WO2005113756A1 - Methode - Google Patents

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Publication number
WO2005113756A1
WO2005113756A1 PCT/EP2005/005262 EP2005005262W WO2005113756A1 WO 2005113756 A1 WO2005113756 A1 WO 2005113756A1 EP 2005005262 W EP2005005262 W EP 2005005262W WO 2005113756 A1 WO2005113756 A1 WO 2005113756A1
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WIPO (PCT)
Prior art keywords
virus
influenza
eggs
vaccine
influenza virus
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PCT/EP2005/005262
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English (en)
Inventor
Emmanuel Hanon
Elisabeth Neumeier
Florence Nozay
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Glaxosmithkline Biologicals S.A.
Saechsisches Serumwerk Dresden Branch Of Smithkline Beecham Pharma Gmbh & Co Kg
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Priority claimed from GB0410872A external-priority patent/GB0410872D0/en
Priority claimed from GB0426737A external-priority patent/GB0426737D0/en
Application filed by Glaxosmithkline Biologicals S.A., Saechsisches Serumwerk Dresden Branch Of Smithkline Beecham Pharma Gmbh & Co Kg filed Critical Glaxosmithkline Biologicals S.A.
Publication of WO2005113756A1 publication Critical patent/WO2005113756A1/fr

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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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention relates to a method to produce influenza virus or influenza virus antigens in embryonated avian eggs.
  • the invention relates to the introduction into the embryonated avian eggs of an influenza virus and any of the following components: a serine protease, such as trypsin, at least one amino acid or derivative thereof, insulin-like growth factor or type I interferon antagonist, to increase the yield of influenza virus or influenza virus antigens produced.
  • a serine protease such as trypsin
  • insulin-like growth factor or type I interferon antagonist insulin-like growth factor or type I interferon antagonist
  • Influenza virus is one of the most ubiquitous viruses present in the world, affecting both humans and livestock. The economic impact of influenza is significant.
  • the influenza virus is an RNA enveloped virus with a particle size of about 125 nm in diameter. It consists basically of an internal nucleocapsid or core of ribonucleic acid (RNA) associated with nucleoprotein, surrounded by a viral envelope with a lipid bilayer structure and external glycoproteins. The inner layer of the viral envelope is composed predominantly of matrix proteins and the outer layer mostly of the host-derived lipid material. The surface glycoproteins neuraminidase (NA) and haemagglutinin (HA) appear as spikes, 10 to 12 nm long, at the surface of the particles. While antigenic differences between type A and B influenza strains is characterized by differences in their matrix or nucleoprotein, the antigenicity of influenza A subtypes is defined by differences in their surface proteins
  • Influenza vaccines are classically trivalent and can take the form of either live attenuated vaccines or inactivated formulations. They usually contain antigens derived from two influenza A virus strains and one influenza B virus strain. A standard injectable dose in most cases contain 15 ⁇ g of haemagglutinin (HA) antigen component from each strain, as measured by single radial immunodiffusion (SRD), usually in a 0.5 ml dose (J.M.
  • Wood et al. An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330).
  • influenza virus strains to be incorporated into influenza vaccine each season are determined by the World Health Organisation in collaboration with national health authorities and vaccine manufacturers.
  • influenza vaccines are either inactivated influenza vaccines or live attenuated influenza vaccines.
  • Inactivated influenza vaccines comprise one of three types of antigen preparation: inactivated whole virus, sub-virions where the membrane of the virus particles is disrupted with detergents or other reagents to solubilise the lipid envelope (so-called 'split' vaccine) or subunits vaccines (produced either recombinantly or purified from disrupted viral particles) in particular HA and NA subunit vaccine.
  • Virosomes are another type of inactivated influenza formulation, where the viral membrane is reconstituted following disruption. These inactivated vaccines are generally given parenterally, in particular intramuscularly (i.m.), but some virosome-based formulations and live attenuated vaccines have been administered intranasally.
  • the currently commercially available influenza vaccines remain the intramuscularly administered split vaccine, whole virus vaccine, subunit injectable or virosomes vaccines.
  • the split and subunit vaccines are prepared by disrupting the virus particle, generally with an organic solvent or a detergent, and separating or purifying the viral proteins to varying extents.
  • Split vaccines are prepared by fragmentation of whole influenza virus, either infectious or inactivated, with solubilizing concentrations of organic solvents or detergents and subsequent removal of the solubilizing agent and some or most of the viral lipid material.
  • Split vaccines generally contain contaminating matrix (M) protein and nucleoprotein (NP) and sometimes lipid, as well as the membrane envelope proteins (such as HA and NA).
  • Split vaccines will usually contain most or all of the virus structural proteins although not necessarily in the same proportions as they occur in the whole virus.
  • Subunit vaccines consist essentially of highly purified viral surface proteins, haemagglutinin (HA) and neuraminidase (NA), which are the surface proteins responsible for eliciting the desired virus neutralising antibodies upon vaccination.
  • Matrix and nucleoproteins are either not detectable or barely detectable in subunit vaccines.
  • the requirements are different for adult populations (18-60 years) and elderly populations (>60 years): a) the following serological assessments should be considered for each strain in adult subjects, aged between 18 and 60, and at least one of the assessments should meet the indicated requirements: • number of seroconversions or significant increase in antihaemagglutinin antibody titre > 40%; • mean geometric increase > 2.5 • the proportion of subjects achieving an HI titre ⁇ 40 or SRH titre >25 mm 2 should be > 70% b) the following serological assessments should be considered for each strain in adult subjects aged over 60, and at least one of the assessments should meet the indicated requirements: • number of seroconversions or significant increase in antihaemagglutinin antibody titre > 30%; • mean geometric increase > 2.0 • the proportion of subjects achieving an HI titre ⁇ 40 or SRH titre >25 mm 2 should be > 60%
  • influenza vaccine For an influenza vaccine to be commercially useful it will not only need to meet those standards, but also in practice it will need to be at least as immunologically efficacious as the currently available vaccines. It will need to be administered using a procedure that is reliable and straightforward for medical staff to carry out. Furthermore, it will also need to be produced by an acceptable process and will of course need to be commercially viable in terms of the amount of antigen and the number of administrations required.
  • the yield of influenza virus in eggs is critical to influenza vaccine production and availability, but the contribution of specific genes to the growth properties of influenza A and B viruses is not well understood.
  • WHO aims to increase routine vaccination of all people at high risk - including at least 50% of the Elderly population - by 2006 (Clayton, et al. Nature Medicine, 2003, 9, 375). This implies that current production capacity of Influenza vaccine has to substantially increase.
  • increasing yield of influenza virus production and/or influenza virus antigens in egg is a strategy of choice.
  • IFNs type I interferons
  • type I interferons can be inhibited by using antagonist such as soluble receptor (B18R protein from Vaccinia virus) (Alcami A, Symons JA, Smith GL J Virol. 2000 Dec; 74 (23): 11230-9).
  • antagonist such as soluble receptor (B18R protein from Vaccinia virus) (Alcami A, Symons JA, Smith GL J Virol. 2000 Dec; 74 (23): 11230-9).
  • Figure 6 Flowchart for the selection of suitable amino acid combinations (choice of the amino acids, optimization of their concentrations, application to other strains)
  • Figure 7 Best predicted combination (higher than 16 263, i.e. higher than the 95% confidence interval upper limit of control mean obtained in the experiment of Example V) for a given number of amino acids.
  • the present inventors have discovered that introducing certain additives in embryonated avian eggs had a positive impact not only on growth of the embryo, but also could lead to an increase of the yield of virus production and/or influenza viral antigen production, in particular to an increase in haemagglutinin production.
  • the additives are selected from the list consisting of at least one amino acid or derivative thereof, a serine protease, insulin-like growth factor, type-1 interferon antagonist, and a combination of one of more of these compounds.
  • a method for producing influenza virus in embryonated avian eggs comprising introducing into said eggs, an influenza virus and at least another component wherein said component is selected from the list consisting of: a serine protease, at least one amino acid or derivative thereof, insulin-like growth factor, type 1 interferon antagonist, and a combination of one or more thereof.
  • Said 'at least one amino acid' may be selected from selected from the list in Table 1 , or may be any derivative of said amino acid.
  • a method for producing enhanced levels of haemagglutinin in embryonated avian eggs comprising introducing into said eggs, an influenza virus and at least another component wherein said component is selected from the list consisting of: a serine protease, at least one amino acid or derivative thereof, insulin-like growth factor, type 1 interferon antagonist, and a combination of one or more thereof.
  • the invention provides a method for producing an influenza virus vaccine comprising the steps of:
  • step (d) formulating the virus of step (c) with a pharmaceutically acceptable carrier or excipient.
  • the invention provides method for prophylaxis of influenza infection or disease in a subject which method comprises administering to the subject a vaccine produced according to the method claimed herein.
  • the present invention provides for a method for producing influenza virus or antigens thereof in embryonated avian eggs, comprising introducing into said eggs, an influenza virus and at least another component wherein said component is selected from the list consisting of: a serine protease, an amino acid or derivative thereof, insulin-like growth factor, type 1 interferon antagonist, and a combination of one or more thereof.
  • the claimed method produces enhanced levels of virus and/or influenza virus antigen yield obtained from embryonated eggs so treated as assessed by comparison with the virus and/or influenza virus antigen yield obtained from similar eggs which are handled in the same manner as the treated eggs but which are not subjected to the same procedure of exposure or contact with any of the additives or components listed above.
  • the virus or virus antigen yield obtained from eggs treated according to the process claimed is higher than the virus or virus antigen yield obtained from similar eggs (so-called the 'control eggs') which have been in contact with a standard solution, i.e. a solution similar to that used for the treated eggs, but which is devoid of any of the described components.
  • the standard solution may be water or a buffered solution such as PBS for example.
  • a method for producing influenza virus in embryonated avian eggs comprising introducing into said eggs, an influenza virus and at least another component wherein said component is selected from the list consisting of: a serine protease, at least one amino acid or derivative thereof, insulin-like growth factor, type 1 interferon antagonist, and a combination of one or more thereof, wherein said eggs produce enhanced levels of influenza virus and/or antigen thereof compared to control eggs which have not been in contact with said additive(s).
  • influenza virus yield obtained from the treated eggs is higher than that obtained from the control eggs. It may be evaluated on the allantoic fluid after clarification (see example I, section 1.3) at different steps during the purification process, as assessed by the haemagglutinin titer (HA titer) and/or total protein content of purified virus at each process step.
  • HA titer haemagglutinin titer
  • influenza virus antigen(s) yield obtained from the treated eggs is higher than that obtained from the control eggs.
  • said influenza virus antigen is haemagglutinin (HA).
  • HA haemagglutinin
  • Another suitable mean to measure the effect of the additive may be performed by multiplying the number of harvested eggs by the protein content of harvested eggs. This product is referred to as 'response' in Example V of the document.
  • higher yield or enhanced level of virus and/or virus antigen is meant a ratio of said virus yield or said virus antigen yield from the treated eggs relative to that from the control eggs higher than 1 as assessed by any suitable method as detailed herein above.
  • the level of virus and/or virus antigen for the control may advantageously be a control mean when several controls have been run simultaneously, as it gives a higher precision to the ratio.
  • a ratio of at least 1.2:1 (treated: control or control mean) is obtained.
  • Particularly suitable is a minimum ratio of 1.5:1.
  • a ratio of at least 2:1 , a ratio of at least 3:1 , a ratio of at least of 5: 1 is obtained.
  • the introduction of the component according to the invention may be done by any suitable way, such as inoculation with a needle or injection using a standardized apparatus.
  • the avian embryonated eggs are usually 9 to 12 days old, typically 10 or 1 1 days old. Suitably they are chicken eggs.
  • the component is introduced in embryonated avian eggs, preferably concomitantly with the influenza virus, typically in the form of a solution, preferably as part of the same solution so as only one inoculation is needed.
  • the embryonated eggs may be injected with the component according to the invention, and then subsequently infected with influenza virus, or vice versa.
  • Both the virus and the other component may be introduced separately or in the form of a mixture either in the allantoic liquid, or in the amniotic liquid, or in other egg compartment or directly in the embryo.
  • the inoculation takes place in the allantoic fluid.
  • influenza virus includes any viruses capable of causing a febrile disease state in animals (including human) characterised by respiratory symptoms, inflammation of mucous membranes and often systemic involvement.
  • the method according to the present invention is especially useful in the production of a variety of influenza viruses, including equine, human, porcine, and avian strains.
  • Influenza virus include any strains, subtypes and types, particularly those recommended by WHO, many of which are available from clinical specimens and from public depositories such as ATCC, ECCAC and BIBSC. Typically type A, type B and/or type C influenza viruses are contemplated in the present invention. Influenza strains which can be consistently propagated to high titers are especially contemplated.
  • reassortant influenza viruses and influenza viruses exhibiting chime c viral surface molecules such as chimeric HA and/or NA surface molecules are contemplated (such as those described in WO 94/29439 for example).
  • Recombinant influenza virus strains are also contemplated (such as those described in WO 91/03552 for example).
  • said at least one amino acid is selected from the list of amino acids given in Table 1 or is a derivative of said amino acid.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen amino acids are present.
  • the minimum number of amino acids can be routinely determined experimentally. In particular, the number of amino acids will be determined so as to lead to a fixed minimum higher yield or enhanced level of virus and/or virus antigen compared to the yield obtained in the absence of said amino acids. What is meant by 'higher yield' is explained herein above.
  • suitable solutions of amino acids are found in Table 9 which exhibits results obtained with tested solutions, and in Table 12 which exhibits solutions predicted by the model detailed in example V.
  • the solution(s) can be chosen by reference to the ratio treated: control or control mean which is desirable to be achieved (i.e. higher than 1) as illustrated in the last column of both tables, and taking into account experimental parameters such as the influenza virus strain, the amino acid concentration in the solution, etc. It may also be advantageous to select, between two solutions giving a similar virus or virus antigen yield, the solution(s) which have only a limited number of amino acids, so as to minimize the interaction between the amino acids to be included in said solution(s).
  • solutions are possible candidates giving a virus yield for treated: control higher than 1.
  • Solutions 2 and 46 give similar yields (ratio treated: control of 1.44) but solution 46 might be preferred as it only contains 1 amino acid by comparison to solution 2 which contains 8 amino acids. If however a maximum yield (for example a ratio treated: control of above 4) is desirable then it may be desirable to select that or those solution(s) which allow(s) achieving said higher yield (such as solution 42, with a ratio of 4.6) even though more amino acids (10 for solution 42) may need to be included in the solution. It is to be understood that the same procedure can routinely be applied to different experimental conditions (change of virus strain, change in amino acid concentrations) and may results into different solution profiles.
  • the serine protease is selected from the list consisting of: trypsin-like protease (such as rProtease, from Invitrogen, 3175 Staley Road, Grand Island, NY 14072, supplier catalogue number 02-106), porcine or bovine trypsin, or recombinant origin (such as Trypzean, a recombinant trypsin produced in corn, Prodigen, 101 Gateway Boulevard, Suite 100 College Station, Texas 77845. Manufacturer code : TRY).
  • trypsin-like protease such as rProtease, from Invitrogen, 3175 Staley Road, Grand Island, NY 14072, supplier catalogue number 02-106
  • porcine or bovine trypsin or recombinant origin (such as Trypzean, a recombinant trypsin produced in corn, Prodigen, 101 Gateway Boulevard, Suite 100 College Station, Texas 77845. Manufacturer code : TRY).
  • a suitable protease is a serine protease such as recombinant trypsin or trypsin-like protease.
  • trypsin is used in the form of a solution, and used at an amount of 6.69 USP per egg.
  • a typical range is from 0.067 to 670 USP, preferably 0.669 - 66.96 USP (standard unit for trypsin activity).
  • serine proteases are from the family of subtilisin-like proteases, such as chymotrypsin, thermolysin, pronase or subtilisin A which are particularly suitable.
  • a suitable Insulin-like growth factor type is IGF-I used at a quantity of 0.4 - 40 ⁇ g per egg, typically at an amount of 4 - 20 ⁇ g per egg, preferably at an amount of 4 ⁇ g or 20 ⁇ g per egg.
  • a suitable type I interferon antagonist is B18R protein from Vaccinia virus (Alcami A, Symons JA, Smith GL J Virol. 2000 Dec; 74 (23): 11230-9) used at a amount of 1 to 1000 ⁇ g per egg, suitably at an amount from 5 to 100, or 100 ⁇ g per egg. Functionally equivalent type I interferon antagonist are also contemplated.
  • the protease, the amino acid, the insulin-like growth factor or the type-1 interferon antagonist are used in the form of an aqueous solution.
  • the amino acid solution for inoculation into the egg exhibits a pH from at least 6 to maximum 11.
  • a pH range of 7 to 1 1 in particular a pH range of 8 -10 is particularly suitable.
  • An amino acid solution with a pH ranging from 9 to 10, Suitably with a pH at around 9.5 is especially contemplated.
  • a pH at 'around of 9.5' is meant to include any pH ranging from 9.25 to 9.75 which will be understood to be contemplated.
  • Combinations of the various components described above are also contemplated in the present invention. In particular combinations of two or more of the components are sought. Typically such a combination will comprise at least one amino acid in admixture with a serine protease, in particular with trypsin. Another combination will comprise at least one amino acid in admixture with either IGF-1 , or type I interferon antagonist or in admixture with both IGF-1 and type I interferon antagonist.
  • the contact with the component according to the invention may be carried out for periods of time varying from an hour to up to several days, suitably from one hour to 96 hours, in particular from 12 hours to 72 hours, typically for 24 hours or alternatively for 48 hours.
  • a typical, although not limiting, concentration of the component is as depicted in Table 1 (third column) for the amino acids, when present, and/or 6.69 USP for the trypsin, and a typical period of incubation in the embryonated eggs is 48 hours, and for production, as a rule it is 72 hours.
  • Enhanced levels of influenza virus may be determined by measuring the increased yield of haemagglutinin (HA).
  • HA haemagglutinin
  • 'increased yield of HA' is meant an amount of HA, as determined by a quantitative assay, including SRD assay, which is higher than that obtained in the absence of injection in the embryonated egg of any of the component selected from the list comprising: a serine protease, at least one amino acid or derivative thereof, insulin-like growth factor, type 1 interferon and a combination of one or more thereof.
  • the yield, and the increase in the yield, of influenza antigen may be determined by measuring the haemagglutinin titer per ml (or in HA units per ml) using suitable methods (Kendal, A.P., Pereira, M.S. and Skehel, J. 1982. Concepts and procedures for laboratory-based influenza surveillance, distributed by the viral diseases unit, WHO, Geneva, or the WHO Collaborating Center for the Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA 30333, U.S.A.).
  • the haemagglutinin antigen component from each influenza strain may be measured by single radial immunodiffusion (SRD) (J.M.
  • Wood et al. An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981 ) 317-330).
  • Also provided by the present invention is a method for producing an influenza virus or virus antigen vaccine, said method comprising the steps of:
  • step (d) formulating the virus or antigen thereof of step (c) with a pharmaceutically acceptable carrier or excipient.
  • virus or antigen thereof may be harvested from the amniotic fluid of the injected embryonated eggs, from the embryo or from the allantoic fluid, which is particularly suitable.
  • the influenza vaccines according to the invention may be either whole, or split or subunit vaccines.
  • Split and subunit vaccines are prepared by disrupting the virus particle, generally with an organic solvent or a detergent, and separating or purifying the viral proteins to varying extents.
  • Split vaccines are prepared by fragmentation of whole influenza virus, either infectious or inactivated, with solubilizing concentrations of organic solvents or detergents and subsequent removal of the solubilizing agent and some or most of the viral lipid material.
  • Split vaccines generally contain residual matrix protein and nucleoprotein and sometimes lipid, as well as the membrane envelope proteins.
  • Split vaccines will usually contain most or all of the virus structural proteins although not necessarily in the same proportions as they occur in the whole virus.
  • Subunit vaccines on the other hand consist essentially of highly purified viral surface proteins, haemagglutinin and neuraminidase, which are the surface proteins responsible for eliciting the desired virus neutralising antibodies upon vaccination.
  • the influenza virus antigen preparation may be produced by any of a number of commercially applicable processes, for example the split flu process described in patent no. DD 300 833 and DD 211 444, incorporated herein by reference.
  • the split flu vaccine is produced according to the methods disclosed in WO 02/097072, the disclosure of which is incorporated herein by reference.
  • split flu was produced using a solvent/detergent treatment, such as tri-n-butyl phosphate, or diethylether in combination with TweenTM (known as "Tween-ether” splitting) and this process is still used in some production facilities.
  • Other splitting agents now employed include detergents or proteolytic enzymes or bile salts, for example sodium deoxycholate as described in patent no. DD 155 875, incorporated herein by reference.
  • influenza virus may be in the form of a whole virus vaccine.
  • This may prove to be an advantage over a split virus vaccine for a pandemic situation as it avoids the uncertainty over whether a split virus vaccine can be successfully produced for a new strain of influenza virus.
  • the conventional detergents used for producing the split virus can damage the virus and render it unusable.
  • this would take time, which may not be available in a pandemic situation.
  • influenza virus preparation is in the form of a purified sub-unit influenza vaccine.
  • Sub-unit influenza vaccines generally contain the two major envelope proteins, HA and NA, and may have an additional advantage over whole virion vaccines as they are generally less reactogenic, particularly in young vaccinees.
  • Sub-unit vaccines can produced either recombinantly or purified from disrupted viral particles.
  • the influenza virus preparation is in the form of a virosome. Virosomes are spherical, unilamellar vesicles which retain the functional viral envelope glycoproteins HA and NA in authentic conformation, intercalated in the virosomes' phospholipids bilayer membrane.
  • Detergents that can be used as splitting agents include cationic detergents e.g. cetyl trimethyl ammonium bromide (CTAB), other ionic detergents e.g. laurylsulfate, taurodeoxycholate, or non-ionic detergents such as the ones described above including Triton X-100 (for example in a process described in Lina et al, 2000, Biologicals 28, 95- 103) and Triton N-101 , or combinations of any two or more detergents.
  • CAB cetyl trimethyl ammonium bromide
  • other ionic detergents e.g. laurylsulfate, taurodeoxycholate
  • non-ionic detergents such as the ones described above including Triton X-100 (for example in a process described in Lina et al, 2000, Biologicals 28, 95- 103) and Triton N-101 , or combinations of any two or more detergents.
  • the preparation process for a split vaccine will include a number of different filtration and/or other separation steps such as ultracentrifugation, ultrafiltration, zonal centrifugation and chromatography (e.g. ion exchange) steps in a variety of combinations, and optionally an inactivation step eg with heat, formaldehyde or ⁇ -propiolactone or UN. which may be carried out before or after splitting.
  • the splitting process may be carried out as a batch, continuous or semi-continuous process.
  • Suitable split flu vaccine antigen preparations comprise a residual amount of Tween 80 and/or Triton X-100 remaining from the production process, although these may be added or their concentrations adjusted after preparation of the split antigen.
  • Tween 80 and Triton X-100 are present.
  • Suitable ranges for the final concentrations of these non-ionic surfactants in the vaccine dose are: Tween 80: 0.01 to 1 %, in particular about 0.1 % (v/v)
  • Triton X-100 0.001 to 0.1 (% w/v), in particular 0.005 to 0.02% (w/v).
  • a micelle modifying excipient in particular ⁇ -tocopherol or a derivative thereof as a haemagglutinin stabiliser may be used in the manufacture of an influenza vaccine.
  • the ⁇ -tocopherol is in the form of an ester, such as the succinate or acetate and in particular the succinate.
  • Suitable concentrations for the ⁇ -tocopherol or derivative are between 1 ⁇ g/ml - 10mg/ml, in particular between 10 ⁇ g/ml - 500 ⁇ g/ml.
  • the vaccine according to the invention generally contains both A and B strain virus antigens, typically in a trivalent composition of two A strains and one B strain.
  • Monovalent vaccines containing antigens from a single type or subtype of influenza virus (such as e.g. H1 N1 , H2N2, H3N2 of type A, type B and type C) may be advantageous in a pandemic situation, for example, where it is important to get as much vaccine produced and administered as quickly as possible.
  • monovalent bulks are produced and subsequently pooled together to produce the divalent or trivalent vaccine.
  • Vaccines containing reassortant influenza viruses and influenza viruses exhibiting chimeric viral surface molecules such as chimeric HA and/or NA surface molecules, as well as vaccine containing recombinant influenza virus strains are also contemplated.
  • the virus strain is associated to a pandemic outbreak or has the potential to be associated to a pandemic outbreak.
  • influenza viruses circulate that are related to those from the preceding epidemic.
  • the viruses spread among people with varying levels of immunity from infections earlier in life.
  • Such circulation over a period of usually 2-3 years, promotes the selection of new strains that have changed enough to cause an epidemic again among the general population; this process is termed 'antigenic drift'.
  • 'Drift variants' may have different impacts in different communities, regions, countries or continents in any one year, although over several years their overall impact is often similar.
  • an influenza pandemics occurs when a new influenza virus appears against which the human population has no immunity.
  • Typical influenza epidemics cause increases in incidence of pneumonia and lower respiratory disease as witnessed by increased rates of hospitalisation or mortality.
  • the elderly or those with underlying chronic diseases are most likely to experience such complications, but young infants also may suffer severe disease.
  • the influenza virus strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • Such strain may also be referred to as 'pandemic strains' in the text below. Suitable strains are, but not limited to: H5N1 , H9N2, H7N7, H2N2 and H1 N1.
  • the vaccine may be administered by any suitable delivery route, such as intramuscular, subcutaneous, intradermal or mucosal e.g. intranasal or oral. Other delivery routes are well known in the art.
  • Needleless syringes that are suitable for subcutaneous administration are for example described in the following patent families: WO 01/05454, WO 01/05453, WO 01/05452, WO 01/05451 , EP1090651 , WO 01/32243, WO 01/41840, WO 01/41839, WO 01/47585, WO 01/56637, WO 01/58512, WO 01/64269, WO 01/78810, WO 01/91835, WO 02/09796, WO 02/34317, WO 04/084977, WO 04/084975, WO 04/093944, WO 04/93947 all incorporated herein by reference. Functional equivalents of these needleless syringe are also contemplated by the present invention.
  • Intradermal vaccines may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO99/34850 and EP1092444, incorporated herein by reference, and functional equivalents thereof.
  • jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in US 5,480,381 , US 5,599,302, US
  • influenza antigen preparation for use in the invention may be selected from the group consisting of split virus antigen preparations, subunit antigens (either recombinantly expressed or prepared from whole virus), inactivated whole virus which may be chemically inactivated with e.g. formaldehyde, ⁇ -propiolactone or otherwise inactivated e.g. UN. or heat inactivated.
  • the antigen preparation is either a split virus preparation, or a subunit antigen prepared from whole virus, particularly by a splitting process followed by purification of the surface antigen.
  • Particularly suitable preparations are split virus preparations.
  • the concentration of haemagglutinin antigen for the or each strain of the influenza virus preparation is 1-1000 ⁇ g per ml, such as 3-300 ⁇ g per ml and in particular about 30 ⁇ g per ml (15 ⁇ g per 0.5 ml dose), as measured by a SRD assay. Lower doses are also contemplated in particular in the pandemics situation.
  • an immunologically effective amount of viral antigen will be used, that is an amount of antigen which induces a protective immune response.
  • the vaccine according to the invention may further comprise an adjuvant or immunostimulant such as but not limited to detoxified lipid A from any source and non- toxic derivatives of lipid A, saponins and other reagents capable of stimulating a TH1 type response.
  • an adjuvant or immunostimulant such as but not limited to detoxified lipid A from any source and non- toxic derivatives of lipid A, saponins and other reagents capable of stimulating a TH1 type response.
  • enterobacterial lipopolysaccharide is a potent stimulator of the immune system, although its use in adjuvants has been curtailed by its toxic effects.
  • LPS enterobacterial lipopolysaccharide
  • MPL monophosphoryl lipid A
  • a further detoxified version of MPL results from the removal of the acyl chain from the 3- position of the disaccharide backbone, and is called 3-O-Deacylated monophosphoryl lipid A (3D-MPL). It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O- deacylated variants thereof.
  • a suitable form of 3D-MPL is in the form of an emulsion having a small particle size less than 0.2 ⁇ m in diameter, and its method of manufacture is disclosed in WO 94/21292.
  • Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO9843670A2.
  • the bacterial lipopolysaccharide derived adjuvants to be formulated in the compositions of the present invention may be purified and processed from bacterial sources, or alternatively they may be synthetic.
  • purified monophosphoryl lipid A is described in Ribi et al 1986 (supra)
  • 3-O-Deacylated monophosphoryl or diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211 and US 4912094.
  • lipopolysaccha des have been described (Hilgers et al., 1986, Int.Arch.Allergy.lmmunol., 79(4):392-6; Hilgers er a/., 1987, Immunology, 60(1 ):141-6; and EP 0 549 074 B1).
  • a particularly suitable bacterial lipopolysaccharide adjuvant is 3D- MPL.
  • the LPS derivatives that may be used in the present invention are those immunostimulants that are similar in structure to that of LPS or MPL or 3D-MPL.
  • the LPS derivatives may be an acylated monosaccharide, which is a sub-portion to the above structure of MPL.
  • Synthetic derivatives of lipid A are also known including, but not limited to:
  • Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363- 386). Saponins are steroid or triterpene glycosides widely distributed in the plant and marine animal kingdoms. Saponins are noted for forming colloidal solutions in water which foam on shaking, and for precipitating cholesterol. When saponins are near cell membranes they create pore-like structures in the membrane which cause the membrane to burst. Haemolysis of erythrocytes is an example of this phenomenon, which is a property of certain, but not all, saponins.
  • Saponins are known as adjuvants in vaccines for systemic administration.
  • the adjuvant and haemolytic activity of individual saponins has been extensively studied in the art (Lacaille-Dubois and Wagner, supra).
  • Quil A derived from the bark of the
  • the haemolytic saponins QS21 and QS17 have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No.5,057,540 and EP 0 362 279 B1.
  • QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant lgG2a antibody response and is a particularly suitable saponin in the context of the present invention.
  • CTLs cytotoxic T cells
  • Th1 cells a predominant lgG2a antibody response
  • Other saponins which have been used in systemic vaccination studies include those derived from other plant species such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10(9):572-577, 1992).
  • An enhanced system involves the combination of a non-toxic lipid A derivative and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
  • the saponins forming part of the present invention may be separate in the form of micelles, or may be in the form of large ordered structures such as ISCOMs (EP 0 109 942 B1 ) or liposomes) when formulated with cholesterol and lipid, or in the form of an oil in water emulsion (WO 95/17210).
  • the saponins may suitably be associated with a metallic salt, such as aluminium hydroxide or aluminium phosphate (WO 98/15287).
  • a particularly potent adjuvant formulation involving QS21 and 3D-MPL in an oil in water emulsion is described in WO 95/17210 and in WO 99/11241 and WO 99/12565, and are particularly suitable formulations.
  • a vaccine comprising an influenza antigen preparation of the present invention adjuvanted with detoxified lipid A or a non-toxic derivative of lipid A, such as a monophosphoryl lipid A or derivative thereof which are particularly suitable.
  • the vaccine additionally comprises a saponin, in particular QS21.
  • the formulation additionally comprises an oil in water emulsion such as that described in WO 95/1720, WO 90/14837 and WO 93/14744.
  • the present invention also provides a method for producing a vaccine formulation comprising mixing an antigen preparation of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.
  • the vaccines according to the invention may further comprise at least one surfactant which may be in particular a non-ionic surfactant.
  • Suitable non-ionic surfactant are selected from the group consisting of the octyl- or nonylphenoxy polyoxyethanols (for example the commercially available Triton TM series), polyoxyethylene sorbitan esters (Tween TM series) and polyoxyethylene ethers or esters of general formula (I):
  • Suitable surfactants falling within formula (I) are molecules in which n is 4-24, more particularly 6-12, and typically 9; the R component is C 1-50 , in particular C 4 -C 20 alkyl and C 12 alkyl is particularly suitable.
  • Octylphenoxy polyoxyethanols and polyoxyethylene sorbitan esters are described in "Surfactant systems” Eds: Attwood and Florence (1983, Chapman and Hall). Octylphenoxy polyoxyethanols (the octoxynols), including t- octylphenoxypolyethoxyethanol (Triton X-100 TM) are also described in Merck Index Entry 6858 (Page 1162, 12 th Edition, Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3).
  • polyoxyethylene sorbitan esters including polyoxyethylene sorbitan monooleate (Tween 80 TM) are described in Merck Index Entry 7742 (Page 1308, 12 th Edition, Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Both may be manufactured using methods described therein, or purchased from commercial sources such as Sigma Inc.
  • non-ionic surfactants include Triton X-45, t-octylphenoxy polyethoxyethanol (Triton X-100), Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, Triton N-101 , Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether (laureth 9) and polyoxyethylene-9-stearyl ether (steareth 9). Triton X-100 and laureth 9 are particularly suitable. Also particularly suitable is the polyoxyethylene sorbitan ester, polyoxyethylene sorbitan monooleate (Tween 80TM).
  • polyoxyethylene ethers of general formula (I) are selected from the following group: polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • polyoxyethylene lauryl ether Alternative terms or names for polyoxyethylene lauryl ether are disclosed in the CAS registry.
  • the CAS registry number of polyoxyethylene-9 lauryl ether is: 9002-92-0.
  • Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 th ed: entry 7717, Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3).
  • Laureth 9 is formed by reacting ethylene oxide with dodecyl alcohol, and has an average of nine ethylene oxide units.
  • Two or more non-ionic surfactants from the different groups of surfactants described may be present in the vaccine formulation described herein.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80 TM) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton) X-100TM is particularly suitable.
  • Another particularly suitable combination of non-ionic surfactants comprises laureth 9 plus a polyoxyethylene sorbitan ester or an octoxynol or both.
  • Non-ionic surfactants such as those discussed above have suitable concentrations in the final vaccine composition as follows: polyoxyethylene sorbitan esters such as Tween 80TM: 0.01 to 1 %, in particular about 0.1% (w/v); octyl- or nonylphenoxy polyoxyethanols such as Triton X-100TM or other detergents in the Triton series: 0.001 to 0.1%, in particular 0.005 to 0.02 % (w/v); polyoxyethylene ethers of general formula (I) such as laureth 9: 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5% (w/v).
  • polyoxyethylene sorbitan esters such as Tween 80TM: 0.01 to 1 %, in particular about 0.1% (w/v)
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100TM or other detergents in the Triton series: 0.001 to 0.1%, in
  • the formulations of the present invention may also comprise a bile acid or a derivative thereof, in particular in the form of a salt.
  • a bile acid or a derivative thereof in particular in the form of a salt.
  • derivatives of cholic acid and salts thereof in particular sodium salts of cholic acid or cholic acid derivatives.
  • bile acids and derivatives thereof include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as glyco-, tauro-, amidopropyl-1- propanesulfonic-, amidopropyl-2-hydroxy-1-propanesulfonic derivatives of the aforementioned bile acids, or N,N-bis (3Dgluconoamidopropyl) deoxycholamide.
  • NaDOC sodium deoxycholate
  • the claimed method, use and vaccine composition relate to viruses other than influenza virus, that require or can be produced by propagation in embryonated eggs, in particular embryonated chicken eggs.
  • Suitable viruses are selected from the list consisting of: orthomyxoviridae (including influenza virus), paramyxoviridae, flaviviridae, togaviridae, rhabdovi dae and coronaviridae families.
  • Example I describes the Improved yield of influenza virus and HA in embryonated chicken eggs in three distinct experiments using a solution of amino acids, or trypsin or a combination of both.
  • Example II describes the large scale preparation of a split influenza vaccine.
  • Example III describes the Preparation of influenza virus antigen preparation for a thiomersal-free vaccine.
  • Example IV describes the SRD Method used to measure haemagglutinin content.
  • Example V describes and illustrates an experiment and a methodology for selecting suitable amino acid solutions.
  • Example I Improved yield of influenza virus and HA in embryonated chicken eggs
  • the inoculation solution contained (a) a solution of trypsin (720000 USP/L, porcine origin, BioWhitaker, catalog number BESP117E), (b) a solution of amino acids 1 a, (c) a solution of amino acids 1 b, (d) a solution of amino acids 2a, (e) a solution of amino acids 2b, (f) no additive.
  • the eggs are inoculated with 0.5 ml of the inoculum solution by hand with a needle and a syringe, but an automatic egg inoculation apparatus is equally suitable.
  • the composition of the amino acid solution is given in Table 2.
  • the ionic concentration may slightly differ from one trypsin lot to another depending on the specific activity of trypsin, and will be adjusted routinely.
  • Amino acid solutions are adapted from Ohta et al., 2001 , Poultry science 80: 1430-1436 (in particular table 1 on page 1431 ), with the following modifications
  • Amino acid solution 1 has a pH of 9.7 and osmolahty of 1550; it does not contain valine.
  • Amino acid solution 2 has a pH of 7.2 and osmolahty of 1033; it does not contain valine nor tyrosine.
  • Solutions 1 b and 2b are a fifth of solutions 1 and 2, respectively.
  • the eggs were inoculated with each solution (0.5 ml) containing Influenza virus and the component according to the invention and prepared as described hereabove, and incubated at 33°C for 48 hours. Before harvest, the embryos were killed by chilling the eggs at 4°C for 24 hours. Typically the cooling temperature may range from 2 to 8°C and the storage time at this temperature may range from 12 to 60 hours. 1.2. Harvest
  • the allantoic fluid was harvested manually (it may be suitable to use an appropriate egg harvesting machine) and clarified by low-speed centrifugation. Usually, 8 to 10 ml of crude allantoic fluid can be collected per egg.
  • Virus was pelleted by ultracent fugation at 17,500 rpm for 2 hours in a Beckman SW19 rotor through a cushion of 20% sucrose.
  • the virus pellet was resuspended in 15 ml of PBS pH 7.4 (PELLET 1) and loaded on a 15 to 55% sucrose gradient. After centrifugation the fraction containing protein was collected and diluted to 35 ml with PBS pH 7.4. After centrifugation for 60 min at 25'200 in a Beckman SW28 rotor the virus pellet was resuspended in 3 ml of PBS, pH 7.4 (PELLET 2).
  • the virus yield was evaluated on the clarified allantoic fluid and at two steps during the purification process (PELLET 1 and PELLET 2).
  • Hemagglutination titer (HA titer) and protein content of purified virus are indicators of the virus yield at each process step.
  • a 2-fold dilution series is prepared in reaction tubes by diluting 250 ⁇ l virus suspension with 250 ⁇ l PBS. 250 ⁇ l chicken red blood cells (RBC) (0.5% in PBS) are then added. The reaction is mixed and the RBCs are allowed to sediment for 1 hour at room temperature.
  • the HA titre corresponds to the inverse of the last dilution that shows complete hemagglutination.
  • Protein assay Protein was measured by photometric determination of the total protein content by BIURET-BCA (Bicinchoninic acid) assay.
  • Sample preparation was done using the Compat-Able(TM) Protein Assay Preparation Reagent Set (0023215, Pierce Chemical Company). Briefly, 50 ⁇ l of each sample was mixed with 500 ⁇ l of reagent 1 and incubated at room temperature for 5 to 10 minutes. 500 ⁇ l reagent 2 were added to each sample and mixed well. After centrifugation at 12,000 rpm for 5 min the supernatant was removed and the pellet was resuspended in 50 ⁇ l of BCA working solution. The working solution consisted of 20 ⁇ l BCA protein assay reagent B (Perbio Nr. 23224) mixed with 1 ml of BCA protein assay reagent A (Perbio Nr. 23222).
  • Exp.2 amino acid solution adapted from Ohta et al., 2001 , Poultry science 80: 1430-1436 (in particular table 1 on page 1431 ), with the following modifications: pH of 9.1 ; it does not contain valine.
  • Exp.4 and 5 solution of trypsin either three times concentrated (exp.4), or used at a third of solution of Exp.1 , respectively.
  • Exp.6 control group
  • the eggs were inoculated with 0.5 ml of a solution containing influenza A/Wyoming/3/ 2003 (H3N2) IVR-147 at a dilution of 10 "5 in PBS, 0.025 mg/jml hydrocortisone and the component according to the invention and prepared as described hereabove, and incubated at 33°C for 72 hours.
  • the embryos were killed by chilling the eggs at 4°C for 24 hours.
  • the cooling temperature may range from 2 to 8°C and the storage time at this temperature may range from 12 to 60 hours.
  • Standard inoculum mixed with an equivalent volume of a solution at pH 9.5 4. Standard inoculum mixed with an equivalent volume of a solution at pH 11
  • the eggs were inoculated with each solution (0.5 ml) containing Influenza virus at a dilution 10 "5 and the component according to the invention and prepared as described hereabove, and incubated at 33°C for 72 hours. Before harvest, the embryos were killed by chilling the eggs at 4°C for 24 hours. Typically the cooling temperature may range from 2 to 8°C and the storage time at this temperature may range from 12 to 60 hours.
  • Example II Large scale preparation of a split influenza vaccine
  • Monovalent split vaccine is prepared, at a large scale, according to the following procedure.
  • a fresh inoculum is prepared by mixing the working seed lot with a phosphate buffered saline containing gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25 ⁇ g/ml (virus strain-dependent).
  • the component according to the invention is added to the solution. 11.2. Inoculation of embryonated eggs
  • the allantoic fluid from the chilled embryonated eggs is harvested. Usually, 8 to 10 ml of crude allantoic fluid is collected per egg. To the crude monovalent virus bulk 0.100 mg/ml thiomersal is optionally added.
  • the harvested allantoic fluid is clarified by moderate speed centrifugation (range: 4000 - 14000 g).
  • Adsorption step To obtain a CaHPO 4 gel in the clarified virus pool, 0.5 mol/L Na 2 HPO 4 and 0.5mol/L CaCI 2 solutions are added to reach a final concentration of CaHPO 4 of 1.5 g to 3.5 g CaHPO 4 /litre depending on the virus strain.
  • the supernatant is removed and the sediment containing the influenza virus is resolubilised by addition of a 0.26 mol/L EDTA-Na 2 solution, dependent on the amount of CaHPO 4 used.
  • the resuspended sediment is filtered on a 6 ⁇ m filter membrane.
  • influenza virus is concentrated by isopycnic centrifugation in a linear sucrose gradient (0 - 55 % (w/v)) containing 100 ⁇ g/ml Thiomersal.
  • the flow rate is 8 - 15 litres/hour.
  • fraction 1 55-52% sucrose fraction 2 approximately 52-38% sucrose fraction 3 38-20% sucrose * fraction 4 20- 0% sucrose * virus strain-dependent: fraction 3 can be reduced to 15% sucrose.
  • fractions 2 and 3 are used for further vaccine preparation. Fraction 3 is washed by diafiltration with phosphate buffer in order to reduce the sucrose content to approximately below 6%. The influenza virus present in this diluted fraction is pelleted to remove soluble contaminants.
  • the pellet is resuspended and thoroughly mixed to obtain a homogeneous suspension.
  • Fraction 2 and the resuspended pellet of fraction 3 are pooled and phosphate buffer is added to obtain a volume of approximately 40 litres. This product is the monovalent whole virus concentrate.
  • the monovalent whole influenza virus concentrate is applied to a ENI-Mark II ultracenthfuge.
  • the K3 rotor contains a linear sucrose gradient (0.2 - 55 % (w/v)) where a sodium deoxycholate gradient is additionally overlayed. Tween 80 is present during splitting up to 0.1 % (w/v).
  • the maximal sodium deoxycholate concentration is 0.7-1.5 % (w/v) and is strain dependent.
  • the flow rate is 8 - 15 litres/hour.
  • sucrose content for fraction limits (47-18%) varies according to strains and is fixed after evaluation:
  • the split virus fraction is filtered on filter membranes ending with a 0.2 ⁇ m membrane.
  • Phosphate buffer containing 0.025 % (w/v) Tween 80 is used for dilution.
  • the final volume of the filtered fraction 2 is 5 times the original fraction volume.
  • the inactivated split virus material is concentrated at least 2 fold in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO.
  • the material is subsequently washed with phosphate buffer containing 0.025 % (w/v) Tween 80 and following with phosphate buffered saline containing 0.01 % (w/v) Tween.
  • the material after ultrafiltration is filtered on filter membranes ending with a 0.2 ⁇ m membrane.
  • the final concentration of haemagglutinin, measured by SRD (method recommended by WHO) should exceed 450 ⁇ g/ml.
  • the monovalent final bulk is stored at 2 - 8°C for a maximum of 18 months.
  • monovalent bulk from different strains are pooled.
  • Example III Preparation of influenza virus antigen preparation for a thiomersal- free vaccine
  • Monovalent split vaccine is prepared at a large scale according to the following procedure.
  • a fresh inoculum is prepared by mixing the working seed lot with a phosphate buffered saline containing gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25 ⁇ g/ml. (virus strain-dependent).
  • the allantoic fluid from the chilled embryonated eggs is harvested. Usually, 8 to 10 ml of crude allantoic fluid is collected per egg.
  • the harvested allantoic fluid is clarified by moderate speed centrifugation (range: 4000 - 14000 g).
  • Saturated ammonium sulfate solution is added to the clarified virus pool to reach a final ammonium sulfate concentration of 0.5 mol/L. After sedimentation for at least 1 hour, the precipitate is removed by filtration on depth filters (0.5 ⁇ m nominal). Alternatively the precipitate may be removed by low speed centrifugation followed by a filtration step.
  • the clarified crude whole virus bulk is filtered on filter membranes ending with a validated sterile membrane (typically 0.2 ⁇ m).
  • the sterile filtered crude monovalent whole virus bulk is concentrated on a cassettes equipped with 1000 kDa MWCO BIOMAXTM membrane at least 6 fold.
  • the concentrated retentate is washed with phosphate buffered saline at least 1.8 times.
  • influenza virus is concentrated by isopycnic centrifugation in a linear sucrose gradient (0-55 % (w/v).
  • the flow rate is 8 - 15 litres/hour.
  • fraction 1 55-52% sucrose fraction 2 approximately 52-38% sucrose - fraction 3 38-20% sucrose* fraction 4 20- 0% sucrose * virus strain-dependent: fraction 3 can be reduced to 15% sucrose.
  • fractions 2 For further vaccine preparation, either only fractions 2 is used or fraction 2 together with a further purified fraction 3 are used.
  • Fraction 3 is washed by diafiltration with phosphate buffer in order to reduce the sucrose content to approximately below 6%.
  • the influenza virus present in this diluted fraction is pelleted to remove soluble contaminants.
  • the pellet is resuspended and thoroughly mixed to obtain a homogeneous suspension.
  • Fraction 2 and the resuspended pellet of fraction 3 are pooled and phosphate buffer is added to obtain a volume of approximately 40 litres. This product is the monovalent whole virus concentrate.
  • the monovalent whole influenza virus concentrate is applied to a ENI-Mark II ultracentrifuge.
  • the K3 rotor contains a linear sucrose gradient (0 - 60 % (w/v)) where a sodium deoxycholate gradient is additionally overlayed.
  • Tween 80 is present during splitting up to 0.1 % (w/v) and Tocopherylsuccinate is added for B-strain viruses up to 0.5 mM.
  • the maximal sodium deoxycholate concentration is 0.7-1.5 % (w/v) and is strain dependent.
  • the flow rate is 8 - 15 litres/hour.
  • the content of the rotor is recovered by three different fractions (the sucrose is measured in a refractometer) Fraction 2 is used for further processing. Sucrose content for fraction limits varies according to strains and is fixed after evaluation.
  • the split virus fraction is filtered on filter membranes ending with a 0.2 ⁇ m membrane.
  • Phosphate buffer containing 0.025 % (w/v) Tween 80 and (for B strains) 0.5 mM Tocopherylsuccinate is used for dilution.
  • the final volume of the filtered fraction 2 is 5 times the original fraction volume.
  • the inactivated split virus material is concentrated at least 2 fold in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO.
  • the Material is subsequently washed with phosphate buffer containing 0.025 % (w/v) Tween 80 and following with phosphate buffered saline containing 0.01 % (w/v) Tween.
  • phosphate buffered saline containing 0.01% (w/v) Tween 80 and 0.1 mM Tocopherylsuccinate is used for washing.
  • the material after ultrafiltration is filtered on filter membranes ending with a 0.2 ⁇ m membrane. Filter membranes are rinsed and the material is diluted if necessary that the protein concentration does not exceed 500 ⁇ g/ml with phosphate buffered saline containing 0.01% (w/v) Tween 80 and, specific for B strains, 0.1 mM Tocopherylsuccinate.
  • the monovalent final bulk is stored at 2 - 8°C for a maximum of 18 months.
  • Example IV SRD Method used to measure haemagglutinin content
  • the plates When completely dry, the plates are stained on Coomassie Brillant Blue solution for 10 min and de-stained twice in a mixture of methanol and acetic acid until clearly defined stained zones become visible. After drying the plates, the diameter of the stained zones surrounding antigen wells is measured in two directions at right angles. Alternatively equipment to measure the surface can be used. Dose-response curves of antigen dilutions against the surface are constructed and the results are calculated according to standard slope-ratio assay methods (Finney, D.J. (1952): Statistical Methods in Biological Assay. London: Griffin, Quoted in: Wood, JM, et al (1977). J. Biol. Standard. 5, 237-247).
  • the different amino acid combination runs carried out experimentally can be analysed by standard statistical or design of experiment software packages (such as the 'design expert 6' software that is used here) to correlate the response obtained to the various components in the combination.
  • experiment software packages such as the 'design expert 6' software that is used here
  • Such a software allows a model to be generated, linking the response obtained to the combination of components in each runs, and can then be used to predict how combinations that have not been experimentally tested might perform in respect of virus yield or antigen production, for example.
  • the objective of the experiment detailed below was to study the effects of 18 amino acids at two levels (presence or absence of the amino acid in the combination) on the 'response' (defined as the total amount of viral protein that would be produced by 100 injected eggs as assessed by the product of i) the percentage of harvested eggs by ii) the protein content per harvested egg - see below).
  • the entire possible solutions that one can prepare from 18 amino acids at two levels are 2 ⁇ 18 solutions, that is to say 262144 solutions (from the solution without any amino acids to the solution containing all the amino acids).
  • the model that was selected is based on the experimentally tested solutions and represents a statistical equation (a mathematical equation to which experimental errors are added) that was defined to express the better the variations of the response by the caused variations of the amino acids.
  • the model is selected so as to be appropriate to closely predict the observed responses of the actually tested solutions, and be used to predict also the response expected with anyone of the solutions that have not actually been tested, or alternatively to rank them by order of efficacy so as to be able to define categories in which several predicted solutions would be expected to lead to a similar range of response (for example a ratio treated: control of a given value, e.g. between 1.2 and 1.5).
  • Design-Expert 6 uses the classical multiple linear regression, coding the absence of an amino acid by a "- 1" and the presence by a "+1".
  • the following example describes an experiment done in order to identify which amino acid, or combination of at least two amino acids, are suitable to lead to improved influenza virus yields, and/or in particular to the improved yield of HA, compared to a standard solution containing no amino acid.
  • amino acids combinations it is often desirable to take into consideration the possible interactions between the amino acids (synergies or inhibitions). Consequently the search for the best amino acids combinations will be done by testing amino acids combinations (containing from 1 to 18 amino acids), and not only amino acid individually tested.
  • the objective of the study was therefore to improve the total amount of viral proteins that would be produced by 100 injected eggs, and consequently the influenza virus yield, using either amino acids taken individually or amino acid combinations.
  • the outcome of the experiment was the total amount of viral protein that would be produced by 100 injected eggs as assessed by the product of i) the percentage of harvested eggs by ii) the protein content per harvested egg. This product was called 'response'. An increase in the total amount of protein produced may be due to a higher percentage of harvested eggs, or to a higher protein content per harvested egg or a combination of both.
  • the factors that were analysed were 18 different amino acids selected from the group consisting of: Phenylalanine, Lysine, Cystein, Asparagine, Glutamic acid, Histidine, Isoleucine, Leucine, Proline, Serine, Valine, Tryptophan, Tyrosine, Alanine, Methionine, Arginine, Threonine, Glycine. They were studied at two levels (i.e. the absence or the presence of the said amino acid in the combination). The concentration for each amino acid was adapted from Ohta et al. (2001 , Poultry science 80: 1430-1436) and used every time the amino acid is present in the combination (but for one combination).
  • Figure 6 presents a flowchart for the selection of suitable amino acid combinations (choice of the amino acids, optimization of their concentrations, implementation to other influenza virus strains).
  • the experiment illustrated in the Example section was performed in influenza virus A/New Caledonia/20/99 (H1N1) IVR-116 strain.
  • Table 7 describes the following experiment: the composition of the tested solutions and the day they were tested. Table 7 also indicates the letters that were used to code, for convenience, the amino acids names during the analysis.
  • Run 8 is a repeat of run 3, and runs 44 and 45 are repeats of run 24; 10 of the 18 amino acids (Cystein, Asparagine, Leucine, Proline, Serine, Valine, Tyrosine, Methionine, Threonine, Glycine) individually tested, at the same concentrations that were used for the 41 combinations (runs 46 to 55);
  • Run 15 consisted in all the amino acids at a concentration different from that used in the other solutions; a control (PBS, 0.025 mg/ml hydrocortisone, containing no exogenous amino acid) every day. As the experiment lasted 11 days, the total number of controls was 11 for this experiment.
  • PBS 0.025 mg/ml hydrocortisone
  • the mean of the eleven controls can be used as a reference to which compare the amino acid combinations in order to define whether the total amount of viral protein that would be produced by 100 injected eggs has improved, and to which extent.
  • Table 8A presents the results of the control for the 11 days that the experiment lasted.
  • the response (product of the percentage of harvested eggs with the protein content per harvested egg) was calculated as described above.
  • Table 8B shows the mean, the standard deviation and the 95% confidence interval around the mean, as calculated from the response of Table 8A data.
  • the mean of the eleven control was 12572. This value has been used as a reference when calculating the ratio of the predicted response of each amino acids combinations relative to the mean of the control. A ratio greater than 1 indicated that the predicted response for this combination was higher than the estimated mean of the control. The 95% confidence interval upper limit of this mean was 16263. Therefore, a first step for selecting the most interesting amino acids combinations was to consider the ones with a predicted response higher than 16263.
  • the software coded the levels of each factor: a "-1" when the amino acid is absent, and a "+1" when it is present.
  • the software fitted a multiple linear regression to the data.
  • the 55 runs of the experiment have been shared between different days. Since there was a high probability that the variability between the days was higher than the variability within the day, a factor was introduced in the model to extract this additional variability due to the day from the residual error. This factor is called a block factor, and the days can be called blocks. The model can then be expressed in function of the amino acids, for an "averaged" block ("averaged" day).
  • Table 10 The analysis of variance table for the model used is shown in Table 10. It shows the importance of using a block factor (the mean square of the block is important).
  • the remaining seventeen amino acids are all included in the model, alone, and many are in one or more 2-factor interactions. They are present in the model because they have an effect on the response. But their effects are not of the same importance.
  • Table 11 gives, for the response, the observed and predicted (by the model) values for the runs of the experiment (but the run 15).
  • the model is now used to maximize the response and, having found the highest values of the response, to look at which amino acids combinations they correspond.
  • Table 12 gives the list of the 249 best combinations (response higher than 25 000, combinations without Phenylalanin) predicted by the model, and the ratio of the predicted response relative to the mean of the twelve controls (12 572).
  • Table 13 and Figure 7 present the best predicted combination (i.e. which give a higher than 16263, 95% confidence interval upper limit of control mean) for a given number of amino acids.
  • Compositions with 7, 8, 9, 10, 11 , 12, 13, 14 and 15 amino acids in Figure 7 correspond to compositions 227, 69, 21 , 10, 12, 2, 1 , 3, 134 in Table 13, respectively.
  • composition 1-6 and 16 are below 16263 and have therefore not been exemplified in
  • Table 14 shows, for these same combinations, the 95% confidence and prediction intervals around the predicted response.
  • the eggs were inoculated manually with 0.5 ml of the inoculation solution. 72 eggs were used for each single experiment. After inoculation the eggs were incubated 72 hours at 33°C. Before harvest the embryos were killed by chilling the eggs at 2 to 8°C for 24 hours.
  • the allantoic fluid was harvested manually and clarified by low-speed centrifugation. Usually 8 to 10 ml crude allantoic fluid can be collected per egg.
  • Virus particles were pelleted by ultracentrifugation at 17,500 rpm for 2 hours in a Beckman SW19 rotor through a 20% sucrose cushion. The virus pellet was resuspended in 15 ml PBS, pH 7.4 and loaded on a 15 to 55% sucrose gradient. After centrifugation the fraction containing the protein peak was collected and diluted to 35 ml with PBS, pH 7.4. Virus particles were pelleted by centrifugation at 25,200 rpm in a Beckman SW28 rotor and the virus pellet was resuspended in 3 ml of PBS, pH 7.4.
  • the virus yield was evaluated by determining the haemagglutination (HA) titer and protein content of purified virus.
  • V.5.1 Confirmatory step
  • the robustness of the model can further be tested in a confirmatory step, where several amino acid combinations predicted by the model are tested in parallel with a control solution.
  • the solutions can be selected using Table 13, which lists the best solution for a given number of amino acids. Only the ones having a predicted response greater of 16000 have been included.
  • Figure 7 plots the predicted response of the Table 13 solutions in function of the number of amino acids contained, as said above.
  • Three combinations can be chosen from Figure 7 and Table 13 (based on the model), and two combinations can be added, chosen independently of the model, among the solutions really performed. A control must also be added. Repeat experiments are ideally made for at least four days.
  • the best combination of seven, ten, and thirteen amino acids can be chosen.
  • concentrations of the amino acids that enter in the best combination selected from the confirmatory step can then be optimized using a response surface study (MYERS R. H., MONTGOMERY D. C. [2002]. Response Surface Methodology: Process and Product Optimization Using Designed Experiments. New York, Wiley, 798 p.).
  • the experimental design normally takes the 'day effect' into account, as a block effect, and will also be built in function of the resources and constraints.
  • a confirmation step will be done, where the optimum will be tested several days in parallel with the control.
  • results may be comparable or not. If the results are not satisfactory enough, additional studies can be performed, and can be planned using the experimental design methodology (COCHRAN W. G., COX G. M. [1957]. Experimental Designs. New York, Wiley, 611 p.; LEWIS G. A., MATHIEU D., PHAN-TAN-LUU R. [1999]. Pharmaceutical Experimental Design. New York, Marcel Dekker, 491 p.), taking into account the resources, the constraints, and the day factor as explained above.
  • Total volume of clarified allantoic fluid 400 ml
  • Total volume of pellet 1 15 ml
  • Total volume of pellet 1 3 ml
  • Table 6 Total content of HA units and protein at three steps of the purification process
  • Table 8A results for the controls (numbers are rounded).
  • Table 8B mean, SD and 95% confidence interval around the mean of the response for the eleven controls (the numbers are rounded).
  • Table 9 results of the experiment (runs and control of each day), and ratio of the response of the runs on the response of the control of the block (numbers are rounded)
  • Table 12 249 best combinations (response > 25 000) predicted by the model, and ratio of the predicted response on the mean of the twelve controls (12 572)
  • Table 13 best predicted combination (higher than 16 263, 95% confidence interval upper limit of control mean) for a given number of amino acids.
  • Table 14 predictions of the response, Cl and PI for each best combination of a given number of amino acids.
  • the variability between the blocks is not included in the intervals.

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Abstract

L'invention concerne une méthode de production de virus de la grippe ou d'antigènes du virus de la grippe dans des oeufs d'oiseaux embryonnés. L'invention concerne, en particulier, l'introduction, dans les oeufs d'oiseaux embryonnés, d'un virus de la grippe et de n'importe lequel des composants suivants: une sérine protéase, telle que la trypsine, au moins un acide aminé ou un dérivé de ce dernier, un facteur de croissance de type insuline ou un antagoniste de l'interféron de type I, afin d'augmenter le rendement du virus de la grippe ou d'antigènes du virus de la grippe produits. L'invention concerne également un virus de la grippe pouvant être obtenu selon ledit procédé, des compositions comprenant ledit virus, leur utilisation en médecine ainsi que des méthodes prophylactiques et/ou thérapeutiques faisant appel audit virus.
PCT/EP2005/005262 2004-05-14 2005-05-12 Methode WO2005113756A1 (fr)

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