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S M Michalek

Publications and source records attributed to S M Michalek.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

ATP-Binding Cassette Transporters↗
PubMed1999

Current status of a mucosal vaccine against dental caries.

The evidence of a specific bacterial cause of dental caries and of the function of the salivary glands as an effector site of the mucosal immune system has provided a scientific basis for the development of a vaccine against this highly prevalent and costly oral disease. Research efforts towards developing an effective and safe caries vaccine have been facilitated by progress in molecular biology, with the cloning and functional characterization of virulence factors from mutans streptococci, the principal causative agent of dental caries, and advancements in mucosal immunology, including the development of sophisticated antigen delivery systems and adjuvants that stimulate the induction of salivary immunoglobulin A antibody responses. Cell-surface fibrillar proteins, which mediate adherence to the salivary pellicle, and glucosyltransferase enzymes, which synthesize adhesive glucans and allow microbial accumulation, are virulence components of mutans streptococci, and primary candidates for a human caries vaccine. Infants, representing the primary target population for a caries vaccine, become mucosally immunocompetent and secrete salivary immunoglobulin A antibodies during the first weeks after birth, whereas mutans streptococci colonize the tooth surfaces at a discrete time period that extends around 26 months of life. Therefore, immunization when infants are about one year old may establish effective immunity against an ensuing colonization attempts by mutans streptococci. The present review critically evaluates recent progress in this field of dental research and attempts to stress the protective potential as well as limitations of caries immunization.

Adhesins, Bacterial↗
PubMed1999

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.

Administration, Intranasal↗
PubMed1999

A controlled clinical study of the effect of nasal immunization with a Streptococcus mutans antigen alone or incorporated into liposomes on induction of immune responses.

Recent attention to mucosal immunization strategies has been focused on the nasal route for vaccine delivery. This study was designed to determine the effectiveness of a liposome-protein vaccine compared to that of a protein-only vaccine in inducing immune responses in humans. Healthy subjects were randomly assigned to two groups and immunized intranasally with a crude antigen preparation rich in glucosyltransferase (C-GTF) from Streptococcus mutans, alone or in liposomes. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals following immunization and were analyzed for anti-C-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A (IgA) anti-C-GTF activity in the nasal wash from both groups after immunization increased to a mean peak of fivefold over the baseline level on day 28. Salivary IgA anti-C-GTF responses were induced to a lesser extent. IgG and IgA anti-C-GTF responses in serum were detected on day 14. The IgA responses were predominantly of the IgA1 subclass. These results show that C-GTF vaccines were more effective in inducing a local secretory IgA antibody response than a salivary or serum response when they were given intranasally. The IgA1 anti-C-GTF response in nasal wash samples for liposomal antigen versus antigen only was the only response which was significantly different (P < 0.04). This suggests that the form of the antigen affects the magnitude of the local mucosal response but not that of a disseminated response. These results provide evidence for the effective use of a nasal protein vaccine in humans for the induction of mucosal and systemic responses.

Administration, Intranasal↗
PubMed1999

Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I.

Glucosyltransferase (GTF) enzymes of mutans streptococci are considered virulence factors due to their ability to synthesize adhesive glucans, which facilitate cell-to-cell adherence and accumulation. In this study we report the cloning, expression, and characterization of the catalytic (CAT) and glucan-binding (GLU) domains of S. mutans GTF-I encoded by gtfB. The CAT and GLU polypeptides represent amino acid residues 253 to 628 and 1183 to 1473, respectively, of S. mutans GTF-I. Antibodies to recombinant CAT and GLU were generated in rabbits and purified by affinity chromatography. Purified anti-CAT antibodies significantly inhibited water-insoluble glucan synthesis by S. mutans and S. sobrinus GTFs (P < 0.0001 and P < 0.05, respectively). The purified anti-GLU antibodies significantly inhibited both water-insoluble and water-soluble glucan synthesis by S. mutans GTFs (P < 0.0001 and P < 0.05, respectively). These results demonstrate that anti-CAT and anti-GLU antibodies are capable of inhibiting a variety of GTF activities. Since antibodies to S. mutans in saliva are implicated in protection against disease, we next assessed the ability of CAT and GLU polypeptides to induce mucosal antibody responses in mice. Intranasal (i.n.) immunization of mice with CAT showed significantly (P < 0.005) elevated levels of specific immunoglobulin G (IgG) antibody activity in serum and specific IgA antibody activity in serum, saliva, vaginal washes, and fecal samples. GLU immunized animals showed significantly (P < 0.005) elevated levels of specific IgA antibody activity in serum and vaginal secretions. Taken together, these results demonstrate that the recombinant CAT and GLU polypeptides are effective in inducing both mucosal and systemic immune responses. The ability of these polypeptides to induce a mucosal IgA immune response in mice after i.n. immunization supports their use as subunit vaccine candidates in the development of an anticaries vaccine.

Animals↗
PubMed1999

Virulence of a spaP mutant of Streptococcus mutans in a gnotobiotic rat model.

Streptococcus mutans, the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in S. mutans. We showed previously that P1 expression is necessary for in vitro adherence of S. mutans to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1, spaP, has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either S. mutans NG8 or an NG8-derived spaP mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of S. mutans, as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.

Animals↗
PubMed1999

Host responses to recombinant hemagglutinin B of Porphyromonas gingivalis in an experimental rat model.

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Several hemagglutinin genes have been identified, cloned, and expressed in Escherichia coli. The purpose of this study was to characterize host responses to purified recombinant hemagglutinin B (rHag B), using the conventional Fischer rat as the experimental animal model. The effectiveness of immunization with rHag B on protection against experimental periodontal bone loss following infection with P. gingivalis was also evaluated. Groups of rats were immunized by the subcutaneous route with rHag B in complete Freund's adjuvant, immunized with rHag B and orally infected with P. gingivalis, nonimmunized and noninfected, or orally infected with P. gingivalis only. Serum and saliva samples were collected throughout the experiment and evaluated for serum immunoglobulin G (IgG) and IgM and salivary IgA antibody activity by enzyme-linked immunosorbent assay. No salivary IgA anti-Hag B activity was detected in the various groups of rats. A slight serum IgM response similar to that seen in preimmune samples was observed. Serum IgG antibody activity to Hag B was detected only in samples from rats immunized with rHag B. This response was primarily of the IgG1 and IgG2a subclasses, followed by IgG2b and low levels of IgG2c. Supernatants from rHag B-stimulated splenic lymphoid cell cultures from immunized rats contained high levels of gamma interferon, followed by interleukin-2 (IL-2), IL-10, and then IL-4. These results are consistent with the induction of T helper type 1 (Th1)- and Th2-like responses. Western blot analysis of sera derived from rHag B-immunized rats reacted with trichloroacetic acid (TCA) precipitates of P. gingivalis 33277, 381, A7A1-28, and W50, revealing a 50-kDa band reflective of Hag B. However, sera derived from rats immunized with P. gingivalis whole cells or from rats infected with P. gingivalis only did not react with rHag B but did react with TCA precipitates of P. gingivalis strains. Finally, radiographic measurements of periodontal bone loss indicated that rats immunized with rHag B had less bone loss than those infected with P. gingivalis only. These results demonstrate the effectiveness of purified rHag B in inducing a protective immune response and support the potential usefulness of this component of P. gingivalis in the development of a vaccine against adult periodontitis.

Adhesins, Bacterial↗
PubMed1999

Secretory immunity in defense against cariogenic mutans streptococci.

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.

Adhesins, Bacterial↗
PubMed1999

Interleukin (IL)-1 and Porphyromonas gingivalis lipopolysaccharide stimulation of IL-6 production by fibroblasts derived from healthy or periodontally diseased human gingival tissue.

BACKGROUND: Human gingival fibroblasts (HGF) play a key role in tissue repair and destruction. These cells express low levels of interleukin (IL)-6 constitutively and increased levels after stimulation with lipopolysaccharide (LPS) or the cytokines IL-1 and tumor necrosis factor-alpha (TNF-alpha). However, little information is available on IL-6 production by fibroblasts derived from diseased tissue. The present study compared constitutive and induced IL-6 production by human gingival fibroblasts (HGF) derived from healthy and diseased periodontal tissue. We also evaluated whether IL-1 acted in a synergistic manner with LPS in inducing IL-6 production by HGF and whether LPS acted via CD14. METHODS: HGF derived from healthy and diseased tissue and foreskin fibroblasts were grown to confluency, photographed, and counted, and their constitutive IL-6 production was quantitated by ELISA. Healthy and diseased HGF were also pretreated with IL-1alpha, followed by incubation with Porphyromonas gingivalis or Escherichia coli LPS. Culture supernatants were assessed for IL-6 protein by ELISA and cell lysates for mRNA by reverse transcription-polymerase chain reaction (RT-PCR). In order to determine if LPS stimulation was mediated through the LPS receptor, CD14, surface receptors on HGF were assessed by flow cytometry and the total RNA CD14 mRNA was assessed. RESULTS: Higher quantities of IL-6 were produced by the diseased HGF before and after stimulation than by the healthy HGF. Pretreatment with IL-1alpha followed by LPS stimulation of the healthy and diseased HGF cell lines resulted in an additive effect on IL-6 production. Pretreatment with IL-1alpha followed by a second incubation with the same stimulant produced higher amounts of IL-6 than cultures incubated with LPS alone or following IL-1alpha pretreatment. Similar amounts of IL-6 mRNA were present in unstimulated HGF from either diseased or healthy tissue and in those incubated with IL-1alpha only. After incubation with IL-1alpha and LPS, diseased HGF produced slightly more mRNA than healthy HGF. CD14 was not expressed by healthy or diseased HGF even after stimulation with either P. gingivalis or E. coli LPS. CD14 message was also undetectable. CONCLUSIONS: Taken together, these results indicate heterogeneity among gingival fibroblasts and an additive effect of IL-1 and P. gingivalis LPS on IL-6 production by HGF. Furthermore, the LPS effect on HGF was not mediated by CD14.

Cell Count↗
PubMed1998

Repetitive adenovirus administration to the parotid gland: role of immunological barriers and induction of oral tolerance.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Adenoviridae↗
PubMed1998

Induction of protective immune responses against Venezuelan equine encephalitis (VEE) virus aerosol challenge with microencapsulated VEE virus vaccine.

Venezuelan equine encephalomyelitis (VEE) virus, a member of the family Togaviridae, genus Alphavirus, causes disease in humans and equids. The virus is normally transmitted by the bite of an infected mosquito however, it can also be highly infectious by aerosol. The purpose of the present study was to determine the effectiveness of formalin-fixed, 60Co-irradiated VEE virus microencapsulated in poly DL-lactide-co-glycolide in inducing immune responses protective against aerosol challenge with virulent VEE virus. Balb/c mice were primed by subcutaneous injection of microencapsulated VEE virus vaccine, followed 30 days later by a single immunization with the same vaccine given via the oral, intratracheal (i.t.) or subcutaneous (s.c.) route. Mice boosted by the i.t. or s.c. route had higher plasma IgG anti-VEE virus levels than orally immunized animals. The responses in the former groups were similar in magnitude to those seen in mice primed and boosted by the i.t. route. Antibody activity was detected in bronchial-alveolar and intestinal washes, fecal extracts and saliva from immunized animals. The levels of IgG and IgA antibody activity in bronchial-alveolar wash fluids from mice boosted by the i.t. route were higher than those seen in animals immunized by the oral or s.c. route with the microsphere vaccine. Mice immunized with the microencapsulated VEE virus vaccine were protected from lethal VEE virus infection following aerosol challenge at approximately three months after the initial immunization. Mucosal immunization via the i.t. route appeared to be the most effective regimen, since 100% of the mice resisted aerosol challenge.

Administration, Inhalation↗
PubMed1998

Effect of immune T cells derived from mucosal or systemic tissue on host responses to Porphyromonas gingivalis.

The purpose of this study was to compare the effects of immune T cells derived from mucosal and systemic tissues on specific humoral responses to the periodontal pathogen Porphyromonas gingivalis and to determine the effect these responses have on periodontal bone loss in an experimental rat model. T cells were derived from either the spleen or Peyer's patches of conventional Fischer rats given live P. gingivalis by the oral route and were adoptively transferred into groups of nude Fischer rats. A group of nude rats to which no T cells were transferred served as control. Within 24 h of the adoptive cell transfer, animals from all groups were orally challenged with live P. gingivalis. Serum and salivary responses were seen in rats given immune T cells, whereas a low, variable serum IgM and no serum IgG or salivary IgA response was detected in the control nude rats, indicating that the major host responses to P. gingivalis whole-cell antigens were T-cell dependent. Rats given immune Peyer's patch-derived T cells exhibited a different serum IgG and salivary IgA immune response pattern than animals given immune splenic T cells. A higher serum IgG, especially IgG2b, antibody response and less horizontal bone loss was seen in rats given splenic than Peyer's patch-derived T cells. Although some differences were seen in the levels of salivary IgA early in the study in the two experimental groups, the levels of IgA were comparable by the termination of the experimental period. Furthermore, no difference was seen in the amount of vertical bone support in these two groups, which was greater than in the controls. These results suggest a role for serum IgG and salivary IgA in periodontal disease protection. The presence of high serum IgG2b antibody activity suggest the involvement of Th1-like cells in the response, whereas salivary IgA antibodies indicates the participation of Th2-like cells. These results suggest that the balance between Th1 and Th2-like cells within the systemic and mucosal compartments and the humoral immune responses to P. gingivalis they mediate are important in determining whether the responses induced are protective against experimental bone loss after oral challenge with the periodontal pathogen P. gingivalis.

Alveolar Bone Loss↗
PubMed1998

Effect of lipopolysaccharide and inflammatory cytokines on interleukin-6 production by healthy human gingival fibroblasts.

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.

Cell Line↗
PubMed1998

Comparison of an adherence domain and a structural region of Streptococcus mutans antigen I/II in protective immunity against dental caries in rats after intranasal immunization.

Previous studies have identified an N-terminal saliva-binding region (SBR) on Streptococcus mutans surface antigen I/II (AgI/II) and suggested its importance in the initial adherence of S. mutans to saliva-coated tooth surfaces and subsequent development of dental caries. In this study, we compared the SBR with a C-terminal structural region of AgI/II (AgII) in their abilities to induce protective immunity against caries in rats. When SBR, AgII, or the whole AgI/II molecule was administered intranasally as a conjugate with the B subunit of cholera toxin (CT), in the presence of CT adjuvant, substantial levels of salivary immunoglobulin A anti-AgI/II antibodies were induced. Evaluation of caries activity showed that the SBR, though not as protective as the parent molecule, was superior to AgII and thus can be further considered as a component in a multivalent caries vaccine.

Administration, Intranasal↗
PubMed1998

Inactivation of the gbpA gene of Streptococcus mutans increases virulence and promotes in vivo accumulation of recombinations between the glucosyltransferase B and C genes.

Glucan-binding protein A (GbpA) of Streptococcus mutans has been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenic gbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutans was grown in the presence of [3H] thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. The gbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB and gtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

Animals↗
PubMed1998

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system.

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.

Adhesins, Bacterial↗
PubMed1997

Nasal immunization of humans with dehydrated liposomes containing Streptococcus mutans antigen.

Five healthy female adult volunteers were intranasally immunized twice (7-day interval) with 250 micrograms of a crude glucosyltransferase (GTF) preparation from Streptococcus mutans in liposomes. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals for 6 weeks following the first immunization for analysis of anti-GTF activity by enzyme-linked immunosorbent assay. The levels of IgA1 anti-GTF activity increased in the nasal wash from all five individuals after immunization. Increases in salivary IgA1 and IgA2 anti-GTF activities were observed to a lesser extent. Increased serum IgM and IgA (but not IgG) anti-GTF activities were seen in immunized subjects. Nasal immunization with a dehydrated liposome-protein vaccine was effective in inducing an apparent secretory IgA antibody response, which was primarily of the IgA1 subclass. These results provide the first evidence of the effective use of a nasal liposome-protein vaccine in humans.

Administration, Intranasal↗