Mucosal vaccination against serogroup B meningococci: induction of bactericidal antibodies and cellular immunity following intranasal immunization with NadA of Neisseria meningitidis and mutants of Escherichia coli heat-labile enterotoxin

Abstract

Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

FIG. 1.

NhhA and NadA, but not App, induce potent serum IgG responses after intranasal immunization with LT or LT mutants. Mice were immunized intranasally with meningococcal protein App (A), NhhA (B), or NadA (C) together with LTwt (1 μg), LTR72 (1 μg), or LTK63 (10 μg). Serum was collected 2 weeks after the second booster immunization, and specific antibody responses were quantified by ELISA. Antibody titers are expressed as the reciprocal of the serum dilution that gave an OD₄₉₂ of 0.3 above that of preimmune serum. Symbols represent values for five or six individual mice per group; bars represent mean values. Results are representative of two experiments from two independent studies carried out in different laboratories. ***, P < 0.001 versus NhhA or NadA alone (ANOVA).

FIG. 2.

Intranasal immunization with NhhA (A) and NadA (B) in the presence of mucosal adjuvants induces IgG1 and IgG2a antibodies. Mice were immunized and data are presented as described in the legend to Fig. 1. Symbols represent values for five or six individual mice per group; bars represent mean values. ***, P < 0.001 versus NhhA or NadA alone (ANOVA).

FIG. 3.

IgA production following immunization with NhhA or NadA and mucosal adjuvants. Mice were immunized intranasally with meningococcal protein NhhA (A) or NadA (B and C) alone or together with LTwt, LTR72, or LTK63. IgA endpoint titers were determined by ELISA on nasal lavage (A and B) and lung homogenate (C) samples recovered 2 weeks after the third immunization. Symbols represent values for five or six individual mice per group; bars represent mean values. ** and ***, P < 0.01 and < 0.001, respectively, versus NhhA or NadA alone (ANOVA); + and +++, P < 0.05 and < 0.001, respectively, versus NadA with LTK63 (Tukey-Kramer test).

FIG. 4.

NadA induces potent bactericidal antibodies after intranasal immunization with LT or LT mutants. Mice were immunized intranasally with PBS, NadA alone, or NadA plus LTwt, LTR72, or LTK63. Bactericidal antibody titers are expressed as the reciprocal of the serum dilution yielding at least 50% killing of the test meningococcal strain. Sera from naïve animals and from animals immunized with NadA alone were tested in pools. Symbols represent values for 12 individual mice per group from two independent experiments; bars represent mean values.

FIG. 5.

Bactericidal antibodies in lungs of mice immunized intranasally with NadA and LT or LT mutants. Mice were immunized intranasally with PBS, NadA alone, or NadA plus LTwt, LTR72, or LTK63. Bactericidal antibody titers are expressed as the reciprocal of the lung homogenate dilution yielding at least 50% killing of the test meningococcal strain. Samples were tested in pools and are mean values for triplicate assays.

FIG. 6.

Induction of systemic Th1- and Th2-type responses in mice immunized with App or NhhA and LTwt, LTR72, or LTK63. Mice were immunized three times (0, 3, and 6 weeks) intranasally with App or NhhA (5 μg) alone or with LTK63 (1 or 10 μg), LTR72 (1 μg), or PBS only as a control. Two weeks after the last immunization spleen cells were stimulated in vitro with antigen (App or NhhA) (1 to 100 μg/ml), and cytokine concentrations in supernatants were determined 3 days later. Cytokine concentrations are expressed as means (+standard deviations) for five mice per group, tested individually in triplicate and are representative of two experiments. *, **, and ***, P < 0.05, < 0.01, and < 0.001, respectively, versus App or NhhA alone; + and +++, P < 0.05 and < 0.001, respectively, versus App or NhhA with LTK63 (1-μg dose) (Tukey-Kramer test).

FIG. 7.

Induction of Th1- and Th2-type responses in mice immunized with NadA and LT or LT mutants. Mice were immunized three times (0, 3, and 6 weeks) intranasally with PBS, NadA (10 μg) alone, or NadA with LTK63 (10 μg), LTR72 (1 μg), or LTwt (1 μg). Two weeks after the last immunization spleen cells from immunized mice were stimulated in vitro with antigen (NadA) (1 to100 μg/ml), and cytokine concentrations in supernatants were determined 3 days later. Cytokine concentrations are expressed as means (+standard deviations) for five mice per group, tested individually in triplicate, and are representative of two experiments from two independent studies. *, **, and ***, P < 0.05, < 0.01, and < 0.001, respectively, versus NadA alone (ANOVA).