2008 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.
A major obstacle to advances in veterinary immunology and disease control is the lack of immunological reagents. This grant established the US Veterinary Immunological Reagents Network (VIRN) (www.vetimm.org) to address the dearth of immunological reagents. BARC APDL scientists provided materials to the consortium including tissue specific cDNAs, plasmids, primers, design of peptides, recombinant proteins, nucleotide and peptide sequences and associated documentation and knowhow. VIRN labs have produced full-length plasmids for 19 genes. Yeast expression vectors that encode swine immune proteins were used to express 5 recombinant chemokines and cytokines at Kingfisher Biotech. To date 2 chemokines and 1 cytokine have been tested for bioactivity at BARC APDL and will be sent to U. Massachusetts for monoclonal antibody (MAb) production. Separately Cornell U. scientists used swine cDNAs for expression of cell surface proteins to produce MAb; to date MAb for 2 swine T cell receptors and one cytokine receptor have been screened for activity. Products generated by the VIRN will be used by animal health researchers, veterinarians, vaccine manufacturers, and other commercial applications.
The VIRN website has been expanded. BARC APDL scientists have provided all documentation and knowhow for the VIRN website www.vetimm.org expansion. They reviewed, and regularly updated, published data and techniques on swine immune reagents and their bioactivity. In addition, they scanned commercial websites for swine immune reagents, and organized the data into easily accessible Tables. As a result researchers worldwide can quickly access lists of available commercial reagents for swine immune and disease studies. Details of Network progress in developing cloned cytokine and chemokine proteins and monoclonal antibodies to them and to cell surface proteins can be easily monitored, and requests for reagents can be honored through the website and the Network’s commercial partner. Additionally, the website provides a forum on which researchers can suggest new priorities for future development. 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.
These projects align with National Program 103 Component II: Genetic and biological determinants of disease susceptibility. Communications for this grant have been supported by ARS by providing support for the monthly conference calls that review Network plans and progress. An ARS Sharepoint site has just been established for the Network for more effective data exchange. Email and phone communication are regularly exchanged between the participating labs as specific reagents are developed and screened.