Bioparticles for the presentation of peanut allergens

Abstract

The present invention pertains to specific bioparticles at the surface of which are expressed specific fragments of the Ara h 2 protein. The bioparticles according to the present invention comprise an envelope consisting of a plasma membrane; and at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising successively a) a peanut allergen, b) a coiled-coil domain or oligomerization sequence; and c) a domain for anchoring in the plasma membrane consisting of a transmembrane segment and a cytosolic segment. Fragments a) and b) are exposed at the surface of the bioparticle. The bioparticles according to the present invention can be used in therapy such as immunotherapy, in particular for preventing / treating peanut allergy. The present invention also pertains to methods for producing such bioparticles.

Description

Title: BIOPARTICLES FOR THE PRESENTATION OF PEANUT ALLERGENSTechnical Field

[0001] The present invention pertains to novel bioparticles for the presentation of specific peanut allergens. The bioparticles according to the present invention can advantageously be used as an immunotherapy for the treatment and prevention of peanut allergies.Background Art

[0002] Peanut allergy is among the most common food allergies and the most common cause of fatal food reactions and anaphylaxis worldwide. Although food allergy-related fatalities are rare, peanut allergy accounts for most of them, even in individuals with a history of mild reactions, making prediction difficult (Lange et al.; Allergo J Int. 2021 ;30(8):261-269). Currently, 18 peanut-derived IgE binding proteins have been identified as allergens (as referenced by the allergen database). Ara h 1 , 2, 3, and 6 are considered major allergens of which Ara h 2 is the clinically most relevant (Hemmings et al. Journal of Allergy and Clinical Immunology 146.3 (2020): 621-630).

[0003] For several decades, several approaches have been tested to develop safe and effective allergen immunotherapy (AIT) for the treatment of peanut allergy. One of the main strategies proposed e.g. comprised subcutaneous AIT with aqueous peanut allergen extract, followed by rectal administration of the main peanut allergens produced in bacteria. Unfortunately, this approach was associated with many severe side-effects and was therefore abandoned (Oppenheimer et al. Journal of Allergy and Clinical Immunology 90.2 (1992): 256-262; Nelson et al. Journal of Allergy and Clinical Immunology 99.6 (1997): 744-751). Other “safer” administration routes, such as sublingual and epicutaneous administrations, were also tested. However, to date, none of them has provided satisfactory results and reached the market.

[0004] The only treatment that did receive market authorization for peanut allergen immunotherapy is oral immunotherapy (OIT). It is effective for desensitization, disappointing for sustained efficacy, and side-effects occur frequently (Castenmiller et al. Clinical & Experimental Allergy 53.5 (2023): 577-581). Furthermore, the most sensitive patients being at risk of severe lifethreatening allergic reactions are unlikely to be helped with this approach, as side-effects may include severe anaphylaxis (Castenmiller et al., 2023).

[0005] Therefore, there is an urgent need to provide all patients, including the severest ones, with a safe and effective treatment.

[0006] Recently, nano- and micro-particulate strategies, such as virus-like particles, have been proposed as potentially safe and effective approaches (see e.g. Krenger et al. Allergy 79.1 (2024): 184-199; Krenger et al. Clinical and Experimental Allergy 53.12 (2023): 1310-1313; Sobczak et al. Allergy 78.7 (2023): 1980-1996).

[0007] It is now well-established that bioparticles (BP), and in particular virus-like particles (VLPs), can be used for the presentation of target antigens to the immune system. There is now accumulatingevidence that delivery of antigens in the form of bioparticles has a profound impact on the skewing of the immune response (De Souza Rebou^as et al. BioMed research international 2012 (2012); Anzaghe, Martina, Stefan Schulke, & Stephan Scheurer. Current allergy and asthma reports 18 (2018): 1-12; Klimek et al. Allergo journal international 27 (2018): 245-255, Gomord et al, pLos One, 15.12 (2020): e0242867). Most bioparticles contain repetitive displays of conformational epitopes that can elicit strong T cell and B cell responses, effectively making them high potency immunization ‘devices’ (Singha et al. ACS nano 12.11 (2018): 10621-10635).

[0008] Ara h 2-presenting bioparticles have been synthesized and tested. These bioparticles, which express copies of the entire mature Ara h 2 protein onto their surfaces, show a remarkable hypo-allergenicity and allow activating dendritic cells and polarizing T cell responses to Th1 (Castenmiller et al., 2023).

[0009] Unfortunately, such Ara h 2-presenting bioparticles show very low immunogenicity and therefore not allow for the efficient desensitization of patients. Furthermore, these bioparticles are difficult to produce and provide very limited purification yields, notably due an undesirable aggregation rate upon recombinant expression.

[0010] There is therefore a need for the provision of efficient immunotherapeutic tools that allow for the treatment / prevention of peanut allergy. Such tools need to be hypo-allergenic while being immunogenic and must be obtainable by means of simple processes that allow high production yields compatible with large-scale applications.Summary

[0011] The invention is defined by the claims.

[0012] The present inventors have managed to develop specific Ara h 2- bioparticles that are hypoallergenic, highly immunogenic and allow for the efficient production of neutralizing antibodies against Ara h 2. Furthermore, the bioparticles according to the present invention can be easily obtained with very high production yields.

[0013] Surprisingly, all these effects are obtained by presenting a very specific fragment of the mature Ara h 2 protein at the surface of the bioparticle. The inventors have shown that presenting this specific fragment allows for the efficient desensitization of peanut allergic patients while allowing for the optimization of the recombinant synthesis of the bioparticle.

[0014] Therefore, according to a first embodiment, the present invention pertains to a bioparticle comprising:

[0015] - an envelope consisting of a plasma membrane; and

[0016] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0017] a) a peanut allergen;

[0018] b) a coiled-coil domain or oligomerization sequence; and

[0019] c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment;

[0020] fragments a) and b) being exposed at the at the surface of the bioparticle;

[0021] wherein said peanut allergen has the amino acid sequence selected from the group consisting of the amino acid sequence as set forth in SEQ ID No. 1 (QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQ); a fragment of at least 27 consecutive amino acids of the sequence of SEQ No. 1 ; and an amino acid sequence having at least 80% of sequence identity with the amino acid sequence as set forth in SEQ ID No. 1 or with a fragment of at least 27 amino acids thereof.

[0022] The present invention further pertains to the use of the of the bioparticle as described above in immunotherapy, in particular for the treatment / prevention of peanut allergy.

[0023] The present invention also pertains to methods for obtaining the bioparticle as defined above.Brief Description of the figures

[0024] Figure 1: Expression and purification of soluble Ara h 2 or H2-BP in plant cells. These extracts after cross-flow filtration and centrifugation at 8000g (supernatant [1 OpL] line 2; pellet [1 OpL] line 3); after size-exclusion chromatography (1stspike [10pL] line 4, 2ndspike [10pL] line 5, exit pools lines 6 and 7); after ion chromatography (end of flowthrough line 8, increasing fractions lines 9, 10 and 11) were separated by SDS-PAGE and immunodetected by immunoserum containing antibodies directed against Ara h 2. Line 1 corresponds to a negative control. Line 12 corresponds to soluble Ara h 2 (1000 ng).

[0025] Figure 2: Schematic representation of the immunogenicity study.

[0026] Figure 3: Graph representing IgG production in rats after H2-BP administration or FD1-BP administration over time.Detailed description of the invention

[0027] The present invention pertains to a bioparticle comprising:

[0028] - an envelope consisting of a plasma membrane; and

[0029] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0030] a) a peanut allergen;

[0031] b) a coiled-coil domain or oligomerization sequence; and

[0032] c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment ;

[0033] fragments a) and b) being exposed at the surface of the bioparticle;

[0034] wherein said peanut allergen has the amino acid sequence selected from the group consisting of the amino acid sequence as set forth in SEQ ID No. 1 (QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQ); a fragment of at least 27 consecutive amino acids of the sequence of SEQ No. 1 ; and an amino acid sequence having at least 80% of sequence identity with the amino acid sequence as set forth in SEQ ID No. 1 or with a fragment of at least 27 amino acids thereof.

[0035] The fusion protein behaves like a viral surface protein when it is expressed in eukaryotic cells. This protein is synthesized in the endoplasmic reticulum and then transported, via the Golgi apparatus, to the plasma membrane. Once it reaches the plasma membrane, this transmembrane fusion protein causes curving of said membrane, which finally forms a bud which separates from the cell membrane and is released into the extracellular space. During the budding, the peanut allergen carried by the coiled-coil domain (or oligomerization sequence) is exposed at the outer surface of the newly formed particle.

[0036] The transmembrane domain remains anchored in the membrane and is not exposed at the surface. A bioparticle is thus obtained, comprising a plasma membrane in which the fusion proteins are anchored at the level of their transmembrane domain, and which exposes at its surface the peanut allergen according to the present invention.

[0037] A “bioparticle” or “BP” refers to a biological nanoparticle consisting of a plasma membrane envelope in which one or more proteins are anchored, which contains no genetic material, which is non-infectious and incapable of multiplying, and which self-assembles to mimic the original structure of a virus. “Virus-like particles” or “VLP” are a specific class of bioparticles in which the structural and immunogenic constituents are of viral origin.

[0038] The bioparticles according to the invention typically have a diameter of between 120 and 200 nm.

[0039] The bioparticles according to the present invention are made of a plasma membrane into which specific type I or II fusion proteins are anchored.

[0040] A “plasma membrane” is a structure well known by the skilled person. A plasma membrane is a lipid bilayer made up of two layers of phospholipids. According to a specific embodiment, a portion of the plasma membrane used in the bioparticles is typical of lipid rafts. The expression "portion of plasma membrane typical of lipid rafts" is intended to mean a phospholipid bilayer (i.e. plasma membrane) found in the microdomains of lipid rafts. Such a bilayer is rich in cholesterol and in phospholipids, preferably in phosphatidylcholine and in phosphatidylethanolamine, and in sphingolipids, such as sphingomyelin, but poor in docosahexaenoic acid. In addition, it has a low density, and is insoluble in mild detergents (for example polysorbates).

[0041] The term “fusion protein” is intended to mean a protein comprising the various fragments a) to c), wherein said fragments are of different origin. In other words, fragments a) to c) are never present fused in the way they exist naturally.

[0042] The expression “type I transmembrane protein anchored in a membrane” is intended to mean a transmembrane protein of which the N-terminal end is extracellular and the C-terminal end is cytosolic. Consequently, the type I transmembrane protein comprises, from the N-terminal to C-terminal end the peanut allergen a), then the coiled-coil domain b) and, finally, the anchoring domain c).

[0043] 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 C-terminal end, the anchoring domain c), then the coiled-coil domain b) and, finally, the peanut allergen a).

[0044] Preferably, the fusion protein according to the invention is a type I transmembrane protein.

[0045] As mentioned above, the fusion protein according to the present invention comprises fragments a), b) and c) successively.

[0046] The term “successively” is intended to mean that fragments a) to c) are present in the order a)-b)-c) (or c)-b)-a)). These various fragments can be directly fused to one another, or else fused to one another via one or more linker(s). Preferably, the fusion protein according to the invention comprises a linker present between the sequences a) and b), and / or between the sequences b) and c).

[0047] Segment a): the peanut allergen

[0048] The “peanut” (Arachis hypogaea) is an annual herbaceous plant of 30 to 50 cm belonging to the botanical family Fabaceae, also known as Leguminosae. The major allergens in peanuts are generally considered to be Ara h 1 and Ara h 3 that are members of the cupin superfamily of proteins, and Ara h 2 and Ara h 6 that are members of the prolamin superfamily (Mueller et al. allergy and asthma reports 14 (2014): 1-9).

[0049] As mentioned above, the peanut allergen according to the present invention is a fragment of the Ara h 2 protein. The complete sequence of the Ara h 2 protein is set forth in SEQ ID No. 2 (which does not include the natural Ara h 2 signal peptide):RQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYERDPYSPSQDPYRQDPYTPSPYDR RGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCG LRAPQRCDLDVESGGRDRY (SEQ ID No. 2).

[0050] In the context of the present invention, the peanut allergen has the amino acid sequence selected from the group consisting of: the amino acid as set forth in SEQ ID No. 1 (QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQ);- a fragment of at least 27 consecutive amino acids of the sequence of SEQ No. 1 ; and- an amino acid sequence having at least 80% of sequence identity with the amino acid sequence as set forth in SEQ ID No. 1 or with fragments of at least 27 amino acids of the amino acid sequence as set forth in SEQ ID No. 1 .

[0051] As used herein, the percentage of sequence identity refers to comparisons among amino acid sequences and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The skilled person will know how to determine the percentage of identity between two amino acid sequences. Various algorithms known from the prior art can be used for measuring the identity between two sequences. For example, sequences can be compared using FASTA, Gap or Bestfit, which are programs from Wisconsin Package™, Genetics Computer Group (GCG), Madison, Wis. Alternatively, the sequences can be compared using the BLAST program (Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Gish and States, Nature Genet. 3: 266-272 (1993); Madden et al., Meth. Enzymol. 266: 131-141 (1996); Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997); Zhang and Madden, Genome Res. 7: 649- 656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)).

[0052] In the context of the present invention, the peanut allergen has a sequence identity of at least 80%, in particular at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% with the amino acid sequence as set forth in SEQ ID No. 1 or with fragments of at least 27 amino acids of the amino acid sequence as set forth in SEQ ID No. 1 .

[0053] According to a specific embodiment, the fragment of at least 27 amino acids of SEQ ID No. 1 comprises amino acid residues 9 to 23 of SEQ ID No. 1 , i.e. comprise the sequence DPYSPSQDPYSPSQ. This specific sequence represents an immunogenic epitope of Ara h 2 (Deak et al. Scientific reports 7.1 (2017): 3981 ; Deak et al. Proceedings of the National Academy of Sciences 116.18 (2019): 8966-8974).

[0054] According to another specific embodiment, the fragment of at least 27 amino acids of SEQ ID No. 1 comprises, in particular consists of, amino acid residues 9 to 36 of SEQ ID No. 1 , i.e. comprise the sequence DPYSPSQDPYSPSQDPDRRDPYSPSPY.

[0055] According to a preferred embodiment, the peanut allergen according to the present invention has the amino acid sequence as set forth in SEQ ID No. 1 .

[0056] Segment b): the coiled-coil domain (or oligomerization sequence) b)

[0057] The coiled-coil domain, or oligomerization sequence, comprises several sense or antisense alpha-helix motifs which are parallel to one another and form an organized matrix that has several well-characterized biological functions. These domains are omnipresent and are found as specific domains for many types of proteins in most organisms. Coiled-coil domains from various sources can assemble to form forms which range from a dimer to a heptamer; some coiled-coil domains will adopt different polymerization levels depending on the point mutations of their amino acid sequence.

[0058] A coiled-coil domain typically consists of a repeat motif of 7 amino acids, of "hxxhcxc" type, wherein "h" is a hydrophobic amino acid, "c" is a charged amino acid, and "x" is any amino acid.

[0059] According to a specific embodiment, the coiled-coil domain used in the context of the invention does not originate from a virus; it is not viral.

[0060] Among the coiled-coil domains that can be used according to the invention, mention will preferably be made ofthose from cortexillin, vimentin, tetrabrachion, golgins, proteins of the "Soluble N-ethylmaleimide-sensitive factor (NSF) Attachment protein REceptor" or SNARE superfamily, or else transcription factors such as GCN4 or a variant thereof, such as GCN4-pLI or GCN4-pll.

[0061] Preferably, the coiled-coil domain is that from the GCN4, GCN4-pLI or GCN4-pll transcription factor.

[0062] According to a preferred embodiment, the coiled-coil domain is the GCNA-pll trimerization sequence of yeast GCN4 transcription factor which has the amino acid sequence as set forth in SEQ ID No. 3: LKQIEDKIEEILSKIYHIENEIARIKKLIGESAA (SEQ ID No. 3).

[0063] The coiled-coil domain can also be chosen from the amino acid sequences as set forth in SEQ ID No. 4:RMKQIEDKLEEILSKLYHIENELARIKKLLGER (GCN4-pLI tetramerization sequence of yeast GCN4 transcription factor); SEQ ID No. 5: VKQLADAVEELASANYHLANAVARLAKAVGER (GCN4-pAA heptamerization sequence of yeast GCN4 transcription factor); SEQ ID No. 6 KQIEDKIENITSKIYNITNEIARIKKLIGNRT (IZN4 glycosylated oligomerization sequence of yeast GCN4 transcription factor), SEQ ID No. 7:INETADDIVYRLTVIIDDRYESLKNL (SNARE oligomerization sequence) and SEQ ID No. 8: LKSRLDTLSQEVALLKEQQALQTVCL (synthetic sequence mimicking a coiled-coil).

[0064] Segment c): the domain for anchoring in the plasma membrane (or transmembrane domain) c)

[0065] The transmembrane domain is a short sequence of lipophilic amino acids which interacts with the specific lipids of the plasma membrane components.

[0066] According to a specific embodiment, the plasma membrane comprises at least one portion typical of lipid rafts.

[0067] These anchoring domains are common (but not through a consensus sequence) to the surface proteins of viruses, but also to proteins which are naturally integrated into the membrane of the living cells. Each transmembrane domain participates in the bending and the budding of the plasma membrane.

[0068] Among the anchoring domains that can be used according to the invention, mention will preferably be made of those from the proteins listed in table 1 A:

[0069] Table 1A: Transmembrane proteins

[0070] Among the anchoring domains that can be used according to the invention, mention will preferably be made of those from the viral envelope proteins listed in table 1 B below:

[0071] Preferably, the anchoring domain that can be used according to the invention is chosen from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin corresponding to the amino acid sequence as set forth in SEQ ID No. 9: YQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI (SEQ ID No. 9) and the anchoring sequence of the PDLP1 protein (A0A0D3D8S3) corresponding to the amino acid sequence as set forth in SEQ ID No. 10:IALAVGGVAVLGFVIVCLLVLKSAMKKKSKYDSY (SEQ ID No. 10).

[0072] Linkers

[0073] The fusion protein according to the invention can advantageously comprise a linker between fragments a) and b), and / or between fragments b) and c).

[0074] Linkers are short sequences of amino acids (2 to 10 amino acids, preferably 2 to 6) which create a flexible arm. They may be useful for creating a flexible space between specific peptide domains, if the fact that the two domains are too close together interferes with correct assembling.

[0075] Preferably, the linker is a sequence of -(GGGS)n-type, wherein n is an integer. Preferably, the linker is chosen from SEQ ID No. 11 (n=1), SEQ ID No. 12 (n=2) and SEQ ID No. 13 (n=3).

[0076] Thus, according to a specific embodiment the fusion protein useful in the context of the present invention comprises successively:

[0077] a) the peanut allergen as defined above;

[0078] b) a coiled-coil domain having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8; and

[0079] c) an anchoring domain selected from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin and the anchoring sequence of the PDLP1 protein.

[0080] Thus, according to a very specific embodiment, the fusion protein comprises, preferably consists of, the amino acid sequence as set forth in SEQ ID No. 14: QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQL KQIEDKIEEILSKIYHIENEIARIKKLIGESAAGGGSYQILSIYSTVASSLALAIMMAGLSLWMCSNGSL QCRICI (SEQ ID No. 14).

[0081] Likewise, preferably, the BP according to the invention comprises:

[0082] - an envelope consisting of a plasma membrane; and

[0083] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0084] a) a peanut allergen as defined above ;

[0085] b) a coiled-coil domain having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8; and

[0086] c) an anchoring domain selected from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin and the anchoring sequence of the PDLP1 protein,

[0087] fragments b) and c) being exposed on the outside of the bioparticle.

[0088] Methods for producing the BP according to the present invention

[0089] According to a further aspect, the present invention also relates to a method for producing a bioparticle as described above, comprising expressing the fusion protein as described above in eukaryotic cells, preferably in plant cells. Surprisingly, the present inventors have shown that expressing the fusion protein described above allows mimicking the natural conformation of the peanut allergen presented. The present inventors have particularly shown that said peanut allergen displays a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues.

[0090] Typically, the nucleotide sequence coding for the fusion protein is placed in an expression vector using conventional methods.

[0091] The selection of a suitable expression vector will depend on the method for 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 instance a promoter, and DNA elements which control the processing of the transcripts, such as termination / polyadenylation sequences, and an expression cassette allowing for the expression of a silencing inhibitor. It also contains sequences such as t-DNAs which are required for the integration of a piece of DNA into the plant or into the plant cell.

[0092] Preferably, the expression vector comprises:

[0093] - at least one nucleotide sequence encoding the fusion protein, preferably functionally linked to a strong promoter, preferably a 35S promoter;

[0094] - an expression cassette allowing the expression of a silencing inhibitor, preferably p19; and

[0095] DNA elements which control the processing of the transcript, such as termination / polyadenylation sequences, preferably the Tnos sequence (nopaline synthase termination sequence).

[0096] The expression vector is preferably pAG01 as described in the international patent application No. WO2013 / 186495.

[0097] In order for the expressed fusion protein to be properly routed in the endoplasmic reticulum, their amino acid sequences can advantageously comprise a signal peptide. The presence of the signal peptide enables correct trafficking of said protein into the endoplasmic reticulum. The signal peptide is then cleaved. Thus, during the budding and the formation of the BPs according to the invention, the fusion protein no longer contains the signal peptide. Consequently, the BPs according to the invention do not contain signal peptides.

[0098] The signal peptide is any signal peptide recognized by a eukaryotic cell.

[0099] Preferably, the signal peptide is chosen from the natural signal peptide of pectate lyase and the signal peptide of tobacco chitinase.

[0100] Preferably, the signal peptide is that of tobacco chitinase, corresponding to the amino acid sequence as set forth in SEQ ID No. 15: MKTNLFLFLIFSLLLSLSSA (SEQ ID No. 15).

[0101] The promoter used for controlling the expression of the fusion protein is a strong promoter, and may be a plant gene promoter, such as for example the ubiquitin promoter, the ribulose-1 , 5- bisphosphate carboxylase small subunit promoter, Agrobacterium tumefaciens promoters, the nopaline synthase and octopine synthase promoters, or else viral promoters such as cauliflower mosaic virus (CaMV) 19S and 35S. Preferably, the strong promoter is 35S.

[0102] The vector is then introduced into a eukaryotic host cell for recombinant expression. The host cell may be a plant cell.

[0103] The general methods for culturing plants, and also the methods for introducing expression vectors into a plant tissue, are available to those skilled in the art. They are various and depend on the plant selected. Preferably, the plants will be cultivated according to the techniques described in the international patent application No. WO2013 / 186495. Such techniques comprise a first step of culturing the plant, under aeroponic or hydroponic conditions and under LED lighting. After this first step, the agroinfiltration of the plants is carried out under vacuum, using agrobacteria comprising DNA fragments encoding the fusion protein. This agroinfiltration step can be carried out by any means for producing a vacuum. Preferably, in the method used according to the invention, it is carried out under vacuum by Venturi effect. Among the agrobacteria that can be used according to the invention, mention is preferably made of the LBA4404, GV3101 , EHA 101 / 105 or C58 strains. Once the agroinfiltration step has been carried out, the plants are put back in culture, typically for 3 to 6 days, ideally while providing frequent misting of said plants for the first 6 hours of culture following the agroinfiltration. The BPs are then extracted and purified.

[0104] The BP extraction can be carried out by enzymatic extraction. This method is an adaptation of the method described in particular in the international application published under reference WO 2014 / 153674. Preferably, the enzymatic extraction of the BPs is carried out by means of the following steps:

[0105] - infiltration under vacuum (in particular as described above for the agroinfiltration) of the aerial part of plants (i.e. the leaves), in an enzymatic solution containing pectocellulosic enzymes, which does not exhibit any proteolytic activity; preferably, a mixture of pectinases and cellulases which is formulated at 4% in a medium comprising 50 mM of sodium citrate, pH 5.2, 0.5 M NaCI and 0.04% metabisulfite. Preferably, the macerozyme is formulated at 0.5% in a medium comprising 50 mM of sodium citrate, pH 5.2, 0.5 M NaCI and 0.04% metabisulfite,

[0106] - the leaves are subsequently sampled and then incubated in the enzymatic solution,

[0107] - the mixture is placed with shaking on an orbital shaker between 20 and 30 rpm at ambient temperature (i.e. approximately 20-23°C) for a period of between 30 minutes and 2 h,

[0108] - the digestate is then filtered, preferably on a 2-3 mm then 250 pm cloth, then optionally continuously centrifuged (for example at 1000 x g for 2-5 minutes), and the supernatant is recovered in order to perform a tangential filtration.

[0109] According to a specific embodiment, the nucleic acid sequence placed into the expression vector for further expression in a cell plant to obtain the bioparticle according to the present invention has the sequence as set forth in SEQ ID No. 16.

[0110] The BP according to the invention may be used in therapy. It may be used as a medicament. It may also be used in allergen immunotherapy (AIT), in particular for the treatment / prevention of peanut allergy in a patient.

[0111] In the context of the present invention, the “patient” or “subject” is a mammal (e.g. a dog, a cat, a pig, a rodent or a primate). In a particular embodiment, the patient is a human.

[0112] In the context of the invention, the term "treating" or "treatment", means reversing, alleviating, inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. A “therapeutically effective amount” is intended for a minimal amount of active agent (e.g., the bioparticle according to the present invention) which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a mammal is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disorder.

[0113] The invention will now be illustrated by means of the following examples.

[0114] ExamplesSynthesis of the bioparticle according to the present invention:

[0115] The cDNA encoding the fusion protein has a nucleic acid sequence as set forth in SEQ ID No. 16: tctagaggtaccatgaagaccaacctgttcttgttcctgatcttcagcctgctgctgagcctgtcatctgctcagcgtgatgaggattcttacgga cgtgatccttactcaccttctcaagatccctatagccctagccaggaccctgatagaagagacccatatagcccctcaccatacgatagaa ggggcgcaggatcatcccaacaccaagagaggtgctgcaacgaactgaatgaattcgagaataatcagcttaagcagattgaggataa gatcgaagagatcctgagcaagatctaccacatcgagaacgagatcgctaggatcaagaagctgatcggagaatctgctgctggtggtg gtagttaccagatcctgtctatctacagcaccgtggcttcatctcttgctctggctattatgatggctggtctgtctctgtggatgtgctctaacggtt ctcttcagtgcaggatctgcatttaaactagtgtcgac (SEQ ID No. 16). The fusion protein has the amino acid sequence as set forth in SEQ ID No. 17: MKTNLFLFLIFSLLLSLSSAQRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQ HQERCCNELNEFENNQLKQIEDKIEEILSKIYHIENEIARIKKLIGESAAGGGSYQILSIYSTVASSLAL AIMMAGLSLWMCSNGSLQCRICI (SEQ ID No. 17). The bioparticle obtained accordingly is referred to as “H2-BP”.

[0116] A soluble Ara h 2 mature protein was also synthesized for comparative purposes.

[0117] The bioparticles thereby produced were purified and analyzed. For soluble Ara h 2, samples were heated at 90° C for 5 min in denaturation buffer A (Tris 62.5 mM pH 6.8, 10% glycerol, 1% SDS and 2% p-mercaptoethanol). For bioparticles, samples were heated at 90° C for 5 min in denaturation buffer B (Tris 62.5 mM pH 6.8, 10% glycerol, 2.5% SDS and 5% p-mercaptoethanol). Both preparations were centrifuged at 8000 g for 15 min before loading on gels. SDS-PAGE was performed on 18% polyacrylamide gels. Following electrophoretic separation, gels were either silver- stained or transferred onto a nitrocellulose membrane (Amersham™ Protran™ 0.45pm NC) for immunodetection. The primary antibody used for immunodetection was a polyclonal rabbit-antisera directed against Ara h 2 at a 1 :5,000 dilution followed by a secondary goat anti-rabbit IgG antibody coupled to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) at a 1 : 30,000- dilution. Western blots were visualized with Amersham™ ECL™ Western Blotting Detection Reagents. The results are shown Figure 1 . These results show that the H2-BP can be synthesized and purified with very high production yields compatible with large-scale applications.Immunogenicity

[0118] The constructs were then tested in vivo to evaluate the production of IgGs in rats. The protocol is detailed in Figure 2. The dose administered was 300pL (10pg) per injection + / - adjuvant ASO3 (1 :1). IgG concentration in samples obtained at Day 0, 14, 28, 42 and 56 after injection was determined and compared to the values obtained with a reference FD1 bioparticle (presenting a fusion form of the Fel d 1 allergen). The results are shown in Figure 3. These results show an excellent immunogenicity which is in the same range than the reference FD1 -bioparticle. These results are significantly higher than those obtained with the complete mature Ara h 2-presenting bioparticle (corresponding to Castenmiller et al.), 2023, which only allowed obtaining an IgG concentration that was more than 200 times lower than the reference FD1 -BP after 28 days (data not shown). In particular, the present results show that the H2-BP allows triggering a humoral response in mice with doses as low as 10pg per injection. The H2-BP therefore provides an excellent immunogenicity (which is further improved upon co-administration of the shark ASO3 adjuvant - data not shown) which makes it an extremely promising candidate for Ara h 2 desensitization. Producing the H2-BP in plant hosts such as N. benthamiana plants allows obtaining a peanut allergen displaying a higher similarity to the natural allergen in terms of basophil activation (data not shown) due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope.Neutralizing antibodies production

[0119] Allergenicity of H2-BP was analyzed using biolayer interferometry. Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. The interactions between the H2-BP and characterized IgE directed Ara h 2 (obtained from patients) were analyzed accordingly. Ag-Ac association measurements are carried out using an “Octet® R8 Protein Analysis System” (Sartorius) equipped with Octet® Streptavidin (SA) Biosensors, for the first tests, or Octet® High Precision Streptavidin 2.0 (SAX2),for actual dosages. The Ag-Ac association reactions are carried out in black ELISA plates, in order to avoid the diffusion of light during the measurement (Greiner plates, ref: 655209). The interferometry data are analyzed using Octet® Analysis studio software. Epitope binning was further used to characterize the binding of mAbs to Ara h 2. In epitope binning, mAbs specific to the same target protein are tested pairwise against all mAbs in a set to assess whether they block one another’s binding to a specific site of the antigen or not. The mAbs that block binding to the same epitope are “binned” together. The results showed that the H2-BP according to the present invention allows for the production of Ara h 2 neutralizing antibodies. Furthermore, the Ara h 2 specific neutralizing IgG obtained upon H2-BP administration were shown to inhibit the crucial immunogenic site comprised in the peanut allergen carried by BP (i.e. comprised in SEQ ID No. 1), but also the major epitopes recognized by patient IgE but absent from the fragment carried by BP (i.e. not comprised in SEQ ID No. 1).

[0120] The bioparticles according to the present invention are therefore a novel and extremely efficient tool for the treatment / prevention of peanut allergies.

Claims

Claims

1. A bioparticle comprising:- an envelope consisting of a plasma membrane; and- at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively: a) a peanut allergen; b) a coiled-coil domain or oligomerization sequence; and c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment; fragments a) and b) being exposed at the at the surface of the bioparticle; wherein said peanut allergen has the amino acid sequence selected from the group consisting of the amino acid sequence as set forth in SEQ ID No. 1 : QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQ; a fragment of at least 27 consecutive amino acids of the sequence of SEQ No. 1 ; and an amino acid sequence having at least 80% of sequence identity with the amino acid sequence as set forth in SEQ ID No. 1 or with a fragment of at least 27 amino acids thereof.

2. The bioparticle according to claim 1 , wherein said peanut allergen has an amino acid sequence having at least 90% of sequence identity with the amino acid sequence as set forth in SEQ ID No. 1 or with fragments of at least 27 amino acids thereof.

3. The bioparticle according to claim 1 or 2, wherein said peanut allergen has an amino acid sequence having at least 95% of sequence identity with the amino acid sequence as set forth in SEQ ID No 1 or fragments of at least 27 amino acids thereof

4. The bioparticle according to any one of claims 1 to 3, wherein said fragment of at least 27 amino acids comprises amino acid residues 9 to 23 of SEQ ID No. 1 which are DPYSPSQDPYSPSQ.

5. The bioparticle according to any one of claims 1 to 4, wherein said fragment of at least 27 amino acids comprises amino acid residues 9 to 36 of SEQ ID No. 1 which are DPYSPSQDPYSPSQDPDRRDPYSPSPY.

6. The bioparticle according to any one of claims 1 to 5, wherein said peanut allergen has the amino acid sequence as set forth in SEQ ID No. 1.

7. The bioparticle according to any one of claims 1 to 5, wherein said fusion protein has the amino acid sequence a set forth in SEQ ID No. 14: QRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQL KQIEDKIEEILSKIYHIENEIARIKKLIGESAAGGGSYQILSIYSTVASSLALAIMMAGLSLWMCSNGSL QCRICI.

8. The bioparticle according to any one of claims 1 to 7, for use in therapy.

9. The bioparticle according to any one of claims 1 to 7, for use in the treatment and / or prevention of peanut allergy.

10. A method for producing the bioparticle according to any one of claims 1 to 7, wherein said fusion protein is expressed in a eukaryotic cell.

11. The method according to claim 10, wherein said eukaryotic cell is a plant cell.

12. The method according to claim 10 or 11, wherein said method comprises introducing the nucleic acid sequence of SEQ ID No. 16 in an expression vector and expressing said vector in a eukaryotic cell.

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