DENTAL CARIES VACCINE

CONTENTS

• INTRODUCTION
• HISTORY
• IMMUNITY - BASICS
• IMMUNISATION
• DENTAL CARIES VACCINE – RATIONALE
• ACTIVE IMMUNISTION (VACCINATION)
• ANTIGENIC DETERMINANTS
• ROUTES OF ADMINISTRATION
• ADVANTAGES
• LIMITATIONS
• PASSIVE IMMUNISATION
• RECENT DEVELOPMENTS
• CONCLUSION
• FREQUENTLY ASKED QUESTIONS

INTRODUCTION

Dental caries is a microbial disease of the calcified tissues of the teeth, characterized

by demineralization of inorganic portion and destruction of organic substances of the tooth

leading to cavity formation. Dental caries is a disease with a multifactorial aetiology, as it is

due to the interaction of various factors: diet, the host’s susceptibility and the presence of

microorganisms over a certain length of time.

Among the oral diseases, dental caries is the most common chronic disease of

mankind. It affects persons of both sexes in all races, all socio-economic strata and every age

group. As children reach school age, they will have an increasing incidence of carious lesions

because of change in dietary habits which includes refined carbohydrates and sweeteners. It is

also profoundly affected by other factors like oral hygiene and saliva.
Although the prevalence of dental caries has declined markedly over the last 20 years in most

countries in the Western world, the disease is still a major problem for both adult and

children especially in developed countries.

Streptococcus mutans, a Gram positive, aciduric and acidogenic bacteria, is considered a

microorganism more associated to this pathology. Dental biofilm, in which S. mutans is

inserted, is a community of bacteria attached to salivary components and embedded in a

matrix of glucan of high molecular weight, produced by this microorganism. The other

microarganisms associated with the caries process are Streptococcus sobrinus, Actinomyces

viscosus, Actinomyces naeslundii and Lactobacillus acidophilus.

Several measures are being used in the prevention and control of caries, as the

disorganization of the biofilm through oral hygiene and use of fluorides. However, the still

high prevalence of such disease in the world population, justifies the search for a new

preventive action, such as the development of a caries vaccine. The main target is the

mechanism of adherence of S. mutans, which can be affected by active or passive

immunization or by DNA vaccines

HISTORY
Modern era regarding vaccine theory began in late 1969 with intravenous immunization

experiments on animals like irus monkeys by William Bowen. Natural history study of oral

streptococcal acquisition in infants revealed that there is colonization of Mutans streptococci

in children between the age of 2nd and 3rd year of life under named circumstances of diet

and other challenges during teeth eruption referred as “WINDOW OF INFECTIVITY”. So

window of vaccine opportunity exist between 12th and 18th months. DNA probe technology

suggests that during first year of life in caries prone patient low level of S.mutans is found in
oral cavity. Goadby was probably, the first to advocate caries control in 1910 by inoculation

of the mouth with organisms which would produce alkaline reaction.

IMMUNITY – BASICS

Immunity is defined as the resistance of the host to pathogens and their toxic effects. The

immune system is a network of cells, tissue and organs that work together to defend the body

against attacks by “foreign” invaders.

CLASSIFICATION OF IMMUNITY

Immunity can be broadly classified into two categories:

1. INNATE IMMUNITY (Non Specific)

2. ACQUIRED IMMUNITY (Specific)

INNATE IMMUNITY

Innate immunity exists because of genetic and constitutional makeup. It has no relationship

with previous infection and immunisation. It acts as the first line of defence against

infections. The mechanisms include anatomical and physical barriers (skin, mucous

membrane), physiological and chemical barriers (milk, secretions) and biological barrier

(mono nuclear phagocytic system – reticuloendothelial system)

ACQUIRED IMMUNITY

Immunity is acquired from outside source. It is more specific. Acquired immunity can be

classified as follows

a. ACTIVE: The individual’s own immune system is the cause of immunity. There is an

active production of antibody by the host. The action is not immediate and rather delayed in

onset. Active immunity possesses a memory component that protects the host from

subsequent infections. Active immunity can be classified into:

 Natural – Antigens are introduced through natural exposure Example: Following

infections

Artificial – Antigens are deliberately introduced through vaccines. Examples MMR,

BCG, Polio vaccines

b. PASSIVE: The immunity is passively transferred. No delay in onset of action and no

memory component to prevent subsequent infections. Preferred during immune-compromised

states. Passive immunity can be classified into:

Natural – Antibodies from mother to child are transferred through milk and across

placenta.

Artificial – Antibodies (produced in another person/organism) are directly introduced

into the host. Example: Tetanus toxoid.

PRIMARY AND SECONDARY IMMUNE RESPONSE

The primary immune response occurs when an antigen comes in contact with the immune

system for the first time. During this time the immune system has to learn to recognize

antigen and how to make antibody against it and eventually produce memory lymphocytes.

The secondary immune response occurs when the second time (3rd, 4th, etc.) the person is

exposed to the same antigen. At this point immunological memory has been established and

the immune system can start making antibodies immediately.

Considering the primary immune response, once an antigen comes in contact with the

immune system, a lag phase is seen prior to the production of antibodies. This lag phase is for

the lymphocytes to recognise the antigen. Following the lag phase that lasts for a few days,

the first antibody to be produced is Ig M, although IgG is produced in small quantities. With
this response produced mainly by the lymph nodes and spleen, the antibody levels decline

rapidly. In the secondary immune response, there exists no lag phase due to the memory

developed following primary immune response and is produced by bone marrow, spleen and

lymph nodes. The antibody produced is mainly IgG and IgM which lasts longer.

IMMUNOGLOBULINS AND DENTAL CARIES VACCINATION

The immunoglobulins namely IgG, IgA, IgM, IgE and IgD are responsible for immune

response. Unlike majority of the vaccinations for prevention of diseases mainly focusing on

IgG and IgM to be delivered via blood, caries, affecting mainly the enamel needs antibody to

be delivered via saliva in the form of secretory immunoglobulin IgA and for root caries as

IgG/ IgM.

DENTAL CARIES VACCINE – RATIONALE

Dental caries being an infectious bacterial disease is a well-established fact since 1924 in the

light of the studies done by Clark and subsequently many other studies have provided

additional proof for this fact. Over past many years, there is a study accumulation of the fact

that the S. mutans is the bacterium most intimately associated with the dental caries.

Slots and Taubaman have correlated the features of the S. mutans and Koch’s postulates as

follows:

• S. mutans is found in the plaque of carious teeth in high numbers and are

comparatively lesser in number in absolutely caries-free mouths.

• The organism can be grown in the pure culture.

• Introduction of this organism in germ-free animals induces caries.

• The organism can then be recovered from the carious lesions and grown in the pure

cultures.
 • Antibodies to this organism are increased in the patients with dental caries.

The scientific basis for the carries vaccine is provided by all the above mentioned features

and especially the last one is characteristic for its scientific basis.

ACTIVE IMMUNISTION (VACCINATION)

The Microbial Aspect of Dental Caries

A) Microbes and their Characteristics S.mutans is the primary aetiologic agent of this disease.

S. sobrinus and lactobacilli are also implicated in this disease. The period which is called”

window of the infectivity” which is between the middle of the second year and the end of the

third year of life, shows more S. mutans colonization in children .

B) Streptococcus mutans and the local immunity. The oral immune system undergoes a rapid

development with the secretory IgA antibody being secreted in the saliva at 1month of age.

Within weeks of the initial exposure to S. mutans, the mucosal IgA is secreted. The saliva of

infants contains the IgM and the IgA1 isotypes in the first month of life and by six to nine

months of age, the adult like distribution of the salivary IgA1 and IgA2 subclasses appear.

C) The adherence of S.mutans and plaque formation

The initial attachment of S mutans to the tooth is through the interaction of the bacterial

protein with lecithin in the dental pellicle. The Streptococcal adhesins [ antigen I/II or PAC]

in S.mutans bind to the tooth pellicle and then secrete glucosyl transferases [GTF] which help

in the accumulation of more S.mutans through an interaction with the bacterial cell associated

glucan-binding proteins. Then, they release lactic acid by their metabolisms, which

demineralize the enamel, thus causing dental caries.

MECHANISM OF ACTION OF A CARIES VACCINE

Protection against dental caries by immunization could be achieved by:

• Immune components from the serum

• IgA antibodies in the salivary secretions

• Combined effect of serum and salivary secretions

Immune components from the Serum:

• Immune antibodies from serum reach the oral cavity through the gingival crevicular

fluid.

• When the teeth erupt, local inflammation is common and during this time, serum

antibodies may stimulate opsonization and phagocytosis of the bacterial cells and,

• Studies have shown that serum antibodies have inhibitory effect on the adherence of

the bacteria and glucosyltransferase enzyme of the bacteria.

• This explains the results of a lower no. of S. Mutans and less dental caries have been

found in the immunized animals than the in the control animals.

• Hence, immunization against the S. Mutans could favour early establishment of a

non-cariogenic flora on the teeth and delays the colonization of the teeth by

pathogenic S. Mutans.

• Such antibodies could inhibit the establishment and metabolic activity of S. Mutans

on the teeth.

Salivary Ig A Antibodies in Saliva (Secretory Ig A):

• In saliva, secretory IgA antibodies predominate and have been found to protect

animals against S. Mutans infections and dental caries.

• S. IgA antibodies in human saliva have been found to inhibit the attachment of

streptococcus to epithelial cells and on hydroxyapatite.

 • Some findings in the human's support the concept that salivary IgA antibodies could interfere

with the establishment of S. Mutans and protect against dental caries.

• Thus some observations support the view that salivary Ig A antibodies could have a protective

effect in humans.

ANTIGENIC DETERMINANTS

The following can be used as antigenic determinants of caries vaccine

1. ADHESINS (WALL ASSOCIATED PROTEINS, ANTIGEN I/II)

To initiate infection, bacterial pathogens must first be able to colonize an appropriate target

tissue of the host. This tropism (ability to gain access to a niche within the body), in

association with the ability of the bacterium to breach mucosal barriers and invade the host,

distinguishes pathogenic from commensal organisms. Colonization begins with the

attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa.

Certain species of bacteria are restricted in terms of the hosts and tissues they infect and the

diseases they cause. Attachment is mediated by adhesin proteins; bacterial lectins are the

most common type of adhesin among both gram-negative and gram positive bacteria. Some

of the most well-characterized colonization factors in gram-positive bacteria the polypeptides

of the antigen I/II family bind to salivary glycoproteins in a lectinlike interaction and promote

adhesion to the tooth surface. These proteins include the original AgI/II from Streptococcus

mutans, also known as SpaP, P1, or PAc, and the Streptococcus sobrinus SpaA and PAg

proteins. Surface proteins of the antigen I/II family contain alanine-rich repeats, which adopt

an α-helical coiled-coil structure, proline rich repeats, and a carboxy-terminal region that

includes the gram-positive cell wall anchor motif. Binding activity to salivary glycoprotein

has been attributed to both the highly conserved alanine rich repeats and the proline-rich

repeating sequences.
2. GLUCOSYL TRANSFERASE ENZYME (GTF):

Glycosyltransferase (Gtf) is one of the crucial virulence factors of Streptococcus mutans, a

major etiological pathogen of dental caries. All the available evidence indicates that

extracellular polysaccharide, particularly glucans produced by S. mutans Gtfs, contribute to

the cariogenicity of dental biofilms. Therefore, inhibition of Gtf activity and the

consequential polysaccharide synthesis may impair the virulence of cariogenic biofilms,

which could be an alternative strategy to prevent the biofilm-related disease.

Glucosyltransferases (Gtfs) play critical roles in the synthesis of Extracellular

polysaccharides, providing sites on dental surfaces for microbial colonization, adherent

glucan for bacterial coherence, and water-insoluble matrix for biofilm formation.. S. mutans

expresses at least three genetically separate Gtfs: GtfB, which synthesizes primarily insoluble

glucans rich in α-1,3 glycosidic linkages; GtfC, which produces a mixture of insoluble and

soluble glucans (with α-1,6-linkages); and GtfD, which synthesizes predominantly soluble

glucans. Each plays a distinct but overlapping role. GtfC has the greatest affinity for saliva-

coated hydroxyapatite (sHA) with enhanced activity when it is adsorbed to sHA, thus

enhancing the adherence of bacterial cells to dental surfaces, whereas GtfB binds with greater

avidity to many oral bacteria including those that do not express Gtfs, thereby converting

them into glucan formers and promoting cell accumulation in the biofilm. GtfD, however,

synthesizes mainly water-soluble glucans, which serve as primers for GtfB.

3. GLUCAN BINDING PROTEIN (GBP)

Streptococcus mutans produces glucan-binding proteins (Gbps), which appear to contribute

to the virulence of S. mutans The first of these glucan-binding proteins, GbpA, was isolated

by Russell GbpA, GbpB was isolated by Smith et al. , GbpC was isolated by Sato et al.. and
most recently, a fourth Gbp, GbpD, was discovered. Given the role of extracellular glucan

in S. mutans sucrose-dependent adhesion, biofilm formation and cariogenicity, it is natural to

speculate upon the roles of S. mutans proteins capable of binding glucan. GbpC may induce

dextran-dependent aggregation of S. mutans cells early in biofilm development but it may be

GbpA and GbpD that maintain microcolony cohesiveness throughout further maturation of

the biofilm. The loss of certain Gbps, individually or in combination, significantly attenuates

the cariogenicity of S. mutans, revealing unique functions for individual Gbps and the

complexity of how they interact with each another

4. DEXTRANASES

Dextran is an important component of early plaque. Dextranase is an enzyme produced by S.

mutans that helps the bacteria invade the dextran rich plaque to adhere to the tooth surface.

ACTIVE IMMUNIZATION:

ROUTES OF ADMINISTRATION

a) Mucosal route.

b) Systemic route (subcutaneous).

c) Active-gingivo salivary route.

a) Common Mucosal Immune route:

This route is mainly used for the production of IgA antibodies. Research indicates that

lymphoid aggregates present in the body, when stimulated by antigens, result in production of

antibodies; predomintantly IgA type, that is secreted through saliva. For prevention of caries

salivary antibodies are more effective than antibodies secreted through blood. The lymphoid

aggregated present in the body include the Gut Associated Lymphoid Tissue (GALT),

mucosa associated lymphoid tissue (MALT) and Nasal Associated lymphoid tissue (NALT).
Included in this mechanism are the following routes:

• Oral Route

• Intranasal

• Tonsillar

• Rectal

• Minor salivary glands

b) Systemic Route Of Immunization:-

This route includes subcutaneous administration of S.mutans antibodies which find their way

to oral cavity. The antibodies predominantly produced are of Ig G and Ig M types that are

reach oral cavity through blood. Hence this mainly protects tooth from cemental caries.

c) Activo-Gingivo Salivary Route:

This route uses gingival crevicular fluid as a vaccine route. Studies show that this mechanism

of vaccine delivery facilitates the production of IgA, IgG and IgM.

ADVANTAGES

• The dosage of caries vaccine can be given along with other vaccine as a part of the

Universal Immunisation Programme. The first dose can be given just before the

eruption of first deciduous teeth and a booster dose prior to the eruption of permanent

teeth.

• It can prevent the disease in children.

LIMITATIONS
 • Studies have shown two possible problems using Streptococcus mutans as an

immunogen - Production of heart reactive antibody, and Need to protect against

several serotypes.

• Another potential dis-advantage is the property of the S. Mutans to change antigenic

nature in its cell wall.

• Dental caries is considered a multifactorial disease of which microorganisms are only

one of the various factors.

• Shift to other cariogenic serotypes or organisms.

• The safety associated with a potential vaccine has come under question because of the

possibility of immunological cross-reactions between streptococcal antigens and

human tissues such as those of Group A, C, and G streptococci and human heart

tissue

• Moreover, antigenic changes can also occur in the cell wall of the streptococcus

mutans due to genetic mutations, these may lead to the change in the antigenic

properties of the Strept. Mutans thus making the caries vaccine ineffective.

PASSIVE IMMUNISATION

Passive Immunization

It is the direct introduction of specific pre-targeted antibodies into the mouth.

These include:

• Monoclonal antibodies topically applied

• Immune bovine milk and whey;
• Egg yolk antibody;
• Transgenic plant antibody.
Monoclonal antibodies

Monoclonal antibodies to S. mutans cell surface antigen I/II have been investigated. The

topical application in human subjects brought a marked reduction in the implanted S.

mutans. Thus, by bypassing the system, less concern exists about the potential side

effects.

Bovine milk and whey

Systemic immunization of cows with a vaccine using whole S. mutans led to the bovine

milk and whey containing polyclonal IgG antibodies

Egg-yolk antibodies

The novel concept of using hen egg-yolk antibodies against the cell-associated

glucosyltransferase of S. mutans was introduced by Hamada. Vaccines used were

formalin killed whole cells and cell associated GTFs. Caries reduction has been found

with both these treatments.

Transgenic plants

The latest in these developments in passive immunization is the use of transgenic plants

to give the antibodies. The researchers have developed a caries vaccine from a genetically

modified (GM) tobacco plant. The vaccine, which is colorless and tasteless, can be

painted onto the teeth rather than injected and is the first plant derived vaccine from GM

plants
RECENT DEVELOPMENTS

ADJUVANTS AND DELIVERY SYSTEMS FOR DENTAL CARIES VACCINES

Various new approaches have been tried out to potentiate aspects of the immune response to

induce sufficient antibodies to achieve a protective effect to overcome the existing

disadvantages.

Synthetic peptides: Any antigen derived from animals or humans has the potential for

hypersensitivity reaction. The chemically synthesized peptides hold an advantage in that this

reaction can be avoided.. The synthetic peptides give antibodies not only in the GCF but also

in the saliva. The synthetic peptide used is derived from the Glucosyltransferase enzyme.

Fusing with salmonella: The avirulent strains of salmonella are an effective vaccine vector;

fusion using recombinant techniques have been used.

Microcapsules and microparticles: Combinations of antigens in or various types of particles

have been used in an attempt to enhance mucosal immune responses.

Liposomes: Liposomes, which are bilayered phospholipids membrane vesicles manufactured

to contain and deliver drugs and antigens, have been used to enhance mucosal responses to

mutans Streptococcal carbohydrate and GTF. Liposomes are thought to improve mucosal

immune responses by facilitating M cell uptake and delivery of antigen to lymphoid elements

of inductive tissue.

Replacement therapy: Replacement therapy consists of using a genetically modified non-

cariogenic strain of S. mutans, unable to metabolize carbohydrates and produce acid,

deploying it in the host’s oral microbiota. Once established, this strain competes with the

cariogenic wild strain, preventing its growth. This type of approach is very promising and

will act similarly to the vaccine, being similar to the use of probiotics and functional foods

used for modification of intestinal microbiota.

CONCLUSION

Vaccination for caries prevention can be a boon in future. Long term human trials with

vaccines having a broad spectrum of action are the future trend in research associated with

caries vaccine. An effective, safe and readily deliverable vaccine may not only help against

pain and health issues associated with caries but also save billions of money spent for

restorative treatment. Considerable caries reduction could be attained if clonisation of

microorganisms could be prevented or reduced at the time of eruption of both deciduous and

permanent teeth. Thus, a successful vaccination directed against S mutans could be a valuable

adjunct to other caries preventive measures.

FREQUENTLY ASKED QUESTIONS

1. Dental caries vaccine

2. Mechanism of action of caies vaccine

3. Advantages and limitations of caries vaccine

4. Routes of administration of caries vaccine

5. Antigenic determinants of dental caries vaccine