Figure 5.

Toxin neutralization and protective efficacy analysis of the antigen D4 vaccination with amorphous and crystalline AH nps. Lethal toxin neutralization assay was performed with anti-D4 sera dilutions of mice groups immunized with either the antigen D4 alone or with amorphous or crystalline AH nps or AH gel. Serial dilutions of sera were mixed with lethal toxin (500 ng/mL PA and 1 μg/mL LF) and incubated with RAW 264.7 cells at 37°C and 5% CO₂ for 4h. The cell viability was measured by MTT assay. Cells without toxin treatment were taken as negative control and their absorbance was used as 100% viability (A). Protective efficacy of the vaccine formulations against B. anthracis spore challenge was evaluated in Swiss albino mice. Fourteen days after the second booster, mice were administered with 0.5 ⨰ 10³ spores of a virulent strain of B. anthracis. Infected mice were kept in an animal isolator in BSL3 and observed for death and morbidity for 14 days (B). The toxin neutralization assay was performed from pooled sera of a single group (n= 5) in triplicate and expressed as mean value with standard deviation (SD).