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
of mankind. Advances in prophylactic measures to deal
with this disease have significantly reduced the overall caries
rate in the United States. However, the Surgeon General’s 2000
report on Oral Health in America stated that a majority of five-
to nine-year-old US children have at least one lesion on the
crowns of their teeth. This percentage increases to 84.7% in
adults who are at least 18 years of age. Nearly 50% of our elder
population (> 75 years old) have root-surface caries. Being
poor is clearly a risk factor for increased decay. More than one-
third of poor two to nine-year-old children have untreated
decayed primary teeth. Poor children from Mexican-American
or non-Hispanic black backgrounds are particularly at risk,
given the fact that over two-thirds of these populations have
untreated decayed teeth.
In developing countries, dental caries is often at epidemic
proportions, especially among the poor. For example, at least
25% of three-year-old children from various areas of Brazil have
detectable caries lesions, many developing lesions within the
first 18 months of life (Mattos-Graner et al., 1996). This high
caries rate continues among the less economically advantaged in
the face of efforts to introduce fluoride at an early age. Similarly,
an oral health survey in China revealed that three-quarters of
five-year-old children studied had evidence of significant dental
decay (Wong et al., 2001). Thus, more effective public health mea-
sures are needed to address this worldwide problem.
Landmark experiments in the 1960s (reviewed in Gibbons
and van Houte, 1975; Loesche, 1986) established that mutans
streptococci are the primary etiologic agents of this disease
and that infection is transmissible. A strong association exists
between level of colonization with mutans streptococci and
dental caries, although other organisms, such as lactobacilli,
have also been implicated in this disease.
Studies of the natural history of mutans streptococcal col-
onization of infants have revealed several interesting features
children become permanently colonized with mutans strepto-
cocci between the middle of the second year and the end of the
third year of life, during a so-called “window of infectivity”
(Caufield et al., 1993). Techniques involving bacteriocins, plas-
mids, and DNA fingerprinting have been used to identify the
source of infection (Berkowitz and Jones, 1985; Caufield et al.,
1993; Li and Caufield, 1995). These studies have shown that
the primary source of infection is maternal, although there is
recent evidence to suggest that non-familial transfer can occur
when environmental conditions favor colonization (Mattos-
Graner et al., 2001). Infection is related to maternal dose
(Kohler et al., 1984; Caufield et al., 1993), in that the higher the
level of maternal mutans streptococcal infection, the higher
the percentage of children who become infected.
Other factors also influence mutans streptococcal colo-
nization. If the environment strongly favors mutans coloniza-
tion—for example, if high maternal infection levels are com-
bined with high dietary sucrose levels—this so-called “win-
dow of infection” shifts to an earlier age. More sensitive tech-
niques for microbial detection, e.g., DNA probe technology,
have also suggested that mutans streptococci can be found in
the oral cavity during the first year of life, especially in caries-
prone populations (Milgrom et al., 2000). However, despite the
influence of maternal dose, children who do not become
infected by approximately three years of age appear to remain
uninfected, or minimally colonized for several years (Caufield
et al., 1993; Smith et al., 1998a), possibly until new opportuni-
ties for colonization occur upon eruption of the secondary
dentition. This suggests that a longer-term benefit could ensue
if mutans streptococcal colonization could be impeded in early
childhood by measures such as immunization.
(II) Ontogeny of Mucosal Immunity
Mucosal applications of dental caries vaccines have been
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
first three years of life. The percentage of children colonized with
mutans streptococci is indicated on the ordinate. Percentages reflect
children under modest maternal challenge (approximately 50% colo-
nized) or children exposed to high maternal levels (approximately
90% colonized). If high maternal MS levels (dose) are combined with
significant exposure to dietary sucrose, initial dental colonization with
MS occurs at a younger age.
nent of major and minor gland salivary secretions and thus
would be considered to be the primary effector of adaptive
immunity in the salivary milieu. Given the natural history of
mutans streptococcal infection described above, immunization
would presumably need to be initiated early in childhood to
interfere with mutans streptococcal colonization. This then
would require that the mucosal immune system be sufficient-
ly mature at this time to respond effectively.
The oral immune environment undergoes rapid, early
development (reviewed in Smith and Taubman, 1992, 1993).
Although secretory IgA antibody in saliva and other secretions is
essentially absent at birth, mature SIgA, i.e., dimeric IgA with a
bound secretory component, is the principal salivary
immunoglobulin secreted in individuals by one month of age.
Consequent to the environmental antigenic challenge, mucosal
IgA antibody to pioneer gut (e.g., Escherichia coli) and oral (e.g.,
Streptococcus mitis and S. salivarius) microbiota appears within
weeks of initial exposure (Fig. 2). In the first months of life, an
infant’s saliva may contain considerable concentrations of IgM
and may, occasionally, be dominated by the IgA1 isotype.
However, by six to nine months of age, most children exhibit a
more adult-like distribution of salivary IgA1 and IgA2 subclass-
es. Salivary antibody to oral commensal microbiota can be
detected in both subclasses at this time. Taken together, this evi-
dence suggests that significant maturation of the mucosal
immune response has occurred by the end of the first year of life.
Natural exposure to mutans streptococci also results in a
mucosal immune response to these organisms (Smith et al.,
1998a). This response often begins to be observed in the sec-
ond and third years of life. Western blot and ELISA analyses
reveal that the major responses appear to be directed primari-
ly to streptococcal components which are considered to be
important in colonization and accumulation, such as antigen
I/II, glucosyltransferase, and glucan-binding protein(s).
336 Crit Rev Oral Biol Med
Figure 2. Appearance of salivary IgA antibody to indigenous oral
flora and antigens of immunization. The appearance of salivary
IgA antibody (Ab) to micro-organisms associated with indigenous
infection or immunization is indicated by arrows. The percentages
of children infected with indigenous oral streptococci (S. mitis, S.
salivarius, S. sanguis, S. mutans) are indicated by the bars. Data
are summarized from Smith and Taubman, 1992, 1993.
Interestingly, in some children, antibody to mutans streptococ-
cal antigens can also be detected independently of the ability
to detect ongoing infection in the second year of life. As is the
case with many bacterial challenges throughout the body, the
threshold of immunological response is lower than that of per-
sistent infection; therefore it is not surprising to observe anti-
body to S. mutans antigens in the absence of its colonization.
Longitudinal studies suggest that antibody reactive with
mutans streptococci results from contact with mutans strepto-
cocci, rather than from earlier colonizing oral streptococci,
since well-developed salivary IgA antibody to pioneer oral
streptococci can be demonstrated prior to the detection of anti-
body reactive with mutans streptococci. Thus colonization, at
least not extensive colonization, with mutans streptococci is
apparently not required for the development of salivary anti-
body to associated mutans streptococcal antigens.
Salivary immune responses to mutans streptococci show
significant individual characteristics in early childhood.
Children respond at different rates following infection, a con-
dition which may be partly the result of the extent of infection
(antigen dose) or age at the time of infection (maturation of
immune response). Even siblings may differ in the amounts or
kinds of IgA antibody specificities appearing in their saliva.
These variations may stem from differences either in the inher-
ent ability of the child to respond or in the characteristics of
genetically different strains of mutans streptococci (thus
potentially differing antigenic challenge) ultimately colonizing
the child. The rate, specificity, and/or extent of the mucosal
immune response to previous encounters with the organism
may also contribute to the success or failure of permanent col-
onization.
Thus, the evidence from salivary IgA responses to com-
mensal oral microbiota indicates that the mucosal immune
system is relatively well-developed by the period during
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-
opment are as yet unclear. GTFs also contain glucan-binding
domains which permit binding to glucans. The interactions of
glucans with cell-associated glucan-binding domains of GTFs
and GBPs combine to cause extensive accumulation of mutans
streptococci in the dental biofilm. Since GTFs and GBPs are
also secreted into the extracellular environment, their specific
or non-specific incorporation into the salivary pellicle would
also provide binding sites for mutans streptococci.
Theoretically, the next phase of pathogenesis results from
the metabolic activities of these masses of accumulated mutans
streptococci (and possibly of other accumulation-associated
micro-organisms). Mutans streptococci are the most prolific
producers of lactic acid in these accumulations (Gibbons and
van Houte, 1975), although other “low pH bacteria” may also
contribute (van Ruyven et al., 2000). Dental caries then ensues,
because the resulting increase in lactic acid synthesis cannot be
sufficiently buffered to prevent enamel dissolution.
(IV) Effective Molecular Targets
for Dental Caries Vaccines
Several stages in the molecular pathogenesis of dental caries
are susceptible to immune intervention. Micro-organisms can
be cleared from the oral cavity by antibody-mediated aggrega-
tion while still in the salivary phase, prior to colonization.
Antibody could also block the receptors necessary for colo-
nization (e.g., adhesins) or accumulation (e.g., glucan-binding
domains of GBPs and GTF), or inactivate GTF enzymes
responsible for glucan formation. Modification of metabolical-
ly important functions may also be targeted. In addition, the
antimicrobial activity of salivary IgA antibody may be
enhanced or redirected by synergism with innate components
of immunity, such as mucin or lactoferrin. This review will
concentrate on adhesins, GTFs, and GBPs as vaccine targets,
since most of the recent experimental effort has been directed
toward these components.
(A) ADHESINS
Adhesins from the two principal human pathogens,
Streptococcus mutans (variously identified as antigen I/II, PAc,
or P1) and Streptococcus sobrinus (SpaA or PAg), have been puri-
fied. Russell and Lehner initially described the S. mutans com-
ponent in 1978. Antigen I/II (AgI/II) was found both in the cul-
ture supernatant as well as on the S. mutans cell surface. This
185-kDa protein is composed of a single polypeptide chain of
approximately 1600 residues (Lee et al., 1988). Significant
sequence homology (66%) exists between S. mutans AgI/II and
S. sobrinus SpaA (Tokuda et al., 1990; LaPolla et al., 1991) as well
as with at least one adhesin from Streptococcus gordonii, a non-
cariogenic oral streptococcus (Demuth et al., 1990). However,
despite the homology between the two mutans streptococcal
adhesins, each appears to bind to separate components in the
pellicle (Gibbons et al., 1986; Lamont et al., 1991).
S. mutans Ag I/II contains an alanine-rich tandem repeat-
ing region in the N-terminal third, and a proline-rich repeat
region in the center of the molecule. These regions have been
associated with the adhesin activity of Ag I/II. Crowley and
colleagues (1993) and Nakai and co-workers (1993) have each
described a region within or near the alanine-rich region that
can bind salivary components in experimental tooth pellicles.
Lehner, Kelly, and co-workers (1994; 1995) suggested that the
338 Crit Rev Oral Biol Med
proline-rich central portion contains an adhesion epitope, ba-
sing their conclusions on adhesion inhibition assays involving
recombinant fragments of Ag I/II.
Immunological approaches support the adhesin-related
function of the AgI/II family of proteins and their repeating
regions. For example, abundant in vitro and in vivo evidence
indicates that antibody with specificity for S. mutans AgI/II or
S. sobrinus SpaA can interfere with bacterial adherence and
subsequent dental caries. Antibody directed to the intact anti-
gen I/II molecule or to its salivary-binding domain blocked
adherence of S. mutans to saliva-coated hydroxyapatite
(Hajishengallis et al., 1992). Furthermore, numerous immu-
nization approaches have shown that active immunization
with intact antigen I/II (Lehner et al., 1981; Katz et al., 1993) or
passive immunization with monoclonal (Ma et al., 1990) or
transgenic antibody (Ma et al., 1998) to putative salivary-bind-
ing domain epitopes within this component can protect
rodents, primates, or humans from dental caries caused by S.
mutans. Immunization of mice with synthetic peptides
(residues 301-319) from the alanine-rich region of antigen I/II
suppressed tooth colonization with S. mutans (Takahashi et al.,
1991). Immunization with S. sobrinus SpaA constructs protect-
ed rats from caries caused by S. sobrinus infection (Redman et
al., 1995). Protection in these experiments could conceivably
occur by antibody blockade of initial colonization events or
antibody-mediated agglutination and clearing of adhesin-
bearing bacteria from the saliva.
(B) GLUCOSYLTRANSFERASES (GTFS)
S. mutans and S. sobrinus each synthesize several glucosyltrans-
ferases. The deduced sequences of these enzymes vary from
1400 to nearly 1600 amino acid residues and contain consider-
able sequence homology, despite differences in the water-solu-
bility and linkages among the glucans synthesized. Genes
responsible for glucan synthesis in S. mutans are gtfB (Shiroza
et al., 1987), which synthesizes an a-1,3-linked insoluble glucan,
gtfC (Pucci et al., 1987), which synthesizes glucan with both a-
1,3 and a-1,6 linkages, and gtfD (Honda et al., 1990), which syn-
thesizes a soluble a-1,6-linked glucan. Similarly, the products
of gtfI (Russell et al., 1987) and gtfS (Gilmore et al., 1990) genes
of S. sobrinus synthesize insoluble and soluble glucan products,
respectively. Mutational inactivation techniques have shown
that each of these gene products is important to the cariogenic-
ity of the respective mutans streptococcal strain. For example,
insertional inactivation of S. mutans gtf genes to replace func-
tional wild-type copies of the gene markedly reduced caries
when gtfB and gtfC genes that coded for GTFs synthesizing
water-insoluble glucan were inactivated. Similar inactivation of
the gtfD gene also resulted in a mutant with lower cariogenici-
ty on smooth surfaces (Yamashita et al., 1993). In addition, the
ability of GTF from initially colonizing S. mutans to synthesize
water-insoluble glucan has been correlated with caries inci-
dence in young children (Mattos-Graner et al., 2000).
The activity of GTF is mediated through both catalytic and
glucan-binding functions. The catalytic activity of GTF appears to
be associated with several, sequentially separate, residues in the
N-terminal third of the molecule. These residues have been iden-
tified by a variety of methods, including the labeling of catalytic
intermediates and site-directed mutagenesis (Mooser et al., 1991;
Funane et al., 1993; Devulapalle et al., 1997; Tsumori et al., 1997;
Monchois et al., 2000). Insight into catalytically important residue
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
native environment is as yet unresolved, biofilm formation on
plastic surfaces by strains of S. mutans is directly correlated
with expression of GbpB (Mattos-Graner et al., 2001), suggest-
ing a role for GbpB in this process.
The third S. mutans non-enzymatic glucan-binding pro-
tein, GpbC, is composed of 583 amino acids. This protein has
a calculated molecular weight of 63.5 kDa. Although GbpC is
unrelated in sequence to GbpA or GTF, it bears some sequence
similarity to the AgI/II adhesin family. The GbpC protein,
detected when S. mutans cultures are stressed during growth,
is associated with dextran-dependent aggregation.
Of the three S. mutans glucan-binding proteins, only GbpB
has been shown to induce a protective immune response to
experimental dental caries (Smith and Taubman, 1996, 1997a).
Protection can be achieved by either subcutaneous injection of
GbpB in the salivary gland region (Smith and Taubman, 1996) or
by mucosal application by the intranasal route (Smith et al.,
1997a). Saliva samples from young children often contain IgA
antibody to GbpB, indicating that initial infection with S. mutans
can lead to natural induction of immunity to this protein (Smith
et al., 1998a). Preliminary studies have shown that GbpA appears
to be less immunogenic than GbpB and that its ability to induce
protective immunity is problematic (Smith et al., 1997a). S. sobri-
nus Gbps have not been evaluated for their protective potential.
(V) Subunit Vaccines
Subunit vaccines, which contain structural elements of the Ag
I/II adhesin family, GTFs or GbpB, have been designed for a
variety of reasons. It had been observed that immune respon-
ses in animals protected by immunization with intact proteins
were associated, at least in part, with in vitro measures of func-
tional inhibition. Thus, more recent studies have attempted to
optimize immune responses to functional epitopes associated
with salivary binding, catalytic processes, or glucan-binding
activities by designing subunit vaccines whose constructs con-
tain single or multiple copies of epitopes from these domains.
In addition, potentially enhanced protection could be achieved
by including, in the subunit vaccine construct, regions of the
virulence component containing strong immunological bind-
ing properties for induction of the desired arm of the immune
response. Furthermore, multivalent subunit vaccines could be
constructed of multiple epitopes which target different func-
tions on the same component (e.g., GTF catalytic and glucan-
binding activities) or functions on different components (e.g.,
AgI/II salivary binding and GTF catalytic activity). Such
approaches would address the variability in mucosal response
characteristics in the target vaccine population. Conjugation of
functionally associated peptides to carbohydrate components
(Wachsmann et al., 1985; Dougan et al., 1987; Lett et al., 1994),
for example glucan, or to other vaccine proteins (e.g., tetanus
toxoid) also would increase the immunogenicity of the peptide
and broaden the reach of the vaccine.
Designing vaccines in this way also permits one to elimi-
nate regions which may induce unwanted antibody specifici-
ties. The Ag I/II family of proteins shares extensive sequence
homology with surface proteins of non-cariogenic S. gordonii
(Demuth et al., 1990), S. intermedius, and S. oralis (Ma et al.,
1991). These homologous sequences may induce cross-reactive
responses that could influence colonization, attachment, or
accumulation of commensal microbiota. Also, cross-reacting
epitopes on serotype f SR protein and human IgG have been
340 Crit Rev Oral Biol Med
reported (Wachsmann et al., 1989; Gangloff et al., 1992). Given
this knowledge, subunit vaccines can be designed to include
the salivary-binding domain(s), but exclude sequence bearing
the potential for induction of unwanted antibody responses.
Subunit vaccines with inherent adjuvant potential could
also be constructed by including some or all of the sequence of
effective immunoadjuvants. Furthermore, this approach could
be adapted to incorporate such effective vaccine constructs into
attenuated expression vectors that have the ability to deliver the
antigen to inductive sites for antibody synthesis. Of the designs
which include mono- and di-epitopic synthetic peptide con-
structs, single recombinant peptides, or peptide chimeras with
immunoadjuvant sequence, used in a variety of applications or
within Salmonella expression systems, each has been reported to
induce caries-protective immunity in experimental systems.
(A) SYNTHETIC PEPTIDE VACCINES
As indicated above, at least two regions of the AgI/II-protein
family appear to be associated with salivary-binding func-
tions. Monoclonal antibody, raised by immunization with
intact Ag I/II, that reacted with the fragment containing the
proline-rich region also inhibited the formation of experimen-
tal dental caries (Lehner et al., 1992). Similarly, workers in
France (Gangloff et al., 1992; Lett et al., 1994) demonstrated that
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
proteins, was reactive with antibody to the native protein.
Synthetic peptide approaches have also shown the alanine-
rich repeat region of Ag I/II to be immunogenic and to induce
protective immunity. For example, subcutaneous immunization
with a synthetic peptide derived from the alanine-rich region of
Ag I/II from S. mutans (residues 301-319: PAcA) induced higher
levels of serum IgG antibody reactive with recombinant Ag I/II
than a synthetic peptide derived from the proline-rich region
(residues 601-629) (Takahashi et al., 1991). Intranasal immuniza-
tion with PAcA, coupled to cholera toxin B subunit, suppressed
colonization of mouse teeth by S. mutans (Takahashi et al., 1991).
Fusion proteins containing PAcA also inhibited sucrose-inde-
pendent adhesion of S. mutans to saliva-coated hydroxyapatide
beads (Yu et al., 1997). Thus, this S. mutans adhesin contains mul-
tiple functionally based epitopes that are sufficiently immuno-
genic to be considered for dental caries vaccines.
B- and T-cell epitopes (summarized in Smith and
Taubman, 1997) have been found in a cell-associated 3.8-kDa
protein component antigen (Lehner et al., 1994). Lehner and his
colleagues (Lehner et al., 1989a,b, 1994) applied free synthetic
peptides containing immunodominant sequences of the 3.8-
kDa antigen of S. mutans to the gingival mucosa of macaques,
resulting in the formation of both salivary IgA and gingival IgG
antibody. Anti-peptide antibody elicited by this topical appli-
cation method prevented colonization of the teeth by S. mutans.
The identification of functionally relevant resi-
dues/domains in glucosyltransferases has led to the design of
several synthetic peptide vaccines. Monoclonal or polyclonal
antibody preparations which were directed to one of several
N-terminal GTF peptides (Fig. 4), each of which contained dif-
ferent catalytically implicated residues, have been shown to
inhibit GTF activity (Dertzbaugh and Macrina, 1990; Chia et
al., 1993, 1997; Smith et al., 1994b, 1997b, 1999; Laloi et al., 1996).
Several of these synthetic peptides which contained strong B-
cell epitopes were synthesized on lysine backbones to contain
four or eight copies of the respective sequence. These con-
13(4):335-349 (2002)
structs induced protective immunity against experimental
dental caries (Taubman et al., 1995; Smith et al., 1997b).
Synthetic peptide constructs have also been based on
sequence derived from the repeating sequences in the C-termi-
nal third of GTF, which has been shown to be associated with
primer-dependent glucan binding (Abo et al., 1991; Lis et al.,
1995; Konishi et al., 1999). A synthetic peptide associated with a
putative glucan-binding site (Fig. 4) was shown to contain both
B- and T-cell epitopes, to induce antibody which could inhibit
the enzymatic activity of GTF (Smith et al., 1994b), and to induce
protective immune responses in the rat caries model (Taubman
et al., 1995). Furthermore, di-epitopic constructs of this peptide
and a peptide from the catalytic domain could be shown to
enhance the protective effect (Taubman et al., 2001), presumably
because antibody was raised to two functional targets and
because the glucan-binding domain peptide provided addition-
al T-cell help for the B-cell epitopes on both peptides.
All of the GTF synthetic peptide sequences which showed
protective effects in the above experiments are highly con-
served among S. mutans and, often, among S. sobrinus GTFs as
well. Antibody directed to these epitopes could therefore be
expected to reduce the activity of many of the GTF isozymes
expressed by these mutans streptococci, thus extending the
protective effect across species lines. In this regard, protection
from dental caries caused by either S. mutans or S. sobrinus
infection in the rat model has been demonstrated after immu-
nization with synthetic peptides from either the catalytic or
glucan-binding domains of one GTF isozyme (Taubman et al.,
1995; Smith et al., 1997b). These studies suggested that protec-
tion could be achieved by immunization with discrete epi-
topes associated with several virulence characteristics.
Combining epitopes from adhesins and GTFs into one con-
struct and enhancing the immune response with additional
sequences (e.g., cholera toxin subunits) could theoretically
increase and possibly extend the protective effect of these sub-
unit vaccines. Some recombinant protein approaches,
described below, have used this design.
(B) RECOMBINANT VACCINES/
ATTENUATED EXPRESSION VECTORS
Recombinant approaches afford the expression of larger por-
tions of functional domains than can be accommodated by syn-
thetic peptides. Also, gene fusions of a functionally relevant
sequence linked to a mucosal adjuvant sequence can result in
chimeric proteins inherently able to enhance immune respon-
ses to the functional epitopes. Furthermore, attenuated mutant
vectors such as Salmonella, which contain plasmids expressing
recombinant peptides, can target the vaccine to appropriate
inductive lymphoid tissue for mucosal responses. Several of
these approaches have successfully induced protective
immune responses for experimental dental caries in rats or
mice by means of chimeric proteins or vectors expressing either
adhesin or GTF epitopes. Redman and co-workers have shown
(1994, 1995) that oral immunization with recombinant
Salmonella typhimurium, expressing surface protein antigen A of
Streptococcus sobrinus, was able to induce persistent mucosal
immune responses which could confer protection after chal-
lenge of Fischer rats with cariogenic S. sobrinus. Hajishengallis
and co-workers have genetically linked the 42-kDa salivary-
binding receptor (SBR) of S. mutans Ag I/II with the A2 and B
subunits of cholera toxin (SBR-CTA2/B) and expressed this
chimeric protein in E. coli BL21 (Hajishengallis et al., 1995).
13(4):335-349 (2002) Crit Rev Oral Biol Med
Intranasal administration of this chimeric protein with CT
resulted in significant reductions in dental caries caused by
infection of Fischer rats with S. mutans UA130 (Hajishengallis
et al., 1998). The SBR-CTA2/B, expressed in an attenuated S.
typhimurium BRD509 vaccine strain containing an nirB promo-
ter (Huang et al., 2000), which was administered intranasally or
intragastrically to antibiotic-pre-treated BALB/c mice, resulted
in salivary antibody to the SBR and a significant reduction in
the number of S. mutans PC3379 recovered from dental plaque
after challenge (Huang et al., 2001). Jespersgaard and co-work-
ers (1999) intranasally immunized BALB/c mice with an E. coli-
expressed recombinant GTF peptide based on a 290-residue
glucan-binding domain sequence, or with a chimeric protein
combining this sequence with thioredoxin. Immunization with
either peptide resulted in protective effects on experimental S.
mutans infection and on resulting dental caries.
Other recombinant strategies involving either adhesin or
GTF constructs, with or without mucosal adjuvant sequences,
have been shown to induce immune responses to these func-
tional domains which could be ultimately protective in caries
vaccine applications. Chimeric proteins, in which short
sequences from predicted catalytically active regions of GTF
were combined with cholera toxin (Dertzbaugh et al., 1990) or the
B subunit of CT (Laloi et al., 1996) and expressed in E. coli HB101,
gave rise to immune responses which could inhibit as much as
50% of GTFB activity. Yu and co-workers (1997) designed a
fusion protein which contained both a 281-residue saliva-bind-
ing alanine-rich region of S. mutans Ag I/II and a 392-residue
glucan-binding domain of GTF-I. A recombinant fusion protein,
expressed in E. coli XL1-Blue, induced IgG antibody in rabbits or
in Holstein cows (Oho et al., 1999) which could inhibit glucan
synthesis by GTF and sucrose-independent and -dependent
adhesion of S. mutans to saliva-coated hydroxyapatite beads.
Constructs involving the attenuated human S. typhi vector
would be expected to have more potential for human vaccine
applications than would S. typhimurium, which is a murine
pathogen. In this regard, attenuated S. typhi CVD908 strains
have been prepared to express peptide chimeras in which GTF
sequences, associated with the glucan-binding domain, are
combined with tetanus toxin fragment C for immunogenicity
(unpublished observations).
(VI) Conjugate Vaccines
Another vaccine approach which may intercept more than one
aspect of mutans streptococcal molecular pathogenesis is the
chemical conjugation of functionally associated protein/peptide
components with bacterial polysaccharides. Added to the value
of including multiple targets within the vaccine is that the con-
jugation of protein with polysaccharide enhances the immuno-
genicity of the T-cell-independent polysaccharide entity. This
principle was first demonstrated by Landsteiner (1936) and
Avery and Goebel (1929) and has been applied with great suc-
cess in the Hemophilus influenzae type b (Hib) conjugate vaccines
to induce protective immunity to the capsular polysaccharide of
H. influenzae in infants and young children. Two groups have
applied this approach to dentally relevant components. Lett and
co-workers (1994) covalently coupled an adhesin-associated 14-
mer synthetic peptide to the serogroup f polysaccharide of S.
mutans strain OMZ 175 by reductive amination. Subcutaneous
injection with the conjugate induced systemic IgM and IgG anti-
body responses to both peptide and polysaccharide which could
be boosted upon subsequent injection. The presence of both B-
341
and T-epitopes in the peptide was required for effective respons-
es. Intragastric intubation of the conjugates associated with lipo-
somes induced primary and secondary salivary IgA antibody to
both components (Lett et al., 1995). In separate studies, Taubman
and co-workers (1998, 1999) have reported that conjugation of
either tetanus toxoid or S. sobrinus GTF to the water-soluble glu-
can synthesized by GTF significantly enhanced serum IgG and
salivary IgA antibody levels to the water-soluble glucan and to
the conjugated protein. Serum GTF inhibitory activity was also
improved by conjugation. The relative protective capacity of
either conjugate approach has yet to be tested. Since initial S.
mutans infection occurs at an age (< 2 yrs) when children are
unable to mount significant anti-polysaccharide responses, these
approaches will be especially important if conjugate vaccines are
shown to enhance the level of protection significantly over that
achieved with protein-based vaccines.
(VII) Routes to Protective Responses
Mucosal applications of dental caries vaccines are generally
preferred for the induction of secretory IgA antibody in the sali-
vary compartment, since this immunoglobulin constitutes the
major immune component of major and minor salivary gland
secretions. Many investigators have shown that exposure of
antigen to mucosally associated lymphoid tissue in the gut,
nasal, bronchial, or rectal site can give rise to immune respon-
ses not only in the region of induction, but also in remote loca-
tions. This has given rise to the notion of a “common mucosal
immune system” (Mestecky, 1993). Consequently, several
mucosal routes have been used to induce protective immune
responses to dental caries vaccine antigens.
(A) ORAL
Many of the earlier studies relied on oral induction of immu-
nity in the gut-associated lymphoid tissues (GALT) to elicit
protective salivary IgA antibody responses. In these studies,
antigen was applied by oral feeding, gastric intubation, or in
vaccine-containing capsules or liposomes. Although the oral
route was not ideal for reasons including the detrimental
effects of stomach acidity on antigen, or because inductive
sites were relatively distant, experiments with this route estab-
lished that induction of mucosal immunity alone was suffi-
cient to change the course of mutans streptococcal infection
and disease in animal models (Michalek et al., 1976; Smith et
al., 1979) and humans (Smith and Taubman, 1987).
(B) INTRANASAL
More recently, attempts have been made to induce protective
immunity in mucosal inductive sites that are in closer anatom-
ical relationship to the oral cavity. Intranasal installation of
antigen, which targets the nasal-associated lymphoid tissue
(NALT) (Brandtzaeg and Haneberg, 1997), has been used to
induce immunity to many bacterial antigens, including those
associated with mutans streptococcal colonization and accu-
mulation. Protective immunity after infection with cariogenic
mutans streptococci could be induced in rats by the IN route
with many S. mutans antigens or functional domains associat-
ed with these components. Protection could be demonstrated
with S. mutans AgI/II (Katz et al., 1993), the SBR of AgI/II
(Hajishengallis et al., 1998), a 19-mer sequence within the SBR
(Takahashi et al., 1991), the glucan-binding domain of S. mutans
GTF-B (Jespersgaard et al., 1999), S. mutans GbpB (Smith et al.,
342 Crit Rev Oral Biol Med
1997a), and fimbrial preparations from S. mutans (Fontana et al.,
1999), with antigen alone or combined with mucosal adjuvants.
(C) TONSILLAR
The ability of tonsillar application of antigen to induce
immune responses in the oral cavity is of great interest.
Tonsillar tissue contains the required elements of immune
induction of secretory IgA responses (van Kempen et al., 2000),
although IgG, rather than IgA, response characteristics are
dominant in this tissue (Boyaka et al., 2000). Nonetheless, the
palatine tonsils, and especially the nasopharyngeal tonsils,
have been suggested to contribute percursor cells to mucosal
effector sites (Brandtzaeg, 1996), such as the salivary glands. In
this regard, Fukuizumi and co-workers (1999) have shown
that topical application of formalin-killed S. sobrinus cells in
rabbits can induce a salivary immune response which can sig-
nificantly decrease the consequences of infection with cario-
genic S. sobrinus. Interestingly, repeated tonsillar application of
particulate antigen can induce the appearance of IgA anti-
body-producing cells in both the major and minor salivary
glands of the rabbit (Inoue et al., 1999).
(D) MINOR SALIVARY GLAND
The minor salivary glands populate the lips, cheeks, and soft
palate. These glands have been suggested as potential routes for
mucosal induction of salivary immune responses (Crawford et
al., 1975; Schroeder et al., 1983), given their short, broad secreto-
ry ducts that facilitate retrograde access of bacteria and their
products (Nair and Schroeder, 1983), and given the lymphatic
tissue aggregates that are often found associated with these
ducts. Experiments in which S. sobrinus GTF was topically
administered onto the lower lips of young adults have suggest-
ed that this route may have potential for dental caries vaccine
delivery. In these experiments, those who received labial appli-
cation of GTF had significantly lower proportions of indigenous
mutans streptococci/total streptococcal flora in their whole sali-
va during a six-week period following a dental prophylaxis,
compared with a placebo group (Smith and Taubman, 1990).
(E) RECTAL
More remote mucosal sites have also been investigated for their
inductive potential. For example, rectal immunization with
non-oral bacterial antigens such as Helicobacter pylori
(Kleanthous et al., 1998) or Streptococcus pneumoniae (Hvalbye et
al., 1999), presented in the context of toxin-based adjuvant, can
result in the appearance of secretory IgA antibody in distant
salivary sites. The colo-rectal region as an inductive location for
mucosal immune responses in humans is suggested from the
fact that this site has the highest concentration of lymphoid fol-
licles in the lower intestinal tract. Preliminary studies have
indicated that this route could also be used to induce salivary
IgA responses to mutans streptococcal antigens such as GTF
(Lam et al., 2001). One could, therefore, foresee the use of vac-
cine suppositories as one alternative for children in whom res-
piratory ailments preclude intranasal application of vaccine.
(VIII) Adjuvants and Delivery Systems
for Dental Caries Vaccines
Mucosal routes of antigen delivery often require additional com-
ponents which can potentiate aspects of the immune response to
induce sufficient antibody to achieve a protective effect.
13(4):335-349 (2002)
 (A) CHOLERA AND E. COLI
HEAT-LABILE ENTEROTOXINS
Cholera toxin (CT) is a powerful mucosal immunoadjuvant
which is frequently used to enhance the induction of mucosal
immunity to a variety of bacterial and viral pathogens in animal
systems. CT is an ADP-ribosylating bacterial toxin with A and B
subunits. The non-toxic pentameric B subunit binds to ganglio-
side receptors on target cells. The toxic A subunit is then translo-
cated into the cell, where this enzymatically active peptide trans-
fers the ADP ribose group of NAD to a GTP-binding protein, ini-
tiating the toxic effects by increasing intracellular levels of cAMP.
The adjuvant effects of CT (reviewed in Freytag and Clements,
1999) are broad-based and can include increased mucosal epithe-
lial cell and macrophage production of pro-inflammatory
cytokines, up-regulation of B7-2 co-stimulatory factors on APCs,
and increased antigen transfer from the mucosal to the systemic
compartment. These effects may be, at least in part, locally man-
ifest and may involve dendritic cells as the predominating anti-
gen-presenting cell (APC) population (Porgador et al., 1997).
Mucosal application of soluble protein or peptide antigen
alone rarely results in elevated or sustained IgA responses.
However, addition of small amounts of CT or the closely relat-
ed E. coli heat-labile enterotoxins (LT) (Takahashi et al., 1996)
can greatly enhance mucosal immune responses to intragastri-
cally or intranasally applied mutans streptococcal antigens
(Russell and Wu, 1991; Martin et al., 2000) or to peptides
derived from these antigens (Smith et al., 2001b). One
approach to reducing or eliminating toxicity while maintain-
ing adjuvanticity was to remove the A subunit from the CT
complex. Mucosal immunization with the B subunit of CT,
mixed with, chemically conjugated to, or genetically fused to
either intact antigen or the presumed functional domains of
mutans streptococcal virulence components (Czerkinsky et al.,
1989; Dertzbaugh et al., 1990; Takahashi et al., 1991; Wu and
Russell, 1993; Laloi et al., 1996; Wu et al., 1997) resulted in sig-
nificant mucosal responses to the complexed antigen (and to
CT), which could be shown to be protective under certain con-
ditions (Katz et al., 1993) and long-lived (Harrod et al., 2001).
Other approaches have been sought to modify CT or LT,
since the increase in CTB-assisted immune response was signif-
icantly less than that achieved with the CT holotoxin, and since,
in some systems, the presence of a small amount of intact CT
was required for response enhancement. Hajishengallis and co-
workers (1995) were able to improve mucosal adjuvanticity by
replacing the toxic A1 (N-terminal portion) of CT with a 42-kDa
portion of the AgI/II containing the salivary-binding region
(SBR), thereby creating a chimeric protein composed of the SBR
linked to the non-toxic C-terminal A2 and B subunits of CT.
Similar enhancement, albeit by potentially different mecha-
nisms, occurred after replacement of the toxic A1 subunit of LT
(serogroup IIa) with SBR (Martin et al., 2001). Responses were
long-lasting (Hajishengallis et al., 1996b), and chimeric SBR-
CTA2/B proteins could be expressed in E. coli and in attenuat-
ed Salmonella typhimurium expression vectors (Hajishengallis et
al., 1996a,b; Huang et al., 2000) which could be shown to induce
protective immune responses in BALB/c mice when adminis-
tered intragastrically or intranasally (Huang et al., 2001).
Alternatively, detoxification of CT and LT has been
attempted with the use of site-directed mutagenesis techniques
to modify amino acid residue areas of the toxic A1 subunit
which are critical to its enzymatic activity or to the required
13(4):335-349 (2002) Crit Rev Oral Biol Med
proteolytic cleavage required for activation. Several detoxified
CT and LT mutants have been prepared which retain signifi-
cant adjuvanticity and may have potential for human use. One
such mutant, LT [LT(R192G)], obtained by substituting glycine
for arginine 192 in the A subunit (Dickinson and Clements,
1995) to interfere with trypsin-mediated cleavage, has been
shown to induce mucosal and systemic immune responses to
intranasally applied GTF peptides which were generally com-
parable with those observed with CT (Smith et al., 2001b).
(B) MICROCAPSULES AND MICROPARTICLES
Combinations of antigen in or on various types of particles have
been used in attempts to enhance mucosal immune responses.
The particularization of antigen achieved by these approaches
may increase the association of antigen with M cells overlying
inductive regions of the secretory immune system (Neutra and
Kraehenbuhl, 1992). Microspheres and microcapsules made of
poly(lactide-co-glycolide) (PLGA) have been used as local
delivery systems (Eldridge et al., 1990; Ermak et al., 1995)
because of their ability to control the rate of release, evade pre-
existent antibody clearance mechanisms, and degrade slowly
without eliciting an inflammatory response to the polymer. Oral
(Challacombe et al., 1992) or topical (Rafferty et al., 1996) immu-
nization with soluble antigen in PLGA microcapsules can lead
to enhanced mucosal responses, although the organic solvents
required for formulation can diminish the biological activity of
the antigen. Denaturation can be significantly diminished by
methods which incorporate antigen into microparticles in an
aqueous phase (Hsu et al., 1994). Intranasal immunization of
aqueously incorporated GTF-PLGA microparticles, which also
contained 1% gelatin as bioadhesive, induced long-lasting sali-
vary immune responses (Smith et al., 2000). Starch microparti-
cles can also be used to increase mucosal responses to soluble
antigens. Montgomery and Rafferty (1998) have shown that top-
ical administration to the oral mucosa of bioadhesive degrad-
able starch microparticles containing dinitrophenyl-bovine
serum albumen, in combination with L-a-lysophosphatidyl-
choline (as a penetration enhancer) and interleukins IL-5 and IL-
6, potentiated long-lived salivary IgA responses, compared with
antigen delivered in soluble form.
(C) LIPOSOMES
Liposomes, which are phospholipid membrane vesicles manu-
factured to contain and deliver drugs and antigens, have been
used to enhance mucosal responses to mutans streptococcal
carbohydrate (Childers et al., 1990-1991) and GTF (Childers et
al., 1996). Liposomes are thought to improve mucosal immune
responses by facilitating M cell uptake and delivery of antigen
to lymphoid elements of inductive tissue (Childers et al., 1990).
Gastric intubation of rats with GTF-liposome vaccines induced
responses which diminished dental caries caused by subse-
quent infection with S. mutans (Childers et al., 1996). Clinical
studies comparing intranasal immunization with GTF-lipo-
somes vs. GTF alone showed that both vaccines increased local
(nasal) and salivary IgA antibody responses to GTF up to five-
fold (Childers et al., 1999). Only the nasal-wash IgA1 antibody
response was higher with the liposome-containing, compared
with the protein-alone, vaccine.
(D) OTHER APPROACHES
Other methods to enhance mucosal responses are being adapt-
ed for dental caries vaccine use. Monophosphoryl lipid A,
343
when administered intranasally to mice as an aqueous formu-
lation with soluble GTF or incorporated into liposomes con-
taining GTF, induced primary and secondary salivary IgA
responses which exceeded those achieved with GTF-lipo-
somes (Childers et al., 2000). Oligodeoxynucleotides contain-
ing unmethylated CpG dinucleotides (CpG ODN) induce pro-
liferation of B-cells and activation of macrophages. Intranasal
or oral administration of CpG ODN with tetanus toxoid (TT)
enhanced murine IgA responses in many mucosal secretions,
including saliva (McCluskie and Davis, 2000). Similarly, intra-
gastric administration of CpG ODN with TT- Al(OH)3
enhanced systemic IgG responses to TT, compared with those
achieved with TT - Al(OH)3 (Eastcott et al., 2001).
(IX) Past, Present, and Future Human Applications
(A) ACTIVE IMMUNIZATION
Few clinical trials have been performed to examine the protective
effect of active immunization with dental caries vaccines contain-
ing defined antigens. However, several studies have shown that
mucosal exposure of humans to immunization with glucosyl-
transferases from S. mutans or S. sobrinus can lead to the forma-
tion of salivary IgA antibody, albeit at modest levels. Childers and
co-workers (1994) orally immunized adults using enteric-coated
capsules filled with crude S. mutans GS-5 GTF antigen prepara-
tions contained in liposomes. Parotid salivary IgA antibody
responses, primarily of the IgA2 subclass, were induced in five of
seven subjects. Similarly, nasal immunization with dehydrated
liposomes containing this GTF preparation induced significant
IgA1 antibody response in nasal washes (Childers et al., 1997,
1999). Parotid salivary antibody levels to GTF were of lower mag-
nitude. In earlier studies, this group (Childers et al., 1990-1991)
showed that oral administration of capsules containing the puri-
fied serotype carbohydrate antigen of S. mutans in liposomes
gave rise to low but detectable levels of salivary antibody.
Smith and Taubman (1987, 1990) reported that mucosal
immunization with GTF could influence the re-emergence of
mutans streptococci in young adults after a dental prophylax-
is. Levels of parotid salivary IgA antibody to GTF increased
after oral immunization with S. sobrinus GTF in enteric cap-
sules, administered together with aluminum phosphate.
Immunization under this protocol delayed the re-accumula-
tion of indigenous oral mutans streptococci, compared with a
placebo group given buffer-filled capsules. A delay in mutans
streptococcal re-emergence was also observed after topical
administration of GTF on the lower lip, although this protocol
did not result in a significant detectable increase in antibody to
the vaccine (Smith and Taubman, 1990). Taken together, these
studies support the hypothesis that mucosal immunization
with dental caries vaccines could be protective, especially in
pediatric populations where mutans streptococci is not yet a
permanent member of the dental biofilm.
(B) PASSIVE IMMUNE APPROACHES
Passive antibody administration has also been examined for
effects on indigenous mutans streptococci. Mouthrinses con-
taining bovine milk (Filler et al., 1991) or hen egg yolk IgY
(Hatta et al., 1997) antibody to S. mutans cells led to modest
short-term decreases in the numbers of indigenous mutans
streptococci in saliva or dental plaque.
Longer-term effects on indigenous flora were observed
344 Crit Rev Oral Biol Med
after topical application of mouse monoclonal IgG or trans-
genic plant secretory SIgA/G antibody, each with specificity
for Ag I/II (Ma et al., 1990, 1998). In these experiments, teeth
were first treated for nine days with chorohexidine. Following
anti-bacterial treatment, antibody was topically applied for
three weeks. Recolonization with mutans streptococci did not
occur for at least two years after treatment of subjects with
mouse monoclonal antibody or at least 4 months after treat-
ment with the transgenic antibody to the Ag I/II epitope. In
contrast, the teeth of all subjects topically treated with non-
specific monoclonals were re-colonized with mutans strepto-
cocci by 82 days in the former experiment and by 58 days in
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-
body may be more efficacious because of its apparent
increased survival time in the oral cavity, compared with IgG,
as well as the increased avidity emanating from its tetravalen-
cy (Ma et al., 1998). The explanation for the long-term effects
on mutans streptococcal colonization after a relatively short
exposure to antibody remains unresolved. Apparently, anti-
body blockage of an important adhesin epitope during the
reconstruction of the dental biofilm following chlorohexidine
treatment places indigenous mutans streptococci at an insur-
mountable competitive disadvantage for recolonization. An
interesting parallel may be the observation that young chil-
dren who do not become naturally infected with mutans strep-
tococci during the “window of infectivity” remain unde-
tectably infected for several years (Caufield et al., 1993; Smith
et al., 1998a), potentially because its niche in the dental biofilm
has been filled by other indigenous flora.
Experimental passive immune protection could also be
achieved with antibody to GTF (Hamada et al., 1991) or GbpB
(Smith et al., 2001a). Thus, topical or dietary administration of
immune reagents with specificity for epitopes on these pro-
teins may also have potential human application.
(C) PROSPECTS AND CONCERNS
Traditional vaccine therapy indicates that immunization
should take place prior to infection. Given the apparent pat-
tern of mutans streptococcal colonization and the association
of these organisms with disease, this would suggest that
immunization for dental caries should begin early in the sec-
ond year of life for those populations under “normal” risk for
infection. Our understanding of the mucosal immune
response indicates that such children are competent to mount
secretory responses to protein antigens, although their ability
to respond to polysaccharide determinants in mucosal appli-
cations of conjugate vaccines is as yet unknown. If bacterial
colonization of the dental biofilm is complete after eruption of
all primary teeth, and if one can, through immunization, pre-
vent mutans streptococcal colonization prior to this period,
then the benefit of early immunization might extend until sec-
ondary teeth begin to erupt, exposing new ecological condi-
tions. Subsequent immunization could then be initiated.
Clearly, several possible dental caries vaccine approaches
may have application in pediatric clinical trials. Several
mutans streptococcal components have been shown to protect
in animal model systems. Protective epitopes of these compo-
nents continue to be identified and incorporated into designs
to increase their protective value. Several routes of adminis-
tration can induce protective immune responses, at least in
13(4):335-349 (2002)
model systems. Also, significant progress is being made in the
adjuvant field to remove the toxic properties of powerful
mucosal adjuvants while maintaining their adjuvant proper-
ties. Animate (attenuated bacterial vectors) and inanimate
(liposomes, microparticles) delivery systems have been identi-
fied to provide more efficient targeting of the vaccine. Both
passive and active immunization approaches have demon-
strated success in animal models and human clinical trials.
With all these apparently effective pre-clinical approaches,
one may ask, “What is the ideal dental caries vaccine
approach?” Ideally, one would favor a vaccine that would give
broadest coverage to intercept infection by all common cario-
genic mutans streptococcal strains, one that would work for
both low- and high-risk populations, one whose immunity
would last through the critical primary and secondary infec-
tion periods, one that could be given with, or as part of, other
immunizations, one that could be given by various routes and
still be effective, one that would be inexpensive, one that could
be delivered by individuals with little training, one that might
provide secondary immunity to others in the population who
were not themselves immunized. Can or should we expect all
of these characteristics in one vaccine?
If we accept that each approach could give a reasonable
level of protection in humans, one still needs to consider for
whom and under what circumstances the dental caries vaccine
is intended. For example, the ideal vaccine application for a
child with asthma, the second most common chronic childhood
ailment, may be at a site (e.g., rectal) and with an adjuvant (e.g.,
detoxified CT or LT) that is quite different from that sufficient to
give a protective response (e.g., intranasal) to a healthy child.
Also, from economic and societal standpoints, a vaccine strate-
gy for children to whom the full advantages of pediatric care are
available may not be the ideal approach for children with limit-
ed health care access. For the former group of children, perhaps
an intranasal application of a GTF or AgI/II-based vaccine may
be best, provided the child is otherwise healthy and given the
ability of parents to make the office visits required to receive
multiple immunizations with a plethora of vaccines. In contrast,
for the child in the barrio, the ideal vaccine may be an attenuat-
ed Salmonella vaccine delivery system that contains vaccine epi-
topes for multiple infectious diseases (e.g., mutans streptococcal,
Salmonella, and rotovirus infections) to induce protective
responses to several childhood diseases and to provide “herd
immunity” by shedding the vaccine strain. Since the thrust of
the WHO vaccine effort is to reduce, rather than increase, the
number of different immunizations that a child receives, an
approach that combines epitopes from several vaccines is likely
to be perceived as more desirable for global application. In fact,
a “living” Salmonella vaccine choice may also have broader
application, given our increasing societal dependence on pedi-
atric daycare and the resulting possibilities for “herd immunity”
in this setting. Thus, features inherent in the vaccine content,
type, and method of application, in vaccine cost to manufacture
and in manpower to deliver, in vaccine acceptance within and
caries risk of the target populations, as well as the local realities
of vaccine therapy—all suggest that more than one vaccine
approach may ultimately be optimal for human use.
What are the unanswered questions? We base our hypothe-
ses about the timing, route, antigen, and delivery vehicles for
response on pre-clinical studies and on studies of human
mucosal (salivary) immune responses to indigenous flora.
However, at present, the nature of the immune response to
13(4):335-349 (2002) Crit Rev Oral Biol Med
mucosal immunization with non-replicating antigens by any
route in the target pediatric population is essentially unknown,
since this is new territory in human vaccine therapy. Dental
caries vaccines would be the first non-living vaccine to be
applied by any mucosal route during the first three years of life.
Given that we would be able to induce a detectable mucosal
response in this hypothetical pediatric clinical trial, we do not
know what the coverage will be. Longitudinal studies in fami-
lies suggest that siblings who are presumably challenged with
the same parental strains of mutans streptococci can demon-
strate different responses to dominant GTF and AgI/II antigens.
Thus, a multi-component vaccine may be needed to ensure
broadest coverage. Also, high-risk populations who have ele-
vated familial exposure to mutans streptococci, coupled with
environmental conditions favoring colonization, may require a
different approach than populations with “normal” risk for
infection. Such high-risk populations may require both active
and passive mechanisms for protection. Pediatric clinical trials
in each of these types of populations with, potentially, more
than one type of vaccine are likely to be required to provide
insights into the best vaccine strategy(ies) for broadest protec-
tion of the respective populations. Also, understanding the sig-
nals for colonization and growth of cariogenic streptococci in
dental biofilms may help us devise more refined and informed
techniques to “lock out” those bacteria that can cause us harm.
Acknowledgments
The author’s research was performed through the generous support of the
National Institute of Dental and Craniofacial Research (DE-01653, DE-
04733, DE/AI-12324). The author is also grateful to Leigh Barnes for assis-
tance with manuscript preparation.
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