Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MEDIUM AND METHOD FOR VIRAL PROPAGATION AND GROWTH
The present invention relates to a medium and
to a method for viral propagation and multiplication on
cells in culture, in particular for producing vaccines.
The production of viral vaccines, whethez~ they
are simply attenuated, inactivated, subunits or
recombinant, involves the large scale production of
viruses.
This production can be carried out, depending
on the nature of the virus, on various supports which
allow the replication of the virus; thus, for example,
for most of the currently commercially available anti-
influenza vaccines, virus proliferation is carried out
on embryonated hens' eggs, whereas for some vaccines
against Japanese encephalitis, this step is carried out
on mouse brains.
However, the use of such supports poses many
problems: availability, reproducibility, but especially
safety because of possible contaminations with viruses,
mycoplasmas or any other undesirable element
originating from the support. Increasingly, attempts
are thus being made to dispense with such supports and,
whenever possible, cell cultures are used which are
infected with a small amount of the virus to be
produced, and which are then maintained under
conditions which promote virus replication inside the
infected cells, as well as the propagation of the
infection from one cell to another. The viruses
produced are then harvested either from the culture
medium in which the cells bathe, when it involves
intracellular viruses-which leave the cell after their
budding (which in particular has the advantage of
making it possible to carry out several virus harvests
with the same culture cells), or by treating the cells
themselves when it involves intracellular viruses.
These techniques entail having cell cultures of
good quality; many developments of the prior art have
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thus related to improving cell cultures and in
particular to the culture media used. In order to limit
the risks of contamination of the cells with
mycoplasmas, viruses, bovine spongiform encephalopathy
agents or any other unconventional transmissible agent,
the use of serum-free, and even protein-free, culture
media has been proposed, as is described in Patent
Application WO 96/15231. Alternatively, application
FR 2,732,347 proposes the use o~f cell culture medium
which is free of animal serum, but which contains plant
extracts.
Thus, due to the use of such media for the cell
culture step, the safety of the products obtained using
the cells thus produced is increased.
However, when it is a matter of producing
viruses forming part of the composition of vaccines,
the steps which are consecutive to this cell culture
phase should themselves also exhibit a maximum degree
of safety.
Specifically, conventionally and as described
in US Patent 4525349, when cells in culture are used
for producing viruses, once the cell culture phase has
been carried out, the medium which was used for the
cell culture is removed and then replaced with a new
medium in which the cells bathe for a moment in order
to be washed; this new medium is then removed; this
cell washing procedure can optionally be repeated a
certain number of times.
Next, a new medium is introduced into the cell
culture, which will allow the cells firstly to survive
and to be infected with the viruses, and secondly to
have at their disposal elements which are sufficient to
be the support for the replication of these viruses.
However, as is described in the article "Protein-free
culture of Vero cells: A substrate for replication of
human pathogenic viruses", Cell Biology International,
Vol. 17 No. 9, 2993 pp. 885-895, even when the cell
culture is carried out in protein-free medium, the
culture medium used in the prior art in this viral
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multiplication and propagation step generally contains
foetal calf serum or, to limit the contamination
problems, at least human albumin. The use of human
albumin at this stage of a method for manufacturing
viral vaccines is considered to be without risk for the
quality of the vaccine. However, with the aim of
completely eliminating all risk, even theoretical, it
would be desirable to have a medium which is suited to
the propagation and multiplication of viruses on a
support consisting of cells in culture, which is free
of human or animal protein, even of recombinant origin,
but which nevertheless allows production yields which
are compatible with industrial requirements so as not
to increase the costs of producing vaccines comprising
viruses produced on cells in culture.
The aim of the invention is in particular to
provide such a medium.
Another aim of the invention is to provide a
medium and a method for viral propagation and
multiplication which then allows, when it is desired, a
virus inactivation step to be carried out.
To achieve these various aims, a subject of the
present invention is a medium for viral propagation and
multiplication on cells in culture, characterized in
that it is free of human or animal proteins, even of
recombinant origin, and in that it comprises elements
of plant origin.
According to one characteristic of the
invention, the medium for viral propagation and
multiplication comprises proteins or glycoproteins
extracted from potatoes or from cucumber, having a
mitogenic potential and having a molecular weight
between 1000 and 200,000 daltons.
According to one particular embodiment, the
medium according to the present invention comprises
plant hydrolysates.
According to one particular characteristic of
the invention, the proteins or glycoproteins are
extracted from potatoes.
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According to another characteristic of the
invention, the proteins or glycoproteins are obtained
in the following way:
- washing and grinding of plants,
- dilution in aqueous medium,
- heat-coagulation of the mixture obtained,
- purification by chromatography.
According to one characteristic of the
invention, the plant hydrolysates are obtained by
chemical and/or enzymatic hydrolysis of raw materials
such as cotton, soybean, wheat or rice. Particularly
good results have been obtained with the products of
the HyPep~ range supplied by the company Quest
International, in particular with the products
comprising a high proportion of peptides with a
molecular mass lower than 1000 daltons.
According to another characteristic, the medium
for viral propagation and multiplication according to
the invention is particularly suited to viral
multiplication on Vero cells.
According to another characteristic, the medium
for viral propagation and multiplication according to
the invention is particularly suited to the
multiplication of Japanese encephalitis viruses,
poliomyelitis viruses and rabies viruses.
A subject of the present invention is also a
method for manufacturing viral vaccines which comprises
a phase of viral production on the cells in culture,
characterized in that the phase of virus propagation
and multiplication is carried out in the presence of a
medium which is free of human or animal protein, even
of recombinant origin, and which comprises plant
extracts.
The present invention will be better understood
upon reading the description which will follow.
The medium and the method according to the
invention, are suited to the production of all viruses
which are able to replicate on cell cultures. They can
in particular be viruses belonging to the following
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families: orthomyxovirus, paramyxovirus, reovirus,
picornavirus, flavivirus, arenavirus, herpesvirus,
poxvirus and adenoviruses. They can also be recombinant
viruses. In particular, the invention is particularly
suited to the production of viruses which can form part
of the composition of vaccines, and in particular of
human vaccines for which the safety requirements are
maximal. They can in particular be the poliomyelitis,
rabies, Japanese encephalitis, yellow fever,
r~.~b~ll ~, mumps, Dengue fever or measles viruses, the
viruses of the various forms of hepatitis, the AIDS or
chickenpox viruses, herpesvirus, viruses of diseases
caused by the respiratory syncytial virus,
cytomegalovirus, EBV, rotaviruses or the influenza.
The present invention is particularly
advantageous for producing the viruses which are
responsible for poliomyelitis, rabies, Japanese
encephalitis, ru b ella , chickenpox, hepatitis A,
influenza, Dengue fever, measles or mumps.
The cells which can be used for virus
production according to the invention are all cells
which can multiply in culture and which are permissive
to the virus whose production is desired. They can in
particular be Vero cells, CV-1 cells, LLC-MK2 cells,
MDCK cells, MDBK cells, WI-38 cells, MRC5 cells (human
fibroblasts) or BHK21 cells. Particularly good results
have been obtained with Vero cell cultures or MRCS cell
cultures.
The cell culture can be carried out according
to the various techniques usually used; in a cell-
cultivator, in roller bottles, in Roux flasks, in
MultitraysT"', in Cell-CubesT"'. For industrial reasons, it
is preferred to carry out the cell culture in cell-
cultivators using microsupports consisting of, for
example, CytodexTM beads.
This cell culture can be carried out under
various conditions, in particular with regard to the
medium used; it is in fact possible to implement the
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present invention even if the culture medium used for
the cell growth phase contained serum or at least human
or animal proteins; in this case, of course, it will be
necessary to carry out thorough washing of the cells
before their infection, so as not to lose the advantage
provided by implementing the invention with regard to
the absence of risk of contamination. However, the use
of cell cultures obtained using a cell culture medium
which is also free of proteins of human or animal
origin is preferred.
Depending on the nature of the cells used and
the viruses to be produced, the most appropriate moment
for carrying out the virus infection with an inoculum
of the virus may vary. Generally, this procedure is
carried out rather in the middle or at the end of the
exponential growth phase; it has in fact been noticed
that, under these conditions, the results obtained are
particularly satisfactory.
According to the invention, the culture medium
used for the step of viral propagation and
multiplication is a medium which is free of human,
animal or recombinant protein, but which comprises
proteins or glycoproteins extracted from potatoes or
from cucumber, having a mitogenic potential and having
a molecular weight between 1000 and 200,000 daltons
and/or hydrolysates of plant extracts. This medium is a
medium which can comprise all or part of the elements
conventionally used for viral multiplication, such as
HAM F 12 medium, but which is supplemented with plant
extracts obtained for example in the following way:
- reduction of the plant pulp,
- treatment of the pulp obtained with a solvent,
- evaporation of the solvent,
- purification of the extract obtained by
chromatography.
The mitogenic potential of the proteins or
glycoproteins which are suitable for the purposes of
the invention can be assessed in particular using
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assays for analysing cell growth and mitogenic activity
such as M.T.T. coloration assays.
The plant extracts which are suitable for the
purposes of the invention are in particular those
described in Patent Application FR 2,732,347. The
products sold by the company Biomedia under the name
GCR1003, TCR1005 or BCR1008 can also be used, alone or
in combination, or the product Prolifix which is also
sold by this company and which comprises all the
nutrient elements of conventional HAM F 12 medium
supplemented with suitable plant extracts.
Alternatively, it is possible to use a
conventional HAM F 12 medium supplemented with plant
hydrolysates such as the HyPep'~ products in a
proportion of 5 g/1.
It is also possible to use a medium comprising
both the proteins or glycoproteins obtained by heat-
coagulation of the plant pulp and plant hydrolysates
such as those mentioned above.
In addition, it is possible to add, to these
media, any other element which is likely to improve or
facilitate one of the steps leading to the production
of a vaccine, just as it is possible to slightly modify
this medium without changing the properties thereof.
The examples which follow illustrate
embodiments of the invention, in a non-limiting way.
EXAMPLE 1: PRODUCTION OF VACCINES AGAINST JAPANESE
ENCEPHALITIS
Vero cells are used to produce the virus which
is responsible for Japanese encephalitis. The cells
used are cells derived from the strain distributed by
ATCC (American Type Culture Collection) under No.
ATCC-CCL 81-VERO F 1415, which is at the 124th passage
and which is taken to the 137th passage in the
conventional way. The virus whose production is desired
is the virus of P3 strain, at the 88th passage, supplied
by NVSI.
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The reservoir of a 15-litre cell-cultivator is
filled with Iscove medium supplemented with 4~ calf
serum containing Cytodex Z~ microcarriers at a
concentration of 3.5 g/1. The medium is seeded with an
inoculum of Vero cells in a proportion of 200,000
cells/ml.
The culture is amplified and subcultured twice
a week every 4 to 5 days. The cell concentrations which
are obtained are between 2 and 3x106 per ml. The cell
suspension obtained is then rinsed with the medium for
viral propagation, with the aim of removing the maximum
amount of residual calf serum.
In order to test a medium according to the
invention comparatively with a medium of the prior art,
the same procedure is carried out in parallel, in
several identical cell-cultivators, with 2 different
media:
- a medium according to the invention consisting of HAM
F 12 medium (supplied by Life Technologies Gibco)
supplemented with Prolifix II medium supplied by the
company Biomedia, at a concentration which is 10
times that indicated in their 1996 catalogue, in a
proportion of 1 volume of 10-fold concentrated
Prolifix II per 10 volumes of HAM F 12,
- a medium according to the prior art consisting of
Iscove medium (supplied by Life Technologies Gibco)
supplemented with human albumin to obtain a final
albumin concentration in the medium of 0.3~.
After rinsing, a virus inoculum is introduced
into each of the cell-cultivators in an amount
corresponding to an MOI (Multiplicity Of Infection) of
1/6000.
The contents of the cell-cultivators are then
kept stirring at a temperature of 37°C for 3 days, and
then the 1St harvest is carried out by recovering the
medium for viral propagation, which has become a viral
suspension, which is replaced in each of the cell-
cultivators with an equal amount of fresh medium of the
same nature; a harvest of the medium for propagation is
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then carried out every 24 hours, until 5 harvests have
been recovered. Each of the harvests is filtered
through a filter with a cut-off threshold of 0.22 ~.m,
and stored at +5°C while waiting to be mixed with the 4
other harvests from the same cell-cultivator.
~nlhen the 5 harvests have been carried out, they
are mixed together and this mixture is then
concentrated by filtration up to a factor of 100. The
mixture consisting of the 5 harvests from the same
cell-cultivator constitutes a batch.
In order to determine the amount of viruses
produced, an infectious activity assay is carried out
for each of the batches produced. The assay is carried
out in 2 different ways: either by infectious activity
measured on a cell layer, or by infectious activity
measured in mice by intracerebral inoculation into the
mice, observation of the animals for 14 days and
calculation of the LD50.
The results are expressed as Log 10 of
infectious particles per litre of cell-cultivator. 4'Then
the titration on cells is considered, the mean of the
results obtained for the various batches produced using
a medium according to the invention is 8.84, whereas
the mean obtained for the batches produced using a
medium according to the prior art is 7.85. If the
titration in mice is considered, the mean for the
batches produced according to the invention is this
time 9.22, whereas the mean for the batches produced
with a medium according to the prior art is 9.30. It is
thus observed, unexpectedly, that the results obtained
with the medium according to the invention are
equivalent to those obtained with a medium of the prior
art, whereas the consequence of the deficit of albumin,
which is a protein considered to play an important role
in transporting nutrient elements inside the cell, was
expected to be a considerable decrease in the amount of
viruses produced.
The batches obtained were then inactivated by
adding a formaldehyde solution in an amount giving a
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final concentration in the mixture of 1/4000 and kept
at room temperature with continuous stirring for 14
days. The verification of the inactivation by means of
a control according to the protocol recommended by the
WHO (Technical Bulletin 771 from 1988) showed that the
batches produced complied with the WHO recommendations.
These inactivated batches were then purified by
ion exchange chromatography and by gel filtration in
order to remove therefrom all the viral proteins and
residual impurities which are not desired in the
vaccines. The mixture obtained was then formulated so
as to allow the production of vaccine doses. The good
quality of the vaccines produced was verified using the
assay of immunogenicity in mice which is recommended by
the WHO for the vaccines against Japanese encephalitis,
and which consists in determining the amount of
neutralizing antibodies produced by immunized mice.
Since the activity of the vaccines produced according
to the method of the invention is not lower than that
of the vaccines of the prior art, this confirms that it
is possible, using the medium and the method according
to the invention, to produce viral vaccines under
conditions of increased safety by dispensing with the
use of albumin.
EXAMPLE 2: COMPARISON OF VARIOUS MEDIA ACCORDING TO THE
TTTS 7L'TTT T rITT
To comparatively test various media, 150-cm3
flasks are used, which are filled with 50 ml of Iscove
medium supplemented with 4~ calf serum. This medium is
seeded with a Vero cell inoculum in a proportion of
250, 000 cells/cm2. The flasks are left for 4 to 5 days
at 37°C and are then rinsed by means of the medium for
viral multiplication and propagation.
The media used are as follows:
- a medium A, which is a medium according to the prior
art, consisting of Iscove medium (supplied by Life
Technologies Gibco) supplemented with human albumin
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to obtain a final albumin concentration in the medium
of 0.3~,
- a medium B consisting of HAM F 12 medium supplemented
with the following mitogenic molecules:
* BCR 1008 in a proportion of 10-3 g/1
* TCR 1005 in a proportion of 10-4 g/1
* GCR 1003 in a proportion of 10-4 g/1
- a medium C consisting of the abovementioned medium
(HAM F 12 supplemented with BCR 1008, TCR 1005 and
GCR 1003), but also comprising 1 g/1 of the HyPep
4602 product which consists of gluten hydrolysate
supplied by the company Questel International,
- a medium C' which is identical to the medium C, but
in which the HyPep 4602 concentration is 5 g/1
- a medium D consisting of HAM F 12 medium supplemented
with HyPep 4602 in a proportion of 1 g/1, but lacking
the mitogenic molecules BCR 1008, TCR 1005 or GCR
1003
a medium D' which is identical to the medium D, but
in which the HyPep 4602 concentration is 5 g/l.
After rinsing, an inoculum of Japanese
encephalitis virus which is identical to that of the
previous example is introduced into each of the flasks
in an amount corresponding to an MOI of 1/6000.
The contents of the flasks are then maintained
for 3 days at 37°C, before the 1St harvest is carried
out. The subsequent harvests are carried out in the
same way as that described in the previous example.
The assays for determining the infectious titre
are carried out on BHK21 cells and give the following
results, which are expressed in TCIDSO/ml (Log 10):
Medium A: 4.6
Medium B: 5.1
Medium C: 5.0
Medium C': 5.6
Medium D: 5.4
Medium D': 6.3.
These results confirm that the media according
to the invention make it possible to obtain yields
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which are as good as, if not better than, the media
according to the prior art which contain albumin.
EXAMPLE 3: PRODUCTION OF VACCINE AGAINST RABIES
A Vero cell culture is prepared in a 2-1 cell-
cultivator in conventional medium and, after washing,
the cells are then infected with rabies virus with an
MOI of 1/1000 and are maintained in a medium which is
identical to that described in Example 1 (i.a. HAM F12
medium supplemented with Profilix~ II); the same
experiment is carried out in an entirely identical
manner, in another cell-cultivator of the same volume,
the only difference being the use of a medium for viral
propagation and multiplication of the prior art
comprising human albumin. Harvests are successively
carried out 4, 5, 6 and 7 days after infection. The
counts carried out on the harvests obtained using the
medium according to the invention and on the harvests
obtained using the medium according to the prior art
made it possible to obtain comparable results with both
media; similarly, the infectious titres of the viruses
obtained were equivalent.
These results thus demonstrate the possibility
of producing virus against rabies on cells.
EXAMPLE 4: PRODUCTION OF VACCINE AGAINST POLIOMYELITIS.
Tests for production of type 1 poliomyelitis
virus on Vero cells in culture were carried out. The
infectious titres of the viruses obtained using a
medium for viral propagation and multiplication
according to the invention having the composition
described in Example 1 were equivalent to the mean of
the infectious titres obtained with the harvests
carried out with the medium used for manufacturing the
currently commercially available vaccines.
