Experimental Animal Models of TB: Chemotherapeutics and Imaging

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

This project encompasses approaches to understand how current anti-tubercular chemotherapy works using modern technologies and to develop new and improved therapies and therapeutic approaches. Individual projects within this framework are (1) developing structural and functional imaging techniques using PET/CT for use in live, M. tuberculosis (Mtb) infected animals, (2) development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by people with active tuberculosis (TB), (3) understanding the activity of various drugs in animal models of TB therapy, (4) correlating responses seen in animal models with the pathology and response to therapy observed in human TB, and (5) developing techniques for assessing drug distribution, penetration, and pharmacokinetics in vivo. Like many bacteria, the virulence factors of Mtb are incompletely understood. In 2024 and into 2025, we have continued to study the effect of disabling the production of certain Mtb secondary metabolites thought to contribute to pathology of the disease in mouse infections. We have investigated the effects of these molecules on host microenvironment through lipid analysis, measuring bacteria replication and tracking host survival post infection as well as various in vitro assays. This project provides an opportunity for the Barry, Barber and Mayer-Barber sections to work together to investigate the immunological implications of bacterial metabolite loss to better characterize the role of these metabolites in disease maintenance/progression. In 2025, several additional murine strains were infected to look for the metabolite’s effect in certain KO mice. Most of our PET/CT studies have used [18F]-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of eukaryotic cells in TB lesions in our tuberculosis animal models. We would prefer a small molecule that could be used to label Mtb endogenously as a PET radiotracer. In 2024 Khan et al. (PMID 38937448), we showed that [18F]FDT could function as a mechanism-based reporter of mycobacteria-selective enzyme activity in vivo in marmosets and in rhesus macaques. Administering [18F]FDT as a PET tracer successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. In 2025, we are completing some further characterization of the tracer to build a case for its use in clinical assessment of TB disease in clinical studies. Toward this goal, we have monitored the Mtb disease progression in marmosets with FDT and seen FDT uptake increase as disease progressed, and we have shown a positive relationship between FDT retention in marmoset lesion tissues and their bacterial content. We have begun monitoring FDT labeling of lesions during drug treatment to document a reduction in signal and to determine a lower limit of detection. Using FDT for detection of relapse of Mtb disease after treatment is in progress. As understanding the metabolic processes active in MTB bacteria in the cavity may lead us to new drug targets, we undertook several experiments to increase the frequency of cavities among the marmoset’s lesions that result from MTB infection. In one approach among several, we BCG-vaccinated a group of the marmosets and monitored them for both disease development and cavity formation. In 2024, as marmosets had not been vaccinated with BCG previously, we undertook several preliminary experiments to confirm that BCG would not cause BCG-osis in the species and that the animals would develop antigen-specific T cells from the exposure. In 2025 as collaboration among T-cell biology section (TBS), and Spatial Immunology Unit (SIU) scientists, we found that marmosets both survived longer and had lower bacterial loads if vaccinated but did not appear to make more cavities. The immunologists found that marmosets made antigen-specific T cells but in a different time course than they usually observe in rhesus macaques. Immunohistochemistry and other assays are ongoing to try to characterize these differences. Treating marmosets with a short anti-TB regimen and then allowing disease to slowly reform has been more successful in yielding more air-filled cavities. The Mtb from these cavities are now being characterized. Mtb infection of rabbits also leads to air-filled cavities after an extended incubation, so a large set of those cavities have also been collected for Mtb characterization. We continued to use the mouse, marmoset and rabbit infection models to evaluate classes of antibiotics from our partners in the Gates Foundation's TB Drug Accelerator and other academic partners including diarylquinolines, quinolines, imidazopyridines, nitroimidazoles, among others. These classes of antibiotics are being explored as composing new regimens for treatment of MTB and understanding the specific contribution of each one to activity including consideration of spatial distribution and the kinetics of accumulation in lesions to avoid temporal and spatial black holes of monotherapy. With each new drug candidate, we test for suitable pharmacokinetics, tolerability with longer term dosing (2 weeks to 2 months), in vivo efficacy, and the candidate’s penetration into granulomas and cavities to correlate the information with any observed efficacy. In 2025, we reported on the penetration of bedaquiline and two next generation diarylquinolines TBAJ587 and TBAJ876, in the necrotic center (caseum) of rabbit cavities in vivo through multiday dosing (1). Laser-capture microdissection of the lesions facilitated mapping of drug concentrations as a function of distance from blood supply in caseous lesions. Simulations revealed that bedaquiline reached steady state and efficacious concentrations in deep caseum after several weeks to months of treatment in comparison only hours or a few days with the newer compounds. The diffusion rates of bedaquiline into and out of lesions where the bacteria are located create regions where the bacteria are exposed to inadequate concentrations to kill the bacteria. The penetration results of TBAJ587 and TBAJ876 predict that Mtb will have less likelihood of developing resistance to these newer compounds since the window of subtherapeutic exposure is likely to be far shorter. In another study investigating anti-Mtb drug activity, we used rabbit cavity caseum in ex vivo assays to investigate the potency of several classes of gyrase inhibitors against Mtb (2). In contrast to fluoroquinolones, many other gyrase inhibitors kill only replicating bacterial cultures but produce negligible cidal activity against Mtb. In contrast, fluoroquinolones were unique in their ability to cleave double-stranded DNA at low concentrations, killing Mtb in both replicating and nonreplicating, persistent states. Several other publications are the direct or indirect result of this project (3,4).