M Leitner, Avigdor Shafferman, Zeev Altboum, I. Mendelson, S. Cohen, Itzhak Inbar, Eytan Elhanany, Yehoshua Gozes, David Kobiler, Morly Fisher, Tamar Bino, Chanoch Kronman, Baruch Velan, Ruth Barak, and H. Rosenberg
The etiological agent of anthrax disease in animals and humans is the spore-forming bacterium Bacillus anthracis. The major factors of virulence of B. anthracis are located on two plasmids, pXO1 and pXO2. pXO2 encodes a poly-d-glutamic acid capsule (19, 41), while pXO1 encodes two binary exotoxins, the lethal toxin (LT) and the edema toxin (ET) (43, 46, 61). These two toxins are composed of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a review, see reference 36). PA is the common receptor binding domain of the toxins and can interact with the two different effector domains, EF and LF, to mediate their entry into target cells (14). EF is a calmodulin-dependent adenylate cyclase (37) responsible for the edema seen at the site of infection in experimental animals (17). The LF is a metalloprotease (34) recently shown to cleave the amino termini of the mitogen-activated protein kinase kinases 1 and 2, which results in their inactivation (13). It remains to be determined whether these are the main physiological substrates for the LT activity in vivo (5, 22). Two types of anthrax vaccines are licensed for use in humans: the spores of the toxigenic, nonencapsulated B. anthracis STI-1 strain (55) and the cell-free PA-based vaccines consisting of aluminum hydroxide-adsorbed supernatant material from cultures of the toxigenic, nonencapsulated B. anthracis strain V770-NPI-R (49) or alum-precipitated culture filtrate from the Sterne strain (6). The use of the live attenuated STI-1 occasionally results in general and local adverse responses, observed both after primary application and revaccination, and the frequency of responses increases with the number of vaccinations (58). Furthermore, it was reported that the STI-1 vaccine has a relatively low immunogenicity (reviewed by Stepanov et al. in reference 58). To increase the immunogenicity, a combined vaccine of live STI-1 supplemented with cell-free PA formulation was evaluated and proposed for veterinary use (1). While the cell-free PA-based vaccines appear to be safer, they require numerous boosters (8) and were shown to have reduced ability to protect laboratory animals against certain virulent strains of B. anthracis (39, 60). In addition, these vaccines contain variable amounts of PA, as well as undefined quantities of LF and EF, adsorbed to aluminum hydroxide (4, 21, 49, 59). It appears, therefore, that there is a need for a safe and more efficient vaccine which could generate stable and prolonged immunity in humans (59). These conclusions led to the evaluation of various adjuvants with purified PA (2, 16, 29, 59) and to the creation of two types of live vaccines: vaccines based on nonvirulent B. anthracis (pXO1+) mutated strains (31, 47) and vaccines expressing PA from a cloned pagA gene using heterologous hosts such as the vaccinia virus, Bacillus subtilis, Salmonella typhimurium (10, 27, 28, 30, 31, 64), or a nontoxinogenic strain of B. anthracis (4). These pioneering studies suggest that recombinant B. anthracis live vaccines may have potential as a future anthrax vaccine. We report here the construction of several recombinant, nonencapsulated, and nontoxinogenic B. anthracis spore-forming strains expressing different levels of PA. We demonstrate that one of these strains, containing the pagA gene under a potent heterologous constitutive promoter, can be safely used to provide efficacious long-lasting immunity in experimental animals following a single immunization dose.