Although the bacterium cannot spread from human to human, B. anthracis
is an attractive choice of bioweaponeers, due to its formation of hardy spores and relatively high morbidity and mortality rates [3
]. Thus, countering a potential anthrax attack using vaccines and prophylactic countermeasures has been a biodefense priority for several decades [5
]. In order to survive and replicate in an infected individual, B. anthracis
must evade host defenses. One mechanism is to selectively kill off cells of the innate immune system [7
]. To accomplish this, B. anthracis
delivers components of the multimeric AT into the bloodstream, where they can encounter host immune cells [9
AT is comprised of 83-kilodalton (kDa) Protective Antigen (PA), which serves as a pore [10
] to translocate the other two components, 89-kDa Edema Factor (EF) and 90-kDa Lethal Factor (LF). Once delivered to the cytosol, EF acts as an adenylate cyclase [12
], adding a cyclic AMP (cAMP) molecule onto critical proteins in neutrophils, leading to inactivation of these cells. The targeting of endothelial cells also results in fluid leakage and excess fluid build-up, known as edema. LF similarly inactivates host protein function via its protease domain, which irreversibly cleaves members of the mitogen-activated protein kinase-kinase (MAPK-K) family [13
]. The impaired immune response combined with edema promotes bacterial replication in the bloodstream, leading to septic shock and possibly death.
The anthrax exotoxin is a critical virulence factor for B. anthracis
]. Because of the requirement for the toxin in disease progression, developing a formulation targeting the toxin has been a priority for pharmaceutical companies interested in a vaccine. The federal government established Project BioShield in 2004, which sought to invest in vaccines and prophylactic agents to employ for defensive purposes. BioShield contracted for 29 million doses of Anthrax Vaccine Absorbed (AVA) [17
], an efficacious first-generation vaccine. AVA is comprised of an impure preparation of cultured supernatants from an avirulent B. anthracis
The PA subunit alone was known to be sufficient for protection against B. anthracis challenge [18
]. Thus, second-generation vaccines were developed that utilized recombinant approaches in a subunit vaccine. Vaccines based on recombinant PA (rPA) have been shown to successfully induce robust antibody-mediated protection [20
]. However, next-generation vaccines also must address technical challenges such as reducing the high rate of dosing and improving stability over time and a range of temperatures.
To differentiate themselves from other rPA formulations, Dr. Collier’s group took a different approach based on their research to develop a third-generation anthrax vaccine. Dr. Collier and colleagues noted that rPA could be optimized to increase immunogenicity and safety during human administration. Their variant takes advantage of structural and functional studies that have elucidated the fundamental organizing principles of the AT pore. The pore is comprised of either a heptameric [21
] or octameric [22
] circular complex of PA proteins. Under conditions of low pH, as occurs in the endosome, the PA “prepore” spontaneously converts into a bonafide membrane-spanning pore [23
]. This is accomplished by the movement of loops that face outward from the pore lumen, moving down into the membrane and forming a 100Å linear pore [24
]. This pore is capable of keeping substrates in an unfolded conformation and stimulating their translocation through the membrane into the cytosolic compartment.
Critical to pore functioning is the action of residue Phe427, which resides on the loops that swing into a pore conformation at low pH. Phe427 forms a ring-like complex called the Φ clamp with other Phe residues in the pore [25
]. The Φ clamp is thought to function as a hydrophobic seal that preserves the local proton gradient in the pore, maintaining the force of directed diffusion during translocation [26
]. Charge reversal mutations near the Φ clamp act as a dominant negative inhibitor of pore function. Dr. Collier’s construct, which contains a double K397D D425K mutation, is capable of binding to other PA subunits but unable to translocate LF or EF through the pore [27
]. The dominant-negative PA is more immunogenic than native PA, potentially due to increased affinity by immune recognition proteins.
Dr. Collier has translated this finding into a proprietary rPA formulation rooted in the dominant negative PA strategy. Initial tests in a rat model of LF toxicity showed protection when immunized with the mutated PA proteins [28
]. Early thermostability studies have been successful [29
], a primary consideration in its ability to be stockpiled. As it may be more efficacious than other vaccines, the vaccine may require fewer administrations than the six AVA currently requires to achieve protection. The vaccine is being commercialized by Soligenix, Inc. through a licensing agreement with Dr. Collier. As of press time, Dr. Collier’s vaccine is being tested as SGX204 for both preventive and prophylactic indications. According to Soligenix, Inc., the proof of concept and animal testing phases were completed, with the compound currently in phase 1 clinical trials [30
]. Thus, this compound has immediate translational applicability to developing medical countermeasures for anthrax infection. Dr. Collier’s basic research into the critical residues in the PA pore led to the solution-identification of dominant negative mutations, opening up immediate commercial and translational applications.