Inhalable vaccine of bacterial culture supernatant extract mediates protection against fatal pulmonary anthrax

Pulmonary anthrax is the most fatal clinical form of anthrax and currently available injectable vaccines do not provide adequate protection against it. Hence, next-generation vaccines that effectively induce immunity against pulmonary anthrax are urgently needed. In the present study, we prepared an attenuated and low protease activity Bacillus anthracis strain A16R-5.1 by deleting five of its extracellular protease activity-associated genes and its lef gene through the CRISPR-Cas9 genome editing system. This mutant strain was then used to formulate a lethal toxin (LeTx)-free culture supernatant extract (CSE) anthrax vaccine, of which half was protective antigen (PA). We generated liquid, powder, and powder reconstituted formulations that could be delivered by aerosolized intratracheal inoculation. All of them induced strong humoral, cellular, and mucosal immune responses. The vaccines also produced LeTx neutralizing antibodies and conferred full protection against the lethal aerosol challenges of B. anthracis Pasteur II spores in mice. Compared to the recombinant PA vaccine, the CSE anthrax vaccine with equal PA content provided superior immunoprotection against pulmonary anthrax. The preceding results suggest that the CSE anthrax vaccine developed herein is suitable and scalable for use in inhalational immunization against pulmonary anthrax.

Automated summary

A low protease activity Bacillus anthracis strain A16R-5.1 was prepared by deleting five extracellular protease activity-associated genes and its lef gene using the CRISPR-Cas9 genome editing system. An anthrax vaccine was formulated from the mutant strain, which contained a lethal toxin-free culture supernatant extract with 50% protective antigen. The vaccine induced strong immune responses and provided full protection against lethal aerosol challenges in mice. Compared to the recombinant protective antigen vaccine, this culture supernatant extract anthrax vaccine showed superior immunoprotection against pulmonary anthrax.