Infected Macrophages as Vaccine Figure 6. Immunization with xIM-1.6.a provides protection against M. tb. Mice vaccinated with xIM-1.6.a were rested for 240 days and then aerosol challenged with M. tb. After 35 days, mice were sacrificed and mycobacterial load was enumerated by CFU plating of diluted lung homogenates. A, data are represented as log10 CFU (n = 4–6 animals/group). B, photomicrographs (106) of H & E stained lungs sections. Control groups of mice were inoculated with uninfected xenogeneic macrophages (xMac). Data are mean 6 SEM of two independent experiments, where ‘*’ and ‘**’ indicate p,0.05 and p,0.01 respectively. doi:10.1371/journal.pone.0016097.g006 Mycobacterial strain and antigens M. tb H37Rv and M. bovis BCG (Danish strain) were kind gifts from Dr. V. M. Katoch, NJIL&OMD, Agra. Mycobacteria were cultured in 7H9 medium containing 0.05% Tween80 supple- mented with 10% ADC enrichment. Short term culture filtrates (STCF) and purified protein derivative (PPD) were prepared by standard protocols [19]. Briefly, the mycobacteria from log phage cultures were grown in 7H9 broth without ADC enrichment and Tween80 for 2 weeks at 37uC. The culture supernatant was harvested by centrifugation and filtered through 0.2 mm membrane filter. Supernatant containing culture filtrate proteins were lyophilized. Lyophilized proteins were dissolved and dialyzed extensively against PBS and stored at 280uC for future use. For PPD preparation, cultures were incubated for 8– 9 weeks at 37uC. Later, cultures were autoclaved at 121uC for 30 min and cooled overnight at room temperature. Bacterial cells were removed by centrifugation followed by filtration. The proteins in the filtrate were precipitated by trichloroacetic acid (4% final concentration) by keeping overnight at room temperature without any agitation. Later, the precipitate was centrifuged at 25006g for 15 min to remove supernatant. The precipitate was washed 26 with 1% TCA and once with 10% NaCl. Then precipitate was dissolved in PBS, filter sterilized and stored at 280uC for future use. Cells and cell lines Peritoneal macrophages. Mice were inoculated with 3 ml of 3% thioglycollate medium intraperitoneally. After 96 h, peritoneal exudates cells (PECs) were harvested and washed 26 with RPMI-1640-FBS-1%. Macrophages were purified by adhering on 90 mm tissue culture dish for 2 h. THP-1. A human macrophage cell line was maintained in RPMI-1640-FBS-5% and used as source of xenogeneic macrophages for vaccine preparation. Bone marrow derived dendritic cells (BMDC). Bone marrow cells were isolated from femur of BALB/c mice and cultured (26106) in 60 mm Petri dish in the presence of GM-CSF (10 ng/ml) and IL-4 (5 ng/ml) in complete RPMI-1640-FBS-10% for 6 days. Cultures were replenished with fresh GM-CSF and IL- 4 after 3 days. Cells were harvested on day 7 for experiments. Preparation of infected macrophages and cytosolic antigenic proteins Infected syngeneic (sIM) and xenogeneic (xIM) macrophage vaccines for immunization and infected macrophages cytosolic (IMC) antigenic proteins for in vitro studies were prepared by culturing live M. tb inside the macrophages as described earlier [5]. Briefly, syngeneic peritoneal macrophages and xenogeneic THP-1 cells were plated overnight at 37uC in antibiotic free RPMI 1640- PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16097