virulence-associated immunomodulatory protein (VIP) which suppresses the host-specific immune response and induces the early production of IL-10. In this study, we evaluated the effects of therapeutic immunization with this VIP on the incidence of caries in S. sobrinus-infected rats. Groups of Wistar rats were orally infected with S. sobrinus and fed with sucrose-sweetened drinking water ad libitum. Five days later, rats were immunized intranasally with active or heat-inactivated VIP plus alum as adjuvant or PBS plus adjuvant (sham-immunized). After 3 wks, all rats were re-immunized as above. Evaluation of dental caries showed that VIP-immunized animals had significantly fewer enamel sulcal and proximal caries lesions than did the sham-immunized animals (p < 0.001). The protective effects following therapeutic VIP immunization were attributed to the induced salivary immunoglobulin A specific to the VIP. These results offer a promising and safe strategy for the development of a vaccine against dental caries.

KEY WORDS: Streptococcus sobrinus • dental caries • vaccination • VIP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dental caries is one of the most prevalent infectious diseases in humans, and a vaccine against this disease has been under intensive investigation. Treatment of dental caries is probably one of the most expensive in the world, due to the wide distribution of this illness (Hanada, 2000). Children and adolescents, as well as adults, could benefit greatly, on both a short- and a long-term basis, from the prevention of the major medical complications of caries. A group of related oral bacteria, known as the mutans streptococci, is implicated as the primary etiological agent of human caries (Coykendall and Gustafson, 1986). Within this group, Streptococcus sobrinus and Streptococcus mutans are the species most commonly isolated from humans (Loesche, 1986; De Soet et al., 1991). Research efforts toward developing an effective and safe caries vaccine have been directed to the functional characterization of virulence factors from the bacteria (Walden and Wilensky, 1982; Newbrun, 1983). As has been reported (Ferreira et al., 1997), S. sobrinus secretes a virulence-associated immunomodulatory protein (VIP) that, like VIP secreted by other micro-organisms, inhibits the host’s specific responses by triggering polyclonal non-specific lymphocyte activation (Arala-Chaves et al., 1988; Ferreira et al., 1988; Lima et al., 1992; Tavares et al., 1993) and inducing the early production of IL-10 in the host (Ferreira et al., 1997; Vilanova et al., 1999; Tavares et al., 2000). Moreover, the VIPs act as virulence factors because their production is correlated with the pathogenicity of the secreting micro-organism, and host treatment with the VIP before colonization increases specific colonization (Santarém et al., 1987; Soares et al., 1990; Lima et al., 1992; Tavares et al., 1993). Thus, the production of these VIPs seems to be a mechanism of immune evasion among pathogens.

Isolation and characterization of these molecules, which play a key role in the survival and interaction of the micro-organism with the host and his/her immune defenses, are fundamental for the development of rational strategies for vaccination and infection therapy. Indeed, as we have demonstrated for systemic candidiasis in mice, VIP can be used as a vaccination target inducing specific protection against the micro-organism (Tavares et al., 1995).

The rat caries model has been extensively used for delineating immune protection against this disease. In these models, there is a discrete period of time, between days 18 and 22, denominated the "window of infectivity" (Caufield, 1997), during which mutans streptococci is implanted into the oral cavities of laboratory rats. After day 22, stable establishment of the bacteria in rats is less probable (Caufield, 1997). On the other hand, induction of an effective salivary IgA response requires at least 2 mucosal immunizations. For these reasons, it is obvious that caries immunity is difficult to establish prior to infection in rats. The present study reports a VIP secreted by S. sobrinus as a novel target for vaccination against caries induced by this bacterium. The vaccination reported here is a therapeutic vaccination because it occurs after the implant of the bacteria into the oral cavity of the rat.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Models
In the caries experiments, we usd 16-day-old male and female Wistar rats, bred at the Faculty of Medicine (Coimbra, Portugal) and kept at the animal facilities of the Instituto Ciências Biomédicas Abel Salazar (Porto, Portugal). The rats were weaned at approximately 20 days of age. The animal experiments were performed according to the European Economic Community animal experimentation guidelines Directive of 24 November 1986 (86/609/EEC) and approved by the Veterinary Ethical Committee of the Instituto Ciências Biomédicas Abel Salazar.

Bacteria
S. sobrinus strain 6715, obtained from the American Type Culture Collection, was stored at -70°C in Brain Heart Infusion broth medium (Difco, Detroit, MI, USA) with 25% (v/v) of glycerol.

Preparation of the VIP
The isolation and purification of VIP have been described in detail (Ferreira et al., 1997). All VIP preparations were lipopolysaccharide (LPS)-free, as assessed by the limulus amebocyte lysate kit (E-Toxate; Sigma, St. Louis, MO, USA) as described (Tavares et al., 2000). The protein content was determined by the methods of Lowry (Lowry et al., 1951).

Protocol for Caries Experiments
    (i) Antigens and adjuvant
The VIP isolated as described above was used as active VIP in a submitogenic dose that was unable to induce immunosuppression in the host (10 µg for the first and 50 µg for the second immunization/rat) or heat-inactivated VIP (10 µg for the first and 50 µg for the second immunization/rat) for the immunoprotection assays. Alum (Alhydrogel® "85", a kind gift of Erik Lindblad, Brenntag Biosector, Federikssund, Denmark),was used as adjuvant.

    (ii) Immunizations
Three experimental groups of Wistar rats were formed, with 10 to 12 animals per group, and treated as follows: At the 16th day after birth, all the rats were given a cariogenic diet with 8% sucrose in the drinking water. At the 18th day and for 4 consecutive days thereafter, all the animals were infected orally with 109 cells of S. sobrinus. Five days later, the animals were immunized intranasally (i.n.) with active or heat-inactivated VIP plus alum as adjuvant or with buffer (PBS) incorporated into the alum (sham-immunized). Each dose volume did not exceed 20 µL. The immunizations were repeated 3 wks later. At the end of the experiment, when the animals were 120 days old, they were killed. The serum and saliva were collected for antibody quantification and the teeth for caries evaluation.

Antibody Analyses
Serum immunoglobulin G (IgG) and salivary immunoglobulin A (IgA) antibodies were tested by ELISA. Polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with 5 µg of VIP per mL, at 4°C overnight. Coated plates were washed in Tween-saline (TS) (0.9% NaCl containing 0.05% Tween 20). The plates were blocked for 1 hr with 200 µL per well of a solution containing 10 µg of bovine serum albumin (Sigma) per mL in PBS, at room temperature. Dilutions of rat serum or saliva samples were added (50 µL/well) in duplicate, and the plates were incubated for 2 hrs at 37°C. After being washed with TS, the bound antibodies were revealed by the addition of 50 µL/well of peroxidase-labeled goat antibody anti-rat IgG (Southern Biotechnology Associates, Birmingham, AL, USA) or peroxidase-labeled mouse anti-rat IgA (Biosource, Nivelles, Belgium) and incubated for 3 hrs at 37°C. After being washed, the plates were incubated with orthophenylenediamine dihydrochloride (Sigma) and H2O2, 100 µL per well for 30 min at room temperature. The reaction was stopped with 10% SDS (50 µL/well), and the colorimetric change was measured with a Biotek Chromoscan at 450 nm. The absorbance values were calculated after subtraction of the background values (no serum or saliva added). Data are reported as ELISA units (EU), which were calculated with saliva or serum obtained from non-immunized and non-infected rats as a reference. Dilutions of 1:2 (A450 of 0.1) were considered 1 EU for reference salivary IgA specific to VIP. Dilutions of 1:10 (A450 of approximately 0.2) were considered 1 EU for reference serum IgG specific to VIP.

Caries Assessment
The extent of enamel caries lesions in the first, second, and third molar teeth of all rats (caries score) was microscopically evaluated by a modified Keyes method as previously described (Keyes, 1958).

Bacterial Recoveries
The mutans streptococcal flora was assessed as previously described (Taubman et al., 2000). Briefly, S. sobrinus infection levels were assessed after systematic swabbing of teeth, sonication, and plating of appropriate dilutions on Todd-Hewitt agar (Difco) with Streptococcus Selective Supplement (0.001 mg of colistin sulphate and 0.5 µg of oxalinic acid per mL) (Oxoid, Hampshire, England). The plates were incubated at 37°C in aerobiose for 48 hrs, and S. sobrinus CFU were then enumerated microscopically.

Statistical Analysis
The level of significance of the results in all groups of rats was determined by one-way ANOVA, calculated with Microsoft Excel 2000 software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Resistance to S. sobrinus-induced Dental Caries in Rats by Intranasal Therapeutic Vaccination with a VIP Secreted by the Bacteria
We conducted these experiments to investigate the protective effect of therapeutic immunization with an active or heat-inactivated VIP in S. sobrinus-induced dental caries in Wistar rats.

The differences in enamel sulcal caries scores between the sham-immunized group and the other two immunized groups were statistically significant (p < 0.001), with a 34% reduction in caries lesions in both immunized groups of rats (Fig. 1Go).



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  		Figure 1. Evaluation of dental caries scores on proximal (A) and sulcal (B) molar surfaces involving enamel lesions in Wistar rats sham-immunized and active- or heat-inactivated-VIP-immunized. Data show means ± SD of 10 to 12 rats per group. Statistical difference in mean score among the 3 groups was assessed by ANOVA. Multiple comparisons among groups indicated a significantly different mean score of active- or inactive-VIP-immunized in comparison with sham-immunized (p < 0.001). No differences were detected between active- and heat-inactivated-VIP-immunized groups. Fig. shows results of 1 of 3 representative independent experiments.

 
The protective effect of VIP immunization on caries lesions was more marked in the enamel proximal caries score. Indeed, the differences in the proximal caries scores between the sham-immunized group and the two VIP-immunized groups were statistically relevant (p < 0.001), with a 60% reduction in the caries lesions in both VIP-immunized groups (Fig. 1Go). No statistical differences were found between active- and heat-inactivated-VIP-immunized groups (p = 1.000). Therefore, immunization with either active or heat-inactivated VIP conferred protection against S. sobrinus-induced dental caries in Wistar rats.

The evaluation of S. sobrinus colonization in the oral cavities of the rats showed that VIP-immunized groups exhibited a significant reduction in S. sobrinus levels, while the sham-immunized group maintained high levels of bacteria throughout the study (TableGo).


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  		Table. Reduction of S. sobrinus Oral Colonization in Active- or Heat-inactivated-VIP-immunized Ratsa
 
Effects of Immunization on Salivary IgA Antibodies
To quantify the antibody responses after i.n. immunization with active or heat-inactivated VIP, we evaluated the titers of serum and salivary antibodies specific for VIP by ELISA in the VIP-immunized and in the sham-immunized groups of animals.

The salivary IgA antibodies specific to VIP in experimental animals were significantly different between sham-immunized and both VIP-immunized groups of rats (p < 0.001, Fig.2Go). The VIP-immunized groups of rats showed similar high levels of specific IgA antibodies (p = 0.3), which means that either active or heat-inactivated VIP immunization induced a localized mucosal immunity. In contrast, no differences in serum levels of IgG antibodies specific for VIP were observed between the sham-immunized group and both VIP-immunized groups of animals (data not shown), which indicated that the intranasal immunization did not induce a systemic immunity. Therefore, the protection observed against dental caries induced by VIP immunization correlates with the production of salivary IgA antibodies specific to VIP.



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  		Figure 2. Comparison among salivary levels, expressed as ELISA Units (EU), of IgA specific for VIP in sham-immunized and active- or heat-inactivated-VIP-immunized rats. Results are means ± SD of 10 to 12 Wistar rats. Statistical difference in mean score among the 3 groups was assessed by ANOVA. Multiple comparisons among groups indicated a significantly different mean score of active- or inactive-VIP-immunized in comparison with sham-immunized rats (p < 0.001). No differences were detected between active- and heat-inactivated-VIP-immunized groups. Fig. shows results of 1 of 3 representative independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated the induction of a salivary IgA antibody response against a VIP secreted by S. sobrinus after i.n. immunization with either an active or heat-inactived VIP in rats which was associated with protection against S. sobrinus-induced dental caries. Therefore, to create a model as similar to humans as possible, we used Wistar rats, an outbred strain, in the experiments. Due to the mutans streptococci window of infectivity (Caufield, 1997), the immunizations occurred after oral bacteria inoculation, i.e., a therapeutic immunization with either active or heat-inactivated VIP was done. The i.n. route was selected because the nasal region has been shown to contain organized nasal-associated lymphoid tissue (NALT) with inductive properties (Michalek et al., 1994; Wu et al., 1997b), which, after antigen exposure, results in the induction of significant levels of salivary IgA antibodies (Wu et al., 1997a; Smith et al., 1999). This route has also been used for human immunization with attenuated influenza (Michalek et al., 1994). All the immunizations were given with the protein or PBS incorporated with alum, since it is an adjuvant approved to be used in humans (O’Hagan et al., 2001). The results presented herein show that efficient therapeutic VIP immunization against S. sobrinus-induced caries was observed in rats. The active VIP was used in a (submitogenic) dose that was unable to induce immunosuppression in the host (unpublished results). However, since this kind of molecule is very dependent on the reactivity of the immune system of the host (Arala-Chaves, 1992), a heat-inactivated VIP was also used in the immunoprotection assays. The results showed that rats immunized i.n. either with submitogenic or heat-inactivated VIP had a significant reduction in caries lesions in comparison with the sham-immunized rats. A significant reduction in the enamel sulcal and proximal caries scores was observed in both immunized groups of rats, when compared with the sham-immunized rats (p < 0.001). We also found that protection induced by active or heat-inactivated VIP immunization was not significantly different (p = 1.00). The similar results obtained with active or heat-inactivated VIP could be explained by the fact that protective antigenic determinants did not depend on protein folding, i.e., are accessible in native and in heat-inactivated VIP, and this will be a safe molecule for the immunization strategy.

Moreover, active or heat-inactivated VIP-immunized animals showed significant inhibition of S. sobrinus colonization, in contrast to sham-immunized animals, which maintained high levels of the bacteria throughout the study.

The finding that the levels of salivary IgA specific for VIP were significantly higher in both VIP-immunized groups in comparison with the sham-immunized group (p < 0.001) indicated that the VIP induced a mucosal immunity. There were no significant differences between the two VIP-immunized groups.

The presence of a slight level of antibodies against VIP in the saliva of sham-immunized rats at the end of the experiment indicates that the bacterial infection can induce an immune response against the VIP that is secreted during the bacterial growth, but that is not enough to reduce the bacterial colonization. This could be explained by the described mitogenic effects of these proteins that induce a polyclonal response in the host but a very mild specific response against them (Arala-Chaves et al., 1988; Minoprio et al., 1991). With the purpose of circumventing the immunobiological dose-dependent effects of the VIP (Arala-Chaves et al., 1979), we used a submitogenic dose of the VIP.

The protection obtained through the VIP immunization supports the involvement of the VIP secreted by S. sobrinus in the pathogenesis of the bacteria, which has also been described for other VIP secreted by Candida albicans (Tavares et al., 2000).

The i.n. VIP immunization did not induce a systemic immunity, since we observed no differences in serum levels of IgG specific for VIP between VIP-immunized groups and sham-immunized groups. This observation could be explained by the fact that we are using alum as adjuvant. However, the role of serum antibodies against mutans streptococci and the degree of protection against caries are still controversial (Hajishengallis and Michalek, 1999). There are several references to the use of structural antigens of mutans streptococci as a target for preventive vaccination against bacteria (Hajishengallis and Michalek, 1999); however, absolute protection has not yet been demonstrated (Hajishengallis and Michalek, 1999). The strategy that we present in this report is based on the use of a target molecule that is important for the survival of the caries-producing micro-organism. This approach has also been successfully assayed by others (Blander and Horwitz, 1991; Horwitz et al., 1995; Reina-San-Martin et al., 2000; Strindelius et al., 2002).

When one takes into account the results obtained in this study, it seems reasonable to hypothesize that the same vaccination approaches could be applied to mucosal therapeutic vaccination against human dental caries.


   ACKNOWLEDGMENTS
 
We thank Nuno Lourenço Gomes (Hospital Geral de Santo António S.A.) for reviewing the English of the manuscript. This research was supported by Fundação para a Ciência e Tecnologia (FCT) from Portugal through grants n° POCTI/33790/MGI/2000 and POCTI/33570/MGI/2000. Patent pending is n° 102907.

Received May 29, 2003; Last revision February 10, 2004; Accepted February 11, 2004


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