Reagents

For the immune assays described below, the following reagents were used for antigenic stimulation. Peptide pools of each of the five antigens included in the constructs are described as follows: for Ag85B, 15 µg/peptide/vial, 69 peptides in total (JPT, MA, USA, Lot: 070813BAR-1); for Rv2626, 15 µg/peptide/vial, 33 peptides in total (Biosynthesis Inc., TX, USA, Lot: T6455-1); for ESAT-6, 15 µg/peptide/vial, 21 peptides in total (JPT, MA, USA, Lot: 200814BAR-2); for RpfD, 15 µg/peptide/vial, 36 peptides in total (JPT, MA, USA, Lot: 12125); for Rv1733, 15 µg/peptide/vial, 50 peptides in total (JPT, MA, USA, Lot: 10741). All peptide pools consisted of 15-mers with 11aa overlap and were synthesized to 80% purity. These antigens were added to a final concentration of 1 µg/ml. Purified Protein Derivative of Tuberculin (PPD; batch T50, SSI, Denmark) was added at a final concentration of 5 µg/ml. As positive control stimuli, a mix of Phorbol 12-Myristate 13-Acetate (PMA; 50 ng/ml, Sigma) and Ionomycin (1 µg/ml, Sigma) was applied for ELISpot assay, Concanavalin A (ConA, 5 µg/ml, Sigma) for T cell proliferation assay (TCPA), and Staphylococcus enterotoxin B (SEB, 1 µg/ml; Sigma) for FACS. R10 complete medium was used as a negative control of stimulation and consisted of Roswell Park Memorial Institute (RPMI) supplemented with 10% FCS, glutamax, and penicillin/streptomycin.