Generation of antibodies against prion protein in wild-type mice via helix 1 peptide immunization

Journal of Neuroimmunology 144 (2003) 38 – 45 www.elsevier.com/locate/jneuroim

Generation of antibodies against prion protein in wild-type mice via helix 1 peptide immunization Michal Arbel, Vered Lavie, Beka Solomon * Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel Received 23 May 2003; received in revised form 31 July 2003; accepted 18 August 2003

Abstract We present here the development of antibodies against prion protein in BALB/C mice using as antigen human helix 1 of PrP. This sequence is suggested to be involved in protein pathological conformational changes, and is distinguished from that of mice by one amino acid. The immune tolerance to an ‘almost-self’ epitope and the poor immunogenicity of short peptides was overcome by using Multiple Antigen Peptide displaying eight copies of helix 1. The generated antibodies recognize the whole prion protein with a high binding constant and the established protocol may lead to an active immunization towards therapeutics of prion disease. D 2003 Elsevier B.V. All rights reserved. Keywords: Prion protein; Active immunization; Anti-PrP antibodies; Helix 1

1. Introduction Prion diseases are rare neurodegenerative disorders in which endogenous glycoprotein, termed ‘‘cellular prion protein’’ (PrPc), mainly expressed in the central nervous system (CNS) and lymph tissues, is refolded to an altered pathogenic isoform enriched with h-sheet structures termed ‘‘scrapie prion protein’’ (PrPSc). All forms of prion diseases share common neurodegenerative histopathologies, resulting in spongiform-like brain tissue, neuronal loss, astrocytic gliosis, and accumulation of abnormal PrPSc in the brain and spleen, which in some cases forms amyloid fibrils. Despite the structural differences between the endogenous PrPc and the scrapie isoform, PrPSc, neither a cellular nor a humoral immune response is developed during the disease.

Abbreviations: PrPc, cellular prion protein; PrPSc, scrapie PrP isoform; CNS, central nervous system; PrP (27 – 30), purified PrPSc protease resistant infective core; SH PrP (27 – 30), Syrian hamster (PrP 27 – 30); mAb, monoclonal antibody; PRNP, prion protein encoding gene; MAP-(Helix 1)8/MAP-Helix 1, Multiple Antigenic Peptide displaying eight copies of helix 1; ELISA, enzyme linked immunosorbent assay; PBS, phosphate buffer saline; BSA, bovine serum albumin; HRP, horseradish peroxidase. * Corresponding author. Tel.: +972-3-6409711; fax: +972-3-6408571. E-mail address: [email protected] (B. Solomon). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.08.036

The immune system does not respond to a protein which it has been educated to regard as ‘‘self’’ and not foreign, and immune tolerance to prion protein isoforms has restricted the production of anti-PrP-specific polyclonal and monoclonal antibodies in the laboratory. Even after purified prion preparations became available, extensive efforts to raise PrP reactive antibodies were unsuccessful (Kasper et al., 1981). Subsequently, large quantities of purified PrPSc protease-resistant infective core (PrP 27– 30), recovered from the brains of scrapie-infected rodents, were used as an antigen for the production of PrP-specific antisera in rabbits (Bendheim et al., 1984; Barry et al., 1985; Bode et al., 1985; Takahashi et al., 1986). Following the generation of antisera to PrP 27 – 30, investigators immunized rabbits with a number of PrP synthetic peptides corresponding to amino acid residues 90– 101, 142– 174, and 220– 233. Typically, antisera raised against synthetic polypeptides were more reactive with the peptides than with PrP 27– 30 (Barry et al., 1986; Wiley et al., 1987). Antibodies recognizing discontinuous (non-linear) epitopes of PrP were tested by binding to longer synthetic peptides that could adopt secondary structure arrangements approximating those found in the full-length protein (Riek et al., 1996; James et al., 1997). Immunizing mice with purified PrP 27 – 30, recovered from Syrian hamster scrapie infected brains (SHa PrP 27– 30), enabled the generation of monoclonal antibody 3F4

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directed to the linear region consisting of four amino acids 109 –112 of the prion protein (Barry and Prusiner, 1986; Kascsak et al., 1987). We have previously reported the production of monoclonal antibodies (mAbs) 2– 40 and 3 –11 by injecting human PrP 106 –126 conjugated to KLH peptide into wild-type mice (Hanan et al., 2001a). The generated antibodies recognized the prion protein only in its native form (under non-denaturating conditions), as well as the pathogenic scrapie structure, supporting the conformational nature of their epitope. Although they are able to prevent PrP 106 – 126 aggregation and resolve already formed aggregates, mAbs 2 –40 and 3– 11 failed to affect the aggregation of the whole protein (Hanan et al., 2001b). Exploiting sequence variations of the PRNP gene between mammals proved to be an effective method of producing anti-PrP antibodies in mice. However, antibody production using this method can be applied only to a small number of epitopes that are different between species and thus are able to stimulate the mice immune system. The production of PrP knockout mice gave an additional opportunity to create anti-PrP antibodies. Monoclonal antibody 6H4 generated in PrP0/0 mice has been shown to prevent scrapie infection of a neuroblastoma cell line, as well as to ‘‘cure’’ chronically infected neuroblastoma cells (Enari et al., 2001). Helix 1 of the prion protein, identified as mAb 6H4 epitope (Korth et al., 1997), is suggested to play a central role in the protein induced conformational changes (Enari et al., 2001; Heppner et al., 2001; Peretz et al., 2001; Winklhofer et al., 2003; White et al., 2003). In this study, we present the development of anti-PrP antibodies directed to the human helix 1 site of the prion protein in BALB/C mice. The sequence of choice for immunization resides in human helix 1 of PrP, identical to that of sheep, cattle and rabbits, and distinguished from the mice sequence by one amino acid (Fig. 1A). In order to overcome immune tolerance to an ‘almost-self’ epitope, as well as the poor immunogenicity of short peptides in

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general, we used as antigen the Multiple Antigen Peptide (MAP) method displaying eight copies of helix 1 [MAP(Helix 1)8; Fig. 1B].

2. Materials and methods All animals were treated according to the resolution of the Animal Care and Use Committee of Tel-Aviv University. 2.1. Immunization protocol BALB/C mice, 8 weeks old, weighing 20 –40 g, were challenged with MAP-Helix1 (100 Ag per immunization), either emulsified with Freund’s adjuvant (Difco; n = 10) or with wild-type filamentous bacteriophage (1010 phages/ml) known as a natural adjuvant (n = 4), five times at 14-day intervals. Blood samples were drawn before the first immunization and 7 days after each injection. Sera were analyzed for anti-MAP-Helix1 as well as for the whole prion protein IgG levels using ELISA. Two N.Z.W. rabbits, 10 weeks old and weighing 2 – 2.5 kg were immunized four times with 2.3 mg of MAP-Helix1 associated with Freund’s adjuvant at 21-day intervals. Blood samples were drawn before the first immunization and after the third and the fourth immunizations. 2.2. ELISA for detection of anti-Helix 1 antibodies Sera raised from mice of both the two immunized groups as well as from the non-immunized group were analyzed for their ability to bind MAP-Helix 1 using ELISA as follows: 1 Ag/well of biotinylated MAP-Helix1 (diluted in PBS) was added to avidin (Sigma) coated microtiter plates (Nalge Nunc International) for 1 h at 37 jC. The plates were washed with PBS (0.05% Tween 20) and then blocked with 3% BSA (Amresco) for 1 h at 37 jC. Serial sera dilutions were added for another hour at

Fig. 1. Prion protein Helix 1 as antigen. (A) Sequence alignment of PrP Helix 1 (amino acids 144 – 153 of human PrP) reveals protein high conservation among species which result in the peptide poor immunogenicity. (B) A schematic illustration of the Multiple Antigen Peptide (MAP) method. A synthetic branching structure of lysine residues coupled to eight copies of helix 1 of the human prion protein.

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37 jC and the sera end point titer, defined as the maximal dilution which can still be observed, was determined using excess of HRP-conjugated secondary antibody (Jackson ImmunoResearch) followed by O-phenylenediamine (Sigma). 2.3. Purification of anti-Helix 1 sera Anti-Helix1 IgG fraction was purified using Protein ASepharose column (Amersham Biosciences). Eluted fraction concentrations were determined and tested for their purity using SDS-PAGE. 2.4. Recognition of the whole prion protein by anti-Helix 1 antibodies 2.4.1. Recognition of recombinant bovine PrP 2.4.1.1. ELISA. Purified IgG antibodies were analyzed for their binding characteristics of the whole PrP using ELISA. A 27 KDa Histidine-tagged full length mature part of bovine PrP (25 – 244) expressed in Escherichia coli and over 95% purified as indicated by SDS-PAGE (Calbiochem, Israel) was used as the coating antigen. ELISA was performed as described above whereas microtiter plates were coated with 1 Ag/well of recombinant bovine-PrP. 2.4.1.2. Western blot analysis. A total of 0.5 Ag of recombinant bovine-PrP was electrophoresed in 12% acrylamide gel under denaturing conditions. Recombinant PrP was detected by electrotransfer to nitrocellulose (Schleicher and Schuell) in 25 mM Tris, 192 mM Glycine and 20% methanol at 350 mA for 90 min. The membrane was blocked for 1 h with 5% milk in TBS (0.3% Tween 20). An overnight incubation at 4 jC with either anti-helix 1 sera (1:1000) or with commercial anti-PrP monoclonal antibody 6H4 (1:5000; Prionics, Zu¨rich, Switzerland) was followed by 1 h incubation with goat anti-mouse IgG Fc specificHRP (Jackson ImmunoResearch). Blots were developed using Enhanced Chemiluminesence system (Pierce) according to the manufacturer’s instructions. 2.4.2. Immunofluorescence N2a-C10 is a stable line expressing MHM2-PrP chimera in addition to endogenous PrP expression (Scott et al., 1992). MHM2-PrP differs from wild-type mouse PrP at positions 108 and 111. Substitution at these positions with the homologous residues from the Syrian hamster PrP sequence (L108M and V111M) creates an epitope for the anti-PrP 3F4 monoclonal antibody. N2a-C10 cells were grown in DMEM, supplemented with 10% fetal calf serum and 1 mg/ml of G-418 (Calbiochem). Live cells were cooled to 4 jC, blocked with 10% goat serum in 3% BSA and incubated either with anti-helix 1 or with non-immune sera (1:2000 dilution in 1% BSA) for 1 h. Secondary antibody goat anti-mouse coupled to Cy3

(Jackson ImmunoResearch) diluted 1:500 in 1% BSA was added for another hour. Cells were then fixed using 4% paraformaldehyde in PBS and rinsed thoroughly before mounting with Prolong anti-fade (Molecular Probes). In order to detect total PrP, cells were fixed (4% paraformaldehyde in PBS), permeabilized using 0.1% Triton X-100 in PBS for 10 min at RT and blocked (3% BSA in PBS) before incubation with the sera and the secondary antibody, as described above. 2.4.3. Immunohistochemistry Anti-MAP-Helix 1 sera binding to the whole prion protein was examined on paraffin sections of normal sheep brains. Sections (4 Am) were prepared from tissue fixed by immersion overnight in 4% paraformaldehyde at 4 jC and processed in paraffin. The sections were deparaffinized and rehydrated, followed by endogenous peroxidase quenching using 3% H2O2 in methanol for 10 min at room temperature, and antigen retrieval (10 min in 0.01 M citrate buffer at 100 jC). Blocking with 3% BSA in PBS was followed by incubation with either anti-MAP-Helix 1 or non-immune sera (1:1000 dilution in blocking solution) for 1 h at room temperature. As second antibody we used Fab conjugated to horseradish peroxidase polymer (Zymed Laboratories) for 30 min at room temperature. Diaminobenzidine (Zymed Laboratories) was used as chromogen. Sections were then counter-stained with hematoxylin, dehydrated and cover-slipped. 2.5. Epitope overlapping between mab 6H4 and anti-Helix 1 sera as measured by additivity test Microtiter plates were coated with 20 ng/well of recombinant-bovine PrP (diluted in PBS). Serial dilutions of both mAb 6H4 (Prionics, Zurich, Switzerland) and anti-Helix 1 sera were added at saturation concentrations either alone or with an equimolar mixed solution of both antibodies (v/v) performing ELISA, as described above. Different concentrations of anti-Helix 1 sera and mAb 6H4 were applied for creation of saturation curves, as follows: 0.5 – 0.05 Ag/ml for anti-helix1 sera and 4 – 0.5 Ag/ml for mAb 6H4. Coating with a double amount of antigen (recombinant PrP) was used as a positive control.

3. Results 3.1. Antibody titer against MAP-Helix 1 Immunoreactivity of sera against MAP-Helix 1 is presented by end point titer defined as the maximal sera dilution in which specific antibodies can be still detected (Fig. 2). The immune response was different in the two regimens of immunization. In the group injected with Freund’s adjuvant, a high level of specific IgG antibodies against MAP-Helix 1 was obtained after the third injection

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Fig. 2. Antibodies end point titer against MAP-Helix 1. Antibodies end point titer against MAP-Helix 1 from the second to fifth immunization, as measured by ELISA. End point titer, defined as the maximal sera dilution which can still be detected, is presented in logarithmic scale. Two regimens of immunization are presented: various mice immunized with MAP-Helix 1 emulsified with Freund’s adjuvant as the stimulus (gray bars) and various mice immunized with MAPHelix 1 associated with non-relevant filamentous phage (white bars). The immunological response for each mouse is maintained in the same order.

(titer 1:50,000) and increased to 1:1,000,000 after the fifth injection. In the group injected with non-relevant filamentous phages, known as good immunogen and natural adjuvant (Meola et al., 1995; Bastien et al., 1997), a strong immune response against the phage was detected after the first immunization (data not shown) but reactivity against MAP-Helix 1, observed after the fourth immunization, was only up to 1:10,000. The untreated group did not show immunoreactivity against MAP-Helix 1. Rabbits bearing an identical sequence of helix 1 to that of humans generated a moderate level of anti MAP-Helix 1 antibodies compared with sera achieved in mice, which reached an end point titer of 1:2000 (data not shown).

3.2. Recognition of the whole prion protein by anti-MAPHelix 1 antibodies The following experiments were performed using sera generated by mice immunized with MAP-Helix1 and adjuvant where the end point titers were the highest. Total IgG was purified from the sera, as described in Materials and methods. The concentration of highly purified eluted fractions (Fig. 3A) was measured by O.D. at 280 nm. Purified anti-Helix 1 sera recognized the full length 27 KDa recombinant bovine-PrP as indicated by Western blot analysis (Fig. 3C) and different sera concentrations were measured for their ability to bind the recombinant bovine-

Fig. 3. Characterization of sera against MAP-Helix 1: purification and recognition of whole recombinant prion protein. Anti-Helix 1 sera was purified using protein A. The purified samples were applied to 12% bis-acrylamid gel and stained with coomassie blue. Antibodies under denaturing conditions showed two separate bands: heavy and light (marked by arrows (A)) IgG fractions were eluted in the first three fractions (lanes 2 – 4) giving the same pattern as normal mouse IgG (lane 5). Lane 1 represents the molecular weight marker. Eluted fractions (lanes 2 – 4) were pooled and IgG concentration was measured. The ability of the purified sera to bind the whole recombinant bovine prion protein was tested using ELISA (B) and western blot analysis (C). Anti-Helix 1 sera is shown to recognize the 27KDa recombinant bovine-PrP under denaturating conditions, similar to monoclonal antibody 6H4 used as positive control.

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Fig. 4. Recognition of full length prion protein by anti-Helix 1 sera. (I) PrP immunofluorescence detection in mouse neuroblastoma cells stably expressing MHM2 PrP (N2a-C10 cells). (a) Native PrP is detected on cell surface of living cells using anti-helix 1 sera (Magnification
1500). (b) Non-immune sera exhibit only low background (Magnification
1100). Total PrP was detected on fixed and permeabilized cells using 4% paraformaldehyde followed by 0.1% Triton X-100, by anti-helix 1 sera administration (c) compared with non-immune sera (d), giving a low background (Magnification
500). (II) Immunohistochemistry detection of PrP in animal brains. Immunolabeling of PrP in sheep brain tissue using anti-Helix 1 sera. Strong immunoreactivity is observed in the granular cell layer of the cerebellum (a – c). Neuronal intracellular vesicle staining is evident (arrow). Corresponding sections immunostained with non-immunized sera did not reveal any labeling (d).

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PrP using ELISA (Fig. 3B). The apparent binding constant towards whole protein was calculated as follows: binding constant ¼

1 antibody conc:ð50% of O:D:max Þ

The apparent binding constant of MAP-Helix 1 sera with recombinant bovine PrP was found to be 5.41
109 M 1. Since the sera bind MAP-Helix1 with higher affinity than the whole recombinant protein, we supposed that helix 1 in the context of the whole protein is not as accessible as in the peptide. Nevertheless, eight copies of helix 1, building the MAP construct, may allow production of antibodies of a conformational nature, some of which does not exist in the whole protein native folding. Sera raised in rabbits were analyzed for their ability to bind the whole prion protein. As mentioned above, four immunizations with MAP-Helix raised a moderate level of IgG antibodies against the self-antigen (titer 1:2000) and an even more moderate level against the whole prion protein (1:500, data not shown). However, this method of immunization generated auto-antibodies without any visible autoimmune symptoms. Sera ability to bind the whole prion protein was evident using recombinant bovine PrP, however it was still remained to be shown that anti-helix 1 sera are able to recognize PrP native folding as expressed in neuronal cell culture and tissue rather than adsorbed to a microtiter plate. For that purpose we used mice neuroblastoma cell line (N2a-C10, stably expressed MHM2-PrP; Fig. 4I), and a healthy sheep brain tissue (Fig. 4II). Specific cell surface staining is observed in cells treated with anti-MAP-Helix 1 sera (Fig. 4Ia) compared with nonimmune sera (Fig. 4Ib). In the permeabilized cells labeling is preferentially perinuclear with scattered staining of the axons (Fig. 4Ic). Permeabilized cells administered with non-immune sera as control show a very low background (Fig. 4Id). Immunohistochemistry of sheep cerebellum sections revealed highly specific neuronal staining in the granular layer (Fig. 4IIA –C). Corresponding sections stained with non-immune sera did not show any labeling (Fig. 4IID). Staining was evident in neuronal intracellular vesicles. 3.3. Recognition of the same or overlapping epitope(s) of PrP by anti-Helix 1 sera and mAb 6H4 Anti-Helix 1 sera and mAb 6H4, either alone or in an equimolar mixed solutions (v/v), were allowed to saturate the pre-coated recombinant prion protein and antibody level was determined by the addition of labeled antimouse IgG secondary antibody. Mixed solutions of both antibodies in saturation concentrations were applied and the bound antibody level was measured. Antibody level can range between a minima of exactly the same antibody level as measured for each antibody alone, where both

Fig. 5. ELISA additivity test. Dilutions of both anti-Helix 1 serum and mAb 6H4 were added to microtiter plates pre-coated with recombinant bovine PrP. Saturation curves were prepared for anti-Helix 1 serum, mAb 6H4 in serial dilutions and the mixed equimolar solution (v/v) of both antibodies (middle curve). The final concentrations of mAb 6H4 and anti-Helix 1 sera either alone or in the mixed solution are 4, 0.5; 2, 0.2; 1, 0.1 and 0.5, 0.05 Ag/well. The three lower curves (continuous lines) represent the binding of the antibodies to 20 ng/well recombinant bovine-PrP, whereas the two upper curves (dashed lines) represent the binding to 40 ng/well (these values can be expected if the two antibodies recognize different epitopes within the protein).

antibodies share the same epitope, and up to a maxima in which a double amount of antibodies is measured where both antibodies bind entirely different epitopes (the same level is achieved by administration of each antibody to a double amount of the coated antigen). As shown in Fig. 5, in the presence of both anti-helix 1 sera and mAb 6H4, the amount of bound antibodies is quite similar to that bound when only mAb 6H4 was applied and is different from that bound when each antibody is added to a double amount of antigen (positive control). This data implies that anti-helix 1 sera and mAb 6H4 share an overlapping epitope. The epitope of mAb 6H4 resides in helix 1 of human PrP and our antigen of choice is constructed to display eight copies of human helix 1. The data obtained support the production of the desired site-directed active immunization protocol.

4. Discussion Since prion protein misfolding and aggregation play a central role in progression of prion disease, inhibition and/or reversal of these conformationally induced pathological changes may be of therapeutic importance. Such intervention can be achieved in several ways, either to stabilize the cellular prion protein (PrPc) structure or to destabilize the prion pathogenic form (PrPSc) and prevent interaction between the two PrP isoforms. Indeed, monoclonal antibodies directed to a key region of the protein, which is not necessarily altered in the refolded protein, can interfere with the conformational changes and prevent progression of the disease (Solomon et al., 1997; Frenkel et al., 1999). Efforts to develop immunization against the transmissible spongiform encephalopathies are largely motivated by the

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recent vaccination of transgenic mice developed as a model of Alzheimer’s disease (Schenk et al., 1999). Anti-PrP antibodies have been reported to inhibit the formation of PrPSc in cultured cells and to clear the cultures of prion infectivity (Gabizon et al.,1988; Enari et al., 2001; Peretz et al., 2001). Site-directed antibodies raised against the epitope which resides in amino acids 95 – 103 and 220– 231 are also effective in vitro, whereas antibodies against epitopes 29– 37 and 72– 86 are ineffective, supporting the importance of epitope localization of the respective antigen (Sigurdsson et al., 2003). Additional studies demonstrate that monoclonal antibodies, as well as recombinant Fab fragments directed to amino acids 132 – 156 comprising helix 1, are able to block PrPSc replication in cell culture (Enari et al., 2001; Peretz et al., 2001). Moreover, in vivo studies with transgenic mice overproducing anti-PrP antibodies (6H4A chain) have shown that a humoral response against PrP might have a protective effect against prion propagation (Heppner et al., 2001). In the present study, we show the feasibility of generating anti-PrP antibodies in wild-type mice, regardless of the immune system tolerance, by using an appropriate immunogen. The consideration of active immunotherapy for prion diseases requires production of high affinity anti-aggregating antibodies in normal rather than knockout animal models. The ability to generate high levels of IgG antibodies directed to helix 1 of the prion protein, overlapping 6H4 epitope, together with the knowledge that antibodies directed against helix 1 prevent PrPSc replication in vitro and in vivo (Enari et al., 2001; Peretz et al., 2001; Heppner et al., 2001), suggests that the established immunization protocol may share these qualities and be useful in developing active immunization against prion diseases. In conclusion, towards developing an active immunization against prion disease we chose helix 1 of the whole human prion as antigen complexed to MAP which display eight copies of selected epitope. A high titer against MAP-Helix 1 was obtained after three immunizations with MAP-Helix 1 associated with Freund’s adjuvant which reached a maximal end point titer of 1:1,000,000. ELISA additivity test has confirmed that the sera and mAb 6H4 bind an overlapping epitope and therefore may have a similar effect on induced conformational changes of the whole protein. The purified fraction of IgG was shown to bind the whole prion protein with high affinity. Nevertheless, using the MAP method for immunization displaying different PrP peptides can be an effective tool eliciting anti-PrP antibodies for diagnostic purposes or even to generate PrPSc specific antibodies. All the above data suggest that the immunological approach can be of great value in the treatment of prion diseases, however, further work is needed to prove clinical relevance.

Acknowledgements We thank Dr. A. Taraboulos and the members of his laboratory for providing of N2a-C10 cell line and performing experiments with the whole prion protein. This work was supported by a generous grant from the Israel Center for the Study of Emerging Diseases.

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