FIG. 4.

Immunity to tuberculosis induced by DNA vaccines constructed from 1818^PE_PGRS and 1818^PE. Mice were immunized with endotoxin-free plasmid DNA purified as previously indicated (8). Groups of C57BL/6 mice were immunized intramuscularly in the hind limbs on days 1, 21, and 42 using 100 μg of plasmid DNA in a total volume of 0.1 ml. (A) The humoral response induced in mice vaccinated with vector only (solid bars), 1818^PE_PGRS (hatched bars), or 1818^PE (open bars) was analyzed by ELISA using sera from mice (five mice each) collected 30 days following the third DNA vaccination. Plates were coated with purified histidine-tagged recombinant proteins r1818^PE_PGRS or r1818^PE as indicated, and results are expressed as the IgG log₁₀ endpoint titer. The dispersion around the mean value for the five mice, for each response was not significant. (B) Comparison of the IFN-γ response generated by immunization of mice with 1818c^PE and 1818c^PE_PGRS DNA vaccines and assessed by cytokine ELISA, following in vitro restimulation of splenocytes with activated macrophages. Murine BMMφ were infected with M. tuberculosis (solid bars) at an MOI of 5:1 or were primed with 1 μg of PPD (open bars) per ml 24 h prior to incubation with splenocytes obtained from vaccinated mice 30 days after the final immunization. Supernatants were collected 72 h later, and the amount of IFN-γ was measured using a cytokine-specific ELISA. The data represent the pooled response from five mice and show the standard deviation from the mean.