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DENTAL CARIES VACCINES:

PROSPECTS AND CONCERNS


D.J. Smith
Department of Immunology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115; dsmith@forsyth.org

ABSTRACT: Dental caries remains one of the most common infectious diseases of mankind. Cariogenic micro-organisms enter
the dental biofilm early in life and can subsequently emerge, under favorable environmental conditions, to cause disease. In oral
fluids, adaptive host defenses aroused by these infections are expressed in the saliva and gingival crevicular fluid. This review
will focus on methods by which mucosal host defenses can be induced by immunization to interfere with dental caries caused
by mutans streptococci. The natural history of mutans streptococcal colonization is described in the context of the ontogeny of
mucosal immunity to these and other indigenous oral streptococci. Molecular targets for dental caries vaccines are explored for
their effectiveness in intact protein and subunit (synthetic peptide, recombinant and conjugate) vaccines in pre-clinical studies.
Recent progress in the development of mucosal adjuvants and viable and non-viable delivery systems for dental caries vaccines
is described. Finally, the results of clinical trials are reviewed, followed by a discussion of the prospects and concerns of human
application of the principles presented.

Key words. Mutans streptococci, secretory IgA, antigen I/II, glucosyltransferase, glucan-binding protein.

(I) Dental Caries, an Infectious Disease (Fig. 1). Under normal circumstances of diet and challenge,

D ental caries remains one of the most widespread diseases children become permanently colonized with mutans strepto-
of mankind. Advances in prophylactic measures to deal cocci between the middle of the second year and the end of the
with this disease have significantly reduced the overall caries third year of life, during a so-called “window of infectivity”
rate in the United States. However, the Surgeon General’s 2000 (Caufield et al., 1993). Techniques involving bacteriocins, plas-
report on Oral Health in America stated that a majority of five- mids, and DNA fingerprinting have been used to identify the
to nine-year-old US children have at least one lesion on the source of infection (Berkowitz and Jones, 1985; Caufield et al.,
crowns of their teeth. This percentage increases to 84.7% in 1993; Li and Caufield, 1995). These studies have shown that
adults who are at least 18 years of age. Nearly 50% of our elder the primary source of infection is maternal, although there is
population (> 75 years old) have root-surface caries. Being recent evidence to suggest that non-familial transfer can occur
poor is clearly a risk factor for increased decay. More than one- when environmental conditions favor colonization (Mattos-
third of poor two to nine-year-old children have untreated Graner et al., 2001). Infection is related to maternal dose
decayed primary teeth. Poor children from Mexican-American (Kohler et al., 1984; Caufield et al., 1993), in that the higher the
or non-Hispanic black backgrounds are particularly at risk, level of maternal mutans streptococcal infection, the higher
given the fact that over two-thirds of these populations have the percentage of children who become infected.
untreated decayed teeth. Other factors also influence mutans streptococcal colo-
In developing countries, dental caries is often at epidemic nization. If the environment strongly favors mutans coloniza-
proportions, especially among the poor. For example, at least tion—for example, if high maternal infection levels are com-
25% of three-year-old children from various areas of Brazil have bined with high dietary sucrose levels—this so-called “win-
detectable caries lesions, many developing lesions within the dow of infection” shifts to an earlier age. More sensitive tech-
first 18 months of life (Mattos-Graner et al., 1996). This high niques for microbial detection, e.g., DNA probe technology,
caries rate continues among the less economically advantaged in have also suggested that mutans streptococci can be found in
the face of efforts to introduce fluoride at an early age. Similarly, the oral cavity during the first year of life, especially in caries-
an oral health survey in China revealed that three-quarters of prone populations (Milgrom et al., 2000). However, despite the
five-year-old children studied had evidence of significant dental influence of maternal dose, children who do not become
decay (Wong et al., 2001). Thus, more effective public health mea- infected by approximately three years of age appear to remain
sures are needed to address this worldwide problem. uninfected, or minimally colonized for several years (Caufield
Landmark experiments in the 1960s (reviewed in Gibbons et al., 1993; Smith et al., 1998a), possibly until new opportuni-
and van Houte, 1975; Loesche, 1986) established that mutans ties for colonization occur upon eruption of the secondary
streptococci are the primary etiologic agents of this disease dentition. This suggests that a longer-term benefit could ensue
and that infection is transmissible. A strong association exists if mutans streptococcal colonization could be impeded in early
between level of colonization with mutans streptococci and childhood by measures such as immunization.
dental caries, although other organisms, such as lactobacilli,
have also been implicated in this disease. (II) Ontogeny of Mucosal Immunity
Studies of the natural history of mutans streptococcal col- Mucosal applications of dental caries vaccines have been
onization of infants have revealed several interesting features sought, since secretory IgA is the principal immune compo-

13(4):335-349 (2002) Crit Rev Oral Biol Med 335


Figure 1. Mutans streptococcal (MS) colonization of humans in the Figure 2. Appearance of salivary IgA antibody to indigenous oral
first three years of life. The percentage of children colonized with flora and antigens of immunization. The appearance of salivary
mutans streptococci is indicated on the ordinate. Percentages reflect IgA antibody (Ab) to micro-organisms associated with indigenous
children under modest maternal challenge (approximately 50% colo- infection or immunization is indicated by arrows. The percentages
nized) or children exposed to high maternal levels (approximately of children infected with indigenous oral streptococci (S. mitis, S.
90% colonized). If high maternal MS levels (dose) are combined with salivarius, S. sanguis, S. mutans) are indicated by the bars. Data
significant exposure to dietary sucrose, initial dental colonization with are summarized from Smith and Taubman, 1992, 1993.
MS occurs at a younger age.

nent of major and minor gland salivary secretions and thus Interestingly, in some children, antibody to mutans streptococ-
would be considered to be the primary effector of adaptive cal antigens can also be detected independently of the ability
immunity in the salivary milieu. Given the natural history of to detect ongoing infection in the second year of life. As is the
mutans streptococcal infection described above, immunization case with many bacterial challenges throughout the body, the
would presumably need to be initiated early in childhood to threshold of immunological response is lower than that of per-
interfere with mutans streptococcal colonization. This then sistent infection; therefore it is not surprising to observe anti-
would require that the mucosal immune system be sufficient- body to S. mutans antigens in the absence of its colonization.
ly mature at this time to respond effectively. Longitudinal studies suggest that antibody reactive with
The oral immune environment undergoes rapid, early mutans streptococci results from contact with mutans strepto-
development (reviewed in Smith and Taubman, 1992, 1993). cocci, rather than from earlier colonizing oral streptococci,
Although secretory IgA antibody in saliva and other secretions is since well-developed salivary IgA antibody to pioneer oral
essentially absent at birth, mature SIgA, i.e., dimeric IgA with a streptococci can be demonstrated prior to the detection of anti-
bound secretory component, is the principal salivary body reactive with mutans streptococci. Thus colonization, at
immunoglobulin secreted in individuals by one month of age. least not extensive colonization, with mutans streptococci is
Consequent to the environmental antigenic challenge, mucosal apparently not required for the development of salivary anti-
IgA antibody to pioneer gut (e.g., Escherichia coli) and oral (e.g., body to associated mutans streptococcal antigens.
Streptococcus mitis and S. salivarius) microbiota appears within Salivary immune responses to mutans streptococci show
weeks of initial exposure (Fig. 2). In the first months of life, an significant individual characteristics in early childhood.
infant’s saliva may contain considerable concentrations of IgM Children respond at different rates following infection, a con-
and may, occasionally, be dominated by the IgA1 isotype. dition which may be partly the result of the extent of infection
However, by six to nine months of age, most children exhibit a (antigen dose) or age at the time of infection (maturation of
more adult-like distribution of salivary IgA1 and IgA2 subclass- immune response). Even siblings may differ in the amounts or
es. Salivary antibody to oral commensal microbiota can be kinds of IgA antibody specificities appearing in their saliva.
detected in both subclasses at this time. Taken together, this evi- These variations may stem from differences either in the inher-
dence suggests that significant maturation of the mucosal ent ability of the child to respond or in the characteristics of
immune response has occurred by the end of the first year of life. genetically different strains of mutans streptococci (thus
Natural exposure to mutans streptococci also results in a potentially differing antigenic challenge) ultimately colonizing
mucosal immune response to these organisms (Smith et al., the child. The rate, specificity, and/or extent of the mucosal
1998a). This response often begins to be observed in the sec- immune response to previous encounters with the organism
ond and third years of life. Western blot and ELISA analyses may also contribute to the success or failure of permanent col-
reveal that the major responses appear to be directed primari- onization.
ly to streptococcal components which are considered to be Thus, the evidence from salivary IgA responses to com-
important in colonization and accumulation, such as antigen mensal oral microbiota indicates that the mucosal immune
I/II, glucosyltransferase, and glucan-binding protein(s). system is relatively well-developed by the period during

336 Crit Rev Oral Biol Med 13(4):335-349 (2002)


which children typically become infect-
ed with mutans streptococci. Most chil-
dren apparently respond immunologi-
cally to transient infection or ongoing
colonization with mutans streptococci
in early childhood. Although the distri-
bution and specificity of children’s
responses are not identical, antibody to
a few major antigens predominates.
Analysis of these data suggests the
possibility that such responses could
be protective if induced prior to critical
colonization events.

(III) Molecular Pathogenesis


of the Disease
The molecular pathogenesis of mutans
streptococci appears to involve several
phases (Staat et al., 1980), each of which
may offer targets for immunological
intervention (Fig. 3). Acidogenic strep-
tococci require the hard surfaces fur-
nished by teeth for sustained coloniza-
tion and accumulation. Initial attach-
ment to the tooth is achieved via the
interaction of bacterial proteins with
lectins in the dental pellicle covering
the tooth surface. This trait is charac-
teristic of a family of streptococcal
adhesins, referred to as antigen I/II or
PAc in Streptococcus mutans, which
have been demonstrated to bind to
salivary components in experimental
tooth pellicles (Hajishengallis et al.,
1992). Lamont and co-workers (1991)
have evidence to suggest that the S.
mutans antigen I/II adherence to sali-
va-coated Streptococcus sanguis and
Actinomyces viscosus is mediated
through an acidic, mucin-like glyco-
protein (agglutinin) found in parotid
and submandibular saliva (Demuth et
al., 1990). Other binding properties
have also been attributed to antigen
I/II (reviewed in Hajishengallis and
Michalek, 1999). At least 2 binding
regions of antigen I/II may be involved
in salivary-component-mediated adhe-
sive activities (Crowley et al., 1993;
Nakai et al., 1993; Kelly et al., 1995).
The ultimate pathogenicity of
mutans streptococci occurs through
erosion of the hydroxyapatite-like min- Figure 3. Models of mutans streptococcal (MS) colonization and accumulation in dental biofilms.
eral in dental enamel by lactic acid, a
bacterial metabolic end-product. However, significantly size insoluble forms of glucan (S. mutans GTF-B and GTF-C)
destructive concentrations of this acid require the substantial have been most closely associated with pathogenicity. These
accumulation of these acidogenic streptococci in dental glucose polymers provide scaffolding for the aggregation of
plaque. This accumulation process is initiated by the activity mutans and other oral streptococci through interaction with
of extracellular glucosyltransferases (GTF), several of which bacterial cell-associated glucan-binding proteins. Several glu-
are constitutively secreted by mutans streptococci. In the pres- can-binding proteins have been described in S. mutans and S.
ence of sucrose, GTFs synthesize several forms of high-molec- sobrinus. Although each of these GBPs has the ability to bind to
ular-weight branched extracellular glucans. GTFs that synthe- certain forms of glucan, and some have been shown to be cell-

13(4):335-349 (2002) Crit Rev Oral Biol Med 337


associated, their unique contributions to in vivo plaque devel- proline-rich central portion contains an adhesion epitope, ba-
opment are as yet unclear. GTFs also contain glucan-binding sing their conclusions on adhesion inhibition assays involving
domains which permit binding to glucans. The interactions of recombinant fragments of Ag I/II.
glucans with cell-associated glucan-binding domains of GTFs Immunological approaches support the adhesin-related
and GBPs combine to cause extensive accumulation of mutans function of the AgI/II family of proteins and their repeating
streptococci in the dental biofilm. Since GTFs and GBPs are regions. For example, abundant in vitro and in vivo evidence
also secreted into the extracellular environment, their specific indicates that antibody with specificity for S. mutans AgI/II or
or non-specific incorporation into the salivary pellicle would S. sobrinus SpaA can interfere with bacterial adherence and
also provide binding sites for mutans streptococci. subsequent dental caries. Antibody directed to the intact anti-
Theoretically, the next phase of pathogenesis results from gen I/II molecule or to its salivary-binding domain blocked
the metabolic activities of these masses of accumulated mutans adherence of S. mutans to saliva-coated hydroxyapatite
streptococci (and possibly of other accumulation-associated (Hajishengallis et al., 1992). Furthermore, numerous immu-
micro-organisms). Mutans streptococci are the most prolific nization approaches have shown that active immunization
producers of lactic acid in these accumulations (Gibbons and with intact antigen I/II (Lehner et al., 1981; Katz et al., 1993) or
van Houte, 1975), although other “low pH bacteria” may also passive immunization with monoclonal (Ma et al., 1990) or
contribute (van Ruyven et al., 2000). Dental caries then ensues, transgenic antibody (Ma et al., 1998) to putative salivary-bind-
because the resulting increase in lactic acid synthesis cannot be ing domain epitopes within this component can protect
sufficiently buffered to prevent enamel dissolution. rodents, primates, or humans from dental caries caused by S.
mutans. Immunization of mice with synthetic peptides
(IV) Effective Molecular Targets (residues 301-319) from the alanine-rich region of antigen I/II
for Dental Caries Vaccines suppressed tooth colonization with S. mutans (Takahashi et al.,
Several stages in the molecular pathogenesis of dental caries 1991). Immunization with S. sobrinus SpaA constructs protect-
are susceptible to immune intervention. Micro-organisms can ed rats from caries caused by S. sobrinus infection (Redman et
be cleared from the oral cavity by antibody-mediated aggrega- al., 1995). Protection in these experiments could conceivably
tion while still in the salivary phase, prior to colonization. occur by antibody blockade of initial colonization events or
Antibody could also block the receptors necessary for colo- antibody-mediated agglutination and clearing of adhesin-
nization (e.g., adhesins) or accumulation (e.g., glucan-binding bearing bacteria from the saliva.
domains of GBPs and GTF), or inactivate GTF enzymes
responsible for glucan formation. Modification of metabolical- (B) GLUCOSYLTRANSFERASES (GTFS)
ly important functions may also be targeted. In addition, the S. mutans and S. sobrinus each synthesize several glucosyltrans-
antimicrobial activity of salivary IgA antibody may be ferases. The deduced sequences of these enzymes vary from
enhanced or redirected by synergism with innate components 1400 to nearly 1600 amino acid residues and contain consider-
of immunity, such as mucin or lactoferrin. This review will able sequence homology, despite differences in the water-solu-
concentrate on adhesins, GTFs, and GBPs as vaccine targets, bility and linkages among the glucans synthesized. Genes
since most of the recent experimental effort has been directed responsible for glucan synthesis in S. mutans are gtfB (Shiroza
toward these components. et al., 1987), which synthesizes an a-1,3-linked insoluble glucan,
gtfC (Pucci et al., 1987), which synthesizes glucan with both a-
(A) ADHESINS 1,3 and a-1,6 linkages, and gtfD (Honda et al., 1990), which syn-
Adhesins from the two principal human pathogens, thesizes a soluble a-1,6-linked glucan. Similarly, the products
Streptococcus mutans (variously identified as antigen I/II, PAc, of gtfI (Russell et al., 1987) and gtfS (Gilmore et al., 1990) genes
or P1) and Streptococcus sobrinus (SpaA or PAg), have been puri- of S. sobrinus synthesize insoluble and soluble glucan products,
fied. Russell and Lehner initially described the S. mutans com- respectively. Mutational inactivation techniques have shown
ponent in 1978. Antigen I/II (AgI/II) was found both in the cul- that each of these gene products is important to the cariogenic-
ture supernatant as well as on the S. mutans cell surface. This ity of the respective mutans streptococcal strain. For example,
185-kDa protein is composed of a single polypeptide chain of insertional inactivation of S. mutans gtf genes to replace func-
approximately 1600 residues (Lee et al., 1988). Significant tional wild-type copies of the gene markedly reduced caries
sequence homology (66%) exists between S. mutans AgI/II and when gtfB and gtfC genes that coded for GTFs synthesizing
S. sobrinus SpaA (Tokuda et al., 1990; LaPolla et al., 1991) as well water-insoluble glucan were inactivated. Similar inactivation of
as with at least one adhesin from Streptococcus gordonii, a non- the gtfD gene also resulted in a mutant with lower cariogenici-
cariogenic oral streptococcus (Demuth et al., 1990). However, ty on smooth surfaces (Yamashita et al., 1993). In addition, the
despite the homology between the two mutans streptococcal ability of GTF from initially colonizing S. mutans to synthesize
adhesins, each appears to bind to separate components in the water-insoluble glucan has been correlated with caries inci-
pellicle (Gibbons et al., 1986; Lamont et al., 1991). dence in young children (Mattos-Graner et al., 2000).
S. mutans Ag I/II contains an alanine-rich tandem repeat- The activity of GTF is mediated through both catalytic and
ing region in the N-terminal third, and a proline-rich repeat glucan-binding functions. The catalytic activity of GTF appears to
region in the center of the molecule. These regions have been be associated with several, sequentially separate, residues in the
associated with the adhesin activity of Ag I/II. Crowley and N-terminal third of the molecule. These residues have been iden-
colleagues (1993) and Nakai and co-workers (1993) have each tified by a variety of methods, including the labeling of catalytic
described a region within or near the alanine-rich region that intermediates and site-directed mutagenesis (Mooser et al., 1991;
can bind salivary components in experimental tooth pellicles. Funane et al., 1993; Devulapalle et al., 1997; Tsumori et al., 1997;
Lehner, Kelly, and co-workers (1994; 1995) suggested that the Monchois et al., 2000). Insight into catalytically important residue

338 Crit Rev Oral Biol Med 13(4):335-349 (2002)


identification has been obtained by sequence alignment tech-
niques (MacGregor et al., 1996; Devulapalle et al., 1997), which
have revealed significant homology between GTFs and alpha
amylase with respect to several invariant residues important to
the catalytic activity of the alpha amylase family, suggesting that
the amylase (b,a)8 barrel element may also be a feature of the GTF
catalytic domain. Collectively, these studies have identified sev-
eral residues within this putative (b,a)8 barrel element which may
be involved in the catalytic activity of GTF. These residues are
underlined in the peptide sequences shown in Fig. 4.
The C-terminal region of the GTF molecule contains a pat-
tern of repeating sequences which have been identified in all
GTFs from mutans streptococci. Evidence that this region has
glucan-binding function is obtained from observations that
large C-terminal, tryptic fragments of GTF retain the ability to
bind alpha 1,6 glucan (Mooser and Wong, 1988; Wong et al.,
1990; Abo et al., 1991), and that recombinant products contain-
ing some or all of the C-terminal third of GTF-I of S. downei or
S. sobrinus also can bind alpha 1,6 glucan (Abo et al., 1991;
Kaseda et al., 2000). The glucan-binding potential of these
repeating GTF sequences is also supported by the observations
that amino acid deletions in this region remove glucan-binding
activity or decrease the efficiency of insoluble glucan synthesis
(Konishi et al., 1999). Also, a glucan-binding protein (GbpA) of
S. mutans contains reiterated sequences that are very similar to
those found in this region of GTF (Banas et al., 1990).
Antibody directed to native GTF or sequences associated
with its catalytic or glucan-binding function interfere with the
synthetic activity of the enzyme and with in vitro plaque for-
mation (Taubman and Smith, 1972; Smith et al., 1978). Since
mutans streptococcal GTFs bear significant sequence homolo-
gy, active immunization with either S. mutans or S. sobrinus
GTFs induced protective immune responses in experimental Figure 4. Association of synthetic peptides with putative regions of glu-
dental caries rodent models after infection with several cosyltransferase function. Lines indicate the approximate location in the
mutans streptococcal species (reviewed in Smith and GTF sequence of synthetic peptides used in multiple antigenic peptide
constructs for immunization and dental caries protection experiments
Taubman, 1997). However, a much lower level of mutans (Smith et al., 1993a, 1994b, 1997b, 1999; Taubman et al., 1995,
streptococcal GTF antibody reactivity was observed with 2000, 2001). Amino acid residues putatively associated with catalytic
GTFs of other oral streptococci (Smith et al., 1981). functions are underlined and in bold. Di-epitopic peptide constructs
Interestingly, at least in the case of S. sobrinus GTF, induction containing two copies of each peptide are indicated by the respective
of anti-GTF antibody was sufficient to have a significant effect line links. The boundaries of the putative (b,a)8 barrel element are indi-
on initial oral colonization with Streptococcus sanguis or S. oralis cated by a bracket. The SAWNSDSEKPFDDHL sequence has been
(Smith et al., 1983). Induction of SIgA antibody in humans by shown to inhibit GTF activity (Dertzbaugh and Macrina, 1990).
oral or topical GTF administration is accompanied by interfer-
ence with accumulation of indigenous mutans streptococci
after dental prophylaxis (Smith and Taubman, 1987, 1990). 1979), GbpB (Smith et al., 1994a), and GbpC (Sato et al., 1997).
Passive administration of antibody to GTF in the diet also can GbpA has a deduced sequence of 563 amino acids (Banas
protect rats from experimental dental caries (Hamada et al., et al., 1990). The molecular weight for the processed protein is
1991). Thus, the presence of antibody to glucosyltransferase in 59.0 kDa. The carboxy-terminal two-thirds of the GbpA
the oral cavity prior to infection can significantly influence the sequence has significant homology with the putative glucan-
disease outcome, presumably by interference with one or more binding regions of mutans streptococcal GTFs. This C-terminal
of the functional activities of the enzyme. region contains 16 repeating units which, together, have been
shown to represent the full glucan-binding domain of this pro-
(C) GLUCAN-BINDING PROTEINS tein (Haas and Banas, 2000). GbpA has a greater affinity for
The ability of mutans streptococci to bind to glucans is pre- water-soluble than for water-insoluble glucan.
sumed to be mediated, at least in part, by cell-wall-associated The expressed GbpB protein is 431 residues long and has
glucan-binding proteins (Gbp). Many proteins with glucan- a calculated molecular weight of 41.3 kDa (Mattos-Graner et
binding properties have been identified in Streptococcus mutans al., 2001). Its sequence is unrelated to those reported for other
and Streptococcus sobrinus (summarized in Smith et al., 1998b). S. mutans GTFs or Gbp’s, paralleling the lack of reaction of
Each glucan-binding protein has the ability to bind a 1-6 glu- anti-GbpB antibody with these proteins. The N-terminal third
can, although other glucan linkages potentially may impart contains several immunodominant regions, which may
higher binding constants. S. mutans secretes at least three dis- explain the significant apparent immunogenicity of this pro-
tinct proteins with glucan-binding activity: GbpA (Russell, tein in humans (Smith et al., 1998a) and animals (Smith and

13(4):335-349 (2002) Crit Rev Oral Biol Med 339


Taubman, 1996). Although the function of this protein in the reported (Wachsmann et al., 1989; Gangloff et al., 1992). Given
native environment is as yet unresolved, biofilm formation on this knowledge, subunit vaccines can be designed to include
plastic surfaces by strains of S. mutans is directly correlated the salivary-binding domain(s), but exclude sequence bearing
with expression of GbpB (Mattos-Graner et al., 2001), suggest- the potential for induction of unwanted antibody responses.
ing a role for GbpB in this process. Subunit vaccines with inherent adjuvant potential could
The third S. mutans non-enzymatic glucan-binding pro- also be constructed by including some or all of the sequence of
tein, GpbC, is composed of 583 amino acids. This protein has effective immunoadjuvants. Furthermore, this approach could
a calculated molecular weight of 63.5 kDa. Although GbpC is be adapted to incorporate such effective vaccine constructs into
unrelated in sequence to GbpA or GTF, it bears some sequence attenuated expression vectors that have the ability to deliver the
similarity to the AgI/II adhesin family. The GbpC protein, antigen to inductive sites for antibody synthesis. Of the designs
detected when S. mutans cultures are stressed during growth, which include mono- and di-epitopic synthetic peptide con-
is associated with dextran-dependent aggregation. structs, single recombinant peptides, or peptide chimeras with
Of the three S. mutans glucan-binding proteins, only GbpB immunoadjuvant sequence, used in a variety of applications or
has been shown to induce a protective immune response to within Salmonella expression systems, each has been reported to
experimental dental caries (Smith and Taubman, 1996, 1997a). induce caries-protective immunity in experimental systems.
Protection can be achieved by either subcutaneous injection of
GbpB in the salivary gland region (Smith and Taubman, 1996) or (A) SYNTHETIC PEPTIDE VACCINES
by mucosal application by the intranasal route (Smith et al., As indicated above, at least two regions of the AgI/II-protein
1997a). Saliva samples from young children often contain IgA family appear to be associated with salivary-binding func-
antibody to GbpB, indicating that initial infection with S. mutans tions. Monoclonal antibody, raised by immunization with
can lead to natural induction of immunity to this protein (Smith intact Ag I/II, that reacted with the fragment containing the
et al., 1998a). Preliminary studies have shown that GbpA appears proline-rich region also inhibited the formation of experimen-
to be less immunogenic than GbpB and that its ability to induce tal dental caries (Lehner et al., 1992). Similarly, workers in
protective immunity is problematic (Smith et al., 1997a). S. sobri- France (Gangloff et al., 1992; Lett et al., 1994) demonstrated that
nus Gbps have not been evaluated for their protective potential. a 14-mer peptide derived from a proline-rich region of the SR
antigen, a member of the S. mutans serotype f Ag I/II family of
(V) Subunit Vaccines proteins, was reactive with antibody to the native protein.
Subunit vaccines, which contain structural elements of the Ag Synthetic peptide approaches have also shown the alanine-
I/II adhesin family, GTFs or GbpB, have been designed for a rich repeat region of Ag I/II to be immunogenic and to induce
variety of reasons. It had been observed that immune respon- protective immunity. For example, subcutaneous immunization
ses in animals protected by immunization with intact proteins with a synthetic peptide derived from the alanine-rich region of
were associated, at least in part, with in vitro measures of func- Ag I/II from S. mutans (residues 301-319: PAcA) induced higher
tional inhibition. Thus, more recent studies have attempted to levels of serum IgG antibody reactive with recombinant Ag I/II
optimize immune responses to functional epitopes associated than a synthetic peptide derived from the proline-rich region
with salivary binding, catalytic processes, or glucan-binding (residues 601-629) (Takahashi et al., 1991). Intranasal immuniza-
activities by designing subunit vaccines whose constructs con- tion with PAcA, coupled to cholera toxin B subunit, suppressed
tain single or multiple copies of epitopes from these domains. colonization of mouse teeth by S. mutans (Takahashi et al., 1991).
In addition, potentially enhanced protection could be achieved Fusion proteins containing PAcA also inhibited sucrose-inde-
by including, in the subunit vaccine construct, regions of the pendent adhesion of S. mutans to saliva-coated hydroxyapatide
virulence component containing strong immunological bind- beads (Yu et al., 1997). Thus, this S. mutans adhesin contains mul-
ing properties for induction of the desired arm of the immune tiple functionally based epitopes that are sufficiently immuno-
response. Furthermore, multivalent subunit vaccines could be genic to be considered for dental caries vaccines.
constructed of multiple epitopes which target different func- B- and T-cell epitopes (summarized in Smith and
tions on the same component (e.g., GTF catalytic and glucan- Taubman, 1997) have been found in a cell-associated 3.8-kDa
binding activities) or functions on different components (e.g., protein component antigen (Lehner et al., 1994). Lehner and his
AgI/II salivary binding and GTF catalytic activity). Such colleagues (Lehner et al., 1989a,b, 1994) applied free synthetic
approaches would address the variability in mucosal response peptides containing immunodominant sequences of the 3.8-
characteristics in the target vaccine population. Conjugation of kDa antigen of S. mutans to the gingival mucosa of macaques,
functionally associated peptides to carbohydrate components resulting in the formation of both salivary IgA and gingival IgG
(Wachsmann et al., 1985; Dougan et al., 1987; Lett et al., 1994), antibody. Anti-peptide antibody elicited by this topical appli-
for example glucan, or to other vaccine proteins (e.g., tetanus cation method prevented colonization of the teeth by S. mutans.
toxoid) also would increase the immunogenicity of the peptide The identification of functionally relevant resi-
and broaden the reach of the vaccine. dues/domains in glucosyltransferases has led to the design of
Designing vaccines in this way also permits one to elimi- several synthetic peptide vaccines. Monoclonal or polyclonal
nate regions which may induce unwanted antibody specifici- antibody preparations which were directed to one of several
ties. The Ag I/II family of proteins shares extensive sequence N-terminal GTF peptides (Fig. 4), each of which contained dif-
homology with surface proteins of non-cariogenic S. gordonii ferent catalytically implicated residues, have been shown to
(Demuth et al., 1990), S. intermedius, and S. oralis (Ma et al., inhibit GTF activity (Dertzbaugh and Macrina, 1990; Chia et
1991). These homologous sequences may induce cross-reactive al., 1993, 1997; Smith et al., 1994b, 1997b, 1999; Laloi et al., 1996).
responses that could influence colonization, attachment, or Several of these synthetic peptides which contained strong B-
accumulation of commensal microbiota. Also, cross-reacting cell epitopes were synthesized on lysine backbones to contain
epitopes on serotype f SR protein and human IgG have been four or eight copies of the respective sequence. These con-

340 Crit Rev Oral Biol Med 13(4):335-349 (2002)


structs induced protective immunity against experimental Intranasal administration of this chimeric protein with CT
dental caries (Taubman et al., 1995; Smith et al., 1997b). resulted in significant reductions in dental caries caused by
Synthetic peptide constructs have also been based on infection of Fischer rats with S. mutans UA130 (Hajishengallis
sequence derived from the repeating sequences in the C-termi- et al., 1998). The SBR-CTA2/B, expressed in an attenuated S.
nal third of GTF, which has been shown to be associated with typhimurium BRD509 vaccine strain containing an nirB promo-
primer-dependent glucan binding (Abo et al., 1991; Lis et al., ter (Huang et al., 2000), which was administered intranasally or
1995; Konishi et al., 1999). A synthetic peptide associated with a intragastrically to antibiotic-pre-treated BALB/c mice, resulted
putative glucan-binding site (Fig. 4) was shown to contain both in salivary antibody to the SBR and a significant reduction in
B- and T-cell epitopes, to induce antibody which could inhibit the number of S. mutans PC3379 recovered from dental plaque
the enzymatic activity of GTF (Smith et al., 1994b), and to induce after challenge (Huang et al., 2001). Jespersgaard and co-work-
protective immune responses in the rat caries model (Taubman ers (1999) intranasally immunized BALB/c mice with an E. coli-
et al., 1995). Furthermore, di-epitopic constructs of this peptide expressed recombinant GTF peptide based on a 290-residue
and a peptide from the catalytic domain could be shown to glucan-binding domain sequence, or with a chimeric protein
enhance the protective effect (Taubman et al., 2001), presumably combining this sequence with thioredoxin. Immunization with
because antibody was raised to two functional targets and either peptide resulted in protective effects on experimental S.
because the glucan-binding domain peptide provided addition- mutans infection and on resulting dental caries.
al T-cell help for the B-cell epitopes on both peptides. Other recombinant strategies involving either adhesin or
All of the GTF synthetic peptide sequences which showed GTF constructs, with or without mucosal adjuvant sequences,
protective effects in the above experiments are highly con- have been shown to induce immune responses to these func-
served among S. mutans and, often, among S. sobrinus GTFs as tional domains which could be ultimately protective in caries
well. Antibody directed to these epitopes could therefore be vaccine applications. Chimeric proteins, in which short
expected to reduce the activity of many of the GTF isozymes sequences from predicted catalytically active regions of GTF
expressed by these mutans streptococci, thus extending the were combined with cholera toxin (Dertzbaugh et al., 1990) or the
protective effect across species lines. In this regard, protection B subunit of CT (Laloi et al., 1996) and expressed in E. coli HB101,
from dental caries caused by either S. mutans or S. sobrinus gave rise to immune responses which could inhibit as much as
infection in the rat model has been demonstrated after immu- 50% of GTFB activity. Yu and co-workers (1997) designed a
nization with synthetic peptides from either the catalytic or fusion protein which contained both a 281-residue saliva-bind-
glucan-binding domains of one GTF isozyme (Taubman et al., ing alanine-rich region of S. mutans Ag I/II and a 392-residue
1995; Smith et al., 1997b). These studies suggested that protec- glucan-binding domain of GTF-I. A recombinant fusion protein,
tion could be achieved by immunization with discrete epi- expressed in E. coli XL1-Blue, induced IgG antibody in rabbits or
topes associated with several virulence characteristics. in Holstein cows (Oho et al., 1999) which could inhibit glucan
Combining epitopes from adhesins and GTFs into one con- synthesis by GTF and sucrose-independent and -dependent
struct and enhancing the immune response with additional adhesion of S. mutans to saliva-coated hydroxyapatite beads.
sequences (e.g., cholera toxin subunits) could theoretically Constructs involving the attenuated human S. typhi vector
increase and possibly extend the protective effect of these sub- would be expected to have more potential for human vaccine
unit vaccines. Some recombinant protein approaches, applications than would S. typhimurium, which is a murine
described below, have used this design. pathogen. In this regard, attenuated S. typhi CVD908 strains
have been prepared to express peptide chimeras in which GTF
(B) RECOMBINANT VACCINES/ sequences, associated with the glucan-binding domain, are
ATTENUATED EXPRESSION VECTORS combined with tetanus toxin fragment C for immunogenicity
Recombinant approaches afford the expression of larger por- (unpublished observations).
tions of functional domains than can be accommodated by syn-
thetic peptides. Also, gene fusions of a functionally relevant (VI) Conjugate Vaccines
sequence linked to a mucosal adjuvant sequence can result in Another vaccine approach which may intercept more than one
chimeric proteins inherently able to enhance immune respon- aspect of mutans streptococcal molecular pathogenesis is the
ses to the functional epitopes. Furthermore, attenuated mutant chemical conjugation of functionally associated protein/peptide
vectors such as Salmonella, which contain plasmids expressing components with bacterial polysaccharides. Added to the value
recombinant peptides, can target the vaccine to appropriate of including multiple targets within the vaccine is that the con-
inductive lymphoid tissue for mucosal responses. Several of jugation of protein with polysaccharide enhances the immuno-
these approaches have successfully induced protective genicity of the T-cell-independent polysaccharide entity. This
immune responses for experimental dental caries in rats or principle was first demonstrated by Landsteiner (1936) and
mice by means of chimeric proteins or vectors expressing either Avery and Goebel (1929) and has been applied with great suc-
adhesin or GTF epitopes. Redman and co-workers have shown cess in the Hemophilus influenzae type b (Hib) conjugate vaccines
(1994, 1995) that oral immunization with recombinant to induce protective immunity to the capsular polysaccharide of
Salmonella typhimurium, expressing surface protein antigen A of H. influenzae in infants and young children. Two groups have
Streptococcus sobrinus, was able to induce persistent mucosal applied this approach to dentally relevant components. Lett and
immune responses which could confer protection after chal- co-workers (1994) covalently coupled an adhesin-associated 14-
lenge of Fischer rats with cariogenic S. sobrinus. Hajishengallis mer synthetic peptide to the serogroup f polysaccharide of S.
and co-workers have genetically linked the 42-kDa salivary- mutans strain OMZ 175 by reductive amination. Subcutaneous
binding receptor (SBR) of S. mutans Ag I/II with the A2 and B injection with the conjugate induced systemic IgM and IgG anti-
subunits of cholera toxin (SBR-CTA2/B) and expressed this body responses to both peptide and polysaccharide which could
chimeric protein in E. coli BL21 (Hajishengallis et al., 1995). be boosted upon subsequent injection. The presence of both B-

13(4):335-349 (2002) Crit Rev Oral Biol Med 341


and T-epitopes in the peptide was required for effective respons- 1997a), and fimbrial preparations from S. mutans (Fontana et al.,
es. Intragastric intubation of the conjugates associated with lipo- 1999), with antigen alone or combined with mucosal adjuvants.
somes induced primary and secondary salivary IgA antibody to
both components (Lett et al., 1995). In separate studies, Taubman (C) TONSILLAR
and co-workers (1998, 1999) have reported that conjugation of The ability of tonsillar application of antigen to induce
either tetanus toxoid or S. sobrinus GTF to the water-soluble glu- immune responses in the oral cavity is of great interest.
can synthesized by GTF significantly enhanced serum IgG and Tonsillar tissue contains the required elements of immune
salivary IgA antibody levels to the water-soluble glucan and to induction of secretory IgA responses (van Kempen et al., 2000),
the conjugated protein. Serum GTF inhibitory activity was also although IgG, rather than IgA, response characteristics are
improved by conjugation. The relative protective capacity of dominant in this tissue (Boyaka et al., 2000). Nonetheless, the
either conjugate approach has yet to be tested. Since initial S. palatine tonsils, and especially the nasopharyngeal tonsils,
mutans infection occurs at an age (< 2 yrs) when children are have been suggested to contribute percursor cells to mucosal
unable to mount significant anti-polysaccharide responses, these effector sites (Brandtzaeg, 1996), such as the salivary glands. In
approaches will be especially important if conjugate vaccines are this regard, Fukuizumi and co-workers (1999) have shown
shown to enhance the level of protection significantly over that that topical application of formalin-killed S. sobrinus cells in
achieved with protein-based vaccines. rabbits can induce a salivary immune response which can sig-
nificantly decrease the consequences of infection with cario-
(VII) Routes to Protective Responses genic S. sobrinus. Interestingly, repeated tonsillar application of
Mucosal applications of dental caries vaccines are generally particulate antigen can induce the appearance of IgA anti-
preferred for the induction of secretory IgA antibody in the sali- body-producing cells in both the major and minor salivary
vary compartment, since this immunoglobulin constitutes the glands of the rabbit (Inoue et al., 1999).
major immune component of major and minor salivary gland
secretions. Many investigators have shown that exposure of
(D) MINOR SALIVARY GLAND
antigen to mucosally associated lymphoid tissue in the gut, The minor salivary glands populate the lips, cheeks, and soft
nasal, bronchial, or rectal site can give rise to immune respon- palate. These glands have been suggested as potential routes for
ses not only in the region of induction, but also in remote loca- mucosal induction of salivary immune responses (Crawford et
tions. This has given rise to the notion of a “common mucosal al., 1975; Schroeder et al., 1983), given their short, broad secreto-
immune system” (Mestecky, 1993). Consequently, several ry ducts that facilitate retrograde access of bacteria and their
mucosal routes have been used to induce protective immune products (Nair and Schroeder, 1983), and given the lymphatic
responses to dental caries vaccine antigens. tissue aggregates that are often found associated with these
ducts. Experiments in which S. sobrinus GTF was topically
(A) ORAL administered onto the lower lips of young adults have suggest-
Many of the earlier studies relied on oral induction of immu- ed that this route may have potential for dental caries vaccine
nity in the gut-associated lymphoid tissues (GALT) to elicit delivery. In these experiments, those who received labial appli-
protective salivary IgA antibody responses. In these studies, cation of GTF had significantly lower proportions of indigenous
antigen was applied by oral feeding, gastric intubation, or in mutans streptococci/total streptococcal flora in their whole sali-
vaccine-containing capsules or liposomes. Although the oral va during a six-week period following a dental prophylaxis,
route was not ideal for reasons including the detrimental compared with a placebo group (Smith and Taubman, 1990).
effects of stomach acidity on antigen, or because inductive
sites were relatively distant, experiments with this route estab-
(E) RECTAL
lished that induction of mucosal immunity alone was suffi- More remote mucosal sites have also been investigated for their
cient to change the course of mutans streptococcal infection inductive potential. For example, rectal immunization with
and disease in animal models (Michalek et al., 1976; Smith et non-oral bacterial antigens such as Helicobacter pylori
al., 1979) and humans (Smith and Taubman, 1987). (Kleanthous et al., 1998) or Streptococcus pneumoniae (Hvalbye et
al., 1999), presented in the context of toxin-based adjuvant, can
(B) INTRANASAL result in the appearance of secretory IgA antibody in distant
More recently, attempts have been made to induce protective salivary sites. The colo-rectal region as an inductive location for
immunity in mucosal inductive sites that are in closer anatom- mucosal immune responses in humans is suggested from the
ical relationship to the oral cavity. Intranasal installation of fact that this site has the highest concentration of lymphoid fol-
antigen, which targets the nasal-associated lymphoid tissue licles in the lower intestinal tract. Preliminary studies have
(NALT) (Brandtzaeg and Haneberg, 1997), has been used to indicated that this route could also be used to induce salivary
induce immunity to many bacterial antigens, including those IgA responses to mutans streptococcal antigens such as GTF
associated with mutans streptococcal colonization and accu- (Lam et al., 2001). One could, therefore, foresee the use of vac-
mulation. Protective immunity after infection with cariogenic cine suppositories as one alternative for children in whom res-
mutans streptococci could be induced in rats by the IN route piratory ailments preclude intranasal application of vaccine.
with many S. mutans antigens or functional domains associat-
ed with these components. Protection could be demonstrated (VIII) Adjuvants and Delivery Systems
with S. mutans AgI/II (Katz et al., 1993), the SBR of AgI/II for Dental Caries Vaccines
(Hajishengallis et al., 1998), a 19-mer sequence within the SBR Mucosal routes of antigen delivery often require additional com-
(Takahashi et al., 1991), the glucan-binding domain of S. mutans ponents which can potentiate aspects of the immune response to
GTF-B (Jespersgaard et al., 1999), S. mutans GbpB (Smith et al., induce sufficient antibody to achieve a protective effect.

342 Crit Rev Oral Biol Med 13(4):335-349 (2002)


(A) CHOLERA AND E. COLI proteolytic cleavage required for activation. Several detoxified
HEAT-LABILE ENTEROTOXINS CT and LT mutants have been prepared which retain signifi-
cant adjuvanticity and may have potential for human use. One
Cholera toxin (CT) is a powerful mucosal immunoadjuvant such mutant, LT [LT(R192G)], obtained by substituting glycine
which is frequently used to enhance the induction of mucosal for arginine 192 in the A subunit (Dickinson and Clements,
immunity to a variety of bacterial and viral pathogens in animal 1995) to interfere with trypsin-mediated cleavage, has been
systems. CT is an ADP-ribosylating bacterial toxin with A and B shown to induce mucosal and systemic immune responses to
subunits. The non-toxic pentameric B subunit binds to ganglio- intranasally applied GTF peptides which were generally com-
side receptors on target cells. The toxic A subunit is then translo- parable with those observed with CT (Smith et al., 2001b).
cated into the cell, where this enzymatically active peptide trans-
fers the ADP ribose group of NAD to a GTP-binding protein, ini- (B) MICROCAPSULES AND MICROPARTICLES
tiating the toxic effects by increasing intracellular levels of cAMP. Combinations of antigen in or on various types of particles have
The adjuvant effects of CT (reviewed in Freytag and Clements, been used in attempts to enhance mucosal immune responses.
1999) are broad-based and can include increased mucosal epithe- The particularization of antigen achieved by these approaches
lial cell and macrophage production of pro-inflammatory may increase the association of antigen with M cells overlying
cytokines, up-regulation of B7-2 co-stimulatory factors on APCs, inductive regions of the secretory immune system (Neutra and
and increased antigen transfer from the mucosal to the systemic Kraehenbuhl, 1992). Microspheres and microcapsules made of
compartment. These effects may be, at least in part, locally man- poly(lactide-co-glycolide) (PLGA) have been used as local
ifest and may involve dendritic cells as the predominating anti- delivery systems (Eldridge et al., 1990; Ermak et al., 1995)
gen-presenting cell (APC) population (Porgador et al., 1997). because of their ability to control the rate of release, evade pre-
Mucosal application of soluble protein or peptide antigen existent antibody clearance mechanisms, and degrade slowly
alone rarely results in elevated or sustained IgA responses. without eliciting an inflammatory response to the polymer. Oral
However, addition of small amounts of CT or the closely relat- (Challacombe et al., 1992) or topical (Rafferty et al., 1996) immu-
ed E. coli heat-labile enterotoxins (LT) (Takahashi et al., 1996) nization with soluble antigen in PLGA microcapsules can lead
can greatly enhance mucosal immune responses to intragastri- to enhanced mucosal responses, although the organic solvents
cally or intranasally applied mutans streptococcal antigens required for formulation can diminish the biological activity of
(Russell and Wu, 1991; Martin et al., 2000) or to peptides the antigen. Denaturation can be significantly diminished by
derived from these antigens (Smith et al., 2001b). One methods which incorporate antigen into microparticles in an
approach to reducing or eliminating toxicity while maintain- aqueous phase (Hsu et al., 1994). Intranasal immunization of
ing adjuvanticity was to remove the A subunit from the CT aqueously incorporated GTF-PLGA microparticles, which also
complex. Mucosal immunization with the B subunit of CT, contained 1% gelatin as bioadhesive, induced long-lasting sali-
mixed with, chemically conjugated to, or genetically fused to vary immune responses (Smith et al., 2000). Starch microparti-
either intact antigen or the presumed functional domains of cles can also be used to increase mucosal responses to soluble
mutans streptococcal virulence components (Czerkinsky et al., antigens. Montgomery and Rafferty (1998) have shown that top-
1989; Dertzbaugh et al., 1990; Takahashi et al., 1991; Wu and ical administration to the oral mucosa of bioadhesive degrad-
Russell, 1993; Laloi et al., 1996; Wu et al., 1997) resulted in sig- able starch microparticles containing dinitrophenyl-bovine
nificant mucosal responses to the complexed antigen (and to serum albumen, in combination with L-a-lysophosphatidyl-
CT), which could be shown to be protective under certain con- choline (as a penetration enhancer) and interleukins IL-5 and IL-
ditions (Katz et al., 1993) and long-lived (Harrod et al., 2001). 6, potentiated long-lived salivary IgA responses, compared with
Other approaches have been sought to modify CT or LT, antigen delivered in soluble form.
since the increase in CTB-assisted immune response was signif-
icantly less than that achieved with the CT holotoxin, and since, (C) LIPOSOMES
in some systems, the presence of a small amount of intact CT Liposomes, which are phospholipid membrane vesicles manu-
was required for response enhancement. Hajishengallis and co- factured to contain and deliver drugs and antigens, have been
workers (1995) were able to improve mucosal adjuvanticity by used to enhance mucosal responses to mutans streptococcal
replacing the toxic A1 (N-terminal portion) of CT with a 42-kDa carbohydrate (Childers et al., 1990-1991) and GTF (Childers et
portion of the AgI/II containing the salivary-binding region al., 1996). Liposomes are thought to improve mucosal immune
(SBR), thereby creating a chimeric protein composed of the SBR responses by facilitating M cell uptake and delivery of antigen
linked to the non-toxic C-terminal A2 and B subunits of CT. to lymphoid elements of inductive tissue (Childers et al., 1990).
Similar enhancement, albeit by potentially different mecha- Gastric intubation of rats with GTF-liposome vaccines induced
nisms, occurred after replacement of the toxic A1 subunit of LT responses which diminished dental caries caused by subse-
(serogroup IIa) with SBR (Martin et al., 2001). Responses were quent infection with S. mutans (Childers et al., 1996). Clinical
long-lasting (Hajishengallis et al., 1996b), and chimeric SBR- studies comparing intranasal immunization with GTF-lipo-
CTA2/B proteins could be expressed in E. coli and in attenuat- somes vs. GTF alone showed that both vaccines increased local
ed Salmonella typhimurium expression vectors (Hajishengallis et (nasal) and salivary IgA antibody responses to GTF up to five-
al., 1996a,b; Huang et al., 2000) which could be shown to induce fold (Childers et al., 1999). Only the nasal-wash IgA1 antibody
protective immune responses in BALB/c mice when adminis- response was higher with the liposome-containing, compared
tered intragastrically or intranasally (Huang et al., 2001). with the protein-alone, vaccine.
Alternatively, detoxification of CT and LT has been
attempted with the use of site-directed mutagenesis techniques (D) OTHER APPROACHES
to modify amino acid residue areas of the toxic A1 subunit Other methods to enhance mucosal responses are being adapt-
which are critical to its enzymatic activity or to the required ed for dental caries vaccine use. Monophosphoryl lipid A,

13(4):335-349 (2002) Crit Rev Oral Biol Med 343


when administered intranasally to mice as an aqueous formu- after topical application of mouse monoclonal IgG or trans-
lation with soluble GTF or incorporated into liposomes con- genic plant secretory SIgA/G antibody, each with specificity
taining GTF, induced primary and secondary salivary IgA for Ag I/II (Ma et al., 1990, 1998). In these experiments, teeth
responses which exceeded those achieved with GTF-lipo- were first treated for nine days with chorohexidine. Following
somes (Childers et al., 2000). Oligodeoxynucleotides contain- anti-bacterial treatment, antibody was topically applied for
ing unmethylated CpG dinucleotides (CpG ODN) induce pro- three weeks. Recolonization with mutans streptococci did not
liferation of B-cells and activation of macrophages. Intranasal occur for at least two years after treatment of subjects with
or oral administration of CpG ODN with tetanus toxoid (TT) mouse monoclonal antibody or at least 4 months after treat-
enhanced murine IgA responses in many mucosal secretions, ment with the transgenic antibody to the Ag I/II epitope. In
including saliva (McCluskie and Davis, 2000). Similarly, intra- contrast, the teeth of all subjects topically treated with non-
gastric administration of CpG ODN with TT- Al(OH)3 specific monoclonals were re-colonized with mutans strepto-
enhanced systemic IgG responses to TT, compared with those cocci by 82 days in the former experiment and by 58 days in
achieved with TT - Al(OH)3 (Eastcott et al., 2001). the later experiment. Bivalent antigen binding appeared to be
required, since Fab fragments did not afford protection. The
authors suggest that the secretory form of the monoclonal anti-
(IX) Past, Present, and Future Human Applications body may be more efficacious because of its apparent
increased survival time in the oral cavity, compared with IgG,
(A) ACTIVE IMMUNIZATION as well as the increased avidity emanating from its tetravalen-
Few clinical trials have been performed to examine the protective cy (Ma et al., 1998). The explanation for the long-term effects
effect of active immunization with dental caries vaccines contain- on mutans streptococcal colonization after a relatively short
ing defined antigens. However, several studies have shown that exposure to antibody remains unresolved. Apparently, anti-
mucosal exposure of humans to immunization with glucosyl- body blockage of an important adhesin epitope during the
transferases from S. mutans or S. sobrinus can lead to the forma- reconstruction of the dental biofilm following chlorohexidine
tion of salivary IgA antibody, albeit at modest levels. Childers and treatment places indigenous mutans streptococci at an insur-
co-workers (1994) orally immunized adults using enteric-coated mountable competitive disadvantage for recolonization. An
capsules filled with crude S. mutans GS-5 GTF antigen prepara- interesting parallel may be the observation that young chil-
tions contained in liposomes. Parotid salivary IgA antibody dren who do not become naturally infected with mutans strep-
responses, primarily of the IgA2 subclass, were induced in five of tococci during the “window of infectivity” remain unde-
seven subjects. Similarly, nasal immunization with dehydrated tectably infected for several years (Caufield et al., 1993; Smith
liposomes containing this GTF preparation induced significant et al., 1998a), potentially because its niche in the dental biofilm
IgA1 antibody response in nasal washes (Childers et al., 1997, has been filled by other indigenous flora.
1999). Parotid salivary antibody levels to GTF were of lower mag- Experimental passive immune protection could also be
nitude. In earlier studies, this group (Childers et al., 1990-1991) achieved with antibody to GTF (Hamada et al., 1991) or GbpB
showed that oral administration of capsules containing the puri- (Smith et al., 2001a). Thus, topical or dietary administration of
fied serotype carbohydrate antigen of S. mutans in liposomes immune reagents with specificity for epitopes on these pro-
gave rise to low but detectable levels of salivary antibody. teins may also have potential human application.
Smith and Taubman (1987, 1990) reported that mucosal
immunization with GTF could influence the re-emergence of (C) PROSPECTS AND CONCERNS
mutans streptococci in young adults after a dental prophylax- Traditional vaccine therapy indicates that immunization
is. Levels of parotid salivary IgA antibody to GTF increased should take place prior to infection. Given the apparent pat-
after oral immunization with S. sobrinus GTF in enteric cap- tern of mutans streptococcal colonization and the association
sules, administered together with aluminum phosphate. of these organisms with disease, this would suggest that
Immunization under this protocol delayed the re-accumula- immunization for dental caries should begin early in the sec-
tion of indigenous oral mutans streptococci, compared with a ond year of life for those populations under “normal” risk for
placebo group given buffer-filled capsules. A delay in mutans infection. Our understanding of the mucosal immune
streptococcal re-emergence was also observed after topical response indicates that such children are competent to mount
administration of GTF on the lower lip, although this protocol secretory responses to protein antigens, although their ability
did not result in a significant detectable increase in antibody to to respond to polysaccharide determinants in mucosal appli-
the vaccine (Smith and Taubman, 1990). Taken together, these cations of conjugate vaccines is as yet unknown. If bacterial
studies support the hypothesis that mucosal immunization colonization of the dental biofilm is complete after eruption of
with dental caries vaccines could be protective, especially in all primary teeth, and if one can, through immunization, pre-
pediatric populations where mutans streptococci is not yet a vent mutans streptococcal colonization prior to this period,
permanent member of the dental biofilm. then the benefit of early immunization might extend until sec-
ondary teeth begin to erupt, exposing new ecological condi-
(B) PASSIVE IMMUNE APPROACHES tions. Subsequent immunization could then be initiated.
Passive antibody administration has also been examined for Clearly, several possible dental caries vaccine approaches
effects on indigenous mutans streptococci. Mouthrinses con- may have application in pediatric clinical trials. Several
taining bovine milk (Filler et al., 1991) or hen egg yolk IgY mutans streptococcal components have been shown to protect
(Hatta et al., 1997) antibody to S. mutans cells led to modest in animal model systems. Protective epitopes of these compo-
short-term decreases in the numbers of indigenous mutans nents continue to be identified and incorporated into designs
streptococci in saliva or dental plaque. to increase their protective value. Several routes of adminis-
Longer-term effects on indigenous flora were observed tration can induce protective immune responses, at least in

344 Crit Rev Oral Biol Med 13(4):335-349 (2002)


model systems. Also, significant progress is being made in the mucosal immunization with non-replicating antigens by any
adjuvant field to remove the toxic properties of powerful route in the target pediatric population is essentially unknown,
mucosal adjuvants while maintaining their adjuvant proper- since this is new territory in human vaccine therapy. Dental
ties. Animate (attenuated bacterial vectors) and inanimate caries vaccines would be the first non-living vaccine to be
(liposomes, microparticles) delivery systems have been identi- applied by any mucosal route during the first three years of life.
fied to provide more efficient targeting of the vaccine. Both Given that we would be able to induce a detectable mucosal
passive and active immunization approaches have demon- response in this hypothetical pediatric clinical trial, we do not
strated success in animal models and human clinical trials. know what the coverage will be. Longitudinal studies in fami-
With all these apparently effective pre-clinical approaches, lies suggest that siblings who are presumably challenged with
one may ask, “What is the ideal dental caries vaccine the same parental strains of mutans streptococci can demon-
approach?” Ideally, one would favor a vaccine that would give strate different responses to dominant GTF and AgI/II antigens.
broadest coverage to intercept infection by all common cario- Thus, a multi-component vaccine may be needed to ensure
genic mutans streptococcal strains, one that would work for broadest coverage. Also, high-risk populations who have ele-
both low- and high-risk populations, one whose immunity vated familial exposure to mutans streptococci, coupled with
would last through the critical primary and secondary infec- environmental conditions favoring colonization, may require a
tion periods, one that could be given with, or as part of, other different approach than populations with “normal” risk for
immunizations, one that could be given by various routes and infection. Such high-risk populations may require both active
still be effective, one that would be inexpensive, one that could and passive mechanisms for protection. Pediatric clinical trials
be delivered by individuals with little training, one that might in each of these types of populations with, potentially, more
provide secondary immunity to others in the population who than one type of vaccine are likely to be required to provide
were not themselves immunized. Can or should we expect all insights into the best vaccine strategy(ies) for broadest protec-
of these characteristics in one vaccine? tion of the respective populations. Also, understanding the sig-
If we accept that each approach could give a reasonable nals for colonization and growth of cariogenic streptococci in
level of protection in humans, one still needs to consider for dental biofilms may help us devise more refined and informed
whom and under what circumstances the dental caries vaccine techniques to “lock out” those bacteria that can cause us harm.
is intended. For example, the ideal vaccine application for a
child with asthma, the second most common chronic childhood Acknowledgments
ailment, may be at a site (e.g., rectal) and with an adjuvant (e.g., The author’s research was performed through the generous support of the
detoxified CT or LT) that is quite different from that sufficient to National Institute of Dental and Craniofacial Research (DE-01653, DE-
give a protective response (e.g., intranasal) to a healthy child.
04733, DE/AI-12324). The author is also grateful to Leigh Barnes for assis-
Also, from economic and societal standpoints, a vaccine strate-
tance with manuscript preparation.
gy for children to whom the full advantages of pediatric care are
available may not be the ideal approach for children with limit-
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