Title: Assessing and defining pre-clinical vCJD infectivity using transmission and protein aggregation models
Funder: Department of Health and Social Care

Abstract: Background Despite widespread BSE exposure the number of vCJD cases in the UK is small, with only 178 cases to date. With one exception all tested cases were homozygous for methionine at codon 129 of the prion protein gene (PRNP). The recently identified heterozygous patient suggests that a second wave of cases is possible. Transfusion transmitted infection from pre-clinical vCJD donors to individuals of the MM and MV genotypes introduces further uncertainties, especially in the context of the prevalence of asymptomatic carriers as inferred from PrP immunohistochemistry (IHC) studies of surgically resected appendices. A prevalence of approximately 1:2000-3000 has been accepted however questions remain about the interpretation of PrP IHC as a prevalence tool. Aims in relation to the issue to be addressed As a result of the Appendix studies two issues arise: a) Do the assumed infectivity and strain properties associated with appendix PrP truly reflect vCJD? b) In light of the prevalence studies and the emergence of vCJD in the MV genotype the need for pre-clinical blood test to identify vCJD is crucial. Research plan and methods of investigation a) Mouse transmissions studies using frozen patient tissues remain the gold standard for infectivity strain typing. However the Appendix studies employed fixed/paraffin embedded tissues which are not normally used for transmission (or biochemical) studies. Before these very scare and valuable Appendix study tissues are tested, we need to ensure that we are able to obtain reliable and meaningful results from fixed tissue. Therefore in vivo transmissions of fixed and frozen central nervous system (CNS) and peripheral tissues, including spleen, from transgenic mice from each PRNP codon-129 genotype infected with vCJD or selected sCJD strains will be carried out. In addition, transmission of fixed and frozen appendix tissue from a definite vCJD case (PRNP-codon-129 MM) will also be carried out. The combination of these studies will allow the effects of tissue fixation to be determined, prior to testing of positive Appendix Study samples. b) Protein amplification and aggregation assays such as PMCA and RT-QuIC have been invaluable in the detection of small amounts of abnormal PrP in biological fluids such as urine in vCJD and cerebrospinal fluid (CSF) in sCJD. A number of PMCA and RT-QuIC techniques which distinguish between sCJD and vCJD and detect vCJD prions in pre-clinical blood samples have been developed (personal communications). However, a direct comparison of these methods has not been undertaken using well defined sCJD and vCJD clinical samples. We will directly compare these techniques using vCJD and sCJD blood, urine and CSF. We will also apply both techniques to frozen and fixed appendix from vCJD cases. In addition the ability of these techniques to identify pre-clinical blood samples will also be assessed using both animal models and human samples. Research team Dr Diack has a proven track record in the undertaking and evaluation of animal transmission studies for the assessment of infectivity and for confirming strain type. Dr Barria and Dr Head have experience in the development and adaptation of PMCA methods for diagnostic and for modelling purposes. Dr Green was instrumental in establishing RT-QuIC as a diagnostic test for sCJD. Drs Ritchie, Knight, Smith and Molesworth will provide clinical, neuropathological and epidemiological support. Potential impact/influence on the relevant policy field, users and wider stakeholders This project will develop and validate in vitro and in vivo approaches to distinguishing between vCJD and sCJD that can be applied to clinical, pre-clinical samples and to the Appendix III samples. In so doing it will improve clinical diagnosis, facilitate preclinical screening and inform on the uncertainties that currently surround Appendix III.

Lay abstract: In 1996 a new form of Creutzfeldt-Jakob disease, termed variant CJD or vCJD, was identified. The most likely cause of vCJD is through consumption of bovine spongiform encephalopathy (BSE) contaminated food products from the UK and Europe. Currently, 178 cases of vCJD have been reported in the UK with over 200 cases worldwide. Three of these cases have resulted from blood transfusions from donors who subsequently developed vCJD. Although reported cases of vCJD have steadily decreased since a peak in 2001 with the last case reported in 2016; retrospective analysis of anonymised fixed appendix samples has discovered the presence of abnormal PrP (the infectious agent) indicating asymptomatic infection. Analysis of the anonymised appendix samples (appendix-III study), suggest that the number of the asymptomatic vCJD carriers in the UK might be approximately 1 in 4000. Understanding this discrepancy between numbers of clinical and asymptomatic individuals is important because it tells us whether there is a threat of further vCJD transmission between people by blood transfusion and what precautions should be put in place to prevent it. Our project aims to a) investigate the nature of the abnormal PrP found in these appendices and b) develop blood tests that may identify those samples that could pose a risk of transmitting vCJD. We aim to inoculate brain tissue and spleen from mice infected with vCJD and sCJD and from human vCJD appendices which have been treated and fixed in the same way as the Appendix III samples into a panel of mice. This will allow us to study nature of the abnormal PrP found in human appendices and whether they are able to transmit disease. The aim is to subsequently undertake these studies with the positive appendices (from the three appendix surveys performed) to see if they too contain infectivity, and if so, determine whether it is vCJD or sCJD. PMCA and RT-QuIC are laboratory techniques that can amplify the small amounts of abnormal PrP found in biological fluid and/or tissue. The two technologies are cheap, fast, and will be used to examine appendix tissue from the above transmission studies and to develop potential diagnostic and screening tests for vCJD in biological fluids such as blood, urine and cerebrospinal fluid.

Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL