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

Rachel Tanner,Emily Hoogkamer,Stephanie A Harris,Charelle Boot,Helen Mcshane,Frank A W Verreck,Matthew K O’Shea,Julia Bitencourt,Charlotte Sarfas,Claudia C Sombroek,Sally A Sharpe,Daniel Wright,Helen A Fletcher,Rachel Wittenberg,Iman Satti,Andrew D White

doi:10.1038/s41541-020-00263-7

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.

Highlights

Tuberculosis (TB) is the leading cause of death due to an infectious pathogen with an estimated 10 million new cases and 1.42 million deaths in 20191
The use of non-human primates (NHPs) in medical research is emotive[5], and assessment of protective TB vaccine efficacy currently necessitates in vivo challenge with virulent Mycobacterium tuberculosis (M.tb), a procedure resulting in disease development and classified in macaques as ‘Moderate Severity’ under UK ASPA licensure and European legislation[6]
Applying the whole blood mycobacterial growth inhibition assay (MGIA), significantly improved control of BCG growth was observed at 8 weeks and 20 weeks post-BCG vaccination compared with baseline (p = 0.0048, mean difference (MD) = 0.60 log[10] CFU, 95% CI 0.23–0.97; and p = 0.0412, MD = 0.37 log[10] CFU, 95% CI 0.02–0.72 respectively; RM one-way ANOVA with Dunnett’s post-test, p = 0.0002, F(1.44, 10.06) = 27.2; Fig. 1a)

Summary

Introduction

Tuberculosis (TB) is the leading cause of death due to an infectious pathogen with an estimated 10 million new cases and 1.42 million deaths in 20191. A major barrier to the development of an efficacious vaccine is the lack of a validated correlate of protection from TB, several potential biomarkers have been proposed[2,3,4]. Novel TB vaccine candidates or regimens are currently evaluated using preclinical models including non-human primates (NHPs). The use of NHPs in medical research is emotive[5], and assessment of protective TB vaccine efficacy currently necessitates in vivo challenge with virulent Mycobacterium tuberculosis (M.tb), a procedure resulting in disease development and classified in macaques as ‘Moderate Severity’ under UK ASPA licensure and European legislation[6]. Sufficient animal numbers are required to ensure reliable conclusions, and experiments are relatively time- and costconsuming requiring high biosafety containment facilities[7]

Objectives

Methods

Results

Conclusion