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Colloquium
Transmission of prions
C. Weissmann*, M. Enarit, P.-C. Klohn, D. Rossit, and E. Flechsig
Medical Research Council Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1 N 3BG,
United Kingdom
The "protein only" hypothesis states that the infectious agent
causing transmissible spongiform encephalopathies is a conforma-
tional isomer of PrP, a host protein predominantly expressed in
brain, and is strongly supported by many lines of evidence. Prion
diseases are so far unique among conformational diseases in that
they are transmissible, not only experimentally but also by natural
routes, mainly by ingestion. A striking feature of prions is their
extraordinary resistance to conventional sterilization procedures,
and their capacity to bind to surfaces of metal and plastic without
losing infectivity. This property, first observed in a clinical setting,
is now being investigated in experimental settings, both in animals
and in cell culture.
transmissible spongiform encephalopathies (TSEs), or prion
~ diseases, are degenerative disorders of the central nervous
system leading to motor dysfunction, dementia, and death. Prion
diseases include scrapie of sheep, bovine spongiform encepha-
lopathy (BSE) in cattle, and human diseases such as Creutzfeldt-
Jakob disease (CJD), Gerstmann-Straussler-Scheinker syn-
drome (GSS) and fatal familial insomnia (FFI). More recently,
variant CJD (vCJD), ascribed to consumption of BSE-
contaminated products (1), has claimed over 120 victims. Nei-
ther humoral nor cellular immunological responses have been
detected in prion diseases.
Transmissibility of scrapie was first demonstrated in 1939 (2~.
The remarkable resistance of the causative agent, later desig-
nated prion, was revealed early on when 10% of a flock of
Scottish sheep came down with scrapie after having been
injected with a vaccine against looping ill prepared from form-
aldehyde-treated sheep brain extract (3~. The agent's unusual
resistance to UV irradiation suggested that it might be devoid of
nucleic acid (44. The "protein only" hypothesis (5) in its updated
version (6) proposes that the prion is a conformational isoform
of the normal host protein prpC (7, 8), which is found predom-
inantly on the outer surface of neurons, attached by a glyco-
sylphosphatidylinositol (GPI) anchor. The abnormal conformer,
when introduced into the organism, is thought to cause the
conversion of prpC into a likeness of itself.
In prion disease, a largely pro/ease-resistant, aggregated form
of PrP designated PrPSc, accumulates, mainly in brain. It is
believed to be the principal or only constituent of the prion (6~.
No differences in the primary structure of prpC and PrPSc were
detected, suggesting that they differ in their conformation (9~.
The tertiary structure of prpC has been elucidated (10), whereas
that of PrPSC has not; however, the I3-sheet content of PrPSC was
shown to be high whereas that of prpC is low (11, 12~. The
conclusion that some form of PrP is the essential, perhaps only,
constituent of the infectious agent is based on compelling
biochemical and genetic evidence (13, 14~. The finding that PrP
knockout (Prnp/) mice are completely protected against
scrapie disease and fail to propagate prions (15, 16) and that
introduction of murine Prnp transgenes into these mice restores
susceptibility to prions (17) is one of the main sunnorts for the
"protein only" hypothesis.
- ~-r
16378-16383 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4
Within the framework of the "protein only" hypothesis, the
"refolding model" (Fig. L4) proposes that prpC unfolds to some
extent and refolds under the influence of a PrPSc molecule and
that the two states are separated by an activation energy barrier
(18~. The "nucleation model" (Fig. 1B) postulates that prpC is in
equilibrium with PrPSc (or a precursor thereof), that the equi-
librium is largely in favor of PrPC, and that PrPSc is stable only
when it forms a multimer. Once such a multimer or seed is
present, monomer addition ensues rapidly (19~. "Breakage" of
aggregates must be postulated to explain the exponential in-
crease of PrPSc during infection (20~. Conversion in vitro of
prpc to a PrPSc-like product has been achieved by incubating
35S-labeled prpC with PrPSc and demonstrating the appearance
of a pro/ease-resistant radioactive product with the mobility of
pro/ease-treated authentic PrPSc (21~. This in vitro conversion
exhibits the species specificity (22) and strain-specificity (23)
observed in vivo. However, because the yield is less than stoi-
chiometric with regard to the PrPSc used as seed, it has not been
possible to determine whether or not there was an increase in
infectivity. Perhaps the "cyclic amplification" procedure re-
ported recently will lead to this goal (24~. Although it has been
possible to convert recombinant prpC into a ,B-sheet-rich, par-
tially pro/ease-resistant structure by physico-chemical proce-
dures (25, 26), there have so far been no reports that such
material gives rise to transmissible prion disease (27-29~. Also,
it has so far not been possible to renature completely denatured
prion preparation to an infectious state (30, 31) although the
infectivity of partially inactivated material can be increased by
renaturation under certain conditions (32, 334. Prusiner and his
colleagues have reported that intracerebral injection of a syn-
thetic 55-residue peptide corresponding to region 89-143 of
mouse PrP with a PlOlL substitution can induce neurological,
prior-like disease, however this is achieved only in transgenic
mice expressing PrP with the same mutation (29~. The caveats
here are that these transgenic mice show spontaneous disease
even without inoculation, albeit only much later, and that
transmissibility has yet to be demonstrated.
"Natural" Transmission of Prions
Although prion diseases are not contagious in the strict sense,
i.e., by direct contact, they are transmissible perorally and
parenterally. The BSE epidemic that emerged in the mid-eighties
This paper results from the Arthur M. Sackier Colioquium of the Nationai Academy of
Sciences, "Self-Perpetuating Structurai States in Bioiogy, Disease, and Genetics," heicl
March 22-24, 2002, at the Nationai Acaclemy of Sciences in Washington, DC.
Abbreviations: BSE, bovine spongiform encephaiopathy; CJD, Creutzleiclt-]akob clisease;
vCJD, variant CJD.
*To whom reprint requests should be acldressecl. E-maii: charies.weissmann~prion.uci.
ac.uk.
"Present acldress: Nationai Cancer Center Research institute, Radiobiology Division, Tsukiji
S-~-1, Chuo-ku, Tokyo 104-0045, Japan.
"Present address: Department of Pharmacologicai Sciences, Center for Exceilence on
Neurociegenerative Diseases, University of Miian, Via Balzaretti 9, 20133 Miian, italy.
~Present adcdress: institut fur Viroiogie und Immunbioiogie, Versbacherstrasse 7, D-97078
Wurzburg, Germany.
www.pnas.org/cgi/cloi/10. ~ 073/pnas.172403799
OCR for page 3
A "Refolding" model
B '~Seeding'' model
O
very,
slow
prpc prpsc
- ,~.
PrP~ prpsc
_ ~ ~
~ ~ . ~
Fig. 1. Models for the conformational conversion of prpC to PrPS'. (A) The
"refolding" model. The conformational change is kinetically controlled, a
high activation energy barrier preventing spontaneous conversion at detect-
able rates. Interaction with exogenously introduced PrPS' causes PrPC to
undergo an induced conformational change to yield PrPS'. This reaction could
be facilitated by an enzyme or chaperone. In the case of certain mutations in
PrPC, spontaneous conversion to PrPS' may occur as a rare event, explaining
why familial CJD or GSS arise spontaneously, albeit late in life. Sporadic CJD
may come about when an extremely rare event (occurring in about one in a
million individuals per year) leads to spontaneous conversion of PrPC to PrPs'.
(B) The "seeding" model. PrPC and PrPS' (or a PrPS'-like molecule, light) are in
equilibrium, with POPE strongly favored. PrPS' is stabilized only when it adds
onto a crystal-like seed or aggregate of PrPS' (dark). Seed formation is rare;
however, once a seed is present, monomer addition ensues rapidly. To explain
exponential conversion rates, aggregates must be continuously fragmented,
generating increasing surfaces for accretion.
and led to about 18O,OOO clinically diagnosed cases (and likely to
many times more nondiagnosed ones) was fueled by the feeding
of BSE-prion-contaminated bone-and-meat meal to cattle (34~.
The kuru epidemic that developed in the first half of the 20th
century in Papua New Guinea was caused by ritualistic canni-
balism (35) and is believed to have originated from a case of
sporadic CJD. Variant CJD is thought to come about by
ingestion of BSE-prion-contaminated foodstuff, and certainly
mice (36), sheep (37), calves (38), and non-human primates (39,
40) can be experimentally infected with the BSE agent by the
oral route. It appears quite likely that sheep scrapie spreads by
ingestion of the infectious agent, although the source has not
been established; infected placenta has been suggested (41), but
scrapie-prion-contaminated feces are a likely possibility that
merits investigation. Perhaps the appearance of vCJD in pre-
dominantly young individuals is due to infection by contami-
nated foodstuff through wounds resulting from teething and
tooth loss between early infancy and adolescence. Experimental
transmission by the dental route has been shown in hamster (424.
Somehow prions make their way from the digestive tract to the
central nervous system (Fig. 2~. The relative resistance of prion
infectivity to protease digestion (43) probably allows a significant
proportion of the infectious agent to survive passage through the
digestive tract (36~. It is not clear how prions pass through the
Weissmann et a/.
BRAIN
intesU-na' PpeaYtchS
Yaseular
sYstem?9
L'cnelo'S'~'c ~ ~:Y~: ~ ~:FDC /
\ Spleen /~;~
. ~ .
Lymph
nodes
Fig. 2. Possible routes of propagation of ingested priors. After oral uptake,
prions may penetrate the intestinal mucosa through M cells and reach Peyer's
patches as well as the enteric nervous system. Depending on the host, prions
may replicate and accumulate in spleen and Iymph nodes. Myeloid dendritic
cells are thought to mediate transport within the Iymphoreticular system.
From the Iymphoreticular system and likely from other sites, prions proceed
along the peripheral nervous system to finally reach the brain, either directly
via the vagus nerve or via the spinal cord, under involvement of the sympa-
thetic nervous system.
intestinal mucosa. M cells, which are portals for antigens and
pathogens (44-46), are able to mediate transport of priors, at
least in an experimental setting (47~. Thus, after oral uptake, the
infectious agent may penetrate the mucosa through M cells and
reach Peyer's patches, where they are found early on (36) as well
as the enteric nervous system (48~. Depending on the host, other
tissues of the lymphoreticular system (LRS), in particular the
spleen but also lymph nodes (49), are sites in which prions
replicate and accumulate; this result is the case in sheep scrapie,
experimental BSE in sheep, vCJD in man, and experimental
mouse scrapie, but not BSE in cattle (50~. Recent reports suggest
that myeloid dendritic cells mediate transport within the lym-
phoreticular system (51, 52~. Interestingly, mature B cells (with
or without prpC expression) are required for amplification of
prions in spleen (53), not, however, because they themselves
harbor or multiply prions (54), but because they are required for
the maturation of follicular dendritic cells, the cells in which
prion amplification and PrPSc accumulation occur (55, 56~.
Nonetheless, neuroinvasion is possible even in the absence of
follicular dendritic cells, suggesting that other cell types in the
periphery also can amplify prions (49, 57~. From the LRS and
likely from other sites, prions proceed along the peripheral
nervous system to finally reach the brain, either directly via the
vagus nerve (58) or via the spinal cord, under involvement of the
sympathetic nervous system (594. If a sufficiently high dose of
prions is administered i.p., neuroinvasion can occur without
participation of the LRS (604. Although prions have not been
detected in muscle of scrapie or BSE-infected animals, infectiv-
ity has been found in some, but not all skeletal muscles of mice
experimentally infected with ME7 or RML prions (61~.
Not only the biosynthesis of priors, but also their spread
depends on PrP-containing cells. This result was demonstrated
by the finding that a PrP-expressing neuroectodermal graft in the
brain of a Prnp/ mouse could be infected by intracerebral
injection of mouse prions but not by intraocular (62) or i.p.
inoculation (63~. Even after irradiation and reconstitution with
a PrP-expressing lymphohemopoietic system, prions failed to
reach the graft after i.p. or i.v. inoculation, showing that neu-
roinvasion, at least in the mouse, was not mediated by prion
transport through the circulation (63) and underlining the
requirement of an interposed PrP-expressing compartment, later
shown to be the peripheral nervous system (60~. In the case of
PNAS | December 10, 2002 | vol. 99 | supp~. 4 | 16379
OCR for page 4
Steel wire (0~15 x 5 mm)
~ ,% ~
Table 1. Transient insertion of infectious wires into brains of
indicator mice
~ ~ ~ Sick/ Incubation time
~ i! . be,,
Scrapie-infected Inoculation total + SD, days
C57B/6mouse , Wires infected by exposure to scrapie brain
/ (2 months before disease)
Of Transient insertion into indicator mice
30 min 4/4* 94 + 10
120min 2/2t 87 + 113
Incubation ime
(no. sick mice/total) Permanent insertion into indicator mice
~ ale. Wires not previously inserted 3/3 71 + 2
5min I wash a/ ~9 ~ ii1 (~5/~5) Wires ahertransientinse~ionfor:
30 min 4/4 71 + 3
120 min 5/5 68 + 1
I I wash ~ ~ 65~4 Controls
30 min | ~ J ~ ~ / (6/6) Wires exposed to brain homogenate 6/6 76 + 3
I Brain homogenate (1%, 0.03 ml) 3/3 69 + 3
1 20 min
wash /~~
'I __
1 0~
69~5
(6/6)
16 h L: ~ ~ p/7)
0% brain
homogenate* ~
1% ~ 6(8/4)
*Titre: 6~8 log LD~o unitstml
Fig. 3. Transmission of mouse scrapie prions by stainless steel wire. Stainless
steel wires were inserted into the brains of scrapie-infected mice for 5, 30, or
120 min. washed exhaustively, and introduced permanently into brains of
indicator mice. Five minutes of contact sufficed for the wire to acquire a
maximum load of infectivity, equivalent to the injection of 30 Al of 1%
homogenate of the same brain. Data from ref. 73.
experimental mouse scrapie, prion infectivity could not be
detected in leukocytes (64), nor was infectivity detected in the
blood of BSE-infected cattle (50) or scrapie-infected sheep (65~.
However, a low but reproducible titer of prions was detected in
blood of scrapie-infected hamsters (66~. Also, 1 of 19 sheep
transfused with blood from experimentally, orally BSE-infected
sheep came down with prion disease (67~. The level of prions in
blood, which in all cases examined appears to be low or unde-
tectable by the mostly not very sensitive methods used, may vary
in different species and/or with different prion strains.
latrogenic Transmission of Prions
Almost 300 cases of involuntary transmission of CJD by medical
interventions have been reported (68~. Most cases are due to
injection of cadaveric human growth hormone or transplantation
of aura mater; however, a few incidents associated with cornea
transplantation have been reported. Four instances of CJD after
neurosurgical intervention have been attributed to surgical
instruments that had previously been used on CJD patients (69~;
however, causality was proven only in one case. An electrode that
had been inserted into the cortex of an unrecognized CJD
patient was subjected to a decontamination procedure involving
treatment with benzene, 70% ethanol, and formaldehyde vapor.
It was then used in succession on two young patients and cleaned
as above after each use. Within 2 yr, both patients came down
with CJD. After these events, the tip of the electrode was
implanted into the brain of a chimpanzee where it too caused
lethal spongiform encephalopathy, proving that the electrode
16380 1 www.pnas.org/cgi/doi/10.1073/pnas.172403799
Infectious wires were prepared by insertion for 5 min into scrapie-infected
mouse brain. After washing, they were inserted into brains of six deeply
anesthetized Tga20 indicator mice for the times indicated. The recovered
wires were washed and implanted into Tga20 mice. As controls, wires incu-
bated with 10% homogenate (6.8 log LD50 units/ml) of the same brain and the
homogenate itself were introduced into indicator mice. Modified from ref. 73.
*Two of six mice died on the day of the intervention.
tFour of six mice died within a day of the intervention.
had retained infectious prions over several years and despite
repeated attempts at sterilization (70, 71~.
Experimental Transmission of Surface-Bound Prions
The electrode described above had a complex structure: a steel
shaft of about 6 mm diameter, with multiple silver contacts
separated by rings of insulating plastic allowing for the existence
of crevices into which infectious material might have penetrated.
To clarify whether prions would bind to a homogeneous surface,
we used fine stainless steel wires as model for a surgical
instrument. In a first experiment, wires were incubated overnight
with brain homogenate from a terminally sick, murine-scrapie-
infected mouse, washed exhaustively with PBS, and permanently
implanted into brains of indicator mice. This procedure resulted
in scrapie disease within about 70 days, an incubation time only
slightly longer than that obtained by injecting 30 Al of 1% brain
homogenate (72~. To mimic more closely real-life conditions,
stainless steel wires were inserted directly into the brains of
scrapie-infected, clinically still healthy mice for various periods
of time, washed exhaustively, and assayed by permanent inser-
tion into brains of indicator mice. Surprisingly, 5 min of contact
sufficed for the wire to acquire a maximum load of infectivity,
equivalent to the injection of 30 Al of 1% homogenate of the
same brain (Fig. 3~. A second important question regards the
length of time an infectious wire must remain in contact with
brain tissue to initiate disease. Rather than leave the infectious
wires permanently in the indicator mouse, they were inserted
transiently, for 30 or 120 min. to mimic the conditions that might
obtain during a surgical operation. As shown in Table 1, a
contact time of 30 min was sufficient to elicit disease, albeit with
lower efficiency than was obtained after permanent insertion, as
evidenced by the longer incubation time. The wires that had been
inserted transiently into indicator mice remained fully infectious
when introduced permanently into a further set of indicator mice
(Table 1; ref. 73), reflecting the persistence of infectivity, as in
the incident with the intracerebral electrode described above.
Why are wires exposed to infected brain or brain homogenates
at least as infectious as injected homogenates, which contain far
more protein than can be bound to a wire? The surfaces of steel
and other metals tightly bind what appears to be a monolayer of
protein (74-76~. The unexpected high infectivity of steel wires
weissmann et a/.
OCR for page 5
% homogenate
Fig. 4. Infection of mouse neuroblastoma cells by plastic-bound priors.
Wells of a polystyrene 96-well microtiter plate were exposed to various
dilutions of a homogenate of scrapie-prion-infected mouse brain, washed
exhaustively, and dried. Ten thousand N2a/Bos2 mouse neuroblastoma cells
(77) were cultured in the wells for 3 days, then transferred to 24-well plates
and cultured 4 wk. splitting 1:10 twice a week. The cells were then transferred
to coverslips and assayed for the presence of PrPS' (78). Optimal infectivity
resulted when plates were coated with 0.0125% homogenate. High concen-
trations of brain proteins bound to the plastic appear inhibitory for cell B
infection (P.-C.K. and C.W., unpublished results).
could be due to selective binding of infectious particles or a
higher potency of surface bound infectivity. It has been shown
that, despite the resistance of PrPSc and scrapie infectivity to
treatment in vitro with proteinase K, prion titers in brain after
intracerebral inoculation decrease below the level of detectabil-
ity within 4 days or less (15~. On the other hand, infectious wires
left for 5 days in brain still retained infectivity (73~. Perhaps
metal-bound prions may be protected against rapid degradation
in the brain, and their apparently high specific infectivity may
therefore be due to the long persistence of relatively low levels
of infectivity. It can be mentioned in passing that prior-coated
gold wires exhibit similar infectivity intracerebrally as steel wires
(73), and that plastic surfaces, such as polystyrene (Fig. 4),
polypropylene, or polyethylene also tightly bind prions and
transmit scrapie infectivity to adherent susceptible cultured cells
(M.E., D.R., P.-C.K. and C.W., unpublished data).
We attempted to elute PrP from infectious steel wires with 2M
NaOH, but failed to detect either protein (detection limit, 50 ng
per wire) or PrP (detection limit 15 pg per wire). On the other
hand, PrP immunoreactivity could be detected at the surface of
prior-coated wires by chemiluminescence (73~. This finding
raises the question as to whether infection of brain tissue elicited
by infected wires comes about by direct contact with irreversibly
surface-bound prions or whether it is due to a slow, so far
undetected release of priors. This question is difficult to answer
experimentally; however, it would seem that intimate contact
between the prior-loaded surface and target cells is a prereq-
uisite for transmission of infectivity. Prion-coated wires were
placed on monolayers of mouse neuroblastoma cells highly
susceptible to mouse prions (77~. After 1 to 14 days, the wires,
to which some cells had adhered, were transferred onto cover-
slips in the wells of a tissue culture plate and incubated for 14
days, allowing the cells to migrate off the wire and multiply. Cells
derived from both the residual monolayer and the wire were
blotted onto nitrocellulose membranes and assayed for the
presence of pro/ease-resistant PrP, the surrogate marker of prion
infection (784. Only the cells derived from the infected wire, but
not from the residual monolayer, were PrPSc positive (Fig. 5) and
contained infectivity (M.E., E.F. and C.W., unpublished data).
This experiment shows that intimate contact between the prion-
carrying surface and susceptible cells greatly promotes infection
or is prerequisite. Similarly, cell-to-cell transmission of infectiv-
ity in cell culture is orders of magnitude more efficient than
transmission by a prion preparation (79~.
weissmann et al.
A :/Steel wire
_
_.. _
1% brain
homogenate
/
j14days
~wire bound ceils
remaining cells
immuno staining cell staining
,, sIL
,_,
Immuno-detection
f PrPSc
~,
contact time (days)
Fig. 5. Neuroblastoma cells are infected by contact with prior-coated
stainless steel wires. (A) Stainless steel wires were exposed to scrapie-infected
brain homogenates, washed, and placed on a confluent layer of neuroblas-
toma cells. After periods ranging from 1 to 14 days, the wire, to which a few
cells had attached, were placed on a coverslip in a separate well and cultured
for further 14 days. Both the cells remaining in the original dish ("remaining
cells") and those derived from the cells clinging to the wire were assayed for
PrPS' by the cell blot assay (78) and the mouse bioassay. (B Left) The cultures
derived from wire-bound cells have been infected, as evidenced by the accu-
mulation of PrPS', whereas residual cells remain uninfected. (Right) The loca-
tion of cells as revealed by ethidium bromidestaining. UN, Blankwire; UB, wire
treated with uninfected brain homogenate (M.E. and C.W., unpublished
results).
The availability of prior-coated steel wires mimicking con-
taminated surgical instruments makes it possible to assess the
efficacy of sterilization conditions on surface-bound priors.
Preliminary results (Table 2) confirm that treatment with form-
aldehyde is insufficient to sterilize infectious wires, whereas
treatment with sodium hydroxide, guanidinium thiocyanate (73),
or autoclaving at 121C for 20 min is efficacious (E.F. and C.W.,
unpublished results). It is, however, not appropriate to derive
from these experiments recommendations for the sterilization of
surgical instruments; it will first be necessary to validate the
procedures scaling up the contact surface between metal and
brain tissue and, importantly, using vCJD prions in a susceptible
host, preferably a non-human primate.
Concluding Remarks
Twenty or more diseases of humans are associated with the
deposition of I3-sheet-rich protein aggregates, or amyloid (80,
PNAS | December ~o, 2002 | vo~. 99 | supp~. 4 | 16381
OCR for page 6
Table 2. Effect of various treatments on the infectivity of
wire-bound prions
Incubation
time + SD,
Sick/total days
Uninfected wires
Untreated
Infected wires
Untreated
NaOH (1 M, 1 h, 25C)
Forma Idehyde (10%, 1 h, 25C)
Guanidinium thiocyanate (4 M, 16 h, 25C)
Autoclaving (121C, 20 min)
0/3
6/6
0/6
6/6
0/6
0/6
Modified from ref. 73 and unpublished results (E.F. and C.W.).
>260
76 + 5
>260
92 + 8
>260
>170
81~. They are frequently designated "conformational diseases"
although it is not in all cases clear whether, or to what extent, the
misfolded proteins are the cause of the disease rather than the
consequence. Prion diseases are so far unique conformational
diseases, because they are transmissible by misfolded protein,
not only under experimental conditions but also naturally,
predominantly by ingestion. Although in certain cases the in-
ception of an experimental amyloidosis can be accelerated by the
injection of amyloid into a predisposed host (82), prions are
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PNA5 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16383
Representative terms from entire chapter:
steel wires
