Oral Immunology DEND 215
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  • Whey from immunized cows, used as a mouthrinse, appeared to decrease S. mutans in volunteers.
  • Xenogeneic, derived from another species Allogeneic, describes tissues that are genetically different and therefore incompatible when transplanted

Transcript

  • 1. Caries Immunology Dr. Aaron Weinberg DMD, PhD Department of Biological Sciences CASE School of Dental Medicine
  • 2. Outline of Lecture
    • Caries Immunology
      • Background
      • Caries and sIgA
      • Mutans streptococci
        • Streptococcus mutans
      • Designing an anticaries vaccine
        • whole bacteria vs targetted virulence factors
        • Active and passive immunization
        • MAb
        • Chimeric MAb
        • CDR-grafted MAb
        • Xenomic mice
  • 3. Background
    • Billions of $$ spent per year in U.S. on treating dental caries
    • Surgeon General’s report on oral health states that caries is a major health problem in U.S.
    • Fluoridation has reduced caries by ½ in children 5-17 yo
    • National anticaries strategy:
      • To combat the microbial agent
      • To increase tooth resistance
      • To modify diet
      • To deliver anticaries measures to the public
    • To combat the microbial agent
      • Clark, 1924 ( Brit. J. Exp. Pathol ) isolated S. mutans; was first to implicate this bacterium to DCs. Was met with resistance.
      • McClure and Hewitt, 1946 ( J. Dent. Res. ) used penicillin, rats and Lactobacillus acidophilus to show bacterial association with DCs.
      • Orland et al, 1954 ( J. Dent. Res. ) used gnotobiotic rats to prove that cariogenic diet alone is not enough to induce DCs; i.e., bacteria!
  • 4. Background cont’d
    • By mid-1960’s, after various epidemiological and etiological studies, S. mutans re-emerged as prime candidate for antimicrobial attack.
    • Tomasi et al , 1965 ( J Exp Med ): IgA found to be important immunological agent in saliva.
    •  dental vaccination approaches targeting a specific pathogen ( S. mutans ) and manipulating a specific humoral immune system (sIgA).
  • 5. Natural development of sIgA
    • At birth : no sIgA in saliva
    • Predentate infants (16-28 wks):
      • Detected against “1 st wave” of strep. organisms: S. mitis , S. salivarius (Smith and Taubman, 1992)
      • These organisms initially colonize mucosal surfaces
      • No Abs to S. mutans detected
    • Dentate children :
      • Tooth eruption brings “2 nd wave” of strep. organisms: S. sanguis , S. mutans
      • Antibodies (Abs) against S. mutans observed in 1 yr old children
      • Abs against: serotype specific carbohydrate, protein I/II, glucosyltransferase, glucans, teichoic acids
      • w/i 10 yrs the child has IgA levels comparable to an adult (adult parotid saliva contains 30-160  g/ml IgA)
  • 6. Caries and sIgA
    • Early correlation studies:
      • Ø rstavik, Brandtzaeg, 1975: low titers of parotid sIgA corresponds with increase in dental caries
      • IgA deficiency
        • Afflicts ~1:1000 people and is associated with dental caries
        • Subjects suffer from chronic rhinitis and sinusitis; leads to habitual mouth breathing; use of sucrose containing medicinal syrups; poor oral hygiene during acute infection; bottle feeding to help with sleep;
        •  difficult to control these studies
        • but, in group with compensatory high anti- S. mutans IgM titers in saliva, caries activity was significantly lower (McGhee, Michalek, 1981)
      • sIgA against S. mutans
        • Parotid sIgA recognizes all major serogroups of S. mutans
        • PsIgA against surface Ag I/II blocks S. mutans adhesion to saliva coated hydroxyapatite (Hajishenagallis et al, 1992)
        •  suggests a mechanism of protection that exists and/or could be exploited
      • Serum antibodies (SAs) and caries resistance
        • Conflicting reports; overall, SAs from gingival crevice may confer modest degree of protection to tooth in cervical area and none in coronal portion.
  • 7. sIgA
    • Major salivary glands produce 70% of total salivary sIgA
    • 30% comes from minor salivary glands
    • sIgA system is what we attempt to manipulate to prevent dental caries and certain microbial infections
    • 3 X as much IgA is produced/day than IgG
    • ~2/3 of IgA produced is sIgA
    • Primary function to prevent
    • microbial adherence
    • Bacterial IgA-specific proteases
    • found in S. sanguis; periodontal
    • pathogens.
    (serum)
  • 8. Specific immunity against DCs
    • Caries correlated with sIgA titers and serum IgM to S. mutans .
    • Elevation in titer is due to exposure
    • Are these antibodies protective??
    • Association between sIgA antibodies and resistance to dental infection by S. mutans has still to be convincingly demonstrated.
  • 9. Naturally induced immunity vs artificially induced hyperimmunization
    • N.I.I. (passive) results in increased titers to a wide spectrum of Ags of an organism; may not be protective .
    • Hyperimmunization (vaccination) results in elevation of Ab to therapeutic/ preventative levels of an organism
    • Aim of vaccine to reduce # of pathogen and/or to interfere with its metabolic activity
    • Criteria for effective hyperimmunization:
      • Identify the bad guy
      • Identify the best target in the bad guy
      • Identify which component of the immune system should be targeted?
      • Is there evidence that hyperimmunization will work?
  • 10. Criteria for cariogenicity
    • An organism must exhibit tropism for teeth
    • An organism must be acidogenic
    • An organism must be aciduric
    • An organism must utilize refined sugar (sucrose) (Newbrun, 1983)
  • 11. Lactic acid bacteria as prime suspects
    • Heterogenous family of bacteria
    • Some good, some bad
    • All ferment sugars and form lactic acid as end product
    • Lactic acid is less volatile than other acids and chelates calcium, facilitating demineralization of enamel
    • All form extracellular glucose polymers (glucans) from sucrose via GTF (glucosyltransferase)
  • 12. Mutans streptococci
    • Group of strep species most closely associated with caries of smooth surfaces, pits, fissures
    • 6 serotypes of ms that are associated with man
    • S. mutans serotype C, predominant group associated with enamel surfaces; 80-87% of cases in U.S.
    • Swedish kids; smooth surface caries, 36% presence of serotype c, 54% serotype d/g
  • 13. Targeted immune systems for hyperimmunization
    • Cellular immune mechanisms not targeted
      • Cells have difficulty functioning in the mouth
      • Most bacterial infections handled by secretory immunity (sIgA) or antibody (IgG)-complement-[neutrophil axis]
    • sIgA and crevicular (serum + gingival) IgG-IgM-IgA systems are targeted.
  • 14. Evidence that an anti-caries vaccine could work
    • Studies in the ’70s showed protection in animals using hyperimmunization
    • Ex; hyperimmunized rats fed a cariogenic diet led to protection against smooth surface caries (buccal, proximal), but not pits and fissures (sulcal) (Michalek et al, 1976)
    •  Results suggest that protection is, at best, location dependent. Sulcal protection requires additional protection ; i.e., sealants
    * *
  • 15. Whole S. mutans cells won’t work as the immunogen
    • Why?
      • S. mutans has antigens that cross-react with heart muscle; cardiolipin (diphosphatidyl glycerol); phospholipid found in mytochondrial membrane
      • Although patient death is one form of caries control, this strategy won’t work! (morbid humor)
  • 16. Alternative means of vaccination
    • Purification of candidate antigens and use of a subunit vaccine
    • Using recombinant DNA methods to place virulence factors from cariogenic bugs into a noncariogenic, non-cross-reactive bug.
    • Candidate antigens selected, based on bug’s pathogenic activities.
  • 17. Glucans
    • Sticky stuff cariogenic bugs use for adherence
    • Tree-like homopolymers of glucose featuring gazillions of branches
    • 2 types:
      • Water-soluble glucans
        • Rich in  -1-6 linkages (dextran)
        • glucosyltransferase-s (GTF-S)
      • Water-insoluble glucans
        • Rich in  -1-3 linkages (mutan)
        • glucosyltransferase-I (GTF-I)
    • Antibodies impeding GTF function are protective in animals
  • 18.  
  • 19. Glucan function
    • Plaque accumulation
    • Molecular sieves
    • Retain water
    • Act as secondary attachment apparatus for bugs
    • Strengthen attachment of producing organism to tooth
    •  enables producing organism to control microenvironment
    •  -dextran antibodies proposed as possible target to confer caries protection
  • 20. Adhesins
    • Surface protein antigens
      • SA I/II, B, P1 in S. mutans; ~185-210 kD
      • SpaA in S. sobrinus; ~160-180 kD
      • Are predominant proteins on surface of bugs; ~35% of all surface proteins.
      • Immunologically related to dextranase
      • “ fuzzy coat” by EM
      • Function:
        • Adhere to tooth in absence of sucrose
      • Mutants lacking SA I/II, lack fuzzy coat, bind poorly to exptal pellicle (Harrington and Russell, 1993)
      •  -surface protein antibodies protective in monkeys
      • antibodies against saliva binding region of SA I/II prevent colonization of S. mutans on mice teeth (Huang et al, 2001)
  • 21. Dextranases
    • 160-175 kD enzymes
    • Break down polymers of glucose in  -1-6 linkages to modify glucan product of GTF
    • May permit extracellular glucans to serve as energy stores
    • May function in sucrose-independent adherence (via SPA-related epitope)
    • Mutants lacking dextranase and SpaA ( S. sobrinus ) are avirulent
  • 22. Serotype-defining carbohydrate antigens
    • Complex carbohydrate heteropolymers w/ galactose, glucose
    • 8 serotypes of mutans streptococci
    • designated a-h
    • Serotypes c, e, f ( S. mutans ); d, g, h ( S. sobrinus ) important in humans.
    • Aside from antibody specificity, these structures bind GTF to cell surface
    •  proposed as targets for a caries vaccine
    • Abs against serotype-carbohydrate Ags are protective and prevent binding of GTF to cell
  • 23. Lipoteichoic acids
    • Amphipathic molecules on surface of Gram-positive bugs
    • Analogous to LPS of Gram-negative bugs
      • Anionic, attract cationic ions for stabilization
      • May be involved in adherence
      • Strong inducers of inflammation; TLR2 vs TLR4 for LPS
    • Consist of linear polymers of polyribitol or polyglycerol ± phosphate groups; the carbohydrate backbone is covalently bound to lipid of cytoplasmic membrane
    • Not a good antigen candidate; epitopes could cross-react with host tissue antigens; heart antigens.
  • 24. Gram positive cell envelope
  • 25. Active anticaries immunization
    • The heart cross-reactivity issue using whole attenuated bugs may be a false concern
    • If vaccine is administered orally to stimulate sIgA rather than IgG  using enteric pathway
      • IgA is more beneficial by immune elimination at mucosal surfaces
      • Eliciting systemic IgA causes binding to antigen and preventing complement fixation
    • Peroral vaccination (po) - po immunization by S. mutans elevates sIgA Abs (Gregory, Filler, 1987) - humans given gelatin-capsules of killed S. mutans whole bugs, 10d - sIgA against GTF and SA I/II found in all cases - reduction in S. mutans from dental plaque Not addressed (1) if this improved the caries situation, (2) if harmful serum IgG Abs against LTA were found
  • 26. Active anticaries immunization cont’d
    • Subunit vaccination
      • Parts of purified bacterial antigens
    • Synthetic peptides
      • Chemically synthesizing a piece of a large protein
      • Ex: Targeting an active domain of GTF (structure-function studies)
        • peptide from glucan binding domain of GTF
          • Abs against this domain inhibit GTF 30%; not good.
        • peptide from an amino-terminal sequence
          • Abs against this domain are 80% inhibitory
  • 27. Molecular genetics and the enteric pathway • introducing antigen genes in harmless enteric bacteria • these bacteria proliferate in gut, exhibiting greater staying power than gelatin capsules w/antigen • currently under investigation • is this microbe totally harmless??? • some plasmid vectors used have genes that encode antibiotic resistance
  • 28. Gingival swabs and “local pathway”
    • • swabbing gingiva elicits immune response
    • • Ex. 3800 dalton low mol wt component of S. mutans , swabbed on monkey gingiva elicits IgG in crevicular fluid
    • and sIgA in saliva (Lehner et al, 1986)
    • - How is there sIgA?
    • - some antigen must be ingested
    • • Therapeutically, this method may be useful
    • - swabbing administered only 10 times/yr resulted in
    • in S. mutans and in caries
  • 29. Liposomes
    • • artificial membrane vesicles containing aqueous-phase solutes inside or intramembranous molecules w/i the membranes
    • • act as adjuvants
    • • S. mutans Ags (GTF) in dessicated liposomes fed to humans (Childers et al, 1994)
    • - salivary IgA2 against GTF
    • -  dehydrated liposomes may be useful in generating specific salivary immunity against target Ags in oral cavity
  • 30. Adjuvants
    • • increase immunogenicity of peptide antigens
    • • traditional ones are toxic (Freund’s; mineral oils)
    • • liposomes offer attractive alternative
    • • cholera toxin (Mike Russell’s group in U of Alabama)
    • - most promising adjuvant to stimulate mucosal sIgA
    • - after 1 boost, persistenly high titers of sIgA (Hajishengalis et al, 1996)
    • - dimer; toxic CTA-subunit and nontoxic CTB-subunit
    • - adjuvant activity found to reside in CTB
    • - replaced the CTA-subunit with Ag (SA I/II) from S. mutans , constructed an enteric bacterial clone in ‘avirulent’ Salmonella typhimurium expressing SA I/II-CTA2/CTB
    • - sIgA titers to SA I/II obtained (Harokopakis et al, 1997)
  • 31. Fluoride as adjuvant
    • • Ingested fluoride found to be potent adjuvant of mucosal immunity in rats (Butler et al, 1990)
    • • intragastric NaF causes size and cellularity Payer’s patches, mesenteric lymph nodes, number plasma cells secreting IgG, IgA to Ags concurrently administered in water
    • • elevated CD4+ T cells in the lymphoid tissues
    • • Not known how fluoride amplifies mucosal immunity to ingested bacteria
    • • argues in favor of fluride administration as part of caries vaccine program
  • 32. Passive anticaries immunization Abs passively administered
    • • Maternal immunization
      • Oral immunization of pregnant rats
      • Milk from immunized mothers confers protection to weanlings
    • • Xenogeneic immunization
      • Cows immunized against
      • cariogenic bacteria have
      • anticariogenic Abs in cow’s milk
      • IgG1, major secreted Ab isotype
      • S. mutans and caries scores
      • reduced in gnotobiotic mice
      • (Michalek et al, 1987)
    • • What’s the problem in this
    • expt?
  • 33. Passive anticaries immunization cont’d
    • • Bovine whey IgG1, as mouthrinse, interferes with glucan formation and S. mutans adherence (Loimaranta et al, 1997)
    • • Bovine whey from cows immunized w/ S. mutans fusion protein [SAI/II fused w/glucan-binding domain of GTF-I] prevented recolonization of S. mutans in 8 volunteers (Shimazaka, et al 2001)
    • • Chicken eggs
      • New frontier for passive anticaries immunization
      • Michelik (U. Alabama) looking at potential therapeutic capacity of egg in mouthrinse
      • Rocky Balboa has volunteered and is eating dozens of raw chicken eggs!!!
  • 34. Passive anticaries immunization cont’d
    • • Monoclonal antibodies (MAb)
      • Single specificity; produced by cells from a single B-cell clone
      • Mostly derived from mice (i.e., xenogeneic)
      • Fusion of mouse plasma cell and myeloma cell results in “hybridoma;” Ab capacity of plasma cell and proliferative property of myeloma cell
      • In tissue culture, hybridomas generate unlimited amount of MAb
      • Diagnostic tools for:
        • Assessing immunocompetence
        • Identifying infectious agents
        • Monitor concentrations of hormones and chemotherapeutic agents in plasma
    • - Also used as immunosuppressive agents
  • 35. Chimeric MAb
    • Limiting therapeutic factor, xenogeneic, leading to rejection
    • Genetic engineering: fusing Fab with human Fc
    • C region confers function to Ab, giving chimeric MAb functional attributes
    • Ex. cMAb having an IgG1 isotype C region is effective in C’ activation and Ab dependent cell-mediated cytotoxicity
    • cMAb of IgA subclass exhibits anti-inflammatory effects
  • 36. CDR-grafted MAb • Complementarity-defining region (CDR) • Areas of Ab that bind to Ag • Variable region of Ig contains 3-4 hypervariable regions and intervening framework regions; these are the CDR • CDR-MAb contains rodent hypervariable sequences, human framework sequences and human constant regions • Used in organ transplant immune suppression (CD3, CD4, IL-2 recpetor) ; rheumatoid arthritis (CD4, CDw52) , Crohn’s disease (CD4) , systemic vasculitis (CDw52) , leukemia and lymphomas (CDw52, IL-2 receptor) , septic shock (TNF  ) , neoplasm (Lewis-Y,hEGFR2) ,viral infection (HIV, herpes simplex)
  • 37. Xenomic mice
    • • Allogeneic Ab therapy developed against a xenogeneic background
    • • xenomic mice, genetically engineered to make human immunoglobulins
    • • Advantage, theoretically, one strain of mice can make polyclonal human Abs against a host of antigenic challenges, circumventing need to form new hybridomas against new antigens; providing polyclonal specificity; can have functional advantage over MAb
  • 38. Root surface caries
    • • Actinomyces viscosus, A. naeuslundii, a. odontolyticus, A. eriksonii, Rothia dentocariosa
    • • Given the gingival localization of these lesions, complement-IgG-neutrophil axis is more important
    • • Suggestive evidence
      • Neutropenia (Mishkin et al, 1976; Pemu et al, 1996)
    • • RSC is not a problem in children; mostly in elderly