Caries Vaccine - A Seminar

•         Introduction

•         Definition

•         History

•         Specific organism

•         Appropriate immunization agents

•         Mechanism of action of vaccine

•         Routes of administration

•         Present scenario for caries vaccine

•         Conclusion

•         References

Introduction

Dental Caries is the most common dental disease of mankind which is prevalent in developed, developing, and underdeveloped countries and is distributed unevenly among the populations. In the modern world, it has reached epidemic proportions. This global increase in dental caries prevalence affects children as well as adults, primary as well as permanent teeth, and coronal as well as root surfaces.

                             Dental caries is still a major oral health problem in most industrialized countries, affecting 60-90% of school children and the vast majority of adults. Dental caries forms through a complex interaction over time between acid-producing bacteria and fermentable carbohydrate, and many host factors including teeth and saliva. The disease develops in both the crowns and roots of teeth, and it can arise in early childhood as an aggressive tooth decay that affects the primary teeth of infants and toddlers.

               A wide group of microorganisms can be isolated from carious lesions of which Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus fermentum, Actinomyces viscosus are the main pathogenic species involved in the initiation and development of carious lesions. These cariogenic bacteria are capable of producing acid by utilizing sugar which is present in the diet. S. mutans is the most prevalent species among all the microorganisms and has been implicated as a causative organism of dental caries.

 Currently various caries preventive strategies are in use like oral health education, chemical and mechanical control of plaque, use of fluorides, application of pit and fissure sealants etc. 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 people on a global level. Since Dental Caries fulfills the criteria for an infectious disease, the possibilities of Caries vaccine have been considered. Preventive dentistry has taken long strides in the direction of eliminating dental diseases. In this endeavor caries vaccine has generated a good deal of enthusiasm. This modality of treatment can prevent the occurrence of caries on large scale.

Definition

The term vaccine derives from Edward Jenner's 1796 use of cow pox  (Latin vacca- cow), to inoculate humans, providing them protection against smallpox. VACCINE:  “A suspension of attenuated or killed microorganisms administered for the prevention, amelioration or treatment of infectious diseases.” (Stedman’s Dictionary, 1990).They may be prepared from live modified organisms, inactivated or killed organisms, cellular fractions, toxoids or combinations of these.

 Caries Vaccine

•         A vaccine currently under development to treat dental caries by inoculating against bacteria commonly known to contribute to their formation, particularly S. Mutans. (Mosby's dental dictionary, 2^nd ed).

History

The possibility of inducing immunity to dental caries has been considered last many years.

•         Underwood & Milles(1881)- Bacteria are involved in pathogenesis of dental caries. 

•         Miller(1890)- Chemico parasitic theory of dental caries.

•         Goadby (1910)- first to advocate caries control by inoculation of the mouth with organisms which would produce alkaline reaction.

•         J.K.Clarke(1924)- Concluded specific micro-organism Streptococcus mutans is associated with dental caries.

•         Write and Jenkins (1953) – leukocyte count in caries free mouth is more in comparison to caries free mouth.

•         Green (1959) found that caries resistance in a group of dental students was associated with an increased gamma globulin fraction in saliva.

•         Sweencey et al (1966) were also unsuccessful in reducing dental decay in caries susceptible rats immunised by gamma globulin.

•         Wagner (1967) was able to protect gnotobiotic rats against caries by inoculating them with a homologus bacteria in an adjuvant.

•         Bowen (1974) and Cole et al (1977): showed that lactoferrin, lactoperoxidase and lysozyme possess antibacterial effects in vitro and therefore would exert considerable effect on bacterial flora in vivo.

•         Challacombe et al (1980) :  Titers of specific serum Ab against S.mutans higher in subjects with low DMF than with high DMF

•         Russell et al (1982) suggested combinations of different purified antigens  to obtain a more complete protection.

•         Bolton & Hlava (1982)  :  Salivary IgA Ab to S. mutans (but not LB) were higher in children with no detectable caries than in a comparable group with lesions.

•         Lehnar et al (1985) found local passive immunization  with topical application of monoclonal antibodies to a  surface protein of  S. mutans to be protective in Rhesus monkey.

•         Ramasdonk et al (1991) studied the effect of passive imunization on the colonization of S. sorbinus in rats.

Specific organism

•         Over the past many years there has been a study accumulation of evidence that S. mutans is the bacterium most intimately associated with initiation and development of carious lesion.

•         The concept of vaccine can be visualised primarily with the recognition of  S. mutans as key microbe in caries development.

•         Thus efforts have been directed at preventing its colonization in the oral cavity.

Streptococcus mutans

•         Facultative anaerobic, non haemolytic acidogenic organism producing intra as well as extracellular polysaccharides.

•         This organism fulfills Koch’s postulates as cause of dental caries.

•         It is separated into 7 serotypes.

•         Serotype c is most common

•         Structure of S. Mutans:-

–        Cell wall

–        Protoplastic membrane enclosing protoplast

•         Surface antigen on cell wall are responsible for immunogenicity.

Appropriate immunization agents

•         For a vaccine against any disease, it is obviously to be desired that there should be no adverse effects of its administrations.

•         The adverse consequences of injecting a crude bacterial vaccine may be many and varied.

•         The best way to ensure this is to use a vaccine consisting of a single carefully defined and highly purified antigen.

•         In case of Caries vaccine, the sub cellular components of S. mutans which are proposed as vacccines are:

–        Glucosyltransferases (GTF)

–        Wall associated proteins.

–        Glucan binding proteins

–        Dextranases

Glucosyltransferases

•         The GTF are group of extracellular enzymes involved in the synthesis of polymers from sucrose.

•         In rodent studies, the use of GTF as an immunizing antigen has resulted concomitantly in an inhibition of S. mutans accumulations in dental plaques and in caries reduction.

»        (Taubman et al 1983)

Wall Associated proteins

•         Two purified proteins from the surface of S. mutans serotype c are currently being suggested for use as dental caries vaccine.

•         Antigen B:  Shows some promise as a basis for caries vaccine.

•         It is found in all serotypes.

•         But it is suggested to be heart cross reactive

•         Although the significance of its reactivity is still questioned but hint of suspicion indicates this to be unacceptable at present.

•         Antigen A: Small molecular weight cell wall protein

·         Shown to be effective in gnotobiotic rats and monkeys.

·         It is quite distinct from heart cross reactive antigen of S. mutans.

·         The vaccine using this antigen has been produced on large scale and initial trial using volunteers is pending.

Glucan binding proteins

•         Glucan binding proteins have also attracted interest as potential vaccine components.

•         This group includes the glucan binding domain found as a part of GTF that has been used as a vaccine on its own (Taubman et al.,1995).

•         Glucan binding domain is also found in GbpA protein and GbpD, which has lipase activity.

•         Since these all are closely related antibody raised against one molecule is likely to react with other targets.

Dextranases

Dextranase, an important enzyme produced by S. mutans, destroys dextran which is an important constituent of early dental plaque so that the bacterium can easily invade dextran- rich early dental plaque. Dextranase when used as an anitigen can prevent colonization of the organism in early dental plaque.

Mechanism of action of vaccine

Proteins called "antigens" which stimulate the immune response.

The resulting immune response is multi-fold to synthesis "antibodies."

 In addition, "memory cells" are also produced in an immune response.

•         Protection against dental caries by immunization would be achieved by immune components from serum, by IgA antibodies in salivary secretions or by a combined effecct serum and salivary components.

•         The 2 main immunological mechanisms involved in protecting the host against dental caries by immunization are:-

–        Production of secretory IgA secreted in the saliva.

–        Systemic immune system and production of anti bodies that travel  through the gingival epithelium  into the crevicular fluid that bathes tooth and plaque.

•         The significance of antibodies in the protection against dental caries lies in that the presence of high levels of antibodies in the gingival fluid has been correlated with low levels of caries.

•         Secretory IgA 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 apart from other immunoglobulins like IgG and IgM which are derived from the gingival circular fluid. In addition to this, gingival sulcus also contains various cellular components of the immune system like lymphocytes, macrophages and neutrophils. Some of the possible ways by which salivary IgA antibodies act against mutans streptococci are given below .

A.      The family of adhesions from Streptococcus mutans and Streptococcus sobrinus has been shown to be effective antigens. The salivary IgA may act as specific agglutinin acting with the bacterial surface receptors and inhibiting colonization and subsequent caries formation. In addition, they may also inactivate surface glucosyltransferase (GTF) which can significantly influence the disease outcome, presumably by interference with one or more of the functional activities of the enzyme resulting in reduce amount of the plaque.

B.      The second important mechanism involves the migration of antigen-sensitized IgA precursor B cells from Gut-Associated Lymphoid Tissues (GALT) to salivary glands. The GALT, including numerous solitary lymphoid nodules and particularly Peyer’s patches, are a rich source of precursor IgA B cells that have the potential to populate distant lymphoid tissues and the salivary glands. These have the potential to inhibit the activity of GTF.

C.      Humoral and cellular components of the systemic immune system are also present at the gingival crevicular level, which may exert its function at the tooth surface also. On the basis of sufficient evidence, it is evident that after a subcutaneous immunization with S. mutans, the organism is phagocytosed and undergoes antigenic processing by macrophages. T and B lymphocytes are sensitized by macrophages in the lymphoid tissue preventing the antigen HLA Class complex and releasing IL-1. Induction of CD-4 helper and CD-8 cytotoxic suppressor cell response takes place. This interaction plays an essential part in modulating the formation of IgG, IgA and IgM antibodies and lymphocytes.

Routes  of  administration

In general two schools of research have evolved.

1. Concerned with IgG and systemic vaccination using a cell wall constituent of S. mutans
2. Concerned with  oral route of vaccination and stimulation of  IgA.

As secretory IgA constitutes a major immune component of major and minor salivary gland secretions, mucosal applications of dental caries vaccine are generally preferred for the induction of secretory IgA antibody in the salivary compartment. Many investigators have shown that exposure of antigen to mucosally associated lymphoid tissue in the gut, nasal, bronchial, or rectal site can give rise to immune responses not only in the region of induction, but also in remote locations. Therefore, a new concept known as the “common mucosal immune system” was put forward by Mestecky. As a result, several routes have been cited by which immunization against S. mutans can be imparted in an individual. The various routes that have been tried out include:

1. Oral route
2. Systemic route
3. Active gingivo-salivary route
4. Intranasal route
5. Tonsilar route
6. Rectal route
7. Active immunization
8. Passive immunization

Oral route

Several of the previous studies relied on oral induction of immunity in the gut-associated lymphoid tissues (GALT) to elicit protective salivary IgA antibody responses. In these studies, antigen was applied by oral feeding, gastric intubation, or in vaccine-containing capsules or liposomes. Various animal trials that were conducted on germ free rats by administering them with killed S. mutans in drinking water resulted in significant reduction of caries related to increased level of salivary IgA antibodies. Oral immunization of 7 adult volunteers with an enteric coated capsule containing 500 micrograms of GTF from S. mutans also resulted in elevating salivary IgA antibodies to the antigen preparation. Although the oral route was not ideal for reasons including the detrimental effects of stomach acidity on antigen, or because inductive sites were relatively distant, experiments with this route established that induction of mucosal immunity alone was sufficient to change the course of mutans streptococcal infection and disease in animal models.

Disadvantages:

Rapid breakdown of proteins or peptides.

Systemic route

Serum IgA, IgG and IgM antibodies were produced as a result of successful subcutaneous administration of S. mutans in monkeys. The antibodies find their way into the oral cavity via the gingival crevicular fluid and are protective against dental caries.

Whole cells, cell walls, and the 185 KD Streptococcal antigen have been administered on various occasions.

                        A subcutaneous injection of killed cells of S. mutans in Freud’s incomplete adjuvant or aluminium hydroxide elicits IgG, IgM, and IgA classes of antibodies. Studies have shown that IgG antibodies are well maintained at high titre, IgM antibodies progressively fall and IgA antibodies increase slowly in titre.

            The development of serum IgG antibodies takes place within months of immunization, reaching a tire of upto 1:1280 with no change in antibodies being found in the corresponding sham-immunized monkeys. Protection against caries was associated predominantly with increased serum IgG antibodies.

Active gingivo-salivary route

In order to limit the potential side effects which are associated with the other routes of vaccine administration, and to localize the immune response, gingival crevicular fluid has been used as the route of administration. Apart of the IgG, it is also associated with increased IgA levels. The various modalities that were tried were as follows-

·         Injecting lysozyme into rabbit gingiva, which elicited local antibodies from cell response.

·         Brushing live S. mutans onto the gingiva of rhesus monkeys failed to induce antibody formation.

·         Using smaller molecular weight Streptococci antigen resulted in better performance probably due to better penetration.

Intranasal route

More recently, attempts have been made to induce protective immunity in mucosal inductive sites that are in closer anatomical relationship to the oral cavity.

                                                                                    Intranasal installation of antigen, which targets the Nasal-Associated Lymphoid Tissue (NALT), has been used to induce immunity to many bacterial antigens, including those associated with mutans streptococcal colonization and accumulation.

                        Protective immunity after infection with cariogenic mutans streptococci could be induced in rats by the IN route with many S. mutans antigens or functional domains associated with these components. Protection could be demonstrated with S. mutans AgI/II, the SBR of AgI/II, a 19- mer sequence within the SBR, the glucan-binding domain of S. mutans GTF-B, S. mutans GbpB and fimbrial preparations from S. mutans with antigen alone or combined with mucosal adjuvants .

Tonsillar route

Great interest has been aroused due to the ability of tonsilar application to induce immune responses in the oral cavity. Tonsillar tissue contains the required elements of immune induction of secretory IgA responses although IgG, rather than IgA, response characteristics are dominant in this tissue.

                        Nonetheless, the palatine tonsils, and especially the nasopharyngeal tonsils, have been suggested to contribute percursor cells to mucosal effector sites, such as the salivary glands.

             In this regard, various trials have shown that topical application of formalin-killed S. sobrinus cells in rabbits can induce a salivary immune response which can significantly decrease the consequences of infection with cariogenic S. sobrinus. Interestingly, repeated tonsillar application of particulate antigen can induce the appearance of IgA antibody- producing cells in both the major and minor salivary glands of the rabbit.

Rectal

More remote mucosal sites have also been investigated for their inductive potential. For example, rectal immunization with nonoral bacterial antigens such as Helicobacter pylori or Streptococcus pneumoniae presented in the context of toxin-based adjuvant can result in the appearance of secretory IgA antibody in distant salivary sites.

            The colo-rectal region as an inductive location for mucosal immune responses in humans is suggested from the fact that this site has the highest concentration of lymphoid follicles in the lower intestinal tract. Preliminary studies have indicated that this route could also be used to induce salivary IgA responses to mutans streptococcal antigens such as GTF. One could, therefore, foresee the use of vaccine suppositories as one alternative for children in whom respiratory ailments preclude intranasal application of vaccine.

Active  immunization

The various approaches for active immunization are:

·         Use of synthetic S. mutans peptides.

Chemically synthesized peptides are better than  parent cell wall

Enhansed immune response.

Avoidance of host tissue reactivity.

·         Coupling  S. mutans antigens to cholera toxin sub units.

·         Fusing S. mutans genes with avirulent Salmonella.

·         Liposome delivery systems.

Passive Immunization

Another approach lies in the development of antibodies suitable for passive oral application against dental caries. This has considerable potential advantage in that it completely avoids any risks that might arise from active immunization.

Conversely, in the absence of any active response on the part of the recipient, there is no induction of immunological memory, and the administered antibodies can persist in the mouth for only a few hours at most or up to 3 days in plaque. Passive antibody administration has also been examined for effects on indigenous mutans streptococci.

Several approaches are tried. These include:-

·         Immune bovine milk

·         Transgenic plant antibody.

·         Monoclonal Antibodies topically applied

·         Egg yolk antibody

Mouthrinses containing bovine milk or hen egg yolk IgY antibody to S. mutans cells led to modest short-term decreases in the numbers of indigenous mutans streptococci in saliva or dental plaque.

            The latest development in the field of passive immunization is the use of transgenic plants to give the antibodies. The researchers have developed a caries vaccine by generating four transgenic Nicotiana tabacum plants that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory component, respectively. The vaccine, which is colourless and tasteless, can be painted onto the teeth rather than injected and is the first plant derived vaccine from GM plants.

                                    Longer-term effects on indigenous flora were observed after topical application of mouse monoclonal IgG or transgenic plant secretory SIgA/G antibody, each with specificity for Ag I/II.Researchers are also working on ways to inject a peptide that blocks the bacterium S. mutans which causes tooth decay into the fruit so that cavities and painful visits to the dentist could  become a thing of the past.

                                                                                                British scientists at Guys Hospital in London have already isolated a gene and the peptide that prevents the bacterium from sticking to the teeth. They are trying to find ways to deliver the peptide into the mouth through apples and strawberries.

Passive administration of preformed exogenous antibodies offers the advantage of evading risks, however small, that are inherent in any active immunization procedure, but the need to provide a continuous source of antibodies to maintain protection over a prolonged time remains a major challenge.

Although new technologies for antibody engineering and production in animals or especially in plants (‘plantibodies’) offer the prospect of reducing the costs sufficiently to enable these materials to be incorporated into products for daily use, such as mouthwashes and dentifrices, long-term efficacy has yet to be reliably demonstrated .

Present scenario

•         Caro Rx^TM

•         SMaRT Replacement THERAPY^TM

         CaroRx

Planet produced the world's first clinically tested Plantibody, CaroRx™. CaroRx™ binds specifically to Streptococcus mutans, and prevents the bacteria from adhering to teeth. CaroRx™ is intended for regular topical preventative administration by both dental hygienists and patients following a thorough cleaning and intervention for any existing decay. CaroRx™ is currently undergoing Phase II U.S. clinical trials under a U.S. FDA-approved Investigational New Drug (IND) application. Clinical trials using CaroRx™ plantibody, funded by Planet and conducted by Planet's collaborators, Drs. Julian Ma and Thomas Lehner at Guy's Hospital, Kings College London, have shown that this treatment can effectively eliminate these decay-causing bacteria for up to two years. Preclinical animal studies have corroborated the antibacterial effect and decay prevention potential of CaroRx™.

How to Use:

CaroRx™ is designed for use by dentists in a program compatible with the normal 6 month to one year interval for periodic check-up and cleaning. The first step in the use of CaroRx™ is to professionally clean the teeth with a commonly used oral antiseptic to temporarily eradicate both benign and decay-causing bacteria. The antisepsis step is immediately followed by applying CaroRx™ to the teeth several times over a two-week period. No further treatments are required for 6 months to 1 year.

The optimum dose of both antibody and antiseptic mouthwash and the required duration of application are expected to be determined in planned Phase II clinical trials.

Mechanism:

The current working hypothesis for the mechanism behind the effectiveness of CaroRx™ is shown below. Many species of bacteria (represented by different shapes and colors) are found on teeth, most of them harmless. Antiseptic treatment kills theS. mutans and many other bacteria. This permits the opening of the "ecological niche" previously occupied by S. mutans. After antisepsis, during CaroRx™ treatment, adhesion of S. mutans to teeth is blocked, while colonization of other oral bacteria occurs unimpeded. After a sufficient period the niche once occupied by S. mutans is occupied by some other organism, or altered in some other way to exclude S. mutans recolonization. At that point no further antibody treatment is needed to excludeS. mutans.

 SMaRT  Replacement  Therapy ™

SMaRT Replacement Therapy™ is designed to be a painless, one-time, five-minute topical treatment applied to the teeth that has the potential to offer lifelong protection against tooth decay caused by S. mutans, the principal cause of this disease.

This therapy technology is based on the creation of a genetically altered strain of S. mutans, called SMaRT, which does not produce lactic acid.

SMaRT strain is engineered to have a selective colonization advantage over native S. mutans strains in that SMaRT produces minute amounts of a lantibiotic that kills off the native strains but leaves the SMaRT strain unharmed. Thus SMaRT Replacement Therapy can permanently replace native lactic acid-producing strains of S. mutans in the oral cavity, thereby potentially providing lifelong protection against the primary cause of tooth decay. The SMaRT strain has been extensively and successfully tested for safety and efficacy in laboratory and animal models

How to use:

SMaRT Replacement Therapy is designed to be applied topically to the teeth by a dentist, pediatrician or primary care physician during a routine office visit. A suspension of the SMaRT strain is administered using a cotton-tipped swab during a single five-minute, pain-free treatment. Following treatment, the SMaRT strain should displace the native, decay-causing S. mutans strains over a six to twelve month period and permanently occupy the niche on the tooth surfaces normally occupied by native S. mutans.

Current Status of this therapy:

They initiated first Phase 1 clinical trial in April 2005, but we found it difficult to find subjects who fit the trials’s highly cautious inclusion and exclusion criteria, particularly with respect to the subjects’ lack of dentition. In August 2007, the FDA issued a clinical hold letter which required revisions to the protocol for offspring of subjects and FDA removed the clinical hold for our Phase 1 trial in the attenuated strain in October 2007 after explanations from ORAGENICS.
                     Then OREGANICS  commenced a second Phase 1 clinical trial for SMaRT Replacement Therapy during the first quarter of 2011. Due to the very restrictive study enrollment criteria required by the FDA, enrollment of candidates meeting the restrictive criteria in the trial has been very slow.
                  The SMaRT strain has been extensively and successfully tested in the laboratory as well as in animal models , and has demonstrated the following:

•No lactic acid creation under any cultivation conditions tested;
•Dramatically reduced ability to cause tooth decay;
•Genetic stability as demonstrated in mixed culture and biofilm studies and in rodent model studies;
•Production of a level of MU1140 that is comparable to its wild-type parent strain, which was previously shown to readily and persistently colonize the human oral cavity;
•Aggressive displacement of native, decay-causing strains of S. mutans and preemptive colonization of its niche on the teeth of laboratory rats.

In addition, during preclinical and early-stage clinical testing of our SMaRT Replacement Therapy, we observed the following:
•No adverse side effects in either acute or chronic testing in rodent models;
•Colonization of the treated subjects following a five-minute application of SMaRT Replacement Therapy in our first Phase 1 study using the attenuated strain;
•No adverse side effects during our first Phase 1 study.

Manufacturing

The manufacturing methods for producing the SMaRT strain of S. mutans are standard Good Manufacturing Practice, or GMP, fermentation techniques. These techniques involve culturing bacteria in large vessels and harvesting them at saturation by centrifugation or filtration. The cells are then freeze dried or suspended in a pharmaceutical medium appropriate for application in the human oral cavity. These manufacturing methods are commonplace and readily available within the pharmaceutical industry.

A single dose of SMaRT Replacement Therapy contains approximately 10 billion S. mutans cells. The SMaRT strain grows readily in a variety of cultivation media and under a variety of common growth conditions including both aerobic and anaerobic incubations. The SMaRT strain can also utilize various carbon and nitrogen sources and is highly acid tolerant. There is no significant limitation to the manufacturing scale of our SMaRT strain other than the size of the containment vessel.

Conclusion:

Is Caries Vaccine Justified From Public Health Point Of View ?

•         Dental caries is declining in most developed countries.

•         In addition it appears by using combination of water fluoridation and weekly fluoride mouth rinsing reduction of 70-80% can be achieved.

•         So this leads to one question that whether a vaccine which carries some risks, is warranted to achieve a further reduction of 20-30%.

•         Encouragement for basic research remains but there are considerable caution about advancing.There appears to be two major hold ups.First: is general but poorly articulated concern over safety associated with introducing a new vaccine against a disease which is not directly life threatening.Secondly: there is a general feeling that caries is under control and hence no novel preventive measure is needed. In some cases special risk groups can be identified and partly explained.

•         Eg:- patients undergoing head and neck radiation therapy.

•         Those with severe xerostomia.

•         Chronologically sick children on continous medication presented in high sucrose syrups

•         Mentally and physically handicapped unable to practice adequate oral hygiene.

•         Children where management of caries is hazardous. Eg congenital or acquired heart disease.

•         In the developing countries where dental caries prevalence is increased caries vaccine could be beneficial.

References:

•         Ole Fejerskov & Edwina Kidd. Dental Caries - The disease and its clinical management; 2^nd Ed.

•         Shobha Tandon. Textbook of Pedodontics; 2^nd Ed.

•         SG Damle. Textbook of Pediatric dentistry; 3^rd Ed.

•         Soben Peter. Essentials of Preventive and Community Dentistry; 3^rd Ed.

•         Ramandeep S. et al (2012). Vaccine against dental caries- an urgent need. Journal Vaccines Vaccination; 3(2).

•         http://www.oragenics.com/?q=cavity-prevention

•         http://www.planetbiotechnology.com/products.html

•         http://www.xenophilia.com/zb0017.htm