A modified mycobacterial growth inhibition assay for the functional assessment of vaccine-mediated immunity.

The Mycobacterial growth inhibition assay (MGIA) is an ex-vivo assay used to measure the overall functional immune response elicited by infection or vaccination. In tuberculosis (TB) vaccine development, MGIA is a potentially important tool for preclinical evaluation of early-stage vaccine candidates to complement existing assays, and to potentially reduce the need for lengthy and costly pathogenic Mycobacterium tuberculosis (Mtb) animal challenge experiments. The conventional method of MGIA in mice entails directly infecting mixed cell cultures, most commonly splenocytes, from immunised mice with mycobacteria. However, this direct infection of mixed cell populations may yield unreliable results and lacks sufficient sensitivity to discriminate well between different vaccines due to the low number of mycobacteria-permissive cells. Here, we modified the assay by inclusion of mycobacteria-infected congenic murine macrophage cell lines as the target cells, and by measuring the total number of killed cells rather than the relative reduction between different groups. Thus, using splenocytes from Mycobacterium bovis BCG immunised mice, and J774 and MH-S (BALB/c background) or BL/6-M (C57Bl/6 background) macrophage cell lines, we demonstrated that the modified assay resulted in at least 26-fold greater mycobacterial killing per set quantity of splenocytes as compared to the conventional method. This increased sensitivity of measuring mycobacterial killing was confirmed using both the standard culture forming unit (CFU) assay and luminescence readings of luciferase-tagged virulent and avirulent mycobacteria. We propose that the modified MGIA can be used as a highly calibrated tool for quantitating the killing capacity of immune cells in preclinical evaluation of vaccine candidates for TB.

Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity.

Tuberculosis (TB) is a major global health threat that claims more than one million lives annually. With a quarter of the global population harbouring latent TB, post-exposure vaccination aimed at high-risk populations that could develop active TB disease would be of great public health benefit. Mucosal vaccination is an attractive approach for a predominantly lung disease like TB because it elicits both local and systemic immunity. However, the immunological consequence of mucosal immunisation in the presence of existing lung immunity remains largely unexplored. Using a mycobacterial pre-exposure mouse model, we assessed whether pre-existing mucosal and systemic immune responses can be boosted and/or qualitatively altered by intranasal administration of spore- and nanoparticle-based subunit vaccines. Analysis of lung T cell responses revealed an increasing trend in the frequency of important CD4 and CD8 T cell subsets, and T effector memory cells with a Th1 cytokine (IFNγ and TNFα) signature among immunised mice. Additionally, significantly greater antigen specific Th1, Th17 and IL-10 responses, and antigen-induced T cell proliferation were seen from the spleens of immunised mice. Measurement of antigen-specific IgG and IgA from blood and bronchoalveolar lavage fluid also revealed enhanced systemic and local humoral immune responses among immunised animals. Lastly, peripheral blood mononuclear cells (PBMCs) obtained from the TB-endemic country of Mozambique show that individuals with LTBI showed significantly greater CD4 T cell reactivity to the vaccine candidate as compared to healthy controls. These results support further testing of Spore-FP1 and Nano-FP1 as post-exposure TB vaccines.