12 august 2003 cjd update latest facts, figures & findings jonathan p clewley tse unit, virus...
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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
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