System for evaluating ADE effect in vitro and application thereof

Abstract

The invention provides a system for evaluating an ADE effect in vitro and application of the system. The system comprises: (i) and (iii); (ii) and (iii); or (i), (ii) and (iii); in one embodiment, the present invention relates to a cell comprising: (i) an SARS-CoV-2 pseudovirus, (ii) an antibody that targets the receptor binding domain of SARS-CoV-2, and (iii) a cell that overexpresses CD32B; the cells are CHO cells. The system provided by the invention can be used for evaluating the ADE effect of the to-be-detected SARS-CoV-2 or the pseudovirus prepared from the to-be-detected SARS-CoV-2 or the antibody targeting the SARS-CoV-2, and has the advantage of higher sensitivity compared with a system based on cells expressing CD32A. The ADE evaluation system has a wide detection range, and can evaluate the ADE effect caused by the action of the SARS-CoV-2 virus or the pseudovirus prepared from the SARS-CoV-2 virus on CHO-CD32B in the presence of the RBD antibody with a wide concentration range (16 ng / mL-10000 ng / mL).

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a system for evaluating ADE effects in vitro and its application. Background Art

[0002] Antibody-dependent enhancement (ADE) refers to the phenomenon that in some cases, antibodies not only fail to effectively neutralize pathogens, but instead enhance the pathogen's ability to infect cells or cause more serious diseases. The ADE effect was first reported by Hawkes in the 1960s. [1] Since then, ADE has been observed for a variety of viruses, most notably flaviviruses (e.g., dengue virus). [2-4] Studies have shown that when the human body produces neutralizing antibodies against a specific virus, when it encounters a mutated virus or other viruses with similar antigens again, the neutralizing antibodies may bind to the new virus. However, this binding is not enough to completely neutralize the virus. Instead, it binds to the immunoglobulin Fc receptors on immune cells and mediates the virus' entry into these cells, thereby enhancing the virus's ability to infect. [5] In many respiratory viruses, including RSV, measles [6] , SARS-CoV-2, and other viruses, including human immunodeficiency virus (HIV) [7] and Ebola virus (EBOV) [8, 9] In the literature, a correlation between antibodies and the severity of disease progression has been found.

[0003] Given that the overall development of SARS-CoV-2 vaccines is still ongoing, ADE has occurred in many other vaccine clinical studies in the past, especially in vaccines for respiratory mucosal infections. In current research, many manifestations and patterns of ADE are not yet fully understood. Although studies have pointed out that the Fc receptor FcγRIIA of immunoglobulin IgG, also known as CD32A

[10] , which can mediate the ADE phenomenon, however, the specific role and influence of other Fc receptors in the ADE effect still need to be further explored and confirmed. At present, Raji and THP-1 cells are commonly used ADE detection cells, but the specific Fc receptor types that play a role on their cell surface are still controversial. Clarifying the key Fc receptor types in the potential ADE of SARS-CoV-2 and building a convenient and reliable ADE evaluation system have become key steps in studying the ADE phenomenon of the new coronavirus.

[0004] References

[0005] [1]HAWKES R A. Enhancement of the infectivity of arboviruses byspecific antisera produced in domestic fowls [J]. Aust J Exp Biol Med Sci,1964, 42: 465-82.

[0006] [2]GUZMAN M G, ALVAREZ M, RODRIGUEZ-ROCHE R, et al. Neutralizingantibodies after infection with dengue 1 virus [J]. Emerg Infect Dis, 2007,13(2): 282-6.

[0007] [3]WHITEHEAD S S, BLANEY J E, DURBIN A P, et al. Prospects for adengue virus vaccine [J]. Nat Rev Microbiol, 2007, 5(7): 518-28.

[0008] [4]KATZELNICK L C, GRESH L, HALLORAN M E, et al. Antibody-dependentenhancement of severe dengue disease in humans [J]. Science, 2017, 358(6365):929-32.

[0009] [5]KIM H W, CANCHOLA J G, BRANDT C D, et al. Respiratory syncytialvirus disease in infants despite prior administration of antigenicinactivated vaccine [J]. Am J Epidemiol, 1969, 89(4): 422-34.

[0010] [6]IANKOV I D, PENHEITER A R, GRIESMANN G E, et al. Neutralization capacity of measles virus H protein specific IgG determines the balance between antibody-enhanced infectivity and protection in microglial cells [J]. Virus Res, 2013, 172(1-2): 15-23.

[0011] [7]BECK Z, PROHáSZKA Z, FüST G. Traitors of the immune system-enhancing antibodies in HIV infection: their possible implication in HIV vaccine development [J]. Vaccine, 2008, 26(24): 3078-85.

[0012] [8]TAKADA A, FELDMANN H, KSIAZEK T G, et al. Antibody-dependent enhancement of Ebola virus infection [J]. J Virol, 2003, 77(13): 7539-44.

[0013] [9]TAKADA A, WATANABE S, OKAZAKI K, et al. Infectivity-enhancing antibodies to Ebola virus glycoprotein [J]. J Virol, 2001, 75(5): 2324-30.

[0014]

[10] WAN Y, SHANG J, SUN S, et al. Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry [J]. J Virol, 2020, 94(5). Summary of the Invention

[0015] In order to better study the ADE phenomenon of the new coronavirus, the present invention provides a system for evaluating the ADE effect in vitro and its application. The system can be used to evaluate the ADE effect of the new coronavirus or the new coronavirus antibody to be tested, provide a basis for the study of the characteristics and mechanism of ADE of the SARS-CoV-2 vaccine, and provide a reference for the research and development of the SARS-CoV-2 vaccine.

[0016] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0017] A first aspect of the present invention provides a system for evaluating ADE effects in vitro, the system comprising:

[0018] (i) and (iii); (ii) and (iii); or (i), (ii) and (iii);

[0019] (i) is a SARS-CoV-2 pseudovirus, (ii) is an antibody targeting the receptor binding domain of SARS-CoV-2, and (iii) is a cell overexpressing CD32B; the cell is a CHO cell.

[0020] In some embodiments of the present invention, the system comprises one or more of the following conditions:

[0021] The CD32B comprises the amino acid sequence shown in NCBI Accession No. NP_003992.3;

[0022] The CD32B is expressed on the surface of the cell;

[0023] The SARS-CoV-2 pseudovirus comprises a gene encoding a protein having an amino acid residue difference at D614G from the Spike protein of SARS-CoV-2; the Spike protein comprises an amino acid sequence as shown in NCBI accession number YP_009724390.1.

[0024] In some specific embodiments of the present invention, the encoding gene comprises NC_045512.2.

[0025] In some embodiments of the present invention, the system comprises one or more of the following conditions:

[0026] The coding sequence of CD32B comprises the nucleic acid sequence shown in NCBI accession number NM_004001;

[0027] The concentration of the SARS-CoV-2 pseudovirus is 1E5-1E6 TU / mL;

[0028] The deposit number of the cell overexpressing CD32B is CCTCC NO: C2024285;

[0029] The concentration of the antibody is 16-10000 ng / mL;

[0030] The concentration of cells overexpressing CD32B is 1E5-2E5 / mL;

[0031] The antibody comprises the amino acid sequence as shown in NCBI Accession No. YP_009724390.1.

[0032] In some preferred embodiments of the present invention, the concentration of the antibody is 80-2000 ng / mL.

[0033] In some specific embodiments of the present invention, the concentration of the SARS-CoV-2 pseudovirus is 7.5E5 TU / mL.

[0034] The second aspect of the present invention provides a method for preparing the system as described in the first aspect, when the system includes (i), (ii) and (iii), the method includes: transfecting CHO cells with an overexpression plasmid containing a nucleic acid sequence encoding CD32B to prepare the cells overexpressing CD32B, obtaining the SARS-CoV-2 pseudovirus and the antibody against the receptor binding domain of SARS-CoV-2.

[0035] In some embodiments of the present invention, the backbone plasmid of the overexpression plasmid is pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro; and / or, the insertion site of the coding sequence in transfection is BamHI-EcoRI.

[0036] The third aspect of the present invention provides a method for evaluating ADE effects in vitro, the method comprising the following steps: (1) when the evaluation object is a virus, a pseudovirus or a preparation containing a virus, adding the evaluation object to the system comprising (ii) and (iii) as described in the first aspect as a sample to be tested; or, replacing (i) in the system comprising (i), (ii) and (iii) as described in the first aspect with the evaluation object as the sample to be tested; or,

[0037] When the subject of evaluation is an antibody or a preparation containing an antibody, the subject of evaluation is added to the system comprising (i) and (iii) in the system as described in the first aspect as the sample to be tested, or the subject of evaluation is substituted for (ii) in the system comprising (i), (ii) and (iii) in the system as described in the first aspect as the sample to be tested;

[0038] (2) detecting the degree of infection of the cells in the sample to be tested;

[0039] (3) Determine the risk of ADE effects.

[0040] In some embodiments of the present invention, in step (2), the fluorescence intensity of Luciferase is detected to characterize the degree of infection.

[0041] In some embodiments of the present invention, when the degree of infection of the cells in the test sample is higher than that of the cells in the negative control group, the subject to be evaluated is determined to have a risk of causing an ADE effect.

[0042] In some optional embodiments of the present invention, in step (3), the negative control group consists of (i) and (iii) in the system described in the first aspect.

[0043] In some embodiments of the present invention, the evaluation object is selected from the group consisting of SARS-CoV-2 virus or pseudovirus prepared therefrom, SARS-CoV-2 vaccine preparations, antibodies targeting SARS-CoV-2, and samples containing antibodies targeting SARS-CoV-2.

[0044] In some optional embodiments of the present invention, the sample is antiserum or plasma from a SARS-CoV-2 recovered patient.

[0045] A fourth aspect of the present invention provides a cell overexpressing CD32B, wherein the cell is a cell with a deposit number of CCTCC NO: C2024285.

[0046] The fifth aspect of the present invention provides the use of the system as described in the first aspect in assessing the risk of causing an ADE effect.

[0047] In some embodiments of the present invention, the evaluation object is selected from the group consisting of SARS-CoV-2 virus or pseudovirus prepared therefrom, SARS-CoV-2 vaccine preparations, antibodies targeting SARS-CoV-2, and samples containing antibodies targeting SARS-CoV-2.

[0048] In some optional embodiments of the present invention, the sample is antiserum or plasma from a SARS-CoV-2 recovered patient.

[0049] The fifth aspect of the present invention provides a cell for use in evaluating the ADE effect caused by SARS-CoV-2 or an antibody targeting SARS-CoV-2, wherein the cell is a cell overexpressing CD32B; the cell is a CHO cell.

[0050] In some embodiments of the present invention, the CD32B comprises the amino acid sequence shown in NCBI Accession No. NP_003992.3.

[0051] In some specific embodiments of the present invention, the coding sequence of CD32B comprises the nucleic acid sequence shown in NCBI Accession No. NM_004001.

[0052] In some specific embodiments of the present invention, the cells overexpressing CD32B are cells with a deposit number of CCTCC NO: C2024285.

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available except for the overexpressing cells.

[0055] The positive and progressive effects of the present invention are:

[0056] In previous studies, it is often believed that CD32A receptor protein is closely related to the ADE effect. However, the present invention finds that CD32B has the ability to mediate the ADE effect, and its ability to mediate ADE in the system is greater than that of CD32A, suggesting that attention should be paid to the risk of ADE when cells expressing CD32B are present. The present invention uses SARS-CoV-2 pseudovirus and cells expressing CD32B exogenously to identify the ADE effect of SARS-CoV-2 antibodies in vitro. The system for evaluating the ADE effect based on exogenously expressing CD32B cells provided by the present invention has the advantage of being more sensitive than the system based on cells expressing CD32A. In addition, the ADE evaluation system has a wide detection range and can evaluate the ADE effect caused by SARS-CoV-2 virus or pseudovirus prepared therefrom acting on CHO-CD32B in the presence of RBD antibodies in a wide concentration range (16 ng / mL-10000 ng / mL). The system provided by the present invention is a very potential system for evaluating the ADE effect of the new coronavirus, providing a basis for the study of the characteristics and mechanism of ADE of new coronavirus vaccines, and providing a reference for the development of SARS-CoV-2 vaccines. Biomaterial Deposit Information

[0057] The cells overexpressing CD32B of the present invention were deposited in the China Center for Type Culture Collection (CCTCC) on October 30, 2024, with the deposit address being Wuhan University, Wuhan, China, Postal Code 430072, with the deposit number being CCTCC NO: C2024285, the culture name being: Stably transfected Chinese hamster ovary cells CHO-CD32B, and the classification name being: Stably transfected Chinese hamster ovary cells CHO-CD32B Chinese Hamster Ovary. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1This is the map of the linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector (digested with BamHI+EcoRI; 8814 bp).

[0059] Figure 2 This is the FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9741 bp).

[0060] Figure 3 This is the FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9723 bp).

[0061] Figure 4 This is the FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9762 bp).

[0062] Figure 5 This is the sequencing map of h-FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0063] Figure 6 This is the sequencing map of h-FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0064] Figure 7 This is the sequencing map of h-FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0065] Figure 8 This is the result of antibody neutralization ability test. Fig. 9 This shows the situation of HIV / SARS-CoV-2-614G pseudovirus infecting CHO, CHO-CD32A, CHO-CD32B, CHO-CD32C, Raji and THP-1 cells in the absence of antibodies.

[0066] Fig.10 This is the infection of CHO, CHO-CD32A, CHO-CD32B, and CHO-CD32C cells by HIV / SARS-CoV-2-614G in the presence of different concentrations of antibodies. Fig.11 The expression of CD32A, CD32B and CD32C in CHO, CHO-CD32A, CHO-CD32B, CHO7-CD32C, Raji and THP-1 cells.

[0067] Fig.12 This is the situation of SARS-CoV-2 infecting Raji or THP-1 cells (immune cells) in the presence of monoclonal antibodies against the new coronavirus.

[0068] Fig.13 The purpose is to detect the ADE effect of commercially available SARS-CoV-2 antibodies using a system for evaluating the ADE effect of the new coronavirus in vitro. DETAILED DESCRIPTION

[0069] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0070] Example 1: Construction of a cell line expressing immunoglobulin IgG receptor FcγR

[0071] 1.1 Construction of overexpression plasmid

[0072] In this part of the experiment, the FCGR2A / FCGR2B / FCGR2C (i.e., CD32A / CD32B / CD32C) genes were inserted into the pLV-CMV-MCS-EF1-ZsGreen1- T2A-Puro vector. The vector plasmid was provided by Fenghui Biotechnology.

[0073] The NCBI accession number of the amino acid sequence of FCGR2A is NP_067674.2; the NCBI accession number of the FCGR2A gene sequence is: NM_021642;

[0074] The NCBI accession number of the amino acid sequence of FCGR2B is NP_003992.3; the NCBI accession number of the FCGR2B gene sequence is: NM_004001;

[0075] The NCBI accession number of the amino acid sequence of FCGR2C is NP_963857.3; the NCBI accession number of the FCGR2C gene sequence is: NM_201563.

[0076] (1) Vector map

[0077] The linearized pLV-CMV-MCS-EF1-ZsGreen1- T2A-Puro vector map is as follows Figure 1 The vector was completely digested with BamHI-EcoRI, and the large fragment was recovered by 1% Agarose gel electrophoresis to obtain the linearized vector.

[0078] (2) Enzyme digestion of pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector

[0079] The small-scale plasmid pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro was double-digested with BamHI-EcoRI. The enzyme digestion system is shown in Table 1:

[0080] Table 1 Enzyme digestion system

[0081]

[0082] Note: After reacting at 37℃ for 5 h, large fragments were recovered by 1% agarose gel.

[0083] (3) Obtaining FCGR2A / FCGR2B / FCGR2C gene fragments

[0084] Select FCGR2A / FCGR2B / FCGR2C gene sequences to design primers (FCGR2A-F / R, FCGR2B-F / R, FCGR2C-F / R are recombination primers) for constructing gene subcloning vectors. The sequences are shown in Table 2:

[0085] Table 2 Primer sequences

[0086]

[0087] The synthesized primers were diluted to a working solution with a final concentration of 10 µmol / L, and PCR amplification was performed using the diluted primers and template. The system is shown in Table 3:

[0088] Table 3 Amplification system

[0089]

[0090] Add the above materials into the thin-walled tube, mix well, and then place it into the PCR instrument. The final reaction program after adjustment is as shown in Table 4.

[0091] Table 4 Amplification procedures

[0092]

[0093] (4) Ligation of target gene fragment and vector

[0094] The recovered and purified target fragment was connected with the recovered and purified vector: pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro. The connection product was named FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro. Figure 2 As shown; FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as Figure 3As shown; FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as Figure 4 shown.

[0095] Since the primers contain homologous arm sequences to the vector, homologous recombinase is used to recombinate the recovered fragments with the linearized vector, completing the recombination process of recovering the target gene and the vector.

[0096] (5) Transformation of ligation products into competent cells

[0097] Take 10 μL of the ligation product to transform 100 μL of DH5a competent cells: mix the product and competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, immediately place on ice for 2 min, add 500 μL of LB medium preheated to room temperature, culture in a constant temperature shaker at 180 rpm and 37°C for 1 h, centrifuge at 5000 rpm for 3 min, discard 500 μL of the culture supernatant, mix the remaining 100 μL with a pipette, and evenly spread it on an LB plate containing 50 μg / mL ampicillin resistance, invert, and culture in a constant temperature incubator at 37°C overnight.

[0098] (6) PCR identification

[0099] Pick 4 single colonies and inoculate them in 5 mL of LB culture medium containing 50 μg / mL ampicillin resistance, and culture them in a constant temperature shaker at 220 rpm and 37℃ for 5 hours. Use the cultured bacterial solution for PCR identification. The sequencing results are compared with the expected sequence, and the consistency of the comparison results is 100%, indicating that the plasmid construction is successful. The test results are as follows Figure 5-Figure 7 shown.

[0100] 1.2 Plasmid transfection cell line process

[0101] This part of the experiment aims to construct a CD32A / CD32B / CD32C gene overexpression stable cell line, so that the overexpression plasmids carrying CD32A / CD32B / CD32C genes are transfected into CHO cells and stably expressed.

[0102] (1) Preparing cells for the experiment

[0103] Determine relevant information of CHO cell lines, including cell culture conditions, cell proliferation rate, and mycoplasma contamination.

[0104] (2) Screening and determination of the dosage of Puromycin

[0105] The lethal dose information of Puro in the stable cell line screening in CHO cell line was consulted, and six drug concentration gradients (0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL) were determined with reference to the obtained data.

[0106] Day 1: CHO cell line cells were plated in 6-well plates so that the cell density was about 90% on the second day;

[0107] Day 2: Add Puro to cells at the set concentration;

[0108] Day 4: Change the medium and add Puro at the set concentration again;

[0109] Day 7: Observe and find the well with the lowest drug concentration when the cell lethality rate is 100%. The drug concentration used in this well is the Puro screening concentration;

[0110] (3) Screening and construction steps of stable cell lines

[0111] Cell plating: CHO cells were seeded in 6-well plates so that the cell density reached about 70% on the second day;

[0112] Virus infection: 200 μL of lentivirus was added with 2×10 5 in cells;

[0113] Replace the medium: Replace the medium according to the actual situation. For some cells with weak tolerance, the medium should be replaced in time; for some cells with strong tolerance, the medium can be replaced 48-72 hours after infection;

[0114] Observe the infection efficiency: 72 hours after infection, observe the infection efficiency, the minimum efficiency should not be less than 40%;

[0115] Puro screening: The best duration of action is between 3-10 days. The commonly used concentration of Puro is in the range of 1-10 μg / mL. After the optimal screening concentration is determined through preliminary experiments, virus infection can be carried out;

[0116] Infection: After 72 h of infection culture (the infection time depends on the specific conditions of the cells and the infection efficiency), add the drug concentration of 2 μg / mL determined in the previous pre-experiment to the 6-well plate;

[0117] Add Puro: Add Puro drug concentration of 2 μg / mL determined in the previous pilot experiment to the 6-well plate;

[0118] Medium replacement: Depending on the color of the culture medium and cell growth, the screening medium should be replaced every 3 to 5 days. When a large number of cells die, the Puro concentration can be halved to maintain screening;

[0119] Observation: Observe the cell status, growth, and gene expression level and proportion every day until the proportion of fluorescent cells under the microscope is above 90%.

[0120] 1.3 q-PCR detection of transfection effect

[0121] method:

[0122] Using q-PCR technology, fluorescent groups are added to the PCR reaction system, and the entire PCR process is monitored in real time using the accumulated fluorescent signals. Finally, the unknown template is quantitatively analyzed using the standard curve. The change in the amount of amplified products in each cycle of the PCR amplification reaction can be detected in real time based on the change in the fluorescent signal, and the starting template can be quantitatively analyzed by analyzing the Ct value and the standard curve.

[0123] (1) Primer design

[0124] The primer sequences are shown in Table 5:

[0125] Table 5 Primer sequences

[0126]

[0127] The internal reference primers are shown in Table 6:

[0128] Table 6 Internal reference primers

[0129]

[0130] (2) RNA extraction

[0131] i. Add 1000 μL Trizol to the 1.5 mL EP tube containing the cell line, mix thoroughly, then add 200 μL chloroform, shake and mix, let stand for 5 min, and then centrifuge at 12,000 rpm, 4°C for 10 min.

[0132] ii. Take out the 1.5 mL EP tube from the centrifuge, and then aspirate the upper colorless and transparent aqueous phase into another clean 1.5 mL EP tube. (The sample will be divided into three layers: the lower organic phase, the middle layer and the upper aqueous phase, and the RNA is in the upper aqueous phase) Add an equal volume of isopropanol, gently invert up and down to mix, let it stand for 10 minutes, and then centrifuge at 12000 rpm, 4℃ for 10 minutes.

[0133] iii. After centrifugation, a gelatinous precipitate can be seen on the wall or bottom of the tube. The precipitate is RNA. Carefully discard the supernatant and keep the precipitate.

[0134] iv. Wash the RNA precipitate with 1 mL of 75% ethanol (prepared with DEPC H2O). Then centrifuge at 7000 rpm for 5 min at 4°C and remove the supernatant as much as possible.

[0135] v. Leave to dry on the operating table for about 5 to 10 minutes. (Over-drying will greatly reduce the solubility of RNA). Add 25 μL of DEPC H2O to all EP tubes, blow several times with a pipette tip to fully dissolve the RNA, and store at -80℃.

[0136] RNA concentration detection: RNA concentration was detected using a micro-nucleic acid detector. The results are shown in Table 7:

[0137] Table 7 RNA concentration

[0138]

[0139] (3) Reverse transcription PCR

[0140] i. Prepare the reverse transcription reaction solution according to the components shown in Table 8:

[0141] Table 8 Reaction system

[0142]

[0143] ii. Perform the following reaction on a PCR instrument: 72°C for 5 min, then cool on ice.

[0144] iii. As shown in Table 9, add the following reverse transcription reaction solution to the above PCR tube:

[0145] Table 9 Reaction system

[0146]

[0147] iv. As shown in Table 10, reverse transcription reaction was performed on a PCR instrument under the following conditions:

[0148] Table 10 Reaction procedure

[0149]

[0150] v. Use cDNA immediately for experiments or store at 4°C.

[0151] (4) Real-time fluorescence quantitative PCR reaction

[0152] i. The configuration of the reaction system is shown in Table 11:

[0153] Table 11 Reaction system

[0154]

[0155] ii. Reaction condition settings:

[0156] The amplification program is shown in Table 12.

[0157] Table 12 Amplification procedures

[0158]

[0159] The melting program is shown in Table 13.

[0160] Table 13 Melting program

[0161]

[0162] In order to establish a more sensitive SARS-CoV-2 ADE detection cell line and identify FcγR molecules with ADE activity, CHO cells stably expressing type IIA, type IIB, and type IIC FcRs were constructed: CHO-CD32A, CHO-CD32B, and CHO-CD32C. Fig.11 As shown, this example verifies the expression of the immunoglobulin IgG receptor FcγR-expressing cell line and the expression of CD32A, CD32B and CD32C molecules in immune cells by RT PCR.

[0163] Example 2: Construction of SARS-CoV-2 pseudovirus (the pseudovirus was purchased from Jiman Biotech Co., Ltd.)

[0164] This pseudovirus is a replication-defective pseudovirus that is constructed based on the HIV lentiviral packaging system by carrying out the D614G mutation of the Spike protein of SARS-CoV-2 (the NCBI accession number of the amino acid sequence is YP_009724390.1) (the mutated encoding nucleotide sequence is the 21563rd to 25384th positions of the nucleotide sequence shown in the NCBI accession number NC_045512.2), and encapsulating the GFP green fluorescence and Luciferase firefly luciferase gene expression plasmids and capable of infecting 293T-ACE2 cells.

[0165] 2.1 Vector construction

[0166] Culture the bacterial solution containing the vector plasmid overnight, and extract the plasmid using 3-5 mL of fresh bacterial solution (DP107-02 High Purity Plasmid Mini-Extraction Kit). Take 1 µg of fresh plasmid and perform double digestion with the corresponding restriction endonuclease. Perform agarose gel electrophoresis on the digested product, and after the electrophoresis, perform gel recovery. Transfer all the above liquids to the filter column, centrifuge, obtain the recovered vector fragment, and determine the concentration. Perform PCR amplification on the diluted primers and template, perform agarose gel electrophoresis after the PCR, and recover the target gene. Use Hieff Clone TM The recombination reaction system connects the overexpression vector and the target fragment, and introduces the connection product into competent cells for transformation. Finally, sequencing is performed to verify the target vector.

[0167] 2.2 Pseudovirus Packaging

[0168] Preparation of recombinant viral plasmid encoding lentiviral particles and its auxiliary packaging vector plasmid, recombinant plasmid vector and auxiliary plasmid vector were extracted with high purity and endotoxin-free, using HG Transgene TM Reagent was co-transfected into 293T cells, and the culture medium was replaced with complete culture medium 18 h after transfection. After culturing for 48 h, the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate, which was aliquoted and stored at -80°C.

[0169] Example 3: Detection of SARS-CoV-2 Antibody Neutralization Ability

[0170] 3.1 Cell subculture and plating

[0171] The stably transfected cell line HEK293T-ACE2 (from Beijing Sino Biological Technology Co., Ltd.) was subcultured in a 5% CO2 incubator at 37°C using complete culture medium (DMEM + 10% FBS). The medium was changed every 4 days and collected into sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in fresh complete culture medium, stained, counted, and 3 × 10 6 The cells were transferred into cell culture flasks and cultured continuously.

[0172] Take cells with good growth status for plating, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add complete culture medium to resuspend the cells, and adjust the cell concentration to 1×10 5 100 µL / well of the cell suspension was added to a 96-well plate and cultured overnight.

[0173] 3.2 SARS-CoV-2 pseudovirus dilution

[0174] Preparation of pseudovirus dilution: Pseudovirus (titer: 3*108 TU / mL from Jiman Biotech Co., Ltd.) was diluted 5000 times with DMEM + 10% FBS.

[0175] 3.3 SARS-CoV-2 Antibody Serial Dilution

[0176] Preparation of antibody dilution solution: RBD antibody (100 μg, from RD SYSTEMS, catalog number: MAB105802-100; the amino acid sequence of the antibody is shown in NCBI accession number YP_009724390.1) was diluted with 1 mL PBS to prepare a 100 μg / mL stock solution, which was then serially diluted with complete culture medium to 7 dilutions: 100,000 ng / mL, 20,000 ng / mL, 4,000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, and 0 ng / mL.

[0177] 3.4 Adding samples

[0178] After the pseudovirus and antibody were mixed in a 1:1 ratio (the final concentrations of the antibodies were 50000 ng / mL, 10000 ng / mL, 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL and 0 ng / mL; the final concentration of the pseudovirus was 3E4 TU / mL), they were incubated at 37°C for 1 hour, the culture medium of the 96-well plate was removed, and 100 μL of the virus-antibody mixture was added to each well. At the same time, a blank control of 100 μL of culture medium was added. After 6 hours, the supernatant was removed and 100 μL of culture medium was added.

[0179] 3.5 Detection of chemiluminescence

[0180] After 48 hours, the culture medium was removed, the cells were washed once with PBS, 25 µL / well lysis buffer was added and incubated for 5-10 minutes, 20 µL was taken to a white plate, 100 µL of luminescent substrate (Promega Luciferase Assay System) was added, and the Luciferase luminescent signal was detected. The signal value of the analyte was the detection signal value minus the blank control signal value, and the analyte signal value was used as the ordinate for plotting.

[0181] The results are as follows Figure 8 As shown in the figure, as the antibody concentration decreases, the fluorescence signal gradually increases, indicating that the antibody has neutralizing ability. The new coronavirus RBD monoclonal antibody can inhibit HIV / SARS-CoV-2-614G pseudovirus from infecting 293 / ACE2 cells, and its neutralizing ability increases with the increase of antibody concentration. The signal value of the positive control well (i.e., when the corresponding antibody titer is 0) is high, indicating that the pseudovirus has a strong ability to infect.

[0182] Example 4 System for in vitro evaluation of the ADE effect of the new coronavirus

[0183] CHO is a cell found in the ovary of the Chinese hamster. HIV / SARS-CoV-2-614G pseudovirus cannot infect CHO cells, and the novel coronavirus RBD monoclonal antibody cannot promote infection within the concentration range tested.

[0184] 4.1 Cell subculture and plating:

[0185] The stable cell lines CHO-CD32A, CHO-CD32B and CHO-CD32C prepared above as well as immune cells Raji and THP-1 were plated and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in complete culture medium. The cell concentration was adjusted to 1×10 5 100 µL / well of the cell suspension was added to a 96-well plate and cultured overnight.

[0186] 4.2 SARS-CoV-2 pseudovirus dilution

[0187] Preparation of pseudovirus dilution: Pseudovirus (titer: 3×10 8 TU / mL from Jiman Biotech Co., Ltd.) was diluted 200 times with DMEM / F-12 + 10% FBS.

[0188] 4.3 Antibodies and SARS-CoV-2 pseudovirus incubation:

[0189] The above RBD antibody was diluted 5-fold to 8 concentration gradients (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 0 ng / mL), mixed with the same volume of pseudovirus dilution (the final concentrations of the antibodies in the system were 50000 ng / mL, 10000 ng / mL, 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL and 0 ng / mL; the final concentration of pseudovirus was 7.5E5 TU / mL), and incubated at 37°C for 1 h.

[0190] 4.4 Adding samples:

[0191] After the antibody and pseudovirus were incubated, 100 µL / well of the virus-antibody mixture was added to a 96-well plate, and 100 µL of complete culture medium was added to the blank control. The plates were cultured at 37°C and 5% carbon dioxide for 48 hours.

[0192] 4.5 Detection of chemiluminescence

[0193] Completely aspirate the supernatant, wash once with PBS, add 25 µL / well lysis buffer and incubate for 5-10 minutes, take 20 µL to a white plate, add 100 µL of luminescent substrate (Promega Luciferase Assay System), and detect the Luciferase luminescent signal. The signal value of the analyte is the detection signal value minus the blank control signal value, and the analyte signal value is used as the ordinate for plotting.

[0194] like Fig. 9 As shown in the figure, in the absence of antibodies, no obvious signals were detected in each experimental group, which was close to the background value. It can be seen that the expression of CD32A\CD32B\CD32C does not promote the infection of the new coronavirus. Among them, THP-1 cells are mononuclear macrophages, and FcγRⅡ is expressed on the cell surface. Raji cells are commonly used ADE detection cells, and Fc receptors are expressed on the cell surface.

[0195] like Fig.10 As shown in the figure, in the presence of SARS-CoV-2 RBD monoclonal antibody, it significantly promoted HIV / SARS-CoV-2-614G infection of CHO-CD32B and CHO-CD32A cells within the sub-neutralizing concentration range, and the ADE effect mediated by CD32B was stronger than that of CD32A. In addition, the antibody concentration range that can trigger the ADE effect in this ADE system is large, and the detection range of the ADE system is wide, which can sensitively and effectively evaluate the ADE effect caused by SARS-CoV-2 virus or pseudovirus prepared therefrom acting on CHO-CD32B in the presence of different concentrations (16 ng / mL-10000 ng / mL) of RBD antibody. Fig.12 As shown, THP-1 cells are mononuclear macrophages that express FcγRⅡ on their cell surface, but SARS-CoV-2 pseudovirus cannot infect THP-1 cells, and SARS-CoV-2 RBD monoclonal antibody cannot promote infection within the concentration range tested.

[0196] Raji cells are commonly used ADE detection cells and express Fc receptors on their cell surface. However, HIV / SARS-CoV-2-614G pseudovirus cannot infect Raji cells, and the novel coronavirus RBD monoclonal antibody cannot promote infection within the concentration range tested.

[0197] Example 5 Application of a system for evaluating the ADE effect of novel coronavirus in vitro

[0198] The system provided by the present invention is used to detect the risk of ADE effect of commercially available antibodies:

[0199] SARS-CoV-2 Spike S1 rabbit mAb, from ABclonal, catalog number: A20834;

[0200] SARS-CoV-2 Spike S1 rabbit pAb, from ABclonal, catalog number: A20136;

[0201] SARS-CoV-2 Spike rabbit pAb, from ABclonal, catalog number: A20137;

[0202] SARS-CoV-2 Spike S2 ​​ECD rabbit pAb, from ABclonal, catalog number: A20138;

[0203] SARS-CoV-2 Spike S2 ​​rabbit pAb, from ABclonal, catalog number: A20284;

[0204] SARS-CoV-2 Spike S2 ​​subunit antibody, from RD SYSTEMS, catalog number: MAB10889;

[0205] SARS-CoV-2 Spike RBD antibody, from RD SYSTEMS, catalog number: MAB105802;

[0206] SARS-CoV-2 membrane antibody, from RD SYSTEMS, catalog number: MAB10690;

[0207] SARS-CoV-2 membrane antibody, from RD SYSTEMS, catalog number: MAB11038;

[0208] SARS-CoV-2 Envelope Antibody, from RD SYSTEMS, Catalog Number: MAB10925

[0209] 5.1 Cell subculture and plating

[0210] The stably transfected cell line CHO-CD32B prepared in Example 1 was plated and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in complete culture medium to adjust the cell concentration to 1×10 5 100 µL / well of the cell suspension was added to a 96-well plate and cultured overnight.

[0211] 5.2 SARS-CoV-2 pseudovirus dilution

[0212] Preparation of pseudovirus dilution: Pseudovirus (titer: 3×10 8TU / mL from Jiman Biotech Co., Ltd.) was diluted 200 times with DMEM / F-12 + 10% FBS.

[0213] 5.3 Incubation of antibodies with SARS-CoV-2 pseudovirus

[0214] The antibodies were diluted 5-fold to 8 concentration gradients (100,000 ng / mL, 20,000 ng / mL, 4,000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, and 0 ng / mL), mixed with the same volume of pseudovirus dilution (the final concentrations of antibodies in the system were 50,000 ng / mL, 10,000 ng / mL, 2,000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, and 0 ng / mL; the final virus concentration was 7.5E5 TU / mL), and incubated at 37°C for 1 h.

[0215] 5.3 Adding samples

[0216] After the antibody and pseudovirus were incubated, 100 µL / well of the virus-antibody mixture was added to a 96-well plate, and 100 µL of complete culture medium was added to the blank control. The plates were cultured at 37°C and 5% carbon dioxide for 48 hours.

[0217] 5.4 Detection of chemiluminescence

[0218] Completely aspirate the supernatant, wash once with PBS, add 25 µL / well lysis buffer and incubate for 5-10 minutes, take 20 µL to a white plate, add 100 µL of luminescent substrate (Promega Luciferase Assay System), and detect the Luciferase luminescent signal. The signal value of the analyte is the detection signal value minus the blank control signal value, and the analyte signal value is used as the ordinate for plotting.

[0219] The results are as follows Fig.13 As shown, SARS-CoV-2 Spike RBD antibody showed a significant effect in promoting SARS-CoV-2 infection of CHO-CD32B cells at sub-neutralizing concentrations, SARS-CoV-2 Spike S1 rabbit pAb showed a weaker ADE effect, and the other antibodies did not show ADE effect.

[0220] As demonstrated in this example, the system for evaluating the ADE effect provided by the present invention can detect whether the antibody targeting SARS-CoV-2 has the risk of causing the ADE effect. The system comprises a SARS-CoV-2 pseudovirus and a cell overexpressing CD32B; or, the system comprises an antibody targeting the receptor binding domain of SARS-CoV-2 and a cell overexpressing CD32B; or, the system comprises SARS-CoV-2, an antibody targeting the receptor binding domain of SARS-CoV-2 and a cell overexpressing CD32B, and the cell is a CHO cell.

[0221] As demonstrated in the examples, the system for evaluating ADE effects provided by the present invention can be used to detect in vitro whether an evaluation subject has a risk of causing ADE effects.

Claims

1. A system for evaluating ADE effects in vitro, characterized in that: The system comprises: (i) and (iii); (ii) and (iii); or (i), (ii) and (iii); (i) is a SARS-CoV-2 pseudovirus, (ii) is an antibody targeting the receptor binding domain of SARS-CoV-2, and (iii) is a cell overexpressing CD32B; the cell is a CHO cell.

2. The system according to claim 1, characterized in that The system includes one or more of the following conditions: The CD32B comprises the amino acid sequence shown in NCBI Accession No. NP_003992.3; The CD32B is expressed on the surface of the cell; The SARS-CoV-2 pseudovirus comprises a gene encoding a protein having a D614G amino acid residue difference compared to the Spike protein of SARS-CoV-2; the Spike protein comprises an amino acid sequence as shown in NCBI Accession No. YP_009724390.

1.

3. The system according to claim 1 or 2, characterized in that The system includes one or more of the following conditions: The coding sequence of CD32B comprises the nucleic acid sequence shown in NCBI accession number NM_004001; The concentration of the SARS-CoV-2 pseudovirus is 1E5-1E6TU / mL; The deposit number of the cell overexpressing CD32B is CCTCC NO: C2024285; The concentration of the antibody is 16-10000 ng / mL; The antibody comprises the amino acid sequence as shown in NCBI Accession No. YP_009724390.

1.

4. A method for preparing a system as claimed in any one of claims 1 to 3, characterized in that: When the system includes (i), (ii) and (iii), the method includes: transfecting CHO cells with an overexpression plasmid containing a nucleic acid sequence encoding CD32B to prepare the cells overexpressing CD32B, obtaining the SARS-CoV-2 pseudovirus and the antibody targeting the receptor binding domain of SARS-CoV-2.

5. A method for evaluating ADE effect in vitro, characterized in that: The method comprises the following steps: (1) when the evaluation object is a virus, a pseudovirus or a preparation containing a virus, adding the evaluation object to the system comprising (ii) and (iii) in any one of claims 1 to 3 as a sample to be tested; or, replacing (i) in the system comprising (i), (ii) and (iii) in any one of claims 1 to 3 with the evaluation object as the sample to be tested; or, When the subject of evaluation is an antibody or a preparation containing an antibody, the subject of evaluation is added to the system comprising (i) and (iii) in the system as described in any one of claims 1 to 3 as a sample to be tested, or the subject of evaluation replaces (ii) in the system comprising (i), (ii) and (iii) in the system as described in any one of claims 1 to 3 as a sample to be tested; (2) detecting the degree of infection of the cells in the sample to be tested; (3) Determine the risk of ADE effects; Preferably, in step (2), the fluorescence intensity of Luciferase is detected to characterize the degree of infection; and / or, In step (3), when the degree of infection of the cells in the test sample is higher than that of the cells in the negative control group, the evaluation object is determined to have a risk of causing an ADE effect; Optionally, in step (3), the negative control group consists of (i) and (iii) in the system according to any one of claims 1 to 3.

6. The method according to claim 5, characterized in that The evaluation object is selected from SARS-CoV-2 virus or pseudovirus prepared therefrom, SARS-CoV-2 vaccine preparation, antibodies targeting SARS-CoV-2, and samples containing antibodies targeting SARS-CoV-2; the sample is, for example, antiserum or plasma of SARS-CoV-2 recovered patients.

7. A cell overexpressing CD32B, characterized in that: The cell is a cell with a deposit number of CCTCC NO: C2024285.

8. Use of the system according to any one of claims 1 to 3 in assessing the risk of causing an ADE effect; Preferably, the evaluation object is selected from SARS-CoV-2 virus or pseudovirus prepared therefrom, SARS-CoV-2 vaccine preparations, antibodies targeting SARS-CoV-2, and samples containing antibodies targeting SARS-CoV-2; the sample is, for example, antiserum or plasma from a SARS-CoV-2 recovered patient.

9. Use of a cell in evaluating the ADE effect caused by SARS-CoV-2 or an antibody targeting SARS-CoV-2, characterized in that: The cells are cells that overexpress CD32B; and the cells are CHO cells.

10. The use according to claim 9, characterized in that The CD32B comprises the amino acid sequence shown in NCBI Accession No. NP_003992.3; Preferably, the coding sequence of CD32B comprises the nucleic acid sequence as shown in NCBI Accession No. NM_004001; and / or, the cell overexpressing CD32B is a cell with a deposit number of CCTCC NO: C2024285.

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