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  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
  • C12N2710/16161—Methods of inactivation or attenuation
  • C12N2710/16164—Methods of inactivation or attenuation by serial passage
• • C—CHEMISTRY; METALLURGY
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16051—Methods of production or purification of viral material
  • C12N2760/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
• • C—CHEMISTRY; METALLURGY
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16151—Methods of production or purification of viral material
  • C12N2760/16152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

• the present invention relates to the use of ferret cell lines for growing Orthomyxoviruses, to methods for the attenuation of Orthomyxoviruses and to methods for growing a virus of the family Orthomyxoviridae.
• cell line is a general term applied to a defined population of cells which has been maintained in a culture for an extended period and which has usually undergone a spontaneous process of transformation conferring an unlimited culture lifespan on the cells.
• Orthomyxoviridiae comprises as its main members the influenza viruses type A, B and C. These are very closely related viruses. Influenza type A infects humans, horses, swine, seals and a large variety of birds. Influenza type B only infects humans. The pattern of disease caused by type A and B is highly comparable.
• Influenza viruses are currently often grown e.g. on eggs.
• the advantage is that specified pathogen free eggs can easily be obtained, and such eggs are a safe growth medium. Apart from this, it is unlikely that they contain viruses that are infectious for humans. Growth on eggs is however difficult, time consuming and expensive compared to cell culture.
• cell lines are used instead of eggs, the choice of cells is mainly restricted to MDCK cells and Vero cells.
• MDCK cells have the disadvantage that they are somewhat heterogenic and, depending on the growth medium, differentiate in different directions as remote as renal tubuli cells.
• Vero cells have the disadvantage that they are to a certain extend tumorigenic.
• a general disadvantage of many of the cell lines currently in use is the relatively low virus titer. The propagation of virus on non- immortalized cells is a very unattractive option, because due to their limited number of divisions it is very difficult to obtain sufficient cells of one specific type for a commercially feasible multiplication of virus.
• ferrets are susceptible to influenza viruses. Ferrets are used for the determination of the level of attenuation of live attenuated influenza viruses, as described by Maassab, H.F. et al., (In: Plotkin, S.A., Mortimer E.A. eds. Vaccines. Philadelphia: Saunders, 1988: 435-357) and by Boudreault, A., (Can. J. Microbiol. 25: 279-284 (1979). For this kind of tests however, whole ferrets or ferret tracheal organ explant cultures are used. The tracheal organs are the organs affected by the influenza virus. (Mostow, S.R. et al., Dev.
• influenza vaccine manufacturers over the years have chosen to use embryonated eggs and well-known non- ferret animal cell lines for the propagation of influenza virus, and to accept their respective disadvantages.
• ferret cell lines are very well capable of supporting influenza virus growth. They offer a significant advantage over growth on eggs which is difficult, time consuming and expensive compared to cell culture. Moreover, they offer a significant advantage over the MDCK cells above in that they are homogeneous and thus less prone to re- differentiation. Furthermore they lack the tumorigenic character of Vero cells. Even more surprisingly, ferret cell lines are very well capable of supporting influenza viruses of very different origins: they equally support growth of avian, human, equine and porcine origin. This means that they provide a universal tool for the growth of influenza viruses in general.
• Ferret cell lines can be obtained along various routes, known in the art.
• a well-established method is the growth of ferret tissue in the presence of high doses of mutagenic agents. Cells that lose their controlled life cycle will immortalize, whereas non-mutated cells will die after several divisions. Serial passaging of cells that were subjected to mutagenesis, therefore selects for immortalized cell lines.
• ferret tumor tissue for the isolation of immortalized cells as a starting material provides a quicker way of obtaining cell lines.
• Raw ferret tumor tissue however comprises several different cell types. Therefore, cells obtained from this kind of tissue will have to be passaged several times in order to get rid of non-immortalized cells. After serial passage for at least ten, preferably more, such as twenty or more times, non-immortalized cells will practically be lost. From that moment on, individual cells can be picked and submitted to further serial passaging.
• the 2N chromosome number of normal ferret cells is 40.
• the number of chromosomes has stabilized after twenty to forty passages. All cell lines thus obtained accommodate influenza growth.
• the details of the methods used for obtaining cell lines from ferret tumor tissue are described in the Examples below.
• Mpf cell line developed by Throwbridge, discussed above, as a cell line for growing influenza virus. This cell line is available through the American Type Culture Collection under number ATCC CRL 1656.
• a first embodiment of the invention thus relates to the use of a ferret cell line for growing a virus of the family Orthomyxoviridae.
• ferret cell lines according to the invention is especially useful for influenza virus type A, B or C.
• a preferred form of this embodiment relates to the use of a ferret cell line for growing a virus of the family Orthomyxoviridae, wherein said virus is an influenza virus of type A, B or C.
• influenza type A and type B virus are most frequently seen as the cause of disease in humans and animals.
• a more preferred form of this embodiment relates to the use of a ferret cell line for growing a virus of the family Orthomyxoviridae, wherein said virus is an influenza virus of type A or B.
• influenza viruses are grown on the ferret cell line Mpf.
• another form of this embodiment relates to the use of a ferret cell line for growing a virus of the family Orthomyxoviridae, wherein said ferret cell line is the cell line Mpf.
• ferret cells evidently are neither of avian, human, equine nor porcine origin, they are ideally suited for the attenuation of influenza virus of avian, human, equine or porcine (or more generally spoken: non-ferret) origin: such viruses become ferret-cell adapted during passaging and therefore lose their specific adaptation to avian, human, equine or porcine cells.
• This loss of specific adaptation to their natural target cell provides for a very suitable means of attenuating Orthomyxoviridae, more specifically influenza viruses, even more specifically types A and B.
• another embodiment of the present invention relates to methods for attenuating a virus of the family Orthomyxoviridae, more specifically influenza viruses, even more specifically types A and B, wherein that method comprises the serial passaging of said virus on a ferret cell line.
• Still another embodiment relates to methods for growing a virus of the family Orthomyxoviridae. Such methods comprise the steps of infecting a ferret cell line with said virus of the family Orthomyxoviridae followed by harvesting the progeny virus.
• the virus of the family Orthomyxoviridae is an influenza type A, B or C.
• Fat and dead tissue were removed from the tumors and the remaining tissue was cut into small pieces and placed into 100 ml pet-flasks.
• Tissue samples were washed 3 times with PBS phenol red as follows: 50 ml PBS was added to the tissue samples and the pet-flask was softly stirred. Thereafter, tissue samples were allowed to sink under 1 * gravity and PBS phenol red was carefully decanted. After washing, 50 ml PBS phenol red with 0.1% trypsin and 0.02% EDTA was added to the tissue samples. The tissue samples were stirred for 5 minutes at 37 degrees. Tissue lumps were allowed to sink under l*gravity and supernatant was decanted into a falcon tube with 2 ml FCS (Biochrom). This procedure was repeated 3 times, The supernatants were pooled and spun down for 10 minutes at 200xg at room temperature.
• the supernatant was decanted and the cell pellet was resuspended in 5 ml medium (RPMI 1640 (Gibco), 10% fetal calf serum (Biochrom), penicillin/streptamycin. The number of cells was counted with a Burker-Turk counting chamber. A total number of 10 4 cells per cm 2 were plated in a culture flask. Cell growth was screened daily and when a confluence of 80-90% was achieved, the cells were trypsinized and propagated as described below.
• Cells were seeded onto a culture flask in a concentration of 10000 cells per cm 2 in RPMI medium with 10% FCS and penicillin/streptamycin and incubated at 5% CC ⁇ and 37°C until a confluence of 80-90% was achieved, then the cells were passed by trypsinization as follows: cells were washed twice with PBS phenol red. Thereafter, 5 ml PBS with 0.1% trypsin and 0.01% EDTA was added and spread so that the bottom was fully covered. After decanting this solution, the culture flasks were incubated for 5-10 minutes at 37°C . Detached cells were resuspended in 10 ml medium en spun down for 5 min at 200*g. 10 ml of medium was added and cells were brought in suspension and seeded onto a new culture flask in a concentration of 10000 cells/cm 2 .
• cells were one day pre infection seeded at concentration of 5*10E4 cells/cm 2 into 25 cm 2 culture flask. Medium was removed and the monolayer was washed with PBS to remove FCS. 1 ml of virus suspension was put onto the monolayer with 10 U/ml recombinant trypsin. After 1 hour at 37°C, 4 ml RPMI medium with antibiotics and with 10 U/ml recombinant trypsin was added. If no CPE occurred the T25 flasks were put at ⁇ -60°C after 2-4 days incubation. The material was thawed, collected in 15 ml tube, centrifuged lOOOxg 10 minutes, the supernatant was tested for HA activity.
• a microtiter plate was filled with 50 ⁇ l PBS/well and 50 ⁇ l of the test sample was added to the first well of a row. 2-fold serial dilutions were made by mixing and transferring 50 ⁇ l from row to row. From the last well 50 ⁇ l was discarded, 50 ⁇ l of 0.5% CRBC was added to each well. The plate was incubated for one hour at room temperature.
• HA titers were read according to standard methods. HA titers were calculated by counting the wells that give agglutination.
• This example describes how a second ferret tumor cell line was made.
• the procedure as describe in Example 1 was repeated. Growth conditions and media were slightly changed as described below.
• Cells were seeded onto a culture flask in a concentration of 20000 cells per cm 2 in animal component free medium and penicillin/streptamycin and incubated at 5% CO 2 and 37°C until a confluence of 80-90% was achieved, then the cells were passed by trypsinization. Cells were washed twice with PBS phenol red. Thereafter, 5 ml PBS with 0.1% trypsin and 0.01% EDTA was added and the bottom was fully covered. After decanting this solution, the culture flask was incubated for 5-10 minutes at 37°C . The detached cells were resuspended in 4 ml soybean trypsin inhibitor solution and spun down for 5 min at 200xg. 10 ml of animal component free medium was added and cells were brought in suspension and seeded onto a new culture flask in a concentration of 20000 cells/cm 2 .
• Haemagglutination assay A microtiter plate was filled with 50 ⁇ l PBS/well and 50 ⁇ l of the test sample was added to the first well of a row. 2-fold serial dilutions were made by mixing and transferring 50 ⁇ l from row to row. From the last well 50 ⁇ l was discarded, 50 ⁇ l of 0.5% CRBC was added to each well. The plate with a lid was incubated for one hour at room temperature. The HA titer was calculated by counting the wells that give agglutination.
• the cells are a very good substrate for a human influenza strain, cpe occurs after one day after infection, the HA activity increased significantly.
• the low oxygen concentration of 2% did not positively affect virus production/ HA production.
• MDCK cells obtained from ATCC CCL-34 were used as a positive control. MDCK cells were cultured in cell growth medium + 5% FCS.
• Mpf cells were sub-cultured for a couple of times in T75 flasks containing medium with 5% FCS. Subsequently at one day post infection, cells were seeded in T25 flasks at 2.5 x 10 4 cells/cm 2 . At day 0 an 80 % confluent monolayer had formed, appropriate for infection. Medium was removed from the cells and cells were washed with PBS
• Freeze-dried virus was resuspended in 1 ml aqua-bidest. This virus dilution was dissolved at a ratio of 1 : 100 in medium or in a 10 U/ml dilution of Trypsin in medium, both without FCS. This was done to see if influenza can grow without addition of Trypsin.
• the monolayer was then infected with 1 ml of the influenza dilution and incubated for 1 hour at 37 °C. Subsequently 4 ml cell growth medium with or without 10 U/ml Trypsin was added to the infected cells. T25 flasks were incubated for 2 days at 37 °C.
• HlNl Dilutions of 1 : 100 and 1 :200 were made in medium and in 10 U/ml Trypsin, both without FCS.
• the monolayer was then infected with 1 ml of both influenza dilutions and incubated for 1 hour at 37°C. Subsequently 4 ml medium with or without 10 U/ml Trypsin was added to the infected cells. T25 flasks were incubated for 2 days at 37°C.
• a haemagglutination test was done for both types of influenza. In order to release the virus from the cells, the cells were frozen first and then thawed.
• Mpf cells Human Influenza infected into Mp/ cells grown in cell growth medium without serum. Mpf cells were sub-cultured a couple of passages in T75 flasks containing serum-free medium.
• % confluent monolayer was constituted, appropriate for infection.
• Infection of influenza was done both with 10 U/ml Trypsin and without Trypsin in serum- free medium.
• a dilution of 1 : 100 was made of influenza in ImI serum- free medium or in 10 U/ml Trypsin in serum- free medium.
• the monolayers were infected with 1 ml of the two dilutions mentioned above and incubated for 1 hour at 37°C.
• HlNl was sub-cultured twice.
• HA-Titers were determined as described in example 1.
• Figure 2 shows the growth curve of swine influenza grown in Mpf cells. After every passage the virus was harvested and this material was used for inoculation of new fresh cells, thus producing the next passage.
• HA titers For swine influenza grown with 10 U/ml Trypsin (upper line) HA titers increase in time. After 5 passages a HA titer of 21og9/50 ⁇ l was measured. This amount of virus is high compared to the amount measured when influenza was grown for 5 passages in MDCK cells (table 1). For swine influenza grown in Mpf cells without Trypsin (bottom line) hardly any virus was measured. A HA titer of 21og2/50 ⁇ l at the 4 th passage is remarkable compared to the other data.
• Figure 3 shows two growth curves of Human Influenza, HlNl, sub-cultured in Mpf cells. Both curves differ in the used concentration of influenza at the first infection. A is dissolved at 1 : 100 and B at 1 :200. Both HA titers increase slightly during time (except from the 2 nd passage of HlNl 1:100 dilution).
• Mpf cells are suitable for cultivation of Influenza provided that Trypsin is added with every passage. Virus growth in Mpf is much more efficient when compared to influenza growth in MDCK cells. This is the case for both HlNl and H3N2, but especially for H3N2. Mpf cells cultivated in animal component free medium turned out to be even more suitable for growing influenza.
• Media Mpf cells were cultured in animal component free cell growth medium.
• HA-Titers were determined as described in example 1.
• Mpf cells were cultured in T75 flasks containing animal component free medium. Subsequently at day -3 cells were seeded in 3 x T25 at 1.0x10 4 cells/cm 2 .
• H5N2 A virus batch was made in eggs with a HA titer of 21og9 /50 ⁇ l. Cells were infected with 21og2
• the monolayer was infected with 1 ml of the influenza dilution and incubated for 1 hour at 3 VC.
• Figure 6 shows HA titers of H5N2 on Mpf cells. Cells were infected with an HA titer of virus of
• H5N2 turns out to be susceptible for H5N2.
• the first passage of H5N2 on Mpf shows an increase in virus quantity. 21og2/50 ⁇ l was added and a HA titer of maximally 21og5/50 ⁇ l was reached after two days. Multiplication of H5N2 has herewith been demonstrated.

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