DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100x LD50 of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations.