WO2008073490A1 - Purification d'antigènes viraux de la grippe - Google Patents

Purification d'antigènes viraux de la grippe Download PDF

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Publication number
WO2008073490A1
WO2008073490A1 PCT/US2007/025487 US2007025487W WO2008073490A1 WO 2008073490 A1 WO2008073490 A1 WO 2008073490A1 US 2007025487 W US2007025487 W US 2007025487W WO 2008073490 A1 WO2008073490 A1 WO 2008073490A1
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influenza
antigen
fluid
gradient
glycerol
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PCT/US2007/025487
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English (en)
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Yawei Ni
Guo Jianhua
Liying Tian
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Carrington Laboratories Inc.,
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Publication of WO2008073490A1 publication Critical patent/WO2008073490A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/16311—Influenzavirus C, i.e. influenza C virus
    • C12N2760/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods of purifying influenza viral antigens, influenza antigens produced by methods of purifying influenza viral antigens, and influenza vaccines comprising purified influenza antigens.
  • Influenza viruses are enveloped viruses that vary greatly in size and shape. Their size can range from 80 to 120 nm and their shape can be round, oval, filamentous or tube- like. The difference in size also leads to the difference in mass. These differences pose a great challenge to purification efforts as a significant loss in purity and quantity can occur if a purification method does not take these variables into consideration.
  • Influenza whole virion vaccines were widely used prior to the 1980s and are still used in some parts of world (Fukuda et al., 2003). They were gradually phased out due to its higher reactogenicity, i.e., a higher frequency of fever and injection site reactions, and replaced with split or subunit vaccines that have a lower reactogenicity (Gross et al., 1977; Fukuda et al., 2003).
  • the whole virion vaccine is known to be more immunogenic (Zei et al., 1991) and also provides a broader cross protection as it also contains viral internal proteins besides HA and NA.
  • the NP and Ml are among the most abundant viral proteins and the primary target of the T-cell immunity. Immunity generated against viral internal proteins such as nucleoprotein has been shown to be protective (Wraith et al., 1987; Tamura et al., 1996). The higher immunogenicity may be in part due to the presence of viral single stranded RNA which can engage TLR 7 (Lund et al. 2004).
  • influenza virus purification method for manufacturing is linear sucrose gradient centrifugation while certain manufacturers use a different method such as chromatography (Palache et al. 1997).
  • chromatography the most widely used influenza virus purification method for manufacturing.
  • the large continuous flow KII or similar centrifuges from Alfa Wassaaerman or Sorvall (previously Kendro) capable of handling > 80 L per hour at a speed of > 40,000 rpm are used.
  • These continuous flow centrifuges first developed in the 1960-7Os have been performing well over the past 35 years (Anderson et al., 2003). This gradient centrifugation step has been gradually improved and is often coupled with other steps including filtration, diafiltration, and concentration to produce the final purified split or subunit antigen products.
  • a linear dipotassium tartrate (PT) gradient has also been used for influenza virus purification in the literature (Compans et al. 1970 and 1977, Newlin et al. 1975, Morrongiello et al. 1977). It is important to note that the current manufacturing processes are for production of the sub virion and subunit vaccines. Therefore, the purity at whole virion stage does not have to meet the specifications for the final products. This however will not be the case if whole virions are to be used as a vaccine antigen. It has become clear that a linear sucrose gradient centrifugation is not sufficient to produce whole virion antigens of high purity.
  • Figure 1 shows an example of a purification process for MDCK cell-based influenza whole virion antigens.
  • the dotted lines indicate possible process step adjustments or options. This process is merely exemplary as one of skill in the art wuld know that variations are possible.
  • Figure 2 shows protein profiles of virus preparations at different stages of the purification process with the avian A/Duck/Singapore-Q/Fl 19-2/97 (H5N3) strain.
  • Lane 1 17K pelleted virus
  • Lane 2 Banded virus from sucrose banding
  • Lane 3 Main virus band from 30-60% linear sucrose gradient
  • Lane 4 Broad virus band from 30-60% linear sucrose gradient
  • Lane 5 Virus band from G-PT/G gradient loaded with banded virus
  • Lane 6 Virus band from G-PT/G gradient loaded with broad virus band from linear sucrose gradient
  • Lane 7 Virus band from G-PT/G gradient loaded with main virus band in sucrose linear gradient
  • Figure 3 shows virus band formation in different linear gradients.
  • G glycerol
  • PT potassium tartrate.
  • Figure 4 shows electron micrographs of influenza virus particles
  • Figure 5 shows a comparison of virus yields following linear sucrose gradient and G-PT/G gradient centrifugation. All samples for gel electrophoresis were volume- controlled based on the amount of starting materials. The banded viruses from sucrose banding step were either directly subjected to G-PT/G gradient centrifugation, or went through linear sucrose gradient centrifugation first before G-PT/G gradient centrifugation.
  • Lane 1 Broad band from linear sucrose gradient loaded with 8 ml banded virus (suspended in 0.8 ml); Lane 2: Main band from linear sucrose gradient loaded with 8 ml of banded virus (suspended in 0.8 ml); Lane 3: Virus band from G-PT/G gradient loaded with four ml of banded virus (tube 1, suspended in 0.4 ml); Lane 4: Virus band from G-PT/G gradient loaded with four ml of banded virus (tube 2, suspended in 0.4 ml).
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • the present invention provides an improved method of purifying influenza viral antigens, including whole influenza virions.
  • Linear sucrose gradient centrifugation has long been the most widely used method for purification of influenza whole virion in research as well as vaccine manufacturing. However, this method is clearly deficient for producing a highly pure whole virion antigen.
  • the present invention provides an improved reverse glycerol-potassium tartrate gradient that can be utilized to purify whole influenza virion, and other influenza antigens. Unexpectedly, the methods of the present invention not only significantly increased purity, but also yield as compared to the linear sucrose gradient.
  • the whole virion antigens produced with the improved purification process was highly immunogenic, inducing a hemagglutination inhibition assay (HAI) titer at least 2 fold higher than current inactivated split vaccine at a 2 fold lower HA dose.
  • HAI hemagglutination inhibition assay
  • the present invention provides a method of purifying an influenza viral antigen comprising: a) obtaining a fluid comprising influenza antigen; and purifying the fluid by ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to obtain a purified influenza viral antigen.
  • an influenza viral antigen can be a whole influenza virus or virion, an influenza viral particle, an influenza surface antigen or an internal influenza antigen.
  • the whole influenza virus can be a live or an inactivated virus.
  • the influenza internal antigens include viral structural proteins such as, but not limited to, a capsid nucleoprotein (NP), a matrix envelope protein (M protein) and a polymerase (PA) protein or fragments thereof.
  • the influenza surface antigens include, but are not limited to, a hemagglutinin (HA) located at the surface of the envelope or a neuraminidase (NA) surface enzyme protein or fragments thereof.
  • the antigen can be from any influenza virus, now known or identified in the future.
  • the virus can be a human influenza virus or a non-human mammal influenza virus, such as, but not limited to, a swine virus, an equine virus, a bovine virus or an ovine virus.
  • the virus can also be an avian influenza virus. Examples of these viruses include, but are not limited to influenza A, influenza B and influenza C.
  • influenza virus type A there are at least 15 subtypes (Hl -H 15) based on the HA subtype.
  • Hl -H 15 subtypes
  • strains that are categorized under certain subtypes include, but are not limited to, HlNl, H3N2, H5N1, H5N3, H7N7, H2N2, and H9N2.
  • HlNl H3N2, H5N1, H5N3, H7N7, H2N2, and H9N2.
  • Within influenza type B there are two lineages, Yamagata and Victoria.
  • a fluid comprising an influenza viral antigen can be, but is not limited to cell culture medium comprising an influenza antigen, fluid from an embyronated egg comprising influenza antigen, baculovirus expression medium comprising an influenza antigen, allantoic fluid comprising an influenza antigen or a biological fluid comprising an influenza antigen.
  • Biological fluids include, but are not limited to, urine, blood, semen, sputum, spinal fluid, pulmonary fluid, bronchial lavage fluid or saliva from any species.
  • the fluid comprising an influenza antigen can be obtained by culturing influenza virus on host cells, such as mammalian cells, for example, but not limited to, human cells, monkey cells, hamster cells, pig cells, ferret cells, rat cells, mouse cells, chicken cells, MDCK cells, etc.
  • the fluid comprising an influenza antigen can also be obtained from the biological fluid of any animal.
  • the fluid comprising an influenza viral antigen can also be a fluid that comprises partially purified influenza viral antigen.
  • the fluid can comprise whole influenza virus, internal influenza antigen or surface influenza antigen that has been partially purified by affinity chromatography, filtration, centrifugation, or any other means.
  • the fluid can comprise partially purified fragments of an internal influenza antigen or a surface influenza antigen.
  • the fluid comprising an influenza viral antigen can be obtained by any means, for example, one of skill in the art can obtain the fluid by culturing host cells with any influenza virus and harvesting cell culture medium comprising influenza virions. One of skill in the art can also obtain culture medium comprising influenza virions from cell cultures prepared by another person of skill in the art.
  • the fluid can also be obtained from embryonated chicken eggs, baculovirus expression medium or a biological fluid.
  • the fluid can also be provided by another individual, a laboratory, a company, a university, the American Type Culture Collection, or any other entity, for purification utilizing the methods of the present invention.
  • One of skill in the art can also resuspend pelleted or lyophilized influenza viral antigen in medium or buffer to obtain a fluid comprising an influenza viral antigen.
  • the methods of the present invention can be applied to a fluid comprising a partially purified viral antigen.
  • One of skill in the art can partially purify an influenza viral antigen by any means and then further purify the influenza viral antigen utilizing the methods of the present invention.
  • One of skill in the art can also obtain a fluid comprising a partially purified viral antigen from any other source such as a another laboratory, company, university another individual, a laboratory, a company, a university, the American Type Culture Collection or any other entity, for purification utilizing the methods of the present invention.
  • the methods of the present invention utilize ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to purify an influenza viral antigen.
  • the linear reverse glycerol-potassium tartrate gradient is made for example, by a gradient pump such as the Bio-Rad Econo gradient pump that mixes two different solutions at various ratios and pumps the mixed solution into a receiving tube, thus forming a concentration gradient of the solute(s) in the solutions.
  • a reverse glycerol-potassium tartrate gradient can be made with a glycerol solution and a potassium tartrate solution.
  • the glycerol solution can be about 10- 40% and the potassium tartrate solution can be about 20%-60%.
  • the glycerol concentration decreases from about 10% -40% to 0% from the top to the bottom of the tube, whereas the potassium tartrate gradient increases from 0% to about 20-60% from the top to the bottom of the tube.
  • concentration gradients of glycerol and potassium tartrate are in reverse direction.
  • the glycerol gradient can comprise about 10%-40% glycerol (10-40% to 0%). Therefore, the glycerol gradient can range from about 40% glycerol to about 0% glycerol, from about 35% glycerol to about 0% glycerol, from about 25% glycerol to about 0% glycerol, from about 20% glycerol to about 0% glycerol, from about 15% glycerol to about 0% glycerol, from about 10% glycerol to about 0% glycerol, or from about any other percentage between 10% and 40% glycerol and 0% glycerol. Every percentage between 10% and 40% glycerol is disclosed herein as an amount of glycerol that can be utilized in the gradients described herein.
  • the potassium tartrate gradient can comprise about 20-60% potassium tartrate (0% to 20-60%). Therefore, the potassium tartrate gradient can range from about 0% to about 20% potassium tartrate, from about 0% to 25% potassium tartrate, from about 0% to 30% potassium tartrate, from about 0% to 35% potassium tartrate, from about 0% to about 40% potassium tartrate, from about 0% to 45% potassium tartrate, 0% to 50% potassium tartrate, from about 0% to 55% potassium tartrate, from 0% to 60% potassium tartrate or from 0% to any other percentage in between 20% and 60% potassium tartrate. Every percentage between 20% and 60% potassium tartrate is disclosed herein as an amount of potassium tartrate that can be utilized in the gradients described herein.
  • potassium tartrate can be substituted with dipotassium tartrate, sodium potassium tartrate, other tartrate salts, cesium chloride, sodium iodide, sodium bromide, cesium sulfate or cesium acetate.
  • the percentages described above for potassium tartrate can also be utilized for making gradients with dipotassium tartrate, sodium potassium tartrate, other tartrate salts, cesium chloride, sodium iodide, sodium bromide, cesium sulfate or cesium acetate.
  • a fluid comprising an influenza viral antigen is layered on the reverse glycerol-potassium tartrate gradient and ultracentifuged to obtain a band that contains the influenza viral antigen.
  • Centrifugation can be performed at about 27,000 rpm for about 5 to 18 hours (for example, for about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 or any amount of time in between these times) to obtain a band comprising the influenza viral antigen of interest.
  • This band can then be excised from the centrifuge tube via techniques known in the art such as by inserting a syringe into the centrifuge and extracting the contents of the band.
  • the purity of the influenza viral antigen can be assessed at any time, before or after performing a method of the present invention by electrophoresing a sample of the influenza viral antigen and detecting the amount of other non-influenza proteins, if any, that are associated with the viral antigen. Electrophoresis is set forth as an example of how to assess purity. Other methods of assessing purity and yield are set forth in the Examples. In addition to the examples provided herein, other methods are available and would be known to those of skill in the art.
  • Also provided by the present invention is a method of purifying an influenza viral antigen comprising: a) obtaining a fluid comprising influenza antigen; and purifying the fluid by ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to obtain a purified influenza viral antigen, wherein the potassium tartrate gradient contains glycerol.
  • the potassium tartrate gradient can comprise about 20-60% potassium tartrate (0% to 20-60%) as described above, and about 5-30% glycerol. Therefore, the potassium tartrate gradient can comprise about 5%, 10%, 15%, 20%, 25%, 30% or any other percentage between about 5% and 30% glycerol. Every percentage between about 5% and about 30% glycerol is disclosed herein as an amount of glycerol that can be utilized in the potassium tartrate gradients described herein.
  • the methods of the present invention can optionally include a step in which the fluid comprising the influenza antigen is subjected to high speed centrifugation in order to remove cellular debris and other unwanted materials.
  • the fluid prior to layering the fluid on any of the gradients described herein, the fluid can be centrifuged to remove cellular debris.
  • the band can be extracted from the centrifugation tube and centrifuged to remove the associated debris prior to ultracentrifuging the fluid again on a gradient described herein.
  • the methods of the present invention can optionally include a step in which the fluid comprising the influenza antigen is subjected to filtration.
  • the fluid prior to layering the fluid on any of the gradients described herein, the fluid can be subjected to a filtration step in order to remove cellular debris.
  • the band can be extracted from the centrifugation tube and subjected to filtration in order to remove the associated debris prior to ultracentrifuging the fluid again on a gradient described herein.
  • the filtration conditions are selected such that unwanted debris or cellular proteins are removed and the filter membrane retains the influenza viral antigen.
  • Tangential filtration through a membrane of desired porosity can be utilized.
  • Diafiltration methods in which the influenza antigen is washed during filtration can also be employed.
  • One of skill in the art will know that diafiltration is a technique that uses ultrafiltration membranes to completely remove, replace, or lower the concentration of salts or solvents from solutions containing proteins, peptides,nucleic acids, and other biomolecules, such as influenza viral antigens.
  • the process selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size.
  • An ultrafiltration membrane retains molecules that are larger than the pores of the membrane while smaller molecules such as salts, solvents and water, which are 100% permeable, freely pass through the membrane.
  • the methods described herein can optionally include a step wherein the fluid comprising the influenza antigen is partially purified by ultracentrifugation on a sucrose cushion comprising a 10-40% sucrose solution. Therefore, the sucrose cushion can comprise about 10%, 15%, 20%, 25%, 30%, 35%, 40% or any percentage sucrose in between 10% and 40% sucrose. Every percentage between about 10% and about 40% sucrose is disclosed herein as an amount of surose that can be utilized in the sucrose cushions described herein.
  • the fluid containing the partially purified influenza viral antigen can be layered on any of the gradients described herein to obtain a purified influenza viral antigen.
  • the methods described herein can optionally include a step of partially purifying an influenza viral antigen by ultracentrifugation on a nonlinear sucrose gradient comprising a 10-40% sucrose solution as the top layer and a 50-70% sucrose solution as the bottom layer.
  • the top layer of the nonlinear sucrose gradient can comprise about 10%, 15%. 20%, 25%, 30%, 35%, 40% or any percentage of sucrose in between 10% and 40% sucrose. Every percentage between about 10% and about 40% sucrose is disclosed herein as an amount of sucrose that can be utilized in the top layer of a nonlinear sucrose gradient.
  • the bottom layer of the nonlinear sucrose gradient can comprise about 50%, 55%, 60%, 65%, 70% or any percentage of sucrose in between 50% and 70% sucrose.
  • sucrose Every percentage between about 50% and about 70% sucrose is disclosed herein as an amount of sucrose that can be utilized in the bottom layer of a nonlinear sucrose gradient.
  • the fluid containing the partially purified influenza viral antigen can be layered on any of the gradients described herein to obtain a purified influenza viral antigen.
  • influenza viral antigen can be inactivated. Inactivation can occur before, during or after any centrifugation process, before, during or after any filtration process, before, during or after any partial purification process or, before, during or after a purification process involved a linear reverse glycerol-potassium tartrate gradient.
  • the present invention contemplates inactivation of a fluid comprising an influenza viral antigen, inactivation of a filtered fluid comprising an influenza viral antigen, inactivation of a concentrated fluid comprising an influenza viral antigen, inactivation of a fragmented influenza viral antigen, inactivation of an influenza viral antigen contained in a pellet or other non-fluid form.
  • the influenza viral antigen in particular, whole influenza virus or particles, can be inactivated by contacting the virus with one or more of formaldehyde, betapropiolactone, gamma-irradiation, ultraviolet light or any other means, now known or discovered in the future that inactivates an influenza viral antigen.
  • Viral inactivation tests can be performed to determine if inactivation causes a decrease in the infectious titre of the virus. If necessary, the influenza virus can undergo inactivation more than once throughout the purification process.
  • the methods described herein can optionally include a step of concentrating a fluid comprising an influenza viral antigen.
  • the present invention also provides influenza viral antigens purified by the methods set forth herein that can be utilized to stimulate an immune response against influenza in a subject. Therefore, the present invention provides a method of making an influenza antigen that stimulates an immune response against influenza in a subject comprising: obtaining a fluid comprising influenza antigen; and purifying the fluid by ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to obtain a purified influenza viral antigen.
  • Also provided by the present invention is a method of making an influenza vaccine comprising: obtaining a fluid comprising influenza antigen; and purifying the fluid by ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to obtain a purified influenza viral antigen.
  • the methods of making an influenza antigen that simulates an immune response against influenza and the methods of making an influenza vaccine set forth herein utilize ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to purify an influenza viral antigen.
  • the linear reverse glycerol-potassium tartrate gradient is made for example, by a gradient pump such as the Bio-Rad Econo gradient pump that mixes two different solutions at various ratios and pumps the mixed solution into a receiving tube, thus forming a concentration gradient of the solute(s) in solution.
  • a reverse glycerol-potassium tartrate gradient can be made with a glycerol solution and a potassium tartrate solution.
  • the glycerol solution can be about 10-40% and the potassium tartrate solution can be about 20%-60%.
  • the glycerol concentration decreases from about 10% -40% to 0% from the top to the bottom of the tube, whereas the potassium tartrate gradient increases from 0% to about 20-60% from the top to the bottom of the tube.
  • the concentration gradients of glycerol and potassium tartrate are in reverse direction.
  • the glycerol gradient can comprise about 10%-40% glycerol (10-40% to 0%). Therefore, the glycerol gradient can range from about 40% glycerol to about 0% glycerol, from about 35% glycerol to about 0% glycerol, from about 25% glycerol to about 0% glycerol, from about 20% glycerol to about 0% glycerol, from about 15% glycerol to about 0% glycerol, from about 10% glycerol to about 0% glycerol, or from about any other percentage between 10% and 40% glycerol and 0% glycerol. Every percentage between 10% and 40% glycerol is disclosed herein as an amount of glycerol that can be utilized in the gradients described herein.
  • the potassium tartrate gradient can comprise about 20-60% potassium tartrate (0% to 20-60%). Therefore, the potassium tartrate gradient can range from about 0% to about 20% potassium tartrate, from about 0% to 25% potassium tartrate, from about 0% to 30% potassium tartrate, from about 0% to 35% potassium tartrate, from about 0% to about 40% potassium tartrate, from about 0% to 45% potassium tartrate, 0% to 50% potassium tartrate, from about 0% to 55% potassium tartrate, from 0% to 60% potassium tartrate or from 0% to any other percentage in between 20% and 60% potassium tartrate. Every percentage between 20% and 60% potassium tartrate is disclosed herein as an amount of potassium tartrate that can be utilized in the gradients described herein.
  • potassium tartrate can be substituted with dipotassium tartrate, sodium potassium tartrate, other tartrate salts, cesium chloride, sodium iodide, sodium bromide, cesium sulfate or cesium acetate.
  • the percentages described above for potassium tartrate can also be utilized for making gradients with dipotassium tartrate, sodium potassium tartrate, other tartrate salts, cesium chloride, sodium iodide, sodium bromide, cesium sulfate or cesium acetate.
  • a fluid comprising an influenza viral antigen is layered on the reverse glycerol-potassium tartrate gradient and ultracentifuged to obtain a band that contains the influenza viral antigen. Centrifugation can be performed at about 27,000 rpm for about 5 to 18 hours (for example, for about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 or any amount of time in between these times) to obtain a band comprising the influenza viral antigen of interest. This band can then be excised from the centrifuge tube via techniques known in the art such as by inserting a syringe into the centrifuge and extracting the contents of the band.
  • Also provided by the present invention is a method of making an influenza antigen that stimulates an immune response against influenza antigen comprising: a) obtaining a fluid comprising influenza antigen; and purifying the fluid by ultracentrifugation on a linear reverse glycerol-potassium tartrate gradient to obtain a purified influenza viral antigen, wherein the potassium tartrate gradient contains glycerol.
  • the potassium tartrate gradient can comprise about 20-60% potassium tartrate (0% to 20-60%), as described above, and about 5-30% glycerol. Therefore, the potassium tartrate gradient can comprise about 5%, 10%, 15%, 20%, 25%, 30% or any other percentage between about 5% and 30% glycerol. Every percentage between about 5% and about 30% glycerol is disclosed herein as an amount of glycerol that can be utilized in the potassium tartrate gradients described herein.
  • the methods of making an influenza vaccine or the methods of making an influenza antigen that stimulates an immune response against influenza can optionally include a step in which the fluid comprising the influenza antigen is subjected to high speed centrifugation in order to remove cellular debris and other unwanted materials.
  • the fluid prior to layering the fluid on any of the gradients described herein, the fluid can be centrifuged to remove cellular debris.
  • the band can be extracted from the centrifugation tube and centrifuged to remove the associated debris prior to ultracentrifuging the fluid again on a gradient described herein.
  • the methods of making an influenza vaccine or the methods of making an influenza antigen that stimulates an immune response can optionally include a step in which the fluid comprising the influenza antigen is subjected to filtration.
  • the fluid prior to layering the fluid on any of the gradients described herein, the fluid can be subjected to a filtration step in order to remove cellular debris.
  • the band can be extracted from the centrifugation tube and subjected to filtration in order to remove the associated debris prior to ultracentrifuging the fluid again on a gradient described herein.
  • the filtration conditions are selected such that unwanted debris or cellular proteins are removed and the filter membrane retains the influenza viral antigen. Tangential filtration through a membrane of desired porosity (for example, from about 30OkDa to about 100OkDa or any size in between these molecular weights) can be utilized. Diafiltration methods in which the influenza antigen is washed during filtration can also be employed.
  • the methods described herein can optionally include a step wherein the fluid comprising the influenza antigen is partially purified by ultracentrifugation on a sucrose cushion comprising a 10-40% sucrose solution. Therefore, the sucrose cushion can comprise about 10%, 15%, 20%, 25%, 30%, 35%, 40% or any percentage sucrose in between 10% and 40% sucrose. Every percentage between about 10% and about 40% sucrose is disclosed herein as an amount of surose that can be utilized in the sucrose cushions described herein.
  • the fluid containing the partially purified influenza viral antigen can be layered on any of the gradients described herein to obtain a purified influenza vaccine or a purified influenza viral antigen that stimulates an immune response against influenza in a subject.
  • the methods described herein can optionally include a step of partially purifying an influenza viral antigen by ultracentrifugation on a nonlinear sucrose gradient comprising a 10-40% sucrose solution as the top layer and a 50-70% sucrose solution as the bottom layer.
  • the top layer of the nonlinear sucrose gradient can comprise about 10%, 15%. 20%, 25%, 30%, 35%, 40% or any percentage of sucrose in between 10% and 40% sucrose. Every percentage between about 10% and about 40% sucrose is disclosed herein as an amount of sucrose that can be utilized in the top layer of a nonlinear sucrose gradient.
  • the bottom layer of the nonlinear sucrose gradient can comprise about 50%, 55%, 60%, 65%, 70% or any percentage of sucrose in between 50% and 70% sucrose.
  • sucrose Every percentage between about 50% and about 70% sucrose is disclosed herein as an amount of sucrose that can be utilized in the bottom layer of a nonlinear sucrose gradient.
  • the fluid containing the partially purified influenza viral antigen can be layered on any of the gradients described herein to obtain an influenza vaccine or a purified influenza viral antigen that stimulates an immune response against influenza in a subject.
  • influenza viral antigen that stimulates an immune response against influenza in a subject or an influenza viral antigen that is an influenza vaccine can be inactivated. Inactivation can occur before, during or after any centrifugation process, before, during or after any filtration process, before, during or after any partial purification process or, before, during or after a purification process involved a linear reverse glycerol-potassium tartrate gradient.
  • the present invention contemplates inactivation of a fluid comprising an influenza viral antigen, inactivation of a filtered fluid comprising an influenza viral antigen, inactivation of a concentrated fluid comprising an influenza viral antigen, inactivation of a fragmented influenza viral antigen, inactivation of an influenza viral antigen contained in a pellet or other non-fluid form.
  • the influenza viral antigen in particular, whole influenza virus or particles, can be inactivated by contacting the virus with one or more of formaldehyde, betapropiolactone, gamma-irradiation, ultraviolet light or any other means, now known or discovered in the future that inactivates an influenza viral antigen.
  • Viral inactivation tests can be performed to determine if inactivation causes a decrease in the infectious titre of the virus. If necessary, the influenza virus can undergo inactivation more than once throughout the purification process.
  • the methods of making an influenza viral antigen that stimulates an immune response and the methods of making an influenza vaccine can optionally include a step of pelleting the purified influenza viral antigen.
  • the methods of making an influenza viral antigen that stimulates an immune response and the methods of making an influenza vaccine can optionally include a step of concentrating the purified influenza viral antigen.
  • the methods of making an influenza viral antigen that stimulates and immune response and the methods of making an influenza vaccine can optionally include a step of disrupting or fragmenting the influenza viral antigen.
  • Disruption or fragmentation of an influenza viral antigen for example, whole influenza virus, can also result in inactivation of the virus. Fragmentation can be carried out at room temperature (20 to 25 0 C) by adding an amphiphilic nonionic detergent to the purified influenza virus.
  • Such detergents include, but are not limited to Triton X-100TM, Triton X-165TM, Triton X-205TM, Triton X-305TM, Triton X-405TM or octoxinol 9.
  • the fragmentation can take place with stirring, for example, during at least thirty minutes of contact, but it is possible to continue stirring for a longer period of time, for example up to 24 hours.
  • the viral structure is dissociated.
  • the internal components of the virus are released into the medium.
  • the NP and M antigens are released, which can be used to stimulate a cell-mediated immune response to influenza virus.
  • Surface antigens such as HA and NA are also released upon fragmentation.
  • the fragmentation process can be stopped at any time by removal of the detergent. After fragmentation, the medium can be filtered to remove unwanted substances and retain the viral fragments.
  • viral fragments can be further purified utilizing the purification methods disclosed herein or other purification methods to obtain purified internal and surface influenza antigens for preparation of an influenza vaccine.
  • Purified internal and surface influenza antigens can be utilized for human or veterinary use. These antigens can also be used in the preparation of products which can be used for diagnostic purposes.
  • the present invention also provides an influenza vaccine comprising a purified influenza viral antigen produced by any of the methods described herein.
  • An influenza vaccine can comprise any of the antigens produced by the methods provided herein, including whole influenza virus, internal influenza viral antigens and surface viral antigens.
  • Also provided by the present invention is a method of ameliorating the signs or symptoms of influenza infection in a subject, comprising administering to the subject an effective amount of an influenza antigen purified by the methods of the present invention.
  • a method of inducing an immune response against influenza virus in a subject comprising administering to the subject an effective amount of an influenza antigen purified by the methods of the present invention.
  • subject is meant an individual.
  • the subject is a mammal such as a primate, and, more preferably, a human.
  • subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.), laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species (for example, chickens, turkeys, ducks, pheasants, pigeons, doves, parrots, cockatoos, geese, etc.).
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.
  • avian species for example, chickens, turkeys, ducks, pheasants, pigeons, doves
  • compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents herein disclosed are conventional. Remington 's Pharmaceutical Sciences, by Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents herein disclosed. In general, the nature of the carrier will depend on the mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, sesame oil, glycerol, ethanol, combinations thereof, or the like, as a vehicle. The carrier and composition can be sterile, and the formulation suits the mode of administration.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, sodium saccharine, cellulose, magnesium carbonate, or magnesium stearate.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Embodiments of the disclosure including medicaments can be prepared with conventional pharmaceutically acceptable carriers, adjuvants and counterions as would be known to those of skill in the art.
  • the amount of a pharmaceutical composition comprising an influenza antigen effective in decreasing or inhibiting influenza infection can depend on the strain of the virus and can be determined by standard clinical techniques. In addition, in vitro assays can be employed to identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • influenza antigens or vaccines comprising influenza antigens can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal, vaginal and intestinal mucosa, etc.) and can be administered together with other biologically active agents, including other agents that ameliorate the signs or symptoms of flu.
  • Such agents include, but are not limited to Oseltamivir (Tamiflu ® ), Zanamivir (Relenza ® ), Amantadine (Symmetrel ® ), Rimantadine (Flumadine ® ), Tylenol, ibuprofen, aspirin and the like.
  • the disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Instructions for use of the composition can also be included.
  • the immune response against influenza virus can be a cell-mediated immune response.
  • the immune response can also be a humoral immune response that results in the production of antibodies against the influenza viral antigen.
  • the immune response generated by the influenza antigens of the present invention can be sufficient to ameliorate a sign or symptom associated with influenza infection. These signs and symptoms include, but are not limited to, fever, chills, body aches, muscle pain, fatigue, loss of appetite, a cough, runny nose, sore throat, diarrhea and lung inflammation.
  • the amelioration does not have to be complete as amelioration can range from a slight decrease in one or more symptoms associated with influenza infection to complete amelioration of one or more symptoms associated with influenza infection.
  • viral load is a sign of influenza infection. Therefore, the influenza antigens purified by the methods can be administered to a subject to decrease influenza viral load in the subject.
  • Also provided by the present invention is a method of treating or preventing influenza infection in a subject, comprising administering to the subject an effective amount of an influenza vaccine comprising an influenza antigen purified by the methods of the present invention.
  • treating means a decrease in the severity of influenza infection.
  • One of skill in the art would know how to assess the severity of influenza infection and monitor its progress. Therefore, one of skill would also know how to assess a decrease in the severity of influenza infection by measuring a decrease any one of more of the following: viral load, fever, chills, body aches, muscle pain, fatigue, loss of appetite, a cough, runny nose, sore throat, diarrhea and lung inflammation. It is understood that these are exemplary and should not be limiting as one of skill in the art would know how to assess additional parameters according to the subject's medical history, previous exposure to influenza, viral strain etc.
  • a vaccine of the present invention can also be administered to prevent influenza infection. Prevention can range from a delay in the onset of signs or symptoms associated with influenza to completely preventing the onset of signs or symptoms associated with influenza infection.
  • the vaccines described herein can be administered to a subject at risk of having an influenza infection.
  • a subject at risk of having an infection is a subject that is predisposed to develop an infection. Such a subject can include, for example, a subject with a known or suspected exposure to an infectious organism or agent.
  • a subject at risk of having an infection also can include a subject with a condition associated with impaired ability to mount an immune response to an infectious organism or agent, for example, a subject with a congenital or acquired immunodeficiency, a subject undergoing radiation therapy or chemotherapy, a subject with a burn injury, a subject with a traumatic injury, a subject undergoing surgery or other invasive medical or dental procedure, or a similarly immunocompromised individual.
  • a subject with a condition associated with impaired ability to mount an immune response to an infectious organism or agent for example, a subject with a congenital or acquired immunodeficiency, a subject undergoing radiation therapy or chemotherapy, a subject with a burn injury, a subject with a traumatic injury, a subject undergoing surgery or other invasive medical or dental procedure, or a similarly immunocompromised individual.
  • MDCK cells were obtained from ATCC (CCL-34) and were adapted to grow under serum-free conditions using serum-free VP-SFM media (Invitrogen Co.).
  • Virus strains used in this study included the three virus strains in 2004-2005 season inactivated trivalent vaccine [A/New Caledonia/20/99 (HlNl); A/Wyoming/03/2003 (H3N2), B/Jiangsu/10/2003], and a low pathogenicity avian H5 strain (A/Duck/Singapore- Q/Fl 19-2/97, H5N3). All viruses were originally grown in embryonated chicken eggs. They were adapted to grow to a high titer (HA titer > 5120/ml) after one or two passages in MDCK cells. The MDCK-adapted H3 strain lost its ability to agglutinate the chicken red blood cells (RBCs), but still could agglutinate the turkey RBCs.
  • Virus propagation A/New Caledonia/20/99 (HlNl); A/Wyoming/03/2003 (H3N2), B/Jiangsu/10/2003]
  • All viruses were originally grown in embryo
  • MDCK cells grown in pleated surface roller bottles (1450 cm 2 ) were infected at a m.o.i. of 0.01 or less. Viruses were harvested after incubation at 33 0 C for 2-3 days post infection or when >70% cells developed CPE (cytopathic effect). Infected cell culture media was pooled and centrifuged at 8,000 rpm for 20 min before storing at -80 0 C. The infected cell culture media had a HA titer of 5120 — 20480/ml, depending on the virus strains.
  • MDCK cell monolayers in 6-well plates were inoculated with serially diluted virus samples (0.5 ml/well). After incubation for 1 hr, the virus inoculum was removed, 3 ml of 1% agarose in culture media containing 2 ⁇ g/ml trypsin was added to each well. Plates were incubated at 33 0 C for 3 days followed by addition 3 ml 10% formalin to each plate. The plaques were visualized by staining with crystal violet.
  • the clarified infected cell culture media was first concentrated 10 times using a Millipore Pellicon II mini tangential flow filtration unit with a 1,000 kDa cartridge at 4 0 C. The use of this cartridge was justified by the absence of any HA activity in the permeate. Concentrated viruses were then pelleted by centrifugation at 17,000 rpm for 3 hrs at 4 0 C in a Beckman type 19 rotor. The pelleted viruses were resuspended and viruses were banded once on a sucrose step gradient (10 ml 30% sucrose and 5 ml 60% sucrose) using Beckman SW32 rotor (equivalent to SW28). The banded viruses were harvested by piercing the wall of centrifuge tubes and stored at 4 0 C.
  • the banded viruses were diluted and then subjected to linear sucrose (30%-60%) gradient centrifugation in Beckman SW32 rotor at 24,000 rpm for 3 hrs at 4 0 C.
  • the linear sucrose gradient was also used at other sucrose concentration ranges (15-60% and 15-45%).
  • the banded viruses are centrifuged in linear PT (5-40%, potassium tartrate), G-PT (30% glycerol - 40% potassium tartrate), G-PT/G (30% glycerol -40% potassium tartrate/ 10% glycerol) gradient in the same rotor for various time lengths (up to 18 hrs) at 4 0 C.
  • virus bands were recovered by piercing the wall of the tube with a 10 ml syringe. Viruses were pelleted down and suspended in NTE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5).
  • Purified viruses at less than 0.5 mg/ml protein concentration were inactivated by adding formaldehyde (37%) to virus suspension at a final dilution of 1 :4,000. Viruses were kept in a rocking shaker at 4 0 C for 3 days or more. Inactivation was confirmed by absence of visible plaque formation in MDCK cell monolayer and a lack of HA activity in allantoic fluids harvested from inoculated embryonated chicken eggs. Inactivated viruses were pelleted down and suspended in PBS at a protein concentration of 1 - 3 mg/ml and stored at 4 0 C prior to use.
  • the Coomassie blue-stained gel images were acquired using GS-5000 digital imaging system (Alpha Infotech Co.) and the optical densities (OD) of protein bands were measured using the NIH ImageJ program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2006.).
  • the inactivated whole virion antigen was evaluated in Sprague-Dawley rats (170 - 199 g).
  • the antigens used were either a current commercial inactivated split (subvirion) trivalent vaccine or the inactivated whole virion antigens of A/New Caledonia/20/99 (HlNl) or A/Duck/Singapore-Q/Fl 19-2/97 (H5N3) strains produced with the improved purification process.
  • Antigens were administered by intramuscular injection in 0.2 ml for rats.
  • HA Hemagglutination
  • HAI hemagglutination inhibition
  • HA tests were performed with chicken or turkey RBCs in V-bottom 96 well plates at room temperature.
  • the antigen preparations were serially 2-fold diluted in a 50 ⁇ l volume and mixed with equal volumes of 0.5% RBCs. The highest antigen dilution that causes full agglutination is considered to contain 1 HA unit.
  • rat serum samples were first absorbed with chicken RBC.
  • the antigens were either clarified infectious cell lysate or infectious allantoic fluids. They were stored at -80 0 C and thawed prior to use. The antigens were all used at 4 HA units. Controls included positive agglutination by the antigen and negative agglutination by serum itself or RBCs alone. The first serum dilution was 10 fold.
  • the PT gradient was first evaluated. Although this method has been used for purification of egg - and cell - based viruses, no well-defined virus bands could be obtained in this gradient. Thus, other methods had to be examined. Thus, we an improved the linear glycerol - PT (G-PT) reverse gradient centrifugation method for purification of MDCK cell-based influenza viruses was used. The virus banding was further improved by introducing glycerol to the PT portion to make the G- PT/G gradient.
  • G-PT linear glycerol - PT
  • the purity of the influenza whole virions was significantly increased with the improved method as compared to linear sucrose gradient centrifugation. This was reflected by nearly complete lack of possible host cell proteins identifiable by gel electrophoresis, much higher specific HA activity, and homogenous morphology under electron microscope. These observations have not been made previously in published reports.
  • the yield was also significantly increased with the G-PT gradient as compared to the linear sucrose gradient centrifugation.
  • This increase in yield in comparison to the sucrose gradient has not been described previously.
  • the increase in yield was achieved in part by recovering a portion of the viruses that were normally lost during the linear sucrose gradient centrifugation. It was found that in the linear sucrose gradient centrifugation, besides the main virus band, the lower portion of the gradient (broad band) also contains a significant amount of viruses (1/3 to 1/2 of the total). The viruses from the broad band which accounts for almost 1/3 of the total gradient volume are normally discarded.
  • the viruses from the broad band are banded at the same position as those from the main virus band in the G-PT gradient centrifugation, and thus can be recovered if the PT gradient is used instead of the linear sucrose gradient, thus increasing the yield.
  • the viruses from the main and broad bands were shown to be the same based on protein composition and morphology (EM) following purification with the linear G-PT gradient. The increase in the yield in comparison to the sucrose gradient has not been described previously.
  • EM protein composition and morphology
  • the improved method has been evaluated with four different virus strains (Hl, H3, and B strains for 2004-2005 season inactivated trivalent vaccines and A/Duck/Singapore- Q/Fl 19-2/97, H5N3) with the same results obtained.
  • a vaccine antigen needs to be as pure as possible whether from a cell culture system or others. At the same time, its immunogenicity needs to be preserved. Thus, no matter what purification method is used, the antigens produced must be demonstrated to be suitable as a vaccine. This is particularly important when a new purification method is used. It should be noted that the influenza viruses purified using PT gradient have not been tested for immunogenicity, and the G-PT gradient has not been used for influenza virus purification.
  • the whole virion antigen purified using the G-PT or G-PT/G methods of the present invention was highly immunogenic, inducing a HAI titer at least 2 fold higher than current inactivated split vaccine at a nearly 2 fold lower HA dose.
  • Glycerol has been a common excipient for oral, topical, or injectable dosage forms, and dipotassium tartrate or sodium potassium tartrate has been used as antiacid agents. Therefore, glycerol, dipotassium tartrate and sodium potassium tartrate have a solid safety profile.
  • Fig.l An example of a purification process of the present invention is shown in Fig.l .
  • the protein profiles of virus preparations at different stages of the purification process are shown with the avian H5N3 strain (A/Duck/Singapore-Q/Fl 19-2/97) in Fig 2.
  • the viruses or viral proteins steadily increased while the host cell proteins steadily decreased along with the progress of purification steps.
  • sucrose banding step significantly enriched the viruses (Fig. 2).
  • concentration a 1,000 kDa cartridge was used in the Pellicon II unit. This pore size was larger than that (300 kDa) used in the literature.
  • concentration efficiency significantly and also removed more cellular proteins as compared to 300 kDa cartridge as shown by gel analysis of 17 K pellet following concentration.
  • the concentrated virus was banded in a 30%— 60% step sucrose gradient. This further enriched viruses or viral proteins (Fig. 2). The banded viruses were then subjected to linear gradient centrifugations.
  • the banded viruses were then subjected to linear sucrose gradient centrifugation.
  • a primary virus band (main virus band) was formed in the gradient following centrifugation (Fig. 3). Gradient regions above and below the virus band were also collected and analyzed. These two regions had no distinct bands formed, but characterized by an increased turbidity.
  • the top gradient region (above the main band) contained little viruses. But the bottom region (broad band; a region below the main virus band) contained a significant amount of viruses, about 1/3 to 1/2 of the combined total from the main and broad virus bands (see below).
  • the broad band usually collected in 10-12 ml of gradient volume, which is ⁇ 1/3 of the total gradient volume, is normally discarded, thus leading to a 1/3 to 1/2 loss in virus yield. This region of the gradient has not been previously examined extensively.
  • Potassium tartrate (PT) gradient centrifugation has been used for purification of many viruses, including the influenza (McCrea et al., 1961; Newlin et al. 1975, Compans et al. 1970 and 1977, Morrongiello et al. 1977).
  • influenza the number of reports on using the PT gradient is limited and no assessment of purity, yield, or immunogenicity has been provided.
  • Potassium tartrate is considered a milder salt with respect to the stability of viruses as compared to CsCl, but still can be used for equilibrium centrifugation within a relative short time (McCrea et al., 1961).
  • sucrose is a low density gradient material and is not suited for equilibrium centrifugation.
  • Reverse G-PT gradient A reverse glycerol-PT (G-PT) gradient was evaluated. Using a 30% glycerol (30% to 0%) and 40% potassium tartrate (0% to 40%) reverse gradient, a distinct major virus band, though flocculent, was obtained after centrifugation at 27,000 rpm for 18 hrs (Fig. 2). The band flocculence disappeared once the virus band was recovered.
  • the virus band was formed as the first band toward denser gradient regions (bottom). This will allow continuous operation while the band remained at the same position and facilitated the virus recovery.
  • glycerol into the bottom portion of the gradient (30% G - 40% PT and 10% G or G-PT/G)
  • the virus banding was further improved, i.e., the virus band was no longer flocculent and better defined (Fig. 3), thus improving virus banding and recovery.
  • the high purity is also demonstrated by the specific HA activity (HA unit/ ⁇ g antigen).
  • the purified viruses had a minimal 500 HA units/ ⁇ g protein, which is much higher than the reported values (Table 1).
  • the purified viruses were also morphologically homogenous as observed under electron microscope (Fig. 4). Nearly all particles were consistently round with only a very small number of particles with irregular shapes. Small knob-like projections were clearly seen on individual virus particles. This can be due to the the high salt concentration in the G-PT/G gradient which lead to the protrusion of viral internal content on the surface of virus particles. This could be advantageous to immunized subjects in inducing more immune responses against viral internal components.
  • viruses from the broad and main bands in linear sucrose gradient were banded at the same position in the G-PT/G gradient. This was confirmed by nearly the same density (1.997 g/ml for main band, 1.2101g/ml for broad band) determined by weighing 1 ml of gradient materials removed from the center of the virus band in the G- PT/G gradient. EM examination showed that these two virus populations had similar sizes and appearance as shown in Fig. 4.
  • gel electrophoresis analysis further showed that there was no difference in protein composition between or among these two virus populations and the virus purified by G-PT/G gradient directly with banded viruses (Fig. 2).
  • the virus yield was significantly increased using G-PT/G gradient in part by recovering the viruses from the broad band in the linear sucrose gradient that is normally discarded.
  • Protein concentration determination of the purified viruses showed that after G- PT/G gradient centrifugation, the amount of viruses directly recovered from banded viruses was more than 2 fold higher than the combined yield from both main and broad virus bands or 4 fold higher than that of the main virus band from linear sucrose gradient (Table 2). This was also clearly evident in Fig. 5.
  • this increase in yield was highly significant even considering that there would be a small amount of loss associated with the additional purification step by G-PT/G gradient centrifugation of the main and broad band viruses from linear sucrose gradient. 4.
  • the 18 hr run time can be used for production, this time can be shortened.
  • the G-PT/G gradient centrifugation was done for various time lengths (3, 5, 8, and 12 hrs). The results showed that similar banding, yield, and purity were achieved with an 8 hr run. Therefore, centrifugation can be performed for at least about 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or any other time interval between at least about 7 hours and 19 hours.
  • sodium potassium tartrate could also be used in the G-PT or G-PT/G gradient.
  • the immunogenicity of the whole virion antigens obtained with improved purification method was evaluated in rats in comparison to an inactivated split subvirion trivalent vaccine.
  • the whole virion antigen was the A/New Caledonia/20/99 (HlNl), the same HlNl strain as in the commercial trivalent vaccine.
  • Eight ⁇ g of this whole virion antigen in 0.2 ml PBS was injected intramuscularly, which corresponds to a 3 ⁇ g HA based on the 37% HA content in the whole virion antigen (see above).
  • the commercial vaccine was used without any adjustment and 0.2 ml, equivalent to two fifth of one human dose or 6 ⁇ g HA, was injected in the same manner.
  • H5N3 whole virion antigen and trivalent split subvirion vaccine induced a HAI titer at least 2 fold higher than that against the HlNl component in the trivalent vaccine at a dose 2 fold lower based on HA content (Table 3). This was true at all time points except for one covering a 10 week period after one immunization.
  • H5N3 whole virion antigen and trivalent split subvirion vaccine induced a HAI titer at least 2 fold higher than that against the HlNl component in the trivalent vaccine at a dose 2 fold lower based on HA content (Table 3). This was true at all time points except for one covering a 10 week period after one immunization.
  • H5N3 whole virion antigen and trivalent split subvirion vaccine induced a HAI titer at least 2 fold higher than that against the HlNl component in the trivalent vaccine at a dose 2 fold lower based on HA content (Table 3). This was true at all time points except for one covering a 10 week
  • the whole virion antigen of the A/Duck/Singapore- Q/Fl 19-2/97 (H5N3) strain was compared to the same commercial trivalent vaccine, also at a dose nearly 2 fold lower based on HA.
  • the results showed that the whole virion antigen induced a HAI titer at least 2 fold higher than those against all three components (HlNl, H3N2, and B) in the trivalent vaccine at all 4 time points covering an 8-week period (Table 4), only with the exception for the H3N2 at weeks 6 and 8 when one animal showed exceptionally higher titer in this group, resulting in a high StDev value.

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Abstract

L'invention concerne des procédés de purification d'antigènes viraux de la grippe, des antigènes de la grippe produits selon ces procédés, ainsi que des vaccins contre la grippe contenant des antigènes de la grippe purifiés.
PCT/US2007/025487 2006-12-12 2007-12-11 Purification d'antigènes viraux de la grippe WO2008073490A1 (fr)

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