Caries Immunology

Dr. Aaron Weinberg DMD, PhDDepartment of Biological Sciences

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

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

• 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 “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)

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.

sIgAMajor salivary glands produce 70% of total salivary sIgA30% comes from minor salivary glandssIgA 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 sIgAPrimary function to prevent microbial adherenceBacterial IgA-specific proteases found in S. sanguis; periodontal pathogens.

(serum)

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 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?

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• 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)

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

• 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)

Gram positive cell envelope

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.

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

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

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.– 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)

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

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

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– 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

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

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 fluidand 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

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

•

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

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 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 haveanticariogenic Abs in cow’s milk- IgG1, major secreted Ab isotype- S. mutans and caries scoresreduced in gnotobiotic mice (Michalek et al, 1987)

• What’s the problem in this expt?

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!!!

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

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

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