Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity

Abstract

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Figure 1

OVA- and CT-B–specific Ab responses after subcutaneous immunization with OVA combined with mCTs or nCT as adjuvants. Serum IgM, IgG, and IgA responses (A) and IgG subclass responses (B) were assessed by endpoint ELISA. Splenic Ag-specific AFCs (C) were determined by ELISPOT assay. Groups of C57BL/6 mice were immunized subcutaneously with 100 μg of OVA alone or together with 10 μg of rCT-B, 1 μg of nCT, or 10 μg of mCTs, S61F, or E112K, on days 0 and 14. All assays were performed on samples from mice taken 1 wk after the last immunization. Bars represent the mean Ab titer and mean number of AFCs ± SEM in each group of 10 mice and the data are representative of three separate experiments.

Figure 2

OVA- and CTB–specific CD4⁺ T cell proliferative responses. Groups of C57BL/6 mice were immunized subcutaneously with 100 μg of OVA alone or together with 10 μg of rCT-B, 1 μg of nCT, or 10 μg of mCTs, S61F, or E112K, on days 0 and 14. Purified splenic CD4⁺ T cells were cultured at a density of 2 × 10⁶ cells/ml in the presence of 1 mg/ml of OVA or 10⁷ CT-B–coated beads/ml, T cell–depleted, irradiated splenic feeder cells (2.5 × 10⁶ cells/ml) and IL-2 (10 U/ml) in complete medium. Bars represent the mean stimulation index ± SEM in each group. Each group contained 10 mice and are representative of three separate experiments.

Figure 3

Cytokine production from OVA-specific splenic CD4⁺ T cells. Molecules of cytokine-specific mRNA were determined by quantitative RT-PCR using rRNA internal standards. Cytokine production was determined by ELISA. The scale of each figure corresponds to mRNA molecules and protein levels produced by nonimmunized CD4⁺ T cells stimulated with anti-CD3 mAb. Bars represent the mean cytokine profile ± SEM in each group. ND indicates that the molecules were not detected. Each group contained five mice and are representative of three separate experiments.