Dental Caries Dental Caries VaccinesVaccines

An update

Caries vaccinesCaries vaccines

The evidence of a specific bacterial cause of dental caries and the function of salivary glands as an effector site of the mucosal immune system has provided a scientific basis for developing a vaccine against this highly prevalent and costly oral disease.

Research for a safe and effective vaccine was facilitated by: progress in molecular biology(cloning virulence factors of S.mutans), in mucosal immunology(sophisticated Ag delivery system,adjuvants stimulating IgA Ab response)

Evidence for S.Mutans as the major Evidence for S.Mutans as the major bacterial etiologic factor in human dental bacterial etiologic factor in human dental

cariescaries It can almost always be isolated from plaque. Most carious lesions have >10% S. Mutans. Most non-infected areas are non-carious. Longitudinal studies show S. Mutans precedes

development of dental caries.There are studies showing that children who do not

become infected in the first 3 years of life,appear to remain uninfected for several years, possibly until a new opportunity for colonization occurs upon eruption of the secondary dentition.

Ig A – principal immune component Ig A – principal immune component of salivary glands’ secretionof salivary glands’ secretion

Immunization would presumably need to be initiated early in childhood to interfere with mutans streptococcal colonization.

This would also require the mucosal immune system be sufficiently mature to respond effectively.

Although secretory IgA is absent in saliva and other secretions at birth, mature IgA is the principal salivary Ig secreted by 1 month of age.

By 6 to 9 months of age, most children exhibit a more adult- like distribution of salivary IgA1 and IgA2 subclasses.

This suggests that significant maturation of the mucosal immune response has occured by the end of the first year of life. The best moment to immunize is around 1yr of age.

Function of salivary IgAFunction of salivary IgA

Inhibition of adherence of microorganisms on epithelial surface or teeth

Neutralization of toxins or enzymes (GTF) Viral neutralization (polio virus) Antigen trapping and antigen exclusion Interaction of sIgA with non-specific defense

mechanisms(mucins,lactoferrin,lysosyme,lactoperoxidase)

Mucosal Immune System: GALT, BALT, NALT, DALT, SALT

Mechanisms involved in S.mutans Mechanisms involved in S.mutans colonization and pathogenesiscolonization and pathogenesis

Sucrose-independent attachment(Ag I/II) Sucrose dependant reaction (GTF) Bacterial metabolic activities with lactic acid

productionColonization of oral cavity:-sterile at birth-S.mitis, S.salivarius- very early (within 24 h)-S.sanguis by 9 months of age-S.mutans, S.sobrinus-in 18-24 months(window of

infectivity)

Streptococcal components important Streptococcal components important in colonization and accumulationin colonization and accumulation

Antigen I/II(family of adhesins, PAc in S.mutans)-initial attachement to the tooth.

Glucosyltransferase (GTF)-involved in accumulation:- synthesizes high-weight glucan in presence of sucrose;

- contains glucan-binding domains Glucan-binding proteins(GBP)of bacteria bind the

glucan.causing accumulation. Next phase of pathogenesis results from the metabolic

activities of these masses of accumulatedS.mutans and others producing lactic acid

Targets for dental caries Targets for dental caries vaccines- several stagesvaccines- several stages

Micro-organisms can be cleared from the oral cavity by antibody-mediated agregation while still in salivary phase, prior to colonization.

Ab could also block the receptors necessary for colonization(adhesins) or accumulation (glucan-binding domains of GTF or GBPs)

Inactivate GTF enzymes, responsable for glucan formation The antimicrobial effect of IgA can be enhanced by

synergism with innate componets of immunity (mucins,lactoferrin)

Most recent efforts concentrated on adhesins, GTF, GBPs as vaccine targets.

AdhesinsAdhesins

S. mutans: Ag I/II, PAc or P1. S.sobrinus:SpaA or PAg. Significant sequence homology exists between them(66%) P1 of S.mutans contains an alanine-rich repeating region in

the N-terminal third and a proline-rich repeating region in the center of the molecule(adhesin activity)

Ab directed to the intact Ag I/II or to its salivary binding domain blocked adherence of S.mutans.

Immunization with S.sobrinus SpaA constructs protected rats from caries caused by S.sobrinus infection.(Redman et al.,1995)

GTFs & GBPsGTFs & GBPsGTFs: S.mutans and S. sobrinus each synthesize several GTFs; contain considerable

sequence homology. The catalytic activity of GTF appears to be associated with residues in the N-

terminal third of the moloecule. The C-terminal region of GTF molecule contains repeating sequences with glucan-

binding function.GBPs: S.mutans secretes at least 3 distinct proteins with glucan-binding activity: GbpA,

GbpB, GbpC. Only GpbB has been shown to induce a protective immune response to

experimental dental caries.(Smith and Taubman, 1996, 1997a). Protection can be achieved by :subcutaneous injection of GbpB in the salivary

region(Smith and Taubman,1996) or by intanasal mucosal application(Smith et al., 1997a).

S.sobrinus Gbps have not been evaluated for their protective potential.

Types of vaccinesTypes of vaccines

Subunit vaccines- contain structural elements of the Ag I/II, GTF or GbpB. Conjugation of functionally peptides to carbohydrate components (glucan) or to

other vaccine proteins(tetanus toxoid) would increase the immunogenicity of the peptide.

Subunit vaccines can be designed to include the salivary binding domain, but exclude sequences bearing potential for unwanted Ab response(cross-reactions)

A) Synthetic peptide vaccines(alanin-rich repeated region of Ag I/II induced protective immunity). Combining epitopes from adhesins and GTFs into one construct and enhance the immune response with additional sequences(cholera toxin subunits) could increase the protective effect.

B) Recombinant vaccines (attenuated mutant vectors as Salmonella, contain plasmids expressing recombinant peptides)

Conjugate vaccines-chemical conjugation of functionally peptide components

with bacterial polysaccharides.

Problems in development of Problems in development of caries vaccinescaries vaccines

Identification of virulence antigens of S. Mutans

Lack of complete understanding of the mechanisms of immune protection

Possiblity of immunopathological complications to immunization.

Approval to test candidate vaccine in young population.

Means to obtain caries Means to obtain caries immunityimmunity

1.Natural immunity:-maternal protection-ontogeny of mucosal immunity-natural caries immunity 2.Active immunization:-local immunization-systemic immunization-oral/mucosal immunization 3.Passive immunization-a safe procedure, has received recently much attention-applying a monoclonal Ab against Ag I/II on the teeth of human

volunteers prevented colonization by S.mutans.

Routes to protective Routes to protective responsesresponses

Mucosal application of caries vaccines are generally prefered for induction of IgA Ab.

Exposure of Ag to mucosally associated lymphoid tissue in the gut, nasal, bronchial or rectal site can generate immune response not only in the region of induction, but also in remote locations(”common mucosal immune system”)

Oral ImmunizationOral Immunization Oral induction of immunity in GALT to generate IgA Ab response Oral feeding, gastric intubation or in vaccine-containing capsules

or liposomes. Not ideal route because of stomach acidity on Ag, inductive site

were relatively distant. Experiments with this route showed change in the course of

mutans streptococcal infection and disesse in animal models Smith et al., 1979) and humans.(Smith and Taubman,1987)

Lactic streptococcci have long been used to produce diary products and because of their safety they might be the most suitable host for oral vaccine production(a recombinant S.lactis strain expressing PAc of S.mutans induced IgA and IgG responses to PAc.). (Ivaki et al.)

Intranasal ImmunizationIntranasal Immunization More recent attempts Sites are in closer anatomical relationship to the oral cavity Intranasal installation of Ag, targeting NALT Protective immunity after infection with cariogenic

S.mutans could be induced in rats by IN route (Kats et al., 1993)

Advantages:-lower doses of Ag needed (low denaturation)-easy administration-induces both systemic and mucosal immunity.

Tonsillar ImmunizationTonsillar Immunization Tonsillar tissue contains the required elements of immune

induction of secretory IgA response Although, IgG,rather than IgA response characteristics are

dominant in this tissue Palatine tonsils(nasopharyngeal tonsils) Topical application of formalin-killed S.sobrinus cells in

rabbits can induce a salivary imune response that can significantly decrease infection with cariogenic S.sobrinus.(Fukuizumi et al.,1999)

Repeated tonsillar application can induce the appearance of IgA antibody-producing cells in major and minor salivary glands of the rabbit.(Inoue et al., 1999)

Minor salivary glands Minor salivary glands immunizationimmunization

They populate lips, cheeks, soft palate. Short, broad secretory ducts associated with

lymphatic tissue. S.sobrinus GTF was topically administered onto

the lower lips of young adults It had significantly reduced proportion of total

streptococcal flora in their whole saliva during 6 week period following a dental prophylaxis, compared to a placebo group.(Smith and Taubman,1990)

Rectal ImmunizationRectal Immunization

A remote mucosal site investigated for its inductive potential.

It has the highest concentration of lymphoid folicles in the lower intestinal tract.

Preliminary studies have indicated that this route could also be used to induce salivary IgA responses to Ag (GTF).(Lam et al., 2001)

Use of suppositories as one alternative for children in whom respiratory problems preclude intranasal application of vaccine.

Subcutaneous or parenteral Subcutaneous or parenteral immunizationimmunization

Initial studies used whole cells of S.mutans as immunogens.

To eliminate the possibility to induce heart-reactive Ab, subunit or peptide vaccines have been used in systemic immunization to control dental caries.

Subcutaneous immunization of rats with the peptide constructs and GTFs near the salivary glands induced high serum IgA and salivary IgA responses and inhibited oral colonization and caries development.(Taubman et al.)

Passive ImmunizationPassive Immunization

Cows have been immunized with a fusion antigenic protein

A high titer against PAc was found in milk Mouth rinsing with the immunized milk inhibited

the number of S.mutans in saliva and dental plaque.(Toshihiko Koga et al., 2002)

Bovine milk contains kappa-casein and lactoferrin which prevent oral colonization by S.mutans

Immunized bovine milk may be a safe mean of passive immunization for preventing dental caries.

Adjuvants and delivery Adjuvants and delivery systems for caries vaccinessystems for caries vaccines

Mucosal routes of Ag delivery often require additional components that can potentiate the immune response to achieve a protective effect.

-Cholera and E.coli heat-labile enterotoxins or detoxified CT and LT or CT subunit B (remove toxicity by mutagenesis techniques).

-Microcapsules and microparicles- combination of Ag in various types of particles :poly(lactide-co-glycolide) (PLGA) have been used as local delivery systems.

-Liposomes-are phospholipid membrane vesicles manufactured to contain drugs or Ag.They improve mucosal immune response by facilitating M cell uptake and delivery of Ag to lymphoid elements of inductive tissue.

-Monophosphoryl lipid A when administrated IN to mice with soluble GTF had better results than liposomes containing GTF.

Potential adverse effects of Potential adverse effects of immunizationimmunization

Polypeptides (62-67kDa) immunologically cross-reactive with human heart tissue and rabbit skeletal muscle myosin are found in cell membranes of S.mutans.

Heart cross-reactive Ab do not develop in Rhesus monkeys or rabbits immunized with purified Ag I/II from S.mutans.

This suggests that heart tissue and Ag I/II are not antigenically similar.

Due to the potential of streptococcal whole cells to induce heart-reactive Ab, the development of subunit vaccine for

caries has been the focus of intense research interest.

Human applicationsHuman applications Active immunization- mucosal immunization with

dental caries vaccines could be protective, especially in pediatric populations where S.mutans is not yet a permanent member of the dental biofilm.

Passive immunization- the explanation for long term effects on streptococcal colonization after relatively short exposure to Ab is not clear; apparently Ab blockage of an important adhesin epitope during the reconstruction of the dental biofilm following treatment places S.mutans at an insurmontable compettitive disadvantage for recolonization.

Steps in developing caries Steps in developing caries vaccinesvaccines

Identify virulence agents Design means to induce protective response Animal model Human testsCaries vaccines response requirements:-interfere with early colonization-not necessarily bactericidal-non-inflamatory response-persistent response-site directed response (oral cavity)

What is the ideal dental caries What is the ideal dental caries vaccines?vaccines?

Broad coverage for all common cariogenic S.mutans strains ( a multi-component vaccine might be needed for broades coverage).

Should work for both high- and low-risk populations(but high-risk population might need both active and passive mechanisms for protection)

Could be given as a part of another immunization (WHO effort is to reduce no of vacinations)

Could be given by various routes and still be effective Inexpensive Delivered by individuals with little trainning Could provide ”herd immunity”But because of different variations more than one vaccine approach may

ultimately be optimal to use.

ConclusionsConclusions Both passive and active immunization approaches have demonstrated succes

in animal models and human clinical trials. The efficacy of active immunization with subunit vaccines from S.mutans has

been proved to prevent dental caries in animal models. However, there are few studies on efficacy in humans. The primary target of such a vaccine would be young children, who are at

high risk at this disease. Risk-free and more effective approach to prevent human dental caries should

be developed. Recent advances in research on mucosal vaccines will lead to a safe and

effective vaccine Local passive immunization with monoclonal Ab specific for S.mutans

antigens has received recently much attention. These immunological approaches for preventing caries should be applicable to

the control of various mucosal diseases.