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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
2
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
3
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.
4
IMMUNITY
It is defined as resistance exhibited by the host against any foreign
antigen including microorganisms.
5
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
6
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
7
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
8
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
9
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
10
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
11
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
12
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.
13
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
14
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)
15
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.
16
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.
17
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.
18
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.
19
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.
20
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.
21
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.
22
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
24
4. Active immunization
5. Passive immunization
6.
25
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.
26
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.
27
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.
28
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.
29
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
30
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.
31
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.
32
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.
33
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.
34
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
35
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
36
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
37
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.
38
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