Nontoxic Shiga toxin derivatives from Escherichia coli possess adjuvant activity for the augmentation of antigen-specific immune responses via dendritic cell activation

Abstract

Shiga toxin (Stx) derivatives, such as the Stx1 B subunit (StxB1), which mediates toxin binding to the membrane, and mutant Stx1 (mStx1), which is a nontoxic doubly mutated Stx1 harboring amino acid substitutions in the A subunit, possess adjuvant activity via the activation of dendritic cells (DCs). Our results showed that StxB1 and mStx1, but not native Stx1 (nStx1), resulted in enhanced expression of CD86, CD40, and major histocompatibility complex (MHC) class II molecules and, to some extent, also enhanced the expression of CD80 on bone marrow-derived DCs. StxB1-treated DCs exhibited an increase in tumor necrosis factor alpha and interleukin-12 (IL-12) production, a stimulation of DO11.10 T-cell proliferation, and the production of both Th1 and Th2 cytokines, including gamma interferon (IFN-gamma), IL-4, IL-5, IL-6, and IL-10. When mice were given StxB1 subcutaneously, the levels of CD80, CD86, and CD40, as well as MHC class II expression by splenic DCs, were enhanced. The subcutaneous immunization of mice with ovalbumin (OVA) plus mStx1 or StxB1 induced high titers of OVA-specific immunoglobulin M (IgM), IgG1, and IgG2a in serum. OVA-specific CD4+ T cells isolated from mice immunized with OVA plus mStx1 or StxB1 produced IFN-gamma, IL-4, IL-5, IL-6, and IL-10, indicating that mStx1 and StxB1 elicit both Th1- and Th2-type responses. Importantly, mice immunized subcutaneously with tetanus toxoid plus mStx1 or StxB1 were protected from a lethal challenge with tetanus toxin. These results suggest that nontoxic Stx derivatives, including both StxB1 and mStx1, could be effective adjuvants for the induction of mixed Th-type CD4+ T-cell-mediated antigen-specific antibody responses via the activation of DCs.

FIG. 1.

Effects of StxB1, mStx1, and nStx1 on the expression of CD80, CD86, CD40, and MHC class II by bone marrow-derived DCs (BMDCs). BMDCs were cultured with Stx1 derivatives (StxB1, 1 μg/ml; mStx1, 1 μg/ml; nStx1, 1 μg/ml) for 48 h since a preliminary study showed that the maximum levels of surface antigen expression occurred between 24 and 48 h. Cell surface Ag expression was analyzed by flow cytometry as described in Materials and Methods. The data are presented as histograms and are expressed as means of three independent experiments. The percentage within each panel indicates the number of cells staining strongly for the indicated marker. *, P < 0.05 compared with the control medium-treated culture. Data were obtained by using the CD11c⁺ gated cell fraction.

FIG. 2.

Activation of OVA-specific CD4⁺ T-cell responses by Stx1 derivative-treated BMDCs. T-cell proliferation (A) and Th1 and Th2 cytokine production (B) by CD4⁺ T cells from DO10.11 Tg mice stimulated with Stx1 derivative-treated BMDCs were examined. BMDCs pretreated with 1 μg/ml Stx1 derivative were washed and then cocultured with purified CD4⁺ T cells (10⁶/ml) from DO11.10 Tg mice in the presence of 0.3 μM OVA_323-329 peptide for 3 days. An aliquot of cell culture was subjected to DNA synthesis by the addition of [³H]thymidine during the last 18 h of incubation. For cytokine analysis, another aliquot of CD4⁺ T cells was harvested and then treated with 50 nM PMA and 500 nM ionomycin overnight. No or little cytokine release was detected for CD4⁺ T cells without PMA and ionomycin. The results are expressed as mean E/C ± standard errors of the means (SEM) for triplicate cultures. *, P < 0.05 compared with the control medium-treated culture. The count for the control culture was 6,880 ± 380 cpm. The results of the T-cell proliferation assay (A) are expressed as mean E/C (experimental, stimulated value/control, nonstimulated value) ± SEM of triplicate cultures.

FIG. 3.

Induction of OVA-specific antibody responses by coadministered Stx1 derivatives. Mice were subcutaneously immunized with OVA plus mStx1, nStx1, or StxB1. Specifically, C57BL/6 mice were subcutaneously immunized with 100 μg of OVA plus 10 μg of the Stx1 mutant (E167R/R170L; mStx1) (hatched bars), 50 ng of nStx1 (dotted bars), or 10 μg of StxB1 (black bars) as an adjuvant or with OVA alone (white bars) on days 0 and 14. OVA-specific serum IgG, IgM, and IgA Ab (A) and splenic OVA-specific antibody-forming cell (AFC) (B) responses were determined by ELISAs and ELISPOT assays, respectively. Furthermore, OVA-specific IgG subclass Ab responses (C) were also analyzed by ELISAs. Serum samples were collected on day 21 and examined for OVA-specific Abs and OVA-specific IgG subclass Ab responses by ELISAs. Mononuclear cells were isolated from the spleens of subcutaneously immunized mice on day 21 and examined by Ag-specific ELISPOT assays. *, P < 0.05 compared with mice immunized with OVA alone. The results are expressed as means ± SEM from a total of three separate experiments, each of which used five or six mice per group.

FIG. 4.

Analysis of OVA-specific CD4⁺ T-cell responses induced by coadministered Stx1 derivatives. OVA-specific CD4⁺ Th-cell proliferative responses (A) and Th1 and Th2 cytokine synthesis (B) by CD4⁺ T cells isolated from the spleens of mice subcutaneously immunized with OVA plus 10 μg of mStx1 (shaded bars), 50 ng of nStx1 (dotted bars), or 10 μg of StxB1 (black bars) or with OVA alone (white bars) were examined. Purified splenic CD4⁺ T cells were cocultured at a density of 2 × 10⁶ cells/ml with 1 mg/ml of OVA and with T-cell-depleted, irradiated splenic feeder cells (4 × 10⁶ cells/ml) in complete medium containing rIL-2 (10 U/ml) for 3 days for proliferation assays and 5 days for cytokine synthesis measurements. A control culture consisting of the splenic CD4⁺ T cells of naïve mice, feeder cells, and rIL-2 (10 U/ml) resulted in the incorporation of 230 ± 42 cpm of [³H]thymidine. Culture supernatants were harvested and then analyzed for the synthesis of secreted cytokines by the use of appropriate cytokine-specific ELISAs. The minimum detection levels for the individual cytokines detected were as follows: IFN-γ, 9.4 pg/ml; IL-4, 7.8 pg/ml; IL-5, 15.6 pg/ml; IL-6, 15.6 pg/ml; and IL-10, 15.6 pg/ml. The results are expressed as means of the stimulation indexes ± SEM or pg/ml ± SEM from a total of three experiments using five or six mice per group. *, P < 0.05 compared with mice immunized with OVA alone. The results for OVA-specific CD4⁺ T-cell proliferative responses (A) are expressed as E/C (experimental, stimulated value/control, nonstimulated value).

FIG. 5.

Induction of TT-specific serum IgG, IgM, and IgA antibody responses by coadministered Stx1 derivatives (A) and protection against fatal challenge with tetanus toxin (B). Mice were subcutaneously immunized with TT plus mStx1, nStx1, or StxB1. Specifically, C57BL/6 mice were subcutaneously immunized with 100 μl of TT plus 1 μg of Stx1 mutant (E167R/R170L; mStx1) or 1 μg of StxB1 (hatched bars), 10 μg of Stx1 mutant or 10 μg of StxB1 (dotted bars), or 25 μg of Stx1 mutant or 25 μg of StxB1 (black bars) as an adjuvant or with TT alone (white bars) on days 0 and 14. Serum samples were collected on day 21 and examined for TT-specific Ab responses by ELISA. One week after the last immunization, mice were challenged on day 21 by the subcutaneous injection of 130 LD₅₀s of tetanus toxin in 0.5 ml of PBS including 0.2% gelatin. *, P < 0.05 compared with mice immunized with OVA alone. The results are expressed as means ± SEM from a total of three separate experiments, each of which used five or six mice per group.