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Optimizing Strongyloides Testing in an At-Risk US Population

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National Institute of Allergy and Infectious Diseases (NIAID)

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Strongyloidiasis, caused by infection with the parasitic nematode Strongyloides stercoralis, remains an important public health problem in tropical and sub-tropical regions, with an estimated 30-100 million infected persons globally. Because S. stercoralis infection is acquired from contact with contaminated soil, those most at risk include the socioeconomically disadvantaged, those with agricultural occupations, and those living in areas with poor sewage control practices. S. stercoralis larval migration during chronic infection is limited to the gastrointestinal tract and lungs and causes few if any symptoms. However, larvae can disseminate and cause severe disease in immunosuppressed individuals. The importance of identifying S. stercoralis infection before immunosuppressive treatments are started has been highlighted by increased use of corticosteroids during the COVID-19 pandemic and related increases in disseminated strongyloidiasis diagnoses. Because disseminated strongyloidiasis is often diagnosed late, after larvae have caused significant and irreversible end-organ damage, the mortality rate of disseminated strongyloidiasis is near 90%. Regional studies and case reports indicate that strongyloidiasis is commonly diagnosed in tropical and sub- tropical regions of the US. We have found 4-10% seropositivity in a Houston solid organ transplant cohort and, more recently, even higher seropositivity (16.5%) in a central Texas community. Other small studies of US immigrants from low- and middle-income countries have indicated infection rates as high as 46.1%. Screening for strongyloidiasis is suboptimal due to poor knowledge of the disease among US healthcare providers and imperfect performance of available diagnostic tests. The clinical gold standard of strongyloidiasis diagnosis is microscopic evaluation of multiple stool specimens for S. stercoralis larva. Unfortunately, it is challenging for patients to submit even one stool specimen in a timely manner, and US clinical laboratory technicians may not have enough training and expertise to reliably identify larvae. Several serologic tests have been developed, but commercially available tests have imperfect test performance and are less sensitive in immunocompromised individuals who may not be able to mount a reliable humoral immune response. Newer serologic and molecular tests based on recombinant antigens have been developed but are not yet commercially available. We propose a prospective study of adult HHS patients undergoing a panel of parasitologic, serologic, and molecular Strongyloides assays, with the following specific aims: (1) to identify a high-performing strongyloidiasis screening strategy for at-risk individuals living in the US and (2) to evaluate each assay as a test-of-cure for strongyloidiasis. Improvement in Strongyloides testing would improve screening and management practices, thereby allowing for timely treatment and reduction of strongyloidiasis related morbidity.

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United States National Institutes of Health

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Targeting Dentate Gyrus to Disrupt Fentanyl-Linked Context Memories

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Addiction is a chronic, relapsing disorder, characterized by persistent drug-taking behavior despite negative consequences. New therapies are needed that prevent relapse.One trigger for relapse is the context where drug use previously occurred. When a rewarding drug is taken in a particular context, a “reward-context memory” can be formed. Reward-context memories are powerful; placing a human or rodent back into a reward-linked context can provoke drug craving. Disruption of reward-context memories is thought to prevent addiction. However, to disrupt reward-context memories, we have to first understand their neural basis.The neural basis of reward-context memories can be informed by literature on fear-context memories. Fear-context memories are reliant on the hippocampal dentate gyrus (DG) and a discrete population of DG context memory cells—termed an “engram”. Inhibition of DG fear-context memory engrams disrupts fear memory recall. It is unknown whether the DG is a site of reward-context engrams or if inhibition of the DG disrupts reward-context memory recall. Stimulation of the DG prior to context-fear memory encoding enhances fear-context memory. It is unknown if stimulation of the DG prior to reward-context memory enhances reward-context memory recall. My objective is to assess if DG reward-context memories can be manipulated, with the long-term goal of diminishing the salience of context cues and preventing relapse. The Aims of this proposal are to 1) test if DG neurons active during reward-context memory encoding can be inhibited during re-exposure to the reward-context, reducing or inhibiting drug-linked context memory recall and 2) determine if stimulation of DG neurons during reward-context memory encoding improves drug-linked context memory recall. When completed in two years, these experiments will determine whether or not the DG can serve a novel therapeutic target for diminishing the salience of drug-context memories. Further, regardless of experimental outcome, we will be able to elucidate novel properties of reward linked memories. Starting my postdoc during Covid-19 and moving into a new field (from context fear memory to addiction), it took time for me to establish a baseline knowledge and generate pilot data in order to develop these aims. Through this fellowship, I will accomplish defined Training Goals: learn hippocampal circuit manipulation, develop expertise in the field of substance abuse in the context of disordered learning/memory, use machine learning for the characterization for reward behaviors, and gain further professional development skills including project and personnel management. I have a supportive and ideal mentoring team (Drs. Amelia Eisch and Julie Blendy) to guide my training of both conceptual and technical skills. At the Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, I have exceptional resources and a stimulating environment in which to accomplish these goals.The completion of these Aims, and the training provided within, will provide the foundation for my path to becoming an independent investigator at an academic institution.

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designer receptors exclusively activated by designer drugs

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The Biospecimen Analysis Core (BAC) of our Vanderbilt-coordinated human Virome Characterization Center (V2C2) is designed to enable the composition and complexity of a host-contextualized human virome (replicating and integrated) to be rapidly and cost-effectively generated with high-quality across multiple tissue types. In Aim 1, the BAC will receive and characterize the virome across a range of biospecimen types from our Biospecimen Collection Core (BCC). This process will include viral sequencing (whole metagenomics/metatranscriptomic sequencing), validation studies (digital PCR and capture-based methods, among them), and host response characterization, including tropism studies (via bulk and single-cell RNA-seq approaches, in vitro infection studies) and assessments of host inflammatory state (targeted proteomics). In Aim 2, the BAC will establish standard operating procedures for metadata, analytics, and quality control and assessment, including consortium-wide benchmarking and reproducibility of critical assays to investigate and limit variability from the collection point to the final analysis. The BAC will synergize with all other V2C2 cores via (1) scientific counsel to the administrative core and the wider HVP on the development of new technologies based on HVP results to enable viral screening at the epidemiologic scale; (2) enabling collaborative projects with other HVP efforts (e.g., tropism studies with Functional Interactions projects; RFA-RM-23-017), augmenting essential human translational biology in the HVP. Our BCC has several fundamental strengths that ensure success, including: (1) leadership structure with extensive expertise in large epidemiologic efforts: Dr. Simon Mallal (a renowned physician-scientist who works on viral-host co-evolution and genetic interactions, Director of the Vanderbilt VANTAGE sequencing core), Dr. Jane Freedman (a leader in sequencing and proteomics and large NIH studies, including Common Fund efforts), and Dr. Suman Das (a human molecular virologist with extensive experience in methods development and discovery in human virology), all of whom have a track-record of collaboration in large NIH-funded human studies; (2) deep expertise in viral sequencing: Dr. Das and Mallal have sequenced many classes of human viruses (e.g., influenza, RSV, HIV, HCV, HBV, EBV, enteroviruses, rotavirus, norovirus, Ebola, Zika, coronaviruses, resulting in >400K Genbank accessions); (3) expertise in methods development in metagenomics: in this process, they have developed many new laboratory and computational methods enabling capture of the human virome (replicating eukaryotic RNA/DNA viruses and phages) and host integration; (5) rapid assessment of host response characteristics (including mucosal immunity, relation with other commensal microbial flora, and emerging types for characterization [e.g., extracellular vesicles, platelets, proteomics]); (6) large, NIH-funded core laboratory structures that enable cost-efficient BAC conduct (e.g., NHLBI TOPMed 2.0 Core; Vanderbilt resources led PD Mallal/PI Das). Successful completion will provide HVP with a comprehensive host-viral characterization for consortium-wide integration.

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National Heart  Lung  and Blood Institute

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United States National Institutes of Health

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Environmental mixtures, immune function, and vaccine antibody response in children

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National Institute of Environmental Health Sciences (NIEHS)

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I am an environmental epidemiologist and exposure scientist by training with a primary research interest in the impact of environmental exposures on immune function and vaccine response – a critical public health issue highlighted by COVID-19. The goal of this proposal is to obtain training and acquire the skills necessary to become an independent investigator at the air pollution-immunology-infectious disease nexus. I have assembled a world-class mentoring team with interdisciplinary expertise in immunology, toxicology, biostatistics, data science, exposure science, exposomics, and epidemiology. The proposed training plan includes formal coursework, field work, clinical observations, laboratory rotations, academic meetings and conferences, and a variety of activities designed for leadership and professional development. I am confident that by the end of the K99 phase of this proposal, my existing expertise combined with the training proposed in this project will allow me to establish myself as an independent investigator in a tenure-track faculty position, with the skillset to build my future independent research program integrating exposure science, epidemiology, and infectious diseases. For this proposal, I will leverage the Programming Research in Growth, Environment and Social Stress (PROGRESS) cohort, an ongoing longitudinal pregnancy/birth cohort based in Mexico City to investigate the impact of ambient environmental pollutants on the antibody response to routine vaccinations. Specifically, I will: 1) build a daily ozone model for Mexico City, which will be integrated with our existing models for fine particulate matter, nitrogen dioxide, and temperature that already exists for the area; 2) assess the association between these ambient environmental pollutants with vaccine antibody levels to routine vaccinations; 3) assess the association between the same pollutants and levels of several specific cell types involved in vaccine response; and 4) explore whether the associations between ambient environmental pollutants and vaccine antibody levels are partially or fully mediated through the immune cell population subsets assessed in #3). This research proposal has several innovations, including the generation of new exposure data (the first of its kind in Mexico City), the use of cutting-edge statistical approaches, and its leverage of both extant data and new measurements that addresses a unique research question while maximizing cost effectiveness. If successful, this work will be able to significantly foster interdisciplinary research and advance our understanding of a critical and urgent public health issue.

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Human Virome Characterization Center for the Oral-Gut-Brain Axis

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Administrative Core - Abstract Knowledge of the bacteria living within our body has grown rapidly over the past decade, spurred in large part by initiatives such as the NIH Human Microbiome Project (HMP). Such collaborative efforts have redefined our understanding of the human bacteriome, opening new avenues of research in the biomedical and behavioral sciences and improving healthcare delivery. In contrast, although viruses are thought to outnumber bacteria 10:1, the human virome remains largely unexplored. The recent coronavirus disease 2019 (COVID-19) pandemic and emerging threats from pathogenic viruses have sounded the collective global alarm regarding our limited knowledge of potential pathogens within the human virome. Moreover, foundational knowledge of what constitutes a healthy human virome and how it affects human health is lacking, representing a significant knowledge gap. The NIH Human Virome Program (HVP) is a grand undertaking that aims to fill this gap by defining all facets of the human virome at different body sites across the life/health span in a broad demographic range of individuals. This program offers a valuable opportunity to advance our understanding of the full spectrum of “healthy” viromes, thereby promoting advances in biomedical research, enabling identification of perturbations associated with disease, and improving our capacity to detect viruses that pose threats to human populations. The proposed Human Virome Characterization Center (HVCC) embraces this opportunity to define the human virome in the oral–gut–brain axis— a recognized functional unit that broadly impacts human health—across the lifespan in diverse cohorts. As the oropharyngeal site is a well-recognized key entry point for pathogenic viruses, defining the healthy virome of the oral–gut–brain axis will help inform a key inflection point for derailing health. Our research and management teams include a multi-PI (MPI) leadership group and five complementary cores: i) Administrative; ii) Biospecimen Collection; iii) Biospecimen Analysis; iv) Data Analysis and Submission; and v) Ethical, Legal, and Social Implications (ELSI). Working together, we will use cutting-edge technologies and bioinformatic tools to elucidate the viromes of unique human oral–gut–brain specimens across the US and broadly share results of these efforts with the public and scientific community. The Administrative Core will organize, lead, and provide monitoring oversight of the HVCC to generate and validate virome sequencing, clinical, and demographic data to define the human virome of the oral–gut–brain axis of diverse populations along the lifespan and health span. The Administrative core will do so by integrating and sharing ideas across our Cores via dynamic, contemporary communication methods; promote refinement and dissemination of best practices between our groups and the scientific community through multiple venues; and evaluate effectiveness of our methods and the HVP initiative. Through these efforts, the Administrative Core will be the central coalescing force propelling our HVCC toward completion of its aims, thereby delivering on the HVP goals and realizing the NIH’s broad vision for the program.

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National Institute of Dental and Craniofacial Research

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United States National Institutes of Health

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BATF as an inducible regulator of natural killer cell expansion and effector function

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National Institute of Allergy and Infectious Diseases (NIAID)

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Project Summary: Natural killer (NK) cells exist in a chronically “primed” state that facilitates rapid cytokine production, chemokine-mediated dendritic cell and macrophage recruitment, and direct target elimination. These cells play a pivotal role in protection against -herpesviruses and coronaviruses, and in the immune response against metastatic tumors and leukemia. Emerging evidence supports an important role for NK cells in the initiation of the immune response against some solid tumors. While NK cell survival and function can be suppressed in the tumor microenvironment, these cells can be restored by environmental manipulation or checkpoint blockade therapy. These findings raise the possibility that NK cells, if manipulated appropriately, could have therapeutic benefits in multiple disease contexts including in solid tumors. Therefore, a comprehensive understanding of the mechanisms controlling NK cell survival, proliferation, and effector function in different disease contexts is needed. In this grant, we present preliminary data identifying BATF as a transcription factor induced by the proinflammatory cytokine IL-12, which is responsible for the priming of NK cells during their development and for NK cell expansion in response to viral infection. We will test the consequences of altered BATF expression, both loss and gain of function, on NK cell maturation and anti-tumor immunity (Aim 1) and the NK cell response to viral infection (Aim 2). We propose a comprehensive characterization of BATF target genes and the impact of BATF on chromatin accessibility, histone modification, and functional output. Taken together, these studies will provide a broad view of the requirements for BATF and the potential for modulating BATF or its target genes for therapeutic intervention in anti-viral and anti-tumor immune responses.

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RNA epigenetic modifications in SARS-CoV-2

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National Institute of Allergy and Infectious Diseases (NIAID)

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Post-transcriptional RNA modifications are widespread and regulates numerous biological processes including RNA metabolism, protein translation, gene expression, and disease. Among the more than 180 types of RNA modifications, N6-methyladenosine (m6A) and pseudouridine (Ψ) are the two most prevalent. The m6A modification is catalyzed by the host RNA methyltransferase complex of METTL3 and METTL14. The Ψ modification is converted from the nucleoside uridine (U) by the host pseudouridine synthases (PUSs). Despite being discovered in the 1950s, the biological functions of the m6A and Ψ modifications in the context of virus infection remain poorly understood. This project is built upon our recent development of high throughput sequencing techniques that have enabled mapping of m6A and ψ sites at a single base resolution. Using these techniques, we discovered that SARS-CoV-2 RNA isolated from well-differentiated primary human bronchial epithelial (HBE) cultures that include their in vivo target cells is heavily modified with m6A and ψ. In addition, we have found that depletion of several m6A and ψ writer proteins decreases SARS-CoV-2 replication in HBE culture. These findings led to our hypothesis that SARS-CoV-2 acquires m6A and Ψ modifications in its RNA to maximize virus replication. Thus, the goal of this project is to determine the mechanisms by which RNA m6A and ψ modifications modulate SARS-CoV-2 replication, gene expression, innate and adaptive immunity, and pathogenesis. In Aim 1, we will use a CRISP-Cas 9 technique to knock out host RNA m6A methyltransferases and PUSs in HBE cultures to determine the role(s) of m6A and Ψ modifications in the SARS-CoV-2 life cycle. We will also use knockout mice to examine the role(s) of m6A and Ψ modification in SARS-CoV-2 replication in vivo. We will also identify the specific PUS enzyme(s) that catalyze pseudouridylation on SARS-CoV-2 RNA. In Aim 2, we will mutate the m6A and/or ψ sites in the SARS-CoV-2 genomic RNA and use the reverse genetics system to generate recombinant SARS-CoV-2 lacking m6A and/or ψ modification sites and use them to determine the roles of m6A and ψ modifications on viral RNA metabolism, encapsidation, RNA replication, viral protein translation, and innate immunity. In Aim 3, we will determine whether m6A and ψ modifications modulate mucosal and adaptive immune responses of SARS-CoV-2 live attenuated vaccines (LAVs) and determine whether LAVs lacking m6A and/or ψ are more immunogenic in golden Syrian hamsters. Upon completion of this project, we expect to have unravelled the mechanisms by which m6A and Ψ modifications modulate the SARS- CoV-2 replication cycle, leading to the development of novel and improved LAVs and therapies for COVID-19 that target these RNA modifications.

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Synergistic Nanobodies for Pandemic Preparedness

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National Institute of Allergy and Infectious Diseases (NIAID)

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Betacoronaviruses (beta-CoVs), including SARS-CoV-1, MERS-CoV, and SARS-CoV-2, have reshaped our understanding of pandemic preparedness. These viruses demonstrate a remarkable ability to mutate and evade defenses, continuing to infect populations worldwide despite extensive vaccination efforts and antiviral therapies. The chameleon-like nature of SARS-CoV-2, particularly its modifications to the Spike protein, consistently outpaces existing countermeasures, necessitating new strategies. This proposal introduces a pioneering class of nanobodies (Nbs), engineered from the immune system of llamas, designed to provide comprehensive protection against all beta-CoVs. These biologics not only advance treatment but also signify a pivotal step in pandemic preparedness, equipping us to outpace the relentless evolution of beta-CoVs. Our innovation lies in developing multivalent, synergistic combinations of broad-spectrum, high-efficacy Nbs. By harnessing these combinations, we amplify their efficacy and scope, concurrently increasing their resistance to viral mutations. Administered intranasally or directly to the lungs, these Nbs serve as both prophylactics and therapeutic agents. Our first Aim is to strategically expand upon our proven repertoires to identify, isolate, and characterize a much larger and more diverse repertoire of Nbs that collectively are strongly neutralizing across the beta-CoVs. We will use cutting-edge methods to produce diverse Nbs from llamas exposed to spike proteins of various beta- CoVs, selecting those with high affinity, specificity, and stability. We aim to discover synergistic, escape-resistant Nb pairs through combination testing and structural analysis. In our second Aim, we will optimize critical parameters important for developing broadly neutralizing Nb combinations and derivatives for human use. We will evaluate the in vivo synergistic potential of Nbs targeting major threats like MERS-CoV and SARS-CoV-2, and engineer Nbs to optimize their properties and efficacy in preparation for clinical trials. Deploying these pre- programmed Nbs at an outbreak's onset will protect first responders and medical personnel, reduce hospital surges, limit transmission, and buy time for new vaccine development and rollout. They will also provide crucial support to immunocompromised individuals, safeguarding the most vulnerable from the start. We hypothesize that our synergistic Nb combinations will introduce new beta-CoV neutralization methods, effectively prevent and treat infections, and maintain efficacy against emerging beta-CoV threats.

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Middle East Respiratory Syndrome Coronavirus

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Harnessing smartphones for real-time detection of affective disturbance and future depression risk in adolescents

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National Institute of Mental Health (NIMH)

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Negative affective (NA) states (e.g., high sadness, anger, and anxiety) increase substantially during adolescence, which may heighten risk for the onset of affective disorders, in particular major depressive disorder (MDD), which surges during the adolescent years. Over the past decade, affective disturbances and MDD have been rising in adolescents, and the COVID-19 pandemic has only exacerbated this alarming trend. As a result, the Surgeon General and national pediatric organizations (American Academy of Pediatrics, American Academy of Child and Adolescent Psychiatry, and Children’s Hospital Association) recently declared a national state of emergency for youth mental health. Accordingly, there is an acute need to develop personalized data-driven approaches to predict and ultimately interrupt states of markedly high NA as they occur in the daily lives of teens. In addition to the immediate benefits of alleviating acute states of affective distress, reducing the frequency and duration of episodes of high NA may serve to reduce the risk of depression onset in youth. Relevant in this context, the majority (88%) of U.S. teens own a smartphone, which can continuously and unobtrusively measure behaviors predictive of affective disturbance, including activity levels, location, phone use, sleep, and proxies of social interaction. In addition, smartphone data may also predict risk of MDD onset. The ability to prospectively predict MDD prior to its onset would have important clinical implications for the early identification of – and targeted deployment of interventions for – at-risk youth, which is strongly aligned with the NIMH Strategic Plan.  To address these gaps, adolescents ages 12-16 (the age range corresponding to the largest developmental increase in depression) will complete repeated ecological momentary assessment (EMA) surveys of NA (i.e., assessing different negative emotional states) over 30 days. During this period, smartphone sensors and a wrist-worn actigraphy band will collect data on activity levels, location, phone/screen use, calls/texts and estimates of relevant sleep variables (e.g., sleep onset, offset, and duration). The project has two aims. First, a personalized machine learning approach recently developed by the study team will test the accuracy of predicting states of high NA in the daily lives of teens from these passively derived features (Aim 1). The ability to accurately predict states of high NA at the individual level could ultimately inform the development of highly scalable and personalized smartphone-delivered interventions matched to the current affective state (e.g., high sadness vs. anger) of a given teen. Second, during a follow-up phase, participants will be contacted every 6 months to assess changes in symptoms, along with bursts of passive sensor data collection and EMA. Machine learning analyses will test whether passive data, in combination with affect dynamics, predict subject-specific risk of future depression onset with sufficiently high sensitivity and specificity to be clinically useful (Aim 2). To the extent that a data-driven approach could be developed to predict individual risk of future depression onset, it could ultimately inform the development and delivery of individualized, targeted, and timely prevention efforts.

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single episode major depressive disorder

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Mitochondrial deubiquitinase USP30 regulates cell metabolism-mediated miRNA biogenesis and microvascular inflammation

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National Heart Lung and Blood Institute (NHLBI)

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Acute inflammatory diseases are life-threatening health conditions that are most often caused by bacterial or viral infection such as Pseudomonas aeruginosa- or SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) and sepsis. ARDS and sepsis-related mortality remains at unexpectedly high levels due to lack of effective pharmacotherapies. Hence, a new therapeutic strategy for ARDS and sepsis is needed. Microvascular inflammation and barrier disruption play critical roles in the pathogenesis of acute inflammatory diseases. A mitochondrial de-ubiquitinating enzyme, USP30, plays a vital role in regulation of mitochondrial outer membrane protein homeostasis. USP30 has been considered as a potential target for treating Parkinson’s disease and cancers; however, the role of USP30 in microvascular endothelial cells (ECs) and acute inflammatory diseases has not been reported. In our preliminary data, we discovered that inhibiting USP30 diminished EC dysfunction and reduced the severity of experimental lung injury. Mechanistically, we discovered a non-canonical pathway of USP30 that links MAT2A stability, the S- adenosylmethionine (SAM) cycle, DNA methylation, miRNA-30a-5p synthesis. Based on our comprehensive preliminary data, we hypothesize that inhibiting USP30 preserves EC function by modulating intracellular signaling cascades implicated in MAT2A stability, SAM production, DNA methylation, and miR-30a-5p expression. We propose testing the hypothesis with the following Specific Aims: Aim 1 is to determine if USP30 in the endothelium is a potential target for treatment of acute lung injury. We will determine if inhibiting EC USP30 diminishes leukocyte cell adhesion to EC and transendothelial migration and preserves EC barrier integrity. Further, we will determine if depletion of USP30 in endothelial cells reduces severity of pseudomonas aeruginosa- or sepsis-induced experimental lung injury. Aim 2 is to determine the molecular mechanisms by which inhibiting USP30 destabilizes MAT2A, decreases the SAM production, and increases miR-30a-5p. We will determine if inhibiting USP30- induced miR-30a-5p occurs through modulation of MAT2A stability in the SAM cycle, reduction of SAM production and pri-miR-30 promoter methylation. Further, we will determine if miR-30a-5p regulates USP30 inhibition-mediated MDM2, MLC, and NFAT5 downregulation. Aim 3 is to determine if the protective effect of USP30 inhibition occurs through modulating miR-30a-5p expression. We will determine if USP30 inhibition preserves EC function by modulating miR-30a-5p expression in HLMVECs. Further, we will determine if EC USP30 depletion reduces severity of experimental lung injury through modulating miR- 30a-5p expression by using a cutting-edge RNA nanoparticle technology. Comprehensive understanding of mitochondrial de-ubiquitinating enzyme inhibition-induced changes of cell metabolisms and EC function is important for development of new therapeutic targets for treatment of acute inflammatory diseases.