Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-08-18
2001-06-19
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S003000, C435S004000, C435S007100
Reexamination Certificate
active
06248514
ABSTRACT:
BACKGROUND OF THE INVENTION
A particular challenge in the delivery of a gene by a viral vector for therapeutic purposes is the preparation and accurate characterization of clinical dosage forms. Total particle measurement can be made by such techniques as electron microscopy of viral preparations or measurement of total DNA by optical density at 260 nm of a sodium dodecyl sulfate (SDS) treated virus suspension. However, infectivity of a viral preparation, i.e., the number of infectious viral particles in a preparation of virus, is more challenging to accurately measure.
Traditionally, infectivity particles are measured in culture by a plaque-forming unit assay (pfu) that scores the number of viral plaques as a function of dilution. An alternative to the pfu assay is the tissue culture infective dose procedure (TCID
50
), which estimates infectivity as a function of intracellular staining for an antigen by direct immunofluorescence. The methods suffer from limitations including a high degree of inter-assay variability and are affected by such factors as virus replication status, vector characteristics, and virus-cell interactions.
More recently, flow cytometry or FACS (fluorescence-activated cell sorter) assays have been used to measure the number of infected cells in cell cultures infected at relatively high multiplicities of infection. For example, Saalmüller and Mettenleiter (
J. Virol. Methods
44:99-108 (1993)) disclose the identification and quantitation of cells infected by recombinant pseudorabies virus mutants by the reaction of intracellular &bgr;-galactosidase expressed during infection with recombinant viruses with a fluorogenic substrate, followed by detection of positive cells in flow cytometry. Morris et al. (
Virology
197(1):339-48 (1993)) studied the process of productive and non-productive recombinant AcMNPV infection in cultured cells by immunostaining cells to detect the reporter CAT gene product.
The instant invention addresses the need for a more accurate method of quantitating infectious viral particles in a population.
SUMMARY OF THE INVENTION
The present invention provides methods for determining the number of infectious virus particles in a population of virus particles. In some embodiments, the methods involve i) infecting cells in a cell population with virus particles by contacting the cells with a preparation of virus particles, ii) reacting a polypeptide expressed by the virus in infected cells with an antibody labeled with a fluorescent tag, the antibody having specificity for a polypeptide expressed by the virus; and iii) measuring immunofluorescence in the product of step (ii) by flow cytometry to determine the number of infected cells, thereby determining the number of infectious virus particles. In preferred embodiments, the virus particles contacting the cells are present at a concentration of about 2×10
7
particles/ml or less.
The methods of the instant invention are based on the unexpected and surprising result that flow cytometry analysis of cells infected at a low virus to cell ratio yields a more accurate measurement of infectious virus titer than traditional titration methods.
In another embodiment, the invention provides a method for determining the number of infectious virus particles in a population of virus particles in which the method involves: i) infecting cells in a cell population at a total particle to cell ratio of less than about 100:1 to about 0.1:1 to generate infected cells; ii) reacting a polypeptide expressed by the virus in infected cells with an antibody labeled with a fluorescent tag, the antibody having specificity for a polypeptide expressed by the virus; and iii) measuring immunofluorescence in the product of step (ii) by flow cytometry to determine the number of infected cells, thereby determining the number of infectious virus particles.
When the virus is a recombinant virus, the viral polypeptide can be encoded an exogenous gene, such as a reporter gene. In some embodiments of the invention, the exogenous gene is a tumor suppressor gene such as p53 or retinoblastoma (RB). The combinant virus can be replication competent or defective, deficient or incompetent.
In some embodiments of the invention, the virus is adenovirus. Thus, when the infected cells are cultured after infection to allow expression of a viral polypeptide, the viral polypeptide can be an adenovirus polypeptide such as hexon.
Typically the viral polypeptide is reacted with at least one antibody, although the antibody can be a mixture of antibodies. The antibody can be polyclonal or monoclonal.
REFERENCES:
patent: 5470730 (1995-11-01), Greenberg et al.
Saalmuller et al.; “Rapid detection and quantification of cells infected by recombinant herpesvirus . . . ”; J. Vir. Meth.; 44; pp. 99-108, 1993.*
Huyghe et al.; “Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography”; Human Gene Therapy; 6; pp. 1403-1416, 1995.*
Bauer, K.D. et al., “Analysis of Intracellular Proteins,”Methods in Cell Biology41:351-376 (1994).
Eyler, Y.L. et al., “Flow cytometric detection of DNA tumor virus nuclear oncogene products in unfixed cells: saponin FACS of viral oncogene products,”J. Virol. Meth.46:23-27 (1994).
Huyghe, B.G. et al., “Purification of a Type 5 Recombinant Adenovirus Encoding Human p53 by Column Chromatography,”Hum. Gene Ther.6:1403-1416 (1995).
Lynn, D.E. et al., “A BASIC Computer Program for Analyzing Endpoint Assays,”Biotechniques12(6):880-881 (1992).
Maizel, J.V. et al., “The Polypeptides of Adenovirus,”Virology36:115-125 (1968).
March, K.L. et al., “Pharmacokinetics of Adenoviral Vector-Mediated Gene Delivery to Vascular Smooth Muscle Cells: Modulation by Poloxamer 407 and Implications for Cardiovascular Gene Therapy,”Hum. Gene Ther.6:41-53 (1995).
Morris, T.D. et al., “Characterization of Productive and non-productive ACMNPV Infection in Selected Insect Cell Lines,”Virology197:339-348 (1993).
Saalmüller, A. et al., “Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based &bgr;-galactosidase assay and flow cytometry,”J. Virol. Meth.44:33-108 (1993).
Wen, S.F. et al., “Retinoblastoma protein monoclonal antibodies with novel characteristics,”J. Immunol. Meth.169:231-240 (1994).
Wills, K.N. et al., “Gene therapy for hepatocellular carcinoma: Chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymiding kinase gene,”Canc. Gene Ther.2(3):191-197 (1995).
Recombinant DNA: A Short Course,eds. Watson, J.D. et al., W.H. Freeman and Company, New York, New York, pp. 189-190 (1983).
Virology,eds. Fields, B.N. et al., Raven Press, New York, New York, pp. 37-38 (1990).
Hutchins Beth M.
Nunnally Mary H.
Sugarman Barry J.
Canji Inc.
Nelson Brett
Stucker Jeffrey
Towsend and Townsend and Crew LLP
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