Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines

Abstract

Anthrax is caused by the unimpeded growth of Bacillus anthracis in the host and the secretion of toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous when used immediately following exposure to B. anthracis. Thus, we have sought to develop a vaccine or therapeutic agent that is safe and eliminates both secreted toxins and bacilli. To that end, we have previously developed a dually active vaccine by conjugating the capsular poly-gamma-d-glutamate (PGA) with PA to elicit the production of antibodies specific for both bacilli and toxins. In the present report, we describe the improved potency of anthrax vaccines through the use of a dominant-negative inhibitory (DNI) mutant to replace PA in PA or PA-PGA vaccines. When tested in mice, DNI alone is more immunogenic than PA, and DNI-PGA conjugate elicits significantly higher levels of antibodies against PA and PGA than PA-PGA conjugate. To explain the enhanced immunogenicity of DNI, we propose that the two point mutations in DNI may have improved epitopes of PA allowing better antigen presentation to helper T cells. Alternatively, these mutations may enhance the immunological processing of PA by altering endosomal trafficking of the toxin in antigen-presenting cells. Because DNI has previously been demonstrated to inhibit anthrax toxin, postexposure use of DNI-based vaccines, including conjugate vaccines, may provide improved immunogenicity and therapeutic activity simultaneously.

FIG. 1.

(A) Serum levels of anti-PA IgG in individual mice after immunization with three doses of DNI and PA. Each point represents one mouse, and bars indicate the mean values for eight mice each. Group means (± standard errors) are, from left to right, 0.018 ± 0.004, 0.28 ± 0.15, 14.6 ± 2.9, 23.6 ± 3.0, 64.2 ± 4.6, and 271.5 ± 27.2 μg/ml anti-PA IgG, respectively. The first to third injections are the injection after which sera were obtained. (B) Competitive ELISA inhibition demonstrating the antigenic specificity of DNI and PA.

FIG. 2.

Serum levels of IgG specific for PA (A) and PGA (B) in mice immunized with PA-PGA or DNI-PGA conjugate. Each point represents one mouse, and bars indicate the mean values for eight mice each. Group means (± standard errors) are, from left to right, (A) 0.001 ± 0.002, 0.002 ± 0.004, 27.4 ± 2.4, 33.9 ± 2.8, 172.6 ± 8.4, and 385.3 ± 40.1 μg/ml anti-PA IgG, respectively, and (B) 0.001 ± 0.005, 0.001 ± 0.002, 7.8 ± 1.5, 30.3 ± 3.2, 44.2 ± 10.0, and 210.1 ± 42.2 μg/ml anti-PGA IgG, respectively. The first to third doses are the doses after which sera were obtained.

FIG. 3.

Molecular model of DNI. The model is modified from the crystal structure of PA. Mutated residues K397D and D425K are illustrated as space-filling models.

FIG. 4.

Models illustrating the different cellular fates of PA and DNI. (Top) Upon binding cellular receptors (step 1), PA is cleaved (step 2). The small fragments diffuse away, and the cell-bound fraction self-assembles into heptameric cores termed (PA₆₃)₇ (step 3). The heptamers then undergo receptor-mediated endocytosis (step 4). Once inside the acidic endosomal compartment, the PA heptamers change conformation and insert themselves into the endosomal membrane (step 5). (Bottom) DNI, similar to PA, enters the endosome (steps 1 to 4). However, DNI heptamers do not undergo the necessary conformational changes for insertion into the membrane and therefore remain trapped inside the endosome. The part of DNI that carries the two point mutations is shown in grey.