Hardiman: Role of research in developing anthrax countermeasures 287 (AVA) [17], an efficacious first-generation vaccine. AVA is comprised of an impure preparation of cultured supernatants from an avirulent B. anthracis strain. The PA subunit alone was known to be sufficient for protection against B. anthracis challenge [18,19]. Thus, second-generation vaccines were developed that utilized re- combinant approaches in a subunit vaccine. Vaccines based on recombinant PA (rPA) have been shown to successfully induce ro- bust antibody-mediated protection [20]. However, next-generation vaccines also must address technical challenges such as reducing the high rate of dosing and im- proving 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 immuno- genicity and safety during human adminis- tration. Their variant takes advantage of structural and functional studies that have elucidated the fundamental organizing prin- ciples of the AT pore. The pore is comprised of either a heptameric [21] or octameric [22] circular complex of PA proteins. Under con- ditions of low pH, as occurs in the endo- some, the PA “prepore” spontaneously converts into a bonafide membrane-span- ning pore [23]. This is accomplished by the movement of loops that face outward from the pore lumen, moving down into the mem- brane 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, main- taining the force of directed diffusion dur- ing translocation [26]. Charge reversal mutations near the Φ clamp act as a domi- nant negative inhibitor of pore function. Dr. Collier’s construct, which contains a double K397D D425K mutation, is capable of bind- ing to other PA subunits but unable to translocate LF or EF through the pore [27]. The dominant-negative PA is more im- munogenic 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 mu- tated 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 vac- cines, the vaccine may require fewer admin- istrations 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 Soli- genix, 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 infec- tion. Dr. Collier’s basic research into the crit- ical residues in the PA pore led to the solution-identification of dominant negative mutations, opening up immediate commer- cial and translational applications. iDe n TiFy in G Ce l l u l AR Co n SPiRATo RS o F DiSe ASe Cellular cofactors of anthrax toxicity may represent an additional area that can be translated for prophylactic purposes. Recent evidence shows that while anthrax toxin is sufficient to form a membrane pore and di- rect the toxic proteins LF and EF into the cell, this process may be enhanced by cellu- lar cofactors. This is compelling not only for the previously underappreciated role for cel-