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* Department of Microbiology and
Department of Pediatric Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294;
Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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) (9, 10, 11) and dendritic cells (12). Anthrax toxin-fusion protein derivatives consisting of PA and the N-terminal domain of LF (LF1–254) have been used to deliver Ags into the cytosol for presentation via MHC class I molecules and induction of CTL responses (13, 14, 15, 16). A recent report suggested that PA may not be needed for intracellular delivery of proteins by the LF N-terminal fragment (17). Intradermal coimmunization with a DNA plasmid encoding the N-terminal fragment of LF, which shares homology with the N-terminal fragment of EF, was reported to induce higher anti-PA Ab responses than immunization with a single plasmid encoding PA (18). Although EdTx was reported to induce accumulation of cAMP in lymphocytes (19) and suppress T cell activation (20), little is known about the effect of EdTx on adaptive immune responses. Cholera toxin (CT) and the related heat labile toxin I (LT-I) of Escherichia coli are AB-type toxins made of pentameric-binding B subunits and enzymatic A subunits with ADP-ribosyl transferase activities (21, 22, 23). The B subunits of CT and LT-I bind to GM1 gangliosides on target cells (24), while the more promiscuous B subunit of LT-I also exhibits affinity for GM2 and asialo-GM1 (25, 26, 27). CT and LT-I are the best described mucosal adjuvants and both promote mucosal secretory IgA (S-IgA) and plasma Ab responses to coadministered vaccine Ags. Unfortunately, the watery diarrhea induced by these toxins precludes their use as oral adjuvants in humans. In addition, major safety concerns relative to the potential of nasal enterotoxins to target CNS tissues have been reported (28, 29). Thus, nasal enterotoxin could damage CNS tissues in large part through their ADP-ribosyl transferase activity following binding of the B subunit to the promiscuous gangliosides expressed on cells of the CNS (30, 31).
We investigated whether an EdTx derivative could act as a mucosal adjuvant like the enterotoxin CT and LT-I, and promote S-IgA and systemic Ab responses to nasally coadministered vaccine Ags. We also queried whether this regimen would enhance immunity to the binding B subunit PA itself and perhaps provide an extra bonus for anthrax immunity. We further examined whether the receptor specificity of EdTx would lead to the accumulation of this toxin into CNS tissues after nasal delivery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Female C57BL/6 mice, 6–7 wk of age, were obtained from Charles River Laboratories and were 9–12 wk of age when used in these experiments. All studies were performed in accordance with both National Institutes of Health and University of Alabama at Birmingham institutional guidelines to avoid pain and distress.
Immunization
Mice were nasally immunized three times at weekly intervals with 100 µg of OVA (Sigma-Aldrich) alone, OVA plus 5 µg of rPA only, or OVA plus EdTx (5 µg of rPA together with 5 µg of rEF). The rPA was purified from cultures of a recombinant strain of B. anthracis as previously described (32). The EF was obtained from List Biological Laboratories (product no. 173) and was produced in a recombinant strain of B. anthracis using an expression plasmid constructed by S. H. Leppla. This EF protein contains a S447N mutation and was shown to display 
20-fold less enzymatic activity than the native EF (33). Controls included mice nasally immunized with OVA plus 1 µg of CT (List Biological Laboratories). Mice were lightly anesthetized and given 12.5 µl of vaccine/nostril. Blood and external secretions (fecal extracts, vaginal washes, and saliva) were collected as previously described (34, 35).
Evaluation of OVA- and PA-specific Ab isotypes and IgG subclass responses
Previously described ELISA was used to assess anti-OVA and anti-PA Ab levels in plasma and external secretions (34, 35). Briefly, microtiter plates were coated with OVA (1 mg/ml) or PA (5 µg/ml). The IgM, IgG, or IgA Abs were detected with HRP-conjugated goat anti-mouse µ-, -, or 
-H-chain-specific antisera (Southern Biotechnology Associates). Biotin-conjugated rat anti-mouse IgG1 (clone A85-1; 0.5 µg/ml), IgG2a (clone R19-15; 0.5 µg/ml), IgG2b (clone R12-3; 0.5 µg/ml), or IgG3 (clone R40-82; 0.5 µg/ml) mAbs and HRP-conjugated streptavidin (BD Pharmingen) were used to measure IgG subclass responses. The color was developed with the addition of ABTS substrate (Sigma-Aldrich), and the absorbance was measured at 415 nm. End-point titers were expressed as the log2 dilution giving an OD415 of 
0.1 above those obtained with nonimmunized control mouse samples.
Total and Ag-specific IgE Abs
Total IgE Ab levels were determined by a BD OptEIA Set Mouse IgE (BD Pharmingen), according to instructions from the manufacturer. To prevent interference in the assay, serial dilutions of immune plasma were previously depleted of IgG by overnight incubation in Reacti-Bind Protein G-Coated Plates (Pierce). To detect Ag-specific IgE, the microtiter plates were coated with OVA (1 mg/ml) or PA (5 µg/ml). Serial dilutions of plasma were then added, IgE was detected with the biotinylated anti-mouse IgE Abs, and titers were determined as described above.
Macrophage toxicity assay to assess anti-PA-neutralizing Abs
The protective effects of PA-specific Abs were determined using a previously described assay (35) that measures their capacity to protect the J774 M cell line from LeTx (9, 11). Briefly, J774 M (5 x 104 M/well) were added to 96-well, flat-bottom plates. After 12 h of incubation, plasma or external secretions samples were added together with LeTx (400 ng/ml PA plus 40 ng/ml LF) and incubated for an additional 12 h as described elsewhere (31). Viable M were evaluated after addition of MTT (Sigma-Aldrich) (36).
Effect of EdTx on APCs in vitro
J774 M (5 x 104 cells/ml) or freshly isolated mesenteric lymph node or spleen cells from C57BL/6 mice were incubated in the presence of PA only (5 µg/ml), EF only (5 µg/ml), EdTx (PA + EF; 5 µg/ml), or CT (1 µg/ml). Forty-eight hours later, culture supernatants were collected for evaluation of cytokine responses. Cells were collected and stained for 30 min at 4°C with FITC- or PE-conjugated mAbs (BD Pharmingen). After three washing steps and fixation in 2% paraformaldehyde, the expression of activation and costimulatory molecules was analyzed by flow cytometry.
In vitro restimulation of Ag-specific CD4+ T cells and cytokine-specific ELISA
T cells were isolated from spleen and cervical lymph nodes (CLNs) and restimulated in vitro as previously described (34, 35, 37) with OVA (1 mg/ml) or PA (20 µg/ml) in RPMI 1640 medium containing 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, 5 x 10–5 M 2-ME, 1 mM sodium pyruvate, and 10% FCS. The Th1 and Th2 cytokines in culture supernatants were determined by cytokine-specific ELISA as previously described (34, 35, 38). The mAb couples were from BD Pharmingen. Standard curves were generated using murine rIFN-, rIL-5, rIL-6, and rIL-10, rIL-12 (R&D Systems); rIL-2 and rIL-13 (BD Pharmingen); and rIL-4 (Pierce). The ELISAs were capable of detecting 5 pg/ml for IL-2, IL-4, and IL-5; 15 pg/ml for IFN-; 50 pg/ml for IL-13; 100 pg/ml for IL-6, IL-12; and 200 pg/ml for IL-10.
Tracking studies
The PA and CT were labeled with acridinium using an acridinium C2 NHS ester labeling kit (Assay Designs). The specific activity of PA and CT used in the tracking studies were 2.11 x 107 relative light units (RLU)/ng and 2.06 x 107 RLU/ng, respectively. Mice were given the acridinium-labeled compounds by instilling 5-µl quantities into each nare. Twelve and 24 h following immunization, mice were sacrificed and the olfactory nerves and epithelium (ON/E), olfactory bulbs (OB) and brain (B) were removed as previously described (30). Each tissue was weighed and 200 µl of Cellytic MT lysis buffer (Sigma-Aldrich) was added per 10 mg wet weight of tissue. The ice-cold tissues were homogenized (20,000 rpm for 15–20 s) using a Tissue Tearor (Biospec Products) and frozen at –20°C. After thawing, the homogenates were centrifuged at 10,000 x g for 10 min and the supernatants were tested for light activity in triplicate in 96-well Microlite 2 plates (Thermo Labsystems). Nonimmunized mouse tissues served as controls.
Nasal EdTx-induced cytokine responses in vivo
Mice were given PBS, EdTx or CT by instilling 5-µl quantities into each nare. Twelve and 24 h later, mice were sacrificed and nasal washes were collected in 500 µl of PBS and the cytokine content was analyzed by ELISA. The nasopharyngeal-associated lymphoepithelial tissue (NALT), CLN, ON/E, and OBs were removed and subjected to real time RT-PCR (Lightcycler; Roche). The cytokine mRNA levels are expressed as crossing points (CP) or the cycle at which the fluorescence rises appreciably above the background fluorescence as determined by the Second Derivative Maximum Method (Roche Molecular Biochemicals LightCycler Software). The formula mRNA = 2(–(CPcytokine – CP
-actin)) corrects for differences in cDNA concentration between the starting templates of cytokine and housekeeping (i.e., -actin) genes (39). The simplified formula 20 – (CPcytokine – CP- actin) was used to express relative cytokine mRNA responses in tissues of mice given nasal EdTx.
Statistics
The results are expressed as the mean ± 1 SD. Statistical significance (*, p 
0.05) was determined by Student’s t test and by ANOVA followed by the Fisher least significant difference test. For statistical analysis, cytokine levels below the detection limit were recorded as one-half the detection limit (e.g., IFN- = 7.5 pg/ml).
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Results |
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We first examined whether the adenyl cyclase EdTx, which acts through the ATR, would enhance Ab responses to mucosally administered protein Ags. Coadministration with 1 µg of EdTx (1 µg of PA and 1 µg of EF) enhanced OVA-specific plasma IgG Ab responses (Fig. 1A). Higher Ab responses were seen in mice given OVA and 5 µg of PA and 5 µg of EF although the titers failed to reach the statistical difference (Fig. 1A). The binding of EdTx to its receptor alone did not significantly contribute to the observed adjuvant activity, because neither PA alone (Fig. 1B) nor EF alone (data not shown) significantly increased OVA-specific IgG or promoted OVA-specific IgA Ab responses. Coimmunization with PA and EF (i.e., EdTx) sharply increased OVA-specific IgG and induced high levels of IgA Abs (Fig. 1B). The EdTx-induced IgG subclass responses consisted mainly of IgG1 and IgG2b Abs (Fig. 1C). Plasma samples collected 1 wk after two nasal immunizations with OVA and EdTx contained elevated levels of total IgE Abs and low but significant OVA-specific IgE Abs that were not detected in mice nasally immunized with OVA only or OVA plus PA (Fig. 1D).
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In addition to acting as adjuvants for mucosally coadministered Ags, CT and LT-I are good immunogens that induce high Ab responses to their respective cell ganglioside-binding B subunits (40, 41, 42, 43). Thus, we next addressed whether EdTx could enhance immunity to its binding PA subunit. Low levels of PA-specific IgG and IgA Abs were seen in the plasma of mice that received 5 µg of PA. Interestingly, PA-specific IgG and IgA Ab responses were both significantly increased in mice given EdTx (5 µg of PA plus 5 µg of EF), indicating that the enzymatic EF subunit also enhanced PA-specific Ab responses (Fig. 2A). PA-specific IgE Abs were not detected in mice that received PA without EF. However, coadministration of EF induced PA-specific IgE Ab levels (Fig. 2A), which were significantly higher than OVA-specific Abs in the same mice (Fig. 1C). The EdTx-induced PA-specific IgG subclass responses also predominantly consisted of IgG1 and IgG2b Abs followed by IgG2a and IgG3 (Fig. 2B).
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Because nasal administration of EdTx enhanced systemic Ab responses to both the coadministered protein (i.e., OVA) and the binding B subunit (i.e., PA) itself, we next ascertained whether EdTx also induced PA- and OVA-specific IgA and IgG Abs in saliva and mucosal IgA Ab responses in other external secretions. OVA-specific IgA Abs were not detected in the saliva or the vaginal secretions of mice given OVA only or OVA plus PA (Fig. 3A). In contrast, the saliva of mice that received EdTx exhibited both OVA- and PA-specific IgA and IgG Abs (Fig. 3A). In addition, PA- and OVA-specific IgA Abs were detected in the vaginal washes of mice immunized with OVA plus EdTx (Fig. 3B).
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EdTx induces anti-PA-neutralizing Abs
Nasal immunization with OVA and PA resulted in low levels of neutralizing Abs that were seen only in the plasma (Table I). Mice immunized with OVA and EdTx exhibited significantly higher titers of neutralizing Abs in the plasma than mice immunized with OVA plus PA (Table I). More interestingly, saliva of mice given OVA and EdTx contained significant levels of anti-PA neutralizing Abs (Table I). No significant neutralizing Abs were detected in vaginal washes (Table I).
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To characterize CD4+ Th cell cytokine pathways associated with EdTx-induced immunity, we examined the pattern of cytokines secreted by OVA- and PA-specific CD4+ T cell after a 5-day in vitro restimulation. Spleen CD4+ T cells from mice immunized with OVA and EdTx secreted mixed Th1- and Th2-type cytokines after in vitro restimulation with OVA or PA as indicated by high levels of IFN- (Th1) but also IL-5, IL-6, and IL-13 in culture supernatants (Table II). IL-4 levels were below the limit of detection. The same profile of cytokine responses was seen in the culture supernatant of CLN CD4+ T cells restimulated under the same conditions (data not shown).
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, in vitro
We assessed the direct effects of EdTx or CT on the expression of costimulatory molecules (i.e., CD40 and CD86) as well as cytokine secretion by J774 M to address whether EdTx regulates APC functions. Consistent with previous reports (44, 45, 46), CT induced high levels of IL-6 responses in treated cells (Fig. 4A) and increased costimulatory molecule expression (Fig. 4B) by M in vitro. The IL-6 responses after EdTx stimulation were 10-fold lower than those measured in culture supernatants of J774 M cultured in the presence of CT (Fig. 4). However, EdTx up-regulated the expression of both CD40 and CD86 by J774 M to the same extent as the mucosal adjuvant CT. These findings were further confirmed on T cell-depleted spleen cells (data not shown).
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We next explored the possibility that EdTx could target the ON/E or other CNS tissues and induce inflammatory responses. Fig. 5A illustrates the failure of PA to accumulate in the ON/E 24 h following nasal delivery of 5 µg of PA. In addition, there was no PA detectable in the OBs or brain and only minor amounts (< 0.15 ng/10 mg tissue) were observed in the NALT, CLN, or spleen 24 h after nasal delivery (data not shown). Nasal PA given together with EF (EdTx) did not increase PA accumulation in olfactory or brain tissues (Fig. 5A). In contrast to PA or EdTx, nasal delivery of a 0.5-µg dose of CT resulted in significant accumulation in the ON/E which was further increased when the CT dose was 10-fold higher (Fig. 5A). We also found that nasal CT, but not EdTx, up-regulated IL-1 mRNA levels in the ON/E and in the NALT suggesting that CT targeted these two sites (Fig. 5B). Finally, mice given nasal CT, but not those given nasal EdTx, exhibited high levels of IL-6 in nasal washes (Fig. 5C).
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Discussion |
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Anthrax toxin derivatives have been evaluated as molecular syringes for intracellular delivery of peptides (13, 51) or protein Ags (14, 15, 16) for presentation via the MHC class I pathway and induction of cytotoxic CD8+ T cells. Our results show that in vivo delivery of anthrax EdTx provides necessary signals for induction of mucosal and systemic immunity to coadministered protein Ags. Although data summarized in this manuscript only referred to nasal delivery of EdTx, we also found that EdTx is an adjuvant for protein Ags coinjected i.p. in both C57BL/6 and BALB/c mice (data not shown). Studies with the ganglioside-targeting enterotoxins CT and LT-I (52, 53, 54, 55) and derivatives including their chimeras (34, 56) or the B cell-targeting CTA1-DD (31, 57) have demonstrated the importance of receptor binding for controlling the immune responses induced by these ADP-ribosylating adjuvants. Thus, the adjuvant activity of CT appears to be more dependent on IL-4 and CD4+ Th2 cell cytokines (52, 53). In contrast, the more promiscuous LT-I, which binds GM1 gangliosides like CT but also asialo-GM1 and -GM2 gangliosides, promotes a broader spectrum of responses with CD4+ Th cells producing both IFN- and Th2-type cytokines (34, 54). The adjuvant activity of nasal EdTx appears to involve Ab and T cells responses that resemble those induced by LT-I rather than CT. Thus, EdTx induced CD4+ T cells secreting both IFN- and Th2-type cytokines. Further, EdTx as an adjuvant promoted only modest levels of OVA-specific IgE (log2 titers = 3) when compared with those seen after nasal immunization with CT (i.e., log2 titers = 8). Although it has been suggested that ATRs may not be expressed at the apical membrane of epithelial cells (50), there is no information to date on the relative expression of ATRs on immune cells and potential cellular targets of nasally administered EdTx. We have shown here that PA alone or as a component of EdTx does not target olfactory tissues and do not induce IL-1-specific mRNA in ON/E. It is also important to note that EdTx promotes a similar profile of serum Ab responses than CTA1-DD which targets B cells (31).
The mechanisms underlying the induction of mucosal immunity and S-IgA Ab responses by bacterial toxins remain only partially understood. Studies over the past two decades have shown a role for CT-induced cytokines on its mucosal adjuvanticity. Thus, CT was shown to induce both IL-6 and IL-1 secretion by epithelial cells and APCs (44, 58, 59, 60). Both IL-1 and IL-6 were later shown to be adjuvants for systemic immunity to nasally coadministered protein vaccines (38, 61) and IL-1 was also able to promote mucosal IgA Ab responses (61). Other factors thought to contribute to the adjuvanticity of CT include its ability to up-regulate the expression of MHC (44) and costimulatory molecules (45, 46, 62). We have shown that EdTx stimulates IL-6 secretion by M cultures and enhances the expression of costimulatory molecules. We should stress that IL-6 levels induced by EdTx were 10-fold lower than those seen in M cultures stimulated with the same dose of CT. The lower stimulatory effect of EdTx for IL-6 secretion was further confirmed in vivo where nasal delivery of CT but not EdTx induced IL-6 secretion in nasal washes. It is unlikely that the reduced ability of EdTx to induce IL-6 (and IL-1) could alone explain the polarized Ag-specific mucosal IgA Abs responses when compared with those generally induced by CT (34, 46, 63) or LT as adjuvants (34, 46, 54, 56). The role of the adenylate cyclase activity for the adjuvanticity remains to be elucidated. In this regard, the EF used in our studies contains a S447N mutation that could account for its recently reported 20-fold lower ability to induce cAMP when compared with recombinant native EF protein (33). Several mutants of both CT and LT-I, which are devoid of ADP-ribosyl transferase activity, were shown to retain their mucosal adjuvant activity and the ability to promote high mucosal IgA Ab responses. In another system, the mucosal adjuvant activity of CTA1-DD was reported to require both effective B cell targeting and the ADP ribosyl transferase activity (57). Thus, further studies are warranted to establish the mechanisms of mucosal adjuvanticity of EdTx.
Enterotoxin adjuvants are also potent immunogens which induce elevated immune responses to their binding subunits (41, 43). A major finding of this study resides in the fact that EdTx is a potent immunogen and that the presence of EF increased anti-PA responses above levels achieved after administration of PA alone. We have previously reported that high levels of PA-specific Abs could be detected in mouse plasma following nasal immunization with PA doses of up to 25 µg when CT was used as adjuvant (35). The same studies showed that 40 µg of PA/dose were needed to promote PA-specific mucosal IgA Abs. The results summarized here indicated that ATR targeting with much lower doses of PA (i.e., 5 µg) and EF allows the induction of PA-specific S-IgA Abs. Interestingly, increasing the dose of PA given with EF to 40 µg did not increase the levels of PA-specific mucosal IgA Abs, suggesting that codelivery of EF achieved optimal S-IgA Ab responses with low nasal dose of PA. Although, nasal immunization with CT or LT-I as adjuvant primarily promotes IgA Ab responses in the respiratory and genitourinary tracts, low but significant responses are consistently seen in the gastrointestinal tract. PA was previously reported to deliver a functionally active CT-A subunit into mammalian cells using an LF1–254-CT-A fusion protein (64). Therefore, it will be interesting to examine whether the ATR-mediated cellular targeting, the adenylate cyclase, and possibly other activities of EF govern the polarized PA-specific mucosal IgA Ab responses induced by EdTx as adjuvant.
In summary, we have shown that ATR targeting of an adenylate cyclase subunit provides an effective strategy for enhancing the immune response to nasal vaccines. The EdTx as nasal adjuvant does not target the olfactory or other CNS tissues and thus could represent a safer alternative to ganglioside-binding adjuvants. In addition, EdTx very efficiently promoted anti-PA Ab responses both in saliva and in plasma and this could have important implications for improving the efficacy of current PA-based anthrax vaccines.
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI 43197, AI 18958, DC 04976, DE 12242, and DK 44240. 
2 Address correspondence and reprint requests to Dr. Prosper N. Boyaka, Department of Microbiology, University of Alabama at Birmingham, 772 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294-2170. E-mail address: prosper{at}uab.edu
3 Abbreviations used in this paper: PA, protective Ag; LF, lethal factor; EF, edema factor; EdTx, anthrax edema toxin; LeTx, lethal toxin; ATR, anthrax toxin receptor; CT, cholera toxin; LT-I, heat labile toxin-I; S-IgA, secretory IgA; CLN, cervical lymph node; RLU, relative light unit; ON/E, olfactory nerves and epithelium; OB, olfactory bulb; M, macrophage; NALT, nasopharyngeal-associated lymphoepithelial tissue; CP, crossing point.
4 Current address: College of Veterinary Medicine, Auburn University, AL 36849.
Received for publication June 13, 2005. Accepted for publication November 2, 2005.
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and MIP-1 differentially mediate mucosal and systemic adaptive immunity. Blood 101: 807-814. 
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H. F. Nawar, S. Arce, M. W. Russell, and T. D. Connell Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants Infect. Immun., February 1, 2007; 75(2): 621 - 633. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), OVA plus 5 µg of PA (
), or OVA plus 5 µg of EdTx (5 µg of PA plus 5 µg of EF) (
). Plasma levels of OVA-specific Abs were determined 1 wk after the last immunization. The results are expressed as the reciprocal log2 titers ± one SD and are from three (A) or five experiments (B–D) and four mice/group. *, p < 0.05.
