eSymposia | Tuberculosis: Science Aimed at Ending the Epidemic

Dec 2, 2020 ‐ Dec 4, 2020



Sessions

Data-driven Inference of Drug Synergy

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Data-driven Inference of Drug Synergy Combination therapy is an important strategy for treating tuberculosis (TB), which kills almost 2 million people each year. Long treatment durations and growing rates of multi-drug resistant (MDR) TB have underscored the need for entirely new multi-drug regimens rather than single agents to combat the TB pandemic. Because empirically assessing the astronomically immense set of all possible drug combinations is prohibitive, there is pressing need for rational approaches to prioritize new drug regimens for clinical trials. To begin addressing this problem, we adapted a computational tool that we recently created—Inferring Drug Interactions using chemo-Genomics and Orthology, (INDIGO)—which accurately predicts drug synergy/antagonism in E. coli based on the available high resolution chemogenomic data available for that organism. We modified INDIGO to predict synergy/antagonism in 26,106 2-way and over 1 million 3-way interventions involving 164 compounds and 65 perturbations in Mycobacterium tuberculosis (MTB), by leveraging publicly available transcriptomic data. In vitro validation of predicted 2-way and 3-way interactions by checkerboard assay revealed strong correlation with model predictions, and model predictions also correlated significantly with 2-month sputum conversion rates elicited by multidrug regimens as reported from clinical trials. In addition, INDIGO analysis has revealed approximately 250 highly conserved genes that are most predictive of drug interaction outcome. We have integrated this core gene set and MTB network information to identify pathways and regulators that influence drug interaction outcomes, which in turn reveals new targets for combination therapy. INDIGO shows great promise for efficiently selecting novel TB drug regimens by prioritizing combinations based on extent of synergy or antagonism and by identifying mediators that can enhance these interactions.

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RD1 and RD9 Proteins and Peptides of Mycobacterium tuberculosis and Antibody Reactivity

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

RD1 and RD9 Proteins and Peptides of Mycobacterium tuberculosis and Antibody Reactivity RD1 and RD9 Proteins and Peptides of Mycobacterium tuberculosis and Antibody Reactivity Hanif SNM1, Mustafa AS2 1Department of Basic Sciences, University of Pikeville, Kentucky, USA.; 2Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait Abstract Introduction: The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, and ESXB and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. Aims: To determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e. rabbits, mice, and humans. Methods: Sera were obtained from i. rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV, ii. mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes, and iii. tuberculosis patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. Results: The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85) and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355) and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB, and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85) and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355) and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70) P5 (aa 61-85) and P6 (aa 76-94) of ESXV showed positive reactivity with sera from tuberculosis patients and healthy subjects. Conclusions: The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.

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Pyrazinamide inhibits the host enzyme PARP1 to reduce inflammation and accelerate bacterial clearance

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Pyrazinamide inhibits the host enzyme PARP1 to reduce inflammation and accelerate bacterial clearance Stefanie Krug1, Pankaj Kumar1,2, Bong Gu Kang1, Ted M. Dawson1, Valina L. Dawson1, & William R. Bishai1* 1The Johns Hopkins School of Medicine, Baltimore, MD, USA, 2Jamia Hamdard University, New Delhi, New Delhi, India Background: Pyrazinamide (PZA) is an enigma: Despite low bactericidal activity and brief administration, PZA is an irreplaceable sterilizing component of most TB regimens. Interestingly, PZA also has immune-modulatory activity but the relevance and targets of PZA’s host effects are unclear. We hypothesized that PZA inhibits the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1) based on its structural similarity to the PARP inhibitor nicotinamide. PARP1 drives inflammation, and we recently discovered that PARP1 is robustly activated and enhances cytokine production in TB. Since destructive inflammation can antagonize mycobacterial killing, we further hypothesized that PZA might reduce lung damage and accelerate bacterial clearance by inhibiting PARP1. Methods: We evaluated PZA-PARP1 interactions by structural alignment and DSF; PZA’s effects on PARP activity in cells and mice; PZA’s efficacy in PARP1-/- mice; and pharmacologic PARP inhibition as a novel adjunct TB therapy by comparing disease progression, cytokines and inflammation in mice treated with PZA or the FDA-approved PARP inhibitor talazoparib. Results: Here, we identify PARP1 as the first host target of PZA and show that standard PZA treatment reverses PARP activity in TB-infected mice to uninfected levels. Like PZA, adjunctive PARP inhibition with talazoparib reduces IFNγ, IL-1ß, TNFα, IL-6 and MCP-1 levels and lung pathology in a CFU-independent manner. Remarkably, PZA-treated PARP1-/- mice have 0.5 log10 more bacteria and higher cytokine levels than WT mice, indicating that PARP inhibition contributes to PZA’s activity. Conclusions: We identified PZA as a novel PARP inhibitor and demonstrate for the first time that host effects are integral to PZA’s mode of action. PARP inhibition presents an exciting new avenue for TB therapy with the potential of greatly improving patient outcome.

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Targeted nanoparticulate drug delivery system to lungs: A promising approach for pulmonary tuberculosis

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Targeted nanoparticulate drug delivery system to lungs: A promising approach for pulmonary tuberculosis Sujit Kumar Debnath1, Monalisha Debnath2, S. Saisivam3, Rohit Srivastava1,* *,1Department of Biosciences and Bioengineering Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra, India; 2Department of Electrical Engineering, Indian Institute of Technology Kharagpur, West Bengal, India; 3Gujarat Technological University, Ahmedabad, Gujarat, India *Corresponding author Tuberculosis is a deadly disease worldwide. According to the WHO, a total of 1.4 million people died from TB in 2019 worldwide. TB is one of the top 10 death-causing diseases. The rising incidence of drug resistance cases becomes a more challenging task for clinicians. Tuberculosis frequently attacks the lungs through the inhalation route and cause pulmonary tuberculosis. Targeted delivery can be possible successfully with the help of a nanoparticulate form of drugs. Polymeric nanoparticles, lipid-nanoparticles, and liposomes are extensively explored in the targeted delivery. We have optimized two polymeric nanoparticles of prothionamide (A model drug): PLGA (50:50) and Chitosan nanoparticles. There are several conventional dosage forms available in the market. But, they are non-specific, and a small fraction of the administered dose reaches the lung. In the lungs targeted delivery, the inhalation route is the ultimate opportunistic to get the desirable therapeutic action. We modified the above two nanoparticles into a dry powder inhaler to make it suitable for pulmonary administration. The devices available for human administration are not suitable for animal administration. Thus, we fabricated the delivery device for DPI using 3D printing. During animal experiments, this delivery device could able to deliver DPI successfully to the trachea part of the rat. After administration, bio-distribution, and lugs pharmacokinetics were assessed. In-vivo, PLGA (50:50) would be able to release 43% of the content, and chitosan would able to release more than 96% of the delivered amount in 24 hours. Both the polymers showed an initial blast effect that rise the plasma and lung concentration above the MIC. This study also demonstrated the drug concentration in the lungs is comparatively high rather than the concentration present in the plasma. Hence, using a DPI inhaler, the drug targeting can be possible to lungs and additionally restrict drug for bio-distribution.

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A fluorogenic trehalose probe for tracking phagocytosed mycobacterium tuberculosis

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

A fluorogenic trehalose probe for tracking phagocytosed mycobacterium tuberculosis Tingting Dai1, Jinghang Xie2, Qihua Zhu2,3,4, Mireille Kamariza5, Ke Jiang2, Carolyn R. Bertozzi1,6, Jianghong Rao1,2* 1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.; 2Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA 94305, USA.; 3State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 211198, China.; 4Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China.; 5Department of Biology, Stanford University, Stanford, CA 94305, USA.; 6Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.6 million deaths every year. Metabolic labeling studies that provide on the intracellular replication status of viable Mtb are crucial for the elucidation of host-pathogen interactions. We have here developed a cephalosphorinase-digestable green trehalose (CDG-Tre) fluorogenic probe that enabled the visualization of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 µM. Upon activation by BlaC, the -lactamase uniquely expressed by Mtb, CDG-Tre releases a fluorescent trehalose analogue that will be subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed better selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The designed CDG-Tre probe offers a unique metabolism-based strategy to label BCG. It enables to track a single Mtb cell in both pre- and post-phagocytosis and potentially could be used to help elucidate fundamental physiological and pathological process related to the mycomembrane.

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Host immune regulator PARP1 drives TB sex differences

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Host immune regulator PARP1 drives TB sex differences Stefanie Krug, Bong Gu Kang, Ted M. Dawson, Valina L. Dawson, & William R. Bishai The Johns Hopkins School of Medicine, Baltimore, MD, USA Background: TB has a male bias but the underlying biological factors are poorly understood. Men not only develop active TB more frequently but also display worse severity than women, potentially due to the male-specific exaggerated inflammatory response early in infection. Elucidating the basis of the male bias in TB may enable precision medicine interventions for TB treatment and prevention. The conserved eukaryotic enzyme, Poly(ADP-ribose) Polymerase 1 (PARP1), regulates many cellular functions, including inflammatory responses, but its role in TB is so far unknown. Since PARP1 regulates sexually divergent immune responses and drives disease severity in numerous inflammatory conditions, we hypothesized that PARP1 similarly regulates the immune responses associated with TB sex differences. Methods: We quantified PARP1 activity in uninfected and TB-infected mice by PAR Western blot and characterized the role of PARP1 in TB by comparing survival, disease progression, cytokine levels and immunopathology in PARP1-/- and WT (129S1) mice. Results: TB infection robustly activates PARP1 in mouse lungs. Surprisingly, PARP1 deletion was profoundly protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden. In contrast, PARP1 deletion was detrimental in male mice, as indicated by significantly higher bacterial burden and lung inflammation. Remarkably, PARP1 deletion abolished sex differences in TB cytokine responses, including in TNFα, IL-1ß and IFNγ production, suggesting that PARP1 promotes proinflammatory responses specifically in males to control TB infection. Conclusions: Here, we identify the master regulator PARP1 as a driver of sexually divergent TB immune responses and propose that PARP1 contributes TB sex differences by differentially regulating male and female immune processes. In addition, we uncovered a new role of PARP1 as a host factor that contributes to the genetic susceptibility to TB.

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Improving tuberculosis patients’ treatment adherence via electronic monitors and an app versus usual care in Tibet: a pragmatic randomised controlled trial

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Improving tuberculosis patients’ treatment adherence via electronic monitors and an app versus usual care in Tibet: a pragmatic randomised controlled trial Background: Poor treatment adherence is a serious challenge to effective tuberculosis (TB) control in Tibet. This study aims to evaluate the effectiveness of using new technologies, including electronic monitors (e-monitors) and a smartphone app, to improve treatment adherence among new pulmonary TB patients in Tibet, China. Methods: This is a prospective, pragmatic, multicentre, individual-randomized controlled trial with blinded outcome evaluation, and unblinded treatment. New pulmonary TB patients of three counties/districts in Shigatse, Tibet have been randomized to either the intervention or control arm in a 1:1 ratio at the time of their diagnoses since December 2018. All patients in both arms were treated according to the WHO standard TB treatment regimens and China National TB program guidelines, and received their medicines in e-monitors which could report their medication adherence history to the server every day. Additionally, in the intervention arm, e-monitors could remind patients of taking medicines with recorded human voice and share the medication adherence history with health staff via a smartphone app so that video observed treatment could be provided when adherence was problematic. Medication adherence data were collected through the server for interim analysis. Results: We here report the trial progress by the end of March 2020. We have recruited 123 eligible patients, of which 73 were allocated in the intervention arm and 50 in the control arm, with 46/27 patients (intervention/control) still on treatment. Total planned doses were 10,203 in the intervention arm and 7,295 in the control arm. Medication adherence rate was significantly higher in intervention arm (98.9%) than in control arm (40.0%) with p value less than 0.05. We report the responses of applying the e-application for TB patients during the COVID outbreaks. Conclusion: Interim analysis results implied that electronic monitor and its app function improves medication adherence among TB patients and becomes better accepted during the COVID outbreaks.

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Scope for scalable smart formulations for enhanced drug delivery in Tuberculosis

Dec 2, 2020 12:00am ‐ Dec 2, 2020 12:00am

Scope for scalable smart formulations for enhanced drug delivery in Tuberculosis Dr. Yolandy Lemmer Council for Scientific and Industrial Research, Pretoria, South Africa Background: The complexity of drug delivery to pathogenic bacilli that are located within granulomas poses a pronounced challenge for the development of novel treatment programmes to combat tuberculosis. In general, high drug dose levels need to be administered to compensate for the progressive decrease in drug concentrations from the plasma to the macrophage and then finally reaching the bacilli, with concomitant drug induced toxicities and poor patient compliance. Nanoencapsulation of existing drugs with or without targeting ligands have shown the potential to solve this problem, but is challenged by costs of large scale production incurred by sourcing of raw materials and the encapsulation technology itself. Methods: PLGA nanoencapsulation of existing TB drugs has been achieved, with various approaches to reduce cost and improve production by smart formulations and innovative manufacturing strategies. Results: We disclose a successful attempt at drug nanoencapsulation with technologies including unique formulation and spray drying, which is anticipated to make it fully scalable to the industrial level, allowing for an easier entry into large scale production for delivery to the market. Conclusion: Pre-clinical studies clearly demonstrated that our encapsulated formulations showed improved comparative efficacy of treatment when compared to the free drugs alone. These results indicate the sustained efficacy of encapsulated drugs and potential for reducing the dosing frequency to treat uncomplicated TB. The most prominent difference compared to existing state of the art is the easily scalable spray drying technology that we describe. The results emphasize the importance of innovation planning when considering new smart nanomedicine formulations for treatment: scalability of the process must always be considered for a feasible product outcome.

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Taking Science‑Based Solutions to Scale

Dec 2, 2020 10:00am ‐ Dec 2, 2020 10:40am

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Welcoming Remarks and Keynote Address

Dec 2, 2020 10:00am ‐ Dec 2, 2020 10:40am

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