CJD Update

1. CJD Update Latest facts, figures & findings Jonathan P Clewley TSE Unit, Virus Reference Division, Centre for Infections 20 May 2005

2. 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

3. 1. Introduction How we got to where we are: the BSE epidemic

4. Prevalence of neurodegenerative disorders worldwide

5. 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)

6. From BSE, early 1980s to vCJD, early 2000s

7. 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

8. 2. The prion hypothesis The prion protein: PrPC and PrPSc

9. Prion concepts • Proteinaceous infective particle that lacks nucleic acid • Protein + infectious + on • Underlie inherited as well as communicable diseases • Abnormal prions (PrPSc) convert normal ones (PrPC) by shape shifting Prusiner, Sci Am, 1995

10. Normal PrP (30-40 kDa) Referred to as PrPC, PrPsen Sensitive to proteases, detergents Consists mostly of alpha-helix and loops, monomeric Non-infectious, cell surface glycoprotein, function? Many cells & tissues Coded by PRNP Abnormal PrP (27-30 kDa) Referred to as PrPSc, PrPCJD, PrPres Relatively resistant to proteinase K, detergents Consists mostly of ß-sheet, polymeric, forms amyloid Isoform of PrPC, infectious, involved in pathogenesis CNS, spleen, lymph nodes Coded by PRNP Summary of differences between normal & abnormal prions

11. Normal cellular PrP PrPC protein Normal PrPC PrPC in cell membrane

12. Structures of normal and abnormal PrP Normal - PrPC Abnormal - PrPSc, monomeric Abnormal PrPSc fibrils Abnormal - PrPSc, trimeric

13. Protein conversion of alpha PrPC to beta PrPSc

14. Models for the conversion of PrPC to PrPSc Weismann, 2002

15. 3. Secondary spread of CJD:The number of future cases? Tests for BSE and CJD

16. 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?

17. Codon 129 M/V polymorphisms (%)

18. Evidence for transfusion (& other) transmission of vCJD • Level of Evidence Evidence Year • Biological models Oral transmission 1996 • Prions in lymphoid tissue 1997 • Animal evidence Mouse model 1998 • Sheep transfusion 2000 • BSE to primates 2004 • Case reports Human transmission 2004 • Cohort studies etc. None ---- Wilson & Ricketts Lancet ‘04

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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?

27. The End Thank you for listening

28. Irradiation target size too small for a virus; no virus found Amyloid fibrils induce polymerisation of precursors PrP-sen to PrP-res in vitro Infectivity in vitro ? No disease in PrP-null mice Genetic & infectious PrP mutations Over-expression of mutant PrP in transgenic mice causes disease Could be a small virus; prion strains Amyloid diseases not transmissible by fibrils No infectivity in vitro ? PrP merely a co-factor in PrP-null mice Retroviruses are genetic & infectious No transmission or +ve WB in transgenic mice Outstanding question?Is abnormal PrP the transmissible agent? For Against Caughey & Chesebro, 1997

29. Histology and immunohistochemistry of a vCJD case Cortex Haematoxylin & eosin Anti-PrP antibody & haematoxylin Spleen Anti-PrP antibody & haematoxylin Tonsil Ironside & Head, 2004

30. PrPres analysis of spleen by western blot Peden et al, 2004

31. Surgical incidents reported to the CJD incidents panel CDR May 2005

32. NHS trusts sending tonsils to the NATA CDR May 2005

33. Tonsils collected for the NATA CDR May 2005