6-8 week old female CB6F1 mice were purchased from The Jackson Laboratory and maintained in Specific Pathogen Free conditions. After infection animals were maintained in BL3 containment according to the regulations and guidelines of the IDRI Institutional Animal Care and Use Committee. For vaccine efficacy studies mice were immunized one, two, or three times three weeks apart by intramuscular injection. Each immunization contained 5 pmol of recombinant protein and 5 μg of GLA-SE.
Four weeks after the last immunization, mice (n = 7/group) were aerogenically infected with Mtb strain H37Rv (ATCC No. 35718; American Type Culture Collection) using a GlasCol aerosol generator calibrated to deliver 50–100 bacteria into the lungs. To confirm the amount of bacteria delivered an additional three unimmunized animals per infection were euthanized one day later and bacterial burden in the lungs was enumerated. Protection was determined three to six weeks after challenge by harvesting the lungs and spleens from the infected mice, homogenizing the tissue in 0.1% PBS–Tween 80, and plating 5-fold serial dilutions on7H10 agar plates (Molecular Toxicology) for bacterial growth. Bacterial colonies were counted after incubation at 37°C for 14-21 days.
Four weeks after the last immunization, mice (n = 7/group) were aerogenically infected with Mtb strain H37Rv (ATCC No. 35718; American Type Culture Collection) using a GlasCol aerosol generator calibrated to deliver 50–100 bacteria into the lungs. To confirm the amount of bacteria delivered an additional three unimmunized animals per infection were euthanized one day later and bacterial burden in the lungs was enumerated. Protection was determined three to six weeks after challenge by harvesting the lungs and spleens from the infected mice, homogenizing the tissue in 0.1% PBS–Tween 80, and plating 5-fold serial dilutions on7H10 agar plates (Molecular Toxicology) for bacterial growth. Bacterial colonies were counted after incubation at 37°C for 14-21 days.