Title: Development of a cell-based bioassay to accurately measure variant Creutzfeldt-Jakob disease prions in human tissues and biofluids
Funder: Department of Health and Social Care

Abstract: Background Variant Creutzfeldt-Jakob disease (vCJD) is a fatal, progressive human neurodegenerative disease, acquired upon dietary exposure to the infectious agent of Bovine Spongiform Encephalopathy (BSE). During the BSE epidemic (1986-1998), more than 186,000 cattle were infected, leading to the widespread exposure of the UK population to BSE prions. The implementation of robust food safety measures, in 1996, severely reduced dietary exposure to BSE, and led to vCJD becoming a rare disease. However, the death last year of an individual from vCJD, with a MV (methionine/valine) genotype at codon 129 rather than MM genotype, has raised concerns that there may be a second wave of vCJD cases following a longer incubation period, involving less susceptible genotypes. Furthermore, concerns have been raised by the Appendix-III prevalence study of abnormal PrP deposition, in archived appendix tissue samples of people, who were exposed to BSE prions in contaminated beef. Aims The aim of this study is to develop a specific and highly sensitive bioassay which can detect pre-clinical levels of vCJD infection in human tissues and biofluids, particularly an assay that directly measures infectious prions. This will enable us to confirm the results of the prevalence study of abnormal PrP deposition in the UK population and also to guard against the possibility of further inadvertent exposure of the general public, through blood transfusions and surgical procedures. Research Plan Cell lines have proven to be invaluable for in vitro studies of many complex processes and diseases, including studying aspects of prion biology. As there are no cell lines that can stably propagate human vCJD prions, despite worldwide efforts, our objective is to develop cells that can stably propagate vCJD prions and use them to develop a rapid and accurate cell-based bioassay. The development of propagating cells is hindered by the lack of knowledge of the factors that influence susceptibility and is thus absolutely dependent upon high throughput screening for cells that can support vCJD propagation. We will utilise two approaches: 1. Engineer mouse tumour cells by Silencing followed by reconstitution , followed by a screen for susceptible cells. 2. Use a human foetal glial cell line that has been found to support vCJD propagation. Cells that are found to reproducibly support vCJD propagation will be used as the starting point for sequential single cell cloning, to derive a highly susceptible cell line, capable of supporting a robust infection. If this does not produce a sufficiently sensitive cell line, a functional forward genetic screen will be undertaken to increase susceptibility. Research Team The research will be led by Profs. Parmjit Jat and John Collinge. It will be undertaken by a Postdoctoral Associate who will be trained and assisted by Mrs Parineeta Arora. Expert advice, help and guidance will be provided by Drs. Peter Kloehn (Prion Unit) and Holger Hummerich (Prion Unit) and Philip Sharp (Massachusetts Institute of Technology, USA). Potential Impact The ability to directly monitor infectious prions in biofluids will provide a &ldquo;gold standard&rdquo; for diagnosis of prion infection and validation of other diagnostic assays, such as the blood and protein misfolding cyclic amplification assays and provide a biomarker for studying response to treatment. The assay will also enable confirmation of the results of the prevalence study of abnormal PrP deposition in the UK population and increase health protection by guarding against the possibility of further inadvertent exposure of the general public, through blood transfusions and surgical procedures. The automated assay also has the potential to be scaled up, to process thousands of samples per year, as has already been done for the automated cell-based bioassay, for mouse prions.

Lay abstract: Variant Creutzfeldt-Jakob disease (vCJD) in humans is a severe, rapidly progressive, ultimately fatal, degenerative brain disorder, caused by an infectious agent derived from the brains and spinal cords of cows suffering from mad cow disease . The infectious agent, called a prion, is a misfolded version of a normal protein, called the prion protein. It replicates by converting the normal host prion protein, to a misfolded disease state. During the mad cow epidemic, a large number of infected cattle entered the food chain, leading to wide spread exposure of the UK population to the infectious agent. This led to a total of 178 confirmed cases of human vCJD. The number would have been much higher, had the UK government not implemented robust safety measures, to minimise entry of contaminated beef into the food chain and slaughtered 4.4 million cattle during the eradication program. Nevertheless, the death this year of an individual from vCJD, with a different genotype, has raised concerns that there may be a second wave of cases following a longer latency period. These concerns were further highlighted by the third national survey of abnormal prion prevalence, using archived appendix specimens, which found that 1:2000 people born before 1996, and 1:3000 people born after 1996, were positive. To confirm the survey results and also guard against further inadvertent exposure of the general public through blood transfusions and surgical procedures, it is imperative to develop a specific and highly sensitive assay, to monitor pre-clinical levels of vCJD infection, in the UK population. The aim of this project is to develop a highly sensitive and accurate automated cell-based bioassay, which can be used to measure infectious prions in a range of human tissues and biofluids from patients. This assay can be a &ldquo;gold standard&rdquo; for diagnosis of prions and validation of other diagnostic assays and provide a biomarker for studying response to treatment. It also has the potential to be scaled up to process large numbers of samples per year, as has already been done for the mouse prion bioassay.

Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: NEUROLOGICAL