Recombinant Modified <i>Bacillus anthracis</i> Protective Antigen for Use in Vaccines

This invention relates to improved methods of preparing Bacillus anthracis protective antigen (PA) for use in vaccines. PA is a secreted, non-toxic protein with a molecular weight of 83 KDa. PA is a major component of the currently licensed human vaccine (Anthrax Vaccine Adsorbed, AVA). Although the licensed human vaccine has been shown to be effective against cutaneous anthrax infection in animals and humans and against inhalation anthrax in rhesus monkeys, the licensed vaccine has several limitations: (1) AVA elicits a relatively high degree of local and systemic adverse reactions, probably mediated by variable amounts of undefined bacterial products, making standardization difficult; (2) the immunization schedule requires administration of six doses within an eighteen (18) month period, followed by annual boosters; (3) there is no defined vaccine-induced protective level of antibody to PA by which to evaluate new lots of vaccines; and (4) AVA is comprised of a wild-type PA. It has been suggested that a vaccine comprising a modified purified recombinant PA would be effective, safe, allow precise standardization, and require fewer injections.

This invention claims methods of producing and recovering PA from a cell or organism, particularly a recombinant cell or microorganism. The invention claims production and purification of modified PA from a non-sporogenic strain of Bacillus anthracis. In contrast to other previously described methods, greater quantities of PA are obtainable from these cells or microorganisms. Specifically, a scalable fermentation and purification process is claimed that is suitable for vaccine development, and that produces almost three times more product than earlier-reported processes. This is accomplished using a biologically inactive protease-resistant PA variant in a protease-deficient non-sporogenic avirulent strain of B. anthracis (BH445). One of the PA variants described in the patent application lacks the furin and chymotrypsin cleavage sites.

The invention relates to improved methods of producing and recovering sporulation-deficient B. anthracis mutant stains, and for producing and recovering recombinant B. anthracis protective antigen (PA), especially modified PA which is protease resistant, and to methods of using of these PAs or nucleic acids encoding these PAs for eliciting an immunogenic response in humans, including responses which provide protection against, or reduce the severity of, B. anthracis bacterial infections and which are useful to prevent and/or treat illnesses caused by B. anthracis, such as inhalation anthrax, cutaneous anthrax and gastrointestinal anthrax.