INTRODUCTIONDental caries is an infectious microbiologic disease of the teeth that

results in localized dissolution and destruction of the calcified tissue.

Dental caries is one of the most common diseases in humans. In modern

times, it has reached epidemic proportions. The prevalence of dental caries

in developed countries varies greatly and can reach over 90%. The rate of

Caries has been increasing in developing countries with the increase in the

popularity of highly refined sugars. The development of dental caries

requires the presence of cariogenic bacteria that are capable of producing

acid and a sugar present in the diet which favors the colonization of these

bacteria to form acid. Dental caries appears to be a major public health

problem which if left untreated can cause considerable pain, discomfort,

and treatment costs are very high. Dental caries results from the

interaction between the host, the hosts diet, and the microoraganism on

the tooth surface bounded by the time factor. A wide group of

microorganisms are identified from carious lesions of which Streptococcus

mutans ( S. mutans) , Lactobacillus acidophilus , and Actinomyces viscosus

are the main pathogenic species involved in the initiation and development

of dental caries. S. mutans has been implicated as a causative organism of

dental caries. S. mutans accounts for seven distinct species isolated from

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animals and humans; Streptococcus cricetus, Streptococcus ferus,

Streptococcus

macacae, Streptococcus rattus, Streptococcus downey, S. mutans, and

Streptococcus sobrinus . S. mutans and Streptococcus sobrinus are

exclusively isolated from humans and S. mutans is the most prevalent

species.

The traditional way of managing dental caries was by a surgical

approach of drill and fill. This approach has slowly evolved into a more

conservative mode. Various preventive measures have been implicated

for the prevention of dental caries, among which is immunization of

the population against the disease. Many studies have been conducted

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on the development of an effective vaccine to prevent the occurrence

of dental caries.

Of the oral bacteria, mutans streptococci, such as Streptococcus

mutans and S. sobrinus, are considered to be causative agents of dental

caries in humans. There have been numerous studies of the

immunology of mutans streptococci. To control dental caries, dental

caries vaccines have been produced using various cell-surface antigens

of these organisms. Progress in recombinant DNA technology and

peptide synthesis has been applied to the development of recombinant

and synthetic peptide vaccines to control dental caries. Significant

protective effects against dental caries have been shown in

experimental animals, such as mice, rats and monkeys, which have

been subcutaneously, orally, or intra-nasally immunized with these

antigens.

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IMMUNITY

It is defined as resistance exhibited by the host against any foreign

antigen including microorganisms.

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IMMUNITY

Innate

Skin, tears, low pH of stomach,

respiratory cilia, normal flora of

gut

Acquired

Natural

Natural active

During exposure to disease ,

antibobies are produced

Natural passive

Transmission from mother to

child

Artificial

Artificial active

Vaccination (low dose of antigen

introduced in the body)

Artificial passive

Immunization (introduction of antibodies in the

body)

THE IMMUNE RESPONSE

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IMMUNE RESPONSE

CELL MEDIATED

Activation of T lymphocytes,

PMN’s

Production of Tc, TH, Ts and

memory T cells

Phagocytosis

HUMORAL

Production of B lymphocytes

Plasma cells

Produces Ab leading to Ag-Ab

reaction

Memory B cells

Helps in secondary attack

Antibodies (Immunoglobulin)

Definition: An immunoglobulin, a specialized immune protein,

produced because of the introduction of an antigen into the body, and

which possesses the remarkable ability to combine with the very

antigen that triggered its production.

Types of Immunoglobulins:

IgG - protects the body fluids

IgA - protects the body surfaces

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IgM - protects the blood stream

IgD - serves as recognition receptors for antigens

IgE - mediates allergic response or hypersensitivity

VACCINE

‘Vaccine’- Latin word which means a suspension of attenuated or

killed microorganisms administered for the prevention, amelioration or

treatment of infectious disease.‘vaccine’ is an immunobiological

substance designed to produce specific protection against a given

disease. The concept of vaccination against dental caries was

strengthened because of:

The transmission & infectious nature of dental caries.

The discovery & understanding of the secretory immune system.

It stimulates the production of a protective antibody and other immune

mechanisms. Vaccines are prepared from live modified organisms,

inactivated or killed organisms, extracted cellular fractions, toxoids, or a

combination thereof.

THE IMMUNE RESPONSE

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a) The primary response: When an antigen is administered for the rest

time to an animal or human, there is a latent period of induction of 3 to

10 days before antibodies appear in the blood. The antibody that is

elicited first is entirely of the IgM type. The IgM antibody titer rises

steadily during the next 2 to 3 days, reaches a peak level, and then

declines almost as fast as it developed. Meanwhile, if the antigenic

stimulus was sufficient, the IgG antibody appears in a few days. IgG

reaches a peak in 7 to 10 days and then gradually falls over a period of

weeks or months. An important outcome of the primary antigenic

challenge is the education of the reticulo- endothelial system of the

body. Both B and T lymphocytes produce what are known as memory

cells or primed cells. These cells are responsible for the immunological

memory that is established after immunization.

b) Secondary (Booster response): The response to a booster dose differs

in a number of ways from the primary response. The secondary

response also involves the production of IgM and IgG antibodies. A

collaboration between B and T cells is necessary to initiate a secondary

response. There is a brief production of the IgM antibody and a much

larger and more prolonged production of the IgG antibody. This

accelerated response is attributed to immunological memory. The

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immune response (primary and secondary) and immunological memory

are the basis of vaccination and revaccination.

TYPES OF VACCINE

1. Bacterial eg: live BCG for TB

2. Viral:

a. live eg:- oral poliomyelitis vaccine

b. Killed eg:- salk for poliomyelitis

3. Bacterial products: eg :-toxoid for diphtheria

PROPOSED MECHANISM OF ACTION OF CARIES

VACCINE

Saliva contains approximately 13% of immunoglobulin

concentration, a majority of which is secretary IgA. However, saliva also

contains the humoral immunoglobulin IgG and IgM from the gingival

sulcular fluid. In addition, cellular components of the immune system such

as lymphocytes, macrophages, and neutrophils are also present in gingival

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sulcus. Some of the possible ways antibodies might control bacterial

growth are listed below:

1. The salivary immunoglobulin may act as a specific agglutinin

interacting with the bacterial surface receptors and inhibiting

colonization and subsequent caries formation. They might also

inactivate surface glucosyltransferase, which would then reduce the

synthesis of extra cellular glucans resulting in reducing plaque

formation.

2. The salivary glands produce secretory IgA antibodies by direct

immunization of the gut associated lymphoid tissue (GALT), from

where sensitized B-cells may be home to the salivary glands. The

salivary IgA antibodies have, of course, direct access to the tooth

surface. They may prevent S. mutans from adhering to the enamel

surface or they may prevent formation of dextran by inhibiting the

activity of glucosyltransferase (GTF).

3. The gingival crevicular mechanism involves all the humoral and

cellular components of the systemic immune system, which may

exert its function at the tooth surface. There is now sufficient

evidence to postulate what may happen after subcutaneous

immunization with S. mutans. The organism is phagocytosed and

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undergoes antigenic processing by macrophages. In the lymphoid

tissue, T and B-lymphocytes are sensitized by the macrophages

preventing the antigen HLA Class- II complex and releasing IL-I. This

induces the CD-4 helper and CD-8 cytotoxic suppressor cell response

with the activation of IL-2 receptors and the release of IL2. The

interaction between the cells play an essential part in modulating

the formation of IgG, IgA, and IgM classes of antibodies and B-

lymphocytes.

CRITERIA FOR EFFECTIVE IMMUNIZATION

Identification of factor responsible

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Identification of best target in microorganism

Identification of which component of immune system should be

targeted

One has to produce the evidence that hyper-immunization still work

STREPTOCOCUS MUTANS

Most intimately associated bacterium with initiation & development of

carious lesion. Facultative anaerobic, non-haemolytic, acidogenic

organism, producing extra cellular &

intracellular polysaccharides. The

minimum ineffective dose in man is

10⁴ to 10⁵ S. Mutans per ml of saliva.

The organism fulfils Koch’s postulates as

a cause of dental caries.

1. S.mutans is found in the plaque of carious teeth & cannot usually be

isolated in the absence of caries.

2. The organism can be grown in pure culture.

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3. Infection of germ- free rats or normal hamsters with S.mutans has

induced caries.

4. The organism can be recovered from the carious lesion & grown in pure

culture.

5. Antibodies to this organism are increased in patients with caries.

Carcinogenicity of S.mutans has been related to its ability to:

Colonise on the teeth.

Produce extra & intra cellular polysaccharides.

Produce large amount of acids even at low pH.

Utilize salivary glycoproteins

MOLECULAR PATHOGENESIS OF DENTAL CARIES

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MECHANISM INVOLVED IN S .MUTANS COLONIZATION AND

PATHOGENESIS

1. Sucrose-independent attachment (Ag I/II)

- Initial attachment to the tooth is achieved via the interaction of

bacterial proteins with lectins in the dental pellicle covering the tooth

surface. This trait is characteristic of a family of streptococcal adhesins,

referred to as antigen III or Pac in Streptococcus mutans

2. Sucrose dependant reaction (GTF)

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Glucosyltransferaces (GTF) Synthesize several forms of high-

molecular-weight branched extra cellular glucans.

These glucose polymers provide scaffolding for the aggregation of

mutans and other oral streptococci through interaction with

bacterial cell-associated glucan- binding proteins.

3. Bacterial metabolic activities with lactic acid production

Next phase of pathogenesis results from the metabolic activities of

these masses of accumulated mutans streptococci (and possibly of

other accumulation-associated micro- organisms).

Mutans streptococci are the most prolific producers of lactic acid in

these accumulations.

The resulting increase in lactic acid synthesis cannot be sufficiently

buffered to prevent enamel dissolution.

APPARENT FAILURE OF NATURAL IMMUNITY IN

PROTECTION AGAINST DENTAL CARIES

Strep. mutans is a poor immunogen, particularly as it preferentially

colonizes enamel surface.

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Sensitization to the organism might depend on the entry of a

sufficient dose of antigenic material through the junctional

epithelium of the gingiva to immunological!y competent cells. The

efficiency of this route of immunization is Questionable.

Indeed, natural immunization induces low antibody titer which is

relative high for IgM than IgG antibodies and these may not be

directed against Strep. mutans.

The T-cell response to Strep. mutans also appears to be of a low

order of magnitude and may need boosting for the sensitization to

be detected.

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EFFECTIVE MOLECULAR TARGETS FOR DENTAL CARIES

VACCINES

Micro-organisms can be cleared from the oral cavity by antibody

mediated aggregation while still in salivary phase, prior to

colonization.

Antibody could also block the receptors necessary for colonization

(adhesins) or accumulation (glucan-binding domains of GTF or GBPs)

Inactivate GTF enzymes, responsible for glucan formation

The antimicrobial effect of IgA can be enhanced by synergism with

innate components of immunity (mucins,lactoferrin)

The surface antigens of the cells wall are involved in the

immunogenicity of the organism. A large no: of antigens have been

identified, of which the most important are:

1. ADHESINS

185-kDa protein is composed of a single polypeptide chain of

approximately 1600 residues.

S. mutans: variously identified as Ag I/II, PAc or PI.

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S.sobrinus: variously identified as SpaA or PAg.

Four mechanism of bacterial adherence where anti adhesion vaccine

could potentially block colonization and infection have been

identified. They are,

1. Adhesin protein located at distal tip of pilus/fimbriae organelle.

2. Afibrillar adhesion protein.

3. Blocking intimin proteins that helps in intimate association of bacterial

cell with eukaryotic cells.

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4. Bacteria secrete their own receptor protein, internalized by target host

ell phosphorylated and embedded in the eukaryotic cell as well as new

receptor for tight binding by the bacteria.

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2. GLUCOSYLTRANSFERASES

S. mutans has three forms of GTFs:-

1. A water-insoluble glucan-synthesizing enzyme, GTF-I

2. A water-insoluble and water-soluble glucan-synthesizing enzyme(

GTF-SI),

3. A water-soluble glucan-synthesizing enzyme, GTFS

The genes encoding GTF-I, GTF-SI, and GTF-S are called gtfB, the

gtfC, and the gtfD genes, respectively

The catalytic activity of GTF appears to be associated with residues in

the N-terminal of the molecule.

The C-terminal region of GTF molecule contains repeating

sequences with glucan-binding function.

3. GLUCAN BINDING PROTEINS

S.mutans secretes at least 3 distinct proteins with glucan- binding

activity: GbpA, GbpB, and GbpC.

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Only GpbB has been shown to induce a protective immune response

to experimental dental caries.

Protection can be achieved by: subcutaneous injection of GbpB in

the salivary region or by intranasal mucosal application.

4. SURFACE PROTEIN ANTIGEN

Large protein molecule (160-180 KD). Cell surface protein.

Main function sucrose independent adherence of bacteria to

hydroxy apatite surface.

5. DEXTRANASE

Large protein molecule (160-175 KD).

Break polymers of dextrose into glucan.

These enzymes are used by oral streptococci to modify glucan

products.

6. GLUCAN

Tree like homoploymer of glucose with many branches.

Two types- water soluble & water insoluble.

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Act as molecular barrier retain water and do not play a role in intial

colonization.

Greatly helps inadhesion of microorganism to tooth surface.

Functions

o Plaque accumulation

o Molecular sieves

o Retain water

o Act as secondary attachment apparatus

o Strengthen attachment of reproducing organism to tooth.

o Alpha dextran antibody produced as possible target to confer

caries protection.

7. LIPOTEICHOIC ACID

Molecules seen in gram positive bacteria and analogous to

lipopolysaccharide of gram negative bacteria.

Exact role in caries pathogenesis is not clear but propogate adhesin

by surface interaction.

WHEN TO IMMUNIZE 23

• Under normal circumstances of diet and challenge children become

permanently colonized with mutans streptococci between middle of the

2nd year and end of 3rd year.

• Caufield et. Al. referred to this period as the window of infectivity.

• It was found that children who do not become infected by S. mutans by

approx. 3yrs of age appear to remain unaffected or minimally colonized

for several years, possibly until new opportunities for colonization occur

upon eruption of permanent dentition.

• This suggests that - Children Should be immunized before window of

infectivity (immunize at 6-18 months of age)

• Booster dose at time of eruption of first permanent teeth.

ROUTES OF ADMINISTRATION

OR

STRATEGIES OF IMMUNIZATION FOR DENTAL CARIES

1. Induction of common mucosal immune system

2. Systemic route

3. Active gingiva-salivary route

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4. Active immunization

5. Passive immunization

6.

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INDUCTION OF COMMON MUCOSAL IMMUNE SYSTEM

Mucosal application of caries vaccines are generally preferred for

induction of IgA Ab. Exposure of Ag to mucosal 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”)

1. Oral Immunization

Relies on oral induction of immunity in GALT to generate IgA Ab

response.

Antigens applied by oral feeding, gastric intubation or in vaccine-

containing capsules or liposomes.

The rise in secretory IgA is small and of short duration

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 infection with S. mutans and

disease in animal models and in humans.

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2. 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 the antigen, 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.

3. Tonsillar route

The ability of tonsillar application of antigens to induce immune

responses in the oral cavity is of great interest.

The 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.

Repeated tonsillar application of a particulate antigen can induce the

appearance of IgA antibodies producing cells in both the major and

minor salivary glands of the rabbit.

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4. Minor salivary gland

The minor salivary glands populate the lips,

cheeks, and soft palate.

These glands have been suggested as potential

routes for mucosal induction of salivary

immune responses, given their short, broad

secretory ducts that facilitate retrograde access of bacteria and their

products and give the lymphatic tissue aggregates that are often

found to be associated with these ducts.

5. Rectal

More remote mucosal sites have also been investigated for their

inductive potential.

For example, rectal immunization with non

oral 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 antibodies in

distant salivary sites.

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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.

SYSTEMIC ROUTE OF IMMUNIZATION

Subcutaneous administration of S. mutans was used successfully in

monkeys and elicited predominantly serum IgG, IgM, and IgA

antibodies.

The antibodies find their way into the oral cavity via gingival crevicular

fluid and are protective against dental caries.

Whole cells, cell walls, and the 185 KD Streptococcal antigen have been

administered on 2 to 4 occasions.

ACTIVE GINGIVO-SALIVARY ROUTE

In order to limit the potential side effects of other routes, and to

localize the immune response, gingival crevicular uid has been used as

the route of administration. Apart from the IgG, it is also associated

with increased IgA levels.

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The various modalities tried were as follows:

• Injecting lysozyme into rabbit gingival, which elicited local antibodies

from cell response.

• Brushing live S. mutans onto the gingiva of rhesus monkeys, which

failed to induce antibody formation.

• Using smaller molecular weight Streptococci antigen, which resulted

in better performance probably due to better penetration.

PASSIVE IMMUNIZATION

As the name suggests, passive immunization involves passive or external

supplementation of the antibodies. This carries the disadvantage of

repeated applications, as the immunity conferred is temporary. Several

approaches tried were:

• Monoclonal antibodies

Monoclonal antibodies to S. mutans cell surface antigen I/ II have been

investigated. The topical application in human subjects brought a marked

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reduction in the implanted S. mutans. Thus, by bypassing the system, less

concern exists about the potential side effects.

• Bovine milk and whey

Systemic immunization of cows with a vaccine using whole S. mutans led

to the bovine milk and whey containing polyclonal IgG antibodies. This was

then added to the diet of a rat model. The immune whey brought a

reduction in the caries level. This whey was also used in a mouth rinse,

which resulted in a lower percentage of S. mutans in the plaque.

• Egg-yolk antibodies

The novel concept of using hen egg-yolk antibodies against the cell-

associated glucosyltransferase of S. mutans was introduced by Hamada.

Vaccines used were formalin killed whole cells and cell associated GTFs.

Caries reduction has been found with both these treatments.

• Transgenic plants

The latest in these developments in passive immunization is the use of

transgenic plants to give the antibodies. The researchers have developed a

caries vaccine from a genetically modified (GM) tobacco plant. The vaccine,

which is colorless and tasteless, can be painted onto the teeth rather than

injected and is the first plant derived vaccine from GM plants.

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The advantages are listed below:

• The genetic material can be easily exchanged.

• It is possible to manipulate the antibody structure so that while the

specificity of the antibody is maintained, the constant region can be

modified to adapt to human conditions, thus avoiding cross reactivity.

• Large scale production is possible as it would be quite inexpensive.

ACTIVE IMMUNISATION

Active immunization entails the introduction of a foreign molecule into

the body, which causes the body itself to generate immunity against

the target.

Various new approaches have been tried out to potentiate aspects

of the immune response to induce sufficient antibodies to achieve a

protective effect to overcome the existing disadvantages.

1. Synthetic peptides:

Any antigen derived from animals or humans has the potential for

hypersensitivity reaction.

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The chemically synthesized peptides hold an advantage in that this

reaction can be avoided.

This has been found to enhance the immune response. In humans,

synthetic peptides elicited both IgG and T-cell proliferative

responses, and the antibodies were both anti-peptide and anti-

native.

The synthetic peptides give antibodies not only in the GCF but also

in the saliva. The synthetic peptide used is derived from the

Glucosyltransferase enzyme.

2. Coupling with Cholera Toxin Subunits:

Cholera toxin (CT) is a powerful mucosal immune adjuvant, which is

frequently used to enhance the induction of mucosal immunity to a

variety of bacterial and viral pathogens in animals systems.

Mucosal application of a soluble protein or peptide antigen alone rarely

results in elevated or sustained IgA responses.

However, the addition of small amounts of CT or the closely related E.

coli heat-labile enterotoxins (LT) can greatly enhance mucosal immune

responses to intragastrically or intranasally applied mutans

Streptococcal antigens or to peptides derived from these antigens.

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The coupling of the protein with the nontoxic unit of the cholera toxin

was effective in suppressing the colonization of s. mutans.

3. Fusing with salmonella:

The avirulent strains of salmonella are an effective vaccine vector; fusion

using recombinant techniques have been used.

4. Microcapsules and microparticles:

Combinations of antigens in or various types of particles have been

used in an attempt to enhance mucosal immune responses. The

microcapsules and microparticles made of poly lactide-co-glycolide

(PLGA) have been used as local delivery systems 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.

5. Liposomes:

Liposomes, which are bilayered phospholipids membrane vesicles

manufactured to contain and deliver drugs and antigens, have been

used to enhance mucosal responses to mutans Streptococcal

carbohydrate and GTF.

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Liposomes are thought to improve mucosal immune responses by

facilitating M cell uptake and delivery of antigen to lymphoid elements

of inductive tissue.

LIMITATIONS

Clinical trials are few and concentrated only on S. mutans. As

caries is multifactorial, it’s effectiveness is questionable

Risk of hypersensitivity

Cross reactivity of certain antigenic components of S. mutans

with heart tissue (structurally similar to myosin)

Microbial resistance

INDICATIONS

Rampant caries

Immunocompromised patients- organ transplant, AIDS,

sjogrens ,radiation therapy

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Individuals in developing countries who do not have access to

fluoride treatment & regular dental care

CURRENT STATUS OF CARIES VACCINE

Current information suggest that the general clinical use of dental

caries vaccine is several decades away from reality because the need

for caries vaccine could change because of the fluctuating

demographics of disease pattern in pediatric population.

RECENT ADVANCES

1. Subunit vaccine,

2. DNA Vaccine

3. Adjuvant

4. Liposomes

5. ISCOM

6. Biodegradable microsphere

7. Bio adhesive

8. Plantigen & plantibodies

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1. Subunit vaccine

Previously whole vaccine was introduced in to the host to

produce an antibody response.

It had the potential disadvantage of cross reaction with hea,rt

muscles.

Here a particular protein unit of organism is used as antigen.

Synthetic peptide vaccine based on putative functional domains

pf GTF are developed as subunit vaccine.

Advantage –specifically attack antigen surface

Types – synthetic peptide vaccine, recombinant vaccine

2. DNA Vaccine

Purpose is to make antigenicity more specific and long lasting.

Basis – when a specific DNA is administered in to the system the

host can synthesise protein component coded by the DNA

Anti caries DNA developed to express cell wall protein

3. Adjuvant

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4. Liposomes

5. ISCOM

6. Biodegradable microsphere

7. Bio adhesive

8. Plantigen & plantibodies

CONCLUSION

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. The latest approach for combating dental caries is through the development of an effective vaccine that is well suited for public health applications especially in environments that do not lend themselves to regular health care. The focus of the present review is on the development of suitable vaccine to prevent dental caries.

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