A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores

Abstract

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 LD(50) 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.

Fig. 1.

Immune responses in vaccinated rabbits. Shown are group geometric mean titers (GMT) at 2 weeks (□), 6 weeks (▒), and 10 weeks (▪). Shown are anti-PA IgG titer (A), anti-LF IgG titer (B), and Letx neutralization titer (C). Group 9 (AVA) was vaccinated on weeks 4 and 8; therefore, the 10-week value is 2 weeks after the second vaccination, an equivalent time point to the 6-week values for groups 1–8. No titer was detected in vector-immunized rabbits (group 8). *, Group 7 (LF[I-III]) was statistically lower than all other groups given three injections (groups 1, 2, and 6).

Fig. 2.

Immune responses in rabbits post-anthrax spore challenge. GMTs for pDNA and AVA vaccinated rabbits pre- (□), 1-week post- (▒), and 3-weeks post- (▪) B. anthracis inhalation spore challenge. Shown are anti-PA IgG titers (A), anti-LF IgG titers (B), and Letx neutralization titers (C). The GMT of the five rabbits in the LF[I-III] group that survived challenge are graphed separately from the four nonsurvivors. *, Statistically significant increase in 3-week postchallenge titers.

Fig. 3.

Long-term persistence of immune response and postchallenge response. Group 4 rabbits were vaccinated with PA pDNA (designated by arrows) and monitored for anti-PA (▪) and Letx neutralization (□) GMT. All rabbits were challenged on week 40 (32 weeks after the last immunization) and survived.