Caries Immunology

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Caries Immunology

• Dr. Aaron Weinberg DMD, PhD
• Department of Biological Sciences
• CASE School of Dental Medicine

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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

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Background

• Billions of spent per year in U.S. on treating
  dental caries
• Surgeon Generals 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!

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Background contd

• By mid-1960s, 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).

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Natural development of sIgA

• At birth no sIgA in saliva
• Predentate infants (16-28 wks)
• Detected against 1st 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 2nd 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)

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Caries and sIgA

• Early correlation studies
• Ørstavik, Brandtzaeg, 1975 low titers of parotid
  sIgA corresponds with increase in dental caries
• IgA deficiency
• Afflicts 11000 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.

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sIgA
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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.

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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?

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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)

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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)

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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

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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-neut
  rophil axis
• sIgA and crevicular (serum gingival)
  IgG-IgM-IgA systems are targeted.

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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

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Whole S. mutans cells wont 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 wont work! (morbid humor)

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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 bugs
  pathogenic activities.

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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

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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

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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)

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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

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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

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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.

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Gram positive cell envelope
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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

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Active anticaries immunization contd

• 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

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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
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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

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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

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Adjuvants

• increase immunogenicity of peptide antigens
• traditional ones are toxic (Freunds mineral
  oils)
• liposomes offer attractive alternative
• cholera toxin (Mike Russells 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)

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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 Payers 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

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Passive anticaries immunizationAbs 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 cows milk
• IgG1, major secreted Ab isotype
• S. mutans and caries scores
• reduced in gnotobiotic mice
• (Michalek et al, 1987)
• Whats the problem in this
• expt?

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Passive anticaries immunization contd

• 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!!!

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Passive anticaries immunization contd

• 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

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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

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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), Crohns
disease (CD4), systemic vasculitis (CDw52),
leukemia and lymphomas (CDw52, IL-2 receptor),
septic shock (TNF?), neoplasm (Lewis-Y,hEGFR2),vir
al infection (HIV, herpes simplex)
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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

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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