Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinan t protective antigen (rPA) were constructed. Biochemical analyses (includin g electrospray mass spectroscopy and N terminus amino acid sequencing) as w ell as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs wit h 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (greater than or equal to 100 mu g/ml) from a constitut ive heterologous promoter induced high titers of neutralizing anti-PA antib odies. This immune response was long lasting (at least 12 months) and provi ded protection against a lethal challenge of virulent (Vollum) anthrax spor es. The recombinant B. anthracis spore vaccine appears to be more efficacio us than the vegetative cell vaccine. Furthermore, while results clearly sug gest a direct correlation between the level of expression of PA and the pot ency of the vaccine, they also suggest that some B. anthracis spore-associa ted antigen(s) may contribute in a significant manner to protective immunit y.