TB DNA Cocktail

Evaluation of the effectiveness of the DNA cocktail against an aerogenic challenge with M. tuberculosis. The TB DNA cocktail consisted of eight individual vaccines expressing the following TB antigens: antigen 85B, ESAT-6, KatG, MPT8.4, MPT12, MPT63, MPT64, and MPT83.

In previous studies, each of these single vaccines induced significant protective responses in a mouse aerosol challenge model (20, 24). Endotoxin-free plasmid DNA was prepared for each individual construct by using an Endo-Free maxi kit (Qiagen, Valencia, Calif.).

The eight-component DNA vaccine combination was prepared by combining equal amounts of the plasmids to a final concentration of 1 mg of DNA/ml in phosphate-buffered saline (PBS). For these experiments, pathogen-free WT C57BL/6, CD4-deficient (B6.129S2-Cd4tm1Mak), CD8-deficient (B6.129S2-Cd8tm1-Mak), gamma interferon (IFN-γ)-deficient (B6.129S7-Ifngtm1), and tumor necrosis factor (TNF) receptor (p55)-deficient (B6.129-Tnfrsflatm1) female mice were obtained from the Jackson Laboratory (Bar Harbor, Maine).

Groups of mice were injected intramuscularly in all four limbs on days 1, 21, and 42 with 200 μg of the cocktail (25 μg of each individual constituent) or a single plasmid vaccine. The BCG Pasteur immunization was given subcutaneously on day 1 with 106 organisms. Five weeks after the final DNA vaccination, the mice were challenged aerogenically with 200 CFU of M. tuberculosis Erdman per mouse in a Middlebrook-type chamber (Glas-Col, Terre Haute, Ind.). Five mice were sacrificed after 24 h to confirm the size of the challenge dose.