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To evaluate potential modulators of innate immunity and select novel therapeutic adjuvants for their ability to optimize vaccines for avian health.
Approach: In vitro and in vivo assays developed in Dr. Koguts laboratory (Kogut et al., 2003, 2005) will be conducted to evaluate modulators of innate immunity, which will be supplied by Diversa. Material will be injected in ovo and/or intramuscularly into one day-old birds and heterophils will be isolated from peripheral blood for in vitro assays. In addition, recruitment and activation of heterophils by potential immune modulators could be evaluated in the neonatal chicken peritoneal model system. Heterophil functions to be assessed in vitro will include secretion of cytokines, expression of surface molecules, oxidative burst, degranulation, phagocytosis, chemotaxis, bactericidal activity, non-specific and receptor-mediated adherence. Biological assays will be used whenever practical. Binding of immune modulators to potentially relevant heterophil'for IFN- receptors will be determined by using appropriate polyclonal antibodies and agonists of these receptors (competitive inhibition) whenever practical. Changes in heterophil cell morphology will be assessed by microscopy. Lymphokine crude extract produced by stimulated T-cells in vitro will be provided to Diversa for proteomics profiling aimed to identify novel immune modulators. Material will be fractionated using a novel quantitative profiling method that compares the separation and mass profiles of samples to determine the differences between complex mixtures. Since Diversas novel quantitative method is very sensitive, it will also quantify differences of low abundant proteins and will be used to find proteins in serum that are unique to Salmonella-challenged birds. Several assays could be used for initial n vivo evaluation of novel immune modulators, including determination of body weight, feed conversion, Salmonella shedding and organ invasion, and antibody titer (if applicable). <P>Phase II: Several in vivo models could be set up to evaluate immune modulators identified in Phase I. Selected candidates will be evaluated in combination with antigens relevant to virulence and/or important biological functions of poultry entheropatogens. In vivo assays will be chosen based on protective antigens and disease models that will be used to evaluate optimized vaccines. To fully validate selected candidates, additional poultry in vivo models will be considered in addition to Salmonella and may include E. coli, Shigella, Clostridium, Eimeria, Enterococcus, and Campylobacter.