Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells

Hannah Painter,Helen A Fletcher,Andrea Zelmer,Samuel Willcocks,Satria A Prabowo,Rajko Reljic,Rachel Tanner,Felipe Cia,Lisa Stockdale

doi:10.1038/s41598-020-60223-y

Abstract

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette–Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log10 CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log10 reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log10 reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease.

Highlights

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified
A reduction in colony forming unit (CFU) burden after in vivo Mycobacterium tuberculosis (MTB) challenge has been demonstrated in the murine lung in animals administered with Bacille Calmette–Guérin (BCG) and/or various candidate TB vaccines compared with unvaccinated animals[23,24,25]
To determine whether the lung mycobacterial growth inhibition assay (MGIA) could be utilised as an ‘ex vivo challenge model’ to assess vaccine efficacy in animal models, C57BL/6 mice received either BCG Pasteur Aeras subcutaneous (s.c.) or intranasal (i.n.) at week 0, BCG Pasteur Aeras s.c. at week 0 followed by an i.n. boost with the candidate vaccine spore-FP1 at week 3, or received no treatment

Summary

Introduction

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. Differences in virulence, fitness and T-cell subset responses in animal models challenged with diverse clinical strains of MTB have been reported[11,12,13], and there is growing interest in preclinical testing of vaccines against MTB isolates representative of the global diversity of the MTB complex (MTBC). Since each animal provides sufficient cells for multiple assay inputs, the MGIA offers the potential to analyse multiple lineages of the MTBC in parallel, using considerably fewer animals than a challenge study of the same design[16]. These concepts are in line with the ‘Refinement’ and ‘Reduction’ criteria defined by the UK National Centre for the 3Rs17

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Discussion

Conclusion