Note: Descriptions are shown in the official language in which they were submitted.
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Animal cells and processes for the replication of
influenza viruses
The present invention relates to animal cells
which can be infected by influenza viruses and are
adapted to growth in suspension in serum-free medium,
and to processes for the replication of influenza
viruses in cell culture using these cells. The present
invention further relates to the influenza viruses
obtainable by the process described and to vaccines
which contain viruses of this type or constituents
thereof .
All influenza vaccines which have been used
since the 40s until today as permitted vaccines for the
treatment of humans and animals consist of one or more
virus strains which have been replicated in embryonate
hens' eggs. These viruses are isolated from the
allantoic fluid of infected hens' eggs and their
antigens are used as vaccine either as intact virus
particles or as virus particles disintegrated by
detergents and/or solvents - so-called cleaved vaccine
- or as isolated, defined virus proteins - so-called
subunit vaccine. In all permitted vaccines, the viruses
are inactivated by processes known to the person
skilled in the art. The replication of live attenuated
viruses, which are tested in experimental vaccines, is
also carried out in embryonate hens' eggs.
The use of embryonate hens' eggs for vaccine production
is time-, labor- and cost-intensive. The eggs - from
healthy flocks of hens monitored by veterinarians -
have to be incubated before infection, customarily for
12 days. Before infection, the eggs have to be selected
with respect to living embryos, as only these eggs are
suitable for virus replication. After infection the
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eggs are again incubated, customarily for 2 to 3 days.
The embryos still alive at this time are killed by cold --
and the allantoic fluid is then obtained from the
individual eggs by aspiration. By means of laborious
purification processes, substances from the hen's egg
which lead to undesired side effects of the vaccine are
separated from the viruses, and the viruses are
concentrated. As eggs are not sterile (pathogen-free),
it is additionally necessary to remove and/or to
inactivate pyrogens and all pathogens which are
possibly present.
Viruses of other vaccines such as, for example, rabies
viruses, mumps, measles, rubella, polio viruses and
FSME viruses can be replicated in cell cultures. As
1~ cell cultures originating from tested cell banks are
pathogen-free and, in contrast to hens' eggs, are a
defined virus replication system which (theoretically)
is available in almost unlimited amounts, they make
possible economical virus replication under certain
circumstances even in the case of influenza viruses.
Economical vaccine production is possibly also achieved
in that virus isolation and purification from a
defined, sterile cell culture medium appears simpler
than from the strongly protein-containing allantoic
fluid.
The isolation and replication of influenza viruses in
eggs leads to a selection of certain phenotypes, of
which the majority differ from the clinical isolate. In
contrast to this is the isolation and replication of
the viruses in cell culture, in which no passage-
dependent selection occurs (Oxford, J.S. et al.,
J. Gen. Virology 72 (1991), 185 - 189; Robertson, J.S.
et al., J. Gen. Virology 74 (1993) 2047 - 2051). For an
effective vaccine, therefore, virus replication in cell
culture is also to be preferred from this aspect to
- that in eggs.
It is known that influenza viruses can be replicated in
cell cultures. Beside hens' embryo cells and hamster
cells (BHK21-F and HKCC), MDBK cells, and in particular
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MDCK cells have been described as suitable cells for
the in-vitro replication of influenza viruses _
(Kilbourne, E. D., in: Influenza, pages 89 - 110,
Plenum Medicak Book Company - New York and London,
1987). A prerequisite for a successful infection is the
addition of proteases to the infection medium,
preferably trypsin or similar serine proteases, as
these proteases extracellularly cleave the precursor
protein of hemagglutinin [HAo] into active
hemagglutinin [HA1 and HAZ]. Only cleaved hemagglutinin
leads to the adsorption of the influenza viruses on
cells with subsequent virus assimilation into the cells
(Tobita, K. et al., Med. Microbiol. Immunol., 162
(1975), 9 - 14; Lazarowitz, S.G. & Choppin, P.W.,
Virology, 68 (1975) 440 - 454; Klenk, H.-D. et al.,
Virology 68 (1975) 426 - 439) and thus to a further
replication cycle of the virus in the. cell culture.
The Patent US 4 500 513 described the replication of
influenza viruses in cell cultures of adherently
growing cells. After cell proliferation, the nutrient
medium is removed and fresh nutrient medium is added to
the cells with infection of the cells with influenza
viruses taking place simultaneously or shortly
thereafter. A given time after the infection, protease
(e. g. trypsin) is added in order to obtain an optimum
virus replication. The viruses are harvested, purified
and processed to give inactivated or attenuated
vaccine. Economical influenza virus replication as a
prerequisite for vaccine production cannot be
accomplished, however, using the methodology described
in the patent mentioned, as the change of media, the
subsequent infection as well as the addition of trypsin
which is carried out later necessitates opening the
individual cell culture vessels several times and is
thus very labor-intensive. Furthermore, the danger
increases of contamination of the cell culture by
undesirable micro-organisms and viruses with each
manipulation of the culture vessels. A more cost-
effective alternative is cell proliferation in
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fermenter systems known to the person skilled in the
art, the cells growing adherently on microcarriers. The --
serum necessary for the growth of the cells on the
microcarriers (customarily fetal calf serum), however,
contains trypsin inhibitors, so that even in this
production method a change of medium to serum-free
medium is necessary in order to achieve the cleavage of
the influenza hemagglutinin by trypsin and thus an
adequately high virus replication. Thus this
methodology also requires opening of the culture
vessels several times and thus brings with it the
increased danger of contamination.
The present invention is thus based on the
object of making available cells and processes which
make possible simple and economical influenza virus
replication in cell culture.
This object is achieved by the provision of the
embodiments indicated in the patent claims.
The invention thus relates to animal cells which can be
infected by influenza viruses and which are adapted to
growth in suspension in serum-free medium. It was found
that it is possible with the aid of cells of this type
to replicate influenza viruses in cell culture in a
simple and economical manner. By the use of the cells
according to the invention, on the one hand a change of
medium before infection to remove serum can be
dispensed with an on the other hand the addition of
protease can be carried out simultaneously to the
infection. On the whole, only a single opening of the
culture vessel for infection with influenza viruses is
thus necessary, whereby the danger of the contamination
of the cell cultures is drastically reduced. The
expenditure of effort which would be associated with
the change of medium, the infection and the subsequent
protease addition is furthermore decreased. A further
- advantage is that the consumption of media is markedly
decreased.
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The cells according to the invention are
preferably vertebrate cells, e.g. avian cells, in --
particular hens' embryo cells.
In a particularly preferred embodiment, the
S cells according to the invention are mammalian cells,
e.g. from hamsters, cattle, monkeys or dogs, in
particular kidney cells or cell lines derived from
these. They are preferably cells which are derived from
MDCK cells (ATCC CCL34 MDCK (NBL--2)), and particularly
preferably cells of the cell line MDCK 33016. This cell
line was deposited under the deposit number DSM ACC2219
on June 7, 1995 according to the requirements of the
Budapest Convention for the International Recognition
of the Deposition of Micro-organisms for the Purposes
~5 of Patenting in the German Collection of Micro-
organisms (DSM), in Brunswick, Federal Republic of
Germany, recognized as the international deposition
site. The cell line MDCK 33016 is derived from the cell
line MDCK by passaging and selection with respect to
the capability of growing in suspension in serum-free
medium and of replicating various viruses, e.g.
orthomyxoviruses, paramyxoviruses, rhabdoviruses and
flavoviruses. On account of these properties, these
cells are suitable for economical replication of
influenza viruses in cell culture by means of a simple
and cost-effective process.
The present invention therefore also relates to
a process for the replication of influenza viruses in
cell culture, in which cells according to the invention
are used, in particular a process which comprises the
following steps:
(i) proliferation of the cells according to the
invention described above in serum-free medium in
. suspension;
(iii infection of the cells with influenza viruses;
- (iii addition of protease shortly before,
simultaneously to or shortly after infection; and
(iv) further culturing of the infected cells and
isolation of the replicated influenza viruses.
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The cells according to the invention can be
cultured in the course of the process in various serum- --
free media known to the person skilled in the art (e. g.
Iscove's medium, ultra CHO medium (BioWhittaker),
EX-CELL (JRH Biosciences)). Otherwise, the cells for
replication can also be cultured in the customary
serum-containing media (e. g. MEM or DMEM medium with
0.5o to 100, preferably 1.5o to 50, of fetal calf
serum) or protein-free media (e. g. PF-CHO (JRH
Biosciences)). Suitable culture vessels which can be
employed in the course of the process according to the
invention are all vessels known to the person skilled
in the art, such as, for example, spinner bottles,
roller bottles or fermenters.
1~ The temperature for the proliferation of the
cells before infection. with influenza viruses is
preferably 37°C.
Culturing for proliferation of the cells
(step (i)) is carried out in a preferred embodiment of
the process in a perfusion system, e.g. in a stirred
vessel fermenter, using cell retention systems known to
the person skilled in the art, such as, for example,
centrifugation, filtration, spin filters and the like.
The cells are in this case preferably proliferated for
2 to 18 days, particularly preferably for 3 to 11 days.
Exchange of the medium is carried out in the course of
this, increasing from 0 to approximately 1 to 3
fermenter volumes per day. The cells are proliferated
up to very high cell densities in this manner,
preferably up to approximately 2 x 10' cells/ml. The
perfusion rates during culture in the perfusion system
can be regulated both via the cell count, the content
of glucose, glutamine or lactate in the medium and via
other parameters known to the person skilled in the
art.
For infection with influenza viruses, about 85% to 99%,
preferably 93 to 97%, of the fermenter volume is
transferred with cells to a further fermenter. The
cells remaining in the first fermenter can in turn be
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mixed with medium and replicated further in the
perfusion system. In this manner, continuous cell
culture for virus replication is available.
Alternatively to the perfusion system, the
cells in step (i) of the process according to the
invention can preferably also be cultured in a batch
process. The cells according to the invention
proliferate here at 37°C with a generation time of 20
to 30 h up to a cell density of about 8 to 25 x 105
cells/ml.
In a preferred embodiment of the process
according to the invention, the pH of the culture
medium used in step (i) is regulated during culturing
and is in the range from pH 6 . 6 to pH 7 . 8 , preferably
~ 5 in the rancre from pH 6 . 8 to pH 7.3 .
Furthermore, the p0~ value is advantageously regulated
in this step of the process and is preferably between
25o and 95a, in particular between 35o and 600 (based
on the air saturation).
According to the invention, the infection of
the cells cultured in suspension is preferably carried
out when the cells in the batch process have achieved a
cell density of about 8 to 25 x lOs cells/mi or about 5
to 20 x 106 cells/ml in the perfusion system.
In a further preferred embodiment, the
infection of the cells with influenza viruses is
carried out at an m.o.i. (multiplicity of infection) of
about 0.0001 to 10, preferably of 0.002 to 0.5.
The addition of the protease which brings about
the cleavage of the precursor protein of hemagglutinin
[HAo~ and thus the adsorption of the viruses on the
cells, can be carried out according to the invention
shortly before, simultaneously to or shortly after the
infection of the cells with influenza viruses. If the
addition is carried out simultaneously to the
infection, the protease can either be added directly to
the cell culture to be infected or, for example, as a
concentrate together with the virus inoculate. The
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protease is preferably a serine protease, and
particularly preferably trypsin.
In a preferred embodiment, trypsin is added to
the cell culture to be infected up to a final
concentration. of 1 to 200 ~.g/ml, preferably 5 to
50 ~g/ml, and particularly preferably 5 to 30 ug/ml in
the culture medium. During the further culturing of the
infected cells according to step (iv) of the process
according tc the invention, trypsin reactivati~n can be
carried out by fresh addition of trypsin in t~e case of
the batch crocess or in the case of the perfusion
system by continuous addition of a trypsin solution or
by intermittent addition. In the latter case, the
trypsin concentration is preferably in the range from
1 ~g/ml to 6~ ~.g/ml.
After infection, the infected cell c~~lture is
cultured further to replicate the viruses, in
particular until a maximum cytopathic effect or a
maximum amount of virus antigen can be detected.
Preferably, the culturing of the cells is carried out
for 2 to 10 days, in particular for 3 to 7 days. The
culturing can in turn preferably be carried out in the
perfusion system or in the batch process.
In a further preferred embodiment, the cells
are cultured at a temperature of 30°C to 36°C,
preferably of 32°C to 34°C, after infection with
influenza viruses. The culturing of the infected cells
at temperatures below 37°C, in particular in the
temperature ranges indicated above, leads to the
production of influenza viruses which after
inactivatio:: have an appreciably higher activity as
vaccine, i:: comparison with influenza viruses which
have been replicated at 37°C in cell culture.
The culturing of the cells after infection with
influenza viruses (step (iv)) is in turn preferably
carried our at regulated pH and p02. The pH in this
case is p=eferably in the range from 6.6 to 7.8,
particularlw preferably from 6.8 to 7.2, and the p02 in
the range _=om 25o to 150%, preferably from 30o to 75%,
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_ g _
and particularly preferably in the range from 35% to
60% (based on the air saturation).
During the culturing of the cells or virus
replication according to step (iv) of the process, a
substitution of the cell culture medium with freshly
prepared medium, medium concentrate or with defined
constituents such as amino acids, vitamins, lipid
fractions, phosphates etc. for optimizing the antigen
yield is also possible.
After infection with influenza viruses, the cells can
either be slowly diluted by further addition of medium
or medium concentrate over several days or can be
incubated during further perfusion with medium or
medium concentrate decreasing from approximately 1 to 3
to 0 fermenter volumes/day. The perfusion rates can in
this case in turn be regulated by means of the cell
count, the content of glucose, glutamine, lactate or
lactate dehydrogenase in the medium or other parameters
known to the person skilled in the art.
A combination of the perfusion system with a fed-batch
process is further possible.
In a preferred embodiment of the process, the
harvesting and isolation of the replicated influenza
viruses is carried out 2 to 10 days, preferably 3 to
7 days, after infection. To do this, for example, the
cells or cell residues are separated from the culture
medium by means of methods known to the person skilled
in the art, for example by separators or filters.
Following this the concentration of the influenza
viruses present in the culture medium is carried out by
methods known to the person skilled in the art, such
as, for example, gradient centrifugation, filtration,
precipitation and the like.
The invention further relates to influenza
viruses which are obtainable by a process according to
the invention. These can be formulated by known methods
to give a vaccine for administration to humans or
animals. The immunogenicity or efficacy of the
influenza viruses obtained as vaccine can be determined
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by methods known to the person skilled in the art, e.g.
by means of the protection imparted in the loading -
experiment or as antibody titers of neutralizing
antibodies. The determination of the amount of virus or
antigen produced can be carried out, for example, by
the determination of the amount of hemaggiutinin
according to methods known to the person skilled in the
art. It is known, for example, that cleaved
hemagglutinin binds to erythrocytes of various species,
e.g. to hens' erythrocytes. This makes possible a
simple and rapid quantification of the viruses produced
or of the antigen formed.
Thus the invention also relates to vaccines
which contain influenza viruses obtainable from the
arocess according to the invention. Vaccines of this
type can optionally contain the additives customary for
vaccines, in particular substances which increase the
immune response, i.e. so-called adjuvants, e.g.
hydroxide of various metals, constituents of bacterial
cell walls, oils or saponins, and moreover customary
pharmaceutically tolerable excipients.
The viruses can be present in the vaccines as
intact virus particles, in particular as live
attenuated viruses. For this purpose, virus
concentrates are adjusted to the desired titer and
either lyophilized or stabilized in liquid form.
In a further embodiment, the vaccines according
to the invention can contain disintegrated, i.e.
inactivated, or intact, but inactivated viruses. For
this purpose, the infectiousness of the viruses is
destroyed by means of chemical and/or physical methods
(e.g. by detergents or formaldehyde). The vaccine is
then adjusted to the desired amount of antigen and
after possible admixture of adjuvants or after possible
vaccine formulation, dispensed, for example, as
liposomes, microspheres or "slow release" formulations.
In a further preferred embodiment, the vaccines
according to the invention can finally be present as
subunit vaccine, i.e. they can contain defined,
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isolated ~,rirus constituents, preferably isolated
proteins of the influenza virus. These constituents can
be isolated from the influenza viruses by methods known
to the person skilled in the art.
Furthermore, the influenza viruses obtained by
the process according to the invention can be used for
diagnostic purposes. Thus the present invention also
relates to diagnostic compositions which contain
influenza viruses according to the invention or
constituents of such viruses, if appropriate in
combination with additives customary in this field and
suitable detection agents.
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The examples illustrate the invention.
Example 1
Preparation of cell lines which are adapted to growth
in suspension and can be infected by influenza viruses
A cell line which is adapted to growth in
suspension culture and can be infected by influenza
viruses is selected starting from MDCK cells (ATCC
CCL34 MDCK (NBL-2), which had been proliferated by
means of only a few passages or over several months in
the laboratory. This selection was carried out by
proliferation of the cells in roller bottles which were
rotated at 15 rpm (instead of about 3 rprn as is
customary for roller bottles having adherently growing
cells). After several passages of the cells present
suspended in the medium, cell strains growing in
suspension were obtained. These cell strains were
infected with influenza viruses and the strains were
selected which produced the highest virus yield. An
increase in the rate of cells growing in suspension
during the first passages at 16 rpm is achieved over 1
to 3 passages by the addition of selection systems
known to the person skilled in the art, such as
hypoxanthine, aminopterin and thymidine, or alanosine
and adenine, individually or in combination. The
selection of cells growing in suspension is also
possible in other agitated cell culture systems known
to the person skilled in the art, such as stirred
flasks.
Alternatively, highly virus-replicating cell
clones can be established before selection as
suspension cells by cell cloning in microtiter plates.
In this process, the adherently growing starting cells
(after trypsinization) are diluted to a concentration
of about 25 cells/ml with serum-containing medium and
100 ~.1 each of this cell suspension are added to a well
of a microtiter plate. If 100 ul of sterile-filtered
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medium from a 2 to 4-day old (homologous) cell culture
("conditioned medium") are added to each well, the
probability of growth of the cells inoculated at a very
low cell density increases. By means of light-
s microscopic checking, the wells are selected in which
only one cell is contained; the cell lawn resulting
therefrom is then passaged in larger cell culture
vessels. The addition of selection media (e. g.
hyP~xanthine, aminopterin and thympdine, or alanosine
and adenine, individually or in combination) after the
1st cell passage leads over 1 to 3 passages to a
greater distinguishability of the cell clones. The cell
clones resulting in this way were selected with respect
to their specific virus replication and then selected
as suspension cells. The selection of cells which are
adapted to growth in serum-free medium can also be
carried out by methods known to the person skilled in
the art.
Examples of cells which are adapted to growth in serum
free medium in suspension and can be infected by
influenza viruses are the cell lines MDCK 33016 (DSM
ACC2219) and MDCK 13016, whose properties are described
in the following examples.
Example 2
Replication of influenza viruses in the cell line t~CFC
33016
The cell line MDCK 33016 (DSM ACC2219; obtained
from ar~ MDCK cell culture by selection pressure) was
proliferated at 37°C in Iscove's medium with a
splitting rate of 1:8 to 1:12 twice weekly in a roller
bottle which rotated at 16 rpm. Four days after
transfer, a cell count of approximately 7.0 x 105 to
3S 10 x 1:~5 cells/ml was achieved. Simultaneously to the
infection of the now 4-day old cell culture with the
influenza virus strain A/PR/8/34 (m.o.i. - 0.1), the
cell culture was treated with trypsin (25 ~.g/ml final
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concentration) and cultured further at 37°C, and the
virus replication was determined over 3 days (Table I).
Table
Replication of influenza A/PR/8/34 in roller bottles
(cell Line I~CK 33016) after infection of a cell
culture without change of medium, measured as antigen
content (HA units)
HA content after days after infection (dpi)
1 dpi 2 dpi 3 dpi
Experiment 1:64 1:512 1:1024
1
Experiment 1:4 1:128 1:1024
2
Experiment 1:8 1:32 1:512
3
The ratios indicated mean that a l:X dilution
of the virus harvest still has hemagglutinating
properties. The hemagglutinating properties can be
determined, for example, as described in Mayer et al.,
Virologische Arbeitsmethoden, [Virological Working
Methods], Volume 1 (1974), pages 260-261 or in Grist,
Diagnostic Methods in Clinical Virology, pages 72 to
75.
Example 3
Replication of influenza viruses in the cell line I~CR
13016 in spinner bottles
The cell line MDCK 13016 was replicated at 37°C
in Iscove's medium with a splitting rate of 1:6 to 1:10
twice weekly in a spinner bottle (50 rpm). Four days
after transfer, a cell count of 8.0 x 105 cells/ml was
achieved. Simultaneously to the infection of the now 4-
day old cell culture with the influenza virus strain
A/PR/8/34 (m.o.i. - 0.1), the cell culture was treated
with trypsin (25 ~.g/ml final concentration) and
incubated further at 33°C and the virus replication was
determined over 6 days (Table II).
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Replication of influenza A/PR/8/34 in spinner bottles
(cell line I~CK 13016) after infection of a cell
culture without change of medium, measured as antigen
content (HA units)
HA content after days after infection (dpi)
1 dpi 3 dpi 4 dpi 5 dpi 6 dpi
Experiment ~ 1:2 1:128 1:1024 1:1024 _:2048
Experiment 2 1:4 1:512 1:2048 1:2048 1:1024
Example 4
Replication of various influenza strains in the cell
line I~CK 33016 in roller bottles
The cell line MDCK 33016 (DSM ACC2219) was
replicated at 37°C in Iscove's medium with a splitting
rate of 1:8 to 1:12 twice weekly in a roller bottle
which rotated at 16 rpm. Four days after transfer, a
cell count of approximately 7.0 x 105 to
10 x 105 cells/ml was achieved. Simultaneously to the
infection of the now 4-day old cell culture with
various .influenza virus strains (m.o.i. ~ 0.1), the
cell culture was treated with trypsin (25 ~.g/ml final
concentrations and further incubated at 33°C, and the
virus replication was determined on the 5th day after
infection (Table III).
Replication of influenza strains in roller bottles
(cell line I~CK 33016) after infection of a cell
culture without change of medium, measured as antigen
content (HA units)
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HA content 5 days after infection
Influenza strain HA content
A/Singapore/6/86 1:1024
A/Sichuan/2/87 1:256
A/Shanghai/11/87 1:256
A/Guizhou/54/89 1:128
A/Beijing/353/89 1:512
B/Beijing/1/87 1:256
B/Yamagata/16/88 1:512
A/PR/8/34 1:1024
A/Equi i/Prague 1:512
A/Equi 2/Miami 1:256
A/Equi 2 1:128
Fontainebleau
A/Swine/Ghent 1:512
A/Swine/Iowa 1:1024
A/Swine/Arnsberg 1:512
Example 5
Replication of various influenza strains in I~CR 33016
cells in the fermenter
The cell line MDCK 33016 (DSM ACC2219) was inoculated
in Iscove's medium with a cell inoculate of 1 x 105
cells/ml in a stirred vessel fermenter (working volume
l0 8 1). At an incubation temperature of 37°C, a p02 of 50
100 (regulated) and a pH of 7.1 ~ 0.2 (regulated),
the cells proliferated within 4 days to a cell density
of 7 x 105 cells/ml. 8 ml of virus stock solution
(either A/PR/8/34 or A/Singapore/6/86 or
A/Shanghai/11/87 or A/Beijing/1/87 or
B/Massachusetts/71 or B/Yamagata/16/88 or
B/Panama/45/90) and simultaneously 16 ml of a 1.25%
strength trypsin solution were added to these cells and
the inoculated cell culture was incubated further at
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33°C. The virus replication was determined over 6 days
(Table IV). ..
Replication of influenza virus strains in the fermenter
(cell line I~CK 33016) after infection of a cell
culture without change of medium, measured as antigen
content (HA units)
HA content after days after infection (dpi)
1 dpi 3 dpi 4 dpi 5 dpi 6 dpi
A/PR/8/34 1:64 1:512 1:1024 1:2048 1:2048
A/Singapore 1:32 1:512 1:2048 1:2048 _:1024
A/Shanghai 1:8 1:128 1:256 1:256 1:512
A/Beijing 1:16 1:256 1:1024 1:1024 n.d.
B/Yamagata 1:8 1:128 1:512 1:512 n.d.
B/Massachusetts 1:4 1:128 1:256 1:512 n.d.
B/Panama n.d. 1:128 1:256 n.d. 1:1024
Example 6
Influence of the infection dose (m.o.i.) on virus
replication
The cell line MDCK 13016 (obtained from an MDCK
cell culture by selection pressure) was proliferated at
37°C in ultra CHO medium with a splitting rate of 1:8
to 1:12 twice weekly in a roller bottle which rotated
at 16 rpm. Four days after transfer, a cell count of
approximately 7.0 x 105 to 10 x 105 cells/ml was
achieved. The influence of the infective dose (m.o.i.)
on the yield of antigen and infectiousness was
investigated. Simultaneously to the infection of the
now 4 day-old cell culture with the influenza virus
strain A/PR/8/34 (m.o.i. - 0.5 and m.o.i. - 0.005), the
cell culture was treated with trvpsin (25 ~.g/ml final
concentration) and incubated further at 37°C, and the
virus replication was determined over 3 days (Table V).
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Table V
Replication of influenza virus strain PR/8/34 in the
cell line i~CK 13016 in roller bottles after infection
with an m.o.i. of 0.5 or 0.005. The assessment of virus
replication was carried out by antigen detection (HA)
and infectiousness titer (CCIDso cell culture infective
dose 50 o in loglo)
Davs after
infection 2 3 4 5
HA CCIDso HA CCIDSa HA CCIDso HA CCIDse
PP/8/34
m.o.i.= 0.5 128 5.1 256 5.7 512 5.3 1024 5.4
m.c.i. - 0.005 n4 4.9 512 8.0 512 8.3 1024 8.3
The determination of the CCIDso can in this case
be carried out, for example, according to the method
which is described in Paul, Zell- and Gewebekultur
[Cell and tissue cultureJ(1980), p. 395.
Example 7
Influence of media substitution on virus replication
The cell line MDCK 33016 (DSM ACC2219) was
croliferated at 37°C in Iscove's medium with a
splitting rate of 1:8 to 1:12 twice weekly in a roller
bottle which rotated at 16 rpm. Four days after
transfer, a cell count of approximately 7.0 x 105 to
10 x 105 cells/ml was achieved. The influence of a media
substitution on the yield of antigen and infectiousness
was investigated. The now 4-day old cell culture was
infected with the influenza virus strain A/PR/8/34
(m.o.i. - 0.05), the trypsin addition (20 ~g/ml final
concentration in the roller bottle) being carried out
3_0 by mixing the virus inoculum with the trypsin stock
solution. The cell culture was treated with additions
of media and incubated further at 33°C, and the virus
replication was determined over 5 days (Table VI).
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Replication of influenza A/PR/8/34 in roller bottles
(cell line NECK 33016); addition of 5~ (final
concentration) of a triple-concentrated Iscove's
medium, of glucose (final concentration 3~) or glucose
and casein hydrolysate (final concentration 3a or 0.1%)
measured as antigen content (HA units)
HA content after days after infection (dpi)
Addition 1 dpi 3 dpi 4 dpi 5 dpi
--- 1:16 1:256 1:1024 1:1024
(control)
3x Iscove's 1:8 1:128 1:1024 1:2048
Glucose 1:32 1:512 1:2048 1:2048
Glucose/casein 1:8 1:128 1:512 1:1024
hydrolysate
Example 8
Replication of influenza viruses in I~CK 33016 cells in
the f ermenter and obtainment of the viruses
The cell line MDCK 33016 (ACC2219) was
inoculated in Iscove's medium with a cell inoculate of
0.5 x 105 cells/ml in a stirred vessel fermenter
(working volume 10 1). At an incubation temperature of
37°C, a p0z of 55 ~ 10% (regulated) and a pH of
7.1 ~ 0.2 (regulated), the cells proliferated within 4
days to a cell density of 7 x 105 cells/ml. 0.1 ml of
virus stock solution (A/Singapore/6/86; m.o.i. about
0.0015) and simultaneously 16 ml of a 1.250 strength
trypsin solution were added to these cells and the
inoculated cell culture was incubated further at 33°C.
The virus replication was determined after 5 days and
the virus was harvested. Cells and cell residues were
removed by tangential flow filtration (Sarooton Mini-
Microsart Module with 0.45 um pore size; filtration
procedure according to the instructions of the
manufacturer), no loss of antigen (measured as HA)
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being detectable in the filtrate. The virus material
was concentrated from 9.5 1 to 600 ml by fresh
tangential flow filtration tSartocori~' Mini-UltrasartM
Module with 100,000 NMWS (nominal molecular weight
5 separation limit); filtration procedure according to
the instructions of the manufacturer). The amount of
antigen in the concentrate was 5120 HA units (start 256
HA units; concentration factor 20), while the
infectiousness in the concentrate was 9.2 loglo CCIDso
10 (start 8.9 loglo CCIDSO; concentration factor 16); the
loss of antigen and infectiousness was less than 1%,
measured in the filtrate after the 100,000 NMWS
filtration.
15 Example 9
Replication of the influenza viruses in MDCR 33016
cells in the perfusion fermeater
1.6 x 108 cells of the cell line MDCK 33016 (DSM
20 ACC2219) were suspended in UltraCHO medium (0.8 x
105 cells/ml) in the reactor vessel of Biostat MDT~'(Braun
Biotech Int., Melsungen, Germany) with an effective
volume of 2000 ml and proliferated at 37°C in perfusion
operation.with a rising flow rate (entry of oxygen by
hose aeration toxygen regulation 40 ~ 10% p0z); pH
regulation pH <_ 7.2; cell retention by' spin filter
> 95%), The live cell count increased within 11 days by
200-fold to 1?5 x 105 cells/ml (Table VIIIa). 1990 ml of
this cell culture were transferred to a 2nd perfusion
fermenter tworking volume 5 1), while the remaining
cells were made up to 2000 ml again with medium and
cell proliferation was carried out again in perfusion
operation. in the 2nd perfusion fermenter (virus
infection), the cells were infected with the influenza
virus strain A/PR/8/34 (m.o.i. - 0.01) with
simultaneous addition of trypsin (lo ug/ml final
concentration) and incubated for d h. The fermenter was
then incubated further in perfusion operation
tregulation of p0z:40 t 10% and pH:~ 7.2). On the first
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day after infection, incubation was carried out at 37°C
and the virus harvest in the perfused cell culture
supernatant was discarded. From the 2nd day after
infection, virus replication was carried out at 33°C
and the perfusion rate of 2 fermenter volumes/day was
reduced to 0 within 7 days. The trypsin necessary for
virus replication was present in the UltraCHO medium
which was used for the perfusion in a concentration of
~g/ml. The virus harvest (= perfused cell culture
i0 supernatant) was collected at 4°C and the virus
replication over 7 days was determined as the amount of
antigen (Table VIIIb)_
~at~1 a VIIIa
~_5
Replication of I~CR 33016 cells in the perfusion
fermenter
Day Live cell Total cell Perfusion
count count
[x105/ml] [x105/ml] [1/day]
0 0.6 0.6 0
3 8.0 8.3 0
4 14.6 17.5 1.1
5 33.3 34.7 1.1
6 49.8 53.6 2.1
7 84.5. 85.6 3.9
9 82.6 84.9 4.0
9 100.8 104.8 4.1
10 148.5 151.0 4.0
11 175.8 179.6 3.9
Table VIIIb
Replication of influenza A/PR/8/34 in the perfusion
fermenter (cell line I~CR 33016), measured as antigen
content (HA units) in the cumulated perfused cell
culture supernatant
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Day after HA content in Medium Total amount
infection virus harvest addition virus harvest
(perfusion)
1 <4 4 1 0 1
2 8 4 1 4 1
3 64 3 1 7 1
4 256 2 1 9 1
2048 2 1 11 1
6 4096 2 1 12 1
7 4096 0 1 12 1
Example 10
S Preparation of an experimental influenza vaccine
An experimental vaccine was prepared from
influenza virus A/PR/8/34 from Example 2 - A/PR/8
replicated at 37°C - (Experiment 2; vaccine A) and
Example 4 - A/PR/8 replicated at 33°C - (vaccine B).
The influenza viruses in the cell culture medium were
separated from cells and cell fragments by low-speed
centrifugation (2000 g, 20 min, 4°C) and purified by a
sucrose gradient centrifugation (10 to SOo (wt/wt) of
linear sucrose gradient, 30,000 g, 2 h, 4°C). The
influenza virus-containing band was obtained, diluted
1:10 with PBS pH 7.2, and sedimented at 20,000 rpm, and
the pellet was taken up in PBS (volume 500 of the
original cell culture medium). The influenza viruses
were inactivated with formaldehyde (addition twice of
0.0250 of a 35% strength formaldehyde solution at an
interval of 24 h, incubation at 20°C with stirring).
10 NMRI mice each, 18 to 20 g in weight, were
inoculated with 0.3 ml each of these inactivated
experimental vaccines on day 0 and day 28 by
subcutaneous injection. 2 and 4 weeks after the
inoculation and also 1 and 2 weeks after revaccination,
blood was taken from the animals to determine the titer
of neutralizing antibodies against A/PR/8/34. To
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determine the protection rate, the mice were exposed 2
weeks after revaccination (6 weeks after the start of _.
the experiment) by intranasal administration of 1000
LDSO (lethal dose SO%). The results of the experiment
are compiled in Table IX.
Table IX
Efficacy of experimental vaccines: for vaccine A the
influenza virus A/PR/8/34 was replicated at 37°C and
for vaccine B at 33°C. The titer of neutralizing
antibodies against A/PR/8 and also the protection rate
after exposure of the mice were investigated
Titer of neutralizing antibodies/ml* Protection
rate
Number
living/total
2 w pvacc 4 w pvacc 1 w 2 w
prevacc prevacc
Vaccine A <28 56 676 1,620 1/10
Vaccine B 112 1,549 44,670 112,200 9/10
* Weeks after vaccination (w pvacc) and weeks after
1S revaccination (w prevacc)
The experiments confirm that influenza viruses
which had been replicated at 37°C in cell culture with
a high antigen yield (HA titer) only induced a low
neutralizing antibody titer in the mouse and barely
provided protection, while influenza viruses which had
been replicated at 33°C in cell culture also with a
high antigen yield (HA titer) induced very high
neutralizing antibody titers in the mouse and led to
very good protection.
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