MXPA99000396A - Method for measuring inefficiency vi - Google Patents

Abstract

The present invention addresses the need for a more accurate method of quantifying infectious viral particles in a population. The methods of the present invention are based on the result, unexpected and surprising, that the analysis of the flow cytometry of the infected cells in a low ratio of the number of particles to the number of cells, provides a more accurate measurement of the virus titer infectious compared to conventional titration methods

Description

METHOD FOR MEASURING VIRAL INEFFICIENCY BACKGROUND OF THE INVENTION A particular challenge in the delivery of a gene by a viral vector, for therapeutic purposes, is the preparation and exact characterization of the clinical dosage forms. The total measurement of particles can be made by such techniques as the electron microscopy of viral preparations or the measurement of the total DNA by the optical density at 260 nm of a virus suspension treated with sodium dodecyl sulfate (SDS). However, the ineffectiveness of a viral preparation, i.e. the number of infectious viral particles in a virus preparation, is more problematic to measure accurately. The ineffective particles in culture are traditionally measured by an assay of plaque forming units ("pfu"), which classify the number of viral plaques as a function of dilution. An alternative to the pfu assay is the procedure of ineffective doses of tissue cultures (TCID50), which estimate ineffectiveness as a function of the intracellular staining for an antigen by direct immunofluorescence. The method has limitations, which include a high degree of variability. between trials and are affected by such factors as the state of virus response, vector characteristics and virus-cell interactions. More recently, flow cytometry assays or "FACS" (Fluorescence Activated Cell Sorter) have been used to measure the number of infected cells in infected cell cultures at relatively high multiplicities of infection. For example, Saalmüller and Mettenleiter (J. Virol.Methods 44: 99-108 (1993)) disclose the identification and quantification of cells infected by mutants of recombinant pseudorabies viruses, by the reaction of intracellular β-galactosidase, expressed during infection, with recombinant viruses with a fluorogenic substrate, followed by the detection of positive cells in the flow cytometry. Morris et al., (Virology 197 (1): 339-48 (1993)) studied the process of productive and non-productive AcMNPV recombinant infection in cells cultured by immunostained cells, to detect the gene product of chloramphenicol transacetylase ( CAT) of information. The present invention addresses the need for a more accurate method of quantifying infectious viral particles in a population.

COMPENDIUM OF THE INVENTION The methods of the present invention are based on the unexpected and surprising result that analysis of the flow cytometry of infected cells in a low ratio of virus to cell provides a more accurate measurement of the infectious virus titer than the methods of traditional titling. One aspect of the invention relates to a method for determining the number of infectious virus particles in a population of virus particles, comprising: i) infecting the cells in a population of cells in a total ratio of particles to cells smaller than approximately 100: 1 to 0.1: 1, to generate the infected cells; ii) reacting a polypeptide, expressed by the virus in the infected cells with an antibody labeled with a fluorescent tag, this antibody has specificity for a polypeptide expressed by the virus; and iii) measuring the immunofluorescence in the product of step (ii) by flow cytometry, to determine the number of infected cells, thus determining the number of infectious virus particles. Using the virus is a recombinant virus, the viral polypeptide can be encoded by 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 retmoblastoma (RB). The recombinant virus can be a competent or defective, deficient or incompetent response. In some embodiments of the invention, the virus is an adenovirus. A) Yes, when the infected cells are cultured after infection to allow expression of a viral polypeptide, this viral polypeptide can be an adenovirus polypeptide, such as hexon. Typically, the viral polypeptide is reacted with at least one antibody, although this antibody can be a mixture of antibodies. The antibody can be polyclonal or monoclonal. In preferred embodiments of the invention, the total ratio of particles to cells is less than about 100: 1, typically, less than about 10: 1, preferably, less than about 5: 1, and more preferably, less than about 1: 1. In some embodiments, the ratio can be as low as approximately 0.1: 1.

DETAILED DESCRIPTION The present invention provides methods for quantifying infectious viral particles in a population of virus particles. The term "infectious" is used here to refer to the ability of a virus to enter the cells and direct the synthesis of at least one polypeptide encoded by the virus. The ability to reproduce viral nucleic acid is not required, but is included, in this definition. Typically, the virus particles in a preparation are not always infectious. For example, the particles can be damaged in the preparation of the virus, thus not affecting the total number of particles, but decreasing the number of particles capable of infection. Likewise, empty capsids or extracellular virus instability can also contribute to the decrease in ineffectiveness. The range of non-infectious particles to infectious particles in viral preparations can vary from 1: 1 to more than 100: 1. However, even non-infectious viruses can cause cytological changes or damage to exposed cells. Thus, it is advantageous to have an accurate measurement of the number of infectious particles in a population, so as to minimize the number of non-infectious viral particles to which the cells are exposed. Virtually, any virus can be quantified, or titrated, by the methods of the present invention, which include DNA viruses, RNA viruses, competent response viruses, incompetent response viruses, recombinant viruses, viruses carrying transgenes, etc. Preferably, the virus can infect cells in the culture. Some examples of viruses treatable by this technique influence, but are not limited to, adenoviruses, adenococcal viruses, retroviruses, herpes simplex virus, parvovirus, Epstein Barr virus, pnotracheitis virus, parainfluenza virus, bovine diarrhea virus, virus of sindbis, baculovirus, pseudorabies virus, varicella-zoster virus, cytomegalovirus, HIV, hepatitis A, B and C virus and vaccines. In some embodiments of the invention, inefficiency is measured by antibodies directed against a polypeptide expressed by the viruses. This polypeptide may be a structural viral polypeptide, a regulatory polypeptide, a polypeptide such as a polymerase, etc. In some embodiments of the invention, the polypeptide is preferably expressed by an exogenous gene incorporated in the virus, such as a reporter gene. Some examples of reporter genes include ß-galactosidase and chloramphenicol transacetylase (CAT). In further embodiments of the invention, the reporter gene is detected by antibodies directed against a product of the action of the reporter gene, such as the action of an enzyme on a substrate. In other embodiments of such an invention, the exogenous gene is a transgene intended for therapeutic use. Some examples include, but are not limited to, genes tumor suppressors, which include p53 or retinoblastoma (RB); interleukins, which include IL-2, IL-4 and IL-10; interferons, which include alpha-, beta-, and gamma-inter-ferone; other cytokines; thymidine kinase; growth factors, which include GCSF and growth hormone; Factor VIII; adenosine deaminase, etc. The production typically of the polypeptide encoded by a transgene can be measured by an antibody directed against the polypeptide. The antibodies used for detection can be polyclonal, monoclonal or include mixtures of such antibodies. Typically, detection is made directly using a fluorescein conjugated antibody, directed against the viral polypeptide. However, indirect assays are also possible, wherein the antibody directed against the viral polypeptide is then reacted with a fluorescein-labeled antibody. Any fluorescent label compatible with flow cytometry can be used. To carry out the assay of the invention, typically the total number of virus particles in a viral preparation was first measured by any of a number of traditional techniques. For example, an aliquot of a virus preparation can be prepared in a buffer solution containing 0.1% sulfate sodium dodecyl (SDS), after which the optical absorbance was measured at 260 nm (Maizel et al., in Virology 36: 115-125 (1968)). The total particle count can be obtained by preparing a sample of the viral preparation by electron microscopy, and simply counting the number of particles. A further technique for particle enumeration may include the use of anion exchange chromatography (Huyghe et al., In Human Gene Therapy 6: 1403-1416 (1995)). The cells are then infected with dilutions of the viral preparations in total ratios of the number of particles to the number of cells no greater than about 100: 1, typically less than about 10: 1, preferably less than about 5: 1, more preferably less than approximately 1: 1. In some modalities, the ratio is as low as approximately 0.1: 1. Typically, at least one infection will be performed, although, in some embodiments, at least two parallel infections are performed at different ratios of particles to cells. The cells used are typically known to be sensitive to virus infections. Cells are not required to support the response by the virus, but infection is carried out under conditions that allow the expression of the virus polypeptide to be detected.

The total volume of a virus preparation used to the infected cells in cultures is typically determined by an expert artisan taking into account such factors as the total number of cells to be infected, the concentration of particles of the virus preparation and the volume of the container in which the infection is to be carried out. Preferably, the concentration of virus particles used to infect cells in the infection mixture is at least about 10 ^ particles per milliliter, more preferably at least about 106 particles per ml, and especially preferred about 107 particles per ml. ml. Viral preparations are typically prepared under favorable conditions for virus stability. Conditions for infection and, optionally, culture after infection, depend on the particular virus and the viral or reporter gene used for detection. The term "cultures", as used herein, refers to any form of cell culture in which the minimum requirements are supplied to the cells to enable continued survival during the period of interest. Thus, for example, the cultures can refer to the preparation of a cell suspension in a suitable buffer solution, such as a phosphate buffered saline solution, or an incomplete growth medium, over a period of minutes or hours, or refer to the cells that adhere to the culture dishes for minutes to days to weeks, in the presence of a suitable complete growth medium. Typically enough time is provided in the culture for the expression of the desired viral polypeptide, but preferably not enough time is given for the preparation of the infectious virus, which results in further infection of the cells. Thus, it is preferable that only "one round" of infection occurs in these cells. In some modalities, the length of time allowed in "the crop" will be less than 1 hour to several hours. In other preferred embodiments, the length of time will be from 1 to 5 days. Typically, the cells are infected under conditions that favor adsorption of the virus to the cells, although less optimal conditions may be used in some embodiments. Typically, viruses are allowed to adsorb into cells for 1-12 hours. In some embodiments, the cells are infected in a concentrated suspension with concentrated viruses, to increase the rate of infection or the number of infected cells, then diluted to a concentration more favorable for cell or viral growth. In some embodiments of the invention, it may be convenient to wash the cultures of infected cells to remove non-absorbed viruses or components of the medium used for the infection, or to expose the infected cells to means or conditions of more favorable growth for their survival. After sufficient time has elapsed to allow expression of the viral polypeptide, the cells are typically prepared as a suspension of single cells. When the cells are infected as adherent cells in tissue cultures, these cultures are typically treated with a dissociating agent, such as trypsin, to deplete the cells of the substrate. Mechanical means can also be used to detach the cells, such as scraping. The cells are then harvested by centrifugation and prepared in a buffer solution, such as an incomplete or complete growth medium, for reaction with the detection reagents. The cells are typically "fixed" for immunostaining by any of a number of standard techniques. A review of the commonly used binding techniques is provided by Bauer and Jacobberger in Methods in Cell Biology 41: 351-376 (1994)), incorporated herein by reference in its entirety for all purposes. When the polypeptide is detected by its activity, fluorescence reagents can be introduced into the cells to allow detection of activity, such as a substrate labeled with fluorescein for an enzyme.

The populations of infected cells are then subjected to analysis by standard flow cytometry, such as by the methods disclosed by Shapiro in Practical Flow Cytometry 3a Ed., John Wiley and Sons (1994), incorporated herein by reference in their totality for all purposes. The term "FACS" is sometimes used to refer to flow cytometry, although cell sorting is not required in the practice of the present invention. Typically, a minimum of approximately 10,000 events was acquired in the analysis. The death cells were typically excluded from the analysis, or by the input of the forward / lateral scatter or the labeling of Pl and adjustment of the electronic window in the negative fraction of Pl. A variety of commercial software packages (programs) are available to assist in the preparation and analysis of the data, such as the CellQuest ™. The following example attempts to illustrate, but not limit, the invention in any way.

EXAMPLE In this example, the ACNRB, a recombinant response defective adenovirus, was titrated by the TCID50 and by the method of low ratio of the number of particles to the number of cells (low ratio) of the present invention. The exemplary virus used comprised essentially the skeleton of the adenovirus vector disclosed by ills et al., in (Cancer Gene Therapy 2: 191-197 (1995)) with the full-length retinoblast cDNA inserted into the vector. The total number of particles was obtained by the method of "SDS / OD260" and the methods of chromatography. of ion exchange, described above. In both tests, the measured concentration of total particles was 1.0 x 1012 / ml. Infectious particles were titrated with the TCID50 assay, as described by Huyga et al.

(Human Gene Therapy 6: 1403-1416 (1995)). Briefly, 293 cells were placed in a 96-well microtiter plate: 100 μl of 5x10 ^ cells / ml, for each well in serum the complete MEM medium (10% calf bovine serum, 1% glutamine), ( GIBCO BRL). On a separate plate, a 250 μl aliquot of the virus sample, diluted 1: 106 was added to the first column and serially diluted twice through the plate. Seven rows were used for the samples. A row was used for a negative control. An aliquot of 100 μl of each well was transferred to its identical position in plaque 293 planted and allowed to incubate at 37 ° C in a humidified air / 7% CO2 incubator for 2 days. The medium was then decanted by inversion and the cells were fixed with 50% acetone / 50% methanol. After washing with PBS, the fixed cells were incubated for 45 minutes with anti-Ad5 antibody labeled with FITC (Chemicon International # 5016), prepared according to the instructions of the kit. After washing with PBS, the plate was examined * with a fluorescence microscope (490 mm excitation, 520 mm emission) and classified in the presence of the label. The title was determined using the Titerprint Analysis program (Lynn, Biotehcniques 12: 800-881 (1992)). The low ratio assay was performed as follows. 293 cells of 1 x 106 (human embryonic kidney cells, ATCC CRL 1573) were seeded per well in 4 6-well disks. The final volume per well was 1 ml. After about 2 hours, the medium (a Dulbecco's modified Eagle medium (DME high glucose) containing 4500 mg / ml D-glucose, supplemented with 5% beef calf serum supplemented with iron, 2mM L- glutamine and 1 mM sodium pyruvate) in each well, aspirated and replaced with 1.1 ml of the medium (without serum) containing the diluted virus. The adsorption was allowed to occur for 60 minutes, after which an additional 2 ml of the virus-free medium was added to each well. After about 42 hours, the infected cell cultures were processed by flow cytometric analysis.

The cells were detached from the plastic substrate with a solution of tppsma-EDTA (GIBCO-BBL). The detached cells were collected from each well and centrifuged at approximately 200 x g for 10 minutes, at room temperature. The supernatants were removed and the cells were washed in a Dulbecco's phosphate-buffered solution (D-PBS) without calcium or magnesium salts. The pelleted cells were then resuspended in 2 ml of the cold acetone-methanol fixative agent (1: 1), then kept on ice for 15 minutes. 7 ml of D-PBS without calcium or magnesium salts were added to each tube, after which the Leron cells were resuspended in D-PBS with 1% calf serum (vol./vol.). After repeating these last two steps, the cells were resuspended in 50 μl of D-PBS with 1% calf serum 70 μl of the anti-adenovirus antibody conjugated with FITC (Chemicon # 5015) in 2.0 ml of D-PBS were added to each tube. The samples were incubated at 37 ° C for about 50 minutes. The samples were then transferred to flow cytometry analysis tubes, slightly diluted with 0 5 ml of D-PBS, and analyzed by flow cytometry. A flow cytometer system, Becton Dickinson FACScan1M, PN 34011570, 12-00189-01 with FACStation (MAC QUADRA 650 computer, monitor and printer) was used with the software (CellQuest1.

The results are shown in Table 1. By the traditional TCID50 assay, the ratio of the total number of particles to the infectious unit was 63: 1. As is evident in the table, as the ratio of the total number of particles to the number of cells decreases, the ratio of the total number of particles calculated to infectious unit also decreases to as much as L2: 1, thus providing a value for the title of infection that was approximately 5 times higher than in the traditional trial. Thus, this low ratio assay provides an unexpectedly better (ie much more accurate) enumeration of the number of infectious particles in a viral preparation compared to traditional titration methods. The consequences of such exact measurements tested by the present invention are especially important in the calculation of effective doses of recombinant viruses for therapeutic use. All references cited herein are expressly incorporated by reference in their entirety for all purposes.

TABLE 1 DETERMINATION OF THE INFECTIOUS TITLE Calculated Title (Test TCID50 1.6x10 0 IU / ml Concentration of No. of Particles 1.0x10 2NP / ml Ratio NP / IU 63: 1

Claims (16)

1. CLAIMS 1. A method to determine the number of infectious virus particles in a population of virus particles, this method comprises: i) infecting the cells in a population of cells in a total ratio of the particles to the number of cells less than about 100 : 1 to 0.1: 1, to generate the infected cells; ii) reacting a polypeptide, expressed by the virus in the infected cells, with an antibody labeled with a fluorescent tag, this antibody has specificity for a polypeptide expressed by the virus; and iii) measuring the immunofluorescence in the product of step (ii) by flow cytometry, to determine the number of infected cells, thus determining the number of infectious virus particles.
2. 2. The method of claim 1, wherein the virus is the adenovirus.
3. 3. The method of claim 2, wherein the viral polypeptide is hexon.
4. 4. The method of claim 1, wherein the cells are cultured, after infection, to allow expression of the viral polypeptide.
5. 5. The method of claim 1, wherein the virus is a recombinant virus.
6. 6. The method of claim 5, wherein the viral polypeptide is encoded by an exogenous gene.
7. 7. The method of claim 6, wherein the exogenous gene is a reporter gene.
8. 8. The method of claim 6, wherein the exogenous gene is p53.
9. 9. The method of claim 6, wherein the exogenous gene is retinoblastoma.
10. 10. The method of claim 1, wherein the antibody is a mixture of antibodies.
11. 11. The method of claim 1, wherein the antibody is polyclonal.
12. 12. The method of claim 1, wherein the antibody is monoclonal.
13. 13. The method of claim 5, wherein the recombinant virus is defective in the response.
14. 14. The method of claim 1, wherein the ratio is less than about 10: 1 to about 0.1: 1.
15. 15. The method of claim 1, wherein the ratio is less than about 5: 1 to about 0.1: 1.
16. 16. The method of claim 1, wherein the ratio is approximately 0.1: 1.