Caries Vaccine

1. Caries Vaccine Camille Nakamura, DDS

2. Dental Caries (why do we need a vaccine?) Decrease in overall rate in the U.S. Most common chronic disease affecting children in the United States 5x more common than asthma Most prevalent in low socioeconomic population and children Dental caries is costly and prevalent oral disease (US spends almost $40 billion annually to treat caries) A vaccine could target high-risk individuals (physical or emotional disabilities who are unable to practice good oral hygiene) and those with defective enamel structure $40 billion according to the Dental, Oral and Craniofacial data resource center last annual report in 2002$40 billion according to the Dental, Oral and Craniofacial data resource center last annual report in 2002

3. S. mutans 1924 J.K. Clarke isolated a streptococcus from human carious lesions and named the species S. mutans S. mutans has been suggested to have a causative role in dental caries especially since the late 1950s when Orland and Fitzgerald innoculated germfree rats and hamsters with S. mutans and led to the formation of dental caries. Since then the focus of caries research has been on S. mutans.

4. Principals of a Vaccine Must be safe for use in humans Contain appropriate virulence antigens of mutans strep to induce antibodies Given via appropriate route to induce wanted response Cost-effective/practical Vaccine intended for nonlethal disease (caries) must be safer than vaccine for life-threatening infection (where benefits can outweigh adverse reactions).

5. Host response to caries Host/Immunological response Can be mediated by secretory immune system (S-IgA Antibodies in saliva) or systemic immunity w/ IgG or IgM in GCF � mainly S-IgA By 6-9months most children have adult-like distribution of S-Iga (maturation of immune system by 1st year of life) Natural Immunity: Situation remains unclear b/c studies have conflicting results (direct and inverse relationship btw dental caries and S-IgA or S-IgA antibody If S-IgA antbodies were responsible for regulating oral bacteria�would expect that IgA deficiency would have increased susceptibility to dental caries ? differing results S-IgA = blocks ability of S.mutans to bind to hydroxyapatite/tooth surface (binds to bacteria) Natural immunity: redundancy in the immune systemS-IgA = blocks ability of S.mutans to bind to hydroxyapatite/tooth surface (binds to bacteria) Natural immunity: redundancy in the immune system

6. At birth = S. mitis and S. salivarius (bind to gingiva and mucosal structures) After initial eruption of tooth: S. Sanguis begin to colonize Much later see S.mutans Large jump in SIgA levels by the 1st year At birth = S. mitis and S. salivarius (bind to gingiva and mucosal structures) After initial eruption of tooth: S. Sanguis begin to colonize Much later see S.mutans Large jump in SIgA levels by the 1st year

7. Timeline Mucosal immunization of infants before appearance of 1st teeth Study by Caufield et al � �window of infectivity� Children become permanently colonized by mutans strep between middle of 2nd year and end of 3rd year of life If children don�t acquire mutans strep by 26 months of age then unlikely that primary teeth will be colonized Factors can affect window of infectivity: (shift window earlier) High maternal infection levels High dietary sucrose levels -Should immunize before window of infectivity (immunize at 6-18 months of age) Vaccinate early Mother chewing xylitol?Vaccinate early Mother chewing xylitol?

8. Window of Infectivity

9. --Caries vaccine before mutans strep first appears in oral cavity (better chances of immunization than the need to eliminate an established cariogenic flora No current data regarding additional windows of infectivity (when permanent teeth start to erupt?) ? booster immunization at this time may be required to inhibit second attempt for colonization by mutans strep--Caries vaccine before mutans strep first appears in oral cavity (better chances of immunization than the need to eliminate an established cariogenic flora No current data regarding additional windows of infectivity (when permanent teeth start to erupt?) ? booster immunization at this time may be required to inhibit second attempt for colonization by mutans strep

10. Routes of Vaccination 1) Intravenous immunization has been attempted - Involve IgG antibodies from the plasma ? GCF - negative: possibility of inducing tissue-reactive antibodies such as heart-reactive antibodies 2) Mucosal application generally preferred for induction of secretory IgA antibody -S-IgA can be elicited in saliva by stimulating local lymphoid tissue or central inductive sites of CMIS CMIS (common mucosal immune system) IgA induction in lymphoid tissue in gut (GALT), nasal (NALT), bronchial, or rectal site can give rise to immune response Shown that infection of humans with certain M types of Strep pyogenes results in rheumatic fever and acute glomerulonephritis. Due to the ability of certain M types of S. pyogenes to induce autoantibodies in humans that recognize joints, myocardium, valves, kidney and brain. (heart muscles) Evidence of rabbits immunized with S. mutans that produced antibodies that reacted with human sarcolemma and skeletal muscles Strep pyogenes (Protein M � virulence factor) � antibody cross reacts with heart muscle (evidence in past of rabbits who were immunized to strep mutans�)Shown that infection of humans with certain M types of Strep pyogenes results in rheumatic fever and acute glomerulonephritis. Due to the ability of certain M types of S. pyogenes to induce autoantibodies in humans that recognize joints, myocardium, valves, kidney and brain. (heart muscles) Evidence of rabbits immunized with S. mutans that produced antibodies that reacted with human sarcolemma and skeletal muscles Strep pyogenes (Protein M � virulence factor) � antibody cross reacts with heart muscle (evidence in past of rabbits who were immunized to strep mutans�)

11. Mucosal immune system M-cell (M)-containing follicle-associated epithelium through which exogenous antigens are transported actively (M cells extend and surround B-cells, T cells, Dendritic cells and macrophages. to reach APCs (antigen presenting cells), including DCs (dentritic cells), macrophages, B cells, and FDCs (follicular dendritic cells). ALSO: subepithelial DCs may capture antigens at the effector site (exemplified by nasal mucosa in the middle) and migrate via draining lymphatics to local/regional lymph nodes where they become active APCs, which stimulate T cells for productive or downregulatory (suppressive) immune responses. When recognized the B cells then travel to the germinal center and undergo clonal expansion After being primed in lymph nodes to peripheral blood for subsequent extravasation at mucosal effector sites (exemplified by gut mucosa on the right). B cells produce IGA and IGM IgA in plasma is almost entirely monomeric but s-IGA is polymeric (normally in dimer form) � covalently linked to 2 additional chains (1 is J-chain � joining) The J chain allows for the binding of the IgA pIGR (polymeric immunoglobulin receptor found on the basolateral surface of epithelial cells lining mucous membranes and transports J-chain polymers such as IGA to the external secretion. EXTRA BELOW paracellular leakage of smaller amounts (broken arrow) of both locally produced and plasma-derived IgG antibodies into the lumen. There may also be some active transport of IgG mediated by the neonatal Fc receptor (not indicated). Note that IgG cannot interact with J chain to form a binding site for pIgR. The distribution of intraepithelial lymphocytes (mainly T-cell receptor /+CD8+ and some /+ T cells) is also depicted. The inset (lower left corner) shows details of an M cell and its "pocket" containing various cell types. The cartoon is modified from Brandtzaeg and Pabst1 with permission from Elsevier. APCs, antigen-presenting cells; DCs, dendritic cells; FDCs, follicular dendritic cells; HEVs, high endothelial venules; MALT, mucosa-associated lymphoid tissue; mSC, membrane secretory component; pIgR, polymeric Ig receptor; SIgA, secretory IgA; SIgM, secretory IgM. HEV = High endothelial venulesM-cell (M)-containing follicle-associated epithelium through which exogenous antigens are transported actively (M cells extend and surround B-cells, T cells, Dendritic cells and macrophages. to reach APCs (antigen presenting cells), including DCs (dentritic cells), macrophages, B cells, and FDCs (follicular dendritic cells). ALSO: subepithelial DCs may capture antigens at the effector site (exemplified by nasal mucosa in the middle) and migrate via draining lymphatics to local/regional lymph nodes where they become active APCs, which stimulate T cells for productive or downregulatory (suppressive) immune responses. When recognized the B cells then travel to the germinal center and undergo clonal expansion After being primed in lymph nodes to peripheral blood for subsequent extravasation at mucosal effector sites (exemplified by gut mucosa on the right). B cells produce IGA and IGM IgA in plasma is almost entirely monomeric but s-IGA is polymeric (normally in dimer form) � covalently linked to 2 additional chains (1 is J-chain � joining) The J chain allows for the binding of the IgA pIGR (polymeric immunoglobulin receptor found on the basolateral surface of epithelial cells lining mucous membranes and transports J-chain polymers such as IGA to the external secretion. EXTRA BELOW paracellular leakage of smaller amounts (broken arrow) of both locally produced and plasma-derived IgG antibodies into the lumen. There may also be some active transport of IgG mediated by the neonatal Fc receptor (not indicated). Note that IgG cannot interact with J chain to form a binding site for pIgR. The distribution of intraepithelial lymphocytes (mainly T-cell receptor /+CD8+ and some /+ T cells) is also depicted. The inset (lower left corner) shows details of an M cell and its "pocket" containing various cell types. The cartoon is modified from Brandtzaeg and Pabst1 with permission from Elsevier. APCs, antigen-presenting cells; DCs, dendritic cells; FDCs, follicular dendritic cells; HEVs, high endothelial venules; MALT, mucosa-associated lymphoid tissue; mSC, membrane secretory component; pIgR, polymeric Ig receptor; SIgA, secretory IgA; SIgM, secretory IgM. HEV = High endothelial venules

12. Routes of vaccination Oral Nasal Tonsillar Minor Salivary Glands Rectal Oral � traditional method for mucosal immunization (early studies) Via GALT to elicit IgA antibody response Not ideal (detrimental effects of stomach acidity on antigen, inductive site were distant) BUT sufficient to cause change in some animal models Intranasal � advantages over oral route in inducing S-IgA response Via NALT to elicit antibody response Closer anatomical relationship to oral cavity Vaccine applied to environment with less proteolytic enzymes and antigenic competition ? induce local immune responses resulting in antibodies in nasal wash and saliva Successful induction of immunity in many animal models Tonsillar Although IgG rather than IgA response is dominant in tonsillar tissue still able to induce IgA response Topical application of formalin killed S.sobrinus cells in rabbits induced salivary immune response Minor salivary glands Lips, cheeks, soft palate Topical administration to lower lips showed significantly lower proportions of mutans strep during 6 week f/u (Smith and Taubman 1990) Rectal Administration Highest concentration of lymphoid follicles in lower intestinal tract Possible site for alternative for children with respiratory ailments and can�t have intranasal application ** Mucosal immunization to induce S-IgA antibody response has fewer problems than parenteral injection of antigen Oral � traditional method for mucosal immunization (early studies) Via GALT to elicit IgA antibody response Not ideal (detrimental effects of stomach acidity on antigen, inductive site were distant) BUT sufficient to cause change in some animal models Intranasal � advantages over oral route in inducing S-IgA response Via NALT to elicit antibody response Closer anatomical relationship to oral cavity Vaccine applied to environment with less proteolytic enzymes and antigenic competition ? induce local immune responses resulting in antibodies in nasal wash and saliva Successful induction of immunity in many animal models Tonsillar Although IgG rather than IgA response is dominant in tonsillar tissue still able to induce IgA response Topical application of formalin killed S.sobrinus cells in rabbits induced salivary immune response Minor salivary glands Lips, cheeks, soft palate Topical administration to lower lips showed significantly lower proportions of mutans strep during 6 week f/u (Smith and Taubman 1990) Rectal Administration Highest concentration of lymphoid follicles in lower intestinal tract Possible site for alternative for children with respiratory ailments and can�t have intranasal application ** Mucosal immunization to induce S-IgA antibody response has fewer problems than parenteral injection of antigen

13. S. Mutans Virulence factors?

14. Caries vaccine targets( S.mutans virulence factors) Colonization 2-step process: Sucrose independent adherence Adhesin Ag(I/II) and SpaP Initial adherence to pellicle 2) Sucrose dependent accumulation Glucosyltransferase Synthesis of glucans Glucan binding proteins Mediates S. mutans adherence to glucans Dextranase Cleavage of glucans for glycolysis Mutans streptococci requires hard surfaces for colonization and accumulation so target adhesion and colonization properties AgI/II (for S. mutans) or SpaP for S.sobrinus Glucosyltransferase (GTF-S) = synthesizes dextrans with alpha 1,6 linkages Or GTF �I = synthesizes mutans with alpha 1,3 linkages These produce glucans or scaffolding for colonization of S.mutans Mutans streptococci requires hard surfaces for colonization and accumulation so target adhesion and colonization properties AgI/II (for S. mutans) or SpaP for S.sobrinus Glucosyltransferase (GTF-S) = synthesizes dextrans with alpha 1,6 linkages Or GTF �I = synthesizes mutans with alpha 1,3 linkages These produce glucans or scaffolding for colonization of S.mutans

15. Adhesins (Antigen I/II for S.Mutans) � Initial attachment of S. mutans to the tooth surface GTF (Glucosyltransferases) - Accumulation of S.Mutans ? branched extracellular glucans (make scaffolding for aggregation of mutans) 3) Glucan binding proteins � cause extensive accumulation of mutans strep to extracellular glucans. 4)Dextranase- Break down glucans for glycolysis Erosion of hydroxyapatite mineral in dental enamel by lactic acid (bacterial metabolic process) Adhesins (Antigen I/II for S.Mutans) � Initial attachment of S. mutans to the tooth surface GTF (Glucosyltransferases) - Accumulation of S.Mutans ? branched extracellular glucans (make scaffolding for aggregation of mutans) 3) Glucan binding proteins � cause extensive accumulation of mutans strep to extracellular glucans. 4)Dextranase- Break down glucans for glycolysis Erosion of hydroxyapatite mineral in dental enamel by lactic acid (bacterial metabolic process)

16. Adhesins (Ag I/II) Antibody with specificity for S. mutans Ag I/II interferes with bacterial adherence ? prevent dental caries Study: (Hajishengalis et al 1992) Antibody directed to intact antigen I/II molecule or its salivary-binding domain blocked adherence of S. mutans to saliva-coated hydroxyapatite Hajishengalis � U of Alabama sIGA antibodies to Ag I/II inhibit binding of AgI/II with S. mutans to saliva-coated hydroxyapatite Effect of cleavage of s-IGA antibodies by IgAI protease Found: Antibody directed at AgI/II blocked adherence of S. mutans to saliva-coated hydroxyapatite IgAI protease produced by oral bacteria counteract antiadherence activity of s-IGA ? allow for AgI/II adherenceHajishengalis � U of Alabama sIGA antibodies to Ag I/II inhibit binding of AgI/II with S. mutans to saliva-coated hydroxyapatite Effect of cleavage of s-IGA antibodies by IgAI protease Found: Antibody directed at AgI/II blocked adherence of S. mutans to saliva-coated hydroxyapatite IgAI protease produced by oral bacteria counteract antiadherence activity of s-IGA ? allow for AgI/II adherence

17. Glucosyltransferase (GTF) Antibody directed to GTF: interferes with activity of the enzyme and thus plaque formation Study: Taubman and Smith 1977, 1979 Immunization with S.mutans GTFs induce protective immune response in experimental dental caries rodent models Both were designed to investigate ability of anti-GTF antibodies to interfere with glucan formation by glucosyltransferase - One way was by feeding glucosyltransferase preparations to hamsters and inducing immune response Both were designed to investigate ability of anti-GTF antibodies to interfere with glucan formation by glucosyltransferase - One way was by feeding glucosyltransferase preparations to hamsters and inducing immune response

18. Glucan-Binding Proteins mediates ability of mutans strep to bind to glucans S.mutans has 3 different proteins with glucan-binding activity: GbpA,GbpB, GbpC Study: (Smith and Taubman 1996) Only GbpB has shown protective immune response to dental caries (Smith and Taubman 1996) GbpA � Russell 1979 GbpB � Smith 1994 GbpC � Seto et al 1997 Differed in molecular weight and amino acid sequence Only GbpB was able to induce a protective immune response Study showed that induced an increase in sIGA levels and lower s.mutans level in GbpB administered rats EXTRA BELOW After cloning and sequencing, the gbpA gene product was found to share homology with the glucan binding domain of and the gbpA gene was found to encode a constitutively expressed secreted protein (1, 2). Cell surface-associated GbpC was related to the Spa family of streptococcal proteins and was expressed only during conditions of stress (24). GbpB was immunologically distinct from other Gbps expressed by S. mutans and Streptococcus sobrinus and also differed in size and purification properties GbpA � Russell 1979 GbpB � Smith 1994 GbpC � Seto et al 1997 Differed in molecular weight and amino acid sequence Only GbpB was able to induce a protective immune response Study showed that induced an increase in sIGA levels and lower s.mutans level in GbpB administered rats EXTRA BELOW After cloning and sequencing, the gbpA gene product was found to share homology with the glucan binding domain of and the gbpA gene was found to encode a constitutively expressed secreted protein (1, 2). Cell surface-associated GbpC was related to the Spa family of streptococcal proteins and was expressed only during conditions of stress (24). GbpB was immunologically distinct from other Gbps expressed by S. mutans and Streptococcus sobrinus and also differed in size and purification properties

19. Dextranase Converts glucans into glucose for glycolysis Breaks down the glucan matrix (source of energy during nutritional deprivation Studies inducing immune response? 1995 � Colby et al: Genetic alteration of dextranase gene resulted in altered adherence EXTRA BELOW: Cleaves the alpha 1,6 linkage of the glucans Utilization of extra- cellular, soluble dextran as an energy and carbon source during periods of nutritional deprivation is one potential function.1995 � Colby et al: Genetic alteration of dextranase gene resulted in altered adherence EXTRA BELOW: Cleaves the alpha 1,6 linkage of the glucans Utilization of extra- cellular, soluble dextran as an energy and carbon source during periods of nutritional deprivation is one potential function.

20. Problems with caries vaccine Not known how to generate long-term IgA response to antigens Studies of rodents and humans show periods of elevated IgA levels for weeks/months (would require frequent �booster� innoculations) Argument that even delayed infection of S. mutans would be beneficial since newly erupted teeth more susceptible to cariogenic attach than teeth that had more time to mature (calcification)

21. Caries Vaccine?

22. Other research Jeffrey Hillman (Oragenics) � formerly Univ of Florida - patented a genetically modified strain of S. mutans (sMART) 1) Competitive inhibition of native S. mutans 2) Eliminate acid production of S. mutans - produces alcohol (Zymomonas mobilis) Goals: 1) Strain of S. mutans that can outcompete with natural S. mutans strain by binding to the tooth better 2) Delete gene encoding lactate dehydrogenase (and prevent production of lactic acid ? not cariogenic) -Found: Strain would outcompete native S. mutans, but when trying to eliminate lactate dehydrogenase ? toxic to bacteria Overcome this by replacing LDH gene with gene for alcohol dehydrogenase from Zymomonas mobilis ? by product is alcohol Tried on gnotobiotic rat models : successful Currently FDA problems: 1st Phase I clinical trial began in April 2005 but difficult to find patients ? quit Resubmitted 2nd Phase I clinical trial include 10 healthy adult males, but FDA on hold June 2007 ? wanted a plan if serious adverse effects - solution: D-alanine dependent mutant strain (D. alanine not in human diet ? have to administer mouthwash to keep alive) -study scheduled to conclude in 2011 (this year) 3rd Phase I trial : Use original non-attenuated mutated S. mutans strain (that is not alanine dependent)Goals: 1) Strain of S. mutans that can outcompete with natural S. mutans strain by binding to the tooth better 2) Delete gene encoding lactate dehydrogenase (and prevent production of lactic acid ? not cariogenic) -Found: Strain would outcompete native S. mutans, but when trying to eliminate lactate dehydrogenase ? toxic to bacteria Overcome this by replacing LDH gene with gene for alcohol dehydrogenase from Zymomonas mobilis ? by product is alcohol Tried on gnotobiotic rat models : successful Currently FDA problems: 1st Phase I clinical trial began in April 2005 but difficult to find patients ? quit Resubmitted 2nd Phase I clinical trial include 10 healthy adult males, but FDA on hold June 2007 ? wanted a plan if serious adverse effects - solution: D-alanine dependent mutant strain (D. alanine not in human diet ? have to administer mouthwash to keep alive) -study scheduled to conclude in 2011 (this year) 3rd Phase I trial : Use original non-attenuated mutated S. mutans strain (that is not alanine dependent)

23. THANK YOU!