David Russell - US grants

Project Summary / Abstract Due to its extensive penetrance of the human population, Mycobacterium tuberculosis (Mtb) remains a serious health risk to those individuals living with HIV. TB vaccine development programs are hampered by our poor understanding of the immune mechanisms underpinning disease progression. What we propose in this application is the utilization of Mtb reporter strains to provide a functional readout of microbial fitness and replication to enable us to identify and characterize those phagocytes that restrict bacterial growth (controllers) versus those phagocytes the promote bacterial growth (permissive) to understand the basis of disease progression in human tuberculosis. Our hypothesis is that Mtb reporter strains represent a novel route to the identification of the phagocyte populations that best restrict or promote bacterial replication, and that defining these cell populations will provide a rational framework for understanding immune control of tuberculosis. Aim 1: Development and validation of Mtb reporter strains and challenge models in the murine system. We will (a) exploit Mtb reporter strains to identify permissive and controller phagocyte populations; (b) optimize an ex vivo infection protocol for cells recruited to Mtb-infected mouse lung, to be used on NHP granulomas, and human airway and granuloma phagocytes; and (c) perform RNASeq profiling on the different phagocyte populations defined by the Mtb reporter strains for phenotypic analysis and to generate a panel of candidate cell surface markers for NHP and human ex vivo challenge studies. Aim 2. Functional characterization of phagocyte subsets in NHP pulmonary granulomas. We will perform parallel analysis of NHP phagocyte populations infected with reporter bacterial strains following the isolation of individual granulomas from infected monkey lungs. We propose comparable phenotypic characterization using biased and unbiased methods to functionally identify permissive and controller phagocyte populations in NHP tuberculosis in in vivo infections and ex vivo challenge experiments. Specific Aim 3: Determination of human phagocyte phenotypes through ex vivo challenge with Mtb reporter strains. In addition, in collaboration with Drs. Henry Mwandumba (Malawi) and Alasdair Leslie (South Africa) we will determine the functional phenotypes of human airway and human TB granuloma phagocyte subsets by probing the cells ex vivo with the fluorescent Mtb reporter strains.

Project Summary / Abstract Despite several reports of HIV-1-infected alveolar macrophages (AM) in the lungs of HIV-1-infected individuals, the roles played by these cells in the maintenance or persistence of infection remain unresolved. Recent studies in Malawi, conducted on AM from HIV-1-infected individuals that are effectively virally- suppressed by long-term ART, reproducibly detected the presence of HIV-1 mRNA by fluorescent in situ hybridization (FISH). In these studies, we also detected HIV-1 transcripts through single cell sequencing protocols, and have isolated infectious HIV-1 virus from cells recovered by bronchoalveolar lavage from ART- naïve, HIV-1-infected volunteers. The hypothesis we propose to test is that the presence of HIV-1 transcripts in the alveolar macrophages of HIV- infected individuals is indicative of a productive viral infection that has significance for persistence of the virus during ART. R21 Phase: Aim 1. Are HIV-1-infected AM productively infected? We propose co-culture approaches to detect and capture infectious HIV-1 from the AM and peripheral blood of HIV-1-infected volunteers in Malawi. Using permissive, HIV-1-susceptible reporter cells we have already shown that we can acquire infectious virus from ART-naïve individuals in a pilot study on 12 volunteers. We propose expanding this analysis to a larger cohort, including ART-treated, virally-suppressed individuals. R33 Phase: Aim 2. Transcriptional Profiling of Viral and Host Transcripts in HIV-1-Infected Cells. Using methods already established in Malawi, we will generate transcriptional profiles of HIV-infected and uninfected AMs by single-cell Seq-Well and Flow-FISH RNASeq methods to obtain datasets reflecting both single-cell resolution and depth of coverage. We will use these datasets to probe the impact of HIV-1 in cell longevity and to study the cellular tropism of the envs from AM-derived HIV-1 virus. R33 Phase: Aim 3. Perturbation of HIV-1-infected AM Function with Synthetic mRNA and SiRNA. We will use gain-of-function (synthetic mRNA) and loss-of-function (siRNA) approaches to manipulate the phenotype and behavior of HIV-1-infected HMDMs and AMs. The goal is to identify pathways that will drive programmed cell death specifically in those AMs that are HIV-1-infected as a route for selective eradication of this potential viral reservoir. The proposal is based on the contention that AM in virally-suppressed individuals will prove to be productively- infected. The verification of this contention is the major milestone of the R21 period of this phased award.

Project Summary/Abstract Central to the function of macromolecules are the conformational dynamics they undergo in order to carry out biological function. Native mass spectrometry (MS), whereby non-covalent interactions are preserved in the mass spectrometer, is emerging as a powerful technique to study protein stoichiometry, topology, dynamics, and kinetics and protein-ligand interactions. Native MS coupled with ion mobility (IM-MS), which reports on macromolecule shape, is revolutionizing how large conformations of proteins and protein complexes are analyzed and understood. However, existing commercial IM-MS instrumentation is limited by relatively low resolving powers that render their ability to delineate different structures based on differences in their shapes. This proposal describes a program for developing a small, multipass selected overtone mobility spectrometry (M-SOMS) device that can be inserted into commercial platforms commonly used for structural studies. The aim is to improve resolving power in the first year by a factor of 2 to 5 fold; ultimately the resolving power of the M-SOMS device will be tunable, such that high-resolution spectra (having resolving powers that are more than an order of magnitude greater than currently available) will be accessible to any researchers using the commercial platforms. The M-SOMS device will be developed, optimized, and validated using the monomeric and oligomeric ubiquitin system (as well as metallothionein?metal complexes) and applied to tackle larger, more complex protein and protein-ligand systems. Specifically, the high-resolving power will make it possible to discern small conformational differences for the oncoprotein RAS in complex with guanidine nucleotides and analogs, as well as other effector proteins. Lastly, the membrane protein aquaporin, a tetrameric water channel, in complex with lipids will be investigated with the M-SOMS instrument. The latter two studies will represent the first high-resolution mobility study of an intact protein complex. We envisage that M-SOMS will have a significant impact in structural biology and related fields by enabling a number for conformational states to be captured and providing high-resolution mobility restraints for molecular modeling.

? DESCRIPTION (provided by applicant): Recent WHO reports identify Mycobacterium tuberculosis (Mtb) as the single largest cause of death of individuals infected with the Human Immunodeficiency Virus (HIV), a co-infection state highly prevalent in Sub- Saharan Africa. Susceptibility to TB infection appears to be more complex than loss of surveillance through depletion of CD4+ T-cells. In preliminary experiments we show that replication of Mtb in macrophages is enhanced by co-infection with HIV. We propose extensive cellular and molecular analyses of experimental co- infections to be performed at Cornell University, Ithaca, NY. In addition, we have an HIV study cohort at the Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi and will probe the relevance of in vitro phenotypes through analysis of macrophages and lymphocytes from the lungs of HIV-infected individuals. This current study is founded on the hypothesis HIV infection of alveolar macrophages plays a significant role in the increased susceptibility to development of active tuberculosis. Specific Aim #1. How does HIV infection promote the intracellular growth of Mtb? A. What causes the increased permissiveness of the host cell? To elucidate the mechanism(s) by which HIV infection promotes bacterial survival we are targeting the following 3 questions: i. Is the Mtb growth advantage restricted to HIV infected cells? ii. Does HIV infection modulate the microbicidal activities of the macrophage? iii. What HIV ORFs mediate this alteration in the macrophage environment? B. How does Mtb exploit this increased permissiveness? We propose both biased and unbiased genetic screens to identify genes required for enhanced Mtb growth in HIV-infected HMDM. i. Biased approach based on the hypothesis that the altered lipid metabolism favors bacterial replication. ii. Unbiased Transposon-insertion site mapping (TraSH) screen of Mtb in HIV-infected macrophages. Specific Aim #2. How does HIV impact the immune environment of the lung to render it more permissive to infection and/or progression of tuberculosis? We will assess the capacity of HIV to render AM more permissive to infection by Mtb. A. Utilization of HMDMs as surrogates for the development of a Mtb survival assay B. How is the functional phenotype of human alveolar macrophage modulated in the context of HIV infection?

? DESCRIPTION (provided by applicant): Gene therapy based on recombinant adeno-associated virus (rAAV) vectors is showing great clinical promise. Previously, we showed that an intravenous rAAV injection could cause hepatocellular carcinoma (HCC) in newborn mice due to vector integration into and activation of a specific locus on chromosome 12 which we call the AAV-HCC locus in this proposal. Even a single integration event was sufficient to cause HCC in mice. Given that this locus is highly conserved and overexpressed in a subclass of human HCC, these mouse studies raise significant concerns about a possible risk of HCC induction in human gene therapy trials. In order to advance the promising field of liver-directed rAAV therapy, it is important to establish whether rAAV is likely to cause HCC in humans. In this proposal we systematically explore the risk of HCC caused by vector integration at the AAV-HCC locus, using three different animal models to establish the effects of clinically relevant risk factors, as well as vector design on HC induction. In addition, we will assess the risk conferred by random integration of rAAV gene therapy vectors in the liver. Our results will have a significant impact on the clinical practice o liver-directed gene therapy, not only for rAAV vectors, but also for any integrating vector, and may lead to new experimental models of human HCC.

Project Summary Advancements in biophysical techniques, such as X-ray and cryoEM, have undoubtedly accelerated determination of protein structure. However, it still remains challenging to capture snapshots of protein folding intermediates, including non-native states, and breathing motions that protein assemblies undergo to perform their biological function. Moreover, understanding how molecules, such as lipids, modulate protein structure and function is of paramount biological importance. Over the past two decades, mass spectrometry (MS) of intact protein complexes, often referred to as native MS, has emerged as an indispensable biophysical technique whereby non-covalent interactions and protein structure are preserved within the mass spectrometer. Native MS is a rapid and sensitive technique that has already provided invaluable information on subunit stoichiometry and topology, allostery and cooperativity for individual ligand binding events, including their binding thermodynamics. The coupling with ion mobility (IM), a separation technique based on molecule charge and shape, further enhances the capabilities of native MS where it has enabled collision cross section (CCS) measurements for large protein complexes, identification of different conformations for peptides and stabilizing ligands using collision induced unfolding, and insight in folded and denatured structure(s) of proteins. However, low- resolution commercial IM-MS instrumentation has not changed since its introduction 12 years ago. Herein, this proposal seeks to develop transformative native IM-MS technologies with high-resolution IM and MS capabilities that can address modern questions in structural biology, such as conformational dynamics, including those that may have remained ?hidden?, within membrane transporters under turnover conditions. In order to achieve these transformative goals, an interdisciplinary team of researchers whose expertise spans the fields of protein biophysics, expression and purification of proteins inclusive of membrane proteins, as well as traditional protein structure characterization, such as X-ray crystallography, has been assembled. Team members also possess decades of experience in the field of mass spectrometry inclusive of fundamental ion chemistry/physics, seminal contributions that have spawned MS proteomics, and related areas of analytical mass spectrometry and ion mobility- mass spectrometry. Collectively, the background and expertise of this research team is uniquely positioned to transform the field of IM-MS in the area of structural biology. In short, the proposed transformative research will lead to forefront IM-MS instrumentation that is poised to provide unprecedented insights into the structure and assembly of protein complexes and push the field into new frontiers of research.

Our recent advances in the use of fluorescent fitness reporter Mtb strains in the murine TB model has afforded us a unique appreciation of the role of macrophage lineages in both the control and promotion of Mtb growth. Moreover, the extension of these observations through the successful development of Dual RNA-seq protocols for characterization of infected cells isolated directly from the murine lung has provided a further understanding of how host nutritional immunity is central to the control of bacterial growth, at least in early infection. We propose building on our collaboration with Dr. Henry Mwandumba in Malawi and extending these observations through an ex vivo Mtb challenge model with human bronchoalveolar lavage (BAL) cells to functionally phenotype the macrophage lineages present in the human lung airways. Furthermore, we recently demonstrated the persistence of transcriptionally-active HIV-1 genomes in the alveolar macrophages of ART- naïve and ART-suppressed donors in Malawi and believe that we can take advantage of this unique human subjects cohort to characterize the impairment of lung immunity known to occur in people living with HIV-1 that renders them hypersusceptible to both TB and other lower respiratory tract infections. Our hypothesis is that the functional and phenotypic typing of Mtb-infected human lung macrophage subsets from healthy and HIV-1-infected volunteers will generate testable models for immune-mediated control of Mtb growth that will inform future vaccine development programs. Specific Aim 1: Assessment of anti-Mtb immune function in a BAL ex vivo challenge model. This aim will be overseen by Dr. Mwandumba in Malawi. We will use our fluorescent readouts of bacterial fitness to quantify and optimize anti-microbial activities in human BAL cell cultures from HIV-1 uninfected and infected donors challenged with Mtb ex vivo. Specific Aim 2. Utilization of SILAC labeling and single cell RNA-seq to identify soluble modulators of Mtb host macrophage function in HIV-1 negative and HIV-1 positive donors. This aim will be overseen by Dr. Russell at Cornell University on human macrophages and BAL samples from Malawi. We will perform (i) Proteomic analysis of released effector proteins by metabolic labeling studies (SILAC) of secreted proteins from human BAL cells pulsed ex vivo in Malawi. (ii) We will perform single cell (scRNA-seq) RNA-seq analysis of Mtb-challenged BAL cell populations from HIV-1 uninfected and infected donors to identify macrophage- dependent pathways of immune control of Mtb growth and their impairment by HIV-1. Specific Aim 3. The use of Loss of function and Gain of function approaches to assess candidate genes and pathways in successful control of intracellular Mtb infection. We will use siRNA and synthetic mRNA to manipulate host HMDMs and BAL macrophages to validate candidate genes/pathways involved in restriction of bacterial growth, and how it is compromised in the HIV-1 lung environment.