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: PrPC and PrPSc

• 4. Secondary spread of CJD The number of future cases?

• 5. Tests for BSE and CJD

12 August 2003

1. 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

28

24

20

16

12

8

4

0

No of deaths

Year

‘90‘00‘05‘95vCJD deaths

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 scrapieor

BSE came from sporadic cattle TSEwhichever

Subsequent recycling of BSE via feed

12 August 2003

2. The prion hypothesis

The prion protein: PrPC and PrPSc

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)

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

Normal cellular PrP

Normal PrPCPrPC protein

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

Weismann, 2002

Models for the conversion of PrPC to PrPSc

12 August 2003

3. Secondary spread of CJD:The number of future cases?

Tests for BSE and CJD

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?

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 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 ----

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 donationCase 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?

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 & eosin Anti-PrP antibody & haematoxylinCortex

Ironside & Head, 2004

Spleen 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