12 August 2003 CJD Update Latest facts, figures & findings Jonathan P Clewley TSE Unit, Virus Reference Division, Centre for Infections 20 May 2005

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: PrP C and PrP Sc 4. Secondary spread of CJD ­The number of future cases? 5. Tests for BSE and CJD

12 August Introduction How we got to where we are: the BSE epidemic

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

Origin of the BSE epidemic : 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

12 August The prion hypothesis The prion protein: PrP C and PrP Sc

Prion concepts Proteinaceous infective particle that lacks nucleic acid Pr otein + i nfectious + on Underlie inherited as well as communicable diseases Abnormal prions (PrP Sc ) convert normal ones (PrP C ) by shape shifting Prusiner, Sci Am, 1995

Summary of differences between normal & abnormal prions Normal PrP (30-40 kDa) Referred to as PrP C, PrP sen 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 PrP Sc, PrP CJD, PrP res Relatively resistant to proteinase K, detergents Consists mostly of ß-sheet, polymeric, forms amyloid Isoform of PrP C, infectious, involved in pathogenesis CNS, spleen, lymph nodes Coded by PRNP

Normal cellular PrP Normal PrP C PrP C protein PrP C in cell membrane

Structures of normal and abnormal PrP Normal - PrP C Abnormal - PrP Sc, monomeric Abnormal - PrP Sc, trimeric Abnormal PrP Sc fibrils

Protein conversion of alpha PrP C to beta PrP Sc

Weismann, 2002 Models for the conversion of PrP C to PrP Sc

12 August Secondary spread of CJD: The number of future cases? Tests for BSE and CJD

149 vCJD deaths ­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? The need for ante-mortem diagnostic tests for CJD infection

Codon 129 M/V polymorphisms (%)

Evidence for transfusion (& other) transmission of vCJD Wilson & Ricketts Lancet ‘04 Level of EvidenceEvidenceYear Biological modelsOral transmission1996 Prions in lymphoid tissue1997 Animal evidenceMouse model1998 Sheep transfusion2000 BSE to primates2004 Case reportsHuman transmission2004 Cohort studies etc.None ----

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 PrP res 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 PrP Sc in brain, tonsil, appendix Surrogate markers: 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 PrP Sc Use proteinase K (to remove PrP C ) & a sensitive reporter system e.g. DELFIA, FCS, immuno-PCR, tadpoles Use little or no proteinase K but use denaturation to distinguish PrP C and PrP Sc ­the conformational dependent immunoassay (CDI) Capture aggregates of PrP Sc (amyloid fibrils) Protein misfolding cyclic amplification (PMCA) PrP Sc specific antibodies

Why is it hard to get antibodies that can distingush between PrP C and PrP Sc ? Antibodies do not distinguish between PrP C and PrP Sc PrP C and PrP Sc have the same amino acid composition PrP Sc is aggregated, of low immunogenicity and has few exposed specific epitopes PrP C is present at high levels in the body ­& so may swamp antibody recognition of PrP Sc

What sensitivity is needed for PrP CJD 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 = 10 7 molecules) Prion infectivity 5 fg = molecules in vitro Therefore, >1,000 increase in sensitivity of EIAs needed for PrP CJD detection

Finally: Important points & key messages 149 vCJD deaths ­How many more cases? In UK? (tonsil archive at HPA may help answer this), VV & MV cases? Elsewhere? 649 other CJD deaths 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 (PrP Sc /PrP CJD ) is found in muscle What are the prospects for treatment?

12 August 2003 The End Thank you for listening

Outstanding question? Is abnormal PrP the transmissible agent? 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 Caughey & Chesebro, 1997 For Against

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

PrP res 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