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Good morning…….

CARIES VACCINE….
Most common/widespread-Infectious disease
of mankind.
Once acquired-persists throughout life,even if
treated.
Potentially transmissible.

IS CARIES CONQUERED???..
 “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}
 Hence, immunization could be used to protect
the child against caries in this critical period.
Underwood & Milles(1881)-Bacteria are
involved in pathogenesis of dental caries.

 Miller(1890)-chemicoparasitic theory of
dental caries.
 J.K.Clarke(1924)-specific microorganism
[Streptococcus mutans] is associated
with dental caries.
 Since dental caries fulfills the criteria for
infectious disease, possibilities of
vaccination have been considered.
VACCINE:
 Immunobiological substance designed to
produce specific protection against given
disease. It is a suspension of attenuated or
killed microorganisms, administered for the
prevention or treatment of infectious
diseases.
 They may be prepared from live modified
organisms, inactivated or killed organisms,
cellular fractions, toxoids or combinations of
these.
 Antigen – any substance which stimulates
production of antibody with which it reacts
specifically and in an observable manner.
 Antibody – substance which appears in
serum/tissue fluids which react with the
antigen specifically and in an observable
manner.
 Immunity- host resistance towards injury
caused by microbes or products.
Natural host immunity (IgA):-
 Secretory immunity against mutans.
 2nd.most abundant Ig- generated by “common
mucosal immune system.”
 10% of serum Ig= 0.6-4.2mg./ml.
 Half life=6-8 days.
 2 types---Secretory IgA
Serum IgA
-Secretory IgA-115dimer.
-2 IgAs joined by J-chain+one secretory/transport
piece.
-Saliva= 100-300 g/ml.
-Local immunity, Resistant to digestive enzymes.
Functions of IgA:-
 Inhibits adherence of micro-organisms,
colonization.
 Promotes phagocytosis/extracellular killing of
micro-organisms.
 Neutralizes microbial enzymes/toxins.
 Activates alternate complement pathway.
 Specifically eliminates Mutans-phagocytosis
/swallowing.
 2 types---IgA1—60%
IgA2
Mestecky & Russell1986
 Inducing IgA immunizations through new
strategies of mucosal immunization—Induce
Secretory IgA Ab without complication of
parenteral immunization.
MOLECULAR PATHOGENESIS OF CARIES

 Initial attachment to the tooth is


achieved via the interaction of bacterial
proteins with lectins in the dental
pellicle.
 The bacterial proteins being
Streptococcal adhesins (Antigen I/ II or
PAc in Strep. Mutans).
 Lamont and Co workers (1991) – S. mutans
antigen I/II adherence to pellicle is mediated
through an acidic, mucin-like glycoprotein
(agglutinin) found in parotid and
submandibular saliva.
 Atleast 2 binding regions of Ag I/II involved
in adhesive activities( Crowley et al 1993,
Nakai et el 1993, Kelly et el 1995)
 Ultimate pathogenecity of S.mutans – erosion of
the hydroxy apatite in enamel by lactic acid(
bacterial metabolic end-product).
 However, significant destruction by this acid
requires accumulation of streptococci in dental
plaque.
 This accumulation is initiated by the activity of
extra cellular Glucosyltranssferases (GTF),
secreted by S.mutans.
 In the presence of sucrose, GTFs synthesize a
high molecular weight extra cellular glucans.
 GTFs that synthesize insoluble forms of
glucan (GTF-B & GTF-C)- associated with
pathogenecity.
 These glucose polymers aggregate mutans
and other oral streptococci through
interaction with bacterial cell associated
glucan binding proteins (GBP)
 Each GBP binds to certain forms of glucan
 GTFs also contain glucan binding domains
which bind to glucans

 Glucans accumulation of S.
+ mutans in biofilm
(GTFs + GBPs)
NEXT PHASE OF
PATHOGENESIS
 Accumulated Strep.mutans most
prolifically produce lactic acid.(Gibbons
&Van Houte, 1975)
 If this increase in lactic acid synthesis
can’t be sufficiently buffered, Dental
caries ensues.
Effective molecular targets for dental
caries vaccines

 Several stages in the molecular


pathogenesis of D.C are susceptible to
immune intervention viz.,
1. Micro organisms can be cleared by Ab
mediated aggregation prior to colonization
2. Block the receptors necessary for
colonization (Eg- Adhesins) or accumulation
( Eg- Glucan binding domains of GBPs and
GTFs) or inactivate GTF enzymes
responsible for glucan formation.
3. antimicrobial activity of salivary Ig A
Ab may be enhanced or redirected
by synergism with innate
components of immunity, such as
mucin or lactoferrin.
Hence adhesins, GTFs and GBPs
can be used as vaccine targets.
ADHESINS
 Adhesin from S mutans (antigen I/II,
Pac,P1) and S sobrinus ( SpaA OR PAg)
Ag I/II - found on S mutans cell
surface.
- 185-kDa protein, single poly
peptide chain of 1600 residues
- Contains an Alanine–rich
repeating region in the N –terminal third
and proline rich repeat region in the
center.
- Alanine rich region binds to tooth pellicle and
proline rich central portion contains an
adhesion epitope.
- Hence immunological approaches use specific
antibodies against S mutans Ag I/II or S
sobrinus Spa A thereby interfering with
bacterial adherence and subsequent dental
caries.
- Synthetic peptides from the alanine rich
region of Ag I/II supressed tooth colonization
with S mutans.
Glucosyltransferase
GLUCOSYLTRANSFERASES(
GTFs)
 S mutans and S sobrinus synthesize several
GTFs

 Genes responsible for glucan synthesis in S


mutans are gtfB which synthesizes an alfa-
1,3, linked insoluble glucan.

 gtfC which synthesizes glucan with both alfa-


1,3 and alfa-1,6 linkages.
 GTF D which synthesizes soluble alfa-1,6
linked glucan
 When GTF B and GTF C genes were replaced
by functional copies of these genes, it
significantly reduced caries
 The activity of GTF is mediated through
1) catalytic
2) glucan binding functions.
 Catalytic activity of GTF appears to be
associated with separate residues in the
N-terminal third of the molecule.
 The C-terminal region of GTF contains
repeating sequences identified in all S
mutans. This region has the ability to
bind to glucans (alfa 1,6 glucan) mooser
and wong 1988, wong et al 1990.
 Antibody directed to native GTF or sequences
associated with its catalytic or glucan-binding
function interferes with the synthetic activity
of the enzyme and with in-vitro plaque
formation (taubman and smith 1972).
 Hence active immunization with either S
mutans or S sobrinus GTFs induced protective
immune responses in dental caries.
 Induction of SIgA antibody in humans by oral or
topical GTF administration is accompanied by
interference with accumulation of S mutans ( smith
& taubman 1987,1990)

 Passive administration of antibody to GTF in the diet


can protect from dental caries.

 Thus presence of antibody to GTF in the oral cavity


prior to infection can significantly influence the
disease outcome ,presumably by interference with
one or more of the functional activity of the enzyme.
GLUCAN- BINDING PROTEINS
(GBPs)
 S mutans binds to glucans by cell-wall
associated glucan binding proteins
(Gbp)
 EACH GBP has the ability to bind to alfa
1-6 glucan.
 S mutans secretes 3 distinct proteins
with glucan binding activity:
- GbpA,GbpB,GbpC.
 GbpA has greater affinity for water
soluble than for water insoluble glucan.
 GbpB plays a role in bio-film formation
 GbpC is associated with dextran-
dependent aggregation.
 Only GbpB has shown to induce
protective immune response to dental
caries.
SUBUNIT VACCINES
 Contain structural elements of the Ag
I/II adhesin family, gtfs or gbpB
 These vaccines contain single or
multiple copies of epitopes of domains
associated with salivary binding,
catalytic processes or glucan binding
activities.
 These homologous sequences may
induce cross reactive responses
influencing colonization, attachment or
accumulation of commensal microbiota.
 Hence subunit vaccines are designed to
exclude sequences bearing the potential
for induction of unwanted antibody
responses
Types of subunit vaccines

 Synthetic peptide vaccines

 Recombinant vaccines/ attenuated


expression vectors
Synthetic peptide vaccines

 Monoclonal antibody, raised by


immunization with intact Ag I/II inhibits
dental caries
 Alanine rich repeat region of Ag I/II is
immunogenic and induces protective
immunity
 PAcA, coupled to cholera toxin B
subunit suppressed colonization.
 When free synthetic peptides were
applied to the gingival mucosa of
monkeys, salivary IgA and gingival IgG
were formed.
 Hence protection could be achieved
by immunization with discrete epitopes
associated with several virulence
characteristics.
Recombinant vaccines/
attenuated expression vectors
 Recombinant vaccines afford the expression of
larger portions of functional domains that can
be accommodated by synthetic peptides.
 Attenuated mutant vectors such as salmonella,
which contain plasmids can target the vaccine
to appropriate inductive lymphoid tissue for
mucosal responses. Eg: attenuated S. typhi +
gtf sequences + glucan binding domain +
tetanus toxin fragment C.
ROUTES TO PROTECTIVE
RESPONSES
 Mucosal application of caries vaccine are
generally preferred for induction of secretary IgA
antibody, since this Ig constitutes major immune
component of major and minor salivary gland
secretions.
 Exposure of antigen to mucosally associated
lymphoid tissue in the gut, nasal, bronchial or
rectal site arises immune responses even in
remote locations—”common mucosal immune
system’’(mestecky,1993)
(A) ORAL
 Oral induction of immunity in the gut associated
lymphoid tissues (GALT) elicits protective IgA
antibody.
 Antigen was applied by oral feeding, gastric
intubation, vaccine containing capsules or
liposomes.
 Induction of mucosal immunity alone was sufficient
to reduce caries.
 Disadvantages: Effect of stomach acidity on antigen,
Inductive sites were relatively
distant.
(B) INTRA NASAL
 Closer anatomical relationship to the oral
cavity.
 Intranasal installation of antigen targets the
nasal associated lymphoid tissue (NALT)
(Brandtzaeg & Haneberg,1997).
 Induces immunity against streptococcal
colonization & accumulation
 Protection demonstrated with S mutans
Ag I/II,GTF-B.
TONSILLAR
 Contains required elements of immune
induction of secretory IgA responses.
 Palatine tonsils, especially nasopharyngeal
tonsils contribute precursor cells to
mucosal effector sites, such as the salivary
glands.
 Topical application of formalin-killed
S.sobrinus cells can induce salivary immune
responses to decrease caries
MINOR SALIVARY GLANDS
 found in lips, cheeks and soft palate.

 potential routes for mucosal induction


of salivary immune responses due to
short broad secretory ducts that
facilitate retrograde access of bacteria
and also because of the lymphatic
tissue aggregates associated with these
ducts.
 experiments in which s.sobrinus Gtf
topically applied on lower lips- have
potential for dental caries vaccine.-these
subjects had lower proportions of
S.mutans
RECTAL
 Rectal immunization with H.pylori or
S.pneumoniae result in appearance of
secretory IgA antibody in distant
salivary sites.
 Colo-rectal region has highest
concentration of lymphoid follicles in
lower intestinal tract.
 Can induce salivary IgA responses
to mutans antigens (GTF).
 Used in children with respiratory
ailments where intranasal
application is not possible.
ADJUVANTS AND DELIVERY
SYSTEMS FOR D.C.VACCINES

 Mucosal routes often require additional


components which potentiates aspects
of the immune response to induce
sufficient antibodies to achieve a
protective effect.
CHOLERA AND E.COLI HEAT
LABILE ENTEROTOXINS
 Cholera toxins (CT)- powerful mucosal
immuno-adjuvant.
-ADP- bacterial toxin.
 Adjuvant effects of CT include increased

mucosal epithelial cell and macrophage


production of pro-inflammatory cytokines.
 Can greatly enhance mucosal immune

response.
LIPOSOMES
 Phospolipid membrane vesicles are used to
contain and deliver drugs and antigens.
 Improve mucosal immune responses by
felicitating M cell uptake and delivery of
antigen to lymphoid elements of inductive
tissue .
 IgA1 antibody response was higher with
liposome containing compared to protein
vaccine alone.
ACTIVE IMMUZATION
 Immunization with GTFs from S.mutans or
S.sobrinus leads to formation of salivary IgA
antibodies
 Oral immunization using enteric–coated
capsules filled with crude mutans GTF antigen
preparations which are contained in
liposomes.
 Nasal immunization with dehydrated
liposomes containing GTF preparation induces
IgA1 antibody response.
 topical administration of GTF on lower
lip caused a delay in S.mutans
reemergence.
 Mucosal immunization with dental caries
vaccines could be protective, especially
in pediatric population where S mutans
is not yet a permanent member of the
bio-film.
Passive immunization
 Mouth rinses containing bovine milk or hen egg
yolk IgY antibody to S mutans cells led to
decrease in S mutans in saliva and dental plaque.
 Topical application of mouse monoclonal IgG or
transgenic plant secretory SIgA/G antibody, each
with specificity for Ag I/II.
 Following antibacterial treatment, antibody was
topically applied for 3 weeks.Recolonization with
S mutans did not occur for atleast 2 years after
treatment with mouse monoclonal antibody.
PROSPECTS AND CONCERNS
 For populations under normal risk of
infections Immunization for dental caries
should begin early in the second year of life.
 If bacterial colonization of the dental bio-film
is complete after eruption of all primary teeth
and if through immunization we can prevent
S mutans colonization prior to this period
then benefit of early immunization extends
until secondary teeth begin to erupt.
“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.
 Whose immunity would last through primary
and secondary infection periods.
 Given with other immunizations.
 Given by various routes and still be effective.
 Inexpensive.
 Delivered by individuals with little
training.
 Can or should we expect all of these
characteristics in one vaccine?????......
 E.g., ideal vaccination application for a
child with asthma may be at a site e.g.,
rectal and with an adjuvant e.g.,
detoxified CT.
 Dental caries vaccine would be the first
non living vaccine to be applied by any
mucosal route during the first three
years of life.
 High risk populations may require both
active and passive mechanism for
protection.
 Understanding the signals for colonization
and growth of cariogenic streptococci in
dental bio-films may help us devise more
informed and refined techniques to “
LOCK OUT ” those bacteria that can cause
us harm.
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 )
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.
Thank you...