Caries Immunology Dr. Aaron Weinberg DMD, PhD Department of Biological Sciences CASE School of Dental Medicine
Outline of Lecture Designing an anticaries vaccine whole bacteria vs targetted virulence factors
Active and passive immunization
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 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!
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).
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 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)
Caries and sIgA Early correlation studies: Ø rstavik, Brandtzaeg, 1975: low titers of parotid sIgA corresponds with increase in dental caries 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) 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.
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 Bacterial IgA-specific proteases (serum)
found in S. sanguis; periodontal
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.
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 best target in the bad guy Identify which component of the immune system should be targeted?
Is there evidence that hyperimmunization will work?
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)
Lactic acid bacteria as prime suspects Heterogenous family of bacteria 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)
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
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.
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
Whole S. mutans cells won’t work as the immunogen 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)
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.
Glucans Sticky stuff cariogenic bugs use for adherence Tree-like homopolymers of glucose featuring gazillions of branches Rich in -1-6 linkages (dextran) glucosyltransferase-s (GTF-S) Rich in -1-3 linkages (mutan) glucosyltransferase-I (GTF-I)
Antibodies impeding GTF function are protective in animals
Glucan function 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
Adhesins 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 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)
Dextranases 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
Serotype-defining carbohydrate antigens Complex carbohydrate heteropolymers w/ galactose, glucose 8 serotypes of mutans streptococci 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
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.
Gram positive cell envelope
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 • 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
Eliciting systemic IgA causes binding to antigen and preventing complement fixation
Active anticaries immunization cont’d Parts of purified bacterial antigens 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
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
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) - 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
Liposomes • artificial membrane vesicles containing aqueous-phase solutes inside or intramembranous molecules w/i the membranes • 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
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)
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
Passive anticaries immunization Abs passively administered Oral immunization of pregnant rats Milk from immunized mothers confers protection to weanlings • Xenogeneic immunization anticariogenic Abs in cow’s milk IgG1, major secreted Ab isotype S. mutans and caries scores reduced in gnotobiotic mice
• What’s the problem in this
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) 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!!!
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 Assessing immunocompetence Identifying infectious agents Monitor concentrations of hormones and chemotherapeutic agents in plasma
- Also used as immunosuppressive agents
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
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)
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
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 Neutropenia (Mishkin et al, 1976; Pemu et al, 1996)
• RSC is not a problem in children; mostly in elderly