Method of extracting virus from cell culture

Details Method of extracting virus from cell culture Method of extracting virus from cell culture

2002-03-14

2004-10-26

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

Inactivation or attenuation; producing viral subunits

C435S235100, C435S239000, C424S215100

Reexamination Certificate

active

06808916

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of extracting virus from a cell culture. In particular, the method is useful to extract infectious virus which is suitable for clinical administration to mammals, including human.
REFERENCES
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Chandron and Nibert, “Protease cleavage of reovirus capsid protein mul and mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle”,
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Coffey, M. C., et al., “Reovirus therapy of tumors with activated Ras pathway”,
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Strong, J. E. and P. W. Lee, “The v-erbV oncogene confers enhanced cellular susceptibility to reovirus infection”,
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Strong, J. E., et al., “Evidence that the Epidermal Growth Factor Receptor on Host Cells Confers Reovirus Infection Efficiency”,
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All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Due to the vast number of diseases caused by viruses, virology has been an intensively studied field. There has always been the demand to produce viruses efficiently in order to isolate and purify viral proteins, to generate vaccines, or to provide infectious viruses for laboratory studies. Recently, the new development of virus therapy has further necessitated the need for efficient production of infectious viruses.
Reovirus therapy is an example of virus therapy. Reovirus is a double-stranded RNA virus capable of binding to a multitude of cells. However, most cells are not susceptible to reovirus infection and binding of reovirus to its cellular receptor results in no viral replication or virus particle production. This is probably the reason why reovirus is not known to be associated with any particular disease.
It was discovered recently that cells transformed with the ras oncogene become susceptible to reovirus infection, while their untransformed counterparts are not (Strong et al., 1998). For example, when reovirus-resistant NIH 3T3 cells were transformed with activated Ras or Sos, a protein which activates Ras, reovirus infection was enhanced. Similarly, mouse fibroblasts that are resistant to reovirus infection became susceptible after transfection with the EGF receptor gene or the v-erbB oncogene, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Thus, reovirus can selectively infect and replicate in cells with an activated Ras pathway.
The ras oncogene accounts for a large percentage of mammalian tumors. Activating mutations of the ras gene itself occur in about 30% of all human tumors (Bos, 1989), primarily in pancreatic (90%), sporadic colorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia (30%). Activation of factors upstream or downstream of ras in the ras pathway is also associated with tumor. For example, overexpression of HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is common in breast cancer (25-30%), and overexpression of platelet-derived growth factor (PDGF) receptor or EGF receptor is prevalent in gliomas and glioblastomas (40-50%). EGF receptor and PDGF receptor are both known to activate ras upon binding to their respective ligand, and v-erbB encodes a constitutively activated receptor lacking the extracellular domain.
Since a large number of human tumors are accounted for by genetic alteration of the proto-oncogene ras or a high Ras activity, reovirus therapy is a new, promising therapy for such conditions (Coffey et al., 1998). Reovirus therapy is highly selective for Ras-associated tumor cells and leaves normal cells uninfected. This therapy has wide applications and can be used in both human and non-human animals.
In order to produce reovirus suitable for clinical administration, fast and efficient methods of producing reovirus in cultured cells are needed. Moreover, the traditional method of extracting viruses from cultured cells is tedious and time consuming, rendering the cost of virus production too high. Therefore, an improved method for virus extraction is also needed.
SUMMARY OF THE INVENTION
The present invention is directed to a method of extracting virus from a culture of cells. Viruses are traditionally extracted from cells by multiple rounds of freeze-thawing, followed by purification with density gradients and ultracentrifugation. In the present invention, we extracted virus from a culture of cells with a detergent and the resulting yields were better than those obtained by freeze-thawing. Moreover, this extraction step can be performed at or above ambient temperature. In particular, the extraction can take place at a convenient temperature such as 25° C. or 37° C. and still produce high virus titers.
Accordingly, one aspect of the invention provides a method of producing virus from a culture of cells, comprising the steps of:
(a) providing a culture of cells which has been infected by the virus;
(b) extracting the virus from the cells by adding a detergent to the culture; and
(c) collecting the virus.
The detergent may be any detergent commonly used for the disruption of cells. Particularly, the detergent is selected from the group consisting of octoxynol-9 to 10 (Triton X-100), polyethylene glycol sorbitan monolaurate (Tween 20), octylphenoxy polyethoxy ethanol (NP-40) and sodium deoxycholate. The detergent is most preferably Triton X-100, particularly at a final concentration of 1%.
This virus extraction method can be performed at any temperature above freezing, particularly above 4° C. Typically, the extraction can be conveniently performed at ambient temperature, without having to maintain a pre-selected temperature. Preferably, the extraction is performed at 25° C. More preferably the extraction is performed at the same temperature as the cells are cultured, for example 37° C., such that the cell culture and virus extraction can be performed in the same incubator.
The cell culture is incubated with the detergent for a period of time sufficient to disrupt the cells. The incubation period is preferably 60 minutes or less, more preferably 30 minutes or less, and most preferably 10 minutes.
In a preferred embodiment, the virus is a non-enveloped virus. The non-enveloped virus is preferably a reovirus. The present invention can be applied to any reovirus, particularly mammalian reoviruses. The mamma

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