A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA, lef and cya genes

We recently reported the development, of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the Sterne Delta htrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, rye and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (Delta htrA < Delta htrA Delta cya < Delta htrA Delta lef < Delta htrA Delta lef Delta cya) in attenuation - up to 10(8)-fold relative to the parental Sterne vaccine strain. Most importantly, all various Sterne Delta htrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10(9) spores) or double doses (> 10(7) spores) of the most attenuated triple mutant strain Sterne Delta htrAlef(MUT)Delta cya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10(5) spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30 weeks, respectively. (C) 2017 Elsevier Ltd. All rights reserved.