SUBMITTED TO

DR RITU JINDAL
PROFESSOR AND HEAD OF DEPT
DEPT OF PEDODONTICS AND
PREVENTIVE DENTISTRY



SUBMITTED BY
DR SAHIL JINDAL
MDS 1 PROF
INTRODUCTION
Dental caries is one of the most common infectious diseases. 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 intranasally immunized with these
antigens
INTRODUCTION

Immunologically active biological substance design to
protect an individual from disease.
Derived from Latin word meaning suspension of killed or
attenuated organisms administered for prevention of
disease.
Artificial method of disease prevention.

AIM
The aim of the vaccine development is to alter a
pathogen or its toxin in such a way that they become
innocuous without losing antigenicity.

PRINCIPLES
Specificityability to identify the specific organism

Memory - Which allow to mount a much stronger
response on a second encounter with antigen.

TYPES
SUB UNIT VACCINE
comprise only part of pathogen

ADVANTAGES
SAFE
MORE EFFECTIVE
DISADVANTAGE
EXPENSIVE
RECOMBINANT VACCINE
CONJUGATE VACCINES
POLYSACCHARIDE ANTIGEN
+
PROTEIN MOLECULE


CONJUGATE VACCINE
DNA VACCINES
THIRD GENERATION VACCINES.
CARIES VACCINE
It is a vaccine to prevent
and protect against
tooth decay.
Colonization of oral cavity

-sterile at birth
-S.mitis, S.salivarius- 24 Hours
-S.sanguis by 9 months of age
-S.mutans, S.sobrinus-in 18-24 months


ROLE OF S MUTANS
S. mutans is the bacterium most intimately associated
with initiation and development of carious lesion.
Strep. mutans is a facultative anaerobic, nonhaemolYtic,
acidogenic organism, producing extra cellular and
intracellular polysaccharides.
Strep. mutans has been separated into seven serotypes (a
to g) by means of precipitating and immunonfluorescence
techniques
Strep. mutans isolated from man appear to belong to
serotype c; the next most common is serotype d.



KOCH POSTULATES FULFILLMENT

I. Strep mutans is found in the plaque of carious teeth
and cannot usually be isolated in the absence of caries.
2. The organism can be grown in pure culture.
3. Infection of germ-free rats or normal hamsters with
Strep. Mutans has induced caries
4. The organism can then be recovered from the carious
lesion and grown in pure culture.
5. Antibodies to this organism are increased in patients
with caries.

Mechanism involved in s .mutans
colonization and pathogenesis

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

Sucrose dependant reaction
(GTF) -
Glucosyltransferaces (GTF)Synthesize several forms of
high-molecular-weight branchedExtra 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.

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.
IMMUNE RESPONSE TO CARIES
PROGRESSION AND S .MUTANS

the production of secretory IgA secreted in the saliva
The second involves the systemic immune system and
the production of anti bodies that travel through the
gingival epithelium to the crevicular fluid that bathes
tooth and plaque.

APPARENT FAILURE OF NATURAL
IMMUNITY IN PROTECTION
AGAINST DENTAL CARIES
Strep. mutans is a poor immunogen, particularly as it
preferentially colonizes enamel surface.
Sensitization to the organism might depend on the entry
of a sufficient dose of antigenic material throughthe
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 againstStrep. mutans.
The T-cell responseto Strep. mutans also appearesto be of
a low order of magnitude and may need boosting for the
sensitization to be detected.

Streptococcal components
important in 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.


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, responsable for glucan formation
The antimicrobial effect of IgA can be enhanced by
synergism with innate components of immunity
(mucins,lactoferrin)

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.
S.sobrinus: variously identified as SpaA or PAg.

GLUCOSYLTRANSFERASES

S. mutans has three forms of GTFs:-
a water-insoluble glucan-synthesizing enzyme, GTF-I
a water-insoluble and water-soluble glucan-synthesizing
enzyme( GTF-SI),
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.

Glucan binding proteins

S.mutans secretes at least 3 distinct proteins with glucan-
binding activity: GbpA, GbpB, and 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 intranasal mucosalapplication (Smith et aI., 1997a).

WHEN TO IMMUNIZE
The best age is at one , after the deciduous teeth
have erupted and before the mutants bacteria
have started to increase in number
ROUTES TO PROTECTIVE RESPONSES --

Mucosal applications of caries vaccines are generally
preferred for induction Of lgA 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 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.

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 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 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
immunization
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 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
immunization
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 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 systems 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
immunization
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 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 vaccines
Identify virulence agents
Design means to induce protective response
Animal model
Human tests

Caries vaccines response
requirements:

Interfere with early colonization
Not necessarily bactericidal
Non-inflammatory response
Persistent response
Site directed response (oral cavity)

What is the ideal dental caries
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.

Conclusions
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.