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As the nation, scientific community, and poultry industries prepare to meet the challenges posed by the spread of the highly pathogen strains of Avian Influenza (AI), there is a need for innovative strategies to produce critical research reagents and rapidly scalable vaccine components for both human and bird populations. IL-12 is a potent adjuvant of cell-mediated immunity and co-administered IL-12 enhances the efficacy of influenza vaccines. IL-12 is a complex heterodimeric glycoprotein requiring eukaryotic bio-production. Research reagent grade CHO- and insect-cell derived mammalian IL-12s cost as much as $2,500 per 100 g of protein. There is no current commercial source for any avian IL-12. We have developed a transgenic tobacco-based bioproduction system for mouse IL-12 (mIL-12) that provides high yields of recombinant protein with equivalent bioactivity compared to animal-cell derived mIL-12 in both in vitro assays (interferon-C stimulation in splenocytes; cell proliferation) and in vivo mouse vaccination trials. Expression levels in our system are 40-500-fold higher than levels of human IL-12 reported by other groups using plant-based production strategies. We therefore propose to exploit this system for the bio-production of chicken IL-12 (ChIL-12). The specific aims of this SBIR Phase I are as follows: Objective I: To construct expression vectors for chicken IL-12. Genes encoding the single chain form of chicken IL-12 will be developed for expression in plants. In addition to the native single-chain form, constructs will be made that incorporate a histidine tag to aid in initial detection and purification and provide a potentially valuable research reagent. Objective II: To transiently express ChIL-12 constructs in tobacco leaves to assess yield potential and bioactivity of recombinant products. ChIL-12 constructs will be introduced into tobacco leaves utilizing Agrobacterium-mediated infiltration methods for transient expression. Plant-synthesized product will be used to assess size, yield, purification behavior and IL-12 bioactivity in vitro using primary chicken splenocytes. This Phase I feasibility study will establish the ability of plants to produce bioactive chicken IL-12; will provide insights as to potential in planta productivity (yield potential) of the chicken cytokine, will establish key detection, purification and assessment tools, and will create the tools required to establish an efficient bio-production system capable of providing bioactive ChIL-12 to researchers. Phase II research will include production of stable transgenic lines, product characterization and purification, in vitro bioactivity assessment on multiple avian species, and an initial demonstration of Th1 adjuvancy and enhanced protection in poultry vaccine trials.
The goal addressed in this project is to develop new high valued health related agricultural products through the application of biotechnological approaches. Domestic and wild fowl are susceptible to many common diseases which can threaten our food supply and the economic health of our agricultural economy. The purpose of this project is to develop new technologies for producing inexpensive high-quality bird flu vaccine components to control the spread of flu in bird populations and reduce the disease cycle supporting the threat of pandemic flu in both domestic poultry and humans. This project specifically focuses on the bio-production of avian interleukin-12 (IL-12), a powerful adjuvant and key modulator of cell-mediated immunity which greatly enhances the efficacy of influenza vaccines in animal studies. We have developed a plant-based bio-production system that provides high yields of recombinant bioactive mouse IL-12 protein. The goals of this SBIR are to produce chicken IL-12 (ChIL-12) using this production system and assess its feasibility with respect to product yield and bioactivity. The aims of Phase I include developing ChIL-12 gene constructs, characterizing ChIL-12 produced in leaf material, and demonstrating the immune system bioactivity of plant-derived ChIL-12. Plant-based bio-production of ChIL-12 may provide the cost and scale advantages necessary for cost effective production of adjuvants for delivery of new avian influenza vaccine to both domestic and wild bird populations leading to a reduction in the threat of pandemic flu to animals and humans.
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This SBIR targets the production of chicken interleukin-12 (ChIL-12) as a promising vaccine adjuvant for protection against avian flu and other diseases requiring strong cell-mediated immunity. We have developed a transgenic tobacco-based bio-production system for mouse IL-12 that provides higher yields of bioactive protein than other available bio-production systems and we propose to adapt this technology for production of chicken IL-12 which is not currently available for research and vaccine development. Phase I will test the feasibility of expressing bioactive ChIL-12 in this tobacco protein expression system. Objective I is devoted to construction of the IL-12 expression vectors required for subsequent transgenic studies. We have expressed numerous foreign proteins in tobacco using both constitutive and wound-inducible promoters. For this project, we will utilized these optimized gene/vector components but replace the mouse sequences with genes encoding ChIL-12. We will produce ChIL-12 in the single chain form using a gly/ser linker based on observations by others that this form shows analogous bioactivity compared to p70 derived from co-expression of the chicken p40 and p35 genes. Objective II will address transient expression of ChIL-12 in tobacco leaves to assess yield potential and characterize recombinant protein. We have developed an Agrobacterium-mediated transient expression system that permits rapid assessment of plant-synthesized product. Activated cultures of A. tumefaciens carrying IL-12 constructs are infiltrated into intact leaves of Nicotiana benthamiana permittng A. tumefaciens to infect and transfer its T-DNA region into plant cells. This approach supports several rounds of transcription/translation independent of integration into the plant genome. We propose to use this transient system to initiate characterization of plant-derived ChIL-12 as follows: <ul>
<li>1) we will determine if chicken IL-12 sequences drive comparable levels of protein compared to the highly expressed mouse IL-12 and determine if sequence and signal peptide variants can impact yields;
<li>2) we will optimize our analytic detection tools and purification steps for ChIL-12;
<li>3) His-tagged product will be purified by metal affinity and used for raising ChIL-12 antibodies in rabbits, and 4) partially purified recombinant IL-12 (+/- the His-tag) will be used to test IL-12 bioactivity in primary chicken splenocytes </ul>