Fusion-modified virus-like particles of cmv

Abstract

The present invention relates to a modified virus-like particle (VLP) of Cucumber Mosaic Virus (CMV) comprising at least one fusion protein, wherein the at least one fusion protein comprises or preferably consists of b) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of (iii) a CMV polypeptide, wherein the CMV polypeptide comprises a coat protein of CMV; and (iv) an antigen polypeptide, wherein the antigen polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigen polypeptide is between amino acid residues corresponding to position 84 and position 85 of SEQ ID NO: 62 of the CMV polypeptide; and (iii) a T helper cell epitope, wherein the T helper cell epitope replaces an N-terminal region of the CMV polypeptide, and wherein preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201980084939.1, filed on December 20, 2019, entitled "Virus-like particles of CMV modified by fusion".

[0002] This invention relates to modified virus-like particles of the plant virus cucumber mosaic virus (CMV), and more specifically to modified VLPs of CMV comprising a chimeric CMV polypeptide, said chimeric CMV polypeptide including an antigenic polypeptide inserted into the CMV polypeptide at a specific site, and further including a Th cell epitope replacing the N-terminal region of said CMV polypeptide. Furthermore, these modified VLPs preferably serve as vaccine platforms for generating an immune response, particularly an antibody response, against said antigenic polypeptide fused to said CMV polypeptide. Additionally, this invention relates to modified virus-like particles, said modified virus-like particles being mosaic virus-like particles, comprising said chimeric CMV polypeptide containing the fused antigenic polypeptide and additional CMV proteins excluding said antigenic polypeptide. Background Technology

[0003] Plant viruses and their derived virus-like particles (VLPs) have recently attracted considerable attention, primarily due to plants' potential as an economical and rapid alternative platform for producing VLP vaccines. This is because of their ability to provide unique post-translational modifications, cost-effectiveness, beneficial safety profiles, rapid production, and scalability (Chen Q and Lai H, Human Vaccines & Immunotherapeutics (2013) 9:26-49; Zeltins A, Molecular Biotechnol (2013) 53:92-107). Furthermore, plant virus VLPs, in particular, are considered as vector structures on their surfaces that present diverse antigens, designed to elicit the observed strong immune responses similar to those observed with infectious mammalian viruses (Balke I et al., Adv. Drug Deliv. Rev. (2018) https: / / doi.org / 10.1016 / j.addr.2018.08.007).

[0004] Cucumber mosaic virus (CMV, Broccoli mosaic virus family) BromoviridaeThe Cucumber Mosaic Virus (CMP) family, genus Cucumber Mosaic Virus (CMP) is a linear, isoscopic plant virus with a wide host range. The viral genome consists of three single-stranded RNAs (RNA1, RNA2, and RNA3), with the capsid protein (CP) gene present in both genomic RNA3 (approximately 2200 nt) and subgenomic RNA4 (approximately 1000 nt). The capsid comprises 180 copies of a single protein species, approximately 25 kDa. Many different strains of CMV are known to be associated with variant symptoms related to the host plant, such as CMV-B, CMV-C, CMV-D, CMV-L, CMV-S, CMV-T, CMV-WL, CMV-V, CMV-Fny, CMV-Ix, CMV-Q, and CMV-R (Carrère I et al., *Archives of Virology* (1999) 144:1846-1857; Edwards MC et al., *Phytopathology* (1983) 73:1117-1120; www.dpvweb.net ).

[0005] In recent years, CMV-based vaccine platforms that present different antigens on their surfaces using chemical linker conjugation technology have been described. The described CMV VLP is derived from a modified CP of CMV with inserted T-cell stimulating epitopes (A. Zeltins et al., Vaccines 2 (2017) 30; WO2016 / 062720).

[0006] Chimeric forms of CMV have also been engineered to act as a presentation system and express epitopes derived from hepatitis C virus (HCV) on their outer surface. Specifically, the pseudo-recombinant form of CMV, CMV-D / S, has been engineered to carry genomic RNA3 from the CMV-S strain and RNA1 and RNA2 from the CMV-D strain. Following inoculation, this system… XanthiViral symptoms, such as mild mosaicism and venous clearance, appeared in tobacco plants. The CP gene was then engineered at different locations to encode hepatitis C virus (HCV) epitopes. The selected peptide is a so-called R9 mimic epitope, a synthetic peptide of 27 amino acids derived from the hypervariable region 1 (HVR1) sequence of the HCV envelope protein E2. The insertion site for the R9 mimic epitope into the CMV gene was selected considering the following important factors: i) the need to protect the N-terminal region of the CMV capsid protein (containing a high concentration of basic amino acids), known as the internal R domain, which participates in protein-RNA interactions to stabilize CMV (Wikoff WR et al., *Virology* (1997) 232: 91-97), characterized by an anomalous N-terminal helix and additional stabilizing effects within the capsid (Smith TJ et al., *Journal of Virology* (2000) 74: 7578-7686); ii) the surface location of the exogenous epitope to increase its probable immunogenicity; and iii) the availability of mutagenic pathways capable of generating modified clones. Based on these considerations, R9 mimic epitopes have been inserted at different locations within the CP gene of CMV-S RNA3 (AF063610, www.dpvweb.netTo insert a single R9 mimic epitope into the CP gene, R9 mimic epitope nucleotide sequences were inserted into positions 253, 475, and 529 of the CP gene. For the insertion of two R9 mimic epitopes, the R9 mimic epitope nucleotide sequences were inserted into positions 392 and 529. Even so, the chimeric CMV prepared in this way maintained its ability to spread systematically in the host plant, but the virus extraction rate obtained was still low with the first two insertion sites. Therefore, to ensure a higher concentration of virus particles in infected tissues, mosaic CMV containing an R9 mimic epitope inserted at position 529 of the CP gene was selected, and the serological reactivity of HCV patients was tested. Serum samples from 60 patients with chronic hepatitis C showed significant immunoreactivity to crude plant extracts infected with the chimeric CMV (Natilla A et al., Archives of Virology (2004) 149:137-154; Piazzolla G et al., Clin Immunol (2005) 25:142-152; Nuzzaci M et al., Archives of Virology (2007) 152:915-928; Nuzzaci M et al., Journal of Virological Methods (2009) 155:118-121; Nuzzaci M et al., Journal of Virological Methods (2010) 165:211-215; Piazzolla G et al., Clinic Immunology (2012) 32:866-876).

[0007] Furthermore, cucumber mosaic virus-based expression systems have been described as potential vaccines against Alzheimer's disease and for the production of porcine circovirus vaccines (Vitti A et al., *Journal of Virology Methods* (2010) 169:332-340; Gellert A et al., *PLoS ONE* (2012) 7(12): e52688). Specifically, chimeric constructs were created carrying Aβ-derived fragments of varying lengths (11 to 15 amino acids) at positions 248, 392, or 529 of the CMV capsid protein (CP) gene, and the viral products were demonstrated to replicate in their natural hosts. On the other hand, porcine circovirus type 2 (PCV2) capsid protein epitopes of up to 20 amino acids were integrated after amino acid position 131 of the plant virus capsid protein of the cucumber mosaic virus (CMV)-R strain. The insertion sites 131 to 132 are located in the middle of the βE-αEF ring of CMV CP, and it is concluded that this position is advantageous because the inserted epitope forms a trigonal group in the middle of the CMV CP trimer, which makes antibody production more efficient.

[0008] Furthermore, the generation of chimeric virus-like particles (VLPs) of CMV has been described, even though expression of the viral capsid protein (CP) of CMV via widely used conventional E. coli expression systems resulted only in insoluble inclusion bodies or very small amounts of soluble protein (Xu Y et al., Chem Commun (2008) 49-51). On the other hand, chimeric CMV capsid proteins expressed from potato virus X (PVX)-based vectors can assemble into VLPs (Natilla, A et al., Archives of Virology (2006) 151:1373-1386; Natilla, A et al., Protein Expression and Purification (2008) 59:117-121; Chen Q and Lai H, Human Vaccines and Immunotherapy (2013) 9: 26-49). Chimeric CMV coat proteins include epitopes of 17 to 25 amino acids in length from Newcastle disease virus (NDV), which are fused to the CMV CP via gene fusion into the internal βH-βI (motif 5) loop corresponding to amino acid positions 194 to 199 (HeX et al., (1998) J Gen Virol 79: 3145-3153).

[0009] Even with progress in VLP-based vaccine development, additional unique VLP systems are still needed. Specifically, vaccines induce variant antibody responses in immunized subjects and individuals, typically spanning a range of variations exceeding 100-fold. Additionally, some vaccines, such as hepatitis B vaccines, experience a number of non-responders. Non-respondership is associated with certain class II MHC molecules, and the failure to induce a robust T helper (Th) cell response is believed to lead to poor antibody responses in these individuals (Goncalves L et al., *Virology* (2004) 326:20-28). Furthermore, older adults generally have poorer antibody responses, and poorer Th cell responses are again considered a cause of inefficient antibody responses. Therefore, vaccines that induce robust Th cell responses in virtually all subjects and individuals are an important goal in vaccine development. Another crucial related issue that needs to be addressed during vaccine construction is antigen spatial conformation. For vaccine construction, it is important to achieve optimal peptide presentation on the particle surface without compromising the overall viral structure. This is obvious, because only under special circumstances can VLPs accommodate protein domains longer than 50 or 70 amino acids or even longer and retain the typical VLP morphology (I. Balke et al., Advanced Drug Delivery Review (2018), https: / / doi.org / 10.1016 / j.addr.2018.08.007; I. Kalnciema et al., Molecular Biotechnology (Mol. Biotechnol.) 52 (2012) 129-139). Summary of the Invention

[0010] It has now been surprisingly discovered that various and very different antigenic peptides of different lengths and properties can be inserted at specific sites on CMV peptides that have been modified by incorporation of T helper cell epitopes, and the resulting fusion proteins can still form and assemble into additional highly immunogenic modified virus-like particles (VLPs). Preferably, and even more surprisingly, mosaic-modified virus-like particles have been produced, comprising the described fusion protein having a fused antigenic peptide, and further comprising CMV proteins without such a fused antigenic peptide. These mosaic-modified CMVVLPs have been found to be highly beneficial and even allow for the incorporation of very long antigenic peptides, such as those exceeding 200 amino acids in length.

[0011] In a first aspect, the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising at least one fusion protein, wherein the at least one fusion protein comprises a) A chimeric CMV polypeptide or preferably composed thereof, wherein the chimeric CMV polypeptide comprises or preferably comprises the following: (i) A CMV polypeptide, wherein the CMV polypeptide comprises or preferably consists of a CMV coat protein, wherein preferably the CMV coat protein comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide. The insertion of the antigenic polypeptide is located between the amino acid residues corresponding to positions 84 and 85 of SEQ ID NO: 62 in the CMV polypeptide; and (iii) A T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62.

[0012] In another aspect, the present invention provides a modified VLP of CMV comprising at least one fusion protein, wherein the at least one fusion protein comprises a) A chimeric CMV polypeptide or preferably composed thereof, wherein the chimeric CMV polypeptide comprises or preferably comprises the following: (i) A CMV polypeptide, wherein the CMV polypeptide comprises or preferably consists of a CMV coat protein, wherein preferably the CMV coat protein comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with the coat protein and preferably with the SEQ ID NO: 62; and (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide. The insertion of the antigenic polypeptide is located between the amino acid residues corresponding to positions 84 and 85 of SEQ ID NO: 62 in the CMV polypeptide; and iii) A first amino acid linker, preferably a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigenic polypeptide, and wherein preferably the first amino acid linker is selected from the group consisting of: (a.) Polyglycine linkers (Gly) with lengths n = 2–10 n ; (b.) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c.) An amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

[0013] In another aspect, the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV), comprising: (a) at least one fusion protein, wherein the at least one fusion protein comprises a) A chimeric CMV polypeptide or preferably composed thereof, wherein the chimeric CMV polypeptide comprises or preferably comprises the following: (i) A CMV polypeptide, wherein the CMV polypeptide comprises or preferably consists of a CMV coat protein, wherein preferably the CMV coat protein comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide. The insertion of the antigenic polypeptide is located between the amino acid residues corresponding to positions 84 and 85 of SEQ ID NO: 62 in the CMV polypeptide; and (b) At least one CMV protein, wherein the CMV protein comprises or is preferably composed of a shell protein of CMV, wherein preferably the shell protein of CMV comprises or is preferably composed of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and wherein the CMV protein is optionally modified by a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, and wherein preferably the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62.

[0014] As this specification continues, other aspects and embodiments of the invention will become apparent. Attached Figure Description

[0015] Figure 1 Description of the pET-CMV-Ntt830-Ab36 plasmid map containing a single restriction enzyme site. All other maps (pET-CMV-Ntt830-Ab15; pET-CMV-Ntt830-Ab16; pET-CMV-Ntt830-Ab17) have essentially the same sequence and gene organization; they differ only in the nucleotide sequence encoding the amyloid (β) peptide.

[0016] Figure 2A SDS-PAGE gel analysis of purified VLPs derived from CMV-Ntt830-Ab36 expression. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); S - Soluble protein in *E. coli* C2566 cells after 18 hours of culture at 20°C prior to sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fraction (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the corresponding CMV-Ntt830-Ab36 chimeric CMV peptide in the SDS / PAGE gel.

[0017] Figure 2BSDS-PAGE gel analysis of purified VLPs derived from CMV-Ntt830-Ab15 expression. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); S - Soluble protein in *E. coli* C2566 cells after 18 hours of culture at 20°C prior to sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fraction (60% from bottom to 0% at top). Asterisk ( * () indicates the relative position of the corresponding CMV-Ntt830-Ab15 chimeric CMV peptide in the SDS / PAGE gel.

[0018] Figure 2C SDS-PAGE gel analysis of purified VLPs derived from CMV-Ntt830-Ab16 expression. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); S - Soluble protein in *E. coli* C2566 cells after 18 hours of culture at 20°C prior to sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fraction (60% from the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the corresponding CMV-Ntt830-Ab16 chimeric CMV peptide in the SDS / PAGE gel.

[0019] Figure 2D SDS-PAGE gel analysis of purified VLPs derived from CMV-Ntt830-Ab17 expression. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); S - Soluble protein in *E. coli* C2566 cells after 18 hours of culture at 20°C prior to sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fraction (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the corresponding CMV-Ntt830-Ab17 chimeric CMV peptide in the SDS / PAGE gel.

[0020] Figure 3A Electron microscopy analysis of purified VLPs derived from CMV-Ntt830-Ab36. White horizontal bars correspond to 100 nm.

[0021] Figure 3B Electron microscopy analysis of purified VLPs derived from CMV-Ntt830-Ab15. White horizontal bars correspond to 100 nm.

[0022] Figure 3C Electron microscopy analysis of purified VLPs derived from CMV-Ntt830-Ab16. White horizontal bars correspond to 100 nm.

[0023] Figure 3D Electron microscopy analysis of purified VLPs derived from CMV-Ntt830-Ab17. White horizontal bars correspond to 100 nm.

[0024] Figure 4 Monoclonal antibodies containing the variable region sequence of aducanumab bind to CMV-Ntt830-Ab36 VLP, Aβ1-42 peptide, and CMV-Ntt830 VLP.

[0025] Figure 5A CMV-Ntt830-Ab16 VLP, CMV-Ntt830-Ab17 VLP and CMV-Ntt830-Ab36 VLP induce antibodies that recognize the full-length Aβ1-42 peptide.

[0026] Figure 5B CMV-Ntt830-Ab36 VLP induces an antibody that recognizes brain plaques in Alzheimer's disease patients. Figure 6 Description of the pETDu-CMVB2xArah202-CMV-tt plasmid with a single restriction enzyme site. The expression vector ensures the simultaneous synthesis of CMV-Ntt830-Arah202 and unmodified CMV-Ntt830.

[0027] Figure 7A SDS-PAGE gels of purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830. M - PageRuler protein size marker (Thermo Fisher Scientific, No. 26620); T - Total protein in *E. coli* C2566 / pETDu-CMVxArah202-CMVtt cells after 18 hours of incubation at 20°C; S - Soluble protein (20-60%) in cell extracts prior to sucrose gradient; P - Insoluble protein; 1-6 - Sucrose gradient fractions (60% from the bottom of the tube to 0% at the top). Asterisks ( * () indicates the relative position of the CMV-Ntt830-Arah202 chimeric CMV polypeptide in the gel.

[0028] Figure 7BWestern blot analysis of purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); T - Total protein in *E. coli* C2566 / pETDu-CMVxArah202-CMVtt cells after 18 hours of incubation at 20°C; S - Soluble protein (20-60%) in cell extracts prior to sucrose gradient; P - Insoluble protein; 1-6 - Sucrose gradient fraction (60% from bottom to 0% at top). For Western blot, rabbit pAb (1:1000; Indoor Biotechologies, No. PA-AH2) targeting Arah2 was used. Western blot confirmed the presence of Arah2 in the CMV VLP fraction. Asterisk ( * () indicates the relative position of the CMV-Ntt830-Arah202 fusion protein in the blot.

[0029] Figure 8A SDS-PAGE gel analysis of purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620), 1 - Purified CMV-Ntt830 VLP (control sample), 2 - Purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830.

[0030] Figure 8B Electron microscopy analysis of purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830. Electron microscopy images of purified mosaic VLPs including CMV-Ntt830-Arah202 and unmodified CMV-Ntt830. White horizontal bars correspond to 100 nm.

[0031] Figure 9 ELISA for IgG against recombinant Ara-h202 was performed 14 days after immunization with mosaic VLP containing Ara-h202 or CMV-Ntt830-Arah202 and unmodified CMV-Ntt830 (CMV-M-Arah202).

[0032] Figure 10A Experimental design to study the protective effect of CMV-M-Arah202-containing vaccines against systemic and local allergic reactions. Figure 10B: Protects against systemic and localized stimulation. Systemic stimulation is achieved using peanut extract.

[0033] Figure 10C Protects against systemic and localized irritation. Perform a skin prick test using peanut extract.

[0034] Figure 11 Description of the pET28-CMVBxFeld1-CMV-tt plasmid with a single restriction enzyme site. The expression vector ensures the simultaneous synthesis of CMV-Ntt830-Feld12 and unmodified CMVNtt830.

[0035] Figure 12A SDS-PAGE gel for purification of mosaic VLP (i.e., CMV-M-Fel) containing CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein. M - PageRuler protein size marker (Thermo Fisher Scientific, No. 26620); O - Total protein in *E. coli* C2566 / pET28-CMVxFeld1-CMVtt cells before induction; T - Total protein in *E. coli* C2566 / pET28-CMVxFeld1-CMVtt cells after 18 hours of culture at 20°C. S - Soluble protein in cell extracts before sucrose gradient centrifugation; P - Insoluble protein; 1-6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the CMV-Ntt830-Feld12 chimeric CMV polypeptide in the gel.

[0036] Figure 12B SDS-PAGE gel analysis of purified mosaic VLPs (i.e., CMV-M-Fel) including CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620), 1 - Purified mosaic VLPs (i.e., CMV-M-Fel) including CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein.

[0037] Figure 12C Electron microscopy analysis of purified mosaic VLP (i.e., CMV-M-Fel) including CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein. Electron microscopy images of purified mosaic VLP (i.e., CMV-M-Fel) including CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein. White horizontal bars correspond to 100 nm.

[0038] Figure 13 CMV-M-Fel induces an effective antibody response against Fel d1. Anti-Fel d1 IgG is shown as absorbance at 450 nm 14 days after immunization of primitive mice with a VLP containing Feld1, CMV-Ntt830-Feld12, and a mosaic VLP containing CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein (i.e., CMV-M-Fel).

[0039] Figure 14A Experimental design to study the protective effect of CMV-M-Fel-containing vaccines against systemic and local allergic reactions.

[0040] Figure 14B The protective effect of CMV-M-Fel-induced systemic stimulation against Fel d1. Stimulation was performed intravenously with 3 μg of Fel d1 extract and recombinant Fel d1 dimer.

[0041] Figure 15 Description of the pET28-CMVB2x19nanp-CMVtt plasmid map. The plasmid is used to express the mosaic VLP (i.e., CMV-M-CSP) containing CMV-Ntt830-19NANP and unmodified CMV-Ntt830 protein.

[0042] Figure 16 SDS-PAGE gel analysis of purified mosaic VLPs (i.e., CMV-M-CSP) including CMV-Ntt830-19NANP and unmodified CMV-Ntt830 protein. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); S - Soluble protein in *E. coli* C2566 after 18 hours of incubation at 20°C in cell extract prior to sucrose gradient; P - Insoluble protein; 1–6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisks ( * () indicates the relative position of CMV-Ntt830-19nanp in the SDS / PAGE gel.

[0043] Figure 17 Electron microscopy image of purified CMV-M-CSP mosaic VLP. White horizontal bars correspond to 100 nm.

[0044] Figure 18Description of the plasmid clone pET-CMV-Ntt830-egy containing the CMVNtt830 gene with a cloned α-synuclein epitope cDNA. This detailed description of the plasmids is of the plasmid clones pET-CMV-Ntt830-egy, pET-CMV-Ntt830-kne, and pET-CMV-Ntt830-mdv, all of which have substantially the same sequence and gene organization; they differ only in the nucleotide sequence encoding the α-synuclein peptide variant.

[0045] Figure 19A SDS-PAGE gel analysis of purified VLPs derived from the expression of CMV-Ntt830-egy. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); T - Total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; 1-6-sucrose gradient fraction (from 60% at the bottom of the tube to 0% at the top).

[0046] Figure 19B SDS-PAGE gel analysis of purified VLPs derived from the expression of CMV-Ntt830-kne. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); T - Total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; 1-6-sucrose gradient fraction (from 60% at the bottom of the tube to 0% at the top).

[0047] Figure 19C SDS-PAGE gel analysis of purified VLPs derived from the expression of CMV-Ntt830-mdv. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); T - Total protein in E. coli C2566 cells after 18 hours of incubation at 20°C; 1-6-sucrose gradient fraction (from 60% at the bottom of the tube to 0% at the top).

[0048] Figure 20A SDS-PAGE gel analysis of purified CMV-Ntt830 and α-synuclein peptide fusion VLP. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); 1 - Purified CMV-Ntt830B-mdv fusion protein VLP; 2 - Purified CMV-Ntt830B-egy fusion protein VLP; 3 - Purified CMV-Ntt830B-kne fusion protein VLP; 4 - Purified unmodified CMV-Ntt830 VLP.

[0049] Figure 20BElectron microscopy image of the purified CMV-Ntt830B-egy fusion protein VLP. White horizontal bars correspond to 100 nm.

[0050] Figure 20C Electron microscopy image of the purified CMV-Ntt830B-kne fusion protein VLP. White horizontal bars correspond to 100 nm.

[0051] Figure 20D Electron microscopy image of the purified CMV-Ntt830B-kne fusion protein VLP. White horizontal bars correspond to 100 nm.

[0052] Figure 21A Description of the pETDu-CMVB3d-CMVB3d-flIL5-CMVtt plasmid map. The plasmid is used to express a mosaic VLP (i.e., CMV-M-fel-IL-5) containing CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein.

[0053] Figure 21B Description of the pETDu-CMVB3d-CMVB3-flIL5-CMVtt plasmid map. The plasmid is used to express CMV-Ntt830-fel-IL-5. * And the mosaic VLP of unmodified CMV-Ntt830 protein (i.e., CMV-M-fel-IL-5) * ).

[0054] Figure 22A SDS-PAGE gel analysis of purified mosaic VLP (i.e., CMV-M-fel-IL-5) including CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); O - Protein in *E. coli* C2566 / pETDu-CMVB3d-flIL5-CMVtt cells before IPTG induction; T - Total protein in *E. coli* C2566 / pETDu-CMVB3d-flIL5-CMVtt cells after 18 hours of culture at 20°C. S - Soluble protein in cell extracts before sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of CMV-Ntt830-fel-IL-5 in the SDS / PAGE gel.

[0055] Figure 22B: A mosaic VLP including CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein (i.e., CMV-M-fel-IL-5) * The purified SDS-PAGE gel analysis was performed.

[0056] M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); O - Protein in *E. coli* C2566 / pETDu-CMVB3-flIL5-CMVtt cells before IPTG induction; T - Total protein in *E. coli* C2566 / pETDu-CMVB3-flIL5-CMVtt cells after 18 hours of culture at 20°C. S - Soluble protein in cell extracts before sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * (This indicates CMV-Ntt830-fel-IL-5) * Relative positions in SDS / PAGE gels.

[0057] Figure 23A SDS-PAGE gel analysis of purified CMV-M-fel-IL-5 mosaic VLP. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); 1 - Purified soluble CMV-M-fel-IL-5 mosaic VLP after clarification at 13,000 rpm; 2 - Insoluble CMV-Ntt830-fel-IL-5 / CMV-Ntt830 after clarification at 13,000 rpm.

[0058] Figure 23B Electron microscopy image of purified CMV-M-fel-IL-5 mosaic VLP. Horizontal bars correspond to 200 nm.

[0059] Figure 23C Purified CMV-M-fel-IL-5 * SDS-PAGE gel analysis of variegated VLP.

[0060] M - Protein size marker PageRuler (Thermo Fisher Scientific, Serial No. 26620); 1 - Purified soluble CMV-M-fel-IL-5 after clarification at 13000 rpm. * Variegated VLP.

[0061] Figure 23D Purified CMV-M-fel-IL-5 * Electron microscope image of the variegated VLP.

[0062] Figure 24 Description of the pETDu-CMVB3d-CMVB3d-2xflIL5-CMVtt plasmid map. The plasmid is used to express a mosaic VLP (i.e., CMV-M-2xfel-IL-5) containing CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein.

[0063] Figure 25 SDS-PAGE gel analysis of purified CMV-Ntt830-2xfel-IL-5 and unmodified CMV-Ntt830 protein in mosaic VLP (i.e., CMV-M-2xfel-IL-5). M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); O - Protein in *E. coli* C2566 / pETDu-CMVB3d-2xflIL5-CMVtt cells before IPTG induction; T - Total protein in *E. coli* C2566 / pETDu-CMVB3d-2xflIL5-CMVtt cells after 18 hours of culture at 20°C. S - Soluble protein in cell extracts before sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the CMV-Ntt830-2xfel-IL-5 protein in the gel.

[0064] Figure 26A SDS-PAGE gel analysis of purified CMV-M-2xfel-IL-5 mosaic VLP. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); 1 - Purified soluble CMV-M-2xfel-IL-5 mosaic VLP after clarification at 13,000 rpm; 2 - Insoluble CMV-Ntt830-2xfel-IL-5 / CMV-Ntt830 after clarification at 13,000 rpm.

[0065] Figure 26B Electron microscopy image of purified CMV-M-2xfel-IL-5 mosaic VLP. Horizontal bars correspond to 200 nm.

[0066] Figure 27 Description of the pETDu-CMVB3d-CMVB3d-cIL1b-CMVtt plasmid map. The plasmid is used to express a mosaic VLP (i.e., CMV-M-cIL-1b) containing both CMV-Ntt830-cIL-1b and unmodified CMV-Ntt830 protein.

[0067] Figure 28 SDS-PAGE gel analysis of purified mosaic VLP (i.e., CMV-M-cIL-1b) including CMV-Ntt830-cIL-1b and unmodified CMV-Ntt830 protein. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); O - Protein in *E. coli* C2566 / pETDu-CMVB3d-cIL1b-CMVtt cells before IPTG induction; T - Total protein in *E. coli* C2566 / pETDu-CMVB3d-cIL1b-CMVtt cells after 18 hours of culture at 20°C. S - Soluble protein in cell extracts before sucrose gradient (20-60%); P - Insoluble protein; 1-6 - Sucrose gradient fractions (from 60% at the bottom of the tube to 0% at the top). Asterisk ( * () indicates the relative position of the CMV-Ntt830-cIL-1b protein in the gel.

[0068] Figure 29A SDS-PAGE gel analysis of purified CMV-M-cIL-1b mosaic VLP. M - Protein size marker PageRuler (Thermo Fisher Scientific, No. 26620); 1 - Purified soluble CMV-M-cIL-1b mosaic VLP after clarification at 13,000 rpm; 2 - Insoluble CMV-Ntt830-cIL-1b / CMV-Ntt830 after clarification at 13,000 rpm.

[0069] Figure 29B Electron microscopy image of purified CMV-M-cIL-1b mosaic VLP. Horizontal bars correspond to 200 nm. Detailed Implementation

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The embodiments, preferred embodiments, and highly preferred embodiments described and disclosed herein should be applied to all aspects, as well as other embodiments, preferred embodiments, and highly preferred embodiments, whether specifically mentioned again or to avoid repetition for the sake of brevity. As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical object of the article. Unless the context clearly indicates otherwise, the term “or” as used herein should be understood to mean “and / or”.

[0071] Virus-like particles (VLPs): As used herein, the term "virus-like particle (VLP)" refers to a non-replicating or non-infectious viral particle, preferably non-replicating and non-infectious, or a non-replicating or non-infectious structure, preferably a viral capsid, resembling a viral particle. As used herein, the term "non-replicating" means that the genome included in the VLP cannot be replicated. As used herein, the term "non-infectious" means that it cannot enter a host cell. Virus-like particles according to the invention are non-replicating and non-infectious because they lack all or part of the viral genome or genome function. Virus-like particles according to the invention may contain nucleic acids different from their genome. Recombinant virus-like particles typically contain host cell-derived RNA. A typical and preferred embodiment of the virus-like particle according to the invention is a viral capsid composed of the polypeptides of the invention. Virus-like particles are typically macromolecular assemblies composed of viral coat proteins, each virus-like particle typically comprising 60, 120, 180, 240, 300, 360, or more than 360 protein subunits. Typically and preferably, the interaction of these subunits leads to the formation of a viral capsid or viral capsid-like structure with an inherent repeating organization. A characteristic of virus-like particles is the highly ordered and repeating arrangement of their subunits.

[0072] Modified virus-like particles (VLPs) of CMV: The term "modified virus-like particles of CMV" refers to virus-like particles comprising at least one fusion protein including a CMV polypeptide. Typically and preferably, modified virus-like particles of CMV have a structure similar to the capsid of CMV. Modified virus-like particles of CMV are non-replicating and / or non-infectious and lack at least one or more genes encoding the CMV replication mechanism, and typically also lack one or more genes encoding one or more proteins responsible for viral attachment or entry into the host. This definition also includes modified virus-like particles in which the aforementioned one or more genes are present but inactive. Preferably, non-replicating and / or non-infectious modified virus-like particles are obtained through recombinant gene technology and typically and preferably do not include a viral genome. Modified virus-like particles comprising two or more different polypeptides are referred to as "mosaic VLPs" and are particularly covered by this invention. Mosaic modified virus-like particles are a very preferred embodiment and aspect of this invention. Therefore, in one embodiment, the modified virus-like particles according to the invention comprise at least two different types of polypeptides. Most preferably, the mosaic VLP comprises two different types of CMV polypeptides optionally modified according to the invention, thereby producing a mosaic-modified CMC VLP. Preferably, the CMV-modified VLP is a macromolecular assembly composed of CMV polypeptides modified according to the invention, each VLP typically comprising 180 such protein subunits.

[0073] Polypeptide: As used herein, the term "polypeptide" refers to a polymer composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). This refers to the molecular chain of amino acids, not a specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, and proteins are included within the definition of a polypeptide. As used herein, the term "polypeptide" should also generally and preferably refer to a polypeptide as previously defined, and encompass modifications such as post-translational modifications, including but not limited to glycosylation. In a preferred embodiment, the term "polypeptide" as used herein should refer to a polypeptide as previously defined and does not encompass modifications such as post-translational modifications, such as glycosylation. Specifically, for the bioactive peptide, modifications such as glycosylation can even subsequently occur in vivo, for example, through bacteria.

[0074] Cucumber mosaic virus (CMV) polypeptide, CMV polypeptide: As used herein, the term "cucumber mosaic virus (CMV) polypeptide" refers to a polypeptide comprising or preferably consisting of: (i) an amino acid sequence of the coat protein of cucumber mosaic virus (CMV), or (ii) a mutant amino acid sequence, wherein the amino acid sequence to be mutated is an amino acid sequence of the coat protein of CMV, and wherein the mutant amino acid sequence and the amino acid sequence to be mutated, i.e., the coat protein of CMV, exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% sequence identity. Generally and preferably, CMV polypeptides are capable of forming virus-like particles of CMV through self-assembly expression. As used herein, when referred to in the context of polypeptides, the term "chimerism" refers to a polypeptide comprising polypeptide components from two or more different sources. This term is further intended to impart a specific manner in which the polypeptide components are bound or coupled together, i.e., by fusion bonds and peptide bonds, respectively. Thus, the term "chimerism CMV polypeptide" is defined as such, and especially according to the present invention.

[0075] Cucumber mosaic virus (CMV) coat protein (CP): As used herein, the term "cucumber mosaic virus (CMV) coat protein (CP)" refers to the coat protein of naturally occurring cucumber mosaic virus. Because cucumber mosaic virus has a very wide host range, many different CMV strains and isolates are known, and the sequences of the coat proteins of said strains and isolates have been determined and are therefore known to those skilled in the art. The sequences of the CMV coat protein (CP) are described and searchable in known databases, such as GenBank. www.dpvweb.net or www.ncbi.nlm.nih.gov / protein / Specific examples of CMV (Central Mosaic Virus) are described on page 12, line 8 to page 13, line 25 of WO 2016 / 062720, the disclosure of which is expressly incorporated herein by reference. Very preferred examples and embodiments of the CMV coat protein are provided in SEQ ID NO: 62. Therefore, preferably, as used herein, the term "coat protein of cucumber mosaic virus (CMV)" refers to the amino acid sequence of the CMV coat protein, wherein the amino acid sequence comprises or preferably consists of the following: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further preferably at least 95%, still further preferably at least 98%, and still still further preferably at least 99% sequence identity with SEQ ID NO: 62.

[0076] Notably, these strains and isolates exhibit highly similar capsid protein sequences across different protein domains, including the N-terminus of the capsid protein. Specifically, 98.1% of all fully sequenced CMV isolates share over 85% sequence identity within the first 28 amino acids of their capsid protein sequences, and 79.5% of all fully sequenced CMV isolates still share over 90% sequence identity within the first 28 amino acids of their capsid protein sequences.

[0077] N-terminal region of CMV peptide: As used herein, the term "N-terminal region of CMV peptide" refers to the N-terminus of the CMV peptide, and particularly to the N-terminus of the CMV capsid protein, or the region at the N-terminus of the CMV peptide or the CMV capsid protein, but starting with the second amino acid at the N-terminus of the CMV peptide or the CMV capsid protein if the CMV peptide or the capsid protein includes an N-terminal methionine residue. Preferably, in the case where the CMV peptide or the capsid protein includes an N-terminal methionine residue, from a practical point of view, according to the invention, the start codon encoding the methionine is typically deleted and added to the N-terminus of the Th cell epitope. More preferably, for cloning purposes, one, two, or three additional amino acids, preferably one amino acid, may optionally be inserted between the stated methionine and the Th cell epitope. As used herein, the term "N-terminal region of the mutant amino acid sequence of the CMV polypeptide or the CMV coat protein" refers to the N-terminus of the mutant amino acid sequence of the CMV polypeptide or the CMV coat protein, or to the region at the N-terminus of the mutant amino acid sequence of the CMV polypeptide or the CMV coat protein, but starting with the second amino acid at the N-terminus of the mutant amino acid sequence of the CMV polypeptide or the CMV coat protein if the mutant amino acid sequence includes an N-terminal methionine residue. Preferably, in the case where the CMV polypeptide or the coat protein includes an N-terminal methionine residue, from a practical point of view, the start codon encoding methionine is typically deleted and added to the N-terminus of the Th cell epitope. More preferably, for cloning purposes, one, two, or three additional amino acids, preferably one amino acid, may optionally be inserted between the stated methionine and the Th cell epitope.

[0078] Recombinant polypeptide: In the context of this invention, when used in the context of polypeptides, the term "recombinant" refers to a polypeptide obtained by a method including at least one recombinant DNA technology step. Typically and preferably, recombinant polypeptides are produced in prokaryotic expression systems. It will be apparent to those skilled in the art that recombinant polypeptides expressed in prokaryotic expression systems such as *E. coli* may include N-terminal methionine residues. During the maturation of the recombinant polypeptide, the N-terminal methionine residues are typically cleaved from the recombinant polypeptide in the expression host. However, the cleavage of the N-terminal methionine may be incomplete. Therefore, formulations of recombinant polypeptides may include a mixture of other identical polypeptides having and not having N-terminal methionine residues. Typically and preferably, formulations of recombinant polypeptides include less than 10%, more preferably less than 5%, and still more preferably less than 1% of the recombinant polypeptide having N-terminal methionine residues.

[0079] Recombinant modified virus-like particles: In the context of this invention, the term "recombinant modified virus-like particles" refers to modified virus-like particles (VLPs) obtained by a method including at least one step of recombinant DNA technology.

[0080] Mutant amino acid sequence: The term "mutant amino acid sequence" refers to an amino acid sequence obtained by introducing a defined set of mutations into the amino acid sequence to be mutated. In the context of this invention, the amino acid sequence to be mutated is typically and preferably the amino acid sequence of the CMV outer shell protein. Therefore, the mutant amino acid sequence differs from the amino acid sequence of the CMV outer shell protein at least one amino acid residue, wherein the mutant amino acid sequence and the amino acid sequence to be mutated exhibit at least 90% sequence identity. Typically and preferably, the mutant amino acid sequence and the amino acid sequence to be mutated exhibit at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Preferably, the mutant amino acid sequence and the sequence to be mutated differ at most 11, 10, 9, 8, 7, 6, 4, 3, 2, or 1 amino acid residue, wherein more preferably, the difference is selected from insertions, deletions, and amino acid exchanges. Preferably, the mutant amino acid sequence differs from the amino acid sequence of the CMV outer shell protein at at least one amino acid, wherein preferably, the difference is an amino acid exchange.

[0081] The position corresponding to the residue…: The position on an amino acid sequence corresponding to a given residue in another amino acid sequence can be identified by sequence alignment, typically and preferably using the BLASTP algorithm, and most preferably using standard settings. Typical and preferred standard settings are: expected threshold: 10; font size: 3; maximum number of matches in the query range: 0; matrix: BLOSUM62; empty cost: 11 for existence, 1 for expansion; component adjustment: conditional component scoring matrix adjustment.

[0082] Sequence identity: Determining the sequence identity of two given amino acid sequences based on the alignment of two sequences. Algorithms for determining sequence identity are available to those skilled in the art. Preferably, the sequence identity of two amino acid sequences is determined using a publicly available computer homology program, such as the "BLAST" program (http: / / ...). / / blast.ncbi.nlm.nih.gov / Blast.cgi ) or "CLUSTALW" http: / / www.genome.jp / tools / clustalw / ), and preferably through the “BLAST” program available on the NCBI homepage ( http: / / blast.ncbi.nlm.nih.gov / Blast.cgiIf determined, use the default settings provided. Typical and preferred criteria are set as follows: Expected threshold: 10; Font size: 3; Maximum number of matches in the query range: 0; Matrix: BLOSUM62; Empty space cost: 11 for existence, 1 for expansion; Component adjustment: Conditional component scoring matrix adjustment.

[0083] Amino acid exchange: The term amino acid exchange refers to the exchange of a given amino acid residue in an amino acid sequence for any other amino acid residue with a different chemical structure, preferably another proteinogenic amino acid residue. Therefore, unlike the insertion or deletion of amino acids, amino acid exchange does not change the total number of amino acids in the amino acid sequence.

[0084] Epitope: The term epitope refers to an antigen, preferably a continuous or discontinuous portion of a polypeptide, which can be specifically bound by an antibody or T-cell receptor in the context of an MHC molecule. Regarding antibodies, specific binding does not include non-specific binding, but does not necessarily exclude cross-reactivity. Epitopes typically consist of 5-20 amino acids in their spatial conformation, which is unique to the antigenic site.

[0085] T helper (Th) cell epitopes: As used herein, the term "T helper (Th) cell epitope" refers to an epitope that can be recognized by helper Th cells. Generally and preferably, as used herein, the term "Th cell epitope" refers to a Th cell epitope that can bind to at least one, preferably more than one, MHC class II molecule. The simplest way to determine whether a peptide sequence is a Th cell epitope is to measure the peptide's ability to bind to an MHC class II molecule. This can be measured by the peptide's ability to compete with known Th cell epitope peptides for binding to MHC class II molecules. Representative selections of HLA-DR molecules are described, for example, by Alexander J et al., *Immunity* (1994) 1:751-761. The affinity of a Th cell epitope for an MHC class II molecule should be at least 10. -5M. Panina-Bordignon P et al., in the *European Journal of Immunology* (1989) 19:2237-2242, present a representative set of MHC class II molecules present in different individuals. Therefore, the term "Th cell epitope" as used herein preferably refers to Th cell epitopes that generate a measurable T cell response after immunization and enhancement. Furthermore, and even more preferably, as used herein, the term "Th cell epitope" preferably refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, and DR2w2a; and preferably selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, and DR2w2a, wherein the affinity is at least 500 nM (as described in Alexander J et al., *Immunology* (1994) 1:751-761 and the references cited herein); the preferred binding assay for assessing said affinity is the assay described in Sette A et al., *Journal of Immunology* (1989) 142:35-40. In an even more preferred manner, as used herein, the term “Th cell epitope” refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, and DR2w2a, wherein the affinity is at least 500 nM (as described in Alexander J et al., Immunology (1994) 1:751-761 and the references cited herein); the preferred binding assay for assessing said affinity is the assay described in Sette A et al., Journal of Immunology (1989) 142:35-40. Th cell epitopes have been described and are known to those skilled in the art, such as Alexander J et al., Immunology (1994) 1:751-761, Panina-Bordignon P et al., European Journal of Immunology (1989) 19:2237-2242, Calvo-Calle JM et al., Immunology (1997) 159:1362-1373, and Valmori D et al., Immunology (1992) 149:717-721.

[0086] Antigenic polypeptide: As used herein, the term "antigenic polypeptide" refers to a molecule capable of being bound by an antibody or T-cell receptor (TCR), if presented by an MHC molecule. Antigenic polypeptides can also be recognized by the immune system and / or induce humoral and / or cellular immune responses, leading to activation of B and / or T lymphocytes. Antigenic polypeptides may have one or more epitopes (B- and T-epitopes). Antigenic polypeptides as used herein may also be a mixture of several individual antigenic polypeptides. The polypeptides of the present invention, particularly the fusion proteins of the present invention forming the modified virus-like particles of the present invention, include antigenic polypeptides.

[0087] Peanut allergen: As used in this article, the term "peanut allergen" refers to peanuts (… Arachis hypogaea Any protein of the type and its isotypes that shows an allergic reaction in humans. Preferably, as used herein, the term "peanut allergen" refers to any peanut allergen and its isotypes that show an allergic reaction in humans. www.allergen.org Proteins whose amino acid sequences are searchable or have at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with such peanut allergens and their isotypes. More preferably, as used herein, the term "peanut allergen" refers to any of the currently identified 17 peanut allergens and their isotypes, as shown in [the original text]. www.allergen.orgThe protein is a protein whose amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with such peanut allergens and their isotypes. More preferably, as used herein, the term "peanut allergen" refers to any one of the following peanut allergens and their isotypes: h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, and Ara h17, or a protein whose amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with such peanut allergens and their isotypes. More preferably, as used herein, the term "peanut allergen" refers to any of the following peanut allergens and their isotypes: Ara h1, Ara h2, Ara h3, and Ara h6, or a protein whose amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with such peanut allergens and their isotypes. More preferably, as used herein, the term "peanut allergen" refers to any of the following proteins and their isotypes: Ara h1, Ara h2, Ara h3, and Ara h6, or a protein whose amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with such peanut allergens. More preferably, as used herein, the term "peanut allergen" refers to any protein selected from the following: Arah1, Ara h2, Ara h201, Ara h202, Ara h3 and Ara h6, or a protein whose amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 95% and even more preferably at least 98% amino acid sequence identity with such peanut allergens.

[0088] Fel d1 protein: As used herein, the term "Fel d1 protein" refers to a protein comprising Fel d1 chain 1 and Fel d1 chain 2, or alternatively composed thereof. Preferably, Fel d1 chain 1 and Fel d1 chain 2 are covalently linked. In a preferred embodiment, Fel d1 chain 1 and Fel d1 chain 2 are linked by at least one disulfide bond. In another preferred embodiment, chain 1 and chain 2 are fused directly or via a spacer, in which case the Fel d1 protein further comprises or alternatively consists of a spacer. Preferably, as defined herein, the Fel d1 protein comprises a total of up to 300, or more preferably up to 200 amino acids. Generally and preferably, the Fel d1 protein according to the invention is capable of in vivo inducing the production of antibodies that specifically bind to naturally occurring Fel d1.

[0089] Chain 1 of Fel d1: As used herein, the term "chain 1 of Fel d1" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or its homologous sequence, or alternatively composed thereof. As used herein, the term "homologous sequence of SEQ ID NO: 76" refers to a polypeptide having greater than 80%, more preferably greater than 90%, and even more preferably greater than 95% identity with SEQ ID NO: 76. As used herein, the term "chain 1 of Fel d1" should also refer to a polypeptide encompassing at least one post-translational modification of chain 1 of Fel d1 as defined herein, including, but not limited to, at least one glycosylated polypeptide. Preferably, as defined herein, chain 1 of Fel d1 consists of at most 130, and even more preferably at most 100 amino acids in total.

[0090] Chain 2 of Fel d1: As used herein, the term "chain 2 of Fel d1" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79, or a homologous sequence thereof, or alternatively composed thereof. As used herein, the term "homologous sequence of SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79" refers to a polypeptide having greater than 80%, more preferably greater than 90%, and even more preferably greater than 95% identity with SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79. As used herein, the term "chain 2 of Fel d1" should also refer to a polypeptide encompassing at least one post-translational modification of chain 2 of Fel d1 as defined herein, including but not limited to at least one glycosylated polypeptide, preferably, as defined herein, chain 2 of Fel d1 consists of at most 150, even more preferably at most 130, and still more preferably at most 100 amino acids in total.

[0091] Adjuvants: As used herein, the term "adjuvant" refers to a nonspecific stimulant of an immune response or a substance that allows the formation of a reservoir in the host, which, when combined with the vaccine and pharmaceutical compositions of the present invention, can provide an even stronger immune response. Preferred adjuvants are complete and incomplete Freund's adjuvants, aluminum-containing adjuvants, preferably aluminum hydroxide, and modified muramyl dipeptides. Further preferred adjuvants are mineral gels (such as aluminum hydroxide), surfactants (such as lysophosphatidylcholine, Pluronic acid polyol, polyanionic peptides, oil emulsions, keyforaminifera hemocyanin, and dinitrophenol), and human adjuvants (such as BCG and Corynebacterium pumilus). Such adjuvants are also well known in the art. Additional adjuvants that can be administered with the compositions of the present invention include, but are not limited to, monophosphoryl lipid immunomodulators, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (alum), MF-59, OM-174, OM-197, OM-294, and virosomal adjuvant technology. Adjuvants may also comprise mixtures of these substances. Virus-like particles are generally described as adjuvants. However, as used in the context of this application, the term "adjuvant" refers to an adjuvant that is not a virus-like particle of the present invention. Rather, "adjuvant" refers to an additional, distinct component of the compositions, vaccines, or pharmaceutical compositions of the present invention.

[0092] Amino acid linker: As used herein, the term "amino acid linker" refers to a linker consisting solely of amino acid residues. The amino acid residues of an amino acid linker are composed of naturally occurring or non-natural amino acids known in the art, all-L or all-D, or mixtures thereof. Preferably, the amino acid residues of an amino acid linker are naturally occurring amino acids, all-L or all-D, or mixtures thereof.

[0093] GS linker: As used herein, the term "GS linker" refers to a linker consisting only of glycine residues and serine amino acid residues. The GS linker according to the invention comprises at least one glycine residue and at least one serine residue. Typically and preferably, the GS linker according to the invention is up to 50 amino acids in length, and typically and more preferably, the GS linker according to the invention is up to 30 amino acids in length.

[0094] GST linker: As used herein, the term "GST linker" refers to a linker comprising, preferably composed of, glycine, serine, and threonine amino acid residues. The GST linker according to the invention comprises at least one glycine residue, at least one serine residue, and at least one threonine residue. Typically and preferably, the GST linker according to the invention is up to 50 amino acids in length, and typically and more preferably, the GST linker according to the invention is up to 30 amino acids in length.

[0095] GSED linker: As used herein, the term "GSED linker" refers to a linker comprising, preferably, glycine, serine, glutamic acid, and aspartic acid amino acid residues. The GSED linker according to the invention comprises at least one glycine residue, at least one serine residue, at least one glutamic acid residue, and at least one aspartic acid residue. Typically and preferably, the GSED linker according to the invention is at most 50 amino acids in length, and typically and more preferably, the GSED linker according to the invention is at most 30 amino acids in length.

[0096] Immunostimulatory substances: As used herein, the term "immunostimulatory substance" refers to a substance capable of inducing and / or enhancing an immune response. As used herein, immunostimulatory substances include, but are not limited to, Toll-like receptor activators and substances that induce cytokine secretion. Toll-like receptor activators include, but are not limited to, immunostimulatory nucleic acids, peptidoglycans, lipopolysaccharides, lipoteichoic acid, imidazoquinone compounds, flagellins, lipoproteins, and immunostimulatory organic substances such as paclitaxel.

[0097] Immunostimulatory nucleic acid (ISS-NA): As used herein, the term immunostimulatory nucleic acid refers to a nucleic acid capable of inducing and / or enhancing an immune response. Immunostimulatory nucleic acids include ribonucleic acid (RNA), and more particularly deoxyribonucleic acid (DNA), wherein both RNA and DNA can be double-stranded or single-stranded. Preferred ISS-NAs are DNA, wherein, more preferably, the DNA is single-stranded. Preferably, the immunostimulatory nucleic acid contains at least one CpG motif comprising an unmethylated C. Very preferred immunostimulatory nucleic acids comprise at least one CpG motif, wherein the at least one CpG motif comprises or preferably consists of at least one CG dinucleotide, wherein the C is unmethylated. Preferably, but not necessarily, the CG dinucleotide is part of a palindromic sequence. The term immunostimulatory nucleic acid also refers to a nucleic acid containing a modified base, preferably 4-bromocytosine. In the context of this invention, particularly preferred are ISS-NAs capable of stimulating the production of IFN-α in dendritic cells. Immunostimulatory nucleic acids that can be used for the purposes of this invention are described, for example, in WO2007 / 068747A1.

[0098] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a nucleic acid sequence comprising 2 or more nucleotides, preferably from about 6 to about 200 nucleotides, more preferably from 20 to about 100 nucleotides, and most preferably from 20 to 40 nucleotides. Very preferably, an oligonucleotide comprises about 30 nucleotides, more preferably exactly 30 nucleotides, and most preferably, an oligonucleotide consists of exactly 30 nucleotides. The oligonucleotide is a polynucleotide or polydeoxynucleotide, and is preferably selected from (a) unmodified RNA or DNA, and (b) modified RNA or DNA. Modification may include a backbone or nucleotide analogs. Preferably, the oligonucleotide is selected from the group consisting of: (a) single-stranded and double-stranded DNA, (b) DNA as a mixture of single-stranded and double-stranded regions, (c) single-stranded and double-stranded RNA, (d) RNA as a mixture of single-stranded and double-stranded regions, and (e) hybrid molecules comprising single-stranded or more preferably double-stranded DNA and RNA or a mixture of single-stranded and double-stranded regions. Preferred nucleotide modifications / analytes are selected from the group consisting of: (a) peptide nucleic acids; (b) inosine; (c) triphenylmethylated bases; (d) thiophosphate esters; (e) alkyl thiophosphate esters; (f) 5-nitroindole deoxyribosylfuranosyl; (g) 5-methyldeoxycytosine; and (h) 5,6-dihydro-5,6-dihydroxydeoxythymidine. Phosphophosphate-modified nucleotides are protected from degradation in cells or organisms and are therefore preferred nucleotide modifications. Unmodified oligonucleotides consisting only of phosphodiester-bound nucleotides are generally more active than modified nucleotides and are therefore generally preferred in the context of this invention. Most preferred are oligonucleotides consisting only of phosphodiester-bound deoxynucleotides, wherein, more preferably, the oligonucleotides are single-stranded. Further preferred are oligonucleotides capable of stimulating IFN-α production in cells (preferably dendritic cells). Very preferred oligonucleotides capable of stimulating IFN-α production in cells are selected from type A CpG and type C CpG. Further preferred are RNA molecules without cap.

[0099] CpG motif: As used herein, the term "CpG motif" refers to a pattern of nucleotides containing an unmethylated central CpG, i.e., an unmethylated CpG dinucleotide, wherein the C is unmethylated and surrounded by at least one base, preferably one or two nucleotides, flanking the central CpG (located on the 3' and 5' sides). Typically and preferably, as used herein, a CpG motif comprises an unmethylated CpG dinucleotide and two nucleotides at its 5' and 3' ends, or alternatively, consists of them. Unbound by theory, the bases flanking the CpG impart a significant portion of the activity to CpG oligonucleotides.

[0100] Unmethylated CpG-containing oligonucleotides: As used herein, the term "unmethylated CpG-containing oligonucleotide" or "CpG" refers to an oligonucleotide containing at least one CpG motif, preferably an oligodeoxynucleotide. Thus, CpG contains at least one unmethylated cytosine-guanine dinucleotide. Preferred CpGs stimulate / activate, for example, vertebrate bone marrow-derived cells, by promoting mitosis or inducing or increasing the expression of their cytokines. For example, CpG can be used to activate B cells, NK cells, and antigen-presenting cells such as dendritic cells, monocytes, and macrophages. Preferably, CpG relates to an oligodeoxynucleotide, more preferably to a single-stranded oligodeoxynucleotide containing unmethylated cytosine, followed by guanosine, wherein the unmethylated cytosine and the guanosine are linked by a phosphate ester bond, wherein preferably the phosphate ester bond is a phosphodiester bond or a thiophosphate bond, and further preferably, the phosphate ester bond is a phosphodiester bond. CpGs may contain nucleotide analogs, such as those containing phosphate thioester bonds, and may be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have higher immunomodulatory activity. Preferably, as used herein, a CpG is an oligonucleotide of at least about ten nucleotides in length and comprising at least one CpG motif, wherein more preferably the CpG is 10 to 60 nucleotides in length, more preferably 15 to 50 nucleotides, still more preferably 20 to 40 nucleotides, still more preferably about 30 nucleotides, and most preferably exactly 30 nucleotides. A CpG may consist of methylated and / or unmethylated nucleotides, wherein the at least one CpG motif comprises at least one CG dinucleotide, wherein C is unmethylated. A CpG may also comprise methylated and unmethylated sequence segments, wherein the at least one CpG motif comprises at least one CG dinucleotide, wherein C is unmethylated. Very preferably, the CpG relates to a single-stranded oligodeoxynucleotide containing an unmethylated cytosine, followed by a guanosine, wherein the unmethylated cytosine and the guanosine are linked by a phosphodiester bond. The CpG may comprise a nucleotide analog, such as an analog containing a thiophosphate bond, and may be double-stranded or single-stranded. Typically, as indicated below, phosphodiester CpGs are type A CpGs, while thiophosphate-stabilized CpGs are type B CpGs. In the context of this invention, preferred CpG oligonucleotides are type A CpGs.

[0101] Type A CpG: As used herein, the term "Type A CpG" or "Type D CpG" refers to an oligodeoxynucleotide (ODN) comprising at least one CpG motif. Type A CpG preferentially stimulates T cell activation and dendritic cell maturation and is capable of stimulating IFN-α production. In Type A CpG, at least one CpG motif nucleotide is linked by at least one phosphodiester bond. Type A CpG comprises at least one phosphodiester bonded CpG motif, which may be side-attached at its 5' end and / or preferably at its 3' end with a phosphate-thioester-bound nucleotide. Preferably, the CpG motif, and particularly preferably, the CG dinucleotide and its directly side-attached region comprising at least one, preferably two, nucleotides, are composed of phosphodiester nucleotides. Preferred Type A CpG consists only of phosphodiester (PO) bonded nucleotides. Typically and preferably, the polyG motif comprises at least one, preferably at least three, at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen Gs (guanosine), most preferably at least ten Gs, or alternatively composed thereof. Preferably, the type A CpG of the present invention comprises a palindromic sequence or alternatively composed thereof.

[0102] Packaged: As used herein, the term "packaged" refers to the state of the polyanionic macromolecule or immunostimulant relative to the core particle and the VLP, respectively. As used herein, the term "packaged" includes covalent bonding, e.g., by chemical coupling, or non-covalent bonding, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc. The term also includes encapsulation or partial encapsulation of the polyanionic macromolecule. Thus, the polyanionic macromolecule or immunostimulant can be encapsulated by the VLP without actual bonding, particularly covalent bonding. In a preferred embodiment, at least one polyanionic macromolecule or immunostimulant is most preferably packaged non-covalently within the VLP. If the immunostimulant is a nucleic acid, preferably DNA, the term "packaged" means that the nucleic acid is not available for nuclease hydrolysis, preferably not available for DNase hydrolysis (e.g., DNase I or nucleases), wherein the accessibility is preferably as described in Examples 11-17 of WO2003 / 024481A2.

[0103] Effective Amount: As used herein, the term "effective amount" refers to the amount necessary or sufficient to achieve the desired biological effect. The effective amount of a composition, or alternatively a pharmaceutical composition, will be the amount required to achieve this desired result, and such an amount can be routinely determined by those skilled in the art. Preferably, as used herein, the term "effective amount" refers to the amount necessary or effective in reducing the level of the at least one peanut allergen to a level that alleviates at least one symptom caused by peanut allergy. Preferably, as used herein, the term "effective amount" refers to the amount necessary or effective in neutralizing the activity of at least one peanut allergen. Effective amounts can vary depending on the specific composition administered and the body type of the subject. Those skilled in the art can determine the effective amount of a particular composition of the present invention empirically without the need for excessive experimentation.

[0104] Treatment: As used herein, the terms “treatment,” “treat,” “treated,” or “treating” refer to prevention and / or treatment. In one embodiment, the terms “treatment,” “treat,” “treated,” or “treating” refer to therapeutic treatment. In another embodiment, the terms “treatment,” “treat,” “treated,” or “treating” refer to preventive treatment.

[0105] In a first aspect, the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising at least one fusion protein, wherein the at least one fusion protein comprises or preferably consists of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide comprising or preferably consists of a coat protein of CMV, wherein preferably the coat protein of CMV comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is located in the CMV polypeptide at a position corresponding to SEQ ID NO: 62. The amino acid residues corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and (iii) a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62. In a very preferred embodiment, the chimeric CMV polypeptide further comprises a first amino acid linker, preferably a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigenic polypeptide, and wherein the first amino acid linker is selected from the group consisting of: (a) a polyglycine linker (Gly) of length n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

[0106] Preferred amino acid linkers, namely GS linkers or GSED linkers, have been found to be advantageous in overcoming the spherical disorder that hinders the assembly process of the modified VLPs of the present invention and / or in overcoming the aggregation tendency between the modified VLPs formed by the present invention and / or in increasing the flexibility of insertion of even very long antigenic peptides. This is particularly advantageous for the preferred CMV peptides of the present invention if the GS linker or GSED linker is further used as a first amino acid linker and a second amino acid linker, and even further, if the first amino acid linker and the second amino acid linker mimic the amino acid sequence present without the inserted antigenic peptide. Thus, it has been found that mimicking the amino acid sequence present without the inserted antigenic peptide is particularly advantageous for the insertion of the antigenic peptide between the amino acid residues corresponding to positions 84 and 85 of SEQ ID NO: 62; particularly between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5. It has been found that the insertion of an antigenic peptide after GS (i.e., after position 84 of SEQ ID NO: 62 and position 88 of SEQ ID NO: 5, respectively) and before YY (i.e., before position 85 of SEQ ID NO: 62 and position 89 of SEQ ID NO: 5, respectively) by preferably using a GS linker or a GSED linker, and particularly by using an insertion of an antigenic peptide with a second amino acid linker located at the C-terminus of the antigenic peptide and serving as a GS linker or a GSED linker ending in GS, is particularly advantageous.

[0107] In another aspect, the present invention provides a modified VLP of CMV comprising at least one fusion protein, wherein the at least one fusion protein comprises or preferably consists of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide comprising or preferably consists of a CMV coat protein, wherein preferably the CMV coat protein comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with the coat protein, preferably with the SEQ ID NO: 62; and (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is located in the CMV polypeptide at the position of the SEQ ID NO: Between the amino acid residues corresponding to amino acid residues at positions 84 and 85 of position 62; and (iii) a first amino acid linker, preferably a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigenic polypeptide, and wherein preferably the first amino acid linker is selected from the group consisting of: (a) polyglycine linkers (Gly) of length n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , wherein r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu. In a highly preferred embodiment, the chimeric CMV polypeptide further comprises a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62.

[0108] In another aspect, the present invention provides a mosaic-modified virus-like particle (VLP) of cucumber mosaic virus (CMV). In a first aspect, the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising: (a) at least one fusion protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, wherein the CMV polypeptide comprises or preferably comprises a coat protein of CMV, wherein preferably the coat protein of CMV comprises or preferably comprises: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is at a position of the CMV polypeptide corresponding to SEQ ID NO: 62. (a) the amino acid residues corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and (b) at least one CMV protein, wherein the CMV protein comprises or is preferably composed of a shell protein of CMV, wherein preferably the shell protein of CMV comprises or is preferably composed of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62; and wherein the CMV protein is optionally modified by a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, and wherein preferably the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62; wherein preferably the CMV protein comprises, and is preferably composed of, SEQ ID NO: 5. In a highly preferred embodiment, the chimeric CMV polypeptide further includes a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein preferably the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62.In another highly preferred embodiment, the chimeric CMV polypeptide further includes a first amino acid linker, preferably a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigen polypeptide, and wherein the first amino acid linker is selected from the group consisting of: (a) a polyglycine linker (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

[0109] For all aspects of the invention, the insertion of the antigenic polypeptide is located between the amino acid residues of the CMV polypeptide corresponding to the amino acid residues at positions 84 and 85 of SEQ ID NO: 62, and the insertion corresponds to the insertion between the serine (S) residue at position 84 of SEQ ID NO: 62 and the tyrosine (Y) residue at position 85 of SEQ ID NO: 62.

[0110] The embodiments, preferred embodiments, and highly preferred embodiments described and disclosed herein should be applied to all aspects, as well as other embodiments, preferred embodiments, and highly preferred embodiments, whether specifically mentioned again or to avoid repetition for the sake of brevity.

[0111] In a preferred embodiment, the CMV polypeptide comprises an amino acid sequence of the outer shell protein of CMV or a mutated amino acid sequence, preferably composed of the same, wherein the amino acid sequence to be mutated is an amino acid sequence of the outer shell protein of CMV, and wherein the mutated amino acid sequence of CMV and the outer shell protein exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% sequence identity; wherein preferably the mutated amino acid sequence and the amino acid sequence to be mutated differ at at least 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, and wherein further preferably these differences are selected from (i) insertion, (ii) deletion, (iii) amino acid exchange, and (iv) any combination of (i) to (iii).

[0112] In a preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62. In yet another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide comprises the outer shell protein of CMV or an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62. In yet another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV polypeptide consists of the outer shell protein of CMV or an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide is the outer shell protein of CMV or an amino acid sequence having at least 75%, preferably 85%, sequence identity with SEQ ID NO: 62.In a preferred embodiment, the CMV polypeptide is a CMV capsid protein or an amino acid sequence having at least 90%, preferably 95%, sequence identity with SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide is a CMV capsid protein having SEQ ID NO: 62. In a preferred embodiment, the CMV capsid protein comprises SEQ ID NO: 62. In a preferred embodiment, the CMV capsid protein is composed of SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide comprises a CMV capsid protein. In a preferred embodiment, the CMV polypeptide is composed of a CMV capsid protein. In a preferred embodiment, the CMV polypeptide comprises a CMV capsid protein, wherein the CMV capsid protein comprises SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide comprises a CMV capsid protein, wherein the CMV capsid protein is composed of SEQ ID NO: 62. In a preferred embodiment, the CMV polypeptide is composed of a CMV capsid protein, wherein the CMV capsid protein is composed of SEQ ID NO: 62.

[0113] In a preferred embodiment, the CMV polypeptide includes the amino acid sequence region of SEQ ID NO: 63, wherein the amino acid sequence region has at least 75% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide includes the amino acid sequence region of SEQ ID NO: 63, wherein the amino acid sequence region has at least 80% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide includes the amino acid sequence region of SEQ ID NO: 63, wherein the amino acid sequence region has at least 85% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide includes the amino acid sequence region of SEQ ID NO: 63, wherein the amino acid sequence region has at least 90% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide includes the amino acid sequence region of SEQ ID NO: 63, wherein the amino acid sequence region has at least 95% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide includes SEQ ID NO: 63 or an amino acid sequence region, wherein the amino acid sequence region has at least 98% sequence identity with SEQ ID NO: 63. In another preferred embodiment, the CMV polypeptide includes SEQ ID NO: 63 or an amino acid sequence region, wherein the amino acid sequence region has at least 99% sequence identity with SEQ ID NO: 63.

[0114] In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of a CMV shell protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO: 62; or (ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO: 63 or an amino acid sequence region, wherein the amino acid sequence region has at least 90% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of a CMV shell protein, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO: 62; or (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO: 63 or an amino acid sequence region, wherein the amino acid sequence region has at least 95% sequence identity with SEQ ID NO: 63. In a preferred embodiment, the CMV polypeptide comprises or preferably consists of: (i) an amino acid sequence of the outer shell protein of CMV, wherein the amino acid sequence comprises or preferably consists of SEQ ID NO: 62; or (ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62; and wherein the amino acid sequence as defined in (i) or (ii) comprises SEQ ID NO: 63.

[0115] In a preferred embodiment, the number of amino acids in the substituted N-terminal region is equal to or less than the number of amino acids constituting the T helper cell epitope. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 9 to 14 consecutive amino acids. In a preferred embodiment, the substituted N-terminal region of the CMV polypeptide consists of 11 to 13 consecutive amino acids. In a preferred embodiment, the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62. In a preferred embodiment, the T helper cell epitope is a generic T helper cell epitope. In a preferred embodiment, the T helper cell epitope consists of up to 20 amino acids.

[0116] In a preferred embodiment of the present invention, the Th cell epitope is selected from HA 307-319 (SEQ ID NO: 67), HBVnc50-69 (SEQ ID NO: 68), TT 830-843 (SEQ ID NO: 64), CS 378-398 (SEQ ID NO: 69), MT17-31 (SEQ ID NO: 70), TT 947-967 (SEQ ID NO: 71), and PADRE (SEQ ID NO: 65). In a very preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin or a PADRE sequence. In a preferred embodiment, the T helper cell epitope is derived from a human vaccine. In a very preferred embodiment, the Th cell epitope is a Th cell epitope derived from tetanus toxin. In a preferred embodiment, the Th cell epitope is a PADRE sequence. In a very preferred embodiment, the Th cell epitope includes the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65. In a very preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65. In a very preferred embodiment, the Th cell epitope includes the amino acid sequence of SEQ ID NO: 64. In a preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 64. In a very preferred embodiment, the Th cell epitope includes the amino acid sequence of SEQ ID NO: 65. In a very preferred embodiment, the Th cell epitope consists of the amino acid sequence of SEQ ID NO: 65.

[0117] In a preferred embodiment, the CMV polypeptide comprises or preferably consists of the amino acid sequence of the outer shell protein of CMV, wherein the amino acid sequence comprises or preferably consists of the following: SEQ ID NO: 62 or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62; and wherein the amino sequence comprises SEQ ID NO: 63, and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and wherein the substituted N-terminal region of the CMV polypeptide consists of 11 to 13 consecutive amino acids, preferably 11 consecutive amino acids, and wherein, more preferably, the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62.

[0118] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 66.

[0119] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 66, wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 5 at position 88 and position 89 of SEQ ID NO: 5, or wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 66 at position 86 and position 87 of SEQ ID NO: 66.

[0120] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 66, wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 66 at position 86 between amino acid residues 87 of SEQ ID NO: 66.

[0121] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 5 between amino acid residues at position 88 and position 89 of SEQ ID NO: 5.

[0122] In a preferred embodiment, the chimeric CMV polypeptide further includes a first amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigen polypeptide, and wherein the first amino acid linker is selected from the group consisting of: (a) a polyglycine linker (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu. In a preferred embodiment, the chimeric CMV polypeptide further comprises a second amino acid linker, wherein the second amino acid linker is located at the N-terminus or C-terminus of the antigenic polypeptide, and wherein the second amino acid linker is selected from the group consisting of: (a) a polyglycine linker (Gly) with a length of n = 2-10. n(b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu. In a preferred embodiment, the chimeric CMV polypeptide further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker is located at the N-terminus of the antigenic polypeptide, and the second amino acid linker is located at the C-terminus of the antigenic polypeptide, and wherein the first amino acid linker and the second amino acid linker are independently selected from the group consisting of: (a) a polyglycine linker (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

[0123] In a preferred embodiment, the length of the first amino acid linker is at most 30 amino acids. In a preferred embodiment, the length of the first amino acid linker is at most 20 amino acids. In a preferred embodiment, the length of the first amino acid linker is at most 15 amino acids. In a preferred embodiment, the length of the second amino acid linker is at most 30 amino acids. In a preferred embodiment, the length of the second amino acid linker is at most 20 amino acids. In a preferred embodiment, the length of the second amino acid linker is at most 15 amino acids. In a preferred embodiment, the first amino acid linker is located at the N-terminus of the antigenic peptide. In a preferred embodiment, the first amino acid linker is located at the C-terminus of the antigenic peptide. In a preferred embodiment, the chimeric CMV peptide further includes a second amino acid linker. In a preferred embodiment, the first amino acid linker is located at the N-terminus of the antigenic peptide. In a preferred embodiment, the second amino acid linker is located at the C-terminus of the antigenic peptide. In a preferred embodiment, the chimeric CMV peptide includes a first amino acid linker and a second amino acid linker. In a preferred embodiment, the first amino acid linker is located at the N-terminus of the antigenic peptide, and the second amino acid linker is located at the C-terminus of the antigenic peptide. In a preferred embodiment, the first amino acid linker is selected from the group consisting of: (a) polyglycine linkers (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5, and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu, wherein preferably, the amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu is a GST linker or a GSED linker. In a preferred embodiment, the first amino acid linker is a polyglycine linker (Gly) with a length of n = 2-10. n In a preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine. In another preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, and the second amino acid linker has a Gly-Ser sequence at its N-terminus. In yet another preferred embodiment, the amino acid sequence of the GS linker is (GS).r (G s S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5, and u = 0 or 1. In another preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker), and the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) uWhere r = 0 or 1, s = 3 or 4, t = 1, 2 or 3 and u = 0 or 1. In another preferred embodiment, the GS linker is at most 30 amino acids long. In another preferred embodiment, the GS linker is at most 20 amino acids long. In another preferred embodiment, the first amino acid linker is a glycine-serine linker (GS linker), and the amino acid sequence of the GS linker is selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 47. In another preferred embodiment, the first amino acid linker has an amino acid sequence selected from SEQ ID NO: 10 and SEQ ID NO: 30. In a preferred embodiment, the first amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu. In a preferred embodiment, the first amino acid linker is a GST linker comprising at least one Gly, at least one Ser, and at least Thr. In another preferred embodiment, the first amino acid linker is a GSED linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid. In yet another preferred embodiment, the first amino acid linker is a GSED linker comprising at least one Gly, at least one Ser, and at least Thr, and the second amino acid linker has a Gly-Ser at its N-terminus. In yet another preferred embodiment, the first amino acid linker is a GSED linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid, and the second amino acid linker has a Gly-Ser at its N-terminus. In a preferred embodiment, the first amino acid linker is a GS linker comprising at least one glycine and at least one serine, a GST linker comprising at least one Gly, at least one Ser, and at least Thr, or a GSED linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid, and the second amino acid linker has a Gly-Ser at its N-terminus. In another preferred embodiment, the first amino acid linker is a GSED linker, wherein the amino acid sequence of the GSED linker is (DED). x (G s S) t (G) y (DED) z (GS) uWhere s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5, and z = 0 or 1. In another preferred embodiment, the first amino acid linker is a GSED linker, wherein the amino acid sequence of the GSED linker is (DED). x (G s S) t (G) y (DED) z (GS) u Where s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 0, y = 1-5, preferably y = 3 and z = 1. In another preferred embodiment, the GSED linker is up to 30 amino acids long. In another preferred embodiment, the GSED linker is up to 20 amino acids long. In another preferred embodiment, the first amino acid linker is a GSED linker, and the GSED linker comprises, preferably constitutes, the amino acid sequence SEQ ID NO: 126.

[0124] In a preferred embodiment, the second amino acid linker is selected from the group consisting of: (a) polyglycine linkers (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu. In a preferred embodiment, the second amino acid linker is selected from the group consisting of: (a) polyglycine linkers (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) uWhere r = 0 or 1, s = 1-5, t = 1-5, and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu, wherein preferably, the amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu is a GST linker or a GSED linker. In a preferred embodiment, the second amino acid linker is a polyglycine linker (Gly) with a length n = 2-10. n In a preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker) composed of at least one glycine and at least one serine. In another preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, and the second amino acid linker has a Gly-Ser sequence at its N-terminus. In yet another preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker), and the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 3 or 4, t = 1, 2 or 3, and u = 0 or 1. In another preferred embodiment, the GS linker is at most 30 amino acids long. In another preferred embodiment, the GS linker is at most 20 amino acids long. In another preferred embodiment, the second amino acid linker is a glycine-serine linker (GS linker), and the amino acid sequence of the GS linker is selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 47. In another preferred embodiment, the second amino acid linker has an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 31, and SEQ ID NO: 47. In another preferred embodiment, the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1. In a preferred embodiment, the second amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu.

[0125] In a preferred embodiment, the second amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, and at least Thr (GST linker). In another preferred embodiment, the second amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid (GSED linker). In a preferred embodiment, the second amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, and at least Thr (GST linker), and the second amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the second amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid (GSED linker), and the second amino acid linker has Gly-Ser at its N-terminus. In a preferred embodiment, the second amino acid linker is a GS linker comprising at least one glycine and at least one serine, an amino acid linker comprising at least one Gly, at least one Ser, and at least Thr (GST linker), or an amino acid linker comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least aspartic acid (GSED linker), and the second amino acid linker has Gly-Ser at its N-terminus. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the amino acid sequence of the GSED linker is (DED). x (G s S) t (G) y (DED) z (GS) u Where s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5, and z = 0 or 1. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the GSED linker is composed of an amino acid sequence (DED). x (G s S) t (G) y (DED) z (GS) u The composition includes s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5, and z = 0 or 1. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the amino acid sequence of the GSED linker is (DED). x (G s S) t (G) y (DED) z (GS) uWhere s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 1, y = 0-5, preferably y = 0 and z = 0. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the GSED linker is composed of an amino acid sequence (DED). x (G s S) t (G) y (DED) z (GS) u The composition includes s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 1, y = 0-5, preferably y = 0 and z = 0. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the GSED linker comprises an amino acid sequence (TS)(DED). x (G s S) t (G) y (DED) z (GS) u Preferably, it comprises the following: s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5, and z = 0 or 1. In another preferred embodiment, the second amino acid linker is a GSED linker, wherein the GSED linker comprises an amino acid sequence (TS)(DED). x (G s S) t (G) y (DED) z (GS) u Preferably, the GSED linker is composed of the following: s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 1, y = 0-5, preferably y = 0 and z = 0. In another preferred embodiment, the length of the GSED linker is up to 30 amino acids. In another preferred embodiment, the length of the GSED linker is up to 20 amino acids. In another preferred embodiment, the second amino acid linker is a GSED linker, and the GSED linker comprises the amino acid sequence SEQ ID NO: 127, preferably composed of the following.

[0126] In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently selected from the group consisting of: (a) polyglycine linkers (Gly) with a length of n = 2-10. n (b) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the amino acid sequence of the GS linker is (GS). r (Gs S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c) an amino acid linker comprising at least one Gly, at least one Ser, and at least one amino acid selected from Thr, Ala, Lys, Asp, and Glu. In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently polyglycine linkers (Gly) with a length of n = 2-10. n In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine. In another preferred embodiment, the first amino acid linker and the second amino acid linker are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine, and wherein the second amino acid linker has a Gly-Ser sequence at its N-terminus. In yet another preferred embodiment, the amino acid sequence of the GS linker is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 1-5, t = 1-5, and u = 0 or 1. In another preferred embodiment, the first amino acid linker and the second amino acid linker are independently glycine-serine linkers (GS linkers), the amino acid sequence of which is (GS). r (G s S) t (GS) u Where r = 0 or 1, s = 3 or 4, t = 1, 2 or 3 and u = 0 or 1. In another preferred embodiment, the first amino acid linker and the second amino acid linker are independently glycine-serine linkers (GS linkers), and the amino acid sequence of the GS linker is selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 47. In a preferred embodiment, the first amino acid linker and the second amino acid linker are independently amino acid linkers comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

[0127] In a preferred embodiment, the first and second amino acid linkers are independently GSED linkers (GSED linkers) comprising at least one Gly, at least one Ser, at least one glutamic acid, and at least one aspartic acid, and the second amino acid linker has a Gly-Ser sequence at its N-terminus. In another preferred embodiment, the first and second amino acid linkers are independently GSED linkers, wherein the GSED linkers independently have an amino acid sequence (DED). x (G s S) t (G) y (DED) z (GS) u , where s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5 and z = 0 or 1; or (TS)(DED) x (G s S) t (G) y (DED) z (GS) u , where s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5 and z = 0 or 1.

[0128] In another preferred embodiment, the first amino acid linker and the second amino acid linker are independently GSED linkers, wherein the GSED linker independently has an amino acid sequence (DED). x (G s S) t (G) y (DED) z (GS) u Where s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 1, y = 0-5, preferably y = 0, and z = 0; or s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 0, y = 1-5, preferably y = 3 and z = 1; or (TS)(DED) x (G s S) t (G) y (DED) z (GS) u , where s = 3 or 4, t = 1, 2 or 3, u = 0 or 1, x = 1, y = 0-5, preferably y = 0, and z = 0.

[0129] In another preferred embodiment, the first amino acid linker and the second amino acid linker are independently GSED linkers, and the amino acid sequence of the GS linker is selected from SEQ ID NO: 126 and SEQ ID NO: 127.

[0130] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the antigenic polypeptide is inserted into the CMV polypeptide to be positioned between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of the CMV polypeptide of SEQ ID NO: 5.

[0131] In a highly preferred embodiment, the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the antigenic polypeptide is inserted into the CMV polypeptide to lie between amino acid residue 88 (Ser) and amino acid residue 89 (Thr) of the CMV polypeptide of SEQ ID NO: 5, and the chimeric CMV polypeptide further comprises a first amino acid linker and a second amino acid linker, wherein the first amino acid linker and the second amino acid linker are independently glycine-serine linkers (GS linkers) comprising at least one glycine and at least one serine, and wherein the second amino acid linker has a Gly-Ser sequence at its N-terminus.

[0132] In one aspect and in a preferred embodiment, the present invention provides mosaic virus-like particles. Therefore, in a preferred embodiment, the modified VLP of CMV further comprises at least one CMV protein, wherein the CMV protein comprises or is preferably composed of a CMV capsid protein, wherein preferably the CMV capsid protein comprises or is preferably composed of: SEQ ID NO: 62; or an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, further more preferably at least 95%, still further preferably at least 98%, and still further more preferably at least 99% sequence identity with SEQ ID NO: 62. In a preferred embodiment, the modified VLP of CMV further comprises at least one CMV protein, wherein the CMV protein comprises a CMV capsid protein or an amino acid sequence having at least 75%, preferably at least 85%, sequence identity with SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein preferably the CMV capsid protein comprises SEQ ID NO: 62.

[0133] In a preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62. In yet another preferred embodiment, the CMV protein comprises a CMV shell protein or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62. In another preferred embodiment, the CMV protein comprises a CMV capsid protein or an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 62. In a preferred embodiment, the CMV protein comprises a CMV capsid protein. In a preferred embodiment, the CMV protein is composed of a CMV capsid protein. In a preferred embodiment, the CMV protein comprises a CMV capsid protein, wherein the CMV capsid protein comprises SEQ ID NO: 62. In a preferred embodiment, the CMV protein comprises a CMV capsid protein, wherein the CMV capsid protein is composed of SEQ ID NO: 62. In a preferred embodiment, the CMV protein is composed of a CMV capsid protein, wherein the CMV capsid protein is composed of SEQ ID NO: 62. In another preferred embodiment, the CMV protein is modified by a T helper cell epitope, wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, and in another preferred embodiment, the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62. In a very preferred embodiment, the CMV protein comprises SEQ ID NO: 5. In a highly preferred embodiment, the CMV protein is composed of SEQ ID NO: 5.

[0134] In a preferred embodiment, the antigenic polypeptide is at least 3 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 3 amino acids long and at most 225 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 3 amino acids long and at most 200 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 40 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 40 amino acids long and at most 225 amino acids long, preferably at most 200 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 50 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 50 amino acids long and at most 200 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 70 amino acids long. In a preferred embodiment, the antigenic polypeptide is at least 70 amino acids long and at most 200 amino acids long.

[0135] In a preferred embodiment, the antigenic polypeptide is a polypeptide derived from the group consisting of: (a) allergens; (b) viruses; (c) bacteria; (d) parasites; (e) tumors; (h) automolecules; (i) hormones; (k) cytokines; (l) chemokines; and (h) bioactive peptides.

[0136] In another preferred embodiment, the antigenic polypeptide is a polypeptide of an allergen, autoantigen, tumor antigen, or pathogen.

[0137] In another preferred embodiment, the antigenic polypeptide is an allergen, wherein the allergen is preferably derived from the group consisting of: (a) pollen extract; (b) dust extract; (c) house dust mite extract; (d) fungal extract; (e) mammalian skin extract; (f) feather extract; (g) insect extract; (h) food extract; (i) hair extract; (j) saliva extract; and (k) serum extract. In another preferred embodiment, the antigenic polypeptide is an allergen, wherein the allergen is selected from the group consisting of: (a) trees; (b) grass; (c) house dust; (d) house dust mites; (e) Aspergillus; (f) animal hair; (g) animal feathers; (h) bee venom; (i) animal products; (j) plant products; (k) animal dander; and (l) peanut allergen.

[0138] In another preferred embodiment, the antigenic polypeptide is a recombinant polypeptide derived from an allergen selected from the group consisting of: (a) bee venom phospholipase A2; (b) ragweed pollen Amb a 1; (c) birch pollen Bet v I; (d) white-faced bumblebee venom 5 DoI m V; (e) house dust mite Der p 1; (f) house dust mite Der f 2; (g) house dust mite Der p 2; (h) house dust mite Lep d; (i) fungal allergen Alt a 1; (j) fungal allergen Asp f 1; (k) fungal allergen Asp f 16; (l) peanut allergen; (m) cat allergen d1; (n) canine allergens Can f1, Can f2; (o) peanut-derived allergen; or (p) Japanese cedar (Cry J2) allergen.

[0139] In another preferred embodiment, the antigenic polypeptide is a recombinant allergen selected from the group consisting of: (a) bee venom phospholipase A2; (b) ragweed pollen Amb a 1; (c) birch pollen Bet v I; (d) white-faced bumblebee venom 5 DoI m V; (e) house dust mite Der p 1; (f) house dust mite Der f 2; (g) house dust mite Der p 2; (h) house dust mite Lepd; (i) fungal allergen Alt a 1; (j) fungal allergen Asp f 1; (k) fungal allergen Asp f 16; (l) peanut allergen; (m) cat allergen d1; (n) canine allergens Can f1, Can f2; (o) peanut-derived allergen; or (p) Japanese cedar (Cry J2) allergen.

[0140] In another highly preferred embodiment, the antigenic polypeptide is a peanut allergen. Preferably, the antigenic polypeptide is a peanut allergen comprising an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73. In a highly preferred embodiment, the peanut allergen comprises or preferably consists of: a protein having an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73; or a protein having an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% sequence identity with SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73. In another highly preferred embodiment, the peanut allergen comprises or preferably consists of: a protein having an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73; or a protein having an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% sequence identity with SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73. In another highly preferred embodiment, the peanut allergen comprises a protein having an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 72, or SEQ ID NO: 73. In another highly preferred embodiment, the peanut allergen comprises the amino acid sequence of SEQ ID NO: 27. In another highly preferred embodiment, the peanut allergen consists of the amino acid sequence of SEQ ID NO: 27. In another highly preferred embodiment, the antigenic polypeptide comprises the amino acid sequence of SEQ ID NO: 27. In yet another highly preferred embodiment, the antigenic polypeptide consists of the amino acid sequence of SEQ ID NO: 27.

[0141] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a peanut allergen; and wherein the peanut allergen comprises an amino acid sequence selected from: (a) SEQ ID NO: 27; (b) SEQ ID NO: 72; (c) SEQ ID NO: 62; NO:73, and wherein preferably the peanut allergen comprises SEQ ID NO: 27, preferably composed of therein; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein preferably the T helper cell epitope comprises, preferably composed of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64.

[0142] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a peanut allergen; and wherein the peanut allergen comprises or preferably comprises SEQ ID NO: 27; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein preferably the T helper cell epitope comprises SEQ ID NO: 62. 64, preferably composed thereof. In another highly preferred embodiment, the chimeric CMV polypeptide is composed of SEQ ID NO:29. In another highly preferred embodiment and aspect herein, the invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating allergies, preferably peanut allergic reactions.

[0143] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein and further comprises at least one CMV protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a peanut allergen; and wherein the peanut allergen comprises or preferably comprises SEQ ID NO: 27; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein preferably the T helper cell epitope comprises SEQ ID NO: 64, preferably composed thereof; and wherein the CMV protein comprises the outer coat protein of CMV, preferably composed thereof, preferably SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably composed thereof. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 29, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment and aspect herein, the present invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating allergies, preferably peanut allergic reactions.

[0144] In another highly preferred embodiment, the antigenic polypeptide is an allergen derived from Japanese cedar Cry J 2. Preferably, the antigenic polypeptide is derived from Japanese cedar Cry J 2 of SEQ ID NO: 74. Preferably, the antigenic polypeptide is derived from Japanese cedar Cry J 2 and includes the amino acid sequence of SEQ ID NO: 74.

[0145] In another highly preferred embodiment, the antigenic polypeptide is an allergen derived from ragweed pollen Amb a1. Preferably, the antigenic polypeptide is derived from ragweed pollen Amb a1 of SEQ ID NO: 75. Preferably, the antigenic polypeptide is derived from ragweed pollen Amb a1 and includes the amino acid sequence of SEQ ID NO: 75.

[0146] In a highly preferred embodiment of the invention, the antigenic polypeptide is an allergen derived from the feline allergen Fel d1. (Domestic cats (domes ... Felis domesticusCats are a significant source of indoor allergens (Lau, S. et al., (2000) Lancet 356, 1392-1397). Symptoms range in severity from relatively mild rhinitis and conjunctivitis to potentially life-threatening asthma attacks. Although patients are sometimes sensitive to several different molecules in cat dander and fur, the primary allergen is Fel d1. The importance of this allergen has been highlighted in numerous studies. In fact, over 80% of cat allergy sufferers exhibit IgE antibodies against this potent allergen (van Ree, R. et al., (1999) J. Allergy Clin Immunol 104, 1223-1230). Fel d1 is a 35-39 kDa acidic glycoprotein containing 10-20% N-linked carbohydrates and is found in cat fur, saliva, and tear glands. It is formed from two non-covalently linked heterodimers. Each heterodimer consists of a 70-residue peptide (called “chain 1”) and a 78, 85, 90, or 92-residue peptide (called “chain 2”) encoded by a different gene (see Duffort, OA et al., (1991) Molecular Immunology 28, 301-309; Morgenstern, JP et al., (1991) Proceedings of the National Academy of Sciences of the United States of America 88, 9690-9694; and Griffith, IJ et al., (1992) Gene 113, 263-268). Several recombinant constructs of Fel d1 have been described (Vailes LD et al., J Allergy Clin Immunol (2002) 110:757-762; Grönlund H et al., J Biol Chem (2003) 278:40144-40151; 2003; Schmitz N et al., J Exp Med (2009) 206:1941-1955; WO2006 / 097530; WO2017 / 042241).

[0147] Therefore, in another highly preferred embodiment, the antigenic polypeptide is rFel d1. In another highly preferred embodiment, the antigenic polypeptide is the Fel d1 protein, wherein the Fel d1 protein is a fusion protein comprising chain 1 and chain 2 of Fel d1, wherein chain 2 of Fel d1 is fused directly to the N-terminus of chain 1 of Fel d1 via a peptide bond or via a spacer through its C-terminus, wherein the spacer consists of an amino acid sequence having 1 to 20 amino acid residues, wherein preferably the spacer consists of an amino acid sequence having 10 to 20 amino acid residues. Very preferably, the spacer consists of an amino acid sequence of 15 amino acid residues, and more preferably the spacer has the amino acid sequence of SEQ ID NO: 30. In another highly preferred embodiment, the antigenic polypeptide is the Fel d1 protein, wherein the Fel d1 protein is a fusion protein comprising chain 1 and chain 2 of Fel d1, wherein chain 1 of Fel d1 is fused directly to the N-terminus of chain 2 of Fel d1 via a peptide bond or via a spacer through its C-terminus, wherein the spacer consists of an amino acid sequence having 1 to 20 amino acid residues, preferably having an amino acid sequence having 10 to 20 amino acid residues. Preferably, chain 1 of Fel d1 comprises the sequence of SEQ ID NO: 76 or a homologous sequence thereof, wherein the homologous sequence shares greater than 90% identity with SEQ ID NO: 76, or even more preferably greater than 95%. More preferably, the chain 2 of Fel d1 includes the sequence of SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79 or its homologous sequence, wherein the homologous sequence has an identity of more than 90% with SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79 and even more preferably more than 95%.

[0148] In a highly preferred embodiment, the antigenic polypeptide is a Fel d1 protein comprising the amino acid sequence selected from: (a) SEQ ID NO: 38; (b) SEQ ID NO: 80; or (c) SEQ ID NO: 81. In another highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 38, preferably. In a highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 80, preferably. In another highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 81, preferably.

[0149] In another highly preferred embodiment, the Fel d1 protein comprises an amino acid sequence selected from (a) SEQ ID NO: 38; (b) SEQ ID NO: 80; (c) SEQ ID NO: 81.

[0150] In another highly preferred embodiment, the Fel d1 protein comprises, preferably constitutes, the amino acid sequence of SEQ ID NO: 38. In another highly preferred embodiment, the Fel d1 protein comprises, preferably constitutes, the amino acid sequence of SEQ ID NO: 80. In another highly preferred embodiment, the Fel d1 protein comprises, preferably constitutes, the amino acid sequence of SEQ ID NO: 81.

[0151] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a Fel d1 protein; and wherein the Fel d1 protein comprises an amino acid sequence selected from: (a) SEQ ID NO: 38; (b) SEQ ID NO: 80; (c) SEQ ID NO: 81, wherein the Fel d1 protein preferably comprises SEQ ID NO: 38, preferably composed thereof; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably composed of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64.

[0152] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a Fel d1 protein; and wherein the Fel d1 protein comprises or preferably comprises SEQ ID NO: 38; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein preferably the T helper cell epitope comprises SEQ ID NO: 38. 64, preferably composed thereof. In another highly preferred embodiment, the chimeric CMV polypeptide is composed of SEQ ID NO:39. In another highly preferred embodiment and aspect herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in methods of treating allergies, preferably feline allergies.

[0153] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein and further comprises at least one CMV protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is a Feld1 protein; and wherein the Feld1 protein comprises or preferably comprises SEQ ID NO: 38; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: The CMV protein comprises amino acids 2-12 of SEQ ID NO: 62, and preferably the T helper cell epitope includes SEQ ID NO: 64, preferably composed thereof; and the CMV protein comprises the outer coat protein of CMV, preferably composed thereof, preferably SEQ ID NO: 62, and the CMV protein is optionally modified by a T helper cell epitope, and the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62, and preferably the T helper cell epitope includes SEQ ID NO: 64, preferably composed thereof. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 39, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment and aspect herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating allergies, preferably feline allergies.

[0154] In another preferred embodiment, the antigenic polypeptide is a tumor antigen, wherein the tumor antigen is preferably selected from the group consisting of: (a) polypeptides of breast cancer cells; (b) polypeptides of kidney cancer cells; (c) polypeptides of prostate cancer cells; (d) polypeptides of skin cancer cells; (e) polypeptides of brain cancer cells; and (f) polypeptides of leukemia cells.

[0155] In another preferred embodiment, the antigenic polypeptide is a tumor antigen selected from the group consisting of: (a) Her2; (b) ganglioside GD2; (c) EGF-R; (d) carcinoembryonic antigen (CEA); (e) CD52; (f) CD21; (g) human melanoma gp100; (h) human melanoma melanA / MART-1; (i) human melanoma melanA / MART-1 analog; (j) tyrosinase; (k) NA17-A nt; (l) MAGE3; (m) p53 protein; and (n) an antigen fragment of any of the tumor antigens in (a) to (m).

[0156] In another preferred embodiment, the antigenic polypeptide is a polypeptide selected from the group consisting of: (a) IgE; (b) IL-6; (c) receptor activator of nuclear factor kappa ligand (RANKL); (d) vascular endothelial growth factor (VEGF); (e) vascular endothelial growth factor receptor (VEGF-R); hepatocyte growth factor (HGF); (f) interleukin-1α; (g) interleukin-1β; (h) interleukin-5; (i) interleukin-8; (j) interleukin-13; (k) interleukin-15; (l) interleukin-17 (IL-17); (m) IL-23; (n) β-L-1. (o) Angiotensin; (p) Chemokines (CC motif) (CCL21); (q) Chemokines (CX motif) (CXCL12); (r) Stromal cell-derived factor 1 (SDF-I); (s) Macrophage colony-stimulating factor (M-CSF); (t) Monocyte chemoattractant protein 1 (MCP-I); (u) Endothelial glycoprotein; (v) Resistin; (w) Gonadotropin-releasing hormone (GnRH); (x) Growth hormone-releasing hormone (GHRH); (y) Luteinizing hormone-releasing hormone (LHRH); (z) Thyrotropin-releasing hormone (TRH); (aa) Macrophage Cell migration inhibitory factor (MIF); (bb) glucose-dependent insulinotropic peptide (GIP); (cc) eosinophil chemokine; (dd) bradykinin; (ee) Des-Arg bradykinin; (ff) B lymphocyte chemoattractant protein (BLC); (gg) macrophage colony-stimulating factor M-CSF; (hh) tumor necrosis factor α (TNFα); (ii) amyloid-β peptide (Aβ1-42); (jj) amyloid-β peptide (Aβ3-6); (kk) human IgE; (ii) CCR5 extracellular domain; (mm) CXCR4 extracellular domain; (nn) gastrin ; (oo) CETP; (pp) C5a; (qq) Epidermal growth factor receptor (EGF-R); (rr) CGRP; (ss) α-synuclein; (tt) calcitonin gene-related peptide (CGRP); (uu) dextrin; (vv) myostatin; (ww) interleukin-4; (xx) thymic stromal lymphopoietin; (yy) interleukin-33; (zz) interleukin-25; (aaa) interleukin-13 or (bbb) fragments of any one of polypeptides (a) to (aaa); and (ccc) antigenic mutants or fragments of any one of polypeptides (a) to (aaa).

[0157] In another preferred embodiment, the antigenic polypeptide is an autoantigen, wherein the autoantigen is a polypeptide selected from the group consisting of: (a) IgE; (b) IL-6; (c) receptor activator of nuclear factor kappa ligand (RANKL); (d) vascular endothelial growth factor (VEGF); (e) vascular endothelial growth factor receptor (VEGF-R); hepatocyte growth factor (HGF); (f) interleukin-1α; (g) interleukin-1β; (h) interleukin-5; (i) interleukin-8; (j) interleukin-13; (k) interleukin-15; (l) interleukin-17 (IL-17); (m) IL-23; (n) ghrelin; (o) angiotensin; (p) chemokine (CC motif) (CCL21); (q) chemokine (CX motif) (CXCL21). 12); (r) Stromal cell-derived factor 1 (SDF-I); (s) Macrophage colony-stimulating factor (M-CSF); (t) Monocyte chemoattractant protein 1 (MCP-I); (u) Endothelial glycoprotein; (v) Resistin; (w) Gonadotropin-releasing hormone (GnRH); (x) Growth hormone-releasing hormone (GHRH); (y) Luteinizing hormone-releasing hormone (LHRH); (z) Thyrotropin-releasing hormone (TRH); (aa) Macrophage migration inhibitory factor (MIF); (bb) Glucose-dependent insulinotropic peptide (GIP); (cc) Eosinophil chemokine; (dd) Bradykinin; (ee) Des-Arg bradykinin; (ff) B lymphocyte chemoattractant protein (BLC); (gg) Macrophage colony-stimulating factor M-CSF; (hh) Tumor necrosis factor α (TNFα); (i) i) Amyloid β-peptide (Aβ1-42); (jj) Amyloid β-peptide (Aβ3-6); (kk) Human IgE; (ii) CCR5 extracellular domain; (mm) CXCR4 extracellular domain; (nn) Gastrin; (oo) CETP; (pp) C5a; (qq) Epidermal growth factor receptor (EGF-R); (rr) CGRP; (ss) α-synuclein; (tt) Calcitonin gene-related peptide (CGRP); (uu) Dextrin; (vv) Myostatin; (ww) Interleukin-4; (xx) Thymic stromal lymphopoietin; (yy) Interleukin-33; (zz) Interleukin-25; (aaa) Interleukin-13 or (bbb) fragments of any one of polypeptides (a) to (aaa); and (ccc) antigenic mutants or fragments of any one of polypeptides (a) to (aaa).

[0158] In a preferred embodiment, the antigenic polypeptide is interleukin-17 (IL-17), preferably human IL-17. Interleukin-17 is a T-cell-derived cytokine that induces the release of pro-inflammatory mediators in a variety of cell types. Abnormal Th17 responses and overexpression of IL-17 are associated with many autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Molecules that block IL-17, such as IL-17-specific monoclonal antibodies, have been shown to effectively improve disease in animal models. Furthermore, active immunization targeting IL-17 using virus-like particles conjugated with recombinant IL-17 has recently been proposed (Röhn TA et al., *European Journal of Immunology* (2006) 36: 1-11). Immunization with IL-17-VLP induces high levels of anti-IL-17 antibodies, thereby overcoming natural tolerance, even without the addition of adjuvants. In both collagen-induced arthritis and experimental autoimmune encephalomyelitis, mice immunized with IL-17-VLP showed lower disease incidence, slower disease progression, and lower disease severity scores. Therefore, in a preferred embodiment, the antigenic polypeptide comprises or is preferably composed of SEQ ID NO: 82. Furthermore, the modified CMV VLP of the present invention is used in methods for treating inflammatory diseases in animals or humans, preferably chronic inflammatory diseases. Preferably, the inflammatory disease is selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, neurodermatitis (allergic dermatitis), more preferably MS, and further preferably the antigenic polypeptide comprises or is preferably composed of SEQ ID NO: 82.

[0159] In another preferred embodiment, the antigenic polypeptide is IL-5, preferably human IL-5. In yet another preferred embodiment, the antigenic polypeptide comprises, or is preferably composed of, SEQ ID NO: 83. Furthermore, the modified VLP of the present invention is used in a method of treating inflammatory diseases in animals or humans, preferably chronic inflammatory diseases. Preferably, the inflammatory disease is selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, neurodermatitis (allergic dermatitis), and further preferably the antigenic polypeptide comprises, or is preferably composed of, SEQ ID NO: 83.

[0160] In another preferred embodiment, the antigenic polypeptide is canine IL-5. In yet another preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 84, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 84. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 84. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 84.

[0161] In another preferred embodiment, the antigenic polypeptide is feline IL-5. In a very preferred embodiment, the antigenic polypeptide comprises or preferably consists of the following: SEQ ID NO: 85, SEQ ID NO: 125, SEQ ID: 141, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 85, SEQ ID NO: 125, and SEQ ID: 141. In another very preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 85, SEQ ID NO: 125, or SEQ ID: 141. In yet another very preferred embodiment, the antigenic polypeptide is composed of SEQ ID NO: 85, SEQ ID NO: 125, or SEQ ID: 141. In another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of: an amino acid sequence of SEQ ID NO: 85 or having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with the sequence of SEQ ID NO: 85. In another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of: SEQ ID NO: 125 or having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with the sequence of SEQ ID NO: 125. In another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 85. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 85. In another highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 125. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 125.

[0162] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises an amino acid sequence selected from: (a) SEQ ID NO: 85; (b) SEQ ID NO: 125; (c) SEQ ID NO: 141, wherein the antigenic polypeptide preferably comprises SEQ ID NO: 125, preferably constitutes thereof; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably constitutes thereof: the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64.

[0163] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises SEQ ID NO: 125, preferably comprising; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably comprising. In another highly preferred embodiment, the chimeric CMV polypeptide is composed of SEQ ID NO: 128, SEQ ID NO: 132, or SEQ ID NO: 139. In another highly preferred embodiment, the chimeric CMV polypeptide is composed of SEQ ID NO: 128. In another highly preferred embodiment, the chimeric CMV polypeptide is composed of SEQ ID NO: 132. In other highly preferred embodiments and aspects herein, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in methods of treating inflammatory diseases in animals or humans, preferably chronic inflammatory diseases. Preferably, the inflammatory disease is selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, and neurodermatitis (allergic dermatitis).

[0164] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein and further comprises at least one CMV protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises, preferably comprises, SEQ ID NO: 125, and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 125. 64, preferably composed thereof; and wherein the CMV protein comprises the outer coat protein of CMV, preferably composed thereof, preferably SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably composed thereof. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 128, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 132, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment and aspect herein, the invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating inflammatory diseases in animals or humans, preferably chronic inflammatory diseases. Preferably, the inflammatory disease is selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, and neurodermatitis (allergic dermatitis).

[0165] In another highly preferred embodiment, the antigenic polypeptide is IL-4, preferably human IL-4. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 86. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 86.

[0166] In another highly preferred embodiment, the antigenic polypeptide is canine IL-4. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 87, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 87. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 87. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 87.

[0167] In another highly preferred embodiment, the antigenic polypeptide is feline IL-4. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 88, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 88. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 88. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 88. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 88.

[0168] In another highly preferred embodiment, the antigenic polypeptide is IL-13, preferably human IL-13. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 89. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 89.

[0169] In another highly preferred embodiment, the antigenic polypeptide is canine IL-13. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 90, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 90. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 90. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 90.

[0170] In another highly preferred embodiment, the antigenic polypeptide is feline IL-13. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 91, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 91. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 91. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 91.

[0171] In another highly preferred embodiment, the antigenic polypeptide is equine IL-13. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 92, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 92. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 92. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 92.

[0172] In another highly preferred embodiment, the antigenic polypeptide is TNFα.

[0173] In another highly preferred embodiment, the antigenic polypeptide is IL-1α, preferably human IL-1α. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 93. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 93.

[0174] In another highly preferred embodiment, the antigenic polypeptide is canine IL-1α. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 94, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 94. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 94. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 94.

[0175] In another highly preferred embodiment, the antigenic polypeptide is feline IL-1α. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 95, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 95. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 95. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 95.

[0176] In another highly preferred embodiment, the antigenic polypeptide is equine IL-1α. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 96, or an amino acid sequence having at least 90%, preferably at least 96%, sequence identity with SEQ ID NO: 95. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 96. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 96.

[0177] In another highly preferred embodiment, the antigenic polypeptide is IL-33, preferably human IL-33. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 97. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 97.

[0178] In another highly preferred embodiment, the antigenic polypeptide is canine IL-33. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 98, or an amino acid sequence having at least 90%, preferably at least 98%, sequence identity with SEQ ID NO: 95. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 98. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 98.

[0179] In another highly preferred embodiment, the antigenic polypeptide is feline IL-33. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 99, or an amino acid sequence having at least 90%, preferably at least 99%, sequence identity with SEQ ID NO: 95. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 99. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 99.

[0180] In another highly preferred embodiment, the antigenic polypeptide is equine IL-33. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 100, or an amino acid sequence having at least 95%, preferably at least 100%, sequence identity with SEQ ID NO: 90. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 100. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 100.

[0181] In another highly preferred embodiment, the antigenic polypeptide is IL-25, preferably human IL-25. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 101. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 101.

[0182] In another highly preferred embodiment, the antigenic polypeptide is canine IL-25. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 102, or an amino acid sequence having at least 95%, preferably at least 102%, sequence identity with SEQ ID NO: 90. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 102. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 102.

[0183] In another highly preferred embodiment, the antigenic polypeptide is feline IL-25. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 103, or an amino acid sequence having at least 95%, preferably at least 103%, sequence identity with SEQ ID NO: 90. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 103. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 103.

[0184] In another highly preferred embodiment, the antigenic polypeptide is equine IL-25. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 104, or an amino acid sequence having at least 95%, preferably at least 104%, sequence identity with SEQ ID NO: 90. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 104. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 104.

[0185] In another highly preferred embodiment, the antigenic polypeptide is IL-1β, preferably human IL-1β. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 105. In another highly preferred embodiment, the antigenic polypeptide is canine IL-1β. In a highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 134, SEQ ID NO: 143, SEQ ID NO: 144, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 134, SEQ ID NO: 143, SEQ ID NO: 144. In another highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 134, SEQ ID NO: 143, SEQ ID NO: 144. In another highly preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 134, SEQ ID NO: 143, and SEQ ID NO: 144. In another highly preferred embodiment, the antigenic polypeptide comprises or preferably comprises the following: an amino acid sequence of SEQ ID NO: 134 or having at least 90%, preferably at least 92%, more preferably at least 95%, and even more preferably at least 98% amino acid sequence identity with SEQ ID NO: 134. In another highly preferred embodiment, the antigenic polypeptide comprises or preferably comprises SEQ ID NO: 135. In yet another preferred embodiment, the antigenic polypeptide comprises SEQ ID NO: 135.

[0186] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises an amino acid sequence selected from: (a) SEQ ID NO: 134; (b) SEQ ID NO: 143; (c) SEQ ID NO: 144, wherein the antigenic polypeptide preferably comprises SEQ ID NO: 134, preferably composed of therein; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably composed of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64.

[0187] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises SEQ ID NO: 134, preferably comprising; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably comprising. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 135. In further highly preferred embodiments and aspects herein, the invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in methods of treating inflammatory diseases in animals or humans, preferably chronic inflammatory diseases. Preferably, the inflammatory disease is selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, and neurodermatitis (allergic dermatitis).

[0188] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein and further comprises at least one CMV protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises, preferably comprises, SEQ ID NO: 134, and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably composed thereof; and wherein the CMV protein comprises the outer coat protein of CMV, preferably composed thereof, preferably SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably composed thereof. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 135, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment and aspect herein, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in methods of treating inflammatory diseases.

[0189] In another highly preferred embodiment, the antigenic polypeptide is feline IL-1β. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 145.

[0190] In another highly preferred embodiment, the antigenic polypeptide is IL-31, preferably human IL-31. In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 106. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 106.

[0191] In another highly preferred embodiment, the antigenic polypeptide is canine IL-31. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 107, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 107. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 107. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 107.

[0192] In another highly preferred embodiment, the antigenic polypeptide is feline IL-31. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 108, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 108. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 108. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 108.

[0193] In another highly preferred embodiment, the antigenic polypeptide is equine IL-31. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 109, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 109. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 109. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 109.

[0194] In another highly preferred embodiment, the antigenic polypeptide is thymic stromal lymphopoietin (TLSP), preferably human thymic stromal lymphopoietin (TLSP). In yet another highly preferred embodiment, the antigenic polypeptide comprises, or preferably consists of, SEQ ID NO: 110. In yet another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 110.

[0195] In another highly preferred embodiment, the antigenic polypeptide is canine TLSP. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 111, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 111. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 111. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 111.

[0196] In another highly preferred embodiment, the antigenic polypeptide is feline TLSP. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 112, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 112. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 112. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 112.

[0197] In another highly preferred embodiment, the antigenic polypeptide is equine TLSP. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 113, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 113. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 113. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 113.

[0198] In another highly preferred embodiment, the antigenic polypeptide is IgE or a peptide or domain included in IgE.

[0199] In another highly preferred embodiment, the antigenic polypeptide is a peptide derived from the N-terminus of Aβ-1-42 (SEQ ID NO: 114), particularly a fragment of Aβ-1-42 (SEQ ID NO: 114) with a length of up to 7 consecutive amino acids, preferably a fragment of Aβ-1-42 (SEQ ID NO: 114) with a length of up to 6 consecutive amino acids.

[0200] Therefore, in another highly preferred embodiment, the antigenic polypeptide is selected from Aβ-1-6 (SEQ ID NO: 1), Aβ-1-7 (SEQ ID NO: 2), Aβ-3-6 (SEQ ID NO: 3), Aβ-1-5 (SEQ ID NO: 4), Aβ-2-6 (SEQ ID NO: 115), or Aβ-3-7 (SEQ ID NO: 116). In another highly preferred embodiment, the antigenic polypeptide is Aβ-1-6 (SEQ ID NO: 1). In another highly preferred embodiment, the antigenic polypeptide is Aβ-1-7 (SEQ ID NO: 2). In another highly preferred embodiment, the antigenic polypeptide is Aβ-3-6 (SEQ ID NO: 3). In another highly preferred embodiment, the antigenic polypeptide is Aβ-1-5 (SEQ ID NO: 4). In another highly preferred embodiment, the antigenic polypeptide is Aβ-2-6 (SEQ ID NO: 115). In another highly preferred embodiment, the antigenic polypeptide is Aβ-3-7 (SEQ ID NO: 116).

[0201] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein, the at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-6 (SEQ ID NO: 1); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: The amino acid sequence 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64. In other very preferred embodiments and aspects herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0202] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-6 (SEQ ID NO: 1); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 6. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0203] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein, the at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-7 (SEQ ID NO: 2); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: The amino acid sequence 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64. In other very preferred embodiments and aspects herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0204] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-7 (SEQ ID NO: 2); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 7. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0205] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein, the at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-3-6 (SEQ ID NO: 3); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: The amino acid sequence 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64. In other very preferred embodiments and aspects herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0206] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-3-6 (SEQ ID NO: 3); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 8. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0207] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-5 (SEQ ID NO: 4); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: The amino acid sequence 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64. In other very preferred embodiments and aspects herein, the invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0208] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is Aβ-1-5 (SEQ ID NO: 4); and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 9. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating Alzheimer's disease.

[0209] In another highly preferred embodiment, the antigenic polypeptide is α-synuclein or a peptide derived from α-synuclein, wherein preferably the peptide consists of 6 to 14 amino acids, and further preferably the antigenic polypeptide is a peptide derived from α-synuclein, the peptide being selected from SEQ D NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 117. Further preferred peptides derived from α-synuclein are disclosed in WO 2011 / 020133, which is incorporated herein by reference.

[0210] Alpha-synuclein (α-Syn) is a small protein with multiple physiological and pathological functions and is one of the main proteins found in Lewy bodies, a pathological marker of Lewy body syndromes (including Parkinson's disease (PD)). Recently, α-Syn has been found in bodily fluids containing blood and cerebrospinal fluid, and may be produced by both peripheral tissues and the central nervous system. The exchange of α-Syn between the brain and peripheral tissues may have significant pathophysiological and therapeutic implications (Gardai SJ et al., PLOS ONE (2013) 8(8): e71634). Evidence overwhelmingly supports the involvement of α-syn in the pathogenesis of Parkinson's disease (PD). However, there is no clear consensus on how α-syn contributes to the pathology of PD and other conucleoprotein diseases.

[0211] Alpha-synuclein is a major component of the Lewy body (LB), and there is much description of the overexpression of α-synuclein leading to aggregation. Human genetic data suggest that missense mutations and duplication of the α-synuclein gene can lead to familial PD. In the case of gene duplication, it is speculated that elevated α-synuclein protein levels lead to the acquisition of the main function of the pathology. Although elevated α-synuclein levels may lead to aggregation and toxicity, studies in recent years have also shown that elevated α-synuclein may interfere with the formation, localization and / or maintenance of vesicle pools (Gardai SJ et al., PLOS ONE (2013) 8(8): e71634; and references cited therein).

[0212] Therefore, in another highly preferred embodiment, the antigenic polypeptide is selected from any one of the sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 117. In another highly preferred embodiment, the antigenic polypeptide is SEQ ID NO: 49. In another highly preferred embodiment, the antigenic polypeptide is SEQ ID NO: 50. In another highly preferred embodiment, the antigenic polypeptide is SEQ ID NO: 51. In another highly preferred embodiment, the antigenic polypeptide is SEQ ID NO: 117.

[0213] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is SEQ ID NO: 49, and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: 49. The amino acid sequence 2-12 of NO:62, wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:65, more preferably SEQ ID NO:64.

[0214] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is SEQ ID NO: 49; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises or preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 52. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in methods of treating diseases, symptoms, or physiological conditions, wherein the diseases, symptoms, or physiological conditions are selected from Lewy body diseases, and wherein preferably the diseases, symptoms, or physiological conditions are Parkinson's disease.

[0215] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide, and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is SEQ ID NO: 50; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to SEQ ID NO: 50. The amino acid sequence 2-12 of NO:62, wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:65, more preferably SEQ ID NO:64.

[0216] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is SEQ ID NO: 50; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 53. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in methods of treating diseases, symptoms, or physiological conditions, wherein the diseases, symptoms, or physiological conditions are selected from Lewy body diseases, and wherein preferably the diseases, symptoms, or physiological conditions are Parkinson's disease.

[0217] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein, said at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein said CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably said CMV polypeptide comprising or preferably consisting of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein said antigenic polypeptide is inserted into said CMV polypeptide, wherein said insertion of said antigenic polypeptide is between amino acid residues of said CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein said antigenic polypeptide is SEQ ID NO: 51; and wherein said T helper cell epitope replaces the N-terminal region of said CMV polypeptide, wherein said N-terminal region of said CMV polypeptide corresponds to SEQ ID NO: 51. The amino acid sequence 2-12 of NO:62, wherein the T helper cell epitope preferably comprises, preferably consists of, the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:65, more preferably SEQ ID NO:64.

[0218] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is SEQ ID NO: 51; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 54. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in methods of treating diseases, symptoms, or physiological conditions, wherein the diseases, symptoms, or physiological conditions are selected from Lewy body diseases, and wherein preferably the diseases, symptoms, or physiological conditions are Parkinson's disease.

[0219] In another very preferred embodiment, the antigenic polypeptide is dextrin. In another very preferred embodiment, the antigenic polypeptide is angiotensin I or a peptide derived from angiotensin I. In yet another very preferred embodiment, the antigenic polypeptide is angiotensin II or a peptide derived from angiotensin II. In yet another very preferred embodiment, the antigenic polypeptide is GnRH. In yet another very preferred embodiment, the antigenic polypeptide is eosinophil chemokine.

[0220] In another highly preferred embodiment, the antigenic polypeptide is myostatin, preferably bovine myostatin. In yet another highly preferred embodiment, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 118, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 118. Preferably, the antigenic polypeptide comprises or preferably consists of SEQ ID NO: 118. In another preferred embodiment, the antigenic polypeptide consists of SEQ ID NO: 118.

[0221] In another preferred embodiment, the antigenic polypeptide is a polypeptide of a parasite, wherein preferably the pathogen is selected from the group consisting of: (a) *Toxoplasma*; (b) *Plasmodium falciparum*; (c) *Plasmodium vivax*; (d) *Plasmodium ovale*; (e) *Plasmodium malariae*; (f) *Leishmania*; (g) *Schistosoma* and (h) nematodes. Preferably, the antigenic polypeptide is derived from *Plasmodium falciparum* or *Plasmodium vivax* (SEQ ID NO: 119).

[0222] In another highly preferred embodiment, the antigenic polypeptide is derived from Plasmodium falciparum. In yet another highly preferred embodiment, the antigenic polypeptide is derived from Plasmodium falciparum comprising or preferably consisting of SEQ ID NO: 44.

[0223] In a highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein, the at least one fusion protein comprising or preferably consisting of a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably consists of: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably consists of: SEQ ID NO: 62; or an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 62, preferably wherein the CMV polypeptide comprises or preferably consists of SEQ ID NO: 62; (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide is derived from Plasmodium falciparum, and wherein preferably the antigenic polypeptide is derived from a parasite comprising SEQ ID NO: 44 or preferably composed of Plasmodium falciparum, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein preferably the T helper cell epitope comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, more preferably SEQ ID NO: 64.

[0224] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein comprising or preferably comprising a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises or preferably comprises SEQ ID NO: 44; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64. In another highly preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 46. In yet another highly preferred embodiment and aspect thereof, the present invention provides the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) for use in a method of treating malaria.

[0225] In another highly preferred embodiment, the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprises at least one fusion protein and further comprises at least one CMV protein, wherein the at least one fusion protein comprises or preferably comprises a) a chimeric CMV polypeptide, wherein the chimeric CMV polypeptide comprises or preferably comprises: (i) a CMV polypeptide, (ii) an antigenic polypeptide and (iii) a T helper cell epitope; and wherein the CMV polypeptide comprises or preferably comprises SEQ ID NO: 62, (ii) an antigenic polypeptide inserted into the CMV polypeptide, wherein the insertion of the antigenic polypeptide is between amino acid residues of the CMV polypeptide corresponding to amino acid residues at positions 84 and 85 of SEQ ID NO: 62; and wherein the antigenic polypeptide comprises or preferably comprises SEQ ID NO: 44; and wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, wherein the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 44. 64, preferably composed thereof; and wherein the CMV protein comprises the outer coat protein of CMV, preferably composed thereof, preferably SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 62, and wherein the T helper cell epitope preferably comprises SEQ ID NO: 64, preferably composed thereof. In another very preferred embodiment, the chimeric CMV polypeptide comprises SEQ ID NO: 46, and the CMV protein comprises SEQ ID NO: 5. In another very preferred embodiment and aspect herein, the present invention provides the modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in a method of treating malaria.

[0226] In another preferred embodiment, the antigenic polypeptide is a bacterial polypeptide, wherein the bacteria are preferably selected from the group consisting of: (a) Chlamydia; (b) Streptococcus; (c) Pneumococcus; (d) Staphylococcus; (e) Salmonella; (f) Mycobacterium; (g) Clostridium; (h) Vibrio; (i) Yersinia; (k) Neisseria meningitidis; and (l) Treponema pallidum.

[0227] In another preferred embodiment, the antigenic polypeptide is a viral antigen, wherein preferably the viral antigen is a polypeptide selected from the group consisting of: (a) HIV and other retroviruses; (b) influenza virus, preferably influenza A M2 extracellular domain or HA or HA globular domain; (c) hepatitis B virus polypeptide, preferably preS1; (d) hepatitis C virus; (e) HPV, preferably HPV16E7; (f) RSV; (g) SARS and other coronaviruses; (h) dengue and other flaviviruses, such as West Nile virus and hand-foot-and-mouth disease virus; (i) chikungunya and other alphaviruses; (k) CMV and other herpesviruses; (l) rotavirus. In another very preferred embodiment, the antigenic polypeptide is derived from RSV. In another very preferred embodiment, the antigenic polypeptide is derived from dengue virus.

[0228] In a preferred embodiment, the antigenic polypeptide is an extracellular domain of the influenza A virus M2 protein or an antigenic fragment thereof. In a very preferred embodiment, the antigenic polypeptide comprises or preferably consists of an extracellular domain of the influenza A virus M2 protein, wherein preferably the extracellular domain of the influenza A virus M2 protein is SEQ ID NO:120. In another preferred embodiment, the antigenic polypeptide is a globular domain of an influenza virus.

[0229] In another highly preferred embodiment, the chimeric CMV polypeptide is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54.

[0230] In another preferred embodiment, the modified VLP further comprises at least one immunostimulatory substance. In a very preferred embodiment, the immunostimulatory substance is packaged into the modified VLP of the present invention. In yet another preferred embodiment, the immunostimulatory substance is mixed with the modified VLP of the present invention. Immunostimulatory substances that can be used in the present invention are well known in the art and are disclosed, in particular, in WO2003 / 024481A2.

[0231] In another embodiment of the invention, the immunostimulant is composed of DNA or RNA of non-eukaryotic origin. In another preferred embodiment, the immunostimulant is selected from the group consisting of: (a) immunostimulatory nucleic acids; (b) peptidoglycans; (c) lipopolysaccharides; (d) lipoteichoic acid; (e) imidazoquinone compounds; (f) flagellin; (g) lipoproteins; and (h) any mixture of at least one substance from (a) to (g). In another preferred embodiment, the immunostimulant is an immunostimulatory nucleic acid selected from the group consisting of: (a) ribonucleic acid; (b) deoxyribonucleic acid; (c) chimeric nucleic acids; and (d) any mixture of (a), (b), and / or (c). In another preferred embodiment, the immunostimulatory nucleic acid is ribonucleic acid, and wherein the ribonucleic acid is bacterial RNA. In another preferred embodiment, the immunostimulatory nucleic acid is poly(IC) or a derivative thereof. In another preferred embodiment, the immunostimulatory nucleic acid is deoxyribonucleic acid, wherein the deoxyribonucleic acid is an unmethylated CpG-containing oligonucleotide.

[0232] In a highly preferred embodiment, the immunostimulatory substance is an unmethylated CpG-containing oligonucleotide. In another preferred embodiment, the unmethylated CpG-containing oligonucleotide is type A CpG. In another preferred embodiment, the type A CpG comprises a palindromic sequence. In another preferred embodiment, the palindromic sequence is side-joined at its 5' end and at its 3' end by guanosine entities. In another preferred embodiment, the palindromic sequence is side-joined at its 5' end by at least 3 and at most 15 guanosine entities, and wherein the palindromic sequence is side-joined at its 3' end by at least 3 and at most 15 guanine entities.

[0233] In another preferred embodiment, the immunostimulant is an unmethylated CpG-containing oligonucleotide, and preferably the unmethylated CpG-containing oligonucleotide comprises a palindromic sequence, and further preferably the CpG motif of the unmethylated CpG-containing oligonucleotide is part of a palindromic sequence.

[0234] In another aspect, the present invention provides modified virus-like particles of the present invention for use as a medicament.

[0235] In another aspect, the present invention provides a vaccine comprising or alternatively consisting of the modified virus-like particles of the present invention. The vaccine is encompassed in which the modified VLP, alone or in any possible combination, comprises any of the technical features disclosed herein. In one embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine is adjuvant-free. In a preferred embodiment, the vaccine comprises an effective amount of the composition of the present invention.

[0236] In another aspect, the present invention relates to pharmaceutical compositions comprising: (a) the modified VLP of the present invention or the vaccine of the present invention; and (b) pharmaceutically acceptable carriers, diluents, and / or excipients. The diluents comprise sterile aqueous solutions (e.g., physiological saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. The pharmaceutical compositions of the present invention may be in the form of salts, buffers, adjuvants, or other substances desired to improve the efficacy of the conjugate. Examples of materials suitable for preparing pharmaceutical compositions are provided in numerous sources, including Remington's Pharmaceutical Sciences (Osol, A editor, Mack Publishing Co., (1990)). In one embodiment, the pharmaceutical composition comprises an effective amount of the vaccine of the present invention.

[0237] Another aspect of the invention is an immunization method comprising administering to an animal or human the modified VLP of the invention, the vaccine of the invention, or the pharmaceutical composition of the invention. In a preferred embodiment, the method comprises administering to an animal or human the composition of the invention, the vaccine of the invention, or the pharmaceutical composition of the invention.

[0238] Another aspect of the invention is a method for treating or preventing diseases, symptoms, or physiological conditions in animals, the method comprising administering to the animal a modified VLP of the invention, a vaccine of the invention, or a pharmaceutical composition of the invention, wherein preferably the animal may be a human. In another preferred embodiment, the modified VLP, the vaccine, or the pharmaceutical composition is administered to the animal subcutaneously, intravenously, intradermally, intranasally, or orally, intranodally, or transdermally.

[0239] In another highly preferred embodiment, the disease, symptom, or physical condition is selected from the group consisting of: allergies, cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0240] In another highly preferred embodiment, the disease, symptom, or physical condition is selected from the group consisting of: RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, neurodermatitis (allergic dermatitis), Alzheimer's disease, Parkinson's disease, influenza A virus infection, malaria, and RSV infection.

[0241] In another highly preferred embodiment, the disease, symptom, or physical condition is an inflammatory disease. In yet another highly preferred embodiment, the disease, symptom, or physical condition is an inflammatory disease selected from RA, MS, psoriasis, asthma, Crohn's disease, colitis, COPD, diabetes, and neurodermatitis (allergic dermatitis).

[0242] In another highly preferred embodiment, the disease, symptom, or physical symptom is an infectious disease. In another highly preferred embodiment, the disease, symptom, or physical symptom is an inflammatory disease selected from influenza A virus infection, malaria, and RSV infection. In another highly preferred embodiment, the disease, symptom, or physical symptom is malaria.

[0243] In another highly preferred embodiment, the disease, symptom, or physical condition is Alzheimer's disease or Parkinson's disease. In another highly preferred embodiment, the disease, symptom, or physical condition is Alzheimer's disease. In another highly preferred embodiment, the disease, symptom, or physical condition is Parkinson's disease.

[0244] Example Example 1 The Aβ1-42 fragment was cloned into the modified coat protein of cucumber mosaic virus (CMV) that produces the CMV-Aβ-chimeric CMV polypeptide. CMV-Aβ-chimeric CMV polypeptides according to the present invention have been prepared, the polypeptides comprising Aβ protein fragments Aβ1-6 (SEQ ID NO: 1), Aβ1-7 (SEQ ID NO: 2), Aβ3-6 (SEQ ID NO: 3) and Aβ1-5 (SEQ ID NO: 4).

[0245] These Aβ peptides have been inserted between the amino acid residues Ser (88) and Tyr (89) of the highly preferred modified CMV coat protein CMV-Ntt830 (SEQ ID NO: 5), which includes a T helper cell epitope derived from tetanus toxoid. The amino acid sequences of these preferred chimeric CMV polypeptides according to the invention are as follows: The amino acid sequence of “CMV-Ntt830-Ab16” is: SEQ ID NO: 6; The amino acid sequence of “CMV-Ntt830-Ab17” is: SEQ ID NO: 7; The amino acid sequence of “CMV-Ntt830-Ab36” is: SEQ ID NO: 8; The amino acid sequence of “CMV-Ntt830-Ab15” is: SEQ ID NO: 9.

[0246] The amino acid sequences of these preferred chimeric CMV peptides further include glycine-serine linkers of the introduced Aβ peptide at both ends. All preferred fusion proteins of SEQ ID NO: 6 to SEQ ID NO: 9 include a GGGS linker located directly at the N-terminus of the introduced Aβ peptide (SEQ ID NO: 10) and a GGGSGS linker located at the C-terminus of the introduced Aβ peptide (SEQ ID NO: 11).

[0247] The nucleotide sequence corresponding to the preferred chimeric CMV polypeptide is as follows: The nucleic acid sequence of “CMV-Ntt830-Ab16” is: SEQ ID NO: 12; The nucleic acid sequence of “CMV-Ntt830-Ab17” is: SEQ ID NO: 13; The nucleic acid sequence of “CMV-Ntt830-Ab36” is: SEQ ID NO: 14; The nucleic acid sequence of “CMV-Ntt830-Ab15” is: SEQ ID NO: 15.

[0248] To introduce these Aβ peptides or other antigenic polypeptides encoding the DNA sequence into the corresponding CMV DNA sequence of CMV-Ntt830, a sequence containing a BamHI site is introduced at the corresponding position for subsequent cloning. The nucleic acid sequence encoding CMV-Ntt830 is prepared as described in Example 3 of WO2016 / 062720A1 and corresponds to SEQ ID NO: 14 of WO2016 / 062720A1.

[0249] The BamHI site was introduced via two-step PCR mutagenesis using the oligonucleotides listed below and the previously constructed pET-CMV-Ntt830 as a template. The template pET-CMV-Ntt830 was prepared as described in Example 3 of WO2016 / 062720A1.

[0250] PCR 1: Forward-pET-90 primer (annealing pET28a+) (SEQ ID NO: 16) Reverse -RGSYrev (SEQ ID NO: 17) 2nd PCR positive-RGSYdir (SEQ ID NO: 18) Reverse-CMV-AgeR (SEQ ID NO: 19) After both PCR products were purified, the next PCR was performed to ligate the PCR fragments (5 cycles without primers, followed by 25 cycles with primers pET-90 and CMV-AgeR).

[0251] After gene amplification, the obtained PCR product was directly cloned into the pTZ57R / T vector (InsTAclone PCR cloning kit, Fermentas, product number K1214). *E. coli* XL1-Blue cells were used as the host for cloning and plasmid amplification.

[0252] To avoid RT-PCR errors, several pTZ57 plasmid clones containing the CMV-Ntt830 gene were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 Gene Analyzer (Applied Biosystems). Following sequencing, error-free pTZ plasmid clones containing the CMV-Ntt830B gene with the introduced BamHI site were digested with NcoI and AgeI enzymes. The fragment was then subcloned into the NcoI / AgeI site of pET-CMV-Ntt830 to obtain the helper vector pET-CMV-Ntt830B.

[0253] To introduce DNA encoding amyloid-(β) peptide, the following oligonucleotides were used in the PCR reaction (the template in all PCRs was pET-CMV-Ntt830): PCR 1: Forward-C-Ab15 (SEQ ID NO: 20) Reverse-CMcpR (SEQ ID NO: 21) PCR 2: Forward-C-Ab16 (SEQ ID NO: 22) Reverse-CMcpR (SEQ ID NO: 21) 3rd PCR: Forward-C-Ab17 (SEQ ID NO: 23) Reverse-CMcpR (SEQ ID NO: 21) 4th PCR: Forward-C-Ab36 (SEQ ID NO: 24) Reverse-CMcpR (SEQ ID NO: 21) All PCR fragments were directly ligated into the pTZ57R / T vector, and the corresponding plasmid clones containing the inserts were isolated after transformation in *E. coli* XL1 cells. Several plasmid clones containing the PCR product DNA were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 Gene Analyzer (Applied Biosystems). After sequencing, the 3' end fragment of the CMV containing the Ab fragment was ligated into the pET-CMV-Ntt830B helper vector using BamHI and HindIII sites. Correct clones were selected after BamHI / HindIII restriction enzyme testing.

[0254] Furthermore, plasmid clones pET-CMV-Ntt830B-Ab15, pET-CMV-Ntt830B-Ab16, pET-CMV-Ntt830B-Ab17, and pET-CMV-Ntt830B-Ab36 were used to transform *E. coli* C2566 cells. An exemplary plasmid map of pET-CMV-Ntt830B-Ab36 is shown in... Figure 1 middle.

[0255] Example 2 Expression of CMV-Aβ-chimeric CMV polypeptide Generate CMV-Aβ VLP For the isolation of CMV-Aβ VLPs (i.e., CMV-Ntt830-Ab16 VLP, CMV-Ntt830-Ab17 VLP, CMV-Ntt830-Ab36 VLP, or CMV-Ntt830-Ab15 VLP), the corresponding plasmids pET-CMV-Ntt830-Ab15, pET-CMV-Ntt830-Ab16, pET-CMV-Ntt830-Ab17, and pET-CMV-Ntt830-Ab36 were transformed into Escherichia coli C2566 competent cells (New England Biolabs, USA).

[0256] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing kanamycin (25 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and the medium was supplemented with 5 mM MgCl2. The cells were incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C.

[0257] The purification of CMV-Aβ VLP involves the following steps: 1) Suspend 3 g of biomass in 20 ml of 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, 5 mM mercaptoethanol, pH 9.0, and treat the suspension with ultrasound (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 50 mM sodium citrate, 5 mM sodium borate, 2 mM EDTA, and 0.5% TX-100. 4) Cover the sucrose gradient with 5 ml of VLP sample; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25,000 rpm, +18°C).

[0258] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze the gradient levels on SDS-PAGE ( Figure 2A , Figure 2B , Figure 2C , Figure 2D ).

[0259] 8) SDS-PAGE analysis showed the presence of VLP in the third sucrose fraction. Dilute 24 ml of the third fraction with 24 ml buffer (5 mM sodium borate, 2 mM EDTA, pH 9.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 3 ml of 5 mM sodium borate and 2 mM EDTA at pH 9.0; 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 5 mM sodium borate, 2 mM EDTA, pH 9.0 buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 5 mM sodium borate and 2 mM EDTA, pH 9.0, overnight, 4°C; 14) Analyze VLP under EM ( Figure 3A , Figure 3B , Figure 3C , Figure 3D ).

[0260] like Figure 2A , Figure 2B , Figure 2C , Figure 2D As shown, four CMV-Aβ VLPs were successfully expressed in E. coli cells, and most of the obtained samples were present in the soluble fraction. Furthermore, sucrose gradient analysis (…) Figure 2A-2D ) and electron microscopy analysis ( Figures 3A-3D As shown, these proteins are present directly in the E. coli cell extract in the form of isospaced VLPs.

[0261] Example 3 Monoclonal antibodies containing the variable region sequence of aducanib recognize CMV-Aβ VLP. Using Aβ 1-42 The recombinant antibody was coated with either CMV-Ntt830-Ab36 VLP or CMV-Ntt830 VLP and the binding of the recombinant antibody to the variable region displaying the aducanib sequence was tested by ELISA.

[0262] ELISA: The ELISA plate (Nunc Immuno MaxiSorp, Rochester, NY) was incubated at 4°C with 100 µl of Aβ. 1-42CMV-Ntt830-Ab36 VLP or CMV-Ntt830 VLP (1 µg / ml) were coated overnight in PBS at pH 7.4. To avoid nonspecific binding, the ELISA plates were blocked with PBST containing 200 µl of 2% BSA and incubated at room temperature for 2 hours. The supernatant of cells expressing a monoclonal antibody with the variable region sequence of aducanib was transferred to the coated plates. After incubation at room temperature for 2 hours, the ELISA plates were washed 5 times with 200 µl of PBST. The binding of serum antibodies was detected by horseradish peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch). The detection antibody was diluted 1:1000 in 2% BSA / PBST and 100 µl of each sample was transferred. The plates were incubated at room temperature for 1 hour. The ELISA plates were washed as described above. The substrate solution was prepared before washing. For this purpose, one tablet (10 mg) of OPD (1,2-phenylenediamine dihydrochloride) and 9 µl of 30% H2O2 were dissolved in 25 mL of citrate buffer (0.066 M Na2HPO4, 0.035 M citrate, pH 5.0). A 100 µl volume of substrate solution was pipetted onto a plate and incubated precisely at room temperature for 7 minutes. To terminate the reaction, 50 µl of stop solution (H2O containing 5% H2SO4) was pipetted directly onto the plate. The absorbance reading of the 1,2-phenylenediamine dihydrochloride colorimetric reaction was analyzed at 450 nm. Figure 4 The monoclonal antibody with the variable region sequence of aducanib was shown to bind to CMV-Ntt830-Aβ VLP.

[0263] Example 4 Mice were immunized with CMV-Aβ VLP. Four female Balb / c mice in each group were immunized with CMV-Ntt830-Ab16 VLP, CMV-Ntt830-Ab17 VLP, or CMV-Ntt830-Ab36 VLP. The VLPs were prepared in 150 µl of 150 mM PBS at pH 7.4 and administered intravenously at 30 µg on day 0. Mice were exsanguinated on day 0 (pre-immunization) and day 14, and serum was analyzed using Aβ1-42-coated ELISA plates. Antibodies induced by CMV-Ntt830-Ab36 VLP were further analyzed by immunohistochemistry on brain slices from Alzheimer's disease patients.

[0264] ELISA: Antibody responses in mouse serum were analyzed at indicated times. To determine Aβ1-42-specific antibodies, ELISA plates (Nunc Immuno MaxiSorp, Rochester, NY) were coated overnight at 4°C, pH 7.4 in PBS containing 100 µl Aβ1-42 (1 µg / ml). To avoid nonspecific binding, the ELISA plates were blocked with PBST containing 200 µl 2% BSA and incubated at room temperature for 2 hours. Serum samples were diluted in 2% BSA / PBST. Pre-diluted serum was transferred to the coated plates and further serially diluted to obtain antibody titers based on OD50. After incubation at room temperature for 2 hours, the ELISA plates were washed five times with 200 µl PBST. Binding of serum antibodies was detected by horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunological Research Laboratories, Inc.). The detection antibody was diluted 1:1000 in 2% BSA / PBST and transferred in 100 µl volumes for each sample. Incubate the plate at room temperature for 1 hour. Wash the ELISA plate as described previously. Before washing, prepare the substrate solution. For this purpose, dissolve one tablet (10 mg) of OPD (1,2-phenylenediamine dihydrochloride) and 9 µl of 30% H2O2 in 25 mL of citrate buffer (0.066 M Na2HPO4, 0.035 M citrate, pH 5.0). Pipette 100 µl of the substrate solution onto the plate and incubate precisely at room temperature for 7 minutes. To terminate the reaction, pipette 50 µl of the stop solution (H2O containing 5% H2SO4) directly onto the plate. Analyze the absorbance reading of the 1,2-phenylenediamine dihydrochloride colorimetric reaction at 450 nm. Figure 5A Antibodies specific to CMV-Ntt830-Ab16 VLP, CMV-Ntt830-Ab17 VLP, or CMV-Ntt830-Ab36 VLP are shown.

[0265] Immunohistochemistry: Paraffin-embedded brain (hippocampus) tissue sections from Alzheimer's disease patients were prepared using a microtome. Sections were mounted on glass slides and endogenous peroxidase was blocked by incubation with 3% H2O2 for 10 minutes. The slides were then washed with PBS-Tween, followed by three 5-minute washes with PBS alone. The slides were then blocked for 30 minutes at room temperature with PBS containing 3% oat serum, 0.5% casein, and 0.1% NaN3. The slides were incubated with a 1:50 dilution of serum from CMV-Ntt830-Ab36 VLP-immunized mice at 4°C for one hour. The slides were washed with PBS-Tween, followed by three 5-minute washes with PBS alone. The slides were then incubated with a 1:1000 dilution of secondary anti-goat anti-mouse IgG-HRP (number 161) at room temperature for 2 hours, washed with PBS-Tween, followed by three 5-minute washes with PBS alone. The bound antibody was visualized using a DAB substrate (using kit abcamab64238), followed by washing with water. Contrast staining with hematoxylin was performed for 30 seconds, followed by washing in water for 2 minutes. Figure 5B The staining of the plaques was shown by immune serum induced by CMV-Ntt830-Ab36 VLP.

[0266] Example 5 Cloning of CMV including modified shell proteins of Ara-h202 To obtain an antigen-containing mosaic VLP from a single plasmid system according to the present invention, the construction step involves inserting the CMV-Ntt830 gene into a multi-connector of pETDuet-1 (Novagen) under a second T7 promoter. In this paper, the CMV-Ntt830 nucleic acid sequence was prepared as described in Example 3 of WO2016 / 062720A1, and corresponds to SEQ ID NO: 14 of WO2016 / 062720A1. For the CMV structural gene with the corresponding cloning restriction site, the CMV-Ntt830 gene was amplified in a PCR reaction using the following oligonucleotides: Forward: CM-830NdeF (SEQ ID NO: 25) Reverse: CM-cpR (SEQ ID NO: 26) After gene amplification, the corresponding PCR products were directly cloned into the pTZ57R / T vector (InsTAclone PCR Cloning Kit, Fuxintes, product number K1214). *E. coli* XL1-Blue cells were used as the host for cloning and plasmid amplification. To avoid RT-PCR errors, several pTZ57 plasmid clones containing the CMV-Ntt830 gene were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 gene analyzer (Applied Biosystems). After sequencing, error-free pTZ plasmid clones containing the CMV-Ntt830 gene were digested with HindIII, treated with Klenow enzyme, and finally treated with NdeI restriction enzyme. The fragments were then subcloned into the NdeI / EcoRV site of pETDuet-1 to obtain the helper vector pETDu-CMV-Ntt830.

[0267] To insert the Ara-h202 protein-coding DNA sequence into the CMV-Ntt830 nucleic acid, a Gly-Ser adapter coding sequence of 15 and 10 amino acids containing a BamHI site was introduced, as described above, between the positions Ser (88) and Tyr (89) corresponding to SEQ ID NO:5 of CMV-Ntt830. The amino acid sequence of the Ara-h202 protein is described in SEQ ID NO: 27, while the corresponding DNA sequence is described in SEQ ID NO: 28.

[0268] The preferred chimeric CMV polypeptide according to the present invention has an amino acid sequence designated as “CMV-Ntt830-Arah202”, which is SEQ ID NO: 29. This preferred chimeric CMV polypeptide comprises glycine-serine linkers of the introduced Ara-h202 protein at both ends, specifically a 15-amino acid GS linker (SEQ ID NO: 30) directly located at the N-terminus of the introduced Ara-h202 protein and a 10-amino acid GS linker (SEQ ID NO: 31) located at the C-terminus of the introduced Ara-h202 protein. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-Arah202 is described in SEQ ID NO: 32.

[0269] First, the CMV-Ntt830 gene fragment and the Ara-h202 coding sequence (sidebanded with BamHI, without "start" and "stop" codons) were amplified in a PCR reaction using the following oligonucleotides: First PCR of the 5' end of the CMV gene: Forward: 830-NcoF (SEQ ID NO: 33) Reverse: C-5xg4s-R (SEQ ID NO: 34) Template: pET-CMV-Ntt830 was used, prepared as described in Example 3 of WO2016 / 062720A1. Second PCR at the 3' end of the CMV gene: Forward: C-5xg4s-F (SEQ ID NO: 35) Reverse: CMCPR (SEQ ID NO: 21) Template: pET-CMV-Ntt830 was used, prepared as described in Example 3 of WO2016 / 062720A1. The third PCR of Arah202: Forward: Ara-BamF2 (SEQ ID NO: 36) Reverse: Ara-BamR2 (SEQ ID NO: 37) Template: The Ara-h202 gene synthesized from the pUCIDT plasmid was prepared as described in Example 13 of WO 2017 / 186808A1.

[0270] All PCR fragments were directly ligated into the pTZ57R / T vector, and the corresponding plasmid clones containing the inserts were isolated after transformation in *E. coli* XL1 cells. To avoid PCR errors, several plasmid clones containing PCR product DNA were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Piperism 3100 gene analyzer (Applied Biosystems). After sequencing, the fragment at the 3' end of the CMV was ligated into the pTZ57-CMV-5' vector at the BamHI and HindIII sites. This yielded the helper plasmid pTZ-CMVB2, containing a GlySer adapter and a BamHI site, for further subcloning of the Ara-h202 coding sequence. Next, a portion of the BamHI-treated Ara-h202 fragment was subcloned into the pTZ-CMVB2 BamHI site. Using primers Ara-BamF2 / CMcpR, the correct clone containing the correctly oriented Ara-h202 insert was identified in a colony PCR reaction. Plasmid clones with positive PCR signals were re-sequencing. Sequencing results of the pTZ-CMVB2-Ara-h202 plasmid clone confirmed the presence of the Ara-h202 gene fused with CMV.

[0271] To construct the expression vector, the CMVB2-Arah202 insert was excised from the helper plasmid using NcoI and HindIII enzymes, and then subcloned into the constructed helper vector pETDu-CMV-Ntt830. The resulting pETDu-CMVB2xArah202-CMV-tt plasmid map is shown in [image / image / description]. Figure 6 middle.

[0272] Example 6 Expression of CMV-modified outer shell protein and fusion-Ara-h202 mosaic particles (CMV-M-Arah202). To isolate the mosaic CMV-Arah202 VLP, E. coli C2566 (New England Biolabs, USA) competent cells were transformed with plasmid pETDu-CMVB2xArah202-CMV-tt.

[0273] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and 5 mM MgCl2 was added to the medium. The culture was incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C. Biomass output was approximately 12 g wet biomass / L of culture, with an OD(600) of 6.8 at the end of culture.

[0274] The purification of the mosaic VLP comprising CMV-Ntt830-Arah202 and unmodified CMV-Ntt830 protein according to the present invention (the mosaic VLP is referred to as "CMV-M-Arah202") comprises the following steps: 1) Suspend 6 g of biomass in 20 ml of 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, 5 mM mercaptoethanol, pH 9.0, and treat the suspension with ultrasound (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 50 mM sodium citrate, 5 mM sodium borate, 2 mM EDTA, and 0.5% Tx-100. 4) Cover the sucrose gradient with 5 ml of VLP sample. Prepare 4 tubes; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25000 rpm, +18℃).

[0275] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze gradient fractions on SDS-PAGE and Western blot (Figure 7).

[0276] 8) SDS-PAGE analysis showed the presence of mosaic VLPs in the third sucrose fraction. Dilute 24 ml of the third fraction with 24 ml buffer (5 mM sodium borate, 2 mM EDTA, pH 9.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 3 ml of 5 mM sodium borate, 2 mM EDTA, pH 9.0; 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 5 mM sodium borate, 2 mM EDTA, pH 9.0 buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 5 mM sodium borate and 2 mM EDTA, pH 9.0, overnight, 4°C; 14) VLP was analyzed after purification by SDS-PAGE gel (Figure 7) and under EM (Figure 8).

[0277] Example 7 Mice were immunized with CMV-M-Arah202 flower leaf granules. Three female Balb / c mice in each group were subcutaneously immunized with Ara-h202 in total of 10 µg as VLP containing CMV-Ntt830-Arah202 and unmodified CMV-Ntt830 protein (CMV-M-Arah202), or in free form. The VLP was prepared in 150 mM PBS at pH 7.4 and administered subcutaneously at a volume of 150 µl (10 µg). Fourteen days post-immunization, mice were exsanguinated, and all immune sera containing recombinant Ara-h202 were analyzed by ELISA.

[0278] ELISA: Antibody responses in mouse serum were analyzed at indicated times. To determine the titer of Ara-h202-specific antibodies, ELISA plates (Nunc Immuno MaxiSorp, Rochester, NY) were coated overnight at 4°C with 100 µl of Ara-h2 purified from peanut extract (1 µg / ml) in PBS. To avoid nonspecific binding, the ELISA plates were blocked with PBST containing 200 µl of 2% BSA and incubated at room temperature for 2 hours. Serum samples were diluted in 2% BSA / PBST. Pre-diluted serum was transferred to the coated plates and further serially diluted to obtain antibody titers based on OD50. After incubation at room temperature for 2 hours, the ELISA plates were washed five times with 200 µl of PBST. Binding of serum antibodies was detected by horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunological Research Laboratories, Inc.). The detection antibody was diluted 1:1000 in 2% BSA / PBST and transferred in 100 µl volumes for each sample. Incubate the plate at room temperature for 1 hour. Wash the ELISA plate as described previously. Before washing, prepare the substrate solution. For this purpose, dissolve one tablet (10 mg) of OPD (1,2-phenylenediamine dihydrochloride) and 9 µl of 30% H2O2 in 25 mL of citrate buffer (0.066 M Na2HPO4, 0.035 M citrate, pH 5.0). Pipette 100 µl of the substrate solution onto the plate and incubate precisely at room temperature for 7 minutes. To terminate the reaction, pipette 50 µl of the stop solution (H2O containing 5% H2SO4) directly onto the plate. Analyze the absorbance reading of the 1,2-phenylenediamine dihydrochloride colorimetric reaction at 450 nm. Figure 9 An antibody specific to Ara-h2 was shown.

[0279] To determine Ara h202 IgG, 96-well NuncMaxisorp™ ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4°C with 2 µg / ml Ara h2 in PBS buffer. After blocking with PBS / 0.15% casein solution for 2 hours, the plates were washed five times with PBS / 0.05% Tween. Serial dilutions of serum were added to the plates, and the plates were incubated at 4°C for 2 hours. The plates were then washed five times with PBS / 0.05% Tween (PBST). Subsequently, HRP0-labeled goat anti-mouse IgG (Jackson Laboratory, Barthon, Maine, USA) was incubated at 4°C for 1 hour. ELISA was performed with TMB (3,30,5,50-tetramethylbenzidine) and H2O2, and terminated with 1 mol / L sulfuric acid. Optical density was measured at 450 nm. The half-maximum antibody titer is defined as the reciprocal of the dilution, which allows for the measurement of the half-maximum OD under saturation conditions. Figure 9 An antibody specific to Ara-h202 was shown.

[0280] Example 8 Immunization with CMV-M-Arah202 to prevent allergic reactions Sensitization and vaccination Mice were sensitized to peanut allergens by intraperitoneal injection of peanut extract prepared in 200 µl alum on days 0 and 7. Mice were then vaccinated with CMV-M-Arah202 or CMV-Ntt830 VLP as a control group (30 µg in 200 µl PBS on day 21). Figure 10A The experimental design for studying the protective effect of CMV-M-Arah202 vaccination against systemic and local allergic reactions is shown.

[0281] Systemic and local stimulation Challenge sensitized and vaccinated BALB / c patients with 20 μg of intravenous peanut extract in the ear or via skin prick test (180 μg / 20 μL PBS). This was achieved by temperature drop ( Figure 10B For simplicity, CMV-Ntt830 VLP is abbreviated as CMV) or fluid tissue extravasation (the diameter of the point, Figure 10CFor simplicity, CMV-Ntt830 VLP (abbreviated as CMV) was used to determine systemic and local allergic reactions separately. The figure represents two independent experiments. Mean + / - SEM values ​​for 5 mice in each group are shown. Allergic reaction curves were analyzed using a two-way Anova test. Skin prick tests were analyzed using a two-tailed Student's t-test.

[0282] Example 9 Construction and expression of a modified outer shell protein containing CMV and a mosaic particle fused with FEL D1 (CMV-M-Fel). First, the coding sequence of the preferred chimeric CMV polypeptide according to the present invention, referred to as "CMV-Ntt830-Feld12", was prepared.

[0283] CMV-Ntt830-Feld12 comprises the Fel d1 protein of SEQ ID NO: 38, which corresponds to a fusion protein of chain 1 and chain 2 of Fel d1, wherein it has a 15-amino acid GS linker connecting chain 1 and chain 2. The construct of SEQ ID NO: 38 is described in Example 7 of WO 2017 / 042241. Further, in CMV-Ntt830-Feld12, the Fel d1 protein of SEQ ID NO: 38 is side-linked by a glycine-serine linker. Specifically, the Feld1 protein of SEQ ID NO: 38 is directly side-linked at its N-terminus by a 15-amino acid GS linker of SEQ ID NO: 30, and directly side-linked at its C-terminus by a 10-amino acid GS linker of SEQ ID NO: 31. Furthermore, the complete construct described above, namely the construct of SEQ ID NO: 38 with the described glycine-serine linker, is inserted between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5 of CMV-Ntt830, thereby generating CMV-Ntt830-Feld12.

[0284] The amino acid sequence of this preferred chimeric CMV polypeptide according to the present invention is referred to as "CMV-Ntt830-Feld12", which is SEQ ID NO: 39. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-Feld12 is described in SEQ ID NO: 40.

[0285] The CMV-Ntt830-Feld12 gene was prepared similarly to that described in Example 7 of WO 2017 / 042241, the entire contents of which are incorporated herein by reference. To obtain the CMV-Ntt830-Feld12 gene, the Fel d1 corresponding gene was first amplified by PCR using the following oligonucleotides: Forward: FG4S-BamF (SEQ ID NO: 41) Reverse: FG4S-BamR (SEQ ID NO: 42) PCR fragments containing Gly-Ser adapters and BamHI sites at both ends were directly ligated into the pTZ57R / T vector, and plasmid clones containing the inserts were isolated after transformation in *E. coli* XL1 cells. To identify inserts without PCR errors, several plasmid clones containing PCR product DNA were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 Gene Analyzer (Applied Biosystems). After sequencing, the correct Fel d1 fragment was ligated into the helper vector pET-CMV-Ntt830B at the BamHI site. The correct clone containing the correctly oriented Fel d1 insert was identified in the "colony PCR" reaction using primers FG4S-BamF / CMcpR. Plasmid clones with positive PCR signals were re-sequencing, and the correct clones were selected for further cloning. Thus, the helper plasmid pET-CMVB-Feld1 was obtained. Next, the NcoI / HindIII-treated CMVB-Feld1 fragment was subcloned into the helper vector pETDu-CMV-Ntt830 (see Example 5). To construct an expression vector without the Amp resistance gene, the entire cassette containing the CMV-Ntt830-Feld12 fusion and the unmodified CMV-Ntt830 gene was excised and subcloned into the NcoI / XhoI site of pET28a+ (Novagen), resulting in the expression vector pET28-CMVBxFeld1-CMVtt. The plasmid map is shown in... Figure 11 middle.

[0286] The isolation of the mosaic CMV-Feld1 VLP, i.e., the isolation of the mosaic VLP (referred to as "CMV-M-Fel") comprising CMV-Ntt830-Feld12 and unmodified CMV-Ntt830 protein according to the present invention, comprises the following steps: Escherichia coli C2566 competent cells (New England Biolabs, USA) were transformed with plasmid pET28-CMVBxFeld1-CMVtt.

[0287] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing kanamycin (25 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and the medium was supplemented with 5 mM MgCl2. The cells were incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C.

[0288] The purification of variegated CMV-M-Fel VLP involves the following steps: 1) Suspend 6 g of biomass in 20 ml of 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, 5 mM mercaptoethanol, pH 9.0, and treat the suspension with ultrasound (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 50 mM sodium citrate, 5 mM sodium borate, 2 mM EDTA, and 0.5% TX-100. 4) Cover the sucrose gradient with 5 ml of VLP sample; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25,000 rpm, +18°C).

[0289] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze the gradient levels on SDS-PAGE ( Figure 12A ); 8) SDS-PAGE analysis showed the presence of mosaic VLPs in the second sucrose fraction. Dilute 24 ml of the third fraction with 24 ml of buffer (5 mM sodium borate, 2 mM EDTA, pH 9.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 3 ml of 5 mM sodium borate and 2 mM EDTA at pH 9.0; 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 5 mM sodium borate, 2 mM EDTA, pH 9.0 buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 5 mM sodium borate and 2 mM EDTA, pH 9.0, overnight, 4°C; 14) After SDS-PAGE gel purification ( Figure 12B ) and in EM ( Figure 12C VLP is analyzed below.

[0290] Example 10 Mice were immunized with CMV-M-Fel granules. Four female Balb / c mice in each group were immunized with CMV-M-Fel or Fel d1 chemically conjugated to CMVNtt830 VLP or recombinant Fel d1 extract. The VLP was prepared in 150 mM PBS at pH 7.4, and 25 μg was injected subcutaneously at a volume of 150 μL. The recombinant Fel d1 extract was prepared in PBS and 10 μg was injected subcutaneously. Two weeks later, the mice were bled, and antibody levels against Fel d1 were determined by ELISA. All immune sera were tested for recombinant Fel d1 by ELISA, as described by Schmitz N et al., Journal of Experimental Medicine (2009) 206:1941-1955.

[0291] ELISA: Antibody responses in mouse serum were analyzed at indicated times. To determine the titer of Fel d1-specific antibodies, ELISA plates (Nunc Immuno MaxiSorp, Rochester, NY) were coated overnight at 4°C with 100 µl of Fel d1 (1 µg / ml) in PBS. To avoid nonspecific binding, the ELISA plates were blocked with PBST containing 200 µl of 2% BSA and incubated at room temperature for 2 hours. Serum samples were diluted in 2% BSA / PBST. Pre-diluted serum was transferred to the coated plates and further serially diluted to obtain antibody titers based on OD50. After incubation at room temperature for 2 hours, the ELISA plates were washed five times with 200 µl of PBST. Binding of serum antibodies was detected by horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunological Research Laboratories, Inc.). The detection antibody was diluted 1:1000 in 2% BSA / PBST and transferred in 100 µl volumes for each sample. The plates were incubated at room temperature for 1 hour. Wash the ELISA plate as described above. Before washing, prepare the substrate solution. For this purpose, dissolve one tablet (10 mg) of OPD (1,2-phenylenediamine dihydrochloride) and 9 µl of 30% H2O2 in 25 mL of citrate buffer (0.066 M Na2HPO4, 0.035 M citrate, pH 5.0). Pipette 100 µl of the substrate solution onto the plate and incubate precisely at room temperature for 7 minutes. To terminate the reaction, pipette 50 µl of the stop solution (H2O containing 5% H2SO4) directly onto the plate. The absorbance reading of the 1,2-phenylenediamine dihydrochloride colorimetric reaction at 450 nm was analyzed. Figure 13 An antibody specific to Fel d1 was shown. Figure 13 ).

[0292] Example 11 Immunization with CMV-M-Fel to prevent allergic reactions On day 1, mice were sensitized to Fel d1 via intraperitoneal sensitization using 1 μg of natural Fel d1 isolated from cat hair in alum. Mice were immunized with CMV-M-Fel or CMV-Ntt830 VLP (30 μg in PBS on day 14) before determining intravenous anaphylactic response upon temperature drop.

[0293] sensitization Mice were sensitized to Fel d1 on day 0 by intraperitoneal injection of a natural Fel d1 extract (1 μg) prepared in 200 µl alum. Mice were then vaccinated with CMV-M-Fel or CMV-Ntt830 VLP as a control group (30 μg in 200 μl PBS on day 21). Figure 14A The experimental design for studying the protective effect of CMV-M-Fel vaccination against systemic and local allergic reactions is shown.

[0294] Systemic stimulation Intravenous provocation was performed in sensitized and vaccinated BALB / c mice using Fel d1 extract. Systemic anaphylactic response was determined by temperature decrease. Figure 14B The figure represents two independent experiments. The mean + / - SEM values ​​for each group of 5 mice are shown. Allergic reaction curves were analyzed using a two-way Anova test.

[0295] Example 12 Construction and expression of mosaic particles (CMV-M-CSP) containing a modified outer coat protein of CMV and a fusion internal repeat sequence of Plasmodium falciparum cyclosporine protein (CSP). To clone the CS protein fragment from Plasmodium falciparum, specifically the 19 NANP repeat sequences (SEQ ID NO: 43) of SEQ ID NO: 44 (19 nanp) derived from the central repeat region, the sequence of SEQ ID NO: 45 was obtained from a commercial source (gene synthesis): The sequence SEQ ID NO: 45 also encodes the 3' end portion of the CMV-Ntt830 gene and the necessary restriction sites BamHI and HindIII.

[0296] Next, the DNA fragment from the gene synthesis product was treated with BamHI / HindIII and then cloned into the helper vector pTZ-CMVB2 (see Example 5). Restriction enzyme site analysis (BamHI / HindIII) was used to identify the correct clone containing the 19NANP insert. The plasmid clones with the correct restriction enzyme pattern were re-sequencing. Next, the NcoI / HindIII-treated CMVB-19NANP fragment was subcloned into the helper vector pETDu-CMV-Ntt830 (see Example 5). To construct an expression vector without the Amp resistance gene, the entire cassette containing the CMV-19NANP fusion and the unmodified CMV-Ntt830 gene from pETDu-CMV-Ntt830 was excised and subcloned into the NcoI / BlpI site of pET28a+ (Novagen), resulting in the expression vector pET28-CMVB2x19nanp-CMVtt. Plasmid maps and sequence details are shown in... Figure 15 middle.

[0297] The amino acid sequence of this preferred chimeric CMV polypeptide according to the invention is referred to as “CMV-Ntt830-19NANP” (or “CMV-Ntt830-19nanp”, which are used interchangeably herein), and is SEQ ID NO: 46. This amino acid sequence of the preferred chimeric CMV polypeptide includes glycine-serine linkers of the introduced 19nanp protein, SEQ ID NO: 44, at both ends: a 15-amino acid GS linker (SEQ ID NO: 30) directly located at the N-terminus of the introduced 19nanp protein and a 12-amino acid GS linker (SEQ ID NO: 47) located at the C-terminus of the introduced 19nanp protein. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-19NANP is described in SEQ ID NO: 48.

[0298] Furthermore, the resulting plasmid clone pET28-CMVB2x19nanp-CMVtt, used to synthesize the mosaic VLP (i.e., CMV-M-CSP) comprising CMV-Ntt830-19NANP and unmodified CMV-Ntt830 protein, was used to transform *E. coli* C2566 cells. To isolate CMV-M-CSP, *E. coli* cell culture, biomass treatment, and purification were performed as described in Example 6. Analysis of the VLP after sucrose gradient purification on an SDS-PAGE gel is shown in... Figure 16 In the middle. Following electron microscopy analysis, images of the purified CMV-M-CSP showed that... Figure 17 middle.

[0299] Example 13 The protective efficacy of CMV-M-CSP Four female Balb / c mice were immunized with CMV-M-CSP in each group. VLP was prepared in 150 mM PBS at pH 7.4 and 10 μg was injected subcutaneously at a volume of 150 μL. To assess vaccine efficacy, six female BALB / c inbred mice and eight 8-week-old female CD1 outbred mice were purchased from Harlem, UK, and each mouse was intramuscularly (im) injected with 20 µg (50 µL) of either CMV-Ntt830 or CMV-M-CSP. Vaccination was performed on days 0 and 21. Blood samples were collected prior to each vaccination on days 0, 21, and 42, and CSP-specific immune responses were measured by ELISA. On day 42, mice were infected with a Plasmodium berghei substitute expressing CSP protein of Plasmodium falciparum. Parasitemia was assessed daily from day 4 post-challenge until mice reached 1% parasitemia.

[0300] Measurement of CSP-specific antibody response by ELISA To assess antibody production, total IgG, and its subclasses, an enzyme-linked immunosorbent assay (ELISA) was performed. For this purpose, 96-well microtiter ELISA plates (Thermo Fisher Scientific, Nottingham, UK) were coated with 100 µL per well in purified CSP at a concentration of 1 µg / mL, diluted in 50 mM carbonate buffer (CBB) at pH 9.6, and incubated overnight at 4°C. The next day, the plates were filled with 200 µL of 2% BSA-PBS to avoid nonspecific binding and incubated at room temperature for 2 hours. Serum from immunized mice was then diluted in 0.2% BSA-PBS buffer, first at a 100:1 ratio, and then serially diluted eleven times at 1 / 3 ratio in the ELISA plates. For total IgG measurements, 100 µL of goat anti-mouse IgG diluted 1:2000 (secondary antibody, HRP conjugate (Thermo Fisher Scientific, Paisley, UK)) was added to each well and incubated at room temperature for 1 hour. To evaluate IgG subclasses, goat anti-mouse IgG subclasses (HRP-conjugated goat anti-mouse IgG1, IgG2a, and IgG2b, Life Technologies) were used at a 1:2000 dilution and incubated at room temperature for 1 hour. For reaction development, 100 μL / well of TMB substrate (Sigma-Aldrich) was applied and incubated at room temperature for 10 minutes under aluminum foil to protect from light. The reaction was then terminated with 0.5 M H₂SO₄ (100 μL / well), and the plates were read at 450 nm using a microplate reader. Titers are expressed as dilutions resulting in the maximum half-maximum OD (OD₅₀).

[0301] Measurement of protective efficacy The parasite used in this study was *Plasmodium berghei*, which expresses the CSP protein of *Plasmodium falciparum* (Sci Rep. 2015 Jul 3;5:11820. doi: 10.1038 / srep11820.). Female *Anopheles stephensi* mosquitoes were fed to infected Tucker-Turkey (TO) mice. The infected mosquitoes were kept in a humidified incubator at 19–21°C for 21 days under a 12-hour diurnal cycle and fed a fructose-para-aminobenzoic acid (PABA) solution. After 21 days, the salivary glands of the mosquitoes were dissected, placed in Schneider medium (PanBiotech, Aidenbach, Germany), and sporozoites were gently released using a glass homogenizer. The sporozoites were diluted to a concentration of 1000 parasites per 100 µL and injected intravenously into the tail vein of the mice. Parasites. Starting from the fourth day after challenge, check for parasitemia daily until the mice reach 1% parasitemia.

[0302] Example 14 Construction and expression of VLP containing CMV fusion and α-synuclein peptide The CMV-α-synuclein-chimeric CMV polypeptide according to the present invention has been prepared, comprising α-synuclein peptide egy (SEQ ID NO: 49), kne (SEQ ID NO: 50) and mdv (SEQ ID NO: 51).

[0303] These α-synuclein peptides have been inserted between the amino acid residues Ser (88) and Tyr (89) of CMV-Ntt830 (SEQ ID NO: 5). The amino acid sequences of these preferred chimeric CMV peptides according to the invention are as follows: The amino acid sequence of “CMV-Ntt830-egy” is: SEQ ID NO: 52; The amino acid sequence of “CMV-Ntt830-kne” is: SEQ ID NO: 53; The amino acid sequence of “CMV-Ntt830-mdv” is SEQ ID NO: 54.

[0304] The amino acid sequences of these preferred chimeric CMV peptides further include glycine-serine linkers at both ends of the introduced α-synuclein peptide. All preferred fusion proteins of SEQ ID NO: 52 to SEQ ID NO: 54 include a GGGS linker (SEQ ID NO: 10) located directly at the N-terminus of the introduced α-synuclein peptide and a GGGSGS linker (SEQ ID NO: 11) located at the C-terminus of the introduced α-synuclein peptide.

[0305] The nucleotide sequence corresponding to the preferred chimeric CMV polypeptide is as follows: The nucleic acid sequence of “CMV-Ntt830-egy” is: SEQ ID NO: 55; The nucleic acid sequence of “CMV-Ntt830-kne” is: SEQ ID NO: 56; The nucleic acid sequence of “CMV-Ntt830-mdv” is SEQ ID NO: 57.

[0306] To introduce DNA encoding an α-synuclein peptide variant into the expression vector, the following oligonucleotides were used in the PCR reaction, with pET-CMV-Ntt830 as the template in all PCRs: First PCR positive: CM-egyF (SEQ ID NO: 58) Reverse: CMcpR (SEQ ID NO: 59) Second PCR positive: CM-kneF (SEQ ID NO: 60) Reverse: CMcpR (SEQ ID NO: 59) 3rd PCR positive: CM-mdvF (SEQ ID NO: 61) Reverse: CMcpR (SEQ ID NO: 59) All PCR fragments were directly ligated into the pTZ57R / T vector, and the corresponding plasmid clones containing the inserts were isolated after transformation in *E. coli* XL1 cells. Several plasmid clones containing the corresponding PCR products were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 Gene Analyzer (Applied Biosystems). After sequencing, the 3' end fragment of the CMV containing the α-synuclein fragment was excised from the pTZ vector using BamHI and HindIII restriction enzyme sites and ligated into the pET-CMV-Ntt830B helper vector (see Example 1). Correct clones were selected after BamHI / HindIII restriction endonuclease testing.

[0307] Furthermore, plasmid clones pET-CMV-Ntt830B-egy, pET-CMV-Ntt830B-kne, and pET-CMV-Ntt830B-mdv were used to transform *E. coli* C2566 cells. Plasmid maps and sequence details are shown in... Figure 18 middle.

[0308] To isolate the corresponding VLP containing α-synuclein peptide, E. coli cell culture, biomass processing and purification were performed as described in Example 2.

[0309] Analysis of VLPs after sucrose gradient purification on SDS-PAGE gels is shown in... Figure 19A , Figure 19B and Figure 19C as well as Figure 20A The electron microscope image is shown in the middle. Figure 20B , Figure 20C and Figure 20D middle.

[0310] Example 15 Construction and expression of a modified outer shell protein containing CMV and a mosaic particle fused with feline interleukin-5 (CMV-M-fel-IL-5). To clone the modified coat protein of CMV, which includes the feline IL-5 antigen, two different vectors were constructed.

[0311] The first vector was constructed using PCR mutagenesis and oligonucleotides, allowing the introduction of amino acid linkers flanking the fel IL-5 antigen, comprising at least one Gly, at least one Ser, and at least one Glu, and even further, at least one Asp: 1 PCR reaction: Forward: 830-NcoF (SEQ ID NO: 33) Reverse: Cmded-BamR (SEQ ID NO: 121) Template: pETDu-CMVB2xArah202-CMV-tt 2nd PCR reaction: Forward: CMded-BamF (SEQ ID NO: 122) Reverse: CM-cpR (SEQ ID NO: 26) Template: pETDu-CMVB2xArah202-CMV-tt After gene fragment amplification, the corresponding PCR products were directly cloned into the pTZ57R / T vector (InsTAclone PCR cloning kit, Fuxintes, product number K1214). *E. coli* XL1-Blue cells were used as the host for cloning and plasmid amplification. To avoid RT-PCR errors, several pTZ57 plasmid clones containing the CMV-Ntt830 gene were sequenced using the BigDye cycle sequencing kit and an ABI Prism 3100 gene analyzer (Applied Biosystems). After sequencing, pTZ plasmid clones containing the product from the first PCR reaction were digested with NcoI / BamHI restriction enzymes, and clones from the second PCR reaction were digested with BamHI / HindIII. These clones were then ligated with the helper vector pETDu-CMVB2xArah202-CMV-tt, which was digested with NcoI / HindIII. Here, plasmid pETDu-CMVB2xArah202-CMV-tt was used as a helper vector to generate a new CMV-based expression vector. NcoI / HindIII treatment completely removed the CMVB2xArah202 gene. The ligation of the three DNA fragments produced the helper plasmid pETDu-CMVB3d-CMVtt. The vector contains the CMV-Ntt830 gene and a Th cell epitope derived from tetanus toxin in two encoded proteins, along with an introduced sequence encoding an amino acid linker comprising at least one Gly, at least one Ser, and at least Glu, and even further comprising at least one Asp, specifically including a Gly-Ser linker with an additional Asp-Glu-Asp segment and a BamHI / SpeI site for subcloning the antigenic DNA sequence of the CMV-Ntt830 gene under a first T7 promoter.

[0312] Furthermore, a second vector was constructed using PCR mutagenesis and oligonucleotides, allowing the introduction of a GS adapter at the N-terminus of the fel IL-5 antigen and an amino acid adapter comprising at least one Gly, at least one Ser, and at least Thr at the C-terminus of the fel IL-5 antigen: 3rd PCR reaction: Forward: CM-BamSpeF (SEQ ID NO: 137) Reverse: CM-cpR (SEQ ID NO: 26) Template: pETDu-CMVB2xArah202-CMV-tt The PCR product from reaction 3 was also directly cloned into the pTZ57R / T vector; the resulting ligation mixture was used to transform *E. coli* XL1-Blue cells. After isolating the plasmid DNA, several clones were sequenced. Correct plasmid clones were digested with BamHI / HindIII restriction enzymes, and the resulting fragments were subcloned into pETDu-CMVB2xArah202-CMV-tt, which was digested with the same enzymes. The ligation reaction produced the helper plasmid pETDu-CMVB3-CMVtt.

[0313] The feline interleukin-5 (IL5) gene was obtained from Gene Synthesis Services (General Biosystems, USA) in the form of plasmid pET42-felIL5N. For cloning, the felIL5 gene was amplified in a PCR reaction using the following oligonucleotides: Forward: I5-BamF (SEQ ID NO: 123) Reverse: I5-SpeR (SEQ ID NO: 124) Template: pET42-felIL5N The PCR product was directly ligated into pTZ57, and several clones were sequenced after plasmid DNA isolation. After identifying clones without sequence errors, the felIL5 gene was further subcloned into pETDu-CMVB3d-CMV-tt or pETDu-CMVB3-CMV-tt at the BamHI / SpeI site. The resulting plasmid maps of pETDu-CMVB3d-flIL5-CMV-tt and pETDu-CMVB3-flIL5-CMV-tt are shown in [image / image / description]. Figure 21A and Figure 21B The plasmids and expression vectors ensure and are used to express mosaic VLPs (i.e., CMV-M-fel-IL-5) including CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein.

[0314] CMV-Ntt830-fel-IL-5 comprises the feline IL-5 protein of SEQ ID NO: 125, which is side-linked by an amino acid linker comprising at least one Gly, at least one Ser, and at least one Glu. Specifically, the feline IL-5 protein of SEQ ID NO: 125 is directly side-linked at its N-terminus by a GSED linker of 18 amino acids in length of SEQ ID NO: 126, and directly side-linked at its C-terminus by a GSED linker of 15 amino acids in length of SEQ ID NO: 127. Further, the complete construct described above, i.e., the construct of SEQ ID NO: 125 side-linked by the described GSED linker, is inserted between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5 of CMV-Ntt830, thereby producing CMV-Ntt830-fel-IL-5. The amino acid sequence of this preferred chimeric CMV polypeptide according to the present invention is referred to as "CMV-Ntt830-fel-IL-5", which is SEQ ID NO: 128. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-fel-IL-5 is described in SEQ ID NO: 129.

[0315] CMV-Ntt830-fel-IL-5 * The feline IL-5 protein, including SEQ ID NO: 125, is side-linked by a GS linker at the N-terminus of the fel IL-5 antigen and an amino acid linker at the C-terminus comprising at least one Gly, at least one Ser, and at least one Thr. Specifically, the feline IL-5 protein of SEQ ID NO: 125 is directly side-linked at its N-terminus by a 15-amino acid GS linker of SEQ ID NO: 30 and directly side-linked at its C-terminus by an 11-amino acid GST linker of SEQ ID NO: 138. Further, the complete construct described above, i.e., the construct of SEQ ID NO: 125 side-linked by the described GS and GST linkers, is inserted between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5 of CMV-Ntt830, thereby generating CMV-Ntt830-fel-IL-5. * The amino acid sequence of this chimeric CMV polypeptide according to the present invention is referred to as "CMV-Ntt830-fel-IL-5". * ", which is SEQ ID NO: 139. This chimeric CMV polypeptide CMV-Ntt830-fel-IL-5 * The corresponding nucleotide sequence is described in SEQ ID NO: 140.

[0316] Therefore, in order to express and purify CMV-M-fel-IL-5 and CMV-M-fel-IL-5 * Escherichia coli C2566 (New England Biolabs, USA) competent cells were transformed with plasmids pETDu-CMVB3d-flIL5-CMVtt and pETDu-CMVB3-flIL5-CMVtt.

[0317] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and 5 mM MgCl2 was added to the medium. The culture was incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C. Biomass output was approximately 14 g wet biomass / L of culture, with an OD(600) of 7.6 at the end of culture.

[0318] Including CMV-Ntt830-fel-IL-5 or CMV-Ntt830-fel-IL-5 * And the mosaic VLP of unmodified CMV-Ntt830 protein (the mosaic VLP is referred to as CMV-M-fel-IL-5 and CMV-M-fel-IL-5). * The purification process includes the following steps: 1) Suspend 1.5 g of biomass in 10 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and treat the suspension with sonication (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 0.5% TX-100. 4) Cover the sucrose gradient with 5 ml of VLP sample. Prepare 2 tubes; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25000 rpm, +18℃).

[0319] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze the gradient levels on SDS-PAGE ( Figure 22A and Figure 22B ).

[0320] 8) SDS-PAGE analysis showed the presence of mosaic VLPs in the second and third sucrose gradient fractions. The second and third fractions were diluted with equal volumes of buffer (20 mM Tris-HCl, 5 mM EDTA, pH 8.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose. 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and leave overnight at 4°C. 14) Clarify the suspension by centrifugation (5 minutes, 13,000 rpm, Eppendorf 5418). 15) After SDS-PAGE gel purification ( Figure 23A and Figure 23C ) and in EM ( Figure 23B and Figure 23D VLP is analyzed below.

[0321] EM images show that, along with CMV-Ntt830-fel-IL-5 * and unmodified CMV-Ntt830 protein and therefore VLPCMV-M-fel-IL-5 * Compared to mosaic VLPs, mosaic VLPs including CMV-Ntt830-fel-IL-5 and unmodified CMV-Ntt830 protein, and therefore mosaic VLP CMV-M-fel-IL-5, contain fewer aggregated VLPs. This finding was confirmed by dynamic light scattering (DLS) analysis of the two mosaic VLPs according to the invention.

[0322] Example 16 Construction and expression of a CMV-modified outer shell protein and a mosaic particle fused to replicate feline interleukin-5 (CMV-M-2xfel-IL-5). To clone the modified coat protein of the CMV containing two copies of the feline IL-5 antigen, a corresponding vector was constructed using PCR mutagenesis and oligonucleotides, allowing the introduction of amino acid linkers comprising at least one Gly, at least one Ser, and at least Glu, and even further including at least one Asp, flanking the feline IL-5 antigen, as well as an amino acid linker to connect the two feline IL-5 antigens: 1 PCR reaction: Forward: I5-BamF (SEQ ID NO: 123) Reverse: 2xIL5-gsKpnR (SEQ ID NO: 130) Template: pETDu-CMVB3d-flIL5-CMV-tt 2nd PCR reaction: Positive: 2xIL5-gsKpnF (SEQ ID NO: 131) Reverse: I5-SpeR (SEQ ID NO: 124) Template: pETDu-CMVB3d-flIL5-CMV-tt After gene fragment amplification, the corresponding PCR products were directly cloned into the pTZ57R / T vector (InsTAclone PCR Cloning Kit, Fuxintes, product number K1214). *E. coli* XL1-Blue cells were used as the host for cloning and plasmid amplification. To identify plasmids free of PCR errors, several pTZ57 plasmid clones containing the fel-IL5 gene were sequenced using the BigDye Cyclic Sequencing Kit and an ABI Prism 3100 Gene Analyzer (Applied Biosystems). Following sequencing, the correct pTZ plasmid clone containing the product from the second PCR reaction was digested with Kpn2 / EcoRI restriction enzymes, and the resulting fragment was ligated into a pTZ plasmid containing the PCR product from the first PCR reaction, digested with the same restriction enzymes. The ligation reaction produced the helper plasmid pTZ-2xflIL5, which contains two copies of the flIL5 gene ligated to the Gly-Ser adapter. Plasmids containing the replicated felIL5 were purified from XL1 cells. The 2xfl-IL5 gene was further excised using the BamHI / SpeI restriction enzyme and ligated into the expression vector pETDu-CMVB3d-CMVtt at the same restriction site. The resulting vector contains CMV-Ntt830 and introduced sequences encoding both feline IL5 genes, separated by a Gly-Ser linker and side-linked by amino acid linkers comprising at least one Gly, at least one Ser, and at least Glu, and even further comprising at least one Asp, specifically including a Gly-Ser linker with an additional Asp-Glu-Asp segment. The plasmid map of the resulting pETDu-CMVB3d-2xflIL5-CMV-tt is shown in [image / image / description]. Figure 24 The plasmids and expression vectors ensure and are used to express mosaic VLPs (i.e., CMV-M-2xfel-IL-5) comprising CMV-Ntt830-2xfel-IL-5 and unmodified CMV-Ntt830 protein.

[0323] CMV-Ntt830-2xfel-IL-5 comprises two copies of the feline IL-5 protein of SEQ ID NO: 125, which is linked to a Gly-Ser linker (SEQ ID NO: 30) and side-linked by an amino acid linker comprising at least one Gly, at least one Ser, and at least one Glu. Specifically, the sequence of the two copies of the feline IL-5 protein of SEQ ID NO: 125 linked to the Gly-Ser linker (SEQ ID NO: 30) is directly side-linked at its N-terminus by an 18-amino acid GSED linker of SEQ ID NO: 126, and directly side-linked at its C-terminus by a 15-amino acid GSED linker of SEQ ID NO: 127. Furthermore, the complete construct described above, namely the construct of two copies of SEQ ID NO: 125 connected to SEQ ID NO: 30 and side-connected by the described GSED connector, is inserted between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5 of CMV-Ntt830, thereby producing CMV-Ntt830-2xfel-IL-5.

[0324] The amino acid sequence of this preferred chimeric CMV polypeptide according to the present invention is referred to as "CMV-Ntt830-2xfel-IL-5", which is SEQ ID NO: 132. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-2xfel-IL-5 is described in SEQ ID NO: 133.

[0325] Therefore, or expressing and purifying mosaic CMV-M-2xfel-IL-5, and transforming Escherichia coli C2566 (New England Biolabs, USA) competent cells with plasmid pETDu-CMVB3d-2xflIL5-CMVtt.

[0326] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and 5 mM MgCl2 was added to the medium. The culture was incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C. Biomass output was approximately 15 g wet biomass / L of culture, with an OD(600) of 8.0 at the end of culture.

[0327] The purification of the mosaic VLP (referred to as "CMV-M-2xfel-IL-5") comprising CMV-Ntt830-2xfel-IL-5 and unmodified CMV-Ntt830 protein according to the present invention comprises the following steps: 1) Suspend 1.5 g of biomass in 10 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and treat the suspension with sonication (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 0.5% TX-100. 4) Cover the sucrose gradient with 5 ml of VLP sample. Prepare 2 tubes; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25000 rpm, +18℃).

[0328] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze gradient levels on SDS-PAGE ( Figure 25 ).

[0329] 8) SDS-PAGE analysis showed the presence of mosaic VLPs in the second and third sucrose gradient fractions. The second and third fractions were combined and diluted with equal volumes of buffer (20 mM Tris-HCl, 5 mM EDTA, pH 8.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose. 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and leave overnight at 4°C. 14) Clarify the suspension by centrifugation (5 minutes, 13,000 rpm, Eppendorf 5418). 15) After SDS-PAGE gel purification ( Figure 26A ) and in EM ( Figure 26B VLP is analyzed below.

[0330] Example 17 Construction and expression of a CMV-M-cIL-1b mosaic particle containing a modified outer shell protein of CMV and fused with canine interleukin-1b. The canine interleukin-1b gene with BamHI and SpeI sites was obtained from a commercial source (the gene synthesis product of pUCcIL1b from General Biosystems, USA). The BamHI / SpeI fragment was excised from the plasmid pUCcIL1b-BS and ligated into the helper vector pETDu-CMVB3d-CMVtt (with BamHI and SpeI sites). The plasmid was isolated from *E. coli* XL1 cells and re-sequencing was performed to verify the introduced cIL-1b sequence. The plasmid map of pETDu-CMVB3d-cIL1b-CMV-tt is shown below (…). Figure 27 As shown in the figure. The plasmid and expression vector ensure and are used to express the mosaic VLP (i.e., CMV-M-cIL-1b) including CMV-Ntt830-cIL-1b and unmodified CMV-Ntt830 protein.

[0331] CMV-Ntt830-cIL-1b comprises the canine IL-1b protein of SEQ ID NO: 134, which is side-linked by an amino acid linker comprising at least one Gly, at least one Ser, and at least one Glu. Specifically, the canine IL-1b protein of SEQ ID NO: 134 is directly side-linked at its N-terminus by a GSED linker of SEQ ID NO: 126 with a length of 18 amino acids, and directly side-linked at its C-terminus by a GSED linker of SEQ ID NO: 127 with a length of 15 amino acids. Further, the complete construct described above, i.e., the construct of SEQ ID NO: 134 side-linked by the described GSED linker, is inserted between the positions corresponding to Ser (88) and Tyr (89) of SEQ ID NO: 5 of CMV-Ntt830, thereby generating CMV-Ntt830-cIL-1b.

[0332] The amino acid sequence of this preferred chimeric CMV polypeptide according to the present invention is referred to as "CMV-Ntt830-cIL-1b", which is SEQ ID NO: 135. The corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV-Ntt830-cIL-1b is described in SEQ ID NO: 136.

[0333] Therefore, the mosaic CMV-M-cIL-1b was expressed and purified, and then transformed into E. coli C2566 (New England Biolabs, USA) competent cells with the plasmid pETDu-CMVB3d-cIL1b-CMVtt.

[0334] After selecting clones with the highest target protein expression levels, *E. coli* cultures were grown at 30°C on a rotary shaker in 2TY medium (1.6% trypsin, 1% yeast extract, 0.5% NaCl, 0.1% glucose) containing ampicillin (100 mg / L) until an OD(600) value of 0.8–1.0 was achieved. Cells were then induced with 0.2 mM IPTG, and 5 mM MgCl2 was added to the medium. The culture was incubated at 20°C on a rotary shaker for 18 hours. The resulting biomass was collected by low-speed centrifugation and frozen at -20°C. Biomass output was approximately 15 g wet biomass / L of culture, with an OD(600) of 8.8 at the end of culture.

[0335] The purification of mosaic VLPs comprising CMV-Ntt830-cIL-1b and unmodified CMV-Ntt830 protein according to the present invention (the mosaic VLPs are referred to as "CMV-M-cIL-1b") comprises the following steps: 1) Suspend 1.5 g of biomass in 10 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and treat the suspension with sonication (Hielscher UP200S sonicator, 16 minutes, 70% amplitude, 0.5 cycles). 2) Centrifuge the lysate at +4°C at 11,000 rpm for 20 minutes; 3) Prepare a sucrose gradient (20-60%) in a 35 ml test tube in a buffer solution containing 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 0.5% TX-100. 4) Cover the sucrose gradient with 5 ml of VLP sample. Prepare 2 tubes; 5) Centrifuge for 6 hours using a Beckman SW32 rotor (25000 rpm, +18℃).

[0336] 6) Divide the contents of each gradient tube into 6 ml fractions. Combine the corresponding fractions; 7) Analyze gradient levels on SDS-PAGE ( Figure 28 ).

[0337] 8) SDS-PAGE analysis showed the presence of mosaic VLPs in the second and third sucrose gradient fractions. The second and third fractions were combined and diluted with equal volumes of buffer (20 mM Tris-HCl, 5 mM EDTA, pH 8.0); 9) Collect VLP by ultracentrifugation using a Type 70 rotor (Beckman Optima L100XP ultracentrifuge; 4 hours, at 50,000 rpm, 5°C); 10) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose. 11) Cover the top of the 20% sucrose “pad” with the VLP suspension (in 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol buffer). 12) Collect VLP by ultracentrifugation using a rotor TLA100.3 (Beckman; 1 hour, 72,000 rpm, 5°C); 13) Dissolve the precipitate in 2 ml of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM mercaptoethanol, 5% glycerol, and 10% sucrose, and leave overnight at 4°C. 14) Clarify the suspension by centrifugation (5 minutes, 13,000 rpm, Eppendorf 5418). 15) In SDS-PAGE gel ( Figure 29A ) on and under EM ( Figure 29B VLPs were analyzed after purification.

Claims

1. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV), comprising: a) At least one fusion protein, wherein the at least one fusion protein comprises a chimeric CMV polypeptide, the chimeric CMV polypeptide comprising... (i) a CMV polypeptide, wherein the CMV polypeptide comprises a CMV coat protein; and (ii) an antigenic polypeptide, wherein the antigenic polypeptide is inserted into the CMV polypeptide, and b) At least one CMV protein, wherein the CMV protein comprises or is composed of a CMV shell protein.

2. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 1, wherein the insertion of the antigenic polypeptide is located between the amino acid residues corresponding to the amino acid residues at positions 84 and 85 of SEQ ID NO: 62 of the CMV polypeptide.

3. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 1, wherein the chimeric CMV polypeptide further comprises a first amino acid linker, wherein the first amino acid linker is located at the N-terminus or C-terminus of the antigen polypeptide, wherein preferably the length of the first amino acid linker is up to 30 amino acids.

4. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 3, wherein the chimeric CMV polypeptide further comprises a second amino acid linker, wherein the first amino acid linker is located at the N-terminus of the antigenic polypeptide, and the second amino acid linker is located at the C-terminus of the antigenic polypeptide, wherein preferably the length of the second amino acid linker is at most 30 amino acids.

5. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 4, wherein the first amino acid linker and the second amino acid linker are independently selected from the group consisting of: (a.) Polyglycine linkers (Gly) with lengths n = 2–10 n ; (b.) A glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein preferably the GS linker has an amino acid sequence (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (c.) An amino acid linker comprising at least one Gly, at least one Ser and at least one amino acid selected from Thr, Ala, Lys, Asp and Glu.

6. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 4, wherein the first amino acid linker and / or the second amino acid linker are independently selected from: (i) a glycine-serine linker (GS linker) comprising at least one glycine and at least one serine, wherein the GS linker has an amino acid sequence (GS). r (G s S) t (GS) u , where r = 0 or 1, s = 1-5, t = 1-5 and u = 0 or 1; and (ii) a glycine-serine-glutamic acid-aspartic acid linker (GSED linker) comprising at least one glycine, at least one serine, at least one glutamic acid and at least one aspartic acid, wherein the GSED linker comprises an amino acid sequence (DED). x (G s S) t (G) y (DED) z (GS) u , where s = 1-5, t = 1-5, u = 0 or 1, x = 0 or 1, y = 0-5 and z = 0 or 1.

7. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the CMV protein comprises an amino acid sequence having at least 90%, preferably 95%, sequence identity with SEQ ID NO: 62, and wherein the CMV protein is optionally modified by a T helper cell epitope, and wherein preferably the coat protein of the CMV comprises SEQ ID NO:

62.

8. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the antigenic polypeptide is a polypeptide derived from the group consisting of: (a) allergens; (b) viruses; (c) bacteria; (d) parasites; (e) tumors; (f) automolecules; (g) hormones; (h) cytokines; and (i) chemokines.

9. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the antigenic polypeptide is β-amyloid peptide, peanut allergen or peanut-derived allergen, feline allergen Feld1, Plasmodium cyclosporin, α-synuclein or a peptide derived from α-synuclein, IL-5 or IL-1.

10. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the antigenic polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1-4, 27, 38, 44, 49-51, 125 or 134.

11. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the length of the antigenic polypeptide is at least 3 amino acids and at most 225 amino acids, preferably at least 40 amino acids and at most 225 amino acids, more preferably at least 50 amino acids and at most 200 amino acids, and most preferably at least 70 amino acids and at most 200 amino acids.

12. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the chimeric CMV polypeptide comprises a T helper cell epitope.

13. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 12, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, preferably wherein the N-terminal region of the CMV polypeptide replaced by the T helper cell epitope corresponds to amino acids 2-12 of SEQ ID NO:

62.

14. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 12, wherein the T helper cell epitope is derived from tetanus toxin or is a PADRE sequence.

15. The modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to claim 12, wherein the T helper cell epitope comprises the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO:

65.

16. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the CMV polypeptide comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 62, or wherein the CMV polypeptide comprises the amino acid sequence of SEQ ID NO:

62. Preferably, the amino acid sequence of the CMV polypeptide includes SEQ ID NO: 63 or an amino acid sequence region, wherein the amino acid sequence region has at least 90% sequence identity with SEQ ID NO:

63.

17. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 66, wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 5 between amino acid residues at positions 88 and 89 of SEQ ID NO: 5, or wherein the antigenic polypeptide is inserted into the chimeric CMV polypeptide of SEQ ID NO: 66 between amino acid residues at positions 86 and 87 of SEQ ID NO:

66.

18. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the chimeric CMV polypeptide is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 135 or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide is composed of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 129, SEQ ID NO: 13 ... The codes are NO:133, SEQ ID NO:136, or SEQ ID NO:

140.

19. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the chimeric CMV polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 135 or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide is composed of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 139, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 139, or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 139, or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide comprises ... Encoded by NO:129, SEQ ID NO:133, SEQ ID NO:136, or SEQ ID NO:140, and wherein the at least one CMV protein comprises SEQ ID NO:62 optionally modified by a T helper cell epitope, preferably wherein the T helper cell epitope replaces the N-terminal region of the CMV protein, wherein the N-terminal region of the CMV protein corresponds to amino acids 2-12 of SEQ ID NO:62, and wherein the T helper cell epitope is composed of SEQ ID NO:64 or SEQ ID NO:

65.

20. A modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-6, wherein the chimeric CMV polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 135 or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide is composed of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 139, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 135 or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 139, or SEQ ID NO: 139, or wherein the chimeric CMV polypeptide comprises ... The protein is encoded by NO:129, SEQ ID NO:133, SEQ ID NO:136, or SEQ ID NO:140, and wherein the at least one CMV protein therein comprises SEQ ID NO:

5.

21. A vaccine comprising a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-20.

22. A pharmaceutical composition comprising: (a) a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) according to any one of claims 1-20; and (b) a pharmaceutically acceptable carrier, diluent, and / or excipient.

23. Use of the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) according to any one of claims 1-20 in the preparation of a medicament for immunization in animals.

24. Use of the modified virus-like particles (VLPs) of cucumber mosaic virus (CMV) according to any one of claims 1-20 in the preparation of a medicament for the treatment or prevention of diseases, symptoms or physiological conditions in animals.

Images