![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Infection and Immunity, May 2005, p. 2797-2804, Vol. 73, No. 5
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.5.2797-2804.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Immunology, The Forsyth Institute, 140 The Fenway, Boston, Massachusetts 02115
Received 8 October 2004/ Returned for modification 16 November 2004/ Accepted 10 January 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Since both GTF and glucan binding proteins (Gbp) are essential to the pathogenicity of MS, these proteins have been pursued as components of potential dental caries vaccines. Native GTF enzymes from Streptococcus mutans and Streptococcus sobrinus (reviewed in references 8 and 14), as well as GbpB (10, 15) from S. mutans (17), have been effective as vaccines in experimental models for dental caries. Synthetic peptide constructs derived from either the catalytic domain (CAT) or the glucan binding domain (GLU) of mutans streptococcal GTF have been shown to induce protective immunity to experimental infection with cariogenic mutans streptococci (21). Identification of S. mutans GbpB-derived synthetic peptides associated with protein activity was more problematic since functional domains of this protein have yet to be resolved. However, since GbpB is exceptionally immunogenic (17), its sequence was explored for peptides which had the potential to be presented on the surfaces of antigen-presenting cells in the context of major histocompatibility complex (MHC) class II molecules to T lymphocytes in the process leading to antibody formation (16). Immunization of rats with a peptide construct, SYI, whose sequence had been identified through bioinformatic analysis to have this potential, gave rise to protective immunity from S. mutans-associated dental caries.
The value of subunit vaccines lies not only in concentrating the immune response to relevant epitopes, but also in permitting the combination in one vaccine of such epitopes from different regions on the native protein or, indeed, including multiple pathogenesis-associated components in the construct. We have shown previously that immunization with a diepitopic multiple antigenic peptide (MAP) construct that contained peptides from two distinct GTF domains (catalytic and glucan binding activities) enhanced the level of protection from dental caries caused by S. sobrinus, compared to the level of protection by immunization with either construct alone (24). In addition to the presence of complementary GTF functional domains, one of these peptides (GLU) contained a T-cell epitope (18, 22) which was thought to be responsible for some of the improvement in immune response to the B-cell epitope within the other peptide (CAT) component (19, 24) of the diepitopic construct of this subunit GTF vaccine.
Additional studies of subunit dental caries vaccines containing epitopes from more than one pathogenic component, for example, chimeric proteins (26) or fusion DNA vaccines (3), each containing epitopes from S. mutans GTF and an adhesin (PAc or AgI/AgII), have reported protective immunity in rats infected with S. mutans. Others (25) have demonstrated the formation of antibody that modified both adhesion and glucan synthesis in the sera of animals immunized with an adhesin-GTF fusion protein. However, this approach has not been pursued by combining GTF epitopes with those of S. mutans GbpB. Thus, we designed diepitopic synthetic peptide constructs containing sequences from each native protein that had previously been shown individually to give rise to protective responses. Our first aim was to explore the potential for immunological enhancement of immune responses to functional epitopes by including a putative T-cell epitope in the constructs containing peptides from GTF and GbpB. A 20-mer peptide, SYI, was selected based on MHC class II binding characteristics and the ability to induce caries-protective activity after immunization (16). This GbpB peptide was combined with either CAT- or GLU-GTF peptides to form SYI-CAT and SYI-GLU diepitopic constructs. Our second aim was to explore the ability of immunization with the diepitopic construct that gave the broader immune response to GTF and GbpB native protein to protect rats from the cariogenic effects of infection with S. mutans or S. sobrinus.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two diepitopic peptide constructs were also synthesized. One of these, SYI-CAT, contained two SYI peptides and two CAT peptides on the same lysine backbone. Another, SYI-GLU, contained two SYI peptides and two GLU peptides on the same backbone. The purity of all peptide constructs was assessed at >90%, using high-pressure liquid chromatography, amino acid analysis, and molecular weight determination by mass spectrometry.
Proteins. GbpB was purified from S. mutans strain SJ by ion-exchange chromatography on MONO-Q HR 5/5 (Pharmacia) in the presence of urea as described previously (16). Bacteria were cultivated overnight at 37°C under anaerobic conditions in a sucrose-free defined medium containing 1% glucose. GbpB prepared in this manner migrates to a position corresponding to 59 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18).
GTF from S. sobrinus strain 6715 was obtained by affinity and gel permeation chromatographies. After bacterial growth at 37°C under anaerobic conditions in a glucose-containing defined medium, enzymes were isolated from culture medium by affinity chromatography on Sephadex G-100 (Pharmacia) with 3 M guanidine HCl as the eluting solvent. This GTF-rich pool was then subjected to fast protein liquid chromatography on Superose 6 (Pharmacia) with 6 M guanidine HCl for elution. The S. sobrinus GTF obtained after gel filtration on Superose 6 contained a mixture of GTF isozymes, including GTF-I and GTF-S, but was essentially free of other proteins. Approximately 90% of the glucan synthesized by this GTF was water insoluble (19).
ELISA.
Serum immunoglobulin G (IgG) and salivary IgA antibody levels were determined by enzyme-linked immunosorbent assay (ELISA). Polystyrene microtiter plates (Flow Laboratories) were coated with 2.5 µg/ml of monoepitopic or diepitopic peptide, or with 0.5 µg/ml of S. mutans GbpB or S. sobrinus GTF. Antibody activity was then measured by incubation with a 1:200 or 1:2,000 dilution of sera or with a 1:4 dilution of saliva. Plates were then developed for IgG antibody with rabbit anti-rat IgG, followed in sequence by alkaline phosphatase goat anti-rabbit IgG (Biosource Inc.) and p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, Mo.). For measurement of salivary IgA antibody, a mouse monoclonal antibody to rat 
chain (Zymed, South San Francisco, Calif.) was used, followed by sequential incubation with biotinylated goat anti-mouse IgG (Zymed), avidin-alkaline phosphatase (ICN Biomedicals, Inc., Aurora, Ohio), and p-nitrophenylphosphate. Reactivity was recorded as absorbance (A405) by a microplate reader (Biotek Instruments, Winooski, Vt.). Data are reported as ELISA units (EU), which were calculated relative to levels in reference sera from rats immunized twice with the respective peptide or native protein. Reference serum dilutions of 1/1,000 for GbpB, 1/2,000 for GTF, 1/200 for CAT, 1/400 for SYI, 1/200 for SYI-CAT, and 1/800 for GLU, which produced an A405 of approximately 0.8, were considered 100 EU.
Experiment 1. Sprague-Dawley CD strain 40-day-old female rats (Charles River Laboratories, Wilmington, Mass.) were used for injection. Six groups of seven rats each were injected subcutaneously (s.c.) in the vicinity of the salivary glands in order to maximize immune responses for the purposes of comparison. Separate groups were immunized with the CAT, GLU, SYI, SYI-CAT, or SYI-GLU construct. Monoepitopic constructs were given in doses of 30 µg; diepitopic constructs were given in 60-µg doses. Dose differences between diepitopic and monoepitopic constructs represented an effort to equalize the dose of the peptide in the monoepitopic construct with that of the respective peptide in the diepitopic construct. A sham-immunized group was immunized with phosphate-buffered saline (PBS). An additional two groups (four rats per group) were immunized with S. mutans GbpB (6 µg/dose) or S. sobrinus GTF (6 µg/dose). The initial injection included 0.04 ml of complete Freund adjuvant (FA) (Difco Laboratories, Detroit, Mich.) per site; one subsequent injection, given 21 days later, included 0.04 ml of incomplete FA per site. Samples for antibody analysis were collected 42 and 63 days after the initial injection; the results of the 63-day sample are reported herein.
For collection of serum and saliva samples, rats were first momentarily anesthetized with a gas mixture of 50% carbon dioxide and 50% oxygen and then anesthetized by intraperitoneal injection of a mixture (0.65 ml/kg of body weight) of 3 parts ketamine (Ketaset,100 mg/ml; Fort Dodge Laboratories, Ft. Dodge, Iowa) and 7 parts xylazine (Rompun, 20 mg/ml; Bayer Corp., Shawnee Mission, Kans.). Saliva secretion was stimulated by s.c. injection of 0.6 ml of carbachol (containing 0.1 mg/ml of saline; Sigma Chemical Co.) per kilogram of rat weight. After saliva collection, rats were awakened by injection s.c., first with 0.1 ml/kg of atropine sulfate (0.4 mg/ml; American Pharmaceutical Partners, Inc., Los Angeles, Calif.) and then with yohimbine (Yobine, 2.0 mg/ml; Lloyd Laboratories, Shenandoah, Iowa) at a volume equal to 1.4 times that used for anesthesia. Blood was collected from the tail vein during anesthesia. Sera from coagulated and centrifuged blood were stored frozen at –20°C until measurement of antibody activity. Clarified saliva was stored at –70°C.
Experiment 2 (S. mutans infection). Four groups (n = 12 to13/group) of 24-day-old Sprague-Dawley female rats were singly caged. Rats were injected s.c. in the salivary gland vicinity with adjuvant (0.04 ml/site) and either PBS (sham), SYI-CAT (60 µg/dose), SYI (30 µg/dose), or CAT (30 µg/dose). Antigen was incorporated with complete FA. Rats were reinjected 13 days later with PBS or peptides at the same dose in incomplete FA. Nine days after the second injection, serum and saliva samples were collected under anesthesia as described above. Thirteen days after the second injection, rats were placed in tubs (six rats per tub), given cariogenic diet 2000 (56% confectioners' sugar), and orally infected with approximately 108 streptomycin-resistant S. mutans strain SJ32 cells for 3 consecutive days. Rats were again singly caged after the infection protocol was completed and continued on diet 2000 for the duration of the experiment. Blood and saliva were collected under anesthesia 64 days after initial infection, followed by CO2 asphyxiation. In preparation for the scoring of dental caries, rat skulls were defleshed by dermastide beetles, followed by a rinse with 70% ethanol.
Antibody inhibition of glucan synthesis. Preinfection sera from experiment 2 (collected 22 days after initial immunization) were evaluated for their ability to inhibit water-insoluble glucan synthesis catalyzed by S. sobrinus GTF, using a filter assay. This GTF preparation contained a mixture of GTF-I and GTF-S. S. sobrinus GTF-I and S. mutans GTF-B had complete homology with the CAT peptide construct in the respective catalytic region. Twenty microliters of diluted sera (1:20 dilutions in 0.02 M sodium PBS and 0.2% sodium azide [PBSA], pH 6.5) were preincubated with 0.036 µg of GTF for 2 h at 37°C in a total volume of 0.04 ml of PBSA. Then, 0.05 mg of sucrose and 40 nCi of [14C]sucrose (approximately 100,000 cpm) were added to 0.1 ml of PBSA in the absence of primer (21). Incubation proceeded for 3 h at 37°C, after which water-insoluble glucan was collected by passage through Whatman GF/F glass fiber filters. Water-insoluble glucan collected on filters was washed and retained radioactivity, determined as previously reported (21). Under the conditions of this assay, approximately 900 cpm were incorporated into water-insoluble glucan in the presence of sham-immune sera. The percentage of inhibition of enzyme activity was calculated using the mean sham incorporation counts per minute values as the 100% incorporation levels. Positive control sera from rats immunized with S. sobrinus GTF, which were obtained from experiment 1, were also included in the assay.
Experiment 3 (S. sobrinus infection). The protocol for experiment 3 was essentially identical to that of experiment 2 except that, following immunization and serological sampling, all animals were infected with streptomycin-resistant S. sobrinus strain 6715. Animals were immunized as described above when rats were 24 and 37 days old. Rats were then bled and salivated under anesthesia 7 days later and were infected with S. sobrinus strain 6715 9 days after the second injection, using the infection protocol followed for experiment 2. Infection continued for 70 days prior to experiment termination.
All experimental protocols were pursued in The Forsyth Institute's animal facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, only after approval by the Institutional Animal Care and Use Committee at The Forsyth Institute.
Bacterial recoveries. The presence of mutans streptococcal infection was assessed at the end of each experiment. After systematic swabbing of teeth, sonication, and plating appropriate dilutions on mitis salivarius agar alone and on mitis salivarius agar with 0.2 mg/ml of streptomycin sulfate, plates were incubated for 48 h at 37°C in 80% N2, 10% CO2, 10% H2. Streptomycin-resistant S. mutans strain SJr or S. sobrinus strain 6715 colony-forming units were then enumerated microscopically.
Caries assessment. The extent and depth of carious lesions in all rat molar teeth (caries score) were microscopically evaluated by a modified method of Keyes and Jordan (7). Caries scores were determined separately on smooth and on occlusal dental surfaces.
Statistical analysis. The differences in the median values among the treatment groups were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey pairwise multiple-comparison test when data were normally distributed. Alternatively, data were analyzed by Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn's multiple-comparison procedure when nonparametric distributions were encountered.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Experiment 2 (immunization and S. mutans infection). To determine whether protection could be induced by immunization with the diepitopic versus monoepitopic peptide construct, groups of weanling rats were immunized s.c. on day 0 (at 24 days of age) and on day 13 with SYI-CAT or with the component peptide constructs. The IgG responses to peptides and native proteins are shown in Fig. 2 for sera taken prior to infection (day 22). Immunization with the diepitopic peptide construct significantly (P < 0.001) enhanced the detectable serum IgG response to CAT (Fig. 2A). However, the serum IgG response to SYI-CAT itself (Fig. 2B) was not significantly different from the response induced by monoepitopic SYI. In contrast, the SYI-immunized group induced a significantly higher level of antibody to SYI (Fig. 2C) than did the diepitopic peptide construct, although the SYI-CAT-immunized group also demonstrated a significant level of SYI-reactive antibody.
|
The mean levels of IgA antibody to native GTF and GbpB proteins in saliva samples collected on day 22 were modest and did not reach significance (Fig. 2). However, as with the serum IgG antibody levels, saliva from the group immunized with the diepitopic peptide construct had the highest mean IgA antibody level to GTF, while saliva from the group immunized with the SYI peptide construct demonstrated the highest mean IgA antibody reactivity with GbpB.
The preinfection sera from experiment 2 were also analyzed for their ability to inhibit GTF-mediated synthesis of water-insoluble glucan from sucrose (Fig. 3). Paralleling the ELISA observations, sera from the diepitopic SYI-CAT-immunized group showed low but significant (P < 0.01) inhibition of GTF activity compared to other peptide- or sham-immunized groups.
|
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The effect of the diepitopic construct on the immune response to mutans streptococcal GTF was more striking. The contribution of SYI in SYI-CAT not only potentiates the response to CAT, but more significantly, it strongly enhances the portion of that antibody response that reacts with (Fig. 1 and 2) and inhibits (Fig. 3) GTF. The CAT peptide sequence contains an aspartate which, when in the context of S. mutans and S. sobrinus GTF sequences, has been demonstrated by molecular genetic and biochemical approaches to be critical to the catalytic properties of GTF (6, 12). The B-cell epitope within the CAT peptide subsumes this aspartate (12); thus, antibody raised to CAT is expected to, and has previously been demonstrated (20, 25) to, interfere with GTF function. Thus, this diepitopic construct, containing a putative T-cell epitope unrelated to GTF, appears to significantly enhance the serum IgG immune response to this important epitope.
Previously we observed an approximately twofold increase in salivary IgA antibody level to GTF when rats were immunized with the GTF-based dipeptide CAT-GLU, rather than with the monoepitopic CAT peptide (24). In the present experiment, only a modest salivary IgA response to GTF was detected when CAT was presented in the context of the diepitopic SYI-CAT construct. Zhang and coworkers (26) attempted to enhance the mucosal response to the immunologically weak S. mutans salivary binding receptor component by intranasally immunizing with this domain, presented as a chimeric protein with a sequence reflecting most of the glucan binding domain. They observed a 10-fold increase in salivary IgA response to the SBR domain, although the response to the native AgI/AgII adhesin was not reported. The difference in ability to increase the mucosal responses may be related to the significant difference in the relative sizes of the synthetic peptide and the protein chimera or to the route of immunization. However, both studies reinforce the ability to broaden and, in some cases, to enhance the immune response to multiple mutans streptococcal virulence antigens.
Interestingly, addition of the GTF-irrelevant SYI peptide to the diepitopic construct containing the GTF-sequence-based GLU peptide showed no enhancement of a serum IgG response to GTF (Fig. 1). This lack of enhancement was observed despite the fact that SYI-GLU induced high levels of serum IgG and salivary IgA antibody to GLU (data not shown). These responses were more consistently elevated than those induced by the respective monoepitopic MAP antigens. Thus, the diepitopic construct SYI-GLU, which contained T- and B-cell epitopes on both peptide components, improved responses to constituent peptide epitopes but not necessarily to GTF. The lack of a detectable improvement in antibody reactive with GTF is perhaps because the cross-reactive epitopes may be masked in the MAP construct, resulting in a response of low affinity and/or amount. The lack of detectable antibody to GTF in most animals immunized with the GLU construct may be a consequence of using a lower dose of peptide than was used in previous experiments (18, 24).
Groups of rats immunized with monoepitopic SYI or CAT constructs and infected with S. mutans strains had lower levels of molar caries than sham-immunized rats (Fig. 4), as previously observed in experiments using higher doses (50 µg) of the various peptide constructs (17, 22, 24). The protective responses to S. mutans infection were presumably based on antibody to epitopes on GbpB (via SYI) and GTF-B (via CAT), which are native proteins that have been associated with different aspects of the molecular pathogenesis of S. mutans-mediated dental caries. The specificity of protection was confirmed in the S. sobrinus infection experiment, since lower caries scores were observed only in the groups immunized with CAT-containing constructs which share a sequence with GTFs of both mutans streptococcal species. The modest protection achieved in rats immunized with CAT occurred despite the inability to detect serum IgG or salivary IgA antibody to the CAT construct alone or to GTF in most CAT-immunized rats prior to infection. Antibody to CAT could, however, be detected at the end of the experiment (data not shown). As indicated above, the CAT peptide construct appears to be less immunogenic than SYI or GLU, and the use of a lower peptide-immunizing dose presumably influenced antibody detection. Nonetheless, caries reductions following CAT immunization underscore the functional significance of amino acid residues with the respective GTF sequences.
Although the SYI-CAT diepitopic construct induced protective responses after infection with cariogenic mutans streptococci, the extent of protection was similar to that observed with the monoepitopic construct(s). Other workers have recently examined the effect of multivalent caries vaccines coding for or containing major adhesin (AgI/AgII) and glucan binding (GTF) domains. By using the percentage of S. mutans cells per total number of streptococci as the measure of protection, Zhang and coworkers (26) observed a significant reduction after immunization with the chimeric SBR-GLU construct but saw no significant reductions after immunization with constituent 42-kDa SBR or 33.5-kDa GLU proteins. Previously, Jespersgaard et al. (5) demonstrated caries-protective effects with the GLU polypeptide alone. Guo and coworkers (3) found that s.c. immunization with fusion DNA vaccines coding for both the GLU of GTF and the adhesin domains of AgI/AgII gave marginally better protection from enamel caries than did a DNA vaccine containing only the adhesin domains. Variations in the model system, including animal species, infection regimens, antibiotic suppression, dietary sucrose levels, and age of infection limit the comparability of these three approaches. The semiquantitative disease data obtained in these experimental models can present a challenge for reliable measurement of degrees of protection, in that significant differences in in vitro measures of antibody levels may not always be accompanied by commensurate differences in measures of disease.
In summary, the present studies have shown that the serological and functional inhibitory responses to an epitope associated with the catalytic activity of GTF can be significantly enhanced by epitopes from a peptide (SYI) from another virulence component that, by itself, can induce only a specific protective immune response to S. mutans. Conversely, the addition of the GTF-derived CAT epitope to the GbpB-derived SYI peptide broadens the resulting protective immune response to include S. sobrinus. Taken together, the combination of epitopes associated with function or immunogenicity from more than one mutans streptococcal virulence component through recombinant DNA vaccine, chimeric protein, or synthetic peptide approaches is a promising approach in the design of more-effective dental caries vaccines.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| 1. | Abo, H., T. Matsumura, T. Kodama, H. Ohta, K. Fukui, K. Kato, and H. Kagawa. 1991. Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase). J. Bacteriol. 173:989-996. |
| 2. | Banas, J. A., and M. M. Vickerman. 2003. Glucan-binding proteins of the oral streptococci. Crit. Rev. Oral Biol. Med. 14:89-99. |
| 3. | Guo, J. H., R. Jia, M. W. Fan, Z. Bian, Z. Chen, and B. Peng. 2004. Construction and immunogenic characterization of a fusion anti-caries DNA vaccine against PAc and glucosyltransferase I of Streptococcus mutans. J. Dent. Res. 83:266-270. |
| 4. | Hamada, S., and H. D. Slade. 1980. Biology, immunology and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44:331-384. |
| 5. | Jespersgaard, C., G. Hajishengallis, Y. Huang, M. W. Russell, D. J. Smith, and S. M. Michalek. 1999. Protective immunity against Streptococcus mutans infection in mice after intranasal immunization with the glucan-binding region of S. mutans glucosyltransferase. Infect. Immun. 67:6543-6549. |
| 6. | Kato, C., Y. Nakano, M. Lis, and H. K. Kuramitsu. 1992. Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases. Biochem. Biophys. Res. Commun. 189:1184-1188.[CrossRef][Medline] |
| 7. | Keyes, P. H., and H. V. Jordan. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Arch. Oral Biol. 9:377.[CrossRef] |
| 8. | Koga, T., T. Oho, Y. Shimazaki, and Y. Nakano. 2002. Immunization against dental caries. Vaccine 20:2027-2044.[CrossRef][Medline] |
| 9. | Lis, M., T. Shiroza, and H. K. Kuramitsu. 1995. Role of the C-terminal direct repeating units of the Streptococcus mutans glucosyltransferase-S in glucan binding. Appl. Environ. Microbiol. 61:2040-2042.[Abstract] |
| 10. | Mattos-Graner, R. O., S. Jin, W. F. King, T. Chen, D. J. Smith, and M. J. Duncan. 2001. Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates. Infect. Immun. 69:6931-6941. |
| 11. | Merrifield, R. B. 1963. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-2154.[CrossRef] |
| 12. | Mooser, G., S. Hefta, R. J. Paxton, J. E. Shively, and T. D. Lee. 1991. Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases. J. Biol. Chem. 266:8916-8922. |
| 13. | Mooser, G., and C. Wong. 1988. Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus. Infect. Immun. 56:880-884. |
| 14. | Smith, D. J. 2002. Dental caries vaccines: prospects and concerns. Crit. Rev. Oral Biol. Med. 13:335-349. |
| 15. | Smith, D. J., H. Akita, W. F. King, and M. A. Taubman. 1994. Purification and antigenicity of a novel glucan-binding protein of Streptococcus mutans. Infect. Immun. 62:2545-2552. |
| 16. | Smith, D. J., W. F. King, L. A. Barnes, Z. S. Peacock, and M. A. Taubman. 2003. Immunogenicity and protective immunity induced by synthetic peptides associated with putative immunodominant regions of Streptococcus mutans glucan-binding protein B. Infect. Immun. 71:1179-1184. |
| 17. | Smith, D. J., and M. A. Taubman. 1996. Experimental immunization of rats with a Streptococcus mutans 59-kilodalton glucan-binding protein protects against dental caries. Infect. Immun. 64:3069-3073.[Abstract] |
| 18. | Smith, D. J., M. A. Taubman, C. J. Holmberg, J. W. Eastcott, W. F. King, and P. Ali-Salaam. 1993. Antigenicity and immunogenicity of a synthetic peptide derived from a glucan-binding domain of mutans streptococcal glucosyltransferase. Infect. Immun. 61:2899-2905. |
| 19. | Smith, D. J., M. A. Taubman, W. F. King, S. Eida, J. R. Powell, and J. Eastcott. 1994. Immunological characteristics of a synthetic peptide associated with a catalytic domain of mutans streptococcal glucosyltransferase. Infect. Immun. 62:5470-5476. |
| 20. | Tam, J. P. 1988. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 85:5409-5413. |
| 21. | Taubman, M. A., C. J. Holmberg, and D. J. Smith. 1995. Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic regions of mutans streptococcal glucosyltransferase protects against dental caries. Infect. Immun. 63:3088-3093.[Abstract] |
| 22. | Taubman, M. A., C. J. Holmberg, D. J. Smith, and J. W. Eastcott. 1995. T and B cell epitopes from peptide sequences associated with glucosyltransferase function. Clin. Immunol. Immunopathol. 76:s95. |
| 23. | Taubman, M. A., D. J. Smith, C. J. Holmberg, and J. W. Eastcott. 2000. Coimmunization with complementary glucosyltransferase peptides results in enhanced immunogenicity and protection against dental caries. Infect. Immun. 68:2698-2703. |
| 24. | Taubman, M. A., C. J. Holmberg, and D. J. Smith. 2001. Diepitopic construct of functionally and epitopically relevant complementary peptides enhances immunogenicity, reactivity with glucosyltransferase, and protection from dental caries. Infect. Immun. 69:4210-4216. |
| 25. | Yu, H., Y. Nakano, Y. Yamashita, T. Oho, and T. Koga. 1997. Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans. Infect. Immun. 65:2292-2298.[Abstract] |
| 26. | Zhang, P., C. Jespersgaard, L. Lamberty-Mallory, J. Katz, Y. Huang, G. Hajishengallis, and S. M. Michalek. 2002. Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection. Infect. Immun. 70:6779-6787. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
