Platform for monomeric nucleotproteins for single or multi-cargo nanoparticles

Abstract

The present invention provides a monomeric fusion protein comprising: a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b), b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and c) a domain that comprises at least one cargo of interest. Compositions, kits, and uses thereof are also described herein.

Description

[0001] PLATFORM FOR MONOMERIC NUCLEOTPROTEINS FOR SINGLE- OR MULTI-CARGO NANOPARTICLES

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of nanobiotechnology. Particularly, the present invention relates to the field of protein nanoparticles with RNA and their uses.

[0004] BACKGROUND ART

[0005] Nanotechnology involves the direct control of matter at the molecular nanometer scale to build nanofactories, nanomachines, and other devices for potential applications including electronics, alternative fuels, and medicine. While an increasing number of novel drugs and therapeutic agents are being discovered, the problem of combining them to provide multiple functionalities and components on demand has not been solved. In this regard, the nascent use of nucleic acids as a material to coordinate the precise arrangements of specific molecules marked an important milestone in the relatively recent history of nanotechnology. Nucleic-acid nanostructures have been used as scaffolds for different molecules and proteins. In particular, RNA has garnered tremendous interest as a building block for the self-assembly of sophisticated and functional nanostructures by virtue of its ease of synthesis by in vivo or in vitro transcription, its superior mechanical and thermodynamic properties, and its functional roles in nature.

[0006] A protein of interest to produce scaffold fusion proteins is the nucleoproteins (N) from the Mononegavirales. This protein binds to RNA and generates multivalent particles that can act as carriers for moieties of interest. During viral infection, the N protein can oligomerize forming rod-like structures that protect the viral genome. Recombinant expression of N often yields ring-like particles (RLPs) that self-assemble in vivo during expression, prior to protein purification. However, they assemble in a non-controllable fashion in the expression system. When the assembled proteins are purified, various homooligomers encompassing the same proteic subunits but differing on the RNA sequence are obtained, containing non-specific RNA. Currently, this multiphase natural process cannot be reproduced in a robust and controlled way in vitro, thus the possibilities of biotechnological applications or engineering of multicomponent and multivalent nanostructures are severely limited.

[0007] In the last 15 years, a detailed biochemical and structural characterization of various N of Mononegavirales has been achieved. The availability of structures obtained by XRD or CryoEM has made it possible to identify the contacts between protomers, with the RNA and the interaction with the phosphoprotein P or its homologue, which acts as an N chaperone, allowing progress in the study of these viral components. Multiple strategies have been developed to obtain monomeric N, such as the design of truncated proteins, the expression ofseparate domains, point mutations that prevent binding to RNA and the co-expression of N and P. However, all of those strategies failed in providing a versatile platform for the production of monomeric N protein and the formation of heteromultimers in a controllable fashion. Although these strategies allow the obtaining of monomeric species, they present the following disadvantages: 1) they lose their oligomerization capacity, 2) the N protein alone is difficult to purify in soluble form because, when expressed recombinantly, it binds spontaneously and non-specifically to cellular RNA, forming insoluble structures of very high molecule weight, 3) the formation of the RLPs is not controllable, as it occurs spontaneously in the producer cell, 4) combinatorial obtention of heterooligomeric RLPs composed of various N proteins carrying different protein cargoes of interest with a controlled stoichiometry is not feasible.

[0008] Therefore, a methodology that allows the controlled assembly of these RLPs systems to constitute them into an authentic platform with scaffolding potential is needed.

[0009] BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1: Combinatorial design of next-generation nanodevices. The modular, versatile platform based on N nucleocapsid proteins from Mononegavirales allows for generation of libraries of functionalities, consisting of peptides and proteins such as antigens, adjuvants, enzymes, binders and other components that can easily be combined to generate multifunctional MMPs, that can be used in several applications.

[0011] Figure 2 topology, and FIG. 3 production. Naming convention for the MPs according to number of cargoes, topology and obtention procedures. Fig. 2: Examples of different types of closed ring-like (cR), open ring-like (oR), worm-like (W) single (S), multi (M) or no (N) cargo MPs. Fig. 3: Some MPs, like cRSMPs can be produced either in a modular (Mod) or non-modular (noMod) fashion. For multicargo MPs, only the modular pipeline is possible.

[0012] Additionally, incorporation of controlled RNA sequences is only possible in the modular pipeline, either for multi or single cargo MPs. In the non-modular pipeline, different noncontrolled RNA sequences (different colors) are incorporated.

[0013] Figure 4. Gene designs for the generation of monomeric PchM-N-X (upper panel; backbone A) or X-N-PchM molecules (lower panel; backbone B).

[0014] Figure 5. Representative Size Exclusion Chromatography (SEC) profiles of monomeric P-N-X and X-N-P constructs including nucleoproteins from different Mononegavirales viruses and their respective phosphoproteins. The cargoes are the fluorescent proteins sfGFP, mCherry, CFP and EFFP for NEW, NVSV, NM6V and Ne , respectively. The asterisks indicate the elution times of the monomers.Figure 6. Size Exclusion Chromatography profiles (Top) and Dynamic Light Scattering distribution of representative PchM-N-X monomeric construct (A) left panel, PC M / N: EbV; X: mCFP) and A / -XSMPs fusion (B) right panel, N: RSV; X: LCB3 SARS-CoV-2 minibinder).

[0015] Figure 7. Monomer stability analyzed by Size Exclusion Chromatography. Protein samples kept at 4°C for 60 days remain soluble and monomeric. (Left panel, PC M / N: VSV; X: sfGFP). Protein samples subjected to Freeze-Thaw cycles remain soluble and monomeric (Right panel, PchM / N: VSV; X:mCFP).

[0016] Figure 8. Oligomers subjected to Freeze-Thaw cycles remain soluble. Oligomer stability analyzed by DLS. N: EbV; X: mCherry

[0017] Figure 9. Analysis of the PChM : N interaction. We generated 4 variants: WT, V.A(I24A,I27A), V.B (V21AJ27A), and V.C (Y14AJ27A) to weaken the overall PChM : N interaction (Top). Size Exclusion Chromatography profiles of monomeric variants (bottom).

[0018] Figure 10. Effect of His-Tag position on NEW oligomerization. Partial oligomerization is triggered by TEV cleavage, and is completed by the addition of RNA for PchM-N-X_HT (Left). In the case of HT_PchM-N-X (Right), self-assembly is only observed when RNA is added (PchM / N: EbV; X:mCFP).

[0019] Figure 11. Effect of His-Tag position on Nvsv oligomerization. Oligomerization is triggered by the addition of RNA after TEV cleavage for PchM-N-X_HT (Left) or PchM_HT-N-X (Right) constructs. PC M / N: VSV; X: mCherry

[0020] Figure 12. Summary of the effect of the His-Tag position on oligomerization on the P-N-X constructs for different nucleoproteins.

[0021] Figure 13 and Fig. 14. We generated 4 constructs encompassing versions (EbV.v1-4) of NEW that differ on the length of N (v1: NEW 1-450; v2: N EbV 1-430; v3: N EbV 1-389; v4: N EbV 1-367). Fig. 13 shows v4 and v3, and Fig. 14 shows v2 and v1. These versions were evaluated for their ability to oligomerize. The four chromatographic panels show SEC analysis of the untreated monomers (solid lines), the constructs treated with TEV (dashed lines) and the constructs after treatment with TEV and tRNA (dotted lines). The labels M, X, and MPs indicate the chromatographic peaks assigned to the monomer, cleaved cargo and multimeric particles, respectively. Cargo is CFP for v1-3 and GFP for v4.

[0022] Figure 15. The constructs of different length present varying degrees of preservation of selfassembling capabilities and spontaneous cargo cleavage. NEW 'IS the optimal construct as it simultaneously minimizes cargo cleavage while maximizing self-assembling efficiency.Figure 16. Addition of reducing agents favors the obtention of monomeric species during the purification of PchM-N-X constructs. PC M / N: EbV; X: sfGFP

[0023] Figure 17. Oligomerization on the Z axis for nucleoproteins forming rod-like particles of undefined length by establishing contacts with first neighbors and N protomers above and below.

[0024] Figure 18. SEC profiles from NEW expression and purification (left) and NEW-X (right, X:GFP). The presence of cargo limits the presence of low-order oligomers and favors the formation of homogenous MPs.

[0025] Figure 19. Comparison between the Nucleoproteins from, VSV (right, X:GFP) and RSV (left, X: LCB3 / LCB1 SARS-CoV-2 minibinder) regarding their self-assembling capabilities in the context of N-X (full line) orX-N (dotted line) constructs.

[0026] Figure 20. Generation of homooligomeric MPs (SMPs) from monomeric P-N-X using N from VSV. (A) SEC characterization of the untreated monomers (dotted line), monomers treated only with TEV (dashed line) and oligomers obtained after treatment with TEV and RNA (solid line). The asterisk indicates the elution of oligomers, which were further characterized by DLS (B) and TEM (C).

[0027] Figure 21. Generation of homooligomeric MPs (SMPs) from monomeric PchM-N-X (PC M / N: EbV; X: GFP), monitored by Size Exclusion Chromatography (Upper left), Transmission Electron Microscopy (Upper right) and Dynamic Light Scattering (Bottom).

[0028] Figure 22. For constructs based on NEW (A), the removal of PchM triggers reversible oligomerization in absence of RNA. This process is reversibly controlled by the protein concentration, as shown by SEC for 5 pM (B) and 14 pM (C) protein concentrations. Chromatographic peaks corresponding to the the multimeric protein, the monomer and the cargo (GFP) are labelled as MP, M and X, respectively.

[0029] Figure 23. Formation of MPs combining MBP and mCherry. Their functionality was analyzed using an affinity exchange column (left panel) and fluorescence spectroscopy (right panel).

[0030] Figure 24. Formation of MMPs combining CFP and YFP. The resulting MPs were analyzed by SEC (left panel), Fluorescence spectroscopy (middle panel), and TEM (right panel).

[0031] Figure 25. Formation of MMPs combining MBP CFP and YFP. The resulting MPs were analyzed by SEC (top left panel), Fluorescence spectroscopy (top right panel) and DLS (bottom panel).

[0032] Figure 26. Formation of MMPs combining MBP and GFP. The resulting MPs were analyzed by amylose binding chromatography, Fluorescence spectroscopy, DLS and TEM.Figure 27. Formation of MMPs combining MBP, mCherry and GFP. The resulting MPs were analyzed by amylose binding chromatography, SEC, Fluorescence spectroscopy and DLS.

[0033] Figure 28. Formation of RMMPs combining different versions of EbV (6xHis-P-N-X and P-N-X-6xHis), with different backbone lengths (N1-430 and N1-450) Cargos: MBP and CFP. Figure 29. RNA induced oligomerization of TEV cleaved PCHM-NVSV-X and PCHM-NEBV-X monomers into multimeric particles (MPs) through the introduction of complex RNA mixtures. RNAs used to induce the formation of MPs containing RNAs are baker yeast tRNA and yeast torula RNA. 5 pM of either TEV treated PCHM-NVSV-X (A and B) or PCHM-NEBV-X (C and D) monomers are incubated with increasing concentrations of tRNA (A and C) or torula RNA (B and D) ranging from 0 to 100 M. X corresponds to mCherry for PCHM-NVSV-X and mCherry and sfGFP fusion proteins for PCHM-NEBV-X. Upper panels: SEC chromatogram showing the monomers, multimeric particles and RNAs elution volumes upon titration with RNAs. Lower panel: Close-up inspection of the elution volumes of multimeric particles and monomers upon titration with RNAs.

[0034] Figure 30. Specific RNA sequences trigger the formation of multimeric particles of NEBV containing distinct cargos. A to C) PchM- NEW flagellin monomer self-assembly into MPs upon binding to in-vitro transcribed 18-mer and 54-mer poly-A RNAs. Size exclusion chromatography of NEBv-flagellin monomer after TEV digestion containing: no RNA molecules A), in-vitro transcribed 18-mer B) or 54-mer C) Poly-A RNA molecules. Changes in the 260 nm / 280nm ratios are observed upon RNA binding. D) Fluorescence anisotropy titration of a FITC labelled 9-mer RNA molecule with TEV treated or untreated PchM-NE MBP. E) Size exclusion chromatography of PCHM-NEW -sfGFP and PchM-NE mCherry monomers subjected to TEV digestion in the absence or presence of an 18-mer RNA molecule corresponding to the 3'leader sequence of ebola virus RNA genome. F) TEM images of hetero-ring-like particles containing mCherry and sfGFP as cargos, bound to a 18-mer RNA.

[0035] Figure 31 : Formation of MMPs combining mCFP and mYFP. The resulting MPs were analyzed by SEC, Fluorescence spectroscopy and DLS. In addition, the colocalization of the different cargoes in the same MMPs was determined by FRET, with excitation at 420 nm and emission spectra from 460 to 600 nm.

[0036] Figure 32. SEC elution profiles showing efficient assembly of PChM-NEbV-mCFP protomers into RLPs triggered by RNAs of varying lengths and sequences: polyA RNAs (110 and 54 nt), Torula yeast RNA (Ribonucleic acid from Torula yeast Type VI, Sigma-Aldrich R6625), tRNA, and synthetic leader sequences from EbV genome (18nt, seq CGGACACACAAAAAGAAA and 6 nt, seq CGGACA), compared to a non-assembled control without RNA (-RNA). In all cases the oligomerization process was carried out by adding TEV protease (at a 1 / 50 m / m ratio) tocleave the / V-terminal bond with the chaperone module, and RNA at a 5X concentration in TEV buffer (20 mM sodium phosphate pH 8.0, 50 mM NaCI, 1 mM DTT, 0.5 mM EDTA). After 16 hours, the formed oligomers were purified using Size Exclusion Chromatography (SEC) with a Superose 6 Increase column. To prepare Torula RNA stock, approximately 20 mg of RNA extract was dissolved in 1 mL of s200 Buffer (20 mM sodium phosphate pH 8.0, 50 mM NaCI). Since the extract is highly acidic, 100 uL of NaOH 0.5 M was added to bring the solution to pH 8. After incubation at 75°C for 15 minutes, the molar concentration was calculated using an estimated average epsilon of £260= 537500 M'1cm-1. For the tRNA stock, 20 mg of lyophilized tRNA was dissolved in 1 mL of s200 Buffer (20 mM sodium phosphate pH 8.0, 50 mM NaCI) + 5 mM EDTA. The solution was then incubated at 80°C for 20 minutes, and cooled down at room temperature. The molar concentration was estimated with an £260= 625000 M'1cm-1. The 110 and 54 nt polyA and the 6nt and 18nt leaders sequences were purchased from GenScript Figure 33. Structural and biophysical characterization of EbV fusions A. Size-exclusion chromatography (SEC) elution profiles on a Superose 6 column, comparing the retention volumes of the NEbV-2X (blue), NEbV-3X (red) and ChM:NEbV-X (black). B. Hydrodynamic size distribution measured by Dynamic Light Scattering (DLS), demonstrating the monodispersity of the assembled nanoparticles. C. Chromatogram showing the elution profile of the NEbV-3X construct from an amylose affinity column. D. Fluorescence emission spectra.

[0037] E. AlphaFold 3.0 structural model of an RLP reconstituted with 24 protomers, predicting dimensions consistent with experimental results (34 nm external diameter). F. Representative negative-stain Transmission Electron Microscopy (TEM) micrographs showing the formation of discrete, ring-like NEbV-3X nanostructures.

[0038] Figure 34. Structural and biophysical characterization of Measles Virus fusions A. Sizeexclusion chromatography (SEC) elution profiles on a Superose 6 column, comparing the retention volumes of the NMeV-2X (blue) and NMeV-3X (red). B. Fluorescence emission spectra confirming FRET between the fluorophores, indicating the presence of both fluorophores in the same nanoparticle. C. Hydrodynamic size distribution measured by Dynamic Light Scattering (DLS), demonstrating the monodispersity of the assembled nanoparticles. D. AlphaFold predicted structure of the functionalized NMeV-CFP E. Representative negative-stain Transmission Electron Microscopy (TEM) micrographs showing the formation of discrete, ring-like nanostructures (Left: ChM-NMeV-X; Middle and Right: NMeV-3X).

[0039] Figure 35. Structural and biophysical characterization of VSV fusions. A. Size-exclusion chromatography (SEC) elution profiles on a Superose 6 column, comparing the retention volumes of the NVSV-2X (blue) and NVSV-3X (red). B. Fluorescence emission spectra confirming FRET between the fluorophores, indicating the presence of both probes in the samenanoparticle. C. Hydrodynamic size distribution measured by Dynamic Light Scattering (DLS), demonstrating the monodispersity of the assembled nanoparticles. D. AlphaFold prediction of the functionalized NVSV-CFP. E. Representative negative-stain Transmission Electron Microscopy (TEM) micrographs showing the formation of discrete, ring-like nanostructures (Left: ChM-NVSV-X; Middle and Right: NVSV-3X).

[0040] Figure 36. Weight of the mice after three successive inoculations of cargo-free RLPs administered through four different inoculation routes.

[0041] Figure 37: Baseline serology in humans for two different cargo-free RLPs (S1: EbV RLPs, S2: VSV RLPs) and ovoalbumin control (OVA).

[0042] Figure 38. Dendritic cell activation as monitored by the CD86 (left) and CD80 (right) levels. BMDCs were treated ON with 2 pM of RLP, RLP Flagellin, N-Flagellin or 500 ng / ml LPS, and were analyzed by flow cytometry. The CD11c-positive cell population was selected, and CD86 co-stimulatory molecules expression was analysed. Gray: negative control. Frequency of the subpopulation is quantified on the bars graphics. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (ANO A). From left to right: Gray: control. Black: LPS positive control. Yellow: cargo-free RLPs (NEbv). Red: homooligomeric RLPs including flagellin as cargo (red particles). Pink: PChM-NEbV-flagellin monomers.

[0043] Figure 39. Uptake of PChM-NEbV-mCFP monomers and NEbV-mCFP homooligomeric RLPs by dendritic cells, as determined by flow cytometry. Bone marrow-derived dendritic cells (BMDCs) from BalbC / J were incubated with 2pM the different constructs. After one hour, cells were washed, and fluorescence from internalized RLPs was analysed by flow cytometry. The bars represent the mean fluorescence intensity of the cells.

[0044] Figure 40. Top: Representative production workflow using the plasmid encoding the SI I region of the RSV F glycoprotein (FSII) as antigen. The construct was transformed into E. coli, expressed, and purified by nickel-affinity and SEC chromatography. Purified building blocks were then used to assemble multicomponent protein nanoparticles, further purified by FPLC and characterized by UV-Vis, DLS, and TEM. Purity is confirmed by SDS-PAGE. The same workflow was applied to all constructs (Gpep and Flagellin). From these building blocks, we generated a minimal library for vaccine design and screening by combining antigenic sequences from RSV surface glycoproteins as cargo antigens with bacterial flagellin as cargo adjuvant, yielding different RLP formulations.

[0045] Figure 41. Top: heterooligomeric RLPs were produced combining different cargoes at varying rations. In this example, PChM-NEbV-FSIl and PChM-NEbV-Flagellin were mixed and oligomerization was triggered at 1:5, 1:3, 1:1, 3:1 and 5:1 proportions. The resulting RLPs were analyzed by SDS-PAGE and the resulting bands for each heterooligomeric RLP werequantitatively analyzed, enabling the analysis of the average composition of each RLP. The average composition of the RLP was representative of the monomer ratio before triggering oligomerization. These results demonstrate the ability to fine-tune the composition of the RLPs by manipulating the initial mixing stoichiometries. It must be noted that the differences in cargo size do not interfere with the intended final composition. Bottom: 500nM of NEbV-Flagellin, RLP-Flagellin. Both the PChM-NEbV-Flagellin monomer and the heterooligomeric RLP-FSII / flagellin constructs activate TLR5, as assessed with a HEK-TLR5 reporter line (InVivoGen). Briefly, HEK-TLR5 cells were incubated for 24 h at 37°C in a CO2 incubator with 500 nM of NEbV-Flagellin, RLP-Flagellin, RLP-FSI l / Flagellin or RLP-FSII, and with 10 nM of Bacillus subtilis flagellin (positive control). After incubation, reporter activity in the supernatant was measured following the manufacturer’s standard protocol using pNPP substrate. Both the PChM-NEbV-Flagellin monomer and the heterooligomeric RLP-FSI l / Flagellin construct activated TLR5 in the HEK-TLR5 reporter cell line.

[0046] Figure 42. Top: Antibody titers elicited against RSV F protein from mice immunized with MBP-FSI I , MBP-FSII + alum, RLP-FSII and RLP-FSII + Alum. The results clearly show that antibody titers are significantly higher when the antigens are incorporated into RLPs, and further increases with alumina as adjuvant. Bottom: Longitudinal serum antibodies in mice vaccinated with different RSV-F formulations show robust induction and extended persistence compared with controls

[0047] Figure 43. A): Antibody titers elicited against RSV F protein for animals immunized with two doses of RLP-flagellin, RLP-FSII with and without alum and heterooligomeric RLP-FSII / Flagellin. The heterooligomeric RLPs elicited the highest antibody titers. B): Antibody titers elicited against RSV G protein for animals immunized with one or two doses of RLP_flagellin, RLP-Gpep with and without alum and heterooligomeric RLP-Gpep / Flagellin. The heterooligomeric formulations incorporating flagellin elicited the highest antibody titers after a second dose. C) Antibody titers elicited against RSV F protein in mice vaccinated with two doses of RLP FSII with and without alum and heterooligomeric RLP FSII / Gpep with and without alum. Antigen concomitance results in synergistic immunegenic effects that can be further boosted by the addition of adjuvants.

[0048] Figure 44. Production of PChM-NEbV-SC monomers. Upon addition of TEV and RNA, formation of RLPs is observed as assessed by DLS and TEM

[0049] Figure 45. Top panel: Schematic representation of the creation of PChM-NEbV-SC-st-X constructs, by combining PChM-NEbV-SC and st-X. Bottom panel: Using CFP as cargo, we created PChM-NEbV-SC-st-SCFP. The resulting construct was evaluated by SDS-PAGE, showing a molecular weight compatible with the covalent conjugation of both molecules.Figure 46: Top panel: After creation of PChM-NEbV-SC-stYFP, homooligomeric RLPs can be formed by our oligomerization protocol. The RLPs were analyzed by DLS and TEM. Bottom panel: Conjugation of PChM-NEbV-SC with stYFP and stCFP results in the creation of ChM-NEbV-SC-stYFP and ChM-NEbV-SC-stCFP. Upon mixing them and triggering oligomerization by addition of TEV and RNA, heterooligomeric RLPs are formed, as assessed by DLS and TEM

[0050] Figure 47: Top panel: fluorescence spectra of monomeric PChM-NEbV-SC-stYFP (left) and of NEbV-SC-stYFP RLPs (right) obtained by excitation at 510nm. Bottom: fluorescence spectra of the mixture of monomeric PChM-NEbV-SC-stYFP and PChM-NEbV-SC-stCFP (left) and the heterooligomeric NEbV-SC-stYFP / NEbV-SC-stCFP RLPs obtained by excitation at 380 nm (exciting CFP). In the heterooligomeric RLPs, FRET can be observed, demonstrating co-localization of CFP and YFP onto the same RLPs, after monomer conjugation using SpyCatcher-SpyTag and our oligomerization protocol.

[0051] Figure 48: Production of PChM-NEbV-mSA24K1 D results in a functional, soluble monomeric streptavidin. A) PChM-NEbV-mSA is able to oligomerize forming RLPs using our oligomerization protocol. B): PChM-NEbV-mSA24K1 D is able to bind biotinylated FITC (btFITC) (commercial). Streptavidin is used as control). C) The Kd of PChM-NEbV-mSA24K1D was determined by fluorescence anisotropy using a fixed concentration of 5 nM of btFITC and titrating using increasing concentrations of PChM-NEbV-mSA24K1 DA. The experiment was done by triplicate.

[0052] Figure 49: PChM-NEbV- / nSA24K7D can be conjugated with biotinylated YPF (btYFP). After biotinylation, excess free bt is removed by dialysis and PChM-NEbV- / nSA24K7D and btYFP are incubated for 2 hours at room temperature. Then, oligomerization is triggered by addition of TEV and RNA. The resulting oligomeric particles have incorporated btYFP forming PChM-NEbV -mSA24K1D btYFP adducts, as is demonstrated by the fluorescence spectra obtained by excitation at 510 nm of the oligomeric fraction.

[0053] Figure 50: Artistic representation of the platform of the invention for the on-demand combinatorial assembly of hetero-oligomeric ring-like particles from protein and RNA libraries. At the gene level, double fusion proteins are generated of the form PChM-N-X. PChM keeps N in a monomeric state, while X corresponds to different cargoes of interest. A protease site is included between PChM and X to allow for enzymatic cleavage. Each module is expressed and purified individually, generating a library of monomeric protein modules. A library or RNAs may be generated, either by in vitro transcription, RNA synthesis or by purification from different sources. Through combinatorial design, different protein cargoes and RNAs may be combined into homogenous, multivalent, multicomponent RLPs. The number of protomers per particle has been reduced for conceptual clarity.GENERAL DEFINITIONS

[0054] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. If the term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term “about” means the indicated value ± 1% of its value, or the term “about” means the indicated value ± 2% of its value, or the term “about” means the indicated value ± 5% of its value, the term “about” means the indicated value ± 10% of its value, or the term “about” means the indicated value ± 20% of its value, or the term “about” means the indicated value ± 30% of its value; preferably the term “about” means exactly the indicated value (± 0%).

[0055] As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0056] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of", though less preferred.

[0057] When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.The terms “sequence identity” or “percent identity” in the context of two or more nucleotide sequences, polypeptide sequences or proteins sequences refers to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of nucleotide or amino acid residues that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm, preferably BLAST alignment tool, or alternatively, by visual inspection. The “sequence identity” or “percent identity” can be determined by calculating the number of identical nucleotides or amino acids at the same positions in a nucleic acid, polypeptide or protein. Calculation of percent identity includes determination of the optimal alignment between two or more sequences. Alignment can take into account insertions and deletions (i.e. “gaps”) in each of the sequences to be tested, such as, without limitation, in the non-coding regions of nucleic acids and truncations or extensions of polypeptide sequences. Computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST) may be used to determine the percent identity. BLAST is one of the many resources provided by the U.S. National Center for Biotechnology Information. Because the genetic code is degenerate, and more than one codon can encode a given amino acid, coding regions of nucleic acids are considered identical if the nucleic acids encode identical polypeptides. Thus, percent identity could also be calculated based on the polypeptide encoded by the nucleic acid. Percent identity could be calculated based on full length consensus genomic sequences or on a fraction of the genomic sequence, such as for example without limitation on individual open reading frames (ORFs).

[0058] "Percent (%) amino acid sequence identity" with respect to proteins or polypeptides described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence (i.e., the protein or polypeptide from which it is derived), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the sequences being compared. The term “fusion protein” refers to an artificial (i.e., not natural occurring) protein created through the joining of two or more genes or coding regions. A fusion protein comprises two or more domains that are artificial or originally coded by different genes in nature. The domains of a fusion protein have been joined, one after the other, so that they are synthetized or translated as a single unit, and thus the two domains of the fusion protein are part of a singlepolypeptide. The term “domain” refers to distinct functional and / or structural units in a protein, and they are responsible for a particular function or interaction, contributing to the overall role of a protein.

[0059] The term “soluble protein” in the context of the fusion protein of the invention refers to a protein that is not part of the cytosol of a cell, but that is secreted from the cell of the invention to the extracellular media.

[0060] DESCRIPTION OF THE EMBODIMENTS

[0061] We have developed a novel platform that, based on a bottom-up approach, allows for the generation of single-cargo (S) or multi-cargo (M) multimeric particles (MPs) (SMP or MMP, respectively) with defined topologies. These can be: i) open ring-like particles (oR); ii) closed ring-like particles (cR); iii) worm-like particles (W) (Fig. 2), all of nanometric size, where the cargoes can be a variety of peptides and proteins with different functionalities and purposes, such as enzymes, antigens, neoantigens, adjuvants, binding proteins (Z-domain, streptavidin, maltose binding protein, minibinders, nanobodies, spy-catcher, etc.) and fluorescent proteins, among many other possibilities. Some of these cargoes (like monomeric streptavidin or Z- domain) even allow the creation of suprastructures by incorporation of additional ligands (antibodies biotinylated ligands, etc).

[0062] The platform consists of a protein scaffold with self-assembly capabilities that can be elicited by an external trigger. Each module or monomer is composed by this scaffold fused to the cargo of interest. The assembly process can be triggered at will by sequence-specific protease cleavage and RNA addition to produce homo- or hetero-oligomeric MPs (Figure 1).

[0063] The scaffolds used for our MPs are the so called nucleoproteins (N), a family of viral proteins from the order Mononegavirales, which in the context of viral infection oligomerize forming rod¬ like structures of helicoidal symmetry that bind and protect the genomic viral RNA. Recombinant expression of N or N-X constructs results in homo-oligomeric particles that are assembled in the expression system by uptake of nonspecific RNA from the host during protein expression. Depending on the nature of the nucleoprotein and expression system, these assemblies can be partial (2-5 protomers), homooligomeric ring-like particles, worm-like particles, short or long non-soluble rod-like particles with helicoidal symmetry, with little-to- none reproducibility. Thus, the proteins self-assemble in a non-controllable fashion during protein expression in the expression system and upon protein purification, various oligomers are obtained, containing non-specific RNA. Currently, this multiphase natural process cannot be reproduced in a robust and controlled way in vitro, thus the possibilities of biotechnological applications or engineering of multicomponent nanostructures are severely limited.Provided herein is a platform for the generation of modular fusion proteins or chimeras that, in addition to the N module, contain a cargo X and a fragment of a viral phosphoprotein. The viral phosphoproteins (P) from Mononegavirales act as a chaperone in the context of viral infection, keeping N soluble, monomeric and RNA-free. In our platform, we fuse the chaperone module of P (Paw) to N in order to obtain soluble and stable PchM-N-X or X-N- Paw monomers. In between the N and Paw modules we include a TEV enzyme cleavage site, such that in order to trigger either homo- or hetero-oligomerization the desired mixture of Pctw-N-X or X-N-Pctw is treated with TEV (to break the covalent N-PCHM linkage) and with RNA, that stabilizes the oligomers as MPs. Thus, Paw acts as an oligomerization inhibitor and RNA acts as an oligomerization trigger.

[0064] The platform’s main features are:

[0065] Modular: Each component PchM-N-X or X-N-PchM is generated independently; there is no need to constraint optimization parameters to satisfy all the components of the target multicargo MP. They can be optimized and produced in parallel, thus reducing production times.

[0066] Cost effective: Protocols are tailored according to the properties of each PChM-N-X or X-N-PChM module. Easy-to-produce- modules are developed using affordable, scalable and robust methodologies, while only those individual components that require more expensive equipment and / or methodologies are produced in a different pipeline. Versatile: Each module can be combined on demand after it has been obtained and purified. Novel combinations can be designed after production of each module. Applications are truly limitless; multiple modules can be easily combined thus breaking the barrier of a few components, usually two, per fusion protein. In addition, these devices could be used for vaccine design, cancer therapeutics, multienzymatic biocatalisis, imaging and diagnosis.

[0067] Library generation: A library of modules containing a variety of functionalities of interest can be generated, as each module is produced independently. The modules are stored as correctly folded and fully functional proteins. These modules can be combined on- demand and a la carte to generate the desired multifunctional particles. With a library of this kind, there's no need to backtrack to the DNA design of fusion proteins each time, except when expanding the library. This contributes enormously to the cost effectiveness of the technology.

[0068] Facile: Minimum complexity and effortless execution of the design-to-product transition.Smart Combinatorial design: Almost infinite combinations of already available modules allows for limitless creativity ushering a “playing Legos with molecules" approach. Rational combinations of only a few modules may result in multiple possible nanobiodevices with unique properties.

[0069] Previous studies have described the generation of soluble N proteins from the order of Paramyxoviridae family fused in frame with a protein of interest and forming a non-covalent complex with P protein. However, the present invention offers the following differences and associated advantages in view of said studies:

[0070] Difference Advantage associated with the difference They use P for purification purposes Fusion of P allows for the obtention of forming non-covalent N / P interactions, monomeric species. Co-expression of P and N where P interacts with an already does not allow for the formation of P-N contacts formed homooligomeric RLP before the self-assembly process takes place in vivo, while fusion of P to N favors complex formation of N-P before N-N, thus preventing oligomerization.

[0071] N-P 1:1 stechiometry since the P Ability to control the interaction between each domain is fused to N domain N protein with a P and allows the purification of monomeric fusion proteins Addition of the RNA when the P domain Allows to control RNA: particle oligomerization, is cleaved from the N domain. in contrast to spontaneous oligomerization inside the producer cell Addition of the RNA when the formation Allows to generate RNA:particles having the RNA: particle wants to be triggered same RNA sequence of interest.

[0072] Different N proteins with different Formation of RNA:particles that present cargos can be chosen to form different cargoes in each particle, controlled RNA: particles stechiometry of the same Use of the N terminus of the P protein, Avoids the spontaneous formation of comprising the chaperone module RNA:particles as the chaperone module prevents the N protein from forming spontaneous N-N and N:RNA protein

[0073] complexes

[0074]

[0075] Other studies (doi:10.1002 / anie.201602619) describe the formation of a soluble complex between the N and C terminal domains of the N protein with the P protein from Measles virus (order Mononegavirales, family Paramyxoviridae) and their assembly with RNA. However, the present invention provides the following differences and advantages in view of said studies:Difference Advantage associated with this difference

[0076] We include cargoes of interest, which can Cargoes have a dual function: not only be placed in both N or C termini of the N provide functionality to the particles, but protein, as the P domain can be placed in also prevent oligomerization in Z-axis. The the opposite termini RNA: particles generated have a defined size, in contrast to insoluble aggregates The presence of linkers between the Allows to control the spatial orientation of different domains the cargo.

[0077] The formation multimeric particles with The RNA:particels are soluble and have defined topologies, typically composed of uniform and well defined topology and size about 10 protomer units

[0078] Multimeric particles can be single cargo or The RNA:particles combine the multicargo functionalities of the multiple cargoes in a single nanostructure

[0079]

[0080] In view of the above, the present invention provides, in a first aspect, a fusion protein (also called hereinafter the “fusion protein of the invention”) comprising:

[0081] a) a domain that comprises an oligomerization inhibitor of the Nucleoprotein (N), wherein the N protein is from a virus of the order of Mononegavirales,

[0082] b) a domain that comprises a Nucleoprotein (N) from the virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain, and

[0083] c) a domain that comprises at least one cargo of interest.

[0084] Preferably, the fusion protein of the invention comprises:

[0085] a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) from a virus of the order of Mononegavirales,

[0086] b) a domain that comprises a Nucleoprotein (N) from the virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain, and

[0087] c) a domain that comprises at least one cargo of interest.

[0088] Thus, the oligomerization inhibitor comprised in a) is capable of preventing or reducing or inhibiting the oligomerization of the N protein comprised in domain b). Hence, in a first aspect, the fusion protein of the invention comprises:a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) comprised in b)

[0089] b) a domain that comprises a Nucleoprotein (N) from the virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain, and

[0090] c) a domain that comprises at least one cargo of interest.

[0091] Preferably, the fusion protein of the invention comprises:

[0092] a) a domain that comprises the Phosphoprotein (P) from a virus of the order of Mononegavirales,

[0093] b) a domain that comprises a Nucleoprotein (N) from a virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain, and

[0094] c) a domain that comprises at least one cargo of interest,

[0095] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0096] Preferably, the fusion protein of the invention comprises:

[0097] a) a domain that consists of the Phosphoprotein (P) from a virus of the order of Mononegavirales,

[0098] b) a domain that consists of a Nucleoprotein (N) from a virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain, and

[0099] c) a domain that comprises at least one cargo of interest,

[0100] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0101] Due to the interaction between domains a) and b), where domain a) inhibits the oligomerization of domain b), the fusion protein of the invention is always a monomeric protein. By “monomeric protein” is referred herein to a fusion protein that has a single polypeptide chain and does not bind nor form complex with other monomeric fusion proteins. However, when the monomeric fusion protein is cleaved, it becomes a cleaved fusion protein that has the ability of forming multi-protein complexes, also called herein nanoparticles, as will be explained in detail below. Hence, the monomeric protein is a monomeric fusion protein. “Oligomerization”refers to the process by which two or more protein monomers (individual subunits) associate to form a structured complex, known as an oligomer. In the context of the present invention, “oligomerization” preferably refers to the assembly of the N protein invention into oligomers. The “Mononegavirales” order is an order of negative-strand RNA viruses which have nonsegmented genomes. The order includes eleven virus families: Artoviridae, Bornaviridae, Filoviridae, Lispiviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae, and Xinmoviridae.

[0102] “N protein” or “Nucleoprotein" denotes the nucleocapsid protein of the Mononegavirales. The sequences of the N protein of Mononegavirales are known to the person skilled in the art. In the context of the present invention, the terms “N protein" or “Nucleoprotein" refer not only the full length N protein, but also a fragment or functional equivalent of said N protein, with the proviso that said fragment or functional equivalent comprises at least an oligomerization domain and one RNA binding domain. Thus, the expression “N protein” may denote a whole N protein, a truncated N protein or a fragment of the N protein.

[0103] “P protein” or “Phosphoprotein" denotes the viral protein from the order of Mononegavirales that acts as a chaperone for the N protein, inhibiting its oligomerization. The P protein is a cofactor of the viral polymerase (replicase / transcriptase) and can be phosphorylated. The sequences of the P protein of Mononegavirales are known to the person skilled in the art. In the context of the present invention, the terms “P protein” or “Phosphoprotein” refer to the whole P protein, a truncated P protein, or a fragment of the P protein, as long as it is capable of inhibiting the oligomerization of the N protein. That is, in the context of the present invention, the terms “P protein” or “Phosphoprotein” refers to the P protein or a homologue or functional equivalent that acts as an oligomerization inhibitor of the N protein.

[0104] Each of the domains of the fusion protein of the invention is described in detail below:

[0105] Domain a)

[0106] Domain a) comprises or consists of an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) from a virus of the order of Mononegavirales. Preferably, domain a) comprises or consist of an oligomerization inhibitor that is capable of preventing the oligomerization of the N protein comprised in domain b) of the fusion protein of the invention.

[0107] The expression “capable of preventing” refers herein to the ability of the inhibitor comprised in domain a) to inhibit, impede, reduce, avoid, stop or block the oligomerization of the N protein comprised in domain b) with other N proteins. This ability is mediated by the capacity of the inhibitor comprised in domain a) of interacting or binding to the N protein comprised in domain b). Techniques to detect protein oligomerization, and the inhibition of protein oligomerization,are known to the skilled person, and include biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, Western Blot, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and / or binding affinities, among others. As used herein, the terms “reduce”, “reduces”, “reduction”, “diminish”, “inhibit” and similar term mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Thus, domain a) is capable of reducing or inhibiting the oligomerization of the Nucleoprotein (N) from a virus of the order of Mononegavirales, preferably the N protein comprised in domain b)

[0108] In an embodiment, the inhibitor comprised in domain a), preferably the P protein of the order of Mononegavirales, is capable of reducing or preventing the oligomerization of the N protein comprised in domain b), wherein the reduction is in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% as compared, under the same conditions, to the oligomerization of the same N protein comprised in domain b), but without the presence of the oligomerization inhibitor.

[0109] In an embodiment, the oligomerization inhibitor comprised in domain a) is the viral P protein of the order of Mononegavirales, or a fragment or equivalent thereof. Preferably, the oligomerization inhibitor comprises at least the chaperone module of the P protein, more preferably the P protein selected from list consisting of the P proteins of EbV, MeV, VSV, RSV, HMPV, and CuV.

[0110] Preferably, the chaperone module of the P protein comprised in domain a) comprises at least:

[0111] - amino acids 1-51 from P protein of Ebola virus (EbV),

[0112] - amino acids 1-60 from P protein of Vesicular Stomatitis Virus (VSV),

[0113] - amino acids 1-50 from P protein of Measles morbillivirus (MeV),

[0114] - amino acids 1-85 from P protein of Respiratory syncytial virus (RSV),

[0115] - amino acids 2-50 from P protein of Human Cuevavirus (CuV), or

[0116] - amino acids 1-40 from P protein of Human metapneumovirus (HMPV).

[0117] It is noted that here, and thorough the whole document, the positions or locations of the amino acid residues of a protein or a polypeptide sequence are numbered sequentially starting from the first amino acid residue which would then be located at position 1. For example, a protein of 450 amino acids will have those residues numbered 1 (first amino acid residue) until 450(the last amino acid residue). Preferably, the first position or position number 1 corresponds to the first amino acid residue located at the 5-prime (5') end of the polypeptide chain that has a nitrogen atom ora free amino group. Thus, the numbering preferably starts from the first amino acid residue at the N terminal or 5’ end of the protein or polypeptide and ends at the 3’ end or C terminal end of the protein or polypeptide.

[0118] In an embodiment, the P protein comprised in domain a) is from the same virus as the N protein comprised in domain b).

[0119] In an embodiment, the P protein comprised in domain a) comprises at least amino acids 1-51 from the P protein of EbV (also referred to as EbV VP35 protein). In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of EbV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 54, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54. In an embodiment, the P protein comprised in domain a) comprises at least amino acids 1-50 from the P protein of MeV. In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of MeV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 55, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55.

[0120] In an embodiment, the P protein comprised in domain a) comprises at least amino acids 1-60 from the P protein of VSV. In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of VSV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, 58, 59 or 60, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, 58, 59 or 60, respectively.

[0121] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 24 is an alanine (mutation I24A).

[0122] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 27 is an alanine (mutation I27A).Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 21 is an alanine (mutation V21A).

[0123] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 14 is an alanine (mutation Y14A).

[0124] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 24 and 27 is an alanine (mutations I24A and I27A).

[0125] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 21 and 27 is an alanine (mutations V21Aand I27A).

[0126] Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 57, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, with the proviso that the amino acid residues in position number 14 and 27 is an alanine (mutations Y14A and I27A).

[0127] In an embodiment, the P protein comprised in domain a) comprises at least amino acids 1-85 from the P protein of RSV. In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of RSV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 78 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78.

[0128] In an embodiment, the P protein comprised in domain a) comprises at least amino acids 1-40 from the P protein of HMPV. In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of HMPV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 56 or a sequence which is at least 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0129] In an embodiment, the P protein comprised in domain a) comprises at least amino acids 2-50 from the P protein of CuV (also referred to as CuV VP35 protein). In an embodiment, the P protein comprised in domain a) comprises or consists of the full-length P protein of CuV. Preferably, the P protein comprised in domain a) comprises or consists of SEQ ID NO: 61 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61.

[0130] Domain b)

[0131] Domain b) comprises or consists of a Nucleoprotein (N) from the virus of the order of Mononegavirales, or a fragment of said protein comprising at least one oligomerization domain and one RNA binding domain. Since the N protein comprised in this domain comprises at least one oligomerization domain and one RNA binding domain, it is capable of oligomerizing with other N proteins and of binding to RNA. However, due to the presence of domain a), the N protein of domain b) stays in a monomeric form. This is because domain a) comprises an oligomerization inhibitor of the N protein comprised in domain b), as explained above. The fact that domain a) is impeding or inhibiting the oligomerization of domain b) results in the fusion protein of the invention being in a monomeric state, as defined above.

[0132] In an embodiment, the N protein comprised in domain b) is from the same virus as the P protein comprised in domain a).

[0133] In an embodiment, the N protein or the fragment thereof comprising at least one oligomerization domain and one RNA binding domain comprised in domain b) is selected from the list consisting of the N proteins of EbV, MeV, VSV, RSV, HMPV, and CuV.

[0134] In an embodiment, the N protein comprised in domain b) comprises at least amino acids 1-367 from the N protein of EbV. In an embodiment, the N protein comprised in domain b) comprises at least amino acids 1-389 from the N protein of EbV. In an embodiment, the N protein comprised in domain b) comprises at least amino acids 1-430 from the N protein of EbV. In an embodiment, the N protein comprised in domain b) comprises at least amino acids 1-450 from the N protein of EbV. In an embodiment, the N protein comprised in domain b) comprises at least amino acids 2-450 from the N protein of EbV. In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of EbV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 45, 46, 47 or 48 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45, 46, 47, or 48, respectively. In the case of EbV derived fusion proteins, it is preferred that domain a) isplaced in the N-terminal of domain b), and domain c) is placed in the C-terminal of domain b). In the most preferred embodiment, the fusion proteins of the invention, the RNA-nanoparticIs of the invention, and the methods of the invention are performed using the amino acids 1-450 from the N protein of EbV and the chaperone module of the P protein from EbV.

[0135] In an embodiment, the N protein comprised in domain b) comprises at least amino acids 1-405 from the N protein of MeV. In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of MeV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 50 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50.

[0136] In an embodiment, the N protein comprised in domain b) comprises at least amino acids 2-450 from the N protein of CuV. In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of CuV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 53 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

[0137] In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of VSV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 52 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52.

[0138] In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of RSV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 49 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49.

[0139] In an embodiment, the N protein comprised in domain b) comprises or consists of the full-length N protein of HMPV. Preferably, the N protein comprised in domain b) comprises or consists of SEQ ID NO: 51 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51.

[0140] Domain c)

[0141] Domain c) comprises or consists of at least one cargo of interest (COI). Preferably, domain c) comprises two or more cargoes of interest. Preferably, the cargo of interest confers a biological activity to the nanoparticle. As used herein, the term “biologically activity” refers to anyfunctional effect exerted by a molecule in a biological system, including but not limited to binding to a biological target, modulating a biological pathway, catalyzing a biochemical reaction, altering cellular behavior, or interacting with cells, tissues, or biomolecules. Biological activity does not include functions solely related to purification, detection tags, or affinity tags. Preferably, the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle.

[0142] Hence, preferably, the cargo is a functional molecule relevant for diagnostic, therapeutic, immunological, catalytic, targeting, or imaging applications, among others. Preferably, the cargo is a biologically active. Preferably, the cargo is a polypeptide or nucleic acid providing a biological function relevant to the diagnostic, therapeutic, prophylactic, catalytic, or imaging use of the nanoparticle. In an embodiment, the cargo is a polypeptide or nucleic acid capable of exerting a biological effect in a cell, tissue, or organism.

[0143] In an embodiment, the cargo is active in the cell, tissue or organism where it is administrated. By “active” is meant herein that the cargo provides the nanoparticle with a biological function or activity.

[0144] In an embodiment, the cargo is a biologically active polypeptide and is not a purification tag or affinity tag. Preferably, the cargo is not a HisTag.

[0145] In an embodiment, the cargo is a biologically active polypeptide suitable for use in medicine, diagnostics, vaccination, drug delivery, biocatalysis, molecular imaging, or detection methods. In an embodiment, the cargo is a biologically functional polypeptide selected from enzymes, antigens, antibodies, antibody fragments, receptors, signaling peptides, cytokines, hormones, or targeting peptides.

[0146] In an embodiment, the cargo is a biologically active molecule, preferably a polypeptide, that acts as a binder for another molecule of interest. For instance, Spycatcher cargo and monomeric streptavidin used in Example 8.

[0147] In an embodiment, the COI is a protein, nucleic acid, polymer, aptamer, or any biomolecule of interest. Preferably, the COI is a protein of interest (POI), such as enzymes, antigens, neoantigens, adjuvants, binding proteins (Z-domain, streptavidin, maltose binding protein, minibinders, nanobodies, spy-catcher, etc.) and fluorescent proteins, among many other possibilities. Some of these cargoes (like monomeric streptavidin or Z-domain) even allow the creation of suprastructures by incorporation of additional ligands (antibodies biotinylated ligands, etc).In an embodiment, the POI is a protein or polypeptide with a maximum of 50, 75, 100, or 120 kDa. In an embodiment, the POI is a protein or polypeptide with between 1 to 120, preferably between 10 to 120 Kd. In an embodiment, the POI has a maximum of 50, 75, 100, or 120 kDa.

[0148] In an embodiment, the POI is a protein or polypeptide with between 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, or 10 to 1000 amino acids. Preferably, POI is a protein or polypeptide with between 10 to 400.

[0149] In an embodiment, the POI is selected from the list consisting of GFP, MBP, mCherry, mCFP, mYFP, mNeoGreen, Flagellin, Gpep, Z-domain, sitell, mSA, LCB1 SARS-CoV-2 minibinder, and LCB3 SARS-CoV-2 minibinder. Preferably, the POI is selected from the list consisting of SEQ ID NOs: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71-76, 97 or 98, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71-76, 97 or 98, respectively.

[0150] It is noted that, in the context of the present invention, domain a) can be placed in the N-terminal of domain b), and domain c) can be placed in the C-terminal of domain b), or wherein domain c) can be placed in the N-terminal of domain b), and domain a) can placed in the C-terminal of domain b). That is, as shown in the Examples below, the order of domains a) and c) with respect to domain b) can be any order, provided that domain b) is always in between.

[0151] The linker

[0152] As explained above, domains a) and b) are connected by a linker. Similarly, domains b) and c) are connected by a linker. Thus, the fusion protein of the invention comprises at least two linkers.

[0153] The linkers may be any linker as long as it allows enough flexibility so that domains a), b), and c) can be functional domains. That is, the linker should not only connect the different domains, but also provide a cooperative inter-domain interaction, thereby preserving the biological activity of each domain.

[0154] As used herein, the term “linker” refers to a polypeptide comprising of 1-30 amino acids, preferably 3-6 amino acids. The amino acids of the linker may be selected from the group consisting of leucine (Leu, L), isoleucine (lie, I), alanine (Ala, A), glycine (Gly, G), valine (Vai, V), proline (Pro, P), lysine (Lys, K), arginine (Arg, R), Serine (Ser, S), asparagine (Asn, N), and glutamine (Gin, Q), tryptophan (Trp, W), methionine (Met, M) aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, EE), histidine (His, H), phenylalanine (Phe, F), threonine (The, T), and tyrosine (Tyr, Y). Preferably, the linkers are flexible linkers, preferably comprising amino acids Glycine and Serine or Glycine, Proline, and Serine.Suitable linker peptides include (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser-Gly4)n or Gly2(Ser-Gly2)n linker peptides, wherein n is generally a number between 1 and 10, typically between 1 and 4. In a preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence selected from the group consisting of (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser- Gly4)n, Gly2(Ser-Gly2)n, wherein n is a value from 1 to 10. In a more preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence (Gly4-Ser)n, wherein n is a value from 1 to 10. In a yet more preferred embodiment, n is a value from 1 to 4.

[0155] The optimal length of a linker may be determined by several methods, such as by performing screenings with different lengths, or by computational modelling aiming to avoid steric hindrances and clashes. For example, one GGGS repeat at the N-terminus and three GGGS repeats at the C-terminus of the N protein are the preferred linkers choice in the case of the VSV N protein scaffolds.

[0156] Construct design and optimization may be performed by the skilled person by computational modelling and simulation of each cargo in the context of the MPs. In most cases, however, this analysis can be reduced to the design of linkers that guarantee the desired average distances between protein domains to minimize steric clashes. Linkers are typically repeats of a sequence of a few (usually 5 or 6) amino acids, being G and S the most used residues. The number of repeats (n) required to obtain an average end-to-end distance <re> can be easily estimated using the following expression:

[0157] (Eq. 1)

[0158]

[0159] 2lpboaaiinker

[0160] wherein:

[0161] - re refers to the target average distances depending on the size of the cargo of interest. - Ip refers to the persistence length, which is typically between 3 and 7 A, depending on the linker sequence, as summarized in Table 1 for 5 different linkers

[0162] - Bo is 3.8 A,

[0163] - aalinker is the number of amino acids of the linker sequence, preferably between 4-6 amino acids.

[0164] Table 1, reproduced below, summarizes n values calculated using equation 1 for different target average distances <re> and different linker sequences.

[0165] In an embodiment, the linker comprises one or more, preferably between 1-6, repetitions of SEQ ID NO: 77, 79, 80, 81, 82 or 83. In an embodiment, the linker comprises or consists of SEQ ID NO: 83.The cleavage site

[0166] As explained above, the monomeric fusion protein of the invention comprises a cleavage site between domains a) and b). This is essential because the release of domain b) from domain a) will trigger the controllable oligomerization of the N protein comprised in said domain. As used herein a “cleavage site” refers to a specific location between domains a) and b) of the fusion protein of the invention where a peptide bond can be cleaved by enzymatic or chemical means. Cleavable sites may include chemically or enzymatically sensitive sites. Preferably, the peptide bond is cleaved through the specific recognition by an enzyme (peptidase or proteinase), where this recognition triggers the bond's breakdown via enzymatic or chemical mechanisms. This process is known as proteolysis.

[0167] In an embodiment, the cleavage site is placed in the N terminal of the linker connecting domains a) and b). In an embodiment, the cleavage site is placed in the C-terminal of the linker connecting domains a) and b). In an embodiment, the cleavage site is comprised in the linker connected domains a) and b).

[0168] In an embodiment, the cleavage site comprises a region that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or include peptidic regions that are preferentially specific substrates for proteases. Preferably, the cleavage site comprises or consists of a Tev enzyme cleavage site. Preferably, the Tev enzyme cleavage site comprises or consists of SEQ ID NO: 84.

[0169] Preferred fusion proteins of the invention

[0170] The following embodiments represent preferred fusion proteins of the invention:

[0171] In an embodiment, the monomeric fusion protein of the invention comprises:

[0172] a) a domain that comprises the chaperone module of the phosphoprotein (P) from Vesicular Stomatitis Virus (VSV) comprising or consisting of SEQ ID NO: 57, b) a domain that comprises the Nucleoprotein (N) of Vesicular Stomatitis Virus (VSV), comprising or consisting of SEQ ID NO: 52, and

[0173] c) a domain that comprises at least one cargo of interest,

[0174] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0175] In an embodiment, the monomeric fusion protein of the invention comprises:

[0176] a) a domain that comprises the chaperone module of the phosphoprotein (P) from Vesicular Stomatitis Virus (VSV) comprising or consisting of SEQ ID NO: 58,b) a domain that comprises the Nucleoprotein (N) of Vesicular Stomatitis Virus (VSV), comprising or consisting of SEQ ID NO: 52, and

[0177] c) a domain that comprises at least one cargo of interest,

[0178] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0179] In an embodiment, the monomeric fusion protein of the invention comprises:

[0180] a) a domain that comprises the chaperone module of the phosphoprotein (P) from Vesicular Stomatitis Virus (VSV) comprising or consisting of SEQ ID NO: 59, b) a domain that comprises the Nucleoprotein (N) of Vesicular Stomatitis Virus (VSV), comprising or consisting of SEQ ID NO: 52, and

[0181] c) a domain that comprises at least one cargo of interest,

[0182] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0183] In an embodiment, the monomeric fusion protein of the invention comprises:

[0184] a) a domain that comprises the chaperone module of the phosphoprotein (P) from Vesicular Stomatitis Virus (VSV) comprising or consisting of SEQ ID NO: 60, b) a domain that comprises the Nucleoprotein (N) of Vesicular Stomatitis Virus (VSV), comprising or consisting of SEQ ID NO: 52, and

[0185] c) a domain that comprises at least one cargo of interest,

[0186] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0187] In an embodiment, the monomeric fusion protein of the invention comprises, preferably in the N to C-terminal direction:

[0188] a) a domain that comprises P protein from EbV comprising or consisting of SEQ ID NO: 54,

[0189] b) a domain that comprises the Nucleoprotein (N) of EbV, comprising or consisting of SEQ ID NO: 45, andc) a domain that comprises at least one cargo of interest,

[0190] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0191] In an embodiment, the monomeric fusion protein of the invention comprises, preferably in the N to C-terminal direction:

[0192] a) a domain that comprises P protein from EbV comprising or consisting of SEQ ID NO: 54,

[0193] b) a domain that comprises the Nucleoprotein (N) of EbV, comprising or consisting of SEQ ID NO: 46, and

[0194] c) a domain that comprises at least one cargo of interest,

[0195] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0196] In an embodiment, the monomeric fusion protein of the invention comprises, preferably in the N to C-terminal direction:

[0197] a) a domain that comprises P protein from EbV comprising or consisting of SEQ ID NO: 54,

[0198] b) a domain that comprises the Nucleoprotein (N) of EbV, comprising or consisting of SEQ ID NO: 47, and

[0199] c) a domain that comprises at least one cargo of interest,

[0200] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0201] In an embodiment, the monomeric fusion protein of the invention comprises, preferably in the N to C-terminal direction:

[0202] a) a domain that comprises P protein from EbV comprising or consisting of SEQ ID NO: 54,

[0203] b) a domain that comprises the Nucleoprotein (N) of EbV, comprising or consisting of SEQ ID NO: 48, and

[0204] c) a domain that comprises at least one cargo of interest,wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0205] In an embodiment, the monomeric fusion protein of the invention comprises:

[0206] a) a domain that comprises P protein from MeV comprising or consisting of SEQ ID NO: 55,

[0207] b) a domain that comprises the Nucleoprotein (N) of EbV, comprising or consisting of SEQ ID NO: 50, and

[0208] c) a domain that comprises at least one cargo of interest,

[0209] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0210] In an embodiment, the monomeric fusion protein of the invention comprises:

[0211] a) a domain that comprises P protein from CuV comprising or consisting of SEQ ID NO: 61,

[0212] b) a domain that comprises the Nucleoprotein (N) of CuV, comprising or consisting of SEQ ID NO: 53, and

[0213] c) a domain that comprises at least one cargo of interest,

[0214] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0215] In an embodiment, the monomeric fusion protein of the invention comprises:

[0216] a) a domain that comprises P protein from HMPV comprising or consisting of SEQ ID NO: 56,

[0217] b) a domain that comprises the Nucleoprotein (N) of HMPV, comprising or consisting of SEQ ID NO: 51, and

[0218] c) a domain that comprises at least one cargo of interest,

[0219] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0220] In an embodiment, the monomeric fusion protein of the invention comprises:a) a domain that comprises P protein from RSV comprising or consisting of SEQ ID NO: 78,

[0221] b) a domain that comprises the Nucleoprotein (N) of RSV, comprising or consisting of SEQ ID NO: 49, and

[0222] c) a domain that comprises at least one cargo of interest,

[0223] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b).

[0224] In an embodiment, the fusion protein comprises or consists of SEQ ID Nos: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 90-94 or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 90-94 respectively.

[0225] A summary of some features of the invention can be found in Fig. 3, wherein several examples are disclosed.

[0226] As shown in the examples, the cleavage of the cleavage site releases domain b) from domain a) and triggers the oligomerization of the N protein with other N proteins. This results in the formation of nanoparticles that bind RNA. An advantage of the invention is that, when using cleaving the fusion protein of the invention and if an RNA sequence of interest is present, RNA-nanoparticles can be formed in a controllable fashion.

[0227] Included in the first aspect are nucleic acid molecules, such as RNA, DNA, or DNA / RNA hybrids, encoding the fusion protein of the invention. Vectors carrying or encompassing or functionalized with the particle or the fusion protein of the invention are also included herein. For example, cells encoding, expressing, comprising, and / or secreting the fusion protein or the particle of the invention are also included.

[0228] Therefore, in a second aspect, the present invention provides a homogeneous population of RNA-nanoparticles. The term “RNA-nanoparticle” refers to a nanostructure formed by the oligomerization of two or more cleaved fusion proteins of the invention in presence of a plurality of RNA molecules, resulting in protein structures that are bound to said RNA molecules. Each RNA-nanoparticle of the population of RNA-nanoparticles is called herein a “RNA-nanoparticles of the invention”, and is thus characterized by comprising a RNA component and a particle component. The term “homogeneous” is meant herein that that the particle component and the RNA component of each of the RNA-nanoparticle of the population ofRNA-nanoparticles is the same, or substantially the same: the particles have substantially the same particle size or diameter, and the RNA have identical or substantially identical nucleotide sequence. For the avoidance of doubt, as used herein, “homogeneity” refers to the RNA component, and does not necessarily require identity of the cargo moieties carried by the particles. In certain embodiments, individual RNA-nanoparticles comprise a mixture of different cargos because they are assembled from two or more fusion proteins that differ in cargo identity, such that multiple cargos are co-displayed within a single nanoparticle. Accordingly, a population can be homogeneous with respect to RNA identity while being heterogeneous with respect to cargo at the intra-particle level and, optionally or alternatively, at the inter-particle level. Unless expressly stated otherwise, references to homogeneous populations in this specification do not imply homogeneity of cargo. In an embodiment, the population of RNA-nanoparticles is also homogeneous in terms of size and symmetry.

[0229] The particle component of an RNA-nanoparticle of the invention is a nanoparticle formed by the oligomerization of two or more, preferably of five or more, of the cleaved fusion proteins of the invention. The oligomerization is possible due to the presence of the oligomerization domain comprised in the N protein comprised in domain b) and due to the lack of domain a). The term “cleaved fusion protein of the invention” refers to the fusion proteins of the invention once they have been cleaved and thus domain a) has been removed. Thus, a cleaved fusion protein of the invention is a fusion protein of the invention that lacks domain a), and therefore is no longer monomeric because the N protein comprised in domain b) will oligomerize with other cleaved fusion proteins of the invention. Depending on what type of N protein is present in domain b), the oligomerization will occur in different forms or shapes. Also depending on the N protein present in domain b) the nanoparticle will comprise two or more, three or more, four or more, preferably five or more, six or more, seven or more, eight or more, nine or more, or even ten or more, cleaved fusion proteins. In the context of the present invention, each of the cleaved fusion protein that form the particle component of the RNA-nanoparticle of the invention is also referred herein as a “monomer”.

[0230] In an embodiment, at least ten cleaved fusion proteins of the invention are incorporated in the particle. In an embodiment, the RNA-nanoparticle of the invention comprises about 10, about 12, about 24 or about 40 cleaved fusion protein of the invention. In an embodiment, the RNA-nanoparticle of the invention comprises between 10 to 60 or 12 to 60, preferably about 12, 24, or 40, cleaved fusion protein of the invention. Preferably, the RNA-nanoparticle of the invention comprises 12, 24, or 40 cleaved fusion protein (or monomers) of the invention.

[0231] In an embodiment, the monomers forming the RNA-nanoparticle comprise the same cargo. In another embodiment, a RNA-nanoparticle may comprise two or more monomers havingdifferent cargos. Preferably, each RNA-nanoparticle comprises at least two or three monomers that differ in their cargo.

[0232] In an embodiment, the N protein is the N protein of EbV, as defined above, and thus the nanoparticle comprises 24 to 30 monomers or cleaved fusion proteins.

[0233] In an embodiment, the N protein is the N protein of RSV, as defined above, and thus the nanoparticle comprises 8 to 12 monomers or cleaved fusion proteins.

[0234] In an embodiment, the N protein is the N protein of MeV, as defined above, and thus the nanoparticle comprises 11 to 13 monomers or cleaved fusion proteins.

[0235] In an embodiment, the N protein is the N protein of CuV, as defined above, and thus the nanoparticle comprises 24 to 30 monomers or cleaved fusion proteins.

[0236] In an embodiment, the N protein is the N protein of VSV, as defined above, and thus the nanoparticle comprises 10 to 42 monomers or cleaved fusion proteins.

[0237] In an embodiment, the N protein is the N protein of HMPV, as defined above, and thus the nanoparticle comprises 8 to 12 monomers or cleaved fusion proteins.

[0238] It is noted that, although domain a) has been removed from the cleaved fusion protein of the invention, domains b) and c) still remain, and they are still connected by a linker. Thus, the cleaved fusion protein of the invention comprises domain b) and c) of the fusion protein of the invention, connected by a linker. The embodiments and definitions explained above for domains b), c) and linkers also apply herein to the cleaved fusion protein of the invention. Since the cleaved fusion protein comprises domain b), and since domain b) comprises a N protein or a fragment thereof comprising an RNA binding domain and an oligomerization domain (as explained above), the cleaved fusion proteins or monomers are capable of oligomerizing and bind to at least one RNA molecule. Hence, each nanoparticle of the RNA-nanoparticle of the invention is bound to at least an RNA molecule.

[0239] The at least one RNA molecule to which the cleaved fusion protein of the invention binds represents the RNA component of a RNA-nanoparticle of the invention. If the cleavage of the fusion protein of the invention is performed in the presence of a plurality of RNA molecules comprising the same nucleotide sequence, or if a plurality of RNA molecules comprising the same nucleotide sequence are added after their cleavage, the RNA component comprised in the RNA-nanoparticle of the invention will be identical or substantially identical in all RNA-nanoparticles comprised in the population of RNA-nanoparticles, resulting in an homogeneous population of RNA-nanoparticles, as defined above.

[0240] An RNA molecule is “substantially identical” to another RNA molecule when the percentage of nucleobase identity between both regions is at least 98%, at least 99%, at least 99,5%,preferably at least 99,99% or 100%. Thus, the “substantial identity” includes the possible errors (i.e., insertion, deletion or substitution of nucleotides made by polymerase enzymes or by DNA damage, library processing, sequencing or mapping).

[0241] In an embodiment, the RNA molecule is identical, or substantially identical, among the plurality of RNA-nanoparticles, and is of a known sequence.

[0242] In an embodiment, all of the RNA-nanoparticles of the population of the invention comprise the same RNA. In an embodiment, said RNA has at least 5 nucleotides, preferably between 5 to 200 or 5 to 120 nucleotides. Preferably, the RNA has about 6, 18, 54, 60, 64 or 120 nucleotides in length. In an embodiment, the RNA is torula RNA, polyA, polyll, tRNA, etc.

[0243] In an embodiment, the RNA incorporated in the RNA-nanoparticles of the population is a predefined or pre-determine RNA. This means that is not a random or arbitrary RNA, but that it can be chosen and determined. As used herein, “predefined RNA2 refers to an RNA sequence that is designed or selected and thus it is not generated randomly.”

[0244] Preferably, the RNA. nanoparticles of the invention are soluble. Preferably, the RNA-nanoparticles of the invention have a ring or a worm shape. Preferably, the RNA-nanoparticles of the invention are not insoluble, nor have a rod-like shape.

[0245] Therefore, in an embodiment, each RNA-nanoparticle of the invention comprises:

[0246] i) two or more, preferably five or more, cleaved fusion proteins of the invention as defined above, and

[0247] ii) at least one RNA molecule that is bound to each of the cleaved fusion proteins of i), wherein the sequence of said RNA molecule is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA-nanoparticles.

[0248] In an embodiment, each RNA-nanoparticle of the homogeneous population of RNA-nanoparticles of the invention is characterized by comprising:

[0249] i) two or more, preferably five or more, cleaved fusion proteins or monomers that interact with each other thereby forming a nanoparticle, wherein each cleaved fusion protein or monomer comprises a first domain and a second domain that are bound by a linker, and wherein:

[0250] 1) the first domain corresponds to domain b) as defined above, and

[0251] 2) the second domain corresponds to domain c) as defined above, and ii) at least one RNA molecule that is bound to the RNA binding domain of each of the cleaved fusion proteins of i), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA-nanoparticles.The second domain can be placed in the N-terminal of the first domain, or in the C-terminal of the first domain.

[0252] In an embodiment, the homogeneous population of RNA-nanoparticles is obtainable or obtained by a method comprising the steps of:

[0253] 1) cleaving a plurality of fusion proteins of the invention as defined above, thereby removing domain a) in each of them, and

[0254] 2) simultaneously or subsequently to step 1), adding one or more RNA molecules with identical or substantially identical nucleotide sequence, so that the cleaved fusion proteins of the invention of a) oligomerize and binds to said one or more RNA molecules, thereby obtaining a homogeneous population of RNA-nanoparticles.

[0255] Thus, in an embodiment, the homogeneous population of RNA-nanoparticles of the invention comprises:

[0256] i) 10 or more monomers that interact with each other thereby forming a nanoparticle, wherein each monomer comprises a first domain and a second domain that are bound by a linker, wherein:

[0257] 1) the first domain comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and

[0258] 2) the second domain comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle, and

[0259] ii) at least one RNA molecule that is bound to the RNA binding domain of each of the N proteins of the first domain of i.1 ), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA-nanoparticles,

[0260] wherein the homogeneous population of RNA-nanoparticles is obtained or obtainable by a method comprising the steps of:

[0261] 1) providing a plurality of monomeric fusion proteins, each of them comprising:

[0262] a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b),

[0263] b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, andc) a domain that comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle and is not HisTag, wherein the cargo of the monomeric fusion proteins is identical or, preferably, different among the plurality of monomeric fusion proteins, wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and

[0264] wherein a cleavage site is placed between domains a) and b);

[0265] and cleaving said plurality of fusion proteins at the cleavage site to remove domain a), yielding cleaved fusion proteins competent for RNA-mediated oligomerization, and 2) simultaneously or subsequently to step 1), adding one or more RNA molecules with identical or substantially identical nucleotide sequence, so that the cleaved fusion proteins obtained in step 1) oligomerize and bind to said RNA molecules, thereby obtaining a homogeneous population of RNA-nanoparticles.

[0266] In a more embodiment, the homogeneous population of RNA-nanoparticles of the invention comprises:

[0267] i) five or more, preferably 10 or more, monomers that interact with each other thereby forming a nanoparticle, wherein each monomer comprises a first domain and a second domain that are bound by a linker, wherein:

[0268] 1) the first domain comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and

[0269] 2) the second domain comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle, and

[0270] ii) at least one RNA molecule that is bound to the RNA binding domain of each of the N proteins of the first domain of i.1 ), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA-nanoparticles,

[0271] wherein the homogeneous population of RNA-nanoparticles is obtained or obtainable by a method comprising the steps of:

[0272] 1) providing a plurality of monomeric fusion proteins, each of them comprising:

[0273] a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b),b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and

[0274] c) a domain that comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle, wherein the cargo of the monomeric fusion proteins is identical or different among the plurality of monomeric fusion proteins,

[0275] wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and

[0276] wherein a cleavage site is placed between domains a) and b);

[0277] and cleaving said plurality of fusion proteins at the cleavage site to remove domain a), yielding cleaved fusion proteins competent for RNA-mediated oligomerization, and 2) simultaneously or subsequently to step 1), adding one or more RNA molecules with identical or substantially identical nucleotide sequence, so that the cleaved fusion proteins obtained in step 1) oligomerize and bind to said RNA molecules, thereby obtaining a homogeneous population of RNA-nanoparticles.

[0278] In an embodiment, the RNA-nanoparticles of the second aspect are obtained or obtainable with the method as defined in the seventh aspect or any of its embodiments. In an embodiment, the RNA-nanoparticles of the second aspect are obtained or obtainable with the method as defined in the sixth aspect or any of its embodiments.

[0279] In an embodiment, the RNA-nanoparticles of the second aspect are obtained or obtainable with the method as defined in the sixth or seventh aspect, or any of their embodiments, wherein 10 or more monomers are used comprising identical or different cargos.

[0280] In a third aspect, the present invention comprises a composition, also called herein the “composition of the invention”. Said composition of the invention comprises the fusion protein of the invention as defined in the first aspect, or the population of RNA-nanoparticles of the invention as defined in the second aspect, or in any of their embodiments.

[0281] In a fourth aspect, the present invention also provides a kit, also called herein the “kit of the invention”. Said kit of the invention comprises the fusion protein of the invention as defined in the first aspect, or the population of RNA-nanoparticles of the invention as defined in the second aspect, or the composition of the invention as defined in the third aspect, in any of their embodiments. The kit may further comprise other elements such as instructions on how to use or generate the fusion proteins or the population of RNA-nanoparticles or the composition of the invention. For example, the kit may comprise the protease that can cleavage the fusionprotein of the invention, resulting in the cleaved fusion protein of the invention. In another example, the kit may comprise a plurality of RNA molecules characterized in being identical or substantially identical so that they can be used to generate the homogeneous population of RNA-nanoparticles of the invention.

[0282] In a fifth aspect, the present invention provides uses of the fusion protein, the homogeneous population of RNA-nanoparticles, or the composition of the invention. Said uses include, but are not limited to, uses in in the fields of diagnostic methods, medicine, biocatalysts, vaccinology or immunology, personalized medicine, detection methods, spectroscopic methods, drug delivery, theragnostic, biology, bionanotechnology, and / or molecular imaging. Preferably, the use is in vaccine design, spectroscopy, cancer therapeutics, multienzymatic biocatalysis, imaging and / or diagnosis. The use of the fusion protein, the homogeneous population of RNA-nanoparticles, or the composition of the invention will depend on the cargo of interest comprised in domain c) of the fusion protein or cleaved fusion protein of the invention. For instance, if the cargo of interest is a protein used as a vaccine, then the fusion protein, the homogeneous population of RNA-nanoparticles, or the composition of the invention will be useful in methods for the treatment or prevention of a specific disease for which the protein is useful as a vaccine. Since different cargos can be loaded in the RNA-nanoparticles (if different monomers are used), the uses can be combined. Preferably, the RNA-nanoparticles are produced as defined in the sixth or seventh aspect of the invention, or any of their embodiments, wherein 10 or more monomers are used comprising identical or different cargos, and wherein said RNA-nanoparticles are used in the field of diagnostic methods, medicine, biocatalysts, vaccinology or immunology, detection methods, spectroscopic methods, drug delivery, theragnostic, biology, bionanotechnology, or molecular imaging methods. Preferably, the use is in vaccine design, spectroscopy, cancer therapeutics, multienzymatic biocatalysis, imaging and diagnosis

[0283] In an embodiment, a fusion protein comprising or consisting of:

[0284] - SEQ ID NO: 4 (EbV_VP35i.5i_Ni.45o_mCHERRY_6xHis),

[0285] - SEQ ID NO: 5 (EbV_VP35i.5i_Ni.45o_sfGFP_6xHis),

[0286] - SEQ ID NO: 7 (EbV_VP35i.5i_Ni.45o_neoGreen_6xHis),

[0287] - SEQ ID NO: 8 (EbV_6xHis_VP35i.5i_Ni.367_sfGFP),

[0288] - SEQ ID NO: 9 (EbV_6xHis_VP35i.5i_Ni.389_mCFP),

[0289] - SEQ ID NO: 10 (EbV_6xHis_VP35i.5i_Ni.430_mCFP),

[0290] - SEQ ID NO: 11 (EbV_6xHis_VP35i.5i_Ni.45o_mCFP),

[0291] - SEQ ID NO: 8 (EbV_6xHis_VP35i.5i_Ni.367_sfGFP),- SEQ ID NO: 20 (VSV_Pi.60_N_sfGFP_6xHis),

[0292] - SEQ ID NO: 21 (VSV_Pi.6o_N_mCherry_6xHis),

[0293] - SEQ ID NO: 23 (VSV_6xHis_P2.6o_N_sfGFP),

[0294] - SEQ ID NO: 24 (VSV_6xHis_P2-6o_N_mCherry),

[0295] - SEQ ID NO: 26 (VSV_6xHis_P2.6o_N_mCFP),

[0296] - SEQ ID NO: 28 (VSV_mCherry_N_P2.6o_6xHis),

[0297] - SEQ ID NO: 29 (VSV_6xHis P2.6oj24A_i27A_TEV_N_sfGFP),

[0298] - SEQ ID NO: 30 (VSV_6xHis_P2.6o_v2iA_i27A_TEV_N_sfGFP),

[0299] - SEQ ID NO: 31 (VSV_6xHis_P2.6o_Yi4A_i27A_TEV_N_sfGFP),

[0300] - SEQ ID NO: 32 (MeV_Pi-50_Ni-405_mCherry_6xHis),

[0301] - SEQ ID NO: 33 (MeV_Pi.5o_Ni.4o5_sfGFP_6xHis),

[0302] - SEQ ID NO: 26 (VSV_6xHis_P2.6o_N_mCFP),

[0303] - SEQ ID NO: 27 (VSV_6xHis_P2.60_N_YFP),

[0304] - SEQ ID NO: 35 (MeV_Pi.5o_Ni.4o5_YFP_6xHis),

[0305] - SEQ ID NO: 36 (MeV_P1-5O_Ni-4o5_CFP_6xHis),

[0306] - SEQ ID NO: 42 (CuV_6xHis_VP352.5o_N2.45o EGFP),

[0307] - SEQ ID NO: 90 (6xHis_VP35EbV _NEbVi.43o_mYFP),

[0308] - SEQ ID NO: 92 (PVSV_NVSV_mCFP_6xHis),

[0309] - SEQ ID NO: 93 (PVSV_NVSV_mYFP),

[0310] or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 5, 7, 8, 9, 10, 11, 8, 20, 21, 23, 24, 26, 28, 29, 30, 31, 32, 33, 26, 27, 35, 3642, 90, 92 or 93, respectively, may be used in detection methods since their cargo is at least one fluorescent protein.

[0311] In an embodiment, a fusion protein comprising or consisting of:

[0312] - SEQ ID NO: 14 (EbV_6xHis_VP35i.5i_Ni.43o_Sitell),

[0313] - SEQ ID NO: 15 (EbV_6xHis_VP35i-5i_Ni.43o_Flagellin54-23o),

[0314] - SEQ ID NO: 16 (EbV_6xHis_VP35i.5i_Ni.43o_Gpep),

[0315] or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14,15 or 16, respectively, may be used in medicine or in immunology since their cargo are antigens (Sitell and gpep) or adjuvants (Flagellin).

[0316] In an embodiment, a fusion protein comprising or consisting of:

[0317] - SEQ ID NO: 22 (VSV_Pi.6o_N_MBP_6xHis),

[0318] - SEQ ID NO: 25 (VSV_6xHis_P2-6o_N_MBP),

[0319] - SEQ ID NO: 34 (MeV_Pi.5o_Ni.4o5_MBP_6xHis),

[0320] -SEQ ID NO: 91 (6xHis_VP35EbV_NEbVi-43o_MBP),

[0321] - SEQ ID NO: 94 (EbV_Hisx6_VP35_N1-430_SpyCatcher),

[0322] or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO:22, 25, 34, 91, 94, respectively, may be used in binding assays.

[0323] In an embodiment, the fusion protein comprises or consists of SEQ ID NO: 17 (EbV_6xHis_VP35i-5i_Ni.43o_mSA24K1D), or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, 22, 25, or 34 respectively, may be used in Binding of biotinylated ligands.

[0324] In an embodiment, the fusion protein comprises or consists of SEQ ID NO: 6 (EbV_VP351-51_N1-450_MBP_6xHis), SEQ ID NO: 15 (EbV_6xHis_VP351-51_N1-430_Flagellin54-230), SEQ ID NO: 16 (EbV_6xHis_VP351-51_N1-430_Gpep), or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 15 or 16, respectively, may be used in medicine or in immunology, since their cargo are antigens (Sitell and gpep) or adjuvants (Flagellin).

[0325] In a sixth aspect, the present invention provides a method, preferably an in vitro method, for producing or generating an homogeneous population of RNA-nanoparticles of the invention as defined in the second aspect or in any of its embodiments.

[0326] Preferably, the method comprises the steps of:

[0327] 1) providing one or more of the monomeric fusion protein of the invention,

[0328] 2) carrying out the cleavage of the cleavage site,

[0329] 3) optionally, purifying the resultant cleaved fusion proteins from step 2),

[0330] 4) adding one or more RNA molecules with the desired sequence and5) allowing the spontaneous oligomerization of the product of step 3) with the RNA molecules of step 4), thereby providing the homogeneous population of RNA- nanoparticles of the invention.

[0331] Step 1) can be performed by any known method in the art, such as recombinant DNA technology, synthetic DNA synthesis, or CRISPR-based method. As used herein “providing” is synonymous of “generating” or “supplying”.

[0332] Step 2) occurs when the enzyme cleavage sequence of the fusion protein of the invention is contacted with an enzyme that recognises said sequence and thus proceeds to cleavage it. The skilled person knows what means and conditions are needed, depending on the enzyme used, to perform step 2).

[0333] Step 3) is performed by any protein purification method known in the art, such as Affinity Chromatography, or Size Exclusion Chromatography (SEC).

[0334] Step 4) is performed by contacting the purified protein of step 3) with RNA fragments. Preferably, the RNA fragments used in step 4) are of a known sequence. Preferably, the RNA fragments have identical or substantially identical sequence so that the population of RNA-nanoparticles generated are homogeneous. Thus, in one embodiment, the RNA utilized in step 4) comprises a cognate sequence, and all RNA molecules used share the same cognate sequence.

[0335] Lastly, step 5) comprises or consists of providing the conditions that allow the nanoparticles to oligomerize with the RNA. A skilled person understands the conditions that promote or enable oligomerization, which include overnight incubation at room temperature without stirring. To prevent unproductive aggregation, monomer concentrations are typically kept below 25 micromolar during this step. Preferably, after step 5), the RNA-nanoparticles are purified. In a seventh aspect, the present invention provides a further method, preferably an in vitro method, for producing or generating an homogeneous population of RNA-nanoparticles of the invention, as defined in the second aspect or in any of its embodiment. This method comprises the steps of:

[0336] 1) providing one or more monomeric fusion proteins of the invention;

[0337] 2) effecting cleavage of the fusion proteins by adding a cleavage enzyme;

[0338] 3) adding one or more RNA molecules having a desired sequence, thereby triggering the spontaneous oligomerization to form RNA nanoparticles, and

[0339] 4) purifying the RNA nanoparticles,wherein steps 2) and 3) being carried out simultaneously or sequentially, and, when carried out sequentially, step 3) being performed either without intermediate purification or after purifying the cleaved fusion proteins obtained in step b).

[0340] In an embodiment, the method of the seventh aspect comprises the steps of:

[0341] 1) providing one or more monomeric fusion proteins of the invention;

[0342] 2) effecting cleavage of the fusion proteins by adding a cleavage enzyme;

[0343] 3) adding one or more RNA molecules having a desired sequence, thereby triggering the spontaneous oligomerization to form RNA nanoparticles, and

[0344] 4) purifying the RNA nanoparticles,

[0345] wherein steps 1 to 4 are performed sequentially in numerical order, and wherein, preferably prior to step 3, the sample is purified to remove excess cleavage enzyme.

[0346] In an embodiment, the method of the seventh aspect comprises the steps of:

[0347] 1) providing one or more monomeric fusion proteins of the invention;

[0348] 2) effecting cleavage of the fusion proteins by adding a cleavage enzyme;

[0349] 3) adding one or more RNA molecules having a desired sequence, thereby triggering the spontaneous oligomerization to form RNA nanoparticles, and

[0350] 4) purifying the RNA nanoparticles,

[0351] wherein steps 2 and 3 are performed simultaneously.

[0352] Step 1) in the method of the seventh aspect comprises or consists of providing a plurality of one or more monomeric fusion proteins of the invention. Said proteins may be identical to each other, or they may be different, such as, for example, having different cargoes. In an embodiment, each fusion protein may have two or more different cargoes. Thus, in this step, any mixture of fusion proteins and / or cargoes can be provided, as desired.

[0353] Step 2) in the method of the seventh aspect comprises cleaving the P domain from the N domain by supplying to the reaction mixture the corresponding enzyme capable of performing said cleavage. As explained above, the skilled person knows what the conditions are to perform an enzymatic cleavage. In an embodiment, after the cleavage and before step 3), the proteins can be purified so that excess of enzyme is removed. However, this purification step is optional, so that the method can also continue with step 3) without an intermediate purification step. Thus, in this step, the fusion proteins provided in step 1) are contacted with an enzyme that recognizes said sequence and thus proceeds to cleavage it.Step 3 of the method of the seventh aspect comprises triggering oligomerization by adding to the reaction mixture the RNA to be incorporated into the RNA nanoparticles. This RNA comprises a cognate sequence, or at least a sequence that is substantially the same across the RNA nanoparticles generated. In other words, this step does not permit oligomerization with random RNA present in the sample, but instead enables controlled oligomerization by supplying the RNA of interest. Step 3 may be performed concurrently with Step 2 or subsequently. As explained above, when Step 3 is performed after Step 2, an optional intermediate purification may be carried out between the two steps to remove residual cleavage enzyme.

[0354] Step 4 of the method of the seventh aspect comprises purifying the newly formed RNA-nanoparticles. This purification process also removes the excess of RNA. Similar as for the optional purification step after step 2, the skilled person knows purification methods.

[0355] Preferably, the method of the seventh aspect comprises the steps of:

[0356] 1) providing a plurality of monomeric fusion proteins of the invention comprising identical or different cargoes;

[0357] 2) effecting cleavage of the fusion proteins by adding a cleavage enzyme;

[0358] 3) adding one or more RNA molecules having a desired or predetermined sequence, thereby triggering the spontaneous oligomerization to form RNA nanoparticles, and

[0359] 4) purifying the RNA nanoparticles,

[0360] wherein steps 2) and 3) being carried out simultaneously or sequentially, and, when carried out sequentially, step 3) being performed either without intermediate purification or after purifying the cleaved fusion proteins obtained in step b),

[0361] wherein the method does not involve the use of the cargo to purify the particles, and wherein the cargo comprises more than 10 amino acid or confers the RNA-nanoparticles with a biological activity.

[0362] Some preferred embodiments of the method of the sixth or of the seventh aspects are:

[0363] In an embodiment, the fusion proteins provided in the first step comprise one or more cargo, wherein the cargo confers a biological activity to the nanoparticle. Preferably, the monomeric fusion proteins provided in step 1) comprise two or more cargos, wherein each cargo of interest confers a biological activity to the nanoparticle. Preferably, different monomeric fusion proteins, with different cargos, are provided in step 1) so that the RNA-nanoparticle formed comprises different cargoes.Preferably, the fusion protein provided in step 1) comprises or consists of SEQ ID Nos: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 91-94, or a sequence which is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 91-94, respectively.

[0364] In an embodiment, the purification step is performed by any purification method in the art, such as Affinity Chromatography, or Size Exclusion Chromatography (SEC).

[0365] In one embodiment, the methods are conducted under conditions in which the only RNA available for capture by the N protein is the cognate RNA supplied in the corresponding assembly step (step 4 of the method of the sixth aspect, and step 3 of the method of the seventh aspect). In other words, the reaction mixture is free of non-cognate RNA such that oligomerization proceeds exclusively with the RNA of interest, thereby yielding a homogeneous population of RNA nanoparticles with respect to RNA identity. Thus, preferably, the RNA used in the method of the invention is of a predetermined sequence, and not a random RNA.

[0366] In certain implementations, the reaction mixture, or at least the assembly step, is substantially free of non-cognate RNA, for example containing less than 5 percent, 1 percent, 0.5 percent, or 0.1 percent non-cognate RNA by mass relative to the cognate RNA, or less than 1 non-cognate RNA molecule per 100 cognate RNA molecules. Preferably, the only RNA intentionally present in the assembly step is the cognate or pre-defined RNA provided in said step. To achieve these conditions, one or more of the following measures can be used prior to adding the cognate RNA: pre-purification of the fusion protein to remove adventitious nucleic acids, including treatment with a non-specific endonuclease followed by stringent washing and inactivation or removal of the nuclease, high-salt and polyanion washes to strip bound RNA, use of RNase-free reagents, buffers, and consumables, and work in an RNase-free environment. Verification that only the cognate RNA is incorporated can be performed by known assays.

[0367] It is noted that, although the methods defined herein yield a homogeneous population of RNA nanoparticles with respect to the identity of the incorporated RNA, two or more such homogeneous populations can be generated in parallel or sequentially using different RNA sequences or a library of RNA sequences and then combined in predetermined ratios. The resulting composition will comprise a mixed population of RNA nanoparticles that differ in the RNA they carry. Mixing can be performed after the last step to ensure defined composition.Similarly, because each monomeric fusion protein can bear one or more cargo moieties, heterogeneity with respect to cargo content can be achieved in two alternative ways. First, co-assembly can be implemented by providing, in step 1, two or more monomeric fusion proteins that differ in cargo identity, so that the resulting nanoparticles incorporate a mixture of cargo types within individual particles. The relative abundance of each cargo in the assembled nanoparticles can be controlled by the input stoichiometry of the different fusion proteins and by assembly conditions. Second, separate homogeneous subpopulations can be produced in independent reactions using fusion proteins with different cargos, each subpopulation being purified in the last step of the method, and the purified subpopulations then mixed to afford a mixed population that is heterogeneous across particles with respect to cargo. Either strategy can be used independently, or in combination with the RNA strategies above, to produce compositions that are heterogeneous with respect to RNA sequence and / or cargo content while preserving control over the identity and proportion of each component. Figure 50 illustrates an example of a heterogeneous composition and underscores the versatility of the methods of the invention, which enable spatiotemporal control over protein oligomerization. Preferably, the method of the sixth or seventh aspect does not comprise a step of using the cargo to purify the particles or as a purification tag.

[0368] SEQUENCE LISTING

[0369] Full length proteins

[0370] SEQ ID NO 1 : EbV_6xHis_N2-45o MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDD DDDIPFPGPINDDDNPGHQDDDPTDSQLE**

[0371] SEQ ID NO 2: EbV_6xHis_N2-45o_MBP MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDD DDDIPFPGPINDDDNPGHQDDDPTDSQGGSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAF NKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKE FLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTLE**

[0372] SEQ ID NO 3: EbV_6xHis_N2-45o_sfGFP MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDD DDDIPFPGPINDDDNPGHQDDDPTDSQGGGSSKGEELFTGVVPILVELDGDVNGHKFSVR GEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEG YVQERTISFKDDGKYKTRAWKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITA DKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTPIGDPNEK RDHMVLHEYVNAAGITLE

[0373] SEQ ID NO 4: EbV_VP35i-5i_Ni^5o_mCHERRY_6xHis MATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIENNPENLYFQGMD SRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVD FQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSS GKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYP TAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTV LDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGL FPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEK KILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDD DNPGHQDDDPTDSQGGGSGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEG RPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERV MNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDG ALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYKLEHHHHHH*

[0374] SEQ ID NO 5: EbV_VP35i-5i_Ni^5o_sfGFP_6xHis

[0375] MATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIENNPENLYFQGMD SRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVD FQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSS GKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYP TAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTV LDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGL FPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEK KILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDD DNPGHQDDDPTDSQGGGSSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATIGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTPIGDPNEKRDHMVLHEYVNAA GITLEHHHHHH*

[0376] SEQ ID NO 6: EbV_VP35i-5i_Ni^5o_MBP_6xHis MATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIENNPENLYFQGMD SRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVD FQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSS GKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYP TAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTV LDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGL FPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEK KILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDD DNPGHQDDDPTDSQGGGSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDK LEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGY AFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGP WAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLE AVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASG RQTVD EALKDAQTLEH H H H H H*

[0377] SEQ ID NO 7: EbV_VP35i-5i_Ni^5o_neoGreen_6xHis MATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIENNPENLYFQGMD SRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVD FQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSS GKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYP TAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTV LDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGL FPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEK KILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDD DNPGHQDDDPTDSQGGSMVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPND GYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQF EDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK AFTDVM GM DELYKLEH H H H H H*

[0378] SEQ ID NO 8: EbV_6xHis_VP35i.5i_Ni-367_sfGFP MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGASGGGSGGGGSGGGGSGGGGSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGE GDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQE RTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQK NGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTPIGDPNEKRDHM VLHEYVNAAGITLE**

[0379] SEQ ID NO 9: EbV_6xHis_VP35i.5i_Ni-389_mCFPMGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQASGGGSGGGGSGGGGSGGGGSGSMVSKGEELFT GWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE DGNILGHKLEYNYISHNVYITADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLL PDNHYLSTQSKLSKDPKEKRDHMVLLEFVTAARLE**

[0380] SEQ ID NO 10: EbV_6xHis_VP35i.5i_Ni^3o_mCFP MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DI PFPASGGGSGGGGSGGGGSGGGGSGSM VSKGEELFTGVVPI LVELDGDVNGH KFSVS GEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPKEKRD HMVLLEFVTAARLE**

[0381] SEQ ID NO 11: EbV_6xHis_VP35i-5i_Ni^5o_mCFP MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPGPINDDDNPGHQDDDPTDSQASGGGSGGGGSGGGGSGGGGSGSMVSKGEELFT GWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE DGNILGHKLEYNYISHNVYITADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLL PDNHYLSTQSKLSKDPKEKRDHMVLLEFVTAARLE**

[0382] SEQ ID NO 12: EbV_6xHis_N 1-430. _mCFP MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPASGGGSGGGGSGGGGSGGGGSGSMVSKGEELFTGWPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYI TADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPKEK RDHMVLLEFVTAARLE**

[0383] SEQ ID NO 13: EbV_6xHis_Ni.389_mCFP MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQASGGGSGGGGSGGGGSGGGGSGSMVSKGEEL FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQ CFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDF KEDGNILGHKLEYNYISHNVYITADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSTQSKLSKDPKEKRDHMVLLEFVTAARLE**

[0384] SEQ ID NO 14: EbV_6xHis_VP35i-5i_Ni^3o_Sitell MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIV RQQSLE**

[0385] SEQ ID NO 15: EbV_6xHis_VP35i-5i_Ni-43o_Flagellin54-23o MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSGQIRGLEMASKNSQDGISLIQTAEGALTETHAILQ RMRELTVQAGNTGTQQAEDLGAIKDEMDALIEEIDGISNRTEFNGKKLLDGTNSTDGFTFQI GANAGQQLNVKIDSMSSTALGVNALDVTDFAATAFDDQLKSIDTAINTVSTQRAKLGAVQNR LEHTINNLGASGENLTAALE**

[0386] SEQ ID NO 16: EbV_6xHis_VP35i-5i_Ni^3o_Gpep MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSKQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPT CWAICKRIPNKKPGKLE**

[0387] SEQ ID NO 17: EbV_6xHis_VP35i.5i_Ni^3o_mSA24K1D MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSGAEAGITGKWKNQHGSTFKVKAGADGNLTGQY ENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTKWRGQYQGGDEARINT QWNLTYEGGSGPATEQGQDTFTKVKLE**

[0388] SEQ ID NO 18: VSV_6xHis_N MGSHHHHHHEFSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRG YVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVL PDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPE GRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITG MSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFW GQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDST GGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEF DKLE**

[0389] SEQ ID NO 19: VSV_6xHis_sfGFP_N MRGSHHHHHHGTSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTT GKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRA VVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVE DGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKRDHMVLHEYVNAAGITLEL VPRGSMSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGL KSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSD ASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDV WGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDV TTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTAL LLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTN APPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG

[0390] SEQ ID NO 20: VSV_Pi-60_N_sfGFP_6xHis MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSE N LYFQGMSVTVKRI I DNTVI VPKLPAN EDPVEYPADYFRKSKEI PLYI NTTKSLSDLRGYVYQG LKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVS DASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTED VTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTA LLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTT NAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG GSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVN RIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQ QNTPIGDVEDGSVQLADHYQQNTPIGDPNEKRDHMVLHEYVNAAGITLEHHHHHH*

[0391] SEQ ID NO 21 : VSV_Pi-60_N_mCherry_6xHis MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSE N LYFQGMSVTVKRI I DNTVI VPKLPAN EDPVEYPADYFRKSKEI PLYI NTTKSLSDLRGYVYQG LKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVS DASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFD VWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTED VTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTA LLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTT NAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG GSGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGG PLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHY DAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKLEHH HHHH*

[0392] SEQ ID NO 22: VSV_Pi-60_N_MBP_6xHis MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSE N LYFQGMSVTVKRI I DNTVI VPKLPAN EDPVEYPADYFRKSKEI PLYI NTTKSLSDLRGYVYQG LKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVS DASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFD VWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTED VTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTA LLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTT NAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG GSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDII FWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLP NPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVD NAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTV LPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTLEH HHHHH*

[0393] SEQ ID NO 23: VSV_6xHis_P2.6o_N_sfGFP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKP SYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIP LYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIG IFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQC KMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCA ALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSP YSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFC VGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGL REKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKS AMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSH NVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTPIG DPNEKRDHMVLHEYVNAAGITLE**

[0394] SEQ ID NO 24: VSV_6xHis_P2-6o_N_mCherry MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKP SYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIP LYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIG IFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQC KMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCA ALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSP YSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFC VGDNKYTPDDSTGGLTTNAPPQGRDWEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGL REKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSMVSKGEEDNMAIIKEFMRFKVHM EGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKK TMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLD ITSHNEDYTIVEQYERAEGRHSTGGMDELYKLE**

[0395] SEQ ID NO 25: VSV_6xHis_P2.6o_N_MBP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKP SYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIP LYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIG IFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQC KMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCA ALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSP YSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFC VGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGL REKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSMKIEEGKLVIWINGDKGYNGLAEV GKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAF QDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALM FNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDY SIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPN KELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQ MSAFWYAVRTAVINAASGRQTVDEALKDAQTLE**

[0396] SEQ ID NO 26: VSV_6xHis_P2.60_N_mCFP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKP SYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIP LYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIG IFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQC KMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCA ALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSP YSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFC VGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGL REKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSMVSKGEELFTGWPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY ISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLS KDPKEKRDHMVLLEFVTAARLE**SEQ ID NO 27: VSV_6xHis_R2-6o_N_YFP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKP SYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIP LYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIG IFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQC KMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCA ALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSP YSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFC VGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGL REKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSMVSKGEELFTGWPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDF FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKL SKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGKDELSLE**

[0397] SEQ ID NO 28: VSV_mCherry_N_P2-6o_6xHis MGSGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKG GPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSS LQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGH YDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKLEG GGSELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLK SGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDA SRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDV WGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDV TTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTAL LLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTN APPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKASGG GSENLYFQGGGGSEFDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEE HTKPSYFQAADDSHHHHHHS

[0398] SEQ ID NO 29: VSV_6xHis P2.6o_i24A_i27A_TEV_N_sfGFP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAVGEADEAEAQRAEKSNYELFQEDGVEEHTK PSYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEI PLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTI GIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQ CKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKD CAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSK SPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQ FCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQ GLREKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSSKGEELFTGVVPILVELDGDVN GHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFF KSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFN SHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTP IGDPNEKRDHMVLHEYVNAAGITLE*

[0399] SEQ ID NO 30: VSV_6xHis_P2-6O_V21A_I27A. _TEV_N_sfGFP MGSHHHHHHEFDNLTKVREYLKSYSRLDQAAGEIDEAEAQRAEKSNYELFQEDGVEEHTK PSYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEI PLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQ CKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKD CAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSK SPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQ FCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQ GLREKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSSKGEELFTGVVPILVELDGDVN GHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFF KSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFN SHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTP IGDPNEKRDHMVLHEYVNAAGITLE*

[0400] SEQ ID NO 31 : VSV_6xHis_P2-6O_Y14A_I27A. _TEV_N_sfGFP MGSHHHHHHEFDNLTKVREYLKSASRLDQAVGEIDEAEAQRAEKSNYELFQEDGVEEHTK PSYFQAADDSKLGGGSGENLYFQGELSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEI PLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTI GIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQ CKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKD CAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSK SPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQ FCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQ GLREKTIGKYAKSEFDKASGGGSGGGGSGGGGSGGGGSSKGEELFTGVVPILVELDGDVN GHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFF KSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFN SHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTP IGDPNEKRDHMVLHEYVNAAGITLE*

[0401] SEQ ID NO 32: MeV_Pi-5o_Ni^o5_mCherry_6xHis MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCENLYFQGMATLL RSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTG ALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHD DPISSDQSRFGWFEN KEISDI EVQDPEGFNM I LGTI LAQI WVLLAKAVTAPDTAADSELRRWI KYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYI VEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFS AGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASEL GITAEDARLVSEIAMHTTEDGGGSGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEG EGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFK WERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMY PEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQY ERAEGRHSTGGMDELYKLEHHHHHH*

[0402] SEQ ID NO 33: MeV_Pi-5o_Ni^O5_sfGFP_6xHis MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCENLYFQGMATLL RSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTG ALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHD DPISSDQSRFGWFEN KEISDI EVQDPEGFNM I LGTI LAQI WVLLAKAVTAPDTAADSELRRWI KYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYI VEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFS AGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASEL GITAEDARLVSEIAMHTTEDGGGSGSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGD ATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTI SFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDVEDGSVQLADHYQQNTPIGDPNEKRDHMVL HEYVNAAGITLEHHHHHH*

[0403] SEQ ID NO 34: MeV_Pi-5o_Ni^O5_MBP_6xHis MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCENLYFQGMATLL RSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTG ALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHD DPISSDQSRFGWFEN KEISDI EVQDPEGFNM I LGTI LAQI WVLLAKAVTAPDTAADSELRRWI KYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYI VEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFS AGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASEL GITAEDARLVSEIAMHTTEDGGGSGSSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIK VTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTW PLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN YLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVR TAVINAASGRQTVDEALKDAQTLEHHHHHH*

[0404] SEQ ID NO 35: MeV_Pi-5o_Ni^O5_YFP_6xHis MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCENLYFQGMATLL RSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTG ALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHD DPISSDQSRFGWFEN KEISDI EVQDPEGFNM I LGTI LAQI WVLLAKAVTAPDTAADSELRRWI KYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYI VEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFS AGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASEL GITAEDARLVSEIAMHTTEDGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGD ATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF VTAAGITLGMDELYKGGSGGKDELSLEHHHHHH*

[0405] SEQ ID NO 36: MeV_Pi-5o_Ni^O5_CFP_6xHis MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCENLYFQGMATLL RSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSGPKLTG ALIGILSLFVESPGQLIQRITDDPDVSIRLLEVVQSDQSQSGLTFASRGTNMEDEADQYFSHD DPISSDQSRFGWFEN KEISDI EVQDPEGFNM I LGTI LAQI WVLLAKAVTAPDTAADSELRRWI KYTQQRRVVGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYI VEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFS AGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASEL GITAEDARLVSEIAMHTTEDGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGD ATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGI KAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPKEKRDHMVLLEF VTAARLEHHHHHH*

[0406] SEQ ID NO 37: RSV_6xHis_N_LCB3MSHHHHHHHHSENLYFQGSGGGSGGGGSMALSKVKLNDTLNKDQLLSSSKYTIQRSTGD SIDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKANG VDVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCG

[0407] M 11 LCI AALVITKLAAGDRSGLTAVI RRAN N VLKN EM KRYKGLLPKDI ANSFYEVFEKH PH Fl DV FVH FG I AQSSTRGGSRVEGI FAG LFM N AYGAGQVM LRWG VLAKSVKN I M LG H ASVQAEM E QVVEVYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRN QDLYDAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPKENDVELASGGGGSNLDELH MQMTDLVYEALHFAKDEEFQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS*

[0408] SEQ ID NO 38: RSV_6xHis_LCB1_N MSHHHHHHHHSENLYFQGSDKENVLQKIYEIMKELERLGHAEASMQVSDLIYEFMKTKDEN LLEEAERLLEEVKRASGGGSGGGSGGGGSMALSKVKLNDTLNKDQLLSSSKYTIQRSTGDS IDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKANGV DVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGM IILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKHPHFIDVF VH FGI AQSSTRGGSRVEG I FAG LFM N AYGAGQVM LRWG LAKSVKN I M LG H ASVQAEM EQ VVEVYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQ DLYDAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPKENDVELAS*

[0409] SEQ ID NO 39: RSV_6xHis_LCB1_N_LCB3 MSHHHHHHHHSENLYFQGSDKENVLQKIYEIMKELERLGHAEASMQVSDLIYEFMKTKDEN LLEEAERLLEEVKRASGGGSGGGSGGGGSMALSKVKLNDTLNKDQLLSSSKYTIQRSTGDS IDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKANGV DVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGM IILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKHPHFIDVF VH FGI AQSSTRGGSRVEG I FAG LFM N AYGAGQVM LRWG VLAKSVKN I M LG H ASVQAEM EQ VVEVYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQ DLYDAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPKENDVELASGGGGSNLDELHM QMTDLVYEALHFAKDEEFQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS*

[0410] SEQ ID NO 40: 6xHis_N RSV MSHHHHHHHHSENLYFQGSGGGGSMALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTP NYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKANGVDVTT HRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGMIILCI AALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKHPHFIDVFVHF GIAQSSTRGGSRVEGIFAGLFMNAYGAGQVMLRWGVLAKSVKNIMLGHASVQAEMEQVVE VYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLY DAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPKENDVEL*

[0411] SEQ ID NO 41: RSV_6xHis_Streptavidin_N ring_Zdomain MSHHHHHHHHSENLYFQGSAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGC QNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGG SGPATEQGQDTFTKVKASGGGSGGSGGGGSMALSKVKLNDTLNKDQLLSSSKYTIQRSTG DSIDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKAN GVDVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDC GMIILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKHPHFID VFVHFGIAQSSTRGGSRVEGIFAGLFMNAYGAGQVMLRWGVLAKSVKNIMLGHASVQAEM EQVVEVYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVI NYSVLDLTAEELEAI KHQLN PKEN DVELASGGGSGGGSVD NKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKH-AS

[0412] SEQ ID NO 42: CuV_6xHis_VP352-5o_N 2-450 EGFP MGSHHHHHHGGGGSGEFDKVTDAIMTGRLSLDTIPGFQCSAPTSRSRPTGRKCPPWEGPL IEPKTSKLGGGSGEFENLYFQGELNRYLGHGTRTSRENTNLSELHGILSLGLNVDHTIVRKK SIPLFEIGNSDQVCNWIIQIIEAGVDLQDVADSFLTMLCVNHAYQGDPNLFLESPAAHYLKGH GIHFEIQHRDNVDHITDLLGVGSRDKSLRKTLSALEFEPDGSTTAGMFLSFASLFLPKLVVGE RACLEKVQRQIQIHAEQGLIQYPTQWQSVGHMMWFRLIRVNFVLKFLLVHQGMHMMAGH DANDAIIANSISQTRFSGLLIVKTVLEHILQKTEAGVQLHPLARTSKVKGELLAFKSALEALASH REYAPFARLLNLSGVNNLEHGLYPQLSAIALGVATAHGSTLAGVNVSEQYQQLREAATEAE KQLQQHSEMRELETLGLDEQERKILATFHSRKNEINIQQTSSILAIRKERLRKLTEALNEEKNK NALDDEDESEEDDWSPENRGIRSNKGSTKESSSYTASGGGSGGGGSGGGGSGGGGSVS KGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE*

[0413] SEQ ID NO 43: EbV_6xHis_Ni^30_mSA24K1D MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAM TLRKERLAKLTEAITAASLPKTSGHYDD DDDIPFPASGGGSGGGGSGGGGSGGGGSGSGAEAGITGKWKNQHGSTFKVKAGADGNL TGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTKWRGQYQGGDEA RINTQWNLTYEGGSGPATEQGQDTFTKVK

[0414] SEQ ID NO 44: EbV_6xHis_Ni^5o_mSA24K1D MGSHHHHHHEFDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEE ICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDG VKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQI QVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVA QARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLL NLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESR ELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDD DDDIPFPGPINDDDNPGHQDDDPTDSQGGGSGAEAGITGKWKNQHGSTFKVKAGADGNLT GQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTKWRGQYQGGDEA RINTQWNLTYEGGSGPATEQGQDTFTKVK

[0415] SEQ ID NO: 90: 6xHis_VP35EbV _NEbVi^3o_mYFP MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGE GEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYV QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADK QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH MVLLEFVTAAGITLGMDELYKGGSGGKDELSLE SEQ ID NO: 91 : 6xHis_VP35EbV_NEbVi^o_MBP MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGI KVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFT WPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK GETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAV RTAVI NAASGRQTVDEALKDAQTLE

[0416] SEQ ID NO: 92: PVSV_NVSV_mCFP_6xHis MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSE N LYFQGMSVTVKRI I DNTVI VPKLPAN EDPVEYPADYFRKSKEI PLYI NTTKSLSDLRGYVYQG LKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVS DASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFD VWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTED VTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTA LLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTT NAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG GSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPKEKRDHMVLLEFVTAARLEHHHHHH

[0417] SEQ ID NO: 93: PVSV_NVSV_mYFP_6xHis MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSE N LYFQGMSVTVKRI I DNTVI VPKLPAN EDPVEYPADYFRKSKEI PLYI NTTKSLSDLRGYVYQG LKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVS DASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFD VWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTED VTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTA LLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTT NAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDKGG GSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGG KDELSLEHHHHHH SEQ ID NO: 94: EbV_Hisx6_VP35_Ni^o_SpyCatcher MGSHHHHHHEFMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIEN NPENLYFQGMDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEIC QLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVK RLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQV HAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQA RFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNL SGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESREL DHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDD DIPFPASGGGSGGGGSGGGGSGGGGSGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFS KRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITF TVNEQGQVTVNGEATKGDAHTLE SEQ ID NO: 95 SpyTag_mCFP_Hisx6 MGSVPTIVMVDAYKRYKGSGESGGSSMVSKGEELFTGWPILVELDGDVNGHKFSVSGEG EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYV QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQ KNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHM VLLEFVTAAGITLGMDELYKLEHHHHHH SEQ ID NO: 96: SpyTag_mYFP_Hisx6 MGSVPTIVMVDAYKRYKGSGESGGSSMVSKGEELFTGWPILVELDGDVNGHKFSVSGEG EGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQ KNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHM VLLEFVTAAGITLGMDELYKLEHHHHHH

[0418] Modules or domains

[0419] SEQ ID NO 45: EbV_N 1-450 MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAG VDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAV SSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQ YPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVK TVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEH GLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQ EKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPIN DDDNPGHQDDDPTDSQ

[0420] SEQ ID NO 46: EbV_N 1-430 MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAG VDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAV SSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQ YPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVK TVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQ EKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFP

[0421] SEQ ID NO 47: EbV_N 1-389 DSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGV DFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVS SGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQY PTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKT VLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHG LFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQE KKILMNFHQKKNEISFQ

[0422] SEQ ID NO 48: EbV_N 1-367 MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAG VDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAV SSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQ YPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVK TVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEH GLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLG

[0423] SEQ ID NO 49: RSV_N MALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTPNYDVQKHINKLCGMLLITEDANHKFTG LIGMLYAMSRLGREDTIKILRDAGYHVKANGVDVTTHRQDINGKEMKFEVLTLASLTTEIQINI EIESRKSYKKMLKEMGEVAPEYRHDSPDCGMIILCIAALVITKLAAGDRSGLTAVIRRANNVL KNEMKRYKGLLPKDIANSFYEVFEKHPHFIDVFVHFGIAQSSTRGGSRVEGIFAGLFMNAYG AGQVMLRWGVLAKSVKNIMLGHASVQAEMEQWEVYEYAQKLGGEAGFYHILNNPKASLL SLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVINYSVLDLTAE ELEAIKHQLNPKENDV

[0424] SEQ ID NO 50: MeV_N 1-405 MATLLRSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSSITTRSRLLDRLVRLIGNPDVSG PKLTGALIGILSLFVESPGQLIQRITDDPDVSIRLLEWQSDQSQSGLTFASRGTNMEDEADQ YFSHDDPISSDQSRFGWFENKEISDIEVQDPEGFNMILGTILAQIWVLLAKAVTAPDTAADSE LRRWIKYTQQRRWGEFRLERKWLDVVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMIC DIDTYIVEAGLASFILTIKFGIETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSI QNKFSAGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSST LASELGITAEDARLVSEIAMHTTED

[0425] SEQ ID NO 51: HMPV_N MSLQGIHLSDLSYKHAILKESQYTIKRDVGTTTAVTPSSLQQEITLLCGEILYAKHADYKYAAEI GIQYISTALGSERVQQILRNSGSEVQWLTRTYSLGKIKNNKGEDLQMLDIHGVEKSFVEEID KEARKTM ATLLKESSGN I PQNQRPSAPDTPI I LLCVGALI FTKLASTI EVGLETTVRRAN RVLS DALKRYPRMDIPKIARSFYDLFEQKVYHRSLFIEYGKALGSSSTGSKAESLFVNIFMQAYGAG QTMLRWGVIARSSNNIMLGHVSVQAELKQVTEVYDLVREMGPESGLLHLRQSPKAGLLSLANCPNFASVVLGNASGLGIIGMYRGRVPNTELFSAAESYAKSLKESNKINFSSLGLTDEEKEA AEHFLNVSDDSQNDY

[0426] SEQ ID NO 52: VSV_N MSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLKSGNV SIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTS ADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGND SNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDVTTWILN REVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTALLLRSTR ARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTNAPPQGR DVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDK

[0427] SEQ ID NO 53: CuV_N2-45o NRYLGHGTRTSRENTNLSELHGILSLGLNVDHTIVRKKSIPLFEIGNSDQVCNWIIQIIEAGVDL QDVADSFLTMLCVNHAYQGDPNLFLESPAAHYLKGHGIHFEIQHRDNVDHITDLLGVGSRD KSLRKTLSALEFEPDGSTTAGMFLSFASLFLPKLWGERACLEKVQRQIQIHAEQGLIQYPTQ WQSVGHMMVVFRLIRVNFVLKFLLVHQGMHMMAGHDANDAIIANSISQTRFSGLLIVKTVLE HILQKTEAGVQLHPLARTSKVKGELLAFKSALEALASHREYAPFARLLNLSGVNNLEHGLYP QLSAIALGVATAHGSTLAGVNVSEQYQQLREAATEAEKQLQQHSEMRELETLGLDEQERKI LATFHSRKNEINIQQTSSILAIRKERLRKLTEALNEEKNKNALDDEDESEEDDWSPENRGIRS NKGSTKESSSYT

[0428] SEQ ID NO 54: EbV_VP35i-5iMATTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIFCDIENNP

[0429] SEQ ID NO 55: MeV_Pi-50MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATC

[0430] SEQ ID NO 56: HMPV_Pi^0MSFPEGKDILFMGNEAAKLAEAFQKSLRKPGHKRSQSIIG

[0431] SEQ ID NO 57: VSV_Pi-60MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDS

[0432] SEQ ID NO 58: VSV_Pi-60. J24A-I27A MDNLTKVREYLKSYSRLDQAVGEADEAEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDS SEQ ID NO 59: VSV_P1-6O_V21A-I27A MDNLTKVREYLKSYSRLDQAAGEIDEAEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDS SEQ ID NO 60: VSV_Pi-60. Y14A-I27A

[0433] MDNLTKVREYLKSASRLDQAVGEIDEAEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSSEQ ID NO 61: CuV_VP352-5o DKVTDAIMTGRLSLDTIPGFQCSAPTSRSRPTGRKCPPWEGPLIEPKTS

[0434] SEQ ID NO 62: sfGFP MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVT TLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAWKFEGDTLVNRI ELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQ NTPIGDVEDGSVQLADHYQQNTPIGDPNEKRDHMVLHEYVNAAGIT

[0435] SEQ ID NO 63: MBP MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFW AHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNA GAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLP TFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEE LVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

[0436] SEQ ID NO 64: mCherry MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF AWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG EFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAE VKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0437] SEQ ID NO 65: EGFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0438] SEQ ID NO 66: mCFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSTQSKLSKDPKEKRDHMVLLEFVTAAR

[0439] SEQ ID NO 67: mYFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGKD ELS

[0440] SEQ ID NO 68: mNeoGreenMVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSP WILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSH IKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTA RTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

[0441] SEQ ID NO 69: Flagellin B. Subtilis 54-230 MQIRGLEMASKNSQDGISLIQTAEGALTETHAILQRMRELTVQAGNTGTQQAEDLGAIKDEM DALIEEIDGISNRTEFNGKKLLDGTNSTDGFTFQIGANAGQQLNVKIDSMSSTALGVNALDVT DFAATAFDDQLKSIDTAINTVSTQRAKLGAVQNRLEHTINNLGASGENLTAA

[0442] SEQ ID NO 70: Gpep KQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGK

[0443] SEQ ID NO 71: Sitell

[0444] STYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQS

[0445] SEQ ID NO 72: 6xHis

[0446] HHHHHH

[0447] SEQ ID NO 73: Z-Domain MSVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKH

[0448] SEQ ID NO 74: mSA24K_iD MGAEAGITGKWKNQHGSTFKVKAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLE WRVEWNNSTENCHSRTKWRGQYQGGDEARINTQWNLTYEGGSGPATEQGQDTFTKVK

[0449] SEQ ID NO 75: LCB1 SARS-CoV-2 minibinder MDKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER SEQ ID NO 76: LCB3 SARS-CoV-2 minibinder MNDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLER LLS SEQ ID NO 77: poly Glycine-Serine linker (x1): GGGGS

[0450] SEQ ID NO 78: RSV PchM 1-85:

[0451] MEKFAPEFHGEDANNRATKFLESIKGKFTSPKDPKKKDSIISVNSIDIEVTKESPITSNS TIINPTNETDDTAGNKPNYQRKPLV SEQ ID NO: 79-82, see table 1 below.

[0452] SEQ ID NO: 83: Linker GGGSGGGGSGGGGSGGGGS

[0453] SEQ ID NO: 84: Tev enzyme cleavage site: ENLYFQ

[0454] SEQ ID NO: 97: SpyCatcher:MVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISD GHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT SEQ ID NO: 98: SpyTag: VPTIVMVDAYKRYK

[0455] The following items are also encompassed by the present invention:

[0456] 1. A monomeric fusion protein comprising:

[0457] a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b),

[0458] b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and

[0459] c) a domain that comprises at least one cargo of interest,

[0460] wherein domains a) and b) and domains b) and c) are bound by a linker,

[0461] wherein domain b) is placed in between domains a) and c), and

[0462] wherein a cleavage site is placed between domains a) and b).

[0463] 2. The monomeric fusion protein according to item 1, wherein the oligomerization inhibitor comprised in domain a) is the chaperone module of the phosphoprotein (P) from Mononegavirales, or a fragment thereof.

[0464] 3. The monomeric fusion protein according to item 2, wherein the chaperone module of the phosphoproteins (P) comprised in domain a) comprises at least:

[0465] - amino acids 1-51 from P protein of Ebola virus (EbV),

[0466] - amino acids 1-60 from P protein of Vesicular Stomatitis Virus (VSV),

[0467] - amino acids 1-50 from P protein of Measles morbillivirus (MeV),

[0468] - amino acids 1-85 from P protein of Respiratory syncytial virus (RSV),

[0469] - amino acids 2-50 from P protein of Human Cuevavirus (CuV), or

[0470] - amino acids 1-40 from P protein of Human metapneumovirus (HMPV).

[0471] 4. The monomeric fusion protein according to any one of items 1 to 3, wherein the Nucleoprotein (N) comprised in domain b) comprises at least:

[0472] - amino acids 1-367 from the N protein of Ebola virus (EbV),

[0473] - the N protein of Vesicular Stomatitis Virus (VSV),

[0474] - amino acids 1-405 from the N protein of Measles morbillivirus (MeV),

[0475] - the N protein of Respiratory syncytial virus (RSV),

[0476] - amino acids 2-450 from the N protein of Human Cuevavirus (CuV),- the N protein of Human metapneumovirus (HMPV), or

[0477] - any fragment of said N proteins with the proviso that the fragment comprises at least one oligomerization domain and at least one RNA binding domain.

[0478] 5. The monomeric fusion protein according to any one of items 1 to 4, wherein:

[0479] - domain b) comprises at least amino acids 1-367, preferably 1-430, from the N protein of EbV, and domain a) comprises at least amino acids 1-51 of the phosphoprotein (P) of EbV,

[0480] - domain b) comprises the N protein of VSV, and domain a) comprises at least amino acids 1-60 from P protein of VSV,

[0481] - domain b) comprises amino acids 1-450 from the N protein of MeV, and domain a) comprises at least amino acids 1-50 from P protein of MeV,

[0482] - domain b) comprises the N protein of RSV, and domain a) comprises at least amino acids 1-85 from P protein of RSV,

[0483] - domain b) comprises amino acids 2-405 from the N protein of CuV, and domain a) comprises at least amino acids 2-50 from P protein of CuV, or

[0484] - domain b) comprises the N protein of HMPV, and domain a) comprises at least amino acids 1-40 from P protein of HMPV.

[0485] 6. The monomeric fusion protein according to any one of items 1 to 5, wherein:

[0486] - domain b) comprises SEQ ID NO 46: EbV_Ni-43o, and domain a) comprises SEQ ID NO 54: EbV_VP35i-si,

[0487] - domain b) comprises SEQ ID NO 50: MeV_Ni-4os, and domain a) comprises SEQ ID NO 55: MeV_Pi.5o,

[0488] - domain b) comprises SEQ ID NO 49: RSV_N, and domain a) comprises SEQ ID NO 78: RSV PchM 1-85,

[0489] - domain b) comprises SEQ ID NO 51: HMPV_N, and domain a) comprises SEQ ID NO 56: HMPV_PI.4O,

[0490] - domain b) comprises SEQ ID NO 53: CUV_NI-4SO, and domain a) comprises SEQ ID NO 61 : CUV_VP35I.5O, or

[0491] - domain b) comprises SEQ ID NO 52: VSV_N, and domain a) comprises SEQ ID NO 57: VSV_Pi.6o, SEQ ID NO 58: VSV_P 1-60J24A-I27A, SEQ ID NO 59: VSV_P 1-60_ V21A- I27A, or SEQ ID NO 60: VSV_Pi-6o_Yi4A-i27A.7. The monomeric fusion protein according to any one of items 1 to 6, wherein the linker comprises amino acid Gly or Se, and its length or number of repetitions is calculated using the following formula:

[0492] (Eq. 1)

[0493]

[0494] 2lpboaaiinker

[0495] wherein:

[0496] - re refers to the target average distances depending on the size of the cargo of interest. - Ip refers to persistence length, which is typically between 3 and 7 A, depending on the linker sequence,

[0497] - Bo is 3.8 A, and

[0498] - aalinker is the number of amino acids of the linker sequence, preferably between 4-6 amino acids.

[0499] 8. The monomeric fusion protein according to any one of items 1 to 7, comprising or consisting of any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 90-94.

[0500] 9. A homogeneous population of RNA-nanoparticles, wherein each of the RNA-nanoparticles is characterized by comprising:

[0501] i) five or more monomers that interact with each other thereby forming a nanoparticle, wherein each monomer comprises a first domain and a second domain that are bound by a linker, wherein:

[0502] 1) the first domain comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and

[0503] 2) the second domain comprises at least one cargo of interest, and ii) at least one RNA molecule that is bound to the RNA binding domain of each of the N proteins of the first domain of i.1), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA- nanoparticles.

[0504] 10. The homogeneous population of RNA-nanoparticles according to item 9, wherein the Nucleoprotein (N) comprised in the first domain comprises at least:

[0505] - amino acids 1-367 from the N protein of Ebola virus (EbV),

[0506] - the N protein of Vesicular Stomatitis Virus (VSV),- amino acids 1-405 from the N protein of Measles morbillivirus (MeV), - the N protein of Respiratory syncytial virus (RSV),

[0507] - amino acids 2-450 from the N protein of Human Cuevavirus (CuV),

[0508] - the N protein of Human metapneumovirus (51), or

[0509] - any fragment of said N proteins with the proviso that the fragment comprises at least one oligomerization domain and at least one RNA binding domain.

[0510] . The homogeneous population of RNA-nanoparticles according to any one of items 9 or 10, wherein:

[0511] - the first domain is placed in the N-terminal of the second domain, or

[0512] - the first domain is placed in the C-terminal of the second domain.

[0513] . A composition comprising one or more fusion proteins as defined in any one of items 1 to 8, or the population of RNA-nanoparticles as defined in any one of items 9 to 11.

[0514] . The monomeric fusion protein according to any one of items 1 to 8, the population of RNA-nanoparticles as defined in any one of items 9 to 11 , or the composition according to item 12, for use in the fields of diagnostic methods, spectroscopic, medicine, biocatalysts, vaccinology, personalized medicine, detection methods, drug delivery, theragnostic, bionanotechnology, and molecular imaging.

[0515] . A method for producing the homogeneous population of RNA-nanoparticles as defined in any one of items 9 to 11 , the method comprising the steps of:

[0516] 1) providing the monomeric fusion protein as defined in any one of items 1 to 8, 2) carrying out the cleavage of the cleavage site,

[0517] 3) purifying the resultant fusion proteins from step 2),

[0518] 4) adding one or more RNA molecules with identical or substantially identical nucleotide sequence, and

[0519] 5) allowing the spontaneous oligomerization of the product of step 3) with the RNA molecules of step 4), thereby providing the homogeneous population of RNA- nanoparticles.EXAMPLES

[0520] EXAMPLE 1

[0521] 1. MATERIALS & METHODS

[0522] 1.1. FUSION PROTEIN DESIGN

[0523] We designed and generated non-natural standardized backbones consisting of two different gene sequence arrays that allow for the expression and purification of fusion proteins of the types PChM-N-X (backbone A) and X-N-PChM (backbone B), i.e., with a cargo protein X fused at either the C- or the N- terminus of a nucleoprotein N, and inhibitor protein PChM fused to the opposite terminus of N (Figure 4). The selection of one or the other backbone design depends on the desired orientation of the cargo and which parts need to be solvent-exposed to preserve function, which in turn is modulated by tethering of the N- or C- termini of the cargo.

[0524] The fusion proteins obtained with this gene design are soluble and stable monomers that constitute the building blocks of the MPs. Each domain of the designed recombinant genes is flanked with linkers and unique specific recognition sites for different restriction enzymes (RE), enabling discrete modifications without disrupting other components. The constructs also include a STOP sequence and a tag sequence, such as His-Tag, for protein expression and purification, respectively. Note that the HisTag is a cargo in itself, whose functionality is the specific binding of the oligomers to matrices functionalized with suitable receptors, such as Ni-NTA.

[0525] The backbones’ components of the fusion proteins of the invention are:

[0526] 1. Nucleoproteins domain, N: Constitute the scaffold of the MPs. We employ full length or partial sequences of viral nucleoproteins from the Mononegavirales order. We have screened N proteins from several viruses, and tested two of them in detail: the N proteins from Ebola Virus (EbV) and from Vesicular Stomatitis Virus (VSV). Note that although all the results presented here correspond to either EbV or VSV, other nucleoproteins from Mononegavirales can also be used.

[0527] 2. Inhibitor domain, PCHM: Included in the backbones at the opposite end of the cargo X.

[0528] It is a fragment of the viral P phosphoprotein that encompasses at least the chaperone module (e.g. fragments 1-50 from EbV, 1-60 from VSV, 1-50 from Measles, 1-85 from RSV and 1-40 from HMPV), which acts as an oligomerization inhibitor of N. Moreover, PchM impairs RNA binding to N, thus stabilizing RNA-free PchM-N-X and X-N-PC MN monomeric species. Removal or absence of the PchM fragment results in the irreversible formation of homo-oligomeric particles (SMPs) during protein expression, due to the high RNA avidityand their self-assembly capabilities. Thus, presence of the PchM module is essential for the production of monomeric species. Note that co-expression of N and PchM may also allow obtaining monomeric species, but with a minimum yield; the fusion of N and PchM and the subsequent separation to form the MPs becomes mandatory to scale up this technology.

[0529] 3. Cargo domain, X: refers to any peptide, polypeptide whole protein or protein domain that holds a desired functionality. This includes antigens, immunomodulators, binding domains, probes (fluorescent, paramagnetic, redox, etc.) and enzymes, among others. It is important to note that cargoes serve a double function: perform the desired biological activity in the MPs and also hinder polymerization in the Z-axis, thus preventing the formation of unwanted helical rod-like structures.

[0530] 4. Linkers: Each domain of the gene array is linked with a linker. Linkers are widely used in the field of fusion proteins. For example, a linker may be one or more repeats of GGGS sequences. The optimal length of a linker may be determined by several methods, such as by performing screenings with different lengths, or by computational modelling aiming to avoid steric hindrances and clashes. For example, one GGGS repeat at the / V-terminus and three GGGS repeats at the C-terminus are the preferred linkers choice in the case of the VSV N protein scaffolds.

[0531] 5. A protease cleavage site is strategically designed to separate the N-PchM link, resulting in a non-covalent PCHM:N adduct. In all our examples we used TEV protease, but any other protease cleavage site can be used.

[0532] Other elements can be incorporated, such as restriction enzymes (RE) sites can also be used to separate the different elements in the backbone, to provide for a more versatile platform where said elements can be easily changed. RE includes specific recognition sites for the restriction enzymes such as Ncol, EcoRI, Sacl, Nhcl, BamHI, Xhol and Blpl.

[0533] All DNA sequences are designed and optimized for expression in the desired vector. The blueprints shown in Figure 4 are not present in nature. The standardization of the plasmids enables the incorporation and replacement of different cargoes on demand, as well as the substitution of the linkers by improved versions.

[0534] Construct design and optimization may be performed by the skilled person by computational modelling and simulation of each cargo in the context of the MPs. In most cases, however, this analysis can be reduced to the design of linkers that guarantee the desired average distances between protein domains to minimize steric clashes. Linkers are typically repeats of a sequence of a few (usually 5 or 6) amino acids, being G and S the most used residues. Thenumber of repeats (n) required to obtain an average end-to-end distance <re> can be easily estimated using the following expression:

[0535] (Eq. 1)

[0536]

[0537] 2lpboaaiinker

[0538] Equation 1 is a semiempirical expression that is derived from the Worm-Like Chain Model (WMC) (References 1-3) and simplified using empirical correction factor properly validated using <re> values derived from FRET experiments. In this expression Ip is the so-called persistence length, which represents the stiffness of the linker. Therefore, Ip is sequence specific and has been experimentally determined for a large number of linker sequences (Table 1) (References 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13). bO = 3.8 A is the average distance between two consecutive CD atoms in a peptide bond, and aaimker is the number of amino acids of the linker sequence. Table 1 summarizes n values calculated using equation 1 for different target average distances <re> and different linker sequences. The obtained n values are rounded up to the nearest integer to guarantee that the average distance will be equal or larger than the desired one.

[0539] Table 1. Examples of application of equation 1 for 5 different linker sequences and 3 different target distances in each case.

[0540] Desired linker aa linker bO Ip desired <re> n rounded n GGGGS SEQ ID NO: 77 5 3.8 3.04 15 2.16 3

[0541] GGGGS SEQ ID NO: 77 5 3.8 3.04 20 3.84 4

[0542] GGGGS SEQ ID NO: 77 5 3.8 3.04 25 5.99 6 GGSGGS SEQ ID NO: 79 6 3.8 3.7 15 1.48 2 GGSGGS SEQ ID NO: 79 6 3.8 3.7 20 2.63 3 GGSGGS SEQ ID NO: 79 6 3.8 3.7 25 4.10 5 GSSGSS SEQ ID NO: 80 6 3.8 4.5 15 1.21 2

[0543]

[0544] GSSGSS 6 3.8 4.5 20 2.16 3SEQ ID NO: 80

[0545] GSSGSS SEQ ID NO: 80 6 3.8 4.5 25 3.37 4 GSSSSS SEQ ID NO: 81 6 3.8 4.9 15 1.12 2 GSSSSS SEQ ID NO: 81 6 3.8 4.9 20 1.98 2 GSSSSS SEQ ID NO: 81 6 3.8 4.9 25 3.10 4

[0546] SSSSSS SEQ ID NO: 82 6 3.8 6.2 15 0.88 1 SSSSSS SEQ ID NO: 82 6 3.8 6.2 20 1.57 2

[0547] SSSSSS SEQ ID NO: 82 6 3.8 6.2 25 2.45 3

[0548]

[0549] References:

[0550] Kratky, O. & Porod, G. Rdntgenuntersuchung geldster Fadenmolekule. Reel. Trav. Chim. Pays-Bas 68, 1106-1122 (1949).

[0551] 2. van Rosmalen, M., Krom, M. & Merkx, M. Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins. Biochemistry 56, 6565-6574 (2017).

[0552] 3. Evers, T. H., Van Dongen, E. M. W. M., Faesen, A. C., Meijer, E. W. & Merkx, M. Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Linkers. Biochemistry 45, 13183-13192 (2006).

[0553] 4. Sommese, R. F., Sivaramakrishnan, S., Baldwin, R. L. &Spudich, J. A. Helicity of short E-R / K peptides. Protein Sci. 19, 2001-2005 (2010).

[0554] 5. Szabo, B. et al. Intrinsically Disordered Linkers Impart Processivity on Enzymes by Spatial Confinement of Binding Domains. Int. J. Mol. Sci. 20, 2119 (2019).

[0555] 6. Forsberg, Z. et al. The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase. J. Biol. Chem. 299, (2023).

[0556] 7. Arai, R., Wriggers, W., Nishikawa, Y., Nagamune, T. & Fujisawa, T. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. Proteins Struct. Funct. Bioinforma. 57, 829-838 (2004).

[0557] 8. Kjaergaard, M. Estimation of Effective Concentrations Enforced by Complex Linker Architectures from Conformational Ensembles. Biochemistry 61, 171-182 (2022).

[0558] 9. Mondol, T. et al. Aha1 regulates Hsp90’s conformation and function in a stoichiometrydependent way. Biophys. J. 122, 3458-3468 (2023).

[0559] 10. Szilvay, G. R., Blenner, M. A., Shur, O., Cropek, D. M. & Banta, S. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase. Biochemistry 48, 11273-11282 (2009).11. Sivaramakrishnan, S. et al. Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER / K a-Helix. Biophys. J. 97, 2993 (2009).

[0560] 12. Zhou, H.-X. Quantitative Relation between Intermolecular and Intramolecular Binding of Pro-Rich Peptides to SH3 Domains. Biophys. J. 91, 3170-3181 (2006).

[0561] 13. van Rosmalen, M., Krom, M. & Merkx, M. Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins. Biochemistry 56, 6565-6574 (2017).

[0562] 1.2. CLONING, EXPRESSION & PURIFICATION

[0563] The protocols were analytically optimized and included variables such as bacterial (E. co / / ) or yeast (P. pastoris) expression systems, culture media, incubation conditions (times, temperatures), buffers (pH, composition, concentration and ionic strength), addition of specific chemical (reducing agents, prosthetic groups, metals, chelators, etc.), purification conditions, etc.

[0564] The detailed protocol for the expression and purification of PCHM-N-X and X-N-PchM monomeric fusion proteins is based on a multistep sequence, which may need adjustments depending on the specific N, PchM, and X components being fused. The protocols described below correspond to PCHM-N-X and X-N-PchM constructs based on N proteins from EbV, VSV, MeV and CuV (NEW, NVSV, NMev and Ncuv, respectively) and were validated for a variety of cargoes, including 6 fluorescent proteins, binding proteins such as His-Tag, maltose binding protein (MBP) and monomeric streptavidin (mSA4K1D), antigens such as Site II and Gpep, and protein-based adjuvants, such as flagellin.

[0565] (i) Protocol optimized for N constructs (1 L culture)

[0566] 1) Grow the Rosetta™ 2(DE3) cells transformed with the synthetic gene in LB medium containing 50 pg / mL kanamycin and 37 ug / mL chloramphenicol at 37 °C and 200 rpm agitation.

[0567] 2) Inoculate 1:100 dilution culture into a fresh LB medium containing 50 pg / mL kanamycin and 37 ug / mL chloramphenicol, and grow at 37 °C and 200 rpm until reaching an OD600 nm = 0.6. Induce using 0.2 mM IPTG at 18 °C for 16 hs.

[0568] 3) Harvest the cells at 4000 rpm for 10 min at 4 °C. Resuspend the cell pellet (20 mL / L) in lysis buffer (20 mM sodium phosphate pH 8.0, 500 mM NaCI, 1 mM PMSF, 10 ug / mL RNAse)1,2.

[0569] 4) Lyse the cells by sonication on ice (5 sec on / 30 sec off for 7 minutes).5) Centrifuge the lysate at 18000 rpm for 45 min at 4 °C and filter the supernatant with a 0.45 um filter.

[0570] 6) Precipitate the supernatant with 60% m / v ammonium sulfate at 4°C with agitation for 2 hs. Centrifuge at 10000 rpm for 10 min at 4°C and then resuspend it into Wash Buffer (20 mM sodium phosphate pH 8.0, 500 mM NaCI)3

[0571] 7) Dialyze into Buffer A (20 mM sodium phosphate pH 8.0, 500 mM NaCI, 20 mM imidazole).

[0572] 8) Centrifuge the supernatant at 18000 rpm for 45 min at 4 °C.

[0573] 9) Filter the supernatant through 0.45 pm filter and load it into Ni-NTA nickel column pre-equilibrated with Buffer A (20 mM sodium phosphate pH 8.0, 500 mM NaCI, 20 mM imidazole)

[0574] 10) Elute the sample with Buffer B (20 mM sodium phosphate pH 8.0, 500 mM NaCI, 500 mM imidazole).

[0575] 11) Dialyze the sample in S200 Buffer (20 mM sodium phosphate pH 8.0, 150 mM NaCI).

[0576] 12) Centrifuge the sample at 18000 rpm for 45 min at 4 °C.

[0577] 13) Load the sample into a Superdex 200 increase 10 / 300 GL (GE Healthcare) size exclusion column equilibrated in Buffer s200.

[0578] 14) Collect the fraction containing the purified protein and then store it at -80 °C.

[0579] 1The lysis buffer for NEW constructs purification also includes 0.5 mM EDTA.

[0580] 2The lysis buffer for Nvsv constructs purification also includes 0.5 mM B-Me.

[0581] 3The wash buffer for Nvsv constructs purification also includes 0.5 mM B-Me.

[0582] Note that both protocols include the addition of RNAse A to prevent spurious self-assembly along the purification process, and proteases inhibitors to avoid enzymatic cleavage of the constructs. Purification is based on a two-step protocol that includes nickel affinity chromatography followed by size exclusion chromatography (SEC). The SEC purification step provides information regarding the oligomeric state of the construct, as well as on the presence of spurious RNA binding to N, which is determined from the ratio of absorbances recorded at 260 and 280 nm. For most constructs the optimal buffer in terms of monomer stability and solubility is 20 mM phosphate buffer containing 150 mM NaCI, but admit a pH range from 6 and 9 and ionic strengths from 100 to 400 mM.1.3. CONTROLLED SELF-ASSEMBLY

[0583] The generation of homooligomeric or heterooligomeric MPs (SMPs or MMPs) from the different PchM- -X and X- -PchM protomers is done following a general 5-step protocol:

[0584] 1 ) Selection of the desired soluble monomeric PC M- -X and / or X- -PchM fusions from the library

[0585] 2) Mixing of the selected PC M- -X and / or X- -PchM components in the desired stoichiometry

[0586] 3) Digestion with TEV to remove the inhibitor PchM by enzymatic hydrolysis of the PCHM- or -PchM chemical bond. The reaction is performed in a TEV reaction buffer (20 mM sodium phosphate pH 8.0, 50 mM NaCI, 1 mM DTT, 0.5 mM EDTA) in a mass ratio of 1:10 (TEV and fusions respectively).

[0587] 4) Addition of RNA to trigger oligomerization

[0588] 5) Purification of the MPs by SEC to remove non-reactive protomers and RNA molecules

[0589] The specific conditions for steps 2 to 5 may require optimization depending on the PC M- -X and / or X- -PchM monomers employed in the construct.

[0590] So far, we have demonstrated that commercially available tRNA from baker’s yeast (Roche - 10109495001) or RNA from torula yeast Type VI (Sigma-Aldrich - R6625) in 5:1 RNA:N ratio are very efficient in promoting oligomerization for all constructs tested (see examples below). Note that the tRNA used in these proofs of concept is a complex mixture of unknown RNA sequences. For a pharmacologically relevant formulation well defined RNA sequences can be used.

[0591] We have succesfully tested virus-specific RNA sequences corresponding to the leader sequence of the viral genome that binds to the N proteins. For VSV we used the sequence AUG GUAAUA AUG GUU UGU UGU CUU CGU, that includes one repeat of the leader 9-mer sequence UGU CUU CGU. Another important aspect that needs to be considered is the fact that most RNA particles are immunogenic. The possible immunological targets include cell surface or endosome Toll-like receptors and cytosolic sensors; and the immunogenicity can be tuned by sequence, length and shape. An important exception are polyA RNA sequences, which are not immunogenic. We have successfully induced oligomerization of NEbV- and NVSV-based constructs using polyA 7-mer and polyA 9-mer RNA sequences. Therefore, different RNAs of interests can be chosen to trigger oligomerization.2. RESULTS

[0592] 2.1 PchM functions as oligomerization inhibitor

[0593] We assessed whether PchM-N-X and X-N-PchMcar\ be obtained in a monomeric state, implying that PchM can work as an oligomerization inhibitor fused either to the N- or C- termini of the nucleoproteins. To this end, we tested the constructs depicted in Figure 4 using the cargoes shown in Table 2, and analyzed their oligomerization state by Size Exclusion Chromatography (Figure 5). It is observed that both scaffolds elute as monomers (left panel). The purification process allows us to obtain stable and soluble constructs for later processing to form the MPs.

[0594] We tested cargoes of various sizes and functionalities (Table 2 and Figure 5) with N proteins from different viruses, which resulted in successful monomeric and soluble constructs.

[0595] Conclusions: The PChM module can be fused either to the A / - or C- termini of the nucleoproteins and it is functional, keeping the proteins in a monomeric state, even in the presence of cargo proteins at the other end (PchM-N-X and X-N-PchM construct)

[0596] Table 2:Modular SEQ ID

[0597] Viru

[0598] Arrangement N X Seq N° of X NO of s PchM

[0599] each domain Full mCherry, 21 57-52-64; vsv Lenght 1-60 sfGFP, 20 57-52-62;

[0600] (FL) MBP 22 57-52-63 mCherry 4 54-45-64; sfGFP 5 54-45-62; EbV 1-450 1-51

[0601] MBP 6 54-45-63; PchM-N-X-His

[0602] NeoGreen 7 54-45-68 mCherry 32 55-50-64; sfGFP 33 55-50-62; Me

[0603] 1-405 1-50 MBP 34 55-50-63, V

[0604] mCFP 36 55-50-66; mYFP 35 55-50-67 mCherry 24 57-52-64; sfGFP 23 57-52-62; FL 2-60 MBP 25 57-52-63;

[0605] mCFP 26 57-52-66; mYFP 27 57-52-67 VSV

[0606] 2-60_

[0607] FL sfGFP 29 58-52-62 I24A-I27A

[0608] 2-60_

[0609] FL sfGFP 30 59-52-62 V21A-I27A

[0610] 2-60_

[0611] FL sfGFP 31 60-52-62 His-PchM-N-X Y14A-I27A

[0612] 1-367 1-51 sfGFP 8 54-48-62 1-389 1-51 mCFP 9 54-47-66 mCFP 10 54-46-66; Flagellin54-23o 15 54-46-69; EbV

[0613] 1-430 1-51 Gpep 16 54-46-70;

[0614] Sitell 14 54-46-71 ; mSA4KiD 17 54-46-74 1-450 1-51 mCFP 11 54-45-66 CuV 2-450 2-50 EGFP 42 61-53-65 X-N-PchM-His VSV FL 2-60 mCherry 28 57-52-64

[0615]

[0616] 2.3 SCREENING OF NUCLEOPROTEINS AND CARGOES THAT ARE TOLERATED ON THE MPS WITHOUT HINDERING SELF-ASSEMBLY

[0617] We have tested multiple M scaffolds and cargos on the MPs to evaluate whether different combinations inhibit or alter the self-assembly capabilities of M (Table 3). To this end, we have designed and generated N-X orX-N constructs, thus obtaining homo-oligomeric MPs (SMPs) after expression and purification (non-modular approach, see Fig. 3), or expressed soluble monomeric PCHM-N-X or N-X-PchM constructs, followed by TEV cleavage and RNA addition to trigger self-assembly (modular approach, see Fig. 3). In all cases the formation of MPs was assessed by size exclusion chromatography and / or dynamic light scattering (DLS). The size of the MPs is a function of the stoichiometry of ring or worm particles formed and the size of the N protomer. Usually, monomeric N proteins lay within the 40-50kDa range and 8-9 nm (according to DLS), while the MPs are expected to fall in the 400-700 kDa and 30-80 nm ranges.

[0618] Table 3 List of MPS obtained using a variety of N scaffolds and cargoes. It is noted that, as used herein and in the context of this invention, the tag HisTag is not used as a cargo, but as a affinity tag to purify the particles.

[0619] Cargo Size Nucleoprotein N- or C- Function His Mod or Seq (KDa) termini? Tag NoMod? N° (P,

[0620] X or

[0621] N)

[0622] His-Tag* <1 EbV, VSV, N- Binding N NoMod 1, 2,

[0623] RSV 3,

[0624] 12, 13, 18, 19, 37, 40, 43, 44 LCB1 SARS- 7.7 RSV N- SARS-CoV-2 X NoMod 38, CoV-2 neutralization 39 minibinder

[0625] LCB3 SARS- 7.8 RSV C- SARS-CoV-2 N, X NoMod 37, CoV-2 neutralization 39 minibinder

[0626] mCherry 26.7 EbV, VSV, / V- (VSV) Fluorescent X, P Mod 4, MeV c- Probe 21,

[0627] 24,

[0628]

[0629] 28,

[0630] 32 superfolder 28 EbV, VSV, / V- (VSV) Fluorescent N NoMod 3, Green MeV C- Probe 19 Fluorescent

[0631] Protein EbV, VSV, C- Fluorescent X, P Mod 5, 8, (sfGFP) MeV Probe 20,

[0632] 23, 29, 30, 31, 33 Enhanced 28 CuV C- Fluorescent P Mod 42 Green Probe

[0633] Fluorescent

[0634] Protein

[0635] (EGFP)

[0636] Cyan 26 EbV, VSV, C- Fluorescent P Mod 9, Fluorescent MeV Probe 10, Protein 11, (CFP) 26,

[0637] 36 VSV C- X Mod 92

[0638] EbV C- N NoMod 12,

[0639] 13 Yellow 28 VSV, MeV, C- Fluorescent P Mod 27, Fluorescent EbV Probe 35, Protein 90 (YFP)

[0640] VSV c- X Mod 93 mNeonGreen 27 EbV c- Fluorescent X Mod 7 Fluorescent Probe

[0641] Protein

[0642] (mNGFP)

[0643] Flagellin 32.6 c- Adjuvant P Mod 15

[0644] EbV (TLR5

[0645] agonist)

[0646] Maltose 43 EbV c- Binding N, X Both 2, 6 Binding

[0647] Protein VSV, MeV, c- Binding X, P Mod 22, (MBP) EvB 25,

[0648] 34, 91 Z-Domain 6.65 RSV c- Antibody X NoMod 41 binding

[0649]

[0650] Monomeric 12.6 RSV / V- Binding of X NoMod 41 streptavidin biotinylated

[0651] c- ligands

[0652] P Mod 17 c- N NoMod 43, EbV

[0653] 44 Site II 4.4 EbV c- Antigen P Mod 14 Gpep 5.6 EbV c- Antigen P Mod 16 SpyCatcher 12.2 EbV c- Covalent P Mod 94 conjugation

[0654]

[0655] In Figure 6 we show an example of representative SEC and DLS for MPs obtained by triggering the self-assembly of monomeric species PChM-N-X (left) and oligomeric N-X (SMPs) (right). It can be observed that both MPs, regardless of how they were obtained, exhibited similar SEC chromatograms and DLS profiles.

[0656] Conclusions: We have identified multiple M scaffolds that are suitable to generate soluble MPs in a controlled, on demand fashion. These scaffold proteins can be fused to a variety of cargoes of different sizes, without hindering the oligomerization capabilities to form MPs. Cargoes of all sizes can be incorporated as the length of the flexible linker can be modified so that the cargo is orientated as desired and depending on the application. Further, more than one cargo can be placed, as some functional cargoes (Z-domain, monomeric streptavidin) allow for further incorporation of molecules (antibodies, biotinylated ligands) onto the Ps.

[0657] 2.4. THE PROTOMERS CONSTRUCTS ARE STABLE.

[0658] The constructs are soluble and stable. We expressed and purified monomeric PchM-Nvsv- sfGFP. The protein was kept at 4°C during 60 days, and the sample was re-analyzed by Size Exclusion Chromatography. The protein remains monomeric with negligible oligomerization (Figure 7, left panel). Stability is also observed after thawing cycles, as shown in (Figure 7, right panel).

[0659] We then tested if the oligomeric MPs are stable after sample thawing. We produced SMPs of NEbv-mCFP (modular pipeline) and then assessed the size of the fresh sample by DLS. The sample was subjected to freeze-thaw and no precipitation was observed. The size was reassessed by DLS, observing that the MPs remain in an oligomeric state (Figure 8).

[0660] Conclusions: The constructs are monomeric and stable. The molecules remain monomeric and soluble for many months at 4°C, thus allowing for the generation of a stable library offunctionalities. The MPs remain oligomeric and soluble after thawing, allowing their final use when demanded.

[0661] 2.5. MODIFICATION OF THE PCHM SEQUENCE ALTERS THE OLIGOMERIZATION EQUILIBRIUMS OF N IN N-PCHM CONSTRUCTS

[0662] PChM acts as an oligomerization chaperone, keeping N in the monomeric state by hindering the establishment of N-N interactions and / or (depending on the specific N) N-RNA interactions. By analyzing the PChM : N interactions, we set to generate mutations in PChM that can influence the oligomerization state of the constructs. To this end we generated 4 variants: WT, V.A(I24A,I27A), V.B (V21AJ27A), and V.C (Y14AJ27A) that were designed and generated in silico to weaken the overall interaction (Figure 9).

[0663] 2.6. ADDITION OF A TAG

[0664] We have included a His-Tag in all the constructs for purification purposes, or as a cargo of interest. For the modular pipeline, we have tested the effect of the position of the His-Tag exploring two alternatives, fusing the His-Tag either to a) the cargo (X_HT) or b) to PchM (HT_PchM). In the first case the tag is integral part of the cargo, thus endowing the cargo with dual functionality: the intrinsic function of X and the binding properties of the HT moiety. In the second case the HT is cleaved together with PChM upon TEV digestion and, thus, cannot be regarded as a cargo. Thus, the HT_PchM constructs result in a modification of PchM resulting in the addition of 6 histidines. We analyzed whether the location of the His-Tag (HT_PChM or X_HT) has an impact on the oligomerization.

[0665] In the case of Afet>v(Figure 10), the position of the His-Tag affects the self assembling process. Cleavage of PchM in PchM-N-X_HT constructs readily results in partial oligomerization with a 51:49 oligomermonomer proportion. Oligomerization is completed by the addition of RNA (97% MPs). In contrast, cleavage of HT_PchM in HT_PchM-N-X constructs yields 90:10 oligomermonomer proportion, although addition of RNA triggers complete oligomerization as well.

[0666] These results suggest that modification of PChM by addition of 6 histidine residues strengthen the non covalent PChM : N interaction, and that PChM is effectively displaced by RNA to form oligomers.

[0667] In the case of NVSV the effect of the HisTag location is cualitatively similar, albeit quantitatively larger (Figure 11). Cleavage of PchM in PchM-N-X_HT constructs does not trigger selfassembly. Addition of RNA elicits oligomerization in an RNA concentration-dependent fashion, reaching 81% MPs at 10:1 RNA:protein ratio. Likewise, cleavage of HT_PChM does not trigger self-assembly, but addition of RNA induces oligomerization in a concentration-dependentfashion, yielding 19% oligomers at 10:1 RNA:protein ratios (Figure 11). Figure 12 represents a summary of the effect of the His-Tag position on oligomerization on the P-N-X constructs for different nucleoproteins

[0668] Conclusions from sections 2.5 and 2.6;

[0669] 1. Point mutations of the P sequence alter its chaperone function

[0670] 2. Some modifications facilitate the formation of MPs with lower amounts of RNA

[0671] 3. Fusion of the His-tag to the N-terminus of PChM may modulate the oligomerization process, typically lowering the degree of self-assembly upon cleavage with TEV. In all cases the addition of RNA triggers oligomerization, with oligomerization yields ranging from ca. 20 to 100%.

[0672] 2.7. MODIFICATIONS IN THE N PROTEIN

[0673] The N proteins of the Mononegavirales are well defined in existing literature. Nucleoproteins consist, at least, of two well-folded globular domains separated by a hinge where the RNA is bound, and disordered arms at the N- and C- termini. These disordered regions are important for oligomerization, and upon self-assembly they establish contacts with neighbouring protomers in the MPs, both the previous and the following one, in a similar fashion to an interlocking system.

[0674] Therefore, for the fusion protein of the present invention, the N protein preferably has at least one oligomerization domain and at least one RNA binding domain, so that RNA-particles of the invention can be generated upon cleavage of the PchM domain.

[0675] We explored 4 different constructs, incorporating versions of NEW ranging from i) 1-450 (v1), ii) 1-430 (v2), iii) 1-389 (v3) and 1-367 (v4) (Figure 13 and 14) in Po -N-X constructs. The rationale was that trimming of the disordered C-terminus of Afebvwould minimize spontaneous cleavage, but could simultaneously promote undesired steric clashes involving the cargoes thus hampering oligomerization. All variants were expressed and purified, obtaining soluble monomeric protein. Thus, these proteins were screened for i) their ability to self-assemble upon TEV cleavage and addition of RNA and ii) undesired cargo cleavage.

[0676] The constructs using NEbv.v32 and NEW.V exhibit negligible cargo cleavage, while small amounts of cleavage are observed for NEW.VI and NEW.VZ- Constructs NEW.VI and NEW.VZ are able to oligomerize almost completely upon addition of RNA, while NEbv.v3 oligomerizes partially and NEbv.v4 is unable to self-assemble (Figure 15). Thus, this screening process has yielded an optimized soluble monomeric form, NEbv.v2, that minimizes cargo cleavage while retaining the self-assembling properties of N.

[0677] Conclusions:1. The A / - and C- terminal arms of most Ns are disordered and some are susceptible to spontaneous cleavage.

[0678] 2. The A / - and C- terminal arms of the nucleoproteins participate in inter-protomer contacts to form the RLPs.

[0679] 3. We have engineered a variant of EbV N that simultaneously retains optimal self¬ assembly properties while minimizing cargo cleavage.

[0680] 2.8. ADDITION OF REDUCING AGENTS FAVOR MONOMER FORMATION AND PREVENTS SPURIOUS OLIGOMERIZATION DURING PROTEIN PURIFICATION

[0681] The use of reducing agents abolishes intra- and inter-molecular S-S bonding, favoring monomer formation and preventing spurious oligomerization during protein purification The sequence of PchM from VSV encompasses cysteine residues that can generate intercatenary disulphide bonds between P-N-X protomers that can subsequently prompt unwanted oligomerization during protein expression and purification. The addition of reducing agents (B-Mercaptoethanol) during the purification steps is critical for obtaining monomeric species to generate a library of functionalities (Figure 16)

[0682] Conclusions: Addition of reductive agents during the purification step favor the obtention of monomeric species, which is a key step to produce MPs with high efficency

[0683] 2.9. THE PRESENCE OF CARGOES FAVOR THE FORMATION OF HOMOGENEOUS, MONODISPERSED MPS, PREVENTING THE FORMATION OF OTHER INTERMEDIATE OR EXTENDED ASSEMBLIES

[0684] In the context of viral infection, the nucleoproteins are able to self-assemble into rod-like structures, with a helical symmetry involving a characteristic pitch angle (Figure 17). Thus the rod-like structures arise as a consequence of the propagation of self-assembly in the Z-axis, resulting, for each N protomer, in establishing side-by-side contacts with the previous and following first neighbors (NN-I and NN+I) but also establishing axial contacts with N molecules below and above.

[0685] Expression and purification of N results in the formation of multiple species, differing in their oligomeric state including monomers and different oligomerization states including dimers and trimers (Figure 18, left panel) that are likely established by oligomerization on the Z axis The presence of cargoes abolish the axial interactions, with the cargoes themselves acting as “blockers” of possible oligomerization on the Z axis (Figure 18, right panel). Thus, the cargoes limit the total size of the MPs, preventing the formation of non-soluble rod like particles that are formed by oligomerization of N in vivo, thus favouring the formation of homogeneous particles.Conclusions: The presence of cargoes favours the formation of homogeneous MPs in detriment of low-order oligomers or higher order insoluble rod-like structures, by preventing the formation of contacts in the Z axis.

[0686] 2.10. CARGOS CAN BE PLACED IN ANY TERMINI OF THE N PROTEIN

[0687] Cargoes can be fused either to the N- or C- termini of N without hindering the self-assembly capabilities of N. For example (Figure 19), N from VSV and RSV are able to form MPs either from N-XorX-N, as analysed by Size Exclusion Chromatography.

[0688] Conclusions: cargos can be placed in any termini of the N protein.

[0689] 2.11. FORMATION OF SINGLE-CARGO MULTIMERIC PARTICLES (SMPS)

[0690] We have generated a library of functionalities in the form of soluble, monomeric P-N-X orX- N-P. First, we explored the formation of monodisperse homogeneous, single-cargo multimeric proteins (SMPs) by cleavage of PchM and addition of RNA.

[0691] . We triggered homooligomerization of P-N-mCFP using Nvsv, obtaining MPs (WMPs) as observed by SEC, DLS and TEM (Figure 20)

[0692]

[0693] . We triggered homooligomerization of P-N-GFP using NEW (Figure 21) forming RLPs as observed by SEC, DLS and TEM.

[0694] Conclusions: We have attained control of the self-assembly properties of N, being able to keep it monomeric or in an oligomeric state upon removal of PchM and addition of RNA

[0695] 2.12. SELF-ASSEMBLY (WITHOUT RNA) CAN BE REVERSIBLE FOR SOME NUCLEOPROTEINS

[0696] In the case of VSV, removal of Pcwwdoes not elicit significant self-assembly, and it only occurs after addition of RNA (See Section 2.5 and figure 9).

[0697] In the case of EbV, removal of PchM results in reversible self-assembly that is controlled by the protein concentration (Figure 22). The self-assembly can be reversed by protein dilution. Only in the case of addition of RNA, the oligomerization process becomes irreversible.

[0698] Conclusions:

[0699] 1. Some nucleoproteins (EbV) can reversibly self-assemble by removal of PchM even in the absence of RNA. In this case, the oligomerization process is reversible and controlled by protein concentration.

[0700] 2. Other nucleoproteins only self-assemble irreversibly upon addition of RNA (VSV)2.13. FORMATION OF MULTIVALENT, MULTIFUNCTIONAL, HETERO-OLIGOMERIC MPS Having explored and engineering the self-assembling capabilities of the P-N-X and X-N-P constructs to form multivalent monodisperse homooligomeric MPs, we explored the formation heterooligomeric or multicargo MPs (MMPs) by mixing of P-N-X constructs with different cargoes (Xi, X2 X3, etc), TEV cleavage and addition of RNA.

[0701] • vsv

[0702] Example 1: Dual cargo heterooligomers We combined P-N-mCherry and P-N-MBP in an eguimolar 1:1 7.5 pM concentration. After TEV cleavage and addition and 150pM tRNA the samples were incubated ON. The resulting product was subjected to amylose affinity chromatography. The flow-through fraction was collected, and after multiple washing steps, the bound molecules were eluted by addition of maltose. The eluted fraction (indicating presence of MBP) was analyzed in a spectrofluorometer. Upon excitation at 587 nm, corresponding to mCherry’s excitation maximum, a typical mCherry fluorescence spectrum is obtained (Figure 23)

[0703] Example 2: Dual cargo heterooligomers. We combined P-N-CFP and P-N-YFP in an eguimolar 1:1 7.5 uM concentration. After TEV cleavage and 75 uM tRNA addition, samples were incubated overnight (ON) at room temperature (RT). The resulting MPs were analyzed by SEC, fluorescence and TEM. In addition, the colocalization of the different cargoes in the same MMPs was determined by FRET, with excitation at 449 nm and emission spectra from 460 to 600 nm (Figure 24) Fig. 34 shows similar results in forming RLPs but with different cargoes (triple and double cargoes).

[0704] Example 3: Triple cargo heterooligomers: We combined P-N-MBP, P-N-CFP and P-N-YFP in an eguimolar 1:1:1 15 pM concentration. After TEV cleavage and addition and 150pM tRNA the samples were incubated ON at RT. The resulting product was subjected to amylose affinity chromatography. The flow-through fraction was collected, and after multiple washing steps, the bound molecules were eluted by addition of maltose. The eluted fraction (indicating presence of MBP) was analyzed in a spectrofluorometer. FRET between CFP and YFP was observed, thus confirming the presence of triple functionality in the same MPs (Figure 25). Fig.

[0705] 34 shows similar results in forming RLPs but with different cargoes (triple and double cargoes).

[0706] • EbV

[0707] Example 4. We combined PCHM-N-MBP and PchM-N-sfGFP in 1:1 15 pM concentration. After TEV cleavage and addition of specific 18nt RNA (seguence: CGGACACACAAAAAGAAA, corresponding to the 3’ genome of EbV) the samples were incubated ON at room temperature. The resulting product was subjected to amylose affinity chromatography. The flow-through fraction was collected, and after multiple washing steps, the bound molecules were eluted byaddition of maltose. The eluted fraction (indicating presence of MBP) was analyzed by DLS, Absorption and emission spectroscopy and transmission electron microscopy (Figure 26). Absorption maximum at 484 nm corresponds to GFP. Upon excitation at this wavelength, the GFP emission spectrum is obtained (Figure 26). Fig 33, 41, 43 and 46 show more examples with different RNA and or cargoes (including antigens and adjuvants).

[0708] Example 5. Triple cargo heterooligomers We combined PchM-N-MBP, PchM-N-sfGFP and PchM-N-mCherry in 1:2:1 proportions at a final concentration of 37 uM. After TEV cleavage and addition of 200 pM tRNA, the samples were incubated ON at room temperature in buffer 20 mM sodium shosphate and 50 mM NaCI. The resulting product was first purified by size exclusion chromatography (SEC) to isolate the MPs, followed by an amylose affinity chromatography (Figure 27). The flow-through fraction was collected, and after 5 washing steps, the bound molecules were eluted by addition of maltose. The eluted fraction (indicating presence of MBP) was analyzed by DLS, absorption and emission spectroscopy (Figure 27). The absorption spectrum presents maxima at 484 nm and 587 nm, unveiling the presence of both GFP and mCherry. Emission spectra of GFP and mCherry were obtained by excitation of the samples at 484 nm and 587 nm, respectively (Figure 27). Additional examples with different cargoes are shown in Figure 33

[0709] Example 6. Formation of RLPs from different backbones / lengths. We combined different versions of EbV (6xHis-P-N-X and P-N-X-6xHis), with different backbone lengths (N1-430 and N1-450) to test whether different constructs are able to self-assemble. The experiments show that these combinations also lead to correct self assembly (Figure 28).

[0710] • MeV

[0711] Example 7: Dual cargo heterooligomers. We combined P-N-CFP and P-N-YFP in an eguimolar 1:1 7.5 pM concentration. After TEV cleavage and 75 pM tRNA addition, samples were incubated ON at RT. The resulting MPs were analyzed by SEC, fluorescence and DLS. In addition, the colocalization of the different cargoes in the same MMPs was determined by FRET, with excitation at 420 nm and emission spectra from 460 to 600 nm (Figure 31).

[0712] Conclusions:

[0713] 1. It is possible to generate homogeneous monodisperse multifunctional MPs by combination of modules from a library of functionalities

[0714] 2. All cargoes remain functional in the MPs as determined by FRET, Amylose binding and UVvis / Fluorescence spectroscopy

[0715] 3. The cargoes can be easily combined

[0716] 4. Different topologies can be obtained depending on the scaffold and RNA5. The method described herein allows for the formation of hetero-oligomers (RNA- nanoparticles made of monomers having different cargoes) with a desired stoichiometry. For example, Fig. 41 shows that when different monomers are mixed in different proportions, the resulting RNA-nanoparticles maintain the stoichiometry of the monomers mixing.

[0717] 2.14. SPECIFIC RNA SEQUENCES CAN BE USED TO OBTAIN MPS WITH HOMOGENEOUS RNA COMPOSITION

[0718] Non-modular obtention of oligomeric particles by purification of rings and other oligomers from vectors that do not include the PchM module result in oligomeric particles that uptake nonspecific RNA from the host expression system. Our modular approach enables the generation of oligomeric particles containing different RNAs, either from an unknown mixture ora precisely formulated RNA composition (PolyA-RNAs, RNAs from specific viral genomic sequences). Complex heterogenous RNA mixtures that can be used to trigger the formation of MPs containing RNAs are baker yeast tRNA or yeast Torula RNA. The former corresponds to a mixture or purified yeast transfer RNAs and the latter to a heterogeneous mixture of yeast purified RNA molecules ranging from 10 to 120 nucleotides. In the absence of RNA, TEV treated PCHM-NEW-X monomers are in equilibrium with MPs in a protein concentration dependent manner (Figure 22). The addition of 25 pM, 50 pM or 100 pM of either tRNA or torula RNA to TEV treated PCHM-NEW-X completely displaces the equilibrium towards MP assembly, loaded with a complex mixture of RNAs (Figure 29 C and D). In the absence of RNA, TEV cleaved PchM-Nvsv-X behaves as a monomeric species. Gradual incorporation of both, tRNA or torula RNAs, shifts gradually the equilibrium towards the formation of MPs loaded with RNAs (Figure 29 A and B).

[0719] Multimeric particles can also be assembled with a precisely defined sequence and composition of RNAs. For instance, PchM-NEbv-flagellin monomer subject to TEV proteolysis and further incubation with in-vitro transcribed 18-mer or 54-mer polyA-RNAs binds RNA molecules and self-assembly into multimeric particles as assessed by SEC (Figure 30 A, B and C). Moreover, PchM-NEbv-MBP with or without TEV protease treatment, binds a FITC labeled 9-mer polyA-RNA as assessed by fluorescence anisotropy titration studies (Fig 30 D). Uncoupling of RNA binding titration curves revealed that undigested PchM-NEbv-MBP binds 9-mer poly RNA with lower apparent affinity compared to TEV treated PchM-NEbv-MBP, as expected. When PchM- NEbv-sfGFP and PchM-NEbv-mCherry monomers are subjected to TEV treatment, a 18-mer RNA corresponding to the leader sequence of the Ebola virus RNA genome triggers the formation of ring-like multimeric particles (Figure 30 E and F). Altogether, our versatile multimeric particles can be loaded with diverse RNA cargoes, ranging from non-specific towell-defined molecules. This approach enables the formation of precisely controlled protein-RNA complexes with tailored functionalities.

[0720] The advantage of the system’s versatility in terms of RNA binding, is that for some applications where the RNA composition is not important, low-cost nonspecific RNAs can be used. In other applications where a defined RNA sequence are needed, specific RNA sequences can be employed. This is of importance, for example, regarding activation of the innate immune response in the presence of foraneous RNA sequences (by receptors such as TLR7 and TLR8), that are not activated in the presence of polyA but can be stimulated in the presence of specific sequences incorporating different sequence patterns of C, G and II nts.

[0721] In vitro synthesized RNAs, off-the-shelf RNAs, complex RNA mixtures, and short specific leader sequences each trigger efficient PChM-N-X oligomerization after TEV treatment, demonstrating that assembly is driven by the presence, length, or structural features of RNA rather than a specific nucleotide sequence. This sequence-agnostic behavior supports broad applicability of the platform for diverse RNA triggers and strengthens claims directed to RNA-induced self-assembly independent of particular RNA sequences (Figure 32)

[0722] Conclusions:

[0723] 1. Following TEV cleavage of PchM-N-X monomers, multimeric particles can be loaded with a mixture of RNA molecules, such as yeast tRNA or yeast torula RNAs,

[0724] 2. Short-length in-vitro transcribed poly-A RNA sequences bind TEV-treated PCHM-NEBV-X molecules triggering the formation of multimeric particles

[0725] 3. Specific RNA sequences corresponding to the 3'leader sequence of the RNA viral genome bind TEV-treated PchM-NEBv-X molecules triggering the formation of ring-like multimeric particles.

[0726] 4. Multimeric particles enable incorporation of diverse non-specific or specific RNA molecules, leading to the formation of well-defined RNA-protein complexes tailored for various applications.

[0727] EXAMPLE 2:

[0728] Proteins used in this example:

[0729] For EbV: SEQ ID NO: 90, 91, 10

[0730] For VSV: SEQ ID NO: 25, 26, 27, 92, 93

[0731] For MeV: SEQ ID NO: 34, 35, 36All nucleoproteins tested, corresponding to representatives of different families within the order Mononegavirales, enable the generation of stable fusion monomers of the ChM-N-X format which, upon treatment with TEV protease and RNA, assemble into RLPs that are either homo-or hetero-oligomeric, i.e., containing a single type of cargo per RLP or multiple combined cargos. Depending on the nucleoprotein employed, the size of the resulting RLPs — and therefore the number of protomers and cargos per RLP — varies.

[0732] To illustrate this, three examples based on the nucleoproteins of Ebolavirus (EbV), Vesicular stomatitis Indiana virus (VSV), and Measles morbillivirus (MeV) are presented. In all three cases, three different cargos were used: cyan fluorescent protein (mCFP), maltose-binding protein (MBP), and yellow fluorescent protein (mYFP). In each case, homooligomers were generated, as well as heterooligomers containing equal proportions of mCFP and mYFP as cargos (constructs designated N-2X) and heterooligomers containing the three cargos (mCFP, mYFP, and MBP) in similar proportions (constructs designated N-3X).

[0733] Figures 33, 34, and 35 show the production and characterization of N-2X and N-3X RLPs for the three nucleoproteins. In all cases, TEV treatment proteolytically cleaves the ChM-N junction; however, the chaperone remains non-covalently bound to the nucleoprotein, thereby preventing oligomerization. Upon addition of RNA, size-exclusion chromatography (SEC) analysis demonstrates the formation of higher-molecular-weight oligomeric species.

[0734] In the case of the EbV and MeV nucleoproteins, a single predominant oligomeric SEC fraction is obtained with yields on the order of 98%. Dynamic light scattering (DLS) analysis of these fractions reveals relatively monodisperse structures with hydrodynamic radii of 34 ± 10 nm and 16 ± 4 nm for the EbV and MeV constructs, respectively. Transmission electron microscopy (TEM) analysis shows that the EbV oligomers are indeed RLPs, with an external radius of 34 ± 3 nm and an internal radius of 21 ± 3 nm (Figure 33). Modeling of these ring structures using AlphaFold 3.0 indicates that these RLPs contain 24 protomers (and therefore 24 cargos) per RLP.

[0735] Spectroscopic analysis (UV-Vis absorption and fluorescence) further demonstrates that the three cargo types were incorporated in 1:1 or 1:1:1 proportions for the N-2X and N-3X constructs, respectively, consistent with the stoichiometry of the original mixture. Moreover, all constructs exhibit the FRET phenomenon, as evidenced by the appearance of mYFP chromophore fluorescence upon excitation at a wavelength absorbed exclusively by mCFP, indicating that both molecules are present within the RLPs at distances compatible with FRET. The presence of MBP in the N-3X constructs decreases FRET efficiency, suggesting that MBP dilutes the other two cargos, thereby increasing the average donor-acceptor distance of the FRET pair, consistent with a homogeneous distribution of cargos within the RLPs.A similar scenario is observed for the MeV-based N-2X and N-3X constructs (Figure 34), except that in this case the RLPs exhibit hydrodynamic radii of 16 ± 4 nm and external and internal radii of 19 ± 1 nm and 7 ± 1 nm, respectively, as determined by TEM. According to AlphaFold 3.0 modeling, these structures correspond to 12 protomers (and therefore 12 cargos) per RLP. UV-Vis, fluorescence, and FRET spectroscopy analyses likewise indicate that the stoichiometry of the RLPs reflects the composition of the mixture used for their assembly.

[0736] For the VSV-based constructs (Figure 35), oligomerization induced by TEV treatment and RNA addition results in two chromatographic peaks of different sizes. TEM analysis of these peaks indicates that the smaller fraction corresponds to worm-like particles, whereas the larger fraction corresponds to RLPs with an external radius of 31 ± 3 nm, an internal radius of 17 ± 3 nm, and a hydrodynamic radius (DLS) of 39 ± 11 nm. Modeling indicates that these structures consist of 39 protomers (and therefore cargos) per RLP. As in the previous cases, spectroscopic characterization of these RLPs is consistent with incorporation of the different cargos in similar proportions.

[0737] An additional relevant aspect is that, for all constructs, the cargos retain their functionality, as demonstrated by the fluorescent emission of mCFP and mYFP cargos and by the affinity of the incorporated MBP cargo for amylose resin columns.

[0738] EXAMPLE 3:

[0739] Proteins used in this example: SEQ ID NO: 46.

[0740] The platform has been tested for safety in a murine model. Forthat, cargo-free NEW RLPs were inoculated in mice using different administration routes: intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) and intranasal (IN). Three doses of up to 100pg RLPs showed no adverse clinical effects in vivo, as determined by monitoring commonly used clinical parameters such as weight (Figure 36).

[0741] EXAMPLE 4:

[0742] Protein used in this example: SEQ ID NO: 46 and 52.

[0743] We have analysed 13 human blood samples for antibodies against RLPs NEbV and NVSV (S1, and S2, respectively). As shown in Figure 37, antibody titers for the two RLPs tested are similar to those obtained for the control ovalbumin, indicating that there is no baseline serology in humans.

[0744] EXAMPLE 5:

[0745] RLP immunogenicity was assessed in vitro by determining dendritic cell activation quantified by CD80 and CD86 levels after treatment with a negative control, a LPS positive control, and3 different formulations: cargo-free RLPs (NEW SEQ ID NO: 1, PChM-NEbV-flagellin monomers SEQ ID NO: 15 and homooligomeric RLPs of NEbV-flagellin). The results (Figure 38) show that the cargo free RLP are immunogenic, and that this immunogenicity is enhanced for constructs including the protein adjuvant flagellin as cargo.

[0746] EXAMPLE 6:

[0747] Using cyan fluorescent protein as model fluorescent antigen, we observed by flow cytometry that the uptake of PChM-EbV-mCFP monomers is significantly lower than that of NEbV-mCFP homooligomeric RLPs (Figure 39), thus showing that antigen uptake is enhanced by the dense display achieved in the RLP constructs.

[0748] EXAMPLE 7:

[0749] To demonstrate the potential of the platform for formulating vaccines, we have created a minimal library of PChM-NEbv-X monomers, where the cargoes X were antigens from RSV (Site II from the Fusion (FSI I) protein of RSV and central conserved domain of the attachment glycoprotein (Gpep) an antigen for respiratory syncytial virus (RSV) and the protein adjuvant flagellin. With these monomers we generated homooligomeric NEbV-FSIl, NEbV-Gpep and NEbV-Flage RLPs, dual cargo heterooligomeric NEbV-FSIl / Flage and NEbV-FSIl / Gpep, and triple cargo heterooligomeric NEbV-FSIl / Gpep / Flagellin (Figures 40, 41, 42, 43). The RLP composition can be controlled by the mixing stoichiometry, as observed for different proportions of FSII and flagellin (Figure 41). Flagellin is active when incorporated onto the RLPs (Figure 41)

[0750] Different groups of mice were subcutaneously administered with two doses of RLPs containing 5 pg of FSII or 2 pg of Gpep (total mass is adjusted to attain equal molarity of antigen cargo). The second dose was administered 21 days after the first one. The inoculated formulations are: i) negative control, ii) free antigen (MBP-FSII fusion protein), iii) free antigen adjuvated with alumina adjuvant, iv) NEbV-FSIl RLPs v) NEbV-FSIl RLPs adjuvated with alumina, vi) NEbV-Gpep RLPs, vii NEbV_Gpep RLPs adjuvated with alumina, viii) dual cargo NEbV-FSI l / Flagelin, ix) dual cargo NEbV-FSIl / Gpep RLPs RLPs, and x) dual cargo NEbV-Gpep / flagellin. The humoral responses show that:

[0751] 1) The platform is highly immunogenic (compared with soluble monomeric antigens) and this inherent immunogenicity can be further boosted by the use of adjuvants (alum and / or innate immunity activators) (Figure 42)

[0752] 2) The platform elicits long-lasting responses antibody responses in mice (Figure 42)3) Combining antigens and adjuvants (flagellin, a TLR5 agonist) onto the same RLP in heterooligomeric RLPs (using FSII or Gpep as antigens) results in higher antibody titers than those obtained with alum as adjuvant (Figure 43)

[0753] 4) Combining antigens onto the same RLP results in synergistic immunological effects, observed in the boost antibody titers against RSV F when Gpep is also present in the RLPs (Gpep / FSIl heterooligomers). (Figure 43C)

[0754] EXAMPLE 8:

[0755] We have incorporated two different cargoes, SpyCatcher and a monomeric streptavidin, that enable the conjugation of additional cargoes after monomer expression and purification. This enables the creation of PChM-NEbV-X monomers, where X is SpyCatcher (SC) or monomeric streptavidin (mSA24K1D), that allow for covalent conjugation of cargoes incorporating the SpyTag (st) sequence, or the non-covalent incorporation of biotinylated (bt) cargoes, respectively.

[0756] Functionalizable SpyCatcher monomers are compatible with our platform technology.

[0757] Incorporation of SpyCatcher (SC) as a cargo, resulting in PChM-NEbV-SC monomers, are able to oligomerize upon addition of TEV and RNA (Figure 44).

[0758] PChM-NEbV-SC + stX conjugation: PChM-NEbV-SC solution is mixed with the cargo carrying the SpyTag sequence (stX) in a 1:1.5 or 1:2 proportion (excess of stX). The mixture is incubated for 2 hours at room temperature or at 4°C overnight. This results in the creation of PChM-NebV-SC-stX monomers (Figure 45). These can be readily used for oligomerization without removing the excess of stX, since they do not interfere with the oligomerization process and are eliminated during the SEC when the oligomer is purified. This procedure can be used to form either homooligomeric or heterooligomeric RLPs (Figures 46, 47). Cargo colocalization can be assessed by FRET (Figure 47).

[0759] Functionalizable monomeric streptavidin monomers are compatible with our platform technology. Incorporation of mSA34K1D as cargo results in PChM-NEbV-mSA24K1D constructs that are able to self-assemble in the presence of TEV and RNA (Figure 48A). The mSA24K1D is functional, as it is able to bind biotinylated FITC (btFITC), and the affinity can be determined by fluorescence anisotropy (Figure 48C). These modules can be conjugated with biotinylated cargoes (btX), resulting in PChM-NEbV-mSA24K1 D.btX adducts that are competent for self-assembling by addition of TEV and RNA. This procedure can be used to form oligomeric RLPs including biotinylated cargoes (Figure 49).

Claims

CLAIMS1. A homogeneous population of RNA-nanoparticles, wherein each RNA-nanoparticle comprises:i) five or more monomers that interact with each other thereby forming a nanoparticle, wherein each monomer comprises a first domain and a second domain that are bound by a linker, wherein:1) the first domain comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and2) the second domain comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, spectroscopic or imaging activity to the nanoparticle, andii) at least one RNA molecule that is bound to the RNA binding domain of each of the N proteins of the first domain of i.1), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA- nanoparticles,wherein the homogeneous population of RNA-nanoparticles is obtained or obtainable by a method comprising the steps of:1) providing a plurality of monomeric fusion proteins with identical or different cargoes, each of them comprising:a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b), b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and c) a domain that comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, spectroscopic or imaging activity to the nanoparticle,wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b);and cleaving said plurality of fusion proteins at the cleavage site to remove domain a), yielding cleaved fusion proteins competent for RNA-mediated oligomerization, and2) simultaneously or subsequently to step 1), adding one or more RNA molecules with identical or substantially identical nucleotide sequence, so that the cleaved fusion proteins obtained in step 1) oligomerize and bind to said RNA molecules, thereby obtaining a homogeneous population of RNA-nanoparticles.

2. The homogeneous population of RNA-nanoparticles according to claim 1, wherein the cargo is a polypeptide comprising more than 10 amino acids, and preferably less than 1000 amino acids, or wherein the cargo is between 1 to 120 Kda.

3. The homogeneous population of RNA-nanoparticles according to claims 1 or 2, wherein the plurality of fusion proteins provided in step 1) comprises at least two fusion proteins comprising different cargo moieties.

4. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 3, wherein the oligomerization inhibitor comprised in domain a) of the monomeric fusion provided and cleaved in step 1) is the chaperone module of the phosphoprotein (P) from Mononegavirales, or a fragment thereof.

5. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 4, wherein the chaperone module of the phosphoproteins (P) comprised in domain a of the monomeric fusion provided and cleaved in step 1) comprises at least:- amino acids 1-51 from P protein of Ebola virus (EbV),- amino acids 1-60 from P protein of Vesicular Stomatitis Virus (VSV),- amino acids 1-50 from P protein of Measles morbillivirus (MeV),- amino acids 1-85 from P protein of Respiratory syncytial virus (RSV),- amino acids 2-50 from P protein of Human Cuevavirus (CuV), or- amino acids 1-40 from P protein of Human metapneumovirus (HMPV).

6. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 5, wherein the Nucleoprotein (N) comprised in domain b) of the monomeric fusion provided and cleaved in step 1) comprises at least:- amino acids 1-430 from the N protein of Ebola virus (EbV),- the N protein of Vesicular Stomatitis Virus (VSV),- amino acids 1-405 from the N protein of Measles morbillivirus (MeV),- the N protein of Respiratory syncytial virus (RSV),- amino acids 2-450 from the N protein of Human Cuevavirus (CuV),- the N protein of Human metapneumovirus (HMPV), or- any fragment of said N proteins with the proviso that the fragment comprises at least one oligomerization domain and at least one RNA binding domain.

7. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 6, wherein, in the monomeric fusion provided and cleaved in step 1):- domain b) of comprises at least amino acids 1-430, from the N protein of EbV, and domain a) comprises at least amino acids 1-51 of the phosphoprotein (P) of EbV, - domain b) comprises the N protein of VSV, and domain a) comprises at least amino acids 1-60 from P protein of VSV,- domain b) comprises amino acids 1-450 from the N protein of MeV, and domain a) comprises at least amino acids 1-50 from P protein of MeV,- domain b) comprises the N protein of RSV, and domain a) comprises at least amino acids 1-85 from P protein of RSV,- domain b) comprises amino acids 2-405 from the N protein of CuV, and domain a) comprises at least amino acids 2-50 from P protein of CuV, or- domain b) comprises the N protein of HMPV, and domain a) comprises at least amino acids 1-40 from P protein of HMPV.

8. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 7, wherein, in the monomeric fusion provided and cleaved in step 1):- domain b) comprises SEQ ID NO 46: EbV_Ni-43o, and domain a) comprises SEQ ID NO 54: EbV_VP35i-si,- domain b) comprises SEQ ID NO 50: MeV_Ni-4os, and domain a) comprises SEQ ID NO 55: MeV_Pi.5o,- domain b) comprises SEQ ID NO 49: RSV_N, and domain a) comprises SEQ ID NO 78: RSV PchM 1-85,- domain b) comprises SEQ ID NO 51: HMPV_N, and domain a) comprises SEQ ID NO 56: HMPV_PI.4O,- domain b) comprises SEQ ID NO 53: CUV_NI-4SO, and domain a) comprises SEQ ID NO 61 : CUV_VP35I.5O, or- domain b) comprises SEQ ID NO 52: VSV_N, and domain a) comprises SEQ ID NO 57: VSV_Pi.6o, SEQ ID NO 58: VSV_P 1-60J24A-I27A, SEQ ID NO 59: VSV_P 1-60_ V21A- I27A, or SEQ ID NO 60: VSV_Pi-6o_Yi4A-i27A.

9. The homogeneous population of RNA-nanoparticles according to any of claims 1 to 8, wherein the monomeric fusion provided and cleaved in step 1) comprises or consists of any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 90-94.

10. The homogeneous population of RNA-nanoparticles according to any one of claims 1 to 9, for use in the fields of diagnostic methods, medicine, biocatalysis, spectroscopic methods, detection methods, theragnostics, biotechnology, bionanotechnology, or imaging.

11. A homogeneous population of RNA-nanoparticles, wherein each of the RNA-nanoparticles is characterized by comprising:i) five or more monomers that interact with each other thereby forming a nanoparticle, wherein each monomer comprises a first domain and a second domain that are bound by a linker, wherein:1) the first domain comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, and2) the second domain comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, spectroscopic or imaging activity to the nanoparticle, andii) at least one RNA molecule that is bound to the RNA binding domain of each of the N proteins of the first domain of i.1), wherein the sequence of RNA is identical or substantially identical in each of the RNA-nanoparticles of the population of RNA- nanoparticles.

12. The homogeneous population of RNA-nanoparticles according to claim 11, wherein each RNA-nanoparticle comprises at least two monomers comprising different cargo moieties.

13. The homogeneous population of RNA-nanoparticles according to claims 11 or 12, wherein the Nucleoprotein (N) comprised in the first domain comprises at least:- amino acids 1-430 from the N protein of Ebola virus (EbV),- the N protein of Vesicular Stomatitis Virus (VSV),- amino acids 1-405 from the N protein of Measles morbillivirus (MeV),- the N protein of Respiratory syncytial virus (RSV),- amino acids 2-450 from the N protein of Human Cuevavirus (CuV),- the N protein of Human metapneumovirus (51), or- any fragment of said N proteins with the proviso that the fragment comprises at least one oligomerization domain and at least one RNA binding domain.

14. The homogeneous population of RNA-nanoparticles according to any one of claims 11 to 13, wherein:- the first domain is placed in the N-terminal of the second domain, or- the first domain is placed in the C-terminal of the second domain.

15. The homogeneous population of RNA-nanoparticles according to any one of claims 11 to 14, for use in the fields of diagnostic methods, medicine, biocatalysis, spectroscopic methods, detection methods, theragnostics, biotechnology, bionanotechnology, or imaging.

16. A method for producing an homogeneous population of RNA-nanoparticles, the method comprising the steps of:1) providing one or more monomeric fusion proteins with identical or different cargoes, each of them comprising:a) a domain that comprises an oligomerization inhibitor that is capable of preventing the oligomerization of the Nucleoprotein (N) defined in b), b) a domain that comprises the Nucleoprotein (N) from the order of Mononegavirales, or a fragment of said nucleoprotein comprising at least one oligomerization domain and one RNA binding domain, andc) a domain that comprises at least one cargo of interest, wherein the cargo confers a biological, diagnostic, therapeutic, catalytic, targeting, or imaging activity to the nanoparticle,wherein domains a) and b) and domains b) and c) are bound by a linker, wherein domain b) is placed in between domains a) and c), and wherein a cleavage site is placed between domains a) and b);2) cleaving the plurality of fusion proteins at the cleavage site to remove domain (a) from each fusion protein,3) adding one or more RNA molecules having a desired sequence, thereby triggering the spontaneous oligomerization to form RNA nanoparticles, and4) purifying the RNA nanoparticles,wherein steps 2) and 3) can be carried out simultaneously or sequentially, and, when carried out sequentially, optionally further comprise an intermediate purification after step 2); and wherein at least step 3, preferably all steps, are performed in the absence of RNA molecules other than the RNA added in step 3).

17. The method of claim 16, wherein the method is carried out sequentially by performing the steps in numerical order and wherein, optionally, a purification step is included between step 2) and 3).

18. The method of claim 16, wherein steps 2) and 3) are performed simultaneously.

19. The method of any one of claims 16 to 18, wherein the method does not comprise any step of using the cargo as a purification tag.