Transfer of Advances in Science into Dental Education

Caries Vaccines for the Twenty-First Century

Daniel J. Smith, Ph.D.
Abstract: Can infection with the dental caries pathogen, Streptococcus mutans, be intercepted or modified immunologically? Resolving this question requires answers to many questions: What are the pathways by which this cariogenic streptococcus enters and accumulates in the dental biofilm? Can bacterial components associated with virulence induce immune responses? What is the level of maturity of immune pathways in the oral cavity of the young child at the time of infection? Can immune strategies deal effectively with chronic S. mutans infections? Are these vaccines safe? Many such questions have been answered. For example, preclinical application of modern methods of mucosal vaccine design and delivery has routinely resulted in protection from dental caries caused by S. mutans infection, using antigens involved in the sucrose-independent or sucrose-dependent mechanisms of infection by these cariogenic streptococci. Passive administration of antibody to functional epitopes of S. mutans virulence antigens has also provided a degree of protection in preclinical studies and small-scale human investigations. The caries-protective capacity of active immunization with dental caries vaccines now awaits proof of principle in pediatric clinical trials. Dr. Smith is Senior Staff Member, Department of Immunology, The Forsyth Institute. Direct correspondence and requests for reprints to him at Department of Immunology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115; 617-456-7720 phone; 617-456-0742 fax; dsmith@forsyth.org. His research was performed through the generous support of the National Institute of Dental and Craniofacial Research (DE-01653, DE-04733, DE/AI-12324). This article was adapted from his review that appeared in Critical Reviews in Oral Biology and Medicine 2002;13:335-49. Key words: dental caries, SIgA, vaccine, mucosal immunization, Streptococcus mutans, glucosyltransferase, antigen I/II, glucan binding protein Submitted for publication 8/4/03; accepted 8/20/03

s dental caries conquered? Walk into a day care center in Brazil, and chances are that at least 25 percent of the children over three years of age will have active disease. Go to China where epidemiologists report that three-quarters of five-year-old children experience significant dental decay. Stay at home in the United States, and find that being a young kid on the wrong side of the poverty line brings with it the likelihood of untreated decay. Elders are showing up with disease on their root surfaces. In the United States, we spend $70 billion annually in dental services, a significant portion of which pays for dental caries treatment or conditions resulting from tooth decay. Clearly, we have a long way to go before we can declare victory over this disease. We have made great strides in understanding dental caries etiology. Infection as a key component was uncovered more than 110 years ago by Miller who made the link among microorganisms, dietary carbohydrates, and dental disease.1 Early in the last century Clarke isolated the bacterium, Streptococcus mutans (Figure 1), upon which modern dental research has cast its brightest light. In the latter half of the twentieth century, research efforts at NIH and in Scandinavia confirmed the cariogenic properties
1130

of this organism, demonstrated its transmissibility, and described its worldwide distribution. Later, others identified many of its virulence characteristics and unraveled its biochemistry. Ultimately, the complete genome sequence of S. mutans was reported in 2002.2 Although molecular biological and cultural techniques have also incriminated other bacteria in the process and extension of dental caries in various dental habitats, S. mutans continues to be Public Enemy #1, especially for early childhood dental disease.3 How to treat this disease? Use of fluoride in its many forms, use of sugarless products and sealants, and increased access to dental care are among the approaches that have had a significant impact on the amount of disease of the young and economically advantaged. Many of these approaches can be broadly effective. However, economic, behavioral, or cultural barriers to their use have continued the epidemic of dental disease in the mouths of many children in our global village. Vaccine strategies have been invoked often to diminish or prevent the impact of infectious disease, especially among the young. Vaccines are particularly well suited for public health applications, espeJournal of Dental Education Volume 67, Number 10

Figure 1. Scanning electron micrograph of Streptococcus mutans

Image courtesy of Drs. Renata Mattos-Graner and Ziedonis Skobe.

cially in environments that do not lend themselves to regular health care. Given a general appreciation for the infectious component of dental caries, injected vaccines containing lactobacilli were administered with limited success in the 1940s. However, at that time the molecular pathogenesis of S. mutans was unknown, nor was there an understanding of the immune mechanisms that operate in the oral cavity. Most virulence characteristics were unclear, with the exception of the ability of cariogenic bacteria to produce enamel-dissolving acid. Today we have answered many of these questions, permitting us to more knowledgeably explore the potential for vaccine therapy for dental caries associated with S. mutans.

Acquisition of Mutans Streptococci

The modern era of vaccine therapy began in the late 1960s with William Bowens use of S. mutans to intravenously immunize irus monkeys.4 At that time, it was known that most people carried S. mutans in their dental plaque. It became clear from animal studies, however, that once these organisms colonized dental plaque, they were extremely difficult to dislodge. Thus, most (but not all) of the experimental
October 2003

dental caries vaccine approaches attempted to modify initial infection with S. mutans. Translating this approach to humans required that we know when children first become infected with S. mutans and from whom their infection came. Studies of the natural history of oral streptococcal acquisition by infants revealed that, under normal circumstances of diet and challenge, children become permanently colonized with mutans streptococci between the middle of the second year and the end of the third year of life. Page Caufield et al.5 referred to this period as the window of infectivity. Several groups found that the primary source of infection was maternal, although recent evidence suggests that nonfamilial transfer can occur when environmental conditions strongly favor colonization. The window may open even earlier if maternal S. mutans infection levels are high, coupled with frequent exposure to dietary sucrose. More sensitive techniques for microbial detection, e.g., DNA probe technology, have also suggested that low levels of mutans streptococci may be found in the oral cavity during the first year of life, especially in caries-prone populations.6 However, mutans streptococci in such children are not colonizing their preferred ecological habitat at this early age. Thus, the data suggest that a window of vaccine opportunity could exist between twelve and eighteen months for most populations.
1131

Journal of Dental Education

Despite the influence of maternal dose, children who do not become infected by approximately three years of age appear to remain uninfected, or minimally colonized for several years, possibly until new opportunities for colonization occur upon eruption of the secondary dentition. This suggests that a longer-term benefit may ensue if mutans streptococcal colonization could be impeded in early childhood by measures such as immunization.

Molecular Pathogenesis
Thirty years ago British and American scientists demonstrated that experimental protection could be achieved by immunization with mutans streptococci (reviewed by Michalek and Childers7). Attention then focused on immunologically intercepting properties of these organisms that led to disease. The molecular pathogenesis of mutans streptococci involves several phases, each of which offers targets for immunological intervention (Figure 2). These acidogenic streptococci require the hard surfaces furnished by teeth for sustained colonization and accumulation. To colonize the oral cavity, these streptococci must first bind to pre-existing receptors within dental biofilms. Initial attachment to the tooth occurs by the interaction of bacterial proteins with host-derived components in the dental pellicle covering the tooth surface. These bacterial adhesins, first described by Russell and Lehner8 and often referred to as antigen I/II in Streptococcus mutans, bind acidic, mucin-like glycoproteins found in parotid and submandibular saliva. These components are found in salivary pellicles that coat both the tooth surface and early colonizing bacteria such as Streptococcus sanguis and Actinomyces species. The ultimate pathogenicity of mutans streptococci occurs through erosion of the hydroxyapatitelike mineral in dental enamel by lactic acid, a metabolic end product of bacterial growth. However, significantly destructive concentrations of this acid require the substantial accumulation of these acidogenic streptococci in dental plaque. This accumulation process is initiated by the activity of extracellular glucosyltransferases (GTF) of which several are constitutively secreted by mutans streptococci.9,10 In the presence of dietary sucrose, GTFs synthesize several forms of high molecular weight branched extracellular glucans. GTFs that synthesize insoluble

forms of glucan (S. mutans GTF-B and GTF-C) have been most closely associated with pathogenicity. These glucose polymers provide scaffolding for the aggregation of mutans and other oral streptococci through interaction with bacterial cell-associated glucan-binding proteins. Furthermore, glucans modify the porosity of the dental biofilm, thus increasing the availability of nutrients for continued bacterial metabolism. Several glucan-binding proteins have been described in mutans streptococci.11-14 Although each of these glucan-binding proteins (GBPs) has the ability to bind to certain forms of glucan and some have been shown to be cell-associated, their unique contributions to in vivo plaque development are as yet unclear. GTFs also contain glucan-binding domains. The interactions of glucans with cellassociated glucan-binding domains of GTFs and GBPs combine to cause extensive accumulation of mutans streptococci in the dental biofilm (Figure 2). Since GTFs and GBPs are also secreted into the extracellular environment, their specific or nonspecific incorporation into the salivary pellicle would also provide binding sites for mutans streptococci. Theoretically, the next phase of pathogenesis results from the metabolic activities of these masses of accumulated mutans streptococci (and possibly of other accumulation-associated microorganisms). Mutans streptococci are the most prolific producers of lactic acid in these accumulations although other low pH bacteria may also contribute.15 Dental caries ultimately ensues because the resulting increase in lactic acid concentration cannot be sufficiently buffered to prevent enamel dissolution. Thus, several stages in the molecular pathogenesis of dental caries are susceptible to immune intervention. Microorganisms can be cleared from the oral cavity while still in the salivary phase by antibody-mediated aggregation. Antibody could also block the receptors necessary for colonization (e.g., adhesins) or accumulation (e.g., glucan-binding domains of GBPs and GTF) within the dental biofilm. Immune inactivation of GTF enzymes would prevent formation of the glucan matrix. Modification of metabolically important functions may also be targeted. In addition, the antimicrobial activity of salivary antibody may be enhanced or redirected by synergism with innate components of immunity, such as mucins or lactoferrin. As we will see, immunological intervention by many of these components has achieved a measure of experimental success.

1132

Journal of Dental Education Volume 67, Number 10

Ontogeny of Immunity in Saliva

Immunological interception of the initial attempts of mutans streptococci to colonize the tooth surface would seem to be the preferred vaccine strategy since these organisms are exceedingly difficult to displace once they become part of the dental biofilm. Given the natural history of mutans streptococcal infection, this strategy would require yearold children to be sufficiently mature immunologically to form protective levels of antibody in their oral cavity at this time. Secretory IgA (SIgA) is the principal immune component of major and minor gland salivary secretions and thus would be considered to be the primary mediator of adaptive immunity in the salivary milieu. The need to understand the rate and characteristics of salivary immune development triggered a series of studies that now support the rationale for caries vaccine applications in early childhood (Figure 3). These studies revealed that immunity in the oral cavity undergoes rapid, early development (reviewed in Smith and Taubman16). Although SIgA antibody in saliva and other secretions is essentially absent at birth, mature SIgAi.e., dimeric IgA with bound secretory componentis the principal salivary immunoglobulin secreted by one month of age. Induced by the environmental antigenic challenge, mucosal IgA antibody to pioneer gut (e.g., Escherichia coli) and oral (e.g., Streptococcus mitis and S. salivarius) microbiota appears in secretions (including saliva) within weeks of initial microbial exposure. By six to nine months of age most children exhibit an adultlike distribution of salivary IgA subclasses, which include antibody to several antigens of the predominant pioneer oral flora. Can children respond to natural exposure to mutans streptococci? The answer is yes. Salivary antibody to mutans streptococcal antigens is usually first observed in the second and third years of life.17 Salivary responses are often directed to those streptococcal components that are important in colonization and accumulation, such as antigen I/II, GTFs, and GBPs. Longitudinal studies suggest that these antibody specificities result from contact with mutans streptococci, rather than with earlier colonizing oral streptococci, since well-developed salivary IgA antibody to pioneer oral streptococci is present prior to the detection of antibody reactive with mutans strep-

tococci. Interestingly, in some children, antibody to mutans streptococcal antigens can also be detected independently of the ability to detect ongoing infection in the second year of life. As is the case with many bacterial challenges throughout the body, the threshold of immunological response can be lower than that of persistent infection; therefore, it is not surprising to observe antibody to S. mutans antigens in the absence of its colonization. Thus colonization, at least not extensive colonization, with mutans streptococci is apparently not required for the development of salivary antibody to associated mutans streptococcal antigens. Thus, the evidence from salivary IgA responses to commensal oral microbiota indicates that the mucosal immune system is relatively well developed by the period during which children typically become infected with mutans streptococci. Most children apparently respond immunologically to transient infection or ongoing colonization with mutans streptococci in early childhood. Although the distribution and specificity of childrens responses are not identical, antibody to a few major antigens predominates. These data suggest the possibility that such responses could be protective if induced prior to critical colonization events.

Specific Vaccine Targets

What bacterial components make the most effective vaccines? Preclinical studies reveal that several of the protein components involved in the molecular pathogenesis of mutans streptococci can induce protective immunity. Furthermore, protective immunity can be achieved by concentrating the immune response on suspected functional elements of these components either using synthetic peptides or recombinant DNA approaches that permit the expression of complete functional domains (for recent reviews see Russell,18 Koga et al.,19 and Smith20). Adhesins. For example, the family of adhesins from Streptococcus mutans and Streptococcus sobrinus has been shown to be effective antigens, both as intact proteins and as subunit vaccines. These single polypeptide chains are approximately 1600 residues in length and, in S. mutans, contain salivary binding domains associated with an alanine-rich tandem repeating region in the N terminal third and a proline-rich repeat region in the center of the molecule. Abundant in vitro and in vivo evidence, using

October 2003

Journal of Dental Education

1133

a variety of active and passive immunization approaches, indicates that antibody with specificity for mutans streptococcal adhesins can interfere with bacterial adherence and subsequent dental caries caused by S. mutans. Effective subunit vaccines have been designed using synthetic peptides or recombinant proteins to direct the immune response to domains of salivary binding function. Immunization with these constructs is also protective in experimental systems. Protection in these experiments could conceivably occur by antibody blockade of initial colonization events in the dental biofilm or by antibody-mediated aggregation and clearing of adhesinbearing streptococci from the bulk fluid salivary phase. Glucosyltransferases. Mutans streptococci that have lost the ability to make glucan through natural or induced mutations in GTF genes do not produce significant disease in animal models. Growth of mutans streptococci in the presence of antibody to GTF significantly diminishes the amount of biofilm on glass surfaces. Thus it was not surprising that immunization studies using intact GTF vaccines successfully protected animals infected with S. mutans. Passive administration of antibody to GTF in the diet was also protective. GTF is an interesting protein in that it does several things. This enzyme cleaves the bond between the glucose and fructose moieties in sucrose. Activated glucose is then transferred to a growing glucan polymer. Glucans can also bind to an area of repeating sequences in the Cterminal third of the enzyme. These areas of function offered several possibilities for more targeted immune responses. Synthetic peptide or recombinant protein vaccines, designed to include one or more suspected areas of function, protect animal models from experimental dental caries. Since GTFs from the two major cariogenic streptococcal species in humans, S. mutans and S. sobrinus, have very similar sequences in these functional domains, immunization with GTF protein or subunit vaccines from one species can induce a measure of protection for the other species. Thus, the presence of antibody to GTF in the oral cavity, prior to infection, can significantly influence the disease outcome, presumably by interference with one or more of the functional activities of the enzyme. Glucan-binding proteins. Since glucan-binding proteins on the surface of mutans streptococcal cells may provide the receptors for glucan-mediated aggregation, these proteins have also received attention as vaccines. Of the three S. mutans glucan-bind-

ing proteins identified to date, only GbpB has been shown to induce protective immune responses to experimental dental caries. Interestingly, salivas of young children often contain IgA antibody to GbpB, indicating that initial infection with S. mutans can lead to natural induction of immunity to this protein. Bioinformatic methods of identifying molecular regions responsible for this immunogenicity have yielded at least one GbpB subunit vaccine that was effective in preclinical studies. Unlike GTF, however, protection induced by S. mutans GbpB does not extend to S. sobrinus species.

Enhancing Strategies
Routes to Response
Since teeth and the attached biofilms are bathed in antibody-containing saliva, many investigators concentrated on inducing immunity by mucosal routes. Mucosal (SIgA) immune responses can appear in saliva after induction at sites both near and distant from the oral cavity.21 An extreme example of this is the recent demonstration in our laboratory that rectal administration of GTF-induced salivary immunity correlated with experimental dental caries protection. Taking advantage of this characteristic, several mucosal routes have been explored for delivery of dental caries vaccines. Early preclinical studies22 and clinical trials23 used oral delivery of S. mutans antigen to induce immune response in the gut-associated lymphoid tissue. These studies established the principle that protection could be achieved via the mucosal response alone. The oral route is not ideal, however, in part because of the detrimental effects of stomach acid on antigen. Thus, inductive sites in closer anatomical relationship to the oral cavity were explored. Many studies targeted the nasal route to take advantage of the inductive characteristics of nasal-associated lymphoid tissue (reviewed in Russell18). Nasal immunization with mutans streptococcal adhesins, GTF, or GbpB each consistently induced relatively high levels of salivary antibody with concomitant reductions in experimental dental caries. Use of the nasal route in humans is also strengthened by recent successes with a nasally applied influenza vaccine. Tonsillar tissue and minor salivary glands have also been explored as routes to protective immune responses. Although Japanese scientists have reported

1134

Journal of Dental Education Volume 67, Number 10

preclinical protection in rabbits after tonsillar application of mutans streptococci, Noel Childers group24 showed that humans responded better to nasal versus tonsillar application of GTF when salivary antibody was the readout. A clinical trial in our own laboratory suggested that topical application of GTF to labial minor salivary glands could result in delayed reaccumulation of mutans streptococci in young men.25

Immunomodulators and Delivery Systems

Mucosal application of soluble protein or peptide antigens by themselves rarely results in sustained IgA responses. Considerable effort, therefore, has been expended to develop immunomodulators (adjuvants) and delivery systems that enhance mucosal responses, including responses to dental caries vaccines. Enterotoxins such as cholera toxin are powerful mucosal adjuvants. Of course, inherent toxicity precludes human use of the holotoxin. The toxicity issue can be circumvented by using nontoxic parts of the protein or by mutating various residues in the A1 subunit associated with toxic properties. Various degrees of adjuvant activity remain. In some cases, antibody production and protective immunity achieved by combining antigen with modified adjuvants is nearly comparable to that achieved with the holotoxin.26,27 Efforts continue to engineer these enterotoxins for human applications. Improvements have also been made in delivery of vaccine to inductive sites for mucosal immune responses. Liposomes, which are phospholipid membrane vesicles thought to home to mucosally associated lymphoid tissues, can be manufactured to contain vaccine antigens. Preclinical and clinical studies have shown superior salivary responses after mucosal application of GTF-containing liposomes (reviewed in Russell18 and Childers et al.24). Microcapsules and microparticles made of poly(lactide-co-glycolide) (PLGA) have also been used to deliver dental caries vaccines because of their ability to control the rate of release, evade preexistent antibody clearance mechanisms, and degrade slowly without eliciting an inflammatory response to the polymer. Mucosal application of antigens incorporated into PLGA enhance mucosal responses and can facilitate protective salivary immune responses to infectious challenge with mutans streptococci in preclinical studies.28 Starch microparticles can also increase mucosal responses to soluble antigens.29

The pathway to mucosal immune responses in the gut begins by capture of antigens from the lumen by specialized epithelial cells (so-called M cells) that overlie mucosal lymphoid tissue. These cells then pass their captured contents to underlying lymphoid tissue, ultimately resulting in SIgA antibody in secretions. Since Salmonella naturally colonize M cells, these organisms have been engineered for use as replicating antigen delivery systems for which mucosal responses are desired. Attenuated expression vectors can be designed to contain plasmids expressing recombinant peptides that contain mutans streptococcal virulence epitopes alone or in combination with sequences having adjuvant properties. These constructs can induce protective immunity for experimental dental caries after oral, intragastric, or intranasal administration (reviewed in Russell18 and Smith20).

Other Immune Sources of Protective Antibody

Saliva need not be the only source of protective antibody in the oral cavity. Endogenous sources of antibody can also be supplied via the gingival crevicular fluid after systemic injection, chiefly in the form of IgG antibody. Preclinical evidence for the effectiveness of this route was provided in the 1970s by the British group led by Thomas Lehner who showed that subcutaneous injection of primates with mutans streptococcal cells led to significant reductions in smooth surface and fissure caries.30 Later, this group showed that similar protection followed injection of a purified mutans streptococcal adhesin.31 Again, protective immunity was thought to be based on exposure of infecting mutans streptococci to IgG antibody emanating from gingival crevicular fluid. The Lehner group then demonstrated that antibody need not be produced actively by the host, but that protection could be achieved by passive administration of IgG antibody.32 Early studies used mouse monoclonal antibody to salivary binding determinants of the adhesin as the passive antibody source to demonstrate that exogenous antibody of the appropriate specificity could also modify infection and disease. Other groups have added antibody reagents to drinking water, mouthwashes, or the diet and achieved some protective effects in preclinical studies (reviewed in Koga et al.19). Julian Ma et al.33 used transgenic antibody fashioned in tobacco plants to

October 2003

Journal of Dental Education

1135

Figure 2. Molecular pathogenesis of mutans streptococci (MS)

provide a passive form of protection in small-scale clinical trials. In these studies much of the dental biofilm of young adult subjects was first removed by vigorous treatment of the teeth with chlorohexidine-based antibacterials. Repeated topical applications with mouse monoclonal reagents or transgenic antibody were then administered for several weeks. Recolonization with mutans streptococci did not occur for at least two years after treatment of subjects with mouse monoclonal antibody or at least four months after treatment with the transgenic antibody. In contrast, the teeth of all control subjects were recolonized with mutans streptococci within a few months. How does a short-term topical treatment with antibody result in an apparent long-term interference with S. mutans recolonization? The best explanation may be an ecological one. Thus, antibody blockage of an important adhesin epitope during the reconstruction of the dental biofilm following chlorohexidine treatment places indigenous mutans

streptococci at an insurmountable competitive disadvantage for recolonization. An interesting parallel may be the observation that young children who do not become naturally infected with mutans streptococci during the window of infectivity remain undetectably infected for several years, potentially because its habitat in the dental biofilm has been filled by other indigenous flora.

Prospects for the Twenty-First Century

Where do we stand today? Mutans streptococcal GTFs, adhesions, and GbpB each induce cariesprotective immunity in experimental systems, thus are likely candidates for initial pediatric clinical trials. Mucosal application would be expected to be safe since children are already naturally exposed to these components during the first years of life. Re-

1136

Journal of Dental Education Volume 67, Number 10

finements to the vaccine approach, such as identification of additional protective epitopes associated with functional domains, continue to be made and incorporated into single- or multi-component constructs to increase their protective value. Several routes of administration can induce protective immune responses, at least in model systems. Also, significant progress is being made in the adjuvant field to remove the toxic properties of powerful mucosal adjuvants while maintaining their adjuvant properties. Animate (attenuated bacterial vectors) and inanimate (liposomes, microparticles) delivery systems can provide more efficient targeting of the vaccine. Both passive and active immunization approaches have demonstrated success in animal models and human clinical trials in adults. What about tomorrow? Traditional vaccine therapy ideally induces protective immunity prior to infection with the target pathogen. The window of mutans streptococcal infection opens in the middle of the second year of life under normal infectious challenge. To meet this challenge, year-old children would receive a caries vaccine in order to intercept

initial colonization events. Our understanding of the ontogeny of the mucosal immune response indicates that children at this age are competent to mount secretory responses to the proteins that have been suggested as vaccine components. A mucosally applied pediatric dental caries vaccine would be truly novel as the first nonreplicating vaccine applied by any mucosal route. Furthermore, the character of the pediatric response to a nasally applied caries vaccine would tell us about the potential for immunization by mucosal routes, not just for dental caries, but also for the many pathogens that cause disease in the gut or airways. Provided that these trials demonstrate vaccine efficacy, the refinements made to caries vaccine design, delivery, and immune enhancements may provide necessary flexibility in subsequent vaccine applications. For example, the ideal vaccine for a child with asthma, the second most common chronic childhood ailment, may be at a mucosal site and with an adjuvant (e.g., detoxified enterotoxin) that is quite different from that sufficient to give a protective response to a healthy child. Also, from economic and

Figure 3. Association of oral colonization with salivary IgA immune responses (Ab) to indigenous flora

October 2003

Journal of Dental Education

1137

societal standpoints, a vaccine strategy for children to whom the full advantages of pediatric care are available may not be the ideal approach for children with limited health care access. For the former group of children, perhaps an intranasal application of a GTF or AgI/II-based vaccine may be best. In contrast, for children in developing countries, the ideal vaccine may be an attenuated Salmonella expression system that contains vaccine epitopes for multiple infectious diseases (e.g., mutans streptococcal, Salmonella, and rotovirus infections) in order to induce protective responses to several childhood diseases and to provide herd immunity by shedding the vaccine strain. Furthermore, passive immunization with antibody that is mass produced in plants may have particular application in older populations as well as a supplement to active immunization strategies in children. Several challenges face the development of an effective dental caries vaccine. However, the oral health impact on children of the twenty-first century would be enormous, especially for those whose economic or cultural position puts them at increased caries risk. The expected reduction in disease would save tens of billions of dollars annually in expenditures for dental treatment. Associated benefits may accrue if new pathways to intercept some of the most debilitating gastrointestinal diseases of early childhood are uncovered from knowledge gained through clinical trials of mucosally applied dental caries vaccines.

REFERENCES
1. Miller WD. The microorganisms of the human mouth. Philadelphia: S.S. White Dental Mfg. Co., 1890. 2. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 2002;99:14434-9. 3. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80. 4. Bowen WH. A vaccine against dental caries: a pilot experiment in monkeys (Macaca irus). Br Dent J 1969;126:159-60. 5. Caufield PW, Cutter GR, Dasanayake AP. Initial acquisition of mutans streptococci by infants: evidence for a discrete window of infectivity. J Dent Res 1993;72:37-45. 6. Milgrom P, Riedy CA, Weinstein P, Tanner AC, Manibusan L, Bruss J. Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children. Community Dent Oral Epidemiol 2000;28:295-306.

7. Michalek SM, Childers NK. Development and outlook for a caries vaccine. Crit Rev Oral Biol Med 1990;1:3754. 8. Russell MW, Lehner T. Characterization of antigens extracted from cells and culture fluids of Streptococcus mutans serotype c. Arch Oral Biol 1978;23:7-15. 9. Shiroza T, Ueda S, Kuramitsu HK. Sequence analysis of the gtfB gene from Streptococcus mutans. J Bacteriol 1987;169:4263-70. 10. Russell RRB, Gilpin ML, Mukasa H, Dougan G. Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli. J Gen Microbiol 1987;133:935-44. 11. Russell RRB. Glucan-binding proteins of Streptococcus mutans serotype c. J Gen Microbiol 1979;112:197-201. 12. Banas JA, Russell RR, Ferretti JJ Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt. Infect Immun 1990;58:667-73. 13. Smith DJ, Akita H, King WF, Taubman MA. Purification and antigenicity of a novel glucan binding protein of Streptococcus mutans. Infect Immun 1994;62:2545-52. 14. Sato Y, Yamamoto Y, Harutoshi K. Cloning and sequence analysis of the GbpC gene encoding a novel glucan-binding protein of Streptococcus mutans. Infect Immun 1997;65:668-75. 15. van Ruyven FO, Lingstrom P, van Houte J, Kent R. Relationship among mutans streptococci, low-pH bacteria, and iodophilic polysaccharide-producing bacteria in dental plaque and early enamel caries in humans. J Dent Res 2000;79:778-84. 16. Smith DJ, Taubman MA. Ontogeny of immunity to oral microbiota. Crit Rev Oral Biol Med 1992;3:109-33. 17. Smith DJ, King WF, Akita H, Taubman MA. Association of salivary IgA antibody and initial mutans streptococcal infection. Oral Microbiol Immunol 1998;13:278-85. 18. Russell MW. Potential for vaccines in the prevention of caries lesions. Oper Dent 2001(Suppl);6:51-60. 19. Koga T, Oho T, Shimazaki Y, Nakano Y. Immunization against dental caries. Vaccine 2002;20:2027-44. 20. Smith DJ. Dental caries vaccines: prospects and concerns. Crit Rev Oral Biol Med 2002;13:335-49. 21. Mestecky J. Saliva as a manifestation of the common mucosal immune system. Ann N Y Acad Sci 1993;694:184-94. 22. Michalek SM, McGhee JR, Mestecky J, Arnold RR, Bozzo L. Ingestion of Streptococcus mutans induces secretory IgA and caries immunity. Science 1976;192:123840. 23. Smith DJ, Taubman MA. Oral immunization of humans with Streptococcus sobrinus glucosyltransferase. Infect Immun 1987;55:2562-9. 24. Childers NK, Tong G, Li F, Dasanayake AP, Kirk K, Michalek SM. Humans immunized with Streptococcus mutans antigens by mucosal routes. J Dent Res 2002;81:48-52. 25. Smith DJ, Taubman MA. Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci. J Clin Immunol 1990;10:273-81.

1138

Journal of Dental Education Volume 67, Number 10

26. Russell MW, Wu H-Y. Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit. Infect Immun 1991;59:4061-70. 27. Smith DJ, King WF, Barnes LA, Trantolo D, Wise DL, Taubman MA. Facilitated intranasal induction of mucosal and systemic immunity to mutans streptococcal glucosyltransferase peptide vaccines. Infect Immun 2001;69:4767-73. 28. Smith DJ, Lam A, Barnes LA, King WF, Peacock Z, Wise DL, et al. Remote glucosyltransferase-microparticle vaccine delivery induces protective immunity in the oral cavity. Oral Microbiol Immunol 2003;18:240-8. 29. Montgomery PC, Rafferty DE. Induction of secretory and serum antibody responses following oral administration of antigen with bioadhesive degradable starch microparticles. Oral Microbiol Immunol 1998;13:139-49.

30. Lehner T, Challacombe SJ, Wilton JM, Caldwell J. Cellular and humoral immune responses in vaccination against dental caries in monkeys. Nature 1976;264:6972. 31. Lehner T, Russell MW, Caldwell J. Immunisation with a purified protein from Streptococcus mutans against dental caries in rhesus monkeys. Lancet 1980;1:995-6. 32. Lehner T, Russell MW, Challacombe SJ, Scully CM, Hawkes JE. Passive immunisation with serum and immunoglobulins against dental caries in rhesus monkeys. Lancet 1978;1:693-5. 33. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, et al. Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nature Med 1998;4:601-6.

October 2003

Journal of Dental Education

1139