Mutants of Escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic AB complex and enzyme activity on Th1 and Th2 cells

Abstract

Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1-Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive.

FIG. 1

LTK63 enhances proliferation and Th1 and Th2 responses to nasally delivered Pa. BALB/c mice were immunized twice (weeks 0 and 4) i.n. with a two-component Pa consisting of rPT and FHA with or without LTK63 (10 μg) and with or without light anesthesia (anes.). At 6 weeks, spleen cells were isolated and stimulated in vitro with PT, FHA, or killed B. pertussis. After 72 h, supernatants were removed and IFN-γ, IL-4, and IL-5 concentrations were measured by immunoassay. Proliferation was assessed after 4 days. Results are mean cytokine concentrations or counts per minute for proliferation assays on spleen cells from four or five mice per experimental group. Error bars, standard deviations. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 2

LTR72 enhances systemic Th2-type responses to nasally delivered Pa. BALB/c mice were immunized twice (weeks 0 and 4) i.n. with a two-component Pa consisting of rPT and FHA with or without LTR72 (1 μg) and with or without light anesthesia (Anes.). At 6 weeks, spleen cells were isolated and proliferation and cytokine assays were performed as described in the legend to Fig. 1. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 3

LTR72 enhances local lymph node Th2-type responses to nasally delivered Pa. BALB/c mice were immunized twice (weeks 0 and 4) i.n. with a two-component Pa consisting of rPT and FHA with or without LTR72 (1 μg) and with or without light anesthesia (Anes.). At 6 weeks, thoracic lymph node (LN) cells (A) and superficial cervical LN cells (B) were prepared, and proliferation and cytokine assays were performed as described in the legend to Fig. 1. ∗ and +, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 4

Intranasal immunization with Pa formulated with LTK63 (A) or LTR72 (B) as an adjuvant enhances serum IgG and lung IgA responses. Mice were immunized as described in the legends to Fig. 1 and 2. Two weeks after immunization, serum and lung homogenates were prepared and anti-FHA and anti-PT IgG and IgA levels were determined by ELISA. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 5

Effect of toxin dose on the adjuvant effect of LTK63 (K63) and LTR72 (R72). BALB/c mice were immunized twice (weeks 0 and 4) i.n. with a two-component Pa consisting of rPT (5.0 μg), FHA (2.5 μg), and either LTK63 (1 or 10 μg) or LTR72 (1 or 10 μg) under light anesthesia. At 6 weeks, spleen cells were isolated, and proliferation and cytokines were assayed as described in the legend to Fig. 1. Results are mean values for four or five mice per experimental group. Error bars, standard deviations. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 6

Kinetics of B. pertussis clearance from the lungs after respiratory challenge of immunized mice. Groups of 20 BALB/c mice were immunized twice (at weeks 0 and 4) i.n. with a two-component Pa consisting of rPT and FHA with or without LTK63 (10 μg) or with the adjuvant alone (LTK63 in PBS) or PBS alone (A) or with a two-component Pa consisting of rPT and FHA with or without LTR72 (1 μg) and with or without light anesthesia (Anes.) or with the adjuvant alone (LTR72 in PBS) or PBS alone (B). Mice were challenged 2 weeks after the second immunization by aerosol inoculation with B. pertussis. Results are presented as mean lung CFU counts assessed on four individual mice per experimental group at each time point. Error bars, standard errors.

FIG. 7

Intranasal delivery of DTPa with LTK63 generates potent IgG and IgA responses against diphtheria, tetanus, and pertussis antigens. BALB/c mice were immunized i.n. with DTPa containing rPT (5 μg), FHA (2.5 μg), PRN (2.5 μg), TT (10 μg), CRM-197 (10 μg), and LTK63 (10 μg) in a 40-μl volume following light halothane anesthesia or with the same DTPa vaccine adsorbed to alum and injected by the i.m. route. Serum IgG and lung IgA levels against PT, FHA, PRN, TT, and DT were assessed 2 weeks after the second immunization. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus Pa alone (by Student's t test).

FIG. 8

Comparative T-cell responses generated following nasal delivery of DTPa with LTK63 and parenteral delivery of DTPa adsorbed to alum. Mice were immunized as described in the legend to Fig. 7, and T-cell responses were assessed against TT, CRM-197, and killed B. pertussis as described in the legend to Fig. 1. ∗, P < 0.05 versus Pa alone (by Student's t test).

FIG. 9

A mucosally delivered DTPa vaccine with LTK63 as an adjuvant confers protection against B. pertussis infection equivalent to that of a parenterally delivered DTPa formulation adsorbed to alum. Groups of 20 BALB/c mice were immunized as described in the legend to Fig. 7. Control mice were immunized with alum only by the i.m. route. The kinetics of B. pertussis clearance from the lungs after respiratory challenge of immunized mice was assessed as described in the legend to Fig. 6.