WO1998049195A1 - Particules de type coronavirus utilisees comme outils pour la vaccination et le traitement - Google Patents

Particules de type coronavirus utilisees comme outils pour la vaccination et le traitement Download PDF

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WO1998049195A1
WO1998049195A1 PCT/NL1998/000237 NL9800237W WO9849195A1 WO 1998049195 A1 WO1998049195 A1 WO 1998049195A1 NL 9800237 W NL9800237 W NL 9800237W WO 9849195 A1 WO9849195 A1 WO 9849195A1
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virus
protein
particle
coronavirus
particle according
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PCT/NL1998/000237
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Petrus Josephus Marie Rottier
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Universiteit Utrecht
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/20041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/20045—Special targeting system for viral vectors
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/609—Vectors comprising as targeting moiety peptide derived from defined protein from viruses positive strand RNA viruses

Definitions

  • the present invention relates to virus-like particles (VLPs) derived from corona viruses which are modified in various ways, genomically or in their protein composition, thereby exposing at their surface various biological or target molecules and/or carrying within the particles molecules with biological activity which need to be protected or shielded and/or containing genomes from which parts of authentic coronavirus genes or sequences have been removed or altered or into which foreign genes or sequences have been incorporated.
  • VLPs virus-like particles
  • viruses intrinsically represent the most natural delivery systems and seem thus pre-eminently suitable as therapeutic carriers. Their exploitation requires that we can engineer virus-like particles and tailor them to their new function. Such particles must be equipped with specific targeting information and "loaded” with a genetic or non- genetic message. The idea to use viruses for the better is not new but has been limited mainly to retroviruses and adenoviruses which may have limited value as tools for gene therapy .
  • Coronavirions have a rather simple structure. They consist of a nucleocapsid surrounded by a lipid membrane.
  • the helical nucleocapsid is composed of the RNA genome packaged by one type of protein, the nucleocapsid protein N.
  • the viral envelope generally contains 3 membrane proteins: the spike protein (S) , the membrane protein (M) and the envelope protein (E) .
  • S spike protein
  • M membrane protein
  • E envelope protein
  • Some coronaviruses have a fourth protein in their membrane, the hemagglutinin-esterase protein (HE) .
  • HE hemagglutinin-esterase protein
  • the coronavirus nucleocapsids are assembled in the cytoplasm.
  • the nucleocapsids interact with the viral envelope proteins which after their synthesis in the endoplasmic reticulum accumulate in the intermediate compartment, a membrane system localized between the endoplasmic reticulum (ER) and the Golgi complex.
  • This membrane system acts as the budding compartment: the interaction of the nucleocapsids with the viral envelope proteins leads to the pinching off of virions which are then released from the cell by exocytosis .
  • VLP virus-like particles
  • CD CD > ⁇ - ra ra P- P 0 tr t P- P- P- CD 0 TJ CD • --• CD J to CD P 0 PJ 0
  • CD tr ft CD tr TJ CD P tr ⁇ ⁇ CD 0 LQ P- CD 0 P- CD rt ⁇ LQ CD : ra
  • CD CD CD CD 3 0 PJ P- ⁇ PJ tr ⁇ ; P- tr ft CD ⁇ PJ P- 0 P ra ⁇ l PJ 0 CD tr ⁇
  • CD TJ TJ CD P ⁇ > tr CD CD ⁇ PJ P tr P- P tr CD ⁇ P- ft P- J
  • constructs are inserted into plasmids behind a bacteriophage T7 polymerase promoter.
  • the constructs are then co-transfected with plasmids carrying the MHV M and E genes, both also behind the T7 promotor, in OST-7 cells which have been infected with a recombinant vaccina virus expressing the T7 polymerase.
  • the resulting VLPs contain the chimaeric MHV/FIPV S protein.
  • the VLP is provided by the methods used as above with ectodomains of the spike protein of infectious bronchitis coronavirus (IBV) , or the ectodomain (or part thereof) of an envelope protein of any enveloped virus not belonging to the coronaviruses .
  • MHV-based VLPs are provided by the invention which carry at their surface the ectodomain of the pseudorabies virus (PRV) glycoprotein gD instead of the MHV spike ectodomain or the luminal (i.e. amino-terminal) domain (or part thereof) of any nonviral type I membrane protein.
  • PRV pseudorabies virus
  • VLPs are provided that have a cell specificity for chicken cells, or pig cells, or cells reactive with the type I membrane protein.
  • VLPs are produced with modifications that are contained within the particles. This is achieved by the incorporation of modified constructs of any of the corona viral proteins S, M, E and HE. In corona virus particles these proteins have their carboxy-terminal domain enclosed within the interior of the viral envelope. Thus, foreign protein sequences incorporated within, appended to or replacing the carboxy- terminal domain are enclosed as well.
  • VLPs can be provided that contain protein moieties, or fragments thereof, from another virus, or non-viral proteins such as hormones, such as erytrhopoietin.
  • VLPs containing a biological active protein or fragments thereof which is/are shielded by the viral envelope and can be released and/or retrieved later, when the viral membrane is degraded or fused with another membrane.
  • This allows the in vi tro production in cells, or the in vivo production in secretory glands such as milk glands of biologically active substance which are otherwise harmful or toxic to the producing cells, or which for other reasons need to be produced in a shielded form.
  • MHV-based VLPs are provided carrying on their surface or inside an enzy atically active molecule like furin, or a cytokine, or a hormone receptor, or another viral or nonviral polypeptide with biological activity.
  • VLPs are provided with (additional) targeting means that serve to direct the VLP to cells otherwise not accessible to the original corona virus .
  • the invention provides VLPs which are modified at the ectodomain and/or the ectodomain of any of the viral proteins.
  • the VLPs are provided with modified biological molecules as targeting means that serve to direct the VLP to interact with other biological molecules that mirror or can interact with the target means, such as receptor proteins on cells, be it hormone receptors, specific immunoglobulines on B- cells, MHC and MHC associated molecules present on T-cells and other cells, transfer proteins or other receptor molecules known to the person skilled in the field of cell surface receptors.
  • the targeting means can also be provided to interact with known binding sites of selected enzymes on proteins or other molecules that serve as substrate for the selected enzyme.
  • MHV-based VLPs are provided exposing an immunogenic determinant of a bacterial toxin.
  • the VLPs serve as immunogen or vaccine, here directed against the bacterial toxin.
  • B-lymfocytes carrying the corresponding immunoglobuline at their surface are in this case the target cells for the VLPs, once recognozed by the B- lymfocyte, this cell(s) will multiply and produce the appropriate antibody.
  • Preparation of VLPs or coronaviruses with modified spikes can be achieved genetically by modification of the viral genome such that it expresses the modified S protein in infected cells.
  • coronaviruses containing altered spikes in a different way by expressing modified S genes in cells which are in addition infected with coronavirus .
  • the co-incorporation of the mutant spike provides the virus with new targeting means.
  • MHV particles containing the chimaeric MHV/FIPV S protein.
  • the chimaeric S gene construct is expressed in L cells which are subsequently infected with wild-type MHV strain A59 (MHV-A59) or a mutant thereof.
  • the progeny virus released by the cells contains the modified S protein.
  • the altered targeting was used to infect feline cells which are naturally not susceptible to MHV.
  • the cells are now infected as shown by immunofluorescence and produce normal MHV.
  • MHV containing chimaeric S proteins in which part of the S ectodomain has been replaced by the corresponding part (i.e. the luminal or amino-terminal domain) of the human CD4 molecule, as an example of a nonviral protein.
  • modified coronaviruses have acquired the property to infect HIV-infected cells and cells expressing HIV envelope glycoprotein through the specific recognition of the CD4 and HIV gpl20 complex.
  • the HIV-infected cells will undergo a lytic infection, effectively reducing the number of HIV-infected cells in the body and thereby reducing the severity of the disease or even terminating the infection.
  • deletion or mutation can be achieved with a cDNA clone or by recombination.
  • Attenuation is provided by the preparation of an MHV mutant from which an essential gene has been deleted by recombination.
  • a mouse cell line is provided in which the MHV E gene has been chromosomally integrated allowing the E protein to be produced by the expression of the gene.
  • MHV lacking an E gene has been produced in normal mouse cells by recombination using a synthetic RNA containing a perfect copy of the MHV genomic 3 ' -end except for the lack of an intact E gene.
  • the E- defective virus is able to grow only in the cells complementing the defect.
  • the virus produced is attenuated such that it can infect other mouse cells, but non- productively: the lack of an E protein prevents the assembly of progeny.
  • MHV derived VLP is provided into which a reporter gene such as LacZ or green fluorescent protein has been recombined and one in which the chimaeric MHV/FIPV S gene has been incorporated.
  • the expression of the genes is shown by blue or green- fluorescent staining of VLP infected cells and by the acquired ability to infect feline cells, respectively.
  • the other way to obtain coronavirus-based delivery vehicles uses VLPs comprising foreign RNA sequences. Incorporation of foreign RNA sequences into these particles requires their packaging into nucleocapsids.
  • N protein molecules Viral RNA-packaging by nucleocapsid (N) protein molecules occurs by the recognition of specific sequences, packaging signal (s) by the N protein.
  • packaging signal includes a 69 nucleotides long region in gene IB.
  • RNAs containing the coronavirus packaging signal (s), or defective coronaviral genomes in which these signal (s) have been retained but into which foreign sequences have been incorporated, are assembled into VLPs when introduced into cells expressing the N, M and E ( ⁇ S) genes .
  • the VLP can introduce into a target cell a defined RNA that may have one of several functions.
  • An example provided by the invention is a RNA acting as mRNA and specifying a particular protein such as a toxin or an inducer of apoptosis or an antibody fragment .
  • Another example is an antisense RNA or an RNA with ribozyme activity.
  • VLPs which will only carry one or a few pseudo-NC.
  • the invention thus provides the RNAs with amplification signals such that they will be multiplied in the target cell.
  • Semliki Forest virus (SFV) replication sequences are used as the basis of the RNA construct.
  • SFV-derived mRNA further comprising the coronavirus encapsidation sequences and specifying a reporter protein are assembled into VLPs.
  • the SFV-driven amplification allows synthesis of the reporter protein in cells; in animals the appearance of antibodies to the reporter protein testifies to the productive delivery of the VLPs ' content .
  • the invention also provides a VLP which is an antigen or epitope delivery vehicle meant for the induction of specific immune responses, cellular and/or humoral, systemic and/or local, including the induction and production of specific antibodies against proteins, to achieve protection against infection by pathogens, of viral and nonviral origin.
  • VLP which is an antigen or epitope delivery vehicle meant for the induction of specific immune responses, cellular and/or humoral, systemic and/or local, including the induction and production of specific antibodies against proteins, to achieve protection against infection by pathogens, of viral and nonviral origin.
  • the invention provides the induction of antibodies against the reporter protein derived from SFV- derived mRNA further comprising the coronavirus encapsidation sequences and specifying a reporter protein, as described above.
  • the induction of antibodies is demonstrated in mice to the FIPV spike and to PRV gD by immunization with the VLPs, also described above.
  • immune responses can be elicited both against proteins which are encoded by the altered genome of the VLP and/or against proteins which have been incorporated as targeting means in the VLP, thereby partly or wholly replacing the original spike protein.
  • the examples illustrate the applicability of the approach for the induction of immune responses against proteins as diverse as for instance viral, bacterial, parasitic, cellular and hormonal origins.
  • the induction of protective immunity in mice against PRV by the gD exposing VLPs is provided.
  • the example illustrates the applicability of the approach in principle for vaccination against viral, bacterial and parasitic pathogens including for instance human coronaviruses .
  • the invention also provides VLPs for diagnostic purposes. In immunoassay always a great need exists for a well- defined, specific and sensitive antigen that can be prepared in large quantities.
  • the use of MHV- based VLPs carrying the PRV gD ectodomain in an ELISA to detect gD antibodies is provided.
  • the invention also provides VLPs which have fully maintained the original spike protein but which are altered genomically to attenuated the VLP and/or to encode nucleotide sequences that need to be delivered at the cells to which the original coronavirus was targeted.
  • VLPs which have fully maintained the original spike protein but which are altered genomically to attenuated the VLP and/or to encode nucleotide sequences that need to be delivered at the cells to which the original coronavirus was targeted.
  • intestinal epithelial cells, or respiratory epithelial cells that are normally infected by TGEV, or PRCV, respectively, can now interact with VLPs derived from TGEV or PRCV, or other cell-specific coronaviruses if needed, to express proteins normally not expressed by said viruses.
  • respiratory epithelial cells of cystic fibrosis patients can be induced to express lung surfactant molecules that are encoded by the altered genome of the VLP.
  • Coronaviruses are assembled intracellularly by budding into the intermediate compartment and, later in infection, into the endoplasmic reticulum (ER; Klumperman et al . , 1994; Krijnse Locker et al . , 1994).
  • the cytoplasmically synthesized nucleocapsid (NC) - the viral genomic RNA packaged by nucleocapsid protein (N) molecules - interacts with cytoplasmically exposed domains of viral membrane proteins accumulated in the pre-Golgi membranes. Subsequent budding results in the formation of virions that follow the exocytic pathway out of the cell.
  • VLPs coronavirus-like particles
  • S spike-like particles
  • VLP assembly provides us with an extremely valuable and convenient tool to study aspects of coronavirus assembly. This was demonstrated very clearly in a study of the structural requirements of the M protein for assembly. In this study we showed by co-expression of mutated M proteins with the E protein that particle formation is sensitive to changes in all domains of the M protein, i.e. the luminal N-terminal domain, the transmembrane domains and the cytoplasmic C-terminal domain. Particularly the identity of the extreme C-terminus appeared to be very important; substitutions of the terminal residue can abolish VLP assembly nearly completely as does its deletion; deletion of the last two residues or more is fully fatal. To further demonstrate that the VLP assembly system is a faithful model for coronavirion assembly, we introduced several of these mutations into the viral genome by RNA recombination. The results were essentially fully concordant.
  • the aim of the further examples was to study the incorporation of the S protein into viral particles.
  • Ascitis G73 was obtained from a cat infected with feline infectious peritonitis virus (FIPV) and contained antibodies to this virus 1 spike protein.
  • Two expression constructs were prepared encoding mirror image chimaeric MHV/FIPV S proteins : one protein (designated S * ) has the transmembrane + cytoplasmic domain of MHV S and the luminal domain (i.e. ectodomain) of FIPV
  • the construction made use of a convenient Styl site occurring at an identical position in both the MHV S gene and the FIPV S gene; this Styl site marks the location where in the S protein the ectodomain turns into the transmembrane domain.
  • pTFMS encoding the S * gene
  • pBl cDNA clone de Groot et al . , 1989
  • FIPV strain 79-1146 S sequences FIPV strain 79-1146 S sequences.
  • a chimaeric S gene construct was prepared consisting of the 3' Styl/BamHI fragment of the MHV S gene (Vennema et al . , 1990) and the 5' coding sequence of the
  • FIPV S gene spanning the AUG initiation codon down to the corresponding Styl site.
  • the chimaeric gene was ligated as a BamHI fragment into the vector pTUG3 (Vennema et al . ,
  • the reverse chimaeric construct, encoding the S* gene was prepared from the complementary 5' BamHl/Styl MHV S gene fragment and the 3 ' Styl/Sall FIPV S gene fragment and was cloned into pTUC (Vennema et al . , 1991) .
  • the plasmid was designated pMFS .
  • RNA donor construct pFVl described by Fischer et al . (1997) was extended in the upstream direction of the S gene by incorporating 1. Ikb sequences of the MHV gene 2.
  • the chimaeric S gene was introduced into this plasmid as follows. Convenient restriction sites were first introduced into the modified donor construct at the 5 ' end of the S gene and just downstream of the 3 ' end of this gene.
  • an Avrll site was engineered by mutating nucleotides 36 and 37 (both thymidines) in the S signal sequence encoding region into cytidines.
  • the sequence TCTCCTGG was changed into the Sse8387I restriction enzyme recognition sequence CCTGCAGG.
  • a chimaeric S * construct was prepared bordered by the same restriction sites.
  • the Avrll site was engineered using the following PCR primer: 5 ' -CCTAGGGTATATTGGTGATTTTAGATGCATACAAGTTAACGTAACAC-3 ' .
  • the Sse83871 site was created using the PCR primer TCTGTCTTTCCTGCAGGGGCTGTGAT .
  • chimaeric gene construct was retrieved using Avrll and Sse83871 and ligated into the donor plasmid that had been treated previously with the same enzymes.
  • Capped RNA was transcribed from the resulting donor plasmid and used for electroporation into MHV-infected L2 cells to allow recombination (Fischer et al . , 1997).
  • the cells were plated onto a monolayer of FCWF cells to enable multiplication of the recombinant virus .
  • the genes were (co) transfected into OST-7 cells infected with the recombinant vaccinia virus vTF7-3.
  • the proteins were labeled with 35 S-amino acids for 3h starting at 5h post infection (p.i.) .
  • Cells were then lysed, lysates were cleared by centrifugation and prepared for immun- oprecipitation using various antibodies. The analysis of the precipitated proteins is shown in Fig.l.
  • the M and S proteins were correctly expressed as shown by the immun- oprecipitates obtained with the anti-MHV antiserum (K134) raised against purified MHV (lanes 1 and 3) .
  • the E protein (which requires a higher gel percentage to be resolved) is poorly recognized by this serum (not shown) .
  • the monoclonal antibody J 1.3 specifically precipitated the M protein (lanes 4 and 8) , though with a lower efficiency than did K134.
  • Another monoclonal antibody, A3.10 precipitated the S protein with high specificity and efficiency (lane 5) .
  • the antibodies in the feline ascitic fluid G73 recognized the chimaeric protein S * but not the MHV S protein (or the M protein; lanes 2,6 and 7) .
  • VLPs produced by the combination of M, E and S * (lane 3) , not those resulting from M + E + S coexpression (lane 6), could be immunoisolated by the G73 antibodies. That this result (lane 3) was indeed specific was additionally shown by the lack of M protein isolation by these feline antibodies after coexpression of only the M and S * proteins (lane 4) . It should be noted that the direct demonstration of the S * protein in gels was was impaired by the strong copurification of an apparently secreted host cell protein with similar electrophoretic mobility.
  • a derivative of the chimaeric S * gene was incorporated into MHV by homologous RNA recombination.
  • Synthetic capped RNA transcribed from a donor DNA construct containing the S * gene was transfected into murine L2 cells that had been infected with MHV.
  • the cells were plated onto monolayers of FCWF-D cells.
  • the effect of recombination was evident from the formation of huge syncytia by the feline cells. No syncytia were formed when no RNA was transfected nor with any other donor RNA lacking the FIPV sequences .
  • the recombinant virus harvested from the culture media was used to infect fresh FCWF-D cells.
  • CD 0 0 LQ ra ft tr CD CD CD CD PJ a. 0 P- 0 CD CD
  • FIG. 1 Biochemical analysis of expressed viral proteins.
  • OST-7 cells grown in parallel in 3.5cm culture dishes were infected with VTF7-3 and transfected after lh with one or more of the plasmids pTUMM, pTU S, pTM5ab and pTFMS (5mg per plasmid) . They were incubated at 37°C.
  • Lysates were cleared by centrifugation in an Eppendorf centrifuge for 10 min at 10,000 rpm. Immunoprecipitations were done using 200ml-aliquots of cleared lysate which were diluted with 800ml immunoprecipitation buffer (20mM Tris-HCl [pH7.6], 150mM NaCl, 5m EDTA, 0.5% sodium deoxycholate, 0.1% SDS, lmg of protease inhibitors per ml) before adding the antibodies (2ml K134, 3ml G73, 150ml Jl .3 or 20ml A3.10) . After overnight incubation at 4°C 30ml Pansorbin (Calbio- chem) suspension was added and incubation continued for lh.
  • immunoprecipitation buffer 20mM Tris-HCl [pH7.6], 150mM NaCl, 5m EDTA, 0.5% sodium deoxycholate, 0.1% SDS, lmg of protease inhibitors per ml
  • FIG. 1 Analysis of VLPs by affinity purification.
  • CD 3 TJ 2 CD PJ 0 P 3 cJ Hi P, PJ P P h-- rt 3 c
  • CD td CO 0 CD ⁇ o CD rt ⁇ Hi P- 0 >-3 TJ CD P.

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Abstract

L'invention concerne des particules viriformes (VLP) dérivées des coronavirus, qui sont diversement modifiées en ce qui concerne leur génome ou leur composition protéique. Les particules exposent à leur surface diverses molécules biologiques ou cibles et/ou présentent au niveau de leurs molécules une activité biologique qu'il est nécessaire de protéger ou de masquer et/ou contiennent des génomes dont des parties de gènes ou de séquences authentiques de coronavirus ont été éliminées ou modifiées, ou bien dans lesquels des gènes ou des séquences étrangers ont été incorporés. Ces VLP peuvent être utilisés, par exemple, comme systèmes permettant l'administration ciblée d'agents thérapeutiques dans l'organisme, comme vaccins ou comme antigènes dans des analyses à visée diagnostique.
PCT/NL1998/000237 1997-04-29 1998-04-29 Particules de type coronavirus utilisees comme outils pour la vaccination et le traitement WO1998049195A1 (fr)

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WO2001002551A2 (fr) * 1999-06-30 2001-01-11 Evotec Oai Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
EP1219705A1 (fr) * 2000-12-29 2002-07-03 Evotec OAI AG Particules du type virus, leur préparation et leur utilisation en criblage pharmaceutique et en analyse fonctionelle de génomes
WO2002092827A2 (fr) * 2001-05-17 2002-11-21 Universiteit Utrecht Particules de type virus corona comprenant des genomes a deletions fonctionnelles
WO2004078203A2 (fr) * 2003-03-03 2004-09-16 Akzo Nobel N.V. Virus de la bronchite infectieuse comprenant un gene additif modifie
WO2004084940A1 (fr) * 2003-03-26 2004-10-07 Cytos Biotechnology Ag Encapsulation d'oligonucleotides immunostimulateurs dans des particules pseudovirales, procedes de preparation et utilisations
WO2005080419A1 (fr) * 2003-05-16 2005-09-01 Institute Of Microbiology, Chinese Academy Of Sciences Agent polypeptide d'inhibition du coronavirus sars et ses derives et utilisations
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US7597905B2 (en) 2000-02-28 2009-10-06 Beacle Inc. Hollow protein nano-particles and method of introducing substances into cells
WO2012117045A1 (fr) 2011-03-02 2012-09-07 Intervet International B.V. Protéine de spicule du virus de la bronchite infectieuse (ibv) en tant que vaccin sous-unité
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US8586728B2 (en) 2006-12-12 2013-11-19 Cytos Biotechnology Ag Oligonucleotides containing high concentrations of guanine monomers
US8691209B2 (en) 2001-09-14 2014-04-08 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
CN113866420A (zh) * 2020-06-30 2021-12-31 洛阳普泰生物技术有限公司 包被猪伪狂犬病病毒中和表位蛋白片段的elisa抗体检测试剂盒、含有其的疫苗组合物

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WO2001002551A2 (fr) * 1999-06-30 2001-01-11 Evotec Oai Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
US7476517B2 (en) 1999-06-30 2009-01-13 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7419802B2 (en) 1999-06-30 2008-09-02 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7597905B2 (en) 2000-02-28 2009-10-06 Beacle Inc. Hollow protein nano-particles and method of introducing substances into cells
EP1219705A1 (fr) * 2000-12-29 2002-07-03 Evotec OAI AG Particules du type virus, leur préparation et leur utilisation en criblage pharmaceutique et en analyse fonctionelle de génomes
WO2002092827A2 (fr) * 2001-05-17 2002-11-21 Universiteit Utrecht Particules de type virus corona comprenant des genomes a deletions fonctionnelles
AU2002311668B2 (en) * 2001-05-17 2007-10-04 Stichting Voor De Technische Wetenschappen Corona-virus-like particles comprising functionally deleted genomes
WO2002092827A3 (fr) * 2001-05-17 2004-05-27 Univ Utrecht Particules de type virus corona comprenant des genomes a deletions fonctionnelles
US7556957B2 (en) 2001-05-17 2009-07-07 Stichting Voor De Technische Wetenschappen Coronavirus-like particles comprising functionally deleted genomes
US8691209B2 (en) 2001-09-14 2014-04-08 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US9950055B2 (en) 2001-09-14 2018-04-24 Kuros Biosciences Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
WO2004078203A3 (fr) * 2003-03-03 2004-11-25 Akzo Nobel Nv Virus de la bronchite infectieuse comprenant un gene additif modifie
WO2004078203A2 (fr) * 2003-03-03 2004-09-16 Akzo Nobel N.V. Virus de la bronchite infectieuse comprenant un gene additif modifie
US7445785B2 (en) 2003-03-03 2008-11-04 Intervet International B.V. Infectious bronchitis virus with an altered spike gene
US7517520B2 (en) 2003-03-26 2009-04-14 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
AU2004224762B2 (en) * 2003-03-26 2009-12-24 Kuros Us Llc Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
WO2004084940A1 (fr) * 2003-03-26 2004-10-07 Cytos Biotechnology Ag Encapsulation d'oligonucleotides immunostimulateurs dans des particules pseudovirales, procedes de preparation et utilisations
WO2005080419A1 (fr) * 2003-05-16 2005-09-01 Institute Of Microbiology, Chinese Academy Of Sciences Agent polypeptide d'inhibition du coronavirus sars et ses derives et utilisations
SG152020A1 (en) * 2003-08-06 2009-05-29 Nat Environment Agency Diagnostic methods
WO2006056103A1 (fr) * 2004-11-26 2006-06-01 Dna Shuttle Biopharm Co., Ltd. Vecteur d'expression codant des particules de type coronavirus
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US9902972B2 (en) 2006-06-12 2018-02-27 Kuros Biosciences Ag Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
US10358656B2 (en) 2006-06-12 2019-07-23 Kuros Biosciences Ag Oligonucleotides packaged into virus-like particles of RNA bacteriophages
US8586728B2 (en) 2006-12-12 2013-11-19 Cytos Biotechnology Ag Oligonucleotides containing high concentrations of guanine monomers
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