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CJD Update Latest facts, figures & findings Jonathan P Clewley TSE Unit, Virus Reference Division, Centre for Infections 20 May 2005 12 August 2003 Transmissible spongiform encephalopathies & prions Key Points • 1. How we got to where we are - the BSE epidemic • 2. The prion hypothesis • 3. The prion protein: PrPC and PrPSc • 4. Secondary spread of CJD - The number of future cases? • 5. Tests for BSE and CJD 1. Introduction How we got to where we are: the BSE epidemic 12 August 2003 Prevalence of neurodegenerative disorders worldwide What are the transmissible spongiform encephalopathies (TSEs)? Animal Scrapie (sheep), BSE (cattle), CWD (deer), TME (mice), FSE (cats) Human Creutzfeldt-Jakob Disease: sporadic, familial, iatrogenic, variant Gerstmann-Sträussler-Scheinker syndrome (GSS) From BSE, early 1980s to vCJD, early 2000s vCJD deaths 28 N 24 o o f 20 16 d 12 e a 8 t h 4 s 0 ‘90 ‘95 Ye ar ‘00 ‘05 Origin of the BSE epidemic 1980-1983: Change in rendering process for production of cattle feed meat & bone meal (heat & solvents down) either BSE came from sheep scrapie or BSE came from sporadic cattle TSE whichever Subsequent recycling of BSE via feed 2. The prion hypothesis The prion protein: PrPC and PrPSc 12 August 2003 Prion concepts Proteinaceous infective particle that lacks nucleic acid Pr otein + i nfectious + on Underlie inherited as well as communicable diseases Abnormal prions (PrPSc) convert normal ones (PrPC) by shape shifting Prusiner, Sci Am, 1995 Summary of differences between normal & abnormal prions Normal PrP (30-40 kDa) Abnormal PrP (27-30 kDa) Referred to as PrPC, PrPsen Referred to as PrPSc, PrPCJD, PrPres Sensitive to proteases, detergents Relatively resistant to proteinase K, detergents Consists mostly of alpha-helix and loops, monomeric Non-infectious, cell surface glycoprotein, function? Many cells & tissues Coded by PRNP Consists mostly of ß-sheet, polymeric, forms amyloid Isoform of PrPC, infectious, involved in pathogenesis CNS, spleen, lymph nodes Coded by PRNP Normal cellular PrP Normal PrPC protein PrPC PrPC in cell membrane Structures of normal and abnormal PrP Normal - PrPC Abnormal - PrPSc, monomeric Abnormal - PrPSc, trimeric Abnormal PrPSc fibrils Protein conversion of alpha PrPC to beta PrPSc Models for the conversion of PrPC to PrPSc Weismann, 2002 3. Secondary spread of CJD: The number of future cases? Tests for BSE and CJD 12 August 2003 The need for ante-mortem diagnostic tests for CJD infection 149 vCJD deaths 1995-2005 - How many more cases? • By statistics, epidemic is declining • By tissue surveys, thousands incubating disease • Codon 129 M/V genotype Secondary spread of vCJD - Blood transfusion, surgery, sex? other? Codon 129 M/V polymorphisms (%) Evidence for transfusion (& other) transmission of vCJD Level of Evidence Evidence Year Biological models Oral transmission 1996 Prions in lymphoid tissue 1997 Mouse model 1998 Sheep transfusion 2000 BSE to primates 2004 Case reports Human transmission 2004 Cohort studies etc. None ---- Animal evidence Wilson & Ricketts Lancet ‘04 Blood transfusion acquired vCJD & preclinical vCJD Of 48 people who received blood from 15 donors who went on to develop vCJD: Case 1 Recipient died of vCJD 7.5 years after donation Donor developed vCJD 10 years after donation Case 2 Donor died of vCJD 2 years after donation Recipient died of an anuerysm (not CJD) 5 years after donation On autopsy, evidence of PrPres in spleen Patient was M/V at codon 129 of PRNP Both were non-leucodepleted donations Llewelyn et al, 2004; Peden et al, 2004 vCJD diagnostic problems Diagnosis is by clinical criteria, & by western blotting & immunohistochemistry at PM There are no known preclinical serological or PCR markers of infection As there are EIAs for animal TSEs, why aren’t there any for CJD? Western blots & EIAs for BSE, scrapie and CWD from Enfer, Bio-Rad, Iddex & Prionics, typically use brain stem & obex homogenates But Animals are killed or already dead WB/EIA up to a billion times less sensitive than PCR What laboratory diagnostic tests are there for CJD? Western blot ELISA, DELFIA, CDI (in development) Capillary electrophoresis?? Immunopathology: detection of PrPSc in brain, tonsil, appendix Surrogate markers: 14-3-3 protein; various mRNAs (e.g. EDRF); molecular profiles in blood by Fourier transform infrared spectroscopy Towards the development of a pre-mortem EIA for CJD Detection of PrPSc • Use proteinase K (to remove PrPC) & a sensitive reporter system • e.g. DELFIA, FCS, immuno-PCR, tadpoles • Use little or no proteinase K but use denaturation to distinguish PrPC and PrPSc - the conformational dependent immunoassay (CDI) • Capture aggregates of PrPSc (amyloid fibrils) • Protein misfolding cyclic amplification (PMCA) • PrPSc specific antibodies Why is it hard to get antibodies that can distingush between PrPC and PrPSc? Antibodies do not distinguish between PrPC and PrPSc • PrPC and PrPSc have the same amino acid composition • PrPSc is aggregated, of low immunogenicity and has few exposed specific epitopes • PrPC is present at high levels in the body - & so may swamp antibody recognition of PrPSc What sensitivity is needed for PrPCJD detection e.g. compared with HBsAg EIA? • HBV sAg MW = 25,476 Da, 226 aa • PrP MW = 21,000 Da, 208 aa HBsAg assays • Can detect down to 0.1 ng/ml (if 75 ul serum added to assay = 7.5 pg = 107 molecules) Prion infectivity • 5 fg = 104-105 molecules in vitro Therefore, >1,000 increase in sensitivity of EIAs needed for PrPCJD detection Finally: Important points & key messages • 149 vCJD deaths 1995-2005 - How many more cases? In UK? (tonsil archive at HPA may help answer this), VV & MV cases? Elsewhere? • 649 other CJD deaths 1995-2005 • Diagnosis is by clinical criteria & at post-mortem • There are commercial in vitro laboratory diagnostic tests for BSE, CWD and scrapie, but none for CJD - The animal tests are after death - There is no test for asymptomatic disease applicable to blood • vCJD may be transmissible by blood i.e. secondary cases? • Can other animal TSEs cause human disease? - Atypical scrapie, is BSE in sheep, BASE, CWD? • Pathological prion protein (PrPSc/PrPCJD) is found in muscle • What are the prospects for treatment? The End Thank you for listening 12 August 2003 Outstanding question? Is abnormal PrP the transmissible agent? For Irradiation target size too small for a virus; no virus found Amyloid fibrils induce polymerisation of precursors Against Could be a small virus; prion strains Amyloid diseases not transmissible by fibrils No infectivity in vitro ? PrP-sen to PrP-res in vitro PrP merely a co-factor in PrP-null mice Infectivity in vitro ? Retroviruses are genetic & infectious No disease in PrP-null mice No transmission or +ve WB in transgenic mice Genetic & infectious PrP mutations Over-expression of mutant PrP in transgenic mice causes disease Caughey & Chesebro, 1997 Histology and immunohistochemistry of a vCJD case Cortex Haematoxylin & eosin Spleen Ironside & Head, 2004 Anti-PrP antibody & haematoxylin Anti-PrP antibody & haematoxylin Tonsil PrPres analysis of spleen by western blot Peden et al, 2004 Surgical incidents reported to the CJD incidents panel CDR May 2005 NHS trusts sending tonsils to the NATA CDR May 2005 Tonsils collected for the NATA CDR May 2005