1. What happens at the interface between plaque and subgingival tissue? n Clinical observations: inflammation, apical migration of junctional epithelium, tissue destruction, bone resorption, etc. n Analysis of potential bacterial virulence factors: characterize specific activity, assay behavior of isogenic mutants. n Tissue culture models: mono- or multilayer cultured cells challenged with bacteria or bacterial products.

3. Overview n Periodontal disease in context: a common, chronically progressive polymicrobial disease whose progress is mediated by both bacterial factors and host immune responses.

4. bacterium phagocytic cell TISSUE DAMAGE BONE RESORPTION Inflammatory mediators (cytokines) (prostaglandins) Dys-regulation of host proteinase inhibitors Proteases Leukotoxin Evasion of host defenses Inflammatory mediators (cytokines) Proteases Capsule Leukotoxin Activation of inflammatory response Lysosomal enzymes and free radicals from Marsh, 1999 Surface-associated material LTA, LPS Hyaluronidase Colllagenase Arginine-specific protease Cytotoxins

5. Pattern recognition in periodontal microbiology n Approximately 15-20 cultivable species have been associated with periodontal diseases. n Most (except A.a.) are: u Anaerobic and u Asaccharolytic n Recognizable pattern: u Anaerobic, asaccharolytic bacterial species are associated with periodontal disease n Tentative conclusion / hypothesis to be tested: u Anaerobic, asaccharolytic bacteria cause most forms of periodontal disease.

6. Suspected periodontal pathogens SpeciesClinical entitiyOxygen sensitivity Porphyromonas gingivalis AP, EOP, RP Anaerobic Bacteroides forsythus AP, EOP, RP Anaerobic Treponema denticola AP, EOP, RP Anaerobic Prevotella intermedia AP, ANUG? Anaerobic Fusobacterium nucleatum AP, ANUG? Anaerobic Eubacterium nodatum APAnaerobic Selenomonas noxia APAnaerobic Porphyromonas gracilis APAnaerobic Treponema vincentii AP, ANUG Anaerobic Peptostreptococcus micros AP, RP Anaerobic Eubacteriumspp. APAnaerobic Selenomonas spp. APAnaerobic Streptococcus intermedius APAnaerobic A. actinomycetemcomitans LJP, EOP?, RP? Microaerophilic Wolinella recta APMicroaerophilic Eikenella corrodens AP?Microaerophilic

7. Cluster analysis of 32 subgingival species from 13,261 samples. 5 clusters were formed with >60% similarity and included 29 of the 32 species evaluated. Socransky et al. 1998 (green) (orange) (yellow) (red) (purple)

8. (S. Socransky)

10. Periodontal Diseases: Diagnostic tests and vaccine development Microbial considerations ProspectsLimitations

11. Is Periodontal Disease due to: n The nonspecific overgrowth of bacteria, mostly anaerobes, on the tooth surface? Dirty Mouth Syndrome OR n The overgrowth of certain bacteria, mostly anaerobes, on the tooth surface? Specific Infection OR n Genetic predisposition to periodontal disease?

12. The target population for diagnostic tests Why target specific patients for treatment? You can not expect to treat everyone..You can not expect to treat everyone.. You would like to treat patients by most appropriate means.You would like to treat patients by most appropriate means. Populations you would like to treat: Those most at risk to develop disease.Those most at risk to develop disease. Those at most risk for having refractory disease.Those at most risk for having refractory disease.

13. The target population for diagnostic tests Boyer et al., 1996 3.3

14. Relationship between trypsin-like enzyme activity and abscess formation and lethality: P. gingivalis injected subcutaneously in mice Characteristics of P. gingivalis strains Lesions in mice Abscesslethality W50 Trypsin + +++ +TLCK / PMSF -- +TLCK+- +DTT++++ Mutant strains 3079.03 Trypsin + ++++ NG4819 Trypsin - -- BE1 Trypsin +/- +- SW5 -- Adapted from Kesavalu et al, AADR 1992

15. Comparison of BANA test, DNA probes and immunological probes in detection of periodontal pathogens in plaque samples Reference standard is DNA Probes for P. gingivalis and T. denticola SensitivityAccuracy BANA Test 90%83% ELISA94%88% Reference standard is antibodies to P. gingivalis T. denticola and B. forsythus SensitivityAccuracy BANA Test 91%85% DNA Probe 93%88% There were no significant differences between the DNA probes, antibodies and the BANA test. Loesche et al., 1992, J.Clin.Micro.30:420

16. Accuracy of the BANA test n The BANA test detects the presence of BANA-positive species in plaque samples. Known BANA-positive species are T. denticola, P. gingivalis, and B. forsythus, all of which are anaerobic species and putative periodontal pathogens. The accuracy of the BANA test in detecting these species in plaque samples is about 85%, which is comparable to an 88% accuracy for DNA probes and 87% accuracy for immunologic reagents. This information may aid in diagnosis if clinical signs of periodontitis are present. n Clinicians make a diagnosis using clinical findings, a background history, and interpretation of tests such as the BANA test. It is incorrect to think that the BANA test, or any other test, can be used to make a clinical diagnosis in the absence of the above information.

17. BANA test: not-quite current version

18. Diagnostic Tests to Detect Genetic Susceptibility to Periodontitis n Another strategy: u Test based on detection of a genetic polymorphism in the gene encoding IL-1 that is associated with a higher than normal level of IL-1 response and subsequent inflammation. u This polymorphism is present in about 30% of the general population.

20. n n PST (Medical Science Systems,Inc., San Antonio, Texas). This is the only test to reveal a specific genetic markerthat identifies individuals at high risk for severe periodontitis. A sample of the individual's DNA is collected from a finger stick blood sample in a dentist's office. The sample is then sent to Medical Science Systems, Inc. (MSS) and analyzed in a licensed genetic laboratory using advanced technology to determine if the individual isgenotype-positive or genotype-negative. n n The evidence supports that when genotype-positive individuals are bacterially challenged, they are at least six times more likely to develop severe generalized periodontitis. Theymay therefore need to be managed even more aggressively to keep plaque levels under control.

21. Breaking News March 9, 1999 Medical Science Systems seeks patent on gene sequence Medical Science Systems Inc. announced it has filed for patent protection for a new gene sequence and its function. This sequence is in the chromosome region that regulates interleukin-1 (IL-1) cytokine production and levels, which plays a major role in the body's inflammatory response, immune response, and bone and connective tissue metabolism.

22. CURRENT (?) AND FUTURE (?) COMMERCIAL TESTS n Evalusite (available in Europe and Canada). n Omnigene (OmniGene Laboratory Services, Cambridge, MA). n MicroProbe (under development). n PerioScan (BANA Test)(Oral B Laboratories) n PST (Medical Science Systems,Inc., San Antonio, Texas).

23. CURRENT (?) AND FUTURE (?) COMMERCIAL TESTS n n Evalusite (available in Europe and Canada). This chair side immunoassay detects A. actinomycetemcomitans, P. gingivalis, and P. intermedia by complexing antibody to specific antigens on these bacteria. The plaque sample is mixed with a detergent and placed on a membrane with antibodies to the targeted bacteria. When the antibody- antigen complex forms, a colored enzyme substrate is added to the mixture. A positive test will form a blue dot on the reagent pad. This test takes about 10 minutes. n n Omnigene (OmniGene Laboratory Services, Cambridge, MA, currently available). This DNA probe tests for A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, and C. rectus. Nonviable organisms can be detected. Results are given as negative of low, moderate, or high presence of the targeted bacteria.

24. CURRENT (?) AND FUTURE (?) COMMERCIAL TESTS n n MicroProbe (under development). This DNA probe system can be completed in the dental office in 1 hour.Oligonucleotide-coated beads specific for A actinomycetemcomitans, P gingivalis, and P intermedia arecontained on a small plastic card. The patient's plaque sample is dispersed, and the DNA is extracted with reagentscontained in the kit. The card with the beads is moved through the solutions of the kit, and a positive test resultswhen the white bead changes to blue. This reaction is precipitated by the antigen-antibody complex formation ofthe targeted bacteria. n n PerioScan (BANA Test)(Oral B Laboratories, Edwood City, CA, currently available).N-benzoyl-DL-arginine-2-napthylamide (BANA) is a colorless substrate that is degradated by trypsin-like enzymesproduced by B. forsythus, P. gingivalis, and T. denticola. A blue-black color on a reagent card will formif the patient's plaque sample contains one or more of these organisms. The test can be done chairside, takes 15minutes, and is relatively inexpensive. The BANA test can not detect nonviable organisms, and it detects groups of bacteria, not individual species.

25. Progress Toward a Vaccine Against Periodontal Diseases Factors that make a disease/infection amenable to vaccine development: Factors that make a disease/infection amenable to vaccine development: Factors that make a disease/infection recalcitrant to vaccine development: Factors that make a disease/infection recalcitrant to vaccine development:

26. Factors that make a disease/infection amenable to vaccine development: Limited number of pathogenic agents. Limited number of pathogenic agents. The pathogen has a single major virulence factor. The pathogen has a single major virulence factor. The pathogen appears in the blood. The pathogen appears in the blood. Pathology of the disease is not caused by immune mechanisms. Pathology of the disease is not caused by immune mechanisms.

27. Factors that make a disease/infection recalcitrant to vaccine development A variety of pathogens or serotypes causes the disease. A variety of pathogens or serotypes causes the disease. The pathogen(s) possess a variety of virulence factors. The pathogen(s) possess a variety of virulence factors. The pathogen is restricted to mucus membranes or is intracellular. The pathogen is restricted to mucus membranes or is intracellular. Immunopathology can occur. Immunopathology can occur.

28. Ebersole et al., 2001

29. Progress toward vaccination against periodontal diseases For a non-life threatening disease a vaccine must be entirely safe and cause minimal or no side-effects. For a non-life threatening disease a vaccine must be entirely safe and cause minimal or no side-effects. Caries and periodontal disease are localized to the tooth and gingival area thus a successful vaccine must induce immunity at these sites. Caries and periodontal disease are localized to the tooth and gingival area thus a successful vaccine must induce immunity at these sites. Antibodies that occur in these area are mainly IgA in saliva although some IgG is present from gingival crevicular fluid. Antibodies that occur in these area are mainly IgA in saliva although some IgG is present from gingival crevicular fluid. IgA is only a weak activator of complement and is poorly opsonic. The function of IgA seems to be limited to inhibition of attachment and neutralization of enzymes or toxins. IgA is only a weak activator of complement and is poorly opsonic. The function of IgA seems to be limited to inhibition of attachment and neutralization of enzymes or toxins. Salivary IgA can be stimulated via the GALT thus oral vaccines for caries or periodontal disease may be feasible. Salivary IgA can be stimulated via the GALT thus oral vaccines for caries or periodontal disease may be feasible.

30. Periodontal disease vaccine approaches: Active immunization : Active immunization :  Whole cells  Cellular components fimbriae or other antigens of Porphyromonas gingivalis fimbriae or other antigens of Porphyromonas gingivalis Passive immunization ( with specific antibodies ): Passive immunization ( with specific antibodies ):  Monoclonal antibodies to P. gingivalis antigens  Immune bovine milk “  Egg yolk antibody“

31. Periodontal disease vaccine approaches: Possible future approaches: Possible future approaches:  Replacement therapy with non-pathogenic mutants.  Expression of vaccine proteins in bacteria that colonize the gut such as S. lactis or Salmonella.  Gene therapy, eg. expressing the gene for the P. gingivalis fimbriae in salivary gland cells.

32. Examples of anti-P. gingivalis vaccines under development

33. Feasibility of an HA2 Domain-Based Periodontitis Vaccine n In a rat periodontitis model, preinoculation with the Porphyromonas gingivalis HA2 binding domain for hemoglobin provided protection from disease. n Protection was associated with induced anti-HA2 IgG humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was administered in saline. DeCarlo et al., 2003

34. Domain structure and homologies between the P. gingivalis proteases RGP-1 and KGP DeCarlo et al., 2003 conserved hemagglutinin domain involved in P.g. adherence

35. Feasibility of an HA2 Domain-Based Periodontitis Vaccine Higher anti-HA2 IgG levels resulted in less bone loss in the rat periodontitis model. DeCarlo et al., 2003

36. Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas gingivalis n Using bioinformatics methods, analyze the full P. gingivalis genome sequence for genes encoding likely surface- expressed proteins. n Clone these genes, express as recombinant E. coli proteins. n Screen the cloned proteins to see if they are recognized by anti-P. gingivalis antibodies. n Test the recombinant proteins for ability protect against infection with P. gingivalis in an animal model. Ross et al., 2001

37. Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas gingivalis Ross et al., 2001

38. Factors that make a disease/infection amenable to vaccine development: Limited number of pathogenic agents. Limited number of pathogenic agents. The pathogen has a single major virulence factor. The pathogen has a single major virulence factor. The pathogen appears in the blood. The pathogen appears in the blood. Pathology of the disease is not caused by immune mechanisms. Pathology of the disease is not caused by immune mechanisms.

39. Factors that make a disease/infection recalcitrant to vaccine development A variety of pathogens or serotypes causes the disease. A variety of pathogens or serotypes causes the disease. The pathogen(s) possess a variety of virulence factors. The pathogen(s) possess a variety of virulence factors. The pathogen is restricted to mucus membranes or is intracellular. The pathogen is restricted to mucus membranes or is intracellular. Immunopathology can occur. Immunopathology can occur.

41. Antibiotics in treatment of periodontal diseases n Antibiotics used for their antimicrobial effects: u Metronidazole u Amoxicillin n Antibiotics used for their anti-inflammatory effects: u Tetracyline and derivatives: low-dose doxycycline (Periostat): these compounds are used at doses below which antimicrobial effects are seen. these compounds are used at doses below which antimicrobial effects are seen. at these levels, tetracycline derivatives inhibit activation of host metalloproteinases involved in tissue remodelling (collagenases). at these levels, tetracycline derivatives inhibit activation of host metalloproteinases involved in tissue remodelling (collagenases).