Methods for preparing Bacillus anthracis protective antigen for use in vaccines
Inventors
Shiloach, Joseph • Leppla, Stephen H. • Ramirez, Delia M. • Schneerson, Rachel • Robbins, John B. • Hsu, S. Dana • Rosovitz, Mary Jo
Assignees
GOVERNMENT OF UNITED STATES OF AMERICAS HEALTH AND HUMAN SERVICES THE, Secretary of, Department of • US Department of Health and Human Services
Publication Number
US-7763451-B2
Publication Date
2010-07-27
Expiration Date
2022-11-08
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Abstract
The invention relates to improved methods of producing and recovering 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.
Core Innovation
The invention relates to improved methods of producing and recovering Bacillus anthracis protective antigen (PA), especially modified PA which is protease resistant. It includes methods of using these PAs or nucleic acids encoding these PAs to elicit an immunogenic response in humans, particularly to provide protection against or reduce the severity of B. anthracis bacterial infections such as inhalation anthrax, cutaneous anthrax, and gastrointestinal anthrax.
The problem being solved is that the existing licensed anthrax vaccine (Anthrax Vaccine Adsorbed, AVA) has several limitations including eliciting relatively high local and systemic adverse reactions, requiring multiple doses over an extended period, and lacking a defined vaccine-induced protective level of serum PA. There is a need for a well characterized, standardized, effective, and safe vaccine that requires fewer doses and confers immunity to both inhalational and cutaneous anthrax. The invention aims to provide a method for producing biologically inactive, more stable, and highly immunogenic modified purified recombinant PA to overcome these limitations.
The invention exemplifies the development of a production and purification process for recombinant PA from a non-sporogenic avirulent B. anthracis strain. The process includes controlling fermentation at a pH around 7.5 and dissolved oxygen at 30% saturation, addition of EDTA for proteolysis control, and a three-step purification procedure comprising hydrophobic interaction chromatography, ion exchange chromatography, and gel filtration. The purified PA elicited neutralizing antibodies in mice, demonstrating its suitability for vaccine production.
Claims Coverage
The patent includes multiple independent claims covering methods of producing Bacillus anthracis protective antigen and related exotoxin proteins with specific fermentation conditions and purification steps, using modified forms of PA and specific B. anthracis strains.
Method of producing B. anthracis protective antigen with controlled fermentation conditions
The method involves fermenting a cell or microorganism comprising a nucleotide sequence encoding B. anthracis protective antigen in a fermentation medium maintained at pH about 7 to 8 and dissolved oxygen at 30% saturation throughout fermentation.
Recovery of protective antigen using a three-step purification process
The protective antigen is recovered using hydrophobic interaction chromatography followed by ion exchange chromatography and gel filtration, with hydrophobic interaction chromatography preceding ion exchange chromatography.
Use of a protease-deficient nonsporogenic avirulent B. anthracis strain for production
The method employs a protease-deficient, nonsporogenic, avirulent B. anthracis strain, exemplified by BH445 (pXO1-, pXO2-), for producing the protective antigen.
Modified protective antigen variants resistant to cleavage
The protective antigen is a modified PA variant which cannot be cleaved at the chymotrypsin cleavage site, the furin cleavage site, or both, with specific amino acid substitutions such as RKKR167 to SNKE167 and deletions or substitutions at positions 308 and 313-314, enhancing stability.
Addition of EDTA to control proteolytic activity prior to recovery
EDTA is added to the culture medium prior to the recovery step to reduce extracellular proteolytic activity, aiding in preservation of the protective antigen.
Method extended to production of other Bacillus anthracis exotoxin proteins
The method also covers production of other B. anthracis exotoxin proteins such as lethal factor, including modified forms with altered catalytic sites, under similar fermentation conditions.
The independent claims cover a method for producing and recovering modified Bacillus anthracis protective antigen and other exotoxin proteins using controlled fermentation conditions with specific pH and dissolved oxygen levels, protease-deficient avirulent strains, addition of protease inhibitors, and a defined purification process. These claims encompass the production of stable, protease-resistant PA variants useful in vaccines and extend to other exotoxin proteins like lethal factor.
Stated Advantages
Improved yield of Bacillus anthracis protective antigen, producing almost three times more product than previous methods.
Production of modified protective antigen variants that are biologically inactive and protease resistant, enhancing stability.
A simplified and efficient purification process that can eliminate centrifugation and filtration by using expanded bed hydrophobic interaction chromatography.
The purified protective antigen induces strong neutralizing antibody responses in mammals, including humans, suitable for vaccines with fewer doses.
The vaccine compositions are expected to be safer, more standardized, and induce protective immunity more effectively than currently licensed vaccines.
Documented Applications
Use of purified Bacillus anthracis protective antigen or modified variants as vaccines to induce serum antibodies that neutralize anthrax toxin and provide protection against inhalation, cutaneous, and gastrointestinal anthrax.
Use of nucleic acids encoding Bacillus anthracis protective antigen in pharmaceutical compositions or vaccines to induce protective immune responses.
Production of antibodies immunoreactive with Bacillus anthracis protective antigen for prevention, treatment, or amelioration of anthrax infection.
Use of vaccine compositions comprising mixtures of PA, nucleic acids, and antibodies as multivalent vaccines for broad coverage against B. anthracis and other pathogens.
Provision of vaccine kits comprising PA, nucleic acids, vectors, or antibodies for immunization against Bacillus anthracis infection in mammals, including humans.
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