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Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs.

Ten million cases of tuberculosis (TB) were reported in 2018 with a further 1.5 million deaths attributed to the disease. Improved vaccination strategies are urgently required to tackle the ongoing global TB epidemic. In the absence of a validated correlate of protection, highly characterised pre-clinical models are required to assess the protective efficacy of new vaccination strategies. In this study, we demonstrate the application of a rhesus macaque ultra-low dose (ULD) aerosol M. tuberculosis challenge model for the evaluation of TB vaccination strategies by directly comparing the immunogenicity and efficacy of intradermal (ID) and aerosol BCG vaccination delivered using a portable vibrating mesh nebulizer (VMN). Aerosol- and ID-delivered Bacille Calmette-Guérin (BCG) induced comparable frequencies of IFN-γ spot forming units (SFU) measured in peripheral blood mononuclear cells (PBMCs) by ELISpot, although the induction of IFN-γ SFU was significantly delayed following aerosol immunisation. This delayed response was also apparent in an array of secreted pro-inflammatory and chemokine markers, as well as in the frequency of antigen-specific cytokine producing CD4 and CD8 T-cells measured by multi-parameter flow cytometry. Interrogation of antigen-specific memory T-cell phenotypes revealed that vaccination-induced CD4 and CD8 T-cell populations primarily occupied the central memory (TCM) and transitional effector memory (TransEM) phenotype, and that the frequency of CD8 TCM and TransEM populations was significantly higher in aerosol BCG-vaccinated animals in the week prior to M. tuberculosis infection. The total and lung pathology measured following M. tuberculosis challenge was significantly lower in vaccinated animals relative to the unvaccinated control group and pathology measured in extra-pulmonary tissues was significantly reduced in aerosol BCG-vaccinated animals, relative to the ID-immunised group. Similarly, significantly fewer viable M. tuberculosis CFU were recovered from the extra-pulmonary tissues of aerosol BCG-vaccinated macaques relative to unvaccinated animals. In this study, a rhesus macaque ULD M. tuberculosis aerosol challenge model was applied as a refined and sensitive system for the evaluation of TB vaccine efficacy and to confirm that aerosol BCG vaccination delivered by portable VMN can confer a significant level of protection that is equivalent, and by some measures superior, to intradermal BCG vaccination.