DEPARTMENT OF PREVENTIVE AND PEDIATRIC DENTISTRY

Seminar on

CARIES VACCINE

Submitted by ,

Risana. k
2018-2021 MDS
 CERTIFICATE

This is to certify that Dr. Risana. k has presented seminar on CARIES

VACCINE as a part of post graduate programme in Department of
preventive and pediatric dentistry in accordance to Kerala University of
Health Sciences.

Signature of Guide Signature of HOD

Introduction

Dental caries is a multifactorial oral disease affecting 60–90% of children in

industrialized countries .It is not a life-threatening disease but has a major impact on
people’s daily lives and well-being.Treatment is expensive therefore has become a public
health problem

Preventive strategies in use include

1. oral health education

2. chemical and mechanical control of plaque
3. use of fluorides
4. application of pit and fissure sealants etc.

However, economic, behavioral, or cultural barriers make dental caries still increase its
prevelance globally.The latest approach of combating dental caries by a vaccine that is
well suited for public health programmes will be of great use to reduce the incidence and
prevelance across the globe..

Pathogenesis of dental caries

The association of S. mutans with dental decay was not recognized until “specific plaque
hypothesis” which proposed. S. mutans and Streptococcus sobrinus as the primary human
pathogens.

Streptococcus mutans in beleived to be the main etiological agent for dental caries
mainly because of the propertoes like

1. Adhesion
sucrose independent adhesion
sucrose dependant adhesion
2. Acidogenecity
3. Aciduricity
Sucrose-independent adhesion

Influenced by antigen I/II, a 185kDa surface protein.

Mutans -Pac or P1
Sobrinus -SPa-A or Pag
The alanine-rich and proline-rich domains are primarily responsible for the interaction
between antigen I/II and salivary components.

Sucrose-Dependent Adhesion

Happens by action of glucosyltransferases (GTFs) by synthesising glucans/ mutans

It has a 1400-1600 aa sequence.

GTFs synthesize

1. watersoluble glycans
2. water-insoluble glucans(mutan)

linear polymer - alpha- 1,6-glycosidic linkages branching and predominantly alpha-

1,3-linkages.

3.forms of glucosyltransferases (GTFs)

Water insoluble glucan synthesizing enzyme: GTF-I

Water insoluble and water-soluble glucan synthesizing ezymes: GTF-SI
Water-soluble glucan synthesizing enzymes: GTF-S

The genes encoding GTF-I, GTF-SI, and GTF-S are GTF-B, GTF-C and GTF-D genes

S. sobrinus, harbor 4 genes encoding GTFs(similar sequence)

Non-enzymatic Glucan-Binding Proteins

In addition to GTFs, non-enzymatic glucan-binding proteins (GBPs) are present.These

are GbpA(563 aa ), GbpB(431 aa) , GbpC(583 aa).Not similar for mutans and sobrinus.

Dextranases

This destroy dextran and thus bacteria can invade dextran-rich early dental plaque.

Dextranase, when used as an antigen, can prevent the colonization of the organism in
early dental plaque

Phosphate binding protein

This facilitates adhesion to abiotic surfaces. Recently vaccines has been developed using
phosphate-binding-protein

(Luz et al. 2012; Ferreira et al. 2016)

Mechanism of Action of Dental Vaccine

The main Ig of saliva is IgA.

IgM and IgG are liberated into saliva from GCF along lymphocytes, macrophages and
neutrophils. Secretory IgA block colonization of Strep. mutans on the tooth.

The presumed modes of activity of antibodies are :

1. Salivary IgA

A. Act as specific agglutinin with the bacterial surface receptors and inhibiting
colonisation
B. Inactivate surface glucosyltransferase (GTF) and prevent adhesion.

2. Migration of antigen-sensitized IgA precursor B cells

from Gut-Associated Lymphoid Tissues (GALT) to Salivary glands and to distant
lymphoid tissues .These inhibit the activity of GTF.

3. Humoral and cellular components of the systemic immune system

After immunization with S. mutans, the organism is phagocytosed and undergoes

antigenic processing by macrophages.T and B lymphocytes are sensitized

Induction of CD-4 helper and CD-8 cytotoxic cells takes place.

This interaction plays an essential part in modulating the formation of IgA antibodies.

Routes of Immunization

1. common mucosal route

oral
nasal
minor salivary gland
 tonsillar
Rectal

2. Systemic (subcutaneous)
3. Active gingivo-salivary

Common Mucosal Immune route

Exposure of an antigen to a mucosally associated lymphoid tissue in the gut, nasal,

bronchial, or rectal site can give rise to immune responses locally and also in remote
locations.

Oral

Antigen can applied by

1. oral feeding
2. gastric intubation
3. vaccine containing capsules or liposome.

A significant reduction in caries was related to an increased level of salivary IgA

antibodies to S. mutans,as the serum antibody titer was minimal.

Smith and Taubman(1987)

Although oral route is not ideal for this approach ,it established that mucosal immunity
alone was sufficient to change the course of mutans infection.

Animal study

Implantation of live S. mutans were inoculated to germ-free rats in drinking water for 45
days .A significant reduction in caries was seen related to an increased level of salivary
IgA antibodies to S. mutans

Eldridge JH, Hammond CJ, Meulbroek JA, Staas JK, Gilley RM, et al. (1990)
Controlled vaccine release in the gut-associated lymphoid tissues. I. Orally
administered biodegradable microspheres target the Peyer’s patches. J Controlled
Release 11: 205-214.

Human study

7 adult volunteers were given enteric coated capsule containing 500 micrograms of GTF
Elevating salivary IgA antibodies to the antigen preparation was seen.

Childers NK, Zhang SS, Michalek SM (1994) Oral immunization of humans with
dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces
salivary immunoglobulin A2 antibody responses. Oral Microbiol Immunol 9: 146–
153.

Oral route was not ideal for reasons including

1. Detrimental effects of stomach acidity on antigen

2. inductive sites were relatively distant
3.Immunological memory in SIgA responses is limited.
4.The rise in secretory antibodies was small and of short duration, even after secondary
immunization.

Intranasal

Result of attempt to induce protective immunity in mucosal inductive sites that are in
closer anatomical relationship to the oral cavity. The Nasal-Associated Lymphoid Tissue
(NALT) gets targeted and aids in the immunization.

Protective immunity could be induced by

1. S. mutans Ag I/II
2. SBR region of Ag I/II
3. 19-mer sequence within the SBR
4. Glucan binding domain of S. mutans
5. GbpB, and fimbrial preparations from S. mutans with antigen alone or combined with
mucosal adjuvants.

The intranasal application of S. mutans Ag I/II and GBPB vaccines has shown protection
against Strep. mutans (Smith et al. 1997).

Advantages

Lower doses of antigen are required

Induces both systemic and mucosal immunity
Administration is relatively easy.

Tonsillar route
Palatine tonsils,especially the nasopharyngeal tonsils contribute precursor cells to
mucosal effector sites, such as the salivary glands.Tonsillar tissue can activate secretory
IgA responses despite the predominance of IgG.

(Van Kempen ,Boyoka et al 2000)

Repeated tonsillar application of a particulate antigen can induce the appearance of IgA
antibodies producing cells in both the major and minor salivary glands.

Animal study
In rabbits, topical application of formalin-killed Strep. sobrinus in tonsillar region elicited
an immuneresponse which significantly reduced the development of caries (Fukuizumi et
al. 1999)

Minor salivary gland

The minor salivary glands of lips, cheeks, and soft palate are suggested as potential
routes for mucosal induction of salivary immune responses

Short, broad secretory ducts facilitate retrograde access of bacteria and their products to
the lymphatic tissue associated with these ducts.

(Crawford,Nair,Schroder,1983)

Human study

Young adults had S. sobrinus GTF topically administered on lower lip for six-week
period following a dental prophylaxis. In the result those who received labial application
of GTF had significantly lower proportions of indigenous mutans streptococci/total
streptococcal flora in their whole saliva .

Smith DJ (2002) Dental caries vaccines: prospects and concerns. Crit Rev Oral Biol
Med 13: 335-349.

Rectal

Remote mucosal sites have also been investigated for their inductive potential. Rectal
immunization can result in the appearance of SIgA antibodies in distant salivary sites
(with non oral bacterial antigens such as Helicobacter pylorior Streptococcus pneumoniae
as toxin-based adjuvant)

(Lam et al 2001)
High concentration of lymphoid follicles in the site can induce salivary IgA responses to
antigens such as GTF.

-Vaccine suppositories may develop as alternative for children in whom respiratory

ailments preclude the intranasal application of the vaccine.

Systemic Route of Immunization

Subcutaneous administration of S. mutans elicit serum IgG, IgM, and IgA antibodies.The
antibodies find their way into the oral cavity through GCF

Antigens used

Whole cells, cell walls, and the 185 KD Streptococcal antigen .

Protection was associated predominantly with increased serum IgG .

A subcutaneous injection of killed cells of S. mutans in Freund’s incomplete adjuvant or

aluminum hydroxide elicits IgG, IgM, and IgA classes of antibodies.

IgG antibodies are well maintained at a high titer

IgM antibodies progressively fall
IgA antibodies increase slowly in titer.

Animal study

Development of serum IgG takes place within months of immunization, reaching upto
1:1280 with no change in antibodies being found in the corresponding non immunized
monkeys.

Shivakumar KM, Vidya SK, Chandu GN.Dental caries vaccine. Indian J Dent Res 20:
99-106.

Active Gingivo-Salivary Route

GCF is the route of administration.Need LMW antigens.

They have limited potential side effects and localize the immune response,

Apart from the IgG, it is also associated with increased IgA levels.

The various modalities tried are,

1. Injecting lysozyme into rabbit gingival which elicited local antibodies from cell
response

3. Brushing live S. mutans onto the gingiva of rhesus monkeys, which failed to induce
antibody formation

Passive immunisation

Development of antibodies suitable for oral application.

Advantage - completely avoids risks from active immunization.

Disadvantage

- No induction of immunological memory

- Antibodies persist in mouth for only a few hours at most or up to 3 days in plaque.
- Less effective than active immunisation.
- Repeated applications of vaccines are required, since the action is purely local.

Bovine milk and whey

Polyclonal IgG antibodies in introduced in bovine milk. Whey,when used as mouth rinse,
resulted in a lower bacterial count of S. mutans in plaque.

Mitoma et al developed antibodies against surface antigen and GTF that significantly
reduced the colonization of Strep. mutans when mixed in milk fed daily to rats.

Mitoma, M., Oho, T., Michibata, N., Okano, K., Nakano, Y., Fukuyama, M. and Koga,
T. (2002) Passive immunization with bovine milk containing antibodies to a cell
surface protein antigen-glucosyltransferase fusion protein protects rats against dental
caries. Infect Immun 70, 2721–2724.

Egg-yolk antibodies

- Introduced by Hamada
- Uses hen egg-yolk antibodies against GTF of S. mutans
Vaccines used were formalin killed whole cells and cell associated GTFs. Caries
reduction has been found with both these treatments.

Smith DJ, Godiska R. Passive immunization approaches for dental caries prevention.
Conference Proceedings. Egg Symposium; 2004. p. 1-6.
 Plantigens and Plantibodies

- Plants cells have protein synthesis similar to humans.

- Can be used to synthesise antigens) & antibodies
- Researchers started in 1983 with use Tobacco Plants.
- Later extended to fruits and vegetables.

Inject a peptide that blocks the S. mutans which causes tooth decay into the fruit.

Guys Hospital in London have isolated a gene and the peptide that prevents the
bacterium from sticking to the teeth.

Advantages

- Genetic material can be easily exchanged.

- Vaccination procedure attractive to the general population.
- Specificity of the antibody is maintained,no cross reactivity.
- Large scale production is possible as its inexpensive.

Disadvantages

Rhizosecretion of antigen may contaminate the soil. Accidental transfer of genes to other
plants via pollen grains is also of great concern.Developed in 4 transgenic Nicotiana
tabacum plants that expressed a murine monoclonal antibody kappa chain, a hybrid
immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory
component, respectively.

The vaccine is colourless and tasteless, can be painted onto the teeth rather than injected

This was the first plant derived vaccine from GM plant.Prevented the biofilm formation
in humans; however, adequate titres of antibodies could notbe maintained in the oral
cavity

Ma, J.K., Hikmat, B.Y., Wycoff, K., Vine, N.D., Chargelegue, D., Yu, L., Hein, M.B.
and Lehner, T. (1998) Characterization of a recombinant plant monoclonal secretory
antibody and preventive immunotherapy in humans. Nat Med 4, 601–606.

CARO Rx TM10

First clinically tested plantibody.

Professional cleaning of teeth with oral antiseptic solution.Apply several times over two
week period.Prevents adhesion of S.mutans , colonization of other oral bacteria occurs
unimpeded.

Subjects treated with this, remained caries free for about 6 months in the phase I clinical
trials. Phase II clinical trials are in progress now.

Longer-term effects on indigenous flora were observed after topical application of mouse
monoclonal IgG or transgenic plant secretory SIgA/G antibody, each with specificity for
Ag I/II.

Subunit vaccines

Initially whole vaccine was introduced to produce a response. This had a potential
disadvantage of cross-reaction with heart muscle. Here a particular protein antigen of the
organism is used as an antigen. They have the advantage of specifically attacking the
antigenic surfaces.Subunit vaccines contain the functional part of genome responsible for
Ag I/II or GTFs or GBP

These vaccines contain single or multiple copies (multivalent) of functional

epitopes associated with either of the virulence proteins (monomeric) or in combinations
(dimeric) .Can target multiple virulence properties and eliminate some of the unwanted
immune responses.

(Cao et al. 2016; Oishi et al. 2001).

Example

N-terminal saliva-binding region (SBR) of antigen I/II is important in initial adherence

of this organism.

Vaccine containing

1. SBR with a C-terminal structural region of Ag I/II

2. SBR with a N-terminal structural region of Ag I/IIAgII and
3. whole Ag I/II were administered to rats intranasally,

They induced salivary IgA anti-Ag I/II antibodies.However, protection against caries was
better with SBR compared to Ag II (Hajishengallis et al. 1998).

Synthetic peptide vaccines

Synthetic peptide approaches have shown the alanine-rich repeat region of Ag I/II to be
immunogenic and to induce protective immunity. Subcutaneous immunization with a
synthetic peptide derived from alanine-rich region of Ag I/II from S. mutans induced
higher levels of serum IgG antibody than a synthetic peptide derived from the proline-
rich region

Smith DJ (2002) Dental caries vaccines: prospects and concerns. Crit Rev Oral Biol
Med 13: 335-349.
Conjugate Vaccines:

Chemical conjugation of functionally associated protein/peptide components with

bacterial polysaccharides.In separate studies, it was reported that conjugation of either
tetanus toxoid or S. sobrinus GTF to the water-soluble glucan synthesized by GTF
significantly increae antibody levels to the water-soluble glucan and to the conjugated
protein.

Takahashi I, Okahashi N, Matsushita K, Tokuda M, Kanamoto T, Munekata E, et al.

(1991). Immunogenicity and protective effect against oral colonization by
Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen.
J Immunol 146:332-336.

DNA VACCINE

Make the antigenicity more specific and long lasting. When a specific DNA is
administered into the system, the host synthesize protein component coded by the
DNA.Lower number of carious lesions and high levels of salivary Ig A and serum
Ig G were observed experimental animals following a targeted salivary gland
administration of this DNA vaccine.

Disadvantages

possibility of the induced DNA to cause damage to the host genetic components.

New Fusion Anti-caries DNA Vaccine

Developed at Wuhan Institute of Virology, China

S. mutans have two important virulence factors:

1. cell surface protein PAc and
2. glucosyltransferases

GTFs have two functional domains:

N-terminal catalytic sucrose-binding domain (CAT) and
C-terminal glucan-binding domain (GLU).

Fusion anti-caries DNA vaccine, pGJA-P/VAX, encodes two important antigenic

domains, PAc and GLU, of S. mutans.Successful in reducing the levels of dental caries
caused by S. mutans in animals .
The fusion vaccine induced accelerated and increased specific antibody
responses in serum and saliva compared with nonfusion DNA vaccine in rabbits.
However, its protective effect against S. sobrinus infection proved to be weak.

Jia R, Guo JH, Fan MW, Bian Z, Chen Z, et al. (2006) Immunogenicity of CTLA4
fusion anti-caries DNA vaccine in rabbits and monkeys. Vaccine 24: 5192-5200.

Recombinant and DNA vaccines

Mucosal route vaccines often require adjuvants to enhance the immune

response.Adjuvants accelerate, prolong or enhance antigen-specific immune responses
when used in combination with specific vaccine antigens.

Adjuvant enhances the antigenecity by,

- Acting as a deposit or reservoir, where the antigen can be released progressively.

- Present the antigen directly to immune competent cells.
- Acts as chemical immune stimulators of lymphoid cells

Prepared by isolating the functional genome/s responsible for the target antigens and
linking them to vectors such as attenuated Salmonella typhimurium, Salmonella typhi, E.
coli or plasmid.

Cholera toxin And E. Coli Heat-Labile Enterotoxin:

Cholera toxin (CT) is a powerful mucosal immunoadjuvant

Functions

Increase generation of mucosal immunity to pathogens.

Application of a soluble antigen results in low IgA responses.

Relevant studies

1. Addition of small amounts of CT or E.coli heat-labile enterotoxins (LT) enhance

mucosal immune responses to intragastrically or intranasally applied mutans antigens.
Enterotoxins from E. coli,Vibrio cholera and Salm. typhimurium has been used in caries
vaccines

(Hajishengallis et al. 1998; Saito et al. 2001; Zhao et al. 2011;Batista et al. 2017).

2. Toxin-related adjuvants may cause adverse effects on intranasal administration, so

non-toxic derivatives of the labile-toxin produced by enterotoxigenic E. coli have been
investigated with promising results
(Batista et al. 2017)

3. Oral immunization with recombinant Salmonella typhimurium, expressing surface

protein antigen A of Streptococcus sobrinus, was able to bring about persistent mucosal
immune responses which could give protection after a challenge on rats with cariogenic
S.sobrinus.

Redman TK, Harmon CC, Lallone RL, Michalek SM. Oral immunization with
recombinant Salmonella typhimurium expressing surface protein antigen A of
Streptococcus sobrinus: dose response and induction of protective humoral responses
in rats. Infect Immun. 1995 May;63(5):2004-11.

Microparticles

Made up of poly lactide-co-glycolide (PLGA) can be used as delivery systems

1. Ability to control the rate of release

2. Elude preexistent antibody clearance mechanisms
3. Degrade slowly without eliciting an inflammatory response to the polymer.

Oral immunization with these microspheres effectively delivered and released vaccine in
the gut associated lympohoid tissue.

Taubman MA, Smith DJ, Holmberg CJ, Lees A (1999). GTF-S. sobrinus
polysaccharide conjugates as potential caries vaccines .J Dent Res 78:453.

Nanoparticles

Incorporation of anionic liposomes in chitosan/DNA complexes a nanoparticle system as

a effective carrier for nasal mucosal immunization .Showed a more efficient elicitation
of mucosal immunity.

Smith DJ et al. Remote glucosyltransferase_microparticle vaccine delivery induces

protective immunity in the oral cavity. Oral Microbiol Immunol. 2003 Aug;18(4):240-
8.

Similar results were found by Li et al. (2016)using trimethyl chitosan nanoparticles. Their
pVAX1-wapA/trimethyl chitosan vaccine elicited greater immuneresponse and the
 rats had fewer carious lesions compared to the animals immunized with naked
pVAX1-wapA.

Cao et al successfully generated self-assembling nanoparticles by linking the GB region

of GTF to the N-terminal domain of ferritin which significantly increased the level
of antibodies and produced lower caries scores compared to naked GTF in mice.

Liposomes

Bilayered phospholipids membrane vesicles manufactured to hold and deliver drugs and
antigens.Improve mucosal immune responses by facilitating M cell uptake and delivery
of antigen to lymphoid elements of inductive tissue. The efficacy using liposomes has
been found to increase two fold in a rat model. In humans increased IgA antibodies have
been found.

Chen L et al. Enhanced nasal mucosal delivery and immunogenicity of anti-caries

DNA vaccine through incorporation of anionic liposomes in chitosan/DNA complexes.
PLoS One. 2013 Aug 20;8(8):e71953.

Strain replacement therapy

Novel approach to prevent microbial diseases where a harmless effector strain is

permanently implanted in the host’s microflora.

Human studies

Risk of using caries vaccine.Sera of some patients with rheumatic fever show serological
cross-reactivity between heart tissue antigens and certain antigens from hemolytic
Streptococci.

Experiments utilizing antisera from rabbits immunized with whole cells of S. mutans and
with a high molecular weight protein of S. mutans were reported to cross react with
normal rabbit and human heart tissues. Polypeptides immunologically cross-
reactive with human heart tissue and rabbit skeleton muscles myosin are found in
the cell membrane of S. mutans and Streptococcus ratti

Harris R. Vaccines for dental caries. Aust Dent J 1983;28:115-6.

Future direction

2 paths can be suggested.

1. First, to search for new target virulence genes or antigenic proteins, develop vaccines,
use a proved adjuvant and administration technique
(Ferreira et al. (2016) studied a completely new protein, PstS, which showed promising
results.)

2. Second, existing best-proved animal trial vaccines can be improved to the required
level. Multi-expert multi-centre studies are required where the different vaccines can be
discussed and compared and produced for the benefit of the community.

When to immunise??

1. “window of infectivity”-

further opportunities for colonization exist, for example, when children enter school
and mix socially with a much larger group of their peers, or when the permanent teeth
erupt.

1) It is necessary to immunize infants or young children to provide immune protection

before initial colonization with mutans streptococci

2) Booster immunization to recall responses might be desirable to forestall colonization

at later time points.

3) Young mothers might be immunized actively or passively with the objective of

reducing their oral load of mutans streptococci

Transmission of mutans streptococci appears to be primarily from mother to infant (Li

and Caufield, 1995)

Limitations of caries vaccine

1. Requires large-scale investments which is not feasible for public health systems.

2. constant challenges regarding best route of administration and possibility of cross-

reactions

3. not easy to get FDA approval

4. Dental caries is a multifactorial disease.

5. Fluctuations in demographics

6. Selected populations

7. Parents choice
 Advantages

1. prevent disease in children

2. Can be incorporated into universal immunisation programme

3. cost effective in long run
4. provide lifelong immunity

Disadvantages

1. risk of hypersensitivity

2. risk of microbial resistance

3. cross reactivity with cardiac tissue.

National institute of dental and craniofacial research primary sponsor for caries
vaccine conveyed on jan 28 2003 recommendations

1. more research is needed in bioimmunology

2. real world barriers would have to be considered and overcome
3.there might be some advantage of passive immunity

Conclusion

Despite the promising results in animal and human studies there is no conclusive
evidence about development of a caries vaccine that can be used for community oral
health care , but still there is an optimism that a vaccine will eventually develop in future
and will be beneficient for the reducing the incidence and prevelanece of the disease.

References

1. Shivakumar KM, Vidya SK, Chandu GN (2009) Dental caries vaccine. Indian J Dent
Res 20: 99-106.
2. Bowen WH (1996) Vaccine against dental caries--a personal view. J Dent Res 75:
1530-1533.
3. Hajishengallis G, Russel MW, Michalek SM. Comparison of an adherence domain
and a structural region of S. mutans antigen I/II in protective immunity against
dental caries in rats after intranasal immunization. Infect Immun 1998;66:1740-3.

4.Krithika AC, Kandaswamy D, Gopikrishna V. Caries vaccine-I todayís myth. J Indian

Assoc Public Health Dent 2004;4:21-5.

5.Russel MW, Childers NK, Michalek SM, Smith DJ, Taubman MA. A caries vaccine?
The state of science of immunization against dental caries.
Caries Res 2004;38:230-5.

6.Guo JH, Jia R, Fan MW, Bian Z, Chen Z, Peng B. Construction and Immunologic
characterization of a fusion anti-caries DNA vaccine against Pac and Glucosyltransferase
- I of S. mutans. J Dent Res 2004;83:266-70.

7. Mattos-Graner RO, Smith DJ. The vaccination approach to control infections leading
to dental caries. Braz J Oral Sci 2004;3:595-608.

8. Bowen WH, Cohen B, Cole M, Colman G. Immunization against dental caries:

Summary. J Dent Res 1976;55:C164-5.

9.Hatta H, Tsuda K, Ozeki M, Kim M, Yamamoto T, Otake S, et al.Passive immunization

against dental plaque formation in humans: Effect of a mouth rinse containing egg yolk
antibodies (IgY) specific to Streptococcus mutans. Caries Res 1997;31:268-74.