Start Date: May 28, 2008
End Date: Apr 30, 2013
1. In vitro activation of bovine NK cells will be accomplished through testing a series of bovine cytokines for activation of NK cells. The activation of NK cells will be assayed by increased killing activity on target cells. This correlates with increased expression of two cell surface proteins. Once these cytokines are identified, a human, replication defective adenovirus will be used as a viral vector to prepare constructs. In vitro testing will then be conducted. 2. In vitro activation of porcine NK cells will be conducted for appropriate biological activity. Once an appropriate vector is made and characterized, it will be tested in vitro for induction of NK activation and the kinetics of induction will be determined. 3. Generation of reagents for NK cell analysis will be conducted through the generation of antibodies to cytokines. 4. Adenovirus constructs will be tested in vivo for cytokine expression for rapid protection against FMDV infection in swine and bovine. An optimal dose for the adenovirus vectors will be determined and kinetics of NK activation documented. This construct will then be tested in FMDV challenge studies. 5. Constructs will be tested in vivo to determine if porcine IL-15 derived from cells infected with huAd5-pIL-15 in vitro can activate porcine NK cells to production of interferon gamma and/or increased killing of FMDV infected cells. 6. Preliminary data indicates that bovine IL-15 likely in combination with other cytokines, activates NK cell to increased target cell killing and interferon gamma production. Other bovine cytokine(s) with the potential to drive NK activation will be cloned, expressed and tested in vitro. Adenovirus vectors expressing those cytokines will be constructed and cells infected with huAd5-bovine cytokine will be tested for production of biologically active protein in vitro. Once the cytokine, or combination of cytokines, is shown to activate porcine NK cells to increase the production of interferon gamma and/or increase FMDV infected cell killing, the constructs will be tested in vivo. 7. In order to track the in vivo expression of cytokines of interest, monoclonal antibodies will be prepared against these porcine and proteins. These include but are not limited to bov IL-15, bov IL-21, bov IL-13, por IL-13 and por IL-15. 8. In the past years, the International Livestock Research Instittue (ILRI) Nairobi, Kenya, has produced over 100 monoclonal antibodies reactive with cell surface proteins expressed by cells of the bovine immune system. These have often been published, yet remain unavailable to researchers in the USA because bovine products from Kenya can not be imported. The cell lines will be tested for viability and antibody production at ILRI and all functioning lines will be shipped to PIADC for expansion, storage and safety testing. Once shown to be free of contaminating infectious agents, the cells will be cleared for distribution in the USA subject to agreements between ILRI and the recipient.