2012 Annual Report
1a.Objectives (from AD-416):
USDA, ARS, PIADC in conjunction with the U.S. Department of Homeland Security (DHS), will implement a research program designed to conduct applied and exploratory research on anti-FMD (Foot-and-Mouth Disease) biotherapeutics targeting the innate response of cattle and swine. This study will focus on the action of natural killer cells (NK cells) and their potential to eliminate virus early in infection. Development and testing of adenovirus vectors expressing cytokines inducing NK cells will be accomplished through 1. In vitro activation of bovine NK cells, 2. In vitro activation of porcine NK cells, 3. Generation of important reagents for analysis of NK cell function in swine and cattle, 4. Testing of adenovirus constructs expressing cytokines in vivo and for rapid protection against FMDV infection.
Additional objectives added February 2009:
5. Testing huAD5-pIL-15 in vivo for NK activity.
6. Testing huAd5-bovine cytokine in vivo for NK activity.
Additional objectives added March 2010:
7. Immunologic reagent production of new monoclonal antibody for bovine cytokines and bovine immune cell markers.
8. Importing and making available monoclonal antibodies for bovine immune cell surface proteins previously produced at the International Livestock Research Institute (ILRI), Kenya, inclusive of; testing for antibody production and specificity and subsequent safety testing for release in North America and Europe.
1b.Approach (from AD-416):
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.
New objectives 7 and 8 added in March, 2010 were the focus of activities in this reporting period. We established a cooperative agreement with Green Mountain Antibodies, Inc., (GMA) of Burlington, VT. In the summer of 2011, we trained an undergraduate intern from University of Vermont (UVM) in the techniques for expressing cytokines in replication defective adenoviruses. During FY 2012, the intern returned to UVM, and has transferred the adenovirus technology to GMA and the company is now efficient at growing the viruses and expressing protein for use in monoclonal antibody production. GMA has immunized mice with three different constructs and tested serum for antibody to these cytokines. We now anticipate doing fusions in this coming fiscal year, which will yield the three to six of the desired monoclonal antibodies to cattle and swine cytokines.
Further, we have now safety tested the first 30 monoclonal antibodies (specific for immune system cells of bovine) imported from the International Livestock Research Institute (ILRI) in Nairobi, Kenya. These antibody producing cell lines are all negative for disease agents of concern as per APHIS policies. We are now submitting permits to share these reagents with bovine researchers in the US and elsewhere in North America and Europe. The additional 70 cell lines producing antibodies specific for immune system cells of bovine have been grown and tested for activity at ILRI. They are now ready for shipment. We have initiated permits for importing the cell lines to PIADC for safety testing.