Patent Abstract:
means and methods are provided for the production of mammalian viruses comprising : infecting a culture of immortalized human cells with the virus , incubating the culture infected with virus to propagate the virus under conditions that permit growth of the virus , and to form a virus - containing medium , and removing the virus - containing medium . the viruses can be harvested and be used for the production of vaccines . advantages are that human cells of the present invention can be cultured under defined serum free conditions , and the cells show improved capability for propagating virus . in particular , methods are provided for producing , in cultured human cells , influenza virus and vaccines derived thereof . this method eliminates the necessity to use whole chicken embryos for the production of influenza vaccines . the method provides also for the continuous or batchwise removal of culture media . as such , the invention allows the large - scale , continuous production of viruses to a high titer .

Detailed Description:
the present invention discloses a novel , human immortalized cell line for the purpose of propagating and harvesting virus , for production of the virus . per . c6 ® cells ( wo 97 / 00326 ) were generated by transfection of primary human embryonic retina cells , using a plasmid that contained the ad serotype 5 ( ad5 ) e1a - and e1b - coding sequences ( ad5 nucleotides 459 - 3510 seq id no : 1 of the incorporated herein sequence listing ) under the control of the human phosphoglycerate kinase ( pgk ) promoter . the following features make per . c6 ®, or a derivative , particularly useful as a host for virus production : it is a fully characterized human cell line ; it was developed in compliance with glp ; it can be grown as suspension cultures in defined serum - free medium , devoid of any human or animal serum proteins ; and its growth is compatible with roller bottles , shaker flasks , spinner flasks and bioreactors , with doubling times of about 35 hours . influenza viruses , members of the family of orthomyxoviridae , are the causative agents of annual epidemics of acute respiratory disease . in the us alone , 50 million americans get the flu each year . estimated deaths worldwide ( 1972 - 1992 ) are 60 , 000 ( cdc statistics ). there have been three major cases of pandemic outbreaks of influenza , namely in 1918 ( spanish flu , estimated 40 million deaths ), in 1957 ( asian flu , estimated one million deaths ), and in 1968 ( hong - kong flu , estimated 700 , 000 deaths ). infections with influenza viruses are associated with a broad spectrum of illnesses and complications that result in substantial worldwide morbidity and mortality , especially in older people and patients with chronic illness . vaccination against influenza is most effective in preventing the often fatal complications associated with this infection ( b . r . murphy and r . g . webster , 1996 ). the production of influenza virus on the diploid human cell line mrc - 5 has been reported ( l . herrero - euribe et al ., 1983 ). however , the titers of influenza virus are prohibitively low . present day flu vaccines contain purified hemagglutinin and neuraminidase of influenza virus a and b . the three viruses that represent epidemiologically important strains are influenza a ( h1n1 ), influenza a ( h3n2 ) and influenza b . the division into a and b types is based on antigenic differences between their nucleoprotein ( np ) and matrix ( m ) protein antigen . the influenza a virus is further subdivided into subtypes based on the antigenic composition ( sequence ) of hemagglutinin ( h1 - h15 ) and neuraminidase ( n1 - n9 ) molecules . representatives of each of these subtypes have been isolated from aquatic birds , which probably are the primordial reservoir of all influenza viruses for avian and mammalian species . transmission has been shown between pigs and humans and , recently , ( h5n1 ) between birds and humans . three types of inactivated influenza vaccines are currently used in the world : whole virus , split product and surface antigen or subunit vaccines . these vaccines all contain the surface glycoproteins , hemagglutinin ( ha ) and neuraminidase ( na ) of the influenza virus strains that are expected to circulate in the human population in the upcoming season . these strains , which are incorporated in the vaccine , are grown in embryonated hens &# 39 ; eggs , and the viral particles are subsequently purified before further processing . the need for the yearly adjustment of influenza vaccines is due to antigen variation caused by processes known as “ antigenic drift ” and “ antigenic shift .” antigenic drift occurs by the accumulation of a series of point mutations in either the h or n protein of a virus resulting in amino acid substitutions . these substitutions prevent the binding of neutralizing antibodies , induced by previous infection , and the new variant can infect the host . antigenic shift is the appearance of a new subtype by genetic reassortment between animal and human influenza a viruses . the pandemic strains of 1957 ( h2n2 ) and 1968 ( h3n2 ) are examples of reassorted viruses by which avian h and / or n genes were introduced in circulating human viruses , which subsequently could spread among the human population . based on the epidemiological surveys by over hundred national influenza centers worldwide , the world health organization ( who ) yearly recommends the composition of the influenza vaccine , usually in february for the northern hemisphere , and in september for the southern hemisphere . this practice limits the time window for production and standardization of the vaccine to a maximum of nine months . in case of an urgent demand of many doses of vaccine , for example , when a novel subtype of influenza a virus arises by antigenic shift and antigenic drift , limited availability of eggs may hamper the rapid production of vaccine . further disadvantages of this production system are the lack of flexibility , the risk of the presence of toxins and the risks of adventitious viruses , particularly retroviruses , and concerns about sterility . this presents a serious problem in today &# 39 ; s practice of influenza vaccine production on embryonated hens &# 39 ; eggs . therefore , the use of a cell culture system for influenza vaccine production would be an attractive alternative . influenza viruses can be grown on a number of primary cells , including monkey kidney , calf kidney , hamster kidney and chicken kidney . yet , their use for vaccine production is not practical because of the need to re - establish cultures from these primary cells for each preparation of a vaccine . therefore , the use of continuous cell lines for influenza vaccine production is an attractive alternative . the use of culture systems was facilitated by the realization that the proteolytic cleavage of ha in its two subunits ( ha1 and ha2 ), which is required for influenza virus infectivity , can be obtained by the addition of trypsin . inclusion of trypsin permits replication and plaque formation in madin - darby canine kidney ( mdck ) cells ( k . tobita et al ., 1975 ). the mdck cell line was recently shown to support the growth of influenza virus for vaccine production ( r . brand et al ., 1996 , 1997 ; a . m . palache et al ., 1997 ). the use of trypsin requires growth of the mdck cells in serum - free tissue culture medium ( mdck - sf1 ). however , mdck cells are currently not approved as a substrate for production of influenza virus . however , any non - human system for production of influenza vaccines has an inherent drawback known as “ adaptation .” human influenza a and b virus both carry mutations in the ha , due to adaptation in embryonated hens &# 39 ; eggs . these mutations result in altered antigenicity ( r . w . newman et al ., 1993 ; s . p . williams and j . s . robertson , 1993 ; j . s . robertson et al ., 1994 ; l . v . gubareva et al ., 1994 ; g . c . schild et al ., 1993 ; j . s . robertson et al ., 1987 ; s . kodihalli et al ., 1995 ). in humans , immunization with vaccines containing an ha bearing an egg - adaption mutation induces less neutralizing antibody to virus that contains a non - egg adapted ha ( r . w . newman et al ., 1993 ). human influenza viruses propagated in canine cells such as mdck cells also show adaptation , albeit to a lesser extent . such viruses resemble the original human isolates more closely than egg - derived viruses ( j . s . robertson et al ., 1990 ). furthermore , there is evidence that host - specific changes in na and host - specific phosphorylation patterns of np can affect the replication of influenza viruses ( j . l . schulman and p . palese 1977 ; a . sugiara and m . ueda , 1980 ; 0 . kistner et al ., 1976 ). therefore , it would clearly be advantageous to avoid adaptation or other host - induced changes of influenza virus . it may result in a more homogeneous population of viruses and render the ultimate vaccine more effective . in certain embodiments , the invention provides human cells as a substrate for the production of high titers of influenza virus , suitable for the development of vaccines . to illustrate the invention , the following illustrative examples are provided , not intended to limit the scope of the invention . cell line per . c6 ® cell ( deposited under no . 96022940 at the european collection of animal cell cultures at the center for applied microbiology and research ), or derivatives thereof , were used ( described in u . s . pat . no . 5 , 994 , 128 to fallaux et al ., which is incorporated herein by this reference ). cell lines were banked by a two tier cell bank system . the selected cell line was banked in a research master cell bank ( rmcb ) which was stored in different locations . from this rmcb , working cell banks were prepared as follows : an ampoule of the rmcb was thawed , and the cells were propagated until enough cells were present to freeze the cells by using dry ice . four hundred to 500 ampoules containing 1 ml ( 1 - 2 × 10 6 cells / ml ) of rmcb were stored in the vapor phase of a liquid nitrogen freezer . one ampoule containing 5 × 10 6 per . c6 ® cells of the wcb was thawed in a water bath at 37 ° c . cells were rapidly transferred into a 50 ml tube and re - suspended by adding 9 ml of the suspension medium excell ™ 525 ( jrh biosciences , denver , pa .) supplemented with 1 × l - glutamin . after three minutes of centrifugation at 1000 rpm , cells were re - suspended in a final concentration of 3 × 10 5 cells / ml and cultured in a t80 cm 2 tissue culture flask , at 37 ° c ., 10 % co 2 . two to three days later , cells were seeded into 490 cm 2 tissue culture roller bottles ( corning costar corporation , cambridge , mass ., us ), with a density of 3 × 10 5 / ml and cultured in continuous rotation at 1 rpm . madin darby canine kidney ( mdck ) cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem , life technologies breda , the netherlands ) containing 10 % heat inactivated fetal bovine serum and 1 × l - glutamine ( gibco - brl ), at 37 ° c . and 10 % co 2 . suspension cultures of per . c6 ® were cultured in excell ™ 525 ( jrh biosciences , denver , pa .) supplemented with 1 × l - glutamin , at 37 ° c . and 10 % co 2 , in stationary cultures in six - well dishes ( greiner , alphen aan de rijn , the netherlands ) or in 490 cm 2 tissue culture roller bottles ( corning costar corporation , cambridge , usa ) during continuous rotation at 1 rpm . direct immunofluorescence assays for the detection of influenza virus infection were carried out using the imagen ™ influenza virus a and b kit ( dako , glostrup , denmark ) according to the standard protocol of the supplier . samples were viewed microscopically using epifluorescence illumination . infected cells are characterized by a bright apple - green fluorescence . cell pellets were re - suspended into 300μ of cold pbs - 0 . 5 % bsa + 5μ of propidium iodide 50 μg / ml in pbs - fcs - azide solution . viable and dead cells were then detected via flow cytofluorometric analysis . to 50 μ / l of two - fold diluted virus solutions in pbs , 25 1 / 4 1 of a 1 % suspension of turkey erythrocytes in pbs was added in 96 - well microtiter plates and incubated at 4 ° c . for one hour . the hemagglutination pattern was examined , and expressed as hemagglutinating units ( hau ). the amount of hau corresponded to the reciprocal value of the highest virus dilution that showed complete hemagglutination . per . c6 ® cells are not known for their ability to sustain influenza virus infection and replication . we , therefore , verified whether per . c6 ® cells are permissive for influenza virus infection in comparison with mdck ( madin darby canine kidney ) cells . the day before infection , 2 × 10 5 mdck cells / well were seeded in six - well plates . twenty - four hours later , 4 × 10 5 per . c6 ® cells / well and mdck were infected with the h1n1 strain a / puerto rico / 8 / 34 ( titer 3 . 6 × 10 7 pfu / ml ), obtained from dr . eric claas , dept . of virology , leiden university medical center , nl . infection was performed at various multiplicities of infection (“ mois ”) ranging from of 0 . 1 to 10 pfu / cell . after about two hours of incubation at 37 ° c ., the inoculum was removed and replaced by fresh culture medium . a direct immunofluorescence assay for the detection of influenza virus infection was performed 24 and 48 hours post - infection . the experiment showed permissivity of per . c6 ® cell for influenza infection , with percentages of positive cells moi - dependent and comparable with mdck ( see table 1 ). we verified whether replication and propagation of influenza virus are supported by per . c6 ® cells . the day of infection , per . c6 ® cells were seeded in 490 cm 2 tissue culture roller bottles , with the density of 2 × 10 5 cells / ml in a final volume of 40 ml , in the presence of 5 μg / ml of trypsin - edta ( gibco - brl ). cells were either mock inoculated or infected with the h3n 2 strain a / shenzhen / 227 / 95 ( titer 1 . 5 × 10 6 pfu / ml ), a kind gift from dr . eric claas , department of virology , leiden university medical center , nl . infections were performed at moi 10 − 4 and 10 − 3 pfu / cell . after one hour of incubation at 37 ° c ., the inoculum was removed by spinning down the cells at 1 , 500 rpm and re - suspending them in fresh culture medium + 5 μg / ml of trypsin - edta . harvest of 1 . 3 ml of cell suspension was carried out each day from day 1 to day 6 post - infection . supernatants were stored at − 80 ° c . and used for hemagglutination assays . cell pellets were used for direct immunofluorescence tests and for propidium iodide staining ( see fig2 ). to further investigate the permissivity of per . c6 ® cell for propagation of various influenza strains , we performed an infection by using the h1n1 vaccine strains a / beijing / 262 / 95 and its reassortant x - 127 obtained from the national institute for biological standards and control ( nibsc ), potters bar , uk . the day of infection , per . c6 ® cells were seeded in 490 cm 2 tissue culture roller bottles , with the density of approximately 1 × 10 6 cells / ml in a final volume of 50 ml . cells were inoculated with 5 μl ( 10 - 4 dilution ) and 50 μl ( 10 - 3 dilution ) of virus in the presence of 5 μg / ml trypsin - edta . in order to establish if trypsin was indeed required , one more infection was carried out by inoculating 5 μl of the strain a / beijing / 262 / 95 in the absence of the protease . after approximately one hour of incubation at 37 ° c ., the inoculum was removed by spinning down the cells at 1 , 500 rpm and re - suspending them in fresh culture medium ± 5 μg / ml of trypsin - edta . at day 2 and day 4 post - infection , more trypsin was added to the samples . harvest of 1 . 3 ml of cell suspension was carried out from day 1 to day 6 post - infection . supernatants were stored at − 80 ° c . and used for hemagglutination assays and further infections ; cell pellets were used for direct immunofluorescence tests . results obtained with the above - mentioned immunofluorescence and hemagglutination assays are shown in fig4 and 5 , respectively , illustrating the efficient replication and release of the viruses . we verified if the viruses grown in per . c6 ® cells were infectious and if adaptation to the cell line could increase virus yields . virus supernatants derived from per . c6 ® infected with the strains a / beijing / 262 / 95 and its reassortant x - 127 ( dil . 10 - 3 ) and harvested at day 6 post - infection , were used . at the day of infection , per . c6 ® cells were seeded in 490 cm 2 tissue culture roller bottles , with the density of approximately 1 × 10 6 cells / ml in a final volume of 50 ml . cells were inoculated with 100 μl and 1 ml of virus supernatant in the presence of 5 μg / ml trypsin - edta . in order to establish if trypsin was still required , one more infection was carried out by inoculating 100 μl of the strain a / beijing / 262 / 95 in the absence of the protease . after approximately one hour of incubation at 37 ° c ., the inoculum was removed by spinning down the cells at 1 , 500 rpm and re - suspending them in fresh culture medium ± 5 μg / ml of trypsin - edta . at day 2 and day 4 post - infection , more trypsin was added to the samples . harvest of 1 . 3 ml of cell suspension was carried out from day 1 to day 6 post - infection . supernatants were stored at − 80 ° c . and used for hemagglutination assays and further infections ; cell pellets were used for direct immunofluorescence tests . results obtained with the above - mentioned immunofluorescence and hemagglutination assays are shown in fig6 and 7 , respectively . data obtained with the present experiment showed infectivity of the viruses grown in per . c6 ® cells as well as an increase in virus yields . intact virus is recovered from the culture medium by ion - exchange chromatography . the virus preparations are further processed to an inactivated surface antigen preparation by formaldehyde inactivation , solubilization with detergent and ultrafiltration and ultracentrifugation ( h . bachmayer , 1975 ). bachmayer h . selective solubilization of hemagglutinin and neuraminidase from influenza virus . intervirology 1975 ; 5 : 260 - 272 . brands r ., a . m . palache , and g . j . m . van scharrenburg . madin darby canine kidney ( mdck ) cells for the production of inactivated influenza subunit vaccine . safety characteristics and clinical results in the elderly . in : l . e . brown , e . w . hampson , and r . g . webster , editors . option for the control of influenza iii . amsterdam , elsevier , 1996 ; pp . 683 - 693 . brands r ., a . m . palache , and g . j . m . van scharrenburg . development of influenza subunit vaccine produced using mammalian cell culture technology . m . j . t . carrondo , b . griffths , and j . l . p . moreira , editors . animal cell technology : from vaccines to genetic medicine . dordrecht : kluwer academic publishers , 1997 ; pp . 165 - 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