Reagents for Immunologic Analysis of Non-mammalian and Underrepresented Mammalian Models

Fast-Track proposals will be accepted. Direct-to-phase II proposals will be accepted. Number of anticipated awards: 3-6 Budget (total costs): Phase I: $300,000/year for up to 2 years; Phase II: $1,500,000 with appropriate justification by the applicant for up to 3 years. Page 112 Background This program addresses the limited availability of reagents (e.g., antibodies, proteins, ligands) for the identification and discrimination of immune cells and the characterization of immune responses in non-mammalian models or in specific underrepresented mammalian models. Non-mammalian and under-represented mammalian models of interest include: arthropods, amphibians, fish (e.g., jawless fish, sharks, zebrafish), nematodes, marine echinoids; and guinea pig, ferret, bat, mink, hamster, bird, cotton rat, pig (including minipigs), cat, rabbit and marmoset, respectively. Non-mammalian models are easily tractable model systems to study basic, conserved immune defense pathways and mechanisms. For example, characterization of the Drosophila Toll signaling pathway facilitated the discovery of mammalian Toll-Like Receptors (TLR), which significantly accelerated progress made in the field of innate immunity. Non-mammalian models can be much more easily adapted to high-throughput screening formats than mammalian organisms. Caenorhabditis elegans has been used for whole-organism, high-throughput screening assays to identify developmental and immune response genes, as well as for drug screening. Many non-mammalian species are natural hosts for human pathogens and share many conserved innate immune pathways with humans, such as the NF- B pathway in mosquitoes, the intermediate hosts for Plasmodia parasites. However, studies to better understand immune regulation within non-mammalian models have been constrained by the limited availability of antibodies and other immune-based reagents for use in scientific studies. Certain mammalian models display many features of human immunity but are similarly underutilized due to the limitations noted above. For example, the progression of disease that follows infection of guinea pigs with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), displays many features of human TB. While this model has been used for more than 100 years as a research tool to understand and describe disease mechanisms, immunologic analyses are constrained by the limited availability of immunological reagents specific for the guinea pig. Another example is the ferret model, one of the best animal models of human influenza infection, where immunologic studies also have been limited by the lack of immunological reagents. In addition, minks and cats are highly susceptible to SARS-CoV-2 infection with potential for zoonotic pathogen transmission. However, there are almost no reagents available for immunological studies in these species. Lastly, although bats are the natural reservoir and vector for several major zoonotic diseases that cause severe human diseases, the lack of reagents has impeded studies of how bat adaptive or innate immune responses control these pathogens. Project Goal This program supports the development and validation of reliable antibodies and reagents for the identification and tracking of immune cells or the analysis of immune function/responses (e.g., cytokines, chemokines, intracellular signaling) in nonmammalian models or underrepresented mammalian models. Non-mammalian models are limited to arthropods, amphibians, fish (e.g., jawless fish, sharks, zebrafish), nematodes, and marine echinoids. Underrepresented mammalian models are limited to guinea pig, ferret, bat, mink, hamster, cotton rat, pig (including minipigs), cat, rabbit and marmoset. Phase I Activities must include the following activities: Selection of targets, which may include: immune cell markers; receptors with immune function; or other molecules important for immune function; Development of antibodies or other reagents against these targets, o If polyclonal antibodies are being developed, the plan also must include the development of monoclonal antibodies; Characterization of antibodies or reagents developed, o Initial determination of affinity/avidity and specificity o Confirmation of binding to the intended native antigen/immunogen by flow cytometry and/or other assays. Phase II Activities must include but are not limited to: Comprehensive evaluation of specificity and sensitivity, functional utility, and cross-reactivity/off-target binding of antibodies/reagents by Western blotting (denatured and native protein); immunoprecipitation; immunohistochemistry; and flow cytometry. o The functional studies must minimally include analysis of primary cells or target antigens from host species; o The off-target binding must minimally include evaluation of non-specific binding to other cells or unrelated molecules from host species; o The cross-reactivity must at least contain a screening of binding to related cells or molecules from other species. Optimization (e.g., secondary modifications/conjugations) of the antibodies/reagents for the intended application. Development of well-established protocols by: o setting up working concentrations, assay linearity, assay validation, and functional activity test; Page 113 o determining quantitative criteria such as binding kinetics and signal versus background, and quality control acceptance criteria (e.g., purity requirement, endotoxin testing, specificity and activity(titer) threshold, molecular mass confirmation, multiple freeze-thaw stability) in assay performance and manufacturing. Scale-up production of the reagents, batch to batch comparison, and backup plan(s) to guard against loss of source material (e.g., hybridoma cells). A commercialization plan for distribution and marketing of the reagents. This SBIR will not support: Identification of immune target molecules and development of antibodies/reagents against immune markers or molecules for animal models not listed in the solicitation; Development of antibodies/reagents for molecules or mechanisms not involved in immune responses; Development of novel or refined animal models