Caries vaccine / endodontics courses

CARIES VACCINE….
INDIAN DENTAL ACADEMY
Leader in continuing Dental
Education
www.indiandentalacademy.co
www.indiandentalacademy.com

CONTENTS
• HISTORY
• DEFINITION OF DENTAL CARIES
• DIET AND DENTAL CARIES
• ETIOLOGY OF CARIES
• ENDOGENOUS THEORIES
• EXOGENOUS THEORIES
• STREPTOCOCCUS MUTANS
• VIRULENCE FACTORS
• MOLECULAR PATHOGENESIS OF THE
DISEASE
• IMMUNOLOGY OF CARIES IN MANwww.indiandentalacademy.com

• ANTIBODIES IN THE MOUTH
• HISTORY OF VACCINES
• PREVENTION OF DENTAL CARIES BY
IMMUNIZATION
• MECHANISM OF ACTION CARIES VACCINE
• MOLECULAR TARGETS FOR DENTAL CARIES
VACCINE
• SUBUNIT VACCINES
• ROUTES OF ADMINISTRATION
• STRATERGIES FOR EFFECTIVE MUCOSAL
IMMUNIZATON
• IDEAL VACCINE APPROACH
• REFERENCES

Caries inevitability of life like
taxes and death
introduction

History
• 5500 BC to 7000 BC - Pakistan teeth with perfect drilled
holes
• Homer and Guy de Chauliac- References to caries in
writings
• 1550 BC - Ebers Papyrus - Egyptian text mentions
diseases of teeth.
• 668 to 626 BC - Sargonid dynasty of Assyria , writings from
the king's physician specify the need to extract a tooth due
to spreading inflammation.
• 1798 – Edward jenner (milk maids)
• 1885 – Louis Pasteur (vaccine) first human vaccine
• 1950 – role of lymphocyte identified

DEFINITION OF DENTAL CARIES
• Shafer - as the microbial disease of calcified tissues of the
teeth, characterized by demineralization of the inorganic
portion and destruction of the organic substance of the
tooth.
• Studervant - dental caries is defined as the multifactorial ,
microbiological disease, that results in localized
dissolution and destruction of calcified tissues of teeth.
• WHO - dental caries is defined as the localized , post-
eruptive pathological process of external origin involving
softening of the hard tissues and proceeding to the
formation of the cavity.

DIET AND
DENTAL CARIES
• Relationship
• Bacterial enzyme + fermentable carbohydrate = acid
• Acid + enamel/ dentin = dental caries.
• Taku Fujiwara in 2005 caries = multifactorial disease =
teeth, cariogenic bacteria and fermentable sugars.www.indiandentalacademy.com

ETIOLOGY OF CARIESComplex, indirect factors & direct cause or causes.
No accepted or universally agreed upon cause but considered multifactorial.
1. ENDOGENOUS THEORIES:-
– HUMORAL THEORY
– VITAL THEORY
2. EXOGENOUS THEORIES:-
– WORM THEORY
– CHEMICAL THEORY
– PARASITIC THEORY
– MILLER’S THEORY
– PROTEOLYTIC THEORY
– PROTEOLYTIC CHELATION THEORY
– SUCROSE CHELATION THEORY
3. OTHER THEORIES:-
– AUTO IMMUNE THEORY
– SULPHATASE THEORY
– LEVINE’ S THEORY

ENDOGENOUS THEORIES
HUMORAL THEORY
• Galen, dental caries is produced by internal actions
and corroding humors. The four humors of the body =
blood, phlegm, black bile and yellow bile.
• Imbalance in humors resulted in the disease process.
• Hippocrates added that accumulated debris helped.
VITAL THEORY
• 18th century - postulated that tooth decay originated
within the tooth itself.
1. Caries histologically observing wider area beneath
and smaller pinpoint area on top.
2. Resorption from within the pulp signifies this concept.

EXOGENOUS THEORIES
WORM THEORY
• earliest reference of tooth decay and
toothache, 14th century B.C. from the
ruins of the Ying dynasty showed caries.
The cause of caries was thought to be
invasion of ‘worms’ into teeth.

CHEMICAL THEORY
• 17th and 18th century - new concept that teeth were
destroyed by acids formed in oral cavity.
• Robertson’s in 1835 that dental decay was caused by
acid formed by fermentation of food particles around
teeth.
PARASITIC THEORY
• Dubos( 1954) postulated that micro organisms can
have toxic effects on tissues.
1. A V Leeuwenhock (1632- 1723) indicated that micro
organisms were associated carious process.
2. Ficinus in 1847 also reported the filamentous
organisms in enamel cuticle.www.indiandentalacademy.com

ACIDOGENIC THEORY Miller(1889)
carbohydrate lodged on the tooth surface
acid production - demineralizes the enamel
demineralized enamel is mechanically
removed by the forces of mastication
organisms and acids penetrate dentinal tubules
and bring about the dissolution of dentin
Then proteolytic enzymes ultimately
digest the organic part.www.indiandentalacademy.com

• The three important factors are:-
1. Micro organisms
2. Carbohydrate substrate over the tooth surface.
3. Production of acids leading to protein degradation
• CHEMICO - PARASITIC POLYMICROBIAL PROCESS
• He concluded that:-
– Several microorganisms were capable of producing
acids.
– Lactic acid was identified in carbohydrate and saliva
mixtures.
– Different micro organisms had the potential to invade
carious dentin.
– Acid was present in deeper carious lesions.www.indiandentalacademy.com

Role of carbohydrates
The cariogenic carbohydrates are dietary in origin
affected by:
1. Frequency of ingestion
2. Physical form
3. Chemical form
4. Route of administration
5. Presence of other food constituents.
J. van Houte (1994) –
caries occurs due to
1) High carbohydrates exposure
2) Diminished salivary flowwww.indiandentalacademy.com

Role of micro organisms:-
• Goadby (1900) isolated a gram positive bacillus =
termed it = B. necrodentalis.
• Clarke (1924) described Streptococcus mutans.
• L. acidophilus seen with high frequency.
• other micro organisms :-
1. Streptococcus mutans
2. Lactobacillus
3. Actinomyces sp.

Streptococcus mutans
• facultative aneaerobic, non hemolytic acidogenic organism that
is capable of producing extra and intracellular polysacchrides.
• Fulfills Koch’s criteria.
1. form acid rapidly from carbohydrates (faster than many
organisms)
2. withstands acidic environment
3. forms insoluble glucans form sucrose

Role of dental plaque
• Suggested by Williams in 1897
• Miller - plaque protected the enamel against the carious
process.
• G V Black (1899) = “the gelatinous plaque of the caries
fungus is a thin , transparent film that usually escapes
observation and which is revealed only by careful search.
It is not a thick mass of material alba so frequently found
upon the teeth, nor it is the whitish gummy material.”
• Consists of salivary components (acquired pellicle – glyco
proteins), desquamated epithelial cells, micro organisms.

• Blayney (1942) concluded that
– acidogenic streptococci = 86% of the plaques
– lactobacilli = 57%.
– Glucan sticks to tooth and acts as a barrier against salivary buffers
– Limitations:-
1. Particular type of micro organisms was not isolated
2. Phenomenon of arrested caries could not be explained.
3. Concept of caries – free population could not be explained.
4. Smooth surface caries could not be explained.
sucrose glucan
S.Mutans - GTF

PROTEOLYTIC THEORY
Gottleib (1944)
proteolytic enzymes attacking the lamellae,
rod sheaths, enamel tufts and walls of the tubules.
caries initiated = slightly alkaline pH produced by the
proteolytic activity
Inorganic portion dissolved after organic degradation

• Limitations :-
• Enamel contains only 1.0-1.5 % organic
matrix, out of which proteins are only
0.6%. Too small a percentage for
breakdown.
Pincus(1949) also determined
• initial caries by proteolytic breakdown of dental cuticle.
• Nasmyth’s membrane and enamel proteins are acted
upon by sulphatase of bacilli = yielding sulfuric acid.

PROTEOLYSIS CHELATION THEORY
• Schatz et al (1955) - simultaneous microbial
degradation of organic part by the process of chelation.
• Limitations:-
• Enamel contains only 1.0-1.5 % organic matrix, thus its
dissolution to produce sufficient amount of chelates to
disintegrate the rest of inorganic part of enamel is
doubtful.
Organic part
destruction enamel
breakdown products
chelate minerals
mineral dissolution

• He's Only Two Microns Tall. And He
Has An Attitude
STREPTOCOCCUS MUTANS

STREPTOCOCCUS MUTANS
Major agent of caries:
1) Found only in the plaque associated with
carious teeth.
2) recovered from carious lesions and can be
grown in pure culture.
3) Infection of gnoto animals induces caries.
4) Caries = greater SIgA antibodies.

Based on DNA homology based into following
species:
1) S. mutans
2) S. sobrinus
3) S. cricetus
4) S. rattus
5) S. ferus
6) S. macacae
7) S. docunei
Further 8 serotypes (a, b, c, d, e, f, g & h).
MAJOR CULPRITS

1. Enzyme Glucosyl transferase (GTF) -
(Guggerheins & Shroeder, 1967)
2. Glucan binding proteins (GBP) - (Mal and
others, 1996)
3. Surface Antigens I / II or Cell wall Antigens
- (Brady et al, 1991)
4. Lipoteichoic acid

Enzyme Glucosyl transferase (GTF)
α- 1, 3 = insoluble = S.mutans
α- 1, 6 = soluble = S.sanguis
α- 1, 3 residues of the glucans helps :
1) Formation of plaque
2) Caries
3) de novo accumulation of more S.mutans
# GTF most potent antigenic component
sucrose
Glucans
(water soluble and insoluble)
GTF
GLUCOSE
+

ENZYME GLUCAN
TYPE
GENE
GTF-I, water
insoluble
gtf-B
GTF-γI water soluble
and insoluble
gtf-C
GTF-S water soluble gtf-F
• GTF-I and GTF-S = smooth surface caries
• SIgA antibody may target GTFs and
prevent accumulation

GLUCAN BINDING PROTEINS
• GBPs are cell wall proteins help S.mutans to bind
to glucans present
TYPE AFFINITY /
ACTION
Gbp A Water soluble
glucans
Gbp B Forms biofilms on
plastic surfaces
Vaccination
against GbpB
induces a
protective immune
response to
EXPERIMENTAL
CARIES
Gbp C dextran dependant
aggregationwww.indiandentalacademy.com

CELL WALL ANTIGENS
• Cell wall proteases, largest Antigen A and Antigen B
• Give bacteria “FUZZY APPEARANCE” – SEM
• Function: adhere to salivary pellicle
Antigen A low molecular
weight (29000
daltons)
Antigen B two antigenic
determinants =
called Antigen I
/ II.
Good antigen
but reacts to
the heart
tissue.

LIPOTEICHOIC ACID
• Cell wall component of virtually all gram +ive cells.
• Polymer of GLYCEROL + PHOSPHATE + GLYCOLIPID
• immunological cross reactions.

MOLECULAR PATHOGENESIS OF
THE DISEASE

hard surface covered by dental pellicle
bacterial ADHESINS may attach to it like Antigen I /II
or Pac of streptococcus mutans
• Antigen I /II cause
1. Bacterial attachment to pellicle
2. S.mutans attachment to other bacteria like S.sanguis
and A.viscosus
Further aggregation of new bacteria on insoluble glucan scaffold
occurs due to
1. GBPs
2. GTF www.indiandentalacademy.com

DENTAL BIOFILM formed due to extracellular GTFs
GTF B & C
SUCROSE INSOLUBLE GLUCANS (pathogenic)
Act as Scaffolds for other Bacteria
large number of bacteria and lactic acid produced
CARIESwww.indiandentalacademy.com

IMMUNOLOGY OF CARIES IN MAN
• The defense of the oral cavity can be of two types:
1. Non Specific
for anti bacterial systems as saliva such as
lysozyme, lactoperoxidase and lactoferrin which
are active against many species of bacteria.
2. Specific
This involves exposing the host to killed or
attenuated forms of the organism in order to
instruct the host’s immunological memory to mount
an effective antibacterial response when fully
virulent organisms are encountered.www.indiandentalacademy.com

Antibodies in the mouth two
types
• :
1. The secretory (salivary) antibodie SIgA.
2. The serum antibodies (IgG, IgM and IgA)
which enter the mouth mostly from the
gingival crevice.

• dimeric, comprising two molecules of IgA united by a
polypeptide, “secretory component” together with a
shorter junctional peptide known as the “J” chain
Monomeric form from parotid
J chain unites = dimeric = epithelium of
the gland
Antigenic stimulation
1) Salivary antibodies

MALT associated specialized cells take up Antigens
ANTIGEN PRESENTING CELLS
B Cells & Plasma cells
Migration to various regions like salivary glands
IgA is secreted and processed into dimeric form
# Secretory immunity lacks an effective memory of
antigens.

• Functions :
1. to bind with and aggregate foreign bodies
2. to inactivate antigens and toxins
3. prevent antigen / bacterial adherence to enamel. done by
– By blocking receptors that bind.
– SIgA binding to the cell wall might increase the
hydrophilicity of the antigen and facilitate the entrapment
of bacteria within salivary mucus and their clearance by
swallowing.

antigen is first bound and concentrated by macrophages
B cells and Helper T cells
IgG secreted
complement activation
chemotactic factors which attract phagocytic PMNs,
cause histamine release and lysis
2) Serum Antibodies

• efficacy of serum IgG ?????????
• since dental caries occurs in areas inaccessible to blood
components
• plaque in the cervical region of the tooth and on the root
surfaces of older subjects is thus subjected to the
influence of both the SIgA and serum immunoglobulinswww.indiandentalacademy.com

HISTORY OF VACCINES
• Edward Jenner the pioneer in the field of immunization
• Louis Pasteur eventually succeeded in developing a
vaccine against anthrax and rabies.
• 1930 - caries immunization experiments performed with
Lactobacillus.
• 1960 - Streptococcus mutans became the target as
major pathogen

• RATIONALE
• Vaccine against SM to prevent initiation.
• Vaccination before 6 months or before deciduous
eruption
• Along with DPT and booster doses could be given.
• WINDOW OF INFECTIVITY” {Caufield et al,1993}
• Source of infection- Maternal.
Environmental conditions.
• Children who do not become infected by 3yrs. appear to
remain uninfected for several yrs.,possibly until eruption
of the secondary dentition.{Caufield et al,1993:Smith et
al,1998}.
PREVENTION OF DENTAL
CARIES BY IMMUNIZATION

• 1) ACTIVE IMMUNIZATION
• 2) PASSIVE IMMUNIZATION
MECHANISM OF ACTION CARIES
VACCINE

1) ACTIVE IMMUNIZATION
• Whole antigen or subunit could be administered.
• Attenuated whole streptococcus mutans – may contain
fimbrial Mproteins which cross reacts with the heart
tissue
• MS cells or purified GTFs injected into the salivary gland
region to induce TgA secretion - altering the function of
the gland.
• ADV: LONG TERM PROTECTION
• DIS: CANT BE GIVEN TO IMMUNOCOMPROMISED
PATIENTS

2) PASSIVE IMMUNIZATION
• Readymade antibodies against SM.
• ADV: immediate protection and is useful for immuno-
compromised patients
• DIS: transient protection
Mouth rinses
containing
bovine milk or
hen egg yolk
have = IgY
very short time.
Mucosal
application in
mouse.
monoclonal IgG
or Transgenic
plant secretory
SIgA / G =
specificity for Ag
I / II
Longer action

• 1) ADHESINS
• 2) GLUCOSYLTRANSFERASES (GTFs)
• 3) GLUCAN BINDING PROTEINS (GBP)
MOLECULAR TARGETS FOR
DENTAL CARIES VACCINE

MOLECULAR TARGETS FOR
DENTAL CARIES VACCINE
• Rationale :
1. Clear the microorganism by antibodies before aggregation
while still in the salivary phase, prior to colonization.
2. Block receptors receptors of the enzyme to prevent further
colonization or accumulation.
3. Action of Salivary IgA may be enhanced or redirected by
synergism with innate components of immunity like mucin
or lactoferrin.

• Adhesins like Antigen B are required for SM attachment
• Antigen I / II or Pac are 2 immunogenic portions of
Antigen B that bind to alanine rich region of dental
pellicle.
• Antibodies against Antigen I / II or passive immunization
with monoclonal or transgenic antibody protective
against S.mutans.
• # Antigen III has also been detected but it occurs only in
S.mutans and not in other strains of streptococcus. little
value
1) ADHESINS

2) GLUCOSYLTRANSFERASES (GTFs)
STRAIN GENE RESPONSIBLE FOR GLUCAN
SYNTHESIS
Streptococcus
mutans
gtf B, gtf C and gtf D
Streptococcus
sobrinus
Gtf I, gtf S
• Mutational inactivation of GTF producing genes like gtf
B and gtf D = beneficial.
• Induction of SIgA antibodies in humans by oral or topical
administration causes interference in S.mutans
attachment.
• GTFs form glucans for attachment of SM

3) GLUCAN BINDING PROTEINS
(GBP)
• GBPs help bacteria to attach, aggregate and
colonize to pellicle and to glucan residues
present.
• Salivary IgA antibodies against Gbp B are
indicates initial infection..
TYPE AFFINITY / ACTION
Gbp A Water soluble glucans
Gbp B
(most
immunogenic)
Forms biofilms on
plastic surfaces
induces a protective
immune response to
EXPERIMENTAL
CARIES
Gbp C dextran dependant
aggregationwww.indiandentalacademy.com

SUBUNIT VACCINES
• Contain structural elements for the Ag I / II, GTFs or Gbp B.
• Functional epitopes should be developed for immune
responses to target salivary binding, catalytic processors or
glucan binding activities.
• Conjugation of functionally associated peptides to
carbohydrate components for example glucan, or to other
vaccine proteins also would increase the immunogenecity of
the peptide and broader the reach for the vaccine.
• Since Ag I / II family of proteins have similar sequences,
vaccines could be effective against many streptococcal
strains.

A) SYNTHETIC PEPTIDE
VACCINES:
• Potential targets for vaccine:
1. Proline and Alanine rich segments of Ag I / II protein
2. Synthetic peptide vaccines directed at one of the
several N-terminal GTF peptides
3. Combining epitopes from adhesins and GTFs
enhancing the immunogenecity with additional
sequences.
4. Pac, coupled to cholera toxin B subunit suppressed
colonization.

• free synthetic peptides were applied to the
gingival mucosa of monkeys, induced salivary
IgA and gingival IgG formation.
• Monoclonal antibody, raised by immunization
with intact Ag I/II inhibits dental caries

B) RECOMBINANT VACCINES /
ATTENUATED EXPRESSION VECTORS
• Recombinant DNA is produced in single celled organisms by gene
hybridization so that they express genes which code for sites,
peptide sequences or proteins of the cariogenic bacteria.
• Salmonella and E.coli with recombinant DNA can be administered
as a passive immunization for caries as well in a live or attenuated
form.
• Chimeric proteins can be developed as these proteins get
expressed by organisms with recombinant DNA e.g. Salmonella,
E.coli. www.indiandentalacademy.com

C) CONJUGATE VACCINES:
• This approach uses conjugation of proteins of the
bacteria with polysacchrides which enhances their
immunogenic potential which can cause these
immunogens to get targeted by the T-Cells.
• Thus it is converting of or conjugation of functionally
associated protein / peptide components with bacterial
polysacchrides.
• These vaccines may be better than protein based
vaccines.

ROUTES OF ADMINISTRATION
1. Oral
2. Systemic (subcutaneous)
3. Active gingivo salivary
4. Intranasal
5. Tonsillar
6. Minor salivary glands
7. Rectal

1) ORAL
• Antigen was applied by oral feeding, gastric intubation or
in the form of vaccine containing capsules or liposomes
to elicit a response by GALT.
• Disadvantage:
• gut acid may influence IT.
• Inductive sites are relatively distant
• The rise in secretory antibodies produced was of a very
short duration

2) INTRANASAL
• targets the nasal associated lymphoid tissue (NALT) by
application near the oral cavity
• S.mutans Ag I / II, GTF-B domain of glucan binding),
Gbp B and fimbrial preparations from S.mutans =
afforded protection
• Closer to the oral cavity

3) MINOR SALIVARY GLANDS
• Minor salivary glands of the lips, cheeks and soft palate
can be potential routes for mucosal induction of salivary
immune responses. Because of their short, broad
secretory ducts that facilitate retrograde access of
bacteria and their products.
• Advantages:
1. Lower doses of antigen are required
2. No effect of GIT acid
3. Induces both systemic and mucosal immunity
4. Administration is relatively easy.

4) TONSILLAR
• Tonsillar tissue contains elements of immune
induction of both secretory IgA and IgG
responses.
• Palatine tonsils especially the nasopharyngeal
tonsils contribute precursor cells to mucosal
effector sites like salivary glands.
• Repeated Topical application of formalin killed
S.sobrinus in rabbits can induce IgA producing
cells in both major and minor salivary glands.

5) RECTAL
• The colo – rectal region as an inductive
site has been suggested because of the
fact that this site has the highest
concentration of lymphoid follicles in the
intestinal tract.
• Thus the use of vaccine suppositories as
one alternative for children with respiratory
ailments may be useful.

6) TOPICAL GINGIVO SALIVARY
IMMUNIZATION
• to exclude any potential systemic side
effects and to localize the immune
response to the oral cavity.
• Small molecular weight antigen required
since high molecular weight antigen cant
penetrate crevicular barrier.

STRATERGIES FOR EFFECTIVE
MUCOSAL IMMUNIZATON

“What is the ideal dental caries
vaccine approach”
• A vaccine giving broadest coverage.
• To intercept infection by all common S- mutans
strains.
• For both low and high risk populations.
• Long lasting
• Given with other immunizations.
• Given by various routes and still be effective.

• Inexpensive.
• Delivered by individuals with little training.
• High risk populations may require both
active and passive mechanism for
protection
• Can or should we expect all of these
characteristics in one vaccine?????......

Why is caries vaccine a dead
issue ???????
• Unlike small pox or measles, S. mutans is
only partial responsible for dental caries.
• Dental caries develops on a non living and
non shedding body surface.
( isolated from the activity of phagocytic
cells and complement )

CONCLUSION

REFERENCES
• CARIOLOGY TODAY. 1983,285 – 292.
• CRIT.REV.ORAL BIO MED. 13(4), 335
-49,2002.
• J. DENT EDU VOL 67,NO. 10,2003.
• CARIES RES,2004,38: 230-35.
• OPER.DENT. 6,2001, 51-60.
• J DENT. RES. 83(3): 266-70, 2004.
• CARIES RES. 1999: 4-15.
• J. DENT. RES. 75(8), 1996.
• JADA1996,127; 1477-1487
• OPER DENT 2005 SUPPLEMENT 6www.indiandentalacademy.com

Thank you...www.indiandentalacademy.com

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