DETECTION OF VIRAL STABILITY

Abstract: Viral induction of caspase 3 activity was found to provide a reliable measure of viral activity. Assaying viral induction of caspase 3 activity can be used, for example, in methods for measuring viral potency and stability, and for evaluating the stability of a virus in different formulations.

Description: TITLE OF THE INVENTION DETECTION OF VIRAL STABILITY FIELD OF THE INVENTION The present invention pertains to assays for measuring viral potency and stability and for evaluating the stability of a virus in different formulations. BACKGROUND OF THE INVENTION The references cited herein are not admitted to be prior art to the claimed invention. In developing live virus vaccines it is important to determine viral potency and stability to assure adequate immunization. The plaque assay is a common method for determining viral potency and stability. The plaque assay is often used to examine the effect of different storage conditions on viral stability. The plaque assay involves inoculating viruses onto a cell sheet at dilutions determined by the expected (estimated) potency. The plates containing the infected cells are incubated for a specified length of time and then stained. Viral infection results in plaque formation, or areas of dead or detached cells. Counting the number of plaques gives the corresponding plaque-forming units (PFU), a measure of potency. Viral stability is determined by examining the change in PFU over time. SUMMARY OF THE INVENTION Viral induction of caspase 3 activity was found to provide a reliable measure of viral activity. Assaying viral induction of caspase 3 activity can be used, for example, in methods for measuring viral potency and stability, and for evaluating the stability of a virus in different formulations. Thus, a first aspect of the present invention describes a method of assaying the potency and stability of a virus by measuring viral induction of caspase 3 activity. The method involves infecting a plurality of cells susceptible to caspase 3 induction with the virus and measuring caspase 3 activity as an indication of viral potency and stability. A plurality of cells is a population of the same strain of cells. Another aspect of the present invention describes a method of identifying a stabilizing formulation for a virus. The method involves infecting a strain of cells susceptible to caspase 3 induction with a virus in different test formulations. The strain of cells is provided as different pluralities of cells where each plurality of cells is infected with the virus. Induction of caspase 3 activity produced from virus stored in the different formulations is measured to identify the formulations that stabilize the virus. Other features and advantages of the present invention are apparent based on the descriptions provided herein. The examples provided herein illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology for practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Figures la and lb illustrate induction times for caspase 3 activity using lyophilized virus and liquid bulk virus (i.e., a concentrated lot of clarified virus). The RFU (relative fluorescent unit) value was adjusted by subtracting background signal (adjusted RFU). Figure la illustrates an induction time using lyophilized mumps virus ("MuV"), lyophilized rubella virus ("RbV"), and lyophilized measles virus ("MeV"). Figure lb illustrates an induction time using bulk measles virus. Figures 2a and 2b illustrate a linear range for induction of caspase 3 activity. Figure 2a provides results using measles virus. Figure 2b provides results using mumps virus. Figure 3 illustrates measles virus induction variation in various samples. "Liquid" indicates a non-lyophilized frozen aliquot. "DO" indicates a lyophilized non-incubated sample stored at -70°C. The one-week sample was initially lyophilized and stored at 37°C for one week. "A", "C", "D" and "F" refer to different formulations of the vaccine. Figure 4 illustrates a comparison of the plaque assay and the caspase 3 assay. Activity was measured using measles virus stored in different environments. "Liq" indicates a non-lyophilized frozen aliquot. "DO" indicates a lyophilized, non- incubated sample stored at -70°C. "D7" indicates a lyophilized sample incubated at 37°C for one week. "A", "B", "C" and "D" refer to different formulations. Figure 5 illustrates a comparison of the plaque assay and the caspase 3 assay. Viral activity was measured using mumps virus stored in different environments. "Liq" indicates a non-lyophilized frozen aliquot. "DO" indicates a lyophilized non- incubated sample stored at -70°C. "D7" indicates a lyophilized sample incubated at 37°C for one week. "A", "B", "C" and "D" refer to different vaccine formulations being tested. Figure 6 illustrates a comparison of the plaque assay and the caspase 3 assay. Viral activity was measured using rubella virus stored in different environments. "Liq" indicates non-lyophilized frozen aliquot. "DO" indicates a lyophilized, non- incubated sample stored at -70°C. "D7" indicates lyophilized sample incubated at 37°C for one week. "H", "I", "J", "K" and "L" refer to different formulations of the vaccine. DETAILED DESCRIPTION OF THE INVENTION Measuring viral induction of caspase 3 activity was found to provide a reliable measure of viral potency and stability. Useful properties of measuring viral induction of caspase 3 activity include: induction of caspase 3 activity is achieved by different viruses including measles, mumps and rubella viruses; induction of caspase 3 activity is reproducible; induction of caspase 3 activity is sensitive to viral dilutions; induction of caspase 3 activity is sensitive to sample differences; and induction of caspase 3 activity correlates with the plaque assay. Thus, the caspase 3 assay can be applied to different formulations and a variety of viruses. Additionally, caspase 3 activity is not substantially effected by freezing and thawing, can be specifically detected, and is quenchable. These advantages allow the assay to be easily implemented for routine laboratory handling. Another advantage of the caspase 3 assay in time. When measuring viral stability, the caspase 3 assay can be performed quicker than the plaque assay. The standard plaque assay takes approximately seven to ten days, but the caspase 3 assay can be performed in about half that time. Caspase 3 Caspase 3 (also known as CPP32 or apopain) is a member of the caspase family of proteases. Caspases are induced by apoptosis, which is an active process of cellular suicide. (Thompson, Science 267: 1456-1462, 1995; Nicholson, Nature Biotechnology 14:291-301, 1996; Lincz, Immunology and Cell Biology 76:1-19, 1998.) Apoptosis has been indicated to be induced by different stimuli including infection by different viruses. (Shen, et ah, Current Opinion in Genetic and Development 5:105-111, 1995; Sadzot-Delvaux, et al, Journal of General Virology 76:2875-2879, 1995; Esolen, et al, Journal of Virology 69:3955-3958, 1995; and Ito, et al., FEMS Immunology and Medical Microbiology 15: 115-122, 1996.) However, certain viral proteins have been indicated to block apoptosis, such as Baculovirus protein p35 which inhibits caspase 3. (Lincz, Immunology and Cell Biology 76: 1-19, 1998.) Caspase 3 is a cysteine protease synthesized from a pro-enzyme by cleavage at an Asp residue to form an active protease. The active caspase 3 protease also cleaves at Asp residues. The specific recognition sequence for caspase 3 is the peptide Asp- Glu-Val-Asp. (Fernandes-Alnemri, et al, Journal of Biological Chemistry 269:30761-30764, 1994; Nicholson, et al., Nature 376:31-43, 1995; Srinivasula, et al, Journal of Biological Chemistry 271:21099-21106, 1996; Schlegel, et al., Journal of Biological Chemistry 277: 1841-1844, 1996; and Casciola-Rosen, et al, J. Exp. Med. 183:1951-1964, 1996; each of these references is hereby incorporated by reference herein.) Selection of Appropriate Viruses and Cells Suitable virus and cells for use in the present invention can be selected based upon the susceptibility of a particular cell to caspase 3 induction when infected with a particular virus. The ability of a virus to infect a particular cell can readily be determined, as can the ability of the virus to induce caspase 3 activity. Examples of procedures that can be used to measure the suitability of a cell and a virus are described in the Example section provided below using measles, mumps and rubella virus. Other cells (e.g., MRC-5) and viruses (e.g., varicella-zoster) are expected to be useful in the present invention. Measuring Caspase 3 Activity "Caspase 3 activity" refers to enzymatic activity able to cleave the caspase 3 substrate Asp-Glu-Val-Asp ("DEVD"). Such activity is known to be produced by caspase 3 and at least one related enzyme, Mch3 . (Fernandes-Alnemri, et al, Cancer Research 55:6045-6052, 1995, which is hereby incorporated by reference herein.) Based on the present disclosure, caspase 3 activity induced by viral infection can be measured using techniques well known in the art. Preferably, caspase 3 activity is measured using colorimetric or fluorimetric labeled substrates. More preferably, the employed substrate is DEVD linked to a colorimetric or fluorimetric moiety. Examples of such a moiety include the colorimetric moiety p-nitroanilide (λmax = 505 nm) and the fluorimetric moiety 7-amino-4-trifluoromethyl coumarin ("AFC", λ max = 400 nm). (Zhang, et al, in Apoptosis Detection and Assay Methods, pages 7-14, Eds. Zhu and Chun, BioTechniques Books, 1998, both of which are hereby incorporated by reference herein). Colorimetric and fluorimetric labeled substrates can be employed using procedures such as those described by Zhang, et al, in Apoptosis Detection and Assay Methods, pages 7-14, Eds. Zhu and Chun, BioTechniques Books, 1998; and ApoAlert™ CPP32/Caspase-3 Assay Kits User Manual (PT3083-1), CLONTECH Laboratories, Inc. 1998; both of which are hereby incorporated by reference herein. The caspase 3 assay is preferably employed on viral vaccine samples either in liquid or lyophilized form. For example, lyophilized viral samples can be reconstituted, diluted, and plated onto a cell sheet; the samples are incubated, the cells are lysed, and the cellular lysate collected and frozen at -70°C; after thawing on wet ice (about 5°C) removal of cellular debris by centrifugation occurs, the supernatant is removed to a new tube containing reaction buffer, and the DEVD-AFC substrate is incubated with the cellular supernatant. EXAMPLES Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention. Methods and Materials Methods and materials generally used in growing cells, inducing caspase 3 activity, and measuring caspase 3 activity to obtain the results provided below were as follows: Growth of Vero Cells and RK-13 Cells Vero cells were grown to confluency in 162 cm 2 flasks in M199 media (Gibco BRL cat# 11150-059) containing 10% fetal bovine serum (FBS) and 0.5% neomycin at 35°C with 5% CO 2 . The flasks were then decanted and 3 ml of lx trypsin-ethylene diamine tetra acetic acid (EDTA) (Gibco/BRL cat# 25200-056) was added to the flasks to neutralize the media. The trypsin was decanted and another 2 mis of trypsin was added and incubated for 10 to 15 minutes at 35°C with 5% CO 2. Cells were then washed to the bottom of the flask with 20 ml of M199 containing 10% FBS and 0.5% neomycin. Cells were plated into 24 well plates at 5.4 X 10 4 cells/ml. Each well received 2 ml of the diluted cells (diluted into M199 containing 10% FBS and 0.5% neomycin). The plates were incubated at 35°C for 2 days and then used for caspase 3 induction. The growth of RK-13 cells is the same as Vero cells with the exception of the media. RK-13 cells were grown in MEM media (Gibco BRL) containing 10% FBS and 0.5% neomycin. The cells were plated at a density of 6.0 x 10 4 cells /ml, 2 ml per well. Induction of Caspase 3 Activity Generally, Vero plates were used for measles virus and mumps virus infections, while RK-13 cells were used for the rubella virus infections. Lyophilized formulations of a combination of measles, mumps, and rubella vaccine were used. Lyophilized formulations were reconstituted with 0.7 ml dH 2 O. Frozen liquid samples were thawed at room temperature prior to use. Following reconstitution or liquid thawing, the samples were diluted 1:2, 1:5, 1: 10 or 1:20 in their respective diluents. The diluents contained antibodies against the viruses not being assayed for among mumps, measles, and rubella viruses (e.g., when assaying for measles virus antibodies for mumps and measles virus were used). Cells (2.0xl0 5 ) were infected with 50 μl of the diluted samples. Each sample was used to infect 6 wells on a 24 well plate. Plates were then incubated at 35°C with 5% CO 2 for 1 hr for plates to be assayed for mumps and rubella viruses and for 2 hrs for plates to be assayed for measles virus. During this attachment phase, the plates were rocked every 15 minutes to ensure proper coverage of the cell sheet with media. Following the attachment phase, the cells were overlayed with 1 ml of M199 media containing 10% FBS and 0.5% neomycin for the Vero cells and 1 ml of MEM containing 10% FBS and 0.5% neomycin for the RK-13 cells. The plates were then incubated at 35°C with 5% CO 2 for 93 to 96 hrs. Caspase 3 Assay Following the induction period, media was aspirated from the wells. Thirty microliters of chilled cell lysis buffer (Clontech: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X -100) was added per well. The samples were incubated on ice for 10 minutes and then two wells were pooled together into one microfuge tube. Samples were either frozen on dry ice and stored at -70°C until time of analysis or were assayed immediately. The samples were spun in at 12,000 rpm for 5 minutes at 4°C. Supernatant (50 μl) was removed to a new microfuge tube and 50 μl of 2x Reaction Buffer (Clontech: 100 mM HEPES, pH 7.4, 150 mM NaCl, 0.2% CHAPS) containing 7 mM dithiothreitol (DTT) was added. Five microliters of the 1 mM DEVD-AFC substrate was added to the reaction and vortexed briefly. The samples were then incubated for 1 hr at 37°C in a circulating water bath. Following the incubation, 20 μl of 10% H 3 PO 4 was used to quench the reaction. Quenched reactions (100 μl) were added to a 96 well plate and read in a TECAN fluorescent plate reader (excitation: 390 nm, emission: 480 nm, gain: 60). Results Detection of Caspase 3 Activity Induced by Measles, Mumps and Rubella Viruses The ability of measles, mumps, and rubella viruses to induce caspase 3 activity in cells was confirmed using Vero and RK-13 cells. Caspase 3 activity was measured as described above in Methods and Materials. Vero cells were induced with measles virus, mumps virus, and rubella virus, while RK-13 cells were induced with rubella virus. The results are shown in Table 1 Table 1 Caspase 3 activity expressed as RFU in induced (i.e. infected) cells and non-induced controls. To ensure that the observed signal was due to the virus, one set of Vero cells were infected with measles virus and another set of Vero cells were infected with a placebo. The placebo produced an adjusted RFU of 50-500, while measles virus produced an adjusted RFU of about 7,750. These results demonstrate that significant changes in caspase 3 activity are due to viral infection rather than a formulation component. Effects of Freeze-Thawing on Caspase 3 Activity To allow an assay to be run at various times after infected, samples may be frozen and later thawed at a convenient time. To determine if freezing and thawing affects caspase 3 activity, samples were frozen on dry ice and stored at -70°C following cell lysis. The samples were then thawed and assayed with a set of samples that were not frozen. Non-frozen cells provided an adjusted RFU of 2,167 while frozen cells produced an adjusted RFU of 2,175.5. Thus, freeze-thawing does not appear to effect caspase 3 activity. Accordingly, many samples can be run and then stored for various lengths of time allowing the actual assay to be run at a later time. Determination of the Optimal Time Required for Viral Bulk Incubation The time required to induce caspase 3 activity with various samples can differ depending on the virus and multiplicity of infection (MOI). To determine preferred times for measles, mumps and rubella virus infection, Vero cells or RK-13 cells were infected with (1) measles virus bulk and (2) different lyophilized measles virus, mumps virus, and rubella virus. Vero cells were infected with mumps and measles, while RK-13 cells were infected with rubella virus. A long-term study was initiated with time points collected at 24, 46, 72, and 96 hours for bulk samples and 26, 47, 75, 94, 95, 97 and 100 hours for lyophilized samples. The results of different points are presented in Figures la and lb. All three viruses in lyophilized formulations show a peak signal around 95 hours (Figure la). The optimal signal observed for the infection with measles virus bulk was around 46 hours (Figure lb). This difference in overall time is expected since a bulk sample contains a much higher titer and infects more cells upon inoculation, thereby decreasing the length of time necessary to ensure an optimal signal. Linearity of the Caspase 3 Enzyme Response from Cells induced with Measles Virus and Mumps Virus When investigating and comparing enzyme activity, it is important to collect data found within the linear range of the assay. Following induction with measles and mumps viruses, the samples were assayed and data collected over a one-hour period for the measles virus and an hour and a half for mumps virus. The data presented in Figure 2a (measles virus) shows the assay is linear for at least one hour, and the data presented in Figure 2b (mumps virus) show the assay is linear for at least 75 minutes. Thus, using a one-hour incubation period for caspase 3 enzyme activity will ensure the data collected will be meaningful. Determination of Non-Specific Substrate Cleavage A concern with using a non-purified cell lysate is the effect of non-specific substrate cleavage derived from other proteases found within the lysate. Using a specific inhibitor for caspase 3, DEVD-CHO, the caspase 3 assay was run and analyzed. If non-specific cleavage were occurring in the assay, the addition of the inhibitor would not completely stop the substrate from being cleaved. The results shown in Table 2 indicate that adding lμM DEVD-CHO inhibitor eliminates the RFU signal. TABLE 2 RFU was adjusted to take background into account. Samples without the caspase 3 inhibitor show a -3,000 fold increase over the samples containing the inhibitor. Thus, non-specific cleavage is not occurring in these cellular lysates and the signal is derived from caspase 3 activity. The Effects of Quenching Caspase 3 Assay using 10% H 3 PO To ensure consistent data when using enzymes, it is useful to be able to quench the reaction. Quenching can be used to ensure that each sample is incubated for the same amount of time. It is useful to examine the quenching conditions to ensure that the quench is not having an effect on the RFU signal that is being generated. In some instances, quenching a fluorescent peptide will provide interference on the overall signal making data interpretation difficult. Following a one hour incubation peπod, samples were quenched with 20 μl of 10% H 3 PO 4 and analyzed on the TECAN Plate reader. Data were compared with that obtained from samples that did not undergo quenching. The results are shown m Table 3. TABLE 3 The results indicate the ratio of the non-induced samples to the induced samples is the same. Thus, quenching the reactions does not effect the relative signal intensity. Reproducibility of the Caspase 3 Assay Using samples induced by measles virus, mumps virus, or rubella virus, the reproducibility of measuπng caspase 3 activity was examined by using three vials of the same sample The data presented in Table 4 show that the assay is reproducible and very consistent. TABLE 4 RFU was adjusted to take into account the background. Effects of Dilutions on Caspase 3 Signal To produce a quantitative assay it is important to detect differences in signal when the sample is diluted to different multiplicity of infections. To address this, measles virus and rubella virus lyophilized samples were diluted 1:2, 1:5 and 1:10 in their respective diluents, and mumps virus was diluted 1:20, 1:40, and 1:80, and the effect on caspase 3 signal was measured. The results are shown in Tables 5 and 6. TABLE 5 RFU was adjusted to take into account the background. TABLE 6 RFU was adjusted to take into account the background. Dilution effects were observed for all three viruses. This is observed by the overall decrease in signal as the dilution is increased. Thus, the induction of caspase 3 activity is affected by the multiplicity of infection used to infect the cells. Because signal output is affected through dilutions, it is important to have the same multiplicity of infection for direct comparisons between vaccine formulations. Detection of Differences in Signal Derived from Various Lyophilized Samples The ability of the caspase 3 assay to detect the effect of different sample and storage conditions was measured to determine the suitability of using the caspase 3 assay to measure the stability of viral preparations stored under different conditions. Measles virus was used to infect cells. The data shown in Figure 3 illustrates that the effect of different conditions on viral stability can be measured by measuring caspase 3 activity. Thus, the caspase 3 assay can detect differences in viral potency and stability due to various formulation composition and storage conditions. Correlation Between the Plaque and Caspase 3 Assays A stability study was performed to examine the correlation between the plaque and caspase 3 assays using measles virus, mumps virus, and rubella virus. The data shown in Figures 4-6 illustrate that as the PFU potency decreased, the corresponding caspase 3 signal also decreased. Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims: WHAT IS CLAIMED 1. A method for assaying potency and stability of a virus comprising the steps of: a) contacting a plurality of cells susceptible to caspase 3 induction with said virus; and b) measuring caspase 3 activity as an indication of viral stability. 2. The method of claim 1, wherein said caspase 3 activity is measured using a caspase 3 substrate linked to a fluorimetric or a colorimetric moiety. 3. The method of claim 2, wherein said substrate is the peptide Asp-Glu-Val- Asp. 4. The method of claim 3, wherein said virus is either measles virus, mumps virus, or rubella virus. 5. The method of claim 4, wherein said plurality of cells is either Vero cells or RK-13 cells. 6. The method of claim 3, wherein prior to said step (a) said virus was lyophilized. 7. The method of claim 3, wherein said step (a) and said step (b) are performed at two or more time intervals. 8. The method of claim 3, wherein after said step (a) and prior to said step (b) said cells were frozen and then thawed. 9. A method of identifying a stabilizing formulation comprising the steps of: a) infecting a first plurality of cells with said virus stored in a first test formulation, and contacting a second plurality of cells with said virus stored in a second test formulation, wherein said first and second plurality of cells are the same strain and are susceptible to caspase 3 induction; and b) measuring caspase 3 activity produced from said virus stored in said first and said second test formulation to identify said stabilizing formulation. 10. The method of claim 9, wherein said step (a) further comprises contacting a third plurality of cells with said virus stored in a third test formulation, and contacting a fourth plurality of cells with said virus stored in a fourth test formulation, wherein said third and fourth plurality of cells are the same strain as said first and second plurality of cells. 11. The method of claim 10, wherein said caspase 3 activity is measured using a caspase 3 substrate linked to a fluorimetric or a colorimetric moiety. 12. The method of claim 11, wherein said substrate is the peptide Asp-Glu- Val-Asp. 13. The method of claim 12, wherein said virus is either measles virus, mumps virus, or rubella virus. 14. The method of claim 13, wherein said first, said second, said third, and said fourth plurality of cells is either Vero cells or RK-13 cells. 15. The method of claim 12, wherein said virus stored in said first formulation, said second formulation, said third formulation and said fourth formulation was lyophilized. 16. The method of claim 12, wherein said step (a) and said step (b) are performed at two or more time intervals. 17. The method of claim 16, wherein after said step (a) and prior to said step (b) at least one of said first, said second, said third, or said fourth plurality of cells are frozen and thawed.

Inventors: BLUE JEFFREY T, BLUE, JEFFREY, T.
Applicants: BLUE JEFFREY T, MERCK & CO INC, BLUE, JEFFREY, T., MERCK & CO., INC.
Application year: 2000
Main classification: C12Q1
IPC codes: C12Q1, C12R1, G01N33