2010 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.
Expanded approach to complete objectives 5 and 6:
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.
This project seeks to establish what cytokines can activate natural killer cells (NK cells) of cattle and swine to kill Foot-and-Mouth Disease Virus (FMDV) infected cells in vitro. Once identified, these cytokines will be analyzed for the ability to activate NK cells in vivo and protect against FMDV infection.
1. Determined that the NK cells of swine can be activated in vitro by a number of cytokines, mostly in combination. IL-15 alone was as effective as any cytokine combination and IL-15 plus other proinflammatory cytokines such as IL-12, was only marginally more effective than IL-15 alone. 2.
Constructed a replication defective adenovirus expressing porcine IL-15 and showed that the recombinant IL-15 was made and is biologically active in vitro. 3. Determined that the infection of swine with FMDV results in inhibition of NK cell function during acute infection. NK cell activity recovers as the animal resolves the vesicular lesions. 4. Showed that the NK cells in cattle are activated during the acute phase of FMDV infection and likely contribute to virus clearance ahead of or at least in addition to induction of early antibody responses. 5. Identified at 3 cytokines, IL-12, IL-15 and IL-21, can induce activation in bovine NK cells. The results indicate that activation by these cytokines is not maximal. Human data indicate that plays a prominent role in NK cell activation and we have cloned bovIL-21 and constructed a replication defective adenovirus expressing this cytokine.
This work is detailed in the publication: Toka, FN, et al., (2009). Natural killer cell dysfunction … foot-and-mouth disease virus (FMDV). Clinical and Vaccine Immunology. 16: 1738-1749.
This collaboration was monitored through email and telephone exchange as well as meetings with DHS S&T staff at PIADC.