Awarded Research

Advancing research through collaboration is at the heart of the JHU TRAC mission. Research is organized around scientific programs and working groups that represent a cross-section of member strengths and disciplines. Learn more about key dates and information for past awards. 2023 award opportunities will be posted early fall.

Deciphering Mitochondrial Responses to Mycobacterium tuberculosis Infection of Macrophages
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2023

Deciphering Mitochondrial Responses to Mycobacterium tuberculosis Infection of Macrophages

Recipient: Anne Hamacher-Brady, PhD

It is generally accepted that Mycobacterium tuberculosis (Mtb) modulates host cell death responses, bearing the potential for host-directed therapies. However, underlying mechanisms are incompletely understood, and a comprehensive characterization of contributing cell death modes is lacking. Mitochondria regulate programmed cell death, inflammatory and innate immune signaling. A recent study of our laboratory demonstrated that direct endolysosomal targeting of mitochondria is integral to mitochondrial outer membrane permeabilization, aka MOMP (Wang et al., Dev Cell, 2020). Based on our preliminary observation of Mtb infection-associated targeting of endolysosomes to mitochondria, albeit in apparent absence of functional MOMP, we propose to investigate the hypothesis that Mtb infection alters endolysosome-mitochondrial interaction-based regulation of mitochondrial cell death signaling.

Specific Aims

1) Targeted investigation of potential regulators of endolysosome-mitochondrial interactions during Mtb-induced macrophage cell death.

2) Proteomic mapping of Mtb infection-induced endolysosome-mitochondrial contact sites through TurboID-based proximity labeling.”

Mycobacterium Tuberculosis Transmission Networks in Urban Uganda: Evidence from Spatial and Genomic Data
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2023

Mycobacterium Tuberculosis Transmission Networks in Urban Uganda: Evidence from Spatial and Genomic Data

Recipient: Tess Ryckman, PhD

The proposed project seeks to characterize Mtb transmission in an urban Ugandan community in terms of (1) recency, (2) spatial distribution, and (3) known risk factors. To accomplish this objective, I plan three aims:

AIM 1: Identify transmission clusters. I will use phylogenetic techniques to identify transmission clusters: groups of individuals likely linked by transmission based on genomic distance. The resulting scale of clustering will provide evidence on the extent to which TB is driven by recent transmission within a densely populated urban community. I hypothesize that most isolates will be related to at least one other isolate (based on a standard genetic distance of 12 SNPs), given the small size of the study area (< 2 km2), intensity of screening, and my hypothesis that recent local transmission drives TB epidemics in high-burden countries like Uganda. Evidence of substantial recent localized transmission could imply that community-based efforts to find and treat people with undiagnosed TB could have major local-level impact in communities heavily affected by TB.

AIM 2: Describe the spatial distribution of transmission. I will combine pairwise genetic distances with GPS data on residential and screening locations to describe the association between genetic relatedness and space. I will also merge the transmission clusters from Aim 1 with GPS data to formally analyze the geographic scope of Mtb transmission, aiming to identify transmission “hotspots” within the study area and whether there is a spatial radius relevant to contact tracing efforts. I hypothesize that the probability of two individuals being in the same transmission cluster will be lower for people residing >1km vs <0.5 km apart. Evidence of spatial clustering could support efforts to augment spatially targeted case finding and prevention efforts.

AIM 3: Assess factors associated with the risk of transmission. I will make use of Bayesian phylogenetic techniques that enable inference regarding transmission directionality and then use statistical models to assess associations between hypothesized risk factors and the likelihood of transmitting Mtb. For clusters in which directionality is most uncertain, I will send a subset of isolates for deep sequencing, which can provide evidence on within-host diversity that can clarify transmission directionality. I hypothesize that key TB risk factors that affect contact patterns (e.g., incarceration history, young age) increase the risk of being a source of transmission, while others (e.g., HIV) may only increase the risk of acquiring TB. Findings can inform strategies for focused case detection (e.g., at release from correctional facilities or at venues frequented by young men).

The proposed research will provide unique insights on the scope of Mtb transmission networks in an urban African community – a starting point for evidence-based design of targeted interventions in these settings. Furthermore, this research will help me secure larger-scale funding by extending and bolstering the rationale for research I recently proposed in an initial K01 submission.

Deciphering the hormonal and genetic mechanisms mediating sex differences in tuberculosis: using a sst1-susceptible ‘Four Core Genotype’ mouse model
JHU TRAC POSTDOCTORAL DEVELOPMENTAL AWARDEE 2023

Deciphering the hormonal and genetic mechanisms mediating sex differences in tuberculosis: using a sst1-susceptible ‘Four Core Genotype’ mouse model

Recipient: Manish Gupta, PhD

TB afflicts more men than women with a global M/F morbidity and mortality ratio of ~1.7 but the underlying biologic basis for this sex bias is largely unknown. Previously we used the ‘Four Core Genotypes’ (FCG) mouse model in the standard C57BL/6 mouse background. FCG mice yield four genotypes that are XX or XY, each genotype with either testes or ovaries. Following Mtb infection we found that gonadal male mice (XX-males or XY-males) died significantly earlier than gonadal female mice (XX-females or XY-females. Of note, XY complemented females were more susceptible to Mtb infection than XX. This suggests that sex hormones have a major influence in the rapid the progression of pulmonary TB but that the sex chromosome complement also plays role with XX genotypes being protective. We further characterized these observations with other modalities (lung bacterial burden, histology, immunophenotyping, hormone/cytokine measurements, and bulk-RNAseq), and we are preparing a manuscript. However, the major limitation of our earlier work is that C57BL/6 mice do not develop human-like necrotic granulomas.

With TRAC funding, I propose to test the Mtb susceptibility of FCG mice in the B6sst1-susceptible (B6.Sst1S) background in which mice develop human-like, caseous, necrotic granulomas. By multiple backcross events, I have successfully generated a stable lineage of B6.Sst1S FCG mice and validated the superinduction of type I interferons in their BMDMs post TNF-α stimulation. Using these sst1-susceptible FCGs, I propose to perform low-dose Mtb infections and measure multiple outcome parameters: time-to-death, lung Mtb burden, CyTOF immunophenotyping, spatial transcriptomics, PET/CT imaging, hormone/cytokine measurements, etc. Further, we will correlate epigenetic profiles (ATAC-Seq) with bulk gene expression levels (RNA-Seq) in order to seek cellular evidence of escape from X chromosome inactivation in the BMDMs of these FCGs post Mtb infection. The results should enable me to discriminate the impact of genetics and sex steroids that might differentially modulate innate and adaptive immune responses in males and females, leading to sex differences in disease susceptibility. Research addressing the biological bases for the male bias is likely to provide novel insights into the pathogenesis of active TB disease and latent TB infection. I also hope to use these findings as preliminary data to apply for a K99-R00 and/or K22 award that will enable me to start an independent research career.

Characterization of immunometabolic drivers of tuberculosis infection
JHU TRAC POSTDOCTORAL DEVELOPMENTAL AWARDEE 2023

Characterization of immunometabolic drivers of tuberculosis infection

Recipient: Sadiya Parveen, PhD

Mtb is known to induce diverse metabolic changes in the host (Russell, Lamprecht, et al. 2019). However, the mechanism driving most of these metabolic changes remains understudied. We recently demonstrated that a novel glutamine metabolism antagonist JHU083, that was discovered by Drs Jonthan Powell and Barbara Slusher at JHU, substantially reduces lung bacillary burden and improves the survival of Mtb-infected mice. Additionally, we have shown that JHU083 modulates level of host metabolites in the whole lung tissue and boosts effector T-cell immunity in murine models of tuberculosis (Parveen et al.; under revision at Nat Comm; BioRxiv 2023). Single-cell RNA sequencing-based analysis revealed that JHU083 treatment affected metabolic pathways in a cell type-dependent fashion. The glutamine metabolism antagonist JHU083 downregulated transcripts associated with glutaminolysis in T-cells but upregulated it in macrophages. However, effect of JHU083 upon the metabolites in specific immune cell types remain to be deciphered.

Delineating the fundamental differences in how JHU083 affects the metabolome of macrophages vs. T-cells may lead to identifying host metabolic pathways crucial to the containment of TB. The present proposal aims to characterize these immune cell type-specific metabolic differences. We hypothesize that JHU083-mediated immunometabolic reprogramming is immune cell type dependent. To test this hypothesis, untargeted metabolomic analysis of macrophages and T-cells in vivo (isolated from lungs by magnetic-bead enrichment), with and without JHU083 treatment will be conducted. The targeted pathways will be glycolysis, glutaminolysis, beta-oxidation, amino acid utilization, Kreb’s cycle, and oxidative phosphorylation. These aims will lead to the identification of the host metabolic pathways that are critical for TB containment. We will also likely identify the fundamental differences in how JHU083 affects macrophages versus T-cells. This analysis will likely lead to identifying novel metabolic targets that will provide unique insights toward developing novel host-directed therapies for TB. Despite being largely descriptive, the results will provide preliminary data crucial for generating novel hypotheses for future R01 grants.

Development of a novel diagnostic assay for pediatric tuberculosis
JHU TRAC DEVELOPMENTAL AWARDEE 2022

Development of a novel diagnostic assay for pediatric tuberculosis

Recipient: Subhra Chakraborty, PhD, MPH

Tuberculosis (TB) is a significant problem in both adults and children. Diagnosis of pediatric TB is challenging. Children fail to produce quality sputum, and therefore sensitivity and specificity of the current sputum-based assays are low. Since children swallow sputum, a stool specimen is a potential alternative for bacteriological confirmation of pulmonary TB. Previous studies with stool using Xpert had limitations, including low analytical and diagnostic sensitivity and a lack of a standard stool processing method.   Previously, we innovated a low-resource-settings-adapted simple and sensitive stool processing method coupled with isothermal amplification assay named “RLDT” to detect enteric pathogens (JHTV C16784). We evaluated this assay in several endemic countries, and has attracted multiple grants, including NIH R21 and R01.  We propose to develop RLDT platform-based “TB-RIF-RLDT,” a simple, rapid, and sensitive assay to detect TB and resistance to rifampin (RIF) from stool in children with the following specific aims: a) Develop TB-RIF-RLDT using spiked stool samples with M. tuberculosis bacteria.  b) Determine performance specifications of TB-RIF-RLDT.  c) Evaluate TB-RIF-RLDT at JHU with stool samples from TB-positive and negative children.   With the preliminary data from this feasibility pilot study, we will apply for NIH grants to evaluate the TB-RIF RLDT in endemic countries.

A therapeutic mRNA vaccine targeting Mycobacterium tuberculosis persisters
JHU TRAC DEVELOPMENTAL AWARDEE 2022

A therapeutic mRNA vaccine targeting Mycobacterium tuberculosis persisters

Recipient: Styliani Karanika, MD

The key stringent response enzyme RelMtb is essential for prolonged Mycobacterium tuberculosis (Mtb) survival and antibiotic tolerance in vivo. We have demonstrated that DNA vaccination targeting RelMtb enhanced the activity of isoniazid in a murine tuberculosis (TB) model. After we fused relMtb to the gene encoding Macrophage Inflammatory Protein-3 alpha (MIP-3α), targeting the antigen to immature dendritic cells, we showed substantially enhanced adjunctive therapeutic efficacy of that vaccine resulting in robust Th1 pathway response. The recently demonstrated safety and efficacy of mRNA vaccines in the current pandemic raise the potential of using that vaccine platform, which is closer to the clinical application than are current DNA vaccine candidates due to the historical difficulties with their effective delivery into human cells. This proposal explores whether a similar mRNA vaccine delivered with biodegradable nanoparticles would show equivalent immunogenicity and therapeutic efficacy in TB preclinical models. This study will help the PI obtain preliminary data to submit an R21 proposal focusing on understanding the immune mechanisms of the MIP-3α/relMtb mRNA vaccine and whether it shortens the curative TB treatment in preclinical models. This project is in collaboration with Markham Lab (Bloomberg School of Public Health) and Green Lab (Biomedical Engineering, Johns Hopkins University).

Exploring Host Determinants of Mycobacterium tuberculosis Control
JHU TRAC DEVELOPMENTAL AWARDEE 2022

Exploring Host Determinants of Mycobacterium tuberculosis Control

Recipient: Eileen Scully, MD, PhD

Mtb infection is associated with a range of clinical outcomes, from latent infection with few clinical indicators through severe, progressive infection. While there are several conditions associated with a heightened risk of transition between latent and active infection, there are fewer known mechanisms for why immune control is lost. Understanding the precise immunologic features associated with development of progressive infection will better inform host-directed therapeutics for TB disease. Two features of increased risk are HIV infection and pregnancy. This proposal will explore these two distinct, but often overlapping, risk conditions. In the first aim, studies will build on preliminary work that has already defined transcriptional and epigenetic shifts in the monocyte compartment within individuals with HIV infection before and after HIV viral suppression. We will use these same samples to define the capacity of these monocytes to control Mtb infection with a goal of linking the changes observed after HIV viral suppression to pathways of immune control. In the second aim, we will explore the impact of pregnancy on the development and differentiation of monocyte derived macrophages. These studies will model the first and third trimester of pregnancy with in vitro conditions and test the impact on Mtb control. For both HIV infection and pregnancy, the drivers of increased risk of progressive disease are unknown, and there is a clear need for optimization of treatment. The work from this proposal will be used to expand on previous, HIV-focused studies to support a new application for funding on the impact of HIV on TB innate immune control, and as data to support an R01 on the immunologic determinants of pregnancy related risk of active TB in women with and without HIV coinfection.

Assessing the role of preventive therapy in advancing goals of TB Free Nepal
JHU TRAC DEVELOPMENTAL AWARDEE 2022

Assessing the role of preventive therapy in advancing goals of TB Free Nepal

Recipient: Sourya Shrestha, PhD

A national prevalence survey completed in 2019 estimated that the prevalence of tuberculosis (TB) in Nepal was 416 per 100,000. This was 1.8 times larger than previously estimated by the World Health Organization (WHO); and it revealed that the majority of incident cases (54%) are not notified. As in many high-burden countries, reducing the burden of TB has been a major challenge in Nepal, despite advances in TB diagnosis and treatment, and more intensive active case finding efforts. This prompted the Ministry of Health to announce a bold vision entitled “TB Free Nepal” in 2020, with a goal of reducing TB incidence by 25% in the next five years, and ultimately eliminating TB by 2050. Scaling up TB preventive therapy (TPT) is a major component of this vision. However, it is unclear (i) how impactful TPT will be in a high-transmission setting (where individuals may have high risk of re-exposure on one hand, whereas only treating diagnosed TB cases may be too little too late); (ii) whether prioritizing scale up of preventive therapy is cost-effective (given challenges associated with testing for latent TB, TPT uptake, and completion of TPT); and (iii) what implementation strategies for TPT scale up should be prioritized to make progress toward the vision of TB Free Nepal.

This pilot study, will augment an ongoing study (2021-2023; IMPACT 2 TB; PI: Dr. Maxine Caws; funded by Nick Simons Foundation) that assesses the feasibility of implementing 3HP (3 months of weekly isoniazid and rifapentine) in two districts in Nepal: a predominantly rural western district, Pyuthan, and densely populated district with large urban center, Chitwan. We propose to conduct two primary research activities: 1. Collection of site-specific data on implemention of preventive therapy from Pyuthan and Chitwan including (a) yield (numbers of people diagnosed with TB and LTBI, using TST); (b) prevalence of LTBI among household contacts (c) uptake of TPT; (d) TPT completion; and (e) costs of WHO screening algorithm. Additionally, we will recruit local study staff to collate programmatic data that are already collected as a part of the ongoing study and to collect additional data on household contacts. 2. Development of transmission models, calibrated to rural and urban settings, to project the epidemiological impact, and cost-effectiveness of TPT. Dr. Shrestha will train a junior researcher currently based at Birat Nepal Medical Trust (BNMT), Mr. Rajan Paudel, to develop these models under his supervision with input from the PI of ongoing IMPACT 2 TB study, Dr Maxine Caws, and mentor Dr David Dowdy.

Determination of the mid-treatment molecular clock for multidrug resistant tuberculosis
JHU TRAC DEVELOPMENTAL AWARDEE 2022

Determination of the mid-treatment molecular clock for multidrug resistant tuberculosis

Recipient: Jeffrey Tornheim, MD, MPH

MDR-TB represents 3% of new and 18% of repeat TB infections, with 70% representing primary infection with resistant Mtb.  Several molecular epidemiology programs use a well-established Mtb “molecular clock” to rule-out transmission between infected people by comparing acquisition of single nucleotide polymorphisms–SNPs–over time. Unfortunately, the Mtb molecular clock was originally defined among laboratory isolates or people with drug-susceptible TB without selective pressure from treatment.  Recently, large mutation rate differences have been found by Mtb clade, undermining the generalizability of established thresholds.  Similarly, selection pressure of multidrug MDR-TB treatment likely increases the rate of genetic polymorphisms identified before culture conversion, with direct implications for contact tracing and transmission inferences. To determine the molecular clock of MDR-TB during effective MDR-TB treatment, we will leverage a biorepository of well-characterized isolates collected through a completed longitudinal cohort study in which participants with MDR-TB completed baseline susceptibility testing, individualized treatment, and monthly sputum collection until symptom resolution. Of 204 study participants with baseline WGS results, 58 additional culture-positive mid-treatment samples are available from 41 participants. We will complete WGS from longitudinal samples to identify the time-normalized average pairwise SNP distances between paired MDR-TB isolates, with secondary stratification by phenotypic resistance and treatment regimen.