Polylactide-Coglycolide Microspheres CoEncapsulating Recombinant Tandem Prion Protein with CpG-Oligonucleotide Break Self-Tolerance to Prion Protein in This information is current as Wild-Type Mice and Induce CD4 and CD8 T of September 30, 2021. Cell Responses Gunnar Kaiser-Schulz, Antje Heit, Leticia Quintanilla-Martinez, Franziska Hammerschmidt, Simone

Hess, Luise Jennen, Human Rezaei, Hermann Wagner and Downloaded from Hermann M. Schätzl J Immunol 2007; 179:2797-2807; ; doi: 10.4049/jimmunol.179.5.2797 http://www.jimmunol.org/content/179/5/2797 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Polylactide-Coglycolide Microspheres CoEncapsulating Recombinant Tandem Prion Protein with CpG-Oligonucleotide Break Self-Tolerance to Prion Protein in Wild-Type Mice and Induce CD4 and CD8 T Cell Responses1

Gunnar Kaiser-Schulz,* Antje Heit,† Leticia Quintanilla-Martinez,‡ Franziska Hammerschmidt,* Simone Hess,§ Luise Jennen,‡ Human Rezaei,¶ Hermann Wagner,† and Hermann M. Scha¨tzl2*

Prion diseases are fatal neurodegenerative diseases that are characterized by the conformational conversion of the normal, mainly ␣-helical cellular prion protein (PrP) into the abnormal ␤-sheet-rich infectious isoform (PrPSc). The immune system Downloaded from neither shows reaction against cellular PrP nor PrPSc, most likely due to profound self-tolerance. In previous studies, we were able to partly overcome self-tolerance using recombinantly expressed dimeric PrP (tandem PrP (tPrP)), in association with different adjuvants. Proof of principle for antiprion efficacy was obtained in vitro and in vivo. In this study, we demonstrate the induction of a specific Th1 T cell response in wild-type mice immunized with tPrP and CpG-oligonucleotide (ODN). Biochemical influences such as refolding conditions, ionic strength, pH, and interaction with CpG-ODN affected antigenic structure and thus improved immunogenicity. Furthermore, s.c. immunization with tPrP and CpG-ODN coen- http://www.jimmunol.org/ capsulated in biodegradable polylactide-coglycolide microspheres (PLGA-MS) enhanced CD4 T cell responses and, more prominent, the induction of CD8 T cells. In this vaccination protocol, PLGA-MS function as endosomal delivery device of Ag plus CpG-ODN to macrophages and dendritic cells. In contrast, PLGA-MS-based DNA vaccination approaches with a tPrP construct generated poor humoral and T cell responses. Our data show that prophylactic and therapeutic immunization approaches against prion infections might be feasible using tPrP Ag and CpG-ODN adjuvant without detectable side effects. The Journal of Immunology, 2007, 179: 2797–2807.

Sc rion diseases are fatal and infectious neurodegenerative ponent of infectious prions (1–4). PrP conversion goes along by guest on September 30, 2021 infectious disorders, including Creutzfeldt-Jakob disease with major structural and biochemical changes: ␣-helix-rich P (CJD)3 in humans, scrapie in sheep, chronic wasting dis- PrPC turns into highly insoluble, mainly ␤-sheeted PrPSc be- ease in deer and elk, and bovine spongiform encephalopathy in coming partially resistant to proteolytic digestion (2, 5, 6). PrPC cattle. These diseases are characterized by the conversion of the is a highly conserved protein of unknown function, which is C cellular prion protein (PrP ) into the abnormally folded isoform posttranslationally modified in the secretory pathway and is at- Sc termed PrP , which accumulates and represents the major com- tached to the outer leaflet of the plasma membrane via a gly- colipid anchor. The subcellular locale of conversion to PrPSc is assumed to be the plasma membrane or a compartment in the *Institute of Virology, Prion Research Group, Technical University of Munich, Mu- C nich, Germany; †Institute of Medical Microbiology, Immunology and Hygiene, Tech- early endocytic pathway, probably rafts (7–9). PrP is widely nical University of Munich, Munich, Germany; ‡Institute of Pathology, GSF National expressed in the body, with notably high levels in neurons. PrPC § Research Center for Environment and Health, Neuherberg, Germany; Department of Sc Chemistry, Institute of Biotechnology, Technical University of Munich, Munich, Ger- is essential for the propagation of PrP , as shown by knockout many; and ¶Unite´ de Virologie et Immunologie Mole´culaires, Biologie Physico- mice (PrP0/0) mice, which are resistant to prion infection (10). chimique des Prions, Jouy-en-Josas, France It was shown some time ago that polyclonal anti-PrP Abs can Received for publication February 15, 2007. Accepted for publication June 25, 2007. reduce prion infectivity of hamster brain homogenates (11) and The costs of publication of this article were defrayed in part by the payment of page that anti-PrP Abs inhibit the formation of protease-resistant PrP charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in a cell-free conversion assay (12). Anti-PrP mAbs (13–15) 1 This work was supported by SFB-576 (Project B12), SFB-596 (Projects A8 and and anti-rPrP-directed Fab (16) were shown to suppress prion B14), Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie replication in cell culture models. Passive immunization with (01KO0108), and the European Union Network of Excellence Neuroprion. mAbs was effective when mice were infected by the i.p. route, 2 Address correspondence and reprint requests to Dr. Hermann M. Scha¨tzl, Institute albeit only when very high amounts of mAbs were applied (17). of Virology, Prion Research Group, Technical University of Munich, Trogerstr. 30, 81675 Munich, Germany. E-mail address: [email protected] Transgenic expression of an anti-PrP Ab showed the most 3 Abbreviations used in this paper: CJD, Creutzfeldt-Jakob disease; AFM, atomic promising proof of principle that protection against prion dis- force microscopy; DC, dendritic cell; FTIR, Fourier transformation infrared spectros- ease by immunization is feasible, interestingly, without induc- copy; mPrP, monomeric prion protein; ODN, oligonucleotide; PLGA-MS, polylac- tide-coglycolide microspheres; PrP, prion protein; PrP0/0, knockout mice; PrPC, cel- ing obvious side effects (18). Presumably, the major obstacle lular PrP; PrPSc, abnormal isoformprion protein; tPrP, tandem PrP; wt, wild type; rec for obtaining specific anti-PrP immune responses represents the PrP, recombinant PrP. pronounced self-tolerance to host-encoded PrP. Of note, the Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 sequence of prions generated de novo in an infected individual www.jimmunol.org 2798 T AND B CELL RESPONSE IN PrP AUTOVACCINATION

always reflects the species of the recipient host and is therefore measured with bicinchoninic acid kit and calculated in percentage of sol- not foreign in its primary structure. uble control sample. Many groups attempted to overcome self-tolerance, and some Preparation of microspheres were able to induce a humoral immune response either by using synthetic peptides with or without modifications (19–22), full- Microspheres were prepared using solvent/evaporation technique (43). Polymer was emulsified in methylene chloride with Ag/adjuvant solution at length rPrP (23–25), or recombinant tandem PrP (tPrP) (24, 26). high speed. This primary organic phase was then mixed with an aqueous Other approaches dealt with DNA vaccines (27, 28), retroviral phase containing 3% polyvinyl alcohol, followed by stirring for 4–5 h, particles (29), or alternative application routes (30, 31), the lat- allowing the methylene chloride to evaporate. Further details are described ter ones being also effective against prion challenge in vivo. elsewhere (42). Some groups were reasonably successful in inducing a PrP- Preparation of DNA vaccination microspheres specific T cell response in PrP0/0 mice (32, 33), and to a much The 3F4-tagged full-length murine PrP DNA sequence was obtained by lower extent in wild-type (wt) animals, by either peptide im- subcloning from a vector construct (44) using restriction enzymes XbaI and munization (19, 20, 34) or using recombinant proteins of a dif- PstI (NEB). tPrP sequence bearing 3F4 tags and flanked by signal peptides ferent species (35). (1–231-linker-23–254) was subcloned using BamHI and XbaI (26). Both Immunostimulatory CpG-oligonucleotide (ODN) was shown to constructs were ligated into the DNA vaccination vector pVAX (Invitrogen be a potent stimulator of innate immune cells, such as macro- Life Technologies) and purified with Qiagen Plasmid Giga kit before co- encapsulation into microspheres with CpG-ODN (42). phages and dendritic cells (DCs) (36–38). In DCs and macro- phages, CpG-DNA activates maturation by binding to TLR9, Atomic force microscopy (AFM) thereby facilitating cross-presentation of exogenous Ags (39–41). Protein samples for AFM were dialyzed and diluted to a concentration Downloaded from As TLR9 is expressed within phago-endosomes, concurrent endo- of 0.1 mg/ml with respective dialysis buffer. Samples were mixed with somal translocation of Ag and CpG-ODN results in a significant CpG to a final concentration of 100 ␮M and immediately placed on enhancement of T cell stimulation (39–41). Endosomal transloca- freshly cleaved mica attached to AFM sample discs (Ted Pella). After 3 min of adsorption at 25°C, the discs were washed five times with tion of Ag and CpG-ODN can also be achieved by coencapsulation ddH2O before allowing to air dry. Contact mode imaging was per- of Ag plus CpG-ODN into biodegradable microparticles that act as formed on a multimode scanning probe microscope (Veeco) by using

endosomal delivery device (42). silicon nitride probes (type DNP-S20, Veeco NanoProbe Tips; spring http://www.jimmunol.org/ In this study, we coencapsulated tPrP and CpG-ODN in biode- constant K ϭ 0.06 N/m). gradable polylactide-coglycolide microspheres (PLGA-MS) in at- Fourier transformation infrared spectroscopy (FTIR) tempts to vaccinate for anti-PrP immune responses. We show that tPrP was dialyzed either with 10 mM NaAc (pH 4.5) or ddH O overnight, the mode of Ag preparation affects the magnitude of immune re- 2 followed by desalting with G25 MicroSpin devices (GE-Healthcare) sponse, influenced by refolding conditions and interaction with against acetate Na/D2O (pD 4.1) or D2O, respectively. The protein con- adjuvant CpG-ODN. We show for the first time that tPrP is able to centrations were 6 mg/ml. FTIR spectra were recorded with a Jasco 810 induce a significant CD4ϩ T cell response in spleen of immunized infrared spectrometer equipped with a thermostated cell holder. Each spec- Ϫ1 wt mice. Furthermore, coencapsulation of tPrP and CpG-DNA in trum was an overage of 20 scans witha4cm resolution. The spectrum

deconvolution was made using homemade software. by guest on September 30, 2021 PLGA-MS caused a significant enhancement of T cell responses, as indicated by increased CD4ϩ responses and also by significant Size exclusion chromatography ϩ PrP-specific CD8 T cell responses. Despite these pronounced im- Size exclusion chromatography was done with Åkta fast protein liquid mune responses, no side effects related to autoimmune diseases chromatography equipment (GE-Healthcare) and a 7 ϫ 600-mm gel fil- were observed. Our results indicate that PLGA-MS-based anti- tration column TSK 4000SW (Interchim). Before use, the column was PrP vaccination strategies may provide a feasible and effective calibrated for molecular mass and Stokes radius with low and high mo- vaccination approach for prevention and therapy of prion-based lecular mass calibration kits (GE-Healthcare). Previously to each measure- ment, the column was equilibrated with a minimum of 4-column vol of diseases. elution buffer. Flow rate was 1 ml/min at 20°C; protein elution was mon- itored with UV absorption at 280 nm. Materials and Methods Immunization Animals NMRI mice were immunized with 100 ␮l containing 1 mg/ml freshly di- Female PrP wt NMRI mice were obtained from Janvier. NMRI mice are of alyzed protein; CpG 1826 was added directly before administration to a prnp-A genotype, as are, for example, CD1 and C57BL/6 mice. All animal final concentration of 100 ␮M. Injections were given in two or more por- experiments were in accordance with German animal experimentation tions s.c. into the dorsal region. Three boost injections were administered regulations. every 3 wk. Blood samples and organs were taken 10–21 days after the final boost. For microsphere immunization, 5 mg of the respective Recombinant tPrP PLGA-MS was diluted in PBS and injected s.c. into the tail base in 4-wk intervals, a total of four injections. DNA vaccination was performed by Murine PrP gene subunit ( prnp-A) for monomeric PrP (mPrP) was am- ␮ plified by PCR, as described (26). tPrP consists of two murine PrP se- i.m. application of PBS-diluted PLGA-MS in two 50- l portions. quences (aa 23–231), lacking the C- and N-terminal signal peptides, and ELISA covalently linked by a 7-aa linker (AGAIGGA). Each PrP moiety contains a 3F4 epitope tag. By cloning into bacterial expression vector pQE30 (Qia- Ninety-six-well plates were coated with 150 ␮l of sodium-carbonate buffer gen), an N-terminal poly-histidine tag for purification was added (26). tPrP (0.1 M (pH 9.5)) containing 1 ␮g of NaAc-dialyzed mPrP or tPrP and expression was done in Escherichia coli strain BL21-Gold(DE3) pLysS incubated overnight at room temperature. After washing six times with (Stratagene). Cell lysis with 6 M guanidinium hydrochloride and purifica- PBST (PBS, 1% Tween 20), the plates were blocked for a minimum of 2 h tion in 8 M urea buffer with ProBond Ni2ϩ columns (Sigma-Aldrich) was with 150 ␮l of PBST, 3% BSA, at 37°C. Sera of mice were diluted (as done, as described previously (26). Protein was refolded by dialysis against indicated) in PBST with 3% serum albumin, and 100 ␮l/well was incubated ␮ ddH2O or 10 mM NaAc buffer (pH 4.5) overnight at 4°C under permanent for1hat37°C. After washing six times with PBST (300 l in a Tecan stirring with precooled buffers (10,000 MWCO Dialysis Cassettes; Pierce). plate washer), plates were incubated with 1/4000 diluted HRP-labeled anti- Protein concentration was measured with the bicinchoninic acid kit mouse IgG Ab (GE-Healthcare) for1hat37°C. The plates were washed (Pierce). For solubility test, protein samples were adjusted to final protein again and incubated with 100 ␮l of ABTS solution (Sigma-Aldrich). After

concentration of 1 mg/ml, mixed with respective CpG amounts, and ana- 10 min at 25°C, the OD405 was measured (Tecan plate reader). Cutoff was lyzed by differential ultracentrifugation in a TL-100 ultracentrifuge (1 h at defined as three times the OD405 value of 1/100 diluted preimmune serum 100,000 ϫ g; TLA-45 rotor). Protein concentration of the supernatant was of respective mice. The Journal of Immunology 2799 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Biochemical features of rtPrP with regard to refolding conditions and interaction with CpG-ODN. A, FTIR spectra, including deconvolution, revealed structural differences between the Ags, indicated by the percentages of ␣-helix (blue) and ␤-sheet (red) content. rmPrP consisted mainly of ␣-helix ␤ after NaAc dialysis. In contrast, tPrP was mainly -sheeted after both dialysis conditions. B, Solubility of tPrP after dialysis against pure H2O or sodium acetate solution, mixed with increasing amounts of CpG-ODN. The soluble tPrP fraction was determined by protein concentration measurement in the supernatant fraction after differential ultracentrifugation. Data are shown as percentage rate of original total tPrP amount (1 mg/ml). C, AFM images of canyon-like structures). D, Overlay of size exclusion ,ء) differently refolded tPrP (0.1 mg/ml) in combination with or without 100 ␮M CpG-ODN chromatography measurements with either low or high ionic strength conditions at neutral pH.

For Ab isotypes, plate preparation was the same as for ELISA. Sera incubation with ABTS solution, OD405 was measured and documented by were diluted 1/100 and incubated with 1/4000 dilution of biotin-labeled scanning or digital photography. secondary Ab for 45 min (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM). After washing, wells were incubated with HRP-labeled streptavidin with 1/1000 Cytokine assay diluted for 30 min. The final wash was followed by 10-min incubation with Spleens of individual mice were harvested, isolated with a 100-␮m cell ABTS solution and measurement of OD . 405 strainer mesh (BD Biosciences), and individually analyzed. Lysis of Epitope mapping erythrocytes was achieved by incubation with NH4Cl (0.15 M (pH 7.4)) for 7 min at 25°C, followed by filtration with 100-␮m mesh. A total of For epitope mapping, a peptide bank encompassing the mature full-length 2 ϫ 107 cells in 2 ml of RPMI 1640 medium, containing 5% FCS and murine PrP consisting of 20 residues with an overlap of 5 residues was penicillin/streptomycin, was plated in 12-well plates and restimulated used (PrP 23–231; depicted in Fig. 2C). Peptide 6 encompasses the 3F4 for 16 h either with 20 ␮g of freshly dialyzed tPrP or mock treated at epitope; peptide 6b represents the corresponding wt murine sequence. The 37°C. Control cells were treated with PMA/ionomycin (25 ng/ml, 1 poly-histidine (MRG SHH HHH HGS CKK RPK PG) and linker region ␮g/ml, respectively) for 6 h. A total of 2 ␮l of Golgi plug (BD Bio- (DGR RSS AGA IGG AKK RPK P) of tPrP was used in addition. As sciences) was added per 3 h before addition of ethidium monoazide full-length protein control nontagged mPrP and tPrP were used. Polyclonal solution (1 ␮l of ethidium monoazide, 10 ␮l of anti-mouse CD16/CD32 Ab A7 was obtained by tPrP immunization in rabbit (26). The assay was (Fc-Block; BD Biosciences) per ml wash buffer (PBS with 2.5% FCS)). performed as described in detail elsewhere (26). In brief, CovaLink NH Incubation on ice with strong light for 20 min was followed by transfer microtiter plates (Nunc) were activated with the bifunctional linker disuc- and separation into 96-well plates. After washing three times with 300 cinimidyl suberate in carbonate buffer and incubated with peptide or re- ␮l wash buffer, cells were surface stained with 1/100 dilution of PE- combinant protein, respectively, overnight at room temperature. The coated Cy5-CD4 and PE-CD8 Abs (BD Biosciences) in wash buffer for 20 min. plates were blocked and incubated with prediluted sera, washed, and in- Washing was performed in accordance with the manufacturer’s instruc- cubated with corresponding HRP-labeled conjugate. Upon washing and tions. ␣-IFN-␥ FITC- or FITC-labeled isotype Ab were diluted 1/500 in 2800 T AND B CELL RESPONSE IN PrP AUTOVACCINATION Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. B cells respond to immunization with rtPrP and CpG-ODN depending on refolding conditions. A, Endpoint dilution ELISA of final sera of individual mice from various experiments. PrP wt mice were immunized s.c. with NaAc or H2O-dialyzed rtPrP and CpG-ODN four times in 21-day intervals. Blood was taken 10–21 days after the last immunization. Titers of individual mice were determined by endpoint dilution in an ELISA. Plates ϫ were coated with tPrP; the cutoff was defined as 3 OD405 value of a 1/100 dilution from preimmune sera of respective mice. B, Ab subtyping was performed from individual titer-positive sera. In this figure, the mean OD450 values of the groups of three tPrP(NaAc)- or eight tPrP(H2O)-treated mice are shown. C, Linear epitope mapping of final titer-positive sera showed no severe differences in Ab specificity to linear PrP epitopes between immunization groups. Peptides were covalently linked to CovaLink plates, including a DSS linker. Peptide bank with respective numbering is shown on top. Selected epitope measurements of each group are illustrated as scanned images below, including a scheme of coated peptides on the left. D, OD values of all epitopes are shown for both immunization groups and control Ab A7. All titer-positive sera were mapped, whereas tPrP(NaAc) samples (n ϭ 3) were measured as duplicates. Cutoff, indicated by a horizontal line at OD 0.2, was defined by 3 ϫ OD value without incubation of first Ab. Control Ab A7 revealed signals to all epitopes except 10–12, including linker region. Within immunization groups, some weak N-terminal epitopes and the very prominent epitope 10 corresponding to aa 159–178 of PrP were found. Neither serum showed reactivity to 3F4 tag, poly-histidine tag, or linker-specific epitopes, indicating the induction of PrP-specific linear epitopes. For full-length protein control non-tagged monomeric recPrP (23-23I) was used, which showed no difference in signal intensity compared to tPrP.

Permwash buffer and incubated 30 min before final washing. Samples ously influenced by the design and preparation of the Ag and of the were dissolved in PBS/1% paraformaldehyde. Samples were measured adjuvant used. Of note, rmPrP was significantly less or not effec- with a Beckman Coulter Epics XL flow cytometer (Beckman Coulter); tive in our hands (24, 26). Therefore, we first analyzed in more resulting data were analyzed by EXPO 32 software. detail the biochemical properties of tPrP. Histology tPrP was expressed in E. coli and purified under denaturing con- The brain, spleen, mesenterial lymph nodes, and small and large intestine ditions, as described previously (26). Analysis of structural fea- were explanted and immediately fixed in Roti-Histofix (Roth) and embed- tures was done by FTIR, because precipitated protein also can be ded in paraffin for histological examination. Sections (3–5 mm thick) were measured. Fig. 1A shows FTIR spectra from measurements of dif- cut and stained with H&E. ferent proteins and preparations, including deconvolution. ␣-Heli- cal structure is characterized by a peak at 1651 cmϪ1 (in blue), and Results ␤-sheet content by a peak at 1619 cmϪ1 (in red); percentage rates Biochemical features of rtPrP are indicated in respective figures. rmPrP, confirming previous Previously, we have shown that tPrP is able to partly overcome CD measurements, contained mainly ␣-helical structure after self-tolerance to PrP in PrP wt mice (26). This feature was obvi- refolding (␣-helix, 41%; ␤-sheet, 5%). In contrast, tPrP showed The Journal of Immunology 2801

FIGURE 3. T cell response after immu- nization with tPrP and CpG-ODN, deter- mined by intracellular IFN-␥ staining of in vitro restimulated splenocytes. Spleen of CpG-ODN-only-treated mice was stimulated with rtPrP; splenocytes from naive mice were stimulated with PMA/ionomycin as controls. A, The scatter blot illustrates the percentages of CD4ϩ splenocytes expressing IFN-␥, sorted by immunization groups. Each spot represents the result of an individual mouse; the mean values are displayed by horizontal lines. Exemplary dot blots from FACS measurements are shown for each group. CD4ϩ splenocytes from tPrP(NaAc)- Downloaded from immunized mice expressed significantly more IFN-␥ compared with CpG-ODN- ,(p Յ 0.05 ,ء) only-treated control mice whereas tPrP(H2O)-immunized mice showed a (p Յ 0.01 ,ءء) very significant activation (Mann-Whitney U test). B, Same design as in ϩ

A, but with CD8 surface staining. No signif- http://www.jimmunol.org/ icant increase of IFN-␥ expression was found.

the formation of mainly ␤-sheet structure with both dialysis buffer, tPrP was found mainly in a monomeric state. In contrast, buffers. tPrP(NaAc) revealed a higher ␤-sheet content (␣-helix, high ionic strength, corresponding to conditions after injection into by guest on September 30, 2021 ␤ ␣ 15%; -sheet, 37%) compared with tPrP(H2O) ( -helix, 18%; mice, showed significant oligomerization (Fig. 1D). ␤-sheet, 25%). Taken together, the biochemical characterization clearly shows ␤ Furthermore, refolding with pure ddH2O resulted in a cloudy the formation of mainly -sheets for refolded tPrP, in contrast to solution, indicating partial precipitation of the protein. In contrast, high ␣-helical content in mPrP. Furthermore, solubility and con- dialysis with 10 mM NaAc (pH 4.5) led to no visible precipitation. formation of rtPrP are influenced by refolding conditions, ionic Solubility of protein was therefore quantified by differential ultra- strength, and interaction with CpG adjuvant. centrifugation and is shown as percentage of originally applied amount of protein. Under these conditions, tPrP(NaAc) was com- Ag preparation influences the induction of PrP autoantibodies ϳ pletely and tPrP(H2O) 70% soluble (Fig. 1B). Interestingly, mix- Having found that various conditions significantly affected the bio- ing of refolded protein with adjuvant CpG-ODN led to very sub- chemical properties of tPrP Ag, we next asked whether this is stantial and immediate precipitation. Virtually all tPrP(NaAc) reflected by variations in humoral immune response in vivo. precipitated after addition of 5 or 10 nmol CpG, whereas 28 and Therefore, we immunized PrP wt mice with different tPrP prepa-

23% tPrP(H2O) remained soluble, respectively (Fig. 1B). From rations and measured Ab titers, isotypes, and linear epitopes of these data we conclude that CpG significantly influenced the ag- individual mice after final boosts. Three times OD of 1/100 diluted gregation behavior of the tPrP immunogen. preimmune sera of respective mice were defined as cutoff in the To gain further insight into the aggregation behavior of tPrP in recombinant PrP (recPrP)-specific endpoint titration ELISA. Coat- the presence or absence of CpG, we applied AFM (Fig. 1C). With- ing plates with either mPrP or tPrP revealed no considerable out CpG, AFM revealed no obvious difference between tPrP(H2O) differences in OD values (data not shown). Fig. 2A shows the and tPrP(NaAc) in structural appearance. Evenly widespread, un- endpoint titers of individual mice of three immunization groups. structured proteins are visible in both preparations. In contrast, None of the CpG-only-treated mice displayed a titer, whereas 3 of protein CpG mixtures showed the formation of two different types 12 tPrP(NaAc)-treated mice clearly showed autoantibody titers. Of of aggregates. Canyon-like structures were of two-dimensional ap- note, in the group of tPrP(H2O)-immunized mice, 8 of 12 mice pearance and seemed to consist of smaller aggregates gathered in exhibited anti-PrP Abs. However, the mean titer values between bigger groups. The second type of aggregate appeared as three- the two groups did not significantly differ. dimensional clumps in tPrP(NaAc) preparations, and smaller and Isotyping of titer-positive sera from our study showed no dif- less frequently in tPrP(H2O) samples, indicating unstructured, oli- ferences within the two protein preparation groups and revealed gomeric aggregates (Fig. 1C). the presence of mainly IgG1, IgG2a, and IgG2b, but not of IgG3, We next used size exclusion chromatography to study the ag- IgA, or IgM Abs (Fig. 2B). To further analyze differences in qual- gregation state of tPrP depending on ionic strength (Fig. 1D). Un- ity of humoral response, we performed a linear epitope mapping der low ionic strength buffer conditions, comparable to our dialysis (Fig. 2, C and D). We found reactive Abs mainly against epitope 2802 T AND B CELL RESPONSE IN PrP AUTOVACCINATION

10 and several N-terminal epitopes, confirming previous results (26). Neither reactivity against epitope 6, representing the 3F4 epitope tag sequence contained in tPrP used for immunization in this study, nor against to the respective wt epitope 6 was found. In addition, no binding to potential poly-histidine tag or linker pep- tide epitopes was detected. We found no differences in signal in- tensity of full-length rmPrP without tags and tPrP controls. The reactivity of a polyclonal antiserum obtained by immunization of rabbits with mouse tPrP is shown for comparison (A7; Fig. 2, C and D). In summary, B cell reaction, upon immunization with different Ag preparations, mainly varies in number of responding animals, and not in the quality of response because isotypes and linear epitopes are similar. Only few mice react after administration of tPrP(NaAc), although with high titers, whereas the majority of tPrP(H2O)-treated mice react with a broad variation of individual titers.

T cell response depending on the structure of tPrP Downloaded from Next, we examined whether intensity of T cell responses is influ- enced by Ag preparation, similarly to the differences in B cell response described above. Therefore, spleen cells of individual immunized mice were harvested and individually restimulated with tPrP or not. Cells were CD4 and CD8 surface labeled to

distinguish T cell subpopulations and stained for intracellular http://www.jimmunol.org/ IFN-␥. Fig. 3A shows the percentage rates of IFN-␥-expressing CD4ϩ splenocytes from all individual mice; horizontal lines indicate the FIGURE 4. T cell response after application of microspheres containing mean values of groups. Two mice of the tPrP(NaAc) group re- rtPrP and CpG-ODN. Spleens of individual mice were restimulated in vitro sponded with a well-defined IFN-␥ expression, whereas the others by tPrP, and expression of IFN-␥ was determined by intracellular staining showed only a weak increase. Overall, the results were statistically in combination with CD4 or CD8 surface staining. A, Summary of CD4ϩ significant compared with the CpG-only control group ( p ϭ splenocytes expressing IFN-␥ after immunization with either PLGA-MS containing tPrP and CpG-ODN in separate microspheres (MS(tPrP) ϩ 0.0159; Mann-Whitney t test). Within the tPrP(H2O) group, four of

ϩ by guest on September 30, 2021 five animals clearly responded with even higher IFN-␥ mean val- MS(CpG)) or both coencapsulated in the same compartment (MS(tPrP ues, thus leading to very significant increase compared with the CpG)). Exemplary dot-blot images of FACS measurements are shown for -p ϭ 0.0109; Mann-Whitney U test). B, Pre ,ء) ϩ each immunization group control group ( p ϭ 0.0079). From CD8 T cell subpopulation, ϩ vious layout with CD8 surface staining. Application of combined micro- only one mouse of the tPrP(H2O) group responded with a slightly spheres revealed a very significant increase of IFN-␥ expression in CD8ϩ ␥ p ϭ ,ء) increased IFN- expression, not leading to significant mean value splenocytes compared with the group with separated microsphere (Fig. 3B). Exemplary results of FACS measurements are shown as 0.0186; Mann-Whitney U test). dot-blot analysis of CD4ϩ or CD8ϩ T cells (Fig. 3, A and B, right panels). Taken together, our data show that a significant T cell re- a consistent IFN-␥ secretion (Fig. 4B), leading to a statistically sponse is induced upon immunization with both tPrP prepara- significant difference compared with the control group ( p ϭ tions. The intensity of response seems to depend on the prep- 0.0186; Mann-Whitney t test). aration of Ag, although to a lesser extent as compared with B In summary, vaccination with PLGA-MS coencapsulating tPrP ϩ cell reactions. plus CpG-ODN causes enhanced the CD4 and, to an even more extent, CD8ϩ T cell responses. Microsphere encapsulation drastically enhances T cell response Encouraged by the promising T cell responses found in some DNA vaccination with encapsulated tPrP-encoding vector fails individual mice, we tried to increase this effect by encapsulation to induce a significant immune response of immunogens in PLGA-MS. We used two different PLGA-MS DNA vaccines have been shown to be very efficient when applied preparations for our immunization trials. PLGA-MS containing encapsulated in microspheres (45). Vaccination of wt mice with either tPrP or CpG-DNA exclusively were mixed together and either pVAX (mPrP) or pVAX (tPrP) in combination with CpG, applied to mice s.c. at the tail base. To the other PLGA-MS applied via microspheres i.m., resulted in very modest immune coencapsulating both protein and CpG-DNA were used. T cell responses of only a few animals (Fig. 5). Only one mouse of eight responses of individual mice from both groups displayed partial from the pVAX (tPrP) group showed an Ab titer in an endpoint IFN-␥ secretion upon in vitro restimulation of primed spleno- titration ELISA not higher than 1:200 (Fig. 5A). Two animals cytes (Fig. 4). The CD4ϩ T cells in the group of coencapsulated showed a slight expression of IFN-␥ in CD4ϩ splenocytes in the PLGA-MS revealed a clear response in seven of eight animals, same group (Fig. 5B). No activation of CD8ϩ cells was seen (data including two high responders (Fig. 4A). These results were not shown). statistically significant compared with the group of mixed Taken together, only a marginal B and T cell response is ob- PLGA-MS ( p ϭ 0.0109; Mann-Whitney t test), therefore rep- tained in some animals when tPrP is used as microencapsulated resenting an ideal control group. Furthermore, CD8ϩ T cells of DNA vaccine. No effect is found in this situation for DNA vacci- individual mice primed with coencapsulated PLGA-MS showed nation using mPrP. The Journal of Immunology 2803 Downloaded from

FIGURE 5. DNA vaccination of wt mice via PLGA-MS application resulted in very low B and T cell responses. pVAX vectors encoding for http://www.jimmunol.org/ mPrP or tPrP were packed in PLGA-MS and injected i.m. A, Endpoint dilution titer ELISA displayed only one serum with a very low Ab titer. B, Intracellular IFN-␥ expression of in vitro restimulated splenocytes showed only marginal activation of CD4ϩ T cells in two cases after pVAX tPrP immunization. C, FACS dot blots of splenocytes of the two positive animals.

Histology and examination of side effects by guest on September 30, 2021 Given the possibility that PrP autoimmunization might result in induction of severe side effects in reactive mice, we performed a FIGURE 6. Histological appearance of lymphatic organs correlates detailed histological examination. This was even more important, with immune responses. Representative H&E-stained samples of lymph because it was reported that repeated application of high CpG nodes and Peyer’s patches in gut lumen of all immunization groups. A, CpG-ODN-only-treated mice did not show severe changes in lymphatic doses lead to major deleterious effects in immunological organs organs, only minimal follicular hyperplasia in the lymph nodes and normal- (46). Histological examinations of representative lymphatic or- sized Peyer’s patches (PP). Both tPrP(H2O) (B) and tPrP(NaAc) (C) dis- gans displayed no severe side effects in the autoimmunization played small- to medium-sized Peyer’s patches with practically no germi- situation described in this study. Neither the application of CpG nal centers. Lymph nodes looked very similar with follicular hyperplasia. alone (Fig. 6A), nor treatment with tPrP(NaAc) (Fig. 6B)or D, MS(tPrP) ϩ MS(CpG)-treated animals (MS, microspheres) showed the tPrP(H2O) (Fig. 6C), both in combination with CpG, revealed presence of Peyer’s patches without germinal centers, as well as some signs of severe changes in the lymphatic organs in any of the follicular and paracortical hyperplasia in some lymph nodes. E, Mice im- analyzed mice. In the gut lumen of ileum, only small-sized or munized with combined microspheres (MS(tPrP ϩ CpG)) revealed the major changes in intestine and lymph nodes. Very prominent Peyer’s no Peyer’s patches were visible after treatment with tPrP(H2O); tPrP(NaAc)-treated mice showed normal- or middle-sized Peyer’s patches with follicular hyperplasia and large germinal centers and very important paracortical hyperplasia in lymph nodes were visible. F, Spleens patches without germinal centers. of CpG-ODN-only-treated mice appear normal in H&E staining. (PF, pri- Animals immunized with separate microspheres showed the mary follicles; GC, germinal centers; PCH, paracortical hyperplasia.) presence of Peyer’s patches without germinal centers, and fol- licular and paracortical hyperplasia was observed in some lymph nodes (Fig. 6D). In contrast, animals receiving combined patches in intestine were normal with little formation of ger- PLGA-MS showed major changes in the intestine and in lymph minal centers. The lymph nodes showed both follicular hyper- nodes (Fig. 6E). Some of the mice had very prominent Peyer’s plasia and paracortical hyperplasia or T-zone hyperplasia. The patches with follicular hyperplasia, including large germinal spleen looked normal. In addition, in two animals an acute/ centers. In the lymph nodes, all examined animals displayed chronic inflammation was found in the muscle (data not shown). paracortical T cell hyperplasia, in addition to follicular hyper- In summary, despite the obvious efficacy of the described auto- plasia with well-developed germinal centers. The spleens of all immunization regimen, we neither found signs of severe autoim- immunized mice appeared normal in H&E staining (Fig. 6F). mune reaction, acute inflammation in lymphatic organs, nor any Besides lymphatic organs, random samples of brains displayed CpG-depending destruction in lymphatic organs. Of note, the his- no noticeable changes upon autoimmunization (Fig. 7). Histo- tological appearance of lymphatic organs correlates with evoked logical examination of both groups receiving DNA vaccination immune responses. Signs of follicular hyperplasia were most revealed very similar changes in the lymphatic organs. Peyer’s prominent in animals receiving microspheres containing both tPrP 2804 T AND B CELL RESPONSE IN PrP AUTOVACCINATION

FIGURE 7. No detection of severe side effects in the brain of immunized mice. Histological examination of H&E-stained brain sections of immunized mice did not show signs of inflammation indicative for meningoen- cephalitis. In this figure, examples from tPrP(H2O)- and tPrP(NaAc)-immunized animals in different magnifica- tions are shown. Downloaded from

Ag and CpG. Furthermore, immunized animals did not show soluble oligomers, also found by others (53, 54). The partial in-

changes in behavior upon treatment. solubility of tPrP(H2O) might be based on a salting-out effect http://www.jimmunol.org/

while dialyzed against ddH2O. Discussion Properties described under in vitro conditions do not necessarily The future spread of variant CJD, initially caused by zoonotic reflect the in vivo situation. Importantly, immunization needs ap- trans-species transmission of bovine spongiform encephalopathy plication of an immunogen to a living organism, thereby signifi- to humans, now also transmitted by human-to-human infection, cantly changing the biochemical environment and eventually also e.g., via blood products (secondary variant CJD), remains elusive. biochemical properties of the immunogen. Interestingly, mixing of Because infection with prions is invariably lethal and no therapeu- highly soluble tPrP(NaAc) with negatively charged adjuvant CpG tic or prophylactic regimens are available, there is an urgent need resulted in an immediate and almost complete aggregation of pro- by guest on September 30, 2021 for developing protective antiprion strategies. Prophylactic or ther- tein. In contrast, less soluble tPrP(H2O) was less influenced by apeutic immunization may be an ideal strategy against a variety of CpG contact. It is known that mouse rPrP has a high DNA-binding infectious agents, but presumably is hampered in prion diseases by capacity and can form aggregates in the presence of high m.w. the pronounced self-tolerance against PrP. In this study, we dem- DNA (55–57). In contrast, contact to DNA ODN, as is basically onstrate the induction of PrP-specific humoral and T cell immune CpG-ODN, resulted apparently in a dimerization of mPrP (58). As responses in PrP-expressing wt mice by active immunization. DNA binds to the structured C-terminal region of PrP, modifica- In previous studies by us (26) and others (23–25, 33, 35), it tion of protein structure is likely (59). became obvious that Ag design and application are major deter- Although we could characterize the biochemical properties of minants in overcoming PrP self-tolerance. For example, we de- tPrP preparations under defined in vitro situations (they seemed to scribed the induction of comparable Ab titers and binding to the differ rather in solubility than in structure), predictions concerning same linear epitopes after mPrP and tPrP immunization in wt mice its immunogenicity in vivo are difficult. (26). From the fact that only tPrP-induced sera led to an efficient Abs directed against PrPC or PrPSc are in principle able to in- blocking of the de novo PrPSc formation in prion-infected cell hibit de novo synthesis of PrPSc in vivo and in vitro (13, 16, 17, 25, culture, we concluded the induction of Abs with conformational 26). In this study, we confirmed the previously demonstrated in- epitopes, eventually specific to PrPSc or a folding intermediate. In duction of PrP-specific Abs in PrP wt mice after immunization this study, we analyzed some biochemical features of the tPrP Ag with tPrP in combination with CpG adjuvant (26). Thereby, tPrP- in attempts to optimize conditions for improved immune re- (NaAc) induced high Ab titers only in a few animals, whereas in sponses. We also examined the T cell responses that might help to immunizations with tPrP(H2O) most mice developed titers with trigger B cells producing conformational Abs, and we tried im- variations in individual endpoints. proved methods for Ag administration by using PLGA-MS and The Ab isotyping revealed the production of mainly IgG1, DNA vaccination approaches. IgG2a, and IgG2b PrP-specific isotypes and did not show differ-

The conformation of rPrPs has been extensively studied (47– ences between immunization using tPrP(H2O) or tPrP(NaAc). 51). Overall, full-length rPrP 23–231 showed virtually identical ELISA-based epitope mapping provides a versatile tool for in- biochemical properties compared with cellular PrPC (52–54). Re- vestigating linear epitopes, although the exposure of covalently folding conditions are a critical factor in the formation of structure linked peptides does not necessarily reflect the respective amino and level of solubility. In contrast to rmPrP with mainly ␣-helical acid side chains as exposed by native authentic proteins. Epitopes structure after NaAc dialysis, in both tPrP preparations ␤-sheet were measured individually for all ELISA titer-positive sera and prevailed, leading to the conclusion that the covalent linkage of showed weak reactivity near cutoff against some N-terminal two PrP monomers intrinsically forces the protein into ␤-sheet epitopes, i.e., epitopes 11 and 12, and the outstanding epitope 10 conformation, with possible similarities to PrPSc structure. More- (Fig. 2, C and D). We can exclude the induction of Abs reactive over, physiological salt conditions caused the formation of distinct against linear epitopes a foreign origin as contained in our protein The Journal of Immunology 2805 sequence, like the 3F4 tag, the poly-histidine tag, or the linker cells with possible cryptic PrP epitopes. At least in PrP0/0 mice, region of tPrP. Control serum A7 of a tPrP-immunized rabbit ex- such variances in T cell response were discussed for ␣-helical and hibited Abs specific to the complete panel of linear epitopes, ␤-sheeted rPrP (33). Besides changes of wt epitopes, xenogenic thereby demonstrating the principal binding ability to the co- epitopes, in our case most likely against the two amino acid ex- valently linked peptides. In particular, the differentiation between changes of the 3F4 tag in the tPrP Ag, might help to break toler- epitopes 6 and 6b, corresponding to 3F4 epitope tag or PrP wt ance and enhance T cell response, as it was elegantly demonstrated sequence, respectively, was clearly demonstrated with this positive for Syrian hamster PrP-immunized mice (35). control experiment. With our actual findings, we are now able to give statements for The quantity of humoral anti-PrP response as usually tested in the obvious lack of functional conformational Abs in mPrP vac- ELISA-based formats is not an appropriate method to determine cinations seen in our previous work. The mainly ␣-helical folded C the efficacy of Ab binding to native, authentic PrP on living cells. monomer might present only conformational epitopes similar to To date, we failed to conclusively show specific binding of auto- PrPC, thereby being recognized as self. The explanation for dif- antibodies to authentic PrPC or PrPSc in immunoprecipitation as- ferences between tPrP(H2O) and tPrP(NaAc) immunization might says using brain homogenates or cell lysates, although we could follow similar mechanistic correlations. Both protein preparations measure some reactivity of final sera as compared with respective ␤ share -sheet conformation, whereas tPrP(H2O) was partially sol- preimmune sera in PrP surface FACS-staining approaches (data uble even after contact to adjuvant CpG. Ag presentation of B cells C not shown). We could show specific binding of some sera to PrP is essential for proper humoral response, and the formation of huge in immunoblot stripe blots with brain homogenate, even under aggregates in tPrP(NaAc) preparations might decrease or hinder denaturing conditions (data not shown). In contrast, the absence of the uptake and processing by B cells. Downloaded from detectable binding in these assays may not be a negative result, In summary, these data together with other reported data provide because concentration of Abs in murine blood might be too small solid experimental evidence that an effective humoral antiprion or because Abs might be able to interfere in prion conversion by response can be evoked in certain experimental autoimmunization other mechanisms, e.g., by reacting with putative folding interme- scenarios. ␤ diates. In fact, the -sheeted tandem version of PrP as used in this To increase CD4ϩ T cell help, we tested a vaccination protocol study is thought to mimic such putative folding intermediates. Im- in which both tPrP and CpG-DNA are coencapsulated in biode- http://www.jimmunol.org/ portantly, in our hands, sera of immunized wt mice have shown gradable microspheres (42, 43). PLGA-MS are taken up and di- significant interference in the de novo PrPSc formation in cell cul- gested mainly by macrophages and DCs after s.c. application in ture, whereas mPrP induced similar linear Ab reactivity, but vivo (61). The efficacy of loaded PLGA-MS seems to be based on mainly failed to prevent PrPSc formation (26). In a promising in the enhanced concurrent uptake of proteinaceous Ag and the ad- vivo pilot vaccination approach using tPrP immunogen, two of juvant into endosomes of DCs and macrophages (62). Exogenous eight mice survived longer than 600 days after otherwise deadly Ags are classically internalized and processed by the MHC class i.p. prion challenge (24). These results are presently under further II-presenting pathway, resulting in APC-activating CD4 T cell (Th investigation (G. Kaiser-Schulz, H. Scha¨tzl, and M. Groschup, un-

cell) response (63). In addition, DCs are able to direct exogenous by guest on September 30, 2021 published observations). Ag into the MHC class I presentation pathway via cross-presen- We propose the induction of conformational Abs by specific tation (64). CpG-ODN motifs are known to specifically activate binding of B cells to mainly soluble, ␤-sheeted tPrP, followed by innate immune cells, including macrophages and DCs via targeting internalization, processing, and presentation of linear tPrP epitopes via MHC II. Epitope-specific CD4ϩ T cells are then able to trigger TLR9. Interaction between CpG-ODN and TLR9 takes place in B cell proliferation and Ab production. We show the induction of late endosomal compartments (65), and receptor-mediated endo- a tPrP-specific T cell response in PrP wt mice using a syngenic cytosis was reported to be essential for cross-priming (40). Via full-length rPrP immunogen with only minor changes (e.g., two PLGA-MS immunization, CpG-ODN and exogenous Ag are ef- amino acid exchanges in the 3F4 tag). The significant IFN-␥ ex- fectively cotranslocated into endosomal compartments, thus en- pression of CD4ϩ splenocytes after restimulation with full tPrP hancing TLR9 interaction and subsequent cross-presentation. This protein together with the absence of Th2-specific IL-4 and IL-10 might explain the very pronounced increase in the response of CD4ϩ cells, in the range of 1% and higher, and, interestingly, also (data not shown) indicate the induction of a Th1 response at the ϩ final stage of immunization, presumably polarized by the adjuvant of a CD8 T cell response. When PLGA-MS containing either CpG-ODN (38). The CpG-ODN-based general adjuvant effect tPrP or CpG-ODN alone are mixed, no detectable immune re- may be further enhanced by the strong binding between CpG and sponse at all is induced. One obvious explanation is that under tPrP, because it is known for covalently linked CpG proteins that these conditions no cointernalization of tPrP and CpG-ODN into promote a strong Th1 response via concomitant uptake of Ag and the same APCs is taking place. The lack of Ab titers, despite the adjuvant (39, 40, 60). PrP-specific T cell responses in wt mice in enhanced CD4 T cell response, might be due to the encapsulation experimental vaccination scenarios and the protective effect of cel- of Ag, thereby preventing direct contact to B cells. We have to lular-based immunity in prion infection are in part characterized evaluate the potential lytic function of induced CD8 T cells, be- (19, 20, 22, 34, 35). Peptide immunizations with adjuvant CpG cause they might cause severe autoimmune responses. demonstrated two immunogenic PrP T cell epitopes (residues 143– Inducing autoimmunity bears the obvious risk of induction of 172; 158–187) in wt mice (C57BL/6) (20). Souan et al. (22) suc- severe side effects. As was the case in anti-PrP immunization ap- cessfully generated a T cell response in various mouse strains by proaches reported by others (18–21, 23–27, 29), we could not find designing peptides optimized to fit into the MHC class II-binding such deleterious effects in our studies. In Alzheimer phase II vac- groove of NOD mice (aa 131–150; 211–230). ␤-sheet-rich rPrP, cination studies, in some cases meningoencephalitis appeared (66). like the tPrP described in this study, is known to be partially pro- To exclude similar side effects in our approach, brain sections of teinase K resistant, in contrast to easily degradable ␣-helical mPrP mice were examined for signs of inflammation. All examined (48). Potentially, this biochemical characteristic of tPrP might lead brains appeared normal without any severe changes as indicative to changes in the lysosomal degradation in APCs and thereby for meningoencephalitis. Recently, it was reported that repeated could lead to different MHC class II peptide loading, inducing T daily administrations of high CpG-ODN doses have deleterious 2806 T AND B CELL RESPONSE IN PrP AUTOVACCINATION effects on the morphology and function of lymphoid organs (46). 22. Souan, L., Y. Tal, Y. Felling, I. R. Cohen, A. Taraboulos, and F. Mor. 2001. Such dramatic side effects can be excluded in our studies. Modulation of proteinase-K resistant prion protein by prion peptide immuniza- tion. Eur. J. Immunol. 31: 2338–2346. In summary, we describe in this work for the first time the in- 23. Koller, M. F., T. Grau, and P. Christen. 2002. Induction of antibodies against duction of a substantial CD8 T cell response in PrP wt mice im- murine full-length prion protein in wild-type mice. J. Neuroimmunol. 132: munized with a rPrP of the same species. We need now to test 113–116. 24. Polymenidou, M., F. L. Heppner, E. C. Pellicioli, E. Urich, G. Miele, N. Braun, whether application of coencapsulated Ag and CpG in PLGA-MS F. Wopfner, H. M. Schatzl, B. Becher, and A. Aguzzi. 2004. Humoral immune provides a new and very promising prophylactic approach against response to native eukaryotic prion protein correlates with anti-prion protection. Proc. Natl. Acad. Sci. USA 101(Suppl. 2): 14670–14676. prion diseases in animals and humans. 25. Sigurdsson, E. M., D. R. Brown, M. Daniels, R. J. Kascsak, R. Kascsak, R. Carp, H. C. Meeker, B. Frangione, and T. Wisniewski. 2002. Immunization delays the Acknowledgments onset of prion disease in mice. Am. J. Pathol. 161: 13–17. 26. Gilch, S., F. Wopfner, I. Renner-Muller, E. Kremmer, C. Bauer, E. Wolf, We thank Monika Hammel for technical support, Frank Schmitz for help G. Brem, M. H. Groschup, and H. M. Schatzl. 2003. Polyclonal anti-PrP auto- with the manuscript, and Ilona Mo␤brugger for excellent work on brain antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation histology. in prion-infected cells. J. Biol. Chem. 278: 18524–18531. 27. Fernandez-Borges, N., A. Brun, J. L. Whitton, B. Parra, F. Diaz-San Segundo, F. J. Salguero, J. M. Torres, and F. Rodriguez. 2006. DNA vaccination can break Disclosures immunological tolerance to PrP in wild-type mice and attenuates prion disease The authors have no financial conflict of interest. after intracerebral challenge. J. Virol. 80: 9970–9976. 28. Muller, S., R. Kehm, M. Handermann, N. J. Jakob, U. Bahr, B. Schroder, and G. Darai. 2005. Testing the possibility to protect bovine PrPC transgenic Swiss References mice against bovine PrPSc infection by DNA vaccination using recombinant

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