Published online before print April 1, 2004
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* INSERM E209, Hospital Saint-Antoine, Paris, France;
SEDAC Therapeutics, Le Galenis, Loos, France; and
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
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1 and 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
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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.
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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 
-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-
(IFN-)-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- 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- release was revealed with a secondary, biotinylated, anti-IFN- 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).
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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.
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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).
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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).
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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.
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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- 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- 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.
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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).
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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).
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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.
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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 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.
Received December 26, 2003; revised February 19, 2004; accepted February 25, 2004.
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