An integrated immunological and cellular strategy for sensitive TSE diagnosis and strain discrimination
Prion infections result in progressive, fatal neurodegeneration. No effective therapies are available, and medical interventions, possibly including blood transfusions, have resulted in human-to-human transmission of prions. However, no biomarkers are available for preclinical diagnosis of prion infection in body fluids. All approved methods of diagnosis rely exclusively on the detection of the pathological prion protein (PrPSc), which may not be present in all Transmissible spongiform encephalopathies (TSEs).
The TSEUR consortium develops, validates, and exploits innovative reagents and technologies that will address the above problems in three areas: (1) enhanced detection of PrPSc, (2) direct measurement of prion infectivity, and (3) validation of new TSE biomarkers in body fluids.
By using and establishing powerful new panels of picomolar-affinity anti-PrP monoclonal antibodies to a variety of PrP domains, this consortium aims at generating new, highly sensitive tools for TSE diagnosis and strain discrimination. Field tests will validate the partners' recently identified secreted surrogate markers, whose levels are profoundly increased in preclinical prion infections, for identifying potentially contaminated body fluids. Immuno- PCR technology will be explored as a means to enhance the sensitivity threshold of each assay. Finally, the partners will extend the scrapie cell assay technology for rapid and sensitive detection of bona fide prion infectivity in a variety of paradigms.
The current need for sensitive tests to detect prions or surrogate markers of the prion diseases in affected individuals before the clinical onset of disease has intensified the search for new technologies and tools. Currently, diagnosis relies exclusively on the detection of PrPSc, which may not be present in all TSEs. Importantly, even by using detection methods specifically for PrPSc, no sensitive tests are available to detect prions in blood or urine of affected individuals (e.g. human CJD patients;
scrapie sick sheep). Therefore, not only are new tools to detect PrPSc in various body fluids or organs at various stages of disease needed, but new surrogate markers that would 'sense' the appearance of prion disease are needed as well.
Therefore, in order to detect affected individuals at the subclinical stage, it will be of great importance to find other surrogate markers than PrPSc. Moreover, the appearance of new prion strains that do not differ in the primary amino acid sequence but are believed to vary in the three-dimensional structure has made it clear that there is also a big demand for tools that enable scientists to differentiate those prion strains. Prions strains have different organ tropisms, can be replicated in different hosts with different efficiency and can induce diseases with varying incubation times, lesion profiles, etc. As a consequence of this, it will be of high importance to generate tools to differentiate various prion strains.
The TSEUR consortium proposes to develop, validate, and exploit innovative reagents and technologies that will address the current problems of prion diagnostics in three areas:
The expected result of Work Package (WP) 1 is to significantly improve the currently available scrapie cell assay technique and develop cell-based assays using heterologous tagged PrP variants for the rapid and sensitive determination of bona fide prion infectivity in biological tissues and fluids. WP2 will exploit a powerful new panel of picomolar-affinity anti- PrP monoclonal antibodies to a variety of PrP domains. These will provide the basis for highly sensitive detection of PrPSc and discrimination of prion strains by the consortium's original technology termed 'epitope coding'. WP2 will deliver PrPSc-based diagnostic reagents and technology with significantly lower limits of detection sensitivity.
WP3 will provide and exploit newly identified biomarkers by one member of the TSEUR consortium. RNA levels of these two biomarkers are profoundly altered as a result of prion disease; early in pathogenesis, encoded proteins are known to be present in a variety of body fluids. Highly sensitive monoclonal antibodies directed against human and ovine cystatin 7 and ovine serpinA3 utilising Virus- Like Particle (VLP) technology should overcome problems of immuno-tolerance of immunised mice. Such biomarkers should prove valuable as tools to monitor efficacy of future therapeutic intervention strategies for prion diseases.
WP 4 will deliver a technological platform in which sensitivity of detection of low levels of antigen is amplified by PCR technology (quantitative immuno-PCR). WP5 utilises an existing experimental set-up whereby several groups of sheep are managed in controlled conditions and have been experimentally infected with scrapie and are either harbouring Maedi-visna virus infection or not. The partners will use highly sensitive and cross-species reactive POM anti-PrP monoclonal antibodies characterised in WP2 to assess PrPSc load in mammary gland and milk of these groups of animals. Additionally, they will use ovine TraSCA technology developed in WP1 to determine whether prion infectivity can be detected in milk and mammary gland tissue from scrapieaffected sheep harbouring Maedi-visna viral infection. WP6 ensures the establishment, maintenance, and quality control of the infrastructure needed for sample collection and clinico-biological correlations in sheep tissues.
Overall, the TSEUR programme will result in the provision of innovative diagnostic technologies with the ultimate goal to enhance the safety of the blood supply, to provide minimally invasive diagnostics of human and animal prion diseases, to develop highly sensitive tools for discrimination of prion strains, and to advance basic prion science.
Many potential applications are planned from the results that emerge from this EU consortium: