WO2021260131A1 - Particules pseudo-virales du cmv modifiées par fusion - Google Patents

Particules pseudo-virales du cmv modifiées par fusion Download PDF

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WO2021260131A1
WO2021260131A1 PCT/EP2021/067380 EP2021067380W WO2021260131A1 WO 2021260131 A1 WO2021260131 A1 WO 2021260131A1 EP 2021067380 W EP2021067380 W EP 2021067380W WO 2021260131 A1 WO2021260131 A1 WO 2021260131A1
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cmv
seq
amino acid
polypeptide
linker
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Andris ZELTINS
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Saiba AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/14011—Bromoviridae
    • C12N2770/14023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/14011—Bromoviridae
    • C12N2770/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a modified virus-like particle of plant virus Cucumber Mosaic Virus (CMV), and in particular to a modified VLP of CMV comprising chimeric CMV polypeptides which comprise an antigenic polypeptide inserted into a CMV polypeptide at a specific position and further comprises a Th cell epitope replacing a N- terminal region of said CMV polypeptide, wherein said antigenic polypeptide is a receptor binding domain (RBD) of a coronavirus (CoV), or a fragment thereof.
  • RBD receptor binding domain
  • CoV coronavirus
  • modified VLPs serve as vaccines for generating immune responses, in particular antibody responses, against said antigenic polypeptides fused into said CMV polypeptides for the prevention and treatment of a disease caused by said coronavirus.
  • modified virus-like particles being mosaic virus-like particle comprising said chimeric CMV polypeptides comprising the in-fused antigenic polypeptides and further CMV proteins not comprising said antigenic
  • VLPs Plant viruses and virus-like particles derived therefrom have recently attracted attention, mainly due to the role of plants as an economical and speedy alternative platform for producing VLP vaccines in the light of their ability to provide distinctive posttranslational modifications, cost-effectiveness, beneficial safety profiles, production speed and scalability (Chen Q and Lai H, Human Vaccines & Immunotherapeutics (2013) 9:26-49; Zeltins A, Mol Biotechnol (2013) 53:92-107; Balke I and Zeltins A, Adv. Drug Deliv. Rev. (2019) 145:119-129; Balke I and Zeltins A, Viruses (2020) 12:270-286).
  • CMV Cucumber Mosaic Virus
  • RNA1, RNA2 and RNA3 the coat protein (CP) gene being present both in the genomic RNA3 (about 2200 nt) and in the subgenomic RNA4 (about 1000 nt).
  • the capsid comprises 180 copies of a single protein species of about 25 kDa.
  • CMV VLPs are derived from modified CPs of CMV with inserted T-cell stimulating epitopes (A. Zeltins, et al. Vaccines 2 (2017) 30; WO2016/062720).
  • Chimeric forms of CMV have also been engineered to function as a presentation system and to express on their outer surface epitopes derived from the hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • a CMV pseudo-recombinant form CMV-D/S has been engineered to carry genomic RNA3 from the CMV-S strain and RNA1 and RNA2 from the CMV-D strain.
  • the CP gene was then engineered in different positions, to encode a Hepatitis C virus (HCV) epitope, called R9 mimotope, a synthetic peptide of 27 amino acids derived from many hypervariable region 1 (HVRl) sequences of the HCV envelope protein E2.
  • HCV Hepatitis C virus
  • the selection of the insertion points of the R9 mimotope into the CMV gene was made taking essential factors into account: i) the need to protect the N-terminal region of the CMV coat protein (containing a high concentration of basic amino acids, known as an internal R-domain involved in protein-RNA interactions stabilizing CMV (Wikoff WR, et al., Virology (1997) 232: 91-97) characterized by an unusual N-terminal helix with an additional stabilizing role in the capsid (Smith TJ, et al., J Virol (2000) 74: 7578-7686); ii) the surface location of the foreign epitope to increase the chance of its putative immunogenic capability; iii) the availability of mutagenesis routes able to produce the modified clones.
  • the insertion of the R9 mimotopes has been effected at different locations within the CP gene of CMV-S RNA3 (AF063610, www. dpyweb . net) .
  • the R9 mimotope nucleotide sequence was inserted in positions 253, 475, 529 of said CP gene, whereas for the insertion of two R9 mimotopes, the R9 mimotope nucleotide sequence was inserted in position 392 and 529.
  • the so prepared chimeric CMVs retained their ability to spread systemically in the host plant, a lower virus extraction yield was obtained in case of the first two insertion sites.
  • the mosaic CMV containing the R9 mimotope inserted at position 529 of the CP gene was selected and tested for HCV patient serum reactivity (Natilla A, et al., Arch Virol (2004) 149:137-154; PiazzollaG, etak, J Clin Immunol (2005) 25:142-152; Nuzzaci M, etak, Arch Virol (2007) 152:915-928; Nuzzaci M, et ah, Journal of Virological Methods (2009) 155:118-121; Nuzzaci M, et ah, Journal of Virological Methods (2010) 165:211-215; Piazzolla G, et ah, J Clin Immunol (2012) 32:866-876).
  • cucumber mosaic virus based expression systems have been described either as potential vaccine against Alzheimer’s disease as well as for the production of porcine circovirus specific vaccines (Vitti A, et al., J Virol Methods (2010) 169:332-340; Gellert A, et al., PloS ONE (2012) 7(12): e52688).
  • chimeric constructs bearing different 11-15 amino acids long Ab-derived fragments in positions 248, 392 or 529 of the CMV coat protein (CP) gene were created and the viral products proved to be able to replicate in their natural host.
  • porcine circovirus type 2 (PCV2) capsid protein epitopes of up to a length of 20 amino acids were integrated into the plant virus coat protein of cucumber mosaic virus (CMV)-R strain after amino acid position 131.
  • This insertion point 131-132 is located in the middle of the bE-aEF loop of the CMV CP and it has been concluded that this position has the advantage that the inserted epitopes form a tripartite group in the middle of the CMV CP trimers allowing the production of antibodies more efficiently.
  • VLPs chimeric virus-like particles
  • VX potato virus X
  • the chimeric CMV coat proteins comprise 17-25 amino acids long epitopes of Newcastle disease virus (NDV) genetically fused into the internal bH-bI (motif 5) loop of the CMV CP which corresponds to amino acids 194-199 thereof (He X, et al (1998) J Gen Virol 79: 3145-3153).
  • NDV Newcastle disease virus
  • VLP based vaccines induce variable antibody responses in immunized subjects and individuals often spanning a range of more than 100-fold variation.
  • some vaccines such as the vaccine against Hepatitis B, suffer from a certain number of non-responders. Non-responsiveness is associated with certain MHC class II molecules and failure to induce good T helper (Th) cell responses is believed to be responsible for poor antibody responses seen in these individuals (Goncalves L, et al., Virology (2004) 326:20-28).
  • Th T helper
  • vaccines inducing good Th cell responses in essentially all subjects and individuals are an important goal in the field of vaccine development.
  • one further associated very important problem yet to be solved during the vaccine construction process is antigen spatial conformation.
  • VLPs are able to accommodate more than 50 or 70 amino acids-long protein domains or even still longer and retain the typical VLP morphology (Balke I and Zeltins A, Adv. Drug Deliv. Rev. (2019) 145:119-129; I. Kalnciema, et al. Mol. Biotechnol. 52 (2012) 129- 139).
  • HCoV-229E and HCoV-NL63 are alpha-CoVs.
  • the other five are beta-CoVs and include beside SARS- CoV, MERS-CoV and SARS-CoV-2, the low-pathogenicity coronaviruses HCoV-OC43 and HCoV-HKUl (A. C. Walls et al. Cell (2020) 180:1-12 and references cited therein; Z - W. Ye et al. Int. J. Biol. Sci. (2020) 16:1686-1697).
  • coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein.
  • S glycoprotein As the coronavirus S glycoprotein is surface-exposed and mediates entry into host cells, it is the main target of neutralizing antibodies upon infection and is the primary determinant of viral tropism.
  • the coronavirus spike protein (S) is a large (approx. 180 kDa) glycoprotein that is present on the viral surface as a prominent trimer, and it is composed of two domains, SI and S2.
  • the SI domain mediates receptor binding and is divided into two sub-domains, with the N-terminal subdomain (NTD) often binding sialic acid and the C- terminal subdomain (also known as C-domain) binding a specific proteinaceous receptor and comprising the receptor binding domain (RBD) and the receptor binding motif (RBM).
  • NTD N-terminal subdomain
  • C-domain C-terminal subdomain
  • RBM receptor binding motif
  • SARS-CoV SARS-CoV
  • SARS-CoV-2 SARS-CoV-2 and several SARS-related coronaviruses (SARSr-CoV) interact directly with angiotensin-converting enzyme 2 (ACE2) to enter target cells.
  • ACE2 angiotensin-converting enzyme 2
  • RBDs receptor-binding domains
  • the S protein of SARS-CoV-2 was found to be approximately 75% homologous to the SARS-CoV spike protein, while solely an average of -30% identity was found between the S proteins of HCoV-HKUl, SARS-CoV and MERS-CoV. Despite the differences at the amino acid level, the overall structure of said beta-CoV S proteins showed a similar folding pattern. Interestingly, when the more defined receptor binding motif (RBM) was analyzed for SARS CoV (i.e.
  • antigenic polypeptides of a receptor binding domain (RBD) of a coronavirus (CoV), or a fragment thereof, of various and very different length can be inserted at a specific position of CMV polypeptides already modified by the incorporation of a T helper cell epitope, and that said resulting fusion proteins are still capable of forming and assembling to modified virus-like particles (VLPs) which are, in addition, highly immunogenic.
  • VLPs virus-like particles
  • mosaic modified virus like particles have been generated comprising said described fusion proteins with the in- fused antigenic polypeptides and further comprising CMV proteins with no such in-fused antigenic polypeptides.
  • the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of
  • a CMV polypeptide comprising, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO: 1; and
  • an antigenic polypeptide wherein said antigenic polypeptide is a receptor binding domain (RBD) of a coronavirus (CoV), or a fragment thereof, and 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 of position 84 and position 85 of SEQ ID NO: 1 ; and
  • T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 1.
  • the present invention provides a modified VLP of CMV comprising at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of
  • a CMV polypeptide comprising, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with said coat protein, preferably with said SEQ ID NO: 1 ; and
  • an antigenic polypeptide wherein said antigenic polypeptide is a receptor binding domain (RBD) of a coronavirus (CoV), or a fragment thereof, 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 of position 84 and position 85 of SEQ ID NO: 1 ; and
  • a first amino acid linker preferably a first amino acid linker and a second amino acid linker, wherein said first amino acid linker is positioned at the N- or at the C-terminus of said antigenic polypeptide, and wherein preferably said first amino acid linker is selected from the group consisting of:
  • 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.
  • the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising (a) at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of
  • a CMV polypeptide comprising, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO: 1; and
  • 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 of position 84 and position 85 of SEQ ID NO: 1;
  • said CMV protein comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l, and wherein said CMV protein is optionally modified by a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV protein, and wherein preferably said N-terminal region of said CMV protein corresponds to amino acids 2-12 of SEQ ID NO: 1.
  • the present invention provides a modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising
  • At least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of
  • a CMV polypeptide comprising, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO: 1; and
  • T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:l, and
  • said CMV protein comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l, and wherein said CMV protein is optionally, but preferably, modified by a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV protein, and wherein preferably said N-terminal region of said CMV protein corresponds to amino acids 2-12 of SEQ ID NO:l.
  • FIG. 1 Description of pETDu-CMVB3d-nCoV-D-CMV-tt plasmid map with single-cut restriction enzyme sites.
  • the expression vector ensures simultaneous synthesis of CMV-Ntt830-nCoV-D and unmodified CMV-Ntt830 leading to the mosaic virus-like particle CMV-M-nCoV-D.
  • FIG. 2A SDS-PAGE gel analysis of the purification of the VLP derived from the expression CMV-M-nCoV-D. 0 - total proteins in E. coli C2566/pETDu-CMVB3d-nCoV- D-CMV-tt cells before induction; T - total proteins in E. coli C2566/pETDu-CMVB3d- nCoV-D-CMV-tt cells after 18h cultivation at 20°C; S - soluble proteins in E. coli C2566 cells after 18h cultivation at 20°C in cell extract before sucrose gradient (20-60%); P - insoluble proteins; 1 - 6 - sucrose gradient fractions (from 60% at the bottom of tube to 0% at the top.
  • the asterisc (*) denotes the relative position of the corresponding CMVB3d- nCoV-D chimeric CMV polypeptide in SDS/PAGE gel.
  • FIG. 2B SDS-PAGE gel analysis of the purification of the VLP derived from the expression of CMV-M-nCoV-D.
  • M protein size marker PageRuler (Thermo Fisher Scientific, # 26620); S - soluble proteins after sucrose «cushion» purification and solubilization; P - insoluble proteins after sucrose «cushion» purification and solubilization.
  • the asterisc (*) denotes the relative position of the corresponding CMVB3d-nCoV-D chimeric CMV polypeptide in SDS/PAGE gel.
  • FIG. 4A SDS-PAGE gel analysis of the purification of the VLP derived from the expression CMV-M-nCoV-M.
  • M - protein size marker PageRuler Thermo Fisher Scientific,
  • FIG. 5 Electron microscopy analysis of purified VLPs derived from the expression of CMV-M-nCoV-M.
  • White horizontal bar corresponds to 100 nm.
  • FIG. 6 Description of pETDu-CMVB3d-MERS-D-CMV-tt plasmid map with single-cut restriction enzyme sites.
  • the expression vector ensures simultaneous synthesis of CMV-Ntt830-MERS-D and unmodified CMVNtt830 leading to the mosaic virus-like particle CMV-M-MERS-D.
  • FIG. 7A SDS-PAGE gel analysis of the purification of the VLP derived from the expression CMV-M-MERS-D.
  • M protein size marker PageRuler (Thermo Fisher Scientific, # 26620); 0 - total proteins in E. coli C2566/ pETDu-CMVB3d-MERS-D-CMV- tt cells before induction; T - total proteins in E. coli C2566/ pETDu-CMVB3d-MERS-D- CMV-tt cells after 18h cultivation at 20°C; S - soluble proteins in E.
  • the asterisc (*) denotes the relative position of the corresponding CMVB3d-MERS-D chimeric CMV polypeptide in SDS/PAGE gel.
  • FIG. 8 Description of pETDu-CMVB3d-MERS-M-CMV-tt plasmid map with single-cut restriction enzyme sites.
  • the expression vector ensures simultaneous synthesis of CMV-Ntt830-MERS-M and unmodified CMVNtt830 leading to the mosaic virus-like particle CMV-M-MERS-M.
  • FIG. 9A SDS-PAGE gel analysis of the purification of the VLP derived from the expression CMV-M-MERS-M.
  • M protein size marker PageRuler (Thermo Fisher Scientific, # 26620); 0 - total proteins in E. coli C2566/pETDu-CMVB3d-MERS-M-CMV- tt cells before induction; T - total proteins in E. coli C2566/pETDu-CMVB3d-MERS-M- CMV-tt cells after 18h cultivation at 20°C; S - soluble proteins in E.
  • the asterisc (*) denotes the relative position of the corresponding CMVB3d-MERS-M chimeric CMV polypeptide in SDS/PAGE gel.
  • FIG. 9B SDS-PAGE gel analysis of the purification of the VLP derived from the expression of expression CMV-M-MERS-M.
  • M - protein size marker PageRuler Thermo Fisher Scientific, # 26620
  • the asterisc (*) denotes the relative position of the corresponding CMVB3d- MERS-M chimeric CMV polypeptide in SDS/PAGE gel
  • FIG. 10 Electron microscopy analysis of purified VLPs derived from the expression of CMV-M-MERS-M.
  • White horizontal bar corresponds to 100 nm.
  • FIG. 11 Recombinant human ACE-2, the viral entry receptor for SARS-CoV-2, binds to the receptor binding motif of the SARS-CoV-2 S protein displayed on the CMV- M-nCoV-M mosaic VLP but not to the control VLP (CMVNtt830).
  • FIG. 12A Balb/c mice (5 per group) were vaccinated on day 0, 7, 14 and 21 with 20 pg CMV-M-nCoV-M or control VLPs (CMVNtt830). Sera were collected at indicated time points and tested for IgG antibodies specific for the RBD (receptor binding domain) of SARS CoV-2 Spike protein by ELISA. RBD-specific IgG titers are shown for day 14 and day 21 after first immunisation.
  • FIG. 12B Balb/c mice (5 per group) were vaccinated on day 0, 7, 14 and 21 with 20 pg CMV-M-nCoV-M or control VLPs (CMVNtt830). Sera were collected at indicated time points and tested for IgG antibodies specific for the Spike protein by ELISA. CoV-2 S protein-specific IgG titers are shown for day 21 and day 28 after first immunisation.
  • FIG. 12D Balb/c mice (5 per group) were vaccinated on day 0, 7, 14 and 21 with 20pg CMV-M-nCoV-M or control VLPs (CMVNtt830). Neutralisation titers of sera collected 35 days after first immunisation were assessed in a SARS-CoV-2 CPE-based assay. Neutralisation of virus infection by 20 fold diluted sera is shown. Sera from mice immunised with CMV-M-nCoV-M but not with control CMVNtt830 were able to neutralise SARS- CoV-2 virus.
  • CMVNtt830 Control VLPs
  • FIG. 13 DPP4 binding of CMV-M-MERS-M and CMVNtt830 as a control. Plates coated with 1 pg/ml of DPP4. Binding revealed with an anti-CMV mAb.
  • FIG. 14 A RBD-specific IgG titer for the groups vaccinated with CMVNtt830 control or CMV-M-MERS-M on days 7, 14, 21, 35 and 49 measured with OD450 nm.
  • FIG. 14B Spike-specific IgG titer for the groups vaccinated with CMVNtt830 control or CMV-M-MERS-M vaccine on days 7, 14, 21, 35 and 49 measured with OD450.
  • FIG. 14C Neutralisation titers (CPE) of the groups vaccinated with CMVNtt830 control and CMV-M-MERS-M.
  • CPE Neutralisation titers
  • virus-like particle refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious virus particle, or refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus.
  • non-replicative refers to being incapable of replicating the genome comprised by the VLP.
  • non-infectious refers to being incapable of entering the host cell.
  • a virus-like particle in accordance with the invention is non- replicative and non-infectious since it lacks all or part of the viral genome or genome function.
  • a virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome.
  • Recombinantly produced virus-like particles typically contain host cell derived RNA.
  • a typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid composed of polypeptides of the invention.
  • a virus-like particle is typically a macromolecular assembly composed of viral coat protein which typically comprises 60, 120, 180, 240, 300, 360, or more than 360 protein subunits per virus-like particle. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization.
  • One feature of a virus-like particle is its highly ordered and repetitive arrangement of its subunits.
  • Modified virus-like particle of CMV refers to a virus-like particle comprising at least one fusion protein which comprises a CMV polypeptide. Typically and preferably, modified virus-like particles of CMV resemble the structure of the capsid of CMV. Modified virus-like particles of CMV are non-replicative and/or non-infectious, and lack at least the gene or genes encoding for the replication machinery of the CMV, and typically also lack the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. This definition includes also modified virus-like particles in which the aforementioned gene or genes are still present but inactive.
  • non-replicative and/or non-infectious modified virus-like particles are obtained by recombinant gene technology and typically and preferably do not comprise the viral genome.
  • Modified virus-like particles comprising two or more different polypeptides are referred to as “mosaic VLPs”, and are in particular encompassed by the invention.
  • Mosaic modified virus-like particle are very preferred embodiments and aspects of the present invention.
  • the modified virus-like particle according to the invention comprises at least two different species of polypeptides, very preferably said mosaic VLPs comprise two different species of CMV polypeptides optionally modified in accordance with the present invention leading to mosaic modified CMC VLPs.
  • a modified VLP of CMV is a macromolecular assembly composed of CMV polypeptides modified in accordance with the present invention typically comprising 180 of such protein subunits per VLP.
  • Polypeptide refers to a polymer composed of amino acid monomers which are linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide.
  • polypeptide as used herein should also refer, typically and preferably to a polypeptide as defined before and encompassing modifications such as post-translational modifications, including but not limited to glycosylations.
  • polypeptide as used herein should refer to a polypeptide as defined before and not encompassing modifications such as post- translational modifications such as glycosylations.
  • modifications such as said glycosylations can occur even in vivo thereafter, for example, by bacteria.
  • CMV polypeptide Cucumber Mosaic Virus (CMV) polypeptide
  • CMV polypeptide The term “cucumber mosaic virus (CMV) polypeptide” as used herein refers to a polypeptide comprising or preferably consisting of: (i) an amino acid sequence of a coat protein of cucumber mosaic virus (CMV), or (ii) a mutated amino acid sequence, wherein the amino acid sequence to be mutated is an amino acid sequence of a coat protein of CMV, and wherein said mutated amino acid sequence and said amino acid sequence to be mutated, i.e. said coat protein of CMV, show a sequence identity of at least 90 %, preferably of at least 95%, further preferably of at least 98% and again more preferably of at least 99%.
  • the CMV polypeptide is capable of forming a virus-like particle of CMV upon expression by self-assembly.
  • the term "chimeric” is intended - when referring in the context of polypeptides - to refer to polypeptides comprising polypeptidic components from two or more distinct sources. This term is further intended to confer to the specific manner in which the polypeptide components are bound or attached together, namely by way of fusion and peptide bonds, respectively.
  • the term “chimeric CMV polypeptide” is thus defined as such and in particular in accordance with the present invention.
  • Coat protein (CP) of cucumber mosaic virus (CMV) refers to a coat protein of the cucumber mosaic virus which occurs in nature. Due to extremely wide host range of the cucumber mosaic virus, a lot of different strains and isolates of CMV are known and the sequences of the coat proteins of said strains and isolates have been determined and are, thus, known to the skilled person in the art as well. The sequences of said coat proteins (CPs) of CMV are described in and retrievable from the known databases such as Genbank, www. dpyweb . net. or www.ncbi.nlm.nih.gov/protein/.
  • CMV coat protein is provided in SEQ ID NO: 1.
  • coat protein of cucumber mosaic virus refers to an amino acid sequence of a coat protein of CMV, wherein said amino acid sequence comprises, or preferably consists of, SEQ ID NO:l or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% of SEQ ID NO: 1.
  • strains and isolates have highly similar coat protein sequences at different protein domains, including the N-terminus of the coat protein.
  • 98.1% of all completely sequenced CMV isolates share more than 85% sequence identity within the first 28 amino acids of their coat protein sequence, and still 79.5% of all completely sequenced CMV isolates share more than 90% sequence identity within the first 28 amino acids of their coat protein sequence.
  • N-terminal region of the CMV polypeptide refers either to the N-terminus of said CMV polypeptide, and in particular to the N-terminus of a coat protein of CMV, or to the region of the N-terminus of said CMV polypeptide or said coat protein of CMV but starting with the second amino acid of the N-terminus of said CMV polypeptide or said coat protein of CMV if said CMV polypeptide or said coat protein comprises a N-terminal methionine residue.
  • the start-codon encoding methionine will usually be deleted and added to the N-terminus of the Th cell epitope in accordance with the present invention.
  • one, two or three additional amino acids, preferably one amino acid may be optionally inserted between the stating methionine and the Th cell epitope for cloning purposes.
  • N-terminal region of the mutated amino acid sequence of a CMV polypeptide or a CMV coat protein refers either to the N-terminus of said mutated amino acid sequence of said CMV polypeptide or said coat protein of CMV, or to the region of the N-terminus of said mutated amino acid sequence of said CMV polypeptide or said coat protein of CMV but starting with the second amino acid of the N-terminus of said mutated amino acid sequence of said CMV polypeptide or said coat protein of CMV if said mutated amino acid sequence comprises a N-terminal methionine residue.
  • the start-codon encoding methionine will usually be deleted and added to the N-terminus of the Th cell epitope.
  • one, two or three additional amino acids, preferably one amino acid may be optionally inserted between the stating methionine and the Th cell epitope for cloning purposes.
  • Recombinant polypeptide in the context of the invention refers to a polypeptide which is obtained by a process which comprises at least one step of recombinant DNA technology. Typically and preferably, a recombinant polypeptide is produced in a prokaryotic expression system. It is apparent for the artisan that recombinantly produced polypeptides which are expressed in a prokaryotic expression system such as E. coli may comprise an N-terminal methionine residue. The N-terminal methionine residue is typically cleaved off the recombinant polypeptide in the expression host during the maturation of the recombinant polypeptide.
  • a preparation of a recombinant polypeptide may comprise a mixture of otherwise identical polypeptides with and without an N-terminal methionine residue.
  • a preparation of a recombinant polypeptide comprises less than 10 %, more preferably less than 5 %, and still more preferably less than 1 % recombinant polypeptide with an N-terminal methionine residue.
  • Recombinant modified virus-like particle refers to a modified virus-like particle (VLP) which is obtained by a process which comprises at least one step of recombinant DNA technology.
  • VLP modified virus-like particle
  • Mutated amino acid sequence refers to an amino acid sequence which is obtained by introducing a defined set of mutations into an amino acid sequence to be mutated.
  • said amino acid sequence to be mutated typically and preferably is an amino acid sequence of a coat protein of CMV.
  • a mutated amino acid sequence differs from an amino acid sequence of a coat protein of CMV in at least one amino acid residue, wherein said mutated amino acid sequence and said amino acid sequence to be mutated show a sequence identity of at least 90 %.
  • said mutated amino acid sequence and said amino acid sequence to be mutated show a sequence identity of at least 91 %, 92 %, 93 % 94 %, 95 %, 96 %, 97 %, 98 %, or 99 %.
  • said mutated amino acid sequence and said sequence to be mutated differ in at most 11, 10, 9, 8, 7, 6, 4, 3, 2, or 1 amino acid residues, wherein further preferably said difference is selected from insertion, deletion and amino acid exchange.
  • the mutated amino acid sequence differs from an amino acid sequence of a coat protein of CMV in least one amino acid, wherein preferably said difference is an amino acid exchange.
  • Position corresponding to residues... The position on an amino acid sequence, which is corresponding to given residues of another amino acid sequence can be identified by sequence alignment, typically and preferably by using the BLASTP algorithm, most preferably using the standard settings. Typical and preferred standard settings are: expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 11, extension 1; compositional adjustments: conditional compositional score matrix adjustment.
  • Sequence identity The sequence identity of two given amino acid sequences is determined based on an alignment of both sequences. Algorithms for the determination of sequence identity are available to the artisan. Preferably, the sequence identity of two amino acid sequences is determined using publicly available computer homology programs such as the “BLAST” program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or the “CLUSTALW” (http://www.genome.ip/tools/clustalw/). and hereby preferably by the “BLAST” program provided on the NCBI homepage at http://blast.ncbi.nlm.nih.gov/Blast.cgi. using the default settings provided therein. Typical and preferred standard settings are: expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 11, extension 1; compositional adjustments: conditional compositional score matrix adjustment.
  • amino acid exchange refers to the exchange of a given amino acid residue in an amino acid sequence by any other amino acid residue having a different chemical structure, preferably by another proteinogenic amino acid residue. Thus, in contrast to insertion or deletion of an amino acid, the amino acid exchange does not change the total number of amino acids of said amino acid sequence.
  • Epitope refers to continuous or discontinuous portions of an antigen, preferably a polypeptide, wherein said portions can be specifically bound by an antibody or by a T-cell receptor within the context of an MHC molecule. With respect to antibodies, specific binding excludes non-specific binding but does not necessarily exclude cross reactivity.
  • An epitope typically comprise 5-20 amino acids in a spatial conformation which is unique to the antigenic site.
  • T helper (Th) cell epitope refers to an epitope that is capable of recognition by a helper Th cell.
  • Th cell epitope refers to a Th cell epitope that is capable of binding to at least one, preferably more than one MHC class II molecules.
  • the simplest way to determine whether a peptide sequence is a Th cell epitope is to measure the ability of the peptide to bind to individual MHC class II molecules. This may be measured by the ability of the peptide to compete with the binding of a known Th cell epitope peptide to the MHC class II molecule.
  • Th cell epitope as used herein preferably refers to a Th cell epitope that generates a measurable T cell response upon immunization and boosting.
  • Th cell epitope preferably refers to a Th cell epitope that is capable of binding to at least one, preferably to at least two, and even more preferably to at least three DR alleles selected from of DR1, DR2w2b, DR3, DR4w4, DR4wl4, DR5, DR7, DR52a, DRw53, DR2w2a; and preferably selected from DR1, DR2w2b, DR4w4, DR4wl4, DR5, DR7, DRw53, DR2w2a, with an affinity at least 500nM (as described in Alexander J, et al., Immunity (1994) 1:751-761 and references cited herein); a preferred binding assay to evaluate said affinities is the one described by Sette A, et al., J Immunol (1989) 142:35-40.
  • Th cell epitope refers to a Th cell epitope that is capable of binding to at least one, preferably to at least two, and even more preferably to at least three DR alleles selected from DR1, DR2w2b, DR4w4, DR4wl4, DR5, DR7, DRw53, DR2w2a, with an affinity at least 500nM (as described in Alexander J, et al., Immunity (1994) 1:751-761 and references cited herein); a preferred binding assay to evaluate said affinities is the one described by Sette A, et al., J Immunol (1989) 142:35-40.
  • Th cell epitopes are described, and known to the skilled person in the art, such as by Alexander J, et al., Immunity (1994) 1:751-761, Panina-Bordumble P, et al., Eur J Immunol (1989) 19:2237- 2242, Calvo-Calle JM, et al., J Immunol (1997) 159:1362-1373, and Valmori D, et al., J Immunol (1992) 149:717-721.
  • Antigenic polypeptide refers to a molecule capable of being bound by an antibody or a T-cell receptor (TCR) if presented by MHC molecules.
  • An antigenic polypeptide is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T- lymphocytes.
  • An antigenic polypeptide can have one or more epitopes (B- and T-epitopes).
  • Antigenic polypeptides as used herein may also be mixtures of several individual antigenic polypeptides.
  • the polypeptides of the invention, in particular said inventive fusion proteins which are forming the inventive modified virus-like particles, comprise the antigenic polypeptide.
  • Receptor binding domain refers to parts of proteins that either occur alone or together with partner domains on the same protein chain. Most domains correspond to tertiary structure elements and are able to fold independently. All domains exhibit evolutionary conservation, and many either perform specific functions or contribute in a specific way to the function of their proteins (Forslund SK et al, Methods Mol Biol. (2019) 1910:469-504). Viral structural proteins, such as Coronavirus S proteins, can contain several functional domains, which are necessary for the cell infection process. One such domain in Coronavirus S protein is the receptor binding domain (RBD) which binds to corresponding cell receptor.
  • RBD receptor binding domain
  • Receptor binding motif is a part of receptor binding domain and represent a linear amino acid sequence and/or a 3D structure located on outer surface of the virus and making direct contact with target cell receptors (Sobhy H, Proteomes (2016) 4(1): 3).
  • RBM receptor binding motif
  • the amino acids sequences of RBMs have low homology due to different target cellular receptors.
  • 16 amino acids of RBM make direct contacts with human ACE2 receptor (Lan et al., Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor, Nature, 2020, 581, 215-220).
  • Adjuvant refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response.
  • Preferred adjuvants are complete and incomplete Freund's adjuvant, aluminum containing adjuvant, preferably aluminum hydroxide, and modified muramyldipeptide.
  • Further preferred adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lyso lecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and human adjuvants such as BCG (bacille Calmette Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM- 174, OM- 197, OM-294, and Virosomal adjuvant technology.
  • the adjuvants may also comprise mixtures of these substances.
  • Virus-like particles have been generally described as an adjuvant.
  • the term "adjuvant” refers to an adjuvant not being the inventive modified virus-like particle. Rather “adjuvant” relates to an additional, distinct component of the inventive compositions, vaccines or pharmaceutical compositions.
  • amino acid linker refers to a linker consisting exclusively of amino acid residues.
  • the amino acid residues of the amino acid linker are composed of naturally occurring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof.
  • the amino acid residues of the amino acid linker are preferably naturally occurring amino acids, all-L or all-D or mixtures thereof.
  • GS-linker refers to a linker solely consisting of glycine and serine amino acid residues.
  • the GS-linker in accordance with the present invention comprise at least one glycine and at least one serine residue.
  • the GS-linker in accordance with the present invention has a length of at most 50 amino acids, and typically and further preferably, the GS-linker in accordance with the present invention has a length of at most 30 amino acids.
  • GST-linker refers to a linker comprising, preferably consisting of, glycine, serine and threonine amino acid residues.
  • the GST-linker in accordance with the present invention comprise at least one glycine, at least one serine and at least one threonine residue.
  • the GST-linker in accordance with the present invention has a length of at most 50 amino acids, and typically and further preferably, the GST-linker in accordance with the present invention has a length of at most 30 amino acids.
  • GSED-linker refers to a linker comprising, preferably consisting of, glycine, serine, glutamic acid and aspartic acid amino acid residues.
  • the GSED-linker in accordance with the present invention comprise at least one glycine, at least one serine, at least one glutamic acid and at least one aspartic acid residue.
  • the GSED-linker in accordance with the present invention has a length of at most 50 amino acids, and typically and further preferably, the GSED-linker in accordance with the present invention has a length of at most 30 amino acids.
  • Immunostimulatory substance refers to a substance capable of inducing and/or enhancing an immune response.
  • Immunostimulatory substances include, but are not limited to, toll-like receptor activating substances and substances inducing cytokine secretion.
  • Toll-like receptor activating substances include, but are not limited to, immunostimulatory nucleic acids, peptideoglycans, lipopolysaccharides, lipoteichonic acids, imidazoquinoline compounds, flagellins, lipoproteins, and immuno stimulatory organic substances such as taxol.
  • Immunostimulatory nucleic acid refers to a nucleic acid capable of inducing and/or enhancing an immune response.
  • Immunostimulatory nucleic acids comprise ribonucleic acids and in particular desoxyribonucleic acids, wherein both, ribonucleic acids and desoxyribonucleic acids may be either double stranded or single stranded.
  • Preferred ISS-NA are desoxyribonucleic acids, wherein further preferably said desoxyribonucleic acids are single stranded.
  • immunostimulatory nucleic acids contain at least one CpG motif comprising an unmethylated C.
  • Very preferred immunostimulatory nucleic acids comprise at least one CpG motif, wherein said at least one CpG motif comprises or preferably consist of at least one, preferably one, CG dinucleotide, wherein the C is unmethylated.
  • said CG dinucleotide is part of a palindromic sequence.
  • immunostimulatory nucleic acid also refers to nucleic acids that contain modified bases, preferably 4-bromo-cytosine.
  • ISS- NA which are capable of stimulating IFN- alpha production in dendritic cells.
  • Immunostimulatory nucleic acids useful for the purpose of the invention are described, for example, in W02007/068747A1.
  • oligonucleotide refers to a nucleic acid sequence comprising 2 or more nucleotides, preferably about 6 to about 200 nucleotides, and more preferably 20 to about 100 nucleotides, and most preferably 20 to 40 nucleotides. Very preferably, oligonucleotides comprise about 30 nucleotides, more preferably oligonucleotides comprise exactly 30 nucleotides, and most preferably oligonucleotides consist of exactly 30 nucleotides.
  • Oligonucleotides are polyribonucleotides or polydeoxribonucleotides and are preferably selected from (a) unmodified RNA or DNA, and (b) modified RNA or DNA. The modification may comprise the backbone or nucleotide analogues. Oligonucleotides are preferably selected from the group consisting of (a) single- and double-stranded DNA, (b) DNA that is a mixture of single- and double-stranded regions,
  • RNA single- and double-stranded RNA
  • RNA that is mixture of single- and double-stranded regions
  • hybrid molecules comprising DNA and RNA that are single-stranded or, more preferably, double- stranded or a mixture of single- and double-stranded regions.
  • Unmodified oligonucleotides consisting exclusively of phosphodiester bound nucleotides typically are more active than modified nucleotides and are therefore generally preferred in the context of the invention. Most preferred are oligonucleotides consisting exclusively of phosphodiester bound deoxinucleo tides, wherein further preferably said oligonucleotides are single stranded. Further preferred are oligonucleotides capable of stimulating IFN-alpha production in cells, preferably in dendritic cells. Very preferred oligonucleotides capable of stimulating IFN-alpha production in cells are selected from A- type CpGs and C-type CpGs. Further preferred are RNA-molecules without a Cap.
  • CpG motif refers to a pattern of nucleotides that includes an unmethylated central CpG, i.e. the unmethylated CpG dinucleotide, in which the C is unmethylated, surrounded by at least one base, preferably one or two nucleotides, flanking (on the 3' and the 5' side of) the central CpG.
  • the CpG motif as used herein comprises or alternatively consists of the unmethylated CpG dinucleotide and two nucleotides on its 5 ' and 3 ' ends.
  • the bases flanking the CpG confer a significant part of the activity to the CpG oligonucleotide.
  • Unmethylated CpG-containing oligonucleotide refers to an oligonucleotide, preferably to an oligodesoxynucleotide, containing at least one CpG motif.
  • a CpG contains at least one unmethylated cytosine, guanine dinucleotide.
  • Preferred CpGs stimulate/activate, e.g. have a mitogenic effect on, or induce or increase cytokine expression by, a vertebrate bone marrow derived cell.
  • CpGs can be useful in activating B cells, NK cells and antigen-presenting cells, such as dendritic cells, monocytes and macrophages.
  • CpG relates to an oligodesoxynucleotide, preferably to a single stranded oligodesoxynucleotide, containing an unmethylated cytosine followed 3' by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphate bond, wherein preferably said phosphate bound is a phosphodiester bound or a phosphothioate bound, and wherein further preferably said phosphate bond is a phosphodiester bound.
  • CpGs can include nucleotide analogs such as analogs containing phosphorothio ester bonds and can be double-stranded or single-stranded. Generally, double- stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity.
  • a CpG is an oligonucleotide that is at least about ten nucleotides in length and comprises at least one CpG motif, wherein further preferably said CpG is 10 to 60, more preferably 15 to 50, still more preferably 20 to 40, still more preferably about 30, and most preferably exactly 30 nucleotides in length.
  • a CpG may consist of methylated and/or unmethylated nucleotides, wherein said at least one CpG motif comprises at least one CG dinucleotide wherein the C is unmethylated.
  • the CpG may also comprise methylated and unmethylated sequence stretches, wherein said at least one CpG motif comprises at least one CG dinucleotide wherein the C is unmethylated.
  • CpG relates to a single stranded oligodesoxynucleotide containing an unmethylated cytosine followed 3' by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphodiester bound.
  • the CpGs can include nucleotide analogs such as analogs containing phosphorothioester bonds and can be double-stranded or single-stranded.
  • phosphodiester CpGs are A-type CpGs as indicated below, while phosphothioester stabilized CpGs are B-type CpGs.
  • Preferred CpG oligonucleotides in the context of the invention are A-type CpGs.
  • A-type CpG As used herein, the term "A-type CpG” or “D-type CpG” refers to an oligodesoxynucleotide (ODN) comprising at least one CpG motif.
  • ODN oligodesoxynucleotide
  • A-type CpGs preferentially stimulate activation of T cells and the maturation of dendritic cells and are capable of stimulating IFN-alpha production.
  • the nucleotides of the at least one CpG motif are linked by at least one phosphodiester bond.
  • A-type CpGs comprise at least one phosphodiester bond CpG motif which may be flanked at its 5' end and/or, preferably and, at its 3' end by phosphorothioate bound nucleotides.
  • the CpG motif and hereby preferably the CG dinucleotide and its immediate flanking regions comprising at least one, preferably two nucleotides, are composed of phosphodiester nucleotides.
  • Preferred A-type CpGs exclusively consist of phosphodiester (PO) bond nucleotides.
  • the poly G motif comprises or alternatively consists of at least one, preferably at least three, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gs (guanosines), most preferably by at least 10 Gs.
  • the A-type CpG of the invention comprises or alternatively consists of a palindromic sequence.
  • the term “packaged” as used herein refers to the state of a polyanionic macromolecule or immunostimulatory substances in relation to the core particle and VLP, respectively.
  • the term “packaged” as used herein includes binding that may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.
  • the term also includes the enclosement, or partial enclosement, of a polyanionic macromolecule.
  • the polyanionic macromolecule or immunostimulatory substances can be enclosed by the VLP without the existence of an actual binding, in particular of a covalent binding.
  • the at least one polyanionic macromolecule or immunostimulatory substances is packaged inside the VLP, most preferably in a non-covalent manner.
  • said immunostimulatory substances is nucleic acid, preferably a DNA
  • the term packaged implies that said nucleic acid is not accessible to nucleases hydrolysis, preferably not accessible to DNAse hydrolysis (e.g. DNasel or Benzonase), wherein preferably said accessibility is assayed as described in Examples 11-17 of W02003/024481A2.
  • the term “effective amount” refers to an amount necessary or sufficient to realize a desired biologic effect.
  • An effective amount of the composition, or alternatively the pharmaceutical composition would be the amount that achieves this selected result, and such an amount could be determined as a matter of routine by a person skilled in the art.
  • the term “effective amount”, as used herein refers to an amount necessary or sufficient to be effective to reduce levels of said at least one peanut allergen to a level that causes the reduction of at least one symptom caused by the peanut allergy.
  • the term “effective amount”, as used herein refers to an amount necessary or sufficient to be effective to neutralize the activity of at least one peanut allergen.
  • the effective amount can vary depending on the particular composition being administered and the size of the subject. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.
  • treatment refers to prophylaxis and/or therapy.
  • treatment refers to a therapeutic treatment.
  • treatment refers to a prophylactic treatment.
  • the present invention provides modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of (i) a CMV polypeptide, wherein said CMV polypeptide comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO: 1; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l; and (ii) an antigenic polypeptide, wherein said antigenic polypeptide is a
  • the preferred amino acid linkers namely the GS-linkers or the GSED-linkers, have been found to be very beneficial to overcome spherical disorders hindering the assembly process forming the modified VLPs of the present invention and/or to overcome aggregation tendencies between formed modified VLPs of the present invention and/or to increase flexibility for insertion of even very long antigenic polypeptides.
  • said GS-linkers or GSED-linkers are further applied as first amino acid linker and as second amino acid linker, and again further, if said first amino acid linker and said second amino acid linker mimic the amino acid sequence situation as present without said inserted antigenic polypeptide, This was in particular beneficial for the preferred CMV polypeptides of the present invention.
  • the present invention provides a modified VLP of CMV comprising at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of (i) a CMV polypeptide, wherein said CMV polypeptide comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with said coat protein, preferably with said SEQ ID NO:l; and (ii) an antigenic polypeptide, wherein said antigenic polypeptide is a receptor
  • said chimeric CMV polypeptide further comprises a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:l.
  • the present invention provides a mosaic modified virus-like particle (VLP) of CMV.
  • VLP mosaic modified virus-like particle
  • the present invention provides modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising (a) at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of
  • a CMV polypeptide comprising, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO: 1; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO: 1; and
  • an antigenic polypeptide wherein said antigenic polypeptide is a receptor binding domain (RBD) of a coronavirus (CoV), or a fragment thereof, 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 of position 84 and position 85 of SEQ ID NO:l; and (b) at least one CMV protein, wherein said CMV protein comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l,
  • said chimeric CMV polypeptide further comprises a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 1.
  • the present invention relates to and provides provides modified virus-like particle (VLP) of cucumber mosaic virus (CMV) comprising (a) at least one fusion protein, wherein said at least one fusion protein comprises, or preferably consists of, a) a chimeric CMV polypeptide, wherein said chimeric CMV polypeptide comprises, or preferably consists of (i) a CMV polypeptide, wherein said CMV polypeptide comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l; or an amino acid sequence having a sequence identity of at least 75%, preferably of at
  • said chimeric CMV polypeptide further comprises a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV polypeptide, and wherein preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:l.
  • said insertion of said antigenic polypeptide between amino acid residues of said CMV polypeptide corresponding to amino acid residues of position 84 and position 85 of SEQ ID NO:l corresponds to an insertion between said serine (S) residue being position 84 of SEQ ID NO:l and said tyrosine (Y) residue being position 85 of SEQ ID NO: 1.
  • said CMV polypeptide comprises, preferably consists of, an amino acid sequence of a coat protein of CMV or a mutated amino acid sequence, wherein the amino acid sequence to be mutated is an amino acid sequence of a coat protein of CMV, and wherein said mutated amino acid sequence and said coat protein of CMV show a sequence identity of at least 90 %, preferably of at least 95%, further preferably of at least 98% and again more preferably of at least 99%; wherein preferably said mutated amino acid sequence and said amino acid sequence to be mutated differ in least one and in at most 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).
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 75% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 85% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 95% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO:l.
  • said CMV polypeptide comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO:l.
  • said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 75% with SEQ ID NO:l. In another preferred embodiment, said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO:l. In another preferred embodiment, said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 85% with SEQ ID NO:l. In another preferred embodiment, said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO:l.
  • said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 95% with SEQ ID NO:l. In another preferred embodiment, said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO:l. In another preferred embodiment, said CMV polypeptide consists of a coat protein of CMV or an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO:l. In a preferred embodiment, said CMV polypeptide is a coat protein of CMV or an amino acid sequence having a sequence identity of at least 75%, preferably 85% with SEQ ID NO: 1.
  • said CMV polypeptide is a coat protein of CMV or an amino acid sequence having a sequence identity of at least 90%, preferably 95% with SEQ ID NO:l.
  • said CMV polypeptide is a coat protein of CMV with SEQ ID NO:l.
  • said coat protein of CMV comprises SEQ ID NO:l.
  • said coat protein of CMV consists of SEQ ID NO: l.
  • said CMV polypeptide comprises a coat protein of CMV.
  • said CMV polypeptide consists of a coat protein of CMV.
  • said CMV polypeptide comprises a coat protein of CMV, wherein said coat protein of CMV comprises SEQ ID NO:l. In a preferred embodiment, said CMV polypeptide comprises a coat protein of CMV, wherein said coat protein of CMV consists of SEQ ID NO:l. In a preferred embodiment, said CMV polypeptide consists of a coat protein of CMV, wherein said coat protein of CMV consists of SEQ ID NO: 1.
  • said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 75% with SEQ ID NO:2. In a preferred embodiment, said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 80% with SEQ ID NO:2. In a preferred embodiment, said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 85% with SEQ ID NO:2.
  • said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 90% with SEQ ID NO:2. In a preferred embodiment, said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 95% with SEQ ID NO:2. In a preferred embodiment, said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 98% with SEQ ID NO:2. In a preferred embodiment, said CMV polypeptide comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 99% with SEQ ID NO:2.
  • said CMV polypeptide comprises, or preferably consists of, (i) an amino acid sequence of a coat protein of CMV, wherein said amino acid sequence comprises, or preferably consists of, SEQ ID NO: 1; or (ii) an amino acid sequence having a sequence identity of at least 90 % of SEQ ID NO:l; and wherein said amino sequence as defined in (i) or (ii) comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 90% with SEQ ID NO:2.
  • said CMV polypeptide comprises, or preferably consists of, (i) an amino acid sequence of a coat protein of CMV, wherein said amino acid sequence comprises, or preferably consists of, SEQ ID NO:l; or (ii) an amino acid sequence having a sequence identity of at least 95 % of SEQ ID NO:l; and wherein said amino sequence as defined in (i) or (ii) comprises SEQ ID NO:2 or an amino acid sequence region, wherein said amino acid sequence region has a sequence identity of at least 95% with SEQ ID NO:2.
  • said CMV polypeptide comprises, or preferably consists of, (i) an amino acid sequence of a coat protein of CMV, wherein said amino acid sequence comprises, or preferably consists of, SEQ ID NO:l; or (ii) an amino acid sequence having a sequence identity of at least 90 % of SEQ ID NO:l; and wherein said amino sequence as defined in (i) or (ii) comprises SEQ ID NO:2.
  • the number of amino acids of said N-terminal region replaced is equal to or lower than the number of amino acids of which said T helper cell epitope consists.
  • said replaced N-terminal region of said CMV polypeptide consists of 5 to 15 consecutive amino acids.
  • said replaced N-terminal region of said CMV polypeptide consists of 9 to 14 consecutive amino acids.
  • said replaced N-terminal region of said CMV polypeptide consists of 11 to 13 consecutive amino acids.
  • said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:l.
  • said T helper cell epitope is a universal T helper cell epitope.
  • said T helper cell epitope consists of at most 20 amino acids.
  • the Th cell epitope is selected from TT 830-843 (SEQ ID NO:3), PADRE (SEQ ID NO:4), HA 307-319 (SEQ ID NO:6), HBVnc 50-69 (SEQ ID NO:7), CS 378-398 (SEQ ID NO:8), MT 17-31 (SEQ ID NO:9) and TT 947- 967 (SEQ ID NO: 12).
  • said Th cell epitope is a Th cell epitope derived from tetanus toxin or is a PADRE sequence.
  • said T helper cell epitope is derived from a human vaccine.
  • said Th cell epitope is a Th cell epitope derived from tetanus toxin.
  • said Th cell epitope is a PADRE sequence.
  • said Th cell epitope comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
  • said Th cell epitope consists of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
  • said Th cell epitope comprises the amino acid sequence of SEQ ID NO:3.
  • said Th cell epitope consists of the amino acid sequence of SEQ ID NO:3.
  • said Th cell epitope comprises the amino acid sequence of SEQ ID NO:4.
  • said Th cell epitope consists of the amino acid sequence of SEQ ID NO:4.
  • said CMV polypeptide comprises, or preferably consists of, an amino acid sequence of a coat protein of CMV, wherein said amino acid sequence comprises, or preferably consists of, SEQ ID NO:l or an amino acid sequence having a sequence identity of at least 95 % of SEQ ID NO:l; and wherein said amino sequence comprises SEQ ID NO:2, and wherein said T helper cell epitope replaces the N-terminal region of said CMV polypeptide, and wherein said replaced N-terminal region of said CMV polypeptide consists of 11 to 13 consecutive amino acids, preferably of 11 consecutive amino acids, and wherein further preferably said N-terminal region of said CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:l.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In a very preferred embodiment, said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 13.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO: 13, wherein said antigenic polypeptide is inserted into said chimeric CMV polypeptide of SEQ ID NO: 5 between amino acid residues of position 88 and position 89 of SEQ ID NO:5 or wherein said antigenic polypeptide is inserted into said chimeric CMV polypeptide of SEQ ID NO: 13 between amino acid residues of position 86 and position 87 of SEQ ID NO: 13.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 13, wherein said antigenic polypeptide is inserted into said chimeric CMV polypeptide of SEQ ID NO: 13 between amino acid residues of position 86 and position 87 of SEQ ID NO: 13.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein said antigenic polypeptide is inserted into said chimeric CMV polypeptide of SEQ ID NO:5 between amino acid residues of position 88 and position 89 of SEQ ID NO: 5.
  • a polyglycine linker Gly
  • a polyglycine linker Gly
  • n
  • said first amino acid linker has a length of at most 30 amino acids. In a preferred embodiment, said first amino acid linker has a length of at most 20 amino acids. In a preferred embodiment, said first amino acid linker has a length of at most 15 amino acids. In a preferred embodiment, said second amino acid linker has a length of at most 30 amino acids. In a preferred embodiment, said second amino acid linker has a length of at most 20 amino acids. In a preferred embodiment, said second amino acid linker has a length of at most 15 amino acids. In a preferred embodiment, said first amino acid linker is positioned at the N-terminus of said antigenic polypeptide.
  • said first amino acid linker is positioned at the C-terminus of said antigenic polypeptide.
  • said chimeric CMV polypeptide further comprises a second amino acid linker.
  • said first amino acid linker is positioned at the N- terminus of said antigenic polypeptide.
  • said second amino acid linker is positioned at the C-terminus of said antigenic polypeptide.
  • said chimeric CMV polypeptide comprises a first amino acid linker and a second amino acid linker.
  • said first amino acid linker is positioned at the N-terminus of said antigenic polypeptide, and said second amino acid linker is positioned at the C-terminus of said antigenic polypeptide.
  • said first amino acid linker is a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine.
  • said first amino acid linker is a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine
  • said second amino acid linker has a Gly-Ser at its N-terminus.
  • said first amino acid linker is a glycine-serine linker (GS- linker)
  • said GS-linker has a length of at most 30 amino acids.
  • said first amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, and at least Thr (GST-linker), In a further preferred embodiment, said first 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, said first amino acid linker is an amino acid linker comprising at least one Gly, at least one Ser, and at least Thr (GST-linker), and said second amino acid linker has a Gly-Ser at its N-terminus.
  • said first 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 said second amino acid linker has a Gly-Ser at its N-terminus.
  • said GSED-linker has a length of at most 30 amino acids.
  • said GSED-linker has a length of at most 20 amino acids.
  • said first amino acid linker is a GSED- linker, and said GSED linker comprises, preferably consists of amino acid sequence SEQ ID NO:17.
  • said second amino acid linker is a glycine-serine linker (GS-linker) consisting of at least one glycine and at least one serine.
  • said second amino acid linker is a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, and said second amino acid linker has a Gly-Ser at its N-terminus.
  • said GS-linker has a length of at most 30 amino acids.
  • said GS-linker has a length of at most 20 amino acids.
  • said second amino acid linker is a glycine-serine linker (GS-linker), and said GS linker has an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
  • said second amino acid linker has an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 16.
  • said 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.
  • said 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 a further preferred embodiment, said 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, said 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 said second amino acid linker has a Gly-Ser at its N-terminus.
  • said 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 said second amino acid linker has a Gly-Ser at its N-terminus.
  • said GSED-linker has a length of at most 30 amino acids.
  • said GSED-linker has a length of at most 20 amino acids.
  • said second amino acid linker is a GSED-linker, and said GSED linker comprises, preferably consists of amino acid sequence SEQ ID NO: 18.
  • said first and said second amino acid linker is independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine.
  • said first and said second amino acid linker is independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, and wherein said second amino acid linker has a Gly-Ser at its N-terminus.
  • said first and said second amino acid linker is independently a glycine-serine linker (GS-linker), and said GS linker has an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
  • said first and said second amino acid linker is independently 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.
  • said first and said second amino acid linker is independently a GSED-linker comprising at least one Gly, at least one Ser, at least one glutamic acid and at least aspartic acid (GSED-linker), and said second amino acid linker has a Gly-Ser at its N-terminus.
  • said first and said second amino acid linker is independently a GSED-linker, and said GS linker has an amino acid sequence selected from SEQ ID NO: 17 and SEQ ID NO: 18.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and said antigenic polypeptide is inserted into said CMV polypeptide between amino acid residues 88 (Ser) and amino acid residue 89 (Thr) of said CMV polypeptide of SEQ ID NO: 5.
  • said chimeric CMV polypeptide comprises the amino acid sequence of SEQ ID NO:5 and said antigenic polypeptide is inserted into said CMV polypeptide between amino acid residues 88 (Ser) and amino acid residue 89 (Thr) of said CMV polypeptide of SEQ ID NO:5, and said chimeric CMV polypeptide further comprises a first and a second amino acid linker, wherein said first and said second amino acid linker is independently a glycine-serine linker (GS-linker) comprising at least one glycine and at least one serine, and wherein said second amino acid linker has a Gly-Ser sequence at its N- terminus.
  • GS-linker glycine-serine linker
  • the invention provides for mosaic virus-like particles.
  • said modified VLP of CMV further comprises at least one CMV protein, wherein said CMV protein comprises, or preferably consists of, a coat protein of CMV, wherein preferably said coat protein of CMV comprises, or preferably consists of, SEQ ID NO: 1; or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% and still again further more preferably of at least 99% with SEQ ID NO:l.
  • said modified VLP of CMV further comprises at least one CMV protein, wherein said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 75%, preferably of at least 85% with SEQ ID NO:l, and wherein said CMV protein is optionally modified by a T helper cell epitope, and wherein preferably said coat protein of CMV comprises SEQ ID NO:l.
  • said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 75% with SEQ ID NO:l. In another preferred embodiment, said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 80% with SEQ ID NO:l. In another preferred embodiment, said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 85% with SEQ ID NO:l. In another preferred embodiment, said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO:l.
  • said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 95% with SEQ ID NO:l. In another preferred embodiment, said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO:l. In another preferred embodiment, said CMV protein comprises a coat protein of CMV or an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO:l. In a preferred embodiment, said CMV protein comprises a coat protein of CMV. In a preferred embodiment, said CMV protein consists of a coat protein of CMV.
  • said CMV protein comprises a coat protein of CMV, wherein said coat protein of CMV comprises SEQ ID NO:l. In a preferred embodiment, said CMV protein comprises a coat protein of CMV, wherein said coat protein of CMV consists of SEQ ID NO:l. In a preferred embodiment, said CMV protein consists of a coat protein of CMV, wherein said coat protein of CMV consists of SEQ ID NO: 1. In another preferred embodiment, said CMV protein is modified by a T helper cell epitope, wherein said T helper cell epitope replaces a N-terminal region of said CMV protein., In another preferred embodiment, said N-terminal region of said CMV protein corresponds to amino acids 2-12 of SEQ ID NO:l. In a very preferred embodiment, said CMV protein comprises SEQ ID NO:5. In a very preferred embodiment, said CMV protein consists of SEQ ID NO:5.
  • said antigenic polypeptide has a length of at least 3 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 3 amino acids and at most 225 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 3 amino acids and at most 200 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 40 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 40 amino acids and at most 225 amino acids, preferably at most 200 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 50 amino acids.
  • said antigenic polypeptide has a length of at least 50 amino acids and at most 200 amino acids, preferably of at most 100 amino acids, and again further preferably of at most 80 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 60 amino acids. In a preferred embodiment, said antigenic polypeptide has a length of at least 60 amino acids and at most 200 amino acids, preferably of at most 100 amino acids, and again further preferably of at most 80 amino acids.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 40 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 50 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 60 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a coronavirus (CoV), or a functional fragment thereof, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a coronavirus
  • the term “functional fragment” as used herein refers to a peptidic fragment of a RBD or RBM of a CoV, preferably of a HCoV, and further preferably of a peptidic fragment of a RBM of a HCoV, which is able to bind to the corresponding cell receptor of said CoV, preferably said HCoV.
  • said functional fragment is a peptidic fragment of a RBD or RBM of a CoV, preferably of a peptidic fragment of a RBM of a HCoV that binds to the corresponding cell receptor of said CoV, preferably said HCoV.
  • a preferred method for determining said binding is the method as described in Example 5 for binding to human ACE2 receptor of the RBD, preferably RBM, of SARS-CoV, SAR.S- CoV-2 or analogous HCoV, and such fragments thereof.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV) or a fragment thereof.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 40 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 50 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV), or a fragment thereof, wherein said fragment has a length of at least 60 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a coronavirus (CoV), or a functional fragment thereof, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding domain (RBD) of a spike (S) protein of a human coronavirus (HCoV), or a fragment thereof.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a human coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 40 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a human coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 50 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a human coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 60 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a human coronavirus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV), or a functional fragment thereof, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • RBD receptor binding domain
  • S spike protein of a human coronavirus
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 40 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 50 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV), or a fragment thereof, wherein said fragment has a length of at least 60 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV), or a functional fragment thereof, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • said coronavirus is selected from an alpha- coronavirus (alpha-CoV) or a beta-coronavirus (beta-CoV).
  • said coronavirus is an alpha-coronavirus (alpha-CoV).
  • said coronavirus is a beta-coronavirus (beta-CoV).
  • coronavirus is selected from a human alpha- coronavirus (alpha-HCoV) or a human beta-coronavirus (beta-HCoV).
  • said coronavirus is a human alpha-coronavirus (alpha-HCoV).
  • said coronavirus is a human beta-coronavirus (beta-HCoV).
  • said coronavirus is a human alpha-coronavirus (alpha-HCoV) selected from HCoV-229E and HCoV-NL63.
  • said coronavirus is a human beta-coronavirus (beta-HCoV) selected from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC43 and HCoV-HKUl.
  • said HCoV is selected from HCoV-229E, HCoV-NL63, SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC43 and HCoV-HKUl.
  • said HCoV is MERS-CoV.
  • said HCoV is SARS-CoV.
  • said HCoV is SARS-CoV-2.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is selected from SARS-CoV, MERS-CoV and SARS- CoV-2.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS, and wherein said fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS, and wherein said fragment has a length of at least 40 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS, and wherein said fragment has a length of at least 50 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS, and wherein said fragment has a length of at least 60 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a functional fragment thereof, wherein said HCoV is MERS, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30.
  • amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of MERS-CoV is SEQ ID NO:30, or a fragment thereof.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:30 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98%, still further preferably of at least 99% with SEQ ID NO:30.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:30.
  • 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 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: 1; or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO: 1, preferably wherein said CMV polypeptide comprises, or preferably consists of, SEQ ID NO:l; (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 of position 84 and position 85
  • 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 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of MERS-CoV, wherein said antigenic polypeptide
  • RBD receptor binding domain
  • said chimeric CMV polypeptide consists of SEQ ID NO:31.
  • the present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by MERS-CoV.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • 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 said at least one fusion protein comprises, or preferably consists 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding domain (RBD) of the spike (RBD) of the spike (
  • said chimeric CMV polypeptide consists of SEQ ID NO:31 and said CMV protein consists of SEQ ID NO:5.
  • said present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by MERS-CoV.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SARS- CoV-2 (SARS-CoV-2 S) is SEQ ID NO:24.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 40 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SARS- CoV-2 (SARS-CoV-2 S) is SEQ ID NO:24.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 50 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SAR.S- CoV-2 (SARS-CoV-2 S) is SEQ ID NO:24.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 60 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SAR.S- CoV-2 (SARS-CoV-2 S) is SEQ ID NO:24.
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a functional fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 200 amino acids of said RBD, and further preferably of at most 100 amino acids of said RBD, and again further preferably of at most 80 amino acids of said RBD.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:24.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SARS-CoV-2 is SEQ ID NO:24.
  • the amino acid sequence of said receptor binding domain (RBD) of the spike (S) protein of SARS-CoV-2 is SEQ ID NO:24, or a fragment thereof.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:24 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98%, still further preferably of at least 99% with SEQ ID NO:24.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:24.
  • 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 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: 1; or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO: 1, preferably wherein said CMV polypeptide comprises, or preferably consists of, SEQ ID NO:l; (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 of position 84 and position 85
  • 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 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of SARS-CoV-2, wherein said antigenic polypeptide
  • said chimeric CMV polypeptide consists of SEQ ID NO:25.
  • the present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by SARS-CoV-2.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • 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 said at least one fusion protein comprises, or preferably consists 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding domain (RBD) of the spike (RBD) of the spike (
  • said chimeric CMV polypeptide consists of SEQ ID NO:25 and said CMV protein consists of SEQ ID NO:5.
  • said present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by SARS-CoV-2.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is selected from SARS-CoV, MERS-CoV and SARS-CoV-2.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV.
  • said antigenic polypeptide is the receptor binding motif (RBM) of a spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV.
  • said antigenic polypeptide is the receptor binding motif (RBM) of a spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:NO:40.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 40 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:NO:40.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 50 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:NO:40.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein said fragment has a length of at least 60 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:NO:40.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a functional fragment thereof, wherein said HCoV is SARS-CoV-2, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV- 2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:NO:40.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:40.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:27, or a fragment thereof.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) is SEQ ID NO:40, or a fragment thereof.
  • the fragment of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) comprises, or preferably consists of, the amino acid sequence of SEQ ID NO:41, or a fragment thereof.
  • the fragment of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV-2 (SARS-CoV-2 S) comprises, or preferably consists of, the amino acid sequence of SEQ ID NO:41.
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:40, SEQ ID NO:41 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:27, SEQ ID NO:40 and SEQ ID NO:41.
  • said antigenic polypeptide comprises, or preferably consists of, SEQ ID NO:27 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:27.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:40 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:40.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:41 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:41.
  • said antigenic polypeptide comprises, or preferably consists of, the amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:40 and SEQ ID NO:41. In a further very preferred embodiment, said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:27. In a further very preferred embodiment, said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:40.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:41.
  • 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 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: 1; or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO: 1, preferably wherein said CMV polypeptide comprises, or preferably consists of, SEQ ID NO:l; (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 of position 84 and position 85
  • 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 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of SARS- CoV-2, wherein said antigenic polypeptide
  • RBM receptor binding
  • said chimeric CMV polypeptide consists of SEQ ID NO:28.
  • the present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by SARS-CoV-2.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • 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 said at least one fusion protein comprises, or preferably consists 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding motif (RBM) of the spike (RBM) of the spike (
  • said chimeric CMV polypeptide consists of SEQ ID NO:28 and said CMV protein consists of SEQ ID NO:5.
  • said present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by SARS-CoV-2.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV, and wherein said fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV, and wherein said fragment has a length of at least 40 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV, and wherein said fragment has a length of at least 50 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is SARS-CoV, and wherein said fragment has a length of at least 60 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a functional fragment thereof, wherein said HCoV is SARS-CoV, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO:42.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of SARS-CoV (SARS-CoV S) is SEQ ID NO: 42, or a fragment thereof.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:42 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:42.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:43 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:43.
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:42 and SEQ ID NO:43. In a further very preferred embodiment, said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:42. In a further very preferred embodiment, said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:43.
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:42, SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:42 and SEQ ID NO:43.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV, and wherein said fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV, and wherein said fragment has a length of at least 40 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV, and wherein said fragment has a length of at least 50 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said HCoV is MERS-CoV, and wherein said fragment has a length of at least 60 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a functional fragment thereof, wherein said HCoV is MERS-CoV, and wherein preferably said functional fragment has a length of at least 30 amino acids, and preferably of at most 70 amino acids of said RBM.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35.
  • the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35. In a further very preferred embodiment, the amino acid sequence of said receptor binding motif (RBM) of the spike (S) protein of MERS-CoV is SEQ ID NO:35, or a fragment thereof.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:35 or an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with SEQ ID NO:35.
  • said antigenic polypeptide comprises, or preferably consists of SEQ ID NO:35.
  • 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 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: 1; or an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO: 1, preferably wherein said CMV polypeptide comprises, or preferably consists of, SEQ ID NO:l; (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 of position 84 and position 85
  • 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 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of MERS- CoV, wherein said antigenic polypeptide
  • RBM receptor binding motif
  • said chimeric CMV polypeptide consists of SEQ ID NO:36.
  • the present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by MERS-CoV.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • 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 said at least one fusion protein comprises, or preferably consists 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: 1, (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 of position 84 and position 85 of SEQ ID NO:l; and wherein said antigenic polypeptide is the receptor binding motif (RBM) of the spike (RBM) of the spike (
  • said chimeric CMV polypeptide consists of SEQ ID NO:36 and said CMV protein consists of SEQ ID NO:5.
  • said present invention provides said modified virus-like particle (VLP) of cucumber mosaic virus (CMV) for use in method of preventing and/or treating a disease, preferably an infection, caused by MERS-CoV.
  • VLP modified virus-like particle
  • CMV cucumber mosaic virus
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, wherein said fragment has a length of at least 30 amino acids, preferably of at least 40 amino acids, further preferably of at least 50 amino acids, preferably of at least 60 amino
  • said antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, wherein said fragment has a length of at least 30 amino acids, preferably of at least 40 amino acids, further preferably of at least 50 amino acids, preferably of at least 60 amino
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said antigenic polypeptide comprises, or preferably consists of, the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said antigenic polypeptide comprises, or preferably consists of, the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:
  • said antigenic polypeptide is the receptor binding domain (RBD) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein said antigenic polypeptide comprises, or preferably consists of, the amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:
  • said antigenic polypeptide comprises, or preferably consists of, the amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.
  • RBM receptor binding motif
  • said antigenic polypeptide is the receptor binding motif (RBM) of the spike (S) protein of a human coronavirus (HCoV) or a fragment thereof, wherein antigenic polypeptide comprises, or preferably consists of the amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 and an amino acid sequence having a sequence identity of at least 70 %, preferably of at least 75%, preferably of at least 80%, more preferably of at least 85%, again further preferably of at least 90 %, again more preferably of at least 95%, still further preferably of at least 98% with any of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, wherein said fragment has a length of at least 30 amino acids, preferably of at least 40 amino acids, further preferably
  • said chimeric CMV polypeptide is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, or SEQ ID NO:36.
  • said modified VLP further comprises at least one immunostimulatory substance.
  • said immunostimulatory substance is packaged into the modified VLPs of the invention.
  • the immunostimulatory substance is mixed with the modified VLPs of the invention.
  • Immunostimulatory substances useful for the invention are generally known in the art and are disclosed, inter alia, in W02003/024481A2.
  • said immunostimulatory substance consists of DNA or RNA of non-eukaryotic origin.
  • said immunostimulatory substance is selected from the group consisting of: (a) immunostimulatory nucleic acid; (b) peptidoglycan; (c) lipopolysaccharide; (d) lipoteichonic acid; (e) imidazoquinoline compound; (f) flagelline; (g) lipoprotein; and (h) any mixtures of at least one substance of (a) to (g).
  • said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide.
  • said unmethylated CpG- containing oligonucleotide is an A-type CpG.
  • said A-type CpG comprises a palindromic sequence.
  • said palindromic sequence is flanked at its 5'- terminus and at its 3 '-terminus by guanosine entities.
  • said palindromic sequence is flanked at its 5 '-terminus by at least 3 and at most 15 guanosine entities, and wherein said palindromic sequence is flanked at its 3 '-terminus by at least 3 and at most 15 guanosine entities.
  • said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, and wherein preferably said unmethylated CpG-containing oligonucleotide comprises a palindromic sequence, and wherein further preferably the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence.
  • the invention provides the modified virus-like particle of the invention for use as a medicament.
  • the invention provides a vaccine comprising or alternatively consisting of the modified virus-like particle of the invention.
  • a vaccine comprising or alternatively consisting of the modified virus-like particle of the invention.
  • said modified VLPs comprise any one of the technical features disclosed herein, either alone or in any possible combination.
  • the vaccine further comprises an adjuvant.
  • the vaccine is devoid of an adjuvant.
  • said vaccine comprises an effective amount of the composition of the invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) a modified VLP of the invention or a vaccine of the invention; and (b) a pharmaceutically acceptable carrier, diluent and/or excipient.
  • Said diluent includes sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions.
  • 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.
  • compositions of the invention may be in a form which contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the conjugate.
  • Examples of materials suitable for use in preparation of pharmaceutical compositions are provided in numerous sources including Remington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co., (1990)).
  • said pharmaceutical composition comprises an effective amount of the vaccine of the invention.
  • a further aspect of the invention is a method of immunization comprising administering a modified VLP of the invention, a vaccine of the invention, or a pharmaceutical composition of the invention to an animal or a human.
  • said method comprises administering a composition of the invention, a vaccine of the invention, or a pharmaceutical composition of the invention to an animal or a human.
  • a further aspect of the invention is a method of treating or preventing a disease, disorder or physiological condition in an animal said method comprising administering a modified VLP of the invention, a vaccine of the invention, or a pharmaceutical composition of the invention to said animal, wherein preferably said animal can be a human.
  • said modified VLP, said vaccine, or said pharmaceutical composition is administered to said animal subcutaneously, intravenously, intradermally, intranasally, orally, intranodal or transdermally.
  • said disease, disorder or physiological condition is selected from the group consisting of an allergy, a cancer, an autoimmune disease, an inflammatory disease, an infectious disease.
  • said disease, disorder or physiological condition is an infectious disease.
  • said disease, disorder or physiological condition is an inflammatory disease selected from SARS-CoV-2 infection, MERS-CoV infection and SARS-CoV infection.
  • said disease, disorder or physiological condition is SARS-CoV-2 infection.
  • the step of the construction was the insertion of CMV-Ntt830 gene in the polylinker of pETDuet-1 (Novagen) under the second T7 promotor.
  • the CMV-Ntt830 nucleic acid sequence was prepared as described in Example 3 of W02016/062720A1 and corresponds to SEQ ID NO: 14 of W02016/062720A1.
  • said CMV-Ntt830 gene was amplified in PCR reaction using following the oligonucleotides:
  • pTZ-plasmid clone containing CMV-Ntt830 gene without sequence errors was cut with Hindlll enzyme, treated with Klenow enzyme and finally with Ndel restrictase. Then the fragment was subcloned into the Ndel/EcoRV sites of the pETDuet-1, resulting in the helper vector pETDu-CMV-Ntt830.
  • helper vector For cloning of a modified coat protein of CMV comprising SARS-CoV2 receptor binding domain antigen, next helper vector was constructed.
  • the vector allowing the introduction of amino acid linkers comprising at least one Gly, at least one Ser, and at least Glu, and even further comprising at least one Asp, on both sides of the SARS-CoV2 receptor binding domain antigen was constructed, using PCR mutagenesis and oligonucleotides as follows: 1 st PCR reaction:
  • pTZ-plasmid clone containing product from the 1 st PCR reaction was cut with NcoI/BamHI restrictases
  • the clone from 2 nd PCR was cut with BamHI/Hindlll and joined in ligation reaction with the helper vector pETDu-CMV-Ntt830, which was cut with Ncol/Hindlll.
  • the ligation of three DNA fragments resulted in helper plasmid pETDu-CMVB3d-CMVtt.
  • the vector contains CMV-Ntt830 gene with the Th cell epitope derived from tetanus toxin in both encoded proteins, and introduced sequences coding for amino acid linkers comprising at least one Gly, at least one Ser, and at least Glu, and even further comprising at least one Asp, in detail comprising Gly-Ser linkers with additional Asp-Glu-Asp stretches and BamHESpel sites for subcloning of antigen DNA sequences in the CMV-Ntt830 gene under the first T7 promotor.
  • SARS-CoV2 receptor binding domain (RBD) gene with flanking BamHI and Spel sites was obtained from commercial source (gene synthesis product in pUCIDT, Integrated DNA Technologies Ltd, Belgium).
  • the BamHESpel fragment was excised from plasmid pUCIDT-nCoV-D and ligated into helper vector pETDu-CMVB3d-CMVtt (sites BamHI and Spel).
  • Resulting plasmid was isolated from E.coli XL1 cells and resequenced to verify the introduced nCoV-D sequence.
  • the plasmid map of the pETDu-CMVB3d-nCoV-D- CMV-tt is shown in (FIG. 1).
  • said nCoV-D protein of SEQ ID NO:24 is directly flanked at its N-terminus by the 18 amino acid long GSED-linker of SEQ ID NO: 17 and directly flanked at its C-terminus by the 15 amino acid long GSED- linker of SEQ ID NO: 18. Further, said aforementioned described entire construct, i.e. the construct of SEQ ID NO:24 flanked by the described GSED-linkers, is inserted between positions corresponding to Ser(88) and Tyr(89) of SEQ ID NO:5 of CMV-Ntt830 leading to CMV-Ntt830-nCoV-D.
  • CMV-Ntt830-nCoV-D The amino acid sequence of this preferred chimeric CMV polypeptide in accordance with the present invention referred to as “CMV-Ntt830-nCoV-D” is SEQ ID NO:25.
  • the corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV- Ntt830-nCoV-D is described in SEQ ID NO:26.
  • E. coli C2566 New England Biolabs, USA competent cells were transformed with the plasmid pETDu- CMVB3d-nCoV-D-CMV-tt.
  • E. coli cultures were grown in 2TY (Trypton 1.6%, yeast extract 1%, 0.5% NaCl, 0.1% glucose) medium containing ampicillin (100 mg/1) on a rotary shaker at 30°C to the OD(600) value of 0.8-1.0. Then, the cells were induced with 0.2 mM IPTG, and the medium was supplemented with 5mM MgCh. Incubation was continued on the rotary shaker at 20°C for 18h. The resulting biomass was collected by low-speed centrifugation and was frozen at - 20°C.
  • mosaic VLPs comprising CMV-Ntt830-nCoV-D and unmodified CMV-Ntt830 proteins (said mosaic VLPs referred to as “CMV-M-nCoV-D”) in accordance with the present invention, includes the following steps:
  • VLPs Collect the VLPs by ultracentrifugation using rotor Type 70 (Beckman Optima, L100XP ultracentrifuge; 4h, at 50 OOOrpm, 5°C);
  • VLPs by ultracentrifugation using rotor TLA100.3 (Beckman; 1 h, at 72000rpm, 5°C);
  • CMV-M-nCoV-M CMV and in-fused SARS-CoV2 receptor binding motif
  • SARS-CoV2 receptor binding motif represents a N- and C-terminally truncated fragment of SARS-CoV2 receptor binding domain, which is directly involved in virus binding to the target cells.
  • the motif coding DNA with flanking BamHI and Spe I sites was obtained from commercial source (gene synthesis product in pUCIDT, Integrated DNA Technologies Ltd, Belgium).
  • the BamHESpel fragment was excised from plasmid pUCIDT-nCoV-M and ligated into helper vector pETDu-CMVB3d-CMVtt (sites BamHI and Spel). Resulting plasmid was isolated from E.coli XL1 cells and resequenced to verify the introduced nCoV-D sequence.
  • the plasmid map of the pETDu-CMVB3d-nCoV-M- CMV-tt is shown in (FIG. 3). Said plasmid and expression vector ensures and serves for expression of mosaic VLPs comprising CMVB3d-nCoV-M and unmodified CMV-Ntt830 proteins, i.e. CMV-M-nCoV-M.
  • CMV-Ntt830-nCoV-M comprises full-length SARS-CoV2 coronavirus receptor binding motif of SEQ ID NO:27 which is flanked by amino acid linkers comprising at least one Gly, at least one Ser and at least one Glu.
  • said nCoV-M protein of SEQ ID NO:27 is directly flanked at its N-terminus by the 18 amino acid long GSED-linker of SEQ ID NO: 17 and directly flanked at its C-terminus by the 15 amino acid long GSED-linker of SEQ ID NO: 18.
  • said aforementioned described entire construct i.e.
  • CMV-Ntt830-nCoV-M amino acid sequence of this preferred chimeric CMV polypeptide in accordance with the present invention referred to as “CMV-Ntt830-nCoV-M” is SEQ ID NO:28.
  • the corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV- Ntt830-nCoV-M is described in SEQ ID NO:29.
  • E. coli C2566 New England Biolabs, USA competent cells were transformed with the plasmid pETDu- CMVB3 d-nCo V -M-CM V -tt.
  • E. coli cultures were grown in 2TY (Trypton 1.6%, yeast extract 1%, 0.5% NaCl, 0.1% glucose) medium containing ampicillin (100 mg/1) on a rotary shaker at 30°C to the OD(600) value of 0.8-1.0. Then, the cells were induced with 0.2 mM IPTG, and the medium was supplemented with 5mM MgCh. Incubation was continued on the rotary shaker at 20°C for 18h. The resulting biomass was collected by low-speed centrifugation and was frozen at - 20°C.
  • CMV-M-nCoV-M The purification of mosaic VLPs comprising CMV-Ntt830-nCoV-M and unmodified CMV-Ntt830 proteins (said mosaic VLPs referred to as “CMV-M-nCoV-M”) in accordance with the present invention, includes the steps described in Example 1.
  • the SDS-PAGE analysis of sucrose gradient fractions is shown in FIG. 4A.
  • CMV-M-MERS-D mosaic particle containing modified Coat Protein of CMV and in-fused MERS-CoV2 receptor binding domain
  • MERS coronavirus receptor binding domain coding gene with flanking BamHI and Spe I sites was obtained from commercial source (gene synthesis product in pUCIDT, Integrated DNA Technologies Ltd, Belgium).
  • the BamEQ/Spel fragment was excised from plasmid pUCIDT-MERS-D and ligated into helper vector pETDu-CMVB3d-CMVtt (sites BamHI and Spel).
  • Resulting plasmid was isolated from E.coli XL1 cells and resequenced to verify the introduced nCoV-D sequence.
  • the plasmid map of the pETDu-CMVB3d-MERS- D-CMV-tt is shown in (FIG. 6).
  • Said plasmid and expression vector ensures and serves for expression of mosaic VLPs comprising CMVB3d-MERS-D and unmodified CMV-Ntt830 proteins, i.e. CMV-M-MERS-D.
  • CMV-Ntt830-MERS-D comprises full-length MERS coronavirus receptor-binding domain of SEQ ID NO:30 which is flanked by amino acid linkers comprising at least one Gly, at least one Ser and at least one Glu.
  • said nCoV-D protein of SEQ ID NO:30 is directly flanked at its N-terminus by the 18 amino acid long GSED-linker of SEQ ID NO: 17 and directly flanked at its C-terminus by the 15 amino acid long GSED-linker of SEQ ID NO: 18.
  • said aforementioned described entire construct i.e.
  • CMV-Ntt830-MERS-D The amino acid sequence of this preferred chimeric CMV polypeptide in accordance with the present invention referred to as “CMV-Ntt830-MERS-D” is SEQ ID NO:31.
  • the corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV- Ntt830-MERS-D is described in SEQ ID NO:32.
  • E. coli C2566 New England Biolabs, USA competent cells were transformed with the plasmid pETDu- CMVB3 d-MERS-D-CMV-tt.
  • E. coli cultures were grown in 2TY (Trypton 1.6%, yeast extract 1%, 0.5% NaCl, 0.1% glucose) medium containing ampicillin (100 mg/1) on a rotary shaker at 30°C to the OD(600) value of 0.8-1.0. Then, the cells were induced with 0.2 mM IPTG, and the medium was supplemented with 5mM MgCh. Incubation was continued on the rotary shaker at 20°C for 18h. The resulting biomass was collected by low-speed centrifugation and was frozen at - 20°C.
  • the purification of mosaic VLPs comprising CMV-Ntt830-MERS-D and unmodified CMV-Ntt830 proteins (said mosaic VLPs referred to as “CMV-M-MERS-D”) in accordance with the present invention, includes the steps described in Example 1.
  • the SDS-PAGE analysis of sucrose gradient fractions is shown in FIG. 7A; analysis of the VLPs after purification on SDS-PAGE gel is shown in FIG. 7B.
  • CM -M-MERS-M mosaic particle containing modified Coat Protein of CMV and in-fused MERS receptor binding motif
  • MERS coronavirus receptor binding motif represents a N- and C-terminally truncated fragment of MERS receptor binding domain, which is directly involved in virus binding to the target cells.
  • the motif coding DNA with flanking BamHI and Spel sites was obtained after PCR using pUCIDT-MERS-D plasmid as a template and oligonucleotides (forward SEQ ID NO:33; reverse SEQ ID NO:34).
  • the PCR product was treated with BamHESpel enzymes and directly ligated into helper vector pETDu-CMVB3d-CMVtt (sites BamHI and Spel).
  • plasmid clones were isolated from E.coli XL1 cells and sequenced to identify the clone with introduced MERS-M sequence without PCR errors.
  • the plasmid map of the pETDu-CMVB3d-MERS-M-CMV-tt is shown in (FIG. 8).
  • Said plasmid and expression vector ensures and serves for expression of mosaic VLPs comprising CMVB3d-MERS-M and unmodified CMV-Ntt830 proteins, i.e. CMV-M-MERS-M.
  • CMV-Ntt830-MERS-M comprises full-length MERS coronavirus receptor-binding motif of SEQ ID NO:35 which is flanked by amino acid linkers comprising at least one Gly, at least one Ser and at least one Glu.
  • said nCoV-D protein of SEQ ID NO:35 is directly flanked at its N-terminus by the 18 amino acid long GSED-linker of SEQ ID NO: 17 and directly flanked at its C-terminus by the 15 amino acid long GSED-linker of SEQ ID NO: 18.
  • said aforementioned described entire construct i.e.
  • CMV-Ntt830-MERS-M The amino acid sequence of this preferred chimeric CMV polypeptide in accordance with the present invention referred to as “CMV-Ntt830-MERS-M” is SEQ ID NO:36.
  • the corresponding nucleotide sequence of this preferred chimeric CMV polypeptide CMV- Ntt830-MERS-M is described in SEQ ID NO:37.
  • E. coli C2566 New England Biolabs, USA competent cells were transformed with the plasmid pETDu- CMVB3 d-MERS-M-CMV-tt.
  • E. coli cultures were grown in 2TY (Trypton 1.6%, yeast extract 1%, 0.5% NaCl, 0.1% glucose) medium containing ampicillin (100 mg/1) on a rotary shaker at 30°C to the OD(600) value of 0.8-1.0. Then, the cells were induced with 0.2 mM IPTG, and the medium was supplemented with 5mM MgCh. Incubation was continued on the rotary shaker at 20°C for 18h. The resulting biomass was collected by low-speed centrifugation and was frozen at - 20°C.
  • the purification of mosaic VLPs comprising CMV-Ntt830-MERS-M and unmodified CMV-Ntt830 proteins (said mosaic VLPs referred to as “CMV-M-MERS-M”) in accordance with the present invention, includes the steps described in Example 1.
  • the SDS-PAGE analysis of sucrose gradient fractions is shown in FIG. 9A; analysis of the VLPs after purification on SDS-PAGE gel and under EM is shown in FIG. 9B and FIG. 10, respectively.
  • ACE2 Angiotensin Converting Enzyme 2
  • ELISA plates (Coming 96 Well Half-Area Microplate, Merck Switzerland) were coated overnight at 4°C with 50 m ⁇ of recombinant human ACE-2 at 1 pg/ml (Sigma-Aldrich, Switzerland) in phosphate buffered saline (PBS, pH 7.4). After washing with PBST (PBS/0.01% Tween) unspecific binding the ELISA plates was blocked with Superblock solution (Therm oFisher, Switzerland) and incubated for 1 hours at room temperature. After removal of the blocking solution, 50m1 of serial diluted CMV-M-nCoV-M or control VLP CMVNtt830 was added and incubated for 1 hour at room temperature.
  • CMV cucumber mosaic viurs
  • mice 8-12 weeks female wild type BALB/cOlaHsd mice were purchased from Harlan. In groups of five mice were immunized subcutaneously with 20 pg of either CMV-M-nCoV- M VLPs or unmodified VLP control CMVNtt830. VLPs were injected in a volume of 150 m ⁇ at day 0, 7, 14 and 21. Mice were bled weekly after immunization and sera were tested by ELISA against recombinant SARS CoV-2 Spike protein (S protein) (FIG. 12B) and the recombinant receptor binding domain (RBD) of the SARS CoV-2 S protein (FIG. 12A). Neutralisation titers of sera were determined in a SARS-CoV-2 pseudovirus assay (FIG. 12C) as well as in a CPE-based assay (FIG. 12D).
  • S protein S protein
  • RBD recombinant receptor binding domain
  • ELISA plates (Corning 96 Well Half-Area Microplate, Merck Switzerland) were coated over night with 2pg/ml recombinant RBD (SARS CoV-2 S protein (YP_009724390.1) (Arg319-Phe541) with C terminal 6 x histidine tag expressed in Expi293F(TM) Cells) (SEQ ID NO:38) or SARS-CoV-2 Spike protein (purchased from SinoBiological as SARS-CoV-2 (2019-nCoV) Spike Protein (S1+S2 ECD) (YP_009724390.1; Vall6-Prol213).
  • ELISA titers were defined as the reciprocals of the dilutions needed to achieve 50% of the optical density of the maximal signal measured at saturation. A four-parameter logistic equation was used to calculate the ELISA titers (GraphPad Prism5). The lower limit of quantification (LLOQ) was set to the half of the lowest dilution (i.e. 20 fold) tested, corresponding to an ELISA titer of 10. ELISA titers below the lowest serum dilution tested (i.e. 20-fold) were set to 10.
  • SARS CoV-2 pseudotype virus PV
  • 5xl0 6 HEK293T/17 cells maintained in DMEM+10% FBS (37°C, 5% CO2), were plated 24 hours prior to transient transfection.
  • a ratio of lpg of DNA to 5pg of polyethyl enimine was used for transfecting the cells with pCMV-A8.91 (lentiviral gag-pol; Zufferey R et ak, Journal of Virology (1998) 72(12):9873-9880), pCSFLW (luciferase reporter transfer plasmid; Zufferey R et ak, Nat Biotechnok (1997) 15(9): 871 -875) and a plasmid (purchased from NIBSC pCAGGS_SARS-CoV-2_Spike) encoding for the human codon optimised SARS-CoV-2 S (Wuhan-Hu-1, MN908947.3, (SEQ ID NO:39
  • TCID50 titres (fifty-percent tissue culture infective dose) were quantified by end-point dilution analysis using HEK293T/17 cells transiently expressing ACE-2 and TMPRSS2. Serum samples were heat inactivated (56 ° C for 30 minutes) and serially diluted 5-fold in supplemented media, after which 1 OOxTCID o of PV stock was added. The plates were incubated at 37 ° C (5% CO2) for 1 hour before 2xl0 4 target cells
  • % neutralisation was calculated by dividing the mean bioluminescence of test sample by mean bioluminescence of negative control sample (wells with no serum added) time 100.
  • CPE-based assay the capacity of the induced antibodies in neutralizing wild-type SARS-CoV-2 (SARS-CoV-2/ABS/NL20) was performed. Serum samples were heat- inactivated for 30min at 56°C. Two-fold serial dilutions were prepared starting at 1:20 up to 1:160. 100 TCID50 of the virus was added to each well and incubated for 37°C for lh. The mixture has been added on a monolayer of Vero cells and incubated again for 37°C for 4 days. Four days later the cells were inspected for cytopathic effect. The titer was expressed as the highest dilution that fully inhibits formation of CPE (FIG. 12D).
  • CMV-M-MERS-M binds to Dipeptidyl peptidase-4 (DPP4), the cell entry receptor for DPP4.
  • DPP4 Dipeptidyl peptidase-4
  • ELISA plates (Coming 96 Well Half-Area Microplate, Merck Switzerland) were coated overnight at 4°C with 50 m ⁇ of recombinant human DPP4 (Sino Biological) at 1 pg/ml in phosphate buffered saline (PBS, pH 7.4).
  • the recombinant human DPP4 comprises the native mature form of human DPP IV (NP 001926.2) extracellular domain (Asn 29-Pro 766).
  • PBST PBS/0.01% Tween
  • Superblock solution ThermoFisher, Switzerland
  • CMV-M-MERS-M or control VLP CMVNtt830 were added and incubated for 1 hour at room temperature.
  • dilution buffer PBS/2% BSA/0.05% Tween
  • CMV cucumber mosaic viurs
  • the mouse monoclonal anti-CMV antibody, clone 1-1 A8, has been produced by a hybridoma cell line which was generated by fusing B lymphocytes from CMV VLP vaccinated mice with immortal B cell cancer cells. 1 hour later plates were washed with PBST and incubated with 50m1 of horse radish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch) diluted 1 : 1000 in dilution buffer. The plates were be incubated for 1 hour at room temperature, then washed prior to addition of 50m1 substrate solution (PierceTM TMB Substrate Kit, ThermoFisher, Switzerland). After 10 minutes an equal volume of stopping solution (1M H2SO4) was be added to stop the enzymatic reaction. Absorbance at 450 nm was be read using a SpectraMAX M5, Molecular Devices (FIG. 13).
  • mice Female wild type BALB/cOlaHsd mice were immunized subcutaneously with 100 pg of CMV-M-MERS-M VLPs or administered vehicle control initially twice 3 weeks apart. An injection volume of 150 m ⁇ was used. Blood was collected weekly and sera were tested by ELISA against recombinant MERS Spike protein (S protein) and the recombinant receptor binding domain (RBD) of the MERS S protein (FIG. 14A, FIG. 14B). Neutralisation titers were be determined in a MERS plaque reduction neutralisation assay (FIG. 14C).
  • ELISA plates (Corning 96 Well Half-Area Microplate, Merck Switzerland) were coated over night with 2pg/ml recombinant RBD (Sino Biological MERS -Co V Spike/RBD Protein fragment (RBD, aa 367-606, His Tag)) or MERS-CoV Spike protein (Sino Biological MERS-CoV Spike/ SI Protein (SI Subunit, aa 1-725, His Tag)). Plates were washed with PBS-0.01%Tween and blocked using IOOmI PBS-Casein 0.15% for 2 hours at room temperature.
  • ELISA titers were defined as the reciprocals of the dilutions needed to achieve 50% of the optical density of the maximal signal measured at saturation. A four-parameter logistic equation was used to calculate the ELISA titers (GraphPad Prism5). The lower limit of quantification (LLOQ) was set to the half of the lowest dilution (i.e. 20 fold) tested, corresponding to an ELISA titer of 10. ELISA titers below the lowest serum dilution tested (i.e. 20-fold) were set to 10.
  • MERS-CoV neutralizing antibodies The presence of MERS-CoV neutralizing antibodies in the sera of vaccinated mice was assessed by reduction of cytopathic effect. Serum samples were heat-inactivated for 30 minutes at 56°C. Subsequently, serum samples were diluted two-fold, started at 1:20 to 1:160. Then 100 TCID50 (Median Tissue Culture Infectious Dose) of MERS- CoV/EMC/2012 was added to each well and incubated at 37°C for lh. Following incubation, the mixtures were added on a monolayer of Vero cells and incubated at 37°C for an additional 4 days. After 4 days wells were inspected for presence of cytopathic effect (CPE). Titer is expressed as the highest serum dilution that fully inhibits formation of CPE.
  • CPE cytopathic effect

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Abstract

La présente invention concerne une particule pseudovirale (VLP) modifiée du virus de la mosaïque du concombre (CMV) comprenant au moins une protéine de fusion, ladite au moins une protéine de fusion comprenant, ou consistant de préférence en, (b) un polypeptide de CMV chimérique, ledit polypeptide chimérique de CMV comprenant, ou de préférence consistant en, (iii) un polypeptide de CMV, ledit polypeptide de CMV comprenant, ou de préférence consistant en, une couche protéique de CMV, ladite couche protéique de CMV comprenant, ou de préférence consistant en, la SEQ ID NO : 1 ; ou une séquence d'acides aminés présentant une identité de séquence d'au moins 75 %, de préférence d'au moins 80 %, plus préférablement d'au moins 85 %, encore plus préférablement d'au moins 90 %, encore plus préférablement d'au moins 95 %, encore plus préférablement d'au moins 98 % et encore plus préférablement d'au moins 99 % avec la SEQ ID NO : 1 ; et (iv) un polypeptide antigénique, ledit polypeptide antigénique étant un domaine de liaison au récepteur (RBD) d'un coronavirus (CoV), ou un fragment de celui-ci, ledit polypeptide antigénique étant inséré dans ledit polypeptide de CMV, ladite insertion dudit polypeptide antigénique se faisant entre les résidus d'acides aminés dudit polypeptide de CMV correspondant aux résidus d'acides aminés de la position 84 et de la position 85 de la SEQ ID No: 1 ; et (iii) un épitope de cellules T auxiliaires, ledit épitope de cellules T auxiliaires remplaçant une région N-terminale dudit polypeptide de CMV, et de préférence ladite région N-terminale dudit polypeptide de CMV correspondant aux acides aminés 2-12 de la SEQ ID No:1.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024047090A2 (fr) 2022-08-30 2024-03-07 Saiba Animal Health Ag Particules de cmv de type virus modifiées
WO2024047091A2 (fr) 2022-08-30 2024-03-07 Saiba Animal Health Ag Compositions vétérinaires de particules de type virus modifiées d'antigènes de cmv et ngf

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