Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-␥-D-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A͞J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 g of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.

No abstract