- Improved vaccine for Bacillus anthracis has elevated resistance to premature
degradation through increased thermal and structural stability.
Bacillus anthracis, more commonly known as Anthrax, is a bacterial toxin and is regarded to be a potential threat for bio-warfare or bioterrorism. Exposure to aerosolized B. anthracis spores is toxic and lethal to humans and mammals, and can have devastating consequences if used as an agent for bioterrorism.
Anthrax has been used as a weapon around the world for nearly a century and is classified by the CDC as a Tier 1 agent. In 2001, powdered Anthrax spores were placed into letters and mailed through the U.S. Postal System. A total of 22 people were infected with Anthrax and five casualties. The spores can easily be found in nature or produced in a lab, and can survive for long periods in harsh environments. The microscopic spores can be easily placed in powders, sprays, food, and water, and are often unable to be seen, smelled, or tasted. The infection can occur in four forms: skin, inhalation, intestinal, and injection. Symptoms are different for each infection type, and range from skin lesions, abscess formation, fever, chest pain, vomiting, and abdominal pain.
LIMITATIONS OF CURRENT VACCINE
The only licensed human vaccine in the U.S. is Anthrax Vaccine Adsorbed (AVA), commercially known as BioThrax. FDA vaccination requirements for pre-exposure to Anthrax involve a primary series of three doses and require a booster series at yearly intervals following the primary series. The vaccine must be stored and refrigerated at 2-8 degrees Celsius (36 -46 degrees Fahrenheit) to retain efficacy.
Potency is reduced each time the vaccine is exposed to improper conditions. Without specialized transport with cold chain storage to keep the vaccine between 2-8 degrees Celsius, wide distribution is a challenge. Any period of temperature excursion will result in the physical or chemical degradation of the protein structures that are critical for inducing protective immunity. Improper storage of the vaccine at the necessary conditions will result in a complete loss of efficacy and be rendered useless.
NEW VACCINE COMPLEX UNDETERED BY PREVIOUS RESTRICTIONS
Protective antigen (PA) is the protein responsible for transporting Lethal Factor (LF) and Edema Factor (EF) into the host cell. Researchers developed an advanced Protective Antigen Complex to elicit an immunogenic response in a subject and induce protective immunity. The complex has elevated resistance to PA folding and pore formation, preventing LF and EF from comprising immunity in the cell. The invented complex also raises the thermal stability by at least 20°C compared to the prior serum. By achieving storage of the vaccine at 25 °C , this eliminates the necessity for specialized transport containers with temperature control. This will allow for the wide distribution of the vaccine, making it easily accessible in underdeveloped areas with scarce medical resources.
PA is the key component for many Anthrax vaccines currently licensed as well as those under development. Using a PA complex as the epitote to elicit an immunogenic response and stimulate the production of antibodies, the structure of PA within the vaccine must resist degradation through storage until administered to a subject. While PA complexes have been made to protect against Anthrax, the significant challenge lies in the storage and stability of the complex. Efforts to develop protective adjutants that do not require the use of a cold chain are of interest.
PA is the protein responsible for forming a membrane-spanning channel through the cellular membrane, a critical step in the trans-location of LF and EF into the host cell. Upon binding to a host cell, PA initiates proteolytic cleavage and receptor-mediated endocytosis, allowing the protein to fold into an endosomal compartment, termed pre-pore. The pre-pore is then converted into a pore, the channel structure allowing LF and EF to enter the cell. LF and EF prevent phagocytes and macrophages from protecting the host cell, resulting in a compromised host immunity.
THE STABILIZED COMPLEX
Established groundwork in this field was performed by Dr. Bann, including a patented PA complex able to prevent key steps of pathogenesis. The complex integrates 2-fluorohistidine with PA, resulting in a complex resistant to protonation. (US 7,731,979 B2) Building upon the patented complex, binding a capillary morphogenesis protein-2 (CGM2) directly along a binding interface in domain 4 of the PA complex achieves a structurally and thermodynamically-stabilized complex. Previous in-vitro experiments have indicated domain 2 and domain 4 are part of a hinge that dictates the oligomeric assembly of PA, providing the flexibility critical for pre-pore formation.
By binding CMG2 to His616 in domain 4, the interface has decreased flexibility and prevents the hinge-like motions of domain 2 and domain 4. Inhibiting protein folding is key to preventing pre-pore formation and the trans-location of LF and EF. Long-range stabilization of the PA-CMG2 complex is also achieved in domains that do not have direct contact with CMG2, observed in domain 1’ at His 211 and His253.
Studies found that CMG2 thermodynamically stabilizes PA, preventing structural perturbations to the protein under long-term storage conditions. The invented complex has shown to slow the rate of proteolysis by thermolysin and prevents premature degradation, possibly allowing for a greater proportion of PA to ultimately be presented on antigen-presenting cells. In an unbound complex, the PA interaction with receptors on the host cells depletes the PA concentration able to interact with the host immune system. The inclusion of CMG2 in a vaccine formulation should prevent the depletion of PA, providing an epitope for the production of antibodies against anthrax.
Dr. James Bann is an associate professor in the Department of Chemistry at Wichita State University. After receiving his Ph.D. from Oregon Health Sciences in 2000, Dr. Bann continues to perform research in Biochemistry. Dr. Bann leads research groups to understand how proteins fold and adopt their three-dimensional, biologically active conformations. Focusing on two biological toxins - Anthrax and E. coli, Dr. Bann performs research to recognize the mechanisms responsible for how these toxins form membrane-spanning channels that transport the proteins critical for disease pathogenesis into the host cell.