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2010 Annual Report

1a. Objectives (from AD-416)
We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments.

1b. Approach (from AD-416)
Transmissible spongiform encephalopathies (TSEs) are a family of fatal diseases caused by exposure of animals to infectious prions. The zoonotic prion disease in cattle (Bovine Spongiform Encephalopathy [BSE]) represents an economic threat to the U.S. beef industry through deleterious changes in meat export, herd management practices, consumer perception and safety. Other TSEs, such as Scrapie in sheep and Chronic Wasting Disease (CWD) in cervids, directly impact the economics and management of those commodities and represent reservoirs of infectious prions with the potential for vertical transmission of disease to other agriculturally important animal species. Consequently, U.S. animal industry stakeholders have identified detection of infectious prions as a priority bio-security research issue essential for prevention of TSE diseases. Our expertise in pathogen diagnostics will be used to develop the methodology and molecular tools necessary for the detection of infectious prions (PrPSc) from biological tissues and environmental substrates. (1) We will generate new high-affinity monoclonal antibodies (MAb) against PrPSc for the development of sensitive immunoassays. (2) We will define new methods for the isolation and enrichment of PrPSc to promote improved detection of diseased animals or contaminated substrates. (3) We will develop a method for the detection of PrPSc by mass spectrometry. Anti-PrPSc immunoassays and analytical MS methods will be validated with prion contaminated samples and detection sensitivity determined. Improved sensitivity of PrPSc detection will be achieved by assay refinement and sample enrichment strategies toward predictive diagnostics of TSE in asymptomatic animals. Replacing 5325-32000-007-00D (3/19/2008).

3. Progress Report
We have developed a method to enrich prions from biological tissues resulting in a significant increase in sensitivity of prion detection by immunoassay. This enrichment strategy is being applied for the detection of prions in biological samples with low-level contamination from asymptomatic animals. To overcome the challenges in making anti-PrPSc antibodies we, in collaboration with our academic partners, generated a transgenic mouse that lacks endogenous PrPC (Prnp0/0 Balbc/J). We demonstrated that wild-type mice fail to respond to a purified PrPSc immunogen whereas Prnp0/0 Balbc/J mice elicit a robust and specific immune response producing anti-prion antisera. This model is currently being used to generate hybridoma cells for the production of monoclonal antibodies (MAb) used in prion immunoassays. The identification of hybridoma cells producing anti-PrPSc MAbs requires a screening method that can distinguish PrPSc from normal PrPC. Current screening methods utilized recombinant PrPC or crude homogenates and lack the capacity to discriminate. Consequently, these methods favor identification of antibodies that recognize both PrPC and PrPSc. We have developed a screen that exploits our prion enrichment method to generate sufficient native PrPC and PrPSc for use as an antigen. By using a comparative ELISA as screen our method favors identification of hybridomas producing PrPSc selective MAbs. We have used this method to successfully identify hybridomas making high-affinity anti-prion MAbs useful for PrPSc detection. Together with our academic partners, we have generated eight new anti-prion MAbs and determined their binding properties to prions from different species. Five antibodies exhibit conformational dependent binding to discontinuous PrP epitopes. The high-affinity F4-31 anti-prion MAb has increased the sensitivity of prion detection and will be useful in a wide range of immunoassays. Mammalian cell lines were positively evaluated for PrPC expression by immunoassay. Prion challenge with infectious hamster, ovine, or deer brain extract was ineffective in converting PrPC to PrPSc in any cell line tested. However, PrPSc challenge (RML strain) of mouse cell lines resulted in time-dependent increase in detection of proteinase-K (PK) resistant PrPSc. Yet, the efficiency of conversion was low, the biochemical profile of cellular PrPSc by Western blot was variable, and infectivity of cellular PK-resistant material was indeterminate. Over-expression of recombinant mouse and ovine PrP in cell lines was ineffectual in promoting conversion of PrPC to PK-resistant PrPSc. We conclude that a cell model for the amplification of low-level prion contaminant is currently impractical for reliable prion detection. Blood plasma from hamsters infected with PrPSc was fractionated to enrich prions and shown to be infectious. Efforts to detect prions from plasma of infected animals by mass spectrometry have been unsuccessful, but we can capture and detect prion with our MAbs by immunoassay. We are currently refining our enrichment of plasma to evaluate prion detection in blood of asymptomatic animals.

4. Accomplishments
1. Prion sample concentration. Early detection of prion diseases such as sheep scrapie requires concentration of the prion proteins that serve as disease markers from dilute biological samples. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with the University of California San Francisco, developed a new method for concentrating prion proteins from animal tissues. We found this method results in a significant increase in prion concentration, allowing more sensitive prion detection. This year we filed a patent application and published a manuscript on this new method, showing how it provides more sensitive and early detection of disease in infected animals.

2. New strain of mouse for anti-prion antibody production. Production of antibodies for detection of prion proteins associated with diseases such as sheep scrapie is limited by the resistance of normal mice to making an immune response against their own proteins. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with our partners at the University of California San Francisco, have made a new strain of mouse that lacks the prion protein. Unlike normal mice, the new mice are highly sensitive to immunization with prions. This year we filed a patent application and published a manuscript showing the use of these mice in making new antibodies that can bind and detect prions. Such antibodies may be used for more sensitive and early detection of disease in infected animals.

3. New method for identification of antibodies that detect prion proteins. Making new monoclonal antibodies for detection of prion proteins in diseases such as sheep scrapie requires selection of the best performing cells from among thousands of candidates. Researchers at the Foodborne Contaminants Research Unit in Albany, California have developed a fast and sensitive method to identify such cells, which are taken from mice that have been immunized to produce antibodies that strongly bind to prion protein. This method was used to identify improved antibodies which are now available for use in detection of disease. Furthermore, the new screening method may be used by other scientists for research to make additional new antibodies for prions.

4. New antibodies for detection of prion disease. Sensitive detection of prion proteins for early diagnosis of disease requires antibodies that are capable of strong binding to prions. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with our partners at the University of California San Francisco, generated eight new antibodies that detect prion disease in many different animals. We have published a manuscript that describes these anti-prion antibodies and shows their value in improving detection of prions. Improved detection methods will help in herd management and control of the spread of prion diseases.

5. Prion protein modification. Changes in the prion protein that may lead to disease are poorly understood. Researchers at the Foodborne Contaminants Research Unit in Albany, California used mass spectrometry to detect and measure a specific chemical modification to the prion protein. In contrast to results published by other scientists, we found no difference in this chemical modification between normal and infected forms of the prion protein. We have published a manuscript describing this work, which helps scientists and veterinarians focus on other changes in the prion protein that might be involved in prion disease.

Review Publications
Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson, M.L., Vensel, W.H., Requena, J., Antaki, E.M., Carter, J.M. 2010. Assessing the Role of Oxidized Methionine at Position 213 in the Formation of Prions in Hamsters. Journal of Biochemistry. (49):1854-1861.

Hnasko, R.M., Serban, A.V., Carlson, G.A., Prusiner, S.B., Stanker, L.H. 2010. Generation of Antisera to Purified Prions in Lipid Rafts. Prion. 4:294-104.