Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis

Abstract

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 10(5) CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

FIG. 1.

PA expression plasmids. (A) Cloning of PA fragments into NheI sites of pSEC10 as fusions to the ClyA gene. (B) General structure of pSEC10-based plasmids encoding PA gene fragments: SL3261/PSECPA1-4 encoding domains 1 to 4 of PA, SL3261/pSECPA1+4 encoding domain 1 and domain 4 of PA, and SL3261/pSECPA4 encoding domain 4 of PA.

FIG. 2.

Expression of PA as ClyA fusions from Salmonella. Normalized cell lysates (3 × 10⁸ CFU/ml) and culture supernatants (either neat or diluted 1:10 as indicated) from S. enterica serovar Typhimurium SL3261 recombinants were subjected to SDS-PAGE and Western blotting with PA4-specific monoclonal antibody. Lane M, Bio-Rad broad-range biotinylated SDS marker; lane 1, SL3261/pSECPA1-4 1:10 cell pellet; lane 2, SL3261/pSECPA1-4 neat supernatant; lane 3, SL3261/pSECPA1+4 1:10 cell pellet; lane 4, SL3261/pSECPA1+4 neat supernatant; lane 5, SL3261/pSECPA4 1:10 cell pellet; lane 6, SL3261/pSECPA4 neat supernatant; lane 7, SL3261/pSEC10 1:10 cell pellet; lane 8, SL3261/pSEC10 neat supernatant; lane 9, 0.1 mg of rPA/ml.

FIG. 3.

Serum immune responses to PA. Serum samples were taken from A/J mice orally immunized three times at 2-week intervals with 1 × 10⁸ to 5 × 10⁹ CFU of S. enterica serovar Typhimurium SL3261 or SL3261 recombinants (A and B) or intramuscularly immunized with 10 μg of PA protein adsorbed to a 20% (vol/vol) solution of Alhydrogel (C and D). Sera were used to determine the IgG (A or C) or TNA (B or D) responses to PA.

FIG. 4.

SDS-PAGE analysis of purified recombinant PA proteins. Purified PA proteins (2 μg per 10-μl sample loaded) were subjected to SDS-PAGE. Lane 1, Bio-Rad Kaleidoscope-prestained standards; lane 2, rPA1; lane 3, rPA4; lane 4, rPA1+4; lane 5, rPA1-4; lane 6, Bio-Rad low-range prestained standards.