A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

Abstract

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

“…M.tb is the pathogen of interest and may be used as the mycobacterial inoculum in the direct NHP MGIA; indeed we have demonstrated a BCG-vaccine induced effect and a correlation with protection from in vivo mycobacterial challenge using whole blood from macaques co-cultured with M.tb H37Rv 37 . However, a similar MGIA kinetic was observed whether BCG or M.tb was used as the inoculum, with a correlation between the two measures 37 . Such an association has also been reported in the human direct MGIA 28,44 .…”

“…Furthermore, the NHP model represents a unique opportunity for biological validation of the assay against direct measures of in vivo protection, as discussed below, permitting bridging to use in other species including humans. We present a protocol for the first example of an NHP MGIA using in vitro cell co-culture, adapted from our direct MGIA methods described in humans and mice, with the aim of refining and expediting early TB vaccine testing [35][36][37] .…”

“…We previously demonstrated that reducing the MOI by increasing cell number rather than reducing mycobacterial inoculum increases ability to detect a BCG vaccine induced response using the NHP direct MGIA 37 . This was consistent with findings using both the mouse and human direct MGIAs 29,32 .…”

“…Comparing outcomes with levels of protection from in vivo M.tb or BCG challenge to determine biological validity 37 .…”

High dose aerosol challenge with Mycobacterium tuberculosis fails to overcome BCG vaccination-induced protection in cynomolgus macaques of Chinese origin: implications of natural resistance for TB vaccine evaluation.

“…The direct PBMC mycobacterial growth inhibition assay (MGIA) was carried out using PBMCs at a concentration of 13x10 6 cells inoculated with ~500 CFU Pasteur Aeras in a total volume of 480 µl RPMI (containing 2mM l-glutamine and 25mM HEPES), plus 120 µl autologous serum matched to animal and time-point in a 48-well plate, as described previously 39 . After incubation at 37°C with 5% CO 2 for 96 hours, co-cultures were transferred to 2ml screw-cap tubes and centrifuged at 15,300g for 10 minutes.…”