Nature Reviews Immunology | AOP, published online 5 November 2013; doi:10.

1038/nri3553 REVIEWS

The immunobiology of prion diseases

Adriano Aguzzi1, Mario Nuvolone1,2 and Caihong Zhu1
Abstract | Individuals infected with prions succumb to brain damage, and prion infections
continue to be inexorably lethal. However, many crucial steps in prion pathogenesis occur
in lymphatic organs and precede invasion of the central nervous system. In the past two
decades, a great deal has been learnt concerning the cellular and molecular mechanisms
of prion lymphoinvasion. These properties are diagnostically useful and have, for example,
facilitated preclinical diagnosis of variant CreutzfeldtJakob disease in the tonsils.
Moreover, the early colonization of lymphoid organs can be exploited for post-exposure
prophylaxis of prion infections. As stromal cells of lymphoid organs are crucial for
peripheral prion infection, the dedifferentiation of these cells offers a powerful means
of hindering prion spread in infected individuals. In this Review, we discuss the current
knowledge of the immunobiology of prions with an emphasis on how basic discoveries
might enable translational strategies.

Protein-only hypothesis
Introduced by Griffith and
formally enunciated by
Prusiner, it states that prions
are unconventional infectious
agents that are devoid of
informational nucleic acids and
that uniquely consist of an
infectious, pathogenic protein.
Prion
The aetiological agent of
prion disease; prion is short
for proteinaceous infectious
particle.
1
Institute of Neuropathology,
University Hospital of Zurich,
Schmelzbergstrasse 12,
CH8091 Zurich, Switzerland.
2
Amyloidosis Research and
Treatment Centre, Foundation
Istituto di Ricovero e Cura a
Carattere Scientifico San
Matteo Hospital and
Department of Molecular
Medicine, University of Pavia,
Institute for Advanced Study,
Pavia I-27100, Italy.
Correspondence to A.A.
email: adriano.aguzzi@usz.ch
doi:10.1038/nri3553
Published online
5 November 2013
Diseases caused by prions are fatal neurodegenera-
tive conditions that affect humans and several other
mammals (TABLE1). The transferral of brain extracts
from affected individuals into permissive host spe-
cies can transmit the disease 1. Transmission among
humans occurred during the kuru epidemic in Papua
New Guinea through cannibalistic rituals2. In addi-
tion, more than 450 cases of iatrogenic Creutzfeldt
Jakob disease (iCJD) have occurred following pituitary
hormone treatment or surgical procedures3. Finally,
bovine spongiform encephalopathy (BSE) has affected
more than180,000 cattle worldwide (see the BSE Portal
on the World Organisation for Animal Health website)
and has caused variant CJD (vCJD) in humans4. vCJD
was shown to be transmitted through blood or blood
derivatives, even from subclinical donors5,6.
According to the protein-only hypothesis, the infec-
tious agent that is, the prion itself consists of scrapie
prion protein (PrPSc), which is an assembly of conform-
ers of cellular prion protein (PrPC)7. A PrPSc aggregate
can recruit PrPC proteins and can perpetuate its own
amplification7 in a similar way to crystal growth and
fragmentation8. When this cycle occurs within the cen-
tral nervous system (CNS) and involves membrane-
anchored PrPC at the neuronal surface, a neurotoxic
signal is triggered, plausibly through PrPC itself 9. This
results in the typical spongiform changes that are seen
in diseased brains. The recognition of the infectious
potential of prion diseases resulted in their designation
as transmissible spongiform encephalopathies (TSEs).
Prion diseases are interesting to immunologists
for three main reasons. First, numerous studies have
suggested that there are physiological roles for PrPC
in cells of the immune system10, which suggests that
clarifying such roles might help us to understand
the molecular mechanisms of prion pathogene-
sis. However, the physiological roles of PrPC in the
immune system, and elsewhere, remain unclear 10.
Second, the immune system has a crucial role in prion
pathogenesis: prions can escape immune surveillance,
colonize the immune system of their hosts, hijack
immune components (a stage known as peripheral
replication) and gain access to the CNS (the neuroin-
vasion stage). Although our understanding of the
underlying peripheral replication and neuroinvasion
stages is advanced, we lack a similar comprehension
of the mechanisms underlying prion toxicity after the
invasion of the CNS. Neuroimmunological phenomena
also have an important role in this final phase of prion
diseases11. Third, manipulation of the immune system
might represent a valid therapeutic strategy for prion
diseases12. Indeed, current antiprion interventions have
prominently focused on immunological strategies,
including immunoprophylactic, immunosuppressive
and immunostimulatory approaches12.
In this Review, we discuss the current state of prion
immunobiology. We examine the role of PrPC in the
immune system, the mechanisms of peripheral prion
replication and neuroinvasion by prions, and the neuro
inflammatory changes that are associated with prion

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Table 1 | Prion diseases*
Disease
Sporadic CJD
Iatrogenic CJD
Familial CJD
Variant CJD
Kuru
Fatal familial insomnia
Sporadic fatal insomnia
GerstmannStrussler
Scheinker syndrome
Scrapie
Atypical scrapie
Chronic wasting disease
BSE
Atypical BSE
Feline spongiform
encephalopathy
Transmissible mink
encephalopathy
Spongiform encephalopathy Zoological ungulates
of zoo animals and bovids
BSE, bovine spongiform encephalopathy; CJD, CreutzfeldtJakob disease; PRNP, gene encoding prion protein; PrPC, cellular prion protein. *Data from REF.12.One
case of somatic mosaicism175.
Scrapie prion protein
(PrPSc). The pathological
version of prion protein that is
present in the central nervous
system and other tissues of
patients with transmissible
spongiform encephalopathies.
It is believed to differ from
cellular PrP only in terms of
post-translational
modifications.
Cellular prion protein
(PrPC). The physiological
version of prion protein, which
is present in the central
nervous system and other
tissues under normal
circumstances.
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Natural host species Route of transmission or Other susceptible species
disease-induction mechanism
Humans Unknown Primates, hamsters, guinea pigs, bank voles, humanized
and chimeric humanmouse transgenic mice, and
wild-type mice
Humans Accidental medical exposure Primates, humanized and chimeric humanmouse
to CJD-contaminated tissues, transgenic mice, and wild-type mice
hormones or blood derivatives
Humans Genetic (germline PRNP Primates, bank voles, chimeric humanmouse
mutations) transgenic mice and wild-type mice
Humans Genetic (germline PRNP Primates, guinea pigs, humanized transgenic mice and
mutations) wild-type mice
Humans Ritualistic cannibalism Primates and humanized transgenic mice
Humans Genetic (germline PRNP Humanized and chimeric humanmouse transgenic
mutations) mice, and wild-type mice
Humans Unknown Chimeric humanmouse transgenic mice
Humans Genetic (germline PRNP Primates, guinea pigs, mutated Prnp transgenic mice
mutations) and wild-type mice
Sheep, goat and Horizontal and possibly vertical Primates, elk, hamsters, raccoons, bank voles, ovinized
mouflon transgenic mice (which express sheep PrPC) and
wild-type mice
Sheep and goat Unknown Ovinized transgenic mice and porcinized transgenic
mice (which express pig PrPC)
Mule deer, white-tailed Horizontal and possibly vertical Primates, ferrets, cattle, sheep, cats, hamsters, bank
deer, Rocky Mountain voles, cervidized transgenic mice (which express deer
elk and moose PrPC) or murine Prnp-overexpressing transgenic mice
Cattle Ingestion of BSE-contaminated Primates, guinea pigs, humanized and bovinized
food transgenic mice, and wild-type mice
Cattle Unknown Primates, humanized and bovinized transgenic mice,
and wild-type mice
Zoological and Ingestion of BSE-contaminated Wild-type mice
domestic felids food
Farmed mink Ingestion of BSE-contaminated Primates, cattle, hamsters and raccoons
food
Ingestion of BSE-contaminated Wild-type mice
food
diseases. We also address the immune responses that different viruses and in the modulation of neuroinflam-
can be initiated against prions and the immunologi- matory changes (reviewed in REF.10) (BOX2). Recently,
cal intervention strategies under investigation for the PrPC has been implicated in the pluripotency and differ-
treatment of prion diseases. entiation of embryonic stem cells15, in intestinal barrier
function16 and in the uptake of B.abortus in intestinal
Physiological functions of PrPC microfold cells (M cells)17. None of these functions has
Immune functions of PrPC. Mice represent a power- been unambiguously elucidated at a molecular level and
ful experimental model for prion research (BOX 1). conflicting results have often been reported.
Despite the availability of mice that are deficient in PrP We have recently identified a crucial caveat to the
(encoded by Prnp) since 1992 (REF.13), the elucidation above claims. All currently available Prnp/ mouse lines
of the physiological functions of PrPC is rudimentary. In were generated in embryonic stem cells from the 129
peripheral nerves, PrPC contributes to myelin mainte- mouse strain. Hence, any loci that are linked to Prnp and
nance14. Many other functions have also been ascribed that are polymorphic between 129 and the backcross-
to PrPC, including immunological ones10. It has been ing strain may represent a confounder when comparing
suggested that PrPC is involved in Tcell development, Prnp/ and Prnp+/+ mice. For example, the polymorphic
activates and interacts with dendritic cells (DCs), inhib- signal regulatory protein- (Sirpa) is linked to Prnp and
its phagocytosis in macrophages and contributes to Prnp/ mice were shown to carry the Sirpa allele from
haematopoietic stem cell self-renewal. Similarly, PrPC the 129 strain (Sirpa129) despite extensive backcross-
has been shown to be involved in the internalization ing18. Indeed, the increased phagocytosis of apoptotic
of Brucella abortus in macrophages, in the replication of cells, which was previously reported in Prnp/ mice and
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Prion diseases
Also known as transmissible
spongiform encephalopathies
(TSEs). These are a group of
transmissible
neurodegenerative diseases
that affect humans and various
mammals.
Prion strains
Natural sources or isolates of
prions that, when inoculated
into genetically homogeneous
hosts, induce a prion disease
with peculiar clinical,
histological and biochemical
features.
Propagons
Proteinaceous aggregates that
are capable of seeding a
self-perpetuating reaction of
templated nucleation within a
biological system. Propagons
are not necessarily identical to
scrapie prion proteins but
might represent a subset of
prion protein conformations,
some of which might not be
resistant to proteolysis.
Propagons could, in principle,
have specific post-translational
modifications.
Prionoids
Self-aggregating proteins that
are capable of transmitting
between cells within one
organism, but not from one
organism to another.
Amyloid, tau, huntingtin and
amyloid A protein are
examples of prionoids.
Synuclein was thought to be a
prionoid, but recent evidence
suggests that it might behave
like a bonafide prion.
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Box 1 | Mice in prion research
Small rodents, including mice, can be infected with prions from various natural sources. Prion inoculation of mice
results in bonafide transmissible spongiform encephalopathies (TSEs). Therefore, prion-infected mice have represented
and still represent an important paradigm to increase our understanding of prion biology.
Early studies identified quantitative trait loci (QTLs) that influence the incubation period of scrapie in mice. These QTLs were
later shown to coincide with Prnp, which is the gene encoding cellular prion protein (PrPC)157. Prnp/ mice, which lack PrPC,
were shown to be healthy13 and resistant to prion infection59 and propagation158. Prnp+/ mice had longer prion incubations
than wild-type mice158, which indicates that Prnp gene dosage controls the speed of disease onset.
Numerous Prnp/ strains have been generated. Although most of the Prnp/ strains seem to be essentially healthy, some
were shown to develop a neurodegenerative disease of the cerebellum159. However, it was found that the disease was
caused by the incidental upregulation of the prion protein dublet prion-like protein doppel (Prnd) gene, which is
located immediately downstream of Prnp160.
Several subtle phenotypes have been reported in Prnp/ mice, with inconsistencies across different laboratories and
mouse lines. Differences in genetic background, gut microbiota and experimental methodologies might explain some
of the reported incongruities.
Reciprocal brain-graft experiments between mice lacking or expressing PrPC (REF.9) and mice with a conditional
deletion of Prnp161 showed that neuronal PrPC mediates prion toxicity.
PrPC-overexpressing mice show accelerated disease, thus expediting bioassays to determine prion titres162,163.
Transgenic mice expressing xenogeneic Prnp sequences can display lowered species-transmissibility barriers, thus
facilitating transmission studies of prions from these species164,165.
Certain deletion mutants of PrPC induce neurodegeneration, which is rescued by wild-type PrPC, confirming that PrPC
can mediate neurotoxicity166.
Finally, mice expressing select point mutants of PrPC spontaneously form prions167, corroborating the protein-only
hypothesis.
which was initially attributed to the absence of Prnp, patches before colonizing the spleen22. Accordingly, sus-
has recently been shown instead to be caused by differ- ceptibility to prion infection following oral challenge in
ences in the Sirpa alleles, indicating that the inhibition mice positively correlates with the number of Peyers
of phagocytosis was previously misattributed to PrPC patches that are present in the small intestine23. These
(REF.19). observations suggest that the follicle-associated epithe-
lium (FAE) of the Peyers patches is a plausible prion
PrPC to PrPSc conversion. PrPC and PrPSc differ in their entry site. Another indication that immune mechanisms
tertiary and quaternary structure. Although PrPC contains are implicated in this process comes from the observation
mostly disordered and helical structures, PrPSc has a that experimentally induced bacterial colitis enhanced
high sheet content20. The supramolecular arrangement prion susceptibility on oral exposure24.
of PrPSc is thought to determine the specific features of
different prion strains. PrPC itself is an innocuous constitu- Entry of prions into Peyers patches. Scattered and
ent of many cell types. On infection, the disease-causing intercalated between classical enterocytes, M cells
PrPSc functions as a template that incorporates PrPC into continuously sample the intestinal lumen to facilitate
aggregates. The conversion might require additional chap- immunosurveillance. This property can be hijacked
eroning molecules, such as lipids21. This self-perpetuating by several pathogens, including prions, to invade the
feature of prion propagons is shared by several other pro- intestinal mucosa. Coculture systems using differ-
teins, some of which (termed prionoids) were found to entiated cell lines with morphological and functional
transmit within, but not necessarily between, individuals. features of bonafide M cells showed efficient trans
cytosis of prions25. These observations were corrobo-
Prion entry sites rated by invivo studies that showed that M cells can
Routes of infection. With the exception of the unfortunate uptake orally administered prions and that oral prion
cases in which prions have been inadvertently introduced pathogenesis can be inhibited when M cells are depleted
into the brain3, in acquired prion diseases the infectious through administration of receptor activator of NFB
agent replicates in the periphery before reaching the ligand (RANKL; also known as TNFSF11)26. Although
brain. But how do prions spread from their portals of Mcells have been indicated in prion transport, alter-
entry? The answer crucially depends on the type of expo- native mechanisms, including transcytosis of prions
sure (FIG.1). For oral exposure, which is the most relevant through enterocytes, could also take place27.
route for acquired prion diseases in nature, prions must After crossing the FAE, prions spread possibly
first cross the wall of the digestive tract. Prions resist through a cell-mediated mechanism. Follicle-associated
exposure to digestive enzymes, and gastric acidity affords lymphocytes are unlikely to be involved in this pro-
only limited protection against oral prion challenge. cess23. Macrophages have been shown to inactivate
Intragastric inoculation of prions in mice led to prion prion infectivity invitro28, but residual infectivity could
disease, and prions showed rapid accumulation in Peyers persist within these cells. Bisphosphonate-mediated
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Box 2 | PrPC as a receptor for amyloid oligomers

A genome-wide unbiased screen has identified cellular prion protein (PrPC) as a potential receptor for amyloid
oligomers, which are believed to be the main cause of Alzheimers disease pathogenesis168. This report generated much
attention because it implied that similar mechanisms might be involved in prion disease and Alzheimers disease, as in
both conditions PrPC seems to be the key mediator of toxicity. Binding of oligomeric amyloid to PrPC results in a poorly
understood signalling pathway, which possibly includes the phosphorylation of FYN, microtubule-associated protein
tau (MAPT) and the N-methyl-d-aspartate receptor subunit 2B, ultimately leading to amyloid-mediated
neurotoxicity168,169. Genetic ablation of PrPC or the administration of PrPC-specific antibodies or PrPC-mimetic
compounds that can interfere with PrPCamyloid binding reduces or prevents amyloid-mediated toxicity in a
plethora of invitro and invivo experimental systems168171. However, in other situations, PrPC was shown to be
dispensable for amyloid-mediated toxicity172174, which indicates a more complex scenario. Although differences in
experimental conditions might explain some of the inter-experimental variation, the discrepancy observed in
amyloid-mediated toxicity could reflect a context-dependent involvement of PrPC in amyloid-dependent
neurotoxicity, and this deserves further analysis. Currently, it is unclear whether interference with PrPCamyloid
binding is of any therapeutic value for Alzheimers disease.

macrophage depletion in mice that were challenged Peripheral replication of prions

orally or intraperitoneally with prions resulted in
increased PrPSc levels in lymphoid tissues, which sug-
gests that macrophages limit the amount of prions that
initiate the infection29. However, whether this effect is
truly dependent on macrophage depletion or whether it
is an indirect effect remains to be established29. Further
studies to determine whether macrophages represent
important prion carriers are alsoneeded.
Uptake of prions by DCs. DCs patrol gut-associated lym-
phoid tissue (GALT) and sample luminal or transcytosed
antigens for presentation to and priming of B cells and
Tcells. DCs can acquire intestinally administered prions
and transfer them to mesenteric lymph nodes after their
migration through the lymphatic system30. Depletion
of CD11c+ DCs invivo impaired prion accumulation
in GALT and spleen and reduced the susceptibility to
orally administered prions, pointing towards a role for
DCs in promoting the spread of prions at these sites31.
However, CD11c is also expressed by mononuclear
phagocytes other than DCs within GALT, and the
respective contribution of DCs and other mononuclear
phagocytes to prion uptake is currently unclear.
Transmission through blood. Prions can be efficiently
transmitted through blood or blood derivatives. BSE and
scrapie were transmitted to sheep through whole-blood
transfusion or buffy coats, even from subclinical donors,
which implies that blood represents an efficient vehicle of
infection32. Similarly, blood transfusion efficiently trans-
mitted the TSE chronic wasting disease (CWD) to deer33,
and vCJD was transmitted from blood donors who sub-
sequently developed vCJD5,6. These tragic episodes were
not previously anticipated on the basis of epidemiological
evidence from casecontrol studies34 and resulted in the
creation of blood-donor deferral criteria and quality-con-
trol measures. Extensive experimental work has also indi-
cated that prions can be transmitted through the skin3537
and aerosols3840 and colonize draining lymph nodes soon
after prion exposure. In aerosol transmission, M cells and
epithelial cells of the nasal mucosa seem to be involved
in prion transport 41. Whether these routes are relevant
to naturally occurring TSEs remains to be established.
Prions and SLOs. In many TSEs, prions accumulate
and replicate within secondary lymphoid organs
(SLOs) before neuroinvasion occurs. This is most
apparent in natural scrapie 42, CWD43, transmissible
mink encephalopathy (TME)44 and vCJD45, which are
regarded as lymphotropic prion diseases. Neurotropic
prions directly invade the CNS without requiring
a peripheral replicative phase46. This dichotomy of
lymphotropic and neurotropic prions is likely to be
an oversimplification because numerous observations
indicate that the extent of lymphotropism of a prion is
the result of a combination of the prion strain, inocu-
lation route, the host species and the gene sequence
encoding the prion protein. It has also been observed
that lymphotropic prions seem to have increased host
ranges when compared with neurotropic prions 47.
Owing to the early colonization of lymphoid tissues
by prions, a tonsil biopsy can be used for preclinical
diagnosis of vCJD 48 and in nationwide prevalence
screening studies15.
Targets of prion infectivity: haematopoietic or stromal
cells? The detection of prion infectivity within lym-
phoid organs of prion-infected mice 49 has encour-
aged the identification of the cells and molecules that
are involved in this process. Genetic asplenia or sple-
nectomy, but not athymia or thymectomy, extend the
survival of mice that are peripherally inoculated with
prions, thereby excluding a relevant role for Tcells in
peripheral prion pathogenesis50. However, if splenec-
tomy is carried out in peripherally inoculated mice when
prions have already invaded the spinal cord, survival
is not extended51. Also, constitutive or acquired lym-
phocyte deficiency impairs prion replication following
peripheral, but not intracerebral, inoculation of rodent-
adapted prions52,53. This indicates that splenic replica-
tion of prions is crucial only in the early phases of the
disease if prions are peripherally administered. Of note,
severe combined immunodeficient (SCID) mice proved
to be partially resistant against intracerebral inocula-
tion of a BSE isolate, which suggests that the periph-
eral immune system plays a part in the species-barrier
phenomenon54.

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Follicular dendritic cells
(FDCs). Stromal cells derived
from platelet-derived growth
factor receptor (PDGFR)+
perivascular precursors and
localized in lymphoid follicles.
FDCs trap and retain immune
complexes to stimulate an
immune response. FDCs also
express milk fat globule
epidermal growth factor 8
(MFGE8) to facilitate the
removal of apoptotic cells in
secondary lymphoid organs.
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Fractionation of splenocytes identified subpopula-
tions of cells with high prion infectivity and suggested
that most infectivity is present in the stromal compart-
ment 55,56. Supporting these observations, sublethal ion-
izing irradiation which depletes mitotically active
haematopoietic cells but not mitotically quiescent stro-
mal cells had no effect on prion pathogenesis in mice
that were challenged by different inoculation routes,
doses and strains of rodent-adapted scrapie prions57.
Role of Bcells in prion disease. The recognition that
Bcells are required for neuroinvasive scrapie58 was sur-
prising and triggered a plethora of followup studies.
Expression of PrPC is necessary to sustain prion replica-
tion59, and this requirement has been exploited to identify
the cells that enable prion replication in SLOs. PrPC is
expressed at moderate levels in circulating lymphocytes,
including in Bcells. However, PrPC expression in Bcells is
not required for prion neuroinvasion60, and PrPC expres-
sion solely in Bcells is not sufficient for prion replica-
tion61. In addition, chimeric Prnp/ mice that have been
reconstituted with wild-type bone marrow or fetal liver
cells can support the replication of the Rocky Mountain
laboratory (RML) prion strain in the spleen62,63, even
though optimal replication requires PrPC expression in
both the haematopoietic and the stromal compartments63.
This property could be prion strain-dependent because
similar experiments carried out with the ME7 strain of
rodent-adapted scrapie prions showed no replication in
chimeric Prnp/ mice that were reconstituted with wild-
type bone marrow64. However, other reports indicate sub-
stantial splenic ME7 prion infectivity in Prnp/ mice that
were reconstituted with wild-type or Prnp-overexpressing
bone marrow 65. Collectively, these data indicate that
splenic prion replication requires a PrPC-expressing cell
of stromal origin that depends, directly or indirectly,
on Bcells, and this is irrespective of PrPC expression
in Bcells.
Role of FDCs. Early observations had identified follicular
dendritic cells (FDCs) as a site of PrPSc accumulation in
the lymphoid tissues of prion-infected mice52. In addi-
tion, SCID mice lacking functional FDCs succumb to
intracerebral, but not to intraperitoneal, infection with
the Fukuoka1 prion strain52.
As FDCs are derived from ubiquitous stromal peri
vascular precursor cells66 and they express high levels
of PrPC, they were thought to represent a candidate for
peripheral prion replication (FIG.2). Indeed, treatment
with a soluble lymphotoxin receptor immunoglobulin
(LTRIg) results in the ablation of mature FDCs from the
spleen, abolishes splenic prion accumulation and slows
neuroinvasion after intraperitoneal scrapie prion inocula-
tion67, but it does not alter prion pathogenesis after intrac-
erebral inoculation68. Similar effects were obtained using
an inhibitor of tumour necrosis factor receptor (TNFR)69.
However, dedifferentiation of FDCs following treatment
with LTRIg was efficient at interfering with prion infec-
tion only when applied before, but not after, intraperito-
neal or oral challenge with prions70. Also, mice lacking
LT, LT, LTR, LT and TNF, all resisted intraperitoneal
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Established route of infection
Potential route of infection
Intracranial
Corneal grafts
Airway
Ingestion
Skin
Intramuscular
Intravenous
Figure 1 | Entry sites for acquired prion diseases.
The solid arrows indicate theNature
recognized routes of prion
Reviews | Immunology
transmission. Cases of iatrogenic CreutzfeldtJakob
disease (iCJD) have occurred through corneal and dura
mater transplantations from diseased cadaveric donors,
through the use of prion-contaminated electroencepha-
lography electrodes and neurosurgical instruments, and
through the intramuscular administration of
contaminated pituitary-derived hormones. Ingestion of
meat from cows with bovine spongiform encephalopathy
and cannibalistic rituals cause variant CJD (vCJD) and
kuru, respectively. vCJD can also be transmitted through
blood products. The dashed arrows indicate potential
routes of prion transmission that have been suggested on
the basis of experimental studies in animal models; their
clinical relevance is currently unknown.
prion infection and contained no prion infectivity in
their spleens and, if present, in their lymph nodes71.
Collectively, these data indicate that FDCs have a crucial
role within lymphoid tissues for early peripheral reten-
tion and replication of lymphotropic strains of prions.
Dedifferentiating FDCs might represent a valid option
in post-exposure prophylaxis against prion infections12.
Role of complement in prion disease. As FDCs trap and
retain opsonized antigens within SLOs through the CD21
and/or CD35 complement receptors, several studies
have investigated the effect of ablation of complement
components on prion pathogenesis. Genetic ablation of
complement receptors and the complement components
C3, C1q, C2 and factorB, individually or in combination,
or the temporary depletion of C3 led to increased resist-
ance to peripheral prion infection in mice72,73. Ablation
of stromal cell, but not of haematopoietic cell, CD21 and/
or CD35 resulted in increased resistance to intraperito-
neal prion inoculation74. These data indicate an impor-
tant role for opsonizing complement components in
prion pathogenesis. There might also be other retention
mechanisms for prions72.
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Tonsils Spleen Peyers patches Lymph nodes

PrPSc B cell

FDC

B cell zone T cell zone

T cell

b Normal Chronic inammation Prion replication

ontogenesis leading to tertiary and accumulation
of SLOs lymphoid organs
PrPC PrPSc PrPSc
aggregate
Marginal
MFGE8 sinus
pre-FDC PrPC
PDGFR TNF
Perivascular
Blood
pre-FDC Mature
vessel FcRIIB
LTR B cell FDC
TNFR
LT12 B cell or
CD21
LTi cell
and/or
CD35

Figure 2 | Peripheral prion replication and the involvement of FDCs. a | Peripherally acquired prions replicate in
lymphoid follicles of secondary lymphoid organs (SLOs; such as tonsils, spleen, Peyers patches in theReviews
Nature intestines and lymph
| Immunology
nodes) and are mainly associated with follicular dendritic cells (FDCs). b | During normal ontogenesis of SLOs, FDCs
emerge from platelet-derived growth factor receptor- (PDGFR)-expressing ubiquitous perivascular pre-FDCs through
an intermediate cell termed the marginal sinus pre-FDC. FDC maturation requires exposure to Bcell or lymphoid tissue
inducer (LTi) cell-derived lymphotoxin- heterotrimers (LT12) for the first transition from the perivascular proFDC
stage to the marginal sinus pre-FDC stage, and exposure to Bcell-derived LT12 and tumour necrosis factor (TNF) for the
second transition from the marginal sinus pre-FDC stage to the mature FDC. This process is accompanied by upregulation
and downregulation of numerous transcripts. Similarly, during chronic lymphocytic inflammation, perivascular pre-FDCs
can differentiate into mature FDCs, thereby favouring the formation of tertiary lymphoid organs (TLOs), potentially at any
site of the body. Mature FDCs express high levels of cellular prion protein (PrPC) and are involved in peripheral prion
replication and accumulation. FcRIIB, low affinity immunoglobulin- Fc region receptor II-B; MFGE8, milk fat globule
epidermal growth factor 8; LTR, LT receptor; PrPSc, scrapie prion protein; TNFR, TNF receptor.

FDCs: prion factory or trap? An important question
is do FDCs contain replicating prions or are they just
trapping prions that are produced in close proximity to
them? Mabbott and colleagues75 devised an elegant way
to study the effect of Prnp expression or ablation specifically
in FDCs on splenic prion replication. Complement recep-
tor type 2 (Cr2), which encodes the complementreceptor
component CD21, is known to be expressed in FDCs and
mature Bcells. Reciprocal bone-marrow transplantation
between mice with the appropriate genotypes (Cd21
CrePrnpstop/stop or Prnpflox/flox mice) generates mice in
which Prnp is selectively expressed or deleted either
in FDCs or in mature Bcells. Interestingly, PrPC expres-
sion on FDCs turned out to be necessary and sufficient to
sustain splenic replication of ME7 prions75. Studies identi-
fying genes that are specific for FDCs will be instrumental
in increasing our understanding of the contributions of
FDCs to health and disease, including to prion diseases.

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Mastitis
Inflammation occurring in the
mammary gland. It can be
caused by infection or by
blockage of milk ducts.
NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 7
Ectopic FDCs and ectopic prions. Chronic lymphocytic
inflammation, resulting in FDC-containing lymphoid
follicles in the parenchyma of affected organs, can enable
ectopic prion replication a process that is dependent
on the presence of LT and its receptor 76. When kidneys
are affected, prion replication can be associated with
prion excretion in urine (known as prionuria), even in
pre-symptomatic animals77. The relevance of these find-
ings, which were originally made in transgenic mice and
were subsequently reproduced in experimentally inocu-
lated deer 78, was further confirmed by the observation
that coincident mastitis and scrapie infection in field
sheep can result in PrPSc deposition within mammary
glands79. Furthermore, it has been shown that prions are
present in the colostrum and milk of sheep80 and can
thereby be vertically transmitted81, a process that can be
favoured by mastitis82. Overall, these observations sug-
gest that chronic lymphocytic inflammation can func-
tion as a modifier of prion diseases by extending the
affected tissue distribution. Another important impli-
cation is that prion excretion in milk, which is probably
exacerbated by concomitant mastitis, could represent a
natural route of scrapie transmission within flocks. It
remains unclear whether this applies also to BSE and,
if so, whether dairy products represent a risk to public
health80.
Despite the key role of FDCs during peripheral repli-
cation of prions, scenarios have been identified in which
prions can replicate and accumulate in the absence of
mature FDCs. These include the lymph nodes of Tnf/
and Tnfr1/ mice71,83 as well as soft-tissue granulomas84.
Neuroinvasion by prions
Peripheral nerves facilitate neuroinvasion. After rep-
lication and accumulation within SLOs, prions enter
the CNS, where they ultimately cause neurotoxicity.
The innervation pattern of SLOs is primarily sym-
pathetic, and experimental models show that prion
agents spread from SLOs to the CNS through the
autonomic nervous system56,8587. Chemical or immu-
nological sympathectomy prevented or significantly
delayed peripheral prion pathogenesis88. Conversely,
sympathetic hyperinnervation of SLOs in transgenic
mice shortened prion incubation, which shows that
sympathetic innervation of SLOs is rate limiting for
prion neuroinvasion88.
Disease-associated PrP and prion infectivity have
been found in the enteric nervous system of sheep with
scrapie89, deer with CWD90 and humans with vCJD91,
which suggests that this could represent another portal
of entry after peripheral exposure.
From FDCs to nerves: mind the gap. Manipulation of the
distance between FDCs and splenic nerve endings has led
to the conclusion that the relative positioning of FDCs to
nerves controls the speed of neuroinvasion92. However,
the sessile nature of FDCs and the differential localiza-
tion of FDCs and nerve endings in microanatomical com-
partments within SLOs raise the question of how prions
spread from FDCs to the nerves. Different scenarios
have been envisaged, including direct celltocell contact,
2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
vesicle-associated infectivity (for example, prion trans-
mission through exosomes93), tunnelling nanotubes94 and
free-floating infectious particles95.
After nerves have been invaded, prions travel through
the spinal cord to ultimately reach the brain. The mecha-
nisms underlying this process are not fully understood.
Incoming PrPSc might convert PrPC at the axolemma
surface, thereby initiating a domino-like cascade or,
alternatively, prions could be internalized at the nerve
endings and be transported in a retrograde manner96.
Peripheral immune responses to prions
From the discussion above, it is clear that components
of the immune system can contribute to the spread of
prions. However, an important question is whether
the immune system can actually mount a protective
response to prions. Toll-like receptors (TLRs) are key
mediators of innate immune responses. Prions or their
components might be able to trigger TLRs. Following
prion infection, TLR signalling seems to be protective
under certain conditions. Mice deficient in functional
TLR4 signalling or in interferon-regulatory factor 3
(IRF3), which is a myeloid differentiation primary-
response protein 88 (MYD88)independent transcrip-
tion factor that is activated downstream of TLRs, showed
accelerated prion pathogenesis following intraperitoneal
infection97,98. Interestingly, Myd88/ mice, which are
defective in most TLR-mediated responses, succumb to
disease to a similar extent to wild-type mice following
intraperitoneal inoculation99.
Prion infection usually does not trigger apparent
adaptive immune responses. This is probably due to self-
tolerance caused by the similar immunogenicity of PrPSc
with PrPC; PrPC is ubiquitously expressed by the host.
Immune tolerance prevents robust immune responses
to prions, and PrP-specific antibodies are not detected in
animals infected with prions100. Although subtle altera-
tions in cellular homeostasis were observed in the lym-
phoid organs of animals that are infected with prions101,
immune system function was not compromised102. The
effect of these alterations on prion pathogenesis might
be negligible, as interleukin6 (IL6)-deficient mice
or CD40 ligand (CD40L)deficient mice, which have
impaired germinal centre development, succumbed
to disease at the same rate as wild-type mice following
intraperitoneal inoculation103,104.
CNS immune responses to prions
Progressive deposition of prions in the brain leads to
fatal spongiform encephalopathies, which manifest
as synaptic and neuronal loss, vacuolation and neuro-
inflammation. Neuroinflammation that is associated
with prion infection is characterized by the activation
of astrocytes and microglia, which are the principal
immune cells in theCNS11.
Microglial cell activation. Microglial cell activation is
evident in patients with TSEs105 and in animal models
of prion diseases106, but investigations into the func-
tion of microglia in prion diseases have been hampered
by technical limitations. The cerebellar organotypic
REVIEWS

A cultured slice (COCS) system, in which invivo prion

Astrocyte pathogenesis can be faithfully reproduced, provides a
PrPC MFGE8 promotes
phagocytosis of powerful tool for studying microglial cell functions in
MFGE8 apoptotic neurons prion infections 107. Depletion of microglia resulted
in markedly enhanced PrPSc deposition and augmented
Microglial cell prion infectivity 108. Similar effects were recorded in
PrPSc mice that were deficient for milk fat globule epidermal
accumulation Microglia growth factor 8 (MFGE8; also known as lactadherin),
clear apoptotic which is secreted by astrocytes and promotes phago-
neurons
Damaged neuron cytic engulfment and clearance. MFGE8deficient mice
showed accelerated prion pathogenesis and increased
Excessive prion accumulation levels of apoptotic cell remnants, which suggests that
leads to neurodegeneration
Healthy neuron MFGE8mediated apoptotic cell clearance by micro-
glia quells prion accumulation109. The effect of MFGE8
B depletion was visible only in certain mouse strains,
a Prnp knockdown which implies that there are further polymorphic deter-
minants of prion removal. These experiments revealed
a protective function for microglia in prion disease and
PrPC production
potential insights about the crosstalk between microglia
and astrocytes in neuroinflammation (FIG.3).
Prnp DNA Prnp mRNA
Ultimately, microglia-mediated prion clearance
b Antibodies prevent prion invivo is insufficient, even after intracerebral lipopol-
conversion ysaccharide (LPS) treatment to prime the immune
PrPC PrPSc conversion system110. The failure to clear prions might convert
microglia from the phagocytic M2 phenotype into the
pro-inflammatory M1 phenotype, consequently con-
tributing to disease pathobiology by the spreading of
c Antibodies prevent prion prions or the secretion of cytotoxic mediators. Perhaps
aggregation manipulating microglia to adopt the M2 phenotype
PrPSc aggregate formation might lower prion levels and ameliorate pathology.
PrPSc
Prion bril d Compounds stabilize e Antibodies or Cytokines induced by prion infection. Prion infection
prion brils compounds promote induces the production of pro-inflammatory cytokines,
protein clearance such as IL1, IL1, TNF and IL6, in patients with
Stabilized Degraded TSEs111 and in some mouse models of prion disease112,113
prion bril prion bril but not in ME7infected C57BL/6 mice114. Intriguingly,
f Compounds interfere the anti-inflammatory cytokine transforming growth
with neurotoxicity
factor (TGF) was also significantly induced in mice
that were infected with prions115. Similarly, in patients
PrPC-mediated PrPSc toxicity
with CJD, levels of the anti-inflammatory cytokines
IL4 and IL10 are increased in the cerebrospinal
Neurotoxicity fluid116.
To elucidate whether the induction of these cytokines
Figure 3 | Prion-induced neurodegeneration and potential therapeutic targets. is involved in prion pathogenesis, various mouse mod-
Nature
A | Prion infection elicits neuronal damage and glial activation. Reviews | Immunology
Astrocyte-released milk els deficient for or overexpressing certain cytokines
fat globule epidermal growth factor 8 (MFGE8) facilitates phagocytosis of apoptotic
have been challenged with prions. Although most of the
neurons by microglia. Overall, microglia function as scavengers and have
neuroprotective roles in prion pathogenesis. However, microglia-mediated clearance cytokines do not seem to be important contributors to
might become overwhelmed by the progressive accumulation of scrapie prion protein prion pathogenesis in the CNS (TABLE2), depletion of
(PrPSc). It is possible that excess prion accumulation in the brain could reprogramme IL-1 receptor 1 (IL1R1), which is the receptor for IL1
microglia into a pro-inflammatory phenotype, which might facilitate the spread of prions and IL1, resulted in a small but significant incubation
within the central nervous system, ultimately leading to worsening of the disease and prolongation117,118. This effect is probably due to delayed
neurodegeneration. B | Select stages of prion pathogenesis can offer therapeutic targets astrocytosis, although augmented microglial activation
for treating or preventing transmissible spongiform encephalopathies. Knockdown of the might contribute by enhancing phagocytosis of pri-
gene encoding prion protein (Prnp) or pharmacological downregulation of cellular prion ons in Il1r1/ mice. The role of the IL1R pathway in
protein (PrPC) can interfere with prion conversion and neurotoxicity (part a). Antibodies prion pathogenesis was also indicated by an association
or other compounds can specifically capture prions or can otherwise prevent the
study on the polymorphisms in the Il1r1 locus and the
conversion of PrPC into PrPSc (part b) or the formation of higher-order aggregates (part c).
Compounds can stabilize prion fibrils, thereby interfering with the process of prion incubation time of mouse prion disease119. Conversely,
replication and neurotoxicity (part d). Antibodies or other compounds can promote deficiency of IL10 rendered mice on a 129/Sv
natural protein-aggregate-clearing mechanisms, thereby interfering with the process of background much more susceptible to prions follow-
prion replication and neurotoxicity (part e). Finally, compounds can interfere with the ing both intraperitoneal and intracerebral inocula-
neurotoxic pathway mediated by PrPC at the neuronal cell membrane (part f). tion120, which implies that IL-10 has a neuroprotective

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 REVIEWS

Table 2 | Role of cytokines and chemokines in prion pathogenesis in the central nervous system
Mouse line Genetic Prion Effects (in comparison with wild-type mice Refs
background strain with the same genetic background)
Tnf/ mice 129/SvC57BL/6 ME7 No 103
C57BL/6 RML No 71,118
Tnfr1/ mice 129/Sv RML No 58
129/SvC57BL/6 RML No 71
B6;129S7/SvEvBrd RML No 118
Tnfr2/ mice B6;129S7/SvEvBrd RML No 118
Il6 mice
/
129/SvC57BL/6 ME7 No 103
Il1r1/ mice C57BL/6 139A Delayed astrocytosis, augmented microglial 117
activation and prolonged incubation time by
2225days
B6;129S1/Sv RML Prolonged incubation time by 21days 118
Tgfb1 mice
+/
NIH/Ola RML No 118
Tgfb1 transgenic mice SJL/J RML No 118
Tgfbr2k tTA Prnp
+/ +/ +/
C57BL/6JFVB/N RML No 118
mice*
Il4/ mice BALB/c RML Shortened incubation time by 29days at 120
low-dose inoculum
Il13/ mice BALB/c RML Shortened incubation time by 39days at 120
low-dose inoculum
Il10/ mice 129/Sv RML Accelerated inflammation and shortened 120
incubation time by 4182days
129S6 RML No 118
C57BL/6J RML Shortened incubation time by 34days 118
Ccl2 mice
/
C57BL/6J ME7 Unaltered early behavioural dysfunction, but 121
delayed incubation time by 23weeks
C57BL/6J RML No 122
Ccr1 mice
/
C57BL/6 RML Enhanced ERK1 and ERK2 activation, shortened 123
incubation time by 15days
Ccr2/ mice C57BL/6J RML No 118
Ccr5/ mice C57BL/6J RML No 118
Cxcr3 mice/
C57BL/6 139A Accelerated PrP accumulation, reduced
Sc
125
microglial activation and pro-inflammatory
cytokine production and delayed incubation
time by 2030days
Cxcr5/ mice 129/Sv RML No 92
Ccl, CCchemokine ligand; Ccr, CCchemokine receptor; Cxcr, CXC-chemokine receptor; ERK, extracellular signal-regulated
kinase; Il, interleukin; Il1r1, IL-1 receptor 1; Prnp, gene encoding prion protein; PrPSc, scrapie prion protein; RML, Rocky Mountain
laboratory; Tgfb1, transforming growth factor1; Tnf, tumour necrosis factor; Tnfr, TNF receptor. *These mice neuronally express a
transdominant-negative kinase-deficient mutant of TGF receptor 2.

M2 phenotype
Activated macrophages or
microglia that show phagocytic
behaviour and express factors
such as interleukin4 (IL4),
IL10 and arginase 1.
M1 phenotype
Activated macrophages or
microglia that show
pro-inflammatory features and
express factors such as
interleukin1, tumour necrosis
factor and inducible nitric
oxide synthase.
function in prion disease. Nevertheless, the role of IL10
in prion pathogenesis seems to be context dependent
because mild prion disease acceleration was observed
in Il10/ mice on a C57BL/6J background but not on a
129S1/SvImJ background118.
Chemokines induced by prion infection. Increased
chemokine expression occurs in various neuro-
degenerative disorders, including prion diseases.
CC-chemokine ligand 2 (CCL2; also known as MCP1)
is progressively upregulated in ME7infected C57BL/6
mice. However, survival time following prion infec-
tion was only slightly prolonged in CCL2deficient
mice, and deletion of this chemokine had no effect
on the levels of microglial cell activation or neuronal
damage observed in response to prion infection 121.
This effect might also be prion-strain dependent
because CCL2deficient mice did not show any delay
in disease progression after intracerebral inoculation
with RML prions122. Similarly, mice deficient for the
CCL2 receptor CCchemokine receptor 2 (CCR2)
had an unaltered disease course compared with
wild-type mice after RML prion infection118.
CCL5 (also known as RANTES) and its recep-
tors CCR1, CCR3 and CCR5 are also upregulated
in mouse prion models 115. Following intracerebral

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REVIEWS
inoculation with RML prions, Ccr1/ mice showed
more robust induction of CCR5 and CCL3 than wild-
type mice, which suggests a compensatory mecha-
nism. This induction resulted in enhanced activation
of extracellular signal-regulated kinase 1 (ERK1)
and ERK2, and accelerated disease progression 123.
However, mice deficient for CCR5 developed prion
disease to a similar extent to wild-type mice following
intracerebral inoculation of RML118. Hence, it is still
unclear how much these components influence prion
pathogenesis.
Levels of the chemokines CXC-chemokine ligand9
(CXCL9) and CXCL10, which signal through CXC-
chemokine receptor 3 (CXCR3), are also increased
in prion diseases117,124. Cxcr3/ mice intracerebrally
infected with 139A prions showed prolonged incuba-
tion time but enhanced PrPSc accumulation125. Further
analysis revealed that deletion of CXCR3 resulted in
attenuated microglial activation and consequently
decreased phagocytosis and degradation of PrPSc after
prion infection, which possibly explains the enhanced
deposition of PrPSc. Absence of CXCR3 caused more
pronounced astrocytosis but reduced the production
of pro-inflammatory cytokines in prion disease, which
possibly accounts for the prolonged incubation time.
The chemokine CXCL13 (also known as BLC) is also
upregulated in prion diseases124,126, but mice deficient
for its receptor, CXCR5, had similar incubation time
compared to wild-type mice following intracerebral
inoculation with RML prions92. Finally, the chemokine
axis CX 3C-chemokine ligand 1 (CX 3CL1)CX 3C-
chemokine receptor 1 (CX 3CR1) which has an
important role in microglial activation and amyloid-
and tau protein-mediated pathology127 is altered in
prion diseases 128,129, although the relevance of these
changes remains to be determined.
NFB signalling in prion infection. The nuclear
factor-B (NFB) signalling pathway is involved in
numerous physiological and pathological conditions,
including in the induction of inflammatory cytokines,
and chemokines, and in the regulation of apopto-
sis. NFB is activated in astrocytes of mice infected
with prions130. Moreover, enhanced binding, but no
transcriptional activity, of NFB was observed in
neuroblastoma cells after prion infection, leading to
mitochondria-mediated apoptosis that is associated with
decreased expression of the anti-apoptotic protein B cell
lymphoma-XL (BCL-XL)131.
To investigate the role of NFB in prion dis-
eases, mice deficient for components of the canoni-
cal NFB pathway (Nfkb1/, p65CNSKO, inhibitor of
NFB kinase subunit- (Ikkb)CNSKO and IkkgCNSKO
mice), of the non-canonical NFB pathway (Nfkb2/
and IkkaAA/AA mice), or of NFB target genes (Bcl3/
mice) were intracerebrally inoculated with prions.
Surprisingly, only the mice with impairments in the
non-canonical pathway showed any reduction in dis-
ease incubation time. Therefore, invivo data suggest
that NFBmediated signalling is not a major deter-
minant of prion pathogenesis131,132.
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Immunotherapy for prion diseases
As discussed above, mice with various states of immuno-
deficiency are resistant to peripheral prion infection58,71.
Conversely, pro-inflammatory conditions increase the
susceptibility to prion infection and promote periph-
eral prion deposition24,76,77,79,133. Furthermore, the lym-
phoid tissue might be more permissive than the brain to
cross-species transmission47.
As it precedes neuroinvasion, the lymphoid replica-
tion phase provides a window for post-exposure prophy-
lactic and therapeutic interventions against peripherally
acquired prions. Disease progression can be impeded
through dedifferentiating FDCs by blocking LTR67,68,70
or TNFR1 (REF.69) signalling, inhibiting prion trapping
by FDCs through the modulation of the complement
system7274,134, or by sympathectomy 88. Encouragingly,
when applied early following infection, these therapeu-
tics can significantly decrease splenic PrPSc accumula-
tion and/or prolong the intraperitoneally inoculated
prion incubation time in mice. Meanwhile, numerous
strategies targeting different stages of prion diseases are
currently being explored (FIG.3).
Targeting innate versus adaptive immune responses.
The role of the innate immune system in prion pathol-
ogy remains unclear. Although some reports suggested
that TLR stimulation might provide some benefits during
prion infection97,98, others reported contradictory find-
ings99. An early study found repeated TLR9 stimulation to
be protective against prion pathogenesis135, but ultimately
this was attributable to iatrogenic alterations in the mor-
phology and function of lymphoid structures136.
The potential of antibody-mediated therapy for
prion disease was first reported in a cell-free model
showing that PrP-specific antisera could neutralize
prion infectivity 137. In cell culture models, the mono
clonal antibody 6H4 or the monovalent antibody frag-
ments (D13, D18, R1 and R2) that are specific for PrP
efficiently suppressed prion replication in mouse neu-
roblastoma cells that were chronically infected with
prions 138,139. These results, together with the initial
success of amyloid immunotherapy in mouse models
of Alzheimers disease140, have encouraged the explora-
tion of antibody-mediated immunotherapy for prion
disease, as discussedbelow.
Active immunization against prions. Active immuni-
zation against prions is challenging because immune
responses are stifled by tolerance to PrP. To provide
proofofprinciple for immunotherapy, transgenic mice
expressing the -chain of the PrP-specific antibody
6H4 (6H4) were generated. Encouragingly, expression
of this transgene antagonized prion pathogenesis 141.
Although transgenesis is impractical as a clinical strategy
for human prion diseases, this pioneering study indi-
cates the potential value of antibodies as prophylactic
and therapeutic treatment strategies for prion diseases.
Various vaccination strategies have attempted to
break self-tolerance to prions with limited success
(TABLE3). Synthetic PrP peptides (PrP3150 and PrP211230)
elicited immune responses and reduced PrPSc levels in
 REVIEWS

Table 3 | Active immunization for prion disease*

Mouse strain Immunogen Adjuvant and immunization Prion strain and Effects on delay Refs
strategy infection route development
CD1 Recombinant mouse PrP23230 CFA and IFA, s.c. 139A, i.p. 16day delay 143
NMRI Peptide PrP105125 covalently linked Montanide IMS1313, i.p. 139A, oral 23day delay 176
to KLH
NMRI Recombinant mouse PrP90230 Montanide IMS1313, i.p. 139A, oral Ineffective 176
C57BL/6 Dimeric mouse recombinant CFA and IFA, in combination with RML, i.p. No effect 144
129/Sv PrP23231 OX40-specific antibodies; s.c. for
CFA and i.p. for IFA
CD1 Salmonella spp. vaccine strain In combination with sodium 139A, oral Full protection of mice with 177
expressing mouse PrP bicarbonate and alum, oral high mucosal anti-PrP titres
BALB/c Recombinant mouse fragment In sodium bicarbonate buffer 139A, oral 9day delay 178
PrP90230 with cholera toxin, intranasal
C57BL/6 PrP141159 or PrP165178 conjugated Mycobacterium avium subsp. RML, i.p. 2125day delay 179
with KLH avium-based adjuvant (AdjuVac;
National Wildlife Research
Center), intramuscular
BALB/c Recombinant bovine PrP25242 CFA and IFA, i.p. Fukuoka1, i.p. 31day delay 180
BALB/c Recombinant mouse PrP23231 CFA and IFA, i.p. Fukuoka1, i.p. No effect 180
C57BL/6 pCG plasmid containing mouse CpG, intradermal and s.c. RML, i.p. No effect 181
PrP cDNA fused with tetanus
toxin (P30)
C57BL/6 Peptide PrP98127 or PrP158187 CpG and IFA, s.c. 139A, i.p. 1520day delay 182
FVB/N Dynabeads adsorbed-native PrP Sc
CFA and IFA, s.c. RML, i.c. and i.p. 22day delay in i.p., and no 183
effect in i.c.
C57BL/6 Dendritic cells expressing human Intramuscular injection 139A, i.p. 37day delay 184
PrP together with adenovirus
C57BL/6 Dendritic cells loaded with i.p. 139A, i.p. 40day delay 185
peptides PrP98127
BALB/c 6H4epitope mimicking bacterial CFA and IFA, i.p. Fukuoka1, i.p. 31day delay 186
succinylarginine dihydrolase
C57BL/6 Aggregated PrP CFA and IFA, s.c. RML, i.p. 28day delay 145
129/Ola Plasmid pCMVUbPrP or Anterior tibial muscle BSE, i.c. 2week delay 146
pCMVPrPLII
BSE, bovine spongiform encephalopathy; CFA, complete Freunds adjuvant; i.c, intracerebral; IFA, incomplete Freunds adjuvant; i.p, intraperitoneal; KLH, keyhole
limpet haemocyanin; pCMVPrPLII, plasmid containing prion protein fused to the lysosomal-targeting signal from lysosomal membrane protein 2 under the control
of the cytomegalovirus promoter; pCMVUbPrP, plasmid containing prion protein fused to ubiquitin under the control of the cytomegalovirus promoter; PrP, prion
protein; RML, Rocky Mountain laboratory; s.c., subcutaneous. *Adapted from REF.187.

prion-infected mouse neuroblastomas transplanted Active immunization typically failed to mitigate

into A/J mice142. Only a slight delay (16days) in disease
onset was observed in mice that were immunized with
recombinant mouse PrP (recPrP23230)143. The incuba-
tion time correlated with PrP-specific antibody titres,
which suggests that the beneficial effect was immune
mediated. Other active immunization attempts resulted
in neither a considerable PrP-specific antibody titre
nor a significant increase in survival time. These
discrepancies might pertain to differences in immu-
nogens, regimens, mouse strains or prion strains
but the prospects of active immunization are dim 144.
Interestingly, the effect of active immunization might
extend beyond PrP to have consequences on immune
cell status. Immunization of C57BL/6 mice with aggre-
gated PrP and complete Freunds adjuvant resulted in
an acute depletion of mature FDCs from the spleen
and consequently a prolongation of incubation time
(28days) after peripheral prion challenge145.
prion disease in cases in which it is caused by intrac-
erebral challenge or in cases in which neuroinvasion
had already occurred. This might be due to the blood
brain barrier limiting penetrance of antibodies into the
CNS. However, immunizing 129/Ola mice with a DNA
construct expressing mouse PrP fused with lysosome
membrane protein 2 (LIMP2; also known as SCARB2)
or ubiquitin resulted in a breakage of host tolerance
to PrP and an induction of PrP-specific antibodies.
Surprisingly, this DNA vaccination delayed disease
onset by 2weeks in mice that had been intracerebrally
inoculated with mouse-adapted BSE prions146.
Passive immunization. Even if it were effective, active
immunization carries potential risks, including the
remote possibility of converting immunogens into infec-
tious prions. However, numerous antibodies against vari-
ous PrP epitopes have been generated by immunizing

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REVIEWS

Prnp/ mice with synthetic PrP peptides, recombinant
PrP or native PrP purified from tissues. Peripheral
administration of the PrP-specific monoclonal antibod-
ies ICSM18 (which recognizes PrP146159) and ICSM35
(which recognizes PrP91110) to FVB/N mice147 inhibited
prion accumulation in the spleen. Continuous treat-
ment with these antibodies prolonged mouse survival
times to 500days post inoculation. By contrast, mice
treated with a control antibody succumbed at 197days
post inoculation. However, antibody treatment did not
prevent disease when it was started at clinical disease
onset (129136days post inoculation) or in mice intrac-
erebrally inoculated with RML prions147. When CD1
mice were intraperitoneally given the PrP-specific anti-
bodies 8B4 (which is specific for PrP3452) or 8H4 (which
is specific for PrP175185) immediately after intraperitoneal
inoculation with ME7 prions, the incubation time was
delayed by 10% (REF.148). Moreover, after intraperitoneal
inoculation with 22L prions, peripheral administration
of the PrP-specific antibody 6D11 (which recognizes the
PrP97100 epitope) to CD1 mice for 4 or 8weeks suppressed
PrPSc replication in lymphoid tissues and prolonged
incubation times149.
Disappointingly, a recent study in which C57BL/6
mice infected with RML prions were administered a high
dose of the PrP-specific antibody W226 or its recombi-
nant single-chain variable fragment (scW226) found
them to have only a minor protective effect150. As W226
and ICSM18 antibodies recognize the same PrP epitope
(PrP145153), these divergent effects are difficult to explain.
Clearly, more research is needed to understand the role of
passive immunization in prion disease.
Risks associated with immunotherapy. Passive immu-
nization has so far been ineffective in slowing disease
progression in mice following intracerebral inoculation
or in mice already showing clinical signs. Unfavourable
pharmacokinetics might account for the lack of suc-
cess, but even the intracerebral delivery of PrP-specific
reagents through osmotic pumps151 or adeno-associated
virus (AAV) vectors152 has had disappointing results.
In addition, the intracerebral injection of certain PrP-
specific antibodies, even in their monovalent form, is
reported to be neurotoxic153,154. Interestingly, the tox-
icity of these PrP-specific antibodies is dependent on
two distinct modules of PrPC; the regulatory globu-
lar domain of PrPC, which is bound by the antibody,
and the executive flexible tail of PrPC, which mediates
toxicity. The binding of PrP-specific antibodies to the
globular domain triggers a cascade of events and even-
tually leads to neuronal cell death that is mediated by
the flexible tail154. Therefore, any clinical trials of pas-
sive immunization will require great caution because
PrP-specific antibodies might cause neurotoxicity and
exacerbate clinical deterioration.
PrPFc2 mediated therapy. Soluble dimeric receptors,
also termed immunoadhesins, consist of the Fc portion
of IgG fused to various binding domains155. Transgenic
mice expressing a prion immunoadhesin (PrPFc2)
showed resistance against prion disease, and PrPFc2 was
not converted into a self-propagating, protease-resistant
isoform. These properties encouraged the investigation
of the interaction of PrPFc2 with PrPC and the role of
PrPFc2 in prion pathogenesis. Mice expressing both
PrPC and PrPFc2 showed a decrease of PrPSc accumula-
tion and a delay in the onset of disease156. PrPFc2 binds
to PrPSc and functions as a dominant-negative prion
antagonist. The peculiar features of PrPFc2 indicate that
soluble PrP derivatives might represent a novel category
of antiprion molecules.
Future directions
Despite intense investigations, some fundamental
aspects of the immunobiology of prions are still unclear.
The physiological function of PrPC in the immune sys-
tem remains enigmatic. The absence of Prnp/ mice on
a pure background and that are devoid of the shortcom-
ings that are caused by gene targeting in embryonic stem
cells has so far hampered research in this field. However,
recent advancements in genome-editing technologies
as well as the implementation of rigorous standards in
carrying out and reporting animal experiments leave
grounds for optimism.
The elucidation of the role of the CNS innate
immune system in prion pathogenesis has just started.
As for other aspects of neurobiology, there are reasons
to believe that progress will continue at a rapid pace.
However, research into immunotherapy against prions
will have to proceed cautiously in light of the recent
realization that certain PrP-specific antibodies, also in
monovalent form, can trigger specific PrPC-mediated
neurotoxic pathways. On a more positive note, the eluci-
dation of mechanisms of prion toxicity at the molecular
level and the availability of robust exvivo models of prion
diseases could be instrumental in developing urgently
needed pharmacological interventions against these
currently fatal conditions.

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Acknowledgements
We apologize to all those colleagues whose work was dis-
cussed without proper quotation owing to space constraints.
We thank T. P. Johnson for critically reading our manuscript.
A.A. is the recipient of an Advanced Grant of the European
Research Council and is supported by grants from the
European Union (PRIORITY and NEURINOX), the Swiss
National Foundation, the Foundation Alliance BioSecure, the
Novartis Research Foundation and the Clinical Research
Priority Program (KFSP) of the University of Zurich,
Switzerland. M.N. received grants from Collegio Ghislieri
(Pavia, Italy) and the Foundation for Research at the Medical
Faculty of the University of Zurich.
Competing interests statement
The authors declare no competing interests.
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