Demonstrating the utility of the ex vivo murine mycobacterial growth inhibition assay (MGIA) for high-throughput screening of tuberculosis vaccine candidates against multiple Mycobacterium tuberculosis complex strains

Human tuberculosis (TB) is caused by various members of the Mycobacterium tuberculosis (Mtb) complex. Differences in host response to infection have been reported, illustrative of a need to test vaccines against multiple Mtb strains in preclinical studies. We have previously shown that the murine lung and spleen mycobacterial growth inhibition assay (MGIA) can be used to assess control of ex vivo mycobacterial growth by host cells. The number of mice required for the assay is lower than in vivo Mtb challenge studies, facilitating testing of multiple strains and/or the incorporation of other cellular analyses which may inform the design of future in vivo studies. Here, we present an optimised mycobacterial growth inhibition assay (MGIA) for testing TB vaccines against multiple Mtb clinical isolates. Using an ancient and modern strain of the Mtb complex, we demonstrate that ex vivo bacillus Calmette–Guérin (BCG)-mediated mycobacterial growth inhibition recapitulates protection observed in the lung and spleen following in vivo infection of mice. Further, cellular and transcriptional correlates of growth inhibition in the lung MGIA were identified. Flow cytometric analysis revealed an increased proportion of dendritic cells and interstitial macrophages in the lung cell input from mice vaccinated parentally with BCG compared with unvaccinated mice. RNA-seq analysis of the lung cell input revealed shared and strain-specific transcriptional correlates of BCG-mediated growth inhibition. The ex vivo MGIA may represent a platform to gain early insight into vaccine performance against a collection of Mtb strains to improve preclinical evaluation of TB vaccines. Female C57BL/6 mice (5–7 weeks old) received 1x10^6 CFU BCG Aeras Pasteur or no treatment. Six weeks post vaccination, mice were humanely culled, and lung was removed and processed to generate single cell suspensions. Lung cells (individual mice) were uninfected/infected overnight for gene expression analysis. Specifically, 1 x 10^6 lung cells per mouse were seeded in duplicate (uninfected/infected) in 24-well plates and cultured overnight at 37°C (5% CO2) in the presence or absence of 1 x 10^6 CFU mycobacteria (N0072, N0052 or N1283). The following day, plates were centrifuged at 500 x g to pellet eukaryotic cells, supernatants were removed and 600 µL RNAprotect cell reagent was added to stabilise transcription and prevent RNA degradation. Samples were stored at –70°C until RNA extraction.