Immunization of Mice and Guinea Pigs

For preparation of vaccine stocks, M. bovis BCG (Pasteur stain 1173P2) was cultured in Middlebrook 7H9 medium supplemented with 10% oleic acid albumin dextrose, 0.05% Tween 80 and 0.5% glycerol. The cultures were grown till mid-log phase, harvested by centrifugation, washed, resuspended in 1× PBS and stored as 1,000 μl aliquots at −80°C till further use. The bacterial viability was determined by plating 10.0-fold serial dilutions on Middlebrook 7H11 plates and plates were incubated at 37°C for 3 to 4 weeks. For animal studies, MK-2206 was purchased from Merck and formulated in 30% w/v captisol solution (Cydex pharmaceuticals) as per manufacturer recommendations and previous reports (35, 36). Captisol is a chemically modified β-cyclodextrin that enhances safety, stability, solubility and dermal absorption of the drug (37, 38). For mice experiments, BALB/c mice (female, 6–8 weeks) were subcutaneously immunized and divided into the following 4 groups: (i) sham immunized, (ii) vaccinated with 10⁶ CFU of M. bovis BCG, (iii) vaccinated with 10⁶ CFU of M. bovis BCG followed by administration of MK-2206 (25 mg/kg) at day 2 and day 3 post-immunization at the same site of vaccination and (iv) administration of MK-2206 at day 2 and day 3 along with the adjuvant. Guinea pigs were immunized via intradermal route and divided into 3 groups; (i) sham immunized group, (ii) vaccinated with 10⁵ CFU of M. bovis BCG and (iii) BCG immunized and boosted with administration of MK-2206, at day 2 and day 3 post-immunization at the same site of vaccination.