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Infection and Immunity, February 2007, p. 621-633, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01009-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants{triangledown}

Hesham F. Nawar, Sergio Arce, Michael W. Russell, and Terry D. Connell*

The Witebsky Center for Microbial Pathogenesis and Immunology, The Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York

Received 27 June 2006/ Returned for modification 26 October 2006/ Accepted 9 November 2006


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ABSTRACT
 
The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.


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INTRODUCTION
 
Most human infections begin at mucosal sites, which include the linings of the respiratory, gastrointestinal, and genitourinary tracts. Thus, it is important that prospective vaccines stimulate strong protective immune responses at these sites to abrogate infections. Unfortunately, due to immunoregulatory mechanisms, it is difficult to evoke strong immune responses at mucosal surfaces (37). In addition, especially when administered orally, mucosal vaccines often induce oral tolerance by transient or permanent suppression of T- and B-cell responsiveness to the immunizing antigen (1). This response may occur via clonal deletion of the specifically responsive lymphocytes (6) or by T-cell anergy (29, 39). Oral tolerance may also result from active suppression of the specific lymphocytes following local production of interleukin-10 (IL-10) or transforming growth factor ß by regulatory T cells (57). These mechanisms may explain why most defined soluble antigens administered via mucosal routes induce poor, short-lived immunological effects (53). Therefore, to generate robust protective immunity and long-lived memory responses to a foreign antigen, the mucosal immune system usually requires the aid of immune-stimulating agents (i.e., adjuvants).

Many mucosal adjuvants have been characterized, including liposomes (11), biodegradable polymer microspheres (20), CpG-containing oligonucleotides (31, 38), macrophage-activating lipopeptide-2 (4), monophosphoryl lipid A (3), the immunostimulating complex (49), bacterial outer membrane proteins (10, 18), and bacterial toxins (e.g., Bordetella pertussis adenylate cyclase toxin [32], Bacillus anthracis edema toxin [13], and Shiga toxin [43]). In addition, the adjuvant properties of the type I heat-labile enterotoxins (HLT) produced by Vibrio cholerae and Escherichia coli (CT and LT-I, respectively) and detoxified mutants of these enterotoxins, each of which exhibits mucosal adjuvant activity, have been investigated exhaustively (for reviews, see references 14-16, 23, 25, 26, 28, 45-47, 53, and 58). In contrast, the immunomodulatory activities of the type II HLT of E. coli (LT-IIa and LT-IIb) have not been evaluated as extensively, although these enterotoxins have been demonstrated to be strong mucosal and systemic adjuvants with unique immunomodulatory properties (9, 23, 34, 42). Both LT-IIa and LT-IIb can be distinguished from CT and LT-I at the antigenic, biochemical, and genetic levels (21, 22). Murine immunization experiments have revealed that LT-IIa and LT-IIb exhibit immunomodulatory properties that are greater than or equivalent to those evoked by CT and LT-I (34, 42).

Both the structures and functions of the type II HLT are related to the structures and functions of type I HLT (27, 54). Type I and type II HLT are oligomeric proteins composed of an A polypeptide which is noncovalently coupled to a pentameric array of B polypeptides. The A polypeptide is enzymatically active and upregulates adenylyl cyclase in susceptible cells by catalyzing the ADP ribosylation of the Gs{alpha} regulatory protein (5, 40). This modification of Gs{alpha} promotes accumulation of intracellular cyclic adenosine 3',5'-monophosphate (cAMP), which indirectly induces the intoxicated cell to secrete chloride ions and likely modulates other metabolic processes for which cAMP is a signaling molecule (27, 52). The B pentamers mediate binding of LT-IIa, LT-IIb, CT, and LT-I to one or more gangliosides, a heterogeneous family of glycolipids located on the surface of mammalian cells (51). CT and LT-I bind with high affinity to ganglioside GM1 and with lower affinity to ganglioside GD1b; LT-IIb binds most avidly to GD1a and with much lower affinity to GM2 and GM3; and LT-IIa binds specifically (in descending order of affinity) to gangliosides GD1b, GM1, GT1b, GQ1b, GD2, GD1a and GM3 (17). This differential binding of CT, LT-IIa, and LT-IIb to gangliosides likely promotes the distinguishable patterns of immunomodulatory properties observed in T cells and B cells (2, 23, 35).

The use of HLT as mucosal adjuvants in human vaccines has been restricted by their intrinsic toxicity and by their propensity to traffic to the brain via the olfactory bulb (55, 56). A variety of methods have been used to reduce or eliminate the toxic activities of the type II HLT in order to facilitate their use in human vaccines. For example, several mutant enterotoxins exhibiting reduced or undetectable toxicities have been engineered (7, 8). LT-IIa(T34I), a mutant type II HLT with highly reduced toxicity, had no detectable binding activity with purified gangliosides, with B and T lymphocytes, or with macrophages (42). LT-IIa(T34I), however, was unable to augment significant levels of mucosal immunoglobulin A (IgA) and serum IgG antibodies against a coadministered antigen. These results indicated that binding of LT-IIa to one or more ganglioside receptors is crucial for its adjuvant activity (42).

Subsequently, three additional mutants of LT-IIa with altered ganglioside-binding activities were engineered in an attempt to produce an adjuvant enterotoxin that lacks the intrinsic toxicity while maintaining the desirable immunomodulatory properties of HLT. LT-IIa(T14S) has diminished binding activity with all ganglioside receptors bound by wild-type (wt) LT-IIa; LT-IIa(T14I) and LT-IIa(T14D) bind only to GM1 (7). In this study, we examined the adjuvant activities of these three mutant enterotoxins. The results of our experiments indicated that a high level of binding of LT-IIa enterotoxin to its known gangliosides receptor(s) is not crucial for adjuvant activity. Furthermore, binding of LT-IIa to GM1 is probably sufficient for promoting the enterotoxin's unique pattern of adjuvant activities but insufficient for promoting toxicity. In addition, our results confirmed that LT-IIa exerts its effect using mechanisms different from those used by CT and LT-I, for which binding to GM1 is required both for toxicity and adjuvanticity (12, 19).


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MATERIALS AND METHODS
 
Engineering and purification of His-tagged enterotoxins. His-tagged versions of LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) were engineered, as previously described (42), using pTDC400(T14S), pTDC400(T14I), and pTDC400(T14D), respectively (7), as templates. The new plasmids encoding the LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) holotoxins engineered with His-tagged B polypeptides were designated pHN11, pHN5, and pHN8, respectively. The His-tagged wt B pentamer (LT-IIaB) was cloned, as previously described (24).

All plasmids were introduced into E. coli DH5{alpha}F'Kan (Life Technologies, Inc., Gaithersburg, MD). Expression of recombinant holotoxins was induced by addition of isopropyl-ß-D-thiogalactoside to the culture medium. Recombinant enterotoxins were extracted from the periplasmic space and purified to homogeneity by nickel affinity chromatography and gel filtration chromatography (Sephacryl-100; Pharmacia, Piscataway, NJ,) using an ÄKTA-FPLC (Pharmacia), as previously described (42). Recombinant wt and mutant enterotoxins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting using polyclonal antibodies directed toward LT-IIa holotoxin to demonstrate that each recombinant enterotoxin was purified to apparent homogeneity.

Purification of AgI/II. AgI/II was purified from the culture supernatants of Streptococcus mutans (48).

Lipopolysaccharide assay. All proteins were analyzed by the quantitative Limulus amebocyte lysate assay (Charles River Endosafe, Charleston, SC) for incidental endotoxin contamination. All enterotoxins and AgI/II preparations were essentially free of lipopolysaccharide (<0.03 ng/µg protein).

Ganglioside-dependent ELISA. Binding of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) to purified GD1a, GD1b, GM1, GM2, GM3, GT1b, or GQ1b ganglioside receptors (Matreya, State College, PA) was measured by an enzyme-linked immunosorbent assay (ELISA), as previously described (42) but with several modifications to increase the resolution. Briefly, polyvinyl 96-well ELISA plates were coated overnight at 4°C with 1 ng of ganglioside. After washing and blocking of nonspecific binding with 10% horse serum, 50 µl of a 0.1-µg/ml solution of enterotoxin was added to wells. The plates were incubated for 3 h at 37°C. Unbound enterotoxins were removed by washing with phosphate-buffered saline (PBS) containing 1% horse serum. Fifty microliters of rabbit anti-LT-IIa (diluted 1:5,000 in PBS containing 10% horse serum) was added to the wells. The plates were incubated for 1 h at 37°C and washed with PBS containing 1% horse serum to remove unbound antibodies. Fifty microliters of a solution containing 1.0 µg/ml alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody was added to each well. The plates were incubated for 1 h at 37°C, after which the wells were washed and immediately developed for 30 min using nitrophenyl phosphate (Amresco, Solon, OH) diluted in diethanolamine buffer (100 ml of diethanolamine, 1 mM MgCl2, and enough deionized H2O to bring the volume to 1 liter; pH 9.8) as a substrate. Color reactions were terminated by adding 50 µl of a 2.0 M solution of NaOH to each well. The optical density at 405 nm of each reaction mixture was determined.

Toxicity bioassay. The toxicities of purified enterotoxins were measured using Y1 adrenal cells (CCL-79; American Type Culture Collection, Manassas, VA), a cell line which is sensitive to heat-labile enterotoxins. The toxicities of wt and mutant enterotoxins were measured using a twofold titration series (1.0 µg/well to 0.125 ng/well). One unit of toxicity was defined as the lowest concentration of enterotoxin that induced rounding of 75% to 100% of the cultured mouse Y1 adrenal cells (7, 8, 42).

Detection of cAMP. The accumulation of cAMP in mouse macrophage RAW 264.7 cells (ATCC TIB-71) was measured using a cAMP enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI) as previously described (42).

Animals and immunization. Female BALB/c mice that were approximately 8 weeks old were immunized by the intranasal (i.n.) route. Groups of six mice were immunized three times at 2-week intervals with AgI/II (10 µg) alone or in combination with 1.0 µg of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). Immunizations were administered in a standardized volume (10.0 µl) that was applied slowly to both external nares. All groups were reimmunized i.n. on day 203 with 10 µg of AgI/II alone. Animal experiments were approved by the Institutional Animal Care and Use Committee of the University at Buffalo.

Collection of secretions and sera. Samples of serum, saliva, and vaginal washes were collected from individual mice 1 week before the initial immunization and at days 21, 35, 49, 63, 189, and 210 after the primary immunization (34). Mucosal secretions and serum samples were stored at –70°C.

Antibody analysis. Levels of isotype-specific antibodies in saliva, sera, and vaginal washes were determined by ELISA using a previously described method (34, 42). When used as coadministered intranasal adjuvants, LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) all induced anti-enterotoxin serum IgG. The mutant enterotoxins LT-IIa(T14I) and LT-II(T14D), however, induced levels of anti-enterotoxin serum IgG that were lower than the levels elicited by intranasal immunization with either LT-IIa or LT-IIa(T14S) (data not shown).

Isolation of lymphoid cells. Superficial cervical lymph nodes (CLN) and spleens were excised from naïve and immunized mice, and single-cell suspensions were prepared as previously described (34). The total cell yield and viability were determined with a hemacytometer using trypan blue (Sigma) staining.

Luminex cytokine assays. Spleen and CLN lymphoid cells were plated in triplicate at a concentration of 5 x 105 cells per well in flat-bottom, 96-well tissue culture plates (Nunc, Roskilde, Denmark) and were cultured for 4 days in the presence of AgI/II (10 µg/ml) or in the absence of antigen. Supernatants were collected after centrifugation and stored at –70°C until they were assayed for the presence of cytokines. The levels of IL-4, IL-10, and gamma interferon (IFN-{gamma}) in culture supernatants were determined by a Luminex cytokine assay, as follows. Capture and detection antibody pairs directed against different noncompeting epitopes of the cytokine and recombinant protein standards for IL-4, IL-10, and IFN-{gamma} were purchased from R&D Systems (Minneapolis, MN). Antibodies were covalently coupled to Multi-Analyte carboxylated microspheres (Luminex Corp., Austin, TX) according to the manufacturer's directions. A mixture of water-soluble 24 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 20 mM N-hydroxysulfosuccinimide (Pierce Biotechnology Inc., Rockford, IL) was used to activate free carboxyl groups on the beads. After activation, the beads were washed in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) (Pierce Biotechnology Inc.), and the antibody to be coupled, dialyzed in MES, was immediately added at a concentration of 250 µg/ml. The mixture was rotated overnight at room temperature. The beads were washed and resuspended in PBS-TBN (phosphate-buffered saline [pH 7.4] containing 0.02% Tween 20, 0.1 bovine serum albumin, and 0.02% sodium azide; Sigma) at a concentration of 8 x 106 beads/ml. Multiplex assays were performed in 96-well microtiter plates (Multiscreen HV plates; Millipore, Billerica, MA) with polyvinylidene difluoride membranes which had been prewetted and washed with PBS-TBN. Bead sets coated with capture antibody were diluted in PBS-TBN and pooled. One thousand beads from each set were added to each well. Recombinant cytokine standards were titrated from 333.3 ng/ml to 0.056 pg/ml using threefold dilution in PBS-TBN. Samples were initially diluted fourfold in PBS-TBN, and both standards and samples (50 µl) were added to wells containing beads. The plates were incubated at room temperature for 20 min on a rocker and then washed twice with PBS using a vacuum manifold for aspiration. Next, 50 µl biotinylated detection antibody to each cytokine, diluted in PBS-TBN, was added, the preparations were incubated for 20 min as described above, and then the beads were washed with PBS. Finally, 50 µl of streptavidin-phycoerythrin (Caltag, Burlingame, CA) was added to each well, and the plates were incubated and washed as described above. The beads were resuspended in 150 µl of PBS-TBN and analyzed with a Luminex 100 (Luminex Corp.). Samples were measured in duplicate and blank values were subtracted from all readings. Sample cytokine concentrations were calculated from the mean fluorescence intensity of the beads by interpolation of calibration curves generated from the bead mean fluorescence intensity for each concentration of recombinant cytokine standard.

Binding of enterotoxins to lymphoid cells. Binding of enterotoxins to lymphoid cells was detected as previously described (2, 42), with some minor modifications. Briefly, 107 cells obtained from spleens of naïve mice were treated in vitro with 1.0 µg of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). After incubation on ice for 1 h, the cells were washed and then incubated on ice for 30 min with a pretitrated concentration of polyclonal rabbit antibody to LT-IIa. After washing, the cells were treated with phycoerythrin-conjugated goat anti-rabbit IgG (0.5 µg/ml) and with fluorescein isothiocyanate-conjugated monoclonal antibody to CD4, CD8, B220, CD11b, or CD11c (PharMingen, San Diego, CA). After incubation for 15 min on ice, the cells were washed and incubated with 1.0 µg/ml of propidium iodide. CD16/CD32 antibodies were used to block Fc receptors (PharMingen, San Diego, CA). Data acquisition and analysis were performed using a FACScalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) and the CellQuest software (Beckton-Dickinson).

Determination of the levels of expression of cell surface molecules. Single-cell suspensions from spleens of naïve female BALB/c mice were prepared as described above. Cells (5 x 107) were incubated at 37°C for 48 h in presence or absence of 1.0 µg/ml enterotoxin in RPMI 1640 supplemented with 10% fetal bovine serum. After incubation, the cells were washed in FACS buffer (PBS containing 3% bovine serum albumin and 0.1% sodium azide) and stained with fluorescein isothiocyanate-conjugated antibody to B220 or CD11c and with phycoerythrin-conjugated antibody to CD40, CD80, CD86, or major histocompatibility complex class II (MHC-II) (PharMingen), as described above. At least 105 events were analyzed.

Statistical analysis. Analysis of variance and the Tukey multiple-comparison test were used for multiple comparisons. Unpaired t tests with Welch correction were performed to analyze differences between two groups. Statistical analyses were performed using InStat (GraphPad, San Diego, CA). Differences were considered significant at a P value of <0.05.


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RESULTS
 
Binding of wt and mutant LT-IIa enterotoxins to gangliosides. Abrogation of binding of LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) to gangliosides was originally defined using periplasmic extracts from recombinant strains of E. coli expressing only the B pentamer (7). To confirm that the ganglioside-binding activities of the purified mutant holotoxins were equivalent to those of the mutant B pentamers in the crude extracts, binding of the purified wt and mutant holotoxins to various gangliosides was measured by a ganglioside-specific ELISA (Fig. 1). As expected, LT-IIa bound to gangliosides GD1a, GD1b, GM1, GM2, GM3, GT1b, and GQ1b (7, 42). LT-IIa(T14S) exhibited less binding activity for these seven gangliosides, which was significantly different (P < 0.01) than the results for wt LT-IIa. LT-IIa(T14I) and LT-IIa(T14D), however, bound to only the GM1 ganglioside. Also, the binding activities of LT-IIa(T14I) and LT-IIa(T14D) with GM1 were significantly (P < 0.001) less than the binding activity of wt LT-IIa (Fig. 1). In addition, LT-IIa(T14I) exhibited higher binding activity with GM1 than LT-IIa(T14D) exhibited (P < 0.01) (Fig. 1).


Figure 1
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  		FIG. 1. Specificity of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) for various gangliosides. Polyvinyl microtiter plates were coated with 10 ng of purified ganglioside or with a mixture of gangliosides. Enterotoxins were incubated in the wells of the ganglioside-coated plates, after which the wells were probed with rabbit anti-LT-IIa polyclonal antiserum. The microtiter plates were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody and nitrophenyl phosphate and were analyzed using a TiterTek microplate reader at 405 nm. The error bars indicate standard errors of the means.

Toxicities of wt and mutant enterotoxins. Previous results obtained using crude periplasmic extracts from recombinants expressing LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) in a Y1 adrenal cell toxicity bioassay indicated that the toxicities of these mutant enterotoxins were severely attenuated (7). To confirm these results using purified wt and mutant enterotoxins, the toxicities of the mutant holotoxins were determined. Comparisons of the toxicities revealed that all mutant enterotoxins were less toxic than the wt toxin LT-IIa (Table 1). While 4.88 ng/ml of wt LT-IIa was sufficient to induce rounding of 100% of Y1 adrenal cells, 19.5 ng/ml of LT-IIa(T14S) was required to elicit the same effect. LT-IIa(T14I) and LT-IIa(T14D) were more than 2,000-fold less toxic than wt LT-IIa holotoxin; these two mutant holotoxins exhibited no detectable toxic activity at concentrations up to 10.0 µg/ml. The toxicities of LT-IIa(T14I) and LT-IIa(T14D) were similar to the toxicity of the purified nontoxic B pentamer of wt LT-IIa (LT-IIaB).


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  		TABLE 1. Toxicities of wt and mutant LT-IIa enterotoxins with Y1 adrenal cells

LT-IIa(T34I) is not capable of intoxicating Y1 adrenal cells, yet it has the capacity to induce accumulation of cAMP in RAW 264.7 mouse macrophage cells (42). To determine whether the LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) mutant enterotoxins induce cAMP accumulation in immunocompetent cells, cAMP levels in RAW 264.7 cells were determined after treatment with wt toxin, with mutant enterotoxin, or with LT-IIaB (Fig. 2). The endogenous level of cAMP in untreated RAW 264.7 cells was 2.67 ± 0.21 pmol/5 x 107 cells. As reported previously, wt LT-IIa induced intracellular accumulation of 18.17 ± 0.18 pmol/5 x 107 cells cAMP in RAW 264.7 cells, a level which was 6.82-fold higher than the level observed in untreated cells. LT-IIa(T14S) induced accumulation of 17.64 ± 0.52 pmol/5 x 107 cells cAMP (a concentration 6.62-fold higher than the concentration in untreated cells), which is not significantly different from the level induced by wt LT-IIa. In contrast, LT-IIa(T14I) and LT-IIa(T14D) induced accumulation of significantly (P < 0.001) lower levels of cAMP in RAW 264.7 cells (7.98 ± 0.09 and 4.58 ± 0.25 pmol/5 x 107 cells, respectively). Surprisingly, the nontoxic molecule LT-IIaB induced accumulation of significant levels (P < 0.05) of cAMP (4.13 ± 0.14 pmol/5 x 107 cells) compared to the level in the untreated cells. The ability of LT-IIaB to induce accumulation of cAMP in RAW 264.7 macrophage cells suggested that the capacities of LT-IIa(T14I) and LT-IIa(T14D) to stimulate accumulation of cAMP are probably triggered by an alternative signaling cascade that is independent of the catalytic properties of the catalytic A polypeptide (24). Overall, these data indicated that compared to the capacity of wt LT-IIa, the capacities of LT-IIa(T14I) and LT-IIa(T14D) to increase cAMP levels in RAW 264.7 cells were significantly diminished.


Figure 2
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  		FIG. 2. Production of cAMP in a RAW 264.7 macrophage cell line after treatment with enterotoxin. RAW 264.7 cells (5 x 107) were incubated at 37°C for 4 h with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), LT-IIa(T14D), or LT-IIaB. The cAMP produced by the cells was measured using a cAMP enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). The bars indicate arithmetic means, and the error bars indicate the standard errors of the means (n = 3). Three asterisks indicate a significant difference (P < 0.001) compared to LT-IIa-treated cells. The fold increases (cAMP in treated cells/cAMP in untreated cells) are indicated above the bars.

Binding of wt and mutant LT-IIa enterotoxins to lymphocytes. While wt LT-IIa binds to T cells (CD4+ and CD8+), to B cells (B220+), and to macrophages (CD11b+), LT-IIa(T34I) exhibited extremely diminished binding activities with all three cell types (42). We concluded, therefore, that the diminished adjuvant activity of LT-IIa(T34I) was a result of diminished binding to one or more types of immunocompetent cells (42). To determine the binding patterns of LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) with immunocompetent cells, T cells, B cells, macrophages, and dendritic cells were treated with each enterotoxin and evaluated by flow cytometry to determine the surface binding of the enterotoxins (Fig. 3 and Table 2). LT-IIa(T14S) bound to CD4+ T cells, CD8+ T cells, B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) in patterns indistinguishable from the patterns observed for cells treated with wt LT-IIa. LT-IIa(T14I) and LT-IIa(T14D) bound to all cell types tested, but they bound to a lower proportion of cells. LT-IIa(T14I), however, bound to a higher proportion of cells than LT-IIa(T14D) bound to. These results, along with results obtained previously using LT-IIa(T34I) (42), suggested that ganglioside binding of wt and mutant LT-IIa enterotoxins is crucial for the ability to bind to different cell types. Also, the abilities of wt and mutant LT-IIa enterotoxins to bind to different cell types directly correlate with their binding activities with gangliosides. These results indicated that despite their diminished ganglioside-binding activities, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) bound to receptor(s) on the surface of immunocompetent cells.


Figure 3
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  		FIG. 3. Patterns of binding of wt and mutant LT-IIa enterotoxins to splenic lymphoid cells. Histograms were gated on CD4+ (helper T cells), CD8+ (cytotoxic T cells), B220+ (B cells), CD11b+ (macrophages), or CD11c+ (dendritic cells). Dead cells were excluded by propidium iodide staining. Green solid histograms, control cells (nonspecific binding of antibodies in the absence of enterotoxin); blue line histograms, wt LT-IIa; red line histograms, LT-IIa(T14S); purple line histograms, LT-IIa(T14I); brown line histograms, LT-IIa(T14D). A shift of the fluorescence intensity to the left indicates a decrease in or absence of binding of a enterotoxin to the cells. PE, phycoerythrin.


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  		TABLE 2. Percentages of T helper cells (CD4+), T cytotoxic cells (CD8+), B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) bound by wt and mutant LT-IIa enterotoxins

Mucosal adjuvant activities of wt and mutant LT-IIa enterotoxins. To compare the adjuvant activities of the mutant enterotoxins with those of wt LT-IIa, female BALB/c mice were intranasally immunized with AgI/II, a surface protein from S. mutans (48), in the presence or absence of either LT-IIa or one of the three mutant LT-IIa enterotoxins. The initial immunizations were followed by booster immunizations at day 14 and day 28. Saliva and vaginal secretions were collected at days 21, 35, 49, 63, and 189 to determine the kinetics of the induced anti-AgI/II immune responses. To evaluate memory responses, all groups were immunized intranasally at day 203 with 10 µg AgI/II in the absence of enterotoxins. Saliva and vaginal secretions were collected at day 210 and analyzed to determine the AgI/II-specific IgA antibodies as a measure of the capacity of the enterotoxins to induce memory responses.

Immunization with AgI/II alone did not elicit a strong salivary IgA response to AgI/II (Fig. 4A). In contrast, mice immunized with AgI/II in the presence of wt or mutant enterotoxin produced elevated levels of AgI/II-specific IgA in the saliva after the second immunization (day 21), the levels peaked after the third immunization at day 35, and the production persisted through day 189 (Fig. 4A). At days 35, 49, and 63, the AgI/II-specific salivary IgA levels in mice that received AgI/II plus LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D) were fivefold to eightfold higher than the levels of antigen-specific IgA in mice immunized with only AgI/II. There were no significant differences between the levels of AgI/II-specific IgA in saliva of mice that received wt enterotoxin as an adjuvant and the levels of AgI/II-specific IgA in saliva of mice that received mutant enterotoxin as an adjuvant (P > 0.05). After immunization with AgI/II at day 203, the levels of salivary AgI/II-specific IgA at day 210 were elevated compared to the levels detected at day 189 in mice which had received LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D) as an adjuvant (Fig. 4A). An increase in salivary AgI/II-specific IgA levels after reimmunization with AgI/II at day 203 was not detected in mice which were initially immunized with AgI/II in the absence of enterotoxin (Fig. 4A).


Figure 4
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  		FIG. 4. Effects of wt and mutant enterotoxin adjuvants on salivary IgA (A) and vaginal IgA (B) antibody responses to AgI/II. Mice were immunized intranasally on days 1, 14, and 28 with AgI/II in the presence or absence of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) as mucosal adjuvants. On day 203, all groups were immunized with AgI/II in the absence of enterotoxin. The data are arithmetic means ± standard errors of the means.

Previous investigations demonstrated that LT-IIa is also capable of inducing strong immune responses to coadministered antigens at a distal mucosal site (34, 42) (Fig. 4B). To determine whether mucosal adjuvant responses were potentiated at a distal mucosal site in mice immunized i.n. with AgI/II and mutant enterotoxins, the levels of AgI/II-specific IgA in samples taken from the vaginal mucosa were determined (Fig. 4B). Immunization with AgI/II in the absence of enterotoxin induced low levels of vaginal anti-AgI/II IgA at all times. Mice immunized with AgI/II in the presence of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D), however, produced high levels of AgI/II-specific vaginal IgA compared to the levels produced by mice immunized with only AgI/II (P < 0.001) at days 35, 49, and 63 (Fig. 4B). The vaginal IgA responses to AgI/II in the mice that received an enterotoxin adjuvant peaked at day 35 or at day 49, slowly decreased at later times, persisted through day 63, and declined by day 189. As observed for salivary anti-AgI/II IgA, there was not a significant difference in the levels of vaginal AgI/II-specific IgA between the groups of mice that received enterotoxins as adjuvants. Reimmunization at day 203 with AgI/II elicited levels of vaginal AgI/II-specific IgA that were higher than the levels at day 189 only in mice that had initially received enterotoxin as an adjuvant and not in mice that were initially immunized solely with AgI/II (Fig. 4B). These results demonstrated that LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) are capable of potentiating high levels of mucosal anti-AgI/II responses at a distal mucosal site.

These observations indicated that the mutant enterotoxins LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) had the capacity to potentiate high levels of mucosal anti-AgI/II responses. In combination with the results obtained using LT-IIa(T34I) as an adjuvant (42), the results generated using LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) are consistent with a model in which binding of LT-IIa to one or more of its ganglioside receptor(s) is required for establishing immune responses at mucosal sites.

Systemic adjuvant activities of wt and mutant LT-IIa enterotoxins. Intranasal administration of LT-IIa has also been shown to induce strong circulating antibody responses to coadministered antigens (34, 42). To examine whether LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) had the capacity to potentiate serum antibody responses, antigen-specific IgA and IgG levels were measured in serum collected at various times from mice immunized i.n. with AgI/II in the presence or absence of mutant or wt enterotoxins.

As expected, LT-IIa potentiated anti-AgI/II serum IgA; this effect peaked at day 35 after the second booster immunization administered at day 28 (Fig. 5A) and persisted through day 189. Compared to the serum IgA levels in mice immunized with AgI/II alone, the serum AgI/II-specific IgA responses in mice immunized with AgI/II plus LT-IIa (P < 0.001), AgI/II plus LT-II(T14S) (P < 0.001), AgI/II plus LT-IIa(T14I) (P < 0.01), and AgI/II plus LT-IIa(T14D) (P < 0.05) were significantly elevated at day 35. LT-IIa and LT-IIa(T14S) were more capable than LT-IIa(T14I) (P < 0.05) and LT-IIa(T14D) (P < 0.01) of potentiating serum IgA anti-AgI/II at day 35 (Fig. 5A). i.n. boosting with 10 µg AgI/II in the absence of enterotoxin at day 203 induced threefold increases in serum AgI/II-specific IgA levels at day 210 in all groups compared to the levels of anti-AgI/II IgA on day 189 (Fig. 5A). These levels of serum IgA anti-AgI/II at day 210 were extremely significant (P < 0.001) for groups that received enterotoxin as an adjuvant when they were compared to mice that received only AgI/II. At all times, the serum IgG responses to AgI/II were also elevated in mice immunized with AgI/II plus wt enterotoxin or AgI/II plus mutant enterotoxin compared to the responses in mice immunized solely with AgI/II. The IgG levels peaked at day 49, and the differences between the levels in mice immunized with LT-IIa (P < 0.0001), LT-IIa(T14S) (P < 0.0001), LT-IIa(T14I) (P < 0.001), or LT-IIa(T14D) (P < 0.01) as the adjuvant and the levels in mice immunized with AgI/II alone were extremely significant (Fig. 5B). As observed for serum IgA anti-AgI/II at day 35, wt LT-IIa and LT-IIa(T14S) potentiated levels of serum IgG anti-AgI/II, which peaked at day 49, that were significantly higher than the levels in mice that received either LT-IIa(T14I) (P < 0.05 and P < 0.01, respectively) or LT-IIa(T14D) (P < 0.01 and P < 0.001, respectively). Also, as observed for serum IgA, i.n. boosting with 10 µg AgI/II at day 203 induced two- to threefold increases in serum IgG anti-AgI/II levels at day 210 in groups that received wt or mutant enterotoxin as an adjuvant. These levels were significantly different (P < 0.01) than the levels of anti-AgI/II IgG at day 189 (Fig. 5B). These results demonstrated that mice that received enterotoxin as an adjuvant exhibited better primary immune responses and robust memory responses than mice that received only AgI/II.


Figure 5
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  		FIG. 5. Effects of wt and mutant enterotoxin adjuvants on serum IgA (A) and IgG (B) antibody responses to AgI/II. Mice were immunized intranasally on days 1, 14, and 28 with AgI/II in the presence or absence of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) as mucosal adjuvants. On day 203, all groups were immunized with AgI/II in the absence of enterotoxin. The data are arithmetic means ± standard errors of the means.

Serum IgG subclass responses. Based on IgG subclass distribution, LT-IIa stimulates a biased T-helper 2 (Th2) immune response (34, 42). To determine if LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D) stimulated IgG subclass distributions that were similar or different from the IgG subclass distribution stimulated by wt enterotoxin, the concentrations of AgI/II-specific IgG1, IgG2a, and IgG2b in sera obtained from immunized mice at days 35, 49 and 210 were determined (Fig. 6). Immunization with AgI/II alone induced low levels of IgG1, IgG2a, and IgG2b; IgG1 was the most prominent IgG subclass at all times. At day 35, LT-IIa induced a higher level of IgG1 than of IgG2a or IgG2b (Fig. 6A). At this time, LT-IIa(T14I) and LT-IIa(T14D) induced a pattern of IgG subclass expression similar to that induced by wt LT-IIa (Fig. 6A). LT-IIa(T14S), however, elicited a higher level of IgG2a than either wt LT-IIa or the other two mutant enterotoxins elicited. At day 49, similar patterns of IgG subclass distribution were observed in the sera of mice immunized with either LT-IIa, LT-IIa(T14I), or LT-IIa(T14D) (Fig. 6B). In contrast, LT-IIa(T14S) induced a higher level of IgG2a at day 49 than at day 35 (Fig. 6B), suggesting that this mutant enterotoxin stimulated a more balanced Th1/Th2 immune response. When IgG subclass distributions were determined at day 210, LT-IIa(T14S) maintained the Th1/Th2 balanced pattern of IgG subclass distribution which was observed at day 49 (Fig. 6C). LT-IIa also exhibited a similar shift of IgG levels at day 210, which was consistent with a more balanced Th1/Th2 immune response (Fig. 6C). IgG subclass distributions did not change in mice immunized with LT-IIa(T14I) or LT-IIa(T14D) at day 210 compared to the IgG subclass distributions at days 35 and 49 (Fig. 6C).


Figure 6
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  		FIG. 6. Effects of wt and mutant enterotoxin adjuvants on serum IgG subclass antibody responses to AgI/II. IgG subclasses from mice immunized with AgI/II were examined in the presence or absence of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) as adjuvants at days 35 (A), 49 (B), and 210 (C). The data are arithmetic means ± standard errors of the means.

Based on the distribution patterns of IgG subclasses, these results indicated that LT-IIa induces a Th2 immune response at early times, which shifts at later times toward a more balanced Th1/Th2 immune response. Also, these results indicated that the mutant enterotoxin LT-IIa(T14S) promoted this shift earlier than wt LT-IIa promoted it. LT-IIa(T14I) and LT-IIa(T14D), on the other hand, failed to induce a balanced Th1/Th2 immune response.

Cytokine production. To complement the IgG subclass distribution experiments for investigating the capacity of LT-IIa and its mutants to shift Th1 and Th2 responses, cytokine production was also evaluated. Specifically, the expression patterns of IL-4, IL-10, and IFN-{gamma} were determined in culture supernatants of lymphoid cells obtained from the draining superficial CLN and from the spleens of immunized mice after in vitro stimulation with AgI/II (Fig. 7). As previously reported, only low levels of IL-4 were detected in culture supernatants of CLN lymphoid cells from mice immunized with LT-IIa (34, 42) (Fig. 7A). Similar levels of IL-4 were also detected in culture supernatants of lymphoid cells isolated from CLN of mice immunized with LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) (Fig. 7A). In contrast, the IL-4 levels in culture supernatants of splenic lymphoid cells isolated from mice immunized with AgI/II plus LT-IIa (P < 0.05), AgI/II plus LT-IIa(T14S) (P < 0.01), and AgI/II plus LT-IIa(T14I) (P < 0.05) were significantly higher than the IL-4 levels in culture supernatants of splenic cells obtained from mice immunized with AgI/II without adjuvant (Fig. 7D). The level of IL-4 was slightly elevated in culture supernatants of splenic cells isolated from mice immunized with AgI/II plus LT-IIa(T14D), but this level was not significantly different from the levels of IL-4 in culture supernatants from splenic cells obtained from mice immunized with AgI/II alone (Fig. 7D).


Figure 7
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  		FIG. 7. Production of IL-4 (A and D), IL-10 (B and E), and IFN-{gamma} (C and F) by AgI/II-responsive lymphoid cells isolated from cervical lymph nodes (A to C) and spleens (D to F) of mice immunized intranasally with AgI/II in the presence or absence of LT-IIa, LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) as adjuvants. Cells were isolated at day 217 and stimulated in vitro for 4 days with 10 µg AgI/II. The data are arithmetic means ± standard errors of the means. For comparisons with the AgI/II group the significance of differences is indicated as follows: three asterisks, P < 0.001; two asterisks, P < 0.01; and one asterisk, P < 0.05.

Very high IL-10 levels were detected in culture supernatants of lymphoid cells isolated from both CLN and spleens of mice immunized with or without an enterotoxin adjuvant (Fig. 7B and E). The level of IL-10 in culture supernatants of lymphoid cells isolated from CLN of mice immunized with AgI/II alone was significantly higher than the IL-10 level in mice immunized with LT-IIa (P < 0.01), LT-IIa(T14S) (P < 0.001), LT-IIa(T14I) (P < 0.01), or LT-IIa(T14D) (P < 0.001) as an adjuvant (Fig. 7B). The IL-10 level in culture supernatants of lymphoid cells isolated from spleens of mice immunized with AgI/II alone was also significantly higher than the IL-10 level in culture supernatants of cells isolated from mice immunized with LT-IIa (P < 0.05), LT-IIa(T14S) (P < 0.05), LT-IIa(T14I) (P < 0.01), or LT-IIa(T14D) (P < 0.01) as an adjuvant (Fig. 7E).

Very high concentrations of IFN-{gamma} were detected in culture supernatants of CLN lymphoid cells isolated from mice that received LT-IIa (P < 0.001), LT-IIa(T14S) (P < 0.001), LT-IIa(T14I) (P < 0.01), or LT-IIa(T14D) (P < 0.05) as an adjuvant compared to the concentrations in mice immunized with AgI/II alone (Fig. 7C). Also, very high levels of IFN-{gamma} were detected in culture supernatants of lymphoid cells isolated from spleens of mice immunized with AgI/II alone or with an enterotoxin as an adjuvant (Fig. 7F). The levels of IFN-{gamma} in culture supernatants of lymphoid cells isolated from spleens of mice that received LT-IIa or LT-IIa(T14S) as an adjuvant were very significantly different (P < 0.001) than the levels of IFN-{gamma} in culture supernatants of lymphoid cells isolated from mice immunized with AgI/II alone or AgI/II plus LT-IIa(T14I) or LT-IIa(T14D).

Effect of wt and mutant enterotoxins on antigen-presenting cells. Full activation of antigen-specific T cells requires interaction between the T-cell receptor and MHC-II, as well as interaction between CD28 and B7 and/or between CD40 and CD40L on the surfaces of T cells and antigen-presenting cells (APC) (30). Since it was previously reported that CT, LT-I, LT-IIa, and LT-IIb modulate the expression of MHC-II and costimulatory molecules (CD40, CD80, and CD86) on the surface of APC (2, 33, 36, 41), the effects of the mutant enterotoxins LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) on the levels of expression of CD40, CD80 (B7-1), CD86 (B7-2), and MHC-II on the surfaces of resting B cells (B220+) and dendritic cells (CD11c+) isolated from the spleens of naïve mice were examined.

After treatment of splenic cells for 48 h with wt or mutant enterotoxin, little or no enhancement of the levels of expression of CD40, CD80, CD86, and MHC-II on the surface of B cells was detected (data not shown). LT-IIa and LT-IIa(T14S) enhanced expression of CD86 and MHC-II, slightly increased expression of CD40 and CD80 on the surface of dendritic cells, and increased the proportion of dendritic cells expressing these molecules (Fig. 8). LT-IIa(T14I) and LT-IIa(T14D), however, did not have the same effects as wt LT-IIa on the levels of expression of CD40, CD80, CD86, and MHC-II on dendritic cells. These results indicated that the wt and mutant LT-IIa enterotoxins had the capacity to upregulate, to varying degrees, expression of relevant costimulatory molecules on the surface of APC that are required for T-cell activation.


Figure 8
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  		FIG. 8. Effects of wt and mutant LT-IIa enterotoxins on the levels of expression on the surface of splenic dendritic cells of the costimulatory molecules CD40, CD80, and CD86 and of MHC-II. Splenic cells isolated from naïve BALB/c mice were incubated in the presence or absence of 1 µg/ml of LT-IIa (A, E, I, and M), LT-IIa(T14S) (B, F, J, and N), LT-IIa(T14I) (C, G, K, and O), or LT-IIa(T14D) (D, H, L, and P) for 48 h, and CD40 (A to D), CD80 (E to H), CD86 (I to L), and MHC-II (M to P) were analyzed. The histograms were gated on CD11c+ cells. Thin lines, level of expression of surface molecules on the surface of untreated cells; thick lines, level of expression of surface molecules on the surface of enterotoxin-treated cells.


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DISCUSSION
 
The adjuvant activities of the type II HLT LT-IIa are dependent upon binding of the enterotoxin to its ganglioside receptors, while LT-IIa(T34I), a mutant enterotoxin that does not bind to the GD1b, GD1a, GM1, GM2, GM3, GQ1b, and GT1b gangliosides in vitro, exhibits extremely diminished adjuvant activity with respect to potentiation of mucosal IgA and serum IgA and IgG. Here, we report that single-point replacement of the amino acid threonine with serine, isoleucine, or aspartic acid at position 14 of the B polypeptide of LT-IIa enterotoxin had little or no effect on the adjuvant activities of the mutant enterotoxins yet reduced their toxicities. In addition, our current experiments using LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D), each of which has a distinctive ganglioside-binding activity, in combination with our previous results obtained using LT-IIa(T34I) as an adjuvant (42), confirmed that binding of the LT-IIa enterotoxin to one or more ganglioside receptors is crucial for its adjuvant activity and that while GM1 can be utilized by LT-IIa as a functional receptor to mediate its adjuvant activity, binding to GM1 alone does not appear to mediate LT-IIa toxicity.

Compared to the toxicity of wt LT-IIa, the toxicities of mutant enterotoxins LT-IIa(T14I) and LT-IIa(T14D) were dramatically reduced. These mutant enterotoxins were unable to intoxicate murine Y1 adrenal cells at concentrations 2,000-fold higher than the concentration of wt LT-IIa needed for intoxication (Table 1), and their capacities to induce intracellular accumulation of cAMP in macrophage cells were also extremely diminished (Fig. 2). The level of cAMP accumulated upon treatment of RAW 264.7 macrophage cells with LT-IIa(T14I) or LT-IIa(T14D) was almost equivalent to the level of cAMP accumulated upon treatment with the nontoxic B pentamer of LT-IIa (LT-IIaB), which lacks the enzymatic A polypeptide. The finding that these levels were still elevated compared to the levels in untreated cells suggested that these enterotoxins may have other biochemical effects on cells. Furthermore, CT and LT-I have been shown to traffic to the brain via the olfactory nerve after intranasal delivery, an activity which limits their usefulness as intranasal adjuvants for human use. Since LT-IIa(T14I) and LT-IIa(T14D) exhibit altered ganglioside-binding activities (reflected in their dramatically reduced toxicities), these two mutant enterotoxins potentially have reduced or abrogated capacities for retrograde transport. If this is true, LT-IIa(T14I) and LT-IIa(T14D) may be more amenable enterotoxins for use in human intranasal vaccines.

The mutant enterotoxins were shown to bind to CD4+ T cells, CD8+ T cells, B cells, macrophages, and dendritic cells (Fig. 3). The percentages of different cell types bound by wt and mutant LT-IIa enterotoxins directly correlated with the binding activities of enterotoxins with gangliosides; e.g., LT-IIa(T14I) and LT-IIa(T14D) showed less binding activity with all cell types than LT-IIa and LT-IIa(T14S) showed. These results suggest either that all of the cell types evaluated express GM1 on the cell surface or that the cells express an alternative ganglioside(s) or receptor(s) that mediates binding of LT-IIa(T14I) and LT-IIa(T14D). A similar pattern of diminished binding to leukocytes was observed for the closely related mutant enterotoxin LT-IIb(T13I). This mutant enterotoxin has no detectible binding activity with the GD1a, GD1b, GM1, GM2, GM3, GT1b, and GQ1b gangliosides in vitro but still binds to CD4+ T cells, CD8+ T cells, B cells, and macrophages and is able to act as a potent mucosal and systemic adjuvant (42).

The results of previous immunization experiments using LT-IIa(T34I), a mutant holotoxin which does not exhibit detectable binding to either gangliosides or immunocompetent cells, suggested that the immunomodulatory activity of LT-IIa requires binding to one or more of its ganglioside receptors (42). This hypothesis was confirmed by the results reported here obtained using the mutant enterotoxins LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D), which exhibit altered ganglioside-binding activities. The ability of a mutant LT-IIa enterotoxin to act as an adjuvant was directly correlated with its binding activities with gangliosides and with immunocompetent cells. LT-IIa(T14S), which binds to gangliosides with slightly diminished activities and to immunocompetent cells with equivalent activities compared to wt LT-IIa, was shown to be a potent mucosal and systemic adjuvant with immunomodulatory activities that are comparable to those of wt LT-IIa (Fig. 4 and 5). LT-IIa(T14I) and LT-IIa(T14D), which bind with lower activities than wt LT-IIa to GM1 and immunocompetent cells, however, induced levels of serum IgA and IgG to coadministered antigen that were lower than those induced by wt LT-IIa or LT-IIa(T14S).

To establish protection against pathogens, vaccines must elicit robust immunity and long-lived memory responses to an administered antigen. Induction of such a memory response often requires the aid of an adjuvant to generate antigen-specific memory T and B cells that persist in various lymphoid compartments after immunization. After intranasal immunization, T cells migrate to and reside in the CLN (59). Use of LT-IIa as an intranasal adjuvant has been correlated with the establishment of antigen-specific cells in both CLN and spleens (34, 42). The results of the current experiments were consistent with the results of the previous investigations. LT-IIa stimulated robust IgA and IgG responses to AgI/II at day 210 after reimmunization with AgI/II. LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) also exhibited a similar capacity to stimulate robust IgA and IgG memory responses. In addition, wt and mutant LT-IIa enterotoxins had the capacity to potentiate anti-AgI/II memory responses at mucosal sites.

LT-IIa induces an IgG1-biased distribution pattern of IgG anti-AgI/II subclasses (34, 42). At day 35, mice immunized with wt and three mutant enterotoxins exhibited similar patterns of biased IgG subclass distribution. At day 49, immunization with LT-IIa(T14S) as the adjuvant altered the IgG subclass distribution to a more balanced IgG1/IgG2 pattern, which persisted at day 210. Surprisingly, LT-IIa also was associated at day 210 with a similar balanced pattern of IgG1/IgG2. Both LT-IIa(T14I) and LT-IIa(T14D) were unable to alter the IgG subclass distribution at all times evaluated. Cytokines have a major role in isotype selection and isotype switching during an immune response. Although no single cytokine appears to regulate IgG subclass responses in vivo, IL-4 and IFN-{gamma} are known to enhance production of IgG1 and IgG2a, respectively (44, 50). Our results showed that wt and mutant enterotoxins enhanced IL-4 production, while only LT-IIa and LT-IIa(T14S) significantly enhanced IFN-{gamma} production compared to the effects of LT-IIa(T14I) and LT-IIa(T14D). The higher levels of IFN-{gamma} could potentially have mediated the altered IgG anti-AgI/II subclass distribution in mice which received LT-IIa and LT-IIa(T14S) as mucosal adjuvants.

Type I and type II enterotoxins bind with different patterns to B cells, T cells, macrophages, and dendritic cells (2, 42). Binding of CT, LT-I, and LT-IIa, but not binding of LT-IIb, to CD8+ T cells induces apoptosis (2, 58). Binding of CT or LT-I to B cells leads to polyclonal activation and to upregulated expression of MHC-II, B7, CD40, CD80, CD86, ICAM-1, and IL-2R{alpha} (2, 36, 41). Type I enterotoxins also upregulate expression of CD80 and CD86 on macrophages and dendritic cells (36). Type II enterotoxins have somewhat similar effects on immunocompetent cells, but the effects are not as great. LT-IIa and LT-IIb upregulated CD80 and CD86 on B cells, yet the levels were lower than the levels observed when cells were treated with CT (2). LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) also bound to T cells, B cells, macrophages, and dendritic cells (Fig. 3). Binding of LT-IIa and LT-IIa(T14S) upregulated, to varying degrees, MHC-II, CD40, CD80, and CD86 on dendritic cells (Fig. 8). This enterotoxin-dependent upregulation of MHC-II and costimulatory molecules on dendritic cells likely enhances their roles in antigen presentation, a factor that may contribute to LT-IIa's ability to augment immune responses to coadministered antigens.

In conclusion, we demonstrated that the immunomodulatory activities and the toxicity of LT-IIa are altered by amino acid substitutions at positions T14 and T34 in the B polypeptide. Binding of LT-IIa to its known ganglioside receptors is essential for its immunostimulation properties, since less augmentation of antibody responses was observed when mice were immunized with antigen in the presence of mutant enterotoxins that exhibit lower binding activities with their ganglioside receptors. The amino acid substitutions, however, did not affect later immune responses, since all three mutant LT-IIa enterotoxins enhanced Ag-specific memory responses. Based on the data presented here, we propose that LT-IIa(T14I) and LT-IIa(T14D) are new mucosal adjuvants with extremely reduced toxicity that may have potential use in clinical settings.


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ACKNOWLEDGMENTS
 
We thank Swastika Majumdar and Robert Nugent for their technical help.

This work was supported by National Institutes of Health research grants DE013833 and DE014357 awarded to T.D.C and by grant DE06746 awarded to M.W.R. S.A. was supported by a Bertha H. & Henry C. Buswell Fellowship awarded by the University at Buffalo.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, the University of Buffalo, SUNY, 138 Farber Hall, 3435 Main St., Buffalo, NY 14214. Phone: (716) 829-3364. Fax: (716) 829-2158. E-mail: connell{at}acsu.buffalo.edu. Back

{triangledown} Published ahead of print on 21 November 2006. Back

Editor: A. D. O'Brien


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Infection and Immunity, February 2007, p. 621-633, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01009-06
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