Caries Vaccine And The Ways To Modfiy Streptococcus Mutans

CONTENTS

INTRODUCTION

HISTORICAL AND CURRENT PROSPECTIVE -NEED FOR CARIES VACCINE

CLASSIFICATION OF MICROORGANISMS IMPLICATED IN CARIES

STREPTOCOCCUS MUTANS

CARIES PROCESS

IMMUNOLOGY IN RELATION TO CARIES VACCINE

REVIEW OF LITREATURE
- REPORTS
- INVITRO
- RATS
- RABBITS
- HAMSTERS
- MONKEYS
- HUMANS
- NEW TECHNIQUES

CONTROVERSIES OF DENTAL CARIES VACCINES

SUMMARY

CONCLUSION

BIBLIOGRAPHY

FIGURES

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THIS BOOK IS DEDICATED TO

MY HUSBAND

INTRODUCTION

Dental caries is a multi factorial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substances of the tooth.[189] Dental caries is also a biosocial disease whose causes are rooted in the culture, technology and economy of our society.[73]

Oral cavity is a nutritionally rich environment and consists of a complex bacterial flora.[74] As the definition suggests “Multi factorial”, means not just microorganism but also other factors such as fluorides, dietary habits, oral hygiene practice, salivary flow rate and anatomy of the teeth may alter the rate of destruction of the tooth.[73]

Caries is not caused by any single organism but rather by a variety of microorganisms. These are representatives of at least twenty two genera of bacteria and greater than sixty individual stains have been classified into species.[73]

The control of dental caries presents one of the greatest challenges that must be met today. Different methods used in the prevention of dental caries are diet and nutritional control as well as mechanical and chemical methods. Alteration of the diet by reducing the intake of sucrose or other refined carbohydrates will reduce dental caries.[74]

The primary antimicrobial approach used by dental personal in the prevention of caries is the removal of plaque by oral hygiene technique. This approach has not been successful because effective methods of plaque removal are too difficult for the average person.[74]

Bacteria in the fissures are sealed from the oral cavity by a dental sealant, by which the bacteria cannot survive. Pit and fissure sealants thus appear to act by blocking the nutrient supply to the cariogenic bacteria in the fissures. The possibility of inadvertently sealing over an incipient lesion is obviously a concern in the use of pit and fissure sealants. As long as a sealant is intact the lesion is unlikely to progress. However teeth that are carious should be treated by conventional removal followed by a restoration that has no leakage of saliva at the tooth restoration interface.[74]

For over 40 years it was known that the administration of fluoride systemically via the water supply could reduce the incidence of caries by at least 50%. The optimum concentration for maximal protection against caries is thought to be 1ppm. But in many water supplies it naturally occurs in a concentration greater than this. Despite its proven value in decreasing caries incidence, the addition of fluoride to the drinking water remains controversial and emotive issue. Fluoride can also be added to table salt, milk and tooth toothpastes. It is also used in the mouthwashes and gels for topical use and in tablets for supplementation of the systemic effect. Recently fluoride has been incorporated into thin films for coating teeth, topical varnishes and slow release capsules. Fluoride is also available naturally in tea and the bones of fish [74]. Topical treatment of stannous fluoride has found to selectively affect S.mutans in saliva and smooth surface plaque. Side effects such as mild staining of the teeth and a metallic after taste can deter some people. Excess of fluoride intake can cause fluorosis and other complications.[74]

The use of antimicrobial agents to control plaque has been advocated. The aim of the product is to be anti plaque rather than anti caries. Although antibiotics can prevent caries, these inhibitors are considered inappropriate for routine unsupervised use in toothpaste or by mouth rinses. Their spectrum of activity is too broad, while their indiscriminate use can lead to the emergence of resistant organisms and to overgrowth by opportunistic pathogens.[73] The most effective agent to date has been Chlorhexidine which has a broad spectrum of activity against yeasts, fungi, and a wide range of gram +ve and gram -ve bacteria. Chlorhexidine can reduce plaque caries and gingivitis.[73] But it has several disadvantages as it reduces the nonpathogenic as well as pathogenic flora, severely stains teeth and has a disagreeable taste.[74]

Immunization against dental caries is an attractive potential public health measure. Voluntary immunization would be particularly attractive if an oral means for antibody stimulation could be found.[74]

It would be beneficial to develop a safe reliable economical means to control the disease. Unlike smallpox or measles the etiologic agent is part of the endogenous flora. Only a certain percentage of dental caries is due to streptococcus mutans and consequently an S.mutans vaccine would only provide partial protection.[16] This organism is acquired during infancy and remains with us for the whole life. The implication is that the immune system must respond to the bacteria on a daily and not on a periodic basis. It is possible that constant exposure to an antigen leads to the development of tolerance or to immune exclusion. The formation of lesion in caries involves destruction of tissues, but does not bring the organism in contact with the immune system, and consequently there is no stimulation of immunoglo bulin synthesis.[16] Cariogenic microorganism primarily resides on body surfaces, which are isolated from the activity of phagocytic cells and complement. Antibodies will work by preventing colonization. The hard tissue does not shed and consequently the tooth is colonized by a population, which is relatively stable and does not have to continuously recolonize as in the case of organisms growing on an epithelial surface.[16], [73]

Vaccination could also be a valuable adjunct for handicapped persons and person with reduced salivary secretion rate. This would probably be more acceptable than trying to alter either human behavior or the social environment.

This approach to the prevention of dental caries should not be regarded as being in competition with other methods such as the use of water fluoridation, topical fluorides in the form of mouth rinses, sealants, topical varnish, antimicrobial chemotherapeutics, or good dietary habits. Immunization in combination with all other techniques which have been mentioned, may contribute towards an even greater reduction of the prevalence of this disease.[74]

The protection against caries by caries vaccine has generated hopes and a significant progress has been made in this field. An attempt has been made to review the literature on immunology and vaccination for prevention of caries.

HISTORIC AND CURRENT PRESPECTIVE- NEED FOR CARIES VACCINE.

Teeth are tools that have evolved to ensure survival of species. Survival of all higher forms of life, dependent upon ingestion of food to fuel, life’s process. Structures present in the oral cavity serve the function of gathering, cutting, grinding and admixing food with saliva in order to prepare it for swallowing and its journey through alimentary system and conversion into energy for bodily needs.[100]

Teeth are evolved uniquely to fulfill its function but even nature has to pay a price for creating a specialized grinding and cutting tool. Enamel tissue by virtue of its unique biological properties is highly susceptible to environmental agents. Enamel tissue is incapable of any natural reparative mechanism; however environmental factors such as saliva serve as a natural defense mechanism.139, 140,141

Dental caries is the most prevalent chronic disease affecting the human race. Once it occurs, its manifestation persists throughout life even though the lesion is treated. It affects sexes, all races, all socioeconomic strata and every age group. It usually begins soon after the teeth erupt into the oral cavity.[139]

Dental caries may probably be considered a disease of modern civilization, since pre-historic man rarely suffered from this form of tooth destruction.[139] There is no evidence of dental caries in the relatively few teeth found in skull fragments of our earliest known direct ancestors the pithencanthropen.[183] Anthropologic studies of Van Ben Hosek revealed that the dolicocephalic skulls of men from pre Neolithic periods (12,000 B.C) did not exhibit dental caries. But skulls from brachycephalic man of the Neolithic period (12,000-3000 B.C) had caries teeth.139, 140,141

Palentological evidence shows that it has affected humans at least from the time that agriculture replaced hunting as the principal source of food.[139] Examination of skulls in Britain suggested that caries prevalence changed little from Anglo- Saxon period (5th-7th centuries) to the end of the middle ages, approximately the year 1500.[139],[140] The modern pattern of caries was not evident in Britain until the 16th century.[141]

Dietary changes during the 17th century principally increased refinement and greater use of sucrose, as sugars became more available, are considered chiefly responsible for the development of the modern pattern of caries.[139], [23],[41] Import duties on sugar in Britain began to be removed in 1845 and were completely eliminated in 1875 a period during which the severity of caries greatly increased.[139], [140] By the end of the 19th century, dental caries was well established as an epidemic disease of massive proportions in most developed countries.[23],[141]

The etiology of dental caries is generally agreed to be a complex problem complicated by many indirect factors, which obscure the direct cause or causes. There is no universally accepted opinion of etiology of dental caries.

CURRENT CONCEPT

Interaction between the primary and secondary factors is essential for the initiation and progression of caries, a susceptible host tissue, the tooth, micro flora with a cariogenic potential and a suitable local substrate to meet the nutritional requirements of the pathologic flora (fig 1). The tooth is the target tissue destroyed in the dental caries process. The cariogenic oral flora that is localized to specific sites on the teeth is the agent that produces and secretes the chemical substance. (Organic acids, chelating agents and proteolytic enzymes) that causes the destruction of the inorganic components and the subsequent break down of the organic matrix of enamel and dentine. The local substrate provides the nutritional requirements for the oral micro flora, thereby permitting them to colonize, grow and metabolize on selective surfaces of the teeth. The third factor, the resistance of the tooth, is obviously important since this determines the overall effect of the attack.[74]

Studies conducted by Fitzgerald in 1968 on gnotobiotic animals of the role of microorganisms in caries came to the conclusion that[75]

-Microorganisms are a prerequisite for caries initiation.
-A single type of organism (enterococcus strain) is capable of inducing caries.
-The ability to produce acid is a prerequisite for caries induction but not all acid-producing organisms are cariogenic.
-Streptococcus strains that are capable of inducing caries are also able to synthesize extra cellular dextran or levans. Not all strains that produce extra cellular polysaccharides are capable of caries induction.
-Organisms vary greatly in their capacity to induce caries; comparative virulence cannot be deduced at present with certainty.[75]

Those members of the bacterial flora of the mouth that are localized to specific site on teeth are significant in the caries process. Localization of bacterial activity is conferred by adherence of organism and the formation of a bacterial plaque. The plaque consists of a heterogenous mass of organisms that implants them upon a salivary derived protein film found on all erupted teeth.[75]

Secondary or predisposing factors such as salivary composition and flow rate, oral hygiene and diet influence the caries process. Secondary factors affect one or a combination of the following

-Increase or decrease the quantitative and qualitative nature of the oral micro flora involved in dental caries.
-Increase or reduce the cariogenicity of the local substrate.
-Increase or reduce the tooth resistance to dental caries.

Saliva has many functions: cleansing effect, buffering capacity, provision of an environment saturated with calcium and phosphate, antibacterial action these characteristics influence the rapidity at which the caries develops.[74]

Fluoride is an important trace element that affects the resistance of the mineral of enamel to the caries process and enhances remineralization of the incipient lesions. Fluoride deficiency potentiates caries since the host factor cannot attain its maximum resistance to caries on a sub optimal intake. Other factors that influence caries are oral hygiene and dental plaque control.[74],[75]

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Fig1: Diagrammatic representation of the interplay between primary and secondary factors in caries etiology.

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Fig-2: Diagrammatic representation of different contributing factors in caries

NEED FOR CARIES VACCINE

Man’s pre occupation with disease is understandable; it has always been our most visible and ultimate enemy. Disease is ever present to attack an individual at any stage of his or her growth and development. Thousands of years ago, physicians placed the highest priority on prevention. Disease is prevented where an optional interplay of three primary and many secondary factors results in effective host resistance, elimination or reduction of noxious agents and attaining a healthy environment. In public health, three main levels of prevention have been defined namely primary, secondary and tertiary [75]

Primary preventive measures are aimed at reducing the occurrence of new cases of a disease in a population. This is accomplished by health promotion or by specific protective measures. Secondary prevention aims at reducing the prevalence of an illness or dysfunction. Tertiary prevention involves a treatment phase aimed at maximum limitation of disability and maximum rehabilitation.[75],[30]

By far the most promising methods of prevention are by physical or chemical means. In practice this means dietary advice, fluoride, fissure sealing and regular tooth brushing.[74] the prevention of caries can be accomplished by the above- mentioned methods. These procedures are relatively simple and inexpensive. It is a reasonable conclusion that the use of fluoride more than other measures has contributed to the recent dramatic decline in caries prevalence. The ethics of vaccinating against caries has stimulated increasing interest in the last decades. The possibility of preventing this disease by stimulating the defense mechanisms of the body has been contemplated. Immunization in combination with other techniques will contribute to the greater reduction of this avoidable non-lethal disease. [109]

Considering the magnitude and almost universal impact of caries, it is a paradox that a public supported program for eradication of this disease have never developed, as did programs against polio and small pox.[109]

The major strategies currently being followed for eradication or reduction of caries are: [75]

Preventive programs for individuals at home Preventive programs for dental office Preventive programs for community

Preventive programs for individuals at home include: -

Maintenance of oral hygiene Tooth brushing

Flossing

Fluoride containing dentifrices Topical fluoride applications

Systemic fluoride in the form of lozenges or tablets Diet modification

Dietary restriction of sucrose

Preventive programs for dental office includes: - Effective teaching by dentist of preventive techniques.

Oral hygiene instructions Diet control

Fluoride therapy Occlusal sealants Odontotomy

Preventive programs for community include

Oral hygiene maintenance Communal Fluoride programs Diet modifications

Despite the major accomplishments of preventive dentistry in reducing caries prevalence, the problem is still very much with us. There is continuing need for improvements in our existing preventive products and techniques and a broadening of our anti caries armamentarium. The need is a worldwide one.[23]

Proliferation of new techniques and scientific technology in molecular biology has created possibilities of fashioning new strategies for caries prevention.

CLASSIFICATION OF MICROORGANISM IMPLICATED IN CARIES

Dental decay is the most common disease of mankind. There are number of cariogenic organisms that have the ability to colonize teeth to reduce the pH of about 4.1 in the presence of suitable sugar substrate and to induce caries in germ free animals.[16]

Those that are most commonly accepted to cause caries are

MUTANS STREPTOCOCI

Streptococus mutans
Streptococus sorbinus
Streptococus rattus
Streptococus cricetus

LACTOBACILLI

Lactobacillus casei
Lactobacillus fermentum
Lactobacillus plantarum
Lactobacillus acidophilus

(POSSIBLE ODONTOPATHIC BACTERIA) STREPTOCOCUS

Streptococcus mitis

ACTINOMYCES

Actinomyces viscosus servosa

The view of ‘MUTANS’ STREPTOCOCCI as the most potent or perhaps the sole caries etiologic agents is based on a number of studies. Mutans streptococci are more numerous in individuals having high caries score than found in caries free individuals.[75]

Microorganisms that are most commonly included as Mutans streptococci.[121]

Streptococcus mutans
Streptococcus sobrinus
Streptococcus rattus
Streptococcus cricetus
Streptococcus ferus
Streptococcus downei
Streptococcus macacae

STREPTOCOCUS MUTANS

Streptococcus mutans was first described by J. Kilian Clarke in 1924. Kilian Clarke, a microbiologist, his task was to study the microbiology of dental caries disease. In deep dentin caries lesions, he found a small, chained coccobacillus that was more oval than spherical in shape. He suggested that these microorganisms were mutant streptococci and called them Streptococcus mutans. Clarke tried to prove the association of these streptococci with dental caries disease, but since other researchers did not support his hypothesis, interest in S. mutans waned. In the 1960s, the recently developed method of gnotobiotic animal research stimulated studies on the microbiology of dental caries disease, and S. mutans was convincingly connected to dental caries disease.[144], [145]

S.Mutans is the primary potent organism involved in the carious process. It is a facultative anaerobe, hemolytic acidogenic organism producing extra cellular & intracellular polysaccharides. The organism fulfills Koch’s postulates as a cause of dental caries.[75]

They are: -

1. S.Mutans is found in the plaque of carious teeth & usually cannot be isolated in the absence of caries.
2. The organism can be grown in suitable culture.
3. Infection of germ free rats or normal hamsters with S.mutans induces caries.
4. The organism can then be recovered from the carious lesion and grown in suitable culture.
5. Antibodies to this organism are increased in patients with caries.

Streptococci are broadly classified into obligate anaerobes (peptostreptococci) & facultative anaerobes. The Aerobic & facultative anaerobic streptococci are further classified based on their hemolytic properties.[121]

Streptococcus has been classified in numerous ways, but the most commonly accepted is given in Fig-3

Fig-3 Classification of streptococci based on O2 requirement: -

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Classification based on hemolysis:-

1. Alpha (a) hemolytic streptococci produce a greenish discoloration with partial hemolysis around the colonies. The zone of lyses is small with indefinite margins and unlisted erythrocytes can be made out microscopically within the zone.

E.g.: - S. Slaivarius, Sanguis, mitior and milleri.

2. Beta (b) hemolytic streptococci produce a sharply defined, clear, colorless zone of hemolysis within red blood cells that lyses completely. S.mutans falls in this category.

Lancefeild classified hemolytic streptococci serologically into groups based on the nature of carbohydrates antigen on the cell wall. 19 groups were present from A-U without I & J.

E.g.: - S. pyogenes.

3. Gamma (g) or no hemolytic streptococci produce no change in the medium. These are sometimes called indifferent streptococci.

E.g.: - Faecal streptococci- S.faecalis, S.faecium, S.durans and

Non-Faecal streptococci- S.bovis and S.equines.

Serological classification of S.mutans: -

Serological techniques have been very useful in classifying B –hemolytic S.mutans on basis of immunological reacting.

Lancifield has classified these organisms into 19 groups (A- U) except I & J.

According to the classification in Bergey's Manual of Determinative Bacteriology Holt et al in 1994, the genus Streptococcus includes the pyogenic, oral and anaerobic groups of streptococci, as well as a group of other streptococci. The cells are spherical or ovoid, 0.5 -2.0 m?m in diameter, occurring in pairs or chains when grown in liquid media, and stain Gram-positive. Streptococci re quire nutritionally rich media for growth. The metabolism is fermentative, producing mainly lactate but no gas. The streptococci are catalase-negative, and they commonly attack red blood cells, with either greenish discoloration (a?- hemolysis) or complete clearing (b-?hemolysis).[169] Optimum temperature for growth is 37 o?C, and growth is usually restricted to 25-45 o?C. Streptococci constitute a major population in the oral cavity, with several different species colonizing the various ecological niches of the mouth. Some of the species exhibit Lancefield serological group antigens. Differentiation between the pyogenic, oral and anaerobic groups may be laborious, and combined information is needed for classification.[100]

Mutans streptococci are also classified by means of serotype & genotype: -[121]

SEROTYPE:-

By means of precipitating & immunofluoresence techniques Mutans streptococci have been separated into 8 serotypes (a-h). This is based on polysaccharide antigen residing in the cell wall of the Mutans streptococci. Mutans streptococci isolated from man appears to belong to serotype c. The next common being serotype d. The important serotype promoting caries in humans are streptococcus mutans containing strains of serotype (c, e, f) & possibly S. Sorbinus (serotype d, g). S. rattus (serotype b), S.ferus (serotype c) & S.macacae (serotype c) are rarely isolated from human dental plaque (Fig-4 and Fig-5)

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Fig-4 serotype of S.Mutans[75]

Fig: - 5 Serotype of mutans streptococci

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GENOTYPES:-

Another method of classifying Mutans streptococci is through presence or absence of certain genes. It can be assumed that lactic acid production by mutans streptococci is an important cariogenic factor; biochemical analysis has suggested that the H+ translating ATPase of Mutan Streptococci play an important role in acidurity of this organism. Besides fermentation of sugars, mutant streptococci strains may also produce lactic acids following degradation of reserve polysaccharides. Fructans synthesized by the ftf gene product, FTF (fructosyl transferase) of mutant streptococci as well as intra cellular glycogen could be potential virulence factors for these organisms.[100]

Colonization of tooth surface by mutans streptococci is dependent on both sucrose dependent & independent mechanisms. The latter is mediated by proteins (adhesions) associated with cell surface of mutant streptococci. Several of gene coding for potential adhesions designated as P1, B, antigen I/II or Pac has been stated by Lee et al 1988, Okahashi et al 1989. Glucosyl transferase GTFs is implicated as virulence factors for the mutant streptococci. There are three distinct GTF genes gtf B, gtf C, and gtf D. The genes gtf B and gtf C require sucrose dependent attachment to smooth surface, where as gtf D does not require sucrose. Mutan streptococci strain UA101 even in presence of sucrose does not colonize smooth surface in vitro where as it induces caries in rats. It contains a single

GTF-1 enzyme from a gene, which is derived from homologous recombination between gtf B and gtf C genes. Glucan binding protein (GBP) in Mutan streptococci also play a role in sucrose dependent colonization of tooth surface. (Fig-6)[100]

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Fig-6 Genotypes of Mutans Streptococci

THE STUCTURE:-

The structure & antigenic composition of Mutans streptococci are of considerable importance in our understanding of immune response. Mutans streptococci contain a cell wall and protoplast membrane, which encloses the protoplast of the organism. The matrix of the cell wall consists of a cross linked peptidoglycan which is composed of N-acetyl amino sugars, N- acetyl muramic acid & other peptides. The surface antigen of the cell wall are involved in the immunogenecity of the organism.[100]

A Large number of streptococcal antigens have been identified. These are: -

1. Enzyme glucosyl transferase, which converts sucrose into dextran.
2. Streptococcal antigens I, II, I/II, III.

These four protein antigens (I, II, I/II, III) are present on Mutans streptococci. Among these streptococcal antigens I and II are two determinants usually present in a single molecule termed antigen I/II. Molecular weights of these antigens are: - antigen I (150,000), antigen II (48,000), antigen I/II (185,000) and antigen III (40,000).

Protein antigens I/II and III of Streptococcus mutans were identified in culture supernatants and subsequently have been purified from both culture fluids and cells. Antigen I/II is a protein of molecular weight 185,000 which has been identified in culture fluids and cells of S.mutans serotypes a, c, d, e, f and g. It is partially susceptible to common proteases, which remove the determinants composing antigen I and leave a resistant protein core of molecular weight 48,000-69,000 called antigen II. Antigen III is a protein which is resistant to most common proteases; it has a molecular weight of approximately 39,000 and is found in culture fluids and cells of S.mutans serotypes b, c, e and f. The biological functions of these proteins have not been elucidated but they are distinct from glucosyltransferase and dextran binding proteins. However antigen I/II, and its component parts antigen II, and I can induce protective immunity against dental caries in monkeys. This property correlates with the ability of antisera to these proteins to opsonize S. mutans for phagocytosis by polymorphonuclear leukocytes. These observations show that Antigen I/II is important in interactions with the host and immune defense system.[100]

3. The group specificity resides in the carbohydrates (a to h) and these are polymers of glucose, rhamnose, galactose or galactosamine. Serotype c, e & f contain only polymers of glucose & rhamnose, where as serotype d has galactose.
4. Lipoteichoic acid (LTA), which is derived from protoplast membrane. LTA has carbohydrates as well as glycerol phosphate, which is responsible for most of the cross-reacting between LTA. (Fig-7)

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Fig-7: Cell wall constituents of S. mutans

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