REFERENCES Ole Fejerskov,Dental caries-the disease & its clinical

management(3rd edition)

KM Shivakumar,SK Vidya,GN Chandu Indian journal of dental research (2009)

Mitzi.R.Becker Molecular analysis of bacterial species associated with dental caries (2002)

DEFINED DISEASE PROCESS

EPIDEMIOLOGY

IDENTIFICATION OF CAUSATIVE ORGANISM

STUDY OF IMMUNE RESPONSESTUDY OF ORGANISM

IDENTIFICATION OF PROTECTIVE ANTIGEN

VACCINE TRAIL IN ANIMAL MODEL

TOXICITY TESTING

HUMAN CLINICAL TRIALS

ST EPS IN DEVELOPIN G VACCIN E

DENTAL CARIES Dental caries is the infectious microbiologic disease of

teeth that results in localized dissolution & destruction of calcified tissues.

It is one of most common diseases in humans.

Its prevalence is more in developed & developing countries & can reach over 90%.

The development of caries requires the presence of cariogenic bacteria that are capable of producing acids,which are responsible for destruction of tooth.

CAUSATIVE ORGANISM The different caustive organisms are :-

1) Streptococcus mutans

2) Streptococcus sobrinus

3) Lactobacillus acidophilus

4) Actinomyces viscosus

5) Bifidobacterium

6) Non-mutans streptococci

STREPTOCOCCUS MUTANS It is gram +ve,facultative

anaerobic,coccus shaped bacteria found in oral cavity.

It is one of the most important micro-organism responsible for dental caries.

There are 6 distinct species associated with dental caries-S.cricetus,S.ferus,S.rattus, S.downey,S.mutans & S.sobrinus.

They are most commonly associated with pit & fissure caries(39%).

A large proportion of infants become colonized by S.mutans during the ‘window of infectivity’ around the time of eruption of first molars at the age of 2 years.

They mainly metabolize sucrose to lactic acid by using enzyme glucansucrase.

Due to the role the S. mutans plays in tooth decay, there have been many attempts to make a vaccine for the organism.

LACTOBACILLUS ACIDOPHILUS It is gram +ve,facultative

anaerobic rod-shaped bacteria found in oral cavity.

They cause carious lesions in coronal part of the tooth.

It is mostly commonly associated with dental caries, but to a lesser extent than streptococci.

ANTIGENIC COMPONENTS OF STREPTOCOCCUS MUTANS S.mutans posses various cell surface substances

including Adhesins,Glucosyltransferase & Glucan binding proteins(GBP).

These substances are used in vaccine preparation.

Adhesins :- it forms the two principle human pathogens of S.mutans & has been purified.

It has a mol. weight of 185 kDa composed of single polypeptide chain of approx. 1600 residues.

It contains proline-rich & alanine-rich regions

These regions have been associated with adhesin activity of Ag I/II.

The antibody directed to intact Ag I/II molecule or to its salivary binding domain blocked adherence of S.mutans of saliva-coated hydroxyapatite.

Immunization of mice with synthetic peptide from alanine-rich region of Ag I/II suppressed tooth colonization with S.mutans.

Glucosyltransferase :- S.mutans has three forms of glucosyltransferase(GTF’s) :-

1) water insoluble glucan synthesizing enzyme(GTF-I)

2) Water insoluble & water soluble glucan synthesizing enzymes(GTF-S-I)

3) Water soluble glucan synthesizing enzymes(GTF-S)

The genes encoding these enzymes are GTF-B,GTF-C & GTF-D genes.

All three genes are important for smooth-surface caries formation in pathogen-free rat model system.

Dextranases:- it is an important constituent of early dental plaque.

Dextranase is an enzyme produced by mutans streptococci.

They destroy dextran & thus bacteria can invade dextran rich dental plaque.

Dextranase when used as an antigen,can prevent the colonization of organism in early dental plaque.

VACCINE Suspension of attenuated

or killed micro-organisms administered for the prevention, amelioration or treatment of infectious diseases.

It stimulates production of protective antibodies & other immune mechanisms.

CONCEPT OF DENTAL VACCINE The traditional way of managing dental caries was

by “Drill & fill”.

This approach has slowly evolved into a more conservative mode.

Various preventive measures have been implicated for prevention of dental caries,among which immunization of population against the disease.

Many studies have been conducted on development of an effective vaccine to dental caries,but a safe & effective vaccine is yet not marketed.

HISTORY OF CARIES VACCINE

Bowen(1969) was the first scientist to achieve successful immunization of experimental animals against dental caries.

Later attempts were made by Russell & Johnson(1987),Koga(2002) & Smith(2002).

Despite extensive experimental support for the caries vaccine,a safe & effective vaccine is yet not marketed anywhere in the world.

MECHANISM OF ACTION OF VACCINE

Saliva contains 1-3% of immunoglobulin concentration, majority of which is IgA.

Saliva also contains IgG & IgM.

Some of the possible ways antibodies might control bacterial growth are :-

1) The salivary Ig may act as a specific agglutinin interacting with the bacterial surface receptors & inhibiting colonization & subsequent caries formation.This might also inactivate surface glucosyltransferase,which would reduce the synthesis of extra-cellular glucan resulting in reduced plaque formation.

2) The salivary glands produce secretory IgA antibodies by direct immunization of gut associated lymphoid tissue(GALT),from where sensitized B-cells are home to salivary glands.

The salivary IgA antibodies have direct access to tooth surface.They may prevent S.mutans from adhering to enamel surface or they may prevent formation of dextran by inhibiting the activity of glucosyltransferase.

The injected antigen is phagocytosed & undergoes antigenic processing by macrophages.This causes sensitization of B & T-cells.This induces helper CD-4 & Cytotoxic CD-8 cell response & results in formation of antibodies.

IMMUNE RESPONSE Primary response :- when an antigen is administered

for first time to humans or animals,there is latent period of induction of 3-10 days before antibodies appear in blood.

The first antibody that appear is IgM type.

The IgM antibody titer rises for 2-3 days,reaches its peak level & then declines as fast as it developed.

If the antigenic stimulus is sufficient,the IgG appears in next few days & reaches its peak level in 7-10 days.

The levels of IgG gradually falls over a period of weeks or months.

The important outcome of primary immune response is education of reticulo-endothelial system of body.

Both B & T-lymphocytes produce memory cells which are responsible for immunological memory that is established after immunization.

Secondary or booster response :- the response of booster dose differs from primary response.

The secondary response also involves production of IgM & IgG antibodies.

There is brief production of IgM antibody,but more prolonged production of IgG antibody.

This accelerated response is attributed to immunological memory.

The immune response & immunological memory are basis of vaccination & re-vaccination.

ANIMAL STUDIES Rodents are attractive as experimental animals

because they are inexpensive & easy to maintain.

Rapid decay of their teeth can be induced by Streptococcus mutans when present during the provision of a sugar containing diet.

The ability to establish large experimental groups & to arrive at an accurate diagnosis of caries by examination of tooth surface also makes rodents a good choice for laboratory animals.

Immunization experiments with cells of S.mutans both in rats & monkeys have consistently resulted in significance decrease in dental caries.

Immunization with whole cells of S.mutans or purified Ag I/II produces a reduction of about 70% in both smooth surface & pit & fissure caries when compared with controls.

CLINICAL TRIALS The main objectives for a successful clinical trial

are :-

1) Preliminary data indicating prospects of success

2) A clear outcome measure of success

3) A compliant with high incidence of disease

4) No confounding external factors likely to come into effect during the trial

ROUTES OF IMMUNIZATIONThere are 4 routes of

immunization used for Streptococcus mutans :-

1) Oral

2) Systemic (sub-cutaneous)

3) Active gingivo-salivary

4) Passive dental immunization

ORAL ROUTE

Daily administration of 10 cells of S.mutans in capsules produced small increase in IgA.

The oral route failed to reduce caries significently,as compared with sub-cutaneous immunization.

Experiments in humans of ingestion of S.mutans in gelatin capsules resulted in an increase in IgA antibodies in saliva,although for a limited time.

Oral route is not ideal because of detrimental effects of acidity on antigen or because inductive sites are relatively distant.

SUB-CUTANEOUS ROUTE Sub-cutaneous

administration of S.mutans was used successfully in monkeys & elicitated pre-dominantly IgA,IgG & IgM.

The antibodies find their way into oral cavity via gingival crevicular fluid & are protective against dental caries.

ACTIVE GINGIVO-SALIVARY ROUTE There has been some concern expressed regarding

the side-effects of using these vaccines with other routes.

In order to limit these potential side-effects & to localize the immune response,gingival crevicular fluid has been used as route of administration.

Its main advantage is that it causes increase in levels of both IgA & IgG.

PASSIVE IMMUNIZATION It involves external or passive

supplementation of antibodies.

This carries the disadvantage of repeated applications, as the immunity acquired is temporary.

1) Monoclonal antibodies

2) Bovine milk & whey

3) Egg-yolk antibodies

4) Transgenic plants

ADJUVANTS FOR CARIES VACCINE

1) Synthetic peptides :- Any antigen derived from animals or humans have the potential to cause hypersensitivity.The chemically synthesized peptides do not cause such reactions.The synthetic peptides are derived from glucosyltransferase enzyme.

2) Coupling with cholera toxin sub-units :- Cholera toxin is a powerful mucosal immunoadjuvant,which is used to enhance the induction of mucosal immunity to variety of bacterial or viral pathogens in animal systems.

3) Fusing with Salmonella :- The non-virulent strains of salmonella are an effective vaccine vector.They are fused with vaccine by recombinant technologies.

4) Microcapsules & micro-particles :- They are made up of polylactide-co-glycoside(PLGA) have been used as local delivery systems because of their ability to control the rate of release & slow degradation of capsule.

5) Liposomes :- They are bilayered phospholipids membrane vesicles manufactured to deliver drugs & antigens.They increase mucosal immune response by facilitating M-cell uptake.

HUMAN CLINICAL TRIALS The rationale is that the immunization with S.mutans

should induce a immune response,which might prevent the organism from colonizing the tooth surface & thereby prevent tooth decay.

The vaccine could be given at the same time as the vaccines against diphtheria & tetanus.

As the vaccine would be administered before deciduous dentition is erupted at about 6 months of age,it would prevent the disease in children who show greater incidence of caries.

Immunity could be boosted at intervals to provide life-long protection.

Clinical trials are underway to test the use of pill of S.mutans for control of caries.

It has been experimented that ingestion of capsules containing S.mutans stimulates the production of S-IgA.

According to some reports,there is negative correlation between S-IgA & caries prevalence.

Stimulation of serum immunoglobulin in humans has produced mixed results & no correlation could be made between caries experience & serum immunoglobulin stimulation.

RISKS IN CARIES VACCINE The most serious risk is that the sera of some patients

with rheumatic fever who show serological cross-activity between heart tissue antigens & certain antigens from hemolytic streptococci.

Experiments from anti-sera from rabbits immunized with whole cells of S.mutans were reported to cross-react with normal rabbit & human heart tissues.

Because of the potential of streptococcal whole cells to produce heart reactive antibodies,the development of sub-unit vaccine for controlling dental caries has been subject of intense research interest.

Glucosyltransferase is also tested for cross-reactivity with human heart tissue & the results are negative.

Research also shows that C-terminal part of Antigen I/II contains an epitope,which is cross-reactive with human IgG,it is potentially harmful for immune system.

The human cross-reactive IgG region is also present in other mutans streptococci such as Streptococcus sobrinus.

CONCLUSION There is strong evidence that S.mutans & S.sobrinus

are closely associated with dental caries.

Fluoride treatment used abroad has successfully limited caries progression,but not sufficient to control this disease even when used together with professional tooth cleaning & dietary counseling in populations highly exposed to these cariogenic bacteria.

Active & passive immunization strategies which targets key elements in molecular pathogenesis in mutans streptococci,hold promise.

Along the established methods of caries prevention, caries vaccine have the potential of making highly valuable contribution to control disease.

Regardless of the mechanism by which immune protection against dental caries is achieved,further advances to make immunization caries practical will depend upon clinical trials aimed at establishing whether the findings from animal experiments can be transferred to humans.

Presented by :-

Name- Amanjot Singh

Roll no. – 5

BDS IIIrd prof.