Mycobacterial growth inhibition assay

The direct PBMC mycobacterial growth inhibition assay (MGIA) was carried out using PBMCs at a concentration of 13 × 10⁶ cells inoculated with ~ 500 CFU Pasteur Aeras in a total volume of 480 µl RPMI (containing 2 mM l-glutamine and 25 mM HEPES), plus 120 µl autologous serum matched to animal and time-point in a 48-well plate, as described previously⁴². After incubation at 37 °C with 5% CO₂ for 96 h, co-cultures were transferred to 2 ml screw-cap tubes and centrifuged at 15,300×g for 10 min. During this time, 500 µl sterile water was added to each well to lyse adherent monocytes and release intracellular mycobacteria. Supernatants were removed from the screw-cap tubes by pipetting, and water from the corresponding well added to the remaining pellet. Tubes were pulse vortexed and the suspension was transferred to Mycobacterium growth indicator tubes (MGIT) supplemented with PANTA antibiotics and OADC (Becton Dickinson, UK) for enumeration of surviving mycobacteria using the BACTEC MGIT instrument (Becton Dickinson, UK). On day 0, duplicate direct-to-MGIT inoculum controls were set up by inoculating supplemented BACTEC MGIT tubes with the same number of mycobacteria as the samples. The time to positivity (TTP) read-out was converted to log10 CFU using stock standard curves of TTP against inoculum volume and CFU. Results were normalised by subtracting log10 CFU of the inoculum control from log10 CFU of each sample, and ‘vaccine response’ calculated as (post-vaccination normalised growth–baseline normalised growth), presented as Δlog10 CFU.