PSEUDO-VIRAL PARTICLES AND USES OF SAME

Abstract

The present invention relates to a type I or II transmembrane fusion protein comprising, successively:

a) optionally, a signal peptide;
b) a protein or a peptide of interest;
c) a coiled-coil domain; and
d) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment.

It also relates to the virus-like particles obtained with this fusion protein.

Claims

1. A virus-like particle comprising: an envelope consisting of a plasma membrane of which at least one portion is typical of lipid rafts; and at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively: b) a protein or a peptide of interest; c) a coiled-coil domain or oligomerization sequence, which does not originate from a virus; and d) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring in a plasma membrane of which at least one portion is typical of lipid rafts, fragments b) and c) being exposed at the surface of the virus-like particle.

2. The virus-like particle according to claim 1, wherein a linker is present between fragments b) and c), and/or between fragments c) and d).

3. The virus-like particle according to claim 1, wherein: b) the protein or the peptide of interest is chosen from: allergens and fragments thereof, cell surface proteins and fragments thereof, proteins and peptides that are accumulated in chronic or neurodegenerative diseases, proteins and peptides involved in hypertension, immunoglobulins and fragments thereof, cytokines and fragments thereof, and hormones and fragments thereof; c) the coiled-coil domain is chosen from SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 33 and SEQ ID NO: 30; and d) the anchoring domain is chosen from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 26) and the anchoring sequence of the PDLP1 protein (SEQ ID NO: 31).

4. The virus-like particle according to claim 1, wherein the protein or the peptide of interest is chosen from allergens and fragments thereof.

5. A type I or type II transmembrane fusion protein comprising the following fragments, successively: a) optionally, a signal peptide; b) a protein or a peptide of interest; c) a coiled-coil domain or oligomerization sequence, which does not originate from a virus; and d) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring in a plasma membrane of which at least one portion is typical of lipid rafts.

6. The transmembrane fusion protein according to claim 5, wherein the protein or the peptide of interest is chosen from allergens and fragments thereof.

7. A nucleic acid encoding a fusion protein as claimed in claim 5.

8. A host cell or a vector comprising at least one nucleic acid according to claim 7.

9. A method of treatment comprising a step of administering, to a patient in need thereof, a therapeutically effective amount of the virus-like particle claim 1.

10. A method of allergen immunotherapy comprising a step of administering, to a patient in need thereof, a therapeutically effective amount of the virus-like particle according to claim 1.

11. A method for producing virus-like particles according to claim 1, comprising the expression of the nucleic acid as claimed in claim 7 in eukaryotic cells.

12. The method according to claim 11, characterized in that the eukaryotic cells are plant cells, and in that it comprises the following steps: a) transformation of agrobacteria with an expression vector comprising a nucleic acid as claimed in claim 5 functionally linked to a strong promoter; and b) transfection of the plant cells with the agrobacteria obtained in step a), said transfection comprising the following steps: b1) culture of the plant cells, under aeroponic or hydroponic conditions, and under LED lighting, preferably for four to six weeks under hydroponic conditions, b2) agroinfiltration of the plant cells obtained in b1) under vacuum, with the agrobacteria obtained in step a), preferably carried out under vacuum by Venturi effect, b3) return to culture of the plant cells obtained in b2), typically for 3 to 6 days, in order to obtain the virus-like particles, then extraction of the virus-like particles obtained and purification, in particular by enzymatic extraction.

Description

[0148] The figure legends are the following:

[0149] FIG. 1: Diagrammatic representation of the various expression cassettes for producing an allergen linked to an oligomerization sequence and to a sequence for anchoring the plasma membrane, preferably at the level of the lipid rafts

[0150] A) The cDNA encoding the optimized, preferably harmonized, Der p2 (DP2, SEQ ID NO: 22) is linked to 1) the cDNA encoding the tobacco chitinase signal peptide (PS Chit, SEQ ID NO: 21), 2) an oligomerization (coiled-coil) sequence from a transcription factor (GCN4-pII/trimeric form, BGCN4-PLI/tetrameric form, CGCN4-IZN4/glycosylated form, EGCN4-pAA/heptameric form, D) or from any other family of proteins having a coiled-coil sequence (SNARE, Golgin, Fibritin, G) or a synthetic sequence mimicking a coiled-coil sequence (F), and finally 3) an anchoring sequence from enveloped virus envelope protein (TM/CT of influenza H5, B to I) or from type I protein anchored in the lipid rafts (lipid raft) (J).

[0151] B) Diagram representing the structure of the oligomerization or coiled-coil sequence. This sequence consists of a repeat motif of 7 amino acids, of hxxhcxc type, wherein h is a hydrophobic amino acid, c is a charged amino acid, and x is any amino acid.

[0152] FIG. 2: AllergoPur platform used for the expression and production of the various forms of VLP

[0153] FIG. 3: Production of the proteins described in FIG. 1A

[0154] The proteins extracted from plants transfected under vacuum for the expression of the DP2 (lane 1), DP2-Tri (lanes 2-3) DP2-Tetra (lanes 4-5) or FD1-Tri (lanes 6-7) proteins were analyzed by immunodetection with an antibody directed against the Der p2 or Fel d1 allergen. The immunodetection analysis demonstrates the specific production of the proteins, the molecular weight of which corresponds to the expected weight. Two clones of agrobacteria (C1.1 and C1.2) were analyzed for each construct.

[0155] FIG. 4: Purification and characterization of the VLPs carrying the allergens by size exclusion chromatography

[0156] Protein extracts of leaves producing DP2-Tri (panel D), DP2-Tetra (panel E), DP2 soluble (panel F) FD1-Tri (panel G), DP2triDGCN4 (GCN4 deletion, panel H), DP2tri-Syn (GCN4 replacement, panel I) and DP2tri-KEI (GCN4 replacement, panel J) were separated by chromatography on a calibrated S-500/HR column. The total soluble protein content of each fraction was evaluated by spectrometry (panel A) and staining with Coomassie blue after separation by SDS-PAGE (panel B). The allergen content of the elution fractions was revealed by immunological detection using anti-Der p2 or anti-Fel d1 antibodies. Protein extracts of leaves producing hemagglutinin in the form of VLPs from H5N1 (panel C) were separated by gel filtration on a calibrated S-500/HR column and are used as controls.

[0157] FIG. 5: Characterization of the VLPs carrying the isolated allergens by means of examination by electron microscopy and negative staining

[0158] The VLPs carrying the allergens have a morphology and a size that are very close to those described for influenza virions.

[0159] The bar represents 50 nm.

[0160] FIG. 6: Reactivity of the allergens produced in the form of VLPs with sera from patients allergic to Der p2

[0161] The proteins extracted from plants transfected under vacuum for the expression of the DP2 (lane 1), DP2-Tri (lane 2) and DP2-Tetra (lane 3) proteins were analyzed by immunodetection with sera from patients allergic to Der p2. The immunodetection analysis demonstrates the recognition of the allergens carried by the VLPs by the IgEs of the patient sera.

[0162] FIG. 7: Method for large-scale VLP production

[0163] FIG. 8: Structure of the VLPs and of the fusion proteins assembled according to the invention

[0164] A) Structure of a VLP according to the invention. The VLP consists of a plasma membrane envelope, in which the fusion proteins according to the invention are attached. The protein or the peptide of interest (for example the allergen) is thus exposed at its surface.

[0165] B) Structure of the fusion proteins according to the invention, assembled within the VLP. The oligomerization sequences allow the fusion proteins to form polymers (for example in this case the allergen, A) at the surface of the VLP.

[0166] FIG. 9: The antigens conjugated to VLP have a very strong immunogenic power but no allergenicity

[0167] Panel A: Evaluation of the hyperactivity of the airways induced by the Der p2 allergen, by the Flexivent method. The mice (n=10/group) was sensitized with the Der p2 allergen in soluble form (DP2-Alum) or in VLP form (DP2-VLP/alum and DP2-VLP/saline) and challenged with a mite extractor. Twenty-four hours after the final challenge, the airway hyperactivity to inhaled methacholine was determined by the Flexivent method. The pulmonary reactivity triggered in the presence of the allergen in VLP form is comparable to the control mice.

[0168] Panel B: Counting of the inflammatory cells in the respiratory pathways, collected by bronchoalveolar lavage (BAL) of the lung. The mice (n=10/group) were sensitized with the Der p2 allergen in soluble form (DP2-Alum) or in VLP form (DP2-VLP/alum and DP2-VLP/saline) and challenged with a mite extract. Twenty-four hours after the final challenge, the BAL cells were collected and the cells were counted (eosinophils; neutrophils; macrophages; lymphocytes). The neutrophils are very predominant in the mice having received the Der p2 allergen in soluble form.

[0169] Panel C: Assaying of the Der p2-specific IgGs. The mice (n=10/group) were sensitized with the Der p2 allergen in soluble form (DP2-Alum) or in VLP form (DP2-VLP/alum and DP2-VLP/saline) and challenged with a mite extract. Twenty-four hours after the final challenge, the IgGs were measured in the BAL fluid and the blood serum that was collected by cardiac puncture. The mice having received injections of DP2-VLP with or without adjuvant have an IgG titer which is one thousand times higher than the mice having received the soluble Der p2.

EXAMPLES

Example 1: Molecular Design and Synthesis of the Genes

[0170] The cDNAs are synthesized by optimizing and then harmonizing the codon usage for their recognition by the plant system. In the context of this invention, the preferred optimization is the optimization for expression in Nicotiana benthamiana.

[0171] The constructs are illustrated in FIG. 1. In particular:

A: cDNA encoding the natural form of the protein (SEQ ID NO: 1). This cDNA may or may not be fused to trafficking signals described in patent WO 2008/056265. The corresponding protein has the sequence SEQ ID NO: 2.
B: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pII trimerization signal of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 3). The corresponding protein has the sequence SEQ ID NO: 4.
C: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pLI tetramerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 5). The corresponding protein has the sequence SEQ ID NO: 6.
D: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pAA heptamerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 7). The corresponding protein has the sequence SEQ ID NO: 8.
E: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the IZN4 glycosylated oligomerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 9). The corresponding protein has the sequence SEQ ID NO: 10.
F: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to a synthetic sequence mimicking a coiled-coil and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 11). The corresponding protein has the sequence SEQ ID NO: 12.
G: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to a SNARE oligomerization sequence and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 13). The corresponding protein has the sequence SEQ ID NO: 14.
H: cDNA encoding two fragments of Der p2 (SEQ ID NO: 22) that are fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 15). The corresponding protein has the sequence SEQ ID NO: 16.
I: cDNA encoding the CH1 chain of the Fel d1 allergen (SEQ ID NO: 32) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin (SEQ ID NO: 17). The corresponding protein has the sequence SEQ ID NO: 18.
J: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the tobacco chitinase signal sequence (Neuhaus, J.-M. 1996), to the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor and to the anchoring sequence of the PDLP1 protein (AOAOD3D8S3) of lipid rafts (SEQ ID NO: 19). The corresponding protein has the sequence SEQ ID NO: 20.

Example 2: Plasma Preparation

[0172] Xba I/kpn I and Sal I/Sac I restriction sites are respectively integrated at the 5 and 3 ends of the cDNA during the synthesis. These sites are then used in order to clone the cDNAs into the pAG01 binary expression vector. The cDNAs are cloned upstream of a 35S promoter (35S) and downstream of a nopaline synthase termination sequence (tnos); the pAG01 vector also contains an expression cassette which makes it possible to express the p19 silencing inhibitor simultaneously with the recombinant protein in order to increase the production yields. The vectors are then used to transform the LBA4404 strain of Agrobacterium tumefaciens.

Example 3: Transient Expression of Der p2 Produced in VLP Form in Nicotiana benthamiana LeavesUse of the AllergoPur Platform

[0173] For the production by transient expression, Agrobacterium tumefaciens LBA4404 is used for the transfer of a cDNA encoding Der p2 linked to an oligomerization sequence and to an anchoring sequence without the gene of interest being integrated into the genome of the plant cell. This is referred to as transfection and not transgenesis. The plants are cultured under hydroponic conditions in the presence of a nutritive medium (GHE, floragrow, floramicro, florabloom, 10 ml/15 ml/5 ml per 10 l of osmosed water) and under LED lighting. The agrobacterium is transferred into the foliar tissue via agroinfiltration according to two methods. For the production of small batches of proteases intended for prototype screening, the agrobacteria are manually injected by means of a syringe applied against the epidermis of the lower face of the leaf. Foliar disks sampled from the leaves 4 to 6 days after the agroinfiltration are used for the analysis of the various VLP prototypes. This screening step makes it possible to define the expression vector that will be used for obtaining the Der p2 allergen anchored in the membrane of optimal quality. The same method is used for large-scale production, but in this case, the agroinfiltration is carried out under vacuum, in chambers containing several liters of a culture of agrobacteria and wherein several tens of plants are simultaneously infiltrated. These plants are then put back into culture for 3-6 days before purification of the VLPs carrying the allergen (FIG. 2).

Example 4: Production of the VLPs Carrying the Der p2 Allergen

[0174] The expression of the proteins produced in example 3 and also the yields are respectively analyzed by Western blotting and ELISA. The results are given for 3 allergens produced in VLP form (DP2-tri; DP2-tetra and FD1-tri) (FIG. 3).

Example 5: Evaluation of the VLP Formation/Size Distribution Analysis

[0175] A size distribution analysis of the structures carrying the Der p2 (DP2-tri/DP2-tetra) or Fel d1 (FD1-Tri) allergens was carried out. After infiltration under vacuum of N. benthamiana plants with the Agrobacterium strain LBA4404, as described in example 3, the total protein extracts were separated by size exclusion chromatography on an S-500 (HR) high-resolution column (GE Healthcare Bio-Science Corporation). The elution fractions were controlled with respect to their total protein content and to their VLP-allergen content by Western blotting with anti-allergen antibodies. For all the extracts analyzed, the soluble protein concentration in the eluate reaches a maximum in fractions 14-16 (FIG. 4). On the other hand, the Western blotting analysis demonstrates an accumulation of allergens in fractions 6 and 7 (that is to say before the elution of the Dextran Blue used as marker) which shows the linking of the allergens to very high molecular weight structures in the 2 MDa zone.

[0176] The 32 ml Sephacryl S-500/HR columns (GE Healthcare Bio-Science Corporation) were equilibrated with 50 mM PBS, pH 7.4, 150 mM NaCl. Samples of total protein extracts of 1.5 ml were loaded and then eluted with the equilibration buffer. Twenty-four 1.5 ml elution fractions were collected and analyzed for the content in proteins measured by absorbance spectrophotometry at 280 nm. The proteins of each fraction were concentrated by precipitation with acetone and then redissolved in one and the same volume of elution buffer before the analysis by SDS-PAGE and Western blotting. The elution profiles of the Dextran Blue 2000 and of the soluble proteins were compared for each chromatogram in order to be sure of the reproducibility of this separation technique.

Example 6: VLP MorphologyAnalysis by Electron Microscopy

[0177] The transmission electron microscopy of the purified product (resulting from the production in example 3 followed by purification) indicates that the high-molecular-weight structures isolated by sieving chromatography are VLPs to which allergens are bound. Both in terms of their size and their morphology, which comprises a phospholipid membrane covered with spikes, these VLPs closely resemble influenza virions (FIG. 5).

Example 7: Production of Allergens or of Hypoallergens Carried by VLPs

[0178] The coupling of an allergen to a VLP considerably reduces its in vivo reactivity in IgEs from patients' serum.

[0179] However, the use of hypoallergens further reduces the reactivity of the IgEs and consequently the risks of anaphylactic reaction. The reduction in reactivity of a hypoallergenic form of the Der p2 allergen carried by VLPs is illustrated in FIG. 6.

Example 8: VLP Production

[0180] The detailed method for producing the VLPs, up to their purification (as described in example 6), is illustrated in FIG. 7.

Example 9: Immunogenic Power of the VLPs in Comparison with the Soluble Allergens

[0181] The presentation of an antigen in a highly ordered and repeating network normally brings about strong immune responses, whereas the same antigen presented as a monomer is non-immunogenic.

[0182] In order to compare the immune response with respect to the allergen when it is presented in the form of a highly ordered network, mice were immunized with the Der p2 allergen in soluble monomer form or in VLP-carried form. The titers of IgG against the Der p2 allergen were determined by ELISA.

Protocol

[0183] The protocol is illustrated as follows:

[0184] Analyses after the sacrifice: [0185] Weight loss and change in behavior [0186] Pulmonary function (flexiVent, plethysmography) [0187] Serological response to the allergen (serum IgG, IgE, blot) [0188] Basophil activation test [0189] Histopathology and other serological results (for example: IgG isoforms) in the subsequent phases.

[0190] This study demonstrated that the VLPs coupled to the Der p2 allergen do not trigger bronchial hypereactivity in mice, contrary to soluble Der p2 (see FIG. 9, panel A). Furthermore, the VLPs bonded to Der p2 trigger a systemic Th1-type response with neutrophil activation (see FIG. 9, panels B and C).

Example 10: Desensitization/Vaccination Using the VLPs

[0191] The key parameters of an effective vaccine are the following: rapid induction of a high antibody titer in the absence of adjuvants, and the absence of major side effects.

Protocol

[0192] The protocol is illustrated as follows:

LITERATURE REFERENCES

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