MuNS fusion proteins capable of forming microspheres and uses thereof

Abstract

The present invention relates to a fusion protein encoding a polypeptide capable of forming a minimum region of microspheres and / or nanospheres based on a positive reovirus muNS protein modified to allow the addition of the polypeptide at the C-terminus.

Description

TECHNICAL FIELD

[0001] The present invention relates to a fusion protein comprising a polypeptide based on the minimal region of the orthoreovirus (Reoviridae) Orthoreovirus muNS protein that is modified to allow the addition of molecules at its C-terminus. BACKGROUND

[0002] Avian reoviruses are members of the Orthoreovirus genus, one of the twelve genera of the Reoviridae family. These viruses are important avian pathogens and cause significant economic losses in the poultry industry. Avian reoviruses are non-lipid enveloped viruses that replicate in the cytoplasm of infected cells and have a genome composed of ten double-stranded RNA segments that are surrounded by a double concentric protein coat with a diameter of 85 nm. The genome segments are divided into three classes based on their electrophoretic mobility: three segments of the L (large) class, three segments of the M (medium) class, and four segments of the S (small) class. Except for the tripartite S1 segment, all other genes are monocistronic. These genome segments are transcribed by RNA-dependent polymerases to produce messenger RNAs (mRNAs) with the same nucleotide sequence as the positive strand of the double-stranded RNA segment. Viral mRNAs have a dual function in infected cells: they program the synthesis of viral proteins in ribosomes and also serve as templates for the synthesis of the negative strands of these genome segments.

[0003] A new system is described that uses the muNS protein inclusion of the mammalian reovirus to form a platform for in vivo detection of protein interactions in mammalian cells (Miller et al. , Mol Cell Proteomics., 2007 6 1027-1038) and has also been adapted for use in yeast (Schmitz et al.In this system, the target protein is fused to the C-terminal region of muNS such that the fusion generates a cytoplasmic inclusion and attracts the target protein ligand to it. In the yeast system, these authors demonstrated that their system was superior to the two-hybrid system in the number and type of interactions detected, at least for the proteins tested in the article. However, this system has several problems, among which the following stand out: i) some proteins can fold incorrectly when fused to muNS-Mi and lose the ability to interact with their ligand; ii) some proteins can interfere with the muNS-Mi inclusion formation capacity and not form this inclusion or generate amorphous intracellular aggregates, greatly altering the detection of interactions; iii) the intracellular localization of the target protein or ligand can not be suitable for detection in the cytoplasmic inclusion.

[0004] Document WO 2011 / 098652 describes a system that uses the formation of inclusion bodies of the orthoreovirus muNS protein as a platform for the purification of proteins and simultaneously for the detection of protein-protein interactions, both in vitro and in vivo. This platform is based on the minimal region of the avian orthoreovirus muNS protein capable of forming inclusion bodies that recruit a peptide tag and a protein bound to this tag to said inclusion bodies. The peptide tag comprises a minimal region of the muNS protein of the orthoreovirus that can be incorporated into the inclusion bodies formed by the muNS protein.

[0005] Therefore, it is necessary to improve this system to reduce its complexity and increase the usability of the muNS system. SUMMARY

[0006] The inventors have developed a system based on a minimal fragment capable of forming inclusion bodies of the muNS protein (muNS-Mi), in which they have successfully overcome the obstacle of modifying the C-terminal end of muNS-Mi by adding a sortase recognition sequence that does not affect the capacity of muNS-Mi to form inclusion bodies.

[0007] In a first aspect, the present application relates to a fusion protein comprising the following components: (i) a polypeptide comprising a sequence selected from the group consisting of: - the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1), - the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2), and - a functional equivalent variant of any of the above sequences having the capacity to form nanospheres and / or microspheres, and (ii) a polypeptide selected from the group consisting of: (a) a polypeptide comprising a sortase recognition motif or a remainder of a sortase recognition motif generated upon a sortase-mediated reaction, wherein component (ii) is fused to the carboxy terminus of component (i), or (b) a polypeptide comprising a sortase acceptor motif, wherein component (ii) is fused to the amino terminus of component (i).

[0008] In another aspect, the present application relates to a nanosphere or microsphere comprising a fusion protein according to the present application.

[0009] In another aspect, the present application relates to a polynucleotide encoding a fusion protein according to the present application.

[0010] In another aspect, the present application relates to an expression cassette comprising a polynucleotide according to the present application.

[0011] In another aspect, the present application relates to a vector comprising a polynucleotide according to the present application or an expression cassette according to the present application.

[0012] In another aspect, the present application relates to a vector composition comprising a vector according to the present application and a second vector comprising a second polynucleotide encoding a polypeptide selected from the group consisting of: a polypeptide comprising the sequence 477-542 of an avian orthoreovirus muNS protein (SEQ ID NO: 25), a polypeptide comprising the sequence 561-622 of a mammalian orthoreovirus muNS protein (SEQ ID NO: 26), a functionally equivalent variant of any of the above polypeptides retaining the ability to be incorporated into a nanosphere.

[0013] In another aspect, the present application relates to a cell comprising a fusion protein according to the present application, a polynucleotide according to the present application, an expression cassette, a vector or a vector composition according to the present application.

[0014] In another aspect, the present application relates to a method for producing a fusion protein according to the present application, hereinafter method I of the present application, comprising: (a) expressing in a cell a first polynucleotide encoding said fusion protein, (b) subjecting said cell to conditions suitable for the formation of nanospheres or microspheres, and (c) concentrating said nanospheres or microspheres.

[0015] In another aspect, the present application relates to a method for producing a protein, hereinafter method II of the present application, comprising: (a) expressing in a cell a first polynucleotide encoding a fusion protein according to the present application and a second polynucleotide encoding a second fusion protein comprising the components: (i) a polypeptide selected from the group consisting of: - a polypeptide comprising the sequence 477-542 (SEQ ID NO: 25) of the muNS protein of an avian orthoreovirus, - a polypeptide comprising the sequence 518-721 (SEQ ID NO: 26) of the muNS protein of a mammalian orthoreovirus, - a functionally equivalent variant of any of the above polypeptides which retains the ability to be incorporated into nanospheres and / or microspheres, and (ii) a second polypeptide of interest, wherein component (ii) is the protein of interest; (b) subjecting the cells to conditions suitable for the formation of nanospheres and / or microspheres, and (c) concentrating the nanospheres and / or microspheres.

[0016] In another aspect, the present application relates to a method for producing a first polypeptide of interest, hereinafter method III according to the present application, wherein said method comprises: (a) producing a fusion protein according to the present application by method I according to the present application, wherein said fusion protein comprises: - component (ii) (a) and component (iii) fused to the amino terminus of component (i), wherein component (iii) is said polypeptide of interest, and - a protease recognition sequence, said protease recognition sequence being between its component (i) and component (iii), (b) subjecting the nanospheres and / or microspheres to conditions which cause their disintegration, thereby causing the separation of the fusion protein from the nanospheres and / or microspheres, (c) contacting the product resulting from step (b) with a protease specific for the recognition sequence linking component (i) and component (iii) of the fusion protein under conditions suitable for the proteolysis of the fusion protein, followed by the separation of component (i) and component (iii) of the fusion protein, (d) subjecting the product of step (c) to conditions suitable for the formation of said nanospheres and / or microspheres, and (e) separating the nanospheres and / or microspheres from component (iii).

[0017] In another aspect, the present application relates to a protein obtainable according to method I, II or III according to the present application.

[0018] In another aspect, the present application relates to a method for producing nanospheres or microspheres according to the present application, hereinafter method IV according to the present application, wherein said method comprises the following steps: (a) producing said fusion protein by method I according to the present application, and (b) subjecting said cells to conditions suitable for the formation of nanospheres and / or microspheres.

[0019] In another aspect, the present application relates to a pharmaceutical or immunogenic composition comprising nanospheres and / or microspheres according to the present application and a pharmaceutically acceptable excipient.

[0020] In another aspect, the present application relates to nanospheres and / or microspheres according to the present application for use in medicine.

[0021] In another aspect, the present application relates to nanospheres or microspheres according to the present application or an immunogenic composition according to the present application for use in the treatment and / or prevention of bluetongue, wherein said fusion protein comprised in said nanospheres or microspheres is characterized by comprising at least one of: Bluetongue virus 4 (BTV) outer capsid protein 2 (VP2); Bluetongue virus 4 (BTV) core protein 7 (VP7); Bluetongue virus 4 (BTV) non-structural protein 1 (NS1); a protein having a sequence functionally equivalent to any of the above proteins.

[0022] In another aspect, the present application relates to nanospheres or microspheres according to the present application or an immunogenic composition according to the present application for use in the treatment and / or prevention of African horse sickness, wherein said fusion protein comprised in said nanospheres or microspheres is characterized by comprising at least one of: African horse sickness virus (AHSV) non-structural protein 1 (NS1); a protein having a sequence functionally equivalent to any of the above proteins.

[0023] In another aspect, the present application relates to nanospheres or microspheres according to the present application or a pharmaceutical composition according to the present application for use in the treatment and / or prevention of type 1 diabetes, wherein said fusion protein comprised in said nanospheres or microspheres is characterized by comprising a glucose-6-phosphatase 2 protein (IGRP).

[0024] In another aspect, the present application relates to a method of inducing type 1 diabetes in an animal model, said method comprising administering to the animal an effective amount of nanospheres or microspheres according to the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Expression of MiST-IC. A. PAGE analysis of uninduced (1) or IPTG induced (2) bacteria transformed with pET Duet 1. MiST. MiST purification is shown in the third lane. MW markers are shown on the left of panel B, while the theoretical position of MiST is shown on the right of panel B. The same set of analyzed bacteria in lane 2a was visualized by TEM.

[0026] Figure 2 Labelling of MiST-IC with AZDye 488-Gly-Gly-Gly. A. Purified NS obtained after expression of the protein indicated on the top of each image was incubated with the fluorescent compound AZDye 488-Gly-Gly-Gly (structure below the image) in the presence of sortase A. Excess dye was removed by dialysis and NS were observed under a fluorescence microscope. B. Samples of muNS-Mi (1 and 3) or MiST (2 and 4) incubated (3, 4) or not (1, 2) with AZDye 488-Gly-Gly-Gly in the presence of sortase A were resolved by PAGE and the unstained and unfixed gels visualized under UV light. Bands corresponding to fluorescently labelled MiST (4) and to excess fluorescent label (3, 4) are indicated with arrows. C. The same samples as analyzed in A were subjected to Western blot analysis with an anti-muNS antibody. Positions of MiST and muNS-Mi are indicated with arrows.

[0027] Figure 3 Labelling of MiST-IC with AZDye 488-Gly-Gly-Gly. A. Extracts of uninduced (1) or IPTG induced bacteria previously transformed with double plasmids expressing AvPAL and muNS-Mi (2) or MiST (3). 4 and 5 correspond to purified NS of extracts corresponding to 2 and 3.

[0028] Figure 4 T transfection of HeLa cells with a plasmid directing the expression of the viral muNS protein (muNS) results in the production of a fusion protein formed by muNS fused at its C-terminus to a hemagglutinin epitope (muNS-HA). Cells were fixed with paraformaldehyde and analyzed by immunofluorescence using an anti-muNS antibody. The addition of HA at the C-terminus of muNS was observed to result in the loss of the ability of muNS to form cytoplasmic inclusions.

[0029] Figure 5Expression and purification of versions of MiST in bacteria. A. Electrophoretic analysis. SDS-PAGE analysis of bacteria transformed with pETDuetl. MiST (1, 2) or PETDuetl. G4S-MiST (3, 4) before (1, 3) or after (2, 4) induction with 1 mM IPTG. B. TEM analysis. Micrographs of induced bacteria showing correct formation of MiST and G4S-MiST NS. C and D. Electrophoretic analysis of purification. Final purification of MiST (C) and G4S-MiST (D) NS is shown. Red and blue arrows show the position of MiST and G4S-MiST, respectively.

[0030] Figure 6 Expression and purification of versions of MiST loaded with IC tags in bacteria Av PAL. A. Electrophoretic analysis. SDS-PAGE analysis of bacteria transformed with pETDuetl. MiST / IC-PAL (1, 2) or PETDuetl. G4S-MiST2 / IC-PAL (3, 4) before (1, 3) or after (2, 4) induction with 1 mM IPTG. B. TEM analysis. Micrographs of induced bacteria showing correct formation of MiST and G4S-MiST NS. C and D. Electrophoretic analysis of purification. Final purification of MiST (C) and G4S-MiST (D) NS is shown. Red and blue arrows show the position of MiST and G4S-MiST, respectively. Av PAL. A. Electrophoretic analysis. SDS-PAGE analysis of bacteria transformed with pETDuetl. MiST / IC-PAL (1, 2) or PETDuetl. G4S-MiST2 / IC-PAL (3, 4) before (1, 3) or after (2, 4) induction with 1 mM IPTG. B. TEM analysis. Micrographs of induced bacteria showing correct formation of MiST and G4S-MiST NS. C and D. Electrophoretic analysis of purification. Final purification of MiST (C) and G4S-MiST (D) NS is shown. Red and blue arrows show the position of MiST and G4S-MiST, respectively. Av PAL. A. Electrophoretic analysis. SDS-PAGE analysis of bacteria transformed with pETDuetl. MiST / IC-PAL (1, 2) or PETDuetl. G4S-MiST2 / IC-PAL (3, 4) before (1, 3) or after (2, 4) induction with 1 mM IPTG. B. TEM analysis. Micrographs of induced bacteria showing correct formation of MiST and G4S-MiST NS. C and D. Electrophoretic analysis of purification. Final purification of MiST (C) and G4S-MiST (D) NS is shown. Red and blue arrows show the position of MiST and G4S-MiST, respectively.

[0031] Figure 7: SrtA-mediated derivatization of MiST-IC NS. A. Fluorescence microscopy analysis. Purified NS obtained after protein expression as indicated on the upper part of each image were incubated with the substrate AZDye 488-Gly-Gly-Gly in the presence of SrtA. They were observed with a 100x lens. B. Fluorescence-labeled SDS-PAGE analysis. NS samples of muNS-Mi (lanes 1 and 2), MiST (lanes 3 and 4) and G4S-MiST (lanes 5 and 6) incubated (2, 4, 6) or not (1, 3, 5) with the mentioned fluorescent substrates in the presence of SrtAd were resolved by SDS-PAGE and the unstained and unfixed gel was visualized under UV light. Fluorescence-labeled bands corresponding to MiST and G4S-MiST (lanes 4 and 6) are indicated with purple arrows. Excess of unbound fluorescent compound is indicated with black triangles at the leading edge (lanes 2, 4 and 6). C. Coomassie staining of proteins resolved by SDS-PAGE. The same polyacrylamide gel as in B was subjected to Coomassie staining. The position of labeled MiST and G4S-MiST is indicated with purple arrows. The position of SrtA is indicated with a green triangle. D. Western blot analysis. The same samples as analyzed in the above gel were subjected to Western blot analysis using an anti-muNS antibody. Lane 7 corresponds to a SrtA sample.

[0032] Figure 8 : SrtA-mediated derivatization of MiST-IC NS loaded with av-PAL. The same analysis as in the previous figure was repeated with MiST or G4S-MiST nanospheres loaded with AvPAL, resulting in the same results.

[0033] Figure 9 : Fluorescence microscopy analysis. Samples of the indicated versions of MiST, loaded or not with AvPAL, were incubated with polyglycine-containing eGFP in the presence (Srt+) or not of sortase A, as indicated on the upper part of each image.

[0034] Figure 10 : PAGE-UV analysis. Samples were mixed with Laemmli but not boiled, thus maintaining a semi-denatured conformation of eGFP (“fluorescent gel”). In this case, the band corresponding to the size of eGFP in all lanes is indicated with a green arrow. Sample 1 is an unreacted eGFP control. Whereas samples 2, 3, 4 and 5 are NS of different versions of MiST reacted with eGFP in the presence of sortase A (1: MiST, 2: G4S-MiST, 3: MiST / IC-AvPAL, 4: G4S-MiST / IC-AvPAL).

[0035] Figure 11SDS-PAGE analysis of Gly-GFP decorated MiST nanospheres. Lane 1 corresponds to Gly- containing GFP. Lane 2 corresponds to AvPAL loaded MiST NS. Lanes 3 and 4 are AvPAL loaded Mist (3) or G4S-MiST (4) NS after sortase A reaction in the presence of Gly- containing GFP. The position of each protein residue is indicated with arrows on the right side of the image.

[0036] Figure 12 Expression of 3G-MiST and TEV-3G-MiST. SDS-PAGE analysis of bacteria transformed with plasmid pDuetl-3G.MiST (left panel) or pDuetl-TEV-3G-MiST (right panel) before (1) or after (2) induction with 1 mM IPTG. Gels were stained with Coomassie blue. The position of molecular weight markers is indicated on the left side of each image.

[0037] Figure 13 Purification and SrtA-mediated derivatization of 3G-MiST and TEV-3G-MiST. A. Fluorescence microscopy analysis. A: Coomassie-stained SDS-PAGE gel containing samples of purified 3G-MiST (left panel) and TEV-3G-MiST (right panel). B. Fluorescently labeled SDS-PAGE analysis. NS samples of TEV-3G-MiSt shown in A were incubated with or without the fluorescent substrate 5-FAM-LPETGG, sortase A or TEV protease as indicated in the lower part of the figure. The gel was observed and a picture was taken under UV light to visualize covalently bound substrate.

[0038] Figure 4 Purification and SrtA-mediated derivatization of AvPAL loaded 3G-MiST and TEV-3G-MiST. A. Fluorescence microscopy analysis. A: Coomassie-stained SDS-PAGE gel containing samples of 3G-MiST (left panel) and TEV-3G-MiST (right panel), both loaded with AvPAL and purified. B. Fluorescently labeled SDS-PAGE analysis. NS samples of TEV-3G-MiSt shown in A were incubated with or without the fluorescent substrate 5-FAM-LPETGG, sortase A or TEV protease as indicated in the lower part of the figure. The gel was observed and a picture was taken under UV light to visualize covalently bound substrate. DETAILED DESCRIPTION

[0039] The authors of the present invention have shown that it is possible to obtain a fusion protein comprising the minimal region of the muNS protein (muNS-Mi) forming nanospheres / microspheres, and a signal at the C-terminus of muNS-Mi which allows post-translational modification of muNS-Mi in monomers and in aggregates in the form of nanospheres / microspheres.

[0040] Fusion proteins of the application In this sense, the first aspect, the present invention relates to a fusion protein, hereinafter referred to as fusion protein of the present invention, comprising the following components: (i) a polypeptide comprising a sequence selected from the group consisting of: - the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1), - the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2), and - a functional equivalent variant of any of the above sequences having the ability to form nanospheres and / or microspheres, and (ii) a polypeptide selected from the group consisting of: (a) a polypeptide comprising a sortase recognition motif or the remainder of a sortase recognition motif generated upon a sortase-mediated reaction, wherein component (ii) is fused to the carboxy terminus of component (i), or (b) a polypeptide comprising a sortase acceptor motif, wherein component (ii) is fused to the amino terminus of component (i).

[0041] The term "protein" used herein interchangeably with "polypeptide" refers to a chain of amino acids of any length, wherein the different amino acids are bound to each other by peptide bonds.

[0042] The term "fusion protein" as used herein refers to a polypeptide comprising two or more regions derived from different or heterologous proteins. By definition, fusion proteins are never found in nature.

[0043] The fusion protein of the present invention comprises two components: Component (i) - polypeptide Component (i) of the fusion protein of the present invention is a polypeptide selected from the group consisting of: - the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1), - the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2), and - a functional equivalent variant of any of the above sequences having the ability to form nanospheres and / or microspheres.

[0044] In one particular embodiment, the polypeptide that is part of the fusion protein of the application comprises or consists of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1).

[0045] The term "avian orthoreovirus" or "avian reovirus" as used herein refers to one of the 12 genera belonging to the Reoviridae family, and in particular to one of the genera that infect birds. They have a double-stranded RNA genome and are therefore viruses of group III.

[0046] The term "avian orthoreovirus muNS protein" as used herein refers to one of the non-structural proteins encoded by the avian reovirus or avian orthoreovirus M3 gene, and is the only reovirus protein that is able to form nanospheres and / or microspheres when expressed in the absence of other factors (Touris-Otero et al. Virology, 319; 94-1069). It is a protein of 635 amino acids, defined by accession number Ay608700 in the NCBI database (version Ay608700.1, August 7, 2004) or accession number AAS78998 in the NCBI database (version AAS78998.1, April 10, 2006). In one particular embodiment, the avian orthoreovirus muNS protein has the sequence SEQ ID NO: 3.

[0047] The sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) is a fragment of the avian orthoreovirus muNS protein, comprising the minimal region of the muNS protein of the orthoreovirus that has the ability to form inclusion bodies when expressed in cells or a functional equivalent variant thereof.

[0048] The term "inclusion body(s)" as used herein refers to nuclear or cytoplasmic (typically proteinaceous) aggregates. In particular, the protein of the orthoreovirus genus that forms inclusion bodies is the muNS or μNS protein, which is able to form inclusion bodies when expressed in the absence of other viral factors (Touris-Otero et al., supra ). When inclusion bodies are formed by muNS-Mi proteins, they are small in size and spherical, forming the so-called "nanospheres" and / or "microspheres". The difference between nanospheres and microspheres lies in their size (see below). In this sense, in the present application, the term "inclusion body" equally encompasses the terms "nanosphere" and "microsphere".

[0049] The ability of an avian orthoreovirus muNS protein to form inclusion bodies is determined by the presence of a minimal region of the avian orthoreovirus muNS protein corresponding to residues 448 to 635 of said protein. However, any fragment of the avian orthoreovirus muNS protein comprising said minimal fragment is able to form said inclusion bodies.

[0050] In this sense, in a particular embodiment, component (i) of the fusion protein of the application comprises the full sequence of an avian orthoreovirus muNS protein, or a fragment of the avian orthoreovirus muNS protein comprised between residues 1 and 635 of said protein, or a fragment of contiguous amino acids from residue 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 447 up to residue 635 of the avian orthoreovirus muNS protein. In another particular embodiment, component (i) of the fusion protein of the application comprises a fragment from residue 447 up to residue 636 of the avian orthoreovirus protein, or a fragment from residue 447 up to residue 640, 650, 660, 670, 680, 690, 700, 710, 720 or up to residue 721 of the avian orthoreovirus protein. In another particular embodiment, component (i) of the fusion protein of the application comprises a fragment of the avian orthoreovirus muNS protein comprised between residues 10 and 720 of said protein, or between residues 10 and 700 of said protein, or between residues 20 and 700 of said protein, or between residues 30 and 700 of said protein, or between residues 40 and 700 of said protein, or between residues 60 and 680 of said protein, or between residues 80 and 660 of said protein, or between residues 100 and 640 of said protein, or between residues 120 and 635 of said protein.

[0051] In another particular embodiment, the polypeptide that is part of the fusion protein of the application comprises or consists of the sequence 518-721 of a mammalian orthoreovirus muNS protein (SEQ ID NO: 2).

[0052] The term "mammalian orthoreovirus" as used herein refers to one of the 12 genera belonging to the Reoviridae family, and in particular to one of the genera that infects mammals. They have a double-stranded RNA genome and thus belong to the group III viruses.

[0053] The term "mammalian orthoreovirus muNS or muNS protein" as used herein refers to one of the non-structural proteins encoded by a mammalian reovirus or a mammalian orthoreovirus and is the only mammalian reovirus protein that is capable of forming microspheres when expressed in the absence of other viral factors (Becker, M. M. et al. 2003. J. Virol. 77:5948-5963). It is a protein of 721 amino acids, defined by accession number ABP48918 in the NCBI database (version ABP48918.1, April 11, 2008). et al. 2003. J. Virol. 77:5948-5963). It is a protein of 721 amino acids, defined by accession number ABP48918 in the NCBI database (version ABP48918.1, April 11, 2008).

[0054] In a preferred embodiment wherein the orthoreovirus muNS protein is a mammalian orthoreovirus muNS protein, the minimal region of the mammalian orthoreovirus muNS protein having the ability to form inclusion bodies when expressed in a cell comprises the region corresponding to residues 518 to 721 (SEQ ID NO: 2). In an even more preferred embodiment, component (i) of the fusion protein of the application comprises the full protein or from residue 2 and up to residue 721 of said protein, or from residue 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 470, 480, 490, 500, 510 of said protein and up to residue 721.

[0055] In a particular embodiment, the polypeptide that is part of the fusion protein of the application comprises or consists of a functional equivalent variant of (i) sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or (ii) sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) having the ability to form nanospheres and / or microspheres.

[0056] In one embodiment, a functionally equivalent variant of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% of the nanosphere / microsphere-forming ability of the sequence from which it is derived.

[0057] A functionally equivalent variant of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) includes functionally equivalent variants that show at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence from which they are derived.

[0058] The degree of identity between a variant and the sequence from which it is derived is determined using algorithms and computer methods known to those skilled in the art. Preferably, the BLASTP algorithm is used to determine the identity between two amino acid sequences [BLAST Manual, Altschul, S., et al. , NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al. , J. Mol Biol, 215: 403 - 410 (1990)]. In a more specific embodiment, the sequence identity between a functionally equivalent variant and the protein sequence from which it is derived is calculated over the full length of the polypeptide.

[0059] A "functionally equivalent variant" is understood to mean all those peptides which are derived from the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or from the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) by modification, insertion, substitution and / or deletion of one or more amino acids, provided that the function of the muNS protein sequence from which they are derived is essentially retained.

[0060] The sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) and the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) have the ability to form nanospheres and / or microspheres in cells. Functionally equivalent variants of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) and the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) substantially retain the ability of said sequences to form nanospheres / microspheres in cells. Methods suitable to determine the ability to form nanospheres / microspheres in cells include, but are not limited to, the method described in Example 1 of patent application WO 2011 / 098652, which is based on the expression of the protein of interest in cells and on the analysis of the formation of nanospheres / microspheres (inclusion bodies) by indirect immunofluorescence using polyclonal antibodies directed against the epitope of interest or fused to the protein of interest, thus allowing to check the formation of nanospheres and / or microspheres. In a particular embodiment, functionally equivalent variants of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or of the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more than 100% of the ability of said sequences to form nanospheres / microspheres in cells.

[0061] In a particular embodiment, functionally equivalent variants of the sequence 448-635 of the avian orthoreovirus muNS protein (SEQ ID NO: 1) or of the sequence 518-721 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 2) include functionally equivalent variants showing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with respect to the sequence from which they are derived and retaining at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more than 100% of the ability of said sequences to form nanospheres / microspheres in cells.

[0062] It will be apparent to the person skilled in the art that it can be useful for the component (i) of the fusion protein of the application to have at its amino terminus another polypeptide of interest. In this sense, in a particular embodiment, the fusion protein of the application further comprises component (iii) fused to the amino terminus of component (i), wherein component (iii) is a first polypeptide of interest.

[0063] As will be appreciated by the skilled person, it can be useful to separate component (iii) from component (i). One possibility to separate these components is to use a peptide whose sequence contains a protease cleavage site, allowing separation of the two components.

[0064] In one embodiment, component (iii) is fused to component (i) via a protease recognition sequence. The term "protease" or "peptidase" as used herein refers to enzymes that break down the peptide bonds of proteins, for which they use water molecules to break, and thus they are classified as hydrolases. Examples of protease cleavage sites suitable for incorporation into the fusion protein of the application include, but are not limited to, enterokinase (cleavage site DDDDK; SEQ ID NO: 5), Factor Xa (cleavage site IEDGR; SEQ ID NO: 6), Thrombin (cleavage site LVPRGS; SEQ ID NO: 7), TEV protease (cleavage site ENLYFQG; SEQ ID NO: 8), PreScission protease (cleavage site LEVLFQGP; SEQ ID NO: 9), Intein, etc. In one embodiment, the protease recognition sequence is an enterokinase recognition sequence or a Factor Xa recognition sequence.

[0065] In another embodiment, component (iii) comprises a secretory pathway signal peptide.

[0066] The term "secretory pathway signal peptide" used herein interchangeably with "signal sequence" or "signal peptide" or "localization signal peptide" refers to a short peptide (5-30 amino acids long) present at the N-terminus that directs the transport of proteins to the secretory pathway, whether they are proteins that reside in certain organelles (ER, Golgi or endosomes), proteins that are secreted by the cell, or proteins that are inserted into the cell membrane. The signal peptide directs translocation of the protein bound to the ER. During or after translocation, the signal peptide is cleaved by signal peptidase, generating a free signal peptide and a mature protein.

[0067] Non-limiting examples of secretory pathway signal peptides include signal peptides found in class I and class II major histocompatibility complex molecules, cytokine or immunoglobulin signal sequences, constant chain signal sequences or Lampl, Tapasin, Erp57, Calreticulin and Calnexin. In one embodiment, the secretory pathway targeting sequence is selected from: the sequence MGWSLILLFLVAVATGVHSQ (SEQ ID NO: 10); the sequence MMSFVSLLLVGILFWATEAEQLTKCEVFQ (SEQ ID NO: 11); Human PTH1R signal peptide (MGTARIAPGLALLLCCPVLSSAYAL, SEQ ID NO: 12); Human cytochrome c oxidase VIII mitochondrial localization sequence (MSVLTPLLLRGLTGSARRLPVPRAK, SEQ ID NO: 13) Human mGluR5 signal peptide (MVLLLILSVLLLKEDVRGSA, SEQ ID NO: 14); Human GABAB2R signal peptide (MASPRSSGQPGPPPPPPPPPARLLLLLLLPLLLPLAPG, SEQ ID NO: 15); Human calreticulin signal peptide (MLLSVPLLLGLLGLAVA, SEQ ID NO: 16); and Human Ig gamma 2b heavy chain signal peptide (MGWSCIILFLVATATGKGLTVAGLRSGHIYG, SEQ ID NO: 17).

[0068] In a preferred embodiment, the secretion pathway signal peptide comprises the sequence MGWSLILLFLVAVATGVHSQ (SEQ ID NO: 10).

[0069] The lack of glycosylation allows for more efficient inclusion body formation in the secretion pathway when the fusion protein comprises a signal peptide and is progressing through the secretion pathway.

[0070] In a particular embodiment, component (i) of the fusion protein of the application comprises a secretion pathway signal peptide and is free of N-glycosylation. Thus, in a particular embodiment, component (i) lacks an N-glycosylation consensus sequence. The term "N-glycosylation consensus sequence" as used herein refers to a sequence consisting of -Asn-X-Ser / Thr, wherein X is not proline, which is the most representative consensus sequence, and the least abundant N-glycosylation consensus sequences, such as the sequences -Asn-Gly-, -Asn-X-Cys and -Asn-X-Val.

[0071] In a preferred embodiment, component (i) comprises a secretion pathway signal peptide and the sequence SEQ ID NO: 1 or a functional equivalent variant thereof, wherein the amino acid at position 57 is not an Asn residue. In an even more preferred embodiment, component (i) comprises the sequence SEQ ID NO: 18.

[0072] In another preferred embodiment, component (i) comprises a secretion pathway signal peptide and the sequence SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the amino acid at position 57 is not an Asn residue. In an even more preferred embodiment, the second component comprises the sequence SEQ ID NO: 19.

[0073] In another preferred embodiment, component (i) comprises a secretion pathway signal peptide and the sequence SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the amino acid at position 113 is not an Asn residue. In an even more preferred embodiment, the second component comprises the sequence SEQ ID NO: 20.

[0074] In another preferred embodiment, component (i) comprises a secretion pathway signal peptide and the sequence SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the amino acids at positions 57 and 113 are not Asn residues. In an even more preferred embodiment, the second component comprises the sequence SEQ ID NO: 21.

[0075] It will be apparent to those skilled in the art that it can be desirable for the fusion protein of the application to further comprise a tag facilitating its purification.

[0076] Thus, in another particular embodiment, component (i) and / or component (iii) comprises a peptide facilitating its purification. In another particular embodiment, component (i) comprises a peptide facilitating its purification, wherein said peptide is fused at the amino-terminal end of component (i).

[0077] The term "peptide facilitating purification" as used herein refers to a peptide used for isolating or purifying component (iii) or the fusion protein of the application. In this sense, the peptide is capable of binding to one or more ligands from an affinity matrix, such as affinity chromatography. An example of such a peptide is a histidine tail (His-tag) which can comprise six histidine residues (His6 or H6) and is capable of binding to a nickel or cobalt column with high affinity. Other examples of such peptides include, but are not limited to, an Arg-tag, a FLAG-tag, a Strep-tag, an epitope capable of being recognized by an antibody, such as a c-myc-tag (recognized by an anti-c-myc antibody), an SBP-tag, an S-tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a glutathione S-transferase-tag, a maltose-binding protein, NusA, TrxA, DsbA, an Avi-tag, etc. The skilled person will understand that a peptide facilitating purification can also be used for detecting the polypeptide to which they are bound. This can be done by conventional techniques (e.g., techniques based on antibodies specifically recognizing the peptide facilitating purification).

[0078] The peptide that promotes its purification may be located at the N-terminal or C-terminal position with respect to component (iii). In a preferred embodiment, the peptide that promotes its purification is located at the N-terminal position with respect to component (iii). In another preferred embodiment, the peptide that promotes its purification is located at the C-terminal position with respect to component (iii).

[0079] Component (ii) (a) - polypeptide comprising a sortase recognition motif or a remnant of said motif As described above, the fusion protein consists of at least two components, wherein the second component may be a polypeptide including a sorting enzyme recognition motif.

[0080] As used herein, the term "sortase" refers to a prokaryotic enzyme possessing a catalytic domain that selectively cleaves peptide bonds in polypeptide chains recognized by the sortase's motif and catalyzes transpeptidation. This transpeptidation results in the formation of an amide bond between the terminal carboxyl group resulting from the cleavage and a protein on the cell wall surface. Sorting enzymes are present in almost all Gram-positive bacteria, as well as some Gram-negative bacteria and archaea.

[0081] Sorting enzymes are classified into four distinct classes (A, B, C, and D). Each of these sorting enzyme classes cleaves a different recognition motif, and some members of a certain class cleave multiple peptide motifs. Class A sorting enzymes typically cleave the recognition motif LPXTG (SEQ ID NO: 22) between threonine and glycine, where X represents any amino acid. The cleaved peptide retains a glycine residue at its amino terminus, while the cleaved protein binds to peptidoglycan at the threonine residue. In one specific embodiment, the sorting enzyme is sorting enzyme A, an enzymatically active fragment, or a variant or derivative thereof. In another specific embodiment, sorting enzyme A is a Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus Wild-type sorting enzymes, evolved sorting enzymes (eSrtA), eSrtA(2A-9), eSrtA(4S-9), or Streptococcus pyogenes ( Streptococcus pyogenes Sorting enzyme A. In one embodiment, the sorting enzyme is a Staphylococcus aureus sorting enzyme. In one embodiment, the sorting enzyme recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 22), where X is any amino acid.

[0082] As used herein, the term "sorting enzyme recognition motif" refers to a small amino acid sequence (3-7 amino acids) recognized by a sorting enzyme and used for transpeptidation. This sequence is typically located at the C-terminus of a protein. In another specific embodiment, the sorting enzyme recognition motif is the amino acid sequence LPETG (SEQ ID NO: 23).

[0083] As will be appreciated by the skilled person, in certain cases, the interaction of the sortase recognition motif, the sortase enzyme and the sortase acceptor motif present in the component of interest can be facilitated by increasing the flexibility of the polypeptide comprising the sortase recognition motif. In this sense, in one embodiment, component (i) and component (ii) (a) comprising the sortase recognition motif are covalently bound by a flexible peptide linker.

[0084] The term "flexible peptide linker" as used herein refers to a spacer amino acid sequence which can act as a hinge region between the two polypeptide components, allowing them to move independently of each other, while maintaining the three-dimensional shape of each individual domain, such that the presence of the peptide linker or spacer sequence does not alter the functionality of either component (i) or component (ii) (a). In this sense, a preferred flexible peptide linker according to the present application will be a hinge region characterized by a structural extensibility that allows such movement. The role of the linking region is to provide space between component (i) and component (ii) (a). Thus, on the one hand, the secondary and tertiary structure of component (i) or component (ii) (a) is ensured not to be affected by the presence of any other component, but also to ensure that the sortase recognition motif, the sortase enzyme and the component having the sortase acceptor motif interact more easily with each other. In one embodiment, the flexible peptide linker preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or about 100 amino acids.

[0085] In another embodiment, the flexible peptide linker comprises at least 2 amino acids selected from the group consisting of serine, glycine, alanine and threonine. In another embodiment, the flexible peptide linker is a polyglycine sequence. In another embodiment, the flexible peptide linker comprises a sequence selected from the group consisting of GGGGS (SEQ ID NO: 29), SGGTSGSTSGTGST (SEQ ID NO: 30), AGSSTGSSTGPGSTT (SEQ ID NO: 31), GGSGGAP (SEQ ID NO: 32), GGGVEGGG (SEQ ID NO: 33), PKPSTPPGSS (SEQ ID NO: 34), AAA and AAALE (SEQ ID NO: 35). In another embodiment, the flexible peptide linker comprises the sequence GGGGS (SEQ ID NO: 29).

[0086] The presence of component (ii)(a) in the fusion protein of the application allows this protein to be modified by covalently binding any molecule comprising a sortase acceptor motif. In one embodiment, the fusion protein further comprises a component of interest covalently bound to a sortase recognition motif or to the remaining portion of a sortase recognition motif generated upon a sortase-mediated reaction.

[0087] As will be apparent to those skilled in the art, modification of a sortase recognition motif by a sortase-mediated reaction results in a "remaining portion of a sortase recognition motif" in which one or more amino acids are cleaved from the C-terminus of the sortase recognition motif sequence, and the fusion protein of the application is modified by cleavage of the sortase recognition motif. In this sense, when the sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 22), in which X is any amino acid, the remaining portion of the motif is LPXT, in that it remains after proteolytic cleavage of the carboxy terminal glycine by sortase.

[0088] In another embodiment, the component of interest is characterized by comprising a sortase acceptor motif.

[0089] As used herein, the term "sortase acceptor motif" refers to a polypeptide sequence that acts as an acceptor for a sortase-mediated transfer of a polypeptide to a sortase recognition motif. In one embodiment, the sortase acceptor motif is located at, near, adjacent to, or toward the N-terminus or C-terminus of the component of interest and comprises a non-polar amino acid sequence, in which the sequence is at least one amino acid long. In yet another embodiment, the non-polar amino acid sequence is at least 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids or any interval thereof) long. In certain embodiments, the non-polar amino acid sequence is or comprises one or more glycines or alanines. Exemplary sortase acceptor motifs include G[G]n (where n = 0-5) and A[A]n (where n = 0-5).

[0090] In one embodiment, the component of interest is characterized by comprising a sortase acceptor motif, in which the sortase acceptor motif is characterized by comprising an oligoglycine sequence of at least 3 glycines.

[0091] In one specific embodiment, the component of interest is selected from: Toll-like receptor (TLR) ligands, monophosphoryl lipid A (MPL A), oligonucleotides selected from CpG islands and polyinosinic acid:polycytidylic acid (poly(I:C) – CAS No. 24939-03-5), saponins, lipid molecules, coating molecules, oligosaccharides, antibodies, nanobodies, affitin, viral antigens, bacterial antigens, fungal antigens, allergens, cell-penetrating peptides, affibody, or environmental antigens, tumor antigens, wherein the component of interest is characterized by an oligoglycine sequence comprising at least 3 glycine residues. Non-limiting examples of the component of interest would include TLR (Toll-like receptor) ligands, MPL (monophosphoryl lipid A), oligonucleotides (CpG, poly(I:C) etc.), saponins, lipid molecules, other lipids, coating molecules for various purposes (such as polyglycerol, polyethylene glycol, chitosan, poly(… Poly(hydroxypropyl methacrylate) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-acryloylmorpholine) (PAcM), etc., oligosaccharides, molecules that can be directed to different targets in vivo such as nanobodies, avidin, and antibodies. It will be apparent to those skilled in the art that any molecule containing at least three glycine molecules with a free N-terminus can be coupled.

[0092] It will be apparent to those skilled in the art that viral antigens, bacterial antigens, fungal antigens, allergens, cell-penetrating peptides, affinity molecules, environmental antigens, and tumor antigens as defined above are equally effective as components of interest, wherein the components of interest include oligoglycine sequences of at least 3 glycine.

[0093] Component (ii) (b) - polypeptide comprising a sortase acceptor motif As described, the fusion protein is formed from at least two components, one of which is a polypeptide comprising the sorting enzyme acceptor motif described above.

[0094] In yet another embodiment, the sortase acceptor motif comprises a non-polar amino acid sequence, wherein the non-polar amino acid sequence comprises at least 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids or any interval thereof) in length. In certain embodiments, the non-polar amino acid sequence is or comprises one or more glycines or alanines. In another embodiment, the sortase acceptor motif comprises a sequence according to G[G]n (where n = 0-2) and A[A]n (where n = 0-2).

[0095] In one embodiment, component (ii)(b) is characterized by comprising a sortase acceptor motif, wherein the sortase acceptor motif is characterized by comprising an oligoglycine sequence of at least 1 glycine (G). In another embodiment, the sortase acceptor motif comprises an amino acid sequence selected from the group consisting of G, A, GG, AA, GGG, and AAA. In another embodiment, the sortase acceptor motif is characterized by consisting of an oligoglycine sequence of 3 glycines (GGG). In another embodiment, the sortase acceptor motif is characterized by not comprising an oligoglycine sequence of more than 3 glycines. In another embodiment, the sortase acceptor motif is characterized by not comprising an oligoglycine sequence of 5 glycines.

[0096] In certain cases, to stabilize production of the fusion protein, it can be desirable to protect the sortase acceptor motif at the N-terminus with a polypeptide sequence that can be subsequently cleaved.

[0097] In this sense, in one embodiment, the fusion protein further comprises a protease recognition sequence covalently bound to the amino-terminal end of the sortase acceptor motif. In one embodiment, the protease recognition sequence is an enterokinase recognition sequence or a factor Xa recognition sequence. The term "protease recognition sequence" has been defined above and the definition and embodiments are equally valid for the current embodiment.

[0098] In one embodiment, the fusion protein comprising component (ii)(b) further comprises a component of interest covalently bound to the sortase acceptor motif.

[0099] In one embodiment of interest, the component of interest is characterized by comprising a sortase recognition motif. In one embodiment of interest, the sortase is sortase A, an enzymatically active fragment or a variant or derivative thereof. In another embodiment of interest, the sortase A is S. aureus wild type sortase, an evolved sortase (eSrtA), eSrtA (2A-9), eSrtA (4S-9) or S. pyogenes sortase A. In one embodiment of interest, the sortase is S. aureus sortase. In one embodiment of interest, the sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 22), wherein X is any amino acid. In another embodiment of interest, the sortase recognition motif is the amino acid sequence LPETG (SEQ ID NO: 23).

[0100] In another embodiment of interest, the component of interest is selected from the group consisting of a toll-like receptor (TLR) ligand, monophosphoryl lipid A (MPL A), an oligonucleotide selected from the group consisting of CpG islands and polyinosinic:polycytidylic acid (poly(I:C) - CAS number 24939-03-5), a saponin, a lipid molecule, a coating molecule, an oligosaccharide, an antibody, a nanobody, an affitin, a viral antigen, a bacterial antigen, a fungal antigen, an allergen, a cell-penetrating peptide, an affimer, or an environmental antigen, a tumor antigen, wherein the component of interest is characterized by comprising a sortase recognition sequence or the remaining part of a sortase recognition motif generated upon a sortase-mediated reaction.

[0101] Nanospheres and / or microspheres of the application As mentioned above, the fusion proteins of the application have the ability to form inclusion bodies when expressed in a cell. The size and shape of the inclusion bodies can vary and are referred to as nanospheres or microspheres.

[0102] In this sense, another aspect of the application relates to a nanosphere or microsphere comprising at least one fusion protein of the application, hereinafter referred to as nanosphere / microsphere of the application.

[0103] All definitions and embodiments of interest described in relation to other aspects of the application are equally valid and applicable to the current aspect.

[0104] As mentioned above, nanospheres and microspheres have different sizes. In this sense, in one embodiment of interest, the size of the nanosphere is between 300 nanometers (nm) and 550 nm. In another embodiment of interest, the size of the microsphere is between 0.55 micrometers (pm) and 4 pm, preferably between 1 pm and 4 pm.

[0105] The structure of the minimal region of orthoreovirus muNS protein includes two helical domain regions at the N- and C-termini (C1 and C2) and an interdomain region called the intercoil region (IC). This domain corresponds to sequences 477-542 of avian orthoreovirus muNS protein (SEQ ID NO: 25) or sequences 561-622 of mammalian orthoreovirus muNS protein (SEQ ID NO: 26). The fragment has the ability to be incorporated into nanospheres and microspheres formed by orthoreovirus muNS protein.

[0106] In this sense, in one particular embodiment, the nanospheres / microspheres of the application comprise a second fusion protein comprising the following components: (a) a polypeptide selected from the group consisting of: a polypeptide comprising sequences 477-542 of avian orthoreovirus muNS protein (SEQ ID NO: 25), a polypeptide comprising sequences 561-622 of mammalian orthoreovirus muNS protein (SEQ ID NO: 26), a functionally equivalent variant of any of the above polypeptides that retains the ability to be incorporated into nanospheres and / or microspheres, and (b) a second polypeptide of interest.

[0107] "Functionally equivalent variant" is understood to mean all peptides derived from sequences 477-542 of avian orthoreovirus muNS protein (SEQ ID NO: 25) or from sequences 561-622 of mammalian orthoreovirus muNS protein (SEQ ID NO: 26) by modification, insertion, substitution and / or deletion of one or more amino acids, provided that the function of the muNS protein sequence from which they are derived is substantially retained.

[0108] The sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25) and the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO 26) have the ability to be incorporated into microspheres / nanospheres formed in cells by the fusion proteins of the application. Functional equivalent variants of the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25) and the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 26) substantially retain the ability of said sequence to be incorporated into microspheres / nanospheres formed in cells by the fusion proteins of the application. Methods suitable to determine the ability to be incorporated into microspheres / nanospheres include, but are not limited to, the method described in Example 3 of patent application WO 2011 / 098652, which is based on the formation of nanospheres / microspheres (inclusion bodies) and the expression of the protein of interest in the form of a fusion protein with the fragment that directs it to the nanospheres / microspheres. Then, indirect immunofluorescence will be performed using polyclonal antibodies against the HA epitope or the epitope of interest, thus being able to check whether the fragment is incorporated into the nanospheres / microspheres.

[0109] Functional equivalent variants of the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25) or the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 26) have the ability to be incorporated into microspheres and / or nanospheres. In a particular embodiment, functional equivalent variants of the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25) or the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 26) retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% of the ability of the sequence from which they are derived to be incorporated into nanospheres and / or microspheres.

[0110] Functional equivalent variants of the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25) or the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 26) include functional equivalent variants that show at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the sequence from which they are derived.

[0111] In one embodiment, functional equivalent variants of the sequence 477-542 of avian orthoreovirus muNS protein (SEQ ID NO: 25) or the sequence 561-622 of mammalian orthoreovirus muNS protein (SEQ ID NO: 26) retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% of the ability of the sequence from which they are derived to be incorporated into nanospheres and / or microspheres and exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence from which they are derived.

[0112] The two components (a) and component (b) of the second fusion protein can be linked in any order, i.e., component (b) can be fused to the amino terminus of component (a), or component (a) can be fused to the amino terminus of component (b). In one embodiment, component (b) is fused to the amino terminus of component (a).

[0113] The term "polypeptide of interest" as used herein refers to any polypeptide of any size that is of interest to the user, wherein fusion to the amino terminus of component (i) does not affect the ability of the fusion protein of the application to form nanospheres and / or microspheres. In one preferred embodiment, the polypeptide of interest can be a viral antigen, a bacterial antigen, a fungal antigen, an allergen, a cell penetrating peptide, an affibody, or an environmental or tumor antigen.

[0114] Viral antigens suitable as the first polypeptide of interest include HIV-1 antigens (such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vif, vpr, vpu, rev), human herpes virus antigens (such as gH, gL, gM, gB, gC, gK, gE or gD or derivatives thereof) or immediate early protein antigens such as SHV1 or SHV2 ICP27, ICP47, ICP4, ICP36, cytomegalovirus antigens, particularly human cytomegalovirus antigens (such as gB or derivatives thereof), Epstein-Barr virus antigens (such as gp350 or derivatives thereof), varicella-zoster virus antigens (such as gpl, II, Ill and IE63), or hepatitis viruses such as hepatitis B virus antigens (e.g., hepatitis B surface antigen or hepatitis core antigen), hepatitis C virus antigens (e.g., core antigen, El, NS3 or NS5), paramyxovirus antigens such as respiratory syncytial virus antigens (such as F and G proteins or derivatives thereof), parainfluenza virus antigens, rubella virus antigens (such as El and E2 proteins), measles virus antigens, human papilloma virus antigens (e.g., HPV6, 11, 16, 18, such as LI, L2, El, E2, E3, E4, E5, E6, E7), flavivirus antigens (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus antigens) or influenza virus infected cells (such as HA, NP, NA or M proteins, or combinations thereof), rotavirus antigens (such as VP7sc and other rotavirus components), and the like.

[0115] Bacterial antigens suitable as the first polypeptide of interest include Neisseria (N. Neisseria spp. ), including Neisseria gonorrhoeae (N. N. gonorrhea ) and Neisseria meningitidis (N. N. meningitidis ) antigens (transferrin binding protein, lactoferrin binding protein, PiIC and adhesins); Streptococcus pyogenes (S. S. pyogenes ) antigens (such as M protein or fragments thereof and protease C5A); Streptococcus agalactiae (S. S. agalactiae ), Streptococcus mutans (S. S. mutans ); Haemophilus ducreyi (H. H. ducreyi ); Moraxella (M. Moraxella spp. ), including Moraxella catarrhalis (M. M catarrhalis ), also known as Branhamella catarrhalis (B. Branhamella catarrhalis ) antigens (such as high and low molecular weight adhesins and invasin); Bordetella (B. Bordetella spp. ), including Bordetella pertussis (B. B. pertussis ), such as parapertussis (B. Parapertussis ) and bronchiseptica (B. B. bronchisepticaAntigens (such as pertactin, pertussis toxin or its derivatives, filamentous hemagglutinin, adenylate cyclase, fimbriae); Mycobacteria spp. ( Mycobacterium spp. ), including Mycobacterium tuberculosis ( M. tuberculosis ), Mycobacterium bovis ( M. bovis Mycobacterium leprae ( M. leprae ), Mycobacterium avium ( M. avium Mycobacterium paratuberculosis ( M. paratuberculosis ), Mycobacterium smegmatis ( M. smegmatis ) antigen; Legionella spp. ( Legionella spp. ), including Legionella pneumophila ( L. pneumophila Antigens (e.g., ESAT6, antigens 85A, 85B or 85C, MPT 44, MPT59, MPT45, HSPIO, HSP65, HSP70, HSP75, HSP90, 19 kDa PPD [Rv3763], 38 kDa PPD [Rv0934]); Escherichia coli spp. ( Escherichia spp. ), including enterotoxigenic Escherichia coli (E. coli) E. coli Antigens (e.g., colonization factors, heat-labile toxins or their derivatives, heat-stable toxins or their derivatives), enterohemorrhagic Escherichia coli (EHEC) E. coli ) and enteropathogenic Escherichia coli E. coli Antigens (e.g., Shiga toxin-like toxins or their derivatives); Vibrio spp. Vibrio spp. ), including Vibrio cholerae ( V. cholera Antigens (e.g., cholera toxin or its derivatives); Shigella spp. Shigella spp. ), including Shigella sonnei ( S. sonnei ), Shigella dysenteriae ( S. dysenteriae ), Shigella freundii ( S. flexnerii Antigen; Yersinia spp. ( Yersinia spp. ), including Yersinia enterocolitica ( Y. enterocolitica Antigens (e.g., Yop protein); Yersinia pestis ( Y. pestis Yersinia pseudotuberculosis ( Y. pseudotuberculosis Campylobacter spp. Campylobacter spp. ), including Campylobacter jejuni ( C. jejuni Antigens (e.g., toxins, adhesins, and infiltrates); Salmonella spp. Salmonella spp. ), including Salmonella typhimurium ( S. typhi Salmonella paratyphi () S. paratyphi Salmonella choleraesuis (Swine choleraesuis) S. choleraesuisSalmonella enteritidis ( ) S. enteritidis ) antigen; Listeria spp. ( Listeria spp. ), including Listeria monocytogenes ( L. monocytogenes ) antigen; Helicobacter genus ( Helicobacter spp. ), including Helicobacter pylori ( H. pylori Antigens (e.g., urease, catalase, vacuole toxin); Pseudomonas spp. ( Pseudomonas spp. ), including Pseudomonas aeruginosa ( P. aeruginosa Antigen; Staphylococcus spp. ( Staphylococcus spp. ), including Staphylococcus aureus ( S. aureus Staphylococcus epidermidis ( S. epidermidis ) antigen; Enterococcus spp. ( Enterococcus spp. ), including Enterococcus faecalis ( E. faecalis ), Enterococcus faecalis ( E. faecium Antigens; Clostridium ( Clostridium spp ), including Clostridium tetani ( C. tetani Antigens (e.g., tetanus toxin and its derivatives); botulinum toxin ( C. botulinum Antigens (e.g., botulinum toxin and its derivatives), Clostridium difficile ( C. difficile Antigens (e.g., Clostridium A or B toxins and their derivatives); Bacillus spp. ( Bacillus spp ), including Bacillus anthracis ( B. anthracis Antigens (e.g., anthrax toxin and its derivatives); Corynebacterium spp. ( Corynebacterium spp. ), including Corynebacterium diphtheriae ( C. diphtheriae Antigens (e.g., diphtheria toxin and its derivatives); spirochetes ( Borrelia spp. ), including Borrelia burgdorferi ( B. burgdorferi Antigens (e.g., OspA, OspC, DbpA, DbpB); Borrelia gallini ( B. garinii Antigens (e.g., OspA, OspC, DbpA, DbpB), Borrelia auriculata ( B. afzelii Antigens (e.g., OspA, OspC, DbpA, DbpB) B. andersonfi Antigens (e.g., OspA, OspC, DbpA, DbpB), Helmholtzia ( B. hermsii ); Ehrlich body ( Ehrlichia spp ), including Maelich body ( E. equi Antigens and human granulocytic ehrlichiosis pathogens; Rickettsia genus Rickettsia spp ), including Rickettsiae ( R. rickettsii ) antigen; Chlamydia genus (Chlamydia spp. ), including Chlamydia trachomatis (Ct) antigens (e.g., MOMP, heparin-binding proteins); Chlamydia pneumoniae (Cpn) antigens (e.g., MOMP, heparin-binding proteins), Chlamydia psittaci (Cps) antigens; Leptospira (Lepto) (Leptospira) species, including Leptospira interrogans (L interrogans) antigens; Treponema (Treponema) species, including Treponema pallidum (Tp) antigens; Toxoplasma (Toxoplasma) species and Toxoplasma gondii (Tg) antigens (e.g., SAG2, SAGS, Tg34); Mycobacterium tuberculosis antigens (e.g., Rv2557, Rv2558, RPFs: Rv0837c, Rv1884c, Rv2389c, Rv2450, Rv1009, aceA (Rv0467), PstS1, (Rv0932), SodA (Rv3846), 16 kDa Rv2031c, Tb Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2, and hTCC1); Chlamydia antigens (e.g., high molecular weight protein (HMWP), ORF3 (document EP 366 412), and possible membrana proteins (Pmp); Streptococcus (Streptococcus) species, including Streptococcus pneumoniae (Sp) antigens (PsaA, PspA, streptolysin, choline binding proteins, pneumolysin protein antigens and detoxified mutant derivatives thereof); antigens derived from Haemophilus (Haemophilus) species, including Haemophilus influenzae (Hi) type B (e.g., PRP and conjugates thereof); non-typeable Haemophilus influenzae antigens (e.g., OMP26, high molecular weight adhesin, P5, P6, protein D and lipoprotein D, and pilin and pilin-derived peptides, or multimeric variants or fusion proteins thereof). C. trachomatis Chlamydia pneumoniae C. psittaci Leptospira spp L. interrogans Treponema spp. T. pallidum T. denticola T. hyodysenteriae Toxoplasma spp. T. gondii Streptococcus spp. S. pneumoniae Haemophilus spp. H. influenzae

[0116] Fungal antigens suitable as the first polypeptide of interest include, but are not limited to, for example, Candida fungal antigen components; Histoplasma fungal antigens, such as heat shock protein 60 (HSP60) and other Histoplasma fungal antigen components; Pneumocystis fungal antigens, such as Pneumocystis jirovecii (Pj) antigens; Aspergillus fungal antigens, such as Aspergillus fumigatus (Af) antigens; and Cryptococcus fungal antigens, such as Cryptococcus neoformans (Cn) antigens.​​​​​​​​​​​​​​​Pneumocystis spp. ), including Pneumocystis carinii ( P. carinii Fungal antigens; Cryptococcus fungal antigens, such as capsular polysaccharides and other Cryptococcus fungal antigen components; Coccidia fungal antigens, such as spherule antigens and other coccidia fungal antigen components; Candida spp. Candida spp. ), including Candida albicans ( C. albicans Antigen; Cryptococcus spp. ( Cryptococcus spp. ), including Cryptococcus neoformans ( C. neoformans Antigens; tinea fungal antigens, such as tinea tinea and other coccidial fungal antigen components.

[0117] Protozoan antigens suitable as the first polypeptide of interest include, but are not limited to, Plasmodium genus ( Plasmodium spp. ), such as Plasmodium falciparum ( P. falciparum Antigens and antigens derived from Plasmodium falciparum (such as RTS.S, TRAP, MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27 / 25, Pfs16, Pfs48 / 45, Pfs230 and their analogues in the Plasmodium genus); as well as merozoite surface antigens, sporozoite surface antigens, cyclosporus antigens, gametophyte / gamete surface antigens, and pf blood stage antigens (pf blood stage). Antigens, 55 / RESA and other pseudoprotoplasmic antigen components; Toxoplasma antigens, such as SAG-I, p30 and other Toxoplasma antigen components; Schistosoma antigens, such as glutathione S-transferase, paramyosin and other schistosoma antigen components; Trichomonas genus ( Trichomonas spp. ), including Trichomonas vaginalis ( T. vaginalis ) antigen; Entamoeba ( Entamoeba spp. ), including Entamoeba histolytica ( E. histolytica Antigens; Babesia ( Babesia spp. ), including the field mouse babesi ( B. microti Antigens; Leishmania antigens and other Leishmania antigens, such as gp63, lipopolysaccharide and its related proteins and other Leishmania antigenic components; Giardia spp., including Giardia lemnis ( G. lamblia Antigens; and Trypanosoma cruzi ( Trypanosoma cruziantigens, such as the 75-77 kDa antigen, the 56 kDa antigen, and other components of the antigenic repertoire of the parasite.

[0118] Environmental antigens or allergens suitable as the first polypeptide of interest include, but are not limited to, antigens derived from naturally occurring allergens, such as pollen allergens (tree, ragweed, weed and grass pollen allergens), insect allergens (inhalant, salivary and venom allergens), animal dander and hair allergens and food allergens. Important tree, grass and ragweed pollen allergens are derived from the following taxonomic orders: Fagales, Oleales, Pinaceae and Platanaceae, including Betula (birch), Alnus (alder), Corylus (hazelnut), Carpinus (hornbeam) and Olea (olive), Cryptomeria and Juniperus (cedar), Platanus (sycamore), Poales, including grasses of the genera Lolium (ryegrass), Phleum (timothy), Poa (bluegrass), Cynodon (bermudagrass), Dactylis (orchardgrass), Holcus (sedges), Phalaris (canarygrass), Secale (rye) and Sorghum (sorghum), Asterales and Urticales, including ragweeds of the genera Ambrosia, Artemisia and Parietaria. Other allergenic antigens that can be used include Dermatophagoides and Euroglyphus house dust mite allergens, storage mite allergens, such as Lepidoglyphys, Glycyphagus and Tyrophagus, cockroach allergens, gnat allergens and flea allergens, such as Blatella, Periplaneta, Chironomus and Ctenocepphalides, mammalian allergens, such as cat, dog and horse, avian, venom allergens, including those derived from insect bites or stings, such as the order Hymenoptera, including bees (superfamily Apidae), wasps and ants (superfamily Formicoidae). Still other allergenic antigens that can be used include fungal inhalant allergens, such as Alternaria and Cladosporium.

[0119] Tumor antigens suitable as the first polypeptide of interest include, but are not limited to, MAGE, MART-1 / Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), Adenosine deaminase binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-0017-1A / GA733, Carcinoembryonic antigen (CEA) and its antigenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate specific antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, Prostate specific membrane antigen (PSMA), T cell receptor / CD3-ς chain, the tumor antigen MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the tumor antigen GAGE family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, Tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, beta-catenin, delta-catenin, gamma-catenin, pl2Octn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis colon protein, EphA3, EphA4, EphB2, EphB3, EphB6, EphrinB2, EphrinB3, EphrinB6, EphrinA1, EphrinA2, EphrinA5, EphA1, EphA2, EphA5, EphA6, EphB1, EphB4, EphB6, EphrinB1, EphrinA3, EphrinA4, EphA7, EphB2R, EphB3R, EphB4R, EphB6R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphrinB4R, EphrinA5R, EphA7R, EphrinA3R, EphrinA4R, EphAPC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, tumor antigens Smad family, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, acute lymphoblastic leukemia (etv6, amll, connexin b), B-cell lymphoma (Ig idiotype), glioma (E-cadherin, a-catenin, 13-catenin, 7-catenin, p120ctn), bladder cancer (p21ras), cholangiocarcinoma (p21ras), breast cancer (MUC family, HER2 / neu, c-erbB-2), cervical cancer (p53, p21ras), colon cancer (p21ras, HER2 / neu, c-erbB-2, MUC family), colorectal cancer (colorectal associated antigen (CRC)-0017-1A / GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (connexin b), gastric cancer (HER2 / neu, c-erbB-2, glycoprotein ga733), hepatocellular carcinoma, Hodgkin's lymphoma (lmp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (connexin b), melanoma (p15 protein, gp75, carcinoembryonic antigen, GM2 and GD2 gangliosides, Melan-A / MART-1, cdc27, MAGE-3, p21ras, gp100Pme1117), myeloma (MUC family, p21ras), non-small cell lung cancer (HER2 / neu, c-erbB-2), nasopharyngeal carcinoma (lmp-1, EBNA-1), ovarian cancer (MUC family, HER2 / neu, c-erbB-2), prostate cancer (prostate specific antigen (PSA) and its antigenic epitopes PSA-1, PSA-2 and PSA-3, PSMA, HER2 / neu, c-erbB-2, glycoprotein ga733), renal cancer (HER2 / neu, c-erbB-2), uterine and esophageal squamous cell carcinoma (viral products such as human papilloma virus proteins), testicular cancer (NY-ES0-1), and T-cell leukemia (VLTH-1 epitope).

[0120] When expressed in a cell, the fusion proteins of the present application have the ability to form nanospheres and / or microspheres, regardless of whether they have a component of interest bound to a sortase recognition sequence or bound to a sortase acceptor motif. In this sense, the present application also contemplates the formation of nanospheres and / or microspheres by the fusion proteins of the present application, wherein the proteins do not include a component of interest bound to a sortase recognition sequence, and the subsequent modification of the nanospheres and / or microspheres by the addition of a component of interest to the sortase recognition sequence or sortase acceptor motif.

[0121] In this sense, in one particular embodiment of the nanospheres / microspheres of the present application, the fusion proteins of the present application comprising component (ii)(a) further comprise a component of interest covalently bound to the remaining portion of the sortase recognition motif generated upon a sortase-mediated reaction. In another particular embodiment of the nanospheres / microspheres of the present application, the fusion proteins of the present application comprising component (ii)(b) further comprise a component of interest covalently bound to the sortase acceptor motif.

[0122] It will be apparent to those skilled in the art that the modification of the nanospheres or microspheres by sortase can result in intermediates wherein the fusion proteins of the present application forming the nanospheres or microspheres are modified by cleavage of the sortase recognition motif. In this sense, in one particular embodiment, the nanospheres / microspheres of the present application are modified by the action of a sortase that recognizes and cleaves the sortase recognition motif. In another particular embodiment, the nanospheres / microspheres of the present application are modified by the action of a sortase that recognizes and cleaves the sortase recognition motif, wherein the sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 22), wherein X is any amino acid, and wherein the sortase cleaves between T and G of the sortase recognition motif.

[0123] Likewise, it will be apparent to those skilled in the art that if the fusion proteins of the present application comprise a sortase acceptor motif, the modification of the nanospheres or microspheres by sortase only involves the addition of a polypeptide to the amino-terminal end of the fusion protein, without cleavage of any polypeptide motif. In this sense, in one particular embodiment, the nanospheres / microspheres of the present application are modified by the action of a sortase that recognizes and covalently binds a polypeptide to the sortase acceptor motif. In another particular embodiment, the nanospheres / microspheres of the present application are modified by the action of a sortase that recognizes the sortase acceptor motif, wherein the sortase recognition motif is an amino acid sequence selected from the group consisting of: G, A, GG, AA, GGG and AAA, preferably GGG.

[0124] In another particular embodiment of the nanospheres / microspheres of the present application, the component of interest is selected from the group consisting of: - Toll-like receptor (TLR) ligands; - Monophosphoryl lipid A (MPL A); - Oligonucleotides selected from CpG islands and polyinosinic acid:polycytidine (poly(I:C) – CAS No. 24939-03-5); - Saponins; - Lipid molecules; - The coating molecules are selected from: polyglycerol, polyethylene glycol, chitosan, and poly( Poly(hydroxypropyl methacrylate) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-acryloylmorpholine) (PAcM); - Oligosaccharides; - Antibody; - Nanobodies; - Affinity; - Viral antigens; - Bacterial antigens; - Fungal antigens; - Allergens; - Cell-penetrating peptides; - Affinity; - Environmental antigens; and - Tumor antigens.

[0125] As used herein, the term "toll-like receptor ligand" or "TLR" ligand refers to a molecule that has the ability to bind to proteins of the toll-like receptor protein family. TLRs constitute a family of proteins that are part of the innate immune system. These receptors are transmembrane receptors and recognize molecular patterns expressed by a variety of infectious agents and stimulate a variety of inflammatory responses.

[0126] As used herein, the term "monophosphoryl lipid A" or the acronym "MPL A" refers to a molecule derived from lipopolysaccharide (LPS) or endotoxin that is highly immunostimulatory and has low toxicity, making it suitable for use as a vaccine adjuvant.

[0127] As used herein, the term “oligonucleotide” refers to a single-stranded or double-stranded DNA or RNA sequence having 50 or fewer base pairs, wherein the bases are natural (i.e., adenine, cytosine, guanine, thymine, and uracil) or synthetic, and wherein the oligonucleotide may contain modifications such as, but not limited to, methylation (e.g., 5-methylcytosine), phosphorylation, glycosylation, oxidation, etc.

[0128] The term "saponin" as used herein refers to a class of steroidal or triterpenoid glycosides, named for their soap-like properties: each molecule consists of a fat-soluble component (a steroidal or triterpenoid compound) and a water-soluble component (a sugar), and they form a lather when shaken in water. Saponins are toxic, and their toxicity is thought to arise from their ability to form complexes with sterols, so they can interfere with the digestive system's assimilation of them, or destabilize cell membranes after being absorbed into the bloodstream.

[0129] The term "lipid molecule" as used herein refers to water-insoluble fatty substances, including fats, oils, waxes and related compounds.

[0130] The expression "coating molecule" as used herein refers to any chemical component that can be used to coat nanospheres / microspheres.

[0131] The term "oligosaccharide" in the context of the present invention refers to a molecule formed by the covalent union of 2 to 10 cyclic monosaccharides, wherein when the molecule has 3 or more monosaccharides, they can be linear or branched by the release of a water molecule from a glycosidic bond, a covalent bond established between the alcohol groups of two monosaccharides.

[0132] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule or a derivative of either, which has the ability to specifically bind an antigen under typical physiological conditions. The variable regions of the heavy and light chains of the immunoglobulin molecule contain the binding domains that interact with the antigen. It will also be understood that the term antibody includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides (such as chimeric antibodies and humanized antibodies), as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind an antigen provided by any known technique, unless otherwise indicated.

[0133] The term "nanobody" in the context of the present invention, also known as nanobodies, single-domain antibodies or VHH antibodies, is a camelid antibody that is much smaller than the ordinary antibodies, which are huge by molecular standards, as each of them is an aggregate of two heavy chains and two light chains that are intricately folded and connected to complex sugars, while nanobodies are relatively simple proteins that are approximately one-tenth the size of their human counterparts and are only a few nanometers long.

[0134] The term "avidin" as used herein refers to an artificial protein with the ability to selectively bind antigens.

[0135] The term "cell penetrating peptide" or its acronym "CPP" is a short peptide that facilitates cellular uptake / absorption of various molecular entities associated with the peptide by means of chemical bonds either through covalent bonds or by means of non-covalent interactions. The associated molecular entities range from nano-sized particles to small chemical molecules and large DNA fragments. The function of the CPP is to introduce the associated entity into the cell, a process that usually occurs by endocytosis. The amino acid composition of the CPP usually contains a relatively high abundance of positively charged amino acids such as lysine or arginine or contains sequences of alternating patterns of polar / charged amino acids with non-polar hydrophobic amino acids. These two types of structures are referred to as polycationic structures or amphipathic structures, respectively. A third class of CPPs are hydrophobic peptides that contain only non-polar residues of low net charge or have hydrophobic amino acid groups that are critical for cellular uptake.

[0136] In the context of the present description, the term "affibody" refers to a mimetic antibody capable of binding a specific target protein (receptor). Generally, an affibody molecule consists of 20 to 150 amino acid residues and can be composed of 2 to 10 alpha helices. Examples of affibodies include, but are not limited to, those described by Renli Luo et al. (RSC Chem. Biol., 2022, 3, 830-847).

[0137] The nanospheres / microspheres of the present application can have a variety of applications such as, but not limited to, drug delivery vehicles, vehicles for expression and purification of proteins, immunogenic agents, and the like. This is possible because both the fusion proteins and the nanospheres and microspheres have a variety of possibilities for modifications and alterations.

[0138] In one embodiment, the component of interest further comprises a protease recognition sequence located at the C-terminal region of the sortase acceptor motif characterizing the component of interest. Protease recognition sequences have been defined above and the definition and embodiment are equally valid for the current embodiment.

[0139] In one embodiment, the component of interest further comprises a signal peptide of the secretory pathway located at the C-terminal region of the sortase acceptor motif characterizing the component of interest. Signal peptides of the secretory pathway have been defined above and the definition and embodiment are equally valid for the current embodiment.

[0140] In one embodiment, the component of interest further comprises a tag that facilitates its purification located at the C-terminal region of the sortase acceptor motif characterizing the component of interest. Purification facilitating tags have been defined above and the definition and embodiment are equally valid for the current embodiment.

[0141] In another embodiment of interest, the component of interest further comprises a sortase recognition sequence. Sortase recognition sequences have been defined above and the definition and detailed description are equally valid for the current embodiment.

[0142] In one embodiment of interest, the component of interest further comprises a tag facilitating its purification, located in the C-terminal region of the sortase recognition sequence characterizing the component of interest.

[0143] In one embodiment of the nanosphere and / or microsphere, the first polypeptide of interest, the component of interest and / or the second polypeptide of interest is a glucose-6-phosphatase 2 protein (IGRP) or a functionally equivalent variant.

[0144] The term “glucose-6-phosphatase 2” or “IGRP” or “islet-specific glucose-6-phosphatase catalytic subunit-related protein” as used herein refers to a protein capable of hydrolyzing glucose-6-phosphate to glucose and phosphate. It is a transmembrane protein, expressed in the pancreas and to a lesser extent in the testis. In humans, it is encoded by the G6PC2 gene. The IGRP protein can be of any origin, for example human, bovine, murine, equine, canine, etc. In one embodiment of interest, the IGRP protein is the human protein identified in the UniprotKB database by accession number Q9NQR9 (entry version 147, December 14, 2022; sequence version 1, October 1, 2000). In humans, this protein has three isoforms, identified by the following UniprotKB accession numbers: Isoform 1, considered the canonical sequence, is 355 amino acids long: Q9NQR9-1.

[0145] Isoform 2 is 102 amino acids long: Q9NQR9-2.

[0146] Isoform 3 is 154 amino acids long: Q9NQR9-3.

[0147] In another embodiment of interest, the IGRP protein is the mouse protein identified in the UniprotKB database by accession number Q9Z186 (entry version 145, December 14, 2022; sequence version 1, May 1, 1999). In mice, this protein has two isoforms, identified by the following UniprotKB accession numbers: Isoform 1, considered the canonical sequence, is 355 amino acids long: Q9Z186-1.

[0148] Isoform 2 is 154 amino acids long: Q9Z186-2.

[0149] According to the application, these isoforms and any other isoform of IGRP protein of any origin are considered to be included in the term "IGRP".

[0150] In another particular embodiment, the IGRP protein is the rat protein encoded by the gene identified in the NCBI Genbank database under accession number GeneID 681817 (version of April 24, 2022).

[0151] The term "functionally equivalent variant" as used herein in reference to an IGRP protein refers to any peptide or protein obtained by deletion, insertion, addition or substitution of one or more amino acid residues with respect to the sequence from which it is derived and which retains the function of said sequence.

[0152] In one particular embodiment, the functionally equivalent variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence of the IGRP protein over its entire length.

[0153] It will be apparent to those skilled in the art that this fact that the nanospheres / microspheres can comprise one or more of the first polypeptide of interest, the component of interest and the second polypeptide of interest does not present any limitation regardless of the composition of the nanospheres / microspheres.

[0154] Thus, in one particular embodiment, the nanospheres and / or microspheres of the application comprise a polypeptide of interest and / or a component of interest. In another particular embodiment, the polypeptide of interest is the same or different from the component of interest.

[0155] Likewise, in one particular embodiment of the nanospheres and / or microspheres of the application, the nanospheres / microspheres of the application are bivalents or trivalents.

[0156] The term "nanosphere / microsphere bivalents" as used herein refers to nanospheres and / or microspheres comprising at least two different compounds / molecules / proteins from among the following: the first polypeptide of interest, the component of interest and the second polypeptide of interest. Similarly, the term "trivalent nanospheres / microspheres" as used herein refers to nanospheres and / or microspheres comprising at least three different compounds / molecules / proteins from among the following: the first polypeptide of interest, the component of interest and the second polypeptide of interest.

[0157] Polynucleotides, vectors and host cells of the invention All the definitions and particular embodiments described above in relation with other aspects of the application apply equally to the current aspect and its particular embodiments.

[0158] The fusion protein of the application can be encoded by a polynucleotide. Therefore, another aspect of the application relates to a polynucleotide encoding the fusion protein of the application, hereinafter referred to as the polynucleotide of the application.

[0159] The term "polynucleotide" as used herein refers to a polymer formed by a variable number of monomers of interest, wherein the monomers are nucleotides, including ribonucleotides and deoxyribonucleotides. Polynucleotides include monomers modified by methylation, as well as unmodified forms. The terms "polynucleotide" and "nucleic acid" are used interchangeably in the present application and include mRNA, cDNA and recombinant polynucleotides.

[0160] The polynucleotide can be part of an expression cassette, which is used without limitation to express the polynucleotide in a cell. Therefore, another aspect of the application relates to an expression cassette comprising a polynucleotide according to the application, hereinafter referred to as the cassette of the application.

[0161] The term "expression cassette" as used herein refers to a polynucleotide comprising a gene and a promoter suitable to control the gene. The expression cassette can optionally comprise other sequences, such as a transcription termination signal. The choice of promoter and other regulatory elements (one or more) is generally dependent on the host cell used. Suitable promoters in the context of the present application include constitutive promoters that facilitate constant expression of sequences associated therewith and inducible promoters that require an external stimulus to facilitate transcription of sequences associated therewith.

[0162] Promoters that can be used in embodiments of the application include: constitutive promoters, such as, for example, the promoter of alcohol dehydrogenase (ADH1), the promoter of elongation factor-1-alpha (TEF) and the promoter of the gene encoding triose phosphate isomerase (TPI), the promoter of glycerolaldehyde 3-phosphate dehydrogenase (GPD) and the promoter of 3-phosphoglycerate kinase (GPK), the MRP7 promoter.

[0163] inducible promoters, such as, for example, the promoter of metallothionein (CUP1) whose expression is regulated by the addition of copper to the culture medium, the promoter of the gene encoding the FUS1 gene or the FUS2 gene whose expression is activated in the presence of pheromone (factor alpha), the TET promoter whose expression is regulated in the presence of tetracyclines, the GAL1-10, GAL, GALS promoters which are activated in the presence of galactose, the estrogen-inducible VP16-ER promoter, the promoter of the phosphatase (PH05) whose expression is activated in the presence of phosphate and the promoter of the heat shock protein HSP150 whose expression is activated at high temperatures.

[0164] In one embodiment of the expression cassette of the application, the cassette comprises a first polynucleotide and a second polynucleotide, wherein the second polynucleotide encodes a second fusion protein. In another embodiment, the polynucleotide of the application or the expression cassette of the application is operably associated with a first promoter and the second polynucleotide is operably associated with a second promoter. In another embodiment, the first promoter and the second promoter are the same promoter or different promoters.

[0165] In one embodiment, when the cell in which the polynucleotide of the application is to be expressed is a bacterium, the promoter is a beta-lactamase and lactose promoter system, a T7 RNA polymerase promoter, a lambda promoter, a trp promoter, or a tac promoter. In a more specific embodiment of the application, the promoter is an inducible promoter. In another more specific embodiment, the promoter is an inducible promoter that can be induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG). In an even more specific embodiment, the inducible promoter that can be induced by IPTG is a T7 RNA polymerase promoter.

[0166] In another aspect, the application relates to a vector comprising the polynucleotide or the expression cassette of the application, hereinafter referred to as the vector of the application.

[0167] The term "vector" as used herein refers to a nucleic acid sequence comprising the necessary sequences so that upon transcription and translation of said sequence in a cell, a fusion protein of the application is generated. Said sequence is operably bound to other fragments providing for its autonomous replication in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector comprising, in addition to the region for autonomous replication in the host cell, a region operably linked to the polynucleotide of the application and capable of enhancing the expression of the product of the polynucleotide according to the application. The vectors of the application can be obtained by techniques well known in the art. In a particular embodiment of the application, the vector of the application is a bacterial expression vector. In another particular embodiment of the application, when the organism is a prokaryote such as a bacterium, a suitable vector according to the application is for example the vector pUC18, pUC19, pUC118, pUC119, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, CoIEl, pCRl, RP4, pNH8A, pNH16a, pNH18a. In a particular embodiment of the application, when the bacterium is Escherichia coli, the vector is the plasmid pET, in particular the plasmid pET Duet-1. Said plasmid comprises two different multiple cloning sites (MCS). Genes cloned into the plasmid are transcribed under the control of a T7 phage promoter when T7 RNA polymerase is activated in the host cell. Expression is induced using IPTG (isopropyl-β-D-thiogalactopyranoside), which removes a repressor from the operator to allow transcription and promote expression of the protein of interest.

[0168] In a particular embodiment, the vector of the application further comprises a second polynucleotide encoding a polypeptide selected from the group consisting of: - a polypeptide comprising the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25), - a polypeptide comprising the sequence 561-622 of the mammalian orthoreovirus muNS protein (SEQ ID NO: 26), - a functional equivalent variant of any of the above polypeptides, which retains the ability to be incorporated into nanospheres and microspheres.

[0169] The different polynucleotides encoding the different components of the application can be expressed by different vectors. Thus, another aspect of the application relates to a vector composition, hereinafter referred to as a vector composition of the application, comprising a vector of the application and a second vector comprising a second polynucleotide encoding a polypeptide selected from the group consisting of: - a polypeptide comprising the sequence 477-542 of the avian orthoreovirus muNS protein (SEQ ID NO: 25), - a polypeptide comprising the sequence 561-622 (SEQ ID NO: 26) of the mammalian orthoreovirus muNS protein, - a functionally equivalent variant of any of the above polypeptides which retains the ability to be incorporated into nanospheres.

[0170] The polynucleotide in the vector composition can also be operably bound to a promoter, such as in the case of a single vector. In one embodiment of the vector composition of the present application, the polynucleotide of the present application or the expression cassette of the present application is operably bound to a first promoter and a second polynucleotide is operably bound to a second promoter. In another embodiment, the first promoter and the second promoter are the same promoter or different promoters.

[0171] In one embodiment of the present application, the vector of the vector composition of the present application is a bacterial expression vector. It will be apparent to those skilled in the art that the embodiments, definitions and embodiments described in relation to the vector of the present application are equally valid for the vector of the vector composition.

[0172] The polynucleotide, expression cassette, vector of the present application or the vector composition of the present application can be comprised in a host cell. Another aspect of the present application relates to a cell comprising the fusion protein of the present application, the polynucleotide of the present application, the expression cassette of the present application, the vector of the present application or the vector composition, hereinafter referred to as the cell of the present application.

[0173] The cell of the present application can be any prokaryotic cell or any eukaryotic cell. In the present application, virtually any cell type can be used. Any host cell that can be transformed with the polynucleotide of the present application, or that can be transformed, transfected or infected with a recombinant vector comprising the polynucleotide of the present application, such as an animal cell (e.g. a mammalian cell, an avian cell, an insect cell, etc.), a plant cell, a yeast, a bacterium, etc. The cell of the present application can be obtained by conventional methods known to the person skilled in the art.

[0174] Methods of the invention All definitions and embodiments described above in relation to the other aspects of the present application apply equally to the current aspect and the embodiments thereof.

[0175] Another aspect of the present application relates to a method for producing the fusion protein of the present application, hereinafter referred to as method I of the present application, comprising: (a) expressing in a cell a first polynucleotide encoding said fusion protein, (b) subjecting said cell to conditions suitable for the formation of nanospheres and / or microspheres, and (c) concentrating the nanospheres and / or microspheres.

[0176] The first step of the method for producing the fusion protein of the application comprises expressing in a cell a first polynucleotide encoding said fusion protein.

[0177] As known by the person skilled in the art, in order to express the first polynucleotide in a cell, the polynucleotide has to be introduced into the cell, for example by transforming said cell with a vector comprising the polynucleotide. In case the polynucleotide is operably bound to an inducible promoter, in order to express said polynucleotide, it will be necessary to bring the cell into contact with an inducer. In the specific case where the promoter can be induced by IPTG, in the case of bacterial cells, in order to express the polynucleotide, IPTG is added to the bacterial culture for a time necessary for the expression of the polynucleotide, for example at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours or more. Preferably, the IPTG induction is performed for a period of about 3 hours. In a particular embodiment, the cells are incubated at a temperature of 18 to 37°C, preferably 25 to 37°C, more preferably 37°C during the induction period. In a particular embodiment, when the induction time is greater than 24 hours, the cells are incubated at a temperature of 18 to 25°C during the induction period. Once the induction is over, the bacteria are collected by centrifugation.

[0178] The second step of the method for producing the fusion protein of the application consists in subjecting the cell to conditions suitable for the formation of nanospheres and / or microspheres.

[0179] The conditions suitable for the formation of nanospheres and / or microspheres can be determined by the person skilled in the art for each cell type. Methods suitable for detecting nanosphere and / or microsphere formation include, but are not limited to, the method described in Example 1 of patent application WO 2011 / 098652, wherein the formation of nanospheres / microspheres (inclusions) is detected by indirect immunofluorescence microscopy using an anti-muNS polyclonal antibody. In a particular embodiment, the conditions suitable for the formation of nanospheres and / or microspheres comprise incubating the cell expressing the first polynucleotide for a period of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, and incubating at a temperature of 25 to 37°C, preferably 37°C.

[0180] In a particular embodiment of method I of the application, the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

[0181] The first and second steps of the method for producing the fusion protein of the application can be performed simultaneously or sequentially, such that the expression of the first polynucleotide is first performed, and then the cell is subjected to conditions suitable for the formation of nanospheres and / or microspheres. In a particular embodiment, the first and second steps are performed simultaneously - taking into account the conditions to which the cell is subjected for the expression of the first polynucleotide and the second polynucleotide are suitable for the formation of microspheres. In a more particular embodiment, said conditions comprise incubating the cell in the presence of an inducer, preferably IPTG, for a period of time of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, and at a temperature of 25 to 37°C, preferably 37°C.

[0182] The third step of method I consists in concentrating the nanospheres and / or microspheres. To concentrate the nanospheres / microspheres, the cells in which the nanospheres / microspheres have formed must be lysed by any method, such as incubation with a lysis buffer or sonication, etc. Then, the nanospheres / microspheres can be sedimented by centrifugation, preferably at a speed of about 2700 g. In a particular embodiment, once the cells are lysed, the nanospheres / microspheres are washed with a buffer comprising a divalent cation. The term "divalent cation" as used herein refers to a positively charged ion of any metal of valence 2 in the periodic table. Suitable divalent cations for the application include, but are not limited to, divalent cations of Mg, Cd, Ca, Co, Cu, Fe, Mn, Ni, Sr and Zn. In a preferred embodiment, the divalent cation is Mg 2+ A suitable concentration of divalent cations to induce the formation of muNS protein aggregates is, for example, at least 0.5 mM, at least 0.8 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM or more. In a particular embodiment, the nanospheres and / or microspheres are washed with a buffer comprising Mg 2+ (e.g. MgCl2), preferably at a concentration of 5 mM. In an even more particular embodiment, the nanospheres and / or microspheres are concentrated following the purification protocol indicated in the examples of this document.

[0183] In a particular embodiment, method I of the application further comprises purifying the fusion protein by separating the fusion protein from the nanospheres and / or microspheres.

[0184] In case the fusion protein of the application comprises component (ii) (b) and a protease recognition sequence fused to the amino terminus of the sortase acceptor motif, it can be useful that cleavage of the protease recognition sequence makes the sortase acceptor motif available for interaction with a sortase. Thus, in one particular embodiment of method I of the application, if the fusion protein comprises component (ii) (b) and a protease recognition sequence fused to the amino terminus of the sortase acceptor motif, the method further comprises contacting the fusion protein with a protease specific for the protease recognition sequence fused to the amino terminus of the sortase acceptor sequence under conditions suitable for proteolysis of said recognition sequence, followed by separation of the protease recognition sequence of the fusion protein. Conditions suitable for proteolysis of said protease recognition sequence are described in the description and exemplified in the examples described herein.

[0185] For isolating the fusion protein from the nanospheres and / or microspheres, they have to be subjected to conditions that lead to their disintegration. Thus, in one particular embodiment, method I of the application further comprises purifying the fusion protein by separating it from the nanospheres and / or microspheres, for which the nanospheres and / or microspheres are subjected to conditions that lead to their disintegration.

[0186] Conditions that lead to disintegration of the nanospheres and / or microspheres include among others: incubation of the nanospheres and / or microspheres with a denaturant (e.g. urea or guanidine hydrochloride), incubation of the nanospheres and / or microspheres with an ionic detergent (e.g. high concentration of SDS), incubation with NaCl at a concentration above 500 mM, incubation with a buffer that does not comprise divalent cations (preferably does not comprise Mg 2+ ).

[0187] In one particular embodiment, the conditions that lead to disintegration of the nanospheres and / or microspheres are incubation with a buffer that does not comprise divalent cations (preferably does not comprise Mg 2+ ).

[0188] In one particular embodiment, once the nanospheres and / or microspheres are disintegrated, the fusion protein can be purified by any known method. The method will depend on the nature of the polypeptide of interest and will be known to the person skilled in the art. Polypeptide purification techniques are well known in the art and include, but are not limited to, affinity chromatography, exclusion chromatography, ion exchange chromatography, adsorption chromatography, immunoprecipitation, etc.

[0189] In addition to being able to be modified by sortases and incorporate components of interest into the fusion proteins of the application, the nanospheres / microspheres of the application can also be used as a platform for the incorporation of a second fusion protein comprising the IC region of a positive reovirus muNS protein. This allows the nanospheres / microspheres of the application to be used for the production of proteins using said second fusion protein.

[0190] Thus, another aspect of the application relates to a method for the production of a protein, hereinafter referred to as method II of the application, comprising: (a) expressing in a cell a first polynucleotide encoding a fusion protein of the application and a second polynucleotide encoding a second fusion protein comprising: (i) a polypeptide selected from: - a polypeptide comprising the sequence 477-542 of a avian positive reovirus muNS protein (SEQ ID NO: 25), - a polypeptide comprising the sequence 518-721 of a mammalian positive reovirus muNS protein (SEQ ID NO: 26), - a functional equivalent variant of any of the above polypeptides that retains the ability to be incorporated into nanospheres and / or microspheres, and (ii) a second polypeptide of interest, wherein component (ii) is the protein produced; (b) subjecting said cell to conditions suitable for the formation of nanospheres or microspheres, and (c) concentrating the nanospheres or microspheres.

[0191] In one particular embodiment, method II of the application comprises purifying the fusion protein by separating it from the nanospheres or microspheres.

[0192] In one particular embodiment of method II of the application, component (ii) is fused to the amino terminus of component (i) or to the carboxy terminus of component (i).

[0193] As will be appreciated by the skilled person, it can be useful to separate component (ii) from component (i) in method II of the application. One possibility to separate said components is to use a peptide whose sequence comprises a protease cleavage target, thereby allowing the separation of the two components.

[0194] In one particular embodiment of method II of the application, the second fusion protein comprises a protease recognition sequence between its component (i) and component (ii).

[0195] The term "protease" or "peptidase" as used herein refers to enzymes that break down the peptide bonds of proteins, for which they use water molecules, and are therefore classified as hydrolases. Examples of protease cleavage sites suitable for incorporation into the fusion protein of the present application include, but are not limited to, enterokinase (cleavage site DDDDK; SEQ ID NO: 5), Factor Xa (cleavage site IEDGR; SEQ ID NO: 6), Thrombin (cleavage site LVPRGS; SEQ ID NO: 7), Protease TEV (cleavage site ENLYFQG; SEQ ID NO: 8), Protease PreScission (cleavage site LEVLFQGP; SEQ ID NO: 9), Intein, and the like. In a particular embodiment, the protease recognition sequence is an enterokinase recognition sequence or a Factor Xa recognition sequence.

[0196] In a particular embodiment of the method II of the present application, the second fusion protein comprises a protease recognition sequence between its component (i) and component (ii), and the method II of the present application further comprises the step of isolating the protein resulting from component (i) of the second protein by incubating the second fusion protein with a protease under conditions leading to the cleavage of the recognition sequence by the protease.

[0197] In another particular embodiment, the method II of the present application further comprises purifying the fusion protein by separating the fusion protein from the nanospheres and / or microspheres by subjecting the nanospheres and / or microspheres to conditions leading to their disintegration.

[0198] In another particular embodiment of the method II of the present application, the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

[0199] In another particular embodiment of the method II of the present application, the protein is an IGRP protein.

[0200] Another aspect of the present application relates to a method for producing a first polypeptide of interest, hereinafter referred to as method III of the present application, wherein the method comprises the following steps: (a) producing a fusion protein of the present application by the method I of the present application, wherein the fusion protein comprises: - component (ii) (a) and component (iii) fused to the amino terminus of component (i), wherein component (iii) is a polypeptide of interest, and - a protease recognition sequence between its component (i) and component (iii), (b) subjecting the nanospheres and / or microspheres to conditions leading to their disintegration, thereby causing the separation of the fusion protein from the nanospheres and / or microspheres, (c) contacting the product obtained from step (b) with a protease specific for the recognition sequence linking component (i) and component (iii) of the fusion protein under conditions suitable for proteolysis of the fusion protein, followed by separation of component (i) and component (iii) of the fusion protein, (d) subjecting the product of step (c) to conditions suitable for the formation of nanospheres or microspheres, and (e) separating the nanospheres or microspheres from component (iii).

[0201] In a particular embodiment, the protease recognition sequence binding component (i) and component (iii) of the fusion protein is an enterokinase recognition sequence (cleavage site DDDDK - SEQ ID NO: 5), a factor Xa recognition sequence (cleavage site IEDGR - SEQ ID NO: 6), a thrombin recognition sequence (cleavage site LVPRGS - SEQ ID NO: 7), a protease TEV recognition sequence (cleavage site ENLYFQG - SEQ ID NO: 8), a protease PreScission recognition sequence (cleavage site LEVLFQGP - SEQ ID NO: 9), an intein recognition sequence or a similar sequence. In a more particular embodiment, the protease recognition sequence is an enterokinase recognition sequence or a factor Xa recognition sequence. In an even more particular embodiment, the protease recognition sequence is the sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 6.

[0202] Conditions suitable for proteolysis of the fusion protein include contacting the fusion protein with a protease specific for the recognition sequence linking component (i) and component (iii) of the fusion protein under conditions depending on the specific protease used, pH conditions, temperature conditions, etc. The person skilled in the art knows how to determine these conditions for each specific protease.

[0203] Conditions suitable for the formation of nanospheres and / or microspheres include contacting the product obtained from step (a) with a buffer comprising a divalent cation as described above. In a preferred embodiment, the divalent cation is Mg 2+ A suitable concentration of divalent cations for inducing the formation of muNS protein aggregates is for example at least 0.01 mM, at least 0.1 mM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM or more. In a particular embodiment, the conditions include incubation with a buffer comprising Mg 2+ (preferably at a concentration of 5 mM).

[0204] According to the method of the present application, when the nanospheres and / or microspheres re-form the component (i) of the fusion protein, they are again integrated into said nanospheres and / or microspheres, wherein the component (iii) of the fusion protein is free. Thus, this is a method suitable for protein purification.

[0205] Another aspect of the present application relates to a protein obtainable according to the method I, II or III of the present application.

[0206] Another aspect of the present application relates to a method for producing the nanospheres or microspheres of the present application, hereinafter referred to as method IV of the present application, comprising the following steps: (a) producing a fusion protein by the method I of the present application, and (b) subjecting said cell to conditions suitable for the formation of nanospheres or microspheres.

[0207] In one embodiment of the method IV of the present application, the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

[0208] The conditions for incubating the nanospheres or microspheres in the presence of a sortase are known to the person skilled in the art.

[0209] In one embodiment, the method IV of the present application further comprises the step of incubating the nanospheres and / or microspheres in the presence of a sortase recognizing the sortase recognition motif of component (ii) (a) of the fusion protein and a substrate comprising a sortase acceptor motif, thereby obtaining nanospheres and / or microspheres comprising a substrate comprising a sortase acceptor motif.

[0210] In one embodiment, the method IV of the present application further comprises the step of incubating the nanospheres and / or microspheres in the presence of a sortase recognizing the sortase acceptor motif in component (ii) (b) of the fusion protein and a substrate comprising a sortase recognition motif, thereby obtaining nanospheres and / or microspheres comprising a substrate comprising a sortase recognition motif.

[0211] In one embodiment of the method IV of the present application, the sortase is sortase A, the recognition motif is the sequence SEQ ID NO: 22 or SEQ ID NO: 23, and the sortase acceptor motif is a polyglycine sequence, preferably the sequence GGG.

[0212] In one embodiment of the method IV of the present application, the incubation in the presence of a sortase is in the presence of an excess of calcium ions (Ca 2+ ) and in the absence of phosphate groups (PO4 3-The step of incubating the nanospheres and / or microspheres in the presence of a sortase enzyme is performed at a pH of 6.5 to 8.5 (preferably at a pH of 7.5), at a temperature of 35 °C to 38 °C (preferably 37 °C) for 3 hours (h) to 5 h (preferably 4 h) in another particular embodiment of method IV of the application.

[0213] The step of incubating the nanospheres and / or microspheres in the presence of a sortase enzyme is performed at a pH of 7.5 for 4 h in the presence of an excess of calcium ions and in the absence of phosphate groups in another particular embodiment of method IV of the application.

[0214] The step of incubating the nanospheres and / or microspheres in the presence of a sortase enzyme is stopped by adding an excess of ethylenediaminetetraacetic acid (EDTA) (CAS number 60-00-4) during the reaction in another particular embodiment of method IV of the application.

[0215] The nanospheres or microspheres are purified between the step (b) and the step of incubating the nanospheres and / or microspheres in the presence of a sortase enzyme in another particular embodiment of method IV of the application.

[0216] Pharmaceutical compositions All definitions and particular embodiments described above in relation to other aspects of the application apply equally to the current aspect and the particular embodiments thereof.

[0217] The fusion protein of the application and the nanospheres or microspheres of the application can be used for the development of pharmaceutical compositions and for therapeutic applications.

[0218] Therefore, another aspect of the application relates to a pharmaceutical composition or an immunogenic composition, hereinafter referred to as pharmaceutical composition of the application or immunogenic composition of the application, comprising the nanospheres and / or microspheres of the application and a pharmaceutically acceptable excipient.

[0219] The term "immunogenic composition" refers to a composition that is capable of eliciting, establishing or inducing an immune response, i.e. a cellular immune response or an antibody-mediated immune response, in a subject upon administration. An "immunogenic composition" includes molecules having antigenic properties, such as dead or attenuated bacteria or viruses, but also immunogenic polypeptides. Immunogenic polypeptides are generally referred to as being antigenic. A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell antigen receptor. It is understood that in the present application, the immunogenic composition of the application includes the fusion protein of the application and optionally the nanospheres or microspheres of the application.

[0220] It will be apparent to those skilled in the art that a vaccine is an immunogenic composition. Thus, in a particular embodiment, the immunogenic composition is a vaccine or vaccine composition. The term "vaccine" or "vaccine composition" as used herein refers to an immunogenic composition capable of eliciting, establishing, inducing or improving an immune response against a particular disease, wherein the immune response is a cellular or antibody-mediated immune response following prophylactic administration to a subject. A vaccine or vaccine composition typically comprises an agent (e.g., a polypeptide) similar to a microorganism or a portion thereof that causes the disease. A vaccine or vaccine composition can be prophylactic or therapeutic. The term "vaccine composition" as used herein refers to the immunogenic composition of the present application supplemented with pharmaceutically acceptable carrier excipients that, when administered to a subject, elicit or are capable of directly or indirectly eliciting a protective immune response against a microorganism that causes a disease in the host or subject.

[0221] A "pharmaceutically acceptable excipient" is understood to mean a therapeutically inert substance that is used to incorporate the active ingredients and that is acceptable from a pharmacological / toxicological point of view for the patient to whom the pharmaceutical composition is to be administered and from a physical / chemical point of view for the pharmaceutical chemist who is manufacturing the pharmaceutical composition. Excipients or vehicles also include any substance used to improve the administration and effectiveness of the active ingredients in the pharmaceutical composition. Examples of pharmaceutically acceptable vehicles include one or more of water, saline, aqueous dextrose, glycerol, ethanol or the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, sugar alcohols (such as mannitol, sorbitol), or sodium chloride in the composition. Pharmaceutically acceptable vehicles can further include a minor amount of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the microspheres or compositions that are part of the pharmaceutical composition. Examples of suitable vehicles are known in the literature (see, for example, Remington's Pharmaceutical Sciences, 19thEd., Mack Publishing Company, Easton, PA, 1995). In certain cases, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate. The quantity and nature of the pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Faulí and Trillo C. (1993) "Tratado de Farmacia Galénica", Luzán 5, S.A. Editions, Madrid). th ed., Mack Publishing Company, Easton, PA, 1995). In certain cases, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate. The quantity and nature of the pharmaceutically acceptable excipients depend on the desired dosage form. Pharmaceutically acceptable excipients are known to those skilled in the art (Faulí and Trillo C. (1993) "Tratado de Farmacia Galénica", Luzán 5, S.A. Editions, Madrid).

[0222] The pharmaceutical compositions of the application can be administered by any suitable route, such as orally, subcutaneously, intravenously, intraperitoneally or intramuscularly.

[0223] Medical applications All definitions and specific embodiments described above in relation to other aspects of the application apply equally to the current aspect and its specific embodiments.

[0224] It will be apparent to those skilled in the art that the fusion protein and / or the nanosphere or microsphere can be used in medicine. Thus, another aspect of the application relates to the nanosphere and / or microsphere of the application for use in medicine.

[0225] Another aspect of the application relates to the nanosphere and / or microsphere of the application or the immunogenic composition of the application for use in the treatment and / or prevention of bluetongue, hereinafter referred to as the first medical use of the application, wherein the fusion protein comprised in the nanosphere / microsphere is characterized in comprising at least one of: the bluetongue virus 4 (BTV) outer capsid protein 2 (VP2); the bluetongue virus 4 (BTV) core protein 7 (VP7); the bluetongue virus 4 (BTV) non-structural protein 1 (NS1); a protein having a sequence functionally equivalent to any of the above-mentioned proteins.

[0226] The term "bluetongue" or "sheep catarrhal fever" is a non-contagious viral disease affecting domestic and wild ruminants (mainly sheep, but also cattle, goats, water buffalo, antelopes, deer or elks) and transmitted by the biting midge (Culicoides) species. The virus causing bluetongue, the bluetongue virus (BTV), was identified as a member of the orbivirus genus of the Reoviridae family. Orbivirus The bluetongue virus species or serogroup includes 24 known serotypes and other atypical serotypes described recently. In many animals, bluetongue virus (BTV) infection can be inapparent, but in some infected ruminants it can cause a fatal disease. Disease severity varies between different species and strains, with the most severe symptoms observed in sheep, leading to death, weight loss and impaired wool growth. In highly susceptible sheep, the morbidity can reach 100% and the mortality ranges between 2 and 30%, but can reach 70%.

[0227] The term "outer coat protein 2" or "VP2" as used herein refers to one of the two proteins that, together with VP5, constitute the outer coat of the viral BTV particle. VP2 is the major immunogenic particle of BTV. VP2 is responsible for the adhesion of the virus to the target host cell, possibly as a result of binding to sialic acid. This binding induces the internalization of the virus within the cell mainly through clathrin-dependent endocytosis. In one particular embodiment of the present invention for the treatment of bluetongue disease, the BTV4 outer coat protein 2 comprises the sequence identified in the UniprotKB database by accession number P12434 (entry version 70, December 14, 2022; sequence version 2, November 1, 1991 ).

[0228] The term "core protein 7" or "VP7" as used herein refers to one of the BTV core proteins that can be obtained from the surface of the complete virus. VP7 appears to be important for the interaction of the virus with insect cells. In one particular embodiment of the present invention for the treatment of bluetongue disease, the BTV4 core protein 7 comprises the sequence identified in the UniprotKB database by accession number P69361 (entry version 77, December 14, 2022; sequence version 1, March 3, 2005).

[0229] The term "non-structural protein 1 " or "NS1 " as used herein refers to a positive viral protein synthesis regulator. In one particular embodiment of the present invention for the treatment of bluetongue disease, the BTV4 core protein 7 comprises the sequence identified in the UniprotKB database by accession number Q1W9P8 (entry version 16, December 14, 2022; sequence version 1, May 2, 2006).

[0230] In the case of the first medical use of the present invention, the expression "functionally equivalent sequence" refers to a variant of the VP2, VP7 or NS1 protein that fully or partially retains its function as an immunogenic polypeptide as described above.

[0231] The functionally equivalent variant of VP2, VP7 or NS1 protein includes a protein variant that exhibits at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence from which they are derived.

[0232] In the context of the present application, "treatment and / or prevention of bluetongue" is understood to mean the administration of the nanospheres and / or microspheres of the present application to prevent or delay the onset of symptoms, complications or biochemical indicators of infection, thereby alleviating the symptoms thereof, or stopping or inhibiting their development and progression, such as, for example, death. The treatment can be a prophylactic treatment to prevent the onset of the disease or the manifestation of its clinical or subclinical symptoms, or a therapeutic treatment to eliminate or alleviate the symptoms after the manifestation of the disease.

[0233] In one embodiment of the first medical use of the application, the treatment is performed in a subject.

[0234] The term "patient" or "subject" as used herein refers to any animal, preferably a mammal and includes therein a domesticated and farm animal, a primate and a human (e.g., a human), a non-human primate, a bovine, equine, porcine, ovine, caprine, canine, feline or rodent (e.g., a rat or mouse). In a preferred embodiment, the subject is a human of any age or ethnicity.

[0235] In one embodiment, the first medical use of the application comprises the administration of a therapeutically effective amount of nanospheres and / or microspheres.

[0236] In another embodiment of the first medical use of the application, the nanospheres and / or microspheres are administered without an adjuvant. Without wishing to be bound by a particular theory, it is believed that the administration of antigens without an adjuvant favors the generation of a tolerogenic rather than an inflammatory response in vivo.

[0237] The expression "therapeutically effective amount" as used herein is understood to mean an amount capable of providing a therapeutic effect and can be determined by those skilled in the art by usual methods. In particular, a therapeutically effective amount of the nanospheres and / or microspheres of the present application is an amount capable of eliciting an immune response in a subject. The amount of nanospheres and / or microspheres of the present application that can be included in a pharmaceutical composition according to the present application will vary depending on the subject and the particular mode of administration. Those skilled in the art will understand that the dosage can also be determined under the guidance of Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pages 1707-1711 and Goodman and Goldman's The Pharmacological Basis of Therapeutics, Twelfth Edition (2001), Appendix II, pages 475-493.

[0238] Another aspect of the application relates to the nanospheres and / or microspheres of the application or the immunogenic composition of the application for use in the treatment and / or prevention of African horse sickness, hereinafter referred to as the second medical use of the application, wherein the fusion protein comprised in the nanospheres is characterized by comprising at least one of the following: an African horse sickness virus (AHSV) non-structural protein 1 (NS1); a protein having a sequence functionally equivalent to any of the above-mentioned proteins.

[0239] The term "African horse sickness virus" or "AHSV" as used herein describes a Reoviridae orbivirus AHSV, which has a naked, 60-80 nm, icosahedral virion, highly similar to the causative agent of bluetongue disease in sheep or epizootic hemorrhagic disease in deer. AHSV is resistant to organic solvents and bile salts, but sensitive to non-neutral pH, heat and putrefaction. It has 9 serotypes, with common complement-fixing antigens, but cross-protection is only between 6 and 9. Its virulence is variable, with serotype 4 being the highest, with mortality rates above 90%, while serotype 9 is the lowest, with mortality rates between 70% and 80%. AHSV causes an acute viral disease in horses, transmitted by arthropods, with high mortality and seasonal occurrence, with acute febrile, cardiac and pulmonary cases, and oedematous and hemorrhagic lesions.

[0240] In a particular embodiment of the second medical use of the application, the AHSV non-structural protein 1 comprises the sequence identified in the UniprotKB database by accession number P87505 (entry version 49, December 14, 2022; sequence version 1, May 1, 1997).

[0241] The causal agent is a Reoviridae orbivirus AHSV, which has a naked, 60-80 nm, icosahedral virion, highly similar to the causative agent of bluetongue disease in sheep or epizootic hemorrhagic disease in deer. AHSV is resistant to organic solvents and bile salts, but sensitive to non-neutral pH, heat and putrefaction. It can be cultivated in HeLa and BHK 21 cells and isolated in chicken embryos and suckling mice. It has 9 serotypes, with common complement-fixing antigens, but cross-protection is only between 6 and 9. Its virulence is variable, with serotype 4 being the highest, with mortality rates above 90%, while serotype 9 is the lowest, with mortality rates between 70% and 80%.

[0242] In the context of the second medical use of the application, the expression "functionally equivalent sequence" refers to a variant of the AHSV NS1 protein that fully or partially retains its function as an immunogenic polypeptide, as described above.

[0243] Functionally equivalent NS1 protein variants include protein variants that exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence from which they are derived.

[0244] In one embodiment of the second medical use of the application, the treatment is performed in a subject, preferably a mammal and wherein is included domestic and farm animals, primates and humans (e.g., humans), non-human primates, bovines, equines, porcines, ovines, caprines, canines, felines or rodents (e.g., rats and mice).

[0245] In one embodiment, the second medical use of the application comprises administering a therapeutically effective amount of nanospheres and / or microspheres. In another embodiment of the treatment of African horse sickness of the application, the nanospheres are administered without adjuvant.

[0246] Another aspect of the application relates to the nanospheres and / or microspheres of the application or the pharmaceutical composition of the application for use in the treatment and / or prevention of diabetes mellitus type 1, hereinafter referred to as the third medical use of the application, wherein the fusion protein comprised in the nanospheres and / or microspheres is characterized by comprising a glucose-6-phosphatase 2 protein (IGRP) according to the sequence of accession number Q9NQR9 in the UniProt database (entry date August 3, 2022, sequence version 1).

[0247] The term “type 1 diabetes” or “diabetes mellitus type I” or “juvenile diabetes” or “insulin-dependent diabetes” as used herein refers to a metabolic disease characterized by the selective destruction of beta cells in the pancreas, causing a complete lack of insulin. It is different from type 2 diabetes because it is a form of diabetes that occurs early in life, usually before the age of 30. Only 1 in 20 people with diabetes has type 1 diabetes, which is more common in young people and children. Type 1 diabetes is divided into autoimmune cases (i.e., the most common form) and idiopathic cases. The term “type 1 diabetes” as used herein includes classic type 1 diabetes, which is usually diagnosed before the age of 30 and requires insulin treatment from the time of diagnosis, as well as adult-onset latent autoimmune diabetes, which is diagnosed after the age of 30 and usually does not require insulin treatment within 3-6 months after diagnosis.

[0248] In a particular embodiment of the third medical use of the application, the type 1 diabetes is an autoimmune type 1 diabetes. The term "autoimmune type 1 diabetes" as used herein refers to an autoimmune disease involving selective destruction of beta cells in the pancreas mediated by activated T lymphocytes in subjects with susceptible HLA haplotypes. After a variable duration of preclinical phase (during which the patient remains asymptomatic), when the mass of insulin-producing cells reaches a critical value, the patient exhibits the typical symptoms: polyuria, polydipsia, polyphagia, weight loss and progressive ketosis, which can lead to ketoacidosis if no exogenous insulin treatment is introduced.

[0249] In a more particular embodiment of the third medical use of the application, the type 1 diabetes is "Latent Autoimmune Diabetes in Adults" or "LADA", and as used herein, it refers to an autoimmune diabetes diagnosed in adulthood, usually after the age of 30, in subjects positive for at least one autoantibody selected from the autoantibodies usually present in patients with typical type 1 diabetes, such as islet cell antibodies (ICA), glutamic acid decarboxylase antibodies (GADA), islet antigen-2 antibodies (IA-A2) or insulin autoantibodies (IAA), and who do not require insulin treatment in the first 3 or 6 months after diagnosis. Adult autoimmune diabetes differs from type 1 diabetes in that the beta cell failure progresses slowly, with subsequent gradual dependence on insulin. Usually, in LADA patients, the function of the beta cells is affected within six years after diagnosis.

[0250] In a preferred embodiment of the third medical use of the application, the subject is a human of any age or ethnicity. In a particular embodiment, the subject is at risk of developing type 1 diabetes. A subject at risk of developing type 1 diabetes is an individual who has a first degree relative (brother / sister) diagnosed with type 1 diabetes and who also has multiple (>2) serum autoantibodies against islet (ICA), glutamic acid decarboxylase 65 (GADA65), islet antigen-2 (IA-2) or ZnT8 transporter (ZnT8).

[0251] A particular embodiment of the third medical use of the application comprises the administration of a therapeutically effective amount of nanospheres and / or microspheres. In another particular embodiment of the treatment of type 1 diabetes of the application, the nanospheres and / or microspheres are administered without adjuvant.

[0252] In another particular embodiment of the third medical use of the application, the administration of nanospheres and / or microspheres is by subcutaneous or intravenous route.

[0253] Another aspect of the application relates to a method of inducing type 1 diabetes in an animal model, hereinafter referred to as method V of the application, said method comprising administering to the animal an effective amount of nanospheres and / or microspheres of the application, wherein the fusion protein comprised in the nanospheres and / or microspheres is characterized by comprising a glucose-6-phosphatase 2 protein (IGRP).

[0254] In a particular embodiment, the glucose-6-phosphatase 2 protein (IGRP) has the sequence with accession number Q9NQR9 in the UniProt database, entry date August 3, 2022, sequence version 1.

[0255] When administered to mice in the form of a DNA vaccine, the IGRP protein is capable of inducing diabetes (Fuchs et al ., Clinical and Experimental Immunology, 2014, 176: 199-206). In a particular embodiment, a functionally equivalent variant of the IGRP protein retains the ability of the IGRP protein from which it is derived to induce diabetes. The ability of a protein to induce diabetes in an animal model can be determined by methods known to the person skilled in the art, for example by determining the urine level or the blood glucose level. For example, Fuchs et al. (ibid.) consider that a murine model presents diabetes when two successive measurements of urine glucose level above 5.5 mmol / L or blood glucose level above 13.9 mmol / L are obtained. Furthermore, the IGRP protein also exhibits glucose 6-phosphatase activity, i.e. it is capable of catalyzing the dephosphorylation of glucose-6-phosphate to glucose. In a particular embodiment of method V of the application, a functionally equivalent variant of the IGRP protein retains the phosphatase activity of the IGRP protein from which it is derived. Glucose-6-phosphatase activity can be determined by methods known to the person skilled in the art, for example based on methods for detecting inorganic phosphate in aqueous solution, such as the malachite green method (Petrolonis et al., ibid.), or based on methods for detecting glucose-6-phosphate, such as, for example, the method based on the glucose oxidase / peroxidase system (Mao, H., et al., Anal. Chem. 2002, 74, 379-385). et al

[0256] In a particular embodiment of method V of the application, the method comprises administering to the animal an effective amount of nanospheres and / or microspheres of the application.

[0257] The term "effective amount" means the amount of nanospheres and / or microspheres that leads to the subject to which the nanospheres and / or microspheres are administered being considered as having diabetes.

[0258] In another particular embodiment of method V of the application, the administration is by subcutaneous or intramuscular route. ​

[0259] In another specific embodiment of method V of the present invention, nanospheres and / or microspheres are administered together with an adjuvant.

[0260] *** The following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention.

[0261] Example Example 1 Materials and methods and results muNS-HA or HA-muNS do not form cytoplasmic inclusions To purify the muNS-Mi protein, a protein tagged with a hemagglutinin epitope (HA) was created, fused to the C-terminus of the protein. Immunofluorescence analysis of its expression in HeLa cells showed that adding the tag to the C-terminus of the muNS-Mi protein resulted in the loss of muNS-Mi's ability to form cytoplasmic inclusions. Figure 4 ).

[0262] Construction of plasmids pET Duet 1 -MiST and pET Duet 1 -MiST / IC-AvPAL The muNS-Mi sequence was obtained by PCR amplification of plasmid pCIneo-muNS (488-635) (Brandariz-Nuñez). et al A 2010, PLoS ONE 5(11) e13785), the positive primer used was 5´-CATG CCATGG CACCAGCCGTACTGCTGTC-3´ (target NcoI underlined and ATG start codon double underlined) (SEQ ID NO: 27), and the negative primer is 5´-TT GCGGCCGC AA TCA GCCGGTTTCCGGCAGCAGATCATCCACC -3´ (target NotI underlined and stop start codon double underlined) (SEQ ID NO: 28), containing a sorting enzyme motif followed by a stop codon. The amplified product was inserted into the first polylinker of plasmid pETDuet1 to generate plasmid pET Duet1-MiST. Sanger sequencing confirmed the sequence correctness and the presence of the sorting enzyme motif. Its N-terminus is labeled with IC (Anabaena variegata). Anabaena variabilis ) Phenylanine aminolysin ( AvPALThe sequence was obtained by digesting the plasmid pETDuet1-muNS-Mi / 2-IC-AvPAL with a restriction enzyme previously obtained in the inventors' laboratory (proprietary result, not published) and inserting it into the second polylinker of plasmid pET Duet1-MiST to obtain plasmid pET Duet1-MiST / 2-IC-AvPAL. The sequence was confirmed to be correct by Sanger sequencing.

[0263] MiST expression and purification Competent BL21 bacteria (DE3) were transformed with plasmid pET Duet1-MiST and protein expression was induced by incubation at 37°C for 3 hours in the presence of 1 mM IPTG. When the total bacterial extract was analyzed by SDS-PAGE, a strong protein band of the apparent protein MW of MiST was observed in the stained gel. Figure 1 Lane 2), while it was absent in the extract of uninduced bacteria ( Figure 1 Lane 1). Then, follow the published NS purification protocol (Barreiro-Piñeiro, N). et al (2018, Scientific Reports 8, 16286), obtained Figure 1 The purified material shown in lane 3 indicates that NS was correctly formed. Furthermore, NS was observed under an optical microscope and characterized by DLS as particles with an average diameter of 460 nm. To further confirm the correct formation of NS, uninduced and IPTG-induced bacteria were precipitated, fixed, and analyzed by TEM. Figure 1 The image in b shows the presence of a typical NS derived from muNS in the induced bacteria, which is absent in the non-transformed bacteria (not shown).

[0264] Sortase-mediated MiST surface derivatization The next step is to examine whether the NS formed by MiST can react with polyG-containing compounds—catalyzed by a sorting enzyme. For this purpose, sorting enzyme 5Δ (SiMPLe Protein Labeling Kit, BPS Bioscience) and the fluorescent substrate AZDye 488-Gly-Gly-Gly (Click Chemistry Tools) were used. Figure 2 (The structural formula in a). In this sense, purified muNS-Mi NS or MiST NS at room temperature were incubated overnight with the substrate in the presence of sorting enzyme A. After incubation, both samples were dialyzed to remove excess substrate and then observed under a fluorescence microscope. When observed under a fluorescence microscope, the NS containing the sorting enzyme motif showed obvious fluorescence ( Figure 2a, Image MiST-IC), while no fluorescence was observed in muNS-Mi NS under the same conditions Figure 3 a, Image muNS-Mi), indicating the reaction was successful.

[0265] To eliminate this possibility that non-specific non-covalent association was responsible for the observed fluorescence labeling, both NS preparations were subjected to SDS-PAGE and the unstained and unfixed gels were observed under UV light, showing clear and intense fluorescent bands corresponding to the molecular weight of MiST ( Figure 2 b, lane 4), while no fluorescence labeling occurred in the lanes corresponding to muNS-Mi ( Figure 2 b, lane 3), also in any case when no sortase and fluorescent substrate were present ( Figure 2 b, lanes 1 and 2). The presence, amount and identity of both proteins were also confirmed in parallel by Western blot analysis using anti-muNS antibodies ( Figure 2 c). These results clearly indicate that covalent binding between the fluorescent substrate and MiST, catalyzed by sortase A, leads to MiST labeling. Western blot analysis also shows a slight difference in size between muNS-Mi and MiST due to the addition of the sortase-target sequence ( Figure 2 c, compare lanes 1 and 2 or 3 and 4).

[0266] The effect of derivatization of MiST-NS surface with AZDye on their size and charge was then analyzed. DLS analysis showed that sortase-mediated modification leads to a significant increase in NS size of about 19 nm. An increase in negative charge, measured by Z-potential, was also observed. Both results are fully consistent with the nature of the added molecule (see structure in Figure 2 ).

[0267] Table 1 - DLS analysis and comparison of MiST-IC, either unlabeled or labeled with AZDye 488-Gly-Gly-Gly Size (nm) PdI Z-potential (mv) MiST NS 459 ± 16 0.44 ± 0,10 -27 ± 2 MiST NS AZDye 488-Gly-Gly-Gly ]]> 508 ± 21 0.50 ± 0,10 -34 ± 2 Co-purification of external proteins in NS with MiST After demonstrating that MiST is able to correctly form NS and that these NS also react with the PolyG substrate through the added C-terminal sortase motif, the next step was to check whether this new construct is also able to capture IC-labeled proteins, like the native muNS-Mi. In this sense, the plasmid pET Duetl-MiST / IC-AvPAL was constructed, as described in point 1. This plasmid drives the simultaneous expression of MiST (based on polylinker 1) and IC-AvPAL (based on polylinker 2). After IPTG induction in BL21 bacteria (DE3), bands corresponding to the molecular weight of AvPAL and MiST were clearly visible in the stained gel ( Figure 3, lanes 1 and 2). After following the NS purification protocol published by the inventors (Barreiro-Piπeiro, N. et al . 2018, Scientific Reports 8, 16286) both proteins were clearly present in the NS and therefore they were still associated (Figure 1, lane 3). When comparing the purified NS with the unmodified muNS-Mi in parallel, both NS preparations were extremely similar (Figure 1, lanes 4 and 5), which indicates that the new construct behaves the same as muNS-Mi, not only in terms of inclusion body formation, but also in terms of association with IC-tagged proteins and this will be applicable to all the described applications of the previously characterized IC-tag methodology. Also, the presence of the sortase target sequence in MiST (Figure 1, lane 5) reduces its migration in the acrylamide gel when compared to unmodified muNS-Mi (Figure 1, lane 4). When observing the NS formed by MiST and containing AvPAL under TEM, the same NS structure was observed as shown in Figure 1 b (not shown), as expected. Figure 3 Figure 3 Figure 3 Figure 3 Figure 1

[0268] Conclusions Despite the expected difficulties, a new modified version of muNS-Mi has been developed (named MiST) that carries a sortase A target sequence at its C-terminus.

[0269] MiST is still able to form NS that capture IC-tagged proteins, mimicking muNS-Mi in the IC-tag system.

[0270] In addition, the NS formed by MiST can be further modified with a compound with an N-terminal polyglycine moiety (at least 3xGly) by incubation with sortase A.

[0271] This new version of muNS significantly improves the versatility of the previously characterized IC-tag methodology, since almost any compound can be added to the surface of these preformed spheres.

[0272] Example 2 Construction of plasmids pETDuetl.G4S-MiST and pETDuetl.G4S-MiST / IC-AvPAL The muNS-Mi sequence was obtained by PCR amplification of the plasmid pCIneo-muNS (488-635) (Brandariz-Nuñez et al ​​​​​(2010) J. Virol. 84:4289-4301), using the forward primer 5'- CATGCCATGGCACCAGCCGTACTGCTGTC-3' (SEQ ID NO: 36). The reverse primer was 5'- TTGCGGCCGCAATCAACCACCAGTCTCCGGGCAGAGAACCACCACCCAGATCATCCACC-3' (SEQ ID NO: 37) containing a sortase recognition motif sequence preceded by a G4S motif (SEQ ID NO: 29) and followed by a stop codon. This design allowed the introduction of a G4S motif at the C-terminus of the muNS-Mi sequence, which functions as a flexible spacer before the LPETG motif (SEQ ID NO: 23). The amplified product was inserted into the first polylinker of plasmid pETDuetl to generate plasmid pETDuetl.G4S-MiST. The sequence was confirmed to be correct by Sanger sequencing. The N-terminal tag of the IC-labeled Avicennia variago phenylalanine ammonia lyase (AvPAL) sequence was obtained by restriction enzyme Ndel and Bglll digestion of plasmid pETDuetl.muNS-Mi 2.IC-AvPAL (unpublished results) previously obtained in the inventor's laboratory and insertion into the second polylinker of plasmid pETDuetl.G4S-MiST to obtain plasmid pETDuetl.G4S-MiST / IC-AvPAL. The sequence was confirmed to be correct by Sanger sequencing.

[0273] Figures 5 to 8 Expression and purification of the "normal" version (MiST) and the linker- bearing version (G4S-MiST) as well as the respective versions loaded with IC-tagged AvPAL enzyme are described. The expression and purification methods of the new version of G4S-MiST are the same as for MiST.

[0274] Table 2 shows the size (nm), Pdl and Z-potential (mV) values of different NSs before and after reaction with a fluorescent compound in the presence of SrtA. Data are expressed as mean ± standard deviation; n = 3. Significant differences at a = 0.05 are indicated with asterisks. Data were tracked and statistical analysis was performed using GraphPad Prism.

[0275] Table 2: DLS and Z-potential analysis of functionalized NSs After demonstrating that MiST NS can be functionalized with small substrates, the possibility of assembling a full-protein covalently mediated by a sorting enzyme was tested. For this purpose, a modified eGFP protein containing an N-terminal polyglycine tail was used and incubated with empty or AvPAL-loaded MiST NS in the presence or absence of (+SrtA) sorting enzyme A. Figure 9 The images show that both MiST and G4S-MiST were only able to be GFP-labeled after incubation with sorting enzyme A. Furthermore, the presence of the linker did not indicate any significant improvement in labeling.

[0276] make Figure 9 The NS samples shown underwent electrophoresis under semi-denaturing conditions to retain the fluorescence ability of GFP. Figure 10 As shown, MiST (lanes 2 and 3) and G4S-MiST (lanes 4 and 5) – used alone (2 and 4) or loaded with AvPAL (3 and 5) – covalently bind to glycine-containing GFP after reaction with the sorting enzyme, as an additional band corresponding to the size of the eGFP-MiST binding product can be observed. In the case of the encapsulated enzyme version, a second band of a larger size is observed (which may correspond to the type of dimer produced by the reaction).

[0277] To complete the analysis, a new reaction was performed using NS loaded with the enzyme to be analyzed, this time by complete SDS-PAGE denaturation. Figure 11 The Coomassie staining gels shown clearly demonstrate that, in the presence of glycine-containing GFP and followed by reaction with sorting enzyme A (lanes 3 and 4), both MiST (lane 3) and G4S-MiST (lane 4) produced bands corresponding to the molecular weight of the GFP-MiST fusion. The presence of the G4S linker did not show any significant improvement in the reaction.

[0278] Example 3 Since it has been demonstrated that MiST nanospheres (NS) can be functionalized with whole proteins (such as GFP) containing oligo-Gly tails at their N-terminus, the inventors sought to construct a novel MiST variant that would be helpful in cases where it is impossible or unsuitable to modify the N-terminus of the protein to which the oligo-Gly tail is to be added, for example, by appropriately orienting the binding surface to prevent folding barriers, etc. In such cases, it might be better to perform the opposite strategy, i.e., adding an oligo-Gly sequence to the N-terminus of muNS-Mi, which, in the presence of sorting enzyme A, can react with molecules carrying sorting enzyme motifs. This strategy carries risks because the successful construction of correctly ordered inclusions, as tested with muNS, depends heavily on the N-terminal added sequence, which often results in amorphous, disordered, and useless aggregates (Brandariz-Nuñez). et al., 2010 J.Virol.84:4289 – 4301). To achieve this goal, initially, a tract of three glycines was added to the N-terminus of muNS-Mi immediately following the initial methionine residue to create the 3G-MiST fusion protein. Additionally, a construct was designed carrying a TEV protease motif (ENLYFQG) at the N-terminus, followed by two additional Gly molecules. Since the TEV protease cleaves between Q and G, the N-terminal Gly 3 molecules required for the sorting enzyme reaction will be available at the N-terminus of the construct after digestion of the ENLYFQG sequence. BL21 bacteria were transformed with the resulting plasmids pDuet1-3G-MiSt and pDuet1-TEV-3G-MiST, and protein extracts were compared before and after IPTG induction. Figure 12 (These are lanes 1 and 2, respectively).

[0279] exist Figure 12 In the analyzed gels, the expression of both constructs was clear. Although the TEV-containing version expressed better than the 3 Gly version, both versions were characterized. First, purification was performed using the protocol described above for nanospheres produced with IC labeling and MiSt-IC. Figure 13 As shown in Figure A, both versions are easily purified using standard procedures, indicating that their behavior is similar to the muNS-Mi or MiST versions. Furthermore, both versions are also susceptible to sorting enzyme modification, as they are only covalently labeled with the fluorescent compound when incubated with the fluorescent substrate 5-FAM-LPETG (SEQ ID NO: 23) (LPETG being the sorting enzyme motif) and sorting enzyme A is included in the reaction. Additionally, for TEV-3G-MiST, TEV digestion is necessary prior to the sorting enzyme reaction to make the 3G motif usable at its N-terminus, as is the case from... Figure 13 The result shown in B is inferred from this.

[0280] The same results were obtained using 3G-MiST and TEV-3G-MiST spheres loaded with exogenous protein (AvPAL), such as Figure 14 As shown. Similarly, the difference is that 3G-MiST has lower expression than TEV-3G-MiST, but both capture IC-tagged proteins, are purified using the inventors' disclosed protocol, and react with ligands having sorting enzyme motifs mediated by sorting enzyme A.

Claims

1. A fusion protein comprising the following components: (i) A polypeptide comprising a sequence selected from the following: - The sequence of the muNS protein of avian orovirus (SEQ ID NO: 1), 448-635. - The sequence 518-721 (SEQ ID NO: 2) of the mammalian ororeovirus muNS protein, and - Functional equivalent variants of any of the above sequences having the ability to form nanospheres and / or microspheres, and (ii) Polypeptides, selected from: (a) A polypeptide comprising a sorting enzyme recognition motif or the remainder of a sorting enzyme recognition motif generated after a sorting enzyme-mediated reaction, wherein component (ii) is fused to the carboxyl terminus of component (i), or (b) A polypeptide comprising a sorting enzyme acceptor motif wherein component (ii) is fused to the amino terminus of component (i).

2. The fusion protein according to claim 1, wherein the sorting enzyme recognition motif is the amino acid sequence LPXTG (SEQ ID NO: 22), where X is any amino acid.

3. The fusion protein according to claim 2, wherein the sorting enzyme recognition motif is the amino acid sequence LPETG (SEQ ID NO: 23).

4. The fusion protein according to any one of claims 1 to 3, wherein the fusion protein comprising component (ii) (a) further comprises component (iii) fused to the amino terminus of component (i), wherein component (iii) is the first polypeptide of interest.

5. The fusion protein according to claim 4, wherein component (iii) is fused to component (i) via a protease recognition sequence.

6. The fusion protein according to claim 5, wherein the protease recognition sequence is an enterokinase recognition sequence or a factor Xa recognition sequence.

7. The fusion protein according to any one of claims 4 to 6, wherein component (iii) comprises a secretion pathway signal peptide.

8. The fusion protein according to claim 7, wherein the signal peptide comprises the sequence SEQ ID NO:

10.

9. The fusion protein according to any one of claims 4 to 8, wherein component (iii) comprises a peptide that facilitates its purification.

10. The fusion protein according to any one of claims 1 to 9, wherein the fusion protein comprising component (ii) (a) further comprises a component of interest covalently bound to the remaining portion of the sorting enzyme recognition motif generated after the sorting enzyme-mediated reaction.

11. The fusion protein of claim 10, wherein the component of interest is selected from: Toll-like receptor (TLR) ligands, monophosphoryl lipid A (MPL A), oligonucleotides selected from CpG islands and polyinosinic acid:polycytidylic acid (poly(I:C) – CAS No. 24939-03-5), saponins, lipid molecules, coating molecules, oligosaccharides, antibodies, nanobodies, avidin, viral antigens, bacterial antigens, fungal antigens, allergens, cell-penetrating peptides, avidins, or environmental antigens, tumor antigens, wherein the component of interest is characterized by comprising an oligoglycine sequence of at least 3 glycines.

12. The fusion protein according to any one of claims 1 to 11, wherein component (i) and component (ii) (a) are covalently linked by a flexible peptide linker.

13. The fusion protein of claim 12, wherein the flexible peptide linker comprises the amino acid sequence according to SEQ ID NO: 29 (GGGGS).

14. The fusion protein of claim 1, wherein the sorting enzyme acceptor motif comprises an amino acid sequence having at least 1 to 20 nonpolar amino acids.

15. The fusion protein of claim 14, wherein the nonpolar amino acid sequence comprises a plurality of glycine (G) or alanine (A).

16. The fusion protein according to claim 15, wherein the amino acid sequence is sequence G[G]. n or A[A] n , where n = 0-2.

17. The fusion protein of claim 16, wherein the amino acid sequence is selected from: G, A, GG, AA, GGG, and AAA.

18. The fusion protein of claim 17, wherein the amino acid sequence comprises the sequence GGG.

19. The fusion protein according to any one of claims 1 and 14 to 17, wherein the fusion protein further comprises a protease recognition sequence covalently bound to the amine terminus of the sorting enzyme acceptor motif.

20. The fusion protein of claim 19, wherein the protease recognition sequence is an enterokinase recognition sequence or a factor Xa recognition sequence.

21. The fusion protein according to any one of claims 1 and 14 to 20, wherein the fusion protein comprising component (ii) (b) further comprises the component of interest covalently bound to the sorting enzyme acceptor motif.

22. The fusion protein of claim 21, wherein the component of interest is selected from: Toll-like receptor (TLR) ligands, monophosphoryl lipid A (MPL A), oligonucleotides selected from CpG islands and polyinosinic acid:polycytidylic acid (poly(I:C) – CAS No. 24939-03-5), saponins, lipid molecules, coating molecules, oligosaccharides, antibodies, nanobodies, avidin, viral antigens, bacterial antigens, fungal antigens, allergens, cell-penetrating peptides, avidins, or environmental antigens, tumor antigens, wherein the component of interest is characterized by comprising a sorting enzyme recognition sequence or the remainder of a sorting enzyme recognition motif generated after a sorting enzyme-mediated reaction.

23. Nanospheres or microspheres comprising the fusion protein according to any one of claims 1 to 22.

24. The nanospheres or microspheres of claim 23, further comprising a second fusion protein, the second fusion protein comprising the following components: (a) Polypeptides, selected from: The polypeptide comprises the sequence 477-542 (SEQ ID NO: 25) of the avian orotraumatic virus muNS protein. The polypeptide comprises the sequence 561-622 of the mammalian ororeovirus muNS protein (SEQ ID NO: 26). Functional equivalent variants of any of the aforementioned peptides that retain the ability to be incorporated into nanospheres, and (b) The second polypeptide of interest.

25. The nanospheres according to claim 23 or 24, wherein the nanospheres have a size of 300 nm to 550 nm.

26. The microspheres according to claim 23 or 24, wherein the microspheres have a size of 0.55 μm to 4 μm.

27. The nanospheres or microspheres according to any one of claims 23 to 26, wherein the fusion protein comprising component (ii) (a) further comprises a component of interest covalently bound to the remaining portion of the sorting enzyme recognition motif generated after the sorting enzyme-mediated reaction.

28. The nanospheres or microspheres according to any one of claims 22 to 25, wherein the fusion protein comprising component (ii) (b) further comprises a component of interest covalently bound to the sorting enzyme acceptor motif.

29. The nanospheres or microspheres according to claim 27 or 28, wherein the component of interest is selected from: - Toll-like receptor (TLR) ligands; - Monophosphoryl lipid A (MPL A); - Oligonucleotides selected from CpG islands and polyinosinic acid:polycytidine (poly(I:C) – CAS No. 24939-03-5); - Saponins; - Lipid molecules; - The coating molecules are selected from: polyglycerol, polyethylene glycol, chitosan, and poly( Poly(hydroxypropyl methacrylate) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-acryloylmorpholine) (PAcM); - Oligosaccharides; - Antibody; - Nanobodies; - Affinity; - Viral antigens; - Bacterial antigens; - Fungal antigens; - Allergens; - Cell-penetrating peptides; - Affinity; - Environmental antigens; and - Tumor antigens.

30. The nanospheres or microspheres according to any one of claims 23 to 29, wherein the first polypeptide of interest, and / or the second polypeptide of interest, and / or the component of interest is glucose-6-phosphatase 2 protein (IGRP).

31. A polynucleotide encoding a fusion protein according to any one of claims 1 to 22.

32. An expression cassette comprising the polynucleotide according to claim 31.

33. A vector comprising the polynucleotide of claim 31 or the expression cassette of claim 32.

34. The vector according to claim 33, further comprising a second polynucleotide encoding a polypeptide selected from: - A polypeptide comprising the sequence 477-542 (SEQ ID NO: 25) of the avian ororeovirus muNS protein. - A polypeptide comprising the sequence 561-622 of the mammalian ororeovirus muNS protein (SEQ ID NO: 26). - Functional equivalent variants of any of the above-mentioned peptides that retain the ability to be incorporated into nanospheres.

35. A carrier composition comprising the carrier according to claim 34 and a second carrier, wherein the second carrier comprises a second polynucleotide encoding a polypeptide selected from the group consisting of: - A polypeptide comprising the sequence 477-542 (SEQ ID NO: 25) of the avian ororeovirus muNS protein. - A polypeptide comprising the sequence 561-622 of the mammalian ororeovirus muNS protein (SEQ ID NO: 26). - Functional equivalent variants of any of the above-mentioned peptides that retain the ability to be incorporated into nanospheres.

36. The vector according to claim 33 or 34 or the vector composition according to claim 35, wherein the polynucleotide according to claim 31 or the expression cassette according to claim 32 is operatively bound to the first promoter, and the second polynucleotide is operatively bound to the second promoter.

37. The carrier or carrier composition according to claim 36, wherein the first promoter and the second promoter are the same promoter or different promoters.

38. The vector or vector composition according to any one of claims 34 to 37, wherein the vector is a bacterial expression vector.

39. A cell comprising the fusion protein according to any one of claims 1 to 22, the polynucleotide according to claim 31, the expression cassette according to claim 32, and the vector or vector composition according to any one of claims 33 to 38.

40. A method for producing a fusion protein according to any one of claims 1 to 22, the method comprising: (a) Expressing the first polynucleotide encoding the fusion protein in the cell, (b) subjecting the cells to conditions suitable for the formation of nanospheres or microspheres, and (c) Concentrate the nanospheres or microspheres.

41. The method of claim 39, further comprising purifying the fusion protein by separating the fusion protein from the nanospheres or microspheres.

42. The method of claim 40 or 41, wherein if the fusion protein comprises component (ii) (b) and a protease recognition sequence fused to the N-terminus of the sorting enzyme acceptor motif, the method further comprises, under conditions suitable for hydrolyzing the recognition sequence protein, contacting the fusion protein with a protease specific to the protease recognition sequence fused to the N-terminus of the sorting enzyme acceptor sequence, and subsequently detaching the protease recognition sequence of the fusion protein.

43. The method according to any one of claims 40 to 42, wherein the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

44. A method for producing proteins, the method comprising: (a) Expressing in cells a first polynucleotide encoding a fusion protein according to any one of claims 1 to 22 and a second polynucleotide encoding a second fusion protein comprising the following components: (i) Polypeptides, selected from: - A polypeptide comprising the sequence 477-542 (SEQ ID NO: 25) of the avian ororeovirus muNS protein. - A polypeptide comprising the sequence 518-721 (SEQ ID NO: 26) of the mammalian ororeovirus muNS protein. - Functional equivalent variants of any of the above-mentioned peptides that retain the ability to be incorporated into nanospheres, and (ii) The second polypeptide of interest, Component (ii) is the protein produced; (b) subjecting the cells to conditions suitable for the formation of nanospheres or microspheres, and (c) Concentrate the nanospheres or microspheres.

45. The method of claim 44, further comprising purifying the fusion protein by separating the fusion protein from the nanospheres or microspheres.

46. ​​The method according to claim 44 or 45, wherein the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

47. The method according to any one of claims 44 to 46, wherein the protein is an IGRP protein.

48. A method for producing a first polypeptide of interest, wherein the method comprises the following steps: (a) The fusion protein is produced by the method according to any one of claims 42 to 45, wherein the fusion protein comprises: - Component (ii) (a) and component (iii) fused to the amino terminus of component (i), wherein component (iii) is the polypeptide of interest, and - A protease recognition sequence, said protease recognition sequence between its components (i) and (iii), (b) subjecting the nanospheres and / or microspheres to conditions that cause them to disintegrate, thereby causing the fusion protein to separate from the nanospheres and / or microspheres. (c) Under conditions suitable for proteolytic hydrolysis of the fusion protein, the product obtained in step (b) is contacted with a protease specific to the recognition sequence of the components (i) and (iii) of the fusion protein, and then the components (i) and (iii) of the fusion protein are separated. (d) subjecting the product of step (c) to conditions suitable for the formation of the nanospheres and / or microspheres, and (e) Separate the nanospheres and / or microspheres from component (iii).

49. A protein or polypeptide that can be obtained by the method according to any one of claims 44 to 48.

50. A method for producing nanospheres or microspheres according to claims 23 to 30, the method comprising the following steps: (a) The fusion protein is produced by the method according to claim 39 or 41, and (b) subject the cells to conditions suitable for the formation of nanospheres or microspheres.

51. The method of claim 50, further comprising the step of incubating the nanospheres and / or microspheres in the presence of a sorting enzyme that recognizes the sorting enzyme recognition motif of component (ii) (a) of the fusion protein and a substrate comprising a sorting enzyme acceptor motif, thereby obtaining nanospheres and / or microspheres comprising the substrate containing a tag recognized by the sorting enzyme.

52. The method of claim 50, further comprising the step of incubating the nanospheres and / or microspheres in the presence of a sorting enzyme that recognizes the sorting enzyme acceptor motif of component (ii) (b) of the fusion protein and a substrate comprising the sorting enzyme recognition motif, thereby obtaining nanospheres and / or microspheres comprising the substrate containing the sorting enzyme recognition motif.

53. The method according to any one of claims 50 to 52, wherein the cell is a bacterial cell or a eukaryotic cell, wherein if it is a bacterial cell, nanospheres are formed in step (b), and if it is a eukaryotic cell, microspheres are formed in step (b).

54. The method according to any one of claims 50 to 53, wherein the nanospheres or microspheres are purified between step (b) and step (c).

55. The method according to any one of claims 50 to 54, wherein the sorting enzyme is sorting enzyme A, the recognition motif is sequence SEQ ID NO: 22 or SEQ ID NO: 23, and the sorting enzyme acceptor motif is a polyglycine sequence, preferably sequence GGG.

56. A pharmaceutical composition or immunogenic composition comprising nanospheres and / or microspheres according to any one of claims 23 to 30 and a pharmaceutically acceptable excipient.

57. The nanospheres and / or microspheres according to any one of claims 23 to 30, for use in medicine.

58. The nanospheres and / or microspheres according to any one of claims 23 to 30, or the immunogenic composition according to claim 56, for the treatment and / or prevention of bluetongue disease, wherein the fusion protein included in the nanospheres is characterized by comprising at least one of the following: - Bluetongue virus 4 (BTV) capsid protein 2 (VP2); - Bluetongue virus 4 (BTV) core protein 7 (VP7); - Bluetongue virus 4 (BTV) nonstructural protein 1 (NS1); - A protein having a sequence that is functionally equivalent to any of the proteins mentioned above.

59. The nanospheres and / or microspheres according to any one of claims 23 to 30, or the immunogenic composition according to claim 56, for the treatment and / or prevention of African equine disease, wherein the fusion protein included in the nanospheres is characterized by comprising at least one of the following: - African horse sickness virus (AHSV) non-structural protein 1 (NS1); - A protein having a sequence that is functionally equivalent to any of the proteins mentioned above.

60. The nanospheres and / or microspheres according to any one of claims 23 to 30 or the pharmaceutical composition according to claim 56, for the treatment and / or prevention of type 1 diabetes, wherein the fusion protein included in the nanospheres is characterized by comprising glucose-6-phosphatase 2 protein (IGRP) according to the sequence of accession number Q9NQR9 in the UniProt database, the entry date of which is August 3, 2022, sequence version 1.

61. The nanospheres and / or microspheres of the application according to claim 60, wherein the type 1 diabetes is latent autoimmune diabetes in adults.

62. The nanospheres and / or microspheres of any one of claims 58 to 61, wherein the nanospheres are administered via a subcutaneous or intravenous route.

63. The nanospheres and / or microspheres of any one of claims 58 to 62, wherein the nanospheres and / or microspheres are applied in the absence of an adjuvant.

64. A method for inducing type 1 diabetes in an animal model, the method comprising administering to the animal an effective amount of the nanospheres according to claim 30.

65. The method of claim 64, wherein the administration is performed via a subcutaneous or intramuscular route.

66. The method according to any one of claims 64 or 65, wherein the nanospheres are administered together with an adjuvant.

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