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Originally published online as doi:10.1189/jlb.1203656 on April 1, 2004

Published online before print April 1, 2004
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(Journal of Leukocyte Biology. 2004;76:125-134.)
© 2004 by Society for Leukocyte Biology

Identification of two immunogenic domains of the prion protein—PrP—which activate class II-restricted T cells and elicit antibody responses against the native molecule

Sylvie Gregoire*,{dagger}, Caroline Logre*, Pat Metharom*, Estelle Loing{dagger}, Jacques Chomilier{ddagger}, Martine Bruley Rosset*, Pierre Aucouturier* and Claude Carnaud*,1

* INSERM E209, Hospital Saint-Antoine, Paris, France;
{dagger} SEDAC Therapeutics, Le Galenis, Loos, France; and
{ddagger} Laboratory of Mineralogy-Crystallography, CNRS, University of Paris 6 and Paris 7, France

1Correspondence: INSERM E209, Hôpital Saint-Antoine, Bâtiment Kourilsky, 184 rue du faubourg Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: carnaud{at}st-antoine.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent reports suggest that immunity against the prion protein (PrP) retards transmissible spongiform encephalopathies progression in infected mice. A major obstacle to the development of vaccines comes from the fact that PrP is poorly immunogenic, as it is seen as self by the host immune system. Additional questions concern the immune mechanisms involved in protection and the risk of eliciting adverse reactions in the central nervous system of treated patients. Peptide-based vaccines offer an attractive strategy to overcome these difficulties. We have undertaken the identification of the immunogenic regions of PrP, which trigger helper T cells (Th) associated with antibody production. Our results identify two main regions, one between the structured and flexible portion of PrP (98–127) and a second between {alpha} 1 and {alpha} 2 helix (143–187). Peptides (30-mer) corresponding to these regions elicit class II-restricted Th cells and antibody production against native PrP and could therefore be of potential interest for a peptide-based vaccination.

Key Words: TSE • prion diseases • epitope mapping • peptide-based vaccine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transmissible spongiform encephalopathies (TSE) constitute a well-defined family of fatal neurodegenerative disorders, which can affect, under natural conditions, various species including humans [1 , 2 ]. A hallmark of the different forms of TSE is the accumulation in the central nervous system (CNS) and in the secondary lymphoid organs of a pathological, self-aggregating, and protease-resistant protein, scrapie prion protein (PrPSc), which results from the post-translational conversion of PrPC, a glycosylphosphatidylinositol-anchored host glycoprotein, constitutively expressed on many tissues including neurones, glial cells, and lymphoreticular structures [3 , 4 ]. Although the issue is still debated, there is strong evidence in favor of a causative role of PrPSc in TSE transmission [5 ].

Unlike conventional agents, PrPSC does not elicit detectable immune responses under natural circumstances of infection [6 ], most probably because the protein is perceived as self by the host T cells, which do not distinguish between the two PrP isoforms. However, recent reports indicate that mice, which have been made intentionally immune to PrP by active immunization [7 , 8 ], passive antibody (Ab) transfer [9 , 10 ], or transgenic acquisition of a rearranged heavy chain encoding an anti-PrP Ab [11 ], manifest increased resistance to scrapie infection. Previous studies had shown that anti-PrP monoclonal Ab (mAb) prevented PrPSc conversion in chronically infected N2a neuroblastoma cell lines, thus providing a possible explanation for the effects observed in vivo [12 13 14 ].

Several issues need to be addressed, however, before safe and effective vaccination can be developed in TSE. One is the poor immunogenicity of PrP as a result of its lack of foreigness. A second concerns the risk of developing aggressive autoimmunity in the CNS, a situation that has been reported recently in Alzheimer’s disease patients vaccinated against the peptide Aß1-42 [15 ]. The third issue that ought to be raised concerns types of responses that should be the most effective for long-lasting remissions. Although Ab seem to be important, other protective mechanisms, including regulatory T cells or innate immunity cells, need to be considered in view of the fact that cell-mediated [16 ] and innate immunity [17 18 19 ] are implicated in TSE pathogenesis.

A peptide-based strategy could provide useful clues to those questions [20 ]. Beside the fact that peptides are safe and easy to produce, they can specifically target the response toward critical portions of a protein; they can orient the response toward the production of humoral, cell-mediated, or cytotoxic effectors, and when associated with the right adjuvants or the right antigen-presenting cells (APC), they can overcome natural tolerance, notably through the activation of helper T (Th) cells. An essential preliminary to any peptide-based strategy is therefore the identification of motifs that are recognized by T and/or B cells. The present study was set up so as to identify those motifs in the PrP molecule and to characterize the cellular and humoral responses elicited through them.

To this end, we have synthesized a library of relatively long, 30-mer peptides [21 ] and screened their capacity to elicit responses in PrP-deficient mice, which are not naturally tolerant to the protein [22 ]. Out of a total of 13 overlapping peptides spanning the entire molecule, three peptides identifying two distinct domains of PrP manifest immunogenic properties regarding T cells, B cells, or both and are therefore of potential interest for future immunointerventions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
PrP-deficient mice were donated by Dr. Charles Weissmann (Institute of Neurology, Medical Research Council Prion Unit, London, UK) [23 ] and since then, have been iteratively back-crossed in our facility with C57Bl/6 progenitors. The mice used in the present study were homozygous offspring derived from third or 10th back-cross, as indicated in the legends of figures. They were bred and maintained under strict, specific, pathogen-free conditions in compliance with European Union guidelines.

Vaccinal DNA
Naked DNA was produced by amplification in Top10 Escherichia coli (Invitrogen, Cergy, France) of a pcDNA3.1 plasmid in which the coding sequence of the Prnp gene had been inserted (a gift from Dr. Sylvain Lehmann, Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Montpellier, France). High-quality, endotoxin-free DNA was purified with an Endofree plasmid Giga-prep kit according to the manufacturer’s recommendations (Qiagen, Courtaboeuf, France). Control DNA was obtained by the same procedure from an empty pcDNA3.1 plasmid. For immunization, mice were injected three times at weekly intervals, and 100 µg DNA was divided between the two tibialis anterior muscles. These were sensitized 5 days before the first inoculation with 50 µl cardiotoxin [10 mM solution in phosphate-buffered saline (PBS)] from Naja nigricollis venom (Latoxan, Rosans, France).

Libraries of synthetic peptides
A first library of 13 overlapping peptides, mostly 30-residues long, beginning just after signal peptide and spanning the entire protein sequence, was synthesized by Neosystem (Strasbourg, France). Peptides were at least of a purity grade of 80%, and all were soluble in water. A second library of 13 peptides, 15-mer long with an overlap of 11 residues aimed at mapping more precisely the T cell epitope common to two adjacent, 30-mer peptides, was synthesized with a purity of 82–100% by Polypeptide Laboratories (Paris, France). Some of the 15-mer peptides required the addition of 1% dimethyl sulfoxide in water for a complete solubilization of the 100x stock solution.

Mice were immunized twice at 10-day intervals, with 50 µg peptide in PBS, emulsified, respectively, in complete and incomplete Freund’s adjuvant (Difco, Detroit, MI) and injected subcutaneously in two different spots.

Recombinant PrP
A recombinant baculovirus transfer plasmid was generated by insertion of the Prnp exon from pcDNA3.1PrP into pBAC4x-1. After polymerase chain reaction (PCR) amplification, the 762-bp DNA fragment was ligated into the plasmid vector between BglII and XbaI.

Transfection in Spodoptera frugiperda Sfg cells and rounds of plaque assay were achieved according to classical procedures. The expression of the sequence and of the protein was monitored, respectively, by PCR and Western immunoblotting using SAF83 anti-PrP mAb (a gift from Dr. Jacques Grassi, Atomic Energy Commission, Saclay, France).

For protein production, Trichopulsia ni cells (Hight Five Cells, Invitrogen) were infected with the recombinant virus. Culture supernatants were collected at day 3 and precipitated twice with (NH4)2SO4 at 75% saturation, and recombinant protein was purified by nickel nitriloacetic acid–agarose affinity chromatography (Qiagen), according to the recommendations of the kit supplier. The purity of recombinant PrP was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining.

T cell proliferation assay
Spleens were collected 10 days after the last immunization. Lymphoid cells were dispersed in PBS containing 3% fetal calf serum (FCS; PAA Laboratories, Les Mureaux, France), incubated in NH4Cl–Tris (0.16 M) solution for red blood cell removal, and finally, resuspended in Dulbecco’s modified Eagle’s medium supplemented with L-glutamine, sodium pyruvate, Hepes buffer (Gibco, Invitrogen), 2-mercaptoethanol (50 µM; Sigma, Saint-Quentin-Fallavier, France), and 10% FCS. In some experiments, responder T cells were enriched by negative magnetic sorting. Spleen cells were incubated with a mixture of 10 µg/ml each anti-CD11b [immunoglobulin G (IgG)2b of rat]- and anti-CD19 (IgG2a of rat)-purified mAb, followed by incubation with sheep anti-rat Ig Dynabeads (Dynal, Compiègne, France) and passage through a magnetic field. T cell purity monitored by fluorescein-activated cell sorter analysis was between 95% and 98%.

Responder cells were plated at a density of 3 x 105 cells/well into flat-bottom 96-well plates (Falcon, Becton Dickinson, Le Pont de Claix, France). APC prepared from spleens of naive PrP-deficient mice and irradiated at 2500 rads were distributed at the same density. Peptides in solution were added at three concentrations: 6 µM, 2 µM, and 0.6 µM (corresponding roughly to 20, 6, and 2 µg/ml). In some experiments, T cells were restimulated with APC plus recombinant PrP at three estimated concentrations of 15, 5, and 1.7 µg/ml. In experiments aimed at demonstrating major histocompatibility complex (MHC) class II restriction, Y3P mAb (mouse IgG1), specific for I-Ab (a gift from Dr. Olivier Lantz, Curie Hospital, Paris, France), was added at different dilutions, right from the beginning of the assay. In one experiment, an irrelevant isotype-matched mAb, clone 16B12, recognizing an epitope of influenza hemagglutinin, was added as control.

Plates were incubated for 4.5 days at 37°C in 5% CO2 and pulsed during the last 18 h with 1 µCi/well [3H]-thymidine (Amersham, Orsay, France). Cultures were harvested on filters (Tomtech MacIII, Perkin Elmer, Villebon-sur-Yvette, France), and radioactivity was measured by scintillation (MicroBeta 1450 Trimux, Wallac, Turku, Finland). To pool data from experiments performed at different moments, a proliferation index (P.I.) was calculated by dividing the mean counts per minute (cpm) in experimental wells (at least triplicates) by the mean cpm in control wells (at least nine replicates) containing the same cells but no peptide.

Antibody detection by enzyme-linked immunosorbent assay (ELISA)
Antibodies against peptides or recombinant PrP were detected by ELISA. Polypropylene 96-well microtiter plates (Maxisorp, Nunc, Denmark) were coated overnight at +4°C with the antigen preparation at 10 µg/ml in carbonate buffer and then blocked for 30 min with PBS 5% bovine serum albumin. Sera to be titrated were distributed as duplicates at three dilutions (1/50, 1/250, 1/2500), left for 2 h at room temperature, and washed out. Anti-PrP Ab binding was revealed with a goat anti-mouse Ig secondary Ab coupled to peroxidase (Roche, Meylan, France), followed by addition of o-phenylenediamine in 0.05 M citrate buffer (pH=5; Sigma). The reaction was stopped with 25 µl 2 N sulfuric acid. SAF83, mAb reactive against mouse PrP, was tested in each assay as a positive standard.

Antibody detection by indirect immunofluorescence
Antibodies to membrane-bound PrPC were detected by cell fluorescence. EL4 T cells, stably transfected with the mouse Prnp gene, were further activated overnight on plastic-bound anti-CD3 mAb (clone 2C11, a gift from Dr. Lucienne Chatenoud, Hospital Necker, Paris, France) to maximize PrPC expression. These cells were then incubated with serum dilutions (1/10, 1/20, 1/40), washed, and exposed to a secondary Ab: a rat anti-mouse {kappa}-chain (Mark1, Biosys, Compiegne, France) coupled to fluorescein isothiocyanate or isotype-specific anti-IgG1 and anti-IgG2a Ab, respectively, coupled to phycoerythrin or biotin, revealed with streptavidin–allophycocyanin, all from PharMingen (Le pont de Claix, France). SAF83 was used as a positive standard in every assay. Cell-surface fluorescence was measured by flow cytometry (FACSCalibur, Becton Dickinson) using CellQuest as software. Results are expressed as the geometric mean fluorescence intensity (MFI) for a 1/10 dilution of serum.

Measure of T cell cytokine release by enzyme-linked immunospot (ELISPOT)
The frequency of interferon-{gamma} (IFN-{gamma})-producing cells among primed T lymphocytes was determined by ELISPOT. Nitrocellulose-bottomed 96-well plates (Millipore, Fontenay-sous-Bois, France) were coated with anti-mouse IFN-{gamma} mAb (clone R4-6A2, Pharmigen) for 2 h at 37°C and then overnight at +4°C. The wells were washed, blocked during 2 h at 37°C with RPMI 1640 plus 10% FCS, and plated with 1 x 106 spleen cells plus no peptide or at least one concentration of peptide (10 µg/ml). Every combination was set up as triplicate. After 24 h incubation at 37°C and 5% CO2, the plates were washed, and IFN-{gamma} release was revealed with a secondary, biotinylated, anti-IFN-{gamma} mAb (clone XMG1.2, Pharmigen) followed by addition of alkaline phosphatase-conjugated streptavidin (Boehringer Mannheim, Meylan, France). Spots were visualized using tetrachloroindolylphosphate/tetrazolium nitroblue as substrate (Promega, Charbonnières, France) and counted in an automated ELISPOT plate counter (Autoimmun Diagnostika GmbH, Strassberg, Germany). The results are given as the mean spot numbers per 1 x 106 spleen cells recalled with a given concentration of peptide (usually 10 µg/ml) with in parallel the number of spots observed with the same cells but in the absence of the recall peptide.

Statistical analyses
Statistics between groups were performed with the nonparametric, two-tailed Mann-Whitney test using Prism 3 software (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Screening the 30-mer peptide library with primed spleen cells from mice immunized with DNA
The library of peptides used for the screening of immunogenic epitopes is presented in Figure 1A . Most (eight out of 13) peptides are 30-residues long with an overlap of 15 residues between each. The remaining five, mainly located at the C terminus, have been shortened for solubility reasons.



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  		Figure 1. Peptide libraries used in this study. (A) List of peptides (mostly 30-mer) with 15-residue overlap, covering the entire PrP sequence. (B) Peptides (15-mer) with 11-residue overlap, focusing on one of the two T cell-immunogenic domains.

 
To raise primed T cells endowed with the broadest repertoire, we used a DNA strategy previously reported as effective against PrP [24 ]. PrP-deficient mice were inoculated in the presensitized tibialis anterior muscles of both hind legs with plasmid DNA encoding the PrP sequence. Preliminary experiments had shown that at least two consecutive injections were necessary to raise detectable amounts of antibodies against PrP (data not shown). To optimize the priming, experimental mice were exposed three times to vaccine DNA in parallel with control mice, which received the same amount of DNA prepared from an empty plasmid. Spleen cells collected 7 days after the last injection were tested for proliferation in a 4.5-day assay against the 13 synthetic peptides at three different concentrations. As can be seen in Figure 2A , which summarizes the results of six individual mice, three peptides emerged: two adjacent ones p143–172 and p158–187, which gave rise to an average P.I. of 3.86 and 4.03, respectively, at a peptide concentration of 6 µM and p98–127, which raised weaker (average P.I.=1.88) but still significant responses (P<0.001 by Mann-Whitney) compared with the responses elicited by the remaining peptides (average P.I.=0.95). No other peptide, including those adjacent to p98–127 triggered proliferation significantly above those background values. Spleen cells from mice, which had been injected with control DNA (Fig. 2B) , showed no proliferation whatsoever, confirming that the responses were exclusively observed with immune spleen cells primed with vaccine DNA and ruling out the possibility of some artifact, as a result of the presence of impurities in the synthetic peptides.



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  		Figure 2. In vitro proliferative responses of spleen cells from mice primed in vivo with vaccine DNA. (A) Mice immunized with plasmid DNA encoding the Prnp exon. (B) Mice receiving control DNA from an empty plasmid. The results represent the pooled poliferation indexes ± SE of six individual mice. Mice were from back-cross 3. Responses to peptides p98–127, p143–172, and p158–187 were significantly different from the responses to the remaining peptides (P<0.001 by two-tailed Mann-Whitney test).

 
Priming and recalling T cell responses directly with peptides
To confirm the immunogenicity of those three peptides, we primed naive PrP-deficient mice with each one of them individually and rechallenged in vitro with the same peptide as well as with the two adjacent ones. As shown in Figure 3A 3B 3C , each peptide could elicit a proliferative response by itself. The two adjacent peptides, p143–172 and p158–187, showed almost complete reciprocity in their capacity to prime and recall, suggesting that they share a common and probably single epitope, as neither the peptide downstream of p143–172 nor the one upstream of p158–187 was effective. Immunizing and recalling with p98–127 (Fig. 3C) generated, as expected from previous experiments, a weaker response (P.I. at 6 µM=1.97), which could not be recalled by the two adjacent peptides p83–112 (P.I.=1.09) and p118–142 (P.I.=1.32), suggesting the existence of a relatively weak T cell epitope, probably centered on position 113–117, a segment which happens to be covered neither by the downstream nor by the upstream, neighboring peptides (see Fig. 1A ). The remaining 10 peptides were assayed through the same protocol, each on at least two individual PrP-deficient mice to be sure that a minor epitope had not been missed. None elicited proliferative responses (data not shown).



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  		Figure 3. Proliferative responses of spleen cells from mice primed with the three immunogenic 30-mer peptides and recalled with the same or the adjacent peptides. The results represent the pooled P.I. ± SE of four to six individual mice. Mice were alternatively from back-cross 3 or 10. Responses of p143–172-primed T cells to recall p143–172 and p158–187 are not statistically different from each other (P=0.22) but are statistically different from recall by p128–157 (P<0.0001). Reciprocally, responses of p158–187-primed T cells to recall by p143–172 and by p158–187 are not statistically different (P=0.43) but are statistically different from recall by p173–189 (P<0.0001). (C) T cells primed and recalled by p98–127 give a response significantly higher than that observed after recall by p83–112 (P<0.001) and recall by p118–142 at 2 µM and 0.6 µM peptide concentrations (P<0.05).

 
Further characterization of the spleen cell-proliferative response
The next experiments were aimed at confirming the presence of a unique epitope located somewhere in the overlap between p143–172 and p158–187. To this end, we synthesized a second library, the details of which can be seen in Figure 1B . This library comprises 10 peptides, 15-residues long, with an overlap of 11 residues between each. As shown in Figure 4A and 4B , respectively, spleen cells primed with p143–172 were restimulated with most efficiency by p156–170 (P.I. at 6 µM=4.27). The two adjacent peptides gave only marginal responses. Spleen cells primed with p158–187 were also recalled with most efficiency by the same short peptide p156–170 (P.I. at 6 µM=9.47) and less so by the two adjacent peptides (P.I.=3.33 and 3.87, respectively). Thus, peptide p156–170, which coincides almost perfectly with the sequence shared by the two 30-mer peptides, is likely to carry this unique epitope.



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  		Figure 4. Refining the position of the epitope common to p143–172 and p158–187 and demonstration that synthetic peptides present epitopes similar to those naturally processed. Proliferative responses to the 15-mer library of spleen cells from mice primed with p143–172 (A) or p158–187 (B). Results represent the pool P.I. ± SE of four individual mice in each case. Responses induced by recall peptide p156–170 are significantly different (P<0.01) from responses induced by all the other 15-mer peptides. Adjacent peptides p152–166 and p160–174 gave marginal responses (P<0.05) with spleen cells primed with p158–187. (C) Proliferation of T-enriched spleen cells of two individual mice each, primed with p98–127, p158–187, p156–170, or with no peptide and recalled in the presence of APC loaded with recombinant PrP. Differences between primed T cells and unprimed T cells are highly significant (P<0.0001) with every priming peptide. Mice were all from back-cross 10.

 
We also wished to verify that the immunogenic peptides revealed under such conditions corresponded to epitopes that were naturally presented to T cells upon PrP processing. Mice were therefore primed with peptides p98–127, p158–187, or p156–170, the 15-mer that contains the epitope common to p143–172 and p158–187. T-enriched spleen cell suspensions prepared from those mice were then challenged in vitro in the presence of APC from PrP-deficient mice pulsed with three concentrations of recombinant PrP. As shown in Figure 4C , APC loaded with recombinant PrP, in contrast to "empty" APC, provided good stimulation to immune spleen cells of all three origins, but not to control spleen cells from mice, which had only received Freund’s adjuvant. Average P.I. were, respectively, 2.57, 3.87, and 4.63 for T cells primed with p98–127, p158–187, and p156–170. Thus, all three synthetic peptides present epitopes that correspond to naturally processed, antigenic determinants.

To obtain a more precise profile of the responding T cells, we examined their MHC restriction and the cytokines produced upon recall. Figure 5A shows the effect of anti-I-Ab mAb upon the proliferation of spleen cells primed and recalled by peptide p143–172 or p158–187, respectively. In both cases, the antibodies inhibited proliferation from 30% at 1 µg/ml mAb down to practically 100% at 10 µg/ml. Thus, the responding cells are mostly, if not totally, restricted to MHC class II and are therefore CD4+ Th cells. The blocking was specific as shown by the fact that an irrelevant, isotype-matched mAb used in parallel, at the same concentrations, had no effect (less than 8% inhibition) upon T cell proliferation.



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  		Figure 5. MHC restriction of proliferating T cells and IFN-{gamma} production. (A) Spleen cells from mice primed and recalled with p143–172 or p158–187 (6 µM) were cultured in the presence of anti-I-Ab mAb. One of three identical experiments with mice from back-cross 10. (B) Spots of IFN-{gamma} developed after overnight culture of immune spleen cells with or without recall peptide at 10 µg/ml. Each value represents the number of spots ± SE of triplicate wells within a single experiment. Mice were from back-cross 3 and were considered as positive, immune spleen cell populations which gave a number of spots above background values plus 3 SD.

 
Cytokine production was ascertained by the sensitive technique of ELISPOT. As seen in Figure 5B , spleen cells from individual mice primed with p143–172 or p158–187 and recalled overnight with the same peptides showed detectable numbers of IFN-{gamma} spots (usually 100–200 spots per 1x106 cells). Conversely, among the three mice immunized with p97–126, only one gave a response above control (107 spots per 1x106 cells), a result in line with the relative weakness of the T cell epitope corresponding to this 30-mer peptide. Attempts at detecting interleukin-4 responders by the same technique gave weak responses (data not shown), indicating that the T cells elicited by PrP peptides are more evidently oriented toward a Th1 profile. Furthermore, no cytotoxic activity was detected with T cells primed in vivo by p158–187, recalled in vitro by the same peptide, and assayed against transfected EL4 (H-2b) target cells overexpressing PrP (data not shown).

Generation of antibodies against total PrP
One reason for using relatively long synthetic peptides was to identify immunogenic motifs that might elicit antibody production against PrP. Peptides (30-mer) seemed to be a good compromise for generating B and T cell responses at the same time.

First, we confirmed that individual peptides p97–126, p143–172, and p158–187 gave rise to antibodies that recognized their own priming antigen. Mice were immunized twice with peptide in Freund’s adjuvant, and their sera, collected 10 days after the second challenge, were probed by ELISA. It can be seen (Fig. 6A ) that all three peptides, including p98–127, were equally effective in eliciting humoral responses against themselves. No Ab binding could be detected under the same conditions in sera of mice injected with Freund’s adjuvant alone.



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  		Figure 6. Demonstration by ELISA of serum Ab against the immunizing peptides (A) or against total recombinant PrP (B). Each point is the optical density (O.D.) value of an individual serum at 1/50 dilution. Solid symbols represent sera of mice immunized twice with peptides in Freund’s adjuvant; open symbols are sera of mice injected with adjuvant alone. Mice were from back-cross 3 or 10, alternatively. Horizontal bars show the median value in a given group. Differences are significant (P<0.001) between immune and control sera (A), but not between sera from mice challenged with the various peptides (B).

 
We then probed those sera against plastic-coated, recombinant PrP to determine whether the immunogenic peptides gave rise to antibodies that would recognize corresponding epitopes on the total molecule. Results in Figure 6B demonstrate that this is indeed the case. Individual differences are observed within a group of mice immunized with a given 30-mer peptide, but all immune sera were significatively above control sera (P<0.001), i.e., sera from mice, which had received Freund’s adjuvant only.

Still, the native structure of membrane-bound PrPC is not preserved after adsorption on polystyrene ELISA plates. To better evaluate the capacity of peptide-raised Ab to recognize membrane-bound, native PrP, we assayed the sera by indirect immunofluorescence on cells that overexpressed PrPC. Figure 7A shows an overlay of histograms generated by representative sera of a mouse hyperimmunized with vaccine DNA, a mouse that had been immunized against p98–127, and one immunized against p143–172. As expected, the pcDNA3.1Prnp immune serum is the most potent, reflecting the fact that it probably contains antibodies against a broader range of epitopes than sera resulting from immunization against single peptides. It is interesting that the antiserum raised against p98–127 was more effective than the one raised against p143–172, in spite of the fact that the T cell responses elicited by the former peptide were generally weaker. This tendency was confirmed in Figure 7B , which recapitulates the data of 10 sera against p98–127, 11 sera against p143–172, and 10 sera against 158–187. Median MFI was 34 in the first group versus 17 in the second (P=0.07). In sharp contrast, none of the sera elicited by p158–187 were above control values, despite the fact that this peptide is at least as effective as p143–172 in eliciting T cell responses and triggers good antibody responses against itself (Fig. 6A) and recombinant PrP (Fig. 6B) .



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  		Figure 7. Demonstration of Ab binding to membrane PrPC by indirect immunofluorescence on transfected and activated EL4 cells. (A) Overlay of three representative immune sera versus one control serum. Sera were tested at three dilutions; here, the 1/10 dilution is presented. (B) Cumulative results of immune and control sera presented at a 1/10 dilution. All mice were from back-cross 10. The horizontal bar represents the median value in each group. The difference between p98–127 and p143–172 immune sera is not statistically significant (P=0.07 by Mann-Whitney test).

 
As p98–127, which is a weak T cell activator, could elicit a good antibody response to cellular PrP, we decided to verify that the remaining peptides of the library, which had been found negative in T cell proliferation assays, would not nevertheless elicit Ab against PrP. At least three mice were immunized against each individual peptide. None of the sera raised under such conditions gave responses above controls.

The isotypes of anti-PrPC antibodies were determined with specific secondary reagents. All positive sera contained IgG1 and IgG2a Ab; the proportions were not different whether sera had been raised against p98–127 or p143–172 (data not shown). We could not quantify the respective proportions of each isotype, but the data clearly demonstrated that an isotypic switch controlled by Th cells had occurred in all the combinations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary objective of this study was to identify the immunogenic domains of PrP, capable of activating specific Th cells. There has been a wealth of information regarding the B cell epitopes of PrP [24 25 26 27 ], but little is known so far about Th cell responses [28 , 29 ]. Yet the success of a peptide-based strategy is highly dependent on the elicitation of helper cells that will determine the nature of the response, overcome natural tolerance to PrP, and possibly regulate autoimmune complications. Our results highlight two independent, immunogenic regions, one of relatively weak strength, between positions 98 and 127, and one of stronger immunogenicity, tentatively mapped between residues 156 and 170. There seems to be no other immunogenic domain, at least within the limits of sensitivity of our screening assay. It is however possible that by multiplying the number of iterative in vitro recalls with peptides and by adding exogenous T cell growth factors, minor epitopes might emerge, as shown by Souan et al. [28 ]. In this latter study aimed at analyzing T cell responses from normal PrP-expressing mice, two immunogenic regions were identified at positions 131–150 and 211–230. The former coincides partially with the domain covered by our immunogenic peptide p143–172, but the epitopes are likely to be different, as the downstream peptide p128–157 is ineffective in recalling p143–172-primed T cells. With regard to the second region, our closest corresponding peptide, p212–232, is devoid of immunogenicity under the present experimental conditions. These differences could be explained by the fact that the responding T cells come from mice with different genotypes: PrP-positive wild-type mice in the Souan et al. [28 ] study and PrP-deficient knock-out mice in ours. It would thus seem that the T cell repertoire, which escapes central and peripheral tolerance against PrP in wild-type mice, must go through profound modifications. Conversely, a recent study of the cell-mediated responses generated in PrP-deficient mice [29 ] highlights the immunogenicity of an octamer sequence from human PrP—NQVYYRPM—which coincides almost perfectly with the mouse sequence, NQVYYRPV, in the middle of our immunogenic 15-mer peptide p156–170. It is interesting to note that the octamer was selected with an approach different from ours, based on its predicted capacity to bind to MHC class I antigens.

The second objective of this study was to characterize the humoral responses elicited in the course of immunization with immunogenic peptides. One of the reasons for constructing a library of 30-mer motifs was the anticipation that long enough motifs might generate Ab, not only against themselves but also against the native molecule. The results of the ELISA against recombinant PrP and the immunofluorescence assay against EL4 confirm this prediction. There is obviously no correlation between the strength of the T cell epitope carried by a given peptide and its capacity to elicit Ab against native PrP. Peptide p98–127 turns out to be the most potent immunogen for raising anti-membrane-bound PrP Ab, whereas paradoxically, p158–187, which is a potent T cell immunogen and generates the best Ab responses against plastic-bound PrP, is completely inefficient for raising Ab against cell-surface PrPC. It is also important to note that anti-p98–127 immune sera contained IgG Ab, indicating that a weak Th cell response is nevertheless sufficient to induce isotypic switch and probably affinity maturation, but that conversely, the peptides that had been negative in the T cell screening assay did not generate detectable anti-PrPC Ab.

The contrasting immunogenic properties of peptides p98–127 and p158–187 are best explained by the respective accessibility of the corresponding regions within the molecule. A peptide coinciding with a buried domain has indeed less chance to be contacted by B cell receptors. A theoretical calculation of individual residues [30 ] based on the chrystallographic model of a human PrP dimer [31 ] confirms that the structured C terminus of p98–127 is more exposed than p143–172 and that p158–187 is significantly less accessible than p143–172 (data not shown).

A second obvious condition for generating Ab against membrane PrPC is that the peptide in solution mimicks epitopes of the native protein. It is not possible to infer from the individual primary sequences, the spatial configuration of the 30-mers. Specific nuclear magnetic resonance analyses will be necessary for confirming the presence of PrPC mimotopes on p98–127 and p143–172.

Antibodies with therapeutic value must presumably contact some critical domain of the PrP molecule. This is particularly clear in vitro, where it has been shown that mAb, which recognized PrPC on plastic wells but not in plasmon cell resonance or on cell surface, were quite ineffective [12 ]. Little is known so far regarding the mechanisms through which Ab cure N2a cells in vitro [12 13 14 , 32 ] or slow down prion dissemination in vivo [7 8 9 10 11 ]. There is however some strong evidence for the existence of a few critical domains, one encompassing residues 130–156 and contacted by the prototypic 6H4 mAb. This region, which corresponds to the first {alpha} helix of PrPC, has been involved in PrP conversion and in the crossing of the species barrier [33 34 35 ]. Ab raised against p143–172 and recognizing a corresponding domain of PrPC could thus be of therapeutic value. Other reported regions of potential interest are 159–178, contacted by polyclonal Ab, raised against dimeric PrP, and corresponding to the second ß strand [14 ], and 91–110, recognized by the in vivo protective mAb ICSM 35 [10 ]. Here too, antibodies raised against our 30-mer peptides p98–127 and p143–172 might be of interest. The identification of immunogenic domains and of corresponding peptides eliciting cellular and humoral responses opens new perspectives regarding peptide-based vaccines. Preliminary data indicate that it might be possible to directly immunize wild-type mice expressing PrP with those peptides, provided they are injected with potent adjuvants such as oligo CpG-DNA motifs of bacterial origin [36 , 37 ]. It will then have to be shown that active or passive conferment of B and/or T cell immunity brings about protection against TSE with no adverse consequence regarding the CNS.


   ACKNOWLEDGEMENTS
 
This work was supported by funds from INSERM, from ATC prion 1999, and from "GIS maladies à prions 2001." S. G. has been the recipient of a fellowship CIFRE in partnership between SEDAC Therapeutics and INSERM and is presently the recipient of a fellowship from the Foundation for Medical Research. We are grateful to Dr. F. Godot for his help in producing recombinant PrP, to Dr. R. Carp for discussion and for reviewing the manuscript, as well as to Ms. I. Renault for the management of the PrP knockout breedings.

Received December 26, 2003; revised February 19, 2004; accepted February 25, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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