Vaccine protection against Bacillus cereus-mediated respiratory anthrax-like disease in mice

Abstract

Bacillus cereus strains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores of B. cereus G9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-type B. cereus G9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized the pagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded by pagA2. AVA immunization protected mice against a lethal challenge with spores from B. cereus G9241 or B. cereus Elc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis of B. cereus anthrax-like disease in mice is dependent on pagA1 and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.

Fig 1

B. cereus G9421 virulence plasmids harbor two protective antigen orthologues. (A) B. cereus G9241 virulence plasmid pBCXO1 harbors anthrax toxin genes pagA1 (protective antigen, PA1), lef1 (lethal factor), and cya (edema factor). The B. cereus G9241 virulence plasmid pBC218 harbors genes for expression of BPS capsule as well as pagA2 (protective antigen, PA2) and lef2 (encoding the ADP-ribosylase CerADPr) (28). (B) The amino acid identities between Bacillus anthracis PA and B. cereus PA1 and PA2 were revealed with BLAST searches (37) and are listed for each of its four domains (D1 to D4) (60). (C) The extracellular medium of B. cereus G9241 wild type or its ΔpagA1 and ΔpagA2 mutants was precipitated with TCA and subjected to immunoblotting with PA-specific antibodies. (D) Purified GST hybrids with the N-terminal end of the D1 or D4 domain of PA2 or the D4 domain of PA were subjected to immunoblotting with PA- or GST-specific antibodies. In the right panel, the concentrations of GST-D1_PA2 and GST-D4_PA2 were 500-fold higher than that of GST-D4_PA.

Fig 2

PA1 contributes to the pathogenesis of B. cereus G9241 anthrax-like disease in mice. (A) Survival of C57BL/6 mice (cohorts of 10 animals) following intraperitoneal injection of 1× 10⁵ spores derived from B. cereus G9241 or its ΔpagA1 and ΔpagA2 mutant strains. (B) The bacterial loads in various organ tissues of dead or moribund C57BL/6 mice infected with either wild-type or ΔpagA1 or ΔpagA2 mutant B. cereus G9241 were quantified. Animals were necropsied, organs (spleen, kidney, liver, and lung) were removed, and tissue homogenates were spread on agar plates to enumerate colony formation. Data are representative of three independent experiments.

Fig 3

Histopathology of B. cereus G9241 wild-type and ΔpagA1 or ΔpagA2 mutant disease in mice. C57BL/6 mice were infected with 1 × 10⁵ spores of B. cereus G9241 (A and D) or its ΔpagA1 (B and E) or ΔpagA2 mutant (C and F). Sections from infected spleen and lung tissues were fixed, thin sectioned, and stained with hematoxylin-eosin. Tissue sections were viewed by light microscopy, and images of the spleen (A, B, and C) and lung (D, E, and F) were captured. Low-magnification views are presented on the left, and higher magnifications of the same image are shown on the right of each panel.

Fig 4

AVA-immunized mice are protected against B. cereus G9241 challenge. (A) Lethal toxin neutralization assay. Serum from mock (PBS)-immunized or AVA-immunized C57BL/6 mice was incubated with 0.1 μg anthrax lethal toxin (PA + LF), and the toxin-serum mix as well as a no-serum control reaction mixture was transferred to 10⁵ J774A.1 cells. Cell viability was monitored with an LDH-based cytotoxicity assay after 3 h of incubation. (B) C57BL/6 mice (n = 10) were immunized with a prime-booster regimen of AVA or mock (PBS) in 14-day intervals. Animals were challenged by intraperitoneal inoculation with 1 × 10⁵ spores derived from the B. cereus G9241 wild type or its ΔpagA1 mutant, and survival was monitored. Data are representative of three independent experiments. (C) Bacterial loads in spleen and lung tissues of immunized mice that had been infected with either B. cereus G9241 wild type or its ΔpagA1 mutant. The organs of mock-immunized mice were removed during necropsy when animals were either dead or moribund. Tissue homogenates were spread on agar plates to enumerate colony formation. Data are representative of three independent experiments.

Fig 5

AVA immunization protects mice against B. cereus Elc4 spore challenge. (A) C57BL/6 mice (n = 10) were immunized with a prime-booster regimen of AVA or mock immunization (PBS) in 14-day intervals. Animals were challenged by intraperitoneal inoculation with 1 × 10⁵ spores derived from B. cereus Elc4 or B. cereus G9241, and survival was monitored. Data are representative of three independent experiments. (B) Bacterial loads in organ tissues of mice infected with B. cereus Elc4 or B. cereus G9241. The organs of mock-immunized mice were removed during necropsy when animals were either dead or moribund. Tissue homogenates were spread on agar plates to enumerate colony formation. Data are representative of three independent experiments.

Fig 6

Histopathology of AVA-immunized mice following challenge with spores of B. cereus Elc4 or B. cereus G9241. C57BL/6 mice were infected with 1 × 10⁵ spores of B. cereus Elc4 (A, B, D, and E) or B. cereus G9241 (C and F). Sections from infected spleen and lung tissues were fixed, thin sectioned, and stained with hematoxylin-eosin. Tissue sections were viewed by light microscopy, and images of the spleens (A, B, and C) and lung (D, E, and F) were captured.

Fig 7

Virulence of B. cereus G9241 spores in an aerosol challenge model of respiratory anthrax-like disease in mice. (A) Survival of C57BL/6 mice following aerosol infection. Cohorts of 10 mice were exposed to aerosols of B. cereus G9241 spores and monitored over 14 days. The actual doses retained in the lungs of animals following exposure was determined for a subset of mice from each challenge group and are denoted in the legend. (B) Dissemination of vegetative forms following aerosol infection of B. cereus G9241 spores. Cohorts of 20 mice were infected with 1 × 10⁶ spores derived from B. cereus G9241. Five mice were euthanized in 24-h intervals and necropsied. The spleen and lung homogenates were diluted and spread on agar plates to enumerate bacterial loads. Each circle represents data from one mouse. The closed circle denotes a mouse that succumbed to infection. The bars represent the arithmetic means of CFU values. Data shown are representative of two independent experiments.

Fig 8

Histopathology of lung and spleen tissues in mice infected with aerosolized spores of B. cereus G9241. C57BL/6 mice (n = 20) were infected with 1 × 10⁶ spores of B. cereus G9241 (see Fig. 7B). Immediately following challenge (A and B, 0 h) or after 24 h (C and D), 48 h (E and F), or 72 h (G and H), animals in each cohort were euthanized and necropsied, and lung or spleen tissues were prepared for histopathology. Thin sections were stained with hematoxylin-eosin and viewed by light microscopy using 400× or 1,000× (insets) magnification. Bar, 10 μm. Red arrows identify vegetative forms of B. cereus G9241. See the text for details.

Fig 9

AVA-immunized mice are protected against aerosol challenge of B. cereus G9241 spores. (A) Survival of mock (PBS)- or AVA-immunized C57BL/6 mice (n = 10) following aerosol challenge with B. cereus G9241 wild-type or ΔpagA1 mutant spores. Doses of 3 × 10⁵ wild-type G9241 spores or 1 × 10⁶ ΔpagA1 mutant spores were delivered into the lungs of C57BL/6 mice. (B) Bacterial loads in various organ tissues of mice that succumbed to disease. (C) Numbers of spores retained in the lungs of AVA- or mock (PBS)-immunized animals that survived aerosol challenge with B. cereus G9241 wild-type or ΔpagA1 mutant spores.