3. 5634 BRIEF REVIEW: MUCOSAL VACCINES AND TOLERANCE
induction of T regulatory cells, and these mechanisms are not in the average American diet at doses of 500 mg per day
mutually exclusive (8 –11). In most reports, potential suppres- ( 180g/year), persist throughout childhood, adolescence, and
sion of Ab responses was not extensively studied. An exception early adulthood, with some decrease in later life. Importantly,
may be the apparent suppression of IgE responses to environ- the levels of Abs to food Ags in sera and external secretions dis-
mental allergens. The induction of secretory IgA or IgG Abs play considerable mutual independence, indicating marked dif-
that would successfully compete with IgE Abs for a given aller- ferences in maturation and regulation of both the magnitude
gen or the selective suppression of IgE Ab responses are at the and the Ig isotype of the response within the systemic and mu-
heart of these efforts. Marked differences in the efficacy of oral cosal compartments (19).
immunotherapy may be ascribed to the animal species and to It is important to understand the context in which the mu-
the type and purity of allergens, the Ag delivery or adjuvant sys- cosal immune system operates. In addition to the large quanti-
tems, and the doses used (9, 12). ties of food Ags ingested daily, the intestine is colonized by a
The ability to prevent or even to reverse experimental T cell- complex microbiota, the members of which are just beginning
mediated autoimmune diseases such as experimental allergic to be identified (23). The total numbers are estimated at 100
encephalomyelitis, collagen type II-induced arthritis, or auto- trillion organisms per person, which represents 10-fold more
immune diabetes in animal models (4, 8, 9) has led to renewed microbial cells than human cells present in the body (24). This
interest in applying the principles of oral tolerance to the ther- is truly an enormous challenge of bacterial Ag and adjuvants for
apeutic treatment of autoimmune diseases (e.g., multiple scle- the mucosal immune system, and it is present for the lifetime of
rosis and rheumatoid arthritis) in humans. As explained below, the host. The mechanisms by which most of us live in peace
these efforts have not been encouraging (9). This limited effec- with this microbiota are just beginning to be understood. It is
tiveness in humans raises questions concerning the reasons for clear that the host is responding to this microbial challenge, as
such a remarkable difference in clinical outcomes compared witnessed by the large numbers of lymphoid cells present in the
Downloaded from www.jimmunol.org on October 20, 2010
with results from the animal models. Although the species, age, intestine and the 3–5 g of IgA produced per day in the normal
and gender under investigation as well as the type and dose of human (25). This complex microbial ecosystem, our “other
Ag, the frequency of exposure, the use of adjuvants and Ag de- self”, is in dynamic communication with the intestinal epithe-
livery systems, and other parameters may greatly influence the lium and with the innate and adaptive lymphoid cells present in
outcome, we propose that prior exposure to the particular Ag the intestine (26). This is the context into which a mucosal vac-
plays a decisive role; i.e., once an immune response is elicited cine or tolerogen is being introduced. Much of the difficulty
that mode of response predominates whenever the same Ag is experienced at inducing mucosal immunity or tolerance at will
encountered in the future. to a given Ag is likely due to our relative ignorance of this mi-
crobiota and the resulting host-microbial interactions that are
Does mucosal tolerance exist in humans? occurring continuously at the mucosal surface.
The number of studies addressing this point is surprisingly lim- Despite the large cellular and humoral response to the micro-
ited despite its enormous medical importance (8 –10). Lowney biota, it is commonly thought that the host is immunologically
(13, 14) observed the reduced incidence and intensity of cuta- tolerant to these bacteria (27). Against this idea is the active
neous sensitization in approximately one-half of humans first adaptive immune response to the microbiota that is present in
given an oral application of 2,4-dinitrobenzene in acetone on mice; this response is compartmented to the intestine, i.e., there
the oral mucosa. is abundant IgA to microbial Ags in the intestine but no serum
It is difficult to identify an experimental Ag never previously IgG Abs or systemic T cell responses to these same Ags (28 –30).
encountered in humans and to which, therefore, the population A recent study found no evidence of immunologic tolerance
has no pre-existing immunity. Such an Ag must also be suitable (anergy, deletion, or regulation) of the murine immune re-
both for mucosal immunization and for subsequent systemic sponse to a set of 20 recombinant microbiota protein Ags; in-
challenge to test the induction of systemic unresponsiveness. stead, the systemic T cells remain naive to these Ags (30). The
With these limitations in mind, we (15, 16), and others (17, 18) intestine also contains IL-10-producing CD4 T cells reactive to
selected keyhole limpet hemocyanin (KLH) because of its high the Ags of the commensal microbiota (31). The mucosal im-
immunogenicity as revealed by both humoral and cell-medi- mune response to these Ags appears to be tightly regulated, and
ated responses to systemic immunization with small doses. Ex- details about how regulatory cells are induced and maintained
tended ingestion or intranasal inhalation of KLH by volunteers in the mucosa are just emerging (32, 33). Indeed, the phenom-
lacking pre-existing immunity followed by systemic challenge enon of mucosal tolerance to protein Ags delivered into the in-
resulted in an unexpected outcome: decreased cell-mediated testine may be related to and be dependent upon the regulation
immunity manifested by diminished DTH reactions and T cell of the immune response to the microbiota. The Ag being deliv-
proliferation in vitro but priming for B cell responses. Muco- ered to a mucosal surface is, after all, encountered along with
sally immunized volunteers responded to the subsequent sys- the endogenous microbes. There is much less known about the
temic KLH injection by higher titers of systemic and secretory normal human mucosal immune response to the microbiota.
Abs than those who received only systemic injection of KLH. Although humans produce IgA Abs to commensal bacteria,
Thus, the initial mucosal immunization with high doses of a such responses do not appear to be confined to the intestine as
glycoprotein Ag did not suppress the humoral arm of the im- tightly as in mice as shown by the presence of serum IgG Abs to
mune response. This finding has important implications for Ags of the commensals.
mucosally delivered vaccines. Furthermore, extensive studies of Certainly, strategies for the development of effective mucosal
humoral and cellular immune responses to food Ags (19 –22) vaccines need to consider the large and continuous response to
clearly demonstrate that humoral immune responses to impor- the Ags of the microbiota. A given vaccine Ag is in essence in
tant components such as bovine -globulin, which is consumed competition for the attention of the mucosal immune system
4. The Journal of Immunology 5635
with the many thousands of these microbial Ags that occur in and properties of the vaccine Ag, contamination of the adjuvant
the gut before exposure to the vaccine Ag and will continue long with endotoxin or, in the case of the B subunit, with holotoxin,
after it has disappeared. The delivery of vaccine Ags by microbes and also the species of animal used. In addition, the B subunits
has been tried in various forms. In some systems, commensal of enterotoxins can serve as coupled delivery agents for vaccines,
bacteria themselves are used to deliver the Ag into the intestine. but the results differ from those obtained when enterotoxins are
If there is already an endogenous regulation to the commensal used as admixed adjuvants. The manner of coupling, the nature
vector, it is likely to be transferred to the vaccine Ag as well. of the coupled Ag, the route of administration, and the species
Attenuated pathogens such as Salmonella have been used to de- affect the outcome. Ags chemically conjugated to cholera toxin
liver vaccine Ags. Such vectors do induce immune responses to B (CTB) induce mucosal and systemic Ab responses to the cou-
vaccine Ags in the short term, but the response to the neoanti- pled Ag when given intragastrically or intranasally in mice, but
gen is overwhelmed by the response to the more immunodom- coadministration of a small adjuvant dose of intact CT may be
inant Ags of the Salmonella itself. necessary especially in rats (38 – 40). In the absence of holo-
In contrast to enhanced or essentially unaltered humoral re- toxin, oral administration of Ags chemically conjugated to re-
sponses, the administration of Ags to human gastrointestinal or combinant CTB induces profound tolerance in mice (41). Even
respiratory tracts induces a profound decrease in cell-mediated previously established systemic immune responses were sup-
immunity as manifested by diminished DTH, T cell prolifera- pressed, and T cell-mediated autoimmune conditions such as
tion, and secretion of cytokines with suppressor activity (15– experimental autoimmune encephalomyelitis could be reversed
18). Although most if not all of the currently available human (42). Regulatory T cells (Treg cells, both Foxp3 and Foxp3 )
vaccines generate their protective effects through the induction were induced in mice orally immunized with OVA (which
of specific Abs, it is not known whether mucosal exposure to readily induces tolerance) conjugated to CTB (43).
large doses of inactivated microorganisms (viruses, bacteria, or In contrast, genetically coupled recombinant chimeric pro-
Downloaded from www.jimmunol.org on October 20, 2010
isolated Ags) inhibits the subsequent induction of CTL by ei- teins of the form Ag-A2/B5, in which Ag is fused to the A2 sub-
ther systemic immunization or infection. However, if that is the unit of the enterotoxin and assembled with B subunits, are im-
case, the efficacy of vaccines whose protective function at least munogenic in mice in the absence of an intact holotoxin
partially depends on CTL activity might be compromised. To adjuvant and no evidence of tolerance induction has been
investigate this, Ilan (34) induced tolerance to adenoviral pro- found (44 – 46). Although the mechanisms by which these dif-
teins by feeding them to experimental animals, which then dis- ferent Ag-enterotoxin constructs interact with APC to induce
played markedly reduced anti-adenoviral humoral as well as cel- tolerance or immunity are not understood, clearly there are dif-
lular immunity as evaluated by increased TGF , IL-4, and ferences between the molecular forms that may be amenable to
IL-10 but decreased IFN- production by lymphocytes upon exploitation for the elicitation of diverse responses. However,
exposure to adenoviral Ags in vitro. This finding is of consid- few trials have been conducted in humans. Oral administration
erable importance for gene therapy using adenoviruses as vec- of a peptide derived from human heat-shock protein-60 fused
tors; extended viral survival due to tolerance to the virus and, to CTB was found to prevent relapses of uveitis in Behcet’s dis-
therefore, longer expression of the desired gene product en- ease (47). Human vaccine trials have commenced using non-
coded in a recombinant adenoviral vector would be of great toxic mutants of LT-I with Helicobacter pylori or an intranasal
benefit for both gene therapy and viral vector-based vaccina- influenza vaccine (48, 49).
tion. Conversely, induction of tolerance to a virus as a vaccine
might be an undesirable outcome. It is possible that vaccines Can an existing immune response to a foreign Ag or an autoantigen be
based on live vectors involving commensal (e.g., Escherichia coli suppressed by mucosal immunization
and Lactobacillus) or pathogenic (e.g., Salmonella) bacteria In animal models of type IV DTH to a hapten or of autoim-
would also show differential vector survival in the gastrointes- mune diseases such as experimental allergic encephalomyelitis,
tinal tract as a result of differences in the immune responses to collagen type II-induced arthritis, and autoimmune diabetes
these two types of bacteria (29). mellitus, prior mucosal exposure to the relevant Ag by the oral
or intranasal route diminishes or prevents the development of a
The role of adjuvants in immunity or tolerance
reaction or disease otherwise induced by systemic immuniza-
The role of immunomodulating adjuvants is extremely impor- tion (4, 8, 9, 41– 43, 50). Based on these findings, the impor-
tant in this context. Among these, the most intensively re- tant question was raised as to whether this desirable effect could
searched are the heat-labile enterotoxins of Gram-negative en- be extended to the therapeutic exploitation of mucosal toler-
terobacteria, such as cholera toxin (CT) and the closely related ance in the treatment of pre-existing autoimmunity or DTH.
type I labile toxin (LT), LT-I, of E. coli plus the type II toxins Early empirical studies on the suppression of DTH reactions to
LT-IIa and LT-IIb also from E. coli. The mucosal adjuvant environmental Ags, such as poison ivy or poison oak, provided
properties of CT were first demonstrated by its ability to pre- conflicting but mostly discouraging results; feeding fresh, dried
vent and even reverse the development of oral tolerance (35), or extracted leaves of these plants proved of no benefit in most
and a voluminous literature has since developed demonstrating trials (51, 52). It has been demonstrated in both animals and
the effectiveness of CT or LT-I as mucosal adjuvants when co- humans that pre-existing systemic immunity effectively pre-
administered with a large variety of Ags delivered orally (intra- cludes the induction of mucosal tolerance. Chase (53) convinc-
gastrically) or intranasally (36). The precise mechanism of ac- ingly demonstrated and explicitly stated in his now rarely cited
tion of these adjuvants has not been completely elucidated, and landmark paper that animals first systemically sensitized by a
controversies exist over the requirements for and roles of the A hapten (2,4-dinitrobenzene) are totally refractory to the bene-
and B subunits of these toxins (37). Factors involved in the di- ficial effect of subsequent oral immunotherapy. Similarly, ani-
verse findings include the route of administration, the nature mals with well-established, progressive autoimmune disease
5. 5636 BRIEF REVIEW: MUCOSAL VACCINES AND TOLERANCE
(experimental allergic encephalomyelitis or collagen type II-in-
duced arthritis) induced by initial systemic immunization re-
spond minimally if at all to oral or intranasal immunotherapy
(4). Initial systemic immunization of human volunteers with
KLH followed by extended ingestion of large doses of the same
Ag failed to generate tolerance (54). In this study, the measure-
ment of Ag-driven T cell proliferation, KLH-specific serum and
salivary Ab levels, and cutaneous DTH responses failed to reveal
significant differences between subjects fed KLH or OVA as a
negative control Ag. Thus, systemically induced primary re-
sponses in humans were not attenuated by subsequent oral in-
gestion. However, whether these data can be extended to other
neo-Ags or vaccines given over a much broader range of doses is
unknown. This point is important for immunization with mu-
cosally administered vaccines as well as the potential treatment
of autoimmune diseases. In the first case, it is unlikely that mu-
cosal immunization of an individual with even a low pre-exist-
ing level of systemic immunity would induce T cell-mediated
FIGURE 1. Possible common regulatory pathways for the induction of mu-
systemic unresponsiveness. In the second situation, it is becom-
cosal tolerance and IgA production. Ags are taken up, processed, and presented
ing apparent that the limited success in the treatment of existing in mucosal inductive sites, leading to the stimulation of regulatory T cells (Treg,
autoimmune diseases by mucosal immunization with the au- Tr1, Th3). The main cytokine products of these cells enhance the differentia-
Downloaded from www.jimmunol.org on October 20, 2010
toantigen (8, 9, 55, 56) can be explained by the failure to sup- tion of B cells into pIgA-producing plasma cells in mucosal tissues (arrows
press a pre-existing systemic response. pointing up) and concomitantly suppress systemic T cell responses (arrows
pointing down). This concerted action achieves the common goal of preventing
Functional complementarity of mucosal Ab responses and mucosal the exhaustion of the immune system by the abundance of Ags that are present
tolerance: common regulatory mechanisms at mucosal surfaces. S-IgA, Secretory IgA.
The IgA isotype switching of B cells and their differentiation
into specific IgA Ab-producing plasma cells (57–59) and sys-
temic T cell hyporesponsiveness, both of which are induced by switching, IgA B cell differentiation (TGF and IL-10), epithe-
mucosal exposure to Ags (8, 9, 60), display complementary lial cell expression of the pIgA receptor and transcytosis (IL-4),
functional effects and may share some common regulatory and the suppression of cell-mediated immunity (TGF , IL-4,
pathways. Although specific Abs of any major Ig isotype in se- and IL-10) (8, 9, 57–59, 64, 66 – 68). Although murine B1 cells
cretions prevent the uptake of inert Ags from mucosal surfaces can differentiate into IgA-producing cells that secrete Abs spe-
and restrict the penetration of microorganisms into the sys- cific for intestinal microbes independently of T cells (28), this
temic compartment, Abs of the IgA isotype display unique may not be the case in humans because B1 cells are not detect-
functional advantages, particularly upon Ag encounter in mu- able in the intestinal lamina propria (59). We speculate that the
cosal tissues. In addition to their pronounced resistance to in- inhibition of systemic T cells and the stimulation of IgA re-
testinal proteolytic enzymes and the beneficial effect of multi- sponses may result concomitantly from the induction of these
valency (four or eight Ag binding sites per dimer or tetramer, “inhibitory” cytokine networks.
respectively), specific IgA Abs exhibit strong anti-inflammatory
activity manifested by the inhibition of complement activation
and of the activation of inflammatory cell types (3, 5, 61– 63).
This in turn reduces potential tissue damage, breakdown of the Although the parallel induction of both mucosal Ab responses
mucosal barrier, and indiscriminate uptake of bystander Ags and mucosal tolerance after Ag ingestion has been demon-
(63). In addition, the integral involvement of mucosal epithelial strated first in mice (60) and then in humans (15, 16), exploi-
cells in receptor-mediated transcytosis of locally produced poly- tation of the potential of this phenomenon will require further
meric IgA (pIgA) facilitates the clearance of Ags complexed with investigation. Ags used to date in humans have included only
a specific IgA Ab (64). contact allergens (13, 14) and KLH (15–18). It is well known
Despite this IgA-mediated prevention of mucosal uptake, that mucosal immunization with biologically relevant complex
minute amounts of undigested Ags (e.g., bovine -globulin Ags such as inactivated viruses, bacteria, and their products in-
from milk) can be detected in the circulation, usually in the duces mucosal as well as systemic Ab responses (1, 2, 4), but its
form of IgA-containing immune complexes (65). In addition, impact on cell-mediated immunity, particularly the induction
the parallel induction of mucosal Ab responses and the suppres- of systemic and mucosal CTL responses, has not been ade-
sion of systemic cell-mediated reactions reduce stimulation of quately investigated. An important consideration in this con-
the immune system. Importantly, all of these pathways, includ- text is that mucosal immunization of immunologically naive
ing isotype switching to IgA and terminal differentiation of IgA subjects not previously exposed to HIV with a potential HIV
plasma cells, epithelial transcytosis of pIgA and the induction of vaccine might have the undesirable effect of diminishing cell-
mucosal tolerance are regulated by common cytokine networks mediated, CTL-dependent immunity. This problem may not
(Fig. 1). Subsets of CD4 CD25 or CD4 CD25 regulatory apply to other mucosal vaccines (e.g., poliovirus, influenza vi-
T cells secrete cytokines, including TGF , IL-4, and IL-10, rus) that, like other currently used vaccines irrespective of the
that participate in surface IgM to surface IgA isotype B cell immunization route, achieve their protective effects through
6. The Journal of Immunology 5637
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