certain Gram-negative bacteria have earned a special place in
mucosal immunology as potent immunomodulating agents or
adjuvants. (Other examples include pokeweed mitogen, keyhole
limpet hemocyanin, cobra venom factor, and serum from an unborn
calf!) Adjuvants are defined as materials that are co-administered
with antigens in vaccines to enhance the desired immune response.
The generation of immune responses in mucosal secretions such as
saliva, where secretory immunoglobulin A (S-IgA) is the dominant
isotype of antibody, requires the stimulation of the mucosal immune
system through its inductive sites. The best-known of these sites are
the intestinal Peyer's patches; others include organized mucosa-
associated lymphoid tissues of similar structure in the large bowel,
and the tonsils and adenoids that form Waldeyer's ring in the human
pharynx. The mechanisms by which antigens applied to these sites
are taken up through specialized epithelial cells (M cells) and
delivered to underlying antigen-presenting cells (APC)—leading to
the stimulation of IgA-committed B-cells that then emigrate and
circulate until homing to the effector sites of mucosal immunity,
such as the gut lamina propria or salivary glands—have been
extensively reviewed elsewhere (Mestecky, 1987; Mestecky et al.,
2005). S-IgA is the product of polymeric (p) IgA-secreting plasma
cells that come to reside in the subepithelial spaces of these effector
sites, after pIgA has been transported through the epithelial cells by
means of the polymeric Ig receptor, which becomes the secretory
component of S-IgA in the process of transcytosis. However, it has
become clear that most foreign antigenic material applied to the
mucosal-inductive sites—the great bulk of which consists of
harmless food and other environmental antigens, as well as
commensal micro-organisms—elicits little or no immune response,
or may even induce specific immune tolerance. Effective induction
of active immune responses requires the stimulation of cells of the
innate immune system, including the major APC, such as dendritic
cells and macrophages, by arrays of molecular entities typically
found in microbes, whether pathogenic or not, and therefore
designated "pathogen- (or, better, microbe-) associated molecular
patterns" (PAMP; MAMP) (Janeway and Medzhitov, 2002). These
engage pattern-recognition receptors (PRR) on the APC and deliver
co-stimulatory signals that result in cellular activation and
generation of responses in T- and B-lymphocytes, ultimately
leading to the production of antibody or cytotoxic T-cells. The
development of vaccines critically depends upon the use of
adjuvants that can mimic the stimulus provided by these MAMPs.
This is especially true for vaccines that must be administered by a
mucosal route to induce S-IgA responses in secretions such as
saliva, since non-replicating antigens delivered orally or
intranasally readily induce tolerance rather than active immunity.
Moreover, the majority of antigenic material consumed orally is
degraded by the digestive processes. Several strategies have been
mong the "witch's kitchen" of natural products exploited by
immunologists, the diarrheagenic heat-labile enterotoxins of
The heat-labile enterotoxins, such as cholera toxin (CT), and
the labile toxins types I and II (LT-I and LT-II) of
Escherichia coli have been extensively studied for their
immunomodulatory properties, which result in the
enhancement of immune responses. Despite superficial
similarity in structure, in which a toxic A subunit is coupled
to a pentameric binding B subunit, different toxins have
different immunological properties. Administration of
appropriate antigens admixed with or coupled to these toxins
by oral, intranasal, or other routes in experimental animals
induces mucosal IgA and circulating IgG antibodies that have
protective potential against a variety of enteric, respiratory, or
genital infections. These include the generation of salivary
antibodies that may protect against colonization with mutans
streptococci and the development of dental caries. However,
exploitation of these adjuvants for human use requires an
understanding of their mode of action and the separation of
their desirable immunomodulatory properties from their
toxicity. Recent findings have revealed that adjuvant action is
not critically dependent upon the enzymic activity of the A
subunits, and that the isolated B subunits may exert different
effects on cells of the immune system than do the intact
toxins. Interaction of the toxins with immunocompetent cells
is not exclusively dependent upon their conventional
ganglioside receptors. Immunomodulatory effects have been
observed on dendritic cells, macrophages, CD4+and CD8+T-
cells, and B-cells. Numerous factors—including the precise
form of the toxin adjuvant, properties of the antigen, whether
and how they are coupled, route of administration, and
species of animal model—affect the outcome, whether this is
enhanced humoral and cellular immunity, or specific induced
tolerance toward the antigen.
KEY WORDS: cholera toxin, heat-labile enterotoxin,
vaccine, adjuvant, immune response.
Received March 31, 2005; Accepted August 3, 2005
Immunomodulation with Enterotoxins for the
Generation of Secretory Immunity or Tolerance:
Applications for Oral Infections
G. Hajishengallis1,5, S. Arce2,4, C.M. Gockel2,4,
T.D. Connell2,4, and M.W. Russell2*,3,4
1Department of Microbiology, Immunology, and Parasitology, and
Center of Excellence in Oral and Craniofacial Biology, Louisiana State
University Health Sciences Center, New Orleans, LA, USA;
Departments of 2Microbiology and Immunology and 3Oral Biology,
4The Witebsky Center for Microbial Pathogenesis and Immunology,
University at Buffalo, 3435 Main Street/Farber 138, Buffalo, NY 14214,
USA; 5present address, University of Louisville, Center for Oral Health
and Systemic Disease, Louisville, KY, USA; *corresponding author,
J Dent Res 84(12):1104-1116, 2005
CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE
J Dent Res 84(12) 2005 Enterotoxin Adjuvants for Mucosal Immunity1105
developed in recent decades to overcome the
inherent unresponsiveness or tolerance of
mucosal immune tissues to non-replicating
antigens and thereby elicit potent mucosal (and
systemic) immune responses (Table 1; Mestecky
et al., 1997; Ogra et al., 2001). Some of the most
effective of these approaches involve the use of
heat-labile enterotoxins, such as cholera toxin
(CT), either as adjuvants or as coupled delivery
agents; these are the subject of this review.
CT produced by Vibrio cholerae, and the
closely related heat-labile toxin-I (LT-I)
produced by enterotoxic strains of Escherichia
coli are examples of type I enterotoxins that share
homologous pentameric B subunits, and a highly
homologous, non-covalently associated A
subunit (Fig.). The type II heat-labile toxins (LT-
IIa and LT-IIb) produced by certain strains of E.
coli differ mainly in their B subunits. The B
subunits in themselves are non-toxic in vivo, but
bind avidly to ganglioside receptors, which are
membrane glycolipids found on most cells, and
deliver the A subunits into the cells. The A
subunits are the toxic moiety and possess ADP-
ribosyltransferase activity, which permanently
activates the Gs? component of adenylate
cyclase, leading to unregulated elevation of
intracellular cyclic AMP (cAMP). In gut
epithelial cells, this results in massive secretion
of electrolytes and water—hence the voluminous
diarrhea typical of cholera, or, to a lesser degree,
traveler's diarrhea due to enterotoxic E. coli. However,
convalescents recovering from these afflictions develop potent
mucosal S-IgA and circulating IgG toxin-neutralizing
antibodies, demonstrating that these toxins are excellent
mucosal immunogens. Oral vaccines against cholera and
traveler's diarrhea have been developed containing the pure
(recombinant) B subunits of CT or LT-I; these have been shown
to be safe and effective in humans (Clemens et al., 1991; Åhrén
et al., 1998). Furthermore, experiments in mice and other
laboratory animals have established that these holotoxins are
potent mucosal adjuvants when admixed with other protein
antigens and administered orally, intranasally, or
transcutaneously, leading to the generation of strong mucosal S-
IgA and circulating IgG antibodies against the co-administered
protein (Gockel et al., 2000; Elson and Dertzbaugh, 2005).
Thus, CT and LT-I have come to be regarded as the "gold
standards" of mucosal immunogens and adjuvants. However, if
they are to be exploited in the development of human vaccines,
it is imperative that the useful immuno-enhancing properties be
separated from the harmful toxic activities. This has been
accomplished in a variety of experimental systems (Table 2),
notably including, in the context of this review, efforts to
develop a vaccine against dental caries using antigens derived
from mutans streptococci (Russell et al., 2004).
TYPE I AND TYPE II ENTEROTOXINS AS
The heat-labile enterotoxins of E. coli and V. cholerae belong to a
family of structurally related proteins that cause diarrhea in
humans and animals, and can be classified into two major groups
based on genetic, biochemical, and immunological characteristics.
Table 1. Strategies for Enhancing Mucosal Vaccination
Mucosal adjuvantsHeat-labile enterotoxins: CT, LT-I, LT-IIa, LT-IIb
Coupling to carriers Enterotoxin B subunits: CT-B, LT-I-B, LT-IIa-B, LT-IIb-B
(chemical conjugates or genetic constructs)
and other physical
Biodegradable polymers [e.g., poly(lactide-co-glycolide)]
Live bacterial vectors Attenuated Salmonella, etc.
Commensals (lactobacilli, oral streptococci)
Live viral vectorsVaccinia
Edible plant vectorsPotatoes
Figure. Molecular structure of heat-labile enterotoxins. A and B
polypeptides are translated from a single mRNA, with an excess of B
polypeptides. The leader sequences of both allow them to be transported
into the periplasm, where B subunits assemble into pentamers. A
disulfide bridge spans the junction of the A1 and A2 segments of the A
polypeptide, where a proteolytic nick occurs. The A2 subunit non-
covalently associates with the central pore in the B pentamer.
1106 Hajishengallis et al. J Dent Res 84(12) 2005
The type I subfamily consists of CT, E. coli LT-I, and
antigenically related enterotoxins from several other enteric
bacteria, whereas the type II subfamily consists of the
antigenically cross-reactive LT-IIa and LT-IIb, which are
expressed by strains of E. coli isolated from food products,
animals, or humans with diarrhea (Holmes et al., 1995).
Significant structural differences between members of the type I
and type II subfamilies are demonstrated in neutralization assays.
Antisera against CT and LT-I will not neutralize LT-IIa or LT-
IIb, and vice versa (Holmes et al., 1995). Both types of
enterotoxins are oligomeric proteins composed of a single A
polypeptide that is non-covalently bound to a pentameric array of
B polypeptides (Fig.) (Mekalanos et al., 1979; Gill et al., 1981).
The A1 subunit, which is formed by proteolytic cleavage and
reduction of an intrachain disulfide bond in the A polypeptide, is
the catalytic moiety, while the A2 subunit, which is the C-
terminal tail of the A polypeptide, is inserted into the central hole
of the ring-shaped B pentamer. Binding of the enterotoxins to
specific receptors on the plasma membrane of target cells is
mediated by the B polypeptides (Mekalanos et al., 1979; Fukuta
et al., 1988; Merritt et al., 1994). A consensus endoplasmic
reticulum (ER) retention signal, e.g., (K/R)D(E/N)L, which is
thought to be important in toxic activity, is located at the C-
terminus of the A2 subunits of CT, LT-I, LT-IIa, and LT-IIb (Gill
et al., 1981). Mutants of CT with altered ER signals exhibit
decreased signal transduction capability and, upon uptake, are
more slowly transported to the basolateral membrane of polarized
human epithelial cells (Lencer et al., 1995; Lencer and Tsai,
2003). Residues within the ER retention signal also stabilize the
oligomeric structure of the type I enterotoxins, probably by
preventing dissociation of the A2 region of the A polypeptide
from the central pore of the B pentamer (Rodighiero et al., 1999).
While the A polypeptides of the type I and type II
enterotoxins are highly homologous, significant divergence in
amino acid sequences is observed among
the B polypeptides (Pickett et al., 1986;
Holmes et al., 1995). The B polypeptides
of CT and LT-I exhibit over 80% amino
acid identity. As a result, the toxicity of
CT can be neutralized by anti-LT-I
antibodies, and vice versa (Guth et al.,
1986; Pickett et al., 1986; Finkelstein et
al., 1987; Holmes et al., 1990, 1995). In
contrast, the B polypeptides of LT-IIa and
LT-IIb have little homology (< 14%
sequence identity) to those of CT or LT-I,
and are also antigenically distinct from,
although cross-reactive with, each other
(Holmes et al., 1995).
The major receptors for the type I and type
II heat-labile enterotoxins are ganglio-
sides, a complex family of glycosphingo-
lipids which are normal components of the
eukaryotic plasma membrane (Sonnino et
al., 1986), and which participate in various
cellular functions, including signal
transduction (Nagai and Iwamori, 1984;
Fishman, 1986; Hannun and Linardic,
1993). Over 100 molecular species of
gangliosides have been identified in numerous mammalian
tissues, and each cell type appears to express a distinct subset of
gangliosides (Nagai and Iwamori, 1984; Sonnino et al., 1986;
Hannun and Linardic, 1993). Initially considered to be structural
components of the plasma membrane, many gangliosides are
now thought to be constituents of signaling pathways that detect
external stimuli and transduce those signals to cytoplasmic
molecules that modulate gene transcription, cell growth, and
differentiation (Nagai and Iwamori, 1984; Sonnino et al., 1986;
Hannun and Linardic, 1993). The binding specificities of CT,
LT-I, LT-IIa, and LT-IIb for gangliosides, however, are
distinctive (Fukuta et al., 1988). CT and LT-I bind to
ganglioside GM1(GM1), although competitive studies have
shown that LT-I also binds to one or more glycoproteins
(Critchley et al., 1981; Yamada et al., 1983; Orlandi et al.,
1994); LT-IIa binds most avidly to ganglioside GD1b(GD1b),
less to ganglioside GD1a(GD1a), and has a low but measurable
affinity for GM1; and LT-IIb binds with high affinity only to
GD1a (Fukuta et al., 1988). Differences in ganglioside-binding
activity and specificity are believed to be important in
determination of the host specificity of the type I and type II
enterotoxins with regard to the animal species, tissues, and cell
types that can be intoxicated (Connell et al., 1995; Holmes et
al., 1995). Our research has revealed that their divergent
ganglioside-binding activities target the enterotoxins to different
lymphoid cell subsets (Arce et al., 2005) and determine, at least
in part, the molecular pathways by which they augment immune
responses to co-administered antigens. Immunomodulation by
heat-labile enterotoxins, therefore, is a complex process that
likely requires multiple interactions between the enterotoxins
and immunocompetent cells via gangliosides or other specific
Holoenterotoxins as Adjuvants
Because intact enterotoxins are too toxic to find application as
Table 2. Immunomodulation with Enterotoxins
Detoxified mutant A subunit holotoxins
[e.g., LT-I(S63K), LT-I(A72R), LT-I(R192G)]
Residual toxicity? Neural uptake
Non-binding mutant B subunit holotoxins
Recombinant B subunit Depends on antigen and route
B subunits used as coupled immunogen carriers:
Chemical conjugatesCumbersome, ill-defined
Peptide fusions to B monomersMay disrupt pentameric configuration
Limitations of peptides as immunogens
Chimeric Ag-A2/B5immunogensPeriplasmic transport and assembly required
J Dent Res 84(12) 2005 Enterotoxin Adjuvants for Mucosal Immunity1107
human adjuvants, it has been important to identify adjuvant
activities that are independent of the ability of their A subunits
to elevate intracellular cAMP and the resulting enterotoxic
effects. In the late 1980s and early 1990s, commercially
available preparations of biochemically purified cholera toxin B
subunit (CT-B) were effectively used as an adjuvant when
mixed with orally administered antigens (Elson and Dertzbaugh,
2005), but initial enthusiasm subsided when it was realized that
these preparations were contaminated with bioactive traces of
intact CT. As little as 0.1-1 percent contamination of CT-B with
intact CT, as typically occurs in these preparations, is sufficient
to exert a synergistic adjuvant effect by the intragastric route
(Wilson et al., 1990). Moreover, a point substitution mutant
(E112K) of LT-I, which lacked ADP-ribosylating activity, was
found to be deficient in adjuvant activity upon peroral
administration in mice (Lycke et al., 1992), suggesting that
adjuvanticity was linked to the catalytic activity of the A
subunit. However, coupling of CT-B to protein antigens was
found to potentiate induction of mucosal immune responses to
the coupled protein, compared with immunization with
uncoupled protein (McKenzie and Halsey, 1984; Czerkinsky et
al., 1989; Russell and Wu, 1991), but co-administration of intact
CT as an adjuvant was necessary, as well as coupling of antigen
to CT-B, for effectiveness by the oral route at low doses. These
pioneering studies led to the concept that the A subunit is
responsible for adjuvant action, which is inseparable from
toxicity, whereas the B subunit can provide receptor-binding
function that targets coupled antigens for uptake by
immunocompetent cells. However, subsequent studies have
shown that when antigen is coupled to CT-B in the form of
recombinant chimeric proteins (see below), these are
immunogenic without a requirement for additional holotoxin
adjuvant. Moreover, several studies have since shown that
recombinant CT-B or LT-I-B, completely lacking the A subunit,
can have adjuvant effects with some antigens, especially when
administered by the intranasal route or even orally (Verweij et
al., 1998; Wu and Russell, 1998; Plant et al., 2003). An
important consideration in the evaluation of these studies is that
recombinant proteins may be contaminated with bacterial
lipopolysaccharide (LPS) derived from the expression host
(usually E. coli), which itself can display adjuvant properties.
We have found that recombinant CT-B, having minimal LPS
content, has adjuvant activity for AgI/II of Streptococcus
mutans administered intranasally, and that 100-fold higher
concentrations of LPS are required to reveal adjuvant activity
(Wu and Russell, 1998). Similar immunomodulatory effects
have been observed by others using recombinant LT-1-B devoid
of detectable LPS (Nashar et al., 1996; Fraser et al., 2003),
including B subunits obtained from recombinant clones of
Bacillus brevis, a Gram-positive organism that does not produce
LPS (Maeyama et al., 2001; Yokomizo et al., 2002). In contrast,
studies on mucosal immunization conducted in conventional
animals cannot readily eliminate the possible synergistic
contribution of LPS, or other MAMPs derived from commensal
micro-organisms, to the adjuvant effects observed.
B Subunits as Coupled Delivery Agents
The rationale for coupling the B subunits of heat-labile
enterotoxins to protein immunogens is to confer on them
ganglioside-binding activity, thereby facilitating their uptake at
mucosal-inductive sites, such as the intestinal Peyer's patches.
Moreover, the ganglioside-binding ability of CT-B-linked
immunogens may also enhance their interactions with APC
present in the inductive sites. Antigens may be coupled to
enterotoxin B subunits in three ways: (i) by means of chemical
cross-linking agents, of which N-succinimidyl-3(2-
pyridinedithio) propionate has been widely used; (ii) by genetic
fusion of peptides directly to the B subunit at either the N or C
terminal, or even internally in an exposed loop; or (iii) by
genetic fusion of a protein to the A2 subunit in place of the A1
subunit and co-expression with the B subunit to form chimeric
proteins of the form Ag-A2/B5.
There is experimental evidence in support of the
applicability of all three strategies. Intragastric or intranasal
immunization with surface protein AgI/II of S. mutans
chemically coupled to CT-B induces significantly higher levels
of salivary IgA antibodies compared with immunization with
unconjugated AgI/II (Russell and Wu, 1991; Wu and Russell,
1993). The structure of chemical conjugates of antigens and B
subunits is not accurately known, and it may vary between
batches, even under controlled conditions. However, this
approach is applicable to a wide variety of antigens, including
polysaccharides as well as proteins (Shen et al., 2001).
Better-defined and more consistent coupling is
accomplished with recombinant DNA technology to create
fusion proteins of peptides with enterotoxin B subunits
(Sanchez et al., 1988; Clements, 1990). However, genetic
fusion of large peptides to CT-B has been found to disrupt the
structure of CT-B, inhibiting pentamer formation and GM1
binding (Dertzbaugh and Elson, 1993). The use of intervening
linkers of 6 to 10 amino acids, including two proline residues,
was proposed as an approach for overcoming this problem.
However, even these fusion proteins, consisting of LT-I-B and
the AgI/II-like protein SpaA or dextranase of Streptococcus
sobrinus, exhibit reduced GM1-binding activity compared with
uncoupled LT-I-B (Jagusztyn-Krynicka et al., 1993).
An alternative strategy for genetically linking large
polypeptides to the B subunits exploits the molecular structure
of the AB5enterotoxins (Fig.). This approach takes advantage
of the ability of the A2 subunit to insert into the ring formed by
the CT-B pentamer, and thereby to mediate non-covalent
association of CT-A with CT-B. Accordingly, other protein
Ags genetically fused to the N-terminus of CT-A2 should be
able to assemble as chimeric molecules with CT-B, thereby
creating novel immunogens. By exploiting the native
conformation of AB5enterotoxins, these holotoxin-like, non-
covalently linked constructs are less disruptive to the B subunit
pentameric structure than is direct fusion of antigens to B
subunit monomers. Such chimeric proteins were originally
made from alkaline phosphatase, mannose-binding protein, and
?-lactamase, but their immunogenicity was not assessed
(Jobling and Holmes, 1992). As a first example of the
productive use of such chimeric protein immunogens, the CT-
A1 subunit was genetically replaced by the saliva-binding
region (SBR) of S. mutans AgI/II, which was thus fused to CT-
A2, and co-expressed with CT-B to form a chimeric protein
that retained GM1-binding activity (Hajishengallis et al.,
1995). Another similar chimeric protein was constructed from
SBR and the A2/B subunits of LT-IIa (Martin et al., 2001a).
Intragastric or intranasal immunization of mice with these Ag-
A2/B chimeric proteins induces high levels of salivary IgA
antibodies to native AgI/II, even in the absence of intact toxin
1108Hajishengallis et al.J Dent Res 84(12) 2005
or other adjuvants. This suggests that ganglioside binding alone
is sufficient for enhancing the immune response to the linked
immunogen, although the extent to which the A2 subunit might
contribute to adjuvant action is currently unknown.
MECHANISMS OF ACTION
Immunomodulatory Mechanisms Mediated by
Holoenterotoxins: Role of the A Subunit
Given the crucial role of DC and other APCs (macrophages and
B-cells) in the induction of adaptive immunity (Itano and
Jenkins, 2003), the potentiation of APC function is a major
aspect of adjuvant action, including that of heat-labile
enterotoxins. Most studies have been performed with CT, which
up-regulates expression of the B7-2 (CD86) co-stimulatory
molecule, and stimulates antigen presentation through
enhancement of MHC class II expression and IL-1 production
(Bromander et al., 1991; Matousek et al., 1996; Cong et al.,
1997; Simmons et al., 2001). CT also interacts with
lymphocytes and promotes B-cell isotype-switch differentiation
toward IgG1 and IgA in mice (Holmgren et al., 1993).
Moreover, CT displays complex stimulatory or inhibitory
effects on T-cell proliferation in vitro (Elson and Dertzbaugh,
2005), which are not readily interpreted in terms of its adjuvant
effects in vivo. It is generally found that CT induces CD4+T-
cell polarization toward the Th2 phenotype (Simmons et al.,
2001), although exceptions to this rule have been reported.
Recent findings from our laboratories show that CT, LT-IIa,
and LT-IIb bind differentially to immunocompetent cells (Arce
et al., 2005), and the precise mechanisms through which these
enterotoxins enhance immune responses are being explored. It
appears that the different ganglioside-binding specificities of the
enterotoxins may be a strong determinant for their distinct
immune-regulatory effects on B-cells. Activation of B
lymphocytes by CT or LT-I stimulates antigen presentation
through induction of enhanced MHC class II expression on B-
cells, and by promoting interactions between B-cells and T-cells
via induction of CD86, LFA-1, and ICAM-1 on B lymphocytes
(Nashar et al., 1997, 2001; Arce et al., 2005). Increased contacts
between B- and T-cells may induce B-cell proliferation and
eventually differentiation of B-cells into plasma cells or
memory cells (Parker, 1993). A mechanism through which CT
possibly mediates B-cell differentiation into plasma cells is
through inhibition of CD40 ligand (CD40L; CD154) expression
on CD4+T-cells (Martin et al., 2001b). Interruption of CD40-
CD40L interactions between B-cells and T helper cells is
essential to suspend B-cell proliferation and initiate their
differentiation into plasma cells (Arpin et al., 1995; Liu and
Banchereau, 1997). LT-I can also interfere with CD40-CD40L
interactions between APC and T-cells, through its ability to
down-regulate CD40 on DC (Petrovska et al., 2003). In
contrast, LT-I can provide CD28-mediated co-stimulatory
function to T-cells through its ability to stimulate B7-1 (CD80)
expression on DC (Petrovska et al., 2003). Since LT-IIa and
LT-IIb do not influence CD40L expression on CD4+T-cells
(Martin et al., 2001b), these type II enterotoxins may promote
mainly the formation of memory B-cells, which depends upon
the continuation of CD40-CD40L interactions (Arpin et al.,
1995; Liu and Banchereau, 1997). Co-stimulation of T-cells by
CD86 expressed on B-cells preferentially elicits IL-4 production
by the T-cells, and hence induces polarization of CD4+T-cells
toward the Th2 phenotype (Freeman et al., 1995). Th2 cytokines
like IL-4, IL-5, IL-6, and IL-10 enhance humoral immune
responses by driving the survival, proliferation, and
differentiation of B-cells (Parker, 1993; Paul and Seder, 1994;
Liu and Banchereau, 1997; Hasbold et al., 1999). In addition,
IL-5 and IL-6 promote the survival of long-lived plasma cells
and the maintenance of serum antibody titers (Cassese et al.,
2003). Another mechanism through which CT induces the Th2
phenotype is by suppressing CD40-CD40L interactions between
DC and T-cells, which results in inhibition of the production of
IL-12 (p70), a cytokine which favors Th1 polarization (Martin
et al., 2001b; Braun et al., 1999). CT also inhibits IL-12 (p70)
production directly from dendritic cells and other antigen-
presenting cells, and, furthermore, it inhibits expression of the
IL-12 receptor on T-cells (Braun et al., 1999). Although Th1
cells can co-operate with B-cells to induce plasma cell
differentiation, they do not usually exhibit the full complement
of cytokines characteristic of Th2 cells, and therefore Th1 cells
are less effective than Th2 cells in providing B-cell help (Smith
et al., 2000). Conversely, the lack of these effects in LT-IIa and
LT-IIb may account for their ability to bias responses more
toward Th1, as observed in vivo especially for LT-IIb (Martin et
al., 2000). Furthermore, CT, LT-I (B subunit), or LT-IIa, but not
LT-IIb, selectively induces apoptosis in CD8+T-cells (Elson et
al., 1995; Nashar et al., 1996; Arce et al., 2005). Although when
differentiated, CD8+cells become cytotoxic T-cells, they are
also a major source of IFN?, a cytokine which is important in
driving cell-mediated immunity and promoting the development
of Th1 cells (Sad et al., 1995). This effect may contribute to the
preferential induction of Th2 responses, especially by CT, and,
in combination with other effects, may explain why LT-IIb is
biased more toward inducing Th1 responses.
The precise role of the enzyme activity of the A1 subunit in
the adjuvant action of enterotoxins is uncertain. Up-regulation
of cAMP in itself is unlikely to account for adjuvant activity,
since other agents that accomplish this (e.g., forskolin) do not
serve as adjuvants (Wilson et al., 1993). Furthermore, the toxic
effect of this activity in epithelial cells, especially in the
intestine (which leads to diarrhea), precludes use of the intact
holotoxins as adjuvants in humans. Many studies have
attempted to identify adjuvant mechanisms which are
independent of the catalytic activity of the enterotoxins by the
construction of CT or LT-I holotoxin mutants that lack ADP
ribosylation and associated toxicity, while retaining adjuvant
action in vivo. Although it is unclear how the enzymatically
inactive A subunit would support adjuvant action, one such
class of mutants, Ser to Lys substitution at position 63 in the A
subunit of LT-I (LTK63) or CT (CTK63), maintained the
ability to bind the ADP-ribosylation factor (ARF), which is
important in vesicular membrane trafficking (Pizza et al.,
2001). This ARF-binding activity is independent of the
catalytic activity of the A subunit, although it has not yet been
linked to any adjuvant mechanism. Conversely, a role for the
ADP-ribosylating activity of the A1 subunit is implied by the
adjuvant activity of a construct, designated CTA1-DD, in
which the toxic A1 subunit of CT is coupled to the Ig-binding
domain of staphylococcal protein A (Ågren et al., 1999).
However, this construct is targeted to B-cells in a manner
different from that of enterotoxin B subunits.
Comparison with intact LT-I showed that LTK63 is unable
to up-regulate surface expression of CD80 or to down-regulate
J Dent Res 84(12) 2005 Enterotoxin Adjuvants for Mucosal Immunity1109
expression of CD40 on DC in vitro, whereas LT-I inhibits the
ability of DC to present protein antigen to cognate T-cells
(Petrovska et al., 2003). In vivo, however, both wild-type LT-I
and LTK63 enhance CD80 expression in peritoneal exudate
cells, although only LT-I up-regulates CD86 expression in the
same system (Ryan et al., 2000). Moreover, LTK63 appeared
to promote Th1 responses, in contrast to wild-type LT-I or a
partially detoxified mutant (LTR72), both of which promote
Th2 responses. Thus, this study (Ryan et al., 2000) suggested
that up-regulation of CD86 and promotion of Th2 responses
require enzymatic activity. Both CT and its non-toxic E112K
mutant were found to up-regulate CD86 on B-cells and
macrophages, in contrast to CT-B (Yamamoto et al., 1999). It
may thus be inferred that up-regulation of CD86 is a property
of the CT-A subunit, independent of its catalytic activity. This
conclusion appears to contradict the study by Ryan and co-
workers (Ryan et al., 2000), although the apparent discrepancy
could be attributable to the use of different enterotoxins (LT-I
vs. CT) in different experimental systems. Moreover, similar to
intact CT, the non-toxic mutant was found to inhibit Th1-type
CD4+T-cell responses selectively, thus promoting Th2-type
immune responses. However, it is notable that the E112K
mutant of CT here seems to behave differently from the
corresponding mutant of LT-1, which lacks adjuvant activity by
the oral route (Lycke et al., 1992). This may be attributable to
different routes of immunization (intranasal vs. peroral), since
an E112K mutant of LT-I, when tested intranasally by an
independent group, also exhibited adjuvant properties (Verweij
et al., 1998). Alternatively, minor differences between the
respective A1 subunits, the properties of their B subunits, or
other extrinsic factors may be involved.
Another point mutation (R192G) in LT-I involves the
junction between the A1 and A2 segments of the A subunit.
Proteolytic cleavage at this junction is necessary to activate the
toxin, and after reduction of the disulfide bridge that spans it,
this permits dissociation of the A1 subunit from the A2/B
subunit to occur within the Golgi/ER of the targeted cells in the
mechanism of intoxication (Fujinaga et al., 2003). R192G is
thereby rendered insensitive to proteolytic activation, but it is
still an effective mucosal adjuvant and is non-toxic at adjuvant
doses (Freytag and Clements, 1999).
Although the A2 subunit in enterotoxins is structurally
important in linking the A1 subunit to the B pentamer, it is
uncertain whether it has any immunomodulating activities.
Interestingly, the A2 subunit contains an ER retention signal,
the alteration of which (in CT or LT-I mutants) interferes with
toxicity (Lencer et al., 1995), and it may therefore contribute to
adjuvant action. It is unknown whether the A2 subunit in the
SBR-CTA2/B chimeric immunogen (Hajishengallis et al., 1995)
adds to the immuno-enhancing effect mediated by the GM1-
binding activity of the B subunit. However, since SBR-CTA2/B
induces CD86 expression on B-cells, whereas SBR or CT-B
alone is inactive (Yamamoto et al., 1999; Martin et al., 2001a),
it can be surmised that CT-A2 or the CT-A2/B complex may be
involved in the observed immunostimulatory effect.
Mediated by the B Subunit
The use of highly purified recombinant B subunits of type I or
type II enterotoxins has generated evidence in support of
distinct immunomodulatory properties of the B subunits,
suggesting that these molecules may offer more than targeting
service in vaccine development. CT-B has been shown to
enhance the antigen-presenting function of macrophages
(Matousek et al., 1996), to up-regulate expression of MHC
class II molecules on B-cells (Francis et al., 1992), and to co-
stimulate IL-4 induction in Th2 cells (Li and Fox, 1996).
However, CT-B does not up-regulate expression of CD80 or
CD86 on B-cells or macrophages (Yamamoto et al., 1999). CT-
B indirectly favors Th2 responses by inducing apoptosis of
CD8+T-cells, which constitute a major source of IFN-?
production. Depletion of CD8+T-cells by apoptosis as well as
up-regulation of MHC class II on B-cells are properties also
shared by LT-I-B (Truitt et al., 1998; Williams et al., 1999).
In contrast to CT-B, however, LT-I-B induces IL-10
production and inhibits IL-12 release in monocytes (Simmons
et al., 2001), which further favors Th2-type immune responses.
In addition, LT-I-B stimulates TNF-? release in monocytes
(Turcanu et al., 2002). We similarly found that CT-B lacks
detectable cytokine-inducing capacity in human monocytic
cells or mouse macrophages, whereas in the same cells, the B
subunits of type II enterotoxins (LT-IIa-B and LT-IIb-B)
induce release of TNF-?, IL-6, and IL-1? (Hajishengallis et al.,
2004). Interestingly, the pro-inflammatory activity of these B
subunits is antagonized by their respective holotoxins, probably
through a cAMP-dependent mechanism. Strikingly, both LT-
IIa-B and LT-IIb-B depend upon Toll-like receptor (TLR) 2 for
inducing NF-?B activation and cytokine release in human cell
lines (Hajishengallis et al., 2005). Moreover, macrophages
from TLR2-deficient mice fail to release cytokines in response
to LT-IIa-B or LT-IIb-B, in contrast to wild-type or TLR4-
deficient cells. Therefore, besides their established binding to
gangliosides, the B subunits of type II enterotoxins also engage
in TLR2 interactions, which may represent a novel mechanism
whereby these molecules exert their immunomodulatory
activities. Thus, the LT-II B subunits may promote adaptive
immune responses through induction of co-stimulatory
molecules and immuno-enhancing cytokines by APC, activities
that are readily induced through TLR2 activation (Akira and
Signaling Receptors used by Enterotoxins
It has generally been assumed that the immunomodulatory
effects of the heat-labile enterotoxins depend upon ganglioside
binding, as demonstrated by the finding that a non-binding
mutant (G33D) of LT-I-B is deficient in immunogenicity
(Nashar et al., 1996; Guidry et al., 1997; Truitt et al., 1998).
However, recent evidence suggests that this may not always be
the case. For example, the binding-defective point mutant
within the B subunit of LT-IIb (T13I) strikingly retains in vivo
mucosal adjuvant activity similar to that of the wild-type
molecule (Nawar et al., 2005). Certain point mutants of CT-B
(H57A) or LT-I-B (H57S) that retain high-affinity binding to
GM1 were found defective in inducing apoptosis of CD8+cells
(Aman et al., 2001; Fraser et al., 2003). This finding conflicts
with the previous demonstration that apoptosis of CD8+cells
induced by CT-B or LT-I-B required GM1 binding (Truitt et
al., 1998; Williams et al., 1999). It could be speculated that the
structural alterations in CT-B (H57A) and LT-I-B (H57S),
while not preventing binding to GM1, may preclude
interactions with additional receptors required for signaling.
Therefore, ganglioside binding alone may not be sufficient to
mediate the immunomodulatory effects of type I B subunits.
These observations are consistent with the recently
1110Hajishengallis et al. J Dent Res 84(12) 2005
developed concept that cellular activation by microbial
molecules involves interactions with several co-operating host
receptors within membrane microdomains known as lipid rafts.
Gangliosides are known to be present in lipid rafts, which are
involved in the formation and function of signaling hetero-
oligomeric PRR-TLR complexes (Triantafilou et al., 2004).
Multiple receptors may also be involved in cellular activation
by LT-IIb-B, which binds to GD1a, leading to NF-?B
activation and cytokine induction; indeed, these activities are
diminished in a GD1a non-binding mutant (T13I) of LT-IIb-B.
NF-?B activation and cytokine induction by LT-IIb-B are also
diminished when TLR2 function is inhibited, either by
antibody or genetic deficiency of the receptor (Hajishengallis et
al., 2005). It is known that TLR signaling function is facilitated
by other PRRs, such as CD14, which mediate direct pathogen
recognition (Akira and Takeda, 2004). Thus, it is possible that
GD1a provides co-receptor function to TLR2 for cellular
activation in response to LT-IIb-B, perhaps by bringing it
within range of interaction with TLR2 in lipid rafts, analogous
to the way in which CD14 facilitates LPS recognition by
TLR4. Alternatively, the propensity of TLR2 to respond to
certain lipids attached to proteins or peptides (Takeda et al.,
2003) may enable it to recognize GD1a-bound LT-IIb-B as a
lipid-protein complex. The concept that gangliosides can co-
operate with TLRs is supported by other independent evidence.
A glycosphingolipid that binds E. coli P fimbriae through a
Gal?1-4Gal? disaccharide facilitates activation of TLR4 by P
fimbriae (Frendéus et al., 2001). Certain gangliosides (GD1a,
GD1b, and GT1b among 8 tested) serve as co-receptors with
TLR5 for the induction of human ?-defensin-2 in Caco-2 cells
by Salmonella enteritidis flagellin (Ogushi et al.,
2004). Moreover, asialoGM1 appears to function as
a co-receptor for TLR2-dependent NF-?B activation
and IL-8 induction by Pseudomonas aeruginosa in
airway epithelial cells (Soong et al., 2004).
Thus, a full understanding of the mechanisms of
heat-labile enterotoxin adjuvant action is not yet
available. Effects leading to enhanced immune
responses have been observed on DC, macrophages,
CD4+, and CD8+T-cells, and in B-cells.
Gangliosides are not the only cellular receptors
involved in host interactions, and TLRs add a new
level of complexity; possibly other receptors remain
to be identified. An absolute requirement for toxic
enzyme activity (ADP ribosylation) by the A1
subunit cannot be sustained. However, it is possible
that holotoxins, whether mutant or wild-type, and
isolated B subunits exert adjuvant activity in
different ways. It is also becoming clear that the
different toxins, especially type I and type II, have
subtly but importantly different adjuvant effects and
modes of action on immunocompetent cells. Some
of the main immunological properties of type I and
type II enterotoxins are summarized in Table 3.
TOLERANCE vs. IMMUNITY
Experiments in animals have extensively
demonstrated the ability of type I and type II
enterotoxins to enhance specific immune responses to
co-administered antigens, by mechanisms described
above, including the expansion of antigen-specific B-
and T-cells, alteration of T-cell cytokine production,
and changes in regulatory T-cells. Because their toxic
A1 moiety renders holotoxins unsuitable for human
application, the non-toxic B subunits have been
proposed as more favorable adjuvant candidates, and
evidence has been obtained to support their utility.
However, intragastric administration of antigens
chemically conjugated to pure, recombinant CT-B
has been found to induce profound tolerance to the
same antigens when subsequently injected
parenterally, whereas the addition of small amounts
of holotoxin abrogated tolerance induction (Sun et
al., 1994). Even a single intragastric dose of CT-B-
conjugated antigen was effective. Suppression of T-
cell-mediated immunity manifested by delayed-type
Table 3. Summary of the Main Immunological Properties of Heat-labile Enterotoxinsa
CT LT-I LT-IIaLT-IIb
Mucosal adjuvant effect++++
Systemic adjuvant effect++++
Major ganglioside receptorGM1GM1 GD1b
Predominant T-cell helpTh2 Th1/2 Th1/2Th1 > 2
CD40L expression on CD4+T-cells
CD40 expression on dendritic cells?
CD25 and CD69 expression on
Apoptosis of CD8+T-cells+++—
(CT and CT-B) (LT-I-B)
CD86 expression on B-cells
B-cell differentiation into plasma cells
MHC class II expression on APC
Inflammatory cytokine release by
macrophages (B subunits only)—
% of B220+cells that bind100%(+)* 30% 70%
% of CD3+cells that bind100%(+)* 65%30%
% of CD11b+and CD11c+cells that bind 100%(+)*90% 90%
Interaction with TLR2 (B subunits only)—?++
See text for discussion and references.
Symbols: + positive/yes; — no effect/none; ? increased; ? decreased;
? unknown; * positive but % of cells not known.
J Dent Res 84(12) 2005 Enterotoxin Adjuvants for Mucosal Immunity1111
hypersensitivity (DTH) reactions was nearly complete, and, most
remarkably, tolerance could be induced in animals that had been
previously sensitized to DTH, i.e., previously induced
responsiveness could be reversed. The clinical importance of this
finding, if applicable to antigens involved in auto-immune
disease, is considerable. For example, in various rodent models,
intranasal delivery of CT-B-type II collagen conjugates protects
against experimental autoimmune arthritis (Tarkowski et al.,
1999); intragastric delivery of CT-B-insulin conjugates can
prevent the induction of type 1 diabetes (Bergerot et al., 1997);
and CT-B-myelin basic protein conjugates suppress experimental
allergic encephalitis (Sun et al., 1996). In rat models of uveitis,
disease was prevented by intragastric delivery of recombinant
CT-B-peptide conjugate, by inducing tolerance through a
regulatory subset of memory cells and a shift from Th1 to Th2
and Th3 cytokines (Phipps et al., 2003). However, other reports
have shown that the general conclusion—that enterotoxin B
subunits are tolerogenic, whereas holotoxins are required to
stimulate active immunity—is unsustainable (Wu and Russell,
1998; Gockel and Russell, 2005). Several factors affect the
outcome, including the properties of the antigen involved, type
of construct, route of administration, and species of animal
model. Furthermore, tolerance can be "split", i.e., some aspects
of an immune response can be “tolerized”, while others proceed
as normal. This has been demonstrated in the concomitant
suppression of T-cell responses and the generation of antibodies,
especially in the mucosal compartment, in both mice and humans
(Challacombe and Tomasi, 1980; Husby et al., 1994). Thus,
whether B subunits are effective mucosal adjuvants when
conjugated with antigens has been a matter of controversy,
further complicated by the degree of purity of the B subunit used.
Tolerance induction requires a recombinant B subunit
devoid of the toxic enzyme activity of the A1 moiety, and is
abrogated by the addition of holotoxin. For example, mice
given ovalbumin intragastrically, with or without recombinant
LT-B or CT-B or their non-toxic mutants, became tolerant to
ovalbumin, whereas native CT or LT blocked tolerance
induction (Bagley et al., 2003). Oral immunization with
ovalbumin conjugated to recombinant CT-B has also been used
to suppress IgE-mediated allergic sensitization to ovalbumin
(Rask et al., 2000b). Many studies have used small amounts of
intact CT with recombinant CT-B or biochemically purified
CT-B containing residual traces of intact toxin to enhance
antibody responses or abrogate tolerance to the co-administered
antigen (McKenzie and Halsey, 1984; Czerkinsky et al., 1989;
Russell and Wu, 1991; Asanuma et al., 1998; Hammond et al.,
2001). However, recombinant B subunit alone has been used
successfully to induce an immune response to antigen,
suggesting that other factors contribute to the tolerogenic
outcome (Verweij et al., 1998; Wu and Russell, 1998; Rask et
al., 2000a; Wang et al., 2003). It is possible that the properties
of the antigen itself contribute to this outcome, since studies
demonstrating tolerance have often used auto-antigens, or
antigens such as ovalbumin, which readily induces tolerance,
whereas microbial antigens result in active immune responses.
Although most studies showing that the recombinant B
subunit alone induces tolerance to antigen have used the
intragastric route of delivery, it is now clear that "oral tolerance"
is a more general phenomenon, since suppression of systemic
responses can be induced by application of antigens not only
orally, but also intranasally (Waldo et al., 1994), or even
intravaginally, at least during estrus (Black et al., 2000).
However, the intranasal route of immunization appears to be
more efficient than the oral (or intragastric), since smaller doses
are required to elicit responses, and the adjuvant effect of
detoxified enterotoxin mutants or of their B subunits is more
readily demonstrable by this route. Whether this is because of
important differences in the uptake and processing of antigens
and adjuvants in NALT compared with GALT, or simply
because the vaccine materials are less subject to digestion and
come into more immediate contact with the inductive site tissues
in the nose than in the gastrointestinal tract, is unclear at present.
Immunization via intragastric, intranasal, intravaginal, or
transcutaneous routes with admixed or coupled B subunits as
adjuvant induces mucosal and systemic immune responses to
antigen across many species. Previously, studies have shown
that intragastric immunization of mice and intranasal
immunization of rats or rhesus monkeys with S. mutans AgI/II,
chemically conjugated to or admixed with CT-B, induces
antigen-specific antibodies in plasma as well as mucosal
secretions like saliva (Russell and Wu, 1991; Katz et al., 1993;
Russell et al., 1996). In humans, intranasal delivery of
recombinant CT-B admixed with whole killed vibrios elicits
CT-B-specific titers in serum, as well as genital and rectal
secretions (Kozlowski et al., 2002). Intravaginal or intranasal
immunization of mice with CT-B or antigen conjugated to CT-
B induces antibodies and antibody-secreting cells in the female
genital tract (Johansson et al., 1998). However, by comparison
with intranasal immunization, intravaginal immunization with
AgI/II-CT-B conjugate plus CT adjuvant is ineffective at
disseminating responses among remote mucosal sites (Wu et
al., 2000). Novel immunization strategies like transcutaneous
immunization have also engaged the B subunits of holotoxins
to induce antigen-specific immunity (Hammond et al., 2001).
Furthermore, the method of co-administering antigen and
adjuvant may affect the immune response to the antigen. For
example, the tolerance observed with the use of antigen
admixed with or chemically coupled to recombinant B subunits
might be overcome by the use of genetic coupling of B subunits
to antigen. Chimeric proteins prepared by genetic replacement
of the toxic A1 moiety of CT with an antigen segment of
choice have been found to be immunogenic by either
intragastric or intranasal routes, without a requirement for
additional toxin adjuvant (Hajishengallis et al., 1995; Sultan et
al., 1998; Martin et al., 2001a; Sheoran et al., 2002; Li et al.,
2004; Gockel and Russell, 2005). Using different techniques,
others have genetically coupled peptides to recombinant CT-B
and have shown that intranasal immunization induces antigen-
specific antibody production in the liver, lung, and serum, as
well as reduces T-cell-mediated pathology in schistosome-
parasitized mice (Lebens et al., 2003).
APPLICATIONS AND SIGNIFICANCE
WITH RESPECT TO ORAL DISEASE
An extensive body of literature exists on the use of
enterotoxins, especially CT and LT-I or their non-toxic
derivatives, as mucosally applied adjuvants for numerous
antigens and experimental vaccines against a wide range of
infections. Among these, and the focus of much of our work, is
the application of these approaches for the development of a
vaccine against dental caries (Russell, 1992; Russell et al.,
1999, 2004; Smith, 2002). Thus, we have demonstrated 'proof
1112Hajishengallis et al.J Dent Res 84(12) 2005
of concept', in rodent and monkey models, that intragastric or
intranasal immunization with AgI/II (or its SBR component),
coupled to CT-B, either chemically or genetically induces
salivary IgA antibodies against AgI/II that have protective
potential, and protects against oral challenge with S. mutans
and inhibits the development of caries (Hajishengallis et al.,
1992, 1998; Katz et al., 1993; Russell et al., 1996). Further
studies have shown that responses induced by these approaches
to immunization can persist and be recalled by booster
immunization during the lifetime of a mouse (Hajishengallis et
al., 1996b; Harokopakis et al., 1997; Harrod et al., 2001;
Gockel and Russell, 2005). Attempts to apply the same
approach to glucosyltransferase (GTF), which is another prime
candidate antigen for a vaccine against caries, have been
limited by the inability of E. coli to express the cloned
components in soluble form (Jespersgaard et al., 1999).
However, expression of GTF and SBR components, possibly
together with CT-B, in a live attenuated vector organism might
permit these constructs to be used effectively in a vaccine
(Hajishengallis et al., 1996a; Jespersgaard et al., 2001).
Smith and colleagues have shown, in rats, that serum IgG
and salivary IgA antibody responses to synthetic peptides
derived from GTF sequences can be induced by the co-
administration of CT, or the R192G mutant of LT-I, as an
intranasal adjuvant (Smith et al., 2001). The same adjuvants,
given together with microencapsulated GTF by the rectal route,
enhanced salivary IgA antibody responses that were associated
with diminished development of caries lesions after oral
challenge with S. sobrinus (Smith et al., 2003).
Others have used CT or LT-I, or their B subunits, either as
adjuvants or as coupling agents or genetic fusion partners for
peptides derived from antigens of mutans streptococci. The
initial demonstration of intranasal immunization by S. mutans
AgI/II (PAc) was augmented by CT-B as an adjuvant
(Takahashi et al., 1990). Subsequently, the same group showed
that intranasal immunization of mice with a synthetic peptide
representing residues 301-319 from S. mutans PAc, coupled to
CT-B, induced serum IgG antibodies to the parent antigen and
suppressed oral colonization by S. mutans (Takahashi et al.,
1991). A fusion protein representing a peptide from GTF
coupled to the N-terminus of CT-B retained the GM1-binding
capacity of CT-B and was orally immunogenic in mice
(Dertzbaugh et al., 1990). Even large protein antigens, such as
SpaA and dextranase from S. sobrinus, have been successfully
fused to the C-terminus of LT-I-B by the interposition of a 6-
residue linker sequence containing 2 prolines, and the fusion
proteins retained the antigenicity of the S. sobrinus antigens as
well as some GM1-binding activity of the LT-I-B (Jagusztyn-
Krynicka et al., 1993).
Similarly, several studies have demonstrated that immune
responses to antigens of periodontal pathogens can be induced
by oral or intranasal immunization with CT, LT-I, or LT-II as
adjuvant. Examples include Porphyromonas gingivalis
fimbriae (Connell et al., 1998; Nagasawa et al., 1999; Yanagita
et al., 1999), outer membrane protein of P. gingivalis
(Namikoshi et al., 2003), and Actinobacillus actinomycetem-
comitans polysaccharide-protein conjugate or fimbrial peptide
(Takamatsu-Matsushita et al., 1996; Honma et al., 1999), but
these studies assessed the outcomes only in terms of antibody
or cellular immune responses. A recent study, however, has
reported that intranasal immunization of mice with a
recombinant hemagglutinin/adhesin domain of P. gingivalis
gingipain (Kgp), either mixed with or conjugated to CT-B,
resulted in enhanced serum and mucosal antibody responses to
Kgp, and that the antibodies could inhibit invasion of epithelial
cells by P. gingivalis (Zhang et al., 2005).
Most applications of enterotoxin-enhanced mucosal
immunization have focused on the generation of IgA antibody
responses in secretions, and an enormous body of literature has
accumulated in which enhanced immune responses to a wide
variety of microbial antigens have been demonstrated in this
way. It should be noted that circulating IgG antibodies are also
effectively stimulated by these approaches, thereby expanding
the range of infections that can potentially be addressed by
mucosal immunization (Lycke et al., 1983; Jackson et al.,
1993). Cell-mediated immunity, including the generation of
cytotoxic T lymphocytes (CTL), has been less frequently
studied in this context; however, it is noteworthy that CTL with
activity against HIV-infected cells can be induced by mucosal
immunization with HIV peptides administered together with CT
or LT(R192G) (Porgador et al., 1997; Belyakov et al., 2000).
Given that enterotoxins and their B subunits profoundly
modulate immune responses, even responses that have already
been established by previous immunization, it is tempting to
speculate that these adjuvants, or antigens coupled to
enterotoxin B subunits, might be used to ameliorate chronic
inflammatory disease (Russell, 2003). Periodontal disease
(Russell, 1998; Russell and Sibley, 1999), inflammatory bowel
disease, and Helicobacter pylori infection (Raghavan et al.,
2002) are examples of infection-driven inflammatory
conditions that might be amenable to such an approach. In
these diseases, pathology arises, at least in part, from an
inappropriate, or dysregulated, immune response to the
provoking bacteria, that not only fails to eliminate the pathogen
but also inflicts and perpetuates inflammatory damage to host
tissues. The validity of this approach is strengthened by the
findings, discussed above, that CT and the type II enterotoxins,
or their B subunits, have differential effects on CD4+, CD8+,
and B lymphocytes that may be amenable to manipulation
aimed at reprogramming the immune response to a mode that
destroys the pathogen and alleviates damage to host tissues.
Whether this can be successfully achieved remains to be
determined. Findings that adverse immune responses in animal
models of auto-immune disease or allergy can be corrected by
the administration of the relevant antigen coupled to CT-B
suggest that it might be possible.
Human application of enterotoxins or their derivatives in
vaccines raises questions of safety, particularly with respect to
the holotoxins themselves. It is noteworthy that oral
administration of pure or recombinant CT-B has a record of
safety and acceptability, having been used as a component of a
licensed oral vaccine against cholera (Van Loon et al., 1996).
However, concerns have arisen recently over intranasal
administration of enterotoxins, because of their potential for
retrograde trafficking through the olfactory nerve, which offers
a direct pathway into the brain without an intervening synapse
(Van Ginkel et al., 2000). An intranasal vaccine against
influenza “adjuvanted” with a small dose of LT-I was
withdrawn because of its association with increased incidence
of Bell's palsy, although the mechanism underlying this adverse
effect was unclear (Mutsch et al., 2004). It remains to be
determined whether these potential problems can be
J Dent Res 84(12) 2005 Enterotoxin Adjuvants for Mucosal Immunity1113
circumvented by the use of B subunits or non-toxic derivatives
of enterotoxins as adjuvants or carriers for intranasal
Studies in the authors' laboratories are supported by US-PHS
grants from the National Institute of Dental and Craniofacial
Research: DE015254 (GH), DE013833 (TDC), and DE006746
(MWR); and in part (SA) by the Ralph Hochstetter Medical
Research Fund in honor of Dr. Henry C. and Bertha H. Buswell.
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