Pseudo-viral particles and uses of same

Abstract

The present invention concerns 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. The invention also concerns the pseudo-viral particles obtained with this fusion protein.

Description

The present invention relates to a fusion protein comprising successively the following fragments: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a coiled-coil domain that does not originate from a virus; and d) a plasma membrane anchoring domain, consisting of a transmembrane segment and a cytosolic segment, preferably a plasma membrane anchoring domain at least part of which is typical of lipid rafts. It is also related to virus-like particles (VLPs) obtained with such a fusion protein, said protein being anchored in their membrane. While allergen immunotherapy was first described over 100 years ago by Noon and Freeman (1,2), very little progress has been made in desensitization, with the exception of the method of administration, which has seen the gradual replacement of injectable treatments with sublingual desensitization. For example, since January 2011, a sublingual tablet for grass pollen allergies has been available in France. The introduction of these desensitization tablets has helped to reduce the invasiveness of allergen immunotherapy, but it has not increased its effectiveness. Indeed, oral treatment still uses "natural" allergenic extracts, which are low in allergen concentration and not representative of the diversity of allergens found in the allergenic source. However, over the past decade, new strategies have been proposed to increase the effectiveness and reduce the duration of allergen immunotherapy treatments. These desensitization strategies are based on the use of: of natural allergenic extracts modified to reduce their allergenicity while retaining their immunogenicity (Henmar et al), of recombinant allergens, native or modified to make them hypoallergenic (Valenta et al), peptides corresponding to the epitopes of T cell allergens, in free form or fused with carrier proteins (Larche M., Patel D. et al, Chen et al), adjuvants, or allergens fused to nanoparticles or viral pseudoparticles (Kundig et al, Bachmann MF, Jennings GT, Henmar et al). Strategies using modified allergenic extracts remain of limited effectiveness due to their poor representativeness of the diversity of allergens contained in the source. Recombinant allergens, whether modified or not, or peptides, are weakly immunogenic in soluble form in the absence of adjuvants. However, many adjuvants are poorly tolerated and, as with vaccines, their use is not recommended in allergen immunotherapy. The use of allergens fused to nanoparticles or virus-like particles (VLPs) is, on the other hand, a particularly attractive strategy for allergen immunotherapy. Virus-like particles self-assemble from viral antigens. They contain no genetic material, are therefore non-infectious, and incapable of replication. However, they mimic the original structure of a virus, which allows them to be easily recognized by the immune system and to very effectively activate immunological memory. VLP-based vaccines against hepatitis B, human papillomavirus infections, and influenza (Garland et al., Paavonen et al., D'Aoust et al.) illustrate the vaccine efficacy of antigens when presented to the immune system on the surface of VLPs or nanoparticles. Thus, VLPs have the potential to be used as antigen-presenting structures, particularly for allergens, enabling the induction of a strong immunogenic response in humans. Virus-like particles (VLPs) are of two main types: those produced from viral capsid proteins (VLP CPs) and those produced from enveloped viruses (VLP Envs). The structure and composition of these two types differ significantly. VLP CPs are generally produced by manufacturing the recombinant capsid protein, which self-assembles in host cells through mechanisms similar to those of native viruses. VLP Envs are produced when an envelope protein Env is synthesized and modified by the endomembrane system of a host cell, then migrates to lipid rafts in the plasma membrane, where it concentrates and triggers extracellular budding of the entire membrane / protein assembly. The resulting particle, the VLP, carries immunogenic epitopes of the Env protein on its surface. The use of allergens in the form of virus-like particles thus appears to be an important criterion for the success of immunotherapy. Results obtained with virus-like particles (VLPs) fused to a peptide of the major house dust mite allergen (Der p 1) or to the major cat allergen (Fel d 1) have illustrated the very high immunogenicity of these fusions in mice and humans (Schmitz et al., Kundig et al.). This immunogenicity is so high that a single injection of these VLPs induces sufficient IgG production to protect against a type I allergic reaction. However, the preparation of these VLPs is unfortunately extremely complex and involves numerous steps, some of which are difficult to standardize. Specifically, the VLPs are produced in E. coli after expression of the Qbeta bacteriophage envelope protein. The allergen is then expressed in recombinant form in E. coli, purified, solubilized, and purified again in several steps. Once these two components are produced and purified, they are coupled in vitro. This production technique is clearly too complex, too expensive, and too difficult to standardize for the final product to ever be available for the treatment of allergic patients. Therefore, there is a need for a non-immunogenic and versatile structure that self-assembles in eukaryotic cells, is easy to synthesize, and can support proteins or peptides on its surface. Such a structure could be used to treat allergic patients, as well as in other clinical settings. The Plaintiff has now developed such a structure, which self-assembles and allows for use in therapy. In particular, the unique characteristics of such a structure are: • undetectable immunogenicity of the structure as such; • its ability to assemble into oligomers, such as trimers or tetramers; • its membrane containing unique lipids, typical of lipid rafts; "its protein-poor membrane of the host cell membrane; • its ability to be expressed at high yields in many types of eukaryotic cells (yeast, insects, or plants); and • its ease of preparation. According to a first aspect, the invention consists of a type I or type II transmembrane fusion protein comprising successively the following fragments: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a coiled-coil domain (or oligomerization sequence) that does not originate from a virus; and d) an anchoring domain in the plasma membrane and more particularly in lipid rafts, consisting of a transmembrane segment and a cytosolic segment. Such a fusion protein behaves like a viral surface protein when expressed in eukaryotic cells. Without being limited by any particular theory, this protein is synthesized in the endoplasmic reticulum and then transported via the Golgi apparatus to the plasma membrane. Once it reaches specialized areas of the plasma membrane, preferably lipid rafts, this transmembrane fusion protein causes the membrane to bend, ultimately forming a bud that detaches from the cell membrane and is released into the extracellular space. During budding, the protein or peptide of interest carried by the coiled-coil domain (or oligomerization sequence) is exposed on the outer surface of the newly formed particle. The transmembrane domain remains anchored to the membrane and is not exposed at the surface.This yields a virus-like particle (VLP), illustrated in Figure 8 A, comprising a plasma membrane whose composition is preferably typical of lipid rafts, in which the fusion proteins according to the invention are fixed at the level of their anchoring domain, and which exposes the protein or peptide of interest on its surface, in oligomerized form (thanks to the oligomerization sequence). The structure of the fusion proteins assembled on the surface of the VLP is illustrated in Figure 8B. According to a second aspect, the invention consists of a virus-like particle (VLP) comprising: - an envelope consisting of a plasma membrane, at least part of which is typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored in said membrane (i.e., said envelope), said fusion protein comprising successively the following fragments: b) a protein or peptide of interest; c) a coiled-coil domain (or oligomerization sequence) that does not originate from a virus; and d) a plasma membrane anchoring domain, consisting of a transmembrane segment and a cytosolic segment, preferably a plasma membrane anchoring domain at least part of which is typical of lipid rafts, fragments b) and c) being exposed to the surface of the VLP. In the VLP according to the invention, the fusion protein is anchored in the membrane (and therefore in the envelope) by means of its anchoring domain d). A virus-like particle (VLP) is a nanoparticle consisting of a plasma membrane envelope in which one or more proteins are anchored. It contains no genetic material, is non-infectious and incapable of replication, and self-assembles to mimic the original structure of a virus. The structure of a VLP is illustrated in Figure 8A: the membrane envelope includes proteins anchored and exposed on its surface. Such a virus-like particle according to the invention exhibits the interesting properties indicated above. Furthermore, when the protein or peptide of interest (b) of the fusion protein is an allergen or an allergen fragment, or more generally an antigen, the virus-like particle is effective in antigen presentation and exhibits a high capacity for activating immune system cells. This allows for effective desensitization of allergic patients. In addition, the virus-like particle according to the invention stimulates the production of allergen-specific IgG while minimizing accessibility to basophils. The pseudo-viral particles according to the invention typically have a diameter between 120 and 200 nm. According to a third aspect, the invention relates to a method for producing a pseudo-viral particle, comprising the expression of the fusion protein according to the invention in eukaryotic cells, preferably in plant cells. Preferably, the developed process comprises the expression, in a plant cell, of the fusion proteins according to the invention. After their synthesis in the endoplasmic reticulum and their transport into the endomembrane secretion system of In plant cells, these fusion proteins have the ability to form vesicles when integrated into the plasma membrane. This process is identical to the budding of a virus onto the surface of the cells it infects. One of the major advantages of this technology is its simplicity, since after expression of the fusion proteins and extraction, the VLPs bearing the allergen of interest on their surface are preferably purified in two steps. These VLPs formed in planta exhibit a constant density of allergens or allergen fragments on their surface. The product quality is therefore easily standardized and its composition is consistent. The type I or II transmembrane fusion protein according to the invention comprises the following fragments successively: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a coiled-coil domain (or oligomerization sequence) that does not originate from a virus; and d) a plasma membrane anchoring domain consisting of a transmembrane segment and a cytosolic segment, preferably a plasma membrane anchoring domain at least part of which is typical of lipid rafts. A "fusion protein" is defined as a protein comprising the various fragments b) to d), and optionally a), these fragments being of different origins. In other words, fragments b) to d), and optionally a), are never found fused as such in nature. By "successively," it is understood that fragments a) to d) (or b) to d)) are present in the order a)-b)-c)-d) (or b)-c)-d) or d)-c)-b)). These different fragments can be directly fused to one another, or fused to one another via one or more linker(s). Preferably, the fusion protein according to the invention comprises a linker present between sequences b) and c), and / or between sequences c) and d). The fusion protein initially contains fragments a) to d): the presence of the signal peptide allows for the correct targeting of said protein to the endoplasmic reticulum. The signal peptide is then cleaved. Thus, during budding and the formation of VLPs according to the invention, the fusion protein no longer contains the signal peptide a), but only fragments b) to d). Consequently, the VLPs according to the invention do not contain the signal peptide a). In contrast, the description of fragments b) to d) that follows is applicable to the VLPs. A membrane-anchored type I transmembrane protein is defined as a transmembrane protein whose N-terminus is extracellular and whose C-terminus is cytosolic. Therefore, a type I transmembrane protein comprises, from the N-terminus to the C-terminus, optionally the signal peptide (a), then the protein or peptide of interest (b), then the coiled-coil domain (c), and finally the anchoring domain (d). A membrane-anchored type II transmembrane protein is defined as a transmembrane protein whose C-terminus is extracellular and whose N-terminus is cytosolic. Therefore, a type II transmembrane protein comprises, from the N-terminus to the C-terminus, the anchoring domain (d), then the coiled-coil domain (c), and finally the protein or peptide of interest (b). Preferably, the fusion protein according to the invention is a type I transmembrane protein. Peptide signal a) The signal peptide a) is any signal peptide recognized by a eukaryotic cell. Preferably, the signal peptide is chosen from the natural signal peptide of pectate lyase and the signal peptide of tobacco chitinase. Preferably, the signal peptide is that of tobacco chitinase, with sequence SEQ ID NO: 21. Protein or peptide of interest b) The protein or peptide of interest usable according to the invention may be any sequence of amino acids that is of interest in therapy or prophylaxis. The protein or peptide of interest usable according to the invention may be any sequence of amino acids which would benefit from being wholly or partially exposed on the surface of a virus-like particle, and capable of being recognized by immune cells, and / or triggering a biological reaction. By "protein of interest" we mean a sequence having at least 51 amino acids, preferably at least 100, preferably at least 200. By "peptide of interest" we mean a sequence comprising 2 to 50 amino acids, preferably 5 to 45 amino acids. The protein or peptide of interest usable according to the invention is preferably chosen from: • allergens and their fragments. The major application of a virus-like particle containing such a protein or peptide is immunotherapy. • Viral proteins and their fragments. The main purpose of a virus-like particle containing such a protein or peptide is vaccination. • cell surface proteins and their fragments. The main benefit of a virus-like particle containing such a protein or peptide may be, in particular, to restore immune activity, • proteins and peptides accumulated in chronic or neurodegenerative diseases, • proteins and peptides involved in hypertension (such as angiotensinogen, angiotensin I and angiotensin II), • immunoglobulins, their fragments (such as Fab) and their derivatives (such as scFv), « cytokines and their fragments, and • hormones and their fragments. Preferably, the protein or peptide of interest usable according to the invention is an allergen. Preferably, it is chosen from among the allergens responsible for respiratory allergies from house dust mites, such as Dermatophagoides farinae, Dermatophagoides pteronyssinus or Euroglyphus manei, storage mite allergens such as Blomia tropicalis, Acarus siro type mite allergens (formerly Tyroglyphus farinae), cockroach allergens, tree or grass pollen allergens, animal allergens (cat, dog, horse), mold allergens, allergens responsible for contact allergies such as those from rubber tree latex or allergens responsible for food allergies (milk, egg, fish, fruit). Among the allergens of Dermatophagoides farinae, we can mention Der f 10, Der fil, Der f 13, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18, Der f 2, Der f 2.0101, Der f 2.0102, Der f 2.0103, Der f 2.0104, Der f 2.0105, Der f 2.0106, Der f 2.0107, Der f 2.0108, Der f 2.0109, Der f 2.0110, Der f 2.0111, Der f 2.0112, Der f 2.0113, Der f 2.0114, Der f 2.0115, Der f 2.0116, Der f 2.0117, Der f 20, Der f 3, Der f 4, Der f 5, Der f 6, Der f 7, Der f 8, Der f 9 and Der f HSP70. Among the allergens of Dermatophagoides pteronyssinus, we can mention Der p 10, Der pli, Der p 14, Der p 15, Der p 18, Der p 2, Der p 2.0101, Der p 2.0102, Der p 2.0103, Der p 2.0104, Der p 2.0105, Der p 2.0106, Der p 2.0107. Animal allergens include those found in the seminal fluid, epithelium, milk, saliva, perspiration, and / or urine of these animals. The most common animals are dogs, cats, or horses. Among the cat (Felis domesticus) allergens, we can mention Fel d 1, Fel d 1.0101, Fel d 2, Fel d 2.0101, Fel d 3, Fel d 3.0101, Fel d 4, Fel d 4.0101, Fel d 5, Fel d 5.0101, Fel d 6, Fel d 6.0101, Fel d 7, Fel d 7.0101, Fel d 8, Fel d 8.0101, Fel d Hp, Fel d IgG or Fel d S100. Among dog allergens (Canis familiaris), we can cite Can f 1, Can f 1.0101, Can f 2, Can f 2.0101, Can f 3, Can f 3.0101, Can f 4, Can f 4.0101, Can f 5, Can f 5.0101, Can f 6, Can f 6.0101, Can f 7, Can f 7.0101, Can f 8, Can f Feldl-like, Can f Homs2-like, Can f Phosvitin or Can f TCTP. Among the horse (Equus caballus) allergens, we can mention Equ c 1, Equ c 1.0101, Equ c 2, Equ c 2.0101, Equ c 2.0102, Equ c 3, Equ c 3.0101, Equ c 4, Equ c 4.0101, Equ c PRVB, Equ c 10, Equ cil, Equ c 12, Equ c 8, Equ c 9, Equ c ALA or Equ c BLG. The sequences of these allergens are known, notably in the Uniprot database. Preferably, the protein or peptide of interest usable according to the invention is an allergen of sequence SEQ ID NO: 22 (mature sequence of Der p 2) or SEQ ID NO: 32 (sequence of the CH1 chain of Γ feline allergen Fel d 1). Preferably, the protein or peptide of interest usable according to the invention is a viral protein or one of its fragments. Among viral proteins, examples include the envelope proteins of the Zika virus, as well as influenza virus proteins such as hemagglutinins and neuraminidases. Preferably, the protein or peptide of interest usable according to the invention is the HAI chain of hemagglutinin with sequence SEQ ID NO: 34 or the Zika virus envelope protein with sequence SEQ ID NO: 35. Preferably, the protein or peptide of interest usable according to the invention is a cell surface protein or one of its fragments. Among surface proteins, tumor surface antigens are a notable example. Such proteins and their fragments are used, in particular, to restore immune activity, for example in tumor treatment. Preferably, the protein or peptide of interest usable according to the invention is a protein accumulated in neurodegenerative or chronic diseases. Among the peptides accumulated in chronic diseases, we can mention in particular the amyloid β peptide involved in Alzheimer's disease, the alpha-synuclein protein involved in Parkinson's disease, as well as the CD 20, TNF-alpha or HLA (human leukocyte antigen) proteins involved in rheumatoid arthritis. coiled-coil domain (or oligomerization sequence) c) The coiled-coil domain, or oligomerization sequence, comprises several sense or antisense alpha helix motifs that are parallel to each other and form an organized matrix with several well-characterized biological functions. These domains are ubiquitous and are found as specific domains in many types of proteins in most organisms. Coiled-coil domains from different sources can assemble to form structures ranging from dimers to heptamers; some coiled-coil domains will adopt different levels of polymerization depending on point mutations in their amino acid sequence. A coiled-coil domain is typically made up of a repeating motif of 7 amino acids, of the type "hxxhcxc", where "h" is a hydrophobic amino acid, "c" is a charged amino acid, and "x" is any amino acid. The coiled-coil domain usable according to the invention does not originate from a virus; it is not viral. Among the coiled-coil domains that can be used according to the invention, preferably those derived from cortexillin, vimentin, tetrabrachion, golgins, proteins of the superfamily of "Soluble N-ethylmaleimide-sensitive factor (NSF) Attachment protein REceptor" or SNARE, or transcription factors such as GCN4 or one of its variants, such as GCN4-pLI or GCN4-pII. Preferably, the coiled-coil domain is that derived from the transcription factor GCN4, GCN4-pLI or GCN4-pII. Preferably, the coiled-coil domain is chosen from SEQ ID NO: 24 (GCNA-pII trimerization sequence of yeast GCN4 transcription factor), SEQ ID NO: 27 (GCN4-pLI tetramerization sequence of yeast GCN4 transcription factor), SEQ ID NO: 28 (GCN4-pAA heptamerization sequence of yeast GCN4 transcription factor), SEQ ID NO: 29 (IZN4 glycosylated oligomerization sequence of the factor transcription GCN4 of yeast), SEQ ID NO: 33 (synthetic sequence mimicking a coiled-coil) and SEQ ID NO: 30 (SNARE oligomerization sequence). d) Anchoring domain in the plasma membrane (or transmembrane domain) The transmembrane domain is a short sequence of lipophilic amino acids that interacts with specific lipids of plasma membrane components. Preferably, the plasma membrane includes at least a typical portion of lipid rafts. These anchoring domains are common (though not via a consensus sequence) to viral surface proteins, as well as to proteins that are naturally integrated into the membrane of living cells. Each transmembrane domain participates in the curvature and budding of the plasma membrane. Among the anchoring domains that can be used according to the invention, those derived from the proteins listed in Table 1 A are preferred: Tableau 1A : protéines transmembranaires Nombre de Protéines transmembranaires (TM) Exemples (références Uniprot) domaines TM Leucine-rich repeat receptor-like protein Q9M7A8 kinase NtTMKl 1 Cavéoline Q03135 1 BRI 1 -associated receptor kinase 1 (BAK1) Q94F62 1 Q9LDG0, Q9ZT08, Q8LD58, Q7XHW7, Receptor kinases Q8H811, Q9SUQ3, Q5ZBN0 i Q6KC54, Q6EE26, Q5EDD1, P28582, Calcium-dependent protein kinases Q9ARI5, Q7XZK4, Q8GSB1, Q9FWF0, Q94KH6 NtRac2 / NTGP3 Q9ZRD2 1 ARFl-like GTP-binding protein Q9M7P4 1 Stomatine Q93VP6, Q60634 1 Ascorbate péroxidase Q8W4V7 1 LAT 043561 1 VIP36 P49256 1 Elicitor-inducible protein (EIG-J7) Q9FXT1 1 Chaperone Hsp90-2 Q6UJX5 1 Syntaxine Q9SF29, Q9SRV7, Q9ZSD5 1 Phragmoplastine Q9SMB6 1 Fasciclin-like arabinogalactan protein 8 022126 (precursor) i Ras-related protein RAB8-3 Q8W3J3 2 Among the anchoring domains that can be used according to the invention, those derived from the viral envelope proteins listed in Table 1B below will preferably be mentioned: Family Examples Envelope proteins the dengue virus the yellow fever virus the St. Louis encephalitis virus Flavivirus, the Japanese encephalitis virus, Protein E West Nile virus the Zika virus the BYD virus (identified in China in 2010, which affects ducks) Togavirus Sindbis GP virus eastern equine encephalitis virus western equine encephalitis virus Ross River's virus Le viras O’nyong’nyong oncoviras (5 genera) Lentiviral retroviruses (such as HIV) GP41 / 120 Foamviruses Canine coronaviruses Feline coronaviruses swine communicable gastroenteritis viruses S protein and HE protein porcine respiratory viruses Coronavirus (hemagglutinin and bovine coronavirus esterase) human coronaviruses murine hepatitis viruses Rat silodacryonitis viruses Ebola Viruses Filovirus glycoprotein The tours of Marburg the turns of rage Viral haemorrhagic fever Rhabdovirus GP VSV-EBOV beetroot disease viral delivery Dugbe viras Bunyaviras Gn / Gc Rift Valley Fever Tomato tan disease viruses Hemagglutinin / Orthomyxovirus Influenza virus (myxoinfluenza) neuraminidase mumps virus the Sendai Viras the SV5 viras Newcastle disease virus Measles virus fusion protein F Paramyxovirus Attachment protein distemper virus (ΗΝ,Η or G) rinderpest virus respiratory syncytial virus (RSV) bovine respiratory syncytial virus (BRSV) parainfluenza Lassa fever Argentine hemorrhagic fever Arenaviras Bolivian hemorrhagic fever GP Brazilian hemorrhagic fever Venezuelan hemorrhagic fever Hepadnavirus hepatitis B virus (HBV) GPL, S or M Herpes simplex viras (HSv) Glycoprotein Herpesvirus EHV-1 (equine herpes virus) g D,gB, gH, gL, gC genital herpes virus Orthopoxviras; species type: Vaccinia viras; Diseases: cowpox, vaccinia, smallpox Parapoxviras; species type: Orf viras Avipoxviras; species type: Fowlpox viras Poxviras Capripoxviras; species type: Sheeppox viras GP41 / 120 Leporipoxviras; species type: Myxoma viras Suipoxviras; species type: Swinepox viras Molluscipoxvirus; species type: Molluscum contagiosum viras Yatapoxviras; species type: Yaba monkey tumor virus Preferably, the anchoring domain usable according to the invention is chosen from the anchoring sequence of the hemagglutinin H5 of the influenza H5N1 virus (SEQ ID NO: 26) and the anchoring sequence of the PDLP1 protein (A0A0D3D8S3) (SEQ ID NO: 31). Linkers Preferably, the fusion protein according to the invention comprises a linker present between fragments b) and c), and / or between fragments c) and d). Linkers are short amino acid sequences (2 to 10 amino acids, preferably 2 to 6) that create a flexible arm. They can be useful for creating flexible spacing between the anchoring domain and the coiled-coil domain if too close a proximity between the two domains interferes with proper assembly. They are not necessary under conditions where a direct link between the two domains (anchoring and coiled-coil) does not interfere with the overall three-dimensional structure of the fusion protein. Preferably, the linker is a sequence of type -(GGGS)n-, where n is an integer. Preferably, the linker is chosen from SEQ ID NO: 23 (n=2) and SEQ ID NO: 25 (n=1). Thus, preferably, the fusion protein according to the invention is such that: a) the optional signal peptide has the sequence SEQ ID NO: 21; b) The protein or peptide of interest is chosen from: • allergens and their fragments, • viral proteins and their fragments, • cell surface proteins and their fragments, • proteins and peptides accumulated in chronic or neurodegenerative diseases, • proteins and peptides involved in hypertension, • Immunoglobulins and their fragments, • cytokines and their fragments, and • hormones and their fragments; 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 H5 hemagglutinin anchoring sequence of the influenza H5N1 virus (SEQ ID NO: 26) and the PDLPl protein anchoring sequence (SEQ ID NO: 31). Thus, preferably, the fusion protein according to the invention comprises, preferably consists of, a sequence selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20. Similarly, preferably, the VLP according to the invention comprises: - an envelope consisting of a plasma membrane, at least part of which has a composition typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising successively the following fragments: b) the protein or peptide of interest selected from: • allergens and their fragments, • viral proteins and their fragments, "Cell surface proteins and their fragments, • proteins and peptides accumulated in chronic or neurodegenerative diseases, • proteins and peptides involved in hypertension, • Immunoglobulins and their fragments, • cytokines and their fragments, and "hormones and their fragments; c) the coiled-coil domain 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 chosen from the H5 influenza virus H5 hemagglutinin anchoring sequence (SEQ ID NO: 26) and the PDLP1 protein anchoring sequence (SEQ ID NO: 31), fragments b) and c) being exposed outside the VLP. The present invention also relates to the nucleic acid (or nucleotide sequence) encoding the fusion protein. Once the nucleotide sequence has been obtained, it is placed into an expression vector using conventional methods. The present invention therefore also relates to a vector comprising the nucleic acid encoding the fusion protein. The selection of a suitable expression vector will depend on the method of introducing the expression vector into host cells. A typical expression vector contains eukaryotic DNA elements such as a transcription initiation sequence for the exogenous gene, such as a promoter, and DNA elements that control transcript processing, such as termination / polyadenylation sequences and an expression cassette allowing the expression of a silencing inhibitor. It also contains sequences such as t-DNA, which are necessary for the integration of a DNA fragment into the plant or plant cell. Preferably, the expression vector includes: - at least one nucleotide sequence encoding the fusion protein, preferably functionally linked to a strong promoter, preferably a 35S promoter; - an expression cassette allowing the expression of a silencing inhibitor, preferably pl9; and DNA elements that control transcript processing, such as termination / polyadenylation sequences, preferably the Tnos sequence (nopaline synthase termination sequence). Preferably, the expression vector is pAGOL. The promoters used to control fusion protein expression are strong promoters and can be plant gene promoters, such as the ubiquitin promoter, the small subunit ribulose-1,5-bis-phosphate carboxylase promoter, Agrobacterium tumefaciens promoters, nopaline synthase and octopine synthase promoters, or viral promoters such as 19S and 35S of the cauliflower mosaic virus (CaMV). Preferably, the strong promoter is 35S. The present invention also relates to a host cell comprising at least one nucleic acid encoding the fusion protein. The host cell may be a plant cell. The present invention also relates to a method for producing virus-like particles (VLP), comprising the expression of the nucleic acid encoding the fusion protein in eukaryotic cells, preferably plant cells. General methods for plant cultivation, as well as processes for introducing expression vectors into plant tissue, are available to those skilled in the art. They are varied and depend on the plant selected. Preferably, the plants will be cultivated using techniques specific to the Allergopur platform. This recombinant protein production process is described in application FR1255510 and comprises a first stage of plant cultivation, either aeroponically or hydroponically under LED lighting. After this first stage, agroinfiltration of the plants is carried out under vacuum using agrobacteria containing a DNA fragment encoding the fusion protein according to the invention. This agroinfiltration stage can be implemented by any means that creates a vacuum. Preferably, in the process used according to the invention, it is carried out under vacuum using the Venturi effect.Among the agrobacteria that can be used according to the invention, the following strains can preferably be mentioned: LBA4404, GV3101, EHA 101 / 105 or C58. Once the agroinfiltration step is completed, the plants are returned to cultivation, typically for 3 to 6 days, ideally with frequent misting during the first 6 hours of cultivation following agroinfiltration. Then, the VLPs are extracted and purified as described below. VLP extraction can be carried out by enzymatic extraction. This method is an adaptation of the method described in particular in application WO2014 / 153674. Preferably, enzymatic extraction of VLP is carried out by the following steps: Vacuum infiltration (particularly as described above for Γ agroinfiltration) of the aerial parts of plants (i.e., the leaves) in an enzymatic solution containing pecto-cellulosic enzymes that do not exhibit proteolytic activity. Preferably, a mixture of pectinases and cellulases formulated at 4% in a medium comprising 50 mM sodium citrate pH 5.2, 0.5 M NaCl, and 0.04% metabisulfite. Preferably, the macerozyme is formulated at 0.5% in a medium comprising 50mM sodium citrate, pH 5.2, 0.5M NaCl and 0.04% metabisulfite, the leaves are then collected and incubated in the enzyme solution, the mixture is placed under agitation on an orbital shaker between 20 and 30 rpm at room temperature (i.e. about 20-23°C) for a period of between 30 minutes and 2 hours, The digestate is then filtered, preferably through a 2-3 mm mesh followed by a 250 μm mesh, and then possibly centrifuged continuously (for example, at 1000 x g for 2-5 minutes). The supernatant is collected for tangential flow filtration. Such a process is illustrated in Figure 7. Preferably, the invention also relates to a method for producing virus-like particles (VLPs) according to the invention in a plant cell or plant, comprising the following steps: a) transformation of Agrobacterium with an expression vector comprising a nucleotide sequence encoding a fusion protein according to the invention functionally linked to a strong promoter; and b) transfection of the plant cell or plant with the agrobacteria obtained in step a). The transformation of step a) is typically done by methods known from the prior art, for example by thermal shocks with successive passages to 4°C, -80°C and 37°C. The transfection in step b) preferably includes the following steps: bl) culture of the plant cell or plant, in aeroponics or hydroponics, and under LED lighting, preferably for four to six weeks, b2) agroinfiltration of the plant cell or plant obtained in bl) under vacuum, by the agrobacteria obtained in step a). This agroinfiltration step is preferably carried out under vacuum by Venturi effect. b3) re-culture of the plant cell or plant obtained in b2), typically for 3 to 6 days to obtain the pseudo-viral particles. Finally, the VLPs obtained are extracted and purified, notably by enzymatic extraction as described above. The present invention also relates to a pseudo-viral particle comprising: - an envelope consisting of a plasma membrane, at least part of which has a composition typical of lipid rafts; and - at least one fusion protein according to the invention without signal peptide a), anchored in said membrane. Such a virus-like particle (VLP) includes: - an envelope consisting of a plasma membrane, at least part of which is typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising successively the following fragments: b) a protein or peptide of interest; c) a coiled-coil domain (or oligomerization sequence) that does not originate from a virus; and d) a plasma membrane anchoring domain, consisting of a transmembrane segment and a cytosolic segment, preferably a plasma membrane anchoring domain at least part of which is typical of lipid rafts, fragments b) and c) being exposed outside the VLP. The term "typical plasma membrane component of lipid rafts" refers to a phospholipid bilayer (i.e., plasma membrane) found in the microdomains of lipid rafts. Such a bilayer is rich in cholesterol and phospholipids, preferably phosphatidylcholine and phosphatidylethanolamine, and sphingolipids, such as sphingomyelin, but poor in docosahexaenoic acid. Furthermore, it has a low density and is insoluble in mild detergents (e.g., polysorbates). The unique characteristics of such a VLP are undetectable immunogenicity (except for the protein or peptide of interest b), its ability to assemble spontaneously, its specific lipid content, preferably typical of lipid rafts, the fact that its membrane is very poor in host cell membrane proteins, its ability to be expressed at high yields in many types of eukaryotic cells (yeasts, insects or plants) and its ease of preparation. The VLP according to the invention can be used in therapy. It can be used as a drug. It can also be used in allergen immunotherapy (AIT). The sequences listed in this application are summarized in the table below: The following examples illustrate, but are not intended to limit the scope of the invention. It will be obvious to those skilled in the art that variations and modifications are possible and fall within the scope and spirit of the invention. The figure legends are as follows Figure 1: Schematic representation of the different expression cassettes allowing the production of an allergen associated with an oligomerization sequence and an anchoring sequence in the plasma membrane, preferably at the level of lipid rafts A) Optimized cDNA encoding the Der p2 sequence (DP2, SEQ ID NO: 22), preferably harmonized, is associated with 1) 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, B - GCN4-PLI / tetrameric form, C - GCN4-IZN4 / glycosylated form, E - GCN4-pAA / heptameric form, D) or from any other protein family exhibiting 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 H5 influenza, B to I) or from type I protein anchored in lipid rafts (“lipid raft”) (J). B) Diagram representing the structure of the oligomerization or coiled-coil sequence. This sequence consists of a repeated motif of 7 amino acids, of the type "hxxhcxc", where "h" is a hydrophobic amino acid, "c" is a charged amino acid, and "x" is any amino acid. Figure 2: The AllergoPur platform used for the expression and production of the different forms of VLP Figure 3: Protein production described in Figure 1A Proteins extracted from plants transfected under vacuum for the expression of DP2 (lane 1), DP2-Tri (lanes 2-3), DP2-Tetra (lanes 4-5), or FDl-Tri (lanes 6-7) proteins were analyzed by immunodetection using an antibody directed against Pallergen Der p2 or Fel dl. Immunodetection analysis reveals the specific production of proteins whose molecular mass corresponds to the expected mass. Two Agrobacterium clones (Cl. 1 and C1.2) were analyzed for each construct. Figure 4: Purification and Characterization of VLPs bearing allergens by size exclusion chromatography Protein extracts from leaves producing DP2-Tri (panel D), DP2-Tetra (panel E), soluble DP2 (panel F), FDl-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 content of Total protein soluble content of each fraction was evaluated by spectrometry (panel A) and Coomassie blue staining after separation by SDS-PAGE (panel B). The allergen content of the eluted fractions was revealed by immunological detection using anti-Der p 2 or anti-Fel d 1 antibodies. Protein extracts from leaves producing hemagglutinin as VLP of H5N1 (panel C) were separated by gel filtration on a calibrated S-500 / HR column and are used as controls. Figure 5: Characterization of VLPs carrying isolated allergens by electron microscopy and negative staining VLPs carrying allergens have a morphology and size very similar to those described for influenza virions. The bar represents 50 nm. Figure 6: Reactivity of allergens produced as VLP with sera from patients allergic to Der p2 Proteins extracted from plants transfected under vacuum for the expression of DP2 (lane 1), DP2-Tri (lane 2), and DP2-Tetra (lane 3) proteins were analyzed by immunodetection using sera from patients allergic to Der p2. Immunodetection analysis demonstrated the recognition of allergens carried by VLPs by IgE antibodies from patient sera. Figure 7: Large-scale VLP production process Figure 8: Structure of VLPs and fusion proteins assembled according to the invention A) Structure of a VLP according to the invention. The VLP consists of a plasma membrane envelope, to which the fusion proteins according to the invention are attached. The protein or peptide of interest (for example, the allergen) is thus exposed on its surface. 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 here the allergen, A) on the surface of the VLP. Figure 9: VLP-conjugated antigens exhibit a very strong ability immunogenic but not allergenic Panel A: Evaluation of airway hyperreactivity induced by the allergen Der p 2 using the Flexivent method. Mice (n = 10 / group) were sensitized to the allergen der p 2 in either the solubility form (DP2-Alum) or the VLP form (DP2-VLP / alum and DP2-VLP / saline) and challenged with a dust mite extract. Twenty-four hours after the last challenge, airway hyperreactivity to inhaled methacholine was determined using the Flexivent method. Pulmonary reactivity triggered in the presence of the allergen in VLP form was comparable to that of control mice. Panel B: Enumeration of inflammatory cells in the airways, collected by bronchoalveolar lavage (BAL). Mice (n = 10 / group) were sensitized to the allergen der p 2 in its soluby form (DP2-Alum) or in its VLP form (DP2-VLP / alum and DP2-VLP / saline) and challenged with a dust mite extract. Twenty-four hours after the last challenge, BAL cells were collected and counted (eosinophils; neutrophils; macrophages; lymphocytes). Neutrophils were predominant in mice that received the allergen der p 2 in its soluby form. Panel C: Der p 2 specific IgG assay. Mice (n = 10 / group) were sensitized to the allergen der p 2 in soluble form (DP2-Alum) or in VLP form (DP2-VLP / alum and DP2-VLP / saline) and challenged with a dust mite extract. Twenty-four hours after the last challenge, IgG levels were measured in BAL fluid and in serum from blood collected by cardiac puncture. Mice receiving DP2-VLP injections with or without adjuvant showed IgG levels a thousand times higher than mice receiving soluble Der p 2. EXAMPLES: Example 1: Molecular design and gene synthesis cDNAs are synthesized by optimizing and then harmonizing codon usage for recognition by the plant system. In the context of this invention, the preferred optimization is for expression in Nicotiana benthamiana. The constructions are illustrated in Figure 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 addressing 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 allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor, and the anchor sequence of the H5 hemagglutinin from the influenza H5N1 virus (SEQ ID NO: 3). The corresponding protein has the sequence SEQ ID NO: 4. C: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), the GCN4-pLI tetramerization sequence of the yeast GCN4 transcription factor, and the H5 hemagglutinin anchor sequence of the influenza H5N1 virus (SEQ ID NO: 5). The corresponding protein has the sequence SEQ ID NO: 6. D: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), the GCN4-pAA heptamerization sequence of the yeast GCN4 transcription factor, and the H5 hemagglutinin anchor sequence of the influenza H5N1 virus (SEQ ID NO: 7). The corresponding protein has the sequence SEQ ID NO: 8. E: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the glycosylated oligomerization sequence IZN4 of the yeast transcription factor GCN4, and to the anchor sequence of hemagglutinin H5 from the influenza H5N1 virus (SEQ ID NO: 9). The corresponding protein has the sequence SEQ ID NO: 10. F: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to a synthetic sequence mimicking a coiled-coil, and to the anchor sequence of hemagglutinin H5 from the influenza H5N1 virus (SEQ ID NO: 11). The corresponding protein has the sequence SEQ ID NO: 12. G: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to a SNARE oligomerization sequence, and to the anchoring sequence of the H5 hemagglutinin from the influenza H5N1 virus (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) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor, and the hemagglutinin anchor sequence H5 of the influenza H5N1 virus (SEQ ID NO: 15). The corresponding protein has the sequence SEQ ID NO: 16. I: cDNA encoding the CH1 chain of the Fel dl allergen (SEQ ID NO: 32) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor, and to the H5 hemagglutinin anchor sequence of the influenza H5N1 virus (SEQ ID NO: 17). The corresponding protein has the sequence SEQ ID NO: 18. J: cDNA encoding the mature form of the allergen Der p2 (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), the GCN4-pII trimerization sequence of the yeast GCN4 transcription factor, and the PDLP1 protein anchor sequence (A0A0D3D8S3) from lipid rafts (SEQ ID NO: 19). The corresponding protein has the sequence SEQ ID NO: 20. Example 2: Plasmid preparation Xba I / kpn I and Sal I / Sac I restriction sites are integrated at the 5' and 3' ends of the cDNA, respectively, during synthesis. These sites are then used to clone the cDNAs into the pAGOl expression binary vector. The cDNAs are cloned upstream of a 35S promoter (35S) and downstream of a nopaline synthase termination sequence (tnos). The pAGOl vector also contains an expression cassette that allows simultaneous expression of the silencing inhibitor pl9 of the recombinant protein to increase production yields. The vectors are then used to transform the Agrobacterium tumefaciens strain LBA4404. Example 3: Transient expression of Der p 2 produced as VLP in Nicotiana benthamiana leaves – Using the AllergoPur platform For transient expression production, Agrobacterium tumefaciens LBA4404 is used to transfer a Der p2-encoding cDNA associated with an oligomerization sequence and an anchoring sequence, without the gene of interest being integrated into the plant cell genome. This is referred to as transfection, not transgenesis. The plants are grown hydroponically in a nutrient medium (GHE, Floragrow, Floramicro, Florabloom, 10 mL / 15 mL / 5 mL per 10 L of reverse osmosis water) and under LED lighting. Agrobacterium is transferred into leaf tissue by agroinfiltration using two methods. For the production of small batches of proteases intended for prototype screening, Agrobacterium is manually injected using a syringe applied against the underside of the leaf. Leaf discs taken from the leaves 4 to 6 days after agroinfiltration are used to analyze the different VLP prototypes. This screening step allows for the selection of the expression vector that will be used to obtain the Der p 2 membrane-anchored allergen of optimal quality. The same method is used for large-scale production, but in this case, agroinfiltration is carried out under vacuum in chambers containing several liters of Agrobacterium culture, where several dozen plants are infiltrated simultaneously.These plants are then put back into culture for 3-6 days before the purification of the VLPs carrying the allergen (Figure 2). Example 4: Production of VLPs carrying the allergen Der p2. The expression of the proteins produced in Example 3, as well as the yields, are analyzed by Western blotting and ELISA, respectively. The results are illustrated for 3 allergens produced as VLPs (DP2-tri, DP2-tetra, and FDl-tri) (Figure 3). Example 5: Evaluation of VLP formation / size distribution analysis An analysis of the size distribution of the structures bearing the allergens Der p2 (DP2-tri / DP2-tetra) or Fel dl (FDl-Tri) was performed. After vacuum infiltration 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 a high-resolution S-500 (HR) column (GE Healthcare Bio-Science Corporation). The elution fractions were monitored for their total protein content and their VLP-allergen content by Western blotting with anti-allergen antibodies. For all the extracts analyzed, the concentration of soluble proteins in the eluate reached a maximum in fractions 14–16 (Figure 4).On the other hand, Western blotting analysis highlights an accumulation of allergens in fractions 6 and 7 (i.e. before the elution of Dextran Blue used as a marker) which shows the association of allergens with very high molecular weight structures in the 2 MDa zone. 32 mL columns of Sephacryl S-500 / HR (GE Healthcare Bio-Science Corporation) were equilibrated with 50 mM PBS, pH 7.4, and 150 mM NaCl. Extract samples of Total protein 1.5 mL volumes were loaded and then eluted with the equilibration buffer. Twenty-four 1.5 mL elution fractions were collected and analyzed for protein content by absorbance spectrophotometry at 280 nm. The proteins from each fraction were concentrated by acetone precipitation and then resuspended in the same volume of elution buffer before analysis by SDS-PAGE and Western blotting. The elution profiles of Dextran Blue 2000 and soluble proteins were compared for each chromatogram to ensure the reproducibility of this separation technique. Example 6: Morphology of VLPs - Analysis by electron microscopy Transmission electron microscopy of the purified product (from the production in Example 3 and then purified) indicates that the high molecular weight structures isolated by screening chromatography are VLPs to which allergens are bound. Both in size and morphology, including a phospholipid membrane covered with spikes, these VLPs closely resemble influenza virions (Figure 5). Example 7: Production of allergens or hypoallergens carried by VLPs Coupling an allergen to a VLP significantly reduces its in vivo reactivity with patient sera IgE. However, the use of hypoallergenic agents further reduces IgE reactivity and thus the risk of anaphylactic reaction. The reduction in reactivity to a hypoallergenic form of the allergen Der p 2 carried by VLPs is illustrated in Figure 6. Example 8: VLP Production The detailed production process of VLPs, up to their purification (as described in Example 6), is illustrated in Figure 7. Example 9: Immunogenic potential of VLPs compared to soluble allergens The presentation of an antigen in a highly ordered and repetitive network normally causes strong immune responses, whereas the same antigen presented as a monomer is non-immunogenic. To compare the immune response to the allergen when presented as a highly ordered network, mice were immunized with the allergen Der p2 as a soluble monomer or as a VLP-derived protein. IgG titers against the allergen Der p2 were determined by ELISA. Protocol The protocol is illustrated as follows: Sacrifice Analyses Analysis after the sacrifice: "Weight loss and behavior change" • Pulmonary function (flexiVent, plesthismography) Serological response to the allergen (serum IgG, IgE, blot) Basophil activation test Histopathology and other serological results (e.g., IgGl isoforms) in later stages This study demonstrated that VLPs coupled to the allergen Der p 2 do not trigger bronchial hyperreactivity in mice, unlike soluble Der p 2 (see Figure 9, Panel A). Furthermore, VLPs bound to Der p 2 trigger a systemic Th1-type response with neutrophil activation (see Figure 9, Panels B and C). Example 10: Desensitization / Vaccination using VLPs The key parameters of an effective vaccine are: rapid induction of a high antibody titer in the absence of adjuvants and the absence of major side effects. Protocol The protocol is illustrated as follows: Analyses Bibliographical references The references cited in this application are as follows: 1-Noon L. Prophylactic inoculation against hay fever Lancet 1911; 1: 1572-3 2- Freeman J. Further observation on the treatment of hay fever by hypodermic inoculations of pollen vaccine. Lancet 191 1;2:814-817 3- Henmar et al Clin Exp Immunol 2008; 153: 316-323 4- Valenta et al J Allergy Clin Immunol 2007; 119:826-830 5-Larche M. J Allergy Clin Immunol 2007;119:906-909 6-Patel D. et al J Allergy Clin Immunol 2013;131:103-109 8- Chen et al Allergy 2012;67:609-621 9- undig et al J Allergy Clin Immunol 2006; 117: 1470-1476 10- BachmannMF, Jennings GT Phil Trans R Soc B Biol SCI 2011;366:2815-2822 11-Klimek et al Am J Rhinol Allergy 2013;27:206-212 12- Henmar et al Clin Exp Immunol 2008;153:316-323. 13- Jegerlehner et al Eur J Immunol2002;32:3305-3314. 14- D'aoust et al Plant Biotech J 2008; 6:930-940 15- Garland SM et al N Engl J Med 2007;356: 1928-1943 16-Paavonen et al Lancet.2007;369:2161-2170 17- Schmitz et al J.Exp.Med. 2009;206:1941-1955 18- Undig et al. J Allerg Clin Immunol; 2006; 117: 1470-1476 19- Cielens et al FEBS Letters 2000;482:261-264

Claims

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

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

3. A pseudo-viral particle according to any one of claims 1 to 2, wherein: b) The protein or peptide of interest is chosen from: • allergens and their fragments, • cell surface proteins and their fragments, • proteins and peptides accumulated in chronic or neurodegenerative diseases, • proteins and peptides involved in hypertension, • Immunoglobulins and their fragments, • cytokines and their fragments, and • hormones and their fragments; 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 H5 hemagglutinin anchoring sequence of the influenza H5N1 virus (SEQ ID NO: 26) and the PDLP1 protein anchoring sequence (SEQ ID NO: 31).

4. Pseudoviral particle according to any one of claims 1 to 3, wherein the protein or peptide of interest is selected from allergens and their fragments.

5. Type I or II transmembrane fusion protein comprising successively the following fragments: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a coiled-coil domain or oligomerization sequence, which is not of viral origin; and d) a plasma membrane anchoring domain, consisting of a transmembrane segment and a cytosolic segment, preferably a plasma membrane anchoring domain at least part of which is typical of lipid rafts.

6. Transmembrane fusion protein according to claim 5, wherein the protein or peptide of interest is selected from allergens and their fragments.

7. Nucleic acid encoding a fusion protein according to claim 5 or 6.

8. Host cell or vector comprising at least one nucleic acid according to claim 7.

9. Viral-like particle according to any one of claims 1 to 4 for its use in therapy.

10. Viral-like particle according to any one of claims 1 to 4 for its use in allergen immunotherapy.

11. Method for producing virus-like particles according to any one of claims 1 to 4, comprising the expression of nucleic acid according to claim 7 in eukaryotic cells.

12. A 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 Agrobacterium with an expression vector comprising a nucleic acid according to claim 5 functionally linked to a strong promoter; and b) transfection of plant cells with the Agrobacterium obtained in step a), said transfection comprising the following steps: bl) culture of plant cells, in aeroponics or hydroponics, and under LED lighting, preferably for four to six weeks in hydroponics, b2) agroinfiltration of the plant cells obtained in bl) under vacuum, by the agrobacteria obtained in step a), preferably carried out under vacuum by Venturi effect, b3) re-culturing the plant cells obtained in b2), typically for 3 to 6 days, to obtain the virus-like particles, then extraction of the pseudo-viral particles obtained and purification, notably by enzymatic extraction.

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