Vaccination with prion peptide-displaying
papillomavirus-like particles induces autoantibodies
to normal prion protein that interfere with pathologic
prion protein production in infected cells
Alessandra Handisurya
1
, Sabine Gilch
2
, Dorian Winter
3,4
, Saeed Shafti-Keramat
1
, Dieter Maurer
3,4
,
Hermann M. Scha
¨tzl
2
and Reinhard Kirnbauer
1
1 Laboratory of Viral Oncology, DIAID, Department of Dermatology, Medical University Vienna, Austria
2 Institute of Virology, Technical University of Munich, Germany
3 Laboratory of Experimental and Clinical Immunology, DIAID, Department of Dermatology, Medical University Vienna, Austria
4 Center of Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria
Prion diseases or transmissible spongiform encephalo-
pathies are untreatable, fatal neurodegenerative dis-
orders caused by proteinaceous infectious pathogens
devoid of nucleic acid, termed prions [1]. These
diseases include scrapie of sheep, bovine spongiform
encephalopathy in cattle and Creutzfeldt–Jakob disease
in humans [1]. Although human prion diseases are
rare, the appearance of a new variant of Creutzfeldt–
Keywords
immunotherapy; papillomavirus-like particles;
prion
Correspondence
R. Kirnbauer, Laboratory of Viral Oncology,
DIAID, Department of Dermatology, Medical
University Vienna, Wa
¨hringer Gu
¨rtel 18–20,
A-1090 Vienna, Austria
Fax: +43 1 4030224
Tel: +43 1 40400-7768
E-mail: reinhard.kirnbauer@meduniwien.ac.at
(Received 29 September 2006, revised 21
December 2006, accepted 31 January 2007)
doi:10.1111/j.1742-4658.2007.05721.x
Prion diseases are fatal neurodegenerative disorders caused by proteina-
ceous infectious pathogens termed prions (PrP
Sc
). To date, there is no pro-
phylaxis or therapy available for these transmissible encephalopathies.
Passive immunization with monclonal antibodies recognizing the normal
host-encoded prion protein (PrP
C
) has been reported to abolish PrP
Sc
infec-
tivity and to delay onset of disease. Because of established immunologic
tolerance against the widely expressed PrP
C
, active immunization appears
to be difficult to achieve. To overcome this limitation, papillomavirus-like
particles were generated that display a nine amino acid B-cell epitope,
DWEDRYYRE, of the murine ⁄rat prion protein in an immunogenic cap-
sid surface loop, by insertion into the L1 major capsid protein of bovine
papillomavirus type 1. The PrP peptide was selected on the basis of its pre-
viously suggested central role in prion pathogenesis. Immunization with
PrP–virus-like particles induced high-titer antibodies to PrP in rabbit and
in rat, without inducing overt adverse effects. As determined by peptide-
specific ELISA, rabbit immune sera recognized the inserted murine ⁄rat
epitope and also cross-reacted with the homologous rabbit ⁄human epitope
differing in one amino acid residue. In contrast, rat immune sera recog-
nized the murine ⁄rat peptide only. Sera of both species reacted with PrP
C
in its native conformation in mouse brain and on rat pheochromocytoma
cells, as determined by immunoprecipitation and fluorescence-activated cell
sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity
antibody that inhibited de novo synthesis of PrP
Sc
in prion-infected cells. If
also effective in vivo, PrP–virus-like particle vaccination opens a unique
possibility for immunologic prevention of currently fatal and incurable pri-
on-mediated diseases.
Abbreviations
Ab, antibody; BPV-1, bovine papillomavirus type 1; FACS, fluorescence-activated cell sorting; PIPL-C, phosphatidylinositolphospholipase C;
PrP
C
, normal prion protein; PrP
Sc
, pathologic prion protein; VLP, virus-like particle.
FEBS Journal 274 (2007) 1747–1758 ª2007 The Authors Journal compilation ª2007 FEBS 1747
Jakob disease, probably due to consumption of bovine
spongiform encephalopathy prion-contaminated prod-
ucts, has become an important public health issue
[2,3]. The possibility of horizontal human transmission
through surgery, organ grafting or blood transfusions
has raised further concern [4,5].
The exact pathogenetic mechanism of prion diseases
has remained uncertain. In the most accepted ‘protein-
only’ hypothesis, the causative agent, the pathologic
prion protein (PrP
Sc
), is proposed to be a conforma-
tional isoform of the host-encoded normal prion pro-
tein (PrP
C
). PrP
C
is expressed ubiquitously on cell
surfaces, in particular by neurons, and also on leuko-
cytes, including T cells and B cells [6,7]. Transforma-
tion of normal PrP
C
into infectious PrP
Sc
is thought to
occur through a template-directed process [1]. PrP
C
appears to be required for prion infectivity, as mice
deficient in PrP
C
(Prnp
0⁄0
) do not propagate the infec-
tious agent and fail to develop scrapie following
experimental inoculation [8].
To date, there are no prophylactic or therapeutic
modalities available against prion diseases. During nat-
ural infection with PrP
Sc
, PrP-specific immune responses
are not generated [1,9,10]. Nevertheless, studies in cell
culture and mice indicate that immunotherapeutic strat-
egies against the cellular form of PrP
C
can antagonize
prion infectivity and disease. Monoclonal antibodies
(mAbs) or recombinant F(ab) fragments recognizing
PrP were effective, in vitro, in preventing prion infection
of susceptible mouse neuroblastoma cells and also in
abrogating PrP
Sc
de novo formation in chronically infec-
ted cells [11,12]. In addition, transgenic expression or
passive transfer of mAb against PrP into scrapie-infec-
ted mice suppressed peripheral prion replication as well
as prion infectivity, and significantly delayed onset of
disease [13,14]. Notably, no obvious side-effects were
observed in these mice.
The immune system has evolved to respond vigor-
ously to viral or bacterial antigens, to protect the host.
Several groups have demonstrated that papillomavirus
virus-like particles (VLPs) can activate innate immune
responses in dendritic cells to regulate adaptive
immune responses [15–17], and also directly activate
B-cell IgG production via a TLR-4 ⁄MyD88-dependent
(and T-helper cell-independent) pathway [17]. Immuni-
zations induced high-titer neutralizing antibodies (Abs)
and potent cell-mediated immune responses to the viri-
on capsid protein [18–20]. In large clinical trials,
human papillomavirus VLP vaccinations were safe and
100% effective in preventing persistent infection and
associated genital disease, and polyvalent human papil-
lomavirus vaccines have been approved recently for
human use [21]. In further studies, chimeric VLPs have
been developed that display foreign or self-polypep-
tides in an ordered and closely packed repetitive array
on the capsid surface [22,23]. Immunizations of experi-
mental animals induced high-titer and high-avidity
IgGs to the surface-displayed (self-)antigens that were
long lasting and functionally active in vitro and in vivo.
Importantly, no adverse effects or induction of auto-
immune disease were observed.
The aim of this study is to develop a safe and effective
vaccine that generates a strong Ab response against the
prion self-antigen, but avoids induction of a T-cell
response, to minimize the risk of cell-mediated auto-
immune disease. Development of an effective PrP vac-
cine has been hampered by immunotolerance to the
ubiquitously expressed endogenous PrP
C
. In the past,
this problem has been circumvented experimentally
either by generation of anti-PrP immunity in PrP knock-
out mice or using PrP of a different species as immuno-
gen [24,25]. Few groups have succeeded in generating a
humoral immune response to PrP by active immuniza-
tion using PrP–PrP polyproteins, PrP-expressing retro-
virus particles, bacterially expressed full-length PrP,
synthetic PrP peptides or polypeptides as antigen [26–
28]. However, synthetic peptides are generally weak
immunogens, and may even induce tolerance in the host.
In addition, only Abs that recognize PrP
C
in its native
conformation exerted prionostatic effects, whereas Abs
to denatured PrP were not effective [27,29,30].
To gain further advantage over previous full-length
or partial-length PrP vaccines, we decided to restrict the
PrP immunogen to a short B-cell epitope comprising a
functionally active peptide, to reduce the possibility of
inducing cell-mediated autoimmune disease. To circum-
vent the problem of low immunogenicity and immuno-
tolerance to endogenous PrP
C
, the use of particulate
PrP–VLPs was chosen as the vaccine strategy. This
immunogen is composed of an ordered and closely
spaced assembly of capsomer subunits displaying a PrP
peptide, to induce a humoral immune response specific
to endogeous PrP
C
[18,28]. A peptide of nine amino acid
residues, DWEDRYYRE, of the murine ⁄rat prion pro-
tein (amino acids 144–152) was incorporated into an
immunogenic surface loop of the L1 major capsid pro-
tein of bovine papillomavirus type 1 (BPV-1) and
expressed by recombinant baculovirus technology. This
peptide resides in Helix 1 of PrP
C
[31,32], and has been
extensively characterized previously, as it is the epitope
recognized by mAb 6H4 (Prionics, Schlieren, Switzer-
land) [33]. Helix 1 of the prion protein is suggested to
play a central role in the protein-induced conformation-
al changes. mAb 6H4 has been well established as one
of the most widely used Abs in prion diseases, and is
employed in routine diagnostic tests for transmissible
Prion-virus-like particle vaccine A. Handisurya et al.
1748 FEBS Journal 274 (2007) 1747–1758 ª2007 The Authors Journal compilation ª2007 FEBS
spongiform encephalopathies. The epitope DWE-
DRYYRE is also recognized by other functionally act-
ive Abs, including Ab.Tg [34]. On the basis of the
existence of these pivotal Abs, and under the assump-
tion that this epitope may represent an effective B-cell
epitope, in both rabbits and mice, we have chosen this
peptide sequence to generate chimeric PrP–VLPs.
Immunization with PrP–VLPs induced high-titer Abs to
PrP in rabbits and in rats, without causing overt adverse
effects. Sera specifically recognized PrP
C
in its native
conformation in mouse brain and on rat pheochromocy-
toma cells, when analyzed by immunoprecipitation and
flow cytometric analysis. Importantly, rabbit Abs to PrP
were of high affinity, and effectively inhibited de novo
synthesis of pathogenetic PrP
Sc
in prion-infected cells.
Results
PrP–L1 fusion protein self-assembles into
virus-like particles (PrP–VLPs) that express the
PrP-DWEDRYYRE epitope
To generate chimeric VLPs that display a PrP epitope
on the particle surface (PrP–VLPs), the murine PrP
peptide DWEDRYYRE (amino acids 144–152)
(Fig. 1A) was engineered into the L1 major capsid
protein of BPV-1 [22]. Following expression of the L1–
PrP protein by recombinant baculoviruses in Sf9 insect
cells, particles were purified by density gradient centrif-
ugation. Analysis by transmission electron microscopy
revealed predominantly spherical structures approxi-
mately 50–55 nm in diameter (Fig. 1B), indicating self-
assembly into VLPs with a morphology similar to that
of wild-type L1–VLPs [35]. In addition, incompletely
assembled particles and individual capsomers (the pen-
tamer subunit of VLPs, consisting of five L1 mole-
cules) were observed. To verify expression of the
inserted PrP epitope DWEDRYYRE, the antigenicity
of PrP–VLPs was analyzed by immunoblotting. The
polyclonal rabbit serum A7 recognized a predominant
band of approximately 55 kDa, corresponding to the
expected size of the PrP–L1 fusion protein (Fig. 1C,
left panel), in the purified PrP–VLP preparation and a
crude Sf9 cell lysate. The faster-migrating bands prob-
ably correspond to proteolytic degradation products.
Specific immunoreactivity with A7 was absent with
parental wild-type L1–VLPs. As expected, mAb AU-1
(Fig. 1C, right panel), directed against a linear epitope
Fig. 1. (A) Amino acid sequences of the rat ⁄mouse and rabbit ⁄human PrP peptides encompassing residues 144–152 of the full-length prion
proteins. The rodent peptide differs at position 145 (bold) from the sequence of the rabbit ⁄human peptide [changing tryptophan (W) to tyro-
sine (Y)]. (B) Transmission electron microscopy of chimeric PrP–VLPs (magnification ·30 000). Scale bar represents 200 nm. (C) Immunoblot
of purified wild-type BPV-1 L1–VLPs (lane 1), PrP–VLPs (lane 2), or crude lysate of recombinant baculovirus-infected Sf9 insect cells expres-
sing PrP–L1 (lane 3), using polyclonal rabbit anti-PrP serum A7 raised against murine dimeric PrP amino acids 23–231 (left), or mAb AU-1
directed against the linear BPV-1 L1 epitope DTYRYI (right). Molecular weight markers are indicated. (D) VLP ELISA under nondenaturing
(native) conditions, using conformation-dependent mAbs to L1, which either recognize both pentamers and VLP (mAb 6), or only fully assem-
bled VLP (mAb 9), or mAb AU-1, which recognizes a linear BPV-1 L1 epitope. Intact VLPs, either PrP–VLPs (black bars) or wild-type BPV-1
L1–VLPs (white bars), were used as the antigens.
A. Handisurya et al.Prion-virus-like particle vaccine
FEBS Journal 274 (2007) 1747–1758 ª2007 The Authors Journal compilation ª2007 FEBS 1749
of BPV-1 L1, reacted with both wild-type BPV-1 L1
and chimeric PrP–L1 proteins.
To determine whether PrP–VLPs retain, at least in
part, the antigenic surface structures of wild-type
L1–VLPs, the immunoreactivity of intact particles was
examined by ELISA, using a conformation-dependent
neutralizing mAb directed against BPV-1 L1. mAb 6
has been shown to bind intact VLPs as well as penta-
meric subunits, whereas mAb 9 requires correctly
assembled VLPs for binding [22,36,37]. In contrast, the
non-neutralizing mAb AU-1 recognizes an internal,
linear epitope of L1 (DTYRYI). As shown in Fig. 1D,
immunoreactivity of mAb 6 and mAb 9 was observed
with chimeric PrP–VLPs (black bars) and, as a control,
wild-type L1–VLPs (white bars), indicating assembly
of chimeric PrP–L1 protein into a complete VLP sim-
ilar to wild-type L1. mAb AU-1 also reacted with both
protein preparations under nondenaturing conditions,
demonstrating nonassembled L1 protein, as observed
regularly in purified VLP preparations. Expression of
the inserted peptide was further tested by ELISA using
rabbit anti-PrP serum A7. The antiserum demonstrated
high absorbance values with PrP–VLPs, but not with
wild-type L1–VLPs, indicating display of the PrP
epitope on the PrP–VLP surface (Fig. 1D).
Immunization with PrP–VLPs induces (auto-)Ab
to the PrP peptide DWEDRYYRE
The chosen murine prion epitope DWEDRYYRE is
situated in the central region (Helix 1) of PrP, which is
highly conserved among species [38], and thus generally
is a poor immunogen. To determine the immuno-
genicity of the inserted epitope presented in the context
of a VLP, rabbits were chosen for immunization, as
this species displays 100% amino acid sequence identity
in the PrP peptide sequence to that in humans, and dif-
fers from the murine sequence in only one amino acid
at position 145 (changing tryptophan W to tyrosine Y)
(Fig. 1A). Importantly, no adverse clinical effects
regarding respiratory function, digestion, weight loss or
behavior were observed in the animals over a period of
3 months following PrP–VLP immunization. None of
the PrP–VLP-immunized animals died until time of sac-
rifice. To determine the PrP-specific humoral immune
response, sera obtained before and after immunization
were tested by ELISA, using the synthetic PrP peptide
DWEDRYYRE as the antigen. The peptide was linked
via two alanine spacers to biotin to ensure complete
accessibility after attachment to streptavidin-coated
microtiter plates. Immune sera showed titers > 1 : 400
by endpoint dilution, as compared to the nonspecific
ELISA reactivity of preimmune sera. Figure 2A (left)
shows a representative experiment. Specificity was
confirmed with rabbit control serum immunized with
wild-type BPV-1 L1–VLPs and by testing anti-PrP–
VLP immune sera using an irrelevant control peptide
(AVLPPVP) as the antigen (data not shown). To fur-
ther corroborate the results, rabbit immune sera were
preabsorbed with either PrP peptide or control peptide
(AVLPPVP), prior to testing by PrP peptide ELISA.
As shown in Fig. 2A (right), preabsorption of the
immune serum with PrP peptide significantly reduced
ELISA reactivity by more than 40% as compared to
immune serum preabsorbed with the control peptide.
These results are in agreement with those of Yokoyama
et al., and indicate that DWEDRYYRE represents a
B-cell epitope also in the rabbit host [34].
Active immunization strategies have been hampered
by immunotolerance to endogenous PrP
C
[9,39,40]. To
Fig. 2. Left: New Zealand White rabbits (A)
or Lewis rats (B) were immunized with PrP–
VLPs, and immune sera (squares) were tes-
ted for immunoreactivity against the PrP
peptide DWEDRYYRE by ELISA. Preim-
mune sera of the same animal (diamonds)
served as the appropriate controls. Right: To
determine specificity, immune sera were
preabsorbed with either PrP peptide DWE-
DRYYRE, or control peptide AVLPPVP, prior
to analysis. Data obtained using serum from
one animal are shown as mean A±SDof
triplicate wells (representative of three inde-
pendent experiments).
Prion-virus-like particle vaccine A. Handisurya et al.
1750 FEBS Journal 274 (2007) 1747–1758 ª2007 The Authors Journal compilation ª2007 FEBS
determine whether tolerance can be overcome with our
chimeric PrP–VLP preparations, Lewis rats were cho-
sen for immunization. Rats share 100% amino acid
sequence identity in the inserted DWEDRYYRE epi-
tope, so the PrP peptide represents a complete self-
antigen. Serum samples from rats were obtained before
and 2 weeks after the last immunization, and subse-
quently subjected to peptide ELISA with PrP peptide
DWEDRYYRE. Immunization with chimeric PrP–
VLPs induced auto-Abs against the PrP peptide with
titers > 400, whereas preimmune serum completely
lacked ELISA reactivity (Fig. 2B, left). The specificity
of the results was further confirmed by overnight pre-
absorption of immune sera. ELISA reactivity was
reduced by 70% with PrP peptide preabsorption, com-
pared to preabsorption with control peptide (Fig. 2B,
right).
We next sought to determine whether PrP–VLP-
induced immune sera were qualitatively different,
whether induced in the rat, for which the PrP peptide
comprised a ‘complete’ prion self-antigen (tryptophan
at position 145), or in the rabbit, for which it com-
prised an ‘incomplete’ self-antigen [one amino acid dif-
ference (tyrosine) at position 145]. Thus immune sera
of rabbits and rats were compared by ELISA for reac-
tivity against synthetic peptides representing either the
murine ⁄rat amino acid sequence (DWEDRYYRE) or
the rabbit ⁄human sequence (DYEDRYYRE) of the
PrP peptide. Purified IgG from rabbit antiserum dem-
onstrated comparable immunoreactivity to both the
murine ⁄rat sequence and the rabbit ⁄human sequence
(Fig. 3A). In contrast, reactivity of IgG isolated from
rat antiserum was directed specifically to the
murine ⁄rat peptide (Fig. 3B), but was absent with the
rabbit peptide. To further examine the Ab affinity of
rabbit and rat anti-PrP immune sera, a modified pep-
tide ELISA was employed. The chaotropic agent
ammonium thiocyanate was added at increasing con-
centrations to dissociate antigen–Ab complexes. Toler-
ance to thiocyanate elution is proportional to the
relative strength of antigen–Ab interactions, thus rep-
resenting a measure of Ab affinity [41]. As shown in
Fig. 3C, addition of approximately 1.35 mand 0.35 m
thiocyanate, respectively, was required to achieve a
50% reduction of rabbit or rat Ab binding. Ab-bind-
ing curves (Fig. 3C) for the rabbit immune sera
revealed an almost identical affinity distribution, when
tested against either the murine ⁄rat or the rab-
bit ⁄human peptide. In contrast, the rat immune serum
showed a significantly (up to four-fold) lower affinity
for the murine ⁄rat peptide over a large molar range of
thiocyanate concentrations, demonstrating a qualitat-
ive difference in the nature of the evoked immune
response. As shown previously (Fig. 3B), rat sera
lacked specific reactivity to the rabbit PrP peptide.
Immune sera recognize native PrP
C
in mouse
brain and on rat pheochromocytoma cells
Recent studies have found an association between the
ability of Abs to recognize native PrP
C
expressed on
the cell surface and their ability to cure prion-infected
cells in vitro and inhibit prion pathogenesis in vivo
Fig. 3. Comparison of ELISA reactivity of purified IgG from PrP–
VLP-immunized rabbit (A) and rat (B) to murine ⁄rat PrP peptide
DWEDRYYRE (white bar), or rabbit ⁄human PrP peptide DYED-
RYYRE (black bar). IgG isolated from preimmune serum of the
same animals served as control. (C) Antibody-binding curves of rab-
bit and rat immune sera in the presence of increasing concentra-
tions of ammonium thiocyanate (NH
4
SCN). Rabbit immune sera
were tested for affinity against the murine ⁄rat DWEDRYYRE pep-
tide (diamonds) or the rabbit ⁄human DYEDRYYRE peptide
(squares). Similarly, rat immune sera (triangles) were tested for
their binding affinity to the murine peptide. As rat sera did not bind
to the rabbit ⁄human peptide (B), they were not further tested. All
experiments were conducted at a serum dilution of 1 : 100. The
results for one representative animal each are shown.
A. Handisurya et al.Prion-virus-like particle vaccine
FEBS Journal 274 (2007) 1747–1758 ª2007 The Authors Journal compilation ª2007 FEBS 1751