SEMINAR ON CARIES VACCINE EVOLUTION, TYPES OF VACCINE AND SCOPE FOR FUTURE RESEARCH

1. Dr Divya Gaur
Dept of Pedodontics and Preventive Dentistry

2. • INTRODUCTION
• ACQUISITION OF STREPTOCOCCUS MUTANS
• BIOFILM
• ROLE OF DENTAL PLAQUE
• IMMUNOLOGY OF DENTAL CARIES IN HUMANS
• TYPES OF IMMUNE RESPONSE
• NEED FOR CARIES VACCINE
• REQUIREMENTS OF DENTAL VACCINE
• MOLECULAR PATHOGENESIS OF DENTAL
CARIES
• MOLECULAR TARGETS
• GLUCOSYLTRANFERASE
• GLUCAN BINDING PROTEINS
• ADHESINS

3.  MECHANISM OF ACTION OF VACCINES
 TYPES OF VACCINES
 ROUTES OF IMMUNIZATION
 LOCAL
 SYSTEMIC
 PASSIVE
 ADJUVANTS AND DELIVERY SYSTEMS
 CLINICAL RESEARCH
 RISKS OF USING CARIES VACCINE
 PUBLIC HEALTH VIEW
 RECENT ADVANCES
 CONCLUSION

4.  Dental caries is an infectious microbiologic disease of the teeth that
results in localized dissolution and destruction of the calcified tissue1.
Clifford MS. The art and science of Operative
dentistry. 4th ed. Mosby
Publications; 1995.

5. DENTAL
CARIES
STREPTOCOCCUS
MUTANS
LACTOBACILLUS
ACIDOPHILUS
ACTINOMYCES
VISCOSUS
EXTRINSIC
CARBOHYRATES
INTRACELLULAR
POLYSACCHARIDES
FOURTH FACTOR
ADDED BY
NEWBRUN
SMOOTH
SURFACES
PITS AND FISSURES
KEYES TRIAD AND NEWBRUN‘S
TETRAD

6. MANAGEMENT OF DENTAL CARIES:
EVOLUTION
•‘DRILL AND FILL’
•EXTENSION FOR
PREVENTION
CONVENTIONAL
APPROACH
•PREVENTION FOR
EXTENSION
•MINIMAL TOOTH
CUTTING
CONSERVATIVE
APPROACH •AIMED AT:
•MICROORGANISMS
•HOST FACTOR
•SUBSTRATE
FACTOR
PREVENTIVE
APPROACH

7. STREPTOCOCCUS MUTANS
•WHAT IS IT?
•A gram + facultative anaerobe
characterized by 8 serotypes.
•PREVALENCE:
•Most prevalent serotype: h
•HOW DOES IT CAUSE DENTAL CARIES?
•BIOFILM formation
•Aciduric and acidogenic.
S. mutans
S.Sobrinus
S.Ratti
S.Criceti
S.Downei
S.Ferus
S.macacae

8.  CAUFIELD et al , « window of infectivity « : children become
permanently colonized with mutans streptococci between the middle of
second year and the end of the third year.
 This window may open even earlier if maternal S. mutans infection
levels are high, coupled with frequent exposures to dietary sucrose.
 DNA probe technology have suggested that low levels of mutans
streptococci may be found in the oral cavity during the first year of life.
 Thus, data suggests that ‘window of vaccine opportunity’ could exist
between 12 and 18 months for most populations.

9.  A COMMUNITY OF BACTERIA ATTACHED TO SALIVARY
COMPONENTS AND EMBEDDED IN A MATRIX OF GLUCAN OF HIGH
MOLECULAR WEIGHT, PRODUCED BY STRETOCOCCUS MUTANS.

10.  Suggested by Williams in 1897.
 GV BLACK (1899):
‘the gelatinous plaque of 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 frequently found upon the teeth, nor it is the
whitish gummy material.’
 Consists of salivary components (acquired pellicle- glycoproteins),
desquamated epithelial cells and micro-organisms.
 Blayney (1942)
 Acidogenic streptococci: 86% of plaque
 Lactobacilli: 57%

11. ORAL CAVITY DEFENSE CAN BE OF 2 TYPES:
 NON- SPECIFIC:
 Lysozyme, lactoperoxidase and lactoferrin: active against many species
of bacteria.
 SPECIFIC:
 Involves exposing the host to killed or attenuated forms of organism to
instruct the host’s immunological memory.

12.  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: It is defined as resistance offered by the host to the harmful
pathogenic microbial infection.
Specific Non-Specific
Active Passive
Natural Artificial Natural Artificial
Innate Acquired

13.  SECRETORY SALIVARY ANTIBODIES : IgA
 SERUM ANTIBODIES : IgG, IgM AND IgA

14.  THE PRIMARY RESPONSE:
 When an antigen is administered for the first time to an animal/ human.
 Latent period of induction : 3-10 days.
 Antibodies appear in the blood.
 First antibody to be elicited: IgM type.
 Antibody titre rises steadily during the next 2-3 days, reaches a peak level, and
then declines almost as fast as it develops.
 if antigenic stimulus was sufficient, IgG appears in a few days.
 IgG reaches peak in 7-10 days and then gradually falls over a period of weeks or
months.
IMPORTANT OUTCOME OF PRIMARY RESPONSE: B and T lymphocytes produce
“memory cells” or “ primed cells” that are responsible for immunological memory
established after immunization.

15.  THE SECONDARY (BOOSTER) RESPONSE:
 Also involves production of IgM and IgG antibodies .
 Brief production of IgM and much larger and prolonged production of IgG antibody.
 This accelerated response is attributed to immunological memory.
Park K. Text book of preventive and social medicine. 17th ed. Bhanotidas
Publication; 2004.
Warren L, Ernest J. Medical microbiology and immunology. 6th ed. Lange
Medical Publishing Division; 2000.

16.  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.
 DENTAL CARIES VACCINE: The evidence of a specific bacterial cause of
dental caries and the function of salivary glands as an effector site of the
mucosal immune system has provided a scientific basis for developing a
vaccine against this highly prevalent oral disease.

17.  EDWARD JENNER: pioneered in the field of immunology.
 LOUIS PASTEUR: developed vaccines against rabies and anthrax.
 1930: Caries immunizaition experiments were performed on lactobacillus.
 1960: Streptococcus mutans became the target .

18.  Streptococcus mutans is a poor immunogen.
 Entry of S.mutans through junctional epithelium of gingiva not
sufficient to elicit an immune response.
 T-cell response to S.mutans is of low order and needs boosting for
sensitization.

19.  IDENTIFY THE TARGET.
 IDENTIFY THE COMPONENT OF IMMUNE SYSTEM THAT
SHOULD BE STIMULATED/ INDUCED.
 SHOULD NOT HARM THE HOST

20. ACQUIRED PELLICLE FORMS ON
THE TOOTH SURFACE
INTERACTION OF BACTERIAL
PROTEINS (STREPTOCOCCAL
ADHESINS) WITH DENTAL
PELLICLE.
FURTHER ACCUMULATION OF
ACIDOGENIC STREPTOCOCCI
ANTIGEN
I/II or Pac
GLUCOSYLTRANS
FERASES
GTF-B AND GTF-C
GTF provide scaffolding for aggregation of mutans and other oral
streptococci through interaction with bacterial call- associated
glucan binding proteins.

21. GLUCAN

24. Moro I, Lehner T. Symposium report: Sixth International congress of mucosal
immunology; Dental caries vaccine. J Dent Res July 23rd 1990. p. 1863-4.
Russell RR. The application of molecular genetics to the microbiology of dental caries.
Caries Res 1994;28:69-82.

25.  Function of Glucans: provide scaffolding for the aggregation of mutans and
other oral streptoccocci through interaction with bacterial cell- associated
glucan binding proteins.
 Also, Glucans modify the porosity of the dental biofilm, thus increasing the
availability of nutrients for continued bacterial metabolism.
Moro I, Lehner T. Symposium report: Sixth International congress of mucosal
immunology; Dental caries vaccine. J Dent Res July 23rd 1990. p. 1863-4.
Russell RR. The application of molecular genetics to the microbiology of dental caries.
Caries Res 1994;28:69-82.
 Three forms of GTF:
 Water insoluble glucan synthesizing enzyme: GTF-I
 Water insoluble and water- soluble glucan synthesizing enzymes: GTF-S-
I
 Water- soluble glucan synthesizing enzymes: GTF-S

26.  3 distinct proteins with glucan binding activity: GBP-A, GBP-B and GBP-C.
 Only GBP-B has been shown to induce a protective immune response to
experimental dental caries.

27.  First described by Russell and Lehner.
 Bind acidic, mucin like glycoproteins found in saliva.
 2 principal human pathogens:
 Antigens I/ II, Pac or P1
 Streptococcus sobrinus: Spa-A or Pag.
 Antigens I/II : found in the culture supernatant as well as in S. mutans cell
surface.
 Contains alanine-rich tandem- repeating region in the N-terminal third and a
proline rich repeat region in the centre of the molecule.
 Antibody directed to the intact Ag I/II molecule or to its salivary binding
domain blocked adherence of S. mutans of saliva-coated hydroxyapatite.
Smith DJ. Dental caries vaccines: Prospects and concerns. Crit Rev Oral
Biol Med 2002;13:335-49.

28.  SALIVARY IMMUNOGLOBULIN may interact with BACTERIAL
SURFACE RECEPTORS, and inhibit colonization .
 May inactivate surface glucosyltransferases, which would reduce synthesis of
extra cellular glucans resulting in reducing plaque formation.
 SECRETORY IgA from salivary glands due to direct immunization of gut
associated lymphoid tissue (GALT):
 May prevent MS from adhering to the enamel surface.
 May also prevent formation of dextran by inhibiting the activity of
glucosyltransferase (GTF).
 GINGIVAL CREVICULAR mechanism : All humoral and cellular
components of systemic immune system, that exert its function at tooth
surface.
Lehner T. Immunology of dental caries. Immunology of oral diseases. 3rd ed. Blackwell scientific publications; 1992.
Wilton JM. Future control of dental disease by immunization: Vaccines and oral health. Int Dent J 1984;34:177-83.
Lehner T, Challacombe SJ, Caldwell J. Immuologic basis for vaccination against dental caries in Rhesus monkeys. J Dent
Res 1976;55:C166-80.

29. Contains structural
elements of either
adhesins or GTF or
GBP.
Also called
attenuated
expression vectors.
Helpful in
targeting vaccine to
appropriate
lymphoid tissue for
mucosal response.
Chemical
conjugation of
functionally
associated peptide
components with
bacterial
polyssacharides.
SUB UNIT VACCINE RECOMBINANT VACCINE CONJUGATE VACCINE

30. ORAL/ MUCOSAL
SYSTEMIC (SUBCUTANEOUS)
ACTIVE GINGIVO- SALIVARY
PASSIVE DENTAL IMMUNIZATION

31. Administration of antigen in the vicinity of
salivary glands
Induction of salivary IgA to S.mutans
Adverse reactions-
Local inflammation
Systemic serum antibody response

32.  Mucosal application of dental caries vaccines is preferred for induction of
secretory IgA antibodies in salivary compartment.
 It has been shown that exposure of an antigen to a mucosally associated
lymphoid tissue in the gut, nasal, bronchial, or rectal site can give rise to
immune response not only in the region of induction but also in remote
location.
 Several mucosal routes used to induce protective immune responses to
dental caries vaccine antigens:
 Oral
 Intra Nasal
 Tonsillar
 Minor salivary gland
 Rectal
Russell MW, Hajishengallis G, Childers NK, Michalek SM.
Secretory Immunity in defense against cariogenic Mutans
streptococci. Caries Res 1999;33:4-15.

33.  Early studies relied on oral induction of immunity in GALT to elicit
protective salivary IgA response.
 Smith DJ (2002): applied antigen by oral feeding, gastric intubation, or in
vaccine containing capsules or liposome.
Smith DJ. Dental caries vaccines: Prospects and concerns. Crit Rev Oral Biol Med 2002;13:335-49.

34.  Oral route failed to reduce caries significantly, as compared with
subcutaneous immunization.
 The rise in secretory antibodies produced was small and of short
duration.
 Immunological memory in secretory IgA responses is rather limited.
 Effect of stomach acidity on antigen,
 Inductive sites were relatively distant.

35.  Recent attempts have been made to induce protective immunity in mucosal
inductive sites that are in close anatomical relationship to the oral cavity.
 Intranasal installation of antigen targets the nasal associated lymphoid
tissue (NALT) (Brandtzaeg & Haneberg,1997).
 NALT is stimulated to produce antibodies against mutans streptococcal
colonization and accumulation.
Curtis R 3rd. 1984 Kreshover lecture: Genetic analysis of S. mutans virulence and prospects
for an anticaries vaccine. J Dent Res 1986;65:1034-45.

36. By nasal spray :
 Vaccine is introduced into the mucosal immune system by nasal spray.
Thus causing increased production of immunoglobulin IgA which fights
tooth decay by interrupting the colonization of the bacterium S.mutans.
Advantage:
 lower doses of ag needed
 easy adm
 induces both systemic & mucosal immunity

37.  Has both IgA and IgG response: IgG is more dominant.
 Palatine tonsils and nasopharyngeal tonsils contribute precursor cells to
mucosal effector sites: such as salivary gland.
 Experiments have shown that formalin- killed Streptococcus sobrinus cells
in rabbit can induce a salivary immune response, which can significantly
decrease the consequences of infection with cariogenic Streptococcus
sobrinus.
 Repeated tonsillar application of a particulate antigen can induce the
appearance of IgA antibodies in both major and minor salivary glands of
rabbit. Smith DJ 2002.
Smith DJ. Dental caries vaccines: Prospects and concerns. Crit
Rev Oral Biol Med 2002;13:335-49.

38.  Populate the lips, cheeks, and soft palate.
 Their short, broad secretory ducts facilitate retrograde access of bacteria and
their products: potential routes for mucosal induction of salivary immune
responses.
 Experiments with topical application of Streptococcus sobrinus GTF on
lower lips of young adults : lower proportion of indigenous S. mutans/ total
Streptoccal flora in their whole saliva during a 6 week period following a
dental prophylaxis, compared with a placebo group. Smith and Taubman,
1990
 Used in children with respiratory ailments where intranasal application is
not possible.
Smith DJ. Dental caries vaccines: Prospects and concerns. Crit Rev Oral Biol
Med 2002;13:335-49.

39.  Colorectal region as an inductive location for mucosal immune responses in
humans: it has the highest concentration of lymphoid follicles in the lower
intestinal tract.
 Preliminary studies have indicated that this route could also be used to
induce salivary IgA responses to mutans streptococcal antigens such as GTF.
Smith DJ. Dental caries vaccines: Prospects and concerns. Crit
Rev Oral Biol Med 2002;13:335-49.

40.  Subcutaneous administration of S. mutans : elicited IgG, IgM and IgA
antibodies.
 Antibodies reach oral cavity via gingival crevicular fluid.
 Whole cells, cell walls and the 185 KD streptococcal antigen have been
administered on 2-4 occasions.
 IgG antibodies are well maintained at high titre, IgM progressively fall and
IgA antibodies increase slowly in titre.
 IgG develop within months of immunization , reaching a titre of upto
1:1280.
Lehner T. Immunology of dental caries. Immunology of
oral diseases. 3rd ed. Blackwell scientific publications;
1992.

41.  To limit potential side effects of other routes and to localize the immune
response.
 Associated with increased IgG and IgA.
 Various modalities:
 Injecting lysozyme into rabbit gingiva: elicited local antibodies
 Brushing live S. mutans onto the gingiva of Rhesus monkeys, which failed to
induce antibody formation.
 Using smaller molecular weight S. antigen, which resulted in better performance
probably due to better penetration.
Tandon S. Textbook of Pedodontics. 1st ed. Paras Publishing; 2001

42.  External supplementation of antibodies.
 Approaches tried:
 Monoclonal antibodies: to surface antigen I/ II
 Bovine milk and whey: systemic immunization of cows with a vaccine using
whole S. mutans led to bovine milk and whey containing polyclonal IgG
antibodies.
 Egg- yolk antibodies: Hamada introduced using hen egg-yolk antibodies against
cell- associated GTF of S. mutans.
 Transgenic plants: caries vaccine developed from a genetically modified (GM)
tobacco plant. Vaccine is colourless and tasteless and can be painted onto the teeth
rather than injected and is the first plant derived vaccine.
Tandon S. Textbook of Pedodontics. 1st ed. Paras Publishing; 2001.
News. Genes .n. Greens: The future of oral medicine? Br Dent J
2002;192:674.

43. • Antigen derived from
animals/ humans has
potential for
hypersentivity.
• Give antibodies in GCF
and saliva.
Synthetic
peptides
•CT is a powerful mucosal immuno
adjuvant.
•Mucosal appl of soluble protein or
peptide antigen alone: not effective.
•Addition of CT or E.coli heat labile
LT: enhances immune response.
Coupling with
Cholera Toxin
subunits.
• Avirulent strains of
salmonella act as effective
vaccine vector: fusion
using recombinant
techniques
Fusing with
salmonella
•Microcapsules and microparticles of polylactide-co-
glycolide (PLGA) used as local delivery systems.
•Advantages: ability to control the rate of release,
evade preexistent antibody clearance mechanisms
and degrade slowly without eliciting an inflammatory
response
Microcapsules and
microparticles
•Liposomes are bilayered phospholipids
membrane vesicles manufactured to contain
and deliver drugs and antigens.
•Are thought to improve mucosal immune
responses by facilitating M cell uptake and
delivery of antigen to lymphoid elements of
inductive tissue.
Liposomes
News. Genes .n. Greens: The future of oral medicine? Br Dent J 2002;192:674.

47.  All vaccines have risks.
 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 monkey or rabbits immunized with
purified Ag I/II from S. mutans.
 Due to potential of S.mutans whole cells to induce heart – reactive Ab, the
development of subunit vaccine for caries has been focused of intense research
interest.

48.  Although the prevalence of dental caries has declined according to a
national epidemiological survey by US national institute of dental research,
this oral disease is still a significant health problem that affects
approximately 50% of 5-17 year old children.
 Developing countries without a water fluoridation system and where access
to dental health education and treatment may not be available to everyone
are in great need of a vaccine.

49.  Caries reduction could be attained if colonization of S. mutans could be
prevented or reduced at the time of eruption of both primary and
permanent teeth.
 Caries vaccine can be a successful adjunct to other caries-preventive
measures.

50.  Identify virulence agents
 Design means to induce protective response
 Animal model
 Human tests
Caries vaccines response requirements:
o -interfere with early colonization
o -not necessarily bactericidal
o -non-inflamatory response
o -persistent response
o -site directed response (oral cavity)

51. Trangenic plants: - Hyatt
• Passive immunization with tobacco plants.
• Genetic materials can easily be exchanged
• Possible to manipulate the antibody structure while preserving its specifity
• Can avoid cross reactivity
• Large scale production possible
• cheap

52.  Wuhan Institute of Virology, China: developed a new DNA vaccine : pGJA-
P/VAX
 pGJA-P/VAX: Encoding two antigenic domains , Pac and GLU of S mutans
 Induced accelerated and increased specific antibody response in serum and
saliva compared with non fusion DNA vaccine in rabbits.
 Limitation:
 Weak protective effect against S sobrinus.

53.  Etiologic agent part of indigenous flora
 Vaccination should be done on periodic basis
 Development of tolerance or immune exclusion
 Caries occurs on non living surface

54.  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