2010 Annual Report
1a.Objectives (from AD-416)
This proposal presents a broad community plan to begin to systematically address the immunological reagent gap for the U.S. veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses, and aquaculture species (concentrating on channel catfish and trout) with a goal of 20 reagents per species group. The reagents will include bioactive recombinant cytokines and chemokine proteins, expressed using mammalian, yeast, and bacterial systems, as well as antibodies to them and their receptors as well as antibodies to immunoglobulin isotypes, T cell receptors and other CD molecules. The goal is to produce antibodies that function in ELISA and ELISpot assays, for intracellular staining, for blocking function and signaling, and that are useful in flow cytometric applications as well as in fixed tissue sections. Antibodies to bioactive molecules will also be tested for their ability to block the recombinant molecules as well as natural molecules if it is possible to produce cell culture systems for the later. This project represents the swine component.
1b.Approach (from AD-416)
Prioritization of reagents (tools) to be produced will be established by surveying the larger scientific community and cooperating with other similar international efforts to avoid redundancy and target the most useful reagents for research scientists. Commercial companies will also be surveyed. Bioactive cytokines and chemokines and cell surface CD molecules, antibodies, and T cell receptors will be produced in expression systems in two main labs for poultry and mammals and by the two co-PDs for fish. The expression of these proteins will depend upon provision of cDNA from appropriate cells or tissues from the co-PD labs who are specialists in particular species. RT-PCR using primers based on available sequences or from homologous genes in other related species will be done to obtain the entire gene sequence. The recombinant molecules will be used to prime mice for production of monoclonal antibodies or rabbits for polyclonal antibodies in a centralized lab. After identifying reactive antibodies initially, both the recombinant molecules and the antibodies to them will be evaluated for efficacy in the species specialist labs in a variety of in vitro assays. This project will have oversight by an advisory board composed of industry partners, USDA national program directors and other international reagent-generating groups. The reagents will be provided to the scientific community by depositing in commercial cell banks and encouraging generation and distribution through commercial vendors.
In this Agreement swine immune reagents were developed to probe immune responses to infectious diseases and to improve vaccine design. The U.S. Veterinary Immunological Reagents Network (VIRN) (www.vetimm.org) was established to address the dearth of immunological reagents for food animal species. ARS Researchers at Beltsville, MD, working with Kingfisher Biotech Inc. www.kingfisherbiotech.com/ have expressed a total of 16 recombinant swine immune proteins in yeast. This year 6 recombinant chemokines and cytokines (CCL2, CCL3L1, CCL4, CCL5, IFNalpha, IL-15) have been proven to be bioactive. A panel of monoclonal antibodies (mAbs) against CCL2 was produced at Cornell Univ., characterized, and a new Luminex bead assay for quantitating CCL2 was jointly developed. One mAb each reactive with IL-4R and IL-13R has been characterized; new hybridoma fusions were screened for anti-T cell specific mAb with Cornell Univ. scientists. Products generated by the VIRN will be used by animal health researchers, veterinarians, vaccine manufacturers, and other commercial sources.
The U.S. Veterinary Immunological Reagents Network (VIRN) website for swine immunological reagents was expanded over the past year. ARS Researchers at Beltsville, MD, have provided all documentation and knowhow for the USDA CSREES grant supported immune toolkit or VIRN website (http://www.umass.edu/vetimm/swine/index.html) swine pages. They scanned commercial websites and publications for swine immune reagents and organized the data into easily accessible Tables of currently available reagents for swine immunology. This list served as the basis for updating the Swine priorities for developing new VIRN immune reagents and for VIRN colleagues and swine collaborators to develop plans for their production. Posted results as well as updated Protocols on the VIRN website. As a result this website enables researchers worldwide to quickly identify available immune tools for their experiments. Overall swine disease and vaccine researchers have improved tools to address pig health and vaccine design.
Communications for this grant have been supported by ARS by providing conference call line access for the monthly calls that review Network plans and progress. Email and phone communication are regularly exchanged between the participating labs as specific reagents are developed and screened.