Bacillus Anthracis Protective Antigen Research Articles

Immune correlates of protection against anthrax

Bacillus anthracis protective antigen (PA) has been produced from a recombinant B. subtilis and its efficacy, when combined with the Ribi adjuvant (MPL-TDW-CWS) or alhydrogel, has been compared with that of the licensed UK human vaccine, in guinea pigs challenged with aerosolized Ames strain spores. Recombinant PA combined with the Ribi adjuvant performed as well as PA from B. anthracis cultures in previous reports (Ivins & Welkos 1986; Ivins et al. 1990; Turnbull et al. 1991; Jones et al. 1996; McBride et al. 1998) giving protection in 100% of animals exposed to the highest challenge dose of the Ames strain of B. anthracis that can be administered practically (retained lung doses of approximately 106 spores). In attempts at identifying markers of protection in immunized individuals, rPA in combination with the Ribi adjuvant induced a marker IgG2 response in guinea pigs with no significant differences in IgG1 levels when compared with other vaccine formulations (McBride et al. 1998). In BALBc mice, rPA with the Ribi adjuvant induced a higher IgG2a response compared with rPA with anhydrogel and the human vaccine. To examine the role of anti-PA-specific antibodies in protection, guinea pig sera is being passively transferred into guinea pigs and SCID mice, followed by protection. Similarly, B- and T-lymphocytes from immunized BALB/c mice are being separately and passively transferred into SCID mice with subsequent challenge. The neutralizing ability of the PA-specific antibodies is being studied using an in vitro macrophage lysis assay.

Soluble expression and one-step purification of recombinant bacillus anthracis protective antigen

Abstract: We have greatly simplified production of the Bacillus anthracis protective antigen (PA), the immunoprotective antigen of the anthrax vaccine. By varying induction conditions, we have expressed stable, soluble PA in E. coli, affinity-tagged to facilitate rapid, single-step purification. Recombinant PA exhibited identical properties to B. anthracis PA. Our approach is an attractive strategy for generating purified vaccine antigens that are difficult to express recombinantly.

Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers

The efficacy of recombinant Bacillus anthracis Protective Antigen (rPA) produced in Bacillus subtilis and formulated in Alhydrogel or MPL-TDM-CWS (Ribi adjuvant) has been tested and compared to the licensed UK human vaccine in guinea pigs challenged by the aerosol route with the Ames strain of B. anthracis. rPA combined with the Ribi adjuvant was found to be only formulation to provide 100% protection from challenge. Analysis of immunological parameters in the individual animals revealed significant differences between the rPARibi vaccine group and rPA/Alhydrogel and human vaccine groups for antigen specific lymphocyte proliferation, PA neutralisation and antigen specific IgG2 levels, but indicated no significant differences in PA-specific IgG1 levels. rPA formulated in Alhydrogel induced a mainly IgG1 response whilst the rPARibi vaccine produced a predominantly IgG2 response.

Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs

The efficacy of several human anthrax vaccine candidates comprised of different adjuvants together with Bacillus anthracis protective antigen (PA) was evaluated in guinea pigs challenged by an aerosol of virulent B. anthracis spores. The most efficacious vaccines tested were formulated with PA plus monophosphoryl lipid A (MPL) in a squalenel lecithin/Tween 80 emulsion (SLT) and PA plus the saponin QS-21. The PA+MPL in SLT vaccine, which was lyophilized and then reconstituted before use, demonstrated strong protective immunogenicity, even after storage for 2 years at 4°C. The MPL component was required for maximum efficacy of the vaccine. Eliminating lyophilization of the vaccine did not diminish its protective efficacy. No significant alteration in efficacy was observed when PA was dialyzed against different buffers before preparation of vaccine. PA+MPL in SLT proved superior in efficacy to the licensed United States human anthrax vaccine in the guinea pig model.

Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters.

The pag gene of Bacillus anthracis, located on plasmid pXO1 (185 kb), encodes protective antigen, a component of the anthrax lethal and edema toxins. Synthesis of protective antigen is enhanced during growth of the organism with elevated levels of CO2. The CO2 effect is at the level of transcription, and pXO1-encoded regulatory factors have been implicated in control of pag expression. We used a Tn917-LTV3 insertion mutant of B. anthracis in which the wild-type pag gene on pXO1 was replaced with a pag-lacZ transcriptional fusion to monitor pag promoter activity. Expression of the pag-lacZ fusion is induced five- to eightfold during growth in 5% CO2 compared with growth in air. Growth in 20% CO2 increases transcription up to 19-fold. By monitoring pag-lacZ expression in atmospheres with different O2 and CO2 concentrations, we demonstrated definitively that the CO2 effect is specific and not simply a result of increased anaerobiosis. The results of 5' end mapping of pag transcripts indicate multiple sites of transcript initiation. We have determined two major apparent start sites, designated P1 and P2, located at positions -58 and -26 relative to the translation initiation codon, respectively. Analysis of total RNA from late-log-phase cells shows comparable initiation from P1 and P2 in wild-type strains grown in aerobic conditions. However, initiation from P1 is increased approximately 10-fold in cultures grown with an elevated level (5%) of CO2. We have identified a locus on pXO1, more than 13 kb upstream from the pag gene, which enhances pag transcription. When added in trans, this locus increases the level of transcripts with 5' ends mapping to P1 but has no effect on the level of transcripts with 5' ends mapping to P2. The CO2 effect on P1 is observed only in the presence of the activator locus.

Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants

The protective efficacy of immunization against anthrax with Bacillus anthracis protective antigen (PA) combined with different adjuvants was tested in Hartley guinea pigs and CBA/J and A/J mice. Adjuvant components derived from microbial products that were tested included threonyl-muramyl dipeptide (threonyl-MDP); monophosphoryl lipid A (MPL); trehalose dimycolate (TDM); and the delipidated, deproteinized, cell wall skeleton (CWS) from either Mycobacterium phlei or the BCG strain of Mycobacterium bovis. Non-microbially derived adjuvants tested included aluminum hydroxide and the lipid amine CP-20,961. In guinea pigs, all adjuvants and adjuvant mixtures enhanced antibody titers to PA as well as survival after a parenteral challenge of virulent B. anthracis Ames spores. In contrast, PA alone or combined with either aluminum hydroxide or CP-20,961 failed to protect mice. Vaccines containing PA combined with threonyl-MDP or MPL-TDM-CWS protected a majority of female CBA/J mice. Statistical analysis of survival data in the guinea pigs indicated that PA-MPL-CWS and PA-MPL-TDM-CWS were more efficacious than the currently licensed human anthrax vaccine.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

Immunosorbent chromatography—a technique to purify Bacillus anthracis protective antigen: Doris K. Larson, Lin Peng, Gary J. Calton, Stephen F. Little, Stephen H. Leppla, and Joseph W. Burnett (Division of Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, and U.S. Army Research Institute of Infectious Diseases, Fort Detrick, MD 21701, U.S.A.)

Immunosorbent chromatography—a technique to purify Bacillus anthracis protective antigen: Doris K. Larson, Lin Peng, Gary J. Calton, Stephen F. Little, Stephen H. Leppla, and Joseph W. Burnett (Division of Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, and U.S. Army Research Institute of Infectious Diseases, Fort Detrick, MD 21701, U.S.A.)