Abstract:
This invention relates to high throughput methods of determining a viral titer. The instant invention addresses the need for a more rapid and cost effective method of quantitating infectious viral particles in a sample.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. provisional application 62/196,886 filed on Jul. 24, 2015. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This invention relates to high throughput methods of determining a viral titer. The instant invention addresses the need for a more rapid and cost effective method of quantitating infectious viral particles in a sample. 
       BACKGROUND OF THE INVENTION 
       [0003]    A particular challenge in the delivery of a gene by a viral vector or a virus for therapeutic purposes is the preparation and accurate quantification of clinical dosage forms. The production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to the ever changing demands and applications. 
         [0004]    Virus titer determination or virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. Traditional methods include viral plaque assays which determine the number of plaque forming units (pfu) in a virus sample and the Tissue Culture Infective Dose (TCID 50 ) or Fluorescence Active Infectious Dose (FAID50) which measures the infectious virus titer. This TCID 50  assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. The traditional methods are generally slow and labor-intensive, and suffer from limitations including a high degree of inter-assay variability. 
         [0005]    Enzyme-Linked Immunosorbent Assay (ELISA) is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme&#39;s ability to convert a reagent to a detectable signal that can be used to calculate the concentration of the antigen in the sample. The plate assays of the virus titer determination that are based on immunofluorescence detection using an ELISA are developed, however they are used to quantify proteins from virus samples and not to quantify infectious viruses. 
         [0006]    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, U.S. Pat. No. 6,248,514 discloses the use of flow cytometry to analyze cells infected using specified ranges of viral particle concentration and adsorption time yields. U.S. Pat. No. 7,476,507 discloses FACS-based methods for the determination of the viral titer of a culture of host animal host cells infected with a circovirus. However, for many applications, the cost, size and complexity of the flow cytometry instruments prevent wider use. 
         [0007]    Recently, personal image cytometers are developed which combine the functions of a digital microscope, an image cytometer, and a cell counter in a single benchtop instrument. The commercially available image cytometers include CellInsight (ThermoFisher) and Cytell Cell Imaging System (GE Healthcare). These image cytometers are used to make individual cell, subcellular and multi-cellular measurements, and provide the opportunity for scientists to capture cellular and subcellular image data and analyze results with ease. 
         [0008]    The instant invention addresses the need for an accurate and high throughput method of quantitating infectious viral particles in a sample. 
       SUMMARY OF THE INVENTION 
       [0009]    This invention provides high throughput methods for the rapid determination and quantification of the infectious virus produced in a sample. The invention also provides high throughput methods for the rapid determination and quantification of the infectious virus produced in a batch culture such that an optimum harvesting point can be obtained. The methods encompass providing a sample containing a virus, preparing serial dilutions of the sample, infecting host cells therewith and incubating the culture at an appropriate temperature, reacting an antigen expressed by the virus in infected cells with an antibody labeled with a fluorescent tag, determining the number of fluorescence tagged cells, thereby determining the virus titer in the sample. The high throughput methods may be carried out in a multiple-well plate format. In one embodiment, CellInsight (ThermoFisher) is used. 
         [0010]    In one aspect of the embodiment, a straight line is plotted using linear regression between number of fluorescence cells and virus titer using a virus reference. 
         [0011]    In another aspect of the embodiment, at least one of the serial dilutions of the sample falls within the range of the liner regression line. 
         [0012]    In yet another aspect of the embodiment, the multi-well plates are used. 
         [0013]    In yet another aspect of the embodiment, the number of infected cells in each well is determined by an image cytometer, such as CellInsight. 
         [0014]    The virus can be wild-type virus, attenuated virus, or recombinant virus. When the virus is a recombinant virus, the antigen can be encoded by an exogenous gene. Typically the antigen reacts with at least one antibody, although the antibody can be a mixture of antibodies. The antibody can be polyclonal or monoclonal. 
         [0015]    The sample can be a sample collected from fields, sample from optimization process, seed sample, main culture sample, active ingredient, or final product. 
         [0016]    These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0017]    The following detailed description, given by way of examples, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, incorporated herein by reference, in which: 
           [0018]      FIG. 1  illustrates a sample of dilutions. 
           [0019]      FIG. 2  illustrates a sample of plate design in a conventional plate assay. 
           [0020]      FIG. 3  illustrates a sample of plate design in high throughput virus titration. 
           [0021]      FIG. 4  illustrates a sample of plate design in high throughput virus titration. 
           [0022]      FIG. 5  depicts the linear regression of CPV2. 
           [0023]      FIG. 6  depicts the comparison between conventional virus titer method and high throughput virus titer method for CPV2. 
           [0024]      FIG. 7  depicts the linear regression of Pox virus (Avian ALVAC) expressing an equine influenza antigen. 
           [0025]      FIG. 8  depicts the comparison between conventional virus titer method and high throughput virus titer method for Pox virus (Avian ALVAC) expressing an equine influenza antigen. 
       
    
    
     DETAILED DESCRIPTION 
       [0026]    It is noted that in this disclosure and particularly in the claims, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. 
         [0027]    The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. 
         [0028]    The term “imaging cytometer” or “Cell imaging system” as used herein refers to any device that will irradiate a particle suspended in a fluid medium with light at a wavelength, and is capable of detecting a light at the same or a different wavelength, wherein the detected light indicates the presence of a cell or an indicator thereon, and take images of the number of cells using optical microscopy. The indicator on the cell surface may be an antibody coupled to a fluorophore such as, but not limited to, FITC, Hoechst stain, DAPI, GFP, TRITC and Cy5. The “imaging cytometer” or “image cytometer” may be coupled to a data visualization, data storage, and data management system for real-time analysis. 
         [0029]    The term “host cell” as used herein refers to an isolated cell that is a host for the infection and replication of a virus. A “host cell” denotes a prokaryotic or eukaryotic cell. The “host cell” may be genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. The “host cell” may be a cell established in stable lineage without any genetic modifications, only by repeated culture passages. The “host cell” may be primary cells that are neither modified nor stable. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof. 
         [0030]    The term “in-process” refers to the monitoring of parameters that are characteristic of cell and virus culture, including a virally infected cell culture, throughout the period of the culture. The monitoring can be continuous, such as monitoring the pH or oxygen content of the culture medium, or can be periodic monitoring wherein samples are withdrawn from the culture at selected time points, parameters such as viability or viral antigen content are detected and measured and the parameters are plotted versus the time of the culture. 
         [0031]    The term “seed culture” or “seed” as used herein refers to a culture of host cells infected with a selected virus and which is then incubated for a period to allow the titer of virus to increase. Typically, but not necessarily, the volume of a seed culture is less than the volume of the subsequent main culture or fermentation medium that receives the seed culture. 
         [0032]    The term “animal” is used herein to include all mammals, birds and fish. The animal as used herein may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), bovine (e.g., cattle, buffalos), swine (e.g., pig), ovine (e.g., sheep), caprine (e.g., goats), camelids (e.g., lamas), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), humans, and fish. The term “animal” also includes an individual animal in all stages of development, including embryonic and fetal stages. The term “pig” or “piglet” means an animal of porcine origin, while “sow” refers to a female of reproductive age and capability. 
         [0033]    Abbreviations: ELISA, enzyme-linked immunosorbant assay; Mab, monoclonal antibody; FITC, Fluorescein isothiocyanate; DAPI: Hoechst stain (blue fluorescent dye); DAPI: 4,6-diamidino-2-phenylindole; GFP: green fluorescent protein; TRITC: Tetramethylrhodamine; Cy5: Cyanine 5. 
         [0034]    The present invention provides high throughput methods for the determination of the viral titer of a sample containing a virus. 
         [0035]    The high throughput methods of the invention comprise the steps of: 1) providing a sample containing a virus; 2) preparing serial dilutions of the sample; 3) infecting host cells with the virus on 96-well plates and incubating the culture; 4) reacting an antigen expressed by the virus in infected cells with an antibody labeled with a fluorescent tag; 5) determining the number of infected cells; and 6) determining the virus titer in the sample. 
         [0036]    Conventional plate assays of the virus titer determination that are based on immunofluorescence detection are time consuming and labour intensive. They involve serial dilutions and many duplicates to count the wells (in 96-well plate) with infected cells. The samples to be tested are limited for each session. 
         [0037]    In one aspect of the embodiment, the number of infected cells is determined by CellInsight (Thermo Fisher Scientific Inc.) using a multi-well plate. The number of infected cells in each well is read using CellInsight. The multi-well plate may be a 48-well plate, or a 96-well plate. 
         [0038]    In another aspect of the embodiment, a straight line is plotted using linear regression between number of fluorescence cells and virus titer using a virus reference. In one aspect, at least one of the serial dilutions of the sample falls within the range of the liner regression line. The number of infected cells per well for the at least one dilution of the sample may preferably fall between 0.1 Log 10  and 6.0 Log 10 . 
         [0039]    The method of the invention allows the detection and monitoring of a virus and infected cells by detecting an antigen expressed by the virus in the host cells until the harvest point. The rapidity with which these data are obtained permits frequent periodic monitoring of the progress of the infection of the cell culture. The culture may then be harvested at a point that gives a high yield of virus suitable for vaccine development and production or for seeding a large batch host cell culture for the ultimate production of virus of sufficient quantity for use in, for example, vaccine development and production. 
         [0040]    The seed cultures developed as a result of the use of the methods of the invention can be used to seed large volume fresh cell culture medium, in the order of 5-1000 liter volumes or more. In this procedure, the seed culture harvested is inoculated into a large-scale culture comprising cell culture medium. 
         [0041]    This invention, therefore, provides a high throughput procedure for the in-process monitoring and rapid determination of the useful harvesting point of a cell culture infected with a virus such that an optimum yield of the virus can be obtained. The methods encompass providing a seed culture of a virus infected host cells and inoculating a batch culture therewith, incubating the culture, removing samples of the cultured cells, preparing serial dilutions of the samples, reacting an antigen expressed by the virus in infected cells with an antibody labeled with a fluorescent tag, determining the number of fluorescence cells, thereby determining the virus titer in the batch culture. It is contemplated that the methods of the invention can be usefully applied for detecting and in-process monitoring of the production of any virus cultured on isolated host cells and for which there are available viral antigen specific antibodies. 
         [0042]    In the various embodiments of this method of the invention, the viruses can be any viruses, including DNA viruses, RNA viruses, recombinant viruses, viruses carrying transgenes, baculoviruses expressing antigens, etc. The viruses may include, but are not limited to, adenovirus, retrovirus, herpes simplex virus, parvovirus, classical swine fever virus, influenza virus, circovirus (including porcine circovirus PCV1 and PCV2), porcine reproductive and respiratory syndrome virus, rotavirus, bovine viral diarrhea virus, porcine epidemic diarrhea virus, sindbis virus, baculovirus, pseudorabies virus, varicella-zoster virus, cytomegalovirus, vesicular stomatitis virus, hepatitis A, B, and C viruses, pox virus, foot-and-mouth disease virus, bluetongue virus, newcastle disease virus, infectious bursal disease virus, Marek&#39;s disease virus, infectious laryngotracheitis virus, avian paramyxovirus, westnile virus, nipah virus, hendra virus, African horse sickness virus, canine distemper virus, leukemia virus, calicivirus, Schmallenberg virus, and recombinant viruses thereof. 
         [0043]    It should be understood that the present invention is not limited to the specific compositions, equipment or methods described herein and that any composition having formula or method steps equivalent to those described falls within the scope of the present invention. It will also be understood that although the form of the invention shown and described herein constitutes preferred embodiments of the invention, it is not intended to illustrate all possible forms of the invention. The words used are words of description rather than of limitation. Various changes and variations may be made to the present invention without departing from the spirit and scope of the invention. 
         [0044]    The invention is further described by the following non-limiting examples: 
       EXAMPLES 
     Example 1 
     Summary of the Current Virus Titration Plate Assay 
       [0045]    The viruses used in this example were canine parvovirus (CPV) and recombinant pox virus (avian canarypox virus) expressing an equine influenza antigen. The host cell line is canine myeloma cells A72. 
       Host Cells Preparation 
       [0046]    A fresh suspension of host cells was prepared in DMEM medium+5% FCS (Fetal Calf Sera) at a concentration of 60 000 cells/ml. 150 μl of this cell suspension was added into each well of a 96-well plate. 
       Sample Dilutions 
       [0047]    An aliquot was taken from a sample containing a virus and serial dilutions were carried out in micro tubes according to, for example, the table shown in  FIG. 1 . The virus control (virus reference) was diluted following the same procedure. 
       Plate Design 
       [0048]    The prepared dilutions that encompass the expected titer were dispensed into the plate as shown in  FIG. 2 . To each well containing 150 μl of the cell suspension, 100 μl of the diluted samples were added. For cell controls (line H) (at least one cell control per session), 100 μl of dilution medium was added to the cell suspension. The number of sample dilutions was 8. The number of virus control dilutions was 7. The number of wells per dilution is 6. In this plate design, for one plate, total of 42 wells were used for virus control (virus reference), 6 wells were used for cell control, and 48 wells were used to assay one sample. In some cases, 144 wells (one and a half 96-well plates) were used to read one sample, and the resulting three results per sample were used to calculate the average reading of the sample. 
         [0049]    In this method, to calculate a titer of a sample, one or two 96-well plates are needed to obtain an accurate titer determination. 
       Virus Infection of Host Cells in 96-Well Plates 
       [0050]    The plates were incubated for 4 days in an incubator at around 37° C. with 5% CO 2 . At the end of the incubation period, the control wells had to be checked under an inverted microscope for the completion of the cell layer without toxicity and pollution. 
       Fixation of Plates for Immunofluorescence Reaction 
       [0051]    The plates were fixed with acetone. Briefly, the plates were washed once with PBS 2−  Buffer Saline Phosphate without calcium and magnesium. 100 μl/well of cold acetone 90% (10% of water) was added to the plates and the plates were incubated at −20° C. for 30 min. The acetone was discarded and the plates were completely air-dried. 
       Immunofluorescence Reaction 
       [0052]    For the immunofluorescence reaction, 100 μl/well monoclonal antibody (anti-CPV2 for CPV or anti-EIV antigen for the recombinant Canarypox-equine influenza antigen) was added to the plates and the plates were incubated at 37° C. for 30 min. The plates then were washed twice using PBS. 100 μl/well anti-mouse FITC was added to the plates and the plates were incubated at 37° C. for 30 min. The plates were washed with PBS twice and followed by a rapid wash in neutral water. 
       Titer Calculation 
       [0053]    The plates were read by UV light with filter 488-519λ. For identification, the well is positive if one or more cells are fluorescent. 
         [0054]    The calculation of the titer is carried out by angular transformation or according to Karber&#39;s method, for which the formula is: 
         [0000]        T=d+[r/N]×[n+N/ 2]+1 
         [0055]    T=titer 
         [0056]    d=dilution with 100% of wells to show a fluorescent focus 
         [0057]    r=dilution ratio 
         [0058]    N=number of wells per rate dilution 
         [0059]    n=number of wells showing a fluorescence for rate dilution above d 
       Example 2 
     Improved High Throughput Method of Virus Titration 
       [0060]    The viruses used in this example were canine parvovirus (CPV) and recombinant pox virus (avian canarypox virus) expressing an equine influenza antigen. The host cell line is canine myeloma cells A72. 
         [0061]    Host cells preparation was carried out as described in Example 1. 
       Sample Dilution 
       [0062]    Sample dilution was carried out as described in Example 1. 
       Plate Design 
       [0063]    A sample plate is shown in  FIGS. 3 and 4 . In these plate designs, the number of sample dilutions was 4. The number of virus control dilutions was 7. The number of wells per dilution is 2. For one plate, total of 14 wells were used for virus control (virus reference), 2 wells were used for cell control, 8 wells were used for validation control per session (each session may contain multiple plates), and 8 wells were used to assay one sample for a total of 9 samples. Virus reference was used to plot the linear regression line and to calculate the titer of the unknown sample. Cells control were wells without any virus to check that the session worked well. Validation control contains a virus (other than the virus reference) with known titer. Validation control was titrated on each plate and its titer was calculated and the results were included in the chart control. Validation control provides quality control of the session and ensures that the session worked well. 
         [0064]    Virus infection of host cells in 96-well plates, Fixation of plates for immunofluorescence reaction, and Immunofluorescence reaction were carried out as described in Example 1. 
       Titer Calculation 
       [0065]    The number of infected cells per well was read using CellInsight. A straight line is plotted using linear regression between number of fluorescence cells per well and virus titer using the virus control (virus reference) as shown in  FIG. 5  (CPV) and  FIG. 7  (recombinant pox virus). The virus titer of a sample is then calculated using the straight line (linear regression of reference virus) based on the reading of the infected cells per well of that sample and its dilution factor. 
       Results 
       [0066]    The virus titer calculated using the improved method was compared to the virus titer calculated using the conventional method.  FIG. 6  shows the results for CPV.  FIG. 8  shows the results for recombinant pox virus. The results demonstrated that the high throughput method which determines the infected cells collates well with the conventional virus titer determination method (conventional plate assay) which counts the wells containing infected cells (fluorescence tagged cells). 
       DISCUSSION 
       [0067]    This high throughput method provides the advantage of reducing the number of dilutions and duplicates that is necessary to accurately calculate the virus titer, and therefore the increase of number of samples to be titrated on one plate. 
         [0068]    Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof. 
         [0069]    All documents cited therein or during their prosecution (“application cited documents”) and all documents cited or referenced in the application cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer&#39;s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention