A method for calculating the infectivity of viral vectors by combining infectivity titer and infectivity constant
By combining infectivity titers and infectivity constants, the problem of inaccurate evaluation of viral infectivity in the prior art is solved, and accurate calculation of infectivity related parameters of viral vectors and accurate prediction of transduction efficiency is achieved.
Patent Information
- Application Number
- CN202510089032.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2045-01-21
AI Technical Summary
The prior art lacks accurate and effective methods to evaluate the infectivity of viruses, including viral vectors, making it difficult to accurately predict the transduction efficiency of viral vectors in applications such as gene therapy and drug-targeted transport.
The infectivity calculation method of viral vectors combining infectious titers and infectivity constants was used to calculate infectivity-related parameters through the quadruple concentration test method (QCDM), including infectivity titers, infectivity constants, infection plural numbers and infection efficiency.
Accurate calculation of viral vector infectivity-related parameters is achieved, which can more accurately predict transduction efficiency, help optimize the dose of viral vectors, and serve as a standard for evaluation of viral vector product quality.
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Figure CN119506480B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of virus vector detection, and relates to a method for measuring the infectivity of cells by virus vector transduction. Background Art
[0002] Viral infectivity is the ability of a virus to enter a cell and establish infection. The classic method for testing and evaluating viral infectivity is to determine the titer of viral infectivity. The viral vector described in the present invention is a special virus that has lost its ability to replicate itself after molecular biological modification, but retains the ability to infect host cells and stably express transduced foreign genes in host cells. It is often used in cutting-edge clinical medicine applications such as research on viral infection mechanisms, vaccine preparation, transgenic cell preparation, gene therapy, and targeted drug delivery. Due to the complexity of the viral infection process, there is still a lack of accurate and effective methods for evaluating the infectivity of viruses, including viral vectors, which has greatly hindered the development of virus-related industries. We use viral vectors as a model for studying viral infectivity, and have achieved a more comprehensive and profound discovery of the cognition and measurement of viral infectivity.
[0003] The relevant parameters involved in the infectivity measurement of the present invention include infection efficiency, infection multiplicity, infectivity constant and infectivity titer (in the application of viral vectors, infection efficiency is generally referred to as transduction efficiency). Among them, infection efficiency (infection efficiency, transduction efficiency) is the ratio of the number of cells infected by the virus to the total number of cells, generally in the range of 0-100%; multiplicity of infection (MOI for short) is the ratio of the number of viruses that can infect cells to the total number of cells in a system. ; The infectivity constant is a parameter characterizing the average viral activity in a specific viral sample, specifically characterizing the ability of the virus to enter cells and complete infection, which is calculated by the ratio of 2 times the MOI to the square of the infection completion time; the infectivity titer is the number of viral particles that can infect cells contained in the viral sample, and the common unit is TU / mL. The time for transduction to be completed is the time when all the inoculated viral vectors enter the cells and complete the genome integration.
[0004] In the application of viral vectors, the multiplicity of infection is calculated by the transduction efficiency, which can be used for the detection of the infectious titer of viral vectors. Conversely, the transduction efficiency is calculated by the multiplicity of infection, which can be used to make theoretical judgments on the dosage of viral vectors in applications such as gene therapy and targeted drug delivery.
[0005] Traditional and commonly used viral vector infectivity testing methods include TCID 50 Test method, plaque test method, qPCR test method, flow cytometry test method.
[0006] The TCID50 test method, based on the principle of endpoint dilution, measures the dilution factor that can successfully infect half of the infected objects; the plaque test method detects the total number of viruses in the virus sample that can infect cells by detecting the number of virus plaques formed after the virus sample is inoculated into a monolayer cell culture. Both methods are difficult to be widely used due to reasons including low throughput, complex operation, and large subjective errors.
[0007] The qPCR test method measures the copy number ratio of the viral vector genome and the host cell genome as the MOI, and then calculates the infectious titer based on the product of the multiplicity of infection, the dilution factor, and the initial number of cells. However, not all exogenous genes carried by the viral genome that enters the cell genome can be successfully expressed, so the qPCR test method cannot accurately measure the effective infectious titer of the viral vector.
[0008] The flow cytometry test detects the proportion of successfully transduced cells to the total number of cells by labeling the exogenous proteins expressed by the cells after viral vector transduction. This is used as the MOI, and the infectious titer is calculated based on the product of the multiplicity of infection, the dilution factor, and the initial number of cells. However, a cell can be infected with multiple viral vectors, so the flow cytometry test cannot accurately measure the infectious titer of the viral vector. If the infectious titer cannot be accurately measured, the cell transduction efficiency cannot be predicted.
[0009] Therefore, in the preparation of transgenic cells related to gene therapy, in order to obtain a higher transduction efficiency, as many viral vectors as possible can only be added. However, due to concerns about carcinogenic insertion mutations, the amount of viral vector added has to be limited. Therefore, it is very important to accurately predict the transduction efficiency of viral vectors.
[0010] The above shows that there is still a lack of an accurate and comprehensive method for testing the infectivity of viruses, especially viral vectors. Summary of the invention
[0011] The purpose of the present invention is to provide a method for calculating the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, so as to accurately and comprehensively calculate the infectivity-related parameters of the viral vector.
[0012] To achieve this object, in a basic embodiment, the present invention provides a method for calculating the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein the method comprises the following steps:
[0013] (11) Cell culture and counting: Add the same number of cells to each well of the cell culture plate and culture them in the same volume of the same culture medium. After culturing for 24 hours, measure the number of cells in multiple wells before viral vector transduction, and calculate the average value N of the number of cells in each well, in units of cells / well, as the initial cell number for the transduction experiment;
[0014] (12) Preparation of diluted viral vector samples: dilute the viral vector sample D times with the culture medium, take volume V1 and volume V2 respectively, and make up to the same volume V0 with the culture medium, to obtain the first viral vector diluted sample and the second viral vector diluted sample, respectively, and V2=4V1, and the units of V1 and V2 are both μL;
[0015] (13) Cell transduction: Remove the culture medium in each well of the cell culture plate obtained in step (11), and then add volume of the first viral vector diluted sample and the second viral vector diluted sample, and culture the cells for a period of time;
[0016] (14) Determination of transduction efficiency: Use appropriate methods to determine the transduction efficiency of the diluted samples of the first viral vector on cells. and the transduction efficiency of the second viral vector dilution sample on cells ;
[0017] (15) Calculation of transduction completion time: The completion time of transduction of cells by the first viral vector dilution sample is calculated using the following formula (I): :
[0018] (I)
[0019] (16) Calculation of multiplicity of infection: Calculate the multiplicity of infection of the first viral vector diluted sample to the cells using the t1 calculated by formula (I) and the following formula (II): The multiplicity of infection of the second viral vector diluted sample to the cells ,
[0020] (II)
[0021] (17) Calculation of infectivity constant: The infectivity constant i of the first viral vector diluted sample and the second viral vector diluted sample to cells is calculated using the following formula (III):
[0022] (III)
[0023] (18) Calculation of infectious titer: The infectious titer T of the viral vector sample was calculated using the following formula (IV), in units of TU / mL:
[0024] (IV)
[0025] (19) Prediction of transduction efficiency of any viral vector inoculation volume: Assume Any volume of the viral vector solution less than or equal to V0 taken from the viral vector solution diluted D times in step (12) is supplemented to volume V0 with the culture medium to obtain a third viral vector dilution sample, and The unit is μL, and the initial number of cells detected is N k ,use The volume of the third viral vector dilution sample was used for the viral transduction experiment in the same manner as in step (13). k and r k The multiplicity of infection and transduction efficiency of the diluted sample of the third viral vector for transduction of cells can be calculated using the following formula (V) and formula (VI) respectively to obtain the multiplicity of infection m of the diluted sample of the third viral vector for transduction of cells: k and transduction efficiency r k ,
[0026] (V)
[0027] (VI).
[0028] The infectivity calculation method of the above-mentioned viral vector is referred to as the quadruple concentration detection method (QCDM).
[0029] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein in step (14), the transduction efficiency of the diluted sample of the first viral vector on the cell is determined. and / or the transduction efficiency of the second viral vector dilution sample on cells The methods used are flow cytometry, fluorescence cell counting, qPCR or in situ immunofluorescence testing.
[0030] The principle and brief steps of the flow cytometer detection method for determining infection efficiency are:
[0031] Principle: Flow cytometry is used to detect the proportion of transduced cells to the total number of cells
[0032] Brief steps:
[0033] 1. Take the cells cultured for 72 hours after transduction, and label the transduced cells with primary and secondary antibodies against the proteins expressed after viral vector transduction (if fluorescent protein is expressed, no labeling is required);
[0034] 2. Use flow cytometry to detect the proportion of transduced cells to the total number of cells.
[0035] The principle and brief steps of the fluorescent cell counter detection method for determining infection efficiency are:
[0036] Principle: Use a fluorescent cell counter to detect the proportion of transduced cells to the total number of cells
[0037] Brief steps:
[0038] 1. Take the cells cultured for 72 hours after transduction, and label the transduced cells with primary and secondary antibodies against the proteins expressed after viral vector transduction (if fluorescent protein is expressed, no labeling is required);
[0039] 2. Use a fluorescent cell counter to detect the proportion of transduced cells to the total number of cells.
[0040] The principle and brief steps of the qPCR detection method for determining transduction efficiency are:
[0041] Principle: Use qPCR instrument to detect the ratio of viral genome to cell genome (double copy needs to be divided by 2)
[0042] Brief steps:
[0043] 1. Take cells cultured 72 hours after transduction and extract the cell genome;
[0044] 2. Use qPCR instrument to detect the ratio of viral genome to cell genome copy number (double copy needs to be divided by 2).
[0045] The principle and brief steps of the in situ immunofluorescence assay for determining transduction efficiency are:
[0046] Principle: Use in situ immunofluorescence test to detect the proportion of transduced cells to total cells
[0047] Brief steps:
[0048] 1. Take the cells cultured for 72 hours after the start of transduction, and label the transduced cells with primary and secondary antibodies against the proteins expressed after virus transduction of cells;
[0049] 2. Use a fluorescence microscope or other fluorescence detection equipment to detect the proportion of transduced cells to total cells.
[0050] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining infectious titer and infectious constant, wherein the first viral vector dilution sample, the second viral vector dilution sample, and the third viral vector dilution sample are prepared from the same viral vector sample or the same batch of viral vector samples under the same storage conditions according to step (12) and / or step (19), and then the cells are transduced under the same culture conditions.
[0051] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein the culturing period in step (13) is specifically culturing for 24-120 hours.
[0052] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein the cells are selected from HEK293T, HEK293, Jurkat or Vero cells.
[0053] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining infectious titer and infectious constant, wherein the viral vector sample is selected from one or more of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.
[0054] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein the culture medium can be DMEM culture medium, RPMI1640 culture medium or other culture medium, the content of fetal bovine serum in the culture medium is (0.5-20)% (v / v), and the content of penicillin / streptomycin antibody solution is 1% (v / v).
[0055] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein in step (11), the number of cells added to each well of the cell culture plate is 1×10 4 ~1×10 6 indivual.
[0056] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein in step (12), D=1-1000.
[0057] In a preferred embodiment, the present invention provides a method for measuring the infectivity of a viral vector by combining the infectivity titer and the infectivity constant, wherein in step (12), V0=50-1000 μl.
[0058] The beneficial effect of the present invention is that the infectivity calculation method of the viral vector of the present invention can accurately and comprehensively calculate the infectivity-related parameters of the viral vector, mainly including the infectivity titer, infectivity constant, multiplicity of infection and infection efficiency.
[0059] The present invention realizes the accurate and comprehensive calculation of the infectivity-related parameters of viral vectors for the first time. The calculation results can be applied to the application and basic research fields of cutting-edge clinical medicine such as basic research on viral infection mechanism, vaccine preparation, transgenic cell preparation, gene therapy and targeted drug delivery; it can also help manufacturers of viral vectors and virus-related products optimize production technology; it can also help adjust the dose of viral vectors required when preparing transgenic cells. In particular, the calculated infectivity constant can be used as a new quality assessment standard for viral vector products. BRIEF DESCRIPTION OF THE DRAWINGS
[0060] Figure 1 These are representative pictures of the automatic identification and counting of HEK293T cells after viral vector transduction by the fluorescent cell counter in Example 1, wherein a is a bright field cell imaging picture; b is a bright field cell automatic identification and counting picture; c is an EGFP fluorescent cell imaging picture; and d is an EGFP fluorescent cell automatic identification and counting picture.
[0061] Figure 2 It is a comparison chart of the transduction efficiency predicted by the previous empirical formula in Example 1, the transduction efficiency predicted by the formula of the present invention, and the actual transduction efficiency detected. DETAILED DESCRIPTION
[0062] In order to better understand the technical solutions and advantages of the present invention, the present invention is further described below through embodiments in conjunction with the accompanying drawings.
[0063] Example 1: Using QCDM to measure the infectivity of lentiviral vectors
[0064] Step 1: Preparation of lentiviral vector test samples expressing fluorescent protein: HEK293T cells were inoculated into a 10 cm cell culture dish with a cell inoculation number of 3 × 10 5The cell culture volume was 10 mL of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin solution). The next day, when the cell confluence reached about 70%, the lentiviral vector packaging plasmids pCMV-dR8.91, pMD2.G and pCDH-EGFP (Qingke Biotechnology) were transfected into HEK293T cells using the transfection reagent Lipo8000 (C0533FT, Bio-Tech) according to the instructions of the transfection reagent. 48 hours after transfection, the culture supernatant was collected and concentrated overnight using PEG8000 (P743072, Aladdin). Then, the precipitate was obtained by centrifugation at 8000 g per minute at 4°C for 1 hour. 500 μL of PBS was taken to dissolve the precipitate to obtain a 20-fold concentrated lentiviral vector test sample expressing fluorescent protein.
[0065] Step 2: Inoculate HEK293T cells in a 24-well plate, with 2×10 cells per well. 5 Then, 500 μl of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin solution) was added to each well, and the 24-well plate was placed in a cell culture incubator at 37°C with a 5% CO2 concentration for culture. After 24 hours of culture, the 24-well plate was taken out of the cell culture incubator, and cells from 5 wells were selected for initial cell count determination: after removing the culture medium, 200 μl of trypsin solution was added to each well for digestion at 37°C for 4 minutes, and then 800 μl of DMEM complete medium was added to each well to terminate digestion, and the cells were dispersed by aspiration. 20 μL was added to the counting tank, and the cells in each well were automatically identified and counted using a fluorescent cell counter. The results are shown in Table 1, and the average number of cells in each well was determined to be N = 421400 / well.
[0066] Table 1 Automatic cell counting results for each well
[0067]
[0068] Step 3: Take a lentiviral vector (LV-EGFP) test sample of unknown concentration that can express green fluorescent protein, dilute it D = 35 times with DMEM containing 1% fetal bovine serum, and take 30μl and 120μl respectively ( and , ), supplemented with DMEM containing 1% fetal bovine serum to 300 μl, and then added to different wells of the 24-well plate with the remaining cells obtained in step 1 after discarding the old culture medium, and then put the 24-well plate back into the cell culture incubator at 37°C with 5% CO2 for culture.
[0069] Step 4: Cultivation, that is, 72 hours after transduction, remove the 24-well plate from the cell culture incubator, discard the culture medium of the transduced cell wells, then add 200μl of trypsin to each well, digest at 37°C for 4 minutes, then add 800μl of DMEM complete culture medium to each well to terminate digestion, disperse the cells in each well by pipetting, and then transfer the cells in each well to different 1.5mL centrifuge tubes, and centrifuge at 3000rpm for 3 minutes. After removing the supernatant from each centrifuge tube, add 900μl of 1×PBS to each tube, and centrifuge at 3000rpm for 3 minutes. After removing the supernatant from each centrifuge tube, add 600μl of 1×PBS to each tube, resuspend the cells, take 20μL and add it to the counting tank, and then use a fluorescent cell counter to detect the transduction efficiency ( ) (Representative images, see Figure 1 ), and the transduction completion time, multiplicity of infection, infectivity constant, and infectivity titer were calculated by formula (I)-(IV). The results are shown in Table 2.
[0070] Table 2 Transduction efficiency determination and calculation results of multiplicity of infection, infectivity constant and infectivity titer
[0071]
[0072] Step 5: The transduction efficiency of HEK293T cells transduced with any inoculation volume of LV-EGFP determined for infectivity in step 2 is predicted using the infectivity function (IF) of the present invention, and the Poisson distribution function (PDF, ) as a control. Carry out cell culture and obtain the initial cell number as in step 1 above, and perform transduction as in step 2. The difference is that after dilution, any volume ( ) were 40, 50, 60, 160, 200, and 240 μL, respectively, and transduction and culture were performed in the same manner as in steps 2 and 3. The infection multiplicity and transduction efficiency were calculated using the infectious titer and infectious constant of the viral vector sample obtained in step (4) by formula (V)-(VI), and the transduction efficiency after transduction was actually tested. Then, the predicted transduction efficiency was compared with the tested transduction efficiency. The results are shown in Tables 3 and Figure 2 .
[0073] Table 3 Calculation results of transduction efficiency of cells after transduction with diluted samples of viral vectors at other concentrations
[0074]
[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the essence and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementations are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementations should be considered illustrative rather than restrictive in any respect. The scope of the present invention shall be described by the appended claims, and any changes equivalent to the intent and scope of the claims shall also be included within the scope of the present invention.
Claims
1. A method for calculating the infectivity of a viral vector by combining infectivity titer and infectivity constant, characterized in that: The calculation method comprises the following steps: (11) Cell culture and counting: Add the same number of cells to each well of the cell culture plate and culture them in the same volume of the same culture medium. After culturing for 24 hours, measure the number of cells in multiple wells before viral vector transduction, and calculate the average value N of the number of cells in each well, in units of cells / well, as the initial cell number for the transduction experiment; (12) Preparation of diluted viral vector samples: dilute the viral vector sample D times with the culture medium, take volume V1 and volume V2 respectively, and make up to the same volume V0 with the culture medium, to obtain the first viral vector diluted sample and the second viral vector diluted sample, respectively, and V2=4V1, and the units of V1 and V2 are both μL; (13) Cell transduction: Remove the culture medium in each well of the cell culture plate obtained in step (11), and then add volume of the first viral vector diluted sample and the second viral vector diluted sample, and culture the cells for a period of time; (14) Determination of transduction efficiency: Use appropriate methods to determine the transduction efficiency of the diluted samples of the first viral vector on cells. and the transduction efficiency of the second viral vector dilution sample on cells ; (15) Calculation of transduction completion time: The completion time of transduction of cells by the first viral vector dilution sample is calculated using the following formula (I): : （I） (16) Calculation of multiplicity of infection: Calculate the multiplicity of infection of the first viral vector diluted sample to the cells using the t1 calculated by formula (I) and the following formula (II): The multiplicity of infection of the second viral vector diluted sample to the cells , (II) (17) Calculation of infectivity constant: The infectivity constant i of the first viral vector diluted sample and the second viral vector diluted sample to cells is calculated using the following formula (III): (III) (18) Calculation of infectious titer: The infectious titer T of the viral vector sample was calculated using the following formula (IV), in units of TU / mL: (IV) (19) Prediction of transduction efficiency of any viral vector inoculation volume: Assume Any volume of the viral vector solution less than or equal to V0 taken from the viral vector solution diluted D times in step (12) is supplemented to volume V0 with the culture medium to obtain a third viral vector dilution sample, and The unit is μL, and the initial number of cells detected is N k ,use The volume of the third viral vector dilution sample was used for the viral transduction experiment in the same manner as in step (13). k and r k The multiplicity of infection and transduction efficiency of the diluted sample of the third viral vector for transduction of cells can be calculated using the following formula (V) and formula (VI) respectively to obtain the multiplicity of infection m of the diluted sample of the third viral vector for transduction of cells: k and transduction efficiency r k , （V） (WE).
2. The calculation method according to claim 1, characterized in that: In step (14), the transduction efficiency of the first viral vector diluted sample on cells is determined and / or the transduction efficiency of the second viral vector dilution sample on cells The methods used are flow cytometry, fluorescence cell counting, qPCR or in situ immunofluorescence testing.
3. The calculation method according to claim 1, characterized in that: The first viral vector dilution sample, the second viral vector dilution sample, and the third viral vector dilution sample are prepared from the same viral vector sample or the same batch of viral vector samples under the same storage conditions according to step (12) and / or step (19), and then the cells are transduced under the same culture conditions.
4. The calculation method according to claim 1, characterized in that: The culturing period in step (13) is specifically 24-120 hours.
5. The measuring method according to any one of claims 1 to 3, characterized in that: The cells are selected from HEK293T, HEK293, Jurkat or Vero cells.
6. The measuring method according to any one of claims 1 to 3, characterized in that: The viral vector sample is selected from one or more of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.
7. The measuring method according to any one of claims 1 to 3, characterized in that: The culture medium can be DMEM culture medium, RPMI1640 culture medium or other culture medium, the content of fetal bovine serum in the culture medium is (0.5-20)% (v / v), and the content of penicillin / streptomycin antibody solution is 1% (v / v).
8. The measuring method according to any one of claims 1 to 3, characterized in that: In step (11), the number of cells added to each well of the cell culture plate is 1×10 4 ~1×10 6 indivual.
9. The measuring method according to any one of claims 1 to 3, characterized in that: In step (12), D=1-1000.
10. The measuring method according to any one of claims 1 to 3, characterized in that: In step (12), V0 = 50-1000 μl.