Abstract
The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.
Highlights
Introduction Approximately1 in 4 people globally are infected with tuberculosis (TB), with 10 million new infections and 1.4 million deaths reported in 20191
We present a protocol for the first example of an non-human primates (NHPs) mycobacterial growth inhibition assay (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]
Conducting a literature search for the time-period 2019–2020 using the Google Scholar search terms ‘tuberculosis’ and ‘macaque’, and excluding results relating to TB diagnostics or drugs, and/or SIV coinfection, we identified 21 publications reporting NHP studies of TB vaccines and/or TB immunology employing a mean of 27 animals, the majority of which involved challenge with M.tb
Summary
1 in 4 people globally are infected with tuberculosis (TB), with 10 million new infections and 1.4 million deaths reported in 20191. This serious public health threat is further exacerbated by the spread of multi- and extensivelydrug resistant strains of the causative agent, Mycobacterium tuberculosis (M.tb)[2]. Development of a successful TB vaccine is severely hampered by the lack of a validated correlate or biomarker of protection[4]. It remains unclear which aspects of the immune response confer protection from TB disease, and which parameters to target with a vaccine and to assess as a reliable measure of protective efficacy
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