1. Dr. Aaron Weinberg DMD, PhD Department of Biological Sciences
Caries Immunology
Dr. Aaron Weinberg DMD, PhD
Department of Biological Sciences

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 yrs
National anticaries strategy:
To combat the microbial agent
To increase tooth resistance
To modify diet
To deliver anticaries measures to the public
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 “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 g/ml IgA)

6. Caries and sIgA Early correlation studies:
Ørstavik, 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)

13. 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

14. 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.

15. 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 * *

16. Whole S. mutans cells won’t work as the immunogen
Why?
S. mutans has antigens that could 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)

17. Gram positive cell envelope

18. Alternative means of vaccination
Purification of candidate antigens and use of a subunit vaccine
Candidate antigens selected, based on bug’s pathogenic activities.
Using recombinant DNA methods to place virulence factors from cariogenic bugs into a noncariogenic, non-cross-reactive bug.
GTF released and binds to serotype CHO antigen

22. 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

24. 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
Anti-dextran antibodies proposed as possible target to confer caries protection

25. Adhesins Surface protein antigens
SA I/II, B, P1 in S. mutans; ~ kD
SpaA in S. sobrinus; ~ kD
Are predominant proteins on surface of bugs; ~35% of all surface proteins.
Some 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)

26. 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
Mutants lacking dextranase and SpaA (S. sobrinus) are avirulent

27. 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

28. 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.

29. Gram positive cell envelope

30. 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
sIgA 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

31. 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

32. 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

33. Gingival swabs and “local pathway”
• swabbing gingiva elicits immune response
• Ex 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

34. 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 IgA against GTF
-  dehydrated liposomes may be useful in generating specific salivary immunity against target Ags in oral cavity

35. TGF release by activated T cells promotes B cell isotype switching from IgG and IgM to IgA

36. 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)

37. 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

38. 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?
Whey from immunized cows, used as a mouthrinse, appeared to decrease S. mutans in volunteers.

39. 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!!!

40. 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

41. 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

42. 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

43. 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)

44. 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
Xenogeneic, derived from another species
Allogeneic, describes tissues that are genetically different and therefore incompatible when transplanted