Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant

Abstract

The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.

FIG. 1.

SDS-PAGE analysis of purified TxA_C314HA. E. coli BL21(DE3) carrying the plasmid pGEX/TxA_C314 was grown for 6 h at 37°C following induction with IPTG; cells were then collected by centrifugation and lysed. Soluble proteins were subjected to affinity chromatography purification on a glutathione-Sepharose 4B resin and then separated through SDS-12% PAGE gels stained with Coomassie blue. Lane A, total cell lysate following 6 h of induction with IPTG and before affinity column purification; lane B, recombinant protein (GST-TxA_C314) obtained following affinity chromatography of cell lysate on glutathione-Sepharose 4B resin and collected by addition of reduced glutathione; lane C, purified GST and TxA_C314 obtained following digestion of recombinant GST-TxA_C314 with thrombin; lane D, purified recombinant TxA_C314 obtained by overnight digestion with thrombin (1 U/20-ml resin bed volume) of cell lysate purified and immobilized on glutathione-Sepharose 4B resin; lane MW, molecular mass markers (apparent molecular masses are shown in daltons).

FIG. 2.

Binding of TxA_C314 to enterocytes in mouse intestine. Closed ileal loops were injected with buffer, either alone or containing 20 μg of TxA_C314. After 10 to 120 min the animals were sacrificed; loops were removed, fixed in 4% buffered paraformaldehyde, and placed in embedding medium for frozen tissue specimens; and 10-μm-thick longitudinal sections were cut. To determine TxA_C314 binding to intestinal epithelial cells, sections were incubated with a rabbit polyclonal antibody against the HA epitope fused at the N terminus of TxA_C314. The immunocomplexes were detected with a FITC-conjugated donkey anti-rabbit IgG, and then sections were analyzed with a Leica TCS-NT/SP2 confocal microscope with a 40× objective. (A) Absence of signal in a loop injected with buffer. (B) Representative section obtained from an ileal loop incubated for 10 min with TxA_C314. With the anti-HA antibody, it is possible to detect the presence of a strong signal at villus tips, indicating the presence of TxA_C314 bound to the epithelium. (C) Representative section obtained from an ileal loop 120 min after injection with TxA_C314. Following incubation with a rabbit antibody against the HA epitope fused to TxA_C314 it is possible to observe the presence of a faint signal within the cytoplasm of the epithelial cells.

FIG. 3.

Levels of systemic KLH-specific antibodies in mice immunized by oral gavage. BALB/c mice (8 to 12 per group) were immunized orally on days 0 and 14 with KLH (5 mg) alone or supplemented with TxA_C314 (0.1 to 10 μg), CT (10 μg), or EtxB (10 μg). At day 35, blood was collected by retro-orbital bleeding and serum concentrations of IgG (A), IgA (B), IgG1 (C), and IgG2a (D) reactive to KLH were measured by ELISA. In some experiments TxA_C314 (10 μg/mouse) was preincubated in vitro with anti-C. difficile toxin A antiserum (anti-TxA_C314) and then coadministered by oral gavage with KLH. The level of specific antibodies reacting to KLH was determined by preparing a reference curve by using mouse IgG, IgA, IgG1, or IgG2a and subtracting the background from each sample (nonimmune mice [n = 8]). Results are means ± standard errors. *, P < 0.01 versus KLH alone; ^, P < 0.01 versus KLH plus EtxB, °, P < 0.01 versus KLH plus CT.

FIG. 4.

Fecal anti-KLH- and anti-HEL-specific antibodies. BALB/c mice (8 to 12 per group) were immunized on days 0 and 14 orally with KLH (5 mg) or intranasally with HEL (5 mg) either alone or supplemented with TxA_C314 (0.1 to 10 μg), CT (10 μg), or EtxB (10 μg). Three weeks after the second inoculation, feces were collected and anti-KLH- (A) or anti-HEL (B)-specific IgA levels were measured by ELISA. Results are means ± standard errors. *, P < 0.01 versus KLH (A) or HEL (B) alone; °, P < 0.05 versus KLH plus EtxB; #, P < 0.05 versus HEL plus CT.

FIG. 5.

Levels of systemic anti-HEL-specific antibodies in mice immunized intranasally. BALB/c mice (8 to 12 per group) were immunized intranasally on days 0 and 14 with HEL (5 mg) alone or supplemented with TxA_C314 (0.1 to 10 μg), CT (10 μg), or EtxB (10 μg). At day 35 blood was collected by retro-orbital bleeding and serum concentrations of IgG (A), IgA (B), IgG1 (C), and IgG2a (D) reactive to HEL were measured by ELISA. The level of specific antibodies reacting to HEL was determined by preparing a reference curve with mouse IgG, IgA, IgG1, or IgG2a and subtracting the background from each sample (nonimmune mice [n = 8]). Results are means ± standard errors. *, P < 0.01 versus HEL alone; °, P < 0.05 versus HEL plus EtxB.

FIG. 6.

Levels of systemic anti-CT, anti-EtxB, and anti-TxA_C314 in mice immunized by oral gavage or nasotracheally. BALB/c mice (8 to 12 per group) were immunized twice orally with KLH (5 mg) or intranasally with HEL (5 mg) supplemented with 10 μg of either TxA_C314, CT, or EtxB or with just buffer, and after 35 days blood was collected by retro-orbital bleeding. Following oral gavage (A) or intranasal (B) immunization, specific IgG anti-CT, anti-EtxB, and anti-TxA_C314 in serum were measured by ELISA. The level of specific antibodies was determined by preparing a reference curve with mouse IgG and subtracting the background from each sample (nonimmune mice [n = 8]). Results are means ± standard errors. **, P < 0.01 versus control; *, P < 0.05 versus control.

FIG. 7.

Cytokine production of spleen cells isolated from mice immunized by oral gavage. BALB/c mice were immunized orally on days 0 and 14 with KLH (5 mg) alone or supplemented with TxA_C314 (10 μg), CT (10 μg), or EtxB (10 μg). On day 35 splenocytes were collected, seeded in culture (10⁶/ml), and in vitro restimulated with KLH (50 μg/ml) (black bars) or left unstimulated (open bars). Culture supernatants collected after 72 h were assayed for cytokines IFN-γ, IL-2, IL-4, and IL-10 by ELISA. Results are means ± standard errors from three to six mice per group. *, P < 0.05 versus the respective nonstimulated cells; °, P < 0.05 versus mice immunized with KLH plus EtxB; ^, P < 0.05 versus mice immunized with KLH plus CT.