EP1534845A1 - Obtention de virus recombines de la grippe a l'aide de vecteurs generateurs de baculovirus - Google Patents

Obtention de virus recombines de la grippe a l'aide de vecteurs generateurs de baculovirus

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Publication number
EP1534845A1
EP1534845A1 EP03750434A EP03750434A EP1534845A1 EP 1534845 A1 EP1534845 A1 EP 1534845A1 EP 03750434 A EP03750434 A EP 03750434A EP 03750434 A EP03750434 A EP 03750434A EP 1534845 A1 EP1534845 A1 EP 1534845A1
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Prior art keywords
gene
baculovirus
influenza
modified
virus
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English (en)
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Reingard Grabherr
Andrej Egorov
Kanokwan Poomputsa
Wolfgang Ernst
Christian Kittel
Hermann Katinger
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Polymun Scientific Immunbiologische Forschung GmbH
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Polymun Scientific Immunbiologische Forschung GmbH
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    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is in the fields of recombinant gene technology and vaccine development and relates to a method for rescuing recombinant viruses, particularly recombinant orthomyxoviruses, using baculovirus as a gene shuttle for delivery of desired viral genes to a mammalian host cell for expression. It further relates to a modified baculovirus genome as used in said method, and further to the manufacture of vaccines using said method for rescuing recombinant viruses.
  • influenza virus One of the most important and best characterized members of the orthomyxoviruses is influenza virus.
  • the genome of influenza virus is single stranded RNA of negative polarity that comprises approximately 13500 nucleotides.
  • the genome of both influenza A and B viruses is distributed into eight different segments, coding for nine structural proteins and two non-structural proteins (NS1, NS2) with regulatory functions.
  • the first 12 nucleotides at the 3' end and the first 13 nucleotides at the 5' end of each genome segment of influenza A viruses are conserved in all eight segments. Due to the segmented nature of the viral genome genetic reassortment takes place when mixed infection of two different influenza strains occurs (i.e. avian influenza A strains can reassort with human influenza A viruses).
  • RNP ribonucleoprotein complex
  • PB1, PB2, PA the three viral polymerase proteins
  • NP nuclear protein
  • Reverse genetics for negative-strand viruses first developed for influenza A virus by Luytjes et al (3), has significantly contributed to our biological understanding of these pathogens and still gives rise to improving the methods of vaccine development.
  • the manipulation of the influenza genome by reverse genetics has led to the possibility of preparing stable laboratory strains which contain site-directed mutations that can be designed in a way such that they confer attenuation in one or more genes.
  • establishment of NS gene engineering methods for influenza A virus allowed to discover the main function of the NS1 protein as interferon antagonist and permitted obtaining genetically stable attenuated strains thatcan be used as a live influenza vaccine (4, 5).
  • Baculovirus as Gene Delivery Tool Current methods of expressing genes in a mammalian cell include the use of viral vectors, such as those which are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis viruses, or adeno-associated viruses.
  • Other methods of expressing an exogenous gene in a mammalian cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand- DNA conjugates, calcium phosphate precipitation, and methods which utilize a liposome- or polycation-DNA complex.
  • viruses of the family Baculoviridae have been used to express exogenous genes in insect cells and mammalian cells.
  • Baculoviridaes viruses of the family Baculoviridae (commonly referred to as baculoviruses) have been used to express exogenous genes in insect cells and mammalian cells.
  • One of the most studied baculoviruses is the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV).
  • AcMNPV Autographa californica multiple nuclear polyhedrosis virus
  • the transduction of mammalian cells by baculovirus particles bears no safety risk for either product or operator.
  • the expression of the exogenous genes is transient, unless a G418-resistance-gene is included and selected for by the addition of G418.
  • the transient expression of a mammalian expression cassette present on the baculovirus genome is gene dosage-dependent, corresponding to the number of virus particles used in
  • the invention relates to the construction of one or more baculovirus clones that are able to express one or more, optionally all, genes necessary for the rescue of infectious influenza virus.
  • the baculovirus is a nuclear polyhedrosis virus and most preferably is AcMNPV.
  • Baculoviruses possess several advantages that make them an attractive method for influenza genetic manipulation. Multiple genes and large inserts (up to 38 kb) can be introduced into the baculovirus genome so that one or more or even all influenza genes can be delivered to Vero cells at once.
  • the baculoviruses are inherently unable to replicate in mammalian cells and are therefore considered biologically safe for therapeutic applications for humans.
  • baculovirus Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is an insect virus with a large double- stranded circular DNA genome.
  • AcNPV baculovirus Autographa calif ornica nuclear polyhedrosis virus
  • baculoviruses have primarily been used to over-express proteins from insect derived host cells, they have been demonstrated to serve as a powerful vector to carry various genes into a variety of mammalian cell types at high frequencies (8).
  • baculovirus as an influenza gene delivery vector. Recombinant baculovirus harboring a truncated influenza A NS gene, resulting in a 38 amino acid long NS1 protein, was constructed.
  • GFP green fluorescent protein
  • subsequent infection of baculovirus-transduced Vero cells with a temperature-sensitive (ts) influenza helper virus whose NS gene segment was found to be responsible for the ts phenotype, and subsequent passages in Vero cells at 40 °C resulted in the isolation of an influenza virus carrying the baculovirus-derived truncated NS1 gene.
  • This virus designated ⁇ NS38, did not grow on Madin-Darby canine kidney (MDCK) cells or in eggs, but gave almost equal titers, compared to the wild type variant, in interferon deficient Vero cells (4).
  • the invention further relates to a baculovirus genome that contains one or more uni- or bidirectional expression cassettes encoding at least one natural or modified gene or gene segment or a combination of a least one natural and at least one modified gene or gene segment, of an orthomyxo virus.
  • the invention relates to Baculovirus genome wherein said at least one natural or modified gene or gene segment is from influenza A or B virus.
  • the invention relates to a baculovirus genome wherein said at least one natural or modified gene or gene segment encodes viral mRNA or viral genomic RNA, or both viral mRNA and viral genomic RNA.
  • the invention relates to a baculovirus genome wherein said at least one natural or modified gene or gene segment is flanked by restriction sites that allow for introduction and exchange of said gene or gene segment by cleavage of said restriction sites and subsequent direct ligation of said gene(s) or gene segment(s) into the baculovirus genome. These restriction sites are unique in baculovirus.
  • the invention relates to a baculovirus genome wherein said at least one natural or modified gene or gene segment comprises a modified NS gene segment of influenza virus encoding an NS1 protein of only 38 amino acids in length ( ⁇ NS38), wherein the 38 amino acids are the N-terminal starting sequence of the NS1 protein.
  • the big C-terminal truncation of the NS1 amino acid sequence renders the influenza virus interferon inducing and, simultaneously, interferon sensitive, which has in effect that the resulting virus is attenuated (at least due to its susceptibility to IFN activity) and thus a promising candidate for making live vaccines.
  • the invention relates to a baculovirus genome wherein said at least one natural or modified gene or gene segment comprises a modified NS gene segment of influenza virus, a hemagglutinin (HA) and/or a neuraminidase (NA) gene segment of an epidemic influenza strain, and all remaining gene segments of the epidemic strain or of one or more other influenza wildtype or laboratory master strains.
  • said at least one natural or modified gene or gene segment comprises a modified NS gene segment of influenza virus, a hemagglutinin (HA) and/or a neuraminidase (NA) gene segment of an epidemic influenza strain, and all remaining gene segments of the epidemic strain or of one or more other influenza wildtype or laboratory master strains.
  • the AcMNPV genome comprises 134 kbp of double-stranded DNA.
  • the capacity of foreign gene insertion into the baculovirus genome has been demonstrated to be at least 40 kbp of DNA. Therefore, it becomes possible to stably integrate more than one gene-expression cassette into the AcNPV genome, e.g. target gene plus reporter gene, protein complexes consisting of multiple subunits.
  • the baculovirus genome can carry an exogenous promoter positioned for expression of the exogenous gene.
  • Preferred promoters include the cytomegalovirus early promoter (CMV) and the human ribosomal RNA polymerase I promoter (Pol-I).
  • the baculovirus genome may also carry a polyadenylation signal, the bovine growth hormone poly adenylation signal (BGH-Poly A) or the hepatitis delta virus genomic ribozyme (HDN), a self-cleaving R ⁇ A sequence which ensures the correct 3 ' end.
  • BGH-Poly A bovine growth hormone poly adenylation signal
  • HDN hepatitis delta virus genomic ribozyme
  • the baculovirus is engineered to contain as a heterologous insertion into its genome at least one", preferably several, optionally all genes or gene segments of influenza virus, or of any other orthomyxovirus.
  • Influenza A as well as B viruses possess a segmented single-stranded, negative-strand R ⁇ A genome.
  • All R ⁇ A-segments must be produced as negative strand viral genomic R ⁇ A (vR ⁇ A) and four of these R ⁇ A segments must be present either as functional proteins or as transcripts encoding proteins that have to be functional for transcription and replication of all segments.
  • the different Orthomyxovirus genes may be engineered in any combination or number into a single baculovirus genome or may be divided into two or more portions and distributed to two or more separate baculovirus genomes.
  • a baculovirus containing just one foreign gene e.g. a modified version of the ⁇ S1 gene, may be used for transduction of Nero cells and rescue of infectious virus, e.g. influenza virus or any orthomyxovirus, may be carried out via superinfection with helper virus.
  • co- transduction of several baculoviruses, each 1 containg one or more different orthomyxovirus genes, may be carried out in order to rescue infectious orthomyxovirus particles, preferably without the aid of helper virus.
  • transduction refers to carrier-mediated transfer of genetic material to a living host cell, wherein the carrier is an enveloped virus and the genetic material is contained within the capsid of said virus, and wherein the transfer is accomplished in a way such that upon contact of the virus with the host cell the capsid containing the genetic material penetrates the host cell and migrates to the host cell's nucleus, while the viral envelope remains outside the host cell.
  • the baculovirus used according to the present invention is such a viral carrier.
  • transfection refers to the transfer, usually carrier-mediated transfer, of naked RNA or DNA, e.g. plasmid RNA or DNA vector constructs, to host cells.
  • Suitable carriers are, for instance, RNP (ribonucleoprotein complex) or lipids (e.g. Lipofectin ® ).
  • infection refers to the exposure of host cells to natural or modified viruses and the subsequent delivery of viral RNA or DNA to the host cell using natural pathways, including, as the case may be, penetration of the host cell by the whole virus.
  • the term applies to the delivery of influenza virus RNA gene segments to a host cell, e.g. a Nero cell, using an influenza wildtype or helper virus.
  • a baculovirus-delivered, truncated NS1 gene from influenza A virus ( ⁇ NS38-gene) was expressed in Vero cells and transcribed vRNA was successfully incorporated into influenza virus particles.
  • RT-PCR with PR8 and helper virus-specific primers was performed after plaque purification (Fig. 2).
  • a mixture of the truncated ⁇ NS38-vRNA and helper virus NS-vRNA was detected after two selective passages at 40°C. After several plaque purification steps, a homogenous virus population could be isolated and the ⁇ NS38 gene was confirmed by nucleotide sequence analysis.
  • Recombinant ⁇ NS38 virus showed similar growth patterns in interferon-deficient Vero cells as compared to the 25 A- 1 helper virus (a reassortant virus containing the NS segment form the cold-adapted influenza strain A/Leningrad/134/47/57 and the remaining genes from influenza A/PR/8/34) but was fully restricted from growth on MDCK cells or in eggs.
  • the baculovirus was shown for its effective delivery of foreign genes into Vero cells. Transduction efficiency of the recombinant baculovirus at different MOIs into Vero cells was evaluated, using GFP as a reporter gene. FACS analysis revealed that the Vero cell transduction was concentration-dependent (FIG.2B). More than 90% of the cells were transduced when recombinant baculovirus was used at MOI 5000, as evidenced by their expression of GFP 24 to 48 hours after transduction. Although high numbers of baculovirus particles may be required, this method is considered feasible and significantly superior to the afore-mentioned methods known in the art, since baculoviruses can be easily generated and grown to high titers in insect cells.
  • the truncated NS1 protein of this virus consisting of only 38 N-terminal amino acids, is responsible for the attenuated phenotype in mice, which is based on the finding that ⁇ NS38 virus has an interferon inducing but also an interferon sensitive phenotype.
  • some or all genes of the desired recombinant Orthomyxovirus are inserted into the genome of a single baculovirus and thereafter transduced into Vero cells for expression of the viral genes and for assembly and release of the desired recombinant Orthomyxoviruses.
  • some or all of the genes or gene segments, respectively, of the desired recombinant virus are divided into two portions and distributed into the genomes of two baculoviruses and thereafter co-transduced into Vero cells for expression and assembly of recombinant virus.
  • the term "desired” virus shall mean any virus having a pre-selected phenotype, preferably having an attenuated (e.g. temperature-sensitive, interferon-sensitive, or otherwise replication-inhibited) phenotype, and/or a pre-selected set of genes or gene segments.
  • the desired virus be an influenza virus that comprises a modified NS gene segment responsible for interferon induction and/or for interferon sensitivity such as the ⁇ NS38 gene segment (coding for a full NEP and a truncated NS1 protein of only 38 amino acids length), a hemagglutinin (HA) and/or a neuraminidase (NA) gene segment of an epidemic influenza virus strain, and some or all of the remaining gene segments of one or more other influenza wildtype or laboratory master strains.
  • a modified NS gene segment responsible for interferon induction and/or for interferon sensitivity such as the ⁇ NS38 gene segment (coding for a full NEP and a truncated NS1 protein of only 38 amino acids length), a hemagglutinin (HA) and/or a neuraminidase (NA) gene segment of an epidemic influenza virus strain, and some or all of the remaining gene segments of one or more other influenza wildtype or laboratory master strains.
  • HA hemag
  • the present invention also encompasses an embodiment, wherein a part or the entirety of genes or gene segments, respectively, of a desired recombinant virus, are split into more than two portions and distributed to more than two baculovirus genomes, each baculovirus genome receiving just one of said more than two portions of genes or gene segments, respectively.
  • the recombinant baculoviruses produced by this method and differing from one another by the inserted portion of foreign genes or gene segments are then co-transduced to the host cells, preferably Vero cells, for expression of the viral genes and assembly and release of the desired recombinant virus particles. Both methods have in common that they do not require the use of a helper virus to rescue the desired recombinant virus.
  • helper viruses wherein the transduced genes or gene segments replace the corresponding genes or gene segments of the helper virus.
  • this method requires the employment of a helper virus and a suitable subsequent selection system for rescuing the desired recombinant reassorted viruses (i.e. the modified helper viruses), it is still more efficient than all other reverse genetic methods known to us so far.
  • Fig. 1 Construction of recombinant NS expressing cassette.
  • the NS gene was cloned in reverse orientation between human polymerase I promoter (Poll) and hepatitis delta virus terminator (HDV) to transcribed NS vRNA.
  • a multiple stop codon cassette was introduced, resulting in a truncated, 38 amino acid NS1 protein and a full size, 121 aa NEP.
  • the non- translated part between the stop codon and the splicing signal of NEP was deleted.
  • Fig. 2 Expression of the reporter gene (GFP) in infected Vero cells depending upon baculovirus concentration.
  • GFP reporter gene
  • Vero cells were transduced with baculovirus carrying a GFP reporter gene at different MOIs. Using FACS analysis, the percentage of GFP expressing cells was measured (i.e. reflecting cells are transduced with baculovirus).
  • Fig. 3 Detection of the recombinant influenza NS gene.
  • Virus-containing supernatant obtained from Vero cell culture after transduction with baculovirus and subsequent infection with influenza helper virus, was passaged twice at 40°C and analyzed by RT-PCR, using two sets of primers which selectively detect only wild-type (lane 1) or only recombinant, truncated gene (lane 2)
  • the supernatant comprises a mixture of helper virus 25 A- 1 and rescued ⁇ NS38 virus (Fig. 3 A).
  • Fig. 3B After several plaque purification steps a pure recombinant ⁇ NS38 virus was isolated (Fig. 3B).
  • Influenza virus 25 A- 1 is a temperature-sensitive (ts) reassortant containing the NS gene from the cold-adapted strain A/Leningrad/134/47/57 (H2N2) and the remaining genes from the A/Puerto Rico/8/34 (H1N1) virus.
  • the NS gene is responsible for the temperature-sensitive phenotype of the helper virus.
  • the virus was amplified in Vero cells in serum free medium.
  • the NS gene was cloned between human ribosomal polymerase I promoter (Poll) and hepatitis delta virus genomic ribozyme (HDV), a self-cleaving RNA sequence which ensures the correct 3' end.
  • a multiple stop codon cassette was introduced after the nuclear localization signal 1 (NLS1) of the NS1 protein.
  • the non-translated part between this stop codon cassette and the splicing signal of the nuclear export protein (NEP) was deleted and resulted in a truncated NS1 protein, consisting of only 38 amino acids but a fully intact NEP protein (6).
  • the NS construct, designated ⁇ NS38 was inserted into a baculovirus transfer vector (pCMVGFP), a modified pBacPA (Clonetech), expressing the green fluorescence protein (GFP) under control of the cytomegalovirus promoter (CMV) and the bovine growth hormone polyadenylation site (BGH) as reporter-gene.
  • pCMVGFP- ⁇ NS38 a baculovirus transfer vector
  • CMV cytomegalovirus promoter
  • BGH bovine growth hormone polyadenylation site
  • the recombinant plasmid (pCMVGFP- ⁇ NS38) and linear AcNPY viral DNA (Baculogold, PharMingen) were co-transfected into Spodoptera frugiperda (S ⁇ ) insect cells and recombinant AcCMVGFP- ⁇ NS38 baculoviruses were generated.
  • the recombinant AcCMVGFP- ⁇ NS38 baculoviruses were purified by
  • Vero cells were transduced with recombinant AcCMVGFP- ⁇ NS38 baculovirus, carrying a GFP reporter gene at different MOIs using the procedure described above. After 48 hours, the transduced cells were trypsinized and resuspended in PBS. GFP expressing cells were analyzed by a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • Influenza B virus genes of strain B Lee 40 were released from the cloning vector backbone pHW2000 by restriction digests and inserted into the baculovirus transfer vector pBacPAK ⁇ . More particularly, the three polymerase genes and the gene encoding the nuclear protein of influenza B virus (B Lee 40), were taken from the constructs pHW-Lee-PBl, pHW-Lee-PB2, pHW-Lee-PA, pHW-Lee-NP, which are based on pHW2000 (Hoffmann et al, 2000), kindly received from Thorsten Wolff (Robert Koch-Institute, Berlin) and were inserted each into a baculovirus transfer vector pBacPAK8 (Clontech).
  • the genes are expressed bidirectionally, transcription (i.e. generation) of the messenger RNA is driven by the cytomegalovirus (CMV) early promoter and terminated by the bovine growth hormone (BGH) polyadenylation signal. Transcription (i.e. generation) of genomic RNA is driven by the human polymerase I (Pol I) promoter and terminated by the hepatitis delta virus (HDV) genomic ribozyme, a self-cleaving RNA sequence to ensure the correct 3' end (Neuman et al., 1994). This set of proteins is required for reverse transcription of influenza virus genes and genes that are flanked by influenza virus non-coding sequences, where the nuclear complex binds and initiates transcription.
  • CMV cytomegalovirus
  • BGH bovine growth hormone
  • HDV hepatitis delta virus
  • Recombinant baculovirus clones were generated via homologous recombination using the above described transfer vectors, and amplified and purified by sucrose gradient centrifugation.
  • a plasmid was constructed, which contained the green fluorescent protein (gfp) in minus sense orientation, flanked by influenza B virus non-coding sequences.
  • the gene encoding minus sense RNA of green fluorescent protein is expressed under control of the human polymerase I promoter (Poll) and terminated by the hepatitis delta virus genomic ribozyme (HDV).
  • the RNP-complex comprising the four influenza B virus proteins (PA, PBl, PB2, NP) binds to the non-coding region and initiates transcription. Only when all four influenza virus proteins comprised in the nuclear complex (PBl, PB2, NP, PA) are functionally active hence successfully delivered and expressed, gfp mRNA is being produced and gfp expression can be detected by a fluorescent signal.
  • HEK293 Freestyle cells Invitrogen were transduced with a pool of the four baculovirus clones, using a total moi of either 50, 500, 1000, 5000 or 7500.
  • cells 48 hours post transduction, cells were transfected with 1 ⁇ g of purified plasmid containing the minus sense gfp gene flanked by influenza B virus non-coding sequences. Analysis was performed 24 hours post transfection by fluorescence activated cell sorting.
  • gfp was functionally expressed in the HEK293 freestyle cells due to transcriptional activity of the influenza B virus nuclear complex which formed and assembled within the cells upon delivery and expression of the four different baculovirus clones.
  • the principle underlying the present invention as claimed herein was fully confirmed. Particularly, it was proven that the present invention allows not only for successful delivery of several different genes of interest to a mammalian host cell using the baculovirus-based transduction system but also for successful expression of the delivered genes.
  • each clone comprising another influenza virus gene or gene segment can likewise result in successful transcription and expression of the delivered genes and subsequent assembly of the expressed proteins to form virus particles.
  • RNA polymerase I mediates expression of influenza virual RNA molecules. Virology, 202, 477-479.

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Abstract

L'invention porte sur une méthode selon laquelle on utilise un système produisant des baculovirus comme alternative au sauvetage du virus recombiné de la grippe. A cet effet, on a utilisé un vecteur de baculovirus recombiné contenant des promoteurs actifs de cellules de mammifère et des terminateurs de transcription pour fournir à des cellules Véro des segments du gène NS de la grippe. En plus des segments du gène NS de la grippe le vecteur du baculovirus recombiné contient une GFP (cassette d'expression génique) permettant de vérifier l'efficacité de la transduction des cellules Véro. Plus de 90 % des cellules Véro exprimaient des GFP 24 à 48 heures après la transduction. Après avoir infecté par des virus auxiliaires de la grippe des cellules transductées par des baculovirus, on a pu isoler la descendance de la grippe recombinée porteuse du gène NS de synthèse dérivant du baculovirus. L'invention porte également sur des baculovirus chimères et leur génome chimère et sur un procédé de fabrication de vaccins recombinés de la grippe recourant à la méthode de transduction de baculovirus.
EP03750434A 2002-09-04 2003-08-23 Obtention de virus recombines de la grippe a l'aide de vecteurs generateurs de baculovirus Withdrawn EP1534845A1 (fr)

Applications Claiming Priority (3)

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US40769502P 2002-09-04 2002-09-04
US407695P 2002-09-04
PCT/EP2003/009375 WO2004022760A1 (fr) 2002-09-04 2003-08-23 Obtention de virus recombines de la grippe a l'aide de vecteurs generateurs de baculovirus

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EP1534845A1 true EP1534845A1 (fr) 2005-06-01

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EP (1) EP1534845A1 (fr)
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WO (1) WO2004022760A1 (fr)

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KR20120039047A (ko) * 2009-07-31 2012-04-24 노파르티스 아게 역유전학 시스템
US8513006B2 (en) 2010-09-14 2013-08-20 University of Pittsburgh—of the Commonwealth System of Higher Education Tetravalent influenza vaccine and use thereof
FR2978456B1 (fr) * 2011-07-27 2015-02-20 Genethon Systeme baculoviral pour l'expression d'un vecteur de therapie genique

Non-Patent Citations (1)

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Title
See references of WO2004022760A1 *

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WO2004022760A1 (fr) 2004-03-18

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