Virus-like particles and their uses

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 peptide of interest; c) a helical domain; d) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment. It also relates to virus-like particles obtained with this fusion protein.

Description

Virus-like particles and their uses

[0001] The present invention relates to a fusion protein comprising the following fragments successively: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a helical domain that does not originate from a virus; and (d) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring to a plasma membrane, of which at least one portion is typical of lipid rafts.

[0002] The present invention also relates to virus-like particles (VLPs) obtained with such fusion protein, said protein being anchored in its membrane.

[0003] Although allergen immunotherapy was described by Noon and Freeman (1, 2) more than 100 years ago, very little progress has been made in terms of desensitization, except for the method of administering treatments, with injection treatments gradually being replaced by sublingual desensitization. Thus, since January 2011, for example, in France, for grass pollen allergies, there was a desensitization in the form of a sublingual tablet. The arrival on the market of these desensitization tablets contributed to reducing the invasiveness of allergen immunotherapy, but did not increase its effectiveness. In fact, they are still natural allergenic extracts, which are low in concentration of allergens and not very effective. Petition 870190009246, dated 01 / 29 / 2019, page 58 / 125 2 / 47 representative of the diversity of allergens contained in the allergenic source, which are used for oral curative treatment.

[0004] However, over the last decade, new strategies have been proposed to increase the effectiveness and reduce the duration of allergen immunotherapy treatments.

[0005] These desensitization strategies are based on the use of: - of natural allergenic extracts modified to reduce their allergenicity while preserving their immunogenicity (Henmar et al.), - of native recombinant allergens or recombinant allergens modified to make them hypoallergenic (Valenta et al.), - of peptides corresponding to the epitopes of T cell allergens, in free form or in the form of a fusion with transporter proteins (Larche M., Patel D. et al., Chen et al.), - of adjuvants, or - of allergens fused to nanoparticles or virus-like particles (Kundig et al., Bachmann MF, Jennings GT, Henmar et al.).

[0006] However, strategies that make use of modified allergenic extracts still have limited effectiveness due to their poor representation of the diversity of allergens contained in the source.

[0007] Modified or unmodified recombinant allergens, or peptides, are weakly immunogenic in Petition 870190009246, dated 01 / 29 / 2019, page 59 / 125 3 / 47 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.

[0008] 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 do not contain genetic material and are therefore non-infectious and incapable of multiplying. On the other hand, they mimic the original structure of a virus, which allows them to be easily recognized by the immune system and to activate immunological memory very efficiently. VLP-based vaccines against hepatitis B, papillomavirus infections, or influenza (Garland et al., Paavonen et al., D'Aoust et al.) illustrate the effectiveness of antigen vaccines when they are presented to the immune system on the surface of VLPs or nanoparticles.

[0009] Thus, VLPs have the potential to be used as structures that present antigens and, in particular, allergens, which make it possible to induce a strong immune response in humans.

[00010] There are two main types of VLPs: those produced from viral capsid proteins (CP VLPs) and those produced from enveloped viruses (Env VLPs). The structure and composition of these two types differ substantially. CP VLPs are generally produced by producing recombinant protein from Petition 870190009246, dated 01 / 29 / 2019, page 60 / 125 4 / 47 capsid that self-assembles in host cells according to mechanisms similar to those of native viruses. The production of Env VLPs occurs when an envelope protein Env is synthesized and modified by the endomembrane system of a host cell, migrates to the lipid rafts of the plasma membrane, where it is concentrated and triggers the extracellular budding of the entire membrane / protein assembly. The resulting particle, the VLP, carries immunogenic epitopes of the Env protein on its surface.

[00011] The use of allergens in the form of virus-like particles appears to be an important requirement for the success of immunotherapy. The results obtained with VLPs fused to a peptide of the major mite allergen (Der p 1) or the major cat allergen (Fel d 1) 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 for protection against a type I allergic reaction.

[00012] However, the preparation of these VLPs is, unfortunately, extremely complex and comprises numerous steps, some of which are difficult to standardize. In particular, VLPs are produced, on the one hand, in E. coli, after expression of the Qbeta bacteriophage envelope protein. On the other hand, the allergen is expressed in recombinant form in E. coli, purified, then solubilized and purified again in several steps. Once these two constituents have been produced and purified, they are coupled in vitro. This production technique is obviously Petition 870190009246, dated 01 / 29 / 2019, page 61 / 125 5 / 47 very complex, very expensive and very difficult to standardize so that the final product can one day be available for the treatment of allergic patients.

[00013] There is therefore a need for a non-immunogenic and polyvalent structure that self-assembles in eukaryotic cells, is easy to synthesize, and can transport proteins or peptides on its surface. Such a structure could be used for the treatment of allergic patients, but also in other clinical contexts.

[00014] The applicant has now developed such a structure, which is self-assembling and allows for use in therapy.

[00015] In particular, the unique characteristics of such a structure are: - an undetectable immunogenicity of the structure as such; - its ability to self-assemble into oligomers, such as trimers or tetramers; - its membrane containing unique lipids, typical of lipid rafts; - its membrane has a low content of host cell membrane proteins; - its ability to be expressed with high yields in various types of eukaryotic cells (yeasts, insects or plants); and - its ease of preparation.

[00016] According to a first aspect, the invention relates to a type I or type II transmembrane fusion protein comprising the following fragments, Petition 870190009246, dated 01 / 29 / 2019, page 62 / 125 6 / 47 successively: a) optionally, a signal peptide; b) a protein or peptide of interest; c) a helical domain (or oligomerization sequence) that does not originate from a virus; and d) a domain for anchoring to the plasma membrane and more particularly to lipid rafts, consisting of a transmembrane segment and a cytosolic segment.

[00017] This fusion protein behaves like a viral surface protein when expressed on eukaryotic cells.

[00018] Without being linked to any theory, this protein is synthesized in the endoplasmic reticulum and then transported, through the Golgi complex, to the plasma membrane. Once it reaches specialized regions of the plasma membrane, preferably the lipid rafts, this transmembrane fusion protein causes the membrane to bend, which finally forms a bud that separates from the cell membrane and is released into the extracellular space. During budding, the protein or peptide of interest transported by the helical 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 on the surface.A virus-like particle (VLP), illustrated in Figure 8A, is thus obtained, comprising a plasma membrane whose composition is preferably typical of lipid rafts, in which the fusion proteins according to the invention are linked at the level of their anchoring domain and which exposes the... Petition 870190009246, dated 01 / 29 / 2019, page 63 / 125 7 / 47 protein or peptide of interest on its surface, in oligomerized form (due to the oligomerization sequence).

[00019] The structure of the fusion proteins assembled on the surface of the VLP is illustrated in figure 8B.

[00020] According to a second aspect, the invention consists of a virus-like particle (VLP) comprising: - an envelope consisting of a plasma membrane in which at least a portion is typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored to said membrane (i.e., said envelope), said fusion protein comprising the following fragments, successively: b) a protein or peptide of interest; c) a helical domain (or oligomerization sequence) that does not originate from a virus; and d) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring to a plasma membrane, of which at least a portion is typical of lipid rafts, the fragments b) and c) being exposed on the surface of the VLP.

[00021] In the VLP according to the invention, the fusion protein is anchored to the membrane (and therefore to the envelope) by means of its anchoring domain d).

[00022] The term “virus-like particle (or VLP)” Petition 870190009246, dated 01 / 29 / 2019, page 64 / 125 8 / 47 is intended to signify a nanoparticle consisting of a plasma membrane envelope in which one or more proteins are anchored, which contains no genetic material, is non-infectious and incapable of multiplying, and which self-assembles to mimic the original structure of a virus. The structure of a VLP is illustrated in Figure 8A: the membrane envelope comprises proteins that are anchored and exposed on its surface.

[00023] Such a virus-like particle according to the invention has the advantageous 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 has 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 IgGs while minimizing accessibility to basophils.

[00024] The virus-like particles according to the invention typically have a diameter between 120 and 200 nm.

[00025] According to a third aspect, the invention relates to a method for producing a virus-like particle, comprising the expression of the fusion protein according to the invention in eukaryotic cells, preferably in plant cells.

[00026] In fact, preferably, the method developed involves the expression, in a plant cell, Petition 870190009246, dated 01 / 29 / 2019, page 65 / 125 9 / 47 of the fusion proteins according to the invention. After their synthesis in the endoplasmic reticulum and their transport in the endomembrane secretory system of the plant cell, these fusion proteins have the ability to form vesicles when they are integrated into the plasma membrane. This process is identical to the budding of a virus on the surface of the cells it infects.

[00027] One of the main advantages of this technology is its simplicity, since, after the expression of the fusion and extraction proteins, the VLPs that carry the allergen of interest on their surface are preferably purified in two steps. These VLPs formed in planta have a constant density of allergens or allergen fragments on their surface. The quality of the product can be easily standardized and its composition is constant.

[00028] The type I or type 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 helical domain (or oligomerization sequence) that does not originate from a virus; and (d) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring to a plasma membrane, of which at least a portion is typical of lipid rafts.

[00029] The term “fusion protein” is intended to mean Petition 870190009246, dated 01 / 29 / 2019, page 66 / 125 10 / 47 a protein comprising the various fragments b) ad), and optionally a), said fragments being of different origin. In other words, fragments b) ad), and optionally a), are never present fused together in the way they naturally exist.

[00030] The term successively is intended to mean that the fragments a) ad) (or b) ad)) are present in the order a)-b)-cd) (or b)-c)-d) or d)-c)-b)). These various fragments can be directly fused to each other, or fused together through one or more linker(s). Preferably, the fusion protein according to the invention comprises a linking agent present between the sequences b) and c), and / or between the sequences c) and d).

[00031] The fusion protein initially contains fragments a) ad): the presence of the signal peptide allows the correct trafficking 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) ad). Consequently, the VLPs according to the invention do not contain the signal peptide a). On the other hand, the description of fragments b) ad) that follows is applicable to VLPs.

[00032] The expression membrane-anchored type I transmembrane protein is intended to mean a transmembrane protein of which the N-terminal end is extracellular and the C-terminal end is cytosolic. Therefore, the type I transmembrane protein comprises, from the N-terminal to the C-terminal end, optionally, the Petition 870190009246, dated 01 / 29 / 2019, page 67 / 125 11 / 47 signal peptide a), then the protein or peptide of interest b), then the helical domain c) and finally the anchoring domain d).

[00033] The expression “type II transmembrane protein anchored in a membrane” is intended to mean a transmembrane protein of which the C-terminal end is extracellular and the N-terminal end is cytosolic. Consequently, the type II transmembrane protein comprises, from the N-terminal to the C-terminal end, the anchoring domain d), then the helical domain c), and finally the protein or peptide of interest b).

[00034] Preferably, the fusion protein according to the invention is a type I transmembrane protein. Signal peptide a)

[00035] Signal peptide a) is any signal peptide recognized by a eukaryotic cell.

[00036] Preferably, the signal peptide is selected from the natural signal peptide of pectate lyase and the signal peptide of tobacco chitinase.

[00037] Preferably, the signal peptide is that of tobacco chitinase, with sequence SEQ ID NO: 21. Protein or peptide of interest b)

[00038] The protein or peptide of interest that can be used, according to the invention, can be any amino acid sequence that is of therapeutic or prophylactic interest.

[00039] The protein or peptide of interest that can be used according to the invention can be any amino acid sequence that would benefit from being Petition 870190009246, dated 01 / 29 / 2019, page 68 / 125 12 / 47 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.

[00040] The term “protein of interest” is intended to mean a sequence with at least 51 amino acids, preferably at least 100, preferably at least 200.

[00041] The term “peptide of interest” is intended to mean a sequence comprising 2 to 50 amino acids, preferably 5 to 45 amino acids.

[00042] The protein or peptide of interest that can be used according to the invention is preferably chosen from: - allergens and fragments thereof. The main application of a virus-like particle containing such a protein or peptide is immunotherapy. - viral proteins and fragments thereof. The main advantage of a virus-like particle containing such a protein or peptide is vaccination. - cell surface proteins and fragments thereof. The main advantage 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), Petition 870190009246, dated 01 / 29 / 2019, page 69 / 125 13 / 47 - immunoglobulins, fragments thereof (such as Fab fragments) and derivatives thereof (such as scFv), - cytokines and fragments thereof, and - hormones and fragments thereof.

[00043] Preferably, the protein or peptide of interest that can be used according to the invention is an allergen. Preferably, it is chosen from allergens responsible for respiratory allergies resulting from house dust mites, such as Dermatophagoides farinae, Dermatophagoides pteronyssinus or Euroglyphus manei, storage dust mite allergens such as Blomia tropicalis, Acarus siro type mite allergens (also known as Tyroglyphus farinae), cockroach allergens, tree or grass pollen allergens, animal allergens (cat, dog, horse), mold allergens, allergens responsible for contact allergies, such as rubber tree latex, or other allergens responsible for food allergies (milk, eggs, fish, fruit).

[00044] Entre os alérgenos de Dermatophagoides farinae, pode-se mentioner Der f 10, Der f 11, 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, The f 2.0105, Derf 2.0106, Derf 2.0107, Derf 2.0108, Derf 2.0109, Derf 2.0110, Derf 2.0111, Derf 2.0112, Derf 2.0113, Derf 2.0114, Derf 2.0115, Derf 2.0116, Derf 2.0117, The f 20, The f 3, The f 4, The f 5, The f 6, The f 7, Der f 8, Der f 9 e Der f HSP70.

[00045] Among the allergens of Dermatophagoides pteronyssinus, we can mention Der p 10, Der p 11, Der p Petition 870190009246, dated 29 / 01 / 2019, p. 70 / 125 14 / 47 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, Der p 2.0108, Der p 2.0109, Der p 2.0110, Der p 2.0111, Der p 2.0112, Der p 2.0113, Der p 20, Der p 21, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8, Der p 9.

[00046] Among animal allergens, one can mention allergens from seminal fluid, epithelium, milk, saliva, perspiration and / or urine of said animals. The animals are preferably dogs, cats or horses.

[00047] Among cat allergens (Felis domesticus), one 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.

[00048] Among dog allergens (Canis familiaris), one can mention 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 Feld1-like, Can f Homs2like, Can f phosvitin or Can f TCTP.

[00049] Among horse allergens (Equus caballus), one 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 c 11, Equ c 12, Equ c 8, Equ c 9, Equ c ALA or Equ c BLG.

[00050] The sequences of these allergens are known, particularly in the Uniprot database.

[00051] Preferably, the protein or peptide of Petition 870190009246, dated 01 / 29 / 2019, page 71 / 125 15 / 47 of interest that can be used according to the invention is an allergen with sequence SEQ ID NO: 22 (mature sequence of Der p 2) or SEQ ID NO: 32 (the CH1 chain sequence of the feline allergen Fel d 1).

[00052] Preferably, the protein or peptide of interest that can be used according to the invention is a viral protein or a fragment thereof.

[00053] Among viral proteins, one can mention, in particular, the envelope proteins of the Zika virus and also the proteins of the influenza virus, such as hemagglutinins and neuraminidases. Preferably, the protein or peptide of interest that can be used according to the invention is the HA1 hemagglutinin chain with sequence SEQ ID NO: 34 or the envelope protein of the Zika virus with sequence SEQ ID NO: 35.

[00054] Preferably, the protein or peptide of interest that can be used according to the invention is a cell surface protein or a fragment thereof.

[00055] Among surface proteins, tumor surface antigens can be mentioned in particular. These proteins and fragments thereof are used, in particular, in restoring immune activity, for example, in the treatment of tumors.

[00056] Preferably, the protein or peptide of interest that can be used according to the invention is a protein that accumulates in neurodegenerative or chronic diseases.

[00057] Among the peptides that accumulate in chronic diseases, the β-peptide can be mentioned in particular. Petition 870190009246, dated 01 / 29 / 2019, page 72 / 125 16 / 47 amyloid involved in Alzheimer's disease, the alpha-synuclein protein involved in Parkinson's disease, and also the CD20, TNF-alpha or HLA (human leukocyte antigen) proteins involved in rheumatoid arthritis. Helical domain (or oligomerization sequence) c)

[00058] The helical domain, or oligomerization sequence, comprises several sense or antisense alpha-helix motifs that are parallel to each other and form an organized array that has several well-characterized biological functions. These domains are ubiquitous and are found as specific domains for many protein types in most organisms. Helical domains from various sources can aggregate to form shapes ranging from a dimer to a heptamer; some helical domains will adopt different levels of polymerization depending on point mutations in their amino acid sequence.

[00059] A helical domain typically consists of a 7-amino acid repeat motif of the type hxxhcxc, where h is a hydrophobic amino acid, c is a charged amino acid, and ex is any amino acid.

[00060] The helical domain that can be used according to the invention does not originate from a virus; it is not viral.

[00061] Among the helical domains that can be used according to the invention, mention may preferably be made of those of cortexilin, vimentin, tetrabrachion, golgins, soluble N-ethylmaleimide sensitive factor (NSF) receptor proteins. Petition 870190009246, dated 01 / 29 / 2019, p. 73 / 125 17 / 47 or the SNARE superfamily, or other transcription factors such as GCN4 or a variant thereof, such as GCN4-pLI or GCN4pII.

[00062] Preferably, the helical domain is that of the transcription factor GCN4, GCN4-pLI or GCN4-pII.

[00063] Preferably, the helical domain is chosen from SEQ ID NO: 24 (GCNA-pII trimerization sequence of the yeast GCN4 transcription factor), SEQ ID NO: 27 (GCN4pLI tetramerization sequence of the yeast GCN4 transcription factor), SEQ ID NO: 28 (GCN4-pAA heptamerization sequence of the yeast GCN4 transcription factor), SEQ ID NO: 29 (IZN4 glycosylated oligomerization sequence of the yeast GCN4 transcription factor), SEQ ID NO: 33 (synthetic sequence mimicking a helical structure) and SEQ ID NO: 30 (SNARE oligomerization sequence). Domain for anchoring to the plasma membrane (or transmembrane domain) d)

[00064] The transmembrane domain is a short sequence of lipophilic amino acids that interacts with specific lipid components of the plasma membrane. Preferably, the plasma membrane comprises at least one typical portion of lipid rafts.

[00065] These anchoring domains are common (but not through a consensus sequence) to the surface proteins of viruses, but also to proteins that are naturally integrated into the membrane of living cells. Each transmembrane domain participates in the bending and budding of the plasma membrane. Petition 870190009246, dated 01 / 29 / 2019, page 74 / 125 18 / 47

[00066] Among the anchoring domains that can be used according to the invention, those of the proteins listed in Table 1A may be mentioned preferentially: Table 1A: Transmembrane proteins Transmembrane (TM) Proteins Examples (Uniprot references) Number of TM domains Leucine-rich repeat receptor-like protein kinase NtTMK1 Q9M7A8 1 Caveolin Q03135 1 BRI1-associated receptor kinase 1 (BAK1) Q94F62 1 Receptor kinases Q9LDG0, Q9ZT08, Q8LD58, Q7XHW7, Q8H811, Q9SUQ3, Q5ZBN0 1 Calcium-dependent protein kinases Q6KC54, Q6EE26, Q5EDD1, P28582, Q9ARI5, Q7XZK4, Q8GSB1, Q9FWF0, Q94KH6 1 NtRac2 / NTGP3 Q9ZRD2 1 ARF1-type GTP-binding protein Q9M7P4 1 Stomatin Q93VP6, Q60634 1 Ascorbate peroxidase Q8W4V7 1 LAT O43561 1 VIP36 P49256 1 Protein induced by Q9FXT1 1 Petition 870190009246, dated 01 / 29 / 2019, page 75 / 125 19 / 47 Elicitor (EIG-J7) Hsp90-2 Chaperone Q6UJX5 1 Syntaxin Q9SF29, Q9SRV7, Q9ZSD5 1 Phragmoplastin Q9SMB6 1 Fasciclin-like arabinogalactan protein 8 (precursor) O22126 1 Ras-related protein RAB8-3 Q8W3J3 2 Ras-related protein RAB8-5 Q8W3J2 2 Flotillin Q501E6, O75955 2 Harpin-inducing protein 1 Q6L7J8, Q6L7J7 2 Pectinesterase-like Q9FHN6 2 Protein kinase Q9SH35 3 Endo-1,4-p-glucanase O04890 3 MAL P21145 4 Synaptophysin Q62277 4 Prominin O43490 5 Aquaporin O09224, O09222, Q40595, Q8W506, O24662, Q9FPZ6, Q9FPZ7 6 NADPH oxidase NtrbohD Q8RVJ9 6 Respiratory burst oxidase homolog Q948T9 6 Specific transporter O24405 8 Petition 870190009246, dated 01 / 29 / 2019, page 76 / 125 20 / 47 Lysine and histidine LHT1 Glucan synthase Q9SJM0, Q8S8D4, Q5VS25, Q9ZT82, Q9SFU6 8 Callose synthase Q8H046, Q9LXT9, Q9LTG5, Q9XEG1 9 Plasma membrane ATPase Q42932, Q08436, Q03194, Q5U9D4, Q9SWH2, Q9SWH0 10 Calcium transporter ATPase Q9LU41 10 Ammonium transporter P58905 11 MDR-like ABC transporter mdr8 Q7FMW3 12 P-glycoprotein-like protein Q9SY12 12 ABC transporter O80725, Q9FWX8 12 Phosphate transporter Q9ST22, Q9LLS5, Q9AYT1 12 Transmembrane protein PT3 Q8W4W9 12 H+ / monosaccharide cotransporter MST1 Q06312 12 High-affinity nitrate transporter protein Q84MZ8 12 PDR-type ABC transporter 1 NtPDR1 Q76CU2 12

[00067] Among the anchoring domains that can be used according to the invention, those of viral envelope proteins may be mentioned preferentially. Petition 870190009246, dated 01 / 29 / 2019, page 77 / 125 21 / 47 listed in Table 1B below: Family Examples Envelope proteins Flaviviruses Dengue virus E protein Yellow fever virus Saint Louis encephalitis virus Japanese encephalitis virus West Nile virus Zika virus BYD virus (identified in China in 2010, affecting ducks) Togavirus Sindbis virus GP Eastern equine encephalitis virus Western equine encephalitis virus Ross River virus O'nyong'nyong virus Retroviruses Oncoviruses (5 genera) GP41 / 120 Lentiviruses (like HIV) Spumavirus Coronavirus Canine coronavirus S protein and HE protein (hemagglutinin esterase) Feline coronavirus Transmissible gastroenteritis virus in swine Porcine respiratory virus Feline coronavirus Human coronavirus Murine hepatitis virus Petition 870190009246, dated 01 / 29 / 2019, page 78 / 125 22 / 47 Rat sialadenitis virus Filovirus Ebola virus Glycoprotein Marburg virus Rhabdovirus Rabies virus GP Viral hemorrhagic virus VSV-EBOV Beetroot disease Buniavirus Hantavirus Gn / Gc Dugbe virus Rift valley fever Tomato spotted wilt virus Orthomyxovirus Influenza virus (myxoinfluenza) Hemagglutinin / neuraminidase Paramyxovirus Mumps virus Fusion protein F Binding protein (HN, H or G) Sendai virus SV5 virus Newcastle disease virus Measles virus Canine distemper virus Rinderpest virus Respiratory syncytial virus (RSV) Bovine Respiratory Syncytial Virus (BRV) Parainfluenza Arenavirus Lassa fever GP Argentine hemorrhagic fever Petition 870190009246, dated 01 / 29 / 2019, p. 79 / 125 23 / 47 Bolivian hemorrhagic fever Brazilian hemorrhagic fever Venezuelan hemorrhagic fever Hepadnavirus Hepatitis B virus (HBV) GPL, S or M Herpesvirus Herpes Simplex virus (HSV) Glycoprotein gD, gB, gH, gL, gC EHV-1 (equine herpesvirus) genital herpesvirus Poxvirus Orthopoxvirus; species type: vaccinia virus; disease: cowpox, smallpox GP41 / 120 Parapoxvirus; species type: Orf virus Avipoxvirus; species type: fowlpox virus Capripoxvirus; species type: sheeppox virus Leporipoxvirus; species type: myxoma virus Suipoxvirus; species type: swinepox virus Molluscipoxvirus; species type: Molluscum contagiosum virus Yatapoxvirus; Species type: Yaba monkey tumor virus

[00068] Preferably, the anchoring domain that can be used according to the invention is chosen from the anchoring sequence of the H5 hemagglutinin of the H5N1 influenza virus (SEQ ID NO: 26) and the anchoring sequence of Petition 870190009246, dated 01 / 29 / 2019, page 80 / 125 24 / 47 PDLP1 protein (A0A0D3D8S3) (SEQ ID NO:31). Binders

[00069] Preferably, the fusion protein according to the invention comprises a linking agent present between fragments b) and c), and / or between fragments c) and d).

[00070] 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 a flexible space between the anchoring domain and the helical domain spiral if the proximity of the two domains interferes with proper assembly. They are not necessary under conditions where a direct link between the two domains (anchoring and helical domains) does not interfere with the overall three-dimensional structure of the fusion protein.

[00071] Preferably, the linker is a sequence of the 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).

[00072] 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 fragments thereof, - viral proteins and fragments thereof, - cell surface proteins and fragments thereof, - proteins and peptides that accumulate in Petition 870190009246, dated 01 / 29 / 2019, page 81 / 125 25 / 47 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 helical 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 docking domain is chosen from the docking sequence of the H5 hemagglutinin of the H5N1 influenza virus (SEQ ID NO: 26) and the docking sequence of the PDLP1 protein (SEQ ID NO: 31).

[00073] Thus, preferably, the fusion protein according to the invention comprises, preferably consists of, a sequence chosen 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.

[00074] Similarly, preferably, the VLP according to the invention comprises: - an envelope consisting of a plasma membrane in which at least one portion has a typical lipid raft composition; and - at least one type I or II transmembrane fusion protein anchored to said membrane, said fusion protein comprising the following fragments, successively: b) the protein or peptide of interest chosen from: - allergens and fragments thereof, - viral proteins and fragments thereof, - cell surface proteins and fragments of Petition 870190009246, dated 01 / 29 / 2019, page 82 / 125 26 / 47 same, - proteins and peptides that accumulate 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 helical domain chosen from the SEQID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQID NO: 33 and SEQ ID NO: 30; and d) the docking domain chosen from the H5 hemagglutinin docking sequence of the H5N1 influenza virus (SEQ ID NO: 26) and the PDLP1 protein docking sequence (SEQ ID NO: 31): the fragments b) and c) being exposed on the outside of the VLP.

[00075] An embodiment of the present invention is also the nucleic acids (or a nucleotide sequence) that encodes the fusion protein.

[00076] Once the nucleotide sequence is obtained, it is placed into an expression vector using conventional methods. An embodiment of the present invention is also a vector comprising the nucleic acid encoding the fusion protein. The selection of an appropriate 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, for example a promoter, and DNA elements that control the Petition 870190009246, dated 01 / 29 / 2019, page 83 / 125 27 / 47 processing of transcripts, such as termination / polyadenylation sequences and an expression cassette that allows the expression of a silencing inhibitor. It also contains sequences such as t-DNAs, which are necessary for the integration of a piece of DNA into the plant or plant cell.

[00077] Preferably, the expression vector comprises: - at least one nucleotide sequence that codes for the fusion protein, preferably functionally linked to a strong promoter, preferably a 35S promoter; - an expression cassette that allows the expression of a silencing inhibitor, preferably p19; and - DNA elements that control transcript processing, such as termination / polyadenylation sequences, preferably the Tnos sequence (nopalin synthase termination sequence).

[00078] The expression vector is preferably pAG01.

[00079] The promoters used to control the expression of the fusion protein are strong promoters, and can be plant gene promoters, such as the ubiquitin promoter, the ribulose-1,5-bisphosphate carboxylase small subunit promoter, Agrobacterium tumefaciens promoters, nopalin synthase and octopin synthase promoters, or other viral promoters, such as cauliflower mosaic virus (CaMV) 19S and 35S. Preferably, the strong promoter is 35S.

[00080] An embodiment of the present invention is also a host cell comprising at least one acid Petition 870190009246, dated 01 / 29 / 2019, page 84 / 125 28 / 47 nucleic acid that codes for the fusion protein. The host cell can be a plant cell.

[00081] One embodiment of the present invention is also a method for producing virus-like particles (VLPs) comprising expressing the nucleic acid encoding the fusion protein in eukaryotic cells, preferably plant cells.

[00082] General methods for cultivating plants, as well as methods 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, plants will be cultivated according to techniques specific to the Allergopur platform. This method for producing recombinant proteins is described in patent application FR 1 255 510, and comprises a first step of cultivating the plant under aeroponic or hydroponic conditions and under LED lighting. After this first step, agroinfiltration of the plants is carried out under vacuum using agrobacteria comprising a DNA fragment encoding the fusion protein according to the invention. This agroinfiltration step can be carried out by any means to produce a vacuum. Preferably, in the method used according to the invention, it is carried out under vacuum by the Venturi effect.Among the agrobacteria that can be used according to the invention, the strains LBA4404, GV3101, EHA 101 / 105 or C58 are preferably mentioned.

[00083] Once the agroinfiltration stage has been completed, the plants are placed back into the crop, normally Petition 870190009246, dated 01 / 29 / 2019, page 85 / 125 29 / 47 for 3 to 6 days, ideally providing frequent misting of said plants for the first 6 hours of cultivation after agroinfiltration. The VLPs are then extracted and purified as described below.

[00084] VLP extraction can be performed by enzymatic extraction. This method is an adaptation of the method described in particular in WO 2014 / 153674. Preferably, the enzymatic extraction of VLPs is performed using the following steps: - Vacuum infiltration (in particular as described above for agroinfiltration) of the aerial parts of plants (i.e., leaves) into an enzymatic solution containing pectocellulosic enzymes that do not exhibit any 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 50 mM sodium citrate, pH 5.2, 0.5 M NaCl, and 0.04% metabisulfite. The leaves are subsequently sampled and then incubated in the enzyme solution. The mixture is placed with agitation in an orbital shaker at 20 to 30 rpm at room temperature (i.e., approximately 20-23°C) for a period between 30 minutes and 2 hours. The digest is then filtered, preferably through a 2-3 mm screen, then 250 µm, then optionally continuously centrifuged (e.g. at 1000 χ² g for 2-5 minutes), and the supernatant is recovered in order to carry out... Petition 870190009246, dated 01 / 29 / 2019, page 86 / 125 30 / 47 a tangential filtration.

[00085] This method is illustrated in figure 7.

[00086] Thus, preferably, an embodiment of the invention is also a method for producing virus-like particles (VLPs) according to the invention in a plant cell or in a plant, comprising the following steps: a) transformation of agrobacteria with an expression vector comprising a nucleotide sequence encoding a fusion protein according to the present invention functionally linked to a strong promoter; and b) transfection of the plant cell or plant with the agrobacteria obtained in step a).

[00087] The transformation in step a) is typically carried out using methods known to those skilled in the art, for example, by means of thermal shocks with successive passages to 4°C, -80°C and 37°C.

[00088] The transfection in step b) preferably comprises the following steps: b1) Culture of the plant cell or plant, under aeroponic or hydroponic conditions, and under LED lighting, preferably for four to six weeks; b2) Agroinfiltration of the plant cell or plant obtained in b1) under vacuum, with the agrobacteria obtained in step a). This agroinfiltration step is preferably carried out under vacuum using the Venturi effect; b3) Returning the plant cell or plant obtained in b2) to the culture, typically for 3 to 6 days, to obtain virus-like particles. Petition 870190009246, dated 01 / 29 / 2019, page 87 / 125 31 / 47

[00089] Finally, the VLPs obtained are extracted and purified, in particular by enzymatic extraction as described above.

[00090] An embodiment of the present invention is also a virus-like particle comprising: - an envelope consisting of a plasma membrane in which at least one portion has a typical lipid raft composition; and - at least one fusion protein according to the invention without a signal peptide (a), anchored to said membrane.

[00091] This virus-like particle (VLP) comprises: - an envelope consisting of a plasma membrane in which at least a portion is typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored to said membrane, said fusion protein comprising the following fragments, successively: b) a protein or peptide of interest; c) a helical domain (or oligomerization sequence) that does not originate from a virus; and d) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring to a plasma membrane, of which at least a portion is typical of lipid rafts, the fragments b) and c) being exposed on the outside of the VLP.

[00092] The expression portion of the plasma membrane Petition 870190009246, dated 01 / 29 / 2019, page 88 / 125 32 / 47 typical of lipid rafts is intended to mean a phospholipid bilayer (i.e., plasma membrane) found in the microdomains of lipid rafts. This bilayer is rich in cholesterol and phospholipids, preferentially phosphatidylcholine and phosphatidylethanolamine, and sphingolipids such as sphingomyelin, but weak in docosahexaenoic acid. Furthermore, it has low density and is insoluble in mild detergents (e.g., polysorbates).

[00093] The unique characteristics of such a VLP are undetectable immunogenicity (unlike the protein or peptide of interest b), its ability to spontaneously self-assemble, its specific lipid content, preferentially typical of lipid rafts, the fact that its membrane is very poor in host cell membrane proteins, its ability to be expressed in high yields in numerous types of eukaryotic cells (leaves, insects or plants) and its ease of preparation.

[00094] The VLP according to the invention can be used in therapy. It can be used as a medicine. It can also be used in allergen immunotherapy (AIT).

[00095] The sequences listed in this application are summarized in the table below: SEQ ID NO: Definition 1 cDNA encoding the natural form of Der p2 2 Der p2 protein 3 to 20 cDNA and fusion proteins of Petition 870190009246, dated 01 / 29 / 2019, page 89 / 125 33 / 47 According to the invention: 21 Tobacco chitinase signal peptide 22 Mature Der p2 sequence 23 Ligand 24 GCN4-pII helical domain 25 Ligand 26 Hemagglutinin H5 anchoring sequence of H5N1 influenza virus 27 GCN4-pLI helical domain 28 GCN4pAA helical domain 29 IZN4 helical domain 30 SNARE helical domain 31 PDLP1 protein anchoring sequence 32 Fel d1 feline allergen CH1 chain sequence 33 Synthetic helical domain 34 Hemagglutinin HA1 chain 35 Zika virus envelope protein

[00096] The following examples illustrate, but are not intended to limit, the scope of the invention. It would be obvious to those skilled in the art that variants and modifications are possible and fit within the context and spirit of the invention. Petition 870190009246, dated 01 / 29 / 2019, pp. 90 / 125 34 / 47 invention.

[00097] The figure captions are as follows: Figure 1: Diagrammatic representation of the various expression cassettes for the production of an allergen linked to an oligomerization sequence and a sequence to anchor to the plasma membrane, preferably at the level of lipid rafts.

[00098] A) The cDNA encoding the optimized, preferably harmonized Der p2 (DP2, SEQ ID NO: 22) is linked to 1) cDNA encoding the tobacco chitinase signal peptide (PS Chit, SEQ ID NO: 21), 2) an oligomerization sequence (helical structure) of a transcription factor (GCN4-pII / trimeric form, B-GCN4-PLI / tetrameric form, C-GCN4-IZN4 / glycosylated form, E-GCN4pAA / heptameric form, D) or from any other protein family possessing a helical sequence (SNARE, Golgin, Fibritin, G) or a synthetic sequence mimicking a helical sequence (F), and finally, 3) an anchoring sequence from the envelope protein of enveloped viruses (TM / CT influenza H5, B to I) or from type I proteins anchored in rafts lipids (lipid balsam) (J).

[00099] B) Diagram representing the structure of the oligomerization or helical sequence. This sequence consists 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 ex is any amino acid. Figure 2: AllergoPur platform used for the expression and production of various forms of VLP. Petition 870190009246, dated 01 / 29 / 2019, pp. 91 / 125 35 / 47 Figure 3: Production of the proteins described in Figure 1A [000100] Proteins extracted from plants transfected under vacuum for the expression of DP2 (band 1), DP2Tri (bands 2-3), DP2Tetra (bands 4-5), or FD1Tri (bands 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, whose molecular weight corresponds to the expected weight. Two Agrobacteria clones (Cl.1 and Cl.2) were analyzed for each construct. Figure 4: Purification and characterization of VLPs carrying allergens by size exclusion chromatography. [000101] Protein extracts from leaves producing DP2Tri (panel D), DP2-Tetra (panel E), soluble DP2 (panel F), FD1-Tri (panel G), DP2triDGCN4 (GCN4 deletion, panel H), DP2tri-Syn (GCN4 substitution, panel I), and DP2tri-KEI (GCN4 substitution, 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 Coomassie blue staining after SDS-PAGE separation (panel B). The allergen content of the eluted fractions was revealed by immunological detection using anti-Der p2 or anti-Fel d1 antibodies. Protein extracts from leaves that produce hemagglutinin in the form of H5N1 VLPs (panel C) were separated by gel filtration on a calibrated S-500 / HR column and are used as controls. Figure 5: Characterization of the VLPs that transport the Petition 870190009246, dated 01 / 29 / 2019, page 92 / 125 36 / 47 allergens isolated by electron microscopy examination and negative staining. [000102] The VLPs that carry allergens have a morphology and size that are very close to those described for influenza virions. [000103] The bar represents 50 nm. Figure 6: Reactivity of allergens produced in the form of VLPs with sera from patients allergic to Der p2. [000104] Proteins extracted from vacuum-transfected plants for the expression of DP2 (band 1), DP2-Tri (band 2), and DP2-Tetra (band 3) proteins were analyzed by immunodetection using sera from patients allergic to Der p2. The immunodetection analysis demonstrates the recognition of the allergens transported by the VLPs by the IgEs in the patients' sera. Figure 7: Method for large-scale VLP production Figure 8: Structure of the VLPs and fusion proteins assembled according to the invention. [000105] 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 (e.g., the allergen) is thus exposed on its surface. [000106] B) Structure of the fusion proteins according to the invention, assembled within the VLP. The oligomerization sequences allow the fusion proteins to form polymers (e.g., in this case, the allergen, A) on the surface of the VLP. Figure 9: VLP-conjugated antigens have a power Petition 870190009246, dated 01 / 29 / 2019, page 93 / 125 37 / 47 very strong immunogenic, but they do not present allergenicity. [000107] Panel A: Evaluation of airway hyperreactivity induced by the Der p2 allergen, using the Flexivent method. 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 solution) and challenged with a mite extractor. Twenty-four hours after the final challenge, airway hyperreactivity to inhaled methacholine was determined using the Flexivent method. Pulmonary reactivity triggered in the presence of the allergen in VLP form is comparable to control mice. [000108] Panel B: Counting of inflammatory cells in the airways, collected by bronchoalveolar lavage (BAL) of the lung. Mice (n = 10 / group) were sensitized with the Der p2 allergen in soluble form (DP2Alum) or in VLP form (DP2-VLP / alum and DP2-VLP / saline solution) and challenged with a mite extract. Twenty-four hours after the final challenge, BAL cells were collected and counted (eosinophils; neutrophils; macrophages; lymphocytes). Neutrophils are very predominant in mice that received the Der p2 allergen in soluble form. [000109] Panel C: Der p2-specific IgG assay. 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 solution) and challenged with a mite extract. Twenty-four hours after the final challenge, IgGs were measured in BAL fluid and blood serum was collected by cardiac puncture. The mice Petition 870190009246, dated 01 / 29 / 2019, pp. 94 / 125 38 out of 47 mice that received DP2-VLP injections with or without adjuvant have an IgG titer that is a thousand times higher than mice that received soluble Der p2. EXAMPLES: Example 1: Molecular design and gene synthesis [000110] cDNAs are synthesized by optimizing and then harmonizing the use of codons for their recognition by the plant system. In the context of this invention, the preferred optimization is optimization for expression in Nicotiana benthamiana. [000111] The constructs are described in Figure 1. In particular: [000112] A: cDNA encoding the natural form of the protein (SEQ ID NO: 1). This cDNA may or may not be fused to traffic signals described in patent WO 2008 / 056265. The corresponding protein has the sequence SEQ ID NO: 2. [000113] 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), the GCN4-pII trimerization signal of the yeast GCN4 transcription factor, and the H5 hemagglutinin anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 3). The corresponding protein has the sequence SEQ ID NO: 4. [000114] C: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the GCN4-pLI tetramerization sequence of the yeast GCN4 transcription factor, and to the H5 hemagglutinin anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 5). The protein Petition 870190009246, dated 01 / 29 / 2019, pp. 95 / 125 39 / 47 corresponds to the sequence SEQ ID NO: 6. [000115] D: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the GCN4-pAA heptamerization sequence of the yeast GCN4 transcription factor and to the H5 hemagglutinin anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 7). The corresponding protein has the sequence SEQ ID NO: 8. [000116] E: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the glycosylated oligomerization sequence of IZN4 of the yeast GCN4 transcription factor and to the anchoring sequence of hemagglutinin H5 of the H5N1 influenza virus (SEQ ID NO: 9). The corresponding protein has the sequence SEQ ID NO: 10. [000117] 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), a synthetic sequence mimicking a helical structure, and the H5 hemagglutinin anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 11). The corresponding protein has the sequence SEQ ID NO: 12. [000118] 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), a SNARE oligomerization sequence, and the H5 hemagglutinin anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 13). The corresponding protein has the sequence SEQ ID NO: 14. [000119] H: cDNA encoding two Der p2 fragments Petition 870190009246, dated 01 / 29 / 2019, pp. 96 / 125 40 / 47 (SEQ ID NO: 22) which are fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the trimerization sequence of GCN4-pII of the yeast GCN4 transcription factor and to the anchoring sequence of hemagglutinin H5 of the H5N1 influenza virus (SEQ ID NO: 15). The corresponding protein has the sequence SEQ ID NO: 16. [000120] I: cDNA encoding the CH1 chain of the Fel d1 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 anchoring sequence of the H5N1 influenza virus (SEQ ID NO: 17). The corresponding protein has the sequence SEQ ID NO: 18. [000121] J: cDNA encoding the mature form of the Der p2 allergen (SEQ ID NO: 22) fused to the signal sequence of tobacco chitinase (Neuhaus, J.-M. 1996), to the trimerization sequence of GCN4-pII of the yeast GCN4 transcription factor and to the anchoring sequence of the PDLP1 protein (A0A0D3D8S3) of lipid rafts (SEQ ID NO: 19). The corresponding protein has the sequence SEQ ID NO: 20. Example 2: Plasma preparation [000122] The Xba I / kpn I and Sal I / Sac I restriction sites are integrated into the 5' and 3' ends of the cDNA during synthesis, respectively. These sites are then used to clone the cDNAs into the pAG01 binary expression vector. The cDNAs are cloned upstream of a 35S promoter (35S) and downstream of a nopalin synthase termination sequence (tnos); the pAG01 vector also contains an expression cassette that allows expression of the p19 silencing inhibitor. Petition 870190009246, dated 01 / 29 / 2019, pp. 97 / 125 41 / 47 simultaneously with the recombinant protein in order to increase product 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 leaves - using the AllergoPur platform. [000123] For transient expression production, LBA4404 from Agrobacterium tumefaciens is used for the transfer of a cDNA encoding Der p2 linked to 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 and not transgenesis. Plants are grown under hydroponic conditions in the presence of a nutrient medium (GEM, floragrow, floramicro, florbloom, 10 ml / 15 ml / 5 ml per 10 l of osmotic water) and under LED lighting. [000124] Agrobacteria are transferred to leaf tissue via agroinfiltration using two methods. For the production of small batches of proteases intended for prototype screening, agrobacteria are manually injected using a syringe applied to the epidermis of the underside of the leaf. Leaf discs sampled from leaves 4 to 6 days after agroinfiltration are used for the analysis of various VLP prototypes. This screening step makes it possible to define the expression vector that will be used to obtain the Der p2 allergen anchored to the optimal membrane quality. The same method is used for large-scale production, but in this case, agroinfiltration is used. Petition 870190009246, dated 01 / 29 / 2019, pp. 98 / 125 42 / 47 is performed under vacuum, in chambers containing several liters of an agrobacterial culture, where several dozen plants are simultaneously infiltrated. These plants are then placed back into culture for 3-6 days before purification of the VLPs that carry the allergen (figure 2). Example 4: Production of VLPs that transport the Der p2 allergen. [000125] The expression of the proteins produced in example 3 and also the yields are analyzed respectively by Western blotting and ELISA. The results are presented for 3 allergens produced in the form of VLP (DP2-tri; DP2tetra and FD1-tri) (figure 3). Example 5: Evaluation of the size distribution / formation analysis of VLP [000126] A size distribution analysis of the structures transporting the allergens Der p2 (DP2-tri / DP2-tetra) or Fel d1 (FD1-Tri) was performed. After vacuum infiltration of N. benthamiana plants with the Agrobacterium LBA4404 strain, as described in Example 3, the total protein extracts were separated by size exclusion chromatography on a high-resolution (HR) S-500 column (GE Healthcare Bio-Science Corporation). The elution fractions were controlled for their total protein content and their allergen-VLP content by Western blotting with anti-allergen antibodies. For all extracts analyzed, the concentration of soluble protein in the eluate reaches a maximum in fractions 14-16 (Figure 4). On the other hand, Western blotting analysis demonstrates an accumulation of allergens in fractions 6 and 7 (i.e., before elution with Dextran Blue). Petition 870190009246, dated 01 / 29 / 2019, page 99 / 125 43 / 47 as a marker) which shows the binding of allergens to very high molecular weight structures in zone 2 MDa. [000127] 32 ml Sephacryl S-500 / HR columns (GE Healthcare Bio-Science Corporation) were equilibrated with 50 mM PBS, pH 7.4, 150 mM NaCl. 1.5 ml samples of total protein extracts were loaded and then eluted with equilibration buffer. Twenty-four 1.5 ml elution fractions were collected and analyzed for protein content measured by absorbance spectrophotometry at 280 nm. The proteins from each fraction were concentrated by precipitation with acetone and then redissolved 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: VLP morphology - analysis by electron microscopy [000128] Transmission electron microscopy of the purified product (resulting from production in example 3 followed by purification) indicates that the high molecular weight structures isolated by sieving chromatography are VLPs to which the allergens are bound. In terms of both their size and morphology, which comprises a phospholipid membrane covered with peaks, these VLPs closely resemble influenza virions (figure 5). Example 7: Production of allergens or hypoallergens transported by VLPs Petition 870190009246, dated 01 / 29 / 2019, pages 100 / 125 44 / 47 [000129] The coupling of an allergen to a VLP considerably reduces its in vivo reactivity in serum IgEs of patients. [000130] However, the use of hypoallergenic products further reduces IgE reactivity and, consequently, the risk of anaphylactic reaction. The reduction in reactivity of a hypoallergenic form of the Der p2 allergen transported by VLPs is illustrated in Figure 6. Example 8: VLP Production [000131] The detailed method for producing VLPs, up to their purification (as described in example 6), is illustrated in figure 7. Example 9: Immunogenic power of VLPs compared to soluble allergens [000132] The presentation of an antigen in a highly ordered and repetitive network typically elicits strong immune responses, whereas the same antigen presented as a monomer is non-immunogenic. [000133] To compare the immune response 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-transported form. IgG titers against the Der p2 allergen were determined by ELISA. Protocol [000134] The protocol is illustrated as follows: Petition 870190009246, dated 01 / 29 / 2019, pages 101 / 125 45 / 47 Sacrifice Aclinnfftrragao |sensibilização D-7 Do D7 D14 □' v ------1·'λ— Challenge / stratum D22 D24 Dm D23 D25 Analyses Parameters studied: Required allergen dose, method of administration, adjuvant. [000135] Analysis after the sacrifice: • Weight loss and behavioral change • Lung function (flexiVent, plethysmography) • Serological response to allergen (serum IgG, IgE, blot) • Basophil activation test • Histopathology and other serological results (e.g., IgG isoforms) in subsequent phases. [000136] This study demonstrated that VLPs coupled to the Der p2 allergen do not trigger bronchial hyperreactivity in mice, unlike soluble Der p2 (see Figure 9, panel A). Furthermore, VLPs bound to Der p2 trigger a systemic Thl-like response with neutrophil activation (see Figure 9, panels B and C). Example 10: Desensitization / vaccination using VLPs [000137] 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 significant side effects. Protocol Petition 870190009246, dated 01 / 29 / 2019, pp. 102 / 125 46 / 47 [000138] The protocol is illustrated as follows: Allergen doses Sensitization pathway Adjuvant Analyses Literature references [000139] 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 hypopotent inoculations of pollen vaccine. Lancet 1911; 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-Kundig et al. J Allergy Clin Immunol 2006; 117:14701476 10-BachmannMF, Jennings GT Phil Trans R Soc B Biol SCI 2011; 366:2815-2822 Petição 870190009246, from 01 / 29 / 2019, pág. 103 / 125 47 / 47 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-Kundig et al. J Allerg Clin Immunol; 2006; 117: 1470-1476 19-Cielens et al. FEBS Letters 2000; 482:261-264

Claims

CLAIMS 1. Virus-like particle characterized in that it comprises: - an envelope consisting of a plasma membrane in which at least one portion is typical of lipid rafts; and - at least one type I or II transmembrane fusion protein anchored to said membrane, said fusion protein comprising the following fragments, successively: b) a protein or peptide of interest that is selected from allergens and fragments thereof; c) a helical domain or oligomerization sequence, which does not originate from a virus, said helical domain having an amino acid sequence selected 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;(ed) a domain for anchoring to the plasma membrane, consisting of a transmembrane segment and a cytosolic segment, preferably a domain for anchoring to a plasma membrane of which at least a portion is typical of lipid rafts, said domain for anchoring to the plasma membrane having the amino acid sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 31, fragments (b) and (c) being exposed on the surface of the virus-like particle.

2. Virus-like particle according to claim 1, characterized in that the linker is present between fragments b) and c), and / or between fragments c) and d). Petition 870260059308, dated 06 / 17 / 2026, page 12 / 16 2 / 2 3. Virus-like particle, according to any one of claims 1 or 2, characterized in that said transmembrane fusion protein comprises the amino acid sequence chosen from SEQ ID NO: 4, 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.

4. Use of a virus-like particle as defined by any one of claims 1 to 3, characterized in that it is for the preparation of a medicament.

5. Use of a virus-like particle as defined by any one of claims 1 to 3, characterized in that it is for the preparation of a medicament for allergen immunotherapy.

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