Evaluation of Novel TB Vaccine Candidates

All TB-MAPS vaccines were formulated the day prior to immunization. MAPS complexes were diluted to the appropriate concentration in saline and then mixed with aluminum hydroxide (Alum) (Brenntag) (1.25-mg/mL final concentration) in 5-mL tubes and incubated at 4°C overnight with rotation (24 rpm). Six-week-old female mice (n = 10) received subcutaneous immunizations on the upper back, once every other week for a total of 3 injections. For MAPS1, mice received 10 μg of each Mtb complex and 5 μg of lipidated rhavi complex per dose per mouse. For MAPS2, mice received 12.5 μg of rhavi-ESAT6/CFP10-MPT64 complex and rhavi-TB9.8/TB10.4-MPT83 complex, 5 μg of rhavi-MPT51 complex, and 2.5 μg of lipidated rhavi complex per dose per mouse. The control groups received Alum (1.25-mg/mL final concentration in saline) alone. For BCG immunization, mice received one subcutaneous injection of 200 μL of BCG vaccine (Merck, diluted to 1 × 10⁵ CFU in 200 μL with saline just before immunization). The control group received 200 μL of saline. For BCG followed by MAPS2, mice received one subcutaneous injection with BCG and, 1 month later, received two boosters of MAPS2 at a 2-week interval. The control group received BCG followed by Alum. For BCG and MAPS2 coadministration, mice received one injection of BCG and one injection of MAPS2 at opposite flanks for the first immunization and, 1 month later, received two boosters of MAPS2 at a 2-week interval. For IL-12p40 neutralization studies, mice received 500 μg anti-IL-12p40 antibody (clone 17.8; BioXcell, Lebanon, NH) or isotype control antibody (clone 2A3; BioXcell, Lebanon, NH) via intraperitoneal injection 1 day before and 3 days after each immunization.