Plant-Based Bioproduction of Chicken IL-12 Adjuvant for Bird Flu Vaccine

NON-TECHNICAL SUMMARY: The primary focus of this project is to develop new high valued health related agricultural products through the application of biotechnological research approaches. Domestic and wild fowl, as the disease reservoir for avian influenza virus, play a central role in the re-emergence of this potentially pandemic disease pathogen. The increasing threat of pandemic flu in domestic fowl and human populations has activated governments, agricultural, and medical health agencies in the US and globally to initiate multifaceted research and development efforts aimed at mitigating this threat. New technologies for producing inexpensive high-quality agricultural vaccines to control avian flu at its source will be critical to successful intervention in the disease cycle supporting the threat of pandemic flu in both domestic poultry and humans. This project focuses on the bioproduction of avian interleukin-12 (IL-12) to address both of these needs. IL-12 is a potent adjuvant and key modulator of cell-mediated immunity and greatly enhances the efficacy of influenza vaccines in animal studies. There are currently no sources for IL-12 from any avian species. Phase I of this SBIR project successfully met its objectives in demonstrating the feasibility of plant-based production of bioactive chicken IL-12. We isolated and tested multiple ChIL-12 gene constructs and demonstrated effective expression in plants. We selected a His-tagged native ChIL-12 construct that provides product yields sufficient to support scaled up production for Phase II research and commercialization for the research reagent market. Most importantly, Phase I demonstrated that plant synthesized ChIL-12 shows excellent immune stimulating bioactivity in signature in vitro bioassays using chicken splenocytes. Phase II will focus on advancing ChIL-12 and related products (antibodies, ELISA) to the research reagent market and establishing large-scale production strategies in a transgenic seed-based plant system. Based on our successful Phase I, Phase II will also focus on production scale-up, demonstration of efficacy in avian vaccine trials, and assessment of ChIL-12 activity across avian species. Co-formulation of this strong immuno-adjuvant with avian flu vaccines will be tested to demonstrate whether these poultry vaccines elicit heightened immunity necessary for preventing cross species avian flu transmission. Plant-based bio-production may provide the cost and scale advantages to enable these benefits to be widely integrated into avian influenza vaccine strategies for both domestic and wild bird populations. Success of this Phase II SBIR in demonstrating significant "dose sparing" activity by ChIL-12 in avian flu vaccines will provide the basis for a strong Phase III commercialization program and a significant contribution to US and global efforts to mitigate the danger to humans and animals of pandemic influenza. An effective IL-12 adjuvant may also have broader applications as a "universal" adjuvant for other diseases and vaccines. <P>

APPROACH: Objective 1: ChIL-12 will be produced in the transient tobacco-leaf expression system and used to optimize bioactivity assays for quality control purposes, to enhance purification strategies, to support chicken vaccination/challenge trials, and to develop monoclonal and polyclonal antibodies against ChIL-12. Antibodies will be used to develop a ChIL-12 ELISA kit and establish QA/QC parameters for ChIL-12-related research reagents. Objective 2: We will compare morbidity and mortality in chickens vaccinated with standard influenza A vaccines +/- chIL-12 and challenged with influenza virus. Experiments will be designed to determine non-toxic adjuvant dosing and assess the vaccine dose-sparing potential of IL-12 with both inactivated virus and HA subunit vaccines. Objective 3: ChIL-12 transgene constructs will be introduced into flax and tobacco and stable transgenic lines will be screened to identify lines with high transgene product yields. Objective 4: We will test the ability of ChIL-12 to stimulate splenocytes of other avian species of issue for US poultry industry and global spread of bird flu (turkey, ducks, and geese). We will also confirm that chicken IL-12 is not bioactive on mammalian splenocytes. EVALUATION: There currently is no commercial source of avian IL-12. As an outcome of this Phase II research, our bioactive ChIL-12 (and related immuno-detection tools) will be available to otjher researchers through commercial research reagent vendors and its utility as a dose sparing adjuvant and its application to broader poultry markets (e.g., turkey) will have been demonstrated. IL-12 is a very potent adjuvant with potential utility in addressing many other poultry disease issues. The potential for IL-12 to provide new adjuvant activities for a broad spectrum of poultry diseases increases its market potential and we anticipate that commercial availability (and directed marketing) will facilitate its testing as a vaccine adjuvant for the key commercially important chicken and turkey diseases. The focus of this SBIR is to develop high-value research and animal health products utilizing crop-based bioproduction. Thus, this project fulfills multiple components of the USDA strategic goals including development of new economic opportunities for the agricultural sector, economic development linked with rural America, and protection of the US agriculture and food supply. This project integrates 1) the PIs extensive experience in plant-based bioproduction of high-value therapeutic proteins, 2) Arkansas strengths in poultry production, commodity agriculture and food science, and 3) the dramatic needs for innovative new vaccine strategies brought about by the global spread of highly pathogenic avian influenza which threatens both domestic and wild avian species and elevates the risk of human pandemic strain emergence.