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INTRODUCTION |
Prion diseases, including Creutzfeldt-Jakob disease
(CJD)1 in humans, scrapie in
sheep, and bovine spongiform encephalopathy (BSE) in cattle, are fatal
and neurodegenerative infectious disorders. All of these diseases are
characterized by the accumulation of PrPSc, the abnormally
folded isoform of the cellular prion protein (PrPc), which
represents the major component of infectious prions (1, 2). The
formation of PrPSc is accompanied by profound changes in
PrPc structure and biochemical properties.
PrPc, which is rich in
-helical regions, is converted
into a molecule with mainly
-sheeted structure. PrPSc
becomes highly insoluble and partially resistant to proteolytic digestion (1, 3, 4). During biogenesis PrPc transits
through the secretory pathway and is modified by the attachment of two
N-linked carbohydrate chains and a glycolipid anchor. The
conversion of PrPc into PrPSc is thought to
occur after PrPc has reached the plasma membrane, either at
the plasma membrane or shortly after internalization in rafts (5-7).
It is known that PrPc biosynthesis is a prerequisite for
PrPSc formation, and studies in transgenic animals favor a
model in which PrPc and PrPSc interact
directly, possibly in combination with auxiliary factors (1, 8).
Recent work has pointed to the pivotal role of the immune system in
prion infection from peripheral sites. This has been shown in various
transgenic mouse models, impaired e.g. in B-cell maturation, FDC maturation, or in complement factors (9-12). In line with this,
recent immunization studies against
A4 peptide in transgenic mouse
models for Alzheimer's disease have shown dramatic and unexpected clinical and pathological improvements (13). Taken together, these data
strongly suggest that a prophylactic vaccination strategy against prion
infections might be a reasonable approach. In fact, anti-PrP monoclonal
antibodies and recombinant Fab fragments have shown pronounced
anti-prion activities in prion-infected cultured cells (14, 15).
Generation of a transgenic mouse model where PrP was expressed in the
presence of a defined anti-PrP antibody showed that these mice had no
obvious side effects and that they were partly protected against prion
infection from peripheral sites (16).
A major obstacle for an active vaccination strategy in prion diseases
is the apparent autotolerance against PrP existing within a given
species, which prohibits for example the production of antibodies
against murine PrP epitopes within the mouse (17). This is not the case
in PrP0/0 mice or when PrP of a different species is used
as immunogen (17, 18). Characteristically, no innate or antigen-induced immune response is observed in natural prion infections (1, 2).
Here we show that it is possible to overcome the autotolerance to PrP
of the own species by application of an improved immunogen, without
inducing side effects that would indicate an autoimmune reaction.
Recombinantly expressed dimeric mouse PrP induces highly effective
polyclonal anti-PrP antibodies in rabbits and auto-antibodies in mice,
which are superior to antibodies induced by monomeric PrP in anti-prion
effects in prion-infected cultured cells. We provide evidence that full
IgG molecules are much more efficient in anti-prion activity than
corresponding Fab fragments, possibly by inducing a cross-linking of
PrP molecules. Finally, we show that only the auto-antibodies are
directed against a PrP epitope, which is usually not recognized
and which is postulated to represent an interaction site involved in
the conversion process of PrP (15, 19, 20).
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EXPERIMENTAL PROCEDURES |
Reagents--
Freund's adjuvants and TiterMax were obtained
from Sigma. CpG-rich oligonucleotides (1826) were synthesized from TIB
Molbiol (Berlin, Germany). Proteinase K (PK) was obtained from Roche
Applied Science. Immunoblotting was done using the enhanced
chemiluminescence blotting technique (ECL plus) from Amersham
Biosciences. mAb 4F2 has been described (21). Monoclonal anti-PrP
antibody 3F4 (Signet Pathology) recognizes amino acids 109-112 of
human and hamster PrP. [35S]Met/Cys (Promix; 1000 Ci/mmol) was obtained from Amersham Biosciences. Protein A-Sepharose
was from Amersham Biosciences. Cell culture media and solutions
were obtained from Invitrogen. Disuccinimidyl suberate (DSS) was from
Perbio (Bonn, Germany). PIPLC and all other chemicals were from Roche
Applied Science.
Recombinant Proteins--
Dimeric PrP consists of a tandem
duplication of murine PrP (amino acids 23-231 = 23-231; not
containing the N- and C-terminal signal peptides; amino acids 1-22 and
232-254, respectively). Murine PrP gene subunits were
amplified by PCR from a 3F4-tagged full-length murine PrP using
appropriate primers comprising the 7-amino acid linker sequence
described before (22). Using PvuI sites DNA fragments were
fused and cloned into the bacterial expression vector pQE30, thereby
providing an N-terminal polyhistidine tag. Expression was done in
E. coli strain BL21. Bacteria were lysed in 6 M
guanidinium hydrochloride, cellular debris was removed by
centrifugation at 6,000 × g for 20 min, and the
soluble fraction was loaded onto a Ni2+-column (ProBond,
Invitrogen) pre-equilibrated with binding buffer (8 M urea,
20 mM sodium phosphate, 500 mM sodium chloride,
pH 7.8). The column was washed several times (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, 80 mM imidazole, pH 6.3), and the His-tagged fusion protein
was eluted (8 M urea, 20 mM sodium phosphate,
500 mM sodium chloride, 500 mM imidazole, pH
6.3). Eluted fractions with highest protein concentrations were pooled, urea was removed, and PrP refolded by dialysis against ultra-filtrated water (pH 4.5). Monomeric PrP-(23-231) was similarly constructed as a
3F4-tagged murine PrP in the same vector. The genotype of both
immunogens was confirmed by sequencing to be
Prnpa/a.
Immunizations--
Female C57Bl/6 mice (Prnp genotype
a/a; 6 weeks old) were immunized by subcutaneous application of 50 µg
of dimeric or monomeric recombinant PrP, respectively. For the
classical approach at day 0 protein was applied with complete Freund's
adjuvants (1:1, v/v), followed by two booster injections at days 21 and
42, using incomplete Freund's adjuvants (1:1, v/v). The immunizations
with CpG (10 nmol per injection), TiterMax (1:1, v/v) and the
combination of CpG and incomplete FA (10 nmol of CpG plus FA, 1:1, v/v)
were only boosted once at day 21. Ten days after the last immunization blood was taken for testing of antibody reactivity. Prebleed samples were taken 7 days before starting the immunizations. Rabbits were immunized three times in the classical way using 500 µg of dimeric or
monomeric recombinant PrP, respectively, and Freund's adjuvants.
Antibody Titer (ELISA)--
Antibody titers were determined by
an ELISA in 96-well format. Wells were coated with 1 µg of monomer or
dimer, respectively, in 150 µl of carbonate buffer (0.1 M, pH 9.5) overnight at room temperature. Wells were washed
with PBST (PBS, 1% Tween) and blocked with 150 µl of blocking buffer
(PBST, 3% bovine serum albumin (w/v)) for 2 h at 37 °C. Wells
were incubated with 100 µl of prediluted polyclonal sera for 1 h
at 37 °C. After thorough washing, incubation with the appropriate
mouse or rabbit conjugate (dilution 1:4,000) was performed for 1 h
at 37 °C. The last washing step was followed by incubation with 100 µl of ABTS
(2,2'-azido-bis[3-ethylbenzthiazidine-6-sulfonic acid]). Reaction
was stopped with 1 N H2SO4, and
optical density measured at 405 nm wavelength. Preimmune sera served as
negative controls at a dilution of 1:100, and the cutoff for positive
sera was calculated with 2.5 times the average extinction of preimmune sera. Titers were evaluated by end-point dilution approach (highest dilution of the sera that was still positive).
Cell Culture and Mode of Antibody Application--
The mouse
neuroblastoma cell lines N2a and ScN2a have been described (expressing
wild-type murine PrP and 3F4-tagged mouse PrP; both Prnp
genotype a/a) (23). Cells were maintained in Opti-MEM medium containing
10% fetal calf serum. Polyclonal auto-antibodies were added at a 1:50
dilution, if not otherwise stated. This dilution was determined by
serial dilution studies as most appropriate for discriminating
effective and non-effective antisera. Fabs were applied at a
concentration of 20 µg/ml (400 nM) for overnight incubation in short term experiments. For long term treatment, medium
changes were done every other day, and antibodies were added fresh with
each medium change.
Metabolic Radiolabeling and Immunoprecipitation
Assay--
Confluent cells were washed twice with PBS and incubated
1 h in RPMI 1640 without methionine/cysteine containing 1% fetal calf serum. The medium was supplemented with 800 µCi of
L-[35S]Met/Cys for 16 h (± antisera)
without a chase period. For competition assay, the antibody (1:40 in
labeling medium) was preabsorbed with increasing amounts of recombinant
dimeric prion protein (0-20 µg) for 1 h at 37 °C and then
added to the cells. After incubation, cells were washed twice in
ice-cold phosphate-buffered saline and lysed in cold lysis buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.8, 10 mM EDTA, 0.5% Triton X-100; 0.5% sodium deoxycholate); insoluble material was removed by centrifugation. For PK treatment samples were divided in half. One-half was incubated with PK (20 µg/ml) for 30 min at 37 °C, and digestion was stopped by addition of proteinase inhibitors. Then lysates with and without PK treatment were subjected to ultracentrifugation in a Beckmann TL-100
ultracentrifuge (1 h at 100,000 × g; TLA-45 rotor) in
the presence of 1% sarcosyl. Pellets were resuspended in 100 µl of
RIPA buffer (0.5% Triton X-100, 0.5% deoxycholate, in PBS) with 1%
SDS, boiled for 10 min, and diluted with 900 µl of RIPA buffer
(supplemented with 1% sarcosyl). The primary antibody (1:300) was
incubated overnight at 4 °C. Protein A-Sepharose beads were then
added for 60 min at 4 °C. The immunoadsorbed proteins were washed in
RIPA buffer supplemented with 1% SDS, subjected to a deglycosylation
step with PNGaseF, and analyzed on 12.5% SDS-PAGE followed by autoradiography.
Immunoblot Analysis--
Confluent cell cultures were lysed in
cold lysis buffer. Postnuclear lysates were subjected to PK digestion
or ultracentrifugation as described above or supplemented with
proteinase inhibitors (5 mM phenylmethylsulfonyl fluoride
and 0.5 mM Pefabloc) and directly precipitated with
ethanol. After centrifugation for 30 min at 3,500 rpm, the pellets were
redissolved in TNE buffer, and gel-loading buffer was added. After
boiling for 5 min an aliquot was analyzed on 12.5% SDS-PAGE followed
by immunoblot as described (23).
Preparation of Fab Fragments--
For preparation of Fab
fragments monomer-induced and dimer-induced polyclonal rabbit antisera
were desalted and total IgGs purified using the Econo Pac Serum IgG
purification kit (Bio-Rad) following the manufacturer's instructions.
Concentration of total IgG was determined (1.5 mg/ml). For comparison
of inhibitory effects between IgG and derived Fabs, this fraction was
added to the cells at a dilution of 1:25 (~400 nM). This
is equivalent to the concentration of Fab fragments of 20 µg/ml (400 nM). Buffer was exchanged to 20 mM
Na3PO4, 10 mM EDTA (pH 7.0) and
volume concentrated to 0.5-ml final volume by using Vivaspin 20 columns
(10,000 MWCO, Vivascience). The Fab fragments were generated by papain
digestion using the ImmoPure Fab preparation kit (Perbio) following the
manufacturer's instructions. The Fab fragments were collected in a
final volume of 3 ml, dialyzed against ultrafiltrated water, and
concentrated to a final concentration of 0.5 (dimer-induced) and 1.0 µg/µl (monomer-induced) using Vivaspin columns.
Epitope Mapping--
We have designed a peptide bank consisting
of 14 peptides of 20 amino acids and 1 peptide of 15 amino acids in
length, respectively, with 5 amino acids overlapping to adjacent
peptides, encompassing full-length mature murine PrP (23-231, shown in
Fig. 5A). For coating, wells (CovaLink NH modules, Nunc)
were activated with the bifunctional linker DSS in carbonate buffer (15 mM Na2CO3, 35 mM
NaHCO3, pH 9.6) and incubated with 100 µg of each peptide or 2 µg of recombinant dimer (control), respectively, overnight at
room temperature. Wells were blocked (200 mM NaCl, 0.05%
Triton X-100, 3% (w/v) bovine serum albumin, in PBS) for 30 min at
room temperature, washed (200 mM NaCl, 0.05% Triton X-100,
in PBS), and incubated with prediluted sera for 2 h at room
temperature. After another washing step wells were incubated with the
corresponding conjugate (diluted 1:4,000 with blocking buffer) for
1 h at room temperature. Upon washing and incubation with ABTS,
the reaction was stopped with 1 N
H2SO4 and documented by optical density
measurement and photography with a digital camera.
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RESULTS |
Induction of Anti-PrP Auto-antibodies in PrP Wild-type
Mice--
In order to overcome autotolerance against murine PrP in
mice we used a covalently linked recombinant tandem duplication of murine PrP (designated as PrP dimer) as an immunogen (Fig.
1A). Linkage was done using a
seven-residue linker (22), resulting in a dimer consisting of two
mature PrP moieties (PrP 23-231 = 23-231). This protein and a
monomeric version (PrP-(23-231)) were expressed in E. coli
as polyhistidine fusion proteins. Upon purification on
Ni2+-columns and refolding we typically obtained proteins
with a purity of more than 94% (Fig. 1B). CD analysis
showed that the secondary structure of both monomeric and dimeric PrP
was in an
-helical conformation (data not shown).

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Fig. 1.
Recombinant prion proteins used as immunogen
and auto-antibody titers. A, recombinant prion proteins were
constructed encompassing mature murine PrP with an N-terminal
polyhistidine tag and insertion of the 3F4 epitope. For PrP-dimer, two
PrP molecules were covalently linked by a 7-residue sequence. Proteins
were expressed in E. coli, purified from inclusion bodies
under denaturing conditions, refolded, and dialyzed against
ultrafiltrated water. B, purity of the dimer
(lane 2) and monomer (lane 3) was determined by
SDS-PAGE followed by Coomassie Blue staining. Lane 1 shows a
molecular weight marker (M). Proteins were obtained at a
purity of ~94% and standardized to a concentration of 1 mg/ml.
C, immunization of PrP wild-type mice with recombinant PrP
dimer resulted in high auto-antibody titers. Four groups of 10 mice
each were immunized s.c. with FA, TiterMax, CpG, or a combination of FA
and CpG (see color code depicted on the right side). Blood
was taken 10 days after the last immunization. Titers of individual
mice were determined by end-point dilution of pAb in ELISA using the
PrP dimer as antigen (x-axis). The y-axis shows
the number of antisera positive at a given dilution. Of note, three
animals of the combination group had to be sacrificed before the
booster immunization because of severe local abscess formation.
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We immunized PrP wild-type mice and rabbits with the recombinant
monomeric and dimeric murine PrP, using initially a classical scheme
with Freund's adjuvants (FA) and 50 µg (mice) or 500 µg (rabbits)
of antigen in 3 subcutaneous (s.c.) injections. More than 90% of
wild-type mice and all rabbits showed clearly detectable anti-PrP
antibodies/auto-antibodies in ELISA. There was no significant difference in titers between dimeric and monomeric PrP immunogens. Of
note, no obvious systemic side effects or auto-immune phenomena were
observed in immunized wild-type mice. Also, when testing e.g. peripheral blood counts, no difference between
immunized and non-immunized mice was detectable (data not shown). To
optimize the antibody response we compared different types of adjuvants for the immunization with dimeric PrP. In Fig. 1C ELISA
end-point titers of a representative study in 40 mice are summarized,
induced with 4 different adjuvant types (10 mice each). FA and TiterMax gave comparable results, whereas the combination of CpG-rich
oligonucleotides (CpG) and incomplete FA is not applicable despite
induction of very high titers because of severe local abscess formation
in some animals. Studies on immunization with CpG alone will be
intensified in the future because no local side effects of the
immunization were observed. The anti-PrP titers were determined in
ELISA by end-point dilution (x-axis). The auto-antisera
generated in PrP wild-type mice showed reactivity up to dilutions of
1:20,000. The antisera generated in rabbits showed reactivity up to
dilutions of >1:50,000 (data not shown). In summary, we show that the
immunogen used by this group is able to effectively induce
auto-antibodies against PrP in wild-type mice without inducing obvious
systemic side effects.
Auto-antibodies Induced by Dimeric PrP Effectively Inhibit
PrPSc Biogenesis in Infected Cells--
Having found that
it was possible to generate polyclonal anti-PrP auto-antibodies in
PrP-expressing mice, we asked whether the induced antibodies can
interfere with endogenous PrPSc synthesis in prion-infected
mouse cells. This should also prove the integrity and functionality of
the induced polyclonal antibodies under physiological and native
conditions. We added polyclonal mouse and rabbit antibodies (pAb) to
the culture medium of persistently prion-infected ScN2a cells. As
readout we initially measured effects on the de novo
synthesis of PrPSc. Cells were metabolically labeled and
either rabbit (rab) or murine (mo) dimer-induced
anti-PrP antibodies were added to the medium overnight at a dilution of
1:25 (Fig. 2A). Cells were
lysed (± PK digestion), subjected to ultracentrifugation,
immunoprecipitated, and analyzed in SDS-PAGE upon deglycosylation with
PNGaseF (only pellet fractions shown). Antibodies induced in rabbits by
dimeric PrP completely blocked PrPSc biogenesis
(lanes 9 and 10), and auto-antibodies generated
in mice also significantly inhibited the de novo synthesis
(lanes 11 and 12). Interestingly, a full-length
insoluble PrP population was induced by the antibody treatment that was
PK-sensitive, apparently representing insoluble PrPc (
PK
panel, lanes 3-6). We then compared effects exerted by
auto-antibodies generated either with monomeric or dimeric PrP. Fig.
2B shows a representative analysis of 4 dimer- and 4 monomer-induced auto-antisera generated in PrP wild-type mice (diluted
1:50). In total, sera from 26 individual mice immunized with dimeric
PrP and from 19 mice immunized with monomeric PrP were analyzed for
their ability to interfere with PrPSc biogenesis in cell
culture. Only sera that completely blocked PrPSc de
novo synthesis, resulting in no detectable signal for
PrPSc (see lanes 8 and 9), were
considered positive. We found pAb induced with dimeric PrP to be far
superior in their inhibitory effect, as 12 of 26 (46.2%) were positive
under these experimental conditions, in contrast to only 1 of 19 (5.3%) raised against monomeric PrP. To further verify the impact of
dimer-induced auto-antibodies on the propagation of PrPSc
we performed long term studies with mouse auto-antisera (Fig. 2C, left panel; dilution 1:25). ScN2a cells were
treated with pAb (mouse) for 7 days, and the amount of
PrPSc in the pellet fraction of the lysates (
/+ PK;
lanes 3 and 4) was analyzed in immunoblot in
comparison to mock-treated control cells (lanes 1 and
2). In auto-antibody treated cells, PrPSc was
completely abolished, indicating that pre-existing PrPSc
could be cleared by the cells when the de novo synthesis of
PrPSc was blocked. Even after further cultivation of
treated cells for 7 days without antibody no PrPSc was
detectable (lanes 7 and 8; lanes 5 and
6, mock-treated control cells), arguing that the cells were
eventually cured from PrPSc propagation by the antibody
treatment. Interestingly, when we analyzed PrPc present in
the soluble fraction, the signal was decreased by the auto-antibody
treatment (lane 10 versus lane 9).
After another 7 days without antibody, soluble PrPc was
again expressed at the same level as in the control cells (lane
12 versus lane 11).

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Fig. 2.
Polyclonal dimer-induced anti-PrP
antibodies/auto-antibodies effectively interfere with PrPSc
biogenesis in prion-infected cells. A, de novo
synthesis of PrPSc is blocked by dimer-induced rabbit
anti-PrP antibodies (rab-di) and reduced by dimer-induced
murine PrP auto-antibodies (mo-di). ScN2a cells were
metabolically labeled with [35S]Met/Cys overnight and
incubated simultaneously with antibodies (at a 1:25 dilution in cell
culture medium). Cells were lysed, treated ± PK, subjected to
ultracentrifugation (only pellet fraction shown), and
immunoprecipitated using a polyclonal anti-PrP antibody. After
deglycosylation immunoprecipitates were analyzed in SDS-PAGE followed
by autoradiography. The left panel shows samples without PK
digestion, the right panel with PK. Control (co)
is without antibody incubation. Cells were analyzed in duplicate.
B, dimer-induced auto-antibodies from mice inhibit the
de novo synthesis of PrPSc. Similar analysis as
in A, all samples were PK-treated. Sera from individual mice
(4 each) were compared (di, dimer-induced; mon,
monomer-induced; pAb dilution 1:50). Control (co) shows
cells without antibody treatment. C, long term treatment
with dimer-induced auto-antibodies completely abolishes
PrPSc generation. ScN2a cells were treated with pAb for 7 days. One plate was lysed (7+), and a second plate was cultivated for
another 7 days without pAb (7+/7 ). Lysates ( /+ PK) were subjected
to ultracentrifugation. PrPSc signal in the pellet fraction
(lanes 1-8), and the amount of PrPc present in
the supernatant (lanes 9-12) was analyzed in immunoblots
and compared with mock-treated control cells (co7 and
co14).
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Taken together, our data indicate that auto-antibodies induced by
dimeric PrP are able to effectively interfere with endogenous PrPSc biogenesis in prion-infected cells. After long term
treatment with auto-antibodies, the amount of soluble PrPc
is significantly reduced, and the cells seem to be cured from prion infection.
Highly Superior Effect of Dimer-induced Full IgG Molecules versus
Fab Fragments--
Treatment of prion-infected cells with
pAb/auto-antibodies resulted in a pronounced reduction of
PrPSc. Assuming that this effect was mediated by a direct
interaction of specific antibodies with surface PrPc we
performed competition assays (Fig.
3A). Dimer-induced pAbs were
preabsorbed (1 h at 37 °C in culture medium) with increasing amounts
of recombinant PrP dimer and then added to ScN2a cells simultaneously
with the metabolic labeling mixture. As readout we compared the amounts
of PrPSc and of insoluble full-length PrP in untreated,
pAb-treated cells, and cells treated with preabsorbed pAbs. Treatment
with pAb (non-preabsorbed) or with pAb after preincubation with only 1 µg of recombinant protein (lanes 8 and 9)
completely abolished de novo synthesis of PrPSc
and induced insoluble PrP (lanes 2 and 3).
Preabsorption of pAb with concentrations between 5 and 20 µg
recombinant dimer was sufficient to prevent the antibody-induced
inhibition of PrPSc de novo synthesis, indicated
by the synthesis of PrPSc in almost equal amounts compared
with the untreated control cells (lanes 10-12 versus lane
7) and the absence of insoluble PrP (lanes 4-6). From
this data we conclude that both the inhibition of PrPSc
generation and the induction of insoluble full-length PrP were due to a
specific binding of antibodies to surface PrPc.

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Fig. 3.
Specific binding of pAb to surface PrP and
highly decreased effect of Fab fragments on de novo
synthesis of PrPSc. A, inhibition of
PrPSc synthesis can be competed by preincubation of pAb
in vitro with recombinant PrP dimer. ScN2a cells were
metabolically labeled in the absence ( pAb) or presence of antibodies
(+pAb) preabsorbed in vitro with rising amounts (0-20 µg)
of recombinant PrP dimer. Cell lysates were either left untreated
( PK) or digested with PK (+PK) and subjected to ultracentrifugation.
The detergent-insoluble fraction was used for immunoprecipitation with
a polyclonal anti-PrP antibody. After deglycosylation samples were
analyzed in SDS-PAGE followed by autoradiography. Bars on
the right indicate PrP-specific bands. B,
generation of Fab fragments from polyclonal rabbit antisera. 5 µg of
Fab fragments were subjected to SDS-PAGE followed by Coomassie Blue
staining. Lane 1 shows the Fab fragment derived from the
monomer-induced serum, lane 2 the dimer-induced Fab. The
bar on the right indicates a Fab
fragment-specific band. C, reduced interference of Fab
fragments with PrPSc de novo synthesis. ScN2a
cells were metabolically labeled for 16 h and simultaneously
treated with preimmune serum (co), monomer-induced
(mon), or dimer-induced (di) polyclonal IgG (IgG;
1:25 (400 nM); lanes 2, 3,
7, and 8) or the corresponding Fab fragments
(Fab; mon, di; 20 µg/ml (400 nM); lanes 4,
5, 9, and 10). Lysates were divided
into two samples with (+PK) or without ( PK) PK treatment. All samples
were subjected to ultracentrifugation, and the insoluble fraction was
used for immunoprecipitation followed by deglycosylation and SDS-PAGE.
Bars on the right indicate PrP- specific
bands.
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Given the bivalent and polyclonal character of IgG molecules and the
strong binding of antibodies to PrP we speculated whether the observed
induction of PrP aggregates might be the result of cross-linking of
PrPc by IgGs. To verify this hypothesis we generated Fab
fragments of both monomer- and dimer-induced antisera. Due to the high
amount of serum needed for the preparation this was done from rabbit sera only. The papain cleavage of IgGs was confirmed by SDS-PAGE of the
Fab fragments followed by Coomassie Blue staining (Fig. 3B).
Only Fab molecules of about 29 kDa were visible; heavy chains (~50
kDa) were completely absent.
The efficiency of the polyclonal Fab fragments in terms of PrP
cross-linking and inhibition of PrPSc synthesis compared
with full IgG molecules was tested by metabolic labeling of ScN2a cells
and immunoprecipitation of insoluble PrP after treatment (400 nM each, Fig. 3C). Without PK digestion, IgG
molecules induced full-length insoluble PrP (lanes 2 and
3) as observed before (Figs. 2A and
3A). Interestingly, after treatment with Fab fragments no
insoluble full-length PrP was detectable, which can be explained by the
inability of Fabs to cross-link PrP molecules. In PK-treated lysates,
only the dimer-induced antisera completely blocked PrPSc
generation (lane 8). The corresponding Fab fragment
exhibited a less pronounced effect on PrPSc synthesis
(lane 10). The monomer-induced antisera, although producing almost equal amounts of insoluble PrPc compared with
dimer-induced antibodies (lane 2), had no effect on
PrPSc synthesis (lane 7). The latter was also
true for the corresponding Fab fragments (lane 9).
We then investigated the effect of IgG antisera and derived Fab
fragments on the total PrPSc content of ScN2a cells in a
long term treatment for up to 7 days. At various time points cells were
lysed, treated or not treated with PK, and all lysates were subjected
to ultracentrifugation. Both detergent insoluble (Fig.
4A) and soluble (Fig.
4B) PrP fractions were analyzed in immunoblot. Only upon
incubation with dimer-induced full IgG molecules the cells completely
eliminated PrPSc after 5 days of treatment (lanes
5-8), with the signal significantly decreased after 3 days
(lanes 3 and 4). Treatment with dimer-induced Fab
fragments (Fig. 4A, lanes 9-16) did not result
in an inhibition of prion conversion. Of note, we used 20 µg/ml Fab
(400 nM) fragments derived from pAb, of which anti-PrP only
represents a tiny fraction of total IgG (in contrast to monoclonal Fab
concentrations, described in Ref. 15). As before, insoluble full-length
PrP was detectable that was degraded by PK treatment (lanes
3, 5, and 7). Even in this long term study,
neither monomer-induced IgGs nor the corresponding Fab fragments
significantly decreased the level of PrPSc (data not
shown). As already shown with the auto-antibody treatment (Fig.
2C), the amount of soluble PrPc in cells treated
with dimer-induced IgGs (Fig. 4B, lanes 3-5) was
decreased compared with mock-treated control cells (lane 1). This effect was not observed upon treatment with monomer-induced IgGs
and dimer- or monomer-induced Fabs (lanes 5-13).

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Fig. 4.
Long term treatment with dimer-induced pAb
eliminates PrPSc and reduces PrPc.
A, effect of long term treatment of ScN2a cells with
polyclonal antisera and Fab fragments. ScN2a cells were treated with
dimer-induced (di) pAb (IgG; 1:25; 400 nM) or
the corresponding Fab fragments (Fab; 20 µg/ml; 400 nM)
and lysed at various time points as indicated (0, 3, 5, 7 days,
respectively). Cell lysates were either treated with PK or not ( /+
PK) and subjected to ultracentrifugation. The detergent-insoluble
fraction was analyzed in immunoblots using mAb 3F4. Amounts of
PrPSc were compared with mock-treated control cells (0 days). B, influence on PrPc expression after
long term treatment with polyclonal antibodies and Fab fragments. ScN2a
cells were treated as described in A. The detergent-soluble
fraction without PK digestion was subjected to an immunoblot assay.
PrPc was detected with mAb 3F4. PrP-specific bands are
indicated by the bars on the right.
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In conclusion, pAbs bind specifically to surface PrPc and
can induce the formation of insoluble PrP. This cross-linking of PrP by
full IgG molecules dramatically enhances the anti-prion activity
compared with the effect of corresponding Fab fragments. The reduced
level of soluble PrPc in cells treated with dimer-induced
IgG might add to the strong impact on the synthesis of
PrPSc over time.
Auto-antibodies Are Directed Against a PrP Epitope Involved in
PrPSc Conversion Process--
To further characterize the
polyclonal antisera we performed an epitope mapping using a peptide
bank encompassing the mature full-length murine PrP (Fig.
5A). pAbs generated in rabbits
and PrP wild-type mice were tested for reactivity against these
peptides in an ELISA (Fig. 5B). We found that the
immunization with the monomer in rabbits resulted in a very different
epitope pattern compared with the pattern induced in dimer-immunized
rabbits, with many more C-terminal epitopes recognized by the latter.
The PrP wild-type mice responded with a similar epitope pattern
regardless of the immunogen used, but with a completely different
pattern compared with the rabbits. Of note, non-linear and
conformational epitopes cannot be detected in our assay. Interestingly,
only in the auto-antibody situation we observed a reactivity against epitope 10, which encompasses the second PrP
-sheet. This epitope is
also absent in antisera of PrP0/0 mice that were immunized
with the PrP dimer (data not shown). In summary, we demonstrate that
anti-PrP auto-antibodies in mice are directed against a PrP epitope,
which is apparently not recognized in rabbit and PrP0/0
antisera.

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Fig. 5.
Different PrP epitopes in wild-type mice and
rabbits. A, peptide bank encompassing the murine PrP.
Peptides consist of 20 residues, with an overlap of 5 residues. Peptide
6 encompasses the 3F4 epitope version, 6b represents the
wild-type murine sequence. The positions of the peptides in the epitope
mapping is indicated on the left side in B.
Recombinant monomeric PrP was used as a control (Co).
B, epitope mapping of representative antibodies
generated by immunization with PrP monomer (mon) or dimer
(di). In rabbits, dimer-induced pAb displayed high
reactivity against many different N- and C-terminal epitopes, whereas
monomer-induced pAb showed only reactivity against some N-terminal
epitopes. Immunization with either monomeric or dimeric PrP gave a
similar epitope reactivity in wild-type mice. Peptide 8 and the
recombinant PrP control are recognized by all investigated pAb. Of
note, peptide 10 was only reactive in wild-type mice.
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DISCUSSION |
The rise and spread of the variant of CJD (vCJD), which is
causally linked to BSE in the United Kingdom and several other countries has fueled the discussion on therapeutic and prophylactic tools against human prion disorders. Of particular interest is that
vCJD, in contrast to other human prion diseases, comes with a
pronounced lymphoreticular tropism, which might pose the risk of
accidental transmission within human population. Therefore, prophylactic approaches are urgently needed. The classical prophylactic strategy against infectious diseases is active immunization. This is
hampered in prion diseases for several reasons, one of which is the
obvious autotolerance against PrPc and PrPSc.
Here we demonstrate that it is possible to induce effectively auto-antibodies directed against surface-located PrPc and
that these auto-antibodies have the potential to interfere with
PrPSc biogenesis in prion-infected cells.
Auto-antibodies Against PrPc Can Be Induced in
Wild-type Mice--
We provide evidence that the dimeric PrP as
introduced by us might represent a prototype immunogen useful for
developing active immunization strategies against prion disorders.
Auto-antibodies directed against
A4 peptide were recently described
as effective against pathogenic events in experimental models of
Alzheimer's disease (AD) (13, 24, 25). These antibodies were
apparently able to cross the blood-brain-barrier in AD animal models
(13). As a note of caution, a recent clinical trial in AD patients had to be stopped because of side effects. Our experimental strategy intends to generate auto-antibodies reacting against and binding to
surface-located PrPc. Importantly, in our studies in
wild-type mice we did not observe any obvious side effects. Studies in
mice have shown that PrPc can be removed without inducing
side effects or neurodegeneration (26, 27). We have shown before that
compromising surface PrPc expression can be useful in
prophylaxis of prion diseases (23). Similar results were obtained
recently in prion-infected cells when using RNA-aptamers directed
against surface PrPc (28). A preceding surface expression
of PrPc is known to be a necessary requirement for cellular
prion biogenesis (5-7), and peripheral PrPc expression is
absolutely indispensable for the transport of prions from peripheral
sites of the body to the central nervous system (29). Our short term
studies show that it is not a net decrease of surface-located
PrPc, needed as substrate for prion conversion, which is
responsible for the inhibition of PrPSc biogenesis. On the
other hand, our long term experiments with the most effective antisera
have demonstrated that the steady-state level of soluble
PrPc can be significantly decreased over time.
Using various adjuvant formulations we found that it is possible to
induce high anti-PrP titers (~1:20,000) without the risk of systemic
side effects. Of note, the genetic PrP background of immunogens, mice
and ScN2a cells employed in this study was confirmed by sequencing to
be identical (Prnpa/a). However, up to now it is
not clear whether a sole antibody response will be sufficient or more
importantly a strong involvement of T-cell and innate immunity will be
needed for a prophylactic effect of immunization (30). Recent data have
shown that co-administration of CpG alone was sufficient to prolong
prion disease development after peripheral infection in a postexposure
approach (31). At present it is not known whether this effect is due to
activation of innate immunity, thereby clearing the prion load, or
whether this is by inducing an anti-PrP antibody or T-cell response.
Why is dimeric PrP a better immunogen than monomeric PrP? Although
dimeric PrP shows a very similar
-helical secondary structure when
compared with monomeric PrP in CD studies, we assume that the covalent
linkage forces the PrP dimer to adopt a slightly different folding
compared with authentic PrPc or monomeric recombinant PrP,
showing similarity to folding intermediates occurring during the
conversion process. This might allow a better recognition of this
molecule by the immune system. Evidence for the impact of the structure
was obtained by using slightly degraded PrP dimers for immunization
which showed, in contrast to similarly treated monomeric PrP, a drastic
reduction in immunogenicity (data not shown). Of note, the difference
between monomeric and dimeric PrP auto-antigen was not reflected by the
antibody titers as measured with denatured PrP in ELISA. ELISA titers
both for monomer- and dimer-induced murine auto-antibodies were almost
equal. On the other hand, 46.2% (12/26) of dimer-induced,
ELISA-positive sera from individual mice were positive in our cell
culture assay compared with 5.3% (1/19) monomer-induced individual
sera, which proves to be highly significant (p value = 0.003,
2-test). These data clearly indicate that
dimer-induced auto-antibodies have a more pronounced antiprion effect,
which is not reflected by the ELISA titer. The obvious independence of
ELISA titers and effects in cell culture was also evident from
comparing antibodies induced in rabbits and PrP wild-type mice. Whereas
much higher ELISA titers were obtained in rabbits (>1:50,000, compared
with ~1:20,000 of auto-antibodies), this fact was not reflected by antiprion effects exerted in cell culture in titration studies (data
not shown).
Taken together, our data demonstrate that it is possible to overcome
the autotolerance against PrPc by an active
auto-immunization strategy. We show that there is no direct correlation
between ELISA titer and anti-prion effect as exerted in prion-infected
cell culture, but rather it depends on the structure of the immunogen
and thereby displayed epitopes.
Only Full IgG Molecules Induce Cross-linking and Reduction of
Soluble PrPc--
Previous studies with recombinant Fab
fragments directed against PrP have shown remarkable antiprion effects
in persistently prion-infected cultured cells (15). The Fab D18
reacting against PrP residues 132-156 could cure the cells. Of note,
long term treatment of cells for up to 3 weeks did not result in a
detectable reduction of PrPc. In line with this are recent
reports using the commercial monoclonal anti-PrP antibody 6H4 in
infected cells (14) and a transgenic mouse model where PrPc
was expressed together with a specific anti-PrP antibody (16). Here
again, no effects on PrPc levels were found. Of note,
concentrations of these specific monoclonal antibodies/Fabs used in
both cell culture studies mentioned above for long term treatment were
quite high (in the microgram range, 10-20 µg/ml). Such
concentrations are far above blood concentrations, which can be
achieved by active immunization or even by passive antibody transfer in mammals.
Our studies demonstrate a striking difference in effects exerted by
polyclonal full IgG molecules compared with derived Fab fragments.
First, application of full IgG antibodies resulted in a pronounced
induction of an insoluble full-length PrP population, which was not
detectable using the homologous Fab fragments. Second, the biological
effect of Fabs in cell culture on biogenesis and levels of
PrPSc was highly reduced. Third, only dimer-induced pAbs
decreased the levels of soluble PrPc in long term studies.
We assume that these differences result from the ability of full IgG
molecules to efficiently cross-link adjacent surface-located PrP
molecules. Whereas Fab fragments do not have this possibility and mAbs
can only dimerize PrP using the single given epitope, pAb can use a
variety of different adjacent epitopes for such a cross-linking
mechanism, yielding eventually to the generation of PrP aggregates.
Obviously, these aggregates are rapidly degraded by the cells. Even
after treatment for 7 days with pAb we did not observe accumulation of
insoluble PrPc. Of note, also monomer-induced rabbit
antisera were able to induce a cross-linking of PrP but were not
effective in reducing PrPSc. In epitope mapping,
monomer-induced pAb were found to be reactive only against several
N-terminal epitopes, whereas dimer-induced sera recognized N- as well
as C-terminal epitopes. This indicates that cross-linking of PrP is
only sufficient to decrease PrPSc when PrPc
sites implicated in prion conversion are targeted. The importance of
C-terminal regions in prion conversion is supported by previous findings in cell-free conversion studies where an antibody against a
C-terminal epitope (amino acids 219-232) inhibited prion conversion not by direct interference, but by sterically blocking neighboring binding sites necessary for PrPc-PrPSc
interaction (32). On the other hand, Fab fragments prepared from
dimer-induced pAb recognize exactly the same epitopes as the full IgGs
but are less effective in cell culture. Therefore we conclude that
cross-linking significantly improves the anti-prion activity. It
remains to be established whether this mechanism comes into force in an
in vivo situation. Taken together, our data demonstrate the
striking difference in molecular mechanisms used by various types of
anti-PrP antibodies and underline the very special potential of
polyclonal anti-PrP antibodies.
Anti-PrP Auto-antibodies Are Directed Against an Epitope Involved
in PrP Interactions--
A decisive factor in the efficacy of anti-PrP
antibodies are the epitopes against which the reactivity is directed.
In studies describing the experimental generation of anti-PrP
monoclonal antibodies it was found that the PrP region encompassing
residues 130-156 is a prominent epitope (21). This region of PrP has been found to be critical to prion propagation and transmission (33,
34), which was reinforced in experiments using recombinant Fab
fragments in prion-infected cells (15).
To explain the molecular mechanism for the efficacy of the above
mentioned Fab D18 the authors argued that the targeted epitope is
exactly on the face of the PrP molecule, which is believed to be
involved in PrP-PrP interaction and which is opposite to the postulated
binding site for a putative cellular cofactor involved in prion
conversion (15). Our epitope mapping data of anti-PrP auto-antibodies
clearly indicate a different molecular mechanism in our studies.
Whereas the epitope mentioned before is found in all anti-PrP
preparations tested by us, we detected reactivity in auto-antibodies
only against an epitope not found in heterologous immunizations,
indicating a low immunogenicity in this situation. Of note, in
PrP0/0 mice this epitope is not represented, arguing
against a species-specific epitope. The epitope encompasses PrP
residues 159-178 harboring the second
-sheet and one portion of the
discontinuous epitope mapped previously as binding site of a putative
cellular cofactor (residues Gln-168 and Gln-172, Ref. 20; numbering
according to Ref. 35). This cofactor has been characterized initially in transgenic animal studies as an auxiliary cellular cofactor critical
to prion propagation and to species barrier (1, 8). Interference with
this putative binding activity has been discussed before as a promising
anti-prion target (36). As an alternative explanation this binding site
has been implicated as a mere PrP-PrP interaction site important during
the seeded aggregation process (4). Experimental evidence for this
hypothesis was obtained in studies using PrP peptides for interference
with PrPSc formation in a cell-free conversion assay, where
a peptide encompassing amino acids 166-179 was found to be a potent
inhibitor by binding to PrPc and blocking the
PrPc-PrPSc interaction (20).
Of note, the auto-antibodies induced by our auto-immunization strategy
seem to target exactly this binding site. Because of the proposed
interactions of this PrP region either with cellular cofactors involved
in the conversion process or with adjacent PrP molecules in prion
conversion it might be usually covered and is therefore not recognized
by antibodies. This might explain the complete absence of autoimmune
reactions upon immunization and the lack of reactivity against epitope
10 in sera obtained from rabbits or PrP0/0 mice. At
present, we do not know why we find this reactivity only in the
autoimmune situation. One possible explanation is the selection of
B-cell clones producing antibodies against PrP folding intermediates or
against epitopes, which are usually covered. On the other hand, we
provided experimental evidence that the induced auto-antibodies are
highly effective in prion-infected cultured cells, indicating that the
antibodies are able to target PrP efficiently under these conditions.
The pronounced anti-prion effects, which we found for dimer-induced
auto-antibodies in cell culture were in sharp contrast to that of
monomer-induced antibodies, even though equal antibody titers were
present. Assuming subtle differences in the folding of monomeric and
dimeric PrP, this might be due to important non-linear epitopes in
dimeric PrP, which we cannot measure in our epitope mapping assay. It
is interesting to note that, when using dimeric PrP for generating
monoclonal antibodies in PrP0/0 mice, we obtained various
highly reactive mAbs but were not able to delineate specific linear
epitopes (data not shown). This lets us argue that the dimeric PrP
immunogen has a special propensity to induce discontinuous and/or
conformational epitopes. One still has to evaluate whether pre-existing
anti-PrP auto-antibodies will have the potential to block or reduce the
invasion process of peripherally applied prions in humans and in
animals. Proof-of-principle for such a potential was recently obtained
by Heppner et al. (16). In their transgenic mouse model
PrPc was expressed in the presence of an anti-PrP antibody
without inducing obvious side or auto-immunity effects (16).
Noteworthy, challenge of these mice by intraperitoneal prion infection
resulted in significant reduction of peripheral prion propagation. In
line with this, recent immunization studies in PrP wild-type mice
followed by prion challenge showed a similar although much less
pronounced outcome (37).
We have described here the potential to induce anti-PrP auto-antibodies
in vivo and their pronounced anti-prion effects in prion-infected cultured cells. Our studies provide a solid and interesting experimental basis for further active immunization and
vaccination studies. Given the scenario of increasing vCJD cases in
future years and the possibility that vCJD might have already been
transmitted within the human population, the development of
prophylactic and therapeutic approaches against prion diseases is mandatory.