2009 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 collaborators for fish. The expression of these proteins will depend upon provision of cDNA from appropriate cells or tissues from the collaborator 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.
A panel of immunological reagents have been developed to improve studies on immune responses to infectious diseases and to improve swine vaccine design. This USDA CSREES grant established the U.S. Veterinary Immunological Reagents Network (VIRN) (www.vetimm.org) to address the dearth of immunological reagents. Working with Kingfisher Biotech Inc., swine immune proteins were express in yeast; 5 recombinant chemokines (CXCL10, CXCL11, CCL2, CCL4 and CCL5) have been proven to be bioactive at BARC and monoclonal antibodies (mAbs) produced to several of these and now being characterized. Separately, Cornell U. scientists used recombinant cell surface proteins to produce mAb for 2 swine cytokine receptors, IL-4R and IL-13R; their reactivity has been affirmed at BARC. 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 has been expanded. BARC APDL scientists 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. With this basis they updated the Swine priorities for new immune reagents, worked with VIRN colleagues to develop them, and posted those results as well as the techniques to assess their bioactivity. Details of Network progress in developing cloned cytokine and chemokine proteins and monoclonal antibodies to them and to cell surface proteins can now be easily monitored, and requests for reagents reviewed. As a result, researchers worldwide can quickly access lists of available commercial and planned VIRN reagents for swine immune and disease studies. This website enables swine disease and vaccine researchers to quickly identify the availability of immune tools for their experiments and thus will help to improve pig health and vaccine design.