Attenuated Nontoxinogenic and Nonencapsulated Recombinant Bacillus anthracis Spore Vaccines Protect against Anthrax

Abstract

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 ؋ 10 7 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>100 g/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.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 i...

Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

“…In brief, PA was added to cultured RAW264.7 cells and incubated for a period of 5,15,25,30,45 and 60 min at 37 °C. PA was removed and the cells were washed by fresh culture medium, followed by the addition of LF and incubation at 37 °C for 4 hr before conducting the MTT assay.…”

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits

“…Briefly, standards and test sera were preincubated in 96-well microtiter plates (Corning Costar, Acton, MA) with purified rPA (50 ng/ml final concentration) and lethal factor (40 ng/ml final concentration) for 1 h at 37 • C. before transferring to a plate containing a monolayer of J774A.1 cells, plated the day before the assay with 5 × 10 4 cells per well. Plates were incubated for 4 h at 37 • C in 5% CO 2 , followed by addition of 25 l of 3-[4,5-dimethyl-thiazol-2-y-]-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Company) at 5 mg/ml in PBS for 2 h before lysing the cells by adding 20% (w/v) SDS in 50% dimethylformamide, pH 4.7. Optical density readings were obtained at 570 nm with a reference wavelength at 690 nm on a BioTek 312e microplate reader (BioTek Instruments).…”

“…Several candidate vaccines based on rPA have been described [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] and have been shown to be highly protective against lethal aerosol or parenteral B. anthracis spore challenge.…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…Recent evidences indicate that germination of B. subtilis spores located into phagosomes is a key step on the activation of immune responses since it permit antigen presenting cells to sample, process and display peptides to lymphocytes (Hoa et al 2001). Similar events seem to occur with the spores of the more immunogenic B. anthracis vaccine strains, which germinate and transiently multiply inside macrophages (Cohen et al 2000). Thus, development of B. subtilis strains with enhanced ability to elicit immune responses to passenger antigens shall explore mimic some B. anthracis features as early and more efficient intracellular spore germination, ability to transiently survive and multiply inside phagocytic cells or in vivo express antigens more efficiently after phagocytosis.…”