[Protective efficacy of a new fusion anti-caries DNA vaccine encoding antigens of both Streptococcus mutans and Streptococcus sobrinus].

OBJECTIVE: To construct a new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus so as to enhance the protective effect of DNA vaccine against S. sobrinus infection. METHODS: The CAT fragment of S. sobrinus OMZ176 gtf-I was amplified by semi-nest PCR and then inserted into the plasmid pGJA-P/VAX to construct the recombinant plasmid pGJGAC/VAX. The CHO cell was transfected and the expression of fusion protein detected using cellular immunohistochemistry and Western blot. Mice were immunized with pGJGAC/VAX and control plasmids via the intramuscular (i.m) or intranasal (i.n) routes. During the experiment, blood and saliva samples were collected at a 2-week interval for antibody assay by ELISA. Rats were orally challenged with S. mutans Ingbritt or S. sobrinus 6715 and then immunized i.n with pGJGAC/VAX, pGJA-P/VAX or pVAX1. The Keyes method was used to determine the caries activity. RESULTS: (1) CAT sequence was identical to the related sequence of gtf-I (OMZ176) in GenBank. The recombinant plasmid pGJGAC/VAX encoded the genes of antigens of both S. mutans and S. sobrinus. The expressed protein could respond to specific anti-PAc, anti-GLU and anti-CAT antibodies respectively. (2) As for antibody reactions, mice in the experiment group had significantly higher levels of anti-PAc, anti-GLU and anti-CAT IgG antibodies than those in the pVAX1 group (P < 0.01). The peak responses of specific anti-CAT antibodies were observed at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 62.13 microg/ml and 11.43 microg/ml respectively. The peak responses of specific anti-CAT IgA antibodies were seen at 8 weeks (GAC/i.m) and 10 weeks (GAC/i.n) and were approximately 0.67% and 0.80% respectively. (3) In the group infected with S. mutans or S. sobrinus, the pGJGAC/VAX-immunized rats showed significantly fewer E, Ds and Dm lesions than pVAX1-immunized rats (P < 0.05) and decreased Ds and Dm levels than pGJA-P/VAX-immunized rats (P < 0.05) while there was no obvious difference in E lesions between the two groups (P > 0.05). CONCLUSION: A new fusion anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S. mutans and S. sobrinus is constructed successfully and expressed correctly in eukaryotic cells. It induces effective mucosal and systematic humoral responses so as to provide a better protection against S. sobrinus.

Streptococcus cristatus attenuates Fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells may be invaded by a polymicrobial intracellular flora, including pathogens together with commensals. Various oral pathogens can induce the production of interleukin-8, a potent neutrophil chemotractant, in oral epithelial cells. Evidence from the gut suggests that commensal species may modulate inflammatory responses to pathogens. The aim of this study was to examine the interleukin-8 responses of oral epithelial cells to an oral pro-inflammatory species, Fusobacterium nucleatum, in combination with an oral commensal, Streptococcus cristatus. MATERIAL AND METHODS: KB, TERT-2, TR146 and SCC15 cells were cocultured with F. nucleatum and S. cristatus, either alone or in combination, at 37 degrees C in 5% CO2 under various conditions. The mRNA expression of interleukin-8 was analyzed by reverse transcription-polymerase chain reaction and protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: F. nucleatum alone evoked a potent interleukin-8 response, whereas S. cristatus alone did not induce significant interleukin-8 expression in oral epithelial cells. When present together, S. cristatus attenuated the F. nucleatum-induced interleukin-8 production in the four oral epithelial cell lines to varying degrees. The inhibitory effect of S. cristatus was independent of its viability and its co-aggregation with F. nucleatum, was not related to soluble bacterial products and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. CONCLUSION: Our data suggest that S. cristatus may exert immunomodulatory effects on the interleukin-8 response of oral epithelial cells to F. nucleatum challenge.

Synthetic antigen-binding fragments (Fabs) against S. mutans and S. sobrinus inhibit caries formation.

Streptococcus mutans and Streptococcus sobrinus are the main causative agents of human dental caries. Current strategies for treating caries are costly and do not completely eradicate them completely. Passive immunization using nonhuman antibodies against Streptococcal surface antigens has shown success in human trials, however they often invoke immune reactions. We used phage display to generate human antigen-binding fragments (Fabs) against S. mutans and S. sobrinus. These Fabs were readily expressed in E. coli and bound to the surface S. mutans and S. sobrinus. Fabs inhibited sucrose-induced S. mutans and S. sobrinus biofilm formation in vitro and a combination of S. mutans and S. sobrinus Fabs prevented dental caries formation in a rat caries model. These results demonstrated that S. mutans and S. sobrinus Fabs could be used in passive immunization strategies to prevent dental caries. In the future, this strategy may be applied towards a caries therapy, whereby Fabs are topically applied to the tooth surface.

The Clinical Pattern and Prevalence of Streptococcus mutans and Streptococcus sobrinus among Adult and Children Patients with Dental Caries

Abstract Objective: To explore the clinical pattern, host factors, and presentation of Streptococcus mutans related to caries incidence among children and adults visiting Universitas Airlangga dental clinic. Material and Methods: This was an observational study with a cross-sectional approach with 50 patients in each group of carious children (6-12 years) and adults (18-35 years). Dental decay samples were taken by sterile excavator, put in a BHI's transport medium, and directly incubated overnight at 37 ºC. The next day, they were sub-cultured microbiologically in Tryptone Yeast Cystine Sucrose Bacitracin (TYCSB) selective medium. Bacterial species and serogroups were examined by PCR. All patient's data were collected from medical records and direct observation. Results: Caries were mostly media type in both children and adults. Oral hygiene (OHIS) in children was higher than in adults but not significantly different according to their DMFT. The highest scores for decay, missed and filled teeth were 16, 8 and 7, with an average of 6.82, 1.22 and 0.63, considered quite high. Conclusion: The prevalence of S. mutans was higher in children's caries than in adults, but among the adult patients the co-incidence of S. mutans and S. sobrinus was associated with higher DMFT. The mutans serotypes e, f, and d were more prevalent among children than adults.

Localization on the cell surface of streptococcal protein antigen A.

The cellular localization of the major surface protein SpaA of Streptococcus sobrinus 6715 was examined by immunoelectron microscopy with rabbit polyclonal antibodies directed against purified SpaA protein. Immunoelectron microscopic analysis of thin sections of S. sobrinus cells revealed that the SpaA protein is associated with the fibrillar fuzzy coat of S. sobrinus cells and appears to be distributed over the entire surface of S. sobrinus cells.

Signal sequence and alanine-rich region of streptococcal protein antigen A of Streptococcus sobrinus can direct localization of alkaline phosphatase to the periplasm of Escherichia coli.

Streptococcal protein antigen A (SpaA) of Streptococcus sobrinus is expressed on the surface of cells and extracellularly. TnphoA which lacks signals for transcription and membrane transport of Escherichia coli alkaline phosphatase was used to analyze the sequences necessary for transport of a SpaA/PhoA fusion protein across the cytoplasmic membrane to the periplasm of E. coli cells. Of 15 alkaline phosphatase-producing isolates analyzed, all were found to localize more than 85% of the SpaA/PhoA hybrid protein to the periplasm of E. coli cells. From DNA sequence analysis, all were found to have TnphoA inserted into an identical site. The insertion site of TnphoA was downstream from the coding sequence that generates four tandemly repeated alanine-rich sequences of 82 amino acid residues. These results suggest that in addition to the signal sequence, mature protein sequences containing alanine-rich repeat sequences may play a role in the export of the SpaA protein across a bacterial membrane.

Cloning and sequencing the endocarditis immunodominant antigen of Streptococcus sobrinus strain MUCOB 263.

Immunoblotting sera from cases of Streptococcus mutans or S. sobrinus endocarditis against an extract from S. sobrinus strain MUCOB 263 had identified three immunodominant antigenic bands at 190, 200 and 220 kDa. A lambda ZAPII DNA library was produced from the sheared genomic DNA of S. sobrinus MUCOB 263 and six identical positive clones were identified when this library was screened with serum from a patient with endocarditis caused by a bacterium from the mutans group of streptococci. On subcloning and sequencing, a protein containing 1548 amino acids was identified with a 99.2% homology to the SpaA antigen of S. sobrinus and 68.4% homology to the PAc antigen of S. mutans.

Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes.

Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody.

A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells (SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei. The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotype h), but not with cells or purified PS antigen from any other serotypes of the mutans group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that beta-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the beta-methylgalactopyranoside molecule.

Production, characterization, and application of monoclonal antibodies which distinguish four glucosyltransferases from Streptococcus sobrinus.

A 1,3-alpha-glucan synthase (GTF-I), a highly branched 1, 6-alpha-glucan synthase (GTF-U) and a 1,6-alpha-glucan synthase (GTF-T) were purified to near homogeneity from the culture fluid of Streptococcus sobrinus strain B13N (serotype d) and characterized. In addition, a crude preparation of a recombinant oligo-isomaltosaccharide synthase (rGTF-S) was prepared from a cell-free extract of Escherichia coli MD124 transformant. Using four homogeneous GTF preparations including previously purified rGTF-S as antigens for immunization, 11 murine hybridomas producing a monoclonal antibody (MAb) were established through the fusion of myeloma cells (P3X63-Ag8-U1) and spleen cells of immunized BALB/c mice. When the immunoreactivities of the resultant MAbs were tested, all five MAbs raised against GTF-I, all three MAbs raised against GTF-T, and two of three MAbs raised against GTF-U reacted specifically with the homologous enzyme alone, while one MAb (B86) raised against GTF-U cross-reacted strongly with all GTFs. Although no MAb monospecific for rGTF-S was obtained, precise recognition of GTF-S was possible using the nonspecific B86 antibody together with the MAbs monospecific for the three glucan synthases. Thus, a set of four typical MAbs (B17, B76, B19 and B86) were successfully used for the identification of gene products expressed in 24 previously constructed E. coli phage clones, and the findings suggested that six phage clones might express a gtfU gene encoding GTF-U which has not been hitherto isolated.

Therapeutic vaccine against Streptococcus sobrinus-induced caries.

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.

Effects of phenytoin on dental caries and plaque in Streptococcus sobrinus-infected rats.

Effects of phenytoin (PHT) administration on dental caries in rats infected with Streptococcus sobrinus 6715 were investigated. Twenty-day-old specific pathogen-free Fischer male rats were infected with S. sobrinus 6715 and fed diet 2000 containing 56% sucrose with or without PHT for 52 days. Antibody responses against anti-S. sobrinus in serum and saliva failed to show a statistical difference between PHT-treated and nontreated rats. These results indicate that PHT treatment increased plaque deposition and dental caries in the rats infected with S. sobrinus 6715 and fed diet 2000 containing PHT (1-2 mg/g), as compared with those similarly infected and fed diet without PHT (P < 0.001).

HLA, salivary IgA and mutans streptococci--is there a relation?

The aim of the present studies was to investigate a possible relationship between the human leukocyte antigen (HLA) complex, colonization of mutans streptococci and salivary immunoglobulin A (IgA) antibodies against mutans streptococcal antigens. In the first study a strong inverse relationship between HLA-DR4 and levels of mutans streptococci was observed for a group of renal transplant patients (I). In a group with healthy blood-donors a similar trend was observed (I). This tendency was also seen for a selected population investigated in the second study (II). Since the HLA molecules regulate the production of antibodies in saliva, the salivary IgA activity to three oral streptococci in a population of HLA-DR4-positive and DR4-negative subjects was investigated in the following study (III). It was found that the HLA-DR4-positive subjects, especially the DRB1*0401 and DRB1*0404 subgroups, showed a weaker IgA activity, in particular to Streptococcus mutans, as compared to the HLA-DR4-negative. However, immune response patterns revealed by Western blotting are often complex and for further studies with larger study populations it was crucial to unravel the nature of the detected antigens. In the fourth study (IV), untreated saliva, as well as saliva, in which cell-surface reactive IgA had been absorbed with whole bacteria cells, were analysed in Western blot against different oral streptococci. The high molecular bands, that were absent after absorption, likely represented cell-surface antigens and were thus of interest as they might be involved in adhesion mechanisms and available for blocking in vivo. In the next study (V), the salivary IgA activity to cell-surface antigens of three oral streptococci in relation to different HLA-DRB1*4 alleles was studied in a larger population. The immunoblots were analysed in a computer program and intensity graphs revealed that the DRB1*0401 and *0404 subgroups, compared to other DRB1*04 types, showed fewer as well as less intense immunoblot bands to antigens from S. mutans, S. sobrinus and streptococcal antigen (SA) I/II, but not S. parasanguis. The main conclusion from this thesis is that the HLA profile of the individual seems to influence the salivary IgA response to mutans streptococcal antigens and might thus also affect the conditions for the bacteria in the oral cavity.

[Immunization with targeted fusion anticaries DNA vaccine via intramuscular route:experiment with murine].

OBJECTIVES: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in muscular in vivo. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and fusion anticaries DNA vaccine pGLUA-P in gnotobiotic rats, and observe the kinetics of antibody responses in BALB/c mice. METHODS: (1) Twelve 28-day-old female Wistar rats were randomly divided into 2 groups of 6 rats to be injected with the plasmid pGJA-P containing the signal peptide and extracellular regions of human CTLA(4), hinge and Fc regions of human IgG, the glu sequence of gtfB gene and A-P fragment of pac gene of Streptococcus mutans or the eukaryotic expression plasmid pCI into the quadriceps muscle of thigh respectively. Three days after the rats were killed and specimens of quadriceps muscles of thigh were taken. Immunohistochemical SP staining was used to examine the in situ expression of pGJA-P. (2) Twenty-four 18-day-old female Wistar rats were randomly divided into 4 groups of 6 rats. The rats were fed with cariogenic food. During the age of 20 - 22 days cariogentic food containing broad-spectrum antibiotics was fed. Then aseptic cotton stick was used to swab the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. During the age of 24 - 26 days, S. mutans Ingbritt cultured anaerobically was swab onto the surface of teeth of the rats twice with an interval of 30 minutes. After the inoculation aseptic cotton stick was used to wipe the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. When the gnotobiotic rats were 28 days old they were injected with pGJA-P, pGLUA-P, fusion anticaries DNA vaccine against both PAc, cell surface protein antigen, and glucosyltransferase (GTF), pCI or normal saline into the quadriceps muscle of thigh respectively, 2 weeks later a booster shot was given. When the rats were 63 days old their saliva and blood samples were collected. The serum IgG and salivary IgA were assayed by using ELISA. The gnotobiotic rats were killed and their maxillary bone the mandibles were isolated. The anticaries effect was evaluated by Keyes caries scores. (3) Twenty-four 4-week-old BALB/c mice were randomly divided into 4 groups of 6 mice: to be injected with pGJA-P, pGLUA-P, pCI, or normal saline respectively into the quadriceps muscles of thigh, 2 weeks later a booster shot was given. Before the injection and every 2 weeks after the immunization specimens of saliva and blood were collected. The serum IgG and salivary IgA were assayed by using ELISA. RESULTS: (1) Recombinant protein could be detected in the quadriceps muscles of the rats immunized with pGJA-P, but not in the muscles of the rats immunized with pCI. (2) The levels of serum anti-PAc IgG (1:200 000) and anti-GTF IgG (1:58 000) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:23 000 and 1:11 000 respectively) (both P < 0.01). The levels of salivary anti-PAc IgA (1:8) and anti-GTF IgA (1:6) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:2 and 1:2 respectively) (both P < 0.01). The Keyes scores of the pGJA-P group were significantly lower than those of the pGLUA-P group and the control groups (all P < 0.01). The effective serum IgG and salivary IgA in the pGJA-P group and effective serum IgG in the pGLUA-P group all persisted to the end of the experiment. (3) Two weeks after the initial immunization the serum anti-PAc IgG level of the mice immunized with pGJA-P increased remarkably, 4 times that of the mice immunized with pGLUA-P, and 33 times those of the mice injected with pCI or normal saline. Two weeks after the booster immunization, the serum anti-PAc IgG level of the mice immunized with pGJA-P was 14 times that of the mice immunized with pGLUA-P, and 117 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG immunized with pGJA-P reached its peak 10 weeks after the initial immunization, 4 times that of the mice immunized with pGLUA-P, and 160 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG of the mice immunized with pGLUA-P reached its peak at 16 weeks, however, significantly lower than the peak of the mice immunized with pGJA-P (P < 0.01). The serum anti-Pac IgG levels of the mice injected with pCI or with normal saline were not significantly different (P > 0.05). Since the second week after the initial immunization, significant difference in the serum anti-PAc IgG level could be seen between the mice immunized with pGJA-P or the mice immunized with pGLUA-P, and between the mice immunized with pGJA-P and the mice immunized with pGLUA-P and those injected with pCI or normal saline (all P < 0.01). Six weeks after the initial immunization the salivary anti-PAc IgA level of the mice immunized with pGJA-P was 18 times those of the mice injected with pCI or with normal saline (both P < 0.01), 10 weeks after the salivary anti-PAc IgA level of the mice immunized with pGJA-P reached its peak, 24 times those of the mice immunized with pCI or normal saline without a significant difference between the latter 2 groups (P > 0.05). No effective salivary IgA response was seen in the mice immunized with pGLUA-P. CONCLUSION: pGJA-P can be expressed in vivo. Immunization with pGJA-P intramuscularly induces effective mucosal and systematic humoral responses. It is an effective DNA vaccine against dental caries.

[Anti-caries vaccines]. / Vaccini anticarie.

Even though the reduction of caries-incidence in developed countries, its increasing has been observed nowadays. The use of a vaccine was object of many researches, going under modifications and evaluations during years. Wallace and McCollum showed the chance to induce experimental cavities, while Clarke and McIntosh were the first underlining the roll of S. mutans and Lactobacilli as efforts of the pathology. Williams was the first working with humans and Zinner and Fitzgerald continued. So since Bowen the research tried to build a vaccine made of single bacterial molecules with antigenic power. We can count about a large number of targets, like: the Ag I/II, the glucosyltransferase enzyme (GTF), the glucan-binding-protein (GBP), the destranase, the fruttosyltransferase and the glucans. Among the substances used to obtain a vaccine cacao revealed its capacity against bacteria able to develop cavities, thanks to its cariostatic and anti-glucosyltransferase activity due to polyphenols, that we can find in green tea too. It's also interesting a technique that gives passive antibodies like cow's milk, but in particular the one of a monoclonal antibody made with biotechnology of plants: the Guy's 13. It does not show substantial differences in comparison with the human Ig and it's able to prevent the installation of micro-organism and to reduce cavities in adult patients already infected. For the setting-up of a vaccine, however, only studies, comparison and research will be able to show precise instruments of defence.

[The local immunity of the oral cavity in dental caries]. / Sostoianie mestnogo immuniteta polosti rta pri kariese zubov.

Functional activity of local immunity with regard to dental caries in children is liable to appreciable individual fluctuations which depend on sex, age, and season. In children aged 4 to 5 no association between the titer of antibodies to cariogenic Streptococcus and number of carious lesions was observed, whereas in those aged 9 to 19 these parameters were in inverse relationship. In children aged 11-12 and 14-16 this association is inverse. Regression analysis of nonspecific and specific characteristics of local immunity of the oral cavity in children aged under 12 suffering from caries revealed functional insufficiency of the mechanisms regulating the local immunity, which disappears after fluoroprophylaxis. Test system for screening of mutant Streptococcus cannot be recommended for wide use to detect subjects sensitive to caries, because it has certain limitations as regards the age of examinees.

Salivary IgA response to probiotic bacteria and mutans streptococci after the use of chewing gum containing Lactobacillus reuteri.

We investigated whether ingestion of probiotic bacteria could influence salivary IgA levels, specific anti-mutans streptococci IgA levels and specific antibodies towards the ingested probiotic bacterium. The study was a randomised, double-blind, placebo-controlled trial, where the test group (n = 11) received twice daily chewing of gum containing Lactobacillus reuteri (2 × 10(8)  CFU per dose) and the control group (n = 12) received placebo. Resting saliva was collected before and after 12 weeks of treatment and 4 weeks after end of treatment. Total salivary IgA concentrations were measured by ELISA. Specific IgA reactivity was determined using a whole-cell ELISA. Results were expressed as % IgA per protein in saliva. The level of total IgA% per protein increased significantly between pretreatment levels (13.5%) and follow-up treatment levels (14.4%) within the test group only (P < 0.05). No changes were seen in the control group during the trial. The level of probiotic-reactive antibodies decreased significantly between pre- and post-treatment samples (from 12.2% to 9.0%, P < 0.05) in the test group. Similarly, the level of specific mutans streptococci antibodies decreased significantly between pre- and post-treatment samples (P < 0.05) in the test group only (for Streptococcus mutans from 20.1% to 15.0%; for Streptococcus sobrinus from 7.4% to 5.3%). Ingestion of probiotic bacteria might influence the adaptive immune response of the host.

rEnolase maternal immunization confers caries protection on offspring.

Therapeutic vaccination with Streptococcus sobrinus recombinant enolase (rEnolase) protects rats from dental caries. Here, we investigated the effect that maternal rEnolase vaccination before pregnancy had on the offspring's immune response to S. sobrinus oral infection and dental caries progression. Female Wistar rats were immunized by intranasal and subcutaneous routes with rEnolase adsorbed onto aluminum hydroxide as adjuvant or similarly treated with the adjuvant alone (sham-immunized). Ten days after the last administration, the immunized females were paired with a male rat. The oral immune responses to S. sobrinus infection and dental caries in the offspring were evaluated. The results showed that pups born from rEnolase-immunized mothers had higher levels of rEnolase-specific salivary IgA and IgG antibodies (indicating a placental antibody transfer) and lower sulcal and proximal enamel caries scores than rats born from sham-immunized mothers. In conclusion, rEnolase maternal immunization before pregnancy provides offspring with protection against S. sobrinus-induced dental caries.

Oral therapeutic vaccination with Streptococcus sobrinus recombinant enolase confers protection against dental caries in rats.

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.