Influence of flanking sequences on presentation efficiency of a CD8+ cytotoxic T-cell epitope delivered by parvovirus-like particles

P. Rueda1, G. Morón2, J. Sarraseca1, C. Leclerc2 and J. I. Casal1,{dagger}

1 Inmunología y Genética Aplicada SA (INGENASA), C/Hnos García Noblejas 41, 28037 Madrid, Spain
2 Unité de Biologie des Régulations Immunitaires, INSERM E 352, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris CEDEX 15, France

Correspondence
P. Rueda
prueda{at}ingenasa.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have previously developed an antigen-delivery system based on hybrid recombinant porcine parvovirus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 protein of PPV carrying a foreign epitope at its N terminus. In this study, different constructs were made containing a CD8+ T-cell epitope of chicken ovalbumin (OVA) to analyse the influence of the sequence inserted into VP2 on the correct processing of VLPs by antigen-presenting cells. We analysed the presentation of the OVA epitope inserted without flanking sequences or with either different natural flanking sequences or with the natural flanking sequences of a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus nucleoprotein, and as a dimer with or without linker sequences. All constructs were studied in terms of level of expression, assembly of VLPs and ability to deliver the inserted epitope into the MHC I pathway. The presentation of the OVA epitope was considerably improved by insertion of short natural flanking sequences, which indicated the relevance of the flanking sequences on the processing of PPV-VLPs. Only PPV-VLPs carrying two copies of the OVA epitope linked by two glycines were able to be properly processed, suggesting that the introduction of flexible residues between the two consecutive OVA epitopes may be necessary for the correct presentation of these dimers by PPV-VLPs. These results provide information to improve the insertion of epitopes into PPV-VLPs to facilitate their processing and presentation by MHC class I molecules.

{dagger}Present address: Centro Nacional de Investigaciones Oncológicas (CNIO), C/Melchor Fernandez Almagro 3, 28029 Madrid, Spain.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus-like particles (VLPs) constitute a new generation of non-replicative vectors for delivery of heterologous epitopes and induction of immune responses. Different VLPs are available (Casal, 2001), but we focused our previous studies on porcine parvovirus (PPV) particles expressed in insect cells as a carrier system for the induction of cytotoxic T lymphocyte (CTL) responses. CTLs recognize short epitopes associated with class I molecules of the major histocompatibility complex (MHC). CTL responses play a major role in control and clearance of viral infections and other pathologies such as tumours (Van Pel et al., 1995). We have engineered parvovirus-like particles (PPV-VLPs), formed by the self-assembly of 60 copies of the major structural protein VP2 (Mr 64 000) of PPV (Martinez et al., 1992; Ranz et al., 1989), to deliver CD8+ T-cell epitopes inserted into the N terminus of the PPV VP2. This system presents the advantages of high levels of expression and relative simplicity because it is based on a single protein. Moreover, PPV-VLPs are very stable and can be stored at 4 °C for months without significant alteration in their properties.

In previous studies, we have demonstrated that PPV-VLPs carrying a CD8+ T-cell epitope from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) self-assemble into 25 nm VLPs and elicit a strong CTL response mediated by class I-restricted CD8+ T cells, which can also induce complete protection against a virulent intracerebral challenge with this virus (Sedlik et al., 1997). Moreover, mice immunized intranasally with the same chimeric particles in the absence of adjuvant are also able to elicit strong peptide-specific CTL responses (Sedlik et al., 1999). The efficiency of this delivery system was recently compared with the capacity of peptides linked to microspheres and showed that PPV-VLPs were 100-fold more efficient than microspheres in the induction of CTL responses. The CTL response induced by PPV-VLPs was characterized by a high frequency of specific T cells of high avidity (Sedlik et al., 2000). Finally, PPV-VLPs, in contrast to other systems, do not require CD4+ T cell help for CTL activation (Sedlik et al., 1997). All these findings suggest the suitability of these VLPs to provide an efficient and safe strategy for vaccination and immunotherapy.

One of the key points in the use of these systems is the definition of general rules for the appropriate insertion of epitopes in VLPs for them to be correctly processed and presented. This implies the definition of the optimal size and sequence of the insert. In the present study, to analyse the influence of the sequences flanking the CTL epitope, we have used a Kb-restricted CD8+ T-cell epitope corresponding to aa 257–264 (SIINFEKL) from chicken ovalbumin (OVA) (Moore et al., 1988; Rotzschke et al., 1991). This Kb-restricted epitope has been extensively studied and characterized. Thus, residues 3, 5 and 8 are important for Kb binding, whereas mutations at positions 4, 6 and 7 affect the T-cell receptor recognition (Jameson & Bevan, 1992). Until recently, the influence of the sequences flanking CTL epitopes on the proteolytic processing and presentation of this epitope remained controversial. Although it was initially established that some flanking sequences could prevent the proteolytic generation of the epitope (Del Val et al., 1991), it is not clear whether the presence of natural flanking sequences is necessary for the correct cleavage of the epitope. In fact, several studies have demonstrated the efficient presentation of CTL epitopes regardless of their context (Gilbert et al., 1997; Thomson et al., 1995).

In this report, several strategies were used to study the influence of the flanking sequences on the presentation of the OVA epitope carried by PPV-VLPs (PPV-VLP-OVA constructs). First, we analysed the influence of the natural flanking sequence of the OVA epitope on its presentation to T cells. Secondly, the influence of flanking sequences corresponding to a CD8+ T-cell epitope from the nucleoprotein of LCMV was also analysed. Thirdly, the presentation of dimers of the OVA epitope was tested after insertion either without linker sequences or after connection by either a rigid proline or two flexible glycines. All of these sequences were inserted into the same N-terminal position of PPV VP2, using the same vectors and following the same procedures. The results presented here provide information of interest for the rational design of recombinant vaccines.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mice.
Female C57BL/6 (H-2b) mice (6–8 weeks old) were from Janvier (Le Genet St Isle, France). All animals were maintained under specific-pathogen-free conditions.

Peptides.
The synthetic peptide p257–264 (SIINFEKL) corresponding to the H-2b-restricted CTL epitope (residues 257–264) from chicken OVA was purchased from Neosystem.

Cell lines.
Spodoptera frugiperda (Sf9) cells were grown as previously described (Martinez et al., 1992). B3Z (Karttunen et al., 1992), a CD8+ T-cell hybridoma specific for the OVA 257–264 peptide in the context of Kb, was a generous gift from N. Shastri (University of California, Berkeley, CA).

Design of OVA epitopes and construction of recombinant PPV-VLPs.
Several constructs of the OVA epitope with different flanking sequences or different lengths were prepared (Table 1). Plasmid pPPV29mod (Sedlik et al., 1995), which contains a unique XhoI restriction site immediately downstream of the initiation codon of the VP2 gene, was the starting material for the cloning experiments. The plasmid was linearized with XhoI (MBI Fermentas) and treated with alkaline phosphatase (Roche Molecular Biochemicals). The minimal OVA epitope was regenerated by allowing the annealing of two complementary oligonucleotides with a XhoI restriction site in their 5' ends and then direct ligation into the plasmid as previously described (Casal et al., 1999). The remaining epitopes were regenerated by PCR using two oligonucleotides overlapping by eight nucleotides. The oligonucleotide sequences were designed to maximize baculovirus expression through preferential codon usage (Table 1). PCR conditions were as follows: denaturation at 94 °C for 3 min and 30 cycles composed of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 30 s. The PCR products were digested with XhoI, isolated on low-melting-point agarose gel and cloned into pPPV29mod. The resulting plasmid was used to transform Escherichia coli DH5{alpha}.


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Table 1. Oligonucleotides and amino acid sequences used for the construction of the epitopes

 
After verification of the sequences, the modified VP2 sequence was excised with BamHI and the insert cloned into the BamHI restriction site of the baculovirus transfer vector pAcYM1 (Matsuura et al., 1987). Recombinant viruses were obtained by cotransfection using the lipofectin technique as described previously (Hurtado et al., 1996).

Characterization and purification of PPV-VLP-OVA particles.
Sf9 cells were infected with each recombinant baculovirus at an m.o.i. of 1. Cells were usually collected at 72–96 h post-infection with a complete cytopathic effect. To characterize the presence of the chimeric PPV VP2, cellular extracts were mixed with loading buffer, heated at 100 °C for 5 min and analysed by 9 % SDS-PAGE. Presence of VP2–OVA protein was analysed by Coomassie blue staining or immunoblotting. For immunoblotting, the proteins were transferred to a PVDF membrane (Millipore) using semi-dry equipment (Bio-Rad Laboratories). Membranes were treated with blocking buffer (3 % skimmed milk, 0·05 % Tween 20 in PBS) for 1 h at room temperature. Then they were incubated with an anti-PPV polyclonal rabbit serum overnight at 4 °C. After several washings with 0·05 % Tween 20 in PBS, bound antibodies were detected with peroxidase-conjugated protein A (Sigma-Aldrich) diluted 1 : 1000 in blocking buffer and 4-chloronapthol as a substrate.

Purification of PPV-VLPs was carried out as previously described (Casal, 1999; Rueda et al., 1999). Briefly, infected Sf9 cells were lysed by hypotonic shock with 25 mM bicarbonate solution at 4 °C. Cell debris was removed by centrifugation and the VLPs in the supernatant were precipitated with 20 % ammonium sulphate, resuspended in PBS and dialysed. The identity and properties of the PPV-VLPs were confirmed by SDS-PAGE, immunoblotting, double-antibody sandwich (DAS)-ELISA and electron microscopy.

Quantification of PPV-VLP-OVA particles.
The determination of PPV-VLP-OVA content in each preparation was performed by densitometry and DAS-ELISA. The densitometric assay was carried out with a Kodak Digital Science program using BSA as a reference. DAS-ELISA is a method that is highly conformation specific, exclusively recognizing PPV-VLPs; therefore it can be used specifically to determine the amount of intact capsids. The DAS-ELISA was performed as previously described (Rueda et al., 2001). Briefly, microtitre plates were coated overnight at 4 °C with 0·5 µg per well of anti-PPV monoclonal antibody (mAb) 15C5 (Casal et al., 1992). Plates were washed and serial twofold dilutions of the PPV-VLPs in blocking buffer (350 mM NaCl, 0·05 % Tween 20 in PBS) were then added. After 1 h at 37 °C, plates were washed and anti-PPV biotinylated mAb 13C6 (Casal et al., 1992) diluted 1 : 50 000 in blocking buffer was added for 1 h at 37 °C. Plates were then incubated with peroxidase-labelled streptavidin diluted 1 : 8000 (Sigma) for 30 min at room temperature. Bound enzyme was detected by adding 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid (ABTS; Sigma) as substrate and the reaction was stopped after 10 min by adding 2 % SDS. To calculate the capsid content in the samples, we used highly purified PPV-VLPs from size exclusion chromatography as a standard reference.

For best comparison with soluble peptide, PPV-VLP concentration was expressed as molarity of the whole particle. Therefore, 1 nM PPV-VLP (Mr 3·8x106) corresponds to 60 nM SIINFEKL epitope (Mr 1088) or 120 nM in the case of recombinant PPV-VLP-OVA carrying two copies of the OVA epitope.

Preparation of dendritic cells (DCs).
Spleens from C57BL/6 mice were removed and perfused with collagenase type IV (400 U ml-1 in RPMI 1640; Boehringer Mannheim) containing DNase I (50 µg ml-1; Boehringer Mannheim). Spleens were cut into small pieces and digested in 5 ml collagenase/DNase for 45 min at 37 °C. After inhibition of collagenase activity with 50 mM EDTA in PBS, spleens were dissociated in Ca2+- and Mg2+-free PBS in the presence of 0·5 % FCS and 2·5 mM EDTA. Single spleen-cell suspensions were prepared and labelled with anti-CD16-32 (Fc{gamma}II/III receptor; Pharmingen) and with colloidal superparamagnetic microbeads conjugated to hamster anti-mouse CD11c, (MACS-antiCD11c, N418 clone; Miltenyi Biotec), following the manufacturer's conditions. The positive cells were obtained after magnetic sorting using AutoMACS (program Possel-d; Miltenyi Biotec) and their purity was always between 95 and 99 %. After sorting, the CD11c+ cells were H-2 Kb+, I-Ab low, CD14- and CD40, CD80 and CD86 low or negative; between 25 and 30 % of them were CD8{alpha}+ and between 60 and 70 % were CD11b+ (which were also CD8{alpha}-).

Antigen-presentation assay.
For in vitro assays, CD11c+ spleen cells (105 cells per well) were first pulsed with PPV-VLPs or SIINFEKL peptide for 4 h in 96-well culture microplates in a final volume of 0·2 ml RPMI 1640 Glutamax-I (Gibco Invitrogen Corporation) plus 5x10-5 M 2-ME, 100 IU penicillin ml-1, 100 µg streptomycin ml-1 and 10 % FCS (Gibco) (RPMI 10 %). The antigen concentration used in each experiment is indicated in the respective figures. A concentration of 0·1 nM VLPs corresponds to 120 000 VLPs DC-1. Antigen-presenting cells (APCs) were then washed three times with RPMI 10 %, and 105 B3Z hybridoma cells per well were added in a final volume of 0·2 ml RPMI 10 % and incubated overnight at 37 °C at 2·5 % CO2. The stimulation of B3Z cells was monitored by IL-2 release in cell supernatants, which were harvested and frozen for at least 1 h in dry ice. Next, 104 cells per well of the CTLL cell line (which proliferates proportionally to the amount of IL-2 present in the supernatant) were cultured with 100 µl supernatant in a final volume of 200 µl. After 2 days, [3H]thymidine (ICN Pharmaceuticals) was added and the cells were harvested 18 h later with an automated cell harvester (Skatron). Incorporated thymidine was detected by cell scintillation counting. In all experiments, each point was done in duplicate. Results are expressed in c.p.m.

Peptide structure.
The secondary structure of epitopes was predicted using the PSIPRED 2.0 method (Altschul et al., 1997).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Recombinant PPV-VLPs carrying the OVA epitope with different natural flanking sequences
To characterize the capacity of PPV-VLPs to accept inserts of different sizes and sequences, we prepared a set of recombinant VP2 proteins carrying the OVA epitope with or without different flanking sequences (Fig. 1). Recombinant baculoviruses expressing each VP2–OVA construct were used to infect Sf9 cells. VP2–OVA recombinant proteins were expressed in insect cells and showed molecular masses predicted by the amino acid sequence (Fig. 2). However, the amounts of protein produced by the different recombinant baculovirus were variable (Table 2). All mutants were expressed at high levels except the mutant with five amino acids flanking both sides of the OVA epitope (5-OVA-5), the mutant with three and five amino acids flanking the N and C terminus of the OVA epitope, respectively (3-OVA-5), and the dimer of the OVA epitope (OVA-OVA). The common characteristic of the first two constructs was the presence of an aromatic residue (Trp) at position 267 plus Thr and Ser. Moreover, in these three constructs, the inserted epitopes had long {alpha}-helix or {beta}-sheet structures in their secondary structure. The assembly of the different mutants was evaluated by DAS-ELISA, since the mAbs employed in this assay were specific for the native conformation of VLPs (Rueda et al., 2001) and also by electron microscopy. As shown in Table 2, none of the three constructs (5-OVA-5, 3-OVA-5 and OVA-OVA) was able to assemble, confirming the relationship between poor expression and lack of assembly (Hurtado et al., 1996).



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Fig. 1. Oligonucleotides and amino acid sequences used for insertion in the N terminus of the PPV VP2 capsid gene. Amino acids in bold constitute the epitope sequence. Amino acids of the different flanking sequences are underlined. Restriction sites are indicated.

 


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Fig. 2. Expression of PPV-VLP-OVA constructs in insect cells. Sf9 cells (107) were infected with each recombinant baculovirus at an m.o.i. of 1. Cell extracts were harvested at 72 h post-infection and VLPs were prepared as described in the Methods. VLPs were resuspended in 50 µl PBS. Eight µl of the chimeric 5-OVA-5, 3-OVA-5, OVA-OVA and OVA-GG-OVA VLP-OVA constructs and 1 µl of the other constructs were separated by 9 % SDS-PAGE and stained with Coomassie blue (A) or transferred to nitrocellulose membranes and incubated with anti-PPV rabbit polyclonal serum in immunoblot analysis (B). The names of the different mutants are indicated on the top. Molecular mass markers are indicated on the left.

 

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Table 2. Structural characteristics of the epitopes in relation to the level of expression and assembly of PPV-VLP-OVA constructs

Sf9 cells (107) were infected with the recombinant baculoviruses at an m.o.i of 1. Cells were harvested at 72 h post-infection and lysed by hypotonic shock as described in Methods. After removing cell debris, VLPs were precipitated with ammonium sulphate and resuspended in 50 µl PBS.

 
The ability of the different PPV-VLPs to be processed and presented by APCs to specific MHC class I-restricted T cells was tested by an antigen-presentation assay using purified spleen DCs as APCs. DCs were incubated in vitro with PPV-VLPs and then cultured overnight with a hybridoma (B3Z) specific for the CD8+ T-cell OVA epitope in the context of Kb. Using this system, we demonstrated that PPV-VLPs carrying the minimal sequence of the OVA epitope were not recognized by B3Z cells, showing that the minimal epitope was not efficiently processed by DCs or/and delivered to MHC class I molecules (Fig. 3). Therefore, PPV-VLPs containing the OVA epitope with natural flanking sequences of various lengths were prepared and tested in the same assay. PPV-VLPs containing the largest natural flanking sequences (5-OVA-5) did not stimulate B3Z cells, which could be due to a lack of assembly of these pseudo-particles. In contrast, PPV-VLPs with shorter natural flanking regions (5-OVA-2 and 3-OVA-2) induced a very good IL-2 production by B3Z cells, with maximal presentation of the OVA epitope at approximately 0·1 nM. Slight differences were observed between the presentation of 5-OVA-2 and 3-OVA-2 constructs. As expected, DCs pulsed with control PPV-VLPs without any inserted epitope did not activate B3Z cells.



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Fig. 3. Antigen-presentation assays of recombinant PPV-VLPs carrying the OVA epitope with different flanking sequences. DCs (105 cells per well) were incubated in vitro with non-recombinant PPV-VLPs ({circ}) or with one of the following recombinant PPV-VLPs ({bullet}) for 4 h: (A) OVA; (B) 5-OVA-5; (C) 5-OVA-2; (D) 3-OVA-2. The DCs were then washed and cultured overnight with 105 B3Z cells per well. The presentation of p257–264 to B3Z cells was monitored by IL-2 production. IL-2 production by B3Z was measured by a CTLL proliferation assay and is expressed as mean±SEM of c.p.m. of duplicate wells.

 
Recombinant PPV-VLPs carrying the OVA epitope with flanking sequences of an LCMV CD8+ T-cell epitope
We previously demonstrated that the LCMV nucleoprotein (aa 118–126) epitope inserted into PPV-VLPs with its natural flanking sequences (aa 118–132) induces strong and protective CTL responses (Sedlik et al., 1997). Since the construct containing the minimal OVA epitope was not efficiently presented to specific MHC class I-restricted hybridoma cells, we then analysed whether the addition of heterologous flanking sequences corresponding to the LCMV CD8+ T-cell epitope resulted in adequate antigen presentation. Two constructs were prepared, one containing the residues corresponding to the N terminus (RP) of the LCMV epitope (2LCMV-OVA) and the other containing the six residues corresponding to the C terminus (GNLTAQ) (OVA-6LCMV) of this epitope (Fig. 1). Insect cells were infected with the corresponding recombinant baculoviruses and the chimeric VLPs were characterized as described above. As previously observed for the natural LCMV epitope (Sedlik et al., 1997), these flanking sequences did not have any detrimental effect on the synthesis of the PPV-VLPs and both VLPs were expressed at high levels and were correctly assembled (Fig. 2, Table 2). However, despite this good expression and assembly, these PPV-VLPs were not capable of stimulating B3Z cells in the presence of DCs (Fig. 4), probably due to incorrect processing, thus showing that only OVA natural flanking sequences allow correct processing of the OVA epitope carried by PPV-VLPs.



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Fig. 4. Antigen-presentation assays of recombinant PPV-VLP-OVA constructs carrying LCMV flanking sequences. DCs (105 cells per well) were incubated in vitro with PPV–5-OVA-2 ({bullet}) as a positive control or with either (A) PPV-OVA-6LCMV ({circ}) or (B) PPV-2LCMV-OVA ({circ}) for 4 h. The DCs were then washed and cultured overnight with 105 B3Z cells per well. The presentation of p257–264 to B3Z cells was monitored by IL-2 production. IL-2 production by B3Z cells was measured by a CTLL proliferation assay and is expressed as mean±SEM of c.p.m. of duplicate wells.

 
Presentation of recombinant PPV-VLP-OVA constructs carrying two copies of the OVA epitope
To study whether the capacity of the OVA epitope to induce T-cell responses could be improved by insertion of several copies of the same epitope, we inserted two copies of the epitope in three different configurations: without any linker or with two different linkers, either one proline or two glycines (Fig. 1). The aim was to analyse the influence of a rigid (Pro) or a flexible (Gly) linker on the presentation of the OVA epitope, which would allow the free rotation of the epitopes. Recombinant baculoviruses were prepared as above and used to infect Sf9 cells. The lowest level of expression corresponded to the dimeric construct without a linker. The dimers with the linker were expressed at much higher levels, especially those connected by a proline (Fig. 2). When these constructs were analysed for their self-assembling capacity, we observed that only the PPV VLP construct carrying OVA-OVA with no linker was unable to assemble, in correlation with its low level of expression (Table 2). Therefore, the presentation of this mutant was not analysed. The two other constructs formed particles correctly assembled as demonstrated by electron microscopy (Table 2).

Analysis of B3Z cell stimulation by the two PPV-VLPs containing linkers demonstrated that when a flexible linker was present (OVA-GG-OVA), the OVA epitope was efficiently processed by DCs (Fig. 5A). In contrast, a rigid proline linker between the two copies of the epitope did not allow the presentation of the OVA epitope to B3Z cells (Fig. 5B). Therefore, these results showed that PPV-VLPs can accept at least two copies of a CD8+ T-cell epitope, although their presentation is dependent on the presence of the correct linker between the epitopes. Moreover, surprisingly, whereas the minimal OVA epitope was not presented to B3Z cells, the insertion of two minimal epitopes with a correct linker induced a strong B3Z cell stimulation.



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Fig. 5. Antigen presentation assays of recombinant PPV-VLPs OVA carrying two copies of the OVA epitope. DCs (105 cells per well) were incubated in vitro with non recombinant PPV-VLPs ({circ}) (negative control) or 5-OVA-2 ({bullet}) (positive control), or with either A) OVA-P-OVA ({blacksquare}) or B) OVA-GG-OVA ({blacksquare}) for 4 h and then they were washed and cultured overnight with 105 cells per well of B3Z cells. The presentation of p257–264 to B3Z cells was monitored by IL-2 production. IL-2 production by B3Z was measured by a CTLL proliferation assay and is expressed as mean±SEM of c.p.m. of duplicate wells.

 
Effect of charge and secondary structure on the ability of the VP2 protein to form pseudo-particles
It has often been suggested that maintaining the charge neutrality of the inserted epitopes is required for the integrity of VLPs. We thus analysed the relationship between the net charge of the various inserts and formation of the VLPs (Table 2). This study clearly showed that there was no effect of the charge of the inserted epitope on the level of expression or on the ability of the various PPV-VLPs to assemble. Indeed, constructs with neutral net charge inserts such as OVA-OVA were unable to make particles, whereas constructs with three negative charges such as 5-OVA-2 produced a large number of particles with the correct morphology, and vice versa. Moreover, the first 19 amino acid of the VP2 protein have a negative net charge. Thus, it seems that there is no correlation between the net charge contributed by the epitope and the formation of VLPs.

Finally, using the PSIPRED method (Altschul et al., 1997), a prediction of the secondary structure adopted by the inserted epitopes and their possible influence on the final structure of the particles was made. The minimal OVA epitope seemed to adopt a coil structure, which was modified to {alpha}-helix or {beta}-sheet structures in OVA inserts with different flanking sequences or different lengths (Table 2). A good correlation was observed between the prediction of a high degree of coil structure and the level of expression and assembly. In contrast, prediction of {alpha}-helix or {beta}-sheet structures correlated with low level of expression and assembly. For instance, when two OVA epitopes were inserted without any linker (OVA-OVA), a long {beta}-strand was predicted and no capsids were found in the preparation. Conversely, when two glycines (OVA-GG-OVA) or one proline (OVA-P-OVA) were used as linker between the two epitopes, the predicted secondary structure had less {beta}-strand or {alpha}-helix structure, respectively, and the expression levels were considerably enhanced. These results indicate that it is possible to establish a correlation between the secondary structure of the epitope and the expression of the recombinant VP2 protein.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The use of PPV-VLPs is a powerful system for delivering T-cell epitopes to APCs (Lo-Man et al., 1998; Sedlik et al., 1997). One of the key points in the development of a new delivery system for CTL induction using vectors with genetically inserted epitopes is the understanding of the influence of the insertion site and flanking sequences of the epitope on its processing and presentation to maximize its immunogenicity. We have previously observed that the ability of PPV-VLPs to deliver CD8+ T-cell epitopes varies considerably with epitope nature and that, in general, the insertion of a minimal CD8+ T-cell epitope sequence is not sufficient for efficient antigen presentation and CTL induction (unpublished observations). The reasons for this variability remain unclear. Thus, in the present work, we studied the influence of various flanking sequences of a natural CD8+ T-cell epitope from ovalbumin on the ability of the PPV particles to assemble and deliver this epitope to the MHC class I pathway. Using a specific CD8+ T-cell hybridoma to monitor the appearance of Kb/OVA complexes, we have demonstrated the importance of the natural flanking sequences on the epitope processing and presentation. Moreover, we have also shown that the epitope structure is a determinant of VLP assembly.

In this study, epitopes were inserted in the N terminus of the PPV VP2 protein. During PPV-VLP morphogenesis, the N terminus remains inside the capsid and is not accessible at the particle surface (Cortes et al., 1993; Tsao et al., 1991). Immediately downstream of the insertion site, there is a flexible glycine-rich region, which contains several putative proteolytic sites. The N-terminal cleavage of VP2 to VP3 by host proteases only occurs in full particles, suggesting that the introduction of DNA into the particle allows the N terminus of VP2 to be externalized. This process can be mimicked in vitro by trypsin treatment (Paradiso et al., 1982). Although the process of cell invasion of porcine parvovirus is not well characterized, studies on other parvoviruses sharing many properties with PPV showed that, during entry into the host cell, many VP2 proteins undergo cleavage, which removes approximately 18 residues from their N termini (Paradiso, 1981). This cleavage seems to be essential for virus infectivity because it exposes the conserved glycine-rich sequence, which may be important in virus interaction with cellular membranes, allowing virion translocation across the plasma cell membrane (Cotmore & Tattersall, 1987).

We have recently shown that, in vivo, PPV-VLPs are captured by DCs with a high efficacy by macropinocytosis and that DCs are the only APCs capable of presenting PPV-VLPs to CD8+ T cells (Morón et al., 2002). After their capture, PPV-VLPs are found in late-endosome vesicles of DCs and their processing is dependent on the acidification of endosomes and on the activity of some proteases (Morón et al., 2003). In this context, the processing of N terminus recombinant VP2 containing different flanking sequences can produce different fragments, which, before or after ulterior processing and by a still unknown mechanism, could be translocated to the cytosol and thus could become accessible to the proteosome that would be the responsible for the final trimming of the peptide into the correct epitope for presentation by MHC class I (Cascio et al., 2001; Del Val & Lopez, 2002; Mo et al., 1999).

In various systems, either replicative such as vaccinia or non-replicative such as VLPs produced in yeast (Ty VLPs) (Gilbert et al., 1997) or bacterial toxins reaching the cytosol of APCs (Fayolle et al., 2001), the use of polyepitope chains containing multiple CD8+ T-cell epitopes has been reported for vaccine design (Thomson et al., 1995; Woodberry et al., 1999). In these studies, no influence of the flanking sequences was observed, since the epitopes were linked together and all seemed to be correctly processed. However, in the present study, the insertion of the minimal OVA epitope was not enough for efficient presentation and the insertion of the natural flanking sequences was required for delivery to MHC class I molecules. All constructs containing the OVA epitope with natural flanking sequences that formed particles were efficiently processed. The length of the flanking sequences did not seem crucial because addition of different natural sequences to the N or C terminus of the epitope did not affect antigen presentation. In fact, our data clearly demonstrated that as few as two to three residues are sufficient for efficient processing of the OVA epitope. Interestingly, the flanking sequences of the LCMV CD8+ T-cell epitope, which worked efficiently for the presentation of this epitope inserted into the same VLPs (Sedlik et al., 1997), were ineffective for the OVA epitope. Another possibility could be that the various PPV-VLP preparations have a different capacity to bind DCs, affecting in consequence their capture and processing. However, so far there is no experimental data supporting the presence of any receptor on DCs allowing capture of PPV-VLPs. Moreover, PPV-VLPs inserted with different CD8+ T epitopes are equally captured by DCs (data not showed), showing that the nature of the insertion does not change the capture of PPV-VLPs by DCs.

Using this experimental system, we also demonstrated that, in addition to the crucial role of the natural flanking sequences, the inserted sequence should not contain residues that could interfere with the process of VP2 synthesis and capsid assembly. Our data illustrate that the sequence Trp-Thr-Ser is detrimental for PPV VP2 expression and for capsid formation. A similar effect, although less evident, was previously observed for the insertion of two CD4+ T-cell epitopes, polio C3:T (Sedlik et al., 1995) and PreST from hepatitis B (Lo-Man et al., 1998), which also contain a Trp residue. In these cases, the particles with an altered morphology assembled with low efficiency. Hydrophobic sequences, such as aromatic residues, could stabilize interactions either with VP2 or with other hydrophobic structures, including membranes. Similar results have been also obtained with other constructs (data not shown). Indeed, the presence of the amino acids Thr and Ser in the inserted sequence did not modify VP2 expression, therefore suggesting that aromatic residues are detrimental to VP2 expression and capsid formation. On the other hand, it is also clear that the secondary structure of the inserted sequence affects the level of expression of the VP2 protein and consequently capsid assembly. The predicted secondary structure of the N-terminal natural sequence of VP2 protein at the site of insertion shows that the first 19 amino acids display a clear coil structure. The results of this study clearly show that the more similar the secondary structure of the inserted epitope to the N terminus of the native protein, the higher the level of expression and assembly obtained. Therefore, this study indicates that the presence of the natural flanking sequences of the epitope and its secondary structure are very important for correct capsid assembly and for the optimal processing and presentation of the CD8+ T-cell epitope. However, it is not possible to establish general rules applicable to the flanking sequences because they will probably depend specifically on the system used.

The site of insertion and the mode of presentation of an epitope on a heterologous carrier can dramatically affect its immunological properties (Taylor et al., 2000). In this way, insertion of two consecutive OVA epitopes without a linker between them led to incorrect capsid assembly, whereas linker insertion between the two epitopes led to particles that were properly assembled. Linker insertion could increase peptide flexibility allowing the adoption of different structures that could stabilize the VP2 protein and facilitate capsid assembly. In contrast to the results obtained with the OVA epitope inserted as a single copy, insertion of two copies of the minimal OVA epitope connected by two glycines showed that the nature of the flanking sequences was not decisive for efficient processing. The flexibility of the epitope and the capacity of the system to adopt multiple conformations, which could facilitate cleavage by host proteases, seem to be important, since the insertion of a proline between the two OVA epitopes prevents its correct antigen presentation. However, the negative result observed with Pro was not totally unexpected, since Pro is well known as a hindrance to many proteases (Vanhoof et al., 1995; Yaron & Naider, 1993) and has also been shown to interfere with TAP (transporter associated with antigen presentation)-dependent transport of antigenic peptides when occupying certain positions within the peptide (Neisig et al., 1995).

The ability of the PPV-VLPs to carry more than one copy of an epitope, as demonstrated by the OVA dimer with two glycines, opens up the possibility of incorporating CTL epitopes covering the HLA spectrum of the target population or including multiple epitopes from a pathogen.


   ACKNOWLEDGEMENTS
 
G. Morón was supported by a post-doctoral fellowship of the Consejo Nacional de Inestigaciones Científicas y Tecnológicas (CONICET, Argentina) and by EEC grant QLK2-CT-1999-00429. This work was carried out as a collaborative project between INGENASA and the Institute Pasteur in an EU-supported project (EEC grant # QLK2-CT1999-0318).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 23 July 2003; accepted 7 November 2003.