Michael J. Kimber, PhD - US grants

DESCRIPTION (provided by applicant): Neglected Tropical Diseases (NTDs) caused by helminths (nematodes and flatworms) perpetuate socioeconomic instability in profoundly impoverished developing countries, inflicting crippling morbidity and/or significant mortality. The prevalence of helminth disease is staggering. Lymphatic filariasis is caused by nematodes including Brugia malayi and afflicts over 120 million people worldwide with over 1.2 billion at risk in 81 disease endemic countries. Schistosomes, the etiological agents of schistosomiasis, are the most pathogenic flatworms, infecting over 200 million with more than 650 million at risk. The lynchpin of NTD control efforts for the foreseeable future will remain the administration of anthelmintic drugs - but worryingly few effective drugs exist, hampering these control strategies. Thus there is a pressing need for new, more effective chemotherapies. Compounding the problem is the reticence of the pharmaceutical industry to engage this need for NTD drugs without the motivation of a financial incentive. This application aims to address this problem by proposing a paradigm shift in the way we discover and develop drugs for NTDs. We propose an innovative Public-Private Partnership between academic laboratories at Iowa State University and McGill University, and Pfizer, Inc. The academic laboratories will invest their time and resources in the molecular identification and validation of potential novel drug targets. Pfizer Animal Health (PAH) will advance selected validated lead targets for mechanism-based screening. The lead targets forming the substrate of this proposal are G protein-coupled acetylcholine receptors and acetylcholine-gated chloride channels in the parasitic nematode B. malayi and the parasitic flatworm Schistosoma mansoni. The first phase of the project will take place in the academic laboratories and will show that drugs acting on these receptors have potential as novel anthelmintics. The experiments will combine bioimaging techniques, RNA interference (RNAi), bioassays, physiology and pharmacology to determine which of these receptors will progress to the next phase of the project at PAH. Here selected receptors will be screened, at PAH expense, against a vast compound library amongst which, we believe, will be small molecules with activity at our receptors. The promise of these active compounds as antiparasitic drugs will be explored both in the academic laboratories and in-house at PAH, subject to material transfers agreement and compound availability. Successful completion of this project will have a truly significant impact on the development of new anthelmintic drugs and has the potential to positively impact the health of hundreds of millions of people worldwide. Further, we will greatly advance our understanding of basic worm biology and address some important knowledge gaps in our understanding of nematode and flatworm physiology and function. PUBLIC HEALTH RELEVANCE: This project forms an innovative Public-Private Partnership to accelerate the discovery of new compounds to treat devastating parasitic worm infections. Academic laboratories will identify and describe potential drug targets in the worms, and Pfizer Animal Health will screen vast libraries of compounds across these targets.

? DESCRIPTION (provided by applicant): Lymphatic filariasis (LF) is a neglected tropical disease caused by parasitic nematodes, including Brugia malayi, which are transmitted through the bite of infected vector mosquitoes. The prevalence of LF is staggering; over 120 million people are infected, with over 1.2 billion at risk in 73 endemic countries. Despite coordinated elimination efforts, LF remains a significant global health concern and there is a recognized need for new strategies to control parasitic nematodes. Progress towards that goal is frustrated, however, because we simply do not have adequate understanding of parasite biology. For example, the interaction between mosquito and parasite during the B. malayi life cycle is delicately balanced and although the parasite manipulates the mosquito to create conditions favorable to parasitism, we know little about how they do this. Host-parasite interactions have long been promoted as novel intervention sites because any mechanism that disrupts the vector-parasite interaction and skews the balance in favor of the vector is likely to prevent infection, parasite development or transmission. This project represents an exciting new direction for investigating the vector-parasite interface. We propose that B. malayi can actively manipulate the mosquito host to the benefit of the parasite by using small, regulatory RNAs (microRNAs, miRNA) delivered via specialized packages called exosomes. In Specific Aim 1 of this application we will characterize exosome secretion by Brugia within the mosquito host and identify exosome cargo. Exosomes will be collected from mosquito-borne stage parasites and visualized, quantified and sized using electron microscopy and NanoSight tracking analysis. We will use deep sequencing techniques to characterize the small RNA cargo of the exosomes. Finally, we will use biomarkers and bioinformatics to reveal profile exosome secretion throughout complete parasite development in the mosquito vector. In Specific Aim 2 we will explore the function of these exosomes at the vector-parasite interface. We will inject mosquitoes with purified exosomes and discrete small RNA cargo, then monitor changes to normal vector biology using deep sequencing techniques. This will identify both novel and explicit parasite effector molecules and the vector pathways that are manipulated. Successful completion of this project will generate new knowledge of B. malayi biology fundamental to parasitism. Furthermore, the mechanisms exposed may apply to other parasitic nematodes as they manipulate their hosts, be they vectors, humans, livestock or indeed plants.

Lymphatic filariasis (LF) is a mosquito-borne Neglected Tropical Disease caused by filarial worms including Brugia malayi; over 120 million people worldwide are infected, with over 1.4 billion at risk in 70 endemic countries. Current control strategies employing mass drug administration have reduced prevalence in many areas, but LF remains a significant global health concern. There is a recognized need for new strategies to control LF and other diseases caused by parasitic nematodes. This project focuses on the host-parasite interface during parasite infection, development and persistence of LF and represents an exciting new direction for investigating this field. We propose a novel mechanism by which B. malayi modulates the host immune system, through small, regulatory RNAs and proteins delivered via a specific type of extracellular vesicle called exosomes. Our preliminary data demonstrate that infective stage larvae of B. malayi secrete exosomes, that these exosomes contain a diverse miRNA and protein cargo and that distinct parasite miRNA potentially target host genes. Further, these exosomes are internalized by host macrophages and elicit a specific modulatory phenotype. The overall goals of this proposal are to define the cargo of parasite exosomes secreted across the intra-mammalian Brugia life cycle and probe the mechanistic basis for their bioactivity. In Specific Aim 1, we propose to profile the small RNA and protein cargo of secreted exosomes across the intra-mammalian B. malayi life cycle using a combination of small RNA deep-sequencing (RNA-Seq) and proteomic profiling to identify the molecular mediators of host manipulation delivered by parasite exosomes. In Specific Aim 2, we will define the mechanisms of exosome bioactivity on cellular mediators of the host immune response. We will examine uptake of parasite exosomes by host macrophages to reveal how these vesicles are internalized by host cells, then leverage the genetic capacity of the murine model by using genetic knock outs to generate mechanistic insight into the modulatory phenotype elicited in host macrophages by exosome internalization. The long-term impact of the project will be new knowledge of B. malayi biology and the exposure of new molecules that may be exploited in novel LF control strategies. Further, the mechanisms we describe here may be conserved across animal, human and plant parasitic nematodes and could be utilized for broad-spectrum control applications.

Liquid biopsy has significant advantages over traditional tumor biopsies, because it is minimally invasive and uses biofluids, such as blood and urine, to diagnose cancer and other diseases in their early stages. Exosomes, which are actively secreted from cancer cells, carry molecular constituents of their originating cells. Because these membranous extracellular vesicles can serve as cellular surrogates, exosomes have emerged as a new type of potent biomarkers. However, conventional exosome analysis methods such as immunoblotting or enzyme-linked immunosorbent assays are costly and require approximately twelve hours and excessive volumes of serum to detect transmembrane proteins on the surface of exosomes. Exosome separation requires complex steps to remove debris or cellular components that will confound downstream analysis. High-throughput molecular profiling of exosomes using miniature label-free biosensors is not available. The goal of this project is to develop a new capability to rapidly screen and profile exosomes based on both molecular and size characteristics. This research will lead to a transformative change in exosome analysis by integrating two state-of-the-art technologies on a single silicon chip. In addition, this research will be integrated with education through adding new lab modules to existing undergraduate biomedical engineering minor program curriculum, recruiting female students, and providing summer internship opportunities to African-American students to participate in the project at Iowa State University, and developing a new undergraduate-level course related to nanobiotechnology at Arizona State University.

The project will lead to an integrated silicon-based nano-opto-fluidic platform for rapidly and continuously profiling of both molecular and size features of exosomes. Cascaded nanoscale deterministic lateral displacement pillar arrays will be developed to simplify the isolation and size profiling of exosomes. The exosomes will be effectively separated from interference molecules present in the fluid sample. High-performance lateral flow-through optical biosensors will be developed to quantify the separated exosomes. The exosome samples can flow through the nanoscale biosensor and be immobilized and enriched on the functionalized sensor surface. Because both the separation and detection modules have the features of lateral flow designs, they can be integrated on a single silicon chip using the nanoimprint lithography process. The integration of these two functions will lead to an unprecedented ability to continuously streamline exosome separation, enrichment and detection processes to profile multi-dimensional molecular and size information for multiple protein markers within one hour. The biological validation plan of the project will be carried out using the proposed device to sort and sense exosomes released from a parasitic nematode and etiological agent of the human disease, Lymphatic Filariasis. The proposed technology is advantageous over the lab-based methods in terms of cost, sample consumption, and throughput, and could be extended to the profiling of circulating exocellular exosomes in human or animal biofluids to diagnose a variety of diseases, identify companion biomarkers that are important for drug discovery, and monitor the progress of a therapy.