Fig. 1. Validation of adenoviral vector encoding EFn by PCR. Lane 1, DNA marker, λ-DNA digested with HindIII; lane 2, plasmid pBHG10X/delta E1, E3Cre as DNA template in PCR (positive control for Ad5 Fiber); lane 3, pDC316/EFn (positive control for EFn); lane 4 is a negative control for PCR, no template DNA were added into the PCR reaction mixture; lanes 5–7, genomic DNA of adenovirus Ad/EFn. The primers used in the PCR are indicated in the figure.

Fig. 2. Neutralization of anthrax edema toxin by sera from immunized mice. The CHO-K1 cells were used for the assay in a 96-well tissue culture plate at a density of 40,000 cells/well. The anti-EF serum pools were from eight mice in week 10 after immunization with Ad/EFn in 0, 4, and 8 weeks. Various dilutions of pooled sera were pre-incubated with recombinant EF and PA, and then added into the CHO cells. Cellular protein concentration was determined by BCA protein assay kit (Pierce, IL). The intracellular cAMP concentrations were detected by Cyclic AMP Immunoassay Kit (R&D System Inc., MN).

Fig. 3. Neutralization of anthrax lethal toxin by sera from immunized mice. Various dilutions of pooled sera (n = 8) were pre-incubated with recombinant LF and PA, and then added to J774A.1 mouse macrophage cells. The cell viability was measured. Panel A: sera from different groups immunized with Ad/EFn 12 weeks after the primary immunization. Mice were immunized one (week 0), two (week 0, 4), three (week 0, 4, 8) times with Ad/EFn as indicated. Mice in control group were injected with adenovirus storage buffer three times (week 0, 4, 8). Panel B: sera pool from the same group at different time points after immunized one to three times in week 0, 4, 8 with Ad/EFn.

Fig. 4. Protection against B. anthracis spore challenge in mice immunized with Ad/EFn. Panel A: mice were immunized one (week 0), two (weeks 0, 4), or three (week 0, 4, 8) times as indicated, and then challenged with 200 × LD₅₀ B. anthracis Sterne spore in week 12. Panel B: mice were immunized three times (week 0, 4, 8) and challenged with 100–600 × LD₅₀ B. anthracis Sterne spore as indicated in week 12. Mice in control groups were injected with adenovirus storage buffer three times (week 0, 4, 8).

Summary of antibody titers in sera from A/J mice immunized with Ad/EFn

Values in columns without the same letters (a, b, c) differ significantly between different time points in the same immunization schedule (P < 0.05, n = 8). Values in rows without the same capital letters (X, Y, Z) differ significantly between different immunization schedules at the same time points (P < 0.05, n = 8); ^*P < 0.05, ^**P < 0.01 in comparison with the negative control group at the same time point. Data from the control group (100–200) are not shown in this table. Data were analyzed using LSD test, ANOVA/MANOVA, STATISTICA 6.0 (StatSoft Inc., Tulsa, OK).