Mucosal adjuvant properties of mutant LT-IIa and LT-IIb enterotoxins that exhibit altered ganglioside-binding activities

Abstract

LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female 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(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

FIG. 1.

(A) SDS-PAGE of purified non-His-tagged LT-IIa, His-tagged LT-IIa, and His-tagged LT-IIa(T34I) (lanes 1, 2, and 3, respectively), and His-tagged LT-IIb, His-tagged LT-IIb(T13I), and non-His-tagged LT-IIb (lanes 4, 5, and 6) dissociated into the A subunit (∼28 kDa) and B subunit monomers (∼12.5 and 13.5 kDa for non-His-tagged and His-tagged B subunits, respectively). (B) Western blot of non-His-tagged LT-IIa, His-tagged LT-IIa, and His-tagged LT-IIa(T34I) (lanes 1, 2, and 3, respectively), and His-tagged LT-IIb, His-tagged LT-IIb(T13I), and non-His-tagged LT-IIb (lanes 4, 5, and 6, respectively) probed with rabbit polyclonal antibodies to LT-IIa and LT-IIb, respectively. Molecular masses are noted in kilodaltons.

FIG. 2.

Binding of LT-IIa, LT-IIa(T34I), LT-IIb, and LT-IIb(T13I) to various gangliosides. Polyvinyl plates were coated with 10 ng of purified ganglioside or a mixture of gangliosides. Enterotoxins were allowed to bind to ganglioside-coated plates followed by probing with rabbit polyclonal antibodies. Plates were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody and nitrophenyl phosphate.

FIG. 3.

Salivary IgA (A) and vaginal IgA (B) antibody responses to AgI/II from mice after i.n. immunization with AgI/II alone or with LT-IIa, LT-IIa(T34I), LT-IIb, LT-IIb(T13I), or CT as adjuvant. Results are reported as the arithmetic means ± standard errors of means obtained from immunized mice (n = 6 to 8 mice per group). *, significant difference at P < 0.05 compared to LT-IIa.

FIG. 4.

Serum IgA (A), IgG (B), and IgG (C) subclass antibody responses to AgI/II after i.n. immunization of mice with AgI/II alone or with LT-IIa, LT-IIa(T34I), LT-IIb, LT-IIb(T13I), or CT as adjuvant. Results are reported as the arithmetic means ± standard errors of means of immunized mice (n = 6 to 8 mice per group). IgG subclasses were examined from mice at day 28. The arrow indicates the time point at which the third booster immunization with 5 μg of AgI/II was administered (day 203). * and **, significant differences at P < 0.05 and < 0.01, respectively, compared to LT-IIa.

FIG. 5.

Production of IFN-γ and IL-4 by AgI/II-specific lymphoid cells isolated from CLN (A and C) and spleens (B and D) of BALB/c mice immunized i.n. with AgI/II alone or with LT-IIa, LT-IIa(T34I), LT-IIb, LT-IIb(T13I), or CT at a point 40 days after the third immunization (day 60). Cells were stimulated in vitro for 4 days with 5 μg of AgI/II. Results are reported as the arithmetic means ± standard errors of means (n = 3). (A) ***, significant difference at P < 0.001 compared to LT-IIa. (B) *, significant difference at P < 0.05 compared to LT-IIa. (C) ****, significant difference at P < 0.0001 compared to LT-IIa; ***, significant difference at P < 0.001 compared to LT-IIb.

FIG. 6.

Binding of wt and mutants LT-IIa and LT-IIb to lymphoid cells isolated from CLN of naïve BALB/c mice. Histograms were gated on CD3⁺ (total T cells), CD4⁺ (helper T cells), CD8⁺ (cytotoxic T cells), B220⁺ (B cells), or CD11b⁺ (macrophages). Dead cells were excluded by propidium iodide staining. Light lines, binding patterns of LT-IIa(T34I) and LT-IIb(T13I); bold lines, binding patterns of LT-IIa and LT-IIb. A shift to the left in fluorescent intensity indicates a decrease or absence of binding of an enterotoxin to the cells.

FIG. 7.

Induction of cAMP production in macrophages after treatment with enterotoxin. cAMP was measured in RAW264.7 cells (5 × 10⁷) after incubation for 4 h with LT-IIa, LT-IIa(T34I), LT-IIb, LT-IIb(T13I), or CT. Results are reported as the arithmetic mean values ± standard errors of means (n = 3). *, significant difference at P < 0.05 compared to untreated cells; **, significant difference at P < 0.01 compared to LT-IIb(T13I); ***, significant difference at P < 0.001 compared to LT-IIa(T34I). The fold increase of cAMP in the treated cells over the untreated cells is denoted at the top of the respective bars.