Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-09-29
2002-09-24
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S350000, C424S209100
Reexamination Certificate
active
06455298
ABSTRACT:
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 hen' 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 hen' eggs.
The use of embryonate hen' 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 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 cell cultures originating from tested cell banks are pathogen-free and, in contrast to hen' 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 hen' embryo cells and hamster cells (BHK21-F and HKCC), MDBK cells, and in particular 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 Medical Book Company—New York and London, 1987). A prerequisite for a successful infection is the addition of proteases to the infection medium, preferably typsin or similar serine proteases, as these proteases extracellularly cleave the precursor protein of hemagglutinin [HA
O
] into active hemagglutinin [HA
1
and HA
2
]. 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 U.S. Pat. No. 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 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.
The cells according to the invention are preferably vertebrate cells, e.g. avian cells, in particular hen' embryo cells.
In a particularly preferred embodiment, the 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 Jun. 7, 1995 according to the requirements of the Budapest Convention for the International Recognition of the Deposition of Micro-organisms for the Purposes of Pat
Gröner Albrecht
Vorlop Jürgen
Blackburn Robert P.
Chiron Behring GmbH & Co.
Hale Rebecca M.
Harbin Alisa A.
Mosher Mary E.
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