Báo cáo khoa học: Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells pptx

Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells Alessandra Handisurya1, Sabine Gilch2, Dorian Winter3,4, Saeed Shafti-Keramat1, Dieter Maurer3,4, Hermann M Schatzl2 and Reinhard Kirnbauer1 ă Laboratory of Viral Oncology, DIAID, Department of Dermatology, Medical University Vienna, Austria Institute of Virology, Technical University of Munich, Germany Laboratory of Experimental and Clinical Immunology, DIAID, Department of Dermatology, Medical University Vienna, Austria Center of Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria Keywords immunotherapy; papillomavirus-like particles; prion Correspondence R Kirnbauer, Laboratory of Viral Oncology, DIAID, Department of Dermatology, Medical University Vienna, Wahringer Gurtel 1820, ă ¨ A-1090 Vienna, Austria Fax: +43 4030224 Tel: +43 40400-7768 E-mail: reinhard.kirnbauer@meduniwien.ac.at (Received 29 September 2006, revised 21 December 2006, accepted 31 January 2007) doi:10.1111/j.1742-4658.2007.05721.x Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrPSc) To date, there is no prophylaxis or therapy available for these transmissible encephalopathies Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrPC) has been reported to abolish PrPSc infectivity and to delay onset of disease Because of established immunologic tolerance against the widely expressed PrPC, active immunization appears to be difficult to achieve To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine ⁄ rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis Immunization with PrP–virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects As determined by peptidespecific ELISA, rabbit immune sera recognized the inserted murine ⁄ rat epitope and also cross-reacted with the homologous rabbit ⁄ human epitope differing in one amino acid residue In contrast, rat immune sera recognized the murine ⁄ rat peptide only Sera of both species reacted with PrPC in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrPSc in prion-infected cells If also effective in vivo, PrP–virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases Prion diseases or transmissible spongiform encephalopathies are untreatable, fatal neurodegenerative disorders caused by proteinaceous infectious pathogens devoid of nucleic acid, termed prions [1] These diseases include scrapie of sheep, bovine spongiform encephalopathy in cattle and Creutzfeldt–Jakob disease in humans [1] Although human prion diseases are rare, the appearance of a new variant of Creutzfeldt– Abbreviations Ab, antibody; BPV-1, bovine papillomavirus type 1; FACS, fluorescence-activated cell sorting; PIPL-C, phosphatidylinositolphospholipase C; PrPC, normal prion protein; PrPSc, pathologic prion protein; VLP, virus-like particle FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS 1747 Prion-virus-like particle vaccine A Handisurya et al Jakob disease, probably due to consumption of bovine spongiform encephalopathy prion-contaminated products, has become an important public health issue [2,3] The possibility of horizontal human transmission through surgery, organ grafting or blood transfusions has raised further concern [4,5] The exact pathogenetic mechanism of prion diseases has remained uncertain In the most accepted ‘proteinonly’ hypothesis, the causative agent, the pathologic prion protein (PrPSc), is proposed to be a conformational isoform of the host-encoded normal prion protein (PrPC) PrPC is expressed ubiquitously on cell surfaces, in particular by neurons, and also on leukocytes, including T cells and B cells [6,7] Transformation of normal PrPC into infectious PrPSc is thought to occur through a template-directed process [1] PrPC appears to be required for prion infectivity, as mice deficient in PrPC (Prnp0 ⁄ 0) not propagate the infectious agent and fail to develop scrapie following experimental inoculation [8] To date, there are no prophylactic or therapeutic modalities available against prion diseases During natural infection with PrPSc, PrP-specific immune responses are not generated [1,9,10] Nevertheless, studies in cell culture and mice indicate that immunotherapeutic strategies against the cellular form of PrPC can antagonize prion infectivity and disease Monoclonal antibodies (mAbs) or recombinant F(ab) fragments recognizing PrP were effective, in vitro, in preventing prion infection of susceptible mouse neuroblastoma cells and also in abrogating PrPSc de novo formation in chronically infected cells [11,12] In addition, transgenic expression or passive transfer of mAb against PrP into scrapie-infected mice suppressed peripheral prion replication as well as prion infectivity, and significantly delayed onset of disease [13,14] Notably, no obvious side-effects were observed in these mice The immune system has evolved to respond vigorously to viral or bacterial antigens, to protect the host Several groups have demonstrated that papillomavirus virus-like particles (VLPs) can activate innate immune responses in dendritic cells to regulate adaptive immune responses [15–17], and also directly activate B-cell IgG production via a TLR-4 ⁄ MyD88-dependent (and T-helper cell-independent) pathway [17] Immunizations induced high-titer neutralizing antibodies (Abs) and potent cell-mediated immune responses to the virion capsid protein [18–20] In large clinical trials, human papillomavirus VLP vaccinations were safe and 100% effective in preventing persistent infection and associated genital disease, and polyvalent human papillomavirus vaccines have been approved recently for human use [21] In further studies, chimeric VLPs have 1748 been developed that display foreign or self-polypeptides in an ordered and closely packed repetitive array on the capsid surface [22,23] Immunizations of experimental animals induced high-titer and high-avidity IgGs to the surface-displayed (self-)antigens that were long lasting and functionally active in vitro and in vivo Importantly, no adverse effects or induction of autoimmune disease were observed The aim of this study is to develop a safe and effective vaccine that generates a strong Ab response against the prion self-antigen, but avoids induction of a T-cell response, to minimize the risk of cell-mediated autoimmune disease Development of an effective PrP vaccine has been hampered by immunotolerance to the ubiquitously expressed endogenous PrPC In the past, this problem has been circumvented experimentally either by generation of anti-PrP immunity in PrP knockout mice or using PrP of a different species as immunogen [24,25] Few groups have succeeded in generating a humoral immune response to PrP by active immunization using PrP–PrP polyproteins, PrP-expressing retrovirus particles, bacterially expressed full-length PrP, synthetic PrP peptides or polypeptides as antigen [26– 28] However, synthetic peptides are generally weak immunogens, and may even induce tolerance in the host In addition, only Abs that recognize PrPC in its native conformation exerted prionostatic effects, whereas Abs to denatured PrP were not effective [27,29,30] To gain further advantage over previous full-length or partial-length PrP vaccines, we decided to restrict the PrP immunogen to a short B-cell epitope comprising a functionally active peptide, to reduce the possibility of inducing cell-mediated autoimmune disease To circumvent the problem of low immunogenicity and immunotolerance to endogenous PrPC, the use of particulate PrP–VLPs was chosen as the vaccine strategy This immunogen is composed of an ordered and closely spaced assembly of capsomer subunits displaying a PrP peptide, to induce a humoral immune response specific to endogeous PrPC [18,28] A peptide of nine amino acid residues, DWEDRYYRE, of the murine ⁄ rat prion protein (amino acids 144–152) was incorporated into an immunogenic surface loop of the L1 major capsid protein of bovine papillomavirus type (BPV-1) and expressed by recombinant baculovirus technology This peptide resides in Helix of PrPC [31,32], and has been extensively characterized previously, as it is the epitope recognized by mAb 6H4 (Prionics, Schlieren, Switzerland) [33] Helix of the prion protein is suggested to play a central role in the protein-induced conformational changes mAb 6H4 has been well established as one of the most widely used Abs in prion diseases, and is employed in routine diagnostic tests for transmissible FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS A Handisurya et al spongiform encephalopathies The epitope DWEDRYYRE is also recognized by other functionally active Abs, including Ab.Tg [34] On the basis of the existence of these pivotal Abs, and under the assumption that this epitope may represent an effective B-cell epitope, in both rabbits and mice, we have chosen this peptide sequence to generate chimeric PrP–VLPs Immunization with PrP–VLPs induced high-titer Abs to PrP in rabbits and in rats, without causing overt adverse effects Sera specifically recognized PrPC in its native conformation in mouse brain and on rat pheochromocytoma cells, when analyzed by immunoprecipitation and flow cytometric analysis Importantly, rabbit Abs to PrP were of high affinity, and effectively inhibited de novo synthesis of pathogenetic PrPSc in prion-infected cells Results PrP–L1 fusion protein self-assembles into virus-like particles (PrP–VLPs) that express the PrP-DWEDRYYRE epitope To generate chimeric VLPs that display a PrP epitope on the particle surface (PrP–VLPs), the murine PrP Prion-virus-like particle vaccine peptide DWEDRYYRE (amino acids 144–152) (Fig 1A) was engineered into the L1 major capsid protein of BPV-1 [22] Following expression of the L1– PrP protein by recombinant baculoviruses in Sf9 insect cells, particles were purified by density gradient centrifugation Analysis by transmission electron microscopy revealed predominantly spherical structures approximately 50–55 nm in diameter (Fig 1B), indicating selfassembly into VLPs with a morphology similar to that of wild-type L1–VLPs [35] In addition, incompletely assembled particles and individual capsomers (the pentamer subunit of VLPs, consisting of five L1 molecules) were observed To verify expression of the inserted PrP epitope DWEDRYYRE, the antigenicity of PrP–VLPs was analyzed by immunoblotting The polyclonal rabbit serum A7 recognized a predominant band of approximately 55 kDa, corresponding to the expected size of the PrP–L1 fusion protein (Fig 1C, left panel), in the purified PrP–VLP preparation and a crude Sf9 cell lysate The faster-migrating bands probably correspond to proteolytic degradation products Specific immunoreactivity with A7 was absent with parental wild-type L1–VLPs As expected, mAb AU-1 (Fig 1C, right panel), directed against a linear epitope Fig (A) Amino acid sequences of the rat ⁄ mouse and rabbit ⁄ human PrP peptides encompassing residues 144–152 of the full-length prion proteins The rodent peptide differs at position 145 (bold) from the sequence of the rabbit ⁄ human peptide [changing tryptophan (W) to tyrosine (Y)] (B) Transmission electron microscopy of chimeric PrP–VLPs (magnification · 30 000) Scale bar represents 200 nm (C) Immunoblot of purified wild-type BPV-1 L1–VLPs (lane 1), PrP–VLPs (lane 2), or crude lysate of recombinant baculovirus-infected Sf9 insect cells expressing PrP–L1 (lane 3), using polyclonal rabbit anti-PrP serum A7 raised against murine dimeric PrP amino acids 23–231 (left), or mAb AU-1 directed against the linear BPV-1 L1 epitope DTYRYI (right) Molecular weight markers are indicated (D) VLP ELISA under nondenaturing (native) conditions, using conformation-dependent mAbs to L1, which either recognize both pentamers and VLP (mAb 6), or only fully assembled VLP (mAb 9), or mAb AU-1, which recognizes a linear BPV-1 L1 epitope Intact VLPs, either PrP–VLPs (black bars) or wild-type BPV-1 L1–VLPs (white bars), were used as the antigens FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS 1749 Prion-virus-like particle vaccine A Handisurya et al of BPV-1 L1, reacted with both wild-type BPV-1 L1 and chimeric PrP–L1 proteins To determine whether PrP–VLPs retain, at least in part, the antigenic surface structures of wild-type L1–VLPs, the immunoreactivity of intact particles was examined by ELISA, using a conformation-dependent neutralizing mAb directed against BPV-1 L1 mAb has been shown to bind intact VLPs as well as pentameric subunits, whereas mAb requires correctly assembled VLPs for binding [22,36,37] In contrast, the non-neutralizing mAb AU-1 recognizes an internal, linear epitope of L1 (DTYRYI) As shown in Fig 1D, immunoreactivity of mAb and mAb was observed with chimeric PrP–VLPs (black bars) and, as a control, wild-type L1–VLPs (white bars), indicating assembly of chimeric PrP–L1 protein into a complete VLP similar to wild-type L1 mAb AU-1 also reacted with both protein preparations under nondenaturing conditions, demonstrating nonassembled L1 protein, as observed regularly in purified VLP preparations Expression of the inserted peptide was further tested by ELISA using rabbit anti-PrP serum A7 The antiserum demonstrated high absorbance values with PrP–VLPs, but not with wild-type L1–VLPs, indicating display of the PrP epitope on the PrP–VLP surface (Fig 1D) Immunization with PrP–VLPs induces (auto-)Ab to the PrP peptide DWEDRYYRE The chosen murine prion epitope DWEDRYYRE is situated in the central region (Helix 1) of PrP, which is highly conserved among species [38], and thus generally is a poor immunogen To determine the immunogenicity of the inserted epitope presented in the context of a VLP, rabbits were chosen for immunization, as this species displays 100% amino acid sequence identity in the PrP peptide sequence to that in humans, and differs from the murine sequence in only one amino acid at position 145 (changing tryptophan W to tyrosine Y) (Fig 1A) Importantly, no adverse clinical effects regarding respiratory function, digestion, weight loss or behavior were observed in the animals over a period of months following PrP–VLP immunization None of the PrP–VLP-immunized animals died until time of sacrifice To determine the PrP-specific humoral immune response, sera obtained before and after immunization were tested by ELISA, using the synthetic PrP peptide DWEDRYYRE as the antigen The peptide was linked via two alanine spacers to biotin to ensure complete accessibility after attachment to streptavidin-coated microtiter plates Immune sera showed titers > : 400 by endpoint dilution, as compared to the nonspecific ELISA reactivity of preimmune sera Figure 2A (left) shows a representative experiment Specificity was confirmed with rabbit control serum immunized with wild-type BPV-1 L1–VLPs and by testing anti-PrP– VLP immune sera using an irrelevant control peptide (AVLPPVP) as the antigen (data not shown) To further corroborate the results, rabbit immune sera were preabsorbed with either PrP peptide or control peptide (AVLPPVP), prior to testing by PrP peptide ELISA As shown in Fig 2A (right), preabsorption of the immune serum with PrP peptide significantly reduced ELISA reactivity by more than 40% as compared to immune serum preabsorbed with the control peptide These results are in agreement with those of Yokoyama et al., and indicate that DWEDRYYRE represents a B-cell epitope also in the rabbit host [34] Active immunization strategies have been hampered by immunotolerance to endogenous PrPC [9,39,40] To Fig Left: New Zealand White rabbits (A) or Lewis rats (B) were immunized with PrP– VLPs, and immune sera (squares) were tested for immunoreactivity against the PrP peptide DWEDRYYRE by ELISA Preimmune sera of the same animal (diamonds) served as the appropriate controls Right: To determine specificity, immune sera were preabsorbed with either PrP peptide DWEDRYYRE, or control peptide AVLPPVP, prior to analysis Data obtained using serum from one animal are shown as mean A ± SD of triplicate wells (representative of three independent experiments) 1750 FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS A Handisurya et al determine whether tolerance can be overcome with our chimeric PrP–VLP preparations, Lewis rats were chosen for immunization Rats share 100% amino acid sequence identity in the inserted DWEDRYYRE epitope, so the PrP peptide represents a complete selfantigen Serum samples from rats were obtained before and weeks after the last immunization, and subsequently subjected to peptide ELISA with PrP peptide DWEDRYYRE Immunization with chimeric PrP– VLPs induced auto-Abs against the PrP peptide with titers > 400, whereas preimmune serum completely lacked ELISA reactivity (Fig 2B, left) The specificity of the results was further confirmed by overnight preabsorption of immune sera ELISA reactivity was reduced by 70% with PrP peptide preabsorption, compared to preabsorption with control peptide (Fig 2B, right) We next sought to determine whether PrP–VLPinduced immune sera were qualitatively different, whether induced in the rat, for which the PrP peptide comprised a ‘complete’ prion self-antigen (tryptophan at position 145), or in the rabbit, for which it comprised an ‘incomplete’ self-antigen [one amino acid difference (tyrosine) at position 145] Thus immune sera of rabbits and rats were compared by ELISA for reactivity against synthetic peptides representing either the murine ⁄ rat amino acid sequence (DWEDRYYRE) or the rabbit ⁄ human sequence (DYEDRYYRE) of the PrP peptide Purified IgG from rabbit antiserum demonstrated comparable immunoreactivity to both the murine ⁄ rat sequence and the rabbit ⁄ human sequence (Fig 3A) In contrast, reactivity of IgG isolated from rat antiserum was directed specifically to the murine ⁄ rat peptide (Fig 3B), but was absent with the rabbit peptide To further examine the Ab affinity of rabbit and rat anti-PrP immune sera, a modified peptide ELISA was employed The chaotropic agent ammonium thiocyanate was added at increasing concentrations to dissociate antigen–Ab complexes Tolerance to thiocyanate elution is proportional to the relative strength of antigen–Ab interactions, thus representing a measure of Ab affinity [41] As shown in Fig 3C, addition of approximately 1.35 m and 0.35 m thiocyanate, respectively, was required to achieve a 50% reduction of rabbit or rat Ab binding Ab-binding curves (Fig 3C) for the rabbit immune sera revealed an almost identical affinity distribution, when tested against either the murine ⁄ rat or the rabbit ⁄ human peptide In contrast, the rat immune serum showed a significantly (up to four-fold) lower affinity for the murine ⁄ rat peptide over a large molar range of thiocyanate concentrations, demonstrating a qualitative difference in the nature of the evoked immune Prion-virus-like particle vaccine Fig Comparison of ELISA reactivity of purified IgG from PrP– VLP-immunized rabbit (A) and rat (B) to murine ⁄ rat PrP peptide DWEDRYYRE (white bar), or rabbit ⁄ human PrP peptide DYEDRYYRE (black bar) IgG isolated from preimmune serum of the same animals served as control (C) Antibody-binding curves of rabbit and rat immune sera in the presence of increasing concentrations of ammonium thiocyanate (NH4SCN) Rabbit immune sera were tested for affinity against the murine ⁄ rat DWEDRYYRE peptide (diamonds) or the rabbit ⁄ human DYEDRYYRE peptide (squares) Similarly, rat immune sera (triangles) were tested for their binding affinity to the murine peptide As rat sera did not bind to the rabbit ⁄ human peptide (B), they were not further tested All experiments were conducted at a serum dilution of : 100 The results for one representative animal each are shown response As shown previously (Fig 3B), rat sera lacked specific reactivity to the rabbit PrP peptide Immune sera recognize native PrPC in mouse brain and on rat pheochromocytoma cells Recent studies have found an association between the ability of Abs to recognize native PrPC expressed on the cell surface and their ability to cure prion-infected cells in vitro and inhibit prion pathogenesis in vivo FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS 1751 Prion-virus-like particle vaccine A Handisurya et al Fig Immunoprecipitation of native PrPC from healthy, uninfected, wild-type mouse brain by anti-PrP sera IgG purified from rabbit PrP–VLP immune serum (lane 1), rabbit preimmune IgG (lane 2), PrP–VLP-immunized rat immune IgG (lane 3) or rat preimmune IgG (lane 4) were tested Rabbit immune serum A7 (lane 5) raised against dimeric murine PrP amino acids 23–231 served as positive control A molecular size marker is indicated [11–14,27,29,30] To determine whether evoked antisera recognize native PrPC, brains of healthy, uninfected wild-type C57Bl ⁄ mice (Prnp genotype a ⁄ a) were homogenized and immunoprecipitated using purified IgG from PrP–VLP-immunized rabbit or rats Preimmune IgG of the same animals or anti-PrP rabbit serum A7 served as appropriate negative and positive controls, respectively Equal volumes of brain homogenate were used as starting material, as well as equal amounts of IgG, to allow semiquantitative comparison of recovered immune complexes Immunoprecipitates were analyzed by western blot with mAb 4H11 As shown in Fig 4, IgG purified from rabbit PrP–VLP immune serum specifically detected PrPC, whereas reactivity with IgG purified from preimmune serum of the same animal was basically not detectable As expected, rabbit immune serum A7 also recognized PrPC in mouse brain In contrast, no specific immunoreactivity could be detected with IgG from immunized rats As a more sensitive assay for the detection of IgG binding to native PrPC, fluorescence-activated cell sorting (FACS) analysis of PC-12 rat pheochromocytoma cells, which express low levels of PrPC on their surface [42], was used An increase in surface binding was observed with PrP–VLP-induced rabbit immune IgG (Fig 5A, lower panel) as compared to preimmune IgG of the same rabbit, demonstrating specific binding to PrPC on the PC-12 cell surface Similarly, rat immune IgG (Fig 5B, lower panel), but not preimmune IgG, reacted with PC-12 cells, albeit to a lesser degree than rabbit IgG 1752 Fig Demonstration of anti-PrP serum binding to cell surface PrPC on PC-12 rat pheochromocytoma cells by flow cytometric analysis (A) Upper panel: Fluorescence obtained with negative controls, PC-12 cells alone (open histogram), or PC-12 cells reacted with second-step anti-rabbit serum (closed histogram) Lower panel: Comparison of fluorescence intensity between PC-12 cells incubated with PrP–VLP-induced rabbit immune IgG (closed histogram) or preimmune IgG (open histogram) of the same rabbit (IgG dilution : 300) (B) Upper panel: Negative controls with PC-12 cells (open histogram) or with second-step anti-rat serum (closed histograms) Lower panel: Fluorescence signal obtained with PC-12 cells after addition of rat immune IgG (closed histogram), as compared to rat preimmune IgG (open histogram) (IgG dilution : 100) PrPC is linked to the plasma membrane via a glycosylphosphatidylinositol anchor, which is sensitive to phosphatidylinositolphospholipase C (PIPL-C) treatment To confirm that IgG binding to cell surface PrPC was specific, PC-12 cells were incubated with PIPL-C prior to FACS analysis with IgG from immune sera or the positive control mAb 4H11 Compared to fluorescence obtained in the absence of PIPL-C treatment, removal of PrPC by PIPL-C digestion induced a significant reduction in binding of rabbit and rat IgG, respectively (data not shown), indicating specificity of the FACS results Furthermore, immune IgG was preabsorbed overnight with either PrP–VLPs or wild-type FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS A Handisurya et al BPV-1 L1–VLPs prior to FACS analysis A decrease of IgG binding was consistently observed with PrP– VLP preabsorption of immune sera, as compared to wild-type VLP preabsorption Reactivity was specifically reduced by 36% following overnight preabsorption of rabbit immune IgG with PrP–VLPs The reactivity of PrP–VLP-preabsorbed rat IgG was diminished by 82%, as compared to prior incubation with wild-type L1–VLPs (data not shown) Taken together, these results confirmed specific recognition of native cell surface PrPC by IgG induced by PrP–VLP immunization PrP–VLP-induced rabbit immune sera are functionally active and inhibit PrPSc biogenesis in living cells To determine the biological relevance of the anti-PrPC humoral immune response, antisera were analyzed for their ability to inhibit PrPSc de novo synthesis in persistently prion-infected mouse ScN2a neuroblastoma cells [43] As shown in Fig 6, ScN2a cells were metabolically labeled with [35S]l-cysteine ⁄ methionine for 16 h, and purified rabbit or rat immune IgG was added in parallel to the culture medium As appropriate negative and positive controls, rabbit and rat preimmune IgG or mAb 4H11 were included in the Prion-virus-like particle vaccine analysis, or cells were left untreated Cells were then lysed, and subjected to proteinase K digestion and ultracentrifugation to separate PrPSc from PrPC; the PrPSc-containing fraction was further subjected to immunoprecipitation, followed by a deglycosylation step with N-glycosidase F (PNGase F) PrPSc was readily detectable in untreated ScN2a cells, demonstrating consistent production of PrPSc by infected ScN2a cells Addition of the positive control mAb 4H11 resulted in lack of detectable immunoreactivity against PrPSc in the cells Incubation of the PrPSc-producing cells with IgG, purified from PrP–VLP-immunized rabbits, completely blocked PrPSc reactivity, demonstrating successful inhibition of de novo synthesis of pathologic PrPSc in persistently prion-infected ScN2a cells In contrast, a strong band at the expected size of PrPSc was observed in cells treated with preimmune IgG Thus, inhibition of PrPSc biogenesis was not attributable to nonspecific serum components, but to the presence of anti-PrP IgG induced by immunization with chimeric PrP–VLPs Addition of rat immune IgG did not completely abolish PrPSc production in infected cells This negative result for rat immune IgG correlated with weaker binding to PrPC by FACS (Fig 5) and lower affinity to PrP peptide by thiocyanate ELISA (Fig 3C), as compared to rabbit sera Discussion Fig Effects of anti-PrP sera on PrPSc conversion in persistently prion-infected mouse ScN2a neuroblastoma cells ScN2a cells were left untreated or incubated with the indicated preimmune or immune IgG, or mAb 4H11 as positive control To detect PrPSc conversion, cells were lysed, and then subjected to proteinase K digestion and immunoprecipitation A deglycosylation step was included to reduce the PrPSc and PrPC glycoforms to one single band, as described in detail in Experimental procedures This study shows that high-density display of a short (nine amino acid residues) PrP B-cell epitope on the papillomavirus VLP surface is sufficient to effectively induce Abs to PrP These Abs recognize PrPC in its native cell-associated conformation, thus overcoming the reported immunotolerance for endogenous, hostencoded PrPC [9,39,40] The murine neuroblastoma cell line ScN2a represents a persistently prion-infected cell culture model, which produces considerable amounts of PrPSc Importantly, immunized rabbit IgGs did not merely bind PrP, but effectively inhibited PrPSc de novo synthesis in living cells This strongly suggests a functionally relevant protective role of the induced Ab to PrP (Fig 6), indicating that even previously infected cells may become cured of infection Considering the long latency phase and limited diagnostic methods available, immunization with PrP–VLPs may offer avenues for further experimentation that could suggest an effective strategy for prevention and perhaps even therapy of the as yet uncurable prion diseases When immunologic antiprion strategies are employed, the issue of safety is critical Whereas our strategy has similarities with a recent study employing retroviral particles for display of prion protein [28], FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS 1753 Prion-virus-like particle vaccine A Handisurya et al papillomavirus VLP appears to be advantageous as a vaccine carrier, comprising nonreplicating subunit vaccines that have proven to be safe and highly effective in thousands of vaccinees [21] Papillomavirus L1 has the intrinsic capacity to self-assemble into pentamers that further multimerize into small or full-size VLPs [35] PrP–VLPs accommodate epitope insertion and display the PrP peptide at high density (up to 360 copies per VLP) in a repetitive, closely spaced and highly immunogenic manner on the particle surface Importantly, VLP-based vaccines that induce continuously present auto-Ab to PrPC could represent a safe and effective alternative to passive administration of Abs or peptide vaccination Possible advantages are a more constant Ab level over time, the need for less frequent administration, and the absence of an inactivating Ab response to therapeutic Ab Auto-Ab against PrPC, which is ubiquitously expressed throughout the organism, in particular in neuronal and immune cells, may induce Ab-mediated autoimmune encephalitis or complement-dependent lysis of immune cells However, we have not observed overt adverse effects in animals following PrP–VLP immunizations The relatively low serum titers induced by our PrP–VLP immunizations may have contributed to this favorable result Immunization of rats did not induce antisera that effectively inhibit PrPSc conversion The weaker binding of rat antiprion Ab to pheochromocytoma cells and the four-fold lower affinity as compared to rabbit Ab may account for the observed difference in efficacy This observation may indicate the existence of a threshold in Ab titer or affinity that dictates therapeutic efficacy Modifications of the PrP peptide immunogen, e.g single amino acid differences from the endogenous PrP sequence (as in the rabbit vaccination), may offer an experimental approach to further evaluate the putative efficacy of PrP–VLP vaccines A study has demonstrated apoptosis of hippocampal and cerebellar neurons following intracerebral injection of mAb to PrPC [44] Neurodegeneration was thought to result from cross-linking and clustering of PrPC, triggering an abnormal signaling pathway However, neuronal loss occurred only when extremely high concentrations of Abs to epitopes encompassing amino acids 95–105 of the PrP were used Injection of mAbs that recognize the central region of the PrPC (residues 133–157) and bind efficiently to cell surface PrPC did not induce neuronal apoptosis Given the absence of overt neurologic abnormalities in our vaccinated animals, we argue that auto-Ab to PrP amino acids 144–152 induced by PrP–VLPs either not cross-link PrPC or sterically mask a region of PrPC that interacts with a putative signaling partner Moreover, to 1754 minimize the risk of induction of T-cell-mediated autoimmune disease, including encephalitis, we have employed a short PrP peptide that is unlikely to comprise a T-cell epitope, as compared to the larger or fulllength PrP proteins that have been used in previous reports Nevertheless, larger animal studies are required to further evaluate the safety of PrP–VLP vaccination Several potential mechanisms may underlie the efficacy of antiprion Ab induced by PrP–VLPs [45] Ab to PrP may bind to and mask PrPC or induce redistribution of PrPC from the cell surface into cellular compartments, where it is not available for conversion into pathogenic PrPSc In vivo, anti-PrP IgG may even bind to the surface of B-lymphocytes and thus directly inhibit prion replication and infectivity On the basis of previously published studies [27,33,34], a peptide from central Helix of the prion protein was chosen as immunogen, as this region has been implicated in prion replication and transmission, and to allow functional testing in mouse models [31] Our results are in agreement with those obtained with mAb to Helix 1, which has been shown to be effective in inhibiting prion pathogenesis [12,14,29] Taken together, the results that we have presented here show that a VLP-based PrP vaccine can circumvent immunotolerance to the widely expressed self-antigen PrPC, and induce a robust Ab response that can protect against PrP infection in vitro Further studies are required to evaluate the potential of the PrP–VLP vaccine to prevent or even cure prion infectivity and disease in experimental animal models If proven effective, PrP–VLP vaccination may open a unique possibility for medically applicable immunologic prevention of currently fatal and uncurable prion-mediated diseases Experimental procedures Generation of recombinant baculoviruses expressing the PrP epitope DWEDRYYRE-BPV-1 L1 fusion protein and purification of chimeric PrP–VLPs To generate recombinant baculoviruses expressing the PrP epitope DWEDRYYRE (corresponding to amino acids 144–152 of the murine PrP) on a predicted surface loop of L1–VLP as a fusion protein, oligonucleotides encoding the epitope were inserted by inverse-touchdown PCR into an immunogenic region (between amino acids 133 and 134) of the BPV-1 L1 major capsid protein, using the pEVmod transfer vector as previously described [22] The forward and reverse primer sequences were 5¢-GCTACTACCGTG AAACCCAAACAACAGATGAC-3¢ and 5¢-GGTCCTCC CAGTCGGTGACTTTTCTATTCAC-3Â, respectively Final FEBS Journal 274 (2007) 17471758 ê 2007 The Authors Journal compilation ª 2007 FEBS A Handisurya et al clones were verified by restriction enzyme digestion and by nucleotide sequencing of the inserted sequence and the junctional L1 region By cotransfection of Sf9 insect cells with transfer vector and linearized baculovirus DNA (BaculoGold; BD Biosciences Pharmingen, San Diego, CA), recombinant baculoviruses were generated by standard methods [35] Sf9 insect cells were infected with baculovirus stocks and lysed, and high molecular mass structures were separated by density gradient ultracentrifugation VLP-containing bands were collected and dialyzed [22,46] BPV-1 L1–VLPs consisting of wild-type L1 major capsid protein were generated in a similar manner [35] Characterization of purified PrP–VLPs To analyze the self-assembly of L1 and the integrity of chimeric PrP–VLPs by transmission electron microscopy [47], purified VLP preparations were absorbed onto glowdischarged carbon-coated copper grids, fixed, negatively stained with 1% uranyl acetate, and analyzed in a JEOL (Eching, Germany) 1010 electron microscope at 80 kV and · 30 000 magnification Purified PrP–VLPs and wild-type BPV-1 L1–VLPs were further analyzed for the presence of PrP or BPV-1 L1 epitopes For western blot analysis, PrP–VLPs, wild-type BPV-1 L1-VLPs or lysates of Sf9 cells infected with recombinant baculoviruses were denatured in SDS sample buffer containing 2% b-mercaptoethanol, electrophoresed on 10% SDS polyacrylamide gel, immunoblotted, and probed with Ab A7 to PrP or mouse mAb AU-1 (BabCo, Berkeley Antibody, Richmond, CA), recognizing a linear BPV-1 epitope DTYRYI Polyclonal Ab A7 specific to PrP was obtained in our laboratories after immunization of rabbits with recombinant dimeric mouse PrP amino acids 23–231 [26,48] Finally, blots were incubated with second-step peroxidase-labeled Ab, either goat anti-rabbit (Kierkegaard and Perry Laboratory Inc., Gaithersburg, MD) or goat anti-mouse (Jackson ImmunoResearch Laboratory Inc., West Grove, PA), and developed using the ECL system Purified PrP–VLPs or wild-type BPV-1 L1–VLPs were used as antigen in an ELISA Native VLPs were coated overnight at °C onto 96-well microtiter plates mAb and mAb are BPV-1neutralizing mouse mAbs directed against conformationdependent and type-specific L1 epitopes [36]; mAb AU-1 against L1 and serum A7 against murine PrP are described above Serial dilutions of mAb or antiserum were added to triplicate wells, and developed by peroxidaselabeled goat anti-mouse serum or goat anti-rabbit serum and the peroxidase substrate di-ammonium 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid) Absorbances were determined at 405 nm Specific absorbances were calculated by subtracting mean values obtained in wells without antigen (NaCl ⁄ Pi) from mean values obtained in antigen-coated wells The replication variation in these assays was less than 5% Prion-virus-like particle vaccine Immunization of animals with PrP–VLPs and induction of a humoral immune response New Zealand White rabbits and Lewis rats (Charles River Laboratory, Kisslegg, Germany) were inoculated with 100 lg or 50 lg of PrP–VLPs, respectively, each four times at 2–4-week interval At each injection, VLPs were administered to New Zealand White rabbits with CpG 2006 (80 nmol) plus incomplete Freund’s adjuvant, and to Lewis rats with CpG 2006 (40 nmol) only, to avoid eventual development of granuloma due to incomplete Freund’s adjuvant in the small-sized animals Serum samples were collected from animals prior to immunization, 10 days after the third injection, and weeks after the final boost When indicated, total IgG was purified using the ImmunoPure IgG Purification Kit A Plus or G, respectively (Pierce Biotechnologies, Rockford, IL) Sera were tested by ELISA for reactivity to synthetic PrP peptide DWEDRYYRE (amino acids 144–152) or control peptide AVLPPVP as a specificity control Peptide AVLPPVP is derived from murine choriongonadotropin-b Streptavidin-coated 96-well plates (Nunc A ⁄ S, Roskilde, Denmark) were coated with lg of peptide linked at the N-terminus via two alanine residues to biotin (JPT Peptide Technologies, Berlin, Germany) in 100 mm Tris ⁄ HCl (pH 7.5) ⁄ 150 mm Nacl ⁄ 0.1% Tween-20 coating buffer overnight at °C, and blocked with NaCl ⁄ Pi ⁄ 1% nonfat dry milk Serial dilutions of sera were added in triplicate and, after incubation, plates were developed as described above Mean A405 values of uncoated (NaCl ⁄ Pi) wells were subtracted to determine specific absorbance values of the samples Mean A405 values of immune sera greater than twice the mean A value (± SD) of preimmune sera (at the same serum dilution) were considered positive To further examine specificity, sera of immunized animals were preabsorbed with either PrP peptide DWEDRYYRE or the irrelevant control peptide AVLPPVP by overnight incubation in streptavidin-coated plates coated with lg of the respective biotinylated peptide Subsequently, absorbed sera were analyzed for remaining immunoreactivity to PrP peptide by ELISA For determination of eventual cross-reactivity, immune sera were tested against the murine ⁄ rat sequence of the PrP peptide, DWEDRYYRE, as well as against the rabbit ⁄ human sequence DYEDRYYRE by peptide ELISA as described above To assess the Ab affinity distribution, immune sera were analyzed by peptide ELISA as described above, with the modification of a thiocyanate elution step to dissociate antigen–Ab complexes [41] After Ab incubation (all at a dilution of : 100), ammonium thiocyanate (NH4SCN) in 0.1 m sodium phosphate (pH 6.0) was added to triplicate wells at molarities ranging from 0.0625 to m for 15 at room temperature Control wells were incubated with 0.1 m sodium phosphate without NH4SCN The Ab content in control wells without thiocyanate represents the total bound anti-PrP; Ab contents in wells incubated FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS 1755 Prion-virus-like particle vaccine A Handisurya et al with increasing molar concentrations of thiocyanate were expressed as proportions of this total Determination of PrPC-specific IgG binding by immunoprecipitation and western blot Wild-type C57Bl ⁄ mice (Prnp genotype a ⁄ a) were killed, and their brains were removed and homogenized in lysis buffer (150 mm NaCl, 50 mm Tris ⁄ HCl, pH 8.0, mm EDTA, pH 8.0, 1% Nonidet P-40, and protease inhibitors); this was followed by incubation on ice for h Homogenates were centrifuged at 10 000 g for 20 at °C using a Hettich Universal 30 RF centrifuge with 1412 rotor; supernatants were then collected and stored at ) 20 °C until further use As polyclonal anti-PrP serum displayed a high background in a direct western blot assay of mouse brain extracts, we decided to use a sequential immunoprecipitation–western blot approach to determine PrPC-specific anti-PrP IgG binding PrPC of brain homogenate supernatants was immunoprecipitated with purified IgG from either PrP–VLP-immunized New Zealand White rabbits or Lewis rats with protein G beads (Pierce Biotechnologies) IgG purified from preimmune sera of the same animals or polyclonal rabbit antiserum A7 served as controls After denaturation in SDS sample buffer containing 2% b-mercaptoethanol, samples were electrophoresed on 15% SDS polyacryamide gels, and western blot was performed using mAb 4H11 (dilution : 1000) as described above mAb 4H11 was generated using a dimeric murine PrP as an immunogen [49] All animal experiments were performed in accordance with the Austrian and EU ethical guidelines and approved by the Federal Ministry of Education, Science and Culture (GZ66.009/124-BrGt/2003) Determination of PrPC-specific IgG binding by flow cytometric (FACS) analysis on rat pheochromocytoma cells The rat pheochromocytoma cell line PC-12 (ATCC CRL1721), which has been reported to express PrPC [42], was maintained in RPMI 1640 supplemented with 5% fetal bovine serum, 10% horse serum, antibiotics, and glutamine PC-12 cells were washed with NaCl ⁄ Pi and detached from the culture flasks by keeping cells at °C in NaCl ⁄ Pi for 10 After detachment, cells were washed twice with cold mm EDTA ⁄ NaCl ⁄ Pi containing 0.5% BSA As primary Ab, purified IgG from PrP–VLP-immunized rabbits (diluted : 300) or rats (diluted : 100) was added Purified IgG from preimmune sera at the same dilution served as a negative control, and mAb 4H11 (dilution : 100) as a positive control Second-step Abs used were either Alexa-488-labeled goat anti-rabbit serum (Molecular Probes, Eugene, OR), phycoerythrin-labeled donkey anti-rat serum (BD Biosciences Pharmingen) or Alexa-488-labeled goat anti-mouse serum (Molecular Probes); all Abs were diluted : 150 1756 To confirm the specificity of the results, cells were treated with PIPL-C (Sigma Chemicals, St Louis, MO) to remove cell surface PrPC After two washes with NaCl ⁄ Pi, cells were incubated in serum-free medium for 30 at 37 °C with or without mL)1 PIPL-C prior to analysis Furthermore, IgG from rabbit or rat immune sera were preabsorbed overnight with either PrP–VLPs or parental wildtype BPV-1 L1–VLPs before being subjected to FACS analysis After exclusion of dead cells by 7-aminoactinomycin D staining, analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) Results are expressed in fluorescence units (log scale) Analysis of PrPSc de novo synthesis The mouse neuroblastoma cell lines N2a (ATCC CCL 131) and prion-infected ScN2a have been described previously [43] Cells were maintained in Opti-MEM medium containing 10% fetal bovine serum, antibiotics, and glutamine For analysis of PrPSc de novo synthesis, ScN2a cells were washed twice with NaCl ⁄ Pi at about 80% confluency, and starved for h in RPMI medium without cysteine ⁄ methionine (Sigma) supplemented with 1% fetal bovine serum Then, 400 lCiỈmL)1 [35S]l-cysteine ⁄ methionine (Amersham Biosciences, Piscataway, NJ) was added and incubated for 16 h without a chase period to allow PrPSc synthesis, either in the presence or the absence of immune sera as indicated at a dilution of : 50 As a control for the inhibition of de novo synthesis, the mAb 4H11 (10 lgỈmL)1) was used Lysis, proteinase K digestion and immunoprecipitation of PrP was performed as described [26,50], using the rabbit polyclonal Ab A7 In order to separate PrPC and PrPSc, lysates were subjected to ultracentrifugation at 100 000 g for h (Beckman TL100; TLA-45 rotor; Beckman Coulter, Fullerton, CA) in the presence of 1% N-laurylsarcosine, and the pellet fraction containing PrPSc was used for immunoprecipitation To facilitate quantification, a deglycosylation step with PNGase F was included to reduce the PrPC and PrPSc glycoforms to one single band [26,48] Both Ab A7 and mAb 4H11 recognize each of the PrP glycoforms The samples were analyzed on 12.5% SDS polyacrylamide gel, and this was followed by autoradiography on X-OMAT AR films (Kodak, Amersham Biosciences) Acknowledgements This work was supported by the SFB-576 (project B12) and the BMBF (KO0108) (H Schatzl), and ă the Austrian Science Foundation, FWF (P18990-B13) (R Kirnbauer), and was performed within the framework of the EU FP6 ‘Viraskin’ (R Kirnbauer) and Network of Excellence Neuroprion (H Schatzl) ă FEBS Journal 274 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3957–3966 FEBS Journal 274 (2007) 1747–1758 ª 2007 The Authors Journal compilation ª 2007 FEBS ... (amino acids 144–152) (Fig 1A) was engineered into the L1 major capsid protein of BPV-1 [22] Following expression of the L1– PrP protein by recombinant baculoviruses in Sf9 insect cells, particles. .. auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prioninfected cells J Biol Chem 278, 18524–18531 27 Heppner FL & Aguzzi A (2004) Recent developments in prion. .. leukocytes, including T cells and B cells [6,7] Transformation of normal PrPC into infectious PrPSc is thought to occur through a template-directed process [1] PrPC appears to be required for prion infectivity,