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

Abstract

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

FIG. 1

Cloning scheme of PA-expression vectors. pGEM-3Z E. coli vector carrying the pag gene (PA) under either the Ppag promoter (pPA20-N) or the Pα-amylase (Pa-amy) promoter (pPA20-α) was used to construct four shuttle vectors for expression of PA in Bacillus. The vectors pAUB and pAαUB were based on a pUB110 plasmid, and the vectors pASC-1 and pASC-α were based on pRIT5. These plasmids carry different origins of replications (ori) of plasmids isolated from gram-positive bacteria and from E. coli. mcs, multiple cloning site; Amp, ampicillin resistance in E. coli; Km and Cm, kanamycin and chloramphenicol resistance genes, respectively, in Bacillus. Only relevant restriction sites for construction are presented. For details, see text.

FIG. 2

Effect of AtxA on rPA expression under different growth conditions. (A) Western blot analysis (using anti-PA antiserum) of culture supernatants from Ppag Atx⁺ (MASC-40) and Ppag Atx⁻ (MASC-20) strains. Cells were grown under semianaerobic conditions to an A₅₅₀ of 1, and equivalent amounts of culture supernatants were applied on SDS-PAGE gels. (B) Coomassie blue-stained SDS-PAGE of samples from culture supernatants of MASC-40, MASC-20, and Pα-amylase Atx⁻ (MASC-10) strains grown to an A₅₅₀ of 11 under aerobic conditions. The concentration of PA in each culture was determined by the cytotoxicity assay. The bands corresponding to PA are indicated by an arrow. Abbreviations: Ppag, pagA promoter region; Pα-amy, α-amylase promoter.

FIG. 3

Production of rPA from protease-deficient strains of B. subtilis and B. anthracis. Western blot analysis (using anti-PA-β-gal antiserum) of culture supernatants obtained from B. subtilis DB104 and WB600 strains carrying pAαUB and from B. anthracis ATCC 14185 (pXO1⁺) (lanes 1, 4, and 7, respectively) or dilutions of 1:10 (lanes 2, 5, and 8), 1:50 (lanes 3 and 6), and 1:25 (lane 9). The bands corresponding to PA are indicated by an arrow. PA levels (as determined by the cytotoxicity assay) in the supernatants of the pAαUB-transformed DB104 and WB600 strains (aerobic cultures) and the ATCC 14185 strain (semianaerobic culture) were 35, 90, and 20 μg/ml, respectively.

FIG. 4

Comparison of the immune responses induced by MASC-10 and MASC-20. Animals were immunized on day zero with 5 × 10⁷ spores of MASC-10 (light bar) or MASC-20 (dark bar). Antibody titers (GMT) were detected at the indicated times postimmunization. Anti-core antibodies are directed towards the B. anthracis vegetative cell extract of Δ14185 (see Materials and Methods).

FIG. 5

Longevity of immunity as a function of immunizing dose of MASC-10 spores. Guinea pigs were immunized with a single dose of either 5 × 10⁷, 1 × 10⁶, or 1 × 10⁵ spores of MASC-10 or with PBS as control animals. At the indicated time intervals postimmunization, animals were challenged or used to determine the titers (GMT) of anti-PA antibodies by ELISA (PA/ELISA) or by neutralization assay (Neutra. Ab.). The survival rates (live/total) of control animals upon challenge were 0, 25, and 12% at 1, 5, and 12 months, respectively.