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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.

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., (© 2013 The Authors. Published by John Wiley & Sons Ltd on behalf of the Federation of European Microbiological Societies.)

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.

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.

Mutans streptococci (MS) are generally considered to be the principal etiological agent of dental caries. MS have two important virulence factors: cell- surface protein PAc and glucosyltransferases (GTFs). GTFs have two functional domains: an N-terminal catalytic sucrose-binding domain (CAT) and a C-terminal glucan-binding domain (GLU). A fusion anti-caries DNA vaccine, pGJA-P/VAX, encoding two important antigenic domains, PAc and GLU, of S. mutans, was successful in reducing the levels of dental caries caused by S. mutans in gnotobiotic animals. However, its protective effect against S. sobrinus infection proved to be weak. Does the DNA vaccine need an antigen of S. sobrinus to enhance its ability to inhibit infection? To answer this question, in this study, we cloned the catalytic (cat) fragment of S. sobrinus gtf-I, which demonstrated its ability to inhibit water-insoluble glucan synthesis by S. sobrinus, into pGJA-P/VAX to produce a new anti-caries DNA vaccine.

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.

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.

Background/aims: The cell surface protein antigen (PAg) and glucosyltransferases (GTFs) produced by Streptococcus sobrinus are considered to be major colonization factors of the organism., Methods: We constructed a fusion gene encoding a protein composed of the alanine-rich region of PAg (PAgA) and the glucan-binding domain (GB) of GTF-I, which catalyzes the synthesis of water-insoluble glucan in S. sobrinus. The fusion protein PAgA-GB was purified from cell extracts of Escherichia coli harboring the fusion gene, and antibodies against the fusion protein were prepared in rabbits., Results: In the presence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus MT8145 and Streptococcus mutans Xc to saliva-coated hydroxyapatite beads, and the inhibitory effect on S. sobrinus was stronger than that on S. mutans. In the absence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus and S. mutans, however the inhibitory effect on S. sobrinus was unexpectedly weaker than that on S. mutans. A similar result was observed with the antibody against the intact recombinant PAg protein (rPAg), while the same antibody reacted more strongly against S. sobrinus than against S. mutans cells., Conclusion: Taken together, these results show that the antibody against S. sobrinus GTF-I may be useful for effective inhibition of the sucrose-dependent adhesion of S. sobrinus. However, PAg of S. sobrinus may not function primarily as a receptor for acquired pellicles, and other cell surface proteins may be involved in the sucrose-independent adhesion of S. sobrinus.

Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.

Background/aims: Mutans streptococci are found in almost all individuals, though there are large differences in colonization levels between individuals. These differences are not readily explained, though several factors are believed to influence the colonization. One factor is the immune response to mutans streptococci, mainly provided by salivary immunoglobulin A (IgA). In a previous study, differences in salivary IgA reactions to oral streptococci were observed between human leukocyte antigen (HLA)-DR4-positive and DR4-negative individuals. A lower salivary IgA activity to Streptococcus mutans in particular was most pronounced for two DR4 subgroups, DRB1*0401 and *0404. The main purpose of this study was to further investigate, in a larger study group, the salivary IgA activity to antigens of three oral streptococci in relation to different HLA-DRB1*04 alleles., Methods: Stimulated saliva was collected from 58 HLA-DRB1*04-positive individuals. Whole cell antigen extracts from S. mutans, Streptococcus sobrinus and Streptococcus parasanguis and the streptococcal antigen (SA) I/II were separated in SDS-PAGE, transblotted and detected with diluted saliva (Western blot), and analyzed in a computer program. All distinct immunoblot bands over 100 kDa were recorded and compared in relation to DRB1*04., Results: The immunoblots revealed lower salivary IgA reactions to S. mutans, S. sobrinus and SA I/II, but not to S. parasanguis, for the DRB1*0401- and *0404-positive individuals compared to other DRB1*04 types. For the *0401 subgroup there was a significant association with a lower IgA response to S. mutans., Conclusion: The results confirm earlier observations and may also support previous demonstrated association between colonization by mutans streptococci and the serologically defined HLA-DR4.

The interrelationship of HIV infection, dental caries and mucosal immune responses remains controversial. In our study population of 40 HIV-infected and 40 healthy control children (ages 2-5 years) there was a significantly higher prevalence of dental caries in HIV-infected children (P<0.05). The extent of caries correlated with the severity of HIV disease. To determine whether the immunosuppression that ensues after HIV infection could contribute to the increased caries prevalence, the concentrations of total IgA and IgA specific to cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus) were determined in whole saliva by enzyme-linked immunosorbent assay. Levels of the same bacteria were also quantified in saliva using checkerboard DNA-DNA hybridization. A significantly increased level of total salivary IgA was found in the HIV-positive population (P < 0.05), but there were comparable titers of specific IgA to cariogenic bacteria in HIV-positive and healthy controls. The microbiological assessment also demonstrated similar levels of cariogenic microorganisms in both groups. We conclude that HIV-positive children appear to maintain the capacity to mount a mucosal immune response to cariogenic microorganisms, at least until late stages of disease.

Background: In the immunoblot technique, using whole bacteria cell extracts as antigens, both intra- and extracellular antigens are detected, which gives a large number of immunoglobulin A (IgA) reactions (immunoblot bands) when incubated with saliva. It is important to distinguish which immunoblot bands represent bacterial cell-surface antigens, since these antigens could be involved in adhesion mechanisms and be available for blocking in vivo., Methods: Bacterial extracts of Streptococcus mutans, Streptococcus sobrinus, Streptococcus parasanguis and the streptococcal antigen I/II were separated using SDS-PAGE. The antigens were detected with saliva in Western blot. Untreated saliva and saliva in which cell-surface reactive IgA had been absorbed with whole bacteria cells were analyzed., Results: Approximately half the number of the bands were absent for saliva absorbed with homologous cells, compared to untreated saliva. The absorption pattern was almost identical for S. mutans and S. sobrinus but not for S. parasanguis. Salivary IgA reactive against streptococcal antigen I/II was absorbed by S. mutans cells, to a lesser extent by S. sobrinus cells, and not at all by S. parasanguis cells., Conclusion: It is likely that the bands that were absent after absorption represented cell-surface antigens. For S. mutans and S. sobrinus, these bands were probably the streptococcal antigen I/II., (Copyright Blackwell Munksgaard, 2004.)

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.

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.

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.

A strategy of Streptococcus sobrinus, a major agent of dental caries, to survive and colonize the host consists of the production of a protein that suppresses the specific antibody responses. We have cloned the gene coding for a protein with immunosuppressive activity. It contains an open reading frame of 1302 base pairs encoding a polypeptide with 434 amino acid residues and a molecular mass of 46910 Da. The gene product is homologous to enolases from several organisms. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in a fluoride-sensitive enzymatically active form. Pretreatment of mice with the S. sobrinus recombinant enolase suppresses a primary immune response against T-cell dependent antigens. This immunosuppressive effect is specific to the antigen used in the immunization, as it is not observed when the immune response against other antigens is analysed. Furthermore, the S. sobrinus recombinant enolase stimulates an early production of interleukin-10, an anti-inflammatory cytokine, and not the pro-inflammatory cytokine IFN-gamma. These observations indicate that enolase acts in the suppression of the specific host immune response against S. sobrinus infection.

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.

To evaluate interleukin 1 (IL-1) as a mucosal adjuvant in the induction of salivary antibodies to Streptococcus sobrinus, S. sobrinus together with IL-1 was applied through the palatine tonsils of rabbits. IL-1 caused approximately 50 and 100% increases in the antibodies reacting against S. sobrinus fragments in the saliva and blood plasma, respectively, compared to the antibodies in those same fluids after tonsillar applications of S. sobrinus alone. In the case of the addition of IL-1, the antibodies reacting to the protein antigens of S. sobrinus increased in each fluid, without affecting the antibodies reacting to saccharide antigens. Delayed-type hypersensitivity to S. sobrinus, characterized by ear swelling and by an increase in IFN-gamma mRNA in RT-PCR analysis, was found to be induced only in rabbits immunized with IL-1. S. sobrinus protein antigens caused ear swelling as intense as that caused by S. sobrinus fragments. Thus, IL-1 induced an antibody response and cell-mediated immunity mainly reacting to protein antigens of S. sobrinus.

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva-coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra-high temperature (UHT)-treated milk or to Lactobacillus rhamnosus GG (LGG)-fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30 degrees C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT-IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistreptococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG-positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG-fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf-life of these products. From the anticariogenic point of view it may be beneficial to add bovine-specific antibodies against mutans streptococci to probiotic LGG-containing milk products.

The aim of this study was to describe and compare salivary immunoglobulin A (IgA) antibody reactions to extracts of strains of three oral streptococci in human leukocyte antigen (HLA)-DR4-positive and -DR4-negative subjects. Whole paraffin-stimulated saliva samples were collected from 27 apparently healthy subjects. Previous HLA typing showed that 20 subjects were DR4 positive and 7 were DR4 negative. HLA-DRB1*04 subtyping was performed among the DR4-positive subjects. Whole-cell antigen extracts from Streptococcus mutans (KPSK 2), Streptococcus sobrinus (OMZ 65) and Streptococcus parasanguis (Nt 62) were separated in SDS-PAGE. The antigens were immunoblotted with diluted saliva (Western blot), scanned and analyzed in a computer system. All immunoblot bands were recorded in DR4-positive and DR4-negative saliva pools, and bands with an optical density >or=0.1 were selected for analysis in individual salivas. The DR4-negative subjects in general had more immunoblot bands and more distinct bands than did the DR4-positive subjects. A higher concentration of total IgA in saliva was correlated with more bands, especially to antigens separated from S. mutans. When the number of bands was calculated per IgA unit, significant differences were observed between DR4-positive and DR4-negative salivas. This was particularly seen for S. mutans and S. parasanguis. As the number of bands was analyzed in relation to DR4 subgroups, DRB1*04, there was a lower salivary IgA activity to S. mutans in the DRB1*0401 and *0404. The variable level of correlation previously demonstrated for S. mutans colonisation and serologically defined DR4 positive subjects might be explained by the heterogeneity in this group, and the relation should be sought on a subgroup level.

The effect of mucosal delivery of Streptococcus sobrinus glucosyltransferase (GTF) in bioadhesive poly (D,L-lactide-co-glycolide) (PLGA) microparticles on induction of salivary IgA and serum IgG antibody responses was measured in Sprague-Dawley rats. Preparations of GTF/PLGA/gelatin microparticles, or PLGA/gelatin microparticles or GTF in alum, were administered four times at weekly intervals by intranasal or intragastric routes. Two subcutaneous injections of GTF in PLGA/gelatin microparticles or in alum were given to separate groups of rats. Significant elevations in salivary IgA antibody levels to S. sobrinus GTF were observed only in the groups immunized intranasally 28 days after immunizations were begun. Five of six rats given the GTF microparticles intranasally had positive salivary IgA antibody responses to GTF, and the mean salivary IgA antibody level of this group was 30-fold higher than any other mucosally or systemically immunized group. Salivary IgA responses in the GTF-microparticle group remained significantly higher than all other mucosally immunized groups for at least 10 weeks after the primary immunization. All rats in this group demonstrated aspects of anamnesis following a more limited secondary course of intranasal administration. Intranasal administration of GTF in microparticles also induced a serum IgG response to GTF in some rats. After secondary intranasal GTF microparticle administration, several rats had sustained serum IgG antibody levels that were within the range of sera from rats subcutaneously injected with GTF in microparticles or in alum. Thus intranasal delivery of GTF-containing bioadhesive microparticles induced the highest and longest lasting salivary immune response of any mucosal or systemic route or vehicle tested and could be expected to be a useful method for induction of mucosal immunity.

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.

We previously found that tonsillar application of antigen induces a strong antibody response to Streptococcus sobrinus in saliva and blood plasma. Rabbits immunized against S. sobrinus by tonsillar application were highly resistant to experimental dental caries triggered by oral inoculation of living S. sobrinus organisms with sucrose. In the present study, we examined the reaction of S. sobrinus antigens to the antibodies induced by the tonsillar application of S. sobrinus AHT-k in rabbits and compared them to those antibodies induced by intramuscular injection. In an enzyme-linked immunosorbent assay using ultrasonic fragments from mutans group streptococci, the saliva and blood plasma selectively reacted to S. sobrinus AHT-k (serotype g) and serologically related streptococci (serotypes a, d, and h) in the sixth week after tonsillar application, whereas the blood plasma in the sixth week after intramuscular injection reacted to the unrelated streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci. The antibody reactivity induced after tonsillar application was not lost after treatment of the antigen with heat or proteinase digestion, whereas these treatments resulted in a 70% decrease of the antibody reactivity induced by intramuscular injection. The inhibition by haptenic sugars and the decrease in immunoreactivity by heat treatment and proteinase digestion suggested that 80% of the antibodies induced by tonsillar application reacted to saccharides. These saccharide antigens appeared to be involved in a specific reaction with S. sobrinus-specific streptococci and a selective reaction with serologically related streptococci. These antigens are probably involved in anticaries reactions in experimental dental caries.

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.

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.

Objective: To investigate the effects of monoclonal antibody on salivary agglutinin-mediated aggregation of Streptococcus sobrinus in vitro., Methods: Bacterial aggregation inhibition was measured under OD650 at 10 minutes intervals for 1 hour., Results: It showed that the McAb ZS2/286 raised against the PAg of S. sobrinus 6715 could inhibit the aggregation by some degree, but the effect was not significant., Conclusion: The McAb ZS2/286 might not bind directly to the functionally important region of PAg related to aggregation.

Colostral products from non-immunized cows (CP) and cows immunized with mutans streptococci (IP) were used as mouth rinses in a short-term human study. The acidogenic potential of the products was tested and found to be negligible in vivo before application to subsequent rinsing tests. At first, all the participants received a professional tooth cleaning, after which they rinsed with one of the solutions (IP; CP; water) three times per day for 3 d. After each rinsing period, the resting pH and decrease in plaque pH after sucrose challenge were determined, the amount of plaque was estimated, and all available plaque was collected. No significant differences were recorded in the composition or in the amounts of accumulated plaque. The resting pH values of plaques with low "innate" pH were increased after the IP rinsing period. Surprisingly, the lowest pH values after the sucrose challenge were recorded in IP plaques. The number of cultivable facultative flora or total streptococci were not affected by different rinsings, but the relative number of mutans streptococci significantly decreased after the IP rinsing period when compared to the CP period. Thus, the short term rinsing indicates favourable effects of bovine immune whey on human dental plaque.

Objective: The purpose of this study was to determine the accessibility of monoclonal antibody (McAb), specific for the major cell surface protein antigen (PAg) of Streptococcus sobrinus, to the surface of its native epitopes., Materials and Methods: An indirect immunogold labeling technique was used to detect the reaction of McAb with S. sobrinus 6715. The reactions of polyclonal antibodies (PcAbs) against S. sobrinus 6715 or PAg with S. sobrinus 6715, S. mutans Ingbritt C and S. rattus BHT were studied as controls., Results: The results indicated that PAg was localized on the outer cell surface of S. sobrinus, and McAb was reactive with only a few epitopes of the cell surface, whereas PcAbs were found to be reactive with more epitopes., Conclusions: McAb was specific for the PAg, but there was cross-reaction with S. mutans. Also there seemed to be an association between the fuzzy coat on the surface of S. sobrinus and PAg.

Objective: To investigate the effect of local application of monoclonal antibody (McAb) raised against the major cell surface protein antigen of streptococcus sobrinus (PAg) on the colonization of rats' teeth by Streptococcus sobrinus (S. sobrinus) and the development of dental caries., Methods: 30 wistar rats were divided into three groups: group I received McAb against PAg; group II received unrelated mice ascites; group III received PBS. They were applied on six occasions. After three times of application, streptomycin resistant S. sobrinus 6715 were inoculated onto the teeth and the rats were fed with caries-inducing diet 2000#. The number of colonized S. sobrinus were counted and the presence of caries was examined by the procedure of Keyes'., Results: There was a significant lower level of colonization by S. sobrinus and lower mean caries scores treated with McAb compared with the control groups., Conclusion: Local passive immunization with McAb against PAg may be an effective way to prevent the colonization by implanted S. sobrinus and the development of dental caries.

Objective: To investigate the effect of local application of monoclonal antibody (McAb) raised against the 210KD cell surface protein antigen (PAg) of Streptococcus sobrinus on the colonization of rats' teeth and the development of dental caries., Materials and Methods: Thirty Wistar rats were divided into 3 groups: group 1 received McAb against PAg; group 2 received unrelated mice ascites; group 3 received pure buffered saline. The bacteria were applied 6 times to the occlusal surfaces of molars and to the labial surfaces of incisors. After three applications, streptomycin-resistant S. sobrinus 6715 was applied to the teeth and the rats were fed with caries-inducing diet 2000. The number of colonized S. sobrinus was counted on the streptomycin-supplemented mutans streptocci agar and expressed as a percentage of the total colonies on BHI agar. The presence of caries was examined and scored following Keyes' procedure., Results: There were significantly lower levels of colonization by S. sobrinus on rats' teeth and lower mean caries scores on the teeth treated with McAb compared with those in the control groups., Conclusion: Local passive immunization with McAb against PAg may be an effective way to prevent colonization by implanted S. sobrinus and development of dental caries.

Specific immune defense against cariogenic mutans streptococci is provided largely by salivary secretory IgA antibodies, which are generated by the common mucosal immune system. This system is functional in newborn infants, who develop salivary IgA antibodies as they become colonized by oral microorganisms. The mechanisms of action of salivary IgA antibodies include interference with sucrose-independent and sucrose- dependent attachment of mutans streptococci to tooth surfaces, as well as possible inhibition of metabolic activities. The goal of protecting infants against colonization by mutans streptococci might be accomplished by applying new strategies of mucosal immunization that would induce salivary IgA antibodies without the complications of parenteral immunization. Strategies of mucosal immunization against mutans streptococci currently under development include the use of surface adhesins and glucosyltransferase as key antigens, which are being incorporated into novel mucosal vaccine delivery systems and adjuvants. The oral application of preformed, genetically engineered antibodies to mutans streptococcal antigens also offers new prospects for passive immunization against dental caries.

Living Streptococcus sobrinus cells were orally inoculated into nonimmune rabbits and rabbits immunized with formalin-killed cells of S. sobrinus through tonsillar application to examine the anticaries potential of this method of immunization. The living S. sobrinus cell numbers and the caries areas in the rabbits immunized by tonsillar application decreased to a level one-fifth of that in nonimmune rabbits.

Three purified glucan binding proteins (GBP-2, GBP-3, and GBP-5) from Streptococcus sobrinus 6715 were compared structurally by mass spectroscopy of tryptic fragments and antigenically by Western blot analysis with rat antisera to each GBP or to peptides containing putative glucan binding epitopes of mutans streptococcal glucosyltransferases. Structural and antigenic analyses indicated that GBP-3 and GBP-5 are very similar but that both are essentially unrelated to GBP-2. None of these S. sobrinus GBPs appeared to have a strong antigenic relationship with GBPs from Streptococcus mutans. Thus, S. sobrinus GBP-2 and GBP-3 appear to be distinct proteins with potentially different functions. S. sobrinus GBP-5 may be a proteolytic fragment of GBP-3, or, alternatively, the genes coding for these proteins may be closely related.

Previous immunofluorescence (IF) studies have indicated that Streptococcus mutans may preferentially colonise specific sub-sites within approximal plaque. The present study aimed to extend these observations to other mutans streptococci and lactobacilli in such gingival margin plaque. Two hundred and seventy approximal plaque samples were taken from 90 teeth (3 from each tooth) in 64 children; three gingival margin sub-sites in relation to the contact area: away from (A), to the side of(S) and below (B) the contact area. Samples were processed by indirect IF using high-titred polyclonal anti-S. mutans 'c', anti-S. sobrinus 'd', anti-L. casei and anti-L. acidophilus antisera. An overall positive association was found between S. mutans 'c' and S. sobrinus 'd' (p < 0.001). Significant differences (p < 0.1) were found between the proportional counts at each sub-site for S. mutans 'c': A = 39%, S = 51% and B = 70%, and for S. sobrinus 'd' 21, 33 and 49%. Mutans streptococci (MS) appeared to preferentially colonise the sub-site below the contact area (B = 81%), compared with sub-sites A and S (48 and 62%, respectively). S. mutans 'c' and S. sobrinus 'd' were detected together at subsites A = 12%, S = 22%, and B = 38%, with proportional counts at B sites being higher than those at A (B > A, p < 0.01, and B > S, p < 0.05). Lactobacillus spp. were isolated rarely, and were usually found together with MS. There was a positive relationship between the presence of lactobacilli or MS and caries (white spot lesions only), although these species could frequently be isolated from noncarious sites. The presence of both S. mutans 'c' and S. sobrinus 'd' were strongly correlated with early caries lesions. In addition, this study confirmed the variation in the microflora at different sub-sites within approximal dental plaque.

Due to potential side-effects of active immunization by cariogenic mutans streptococci, oral administration of passively-derived antibodies could be a more acceptable way to reduce colonization and virulence of these microorganisms in human dentition. The aim of this study was to produce antistreptococcal immunoglobulins into bovine colostrum and explore the possible antibacterial mechanisms of these immunoglobulins against mutans streptococci. Specific serum IgG antibodies to whole cell antigens of both Streptococcus mutans and Streptococcus sobrinus increased rapidly in cows during immunization and were high also in the final whey-product. Low concentration (0.5% w/v) of bovine immune preparation inhibited significantly the incorporation of [14C]glucose by both S. mutans and S. sobrinus. Higher concentration (> 1%) was needed to inhibit the glucosyltransferase or fructosyltransferase activities of these bacteria. No such inhibitory effects were observed with the control preparation from the non-immunized cows. Our results indicate that bovine immune colostrum has a significant inhibitory potential against mutans streptococci, apparently dependent on the presence of specific IgG antibodies against S. mutans and S. sobrinus.

Passive immunization has been suggested as a method to prevent colonization of teeth by mutans streptococci. However, the mechanism of action of antibodies, both polyclonal and monoclonal, is not clear. In this study we investigated the effect of polyclonal antibodies (pAbs) and a monoclonal antibody (OMVU10) on the chain length and growth of Streptococcus sobrinus. During growth in broth S. sobrinus formed significantly longer chains in the presence of pAbs in comparison to pre-immune serum (p < 0.01), but pAbs did not influence the growth rate of S. sobrinus in broth. OMVU10 did not influence the growth rate nor the chain length. In order to study the effect of the antibodies on adhesion and growth on a surface, S. sobrinus was grown on hydroxyapatite discs in the presence of two other bacteria, Streptococcus oralis and Actinomyces viscosus. No effect of the antibodies was found on the total cultivable count of the three bacteria after growth on hydroxyapatite discs. However, the morphology of the S. sobrinus microcolonies was different after growth in the presence of both pAbs and OMVU10. The colonies were less dense and chains of S. sobrinus could be seen using a confocal laser microscope. After growth in the presence of the control antibodies, the colonies were dense and no long chains could be observed. It was concluded that pAbs influenced the chain length, in broth and on hydroxyapatite discs, and the colony morphology of S. sobrinus on hydroxyapatite discs, whereas OMVU10 influenced the colony morphology of S. sobrinus on hydroxyapatite discs.

The effect of a polyclonal antiserum and OMVU10, a monoclonal antibody reactive with Antigen B of Streptococcus sobrinus, on the interaction of polymorphonuclear leukocytes with S. sobrinus was studied, using chemiluminescence and bacterial killing assays. Increased stimulation of neutrophils as measured in the chemiluminescence assays was established when S. sobrinus was preincubated with polyclonal antiserum or when polyclonal antiserum was added to the reaction mixture. Higher counts were measured in comparison to preimmune serum. After 90 min, 52% of S. sobrinus preincubated with polyclonal antiserum was killed. Killing was also increased when polyclonal antiserum was added to the reaction mixture in comparison to the controls. No killing was found when bacteria were preincubated with OMVU10 or when OMVU10 was added to the reaction mixture in comparison to Clone 24, a control antibody.

Recombinant strains of Salmonella typhimurium have been studied as antigen delivery systems to determine their effectiveness as multivalent vaccines. Here we compare the efficacy of two strains of S. typhimurium. chi 4072 (pYA2905) and chi 3987 (pYA2905), expressing SpaA of Streptococcus sobrinus 6715 as oral vaccines for dental caries. Both strains are attenuated delta cya delta crp delta asd mutants with their Asd phenotypes complemented by the Asd+ plasmid pYA2905, which also encodes a peptide fragment of SpaA. S. typhimurium chi 3987 (pYA2905), unlike S. typhimurium chi 4072 (pYA2905), contains the 100 kb S. typhimurium virulence plasmid. Fischer rats were orally immunized with approximately 10(9) S. typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) and then challenged with cariogenic S. sobrinus 6715. Rats orally immunized with either strain of recombinant Salmonella developed salivary IgA anti-SpaA responses and had lower levels of S. sobrinus-induced dental caries than nonimmunized, infected animals. In a second series of experiments, the kinetics and isotype of the serum and salivary antibody responses were determined in rats orally immunized with S. typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) on weeks 0 and 8. IgG and IgM serum antibody responses to SpaA and S. typhimurium were detected after the primary and secondary immunizations, and the secondary immunization boosted serum IgG anti-Salmonella activity. In general, animals immunized with chi 3987 (pYA2905) had higher serum anti-SpaA, as well as serum and salivary anti-Salmonella, responses than animals immunized with chi 4072 (pYA2905). This study demonstrates the effective use of two recombinant S. typhimurium strains as oral vaccines for inducing protective and sustained immune responses against a mucosal pathogen and suggests that the recombinant Salmonella vaccine strain carrying the virulence plasmid induced similar or higher protective immune responses than the strain lacking the virulence plasmid.

An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries.

In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined by microbial adhesion to hydrocarbons were measured in potassium phosphate buffer with a pH ranging from 2 to 9. S. sobrinus preincubated with polyclonal antibodies was positively charged at pH 2, 3, and 4 and had an isoelectric point at pH 4.8. Untreated S. sobrinus cells or cells preincubated with MAbs were negatively charged over the whole pH range. X-ray photoelectron spectroscopy showed a decrease in O/C and P/C ratios for bacteria preincubated with polyclonal antibodies. A combination of the pH-dependent zeta potential and the X-ray photoelectron spectroscopy data of the overall chemical composition of the cell surface suggests that polyclonal antibody adsorption occurs through blocking of surface phosphate. The measurement of hydrophobicity by microbial adhesion to hydrocarbons revealed that S. sobrinus preincubated with polyclonal antibodies was hydrophobic (90% of the bacteria bound to hexadecane), whereas the controls were relatively hydrophilic. S. sobrinus preincubated with OMVU10 was found to be more hydrophobic than the controls at pH 5 and 7. Hydrophobicity as measured by water contact angles showed an increase in hydrophobicity when S. sobrinus was preincubated with polyclonal antibodies. The epitopes to which the antibodies are directed were visualized by immunogold labeling and electron microscopy. The results suggested that OMVU10 is reactive with only a few epitopes of the cell surface, whereas polyclonal antibodies were found to be reactive with many epitopes. In conclusion, adsorption of polyclonal antibodies was found to influence the overall physicochemical surface properties of the organism, probably by forming a coating over the whole cell surface. Adsorption of MAbs was more localized, which could explain their lesser influence on these surface properties.

Analysis of the salivary immunoglobulin A (IgA) antibody activity to antigens separated from oral streptococci was carried out on 49 individuals in 11 families. The aim of this study were to i) study the human salivary IgA activity within families to antigens separated from reference strains of Streptococcus mutans, Streptococcus sobrinus and Streptococcus parasanguis and ii) to search for potential differences in the salivary IgA activity to the streptococcal antigens several years apart. The immunoblot revealed similarity in saliva IgA response within the families. A majority of the bands, approximately 7 (median) for each extract was found in both child and parent. A few bands (approximately 3) were found in parents but not in children. Only one to two bands were found in children's saliva without a corresponding band in any of the parents. The antibody activity of saliva samples obtained several years apart was essentially unchanged, especially considering the total number of bands. Differences in the relative intensity of the bands could be seen, and a few bands appeared only in some immunoblots for a specific subject. For 3 of 4 children participating in the longitudinal study, more bands against mutans streptococci appeared with age.

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.

The aims of this study were i) to characterize and compare the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of reference Streptococcus mutans and Streptococcus sobrinus strains, subcultivated for years, with fresh isolates of the same serotype; ii) to study possible differences between the human salivary immunoglobulin A (IgA) activity to reference strains and to fresh bacterial isolates of the saliva donors; iii) to examine for potential differences in the salivary IgA activity to the streptococcal antigens during 1 week; and iv) to map, in the same individuals, the serum IgA activity against the selected bacteria. S. mutans reference and fresh isolated strains showed a similar protein pattern with few exceptions. The immunoblot also revealed similarity in saliva IgA response, with only one subject's saliva displaying clearly one band's difference. For S. sobrinus a larger discrepancy was seen. The antibody activity during the one week interval was essentially unchanged. When incubated with serum, a different immunoblot profile was seen compared with saliva, although most bands revealed by saliva were also displayed by serum.

Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats.

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.

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.

The purpose of this study was to find whether a glycerolphosphate-containing lipoteichoic acid prepared from Streptococcus sobrinus OMZ 176 cells would activate the classical pathway of complement while in solution. Reference activators were lipopolysaccharide from Escherichia coli 0111:B4 and heat-aggregated immunoglobulin G. Serum samples were taken from healthy students. Analysis through crossed immunoelectrophoresis showed that lipoteichoic acid caused an almost complete dissociation of the C1qrs macromolecule. All activators decreased the area of and slowed the electrophoretic mobility of the C4 protein peaks, with lipoteichoic acid causing the most pronounced alterations. Electroimmunoassays showed that lipoteichoic acid separately, yielded detectable amounts of free C1r2s2 subunits; it also generated significantly more trimer complexes between C1r, C1s and C1 inhibitor (C1INH) than did the other two activators. Lipoteichoic acid was, however, a comparatively weak inducer of tetramer C1INH-C1r-C1s-C1INH complexes. Analysis through Western blotting showed that all activators accelerated consumption of C1r, induced complex formations between C1INH and C1s and produced cleavage products of C2. Altogether, the immunochemical analysis gave clear evidence of classical pathway activation by lipoteichoic acid, but its activation profile differed from those seen with lipopolysaccharide and aggregated immunoglobulin G.

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.