Transcript HPA Presentation 2

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
v CJD deaths
28
N
24
o
o
f
20
16
d 12
e
a
8
t
h
4
s
0
‘90
‘95
Year
‘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