Autoluminescent Mycobacterium tuberculosis for Rapid, Real-Time, Non-Invasive Assessment of Drug and Vaccine Efficacy

Preclinical efforts to discover and develop new drugs and vaccines for tuberculosis are hampered by the reliance on colony-forming unit (CFU) counts as primary outcomes for in vivo efficacy studies and the slow growth of Mycobacterium tuberculosis. The utility of bioluminescent M. tuberculosis reporter strains for real-time in vitro and ex vivo assessment of drug and vaccine activity has been demonstrated but a simple, non-invasive, real-time surrogate marker to replace CFU counts for real-time evaluation of drug and vaccine efficacy in vivo has not been described. We describe the development of a fully virulent and stable autoluminescent strain of M. tuberculosis and proof-of-concept experiments demonstrating its utility for in vivo bioluminescence imaging to assess the efficacy of new drugs and vaccines for tuberculosis in a mouse model. Relative light unit (RLU) counts paralleled CFU counts during the active phase of bacterial growth, with a lower limit of detection of approximately 106 CFU in live, anesthetized mice. Experiments distinguishing active from inactive anti-tuberculosis drugs and bacteriostatic drug effects from bactericidal effects were completed in less than 5 days. The ability of a recombinant BCG vaccine to limit bacterial growth was demonstrated within 3 weeks. Use of this autoluminescent reporter strain has the potential to drastically reduce the time, effort, animals and costs consumed in the evaluation of drug activity in vitro and the in vivo assessment of drug and vaccine efficacy

Mechanistic Modeling of Mycobacterium tuberculosis Infection in Murine Models for Drug and Vaccine Efficacy Studies.

Tuberculosis (TB) drug, regimen, and vaccine development rely heavily on preclinical animal experiments, and quantification of bacterial and immune response dynamics is essential for understanding drug and vaccine efficacy. A mechanism-based model was built to describe Mycobacterium tuberculosis H37Rv infection over time in BALB/c and athymic nude mice, which consisted of bacterial replication, bacterial death, and adaptive immune effects. The adaptive immune effect was best described by a sigmoidal function on both bacterial load and incubation time. Applications to demonstrate the utility of this baseline model showed (i) the important influence of the adaptive immune response on pyrazinamide (PZA) drug efficacy, (ii) a persistent adaptive immune effect in mice relapsing after chemotherapy cessation, and (iii) the protective effect of vaccines after M. tuberculosis challenge. These findings demonstrate the utility of our model for describing M. tuberculosis infection and corresponding adaptive immune dynamics for evaluating the efficacy of TB drugs, regimens, and vaccines

Dormancy Phenotype Displayed by Extracellular Mycobacterium tuberculosis within Artificial Granulomas in Mice

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that relMtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-γ dependent

Daily Dosing of Rifapentine Cures Tuberculosis in Three Months or Less in the Murine Model

Eric Nuermberger and colleagues found that after two months of treatment, mice with lung cultures positive for tuberculosis that received daily doses of rifapentine- and moxifloxacin-containing regimens converted to negative lung cultures. This finding could make possible the development of shorter treatment regimens for humans

Using Dynamic Oral Dosing of Rifapentine and Rifabutin to Simulate Exposure Profiles of Long-Acting Formulations in a Mouse Model of Tuberculosis Preventive Therapy

Administration of tuberculosis preventive therapy (TPT) to individuals with latent tuberculosis infection is an important facet of global tuberculosis control. The use of long-acting injectable (LAI) drug formulations may simplify and shorten regimens for this indication. Rifapentine and rifabutin have antituberculosis activity and physiochemical properties suitable for LAI formulation, but there are limited data available for determining the target exposure profiles required for efficacy in TPT regimens. The objective of this study was to determine exposure-activity profiles of rifapentine and rifabutin to inform development of LAI formulations for TPT. We used a validated paucibacillary mouse model of TPT in combination with dynamic oral dosing of both drugs to simulate and understand exposure-activity relationships to inform posology for future LAI formulations. This work identified several LAI-like exposure profiles of rifapentine and rifabutin that, if achieved by LAI formulations, could be efficacious as TPT regimens and thus can serve as experimentally determined targets for novel LAI formulations of these drugs. We present novel methodology to understand the exposure-response relationship and inform the value proposition for investment in development of LAI formulations that have utility beyond latent tuberculosis infection

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made

Efficient rate-power allocation for OFDM in a realistic fading environment

The implementation of practical adaptive resource allocation scheme remains a key criterion to be satisfied for realising spectrally efficient multitone wireless communications. The ever-increasing demand for spectrally efficient broadband wireless transmission technologies has spurred intensive research leading towards the implementation of adaptive OFDM and adaptive MIMO systems. Efforts in this direction have been frustrated however by the lack of a clear and accurate description of the fading behaviour typically encountered in the broadband wireless transmission environment. This has been partially been overcome by the use of mathematical modelling which captures certain large-scale characteristics of the channel and facilitates theoretical research. The “average” channel parameters gleaned from these processes is typically then used to inform the design and configuration of wireless networking equipment after the broad application of generous safety margins. The resulting solu�tion is therefore quite robust to certain transient channel quality degradation yet the generous safety tolerances render it unable to exploit other transient transmission quality improvements We seek to overcome the problems associated with this ap�proach by applying a theoretically sound novel adaptive resource allocation framework to actual broadband wireless channel development data. The allocation framework is derived from the optimal OFDM allocation scheme for a known channel [1]: the channel development data is obtained from actual measurement of a broadband wireless mobile environment [2]. Prediction tech�niques are employed to overcome the time lag between channel assessment and symbol transmission. We present the details of the predictive resource allocation scheme used and include a broad characterisation of the transmission environment in terms of the time-varying fading processes observed. We provide some results of the application of this scheme as typical performance levels that may be achieved in an actual transmission environment

Activities of Rifampin, Rifapentine and Clarithromycin Alone and in Combination against Mycobacterium ulcerans Disease in Mice

Buruli ulcer (BU) is found throughout the world but is particularly prevalent in West Africa. Until 2004, treatment for this disfiguring disease was surgical excision followed by skin grafting, procedures often requiring months of hospitalization. More recently, an 8-week regimen of oral rifampin and streptomycin administered by injection has become the standard of care recommended by the World Health Organization. However, daily injections require sterile needles and syringes to prevent spread of blood borne pathogens and streptomycin has potentially serious side effects, most notably hearing loss. We tested an entirely oral regimen, substituting the long acting rifapentine for rifampin and clarithromycin for streptomycin. We also evaluated each drug separately. We found that rifapentine alone is as good as rifampin plus streptomycin, but the simultaneous addition of effective clarithromycin doses, at least in the mouse, reduces the activity of both rifampin and rifapentine, making it difficult to assess the efficacy of the oral regimens in the model. Studies of serum drug concentrations indicated that separating treatment times by one hour or reducing the clarithromycin dose to one active in humans should overcome this issue in experimental and clinical BU treatment, respectively

Small animal models for human immunodeficiency virus (HIV), hepatitis b, and tuberculosis: Proceedings of an NIAID workshop

The main advantage of animal models of infectious diseases over in vitro studies is the gain in the understanding of the complex dynamics between the immune system and the pathogen. While small animal models have practical advantages over large animal models, it is crucial to be aware of their limitations. Although the small animal model at least needs to be susceptible to the pathogen under study to obtain meaningful data, key elements of pathogenesis should also be reflected when compared to humans. Well-designed small animal models for HIV, hepatitis viruses and tuberculosis require, additionally, a thorough understanding of the similarities and differences in the immune responses between humans and small animals and should incorporate that knowledge into the goals of the study. To discuss these considerations, the NIAID hosted a workshop on ‘Small Animal Models for HIV, Hepatitis B, and Tuberculosis’ on May 30, 2019. Highlights of the workshop are outlined below

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy