Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that produce protective antigen

Abstract

The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming DeltaANR and DeltaSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.

FIG. 1

Construction of shuttle vectors for expression of PA in B. anthracis. Plasmid pBLKSPPA is a pBluescript vector which contains the anthrax PA gene and its endogenous promoter inserted into a multiple cloning site on a ClaI-BamHI fragment. This vector was digested with BamHI, treated with phosphatase, and ligated to gram-positive vectors pUB110 and pC194, which had been digested with BamHI and BstYI, respectively, to create PA-producing shuttle vectors pJB1, pJB2, and pJB3.

FIG. 2

Immunoblot demonstrating production of PA by recombinant anthrax strains. Sterilely filtered, concentrated culture supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10 to 15% gradient gels, electroblotted to nitrocellulose, and probed with anti-PA antibodies. Panel A lanes: 1, ΔANR(pJB1); 2, ΔANR(pPA102); 3, ΔANR control with no plasmid; 4, ΔSterne(pJB1); 5, ΔSterne(pPA102); 6, ΔSterne control with no plasmid. Panel B lanes: 1, ΔANR control with no plasmid; 2, ΔANR(pJB3); 3, ΔSterne(pJB2); 4, ΔSterne(pJB3); 5, purified PA from the Sterne strain; 6, ΔSterne control with no plasmid. The positions of molecular mass standards (sizes are in kilodaltons) are indicated on the left. Approximately equal volumes were applied to all of the lanes.

FIG. 3

Recovery of B. anthracis from guinea pig muscle after inoculation with 10⁹ spores of three recombinant anthrax strains. Bacteria were assayed by dilution and plating 1, 3, and 7 days after inoculation of 10⁹ spores of strains ΔANR(pPA102) (○), ΔSterne(pPA102) (▵), ΔSterne(pJB2) (□). The values shown are mean CFU per milliliter ± the standard deviation of three animals killed at each time point.

FIG. 4

Survival of guinea pigs vaccinated with recombinant, live anthrax vaccines after challenge with 2,000 LD₅₀ of fully virulent B. anthracis. The challenge dose was administered i.m. on day 0. ○, ΔANR(pPA102); •, ΔSterne(pPA102); ▵, ΔANR(pJB3); ▴, ΔSterne(pJB3); □, ΔSterne(pJB2); ◊, saline control. The total numbers of animals challenged were 20 for the saline control, 17 for ΔANR(pPA102) and ΔSterne(pPA102), and 19 for all other groups.