Optimisation Of A Murine Splenocyte Mycobacterial Growth Inhibition Assay Using Virulent Mycobacterium Tuberculosis

In the absence of a validated correlate of protection or robust animal models for human tuberculosis, Mycobacterial growth inhibition assays (MGIAs) aim to assess vaccines ability to inhibit mycobacterial growth in-vitro. We optimised a reproducible murine splenocyte MGIA based on in-vitro infection with virulent Mycobacterium tuberculosis (M. tb) Erdman. We identified splenocyte viability as a problem in state-of-art MGIA protocols, which can be improved by simple changes in culture conditions (viability increase from 21% to 46% at last day of culture). The growth inhibitory potential in mice immunised with either BCG, H56: CAF01 or H56: CAF01 administered side-by-side with BCG was significantly better compared to placebo in all groups (0.3 log(10) CFU [+/- 0.2, p = 0.049], 0.5 [+/- 0.2, p = 0.016] and 0.6 [+/- 0.1, p = 0.0007], respectively) corresponding to the levels of in-vivo protection. Unexpectedly the CAF01 adjuvant control group also induced significant growth inhibition of 0.3 log(10) CFU (+/- 0.2, p = 0.047). Finally, we explored vaccine-associated T cell effector functions. Despite presence of high levels of vaccine-specific T cells, we found no increase in CD4(+) T cell number or cytokine expression profile, nor a difference in cytokine levels in the supernatant after four days culture with or without M. tb. Spontaneous IFN-gamma release correlated with growth inhibition levels (p = 0.02), however the cellular source was not found.