Immune response in humans to a nasal boost with Streptococcus mutans antigens.

We previously reported that a Streptococcus mutans enriched-glucosytransferase (E-GTF) preparation induces an immune response following intranasal, but not tonsillar, immunization of humans. In this study, we determined whether intranasal immunization of these subjects 2 years later resulted in augmented immune responses compared to those seen in control subjects. Subjects previously immunized via the intranasal (IN, n = 7) or tonsillar (IT, n = 7) route and control (n = 12) subjects were immunized via the intranasal route with E-GTF. Nasal wash, saliva, and serum were collected before immunization and then weekly for 3 months after immunization. Significant (P < 0.05) mucosal and serum immunoglobulin A (IgA) anti-E-GTF responses were observed in all three groups. Nasal and serum IgA anti-E-GTF responses were significantly higher (P < 0.05) in the IN group. The salivary responses in the three groups were, in general, similar. These results indicate that intranasal immunization primes the immune system for a localized secondary response to S. mutans antigens.

Comparative analysis of the antibacterial effects of combined mouthrinses on Streptococcus mutans.

BACKGROUND/AIMS: Chlorhexidine has been proposed as a potent chemotherapeutic agent against oral bacteria. However, there are some inconsistent results regarding the usefulness of chlorhexidine mouthrinse as an antimicrobial for Streptococcus mutans. The purpose of this study was to investigate the effectiveness of combining oral rinses to reduce S. mutans levels in human saliva. METHODS: Sixteen healthy adult subjects were randomly assigned to one of four rinse groups using a 4-cell crossover design. The groups rinsed twice a day for 7 days with one of the following: 0.12% chlorhexidine (PerioGard), 1.5% hydrogen peroxide (Peroxyl), a combined chlorhexidine+hydrogen peroxide, or water (control). Every 5 weeks, each group initiated a different rinse. Saline wash samples were collected on days 7 and 21 for assessment of S. mutans and total streptococci. RESULTS: No significant differences were seen in S. mutans levels among the groups; however, the levels of total streptococci on day 7 samples were significantly lower in the chlorhexidine and chlorhexidine+hydrogen peroxide groups than in the hydrogen peroxide and control groups. There was no additional decrease seen in S. mutans or total streptococci levels in the group receiving chlorhexidine+hydrogen peroxide compared to chlorhexidine alone. CONCLUSIONS: Sample variation was high throughout the study, with a significant trend toward lower counts as the study progressed. Adding hydrogen peroxide to the chlorhexidine mouthrinse did not result in a further decrease in S. mutans levels.

Intranasal immunization of humans with Streptococcus mutans antigens.

To evaluate the effectiveness of a low dose of soluble or liposomal (L) glucosyltransferase-enriched preparation (E-GTF) in inducing mucosal immune responses after intranasal immunization, 12 adults were immunized on days 0 and 7 by the IN route with 62.5 microg of soluble E-GTF or L-E-GTF. An increase in the mean salivary IgA anti-E-GTF response (P < 0.03) was seen in the L-E-GTF but not the soluble E-GTF group. A significant increase (P < 0.05) in the mean specific IgA antibody activity was also seen in nasal wash from both groups. Although the nasal wash responses were higher in the L-E-GTF than in the soluble E-GTF group, they were not significantly different. The soluble E-GTF immunized group showed a higher serum IgG response than the L-E-GTF immunized group on day 90 (P < 0.05). These results indicate that as little as 62.5 microg of E-GTF, when given by the intranasal route, induced an IgA response in secretions.

Effect of age on immunoglobulin A subclass distribution in human parotid saliva.

Two subclasses of immunoglobulin A (IgA) antibodies are produced in humans, IgA1 and IgA2, IgA2 being more resistant to digestion by bacterial proteases than IgA1. The amount of IgA in saliva has been shown to vary with age; however, little is known about the correlation between IgA subclass distribution in saliva and age. The purpose of this study was to determine whether differences exist in the levels and ratio of IgA subclasses in parotid saliva of children and adults. Parotid saliva was obtained from healthy children (age range 6-12 years, n = 14) and adults (age range 22-51 years, n = 20) using Schaefer cups. Samples were analyzed for levels of total IgA, IgA1, and IgA2 by ELISA. IgA and IgA1 levels were significantly higher in adults than in children. However, no differences were seen in the ratio of IgA1 and IgA2 in the two groups of subjects. These findings indicate that levels of IgA increase with age, whereas the IgA subclass ratio is established early in life.

Humans immunized with Streptococcus mutans antigens by mucosal routes.

Strategies aimed at the prevention of Streptococcus mutans infection and dental caries include mucosal immunization, which results in salivary anti-S. mutans responses. The purpose of this study was to evaluate the effectiveness of nasal vs. tonsillar immunization with S. mutans antigens in inducing salivary immune responses. Twenty-one adult subjects were immunized twice, within a seven-day interval, with a glucosyltransferase-enriched preparation (E-GTF) administered by nasal or tonsillar topical spray. Parotid saliva, nasal wash, and serum were collected prior to and at one- to two-week intervals for 3 months following immunization and were assayed by ELISA for anti-E-GTF activity. Results were analyzed by means of the mixed-models procedure with p < 0.05 level of significance. Significantly higher anti-E-GTF responses were detected in saliva and nasal wash samples from the group immunized by the nasal compared with the tonsillar route, indicating that nasal immunization was more effective in inducing mucosal responses in adults.

Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein.

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route with Salmonella expressing an insoluble protein and to study the ability to augment recall responses by boosting with either Salmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor of Streptococcus mutans, was recombinantly expressed in the insoluble phase in S. enterica serovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered in Salmonella at week 1 (groups 1 and 2) and recall responses after a week 15 boost with either Salmonella expressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P < 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who received Salmonella followed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations with Salmonella expressing GLU. On the other hand, group 2 mice, which received Salmonella expressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate that S. enterica serovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.

Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1beta, IL-6, and TNF-alpha upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1beta, but not IL-6 and TNF-alpha production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with < or =100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-alpha, while stimulation of THP-1 cells with > or =1 microg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-alpha levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade I-kappaB-alpha or I-kappaB-beta proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade I-kappaB-beta only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.

A vaccine against dental caries: an overview.

Dental caries continues to be a costly and prevalent oral disease. Research efforts towards developing a well tolerated and effective vaccine against dental caries were initiated following the demonstration of a specific bacterial aetiology for this disease. The cariogenic mutans streptococci are the principal bacteria causing this disease. Specific immune defence against these bacteria is provided mainly by secretory immunoglobulin (Ig) A antibodies present in saliva, which are generated by the common mucosal immune system. Progress in the development of a vaccine against dental caries has increased due to both advancements in molecular biology and our understanding of the mucosal immune system and mucosal vaccines. Advancements in molecular biology have facilitated the cloning and functional characterisation of virulence factors of the mutans streptococci, including the cell-surface fibrillar proteins, which mediate adherence to the tooth surface, and the glucosyltransferase enzymes, which synthesise adhesive glucans and allow microbial accumulation on the teeth. Current strategies for immunisation against dental caries are using these virulence factors as key antigens and incorporating them into novel mucosal vaccine systems and delivering them with or without adjuvants to mucosal IgA inductive sites. The most popular routes of mucosal immunisation are via the oral or nasal route. The mucosal immune system is functional in newborn infants, who develop salivary IgA antibodies as they become colonised by oral micro-organisms. Mucosal immunisation strategies result in the induction of salivary IgA antibody responses and pose fewer problems than parenteral injection of antigen. Therefore, mucosal immunisation of infants prior to the appearance of their first teeth may be a well tolerated and effective way to induce immunity against the colonisation of teeth by mutans streptococci and protection against subsequent dental caries. The purpose of this article is to provide an overview of the recent progress on the development of a vaccine against infection by Streptococcus mutans for the prevention of dental caries, with emphasis on the mucosal immune system and vaccine design.

Human salivary immunoglobulin and antigen-specific antibody activity after tonsillectomy.

The importance of the lymphoid tissue collectively known as Waldeyer's ring, which includes the palatine, lingual and nasopharyngeal tonsils, in the induction and contribution of specific antibody responses in human saliva is not clear. The purpose of this study was to determine whether salivary immunoglobulin A (IgA) levels differ in quantity and quality between subjects who have had a tonsillectomy and age, sex and race-matched controls. Parotid saliva, whole saliva, and blood serum samples were collected from 25 volunteer children who had undergone tonsillectomy (T-) within 6-14 months of sampling and from 25 age, sex and race-matched controls. The levels of total IgA (and subclasses) in saliva, and of antigen-specific salivary IgA and serum IgA and IgG antibodies to 4-9 relevant antigens were analyzed by ELISA. No significant difference was observed in the mean total IgA and IgA subclass levels in parotid and whole saliva, although the mean levels for children with a T- were slightly lower. Children with a T- had significantly higher parotid salivary IgA and IgA1 specific/total activity than controls. The total and specific whole saliva IgA and the specific serum IgA or IgG activities were not significantly different from controls. These results indicate an association between the removal of tonsils and increased levels of specific IgA activity in parotid saliva within the first year after a T-.

Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells.

Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4(+) T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4(+) T cells. In a coculture system, CT-treated CD4(+) T cells induced significantly less TNF-alpha and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4(+) T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.

Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter.

The purpose of the present study was to evaluate the effectiveness of an attenuated Salmonella enterica serovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of the Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically inducible nirB promoter, in inducing a protective immune response against S. mutans infection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 10(9) or 10(10) Salmonella CFU, respectively. The Salmonella vaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection against S. mutans infection, mice were challenged after the second immunization with a virulent strain of S. mutans which was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin. S. mutans was initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized with Salmonella clones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutans present in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonella vector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutans colonization of tooth surfaces.

Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Recombinant antigen-enterotoxin A2/B chimeric mucosal immunogens differentially enhance antibody responses and B7-dependent costimulation of CD4(+) T cells.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR-LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR-LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220(+), CD11b(+), or CD11c(+) cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4(+) T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR-LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4(+) T cells.

Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen.

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 microg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence.

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes.

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.

Construction and characterization of a Salmonella enterica serovar typhimurium clone expressing a salivary adhesin of Streptococcus mutans under control of the anaerobically inducible nirB promoter.

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) of Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the inducible nirB promoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing of Salmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains the nirB promoter. The resulting plasmid was introduced into serovar Typhimurium by electroporation. Production of the SBR-CTA2/B chimeric protein under anaerobic conditions was verified by enzyme-linked immunosorbent assay of whole-cell lysates on plates coated with G(M1) ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium under nirB control. Anaerobic expression of SBR was confirmed by Western blotting of whole-cell lysates probed with anti-SBR antibodies. The resulting serovar Typhimurium strains were administered by either the oral or the intranasal route to mice, and colonization was assessed by microbiologic analysis of dissociated spleens, Peyer's patches (PP), and nasal tissues. High numbers of the recombinant strains persisted in PP and spleen for at least 21 days following oral challenge. A single intranasal administration of the Salmonella clones to mice also resulted in the colonization of the nasal tissues by the recombinant bacteria. Salmonellae were recovered from nasal lymphoid tissues, superficial lymph nodes, internal jugular lymph nodes, PP, and spleens of mice for at least 21 days after challenge. This study provides quantitative evidence for colonization by Salmonella strains expressing a recombinant protein under the control of the inducible nirB promoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.

Construction and characterization of an effector strain of Streptococcus mutans for replacement therapy of dental caries.

An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make the Streptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of the ldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF from Zymomonas mobilis in place of the deleted ldh ORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic- and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10(-3)) or by screening cultures grown in vitro, where no revertants were observed among 10(5) colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.

Comparative analysis of the mucosal adjuvanticity of the type II heat-labile enterotoxins LT-IIa and LT-IIb.

Cholera toxin (CT) and the heat-labile enterotoxin of Escherichia coli (LT-I) are members of the serogroup I heat-labile enterotoxins (HLT) and can serve as systemic and mucosal adjuvants. However, information is lacking with respect to the structurally related but antigenically distinct serogroup II HLT, LT-IIa and LT-IIb, which have different binding specificities for ganglioside receptors. The purpose of this study was to assess the effectiveness of LT-IIa and LT-IIb as mucosal adjuvants in comparison to the prototypical type I HLT, CT. BALB/c mice were immunized by the intranasal (i.n.) route with the surface protein adhesin AgI/II of Streptococcus mutans alone or supplemented with an adjuvant amount of CT, LT-IIa, or LT-IIb. Antigen-specific antibody responses in saliva, vaginal wash, and plasma were assayed by enzyme-linked immunosorbent assay. Mice given AgI/II with LT-IIa or LT-IIb by the i.n. route had significantly higher mucosal and systemic antibody responses than mice immunized with AgI/II alone. Anti-AgI/II immunoglobulin A (IgA) antibody activity in saliva and vaginal secretions of mice given AgI/II with LT-IIa or LT-IIb was statistically similar in magnitude to that seen in mice given AgI/II and CT. LT-IIb significantly enhanced the number of AgI/II-specific antibody-secreting cells in the draining superficial cervical lymph nodes compared to LT-IIa and CT. LT-IIb and CT induced significantly higher plasma anti-AgI/II IgG titers compared to LT-IIa. When LT-IIb was used as adjuvant, the proportion of plasma IgG2a relative to IgG1 anti-AgI/II antibody was elevated in contrast to the predominance of IgG1 antibodies promoted by AgI/II alone or when CT or LT-IIa was used. In vitro stimulation of AgI/II-specific cells from the superficial lymph nodes and spleen revealed that LT-IIa and LT-IIb induced secretion of interleukin-4 and significantly higher levels of gamma interferon compared to CT. These results demonstrate that the type II HLT LT-IIa and LT-IIb exhibit potent and distinct adjuvant properties for stimulating immune responses to a noncoupled protein immunogen after mucosal immunization.

Protective immunity against Streptococcus mutans infection in mice after intranasal immunization with the glucan-binding region of S. mutans glucosyltransferase.

Here we present the construction and characterization of a chimeric vaccine protein combining the glucan-binding domain (GLU) of the gtfB-encoded water-insoluble glucan-synthesizing glucosyltransferase enzyme (GTF-I) from Streptococcus mutans and thioredoxin from Escherichia coli, which increases the solubility of coexpressed recombinant proteins and stimulates proliferation of murine T cells. The protective potential of intranasal (i.n.) immunization with this chimeric immunogen was compared to that of the GLU polypeptide alone in a mouse infection model. Both immunogens were able to induce statistically significant mucosal (salivary and vaginal) and serum responses (P < 0.01) which were sustained to the end of the study (experimental day 100). Following infection with S. mutans, sham-immunized mice maintained high levels of this cariogenic organism ( approximately 60% of the total oral streptococci) for at least 5 weeks. In contrast, animals immunized with the thioredoxin-GLU chimeric protein (Thio-GLU) showed significant reduction (>85%) in S. mutans colonization after 3 weeks (P < 0.05). The animals immunized with GLU alone required 5 weeks to demonstrate significant reduction (>50%) of S. mutans infection (P < 0.05). Evaluation of dental caries activity at the end of the study showed that mice immunized with either Thio-GLU or GLU had significantly fewer carious lesions in the buccal enamel or dentinal surfaces than the sham-immunized animals (P < 0.01). The protective effects against S. mutans colonization and caries activity following i.n. immunization with GLU or Thio-GLU are attributed to the induced salivary immunoglobulin A (IgA) anti-GLU responses. Although in general Thio-GLU was not significantly better than GLU alone in stimulating salivary IgA responses and in protection against dental caries, the finding that the GLU polypeptide alone, in the absence of any immunoenhancing agents, is protective against disease offers a promising and safe strategy for the development of a vaccine against caries.