Recombinant modified Bacillus anthracis protective antigen for use in vaccines
Inventors
Leppla, Stephen H. • Rosovitz, Mary Jo • Robbins, John B. • Schneerson, Rachel • Hsu, S. Dana • Shiloach, Joseph • Ramirez, Delia M.
Assignees
US Department of Health and Human Services
Publication Number
US-8394387-B1
Publication Date
2013-03-12
Expiration Date
2023-08-08
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Abstract
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.
Core Innovation
The invention relates to improved methods of producing and recovering sporulation-deficient Bacillus anthracis mutant strains and recombinant Bacillus anthracis protective antigen (PA), especially modified PA which is protease resistant. It also pertains to methods of using these PAs or nucleic acids encoding these PAs for eliciting an immunogenic response in humans, including responses providing protection against or reducing severity of B. anthracis bacterial infections such as inhalation anthrax, cutaneous anthrax, and gastrointestinal anthrax.
The problem being solved relates to the limitations of current anthrax vaccines, particularly the Anthrax Vaccine Adsorbed (AVA), which has drawbacks including high local and systemic adverse reactions, a lengthy immunization schedule requiring multiple doses and boosters, and lack of a defined protective serum PA level. There is a need for a well-characterized, standardized, effective, and safe vaccine that would require fewer doses to confer immunity to both inhalational and cutaneous anthrax. The invention addresses this by providing modified recombinant PA that is biologically inactive, more stable, and highly immunogenic, produced using improved methods involving sporulation-deficient avirulent B. anthracis strains.
Claims Coverage
The patent includes one independent method claim with multiple dependent claims, covering methods related to inducing neutralizing serum antibodies against B. anthracis toxin by administering a pharmaceutical composition containing a specific PA variant, including methods for production, culture conditions, and composition formulations.
Method for inducing neutralizing antibodies using a specific PA protein
Administering to a mammal a pharmaceutical composition containing an amount of a protein comprising the amino acid sequence of SEQ ID NO: 4 sufficient to elicit production of serum antibodies neutralizing B. anthracis toxin.
Production of the PA protein by controlled culture conditions
Producing the protein by culturing a cell or microorganism comprising a nucleotide sequence encoding SEQ ID NO: 4, maintaining the culture medium at about pH 7 to about pH 8 substantially throughout fermentation.
Recovery of PA protein by specific chromatographic methods
Recovering the protein using a purification process comprising hydrophobic interaction chromatography, ion exchange chromatography, and gel filtration.
Addition of EDTA to culture medium prior to protein recovery
Incorporating EDTA addition to the culture medium before recovery to reduce proteolytic activity and improve stability of the PA protein.
Use of a protease-deficient nonsporogenic avirulent Bacillus anthracis strain
Culturing the nucleotide sequence encoding SEQ ID NO: 4 in a protease-deficient, nonsporogenic, avirulent strain of B. anthracis, exemplified by strain BH445.
Use of defined pH control agents and maintenance
Maintaining pH with HCl and NH4OH, preferably maintaining pH at about 7.5 throughout fermentation to optimize PA production.
Pharmaceutical compositions including adjuvants and formalin
Pharmaceutical compositions comprising the PA protein and optionally including adjuvants such as aluminum hydroxide and/or formalin to enhance immunogenicity and stability.
The claims encompass a method to induce neutralizing antibodies against B. anthracis toxin by administering a pharmaceutical composition with a modified PA protein (SEQ ID NO: 4), produced under controlled pH conditions in a protease-deficient nonsporogenic B. anthracis strain, with recovery by specific chromatography and optional adjuvants and formalin in the composition.
Stated Advantages
The modified recombinant PA is more stable and protease resistant compared to wild-type PA, resulting in improved yield and suitability for vaccine production.
The vaccine production methods allow for higher quantities of PA than previously achievable, enhancing vaccine manufacturing efficiency.
The vaccine compositions are expected to induce strong protective IgG antibody responses comparable or superior to currently licensed anthrax vaccines.
The use of sporulation-deficient, protease-deficient avirulent B. anthracis strains reduces biosafety concerns and simplifies large-scale vaccine production.
Modified PA vaccines are safer, better standardized, and likely require fewer injections compared to existing vaccines.
Documented Applications
Prevention and treatment of infections caused by Bacillus anthracis, including inhalation anthrax, cutaneous anthrax, and gastrointestinal anthrax.
Active immunization of humans and other mammals for protective immunity against anthrax infection.
Use as pharmaceutical compositions or vaccines to induce serum antibodies neutralizing anthrax toxin.
Preparation of immune antibodies and passive immunotherapy using anti-PA sera or isolated antibodies for established B. anthracis infections or post-exposure prophylaxis.
Inclusion in immunization schedules for individuals at risk of B. anthracis exposure, including defense against bioterrorism or biowarfare.
Formulation into kits comprising vaccines, nucleic acids, vectors, host cells, or antibodies for prevention and treatment of anthrax infection.
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