WO2010046335A1 - Production du virus influenza par génétique inverse dans les cellules per.c6 en conditions asériques - Google Patents

Production du virus influenza par génétique inverse dans les cellules per.c6 en conditions asériques Download PDF

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WO2010046335A1
WO2010046335A1 PCT/EP2009/063650 EP2009063650W WO2010046335A1 WO 2010046335 A1 WO2010046335 A1 WO 2010046335A1 EP 2009063650 W EP2009063650 W EP 2009063650W WO 2010046335 A1 WO2010046335 A1 WO 2010046335A1
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cells
virus
per
influenza
influenza virus
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Wouter Koudstaal
Jerôme H.H.V. CUSTERS
Jort Vellinga
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Crucell Holland B.V.
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • 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
    • C12N2760/16151—Methods of production or purification of viral material
    • 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
    • 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
    • C12N2760/16151—Methods of production or purification of viral material
    • C12N2760/16152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16251—Methods of production or purification of viral material
    • 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
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16251—Methods of production or purification of viral material
    • C12N2760/16252—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the invention relates to the field of cell culture and influenza virus production. More particularly, it concerns improved methods for the culturing of cells and production of influenza virus particles therefrom by reverse genetics.
  • Influenza A and B viruses cause annual epidemics in the human population worldwide.
  • subtypes of influenza A viruses previously not circulating among humans occasionally cross from animals reservoirs and cause a pandemic.
  • Vaccination is the primary means to control influenza-associated morbidity and mortality.
  • the genomes of influenza A and B viruses consist of eight negative-sense RNA segments. When two viruses infect a single cell, viruses with new combinations of genomic segments, called reassortants, may arise. This property is used for the production of most influenza vaccines to combine the antigenic properties of target circulating strains with favorable growth characteristics and safety profile of laboratory strains.
  • the egg-adapted A/Puerto Rico/8/34 (PR8) strain and cold-adapted strains such as A/ Ann Arbor/6/60 and B/ Ann Arbor/ 1/66 are typically used to provide the RNA segments that encode the internal proteins of the virion.
  • such reassortants are derived by co infecting embryonated hens' eggs with the circulating strain and the backbone strain, and subsequent screening of the progeny for viruses that have the haemagglutinin (HA) and neuraminidase (NA) segments of the first and at least those internal segments of the second that confer a high growth phenotype. This process is cumbersome, time consuming and cannot be done with viruses that are incapable of egg growth.
  • plasmid-based reverse genetics technology [1-5] which allows for the generation of influenza viruses entirely from cloned viral cDNA. This was initially achieved for A/WSN/33 virus by cotransfection of eukaryotic cells with 8 plasmids encoding the viral sense RNA under control of a human RNA Polymerase I (Pol I) promoter and 4 to 9 plasmids encoding mRNA for different viral proteins under the control of an RNA Polymerase II (Pol II) promoter [6, 7].
  • Poly I human RNA Polymerase I
  • Poly II RNA Polymerase II
  • Vera cells have been used most extensively for the recovery of vaccine strains by reverse genetics [2, 4, 12-15]. However, this process is hampered by the low plasmid transfection efficiency of these cells [16-18], and recovered virus requires amplification in a more permissive substrate like eggs, MDCK cells, or chicken embryo fibroblast cells.
  • RNA polymerase I Due to the species specificity of the human RNA polymerase I, plasmid systems using the human pol I promoter can not be used efficiently on MDCK cells, however a rescue system based on a T7 polymerase vector [19], and canine RNA Pol I-driven systems that allow for reverse genetics on MDCK cells were reported recently [16, 19, 20].
  • a candidate human H7N1 vaccine produced on PER.C6 cells that was based on a reverse genetics derived reassortant has been described previously [15]. However, this virus had been generated using a co-culture method with Vera cells and chicken embryonic fibroblasts.
  • methods for rescuing influenza virus after cell transfection were performed in the presence of serum.
  • Such a method would be of great interest both from a technical and a regulatory perspective. Indeed this last aspect is of substantial importance for the production of a vaccine against influenza.
  • Such methods have not yet been described, presumably because they are very demanding from a technical viewpoint. It is an object of the present invention to provide such methods. Summary of the invention
  • PER.C6 cells are highly permissive for influenza virus infection [21], and it was known that such cells can efficiently be trans fected with one or two plasmids. Here we have assessed the suitability of these cells for the generation of influenza viruses by reverse genetics. We demonstrated in the present invention that PER.C6 cells could be used for trans fection, rescue, infection and propagation of a variety of influenza viruses. 6:2 reassortants were successfully rescued using 12 or
  • the invention provides a method for producing influenza virus, comprising the steps of: a) transfecting cells with nucleic acid comprising cDNA that can be transcribed into viral genomic RNA and cDNA encoding the polymerase and nucleoprotein of influenza virus in expressible format, and b) rescuing said influenza virus, characterized in that said cells are PER.C6 cells in suspension and that said transfecting is carried out under serum free free conditions.
  • the complete method is performed under serum free, more preferably under completely animal component free conditions.
  • fresh medium containing PER.C6 cells is added to the trans fected cells, within 8 hours after trans fection.
  • said nucleic acid is contained in 10-12 separate nucleic acid molecules, such as plasmids.
  • said transfection is performed by electroporation. In another embodiment, said transfection is performed using chemically defined transfection reagents.
  • It is also an aspect of the invention to provide a method for producing a vaccine against influenza virus comprising the method described above and further comprising: c) infecting PER.C6 cells in suspension with said rescued influenza virus for propagation of the virus under animal component free conditions, d) harvesting said virus and/or viral components, and preparing a vaccine.
  • FIG. 1 Comparison of rescue of recombinant A/PR/8/34 virus from 293-T cells in co-culture with MDCK cells (CC) and PER.C6 cells (PER.C6). Cells were transfected with 12 plasmids for the rescue of A/PR/8/34 and supernatants were harvested 1 to 7 days after transfection. Virus titres were determined by plaque assay on MDCK cells. The dotted line indicates the limit of detection.
  • FIG. 2. Robustness of rescue of recombinant A/PR/8/34 virus from PER.C6 cells.
  • Cells were transfected with 10 plasmids based on a PER.C ⁇ -adapted A/PR/8/34 virus, supernatants were harvested 7 days later and virus titres were determined by plaque assay on MDCK cells. As a negative control, cells were transfected with all but one (Poll NP. IRES. PA) plasmids. Data from 6 independent rescue experiments are presented. Horizontal bars indicate geometric mean titres. The dotted line indicates the limit of detection.
  • FIG. 3 Protective efficacy of inactivated vaccine based on a reassortant virus rescued from and produced on PER.C6 cells.
  • a challenge control a group of 8 mice was mock vaccinated with DOE.
  • mice Four weeks after the second vaccination (day 49), blood samples were collected and mice were challenged intranasally with 25 LD50 of A/HK/156/97 and observed daily for clinical signs and weighed daily until 14 days after infection.
  • Graphed are HI titres (A), Kaplan-Meier survival curves (B), and median clinical scores (C) for each group. Each circle in A represents the geometric mean of duplicate HI measurements per animal. Horizontal bars indicate geometric mean titres per group. The dotted line indicates the limit of detection.
  • the present invention describes a new method for the production of influenza virus.
  • the method relies on the use of the same cell for transfection, rescue, infection and propagation of the virus.
  • the cell used in the present invention is a suspension PER.C6 cell and all steps of the method are carried out in serum free and preferably animal component free conditions.
  • the method of transfecting cells with nucleic acid in order to rescue influenza virus also called reverse genetics, is well understood in the art and involves the use of standard molecular virology techniques, such as those described in, for example, Neumann et all 999 [7] and other references mentioned therein.
  • Reverse genetics systems have been established to artificially produce members of several virus families such as the Orthomyxoviridae, which comprises the influenza virus.
  • the method for generating negative-sense RNA viruses like influenza from nucleic acid encompasses the following general steps.
  • Cells are transfected with nucleic acid comprising cDNA that can be transcribed into viral genomic RNA and with nucleic acid comprising cDNA that is transcribed into viral polymerase and nucleoprotein.
  • reverse genetics systems for rescuing influenza virus from cDNA have been extensively described in the art, e.g. in [6-8] and WO 00/60050, and are thus available to the skilled person.
  • the viral genomic RNA which is composed of eight vRNAs, is encoded from the inserted nucleic acid under the control of a cellular (from the host cell) RNA polymerase I.
  • the (preferably human) RNA polymerase I promoter is operably linked to the cDNA of the influenza virus genome segment, linked to a RNA polymerase I transcription termination sequence.
  • RNA polymerase I a nucleolar enzyme, synthesizes ribosomal RNA, which like influenza virus RNA, does not contain 5' cap or 3' poly(A) structures.
  • RNA polymerase I transcription of a cDNA construct containing an influenza viral cDNA, flanked by a RNA polymerase I promoter and terminator sequences results in influenza vRNA synthesis.
  • Eight plasmids each containing one cDNA segment that can be transcribed into viral genomic RNA are used in such a system.
  • the viral RNA polymerase I which consists of three subunits (PBl, PB2 and PA), and for the nucleoprotein (NP), cDNAs encoding these in expressible format are also introduced into the cell during transfection [7].
  • the PBl, PB2, PA and NP encoding cDNAs are preferably under control of a RNA polymerase II promoter, for instance the CMV promoter, and a transcription termination sequence. Transcription by RNA polymerase II and subsequent translation in the cell results in the presence of the viral PBl, PB2, PA and NP proteins in the cell, for instance from four plasmids each containing one of the PBl, PB2, PA and NP protein encoding cDNAs operably linked to RNA polymerase II promoters. Subsequently the eight vRNAs are replicated and transcribed into viral genomic RNA by the viral polymerase and the NP proteins.
  • a RNA polymerase II promoter for instance the CMV promoter
  • a transcription termination sequence Transcription by RNA polymerase II and subsequent translation in the cell results in the presence of the viral PBl, PB2, PA and NP proteins in the cell, for instance from four plasmids each containing one of the
  • the nucleic acid that is transfected into the cells is contained in at least 8, for instance 10-12, separate nucleic acid molecules.
  • the nucleic acid comprising the cDNA that is transfected into the cells can for instance be in the form of plasmid DNA, cosmid DNA, linear DNA fragments such as PCR fragments or restriction enzyme digests, and the like. In preferred embodiments the nucleic acid is in the form of plasmids.
  • trans fecting PER.C6 cells with eight RNA polymerase I plasmids encoding all vRNAs, together with protein expression plasmids for the viral polymerase and NP proteins (yielding a total of 12 plasmids) results in the rescue of influenza virus.
  • the coding regions of the PB2, PBl, PA, and NP proteins are cloned into two expression plasmids: one carrying PBl and PB2 separated by an internal ribosomal entry site (IRES) sequence and another carrying NP and PA also separated by an IRES sequence.
  • IRES internal ribosomal entry site
  • a modified RNA polymerase I system can be used in which both negative-sense vRNA and positive-sense mRNA can be synthesized from the same template.
  • the number of required plasmids can be reduced to for example 8 (e.g. [9-11]). Further reduction of the number of plasmids may be possible by combining certain of the nucleic acid molecules as described above on a lower number of plasmids. Alternatively, additional plasmids may be added beyond the 12 plasmids referred to above (see e.g. WO 00/60050).
  • transfection is performed with chemically defined transfection reagents.
  • Said reagent can be lipid based or non-lipid based.
  • lipid based transfection reagent are commercially available, e.g. FuGENE reagent from Roche or Lipofectamine 2000CD from Invitrogen.
  • Lipid based transfection reagents according to the invention encapsulate DNA in the form of a liposome which fuses with the cell and releases DNA into the cell.
  • the transfection efficiency of PER.C6 cells using lipid-based and non-lipid based transfections depends on the type of medium that is being used during the transfection.
  • transfection is performed with electroporation.
  • Electroporation can be performed with for example the Nucleofector technology from Amaxa or with the electroporation system from Biorad.
  • PER.C6 cells for the purpose of the present application shall mean cells from an upstream or downstream passage or a descendent of an upstream or downstream passage of cells as deposited under ECACC no. 96022940 on 29 February 1996.
  • PER.C6 cells are described in US patent 5,994,128 and in [26]. These cells are very suitable for influenza virus production to produce cell-based influenza vaccines, since they can be infected and propagate the virus with high efficiency, as for instance described in [21] and WO 01/38362.
  • PER.C6 cells are capable of growing in suspension in the absence of serum, as for instance described in [31].
  • Serum free according to the present invention means that the medium used for cell growth, transfection and infection lacks whole serum as an ingredient.
  • the complete method is carried out in the absence of any components that have been directly derived from animals, such as serum or serum-components, eggs, etc.
  • the method of producing a vaccine is performed in animal component free conditions. This means that the medium used for cell growth, transfection and infection is devoid of any animal derived components. Moreover, any additives supplemented to the medium during the process of vaccine production are also free of animal-derived components.
  • the absence of animal components in the process of making said influenza vaccine offers a process that is more controlled and safe. For this reason, PER.C6 cells, which are fully characterized human cells and which were developed in compliance with GLP/GMP are very well suited for the use in vaccine manufacturing.
  • fresh medium with (untransfected) PER.C6 cells is added to the trans fected cells in order to increase virus recovery.
  • This step was advantageously found to increase the efficiency of the system. In certain embodiments, this step is performed within about 8 hours after transfection, preferably within 4 hours after transfection. In certain embodiments, this step is performed within about 2 hours, and in certain embodiments within 1 hour, 30 minutes, 10 minutes or immediately after transfection.
  • the fresh medium used for this step is preferably free from animal components.
  • the culture medium contains a suitable protease to cleave influenza HA into HAl and HA2.
  • a suitable protease is trypsin.
  • the protease can be derived from animal source, e.g. pig blood, but is preferably of non-animal origin, e.g. provided by recombinant expression in the absence of animal components. Such proteases are commercially available (see e.g. WO 01/38362, where Accutase was used). Recombinant trypsin is for instance sold under the name TrypLE select by Invitrogen, and this also works for the present invention.
  • the present invention also provides a method for the production of a vaccine against influenza. This method comprises the method for the production of influenza as described above, followed by the next additional steps. After the rescue of influenza virus from PER.C6 cells, the virus is placed in contact with (non-transfected) cells to allow the virus to infect said cells and to propagate.
  • the method for the infection of PER.C6 cells is well known to the skilled person and is described in for example WO 01/38362 and [21].
  • this step takes place automatically after virus has been produced in the first step, if for instance trypsin is present in the culture medium, since PER.C6 cells are still present and will be infected with released virus particles. For industrial purposes it is however preferred to add further cells and culture medium, simply to scale up after the transfection stage to the point where sufficient virus is produced to generate material for vaccination. During this step, amplification of the number of virus particles takes place.
  • this step is preferably performed in PER.C6 cells that are cultured in suspension in the absence of serum, and preferably under conditions that are completely free of components directly derived from animals. As indicated above, this step entails known methods that have been previously described (WO 01/38362 and [21]). This step can suitably be performed in bioreactors, for instance at scales of between 1-20.000 liters, which scale can easily be adjusted to the demand for the vaccine.
  • This method thus provides a process for making influenza virus by reverse genetics, wherein the same cells are used for transfection, rescue, infection and propagation of the virus.
  • the virus or components thereof are harvested from the cell culture. This can be done by routine methods, which are as such known to the skilled person.
  • Suitable viral components for use in influenza vaccines are for instance the heamagglutinin (HA) and neuraminidase (NA) proteins.
  • the virus produced and released in the cell culture medium can be separated from the cellular biomass by conventional methods, such as centrifugation or ultrafiltration, and harvested.
  • the cell culture contains virus particles in the supernatant and complete virus particles as well as virus proteins associated with the cellular biomass.
  • virus particles from the supernatant are harvested and the virus and/or viral components associated with the cellular biomass can also be isolated.
  • the virus found in the cells of the cellular biomass is released from the cells by lysis.
  • the cells can be lysed by conventional methods, such as treating the cells with a detergent, treating with heat, sonication, French-press or other cell lysing methods.
  • the viruses released from the cells can be harvested, concentrated and purified.
  • Viral components still associated with the cellular biomass or with cell fragments can be extracted from the cells or cell fragments by chemical or mechanical methods known in the art. These methods include ultrasonication or treatment with an appropriate detergent to release the virus antigen from the cell or cell fragments, especially from the membrane.
  • the viral components, including viruses, isolated from the cellular biomass then, can be further subjected to a purification step including for instance separation on a sucrose-gradient, adsorption to a chromatography column, washing and eluting the purified virus or viral components.
  • the chromatography column used can for instance be selected from ion-exchange chromatography, affinity-chromatography or size filtration chromatography.
  • Influenza vaccines are based on live virus or inactivated virus, and inactivated vaccines can be based on whole virus, 'split' virus or on purified surface antigens (including hemagglutinin and neuraminidase).
  • Haemagglutinin (HA) is the main immunogen in inactivated influenza vaccines.
  • split vaccines are obtained by treating virions with detergents to produce subvirion preparations, using methods such as the 'Tween-ether' splitting process.
  • Split vaccines generally include multiple antigens from the influenza virion. Vaccines based on purified surface antigens are also called subunit vaccines. After inactivation, for example by formaldehyde or ⁇ -propio lactone, an inactivated influenza virus vaccine is obtained. Alternatively, or after inactivation, a whole virus can be fragmented in subunits to provide a subunit vaccine.
  • the purified influenza virus or viral component is formulated into a pharmaceutical composition.
  • a pharmaceutical composition comprising the influenza virus and at least a pharmaceutically acceptable excipient.
  • Such a composition may be prepared under conditions known to the skilled person, and in certain embodiments is suitable for administration to humans.
  • compositions comprising influenza virus and aluminium are for instance disclosed in EP 1113816.
  • the invention may employ pharmaceutical compositions comprising the influenza virus and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered.
  • Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A.
  • the purified inactivated influenza virus or immunogenic parts thereof preferably are formulated and administered as a sterile solution.
  • Sterile solutions are prepared by sterile filtration or by other methods known per se in the art.
  • the solutions are then lyophilized or filled into pharmaceutical dosage containers.
  • the pH of the solution generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to 7.5.
  • the influenza virus or immunogenic parts thereof typically are in a solution having a suitable pharmaceutically acceptable buffer, and the solution of influenza virus may also contain a salt.
  • stabilizing agent may be present, such as albumin.
  • detergent is added.
  • the vaccine may be formulated into an injectable preparation. These formulations contain effective amounts of influenza virus or immunogenic parts thereof, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
  • influenza vaccine can for instance be performed intramuscularly, intradermally or intranasally, all according to methods known in the art.
  • the 12 plasmid system for the rescue of A/PR/8/34 has been described before [23].
  • the 10 plasmid system was constructed as follows: after eleven passages of A/PR/8/34 (PR8) on PER.C6 cells, RNA was isolated using the QIAamp ® Viral RNA Mini Kit (Qiagen) and cDNA of all 8 segments was made with the Superscript one- step RT-PCR with Platinum Taq kit (Invitrogen) using universal degenerate primers AGTAGAAACAAGGNNNTTTTT (SEQ ID NO: 1) and AGCRAAAGC AGG (SEQ ID NO. 2).
  • Each segment was subsequently amplified with Pwo DNA polymerase (Roche) using segment-specific primers (sequences available upon request) and cloned into a PCR®4 blunt TOPO® vector (Invitrogen).
  • a DNA fragment containing a human Pol I promoter and a mouse terminator sequence separated by a linker containing two Sapl restriction sites generating different overhang sequences was synthesized by Geneart (Regensburg, Germany) and cloned in pPCR-Script vector.
  • the Sapl site in the backbone was removed by deletion of a Tfil segment, giving rise to a vector named pST which is essentially the same as plasmid pPolISapIT described by Subbarao et al. [14].
  • the cloned PR8 segments were amplified with specific primers containing Sapl sites at the 5'- ends and directionally cloned into the pST vector.
  • PB2, PBl, PA, and NP of PR8 were cloned into two expression plasmids: one carrying PBl and PB2 separated by an internal ribosomal entry site (IRES) sequence of EMCV (Clontech) and another carrying NP and PA also separated by an IRES sequence.
  • IRES internal ribosomal entry site
  • pCDNA3/neo Invitrogen
  • the plasmids used for the rescue of A/Victoria/3/75, B/Beijing/87 and A/Chicken/Italy/ 1347/99 have been described elsewhere [15, 24, 25].
  • the genome segments encoding the haemagglutinin (HA) and neuraminidase (NA) of A/Panama/2007/99 (H3N2), NYMC X- 16 IB, NIBRG 14 (H5N1) and the NA segment of IVR-116, were amplified by RT-PCR and cloned into pST as above, using RNA isolated directly from the reconstituted NIBSC material.
  • RNA from A/HK/156/97 was isolated from allantoic fluid of infected eggs and the HA and NA segments were cloned as above, except that the HA cleavage site was modified as described previously [14].
  • DMEM Eagle's medium
  • AEM Adeno Expression Medium
  • VP-SFM AGTTM VP-SFM AGTTM
  • MDCK cells were obtained from the American Type Culture Collection and were maintained in DMEM containing 10% FBS at 37°C in 10% CO 2 atmosphere.
  • PER.C6 cells were seeded at a density of 1 x 10 6 cells in a 6-well plate in DMEM supplemented with 10% FBS and 10 mM MgCl 2 and incubated o/n at 37 0 C, 10% CO 2 . The next day, medium was replaced with DMEM with 2% FBS, 4 mM L- glutamine and 10 mM MgCl 2 . Twenty five ⁇ l Lipofectamine 2000 (Invitrogen) was mixed with 250 ⁇ l Optimem (Invitrogen) and incubated at room temperature.
  • PER.C6 cells were seeded at a density of 12 x 10 6 cells in a 75 cm 2 flask in DMEM supplemented with 10% FBS and 10 mM MgCl 2 and incubated o/n at 37 0 C, 10% CO 2 .
  • 120 ⁇ l Lipofectamine (Invitrogen) was mixed with DMEM (end volume 300 ⁇ l) and incubated for 5 minutes at room temperature.
  • 300 ⁇ l DMEM containing 24 ⁇ g DNA (2.4 ⁇ g of each of the 10 plasmids) was added and incubated at RT for 40 minutes.
  • Inactivated vaccines were prepared by infecting suspension PER.C6 cells in virus growth medium with wild type A/HK/156/97 and reassortant rgPR8-H5Nl- HK/97 viruses at an MOI of 0.001. Five days post infection, 10 U/ml Benzonase (Sigma) was added together with 2 mM MgCl 2 (final concentration). After incubation for 30 minutes at 37 0 C, the cell suspension was centrifuged for 20 minutes at 3500 RPM and supernatant was harvested.
  • the standardized OIE chicken pathogenicity test was conducted at the Central Veterinary Institute (Lelystad, The Netherlands) at BSL 3. Ten 6-week-old chickens were inoculated intravenously with 0.1 ml of a 1/10 dilution of the egg-grown A/HK/156/97 virus, or an equal dose (determined by TCID50) of the reassortant rgPR8-H5Nl-HK97 virus, and observed over a period of 10 days.
  • the in vitro pathogenicity index (IVPI) was determined according to the OIE's manual of diagnostic tests and vaccines for terrestrial animals.
  • mice Female 7-week-old SPF BALB/c mice (Charles River Laboratories) were used in all experiments. For infections, animals were anesthetized with ketamin/xylazin intraperitoneally and inoculated intranasally with 50 ⁇ l infectious virus diluted in PBS.
  • the LD50 of A/HK/156/97 was determined by inoculation groups of 20 mice with 2, 2.5, 3, 3.5, 4, 4.5, or 5 log TCID 50 of egg grown A/HK/156/97 virus diluted in 50 ⁇ l PBS. One group of 10 mice received an equal volume of PBS. The mice were weighed and observed daily for signs of disease and mortality for 14 days. To evaluate the degree of protection from lethal challenge, vaccinated mice were infected as above 4 weeks after the second vaccination with 25 LD50 of A/HK/156/97 virus and weighed and observed daily for signs of disease and mortality for 14 days.
  • Virus titres in allantoic fluids and culture supernatants were determined by either plaque assay or TCID50 on MDCK cells as described previously [21, 24].
  • the LD50 of A/HK/156/97 in BALB/c mice was determined from the serial dilutions by Probit analysis and found to be 3.1 log TCID 50 (95% CI 2.7-3.5).
  • the log transformed HI titres in serum samples from mice that received vaccines based on HK/97 or PR8-HK/97 were compared with an independent t-test. After challenge the vaccinated mouse groups were compared to the control group for differences in survival proportions using Chi-square tests.
  • Statistical analyses were performed using SPSS 15.0 (SPSS Inc., USA).
  • PER.C6 cells grown adherently in static culture were trans fected with 12 plasmids routinely used for the rescue of PR8 virus from 293T/MDCK co- culture. Daily harvests of cell supernatant were tested for the presence of virus by plaque assay on MDCK cells.
  • a virus rescue using the same plasmids and an established method of 293T/MDCK coculture [24] was performed.
  • Fig. 1 illustrates a typical rescue performed in PER.C6 cells or by the co-culture method.
  • Recombinant PR8 virus was detected at day 2 post transfection using the co-culture method and similar titres of rescued virus were obtained at day 3 after transfection using PER.C6 cells. Peak recovery of virus was obtained 6 days after transfection.
  • a 10 plasmid system for the rescue of a PER.C6-adapted PR8 virus was constructed in which the coding regions of PBl and PB2 and those of NP and PA are separated by an IRES sequence and cloned between a CMV promoter and BGH polyadenylation site.
  • Transfection of adherent PER.C6 cells with these 10 plasmids consistently resulted in high viral yields after seven days.
  • the geometric mean of the titres obtained in 6 independent experiments was 2.5 x 10 7 pfu/ml (Fig. 2).
  • influenza viruses with genome compositions reflective of vaccine strains (Table 1). All viruses were successfully recovered.
  • the viral titres in supernatants of transfected cultures obtained in individual rescues varied from 2.3 x 10 8 pfu/ml for PR8, to less than 10 (below the detection limit of the plaque assay) for PR8-HK/97, a reassortant with the internal segments of A/PR/8/34 and HA and NA segments of A/Hong Kong/156/97. Nevertheless, this virus was recovered after inoculating a fresh culture of PER.C6 cells with this supernatant.
  • PER.C6 cells can grow in suspension in the absence of serum and such cells from qualified Master Cell Banks are used for the propagation of influenza viruses. From practical and regulatory points of view, it is beneficial to generate vaccine seed viruses and propagate these using cells from a single cell bank. Moreover, it would be highly beneficial to carry out the complete process in suspension cells in the absence of serum. Such a system was established herein. Influenza viruses were rescued by direct transfection of suspension PER.C6 cells using a Nucleofector device (Amaxa).
  • influenza virus a 6:2 reassortant A/PR/8/34 + A/Viet Nam/1194/2004 HA & NA (H5N1)
  • rescue influenza virus a 6:2 reassortant A/PR/8/34 + A/Viet Nam/1194/2004 HA & NA (H5N1)
  • the supernatant containing the resulting influenza virus was shown to have a titer of 2.5 x 10 7 pfu/ml.
  • suspension PER.C6 cells can be transfected with 10 plasmids and can be used for the generation of influenza viruses.
  • PR8-HK/97 The PR8-HK/97 virus was used as a model to study the protective efficacy of a vaccine based on a reassortant that was both rescued from and propagated on PER.C6 cells. For safety reasons, the HA cleavage site had been modified to attenuate the virus. Indeed, unlike the wild type A/HK/156/97 virus, PR8-HK/97 was unable to form plaques in a monolayer of MDCK cells in the absence of trypsin. Furthermore, the pathogenicity of both viruses was compared using the standardized OIE chicken pathogenicity test.
  • the IVPI of A/HK/156/97 is 2.9 out of a possible maximum of 3.0, indicating that this virus is highly virulent in chickens.
  • the IVPI of PR8- HK/97 is 0.0 as inoculation with this virus did not result in any signs of infection, indicating that this virus is completely apathogenic in chickens.
  • the reassortant PR8- HK/97 virus was expanded on PER.C6 cells, inactivated with BPL, and concentrated by tangential flow filtration.
  • mice was immunized twice (on day 0 and 21) subcutaneously with 1.6 ⁇ g HA of PR8-HK/97 adjuvanted with double oil emulsion (DOE) and challenged four weeks later (day 49) by intranasal inoculation of 25 LD50 of A/HK/156/97. Clinical signs and weights were recorded daily until 14 days after infection.
  • DOE double oil emulsion
  • a group of 8 mice was immunized as above with 1.6 ⁇ g HA of a similarly prepared vaccine based on wild type A/HK/156/97.
  • As challenge control mice received DOE alone.
  • Figure 3A shows the HI antibody titres in individual serum samples collected immediately prior to challenge.
  • mice Mock vaccinated mice all had a titre below the limit of detection and were assigned a value of 10.
  • the geometric means of the HI titres in serum of mice that received vaccines based on wtHK/97 or PR8-HK/97 were 698 and 453, respectively. These titres were not significantly different (p 0.15).
  • Reverse genetics is already the method of choice for the generation of seed viruses for vaccines against potentially pandemic strains and may be used for seasonal vaccines in the future.
  • a prerequisite for the use of reverse genetics in vaccine production is the use of a suitable cell line.
  • the PER.C6 cell line has been extensively documented and cell banks were prepared and characterized according to FDA and European Medicines Agency (EMEA) guidelines.
  • Rescued viruses include PR8-based reassortants containing the HA and NA segments of avian influenza subtypes H5N1 and H7 (identified by the WHO as most likely to cause a pandemic [29]), A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (HlNl), A/Wisconsin/67/2005 and also an influenza B virus. Transfections were performed with unidirectional plasmid systems comprising of 12 and 10 plasmids, and 10 out of 10 viruses were obtained directly in supernatant fluids.
  • A/PR/8/34 which is traditionally used to confer a high-growth phenotype in eggs [30]. Although this virus replicates well on PER.C6 cells, other viruses may have even more favorable growth characteristics on this substrate and could be used as backbone strain. Recombinant A/Victoria/3/75 and B/Beijing/87 viruses (consisting of all original gene segments of these viruses) have also been rescued from PER.C6 cells using the reverse genetics approach described herein, demonstrating that reverse genetics on PER.C6 cells is not limited to A/PR/8/34 and reassortants based on this strain.
  • recombinant influenza viruses can be generated in suspension PER.C6 cells cultured in chemically defined growth medium, devoid of any animal-derived component.
  • This improved methodology offers advantages over existing methods in terms of process simplicity (only cells from one qualified Master Cell Bank required for both the generation of the vaccine seed strain and vaccine production) and compliance to regulatory guidelines.
  • Fouchier RA A reverse-genetics system for Influenza A virus using T7 RNA polymerase. J Gen Virol 2007 Apr;88(Pt 4): 1281-7. [20] Wang Z, Duke GM. Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells. Virol J

Abstract

La présente invention concerne un procédé de production du virus influenza, comprenant les étapes consistant à : transfecter les cellules avec de l'acide nucléique comprenant de l'ADNc qui peut être transcrit en ARN et en ADNc génomiques viraux codant pour la polymérase et la nucléoprotéine du virus influenza dans un format exprimable, et récupérer ledit virus influenza. Le procédé est caractérisé en ce que lesdites cellules sont des cellules PER.C6 en suspension et que le procédé est réalisé en conditions asériques.
PCT/EP2009/063650 2008-10-21 2009-10-19 Production du virus influenza par génétique inverse dans les cellules per.c6 en conditions asériques WO2010046335A1 (fr)

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WO2011048560A1 (fr) * 2009-10-20 2011-04-28 Novartis Ag Procédés génétiques inverses améliorés pour le sauvetage de virus
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