Location: Animal Parasitic Diseases Laboratory
Project Number: 8042-31000-107-03-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2013
End Date: Aug 31, 2017
There are 3 main objectives on this project: Objective 1: Use a comparative and evolutionary genomic approach to identify and characterize the pannematode secretome. Objective 2: Prioritize the nematode proteins with immunogenic potential for experimental screening. Objective 3: Conduct an experimental screen to confirm the allergy suppressive candidates.
1) The pan-nematode secretome will be characterized using computer predictions of secreted nematode peptides (based on signal peptide for secretion, non-classical secretion, and proteomics data) followed by homologous groupings using Markov Clustering. To identify the nematode proteins that are immune modulators, computer predictions will be performed from primary sequence- and structural protein-derived searches against public databases and support vector machine-derived searches to identify novel allergens. B) To characterize the nematode immune modulators, available and upcoming gene/protein expression data will be used to generate expression profiles for all gene products with immune modulating properties over the life cycles of the key parasites that represent different trophic ecologies and span the phylum (hookworms, ascarids, and whipworms). 2) Proteins with immune modulating properties will be scored based on their similarity to known immuogens, presence of common allergenic motifs, pan-species conservation of orthologous protein groups, exposed hydrophobic regions, gene/protein expression in larval stages (especially lung stage), and physical properties of intact proteins to predict allergenicity or lack thereof. Based on the primary sequence physical properties will be calculated, such as isoelectric point (pI), net charge, molecular weight (MW) and electrostatic potential (EP). The orthologous groups will be classified by the Cooperator into allergenic or non-allergenic proteins, and proteins known to bind IgE. This information will be publically available at Nematode.net via a query engine constructed over a relational database built on MySQL. Dissemination of this information will enable the scientific community to expeditiously development of new vaccine targets. 3) The prioritized parasite-derived immune modulating products will be experimentally screened by ARS scientists in a two-step approach. A primary (high throughput) molecular screen will use pig and sheep explanted and cell-line alveolar macrophages. The parasite products that will either directly induce changes in asthma/allergy-related genes or alter the level of cytokines will be subjected to secondary experimental screen on intestinal epithelium. In vivo vaccine efficacy will be tested in mice followed by pigs and sheep. This information will be used by both ARS and the Cooperators to identify, test, and develop novel vaccine candidates against gastrointetinal nematode parasites. 1. A primary (high throughput) real-time PCR and secondary deep sequencing screen will use pig and mouse explanted and cell line alveolar macrophages to evaluate for parasite-derived immune modulating products. (ARS) 2. A second in vivo screen will use mice, pigs, and sheep. (ARS) 3. Comparative and evolutionary genomic approach to identify and characterize the pannematode secretome. (Cooperator) 2. Prioritization of the nematode proteins with vaccine potential for experimental screening. (Cooperator)