FIG 1.

Subcutaneous vaccination with NEAT domains and CFA. (A) Schematic of the vaccination regimen and collection of serum from vaccinated mice. Animals were injected subcutaneously on day 0, 14, or 28 with either the NEAT domain of Hal (Hal_N) (4 μg) or the NEAT domains of IsdX1, IsdX2, IsdC, BslK, and Hal (4 μg each, for 20 μg total) mixed with CFA. Serum was drawn at day −1 (preimmune), day 29 (vaccinated), and day 70 (postchallenge) and tested for anti-NEAT antibodies by ELISAs (see Materials and Methods). (B) Survival curves for mice vaccinated with adjuvant (CFA) alone, adjuvant with Hal_N, or adjuvant with the NEAT domain cocktail after subcutaneous challenge with 10× LD₅₀ of B. anthracis Sterne strain spores. For CFA versus the NEAT cocktail, the P value was 0.014 as determined by a log rank (Mantel-Cox) test, and the P value was 0.017 as determined by a Wilcoxon test. For CFA versus wild-type Hal, the P value was 0.111 as determined by a log rank (Mantel-Cox) test, and the P value was 0.074 as determined by a Wilcoxon test. (C) CFU of bacilli recovered from mouse organs from the experiments described above for panels A and B (asterisks denote statistically significant values [P < 0.01], as determined by one-way analysis of variance). For CFA versus the NEAT cocktail, P values were 0.186 for spleen, 0.061 for kidney, 0.074 for heart, 0.043 for lung, 0.082 for liver, and 0.186 for gastrointestinal tract. For CFA versus wild-type Hal, P values were 0.186 for spleen, 0.009 for kidney, 0.008 for heart, 0.008 for lung, 0.012 for liver, and 0.186 for gastrointestinal tract.