A system for evaluating ADE effect and its application

Abstract

The present invention discloses a system for evaluating the ADE effect and its application. The system comprises: (i) and (iii); (ii) and (iii); or (i), (ii) and (iii); (i) is a pseudovirus of Middle East respiratory syndrome coronavirus, (ii) is an antibody targeting the receptor binding domain of Middle East respiratory syndrome coronavirus, and (iii) is a cell overexpressing CD32B; the cell is a CHO cell; the pseudovirus comprises the Spike protein encoding gene of Middle East respiratory syndrome coronavirus. The present invention utilizes MERS-CoV pseudovirus and cells exogenously expressing CD32B to identify the ADE activity of Middle East respiratory coronavirus antibodies in vitro. The system provided by the present invention is more sensitive than the ADE evaluation system based on immune cell Raji and cells exogenously expressing CD32A, and is a potential system for evaluating the ADE effect of Middle East respiratory coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a system for evaluating ADE effects 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 patients are infected with one serotype of dengue virus (i.e., primary infection), they produce neutralizing antibodies against the same serotype of virus. However, if they are subsequently infected with another serotype of dengue virus (i.e., secondary infection), the pre-existing antibodies cannot fully neutralize the virus. Instead, the antibodies first bind to the virus and then bind to immunoglobulin Fc receptors on immune cells and mediate the entry of the virus into these cells. [5] In many respiratory viruses, including RSV, measles [6] , MERS-CoV, and other viruses, including human immunodeficiency virus (HIV) [7] and Ebola virus (EBOV) [8,9] In the literature, a correlation between previously available antibodies and the severity of disease progression has been found.

[0003] Since no MERS-CoV vaccine has been approved for marketing, and ADE has occurred in many other vaccine clinical studies in the past, especially in vaccines for respiratory mucosal infections, such as the severe acute respiratory syndrome coronavirus (SARS-CoV) of the same species as MERS-CoV.

[10] In addition, there are corresponding discoveries in the development process of respiratory syncytial virus (RSV) vaccine.

[11] Therefore, it is crucial to pay attention to the ADE effect in the development of MERS-CoV vaccines.

[0004] Based on the current research on ADE, although there are reports

[12] , but some of the manifestations or patterns of ADE are still unclear. Although studies have pointed out that the Fc receptor FcγRIIA of immunoglobulin IgG, also known as CD32A, can mediate the ADE phenomenon, 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 type of Fc receptors that work on their cell surface is still controversial. Clarifying the key Fc receptor types in the potential ADE of MERS-CoV and building a convenient and reliable ADE evaluation system have become key steps in studying the ADE phenomenon of Middle East respiratory coronavirus; providing a reference for the development of MERS-CoV vaccines to help vaccine innovation and development.

[0005] References

[0006] [1] HAWKES R A. Enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls [J]. Aust J Exp Biol Med Sci, 1964, 42: 465-82.

[0007] [2] GUZMAN MG, ALVAREZ M, RODRIGUEZ-ROCHE R, et al. Neutralizingantibodies after infection with dengue 1 virus [J]. Emerg Infect Dis, 2007,13(2): 282-6.

[0008] [3] WHITEHEAD SS, BLANEY JE, DURBIN AP, et al. Prospects for adengue virus vaccine [J]. Nat Rev Microbiol, 2007, 5(7): 518-28.

[0009] [4] KATZELNICK LC, GRESH L, HALLORAN ME, et al. Antibody-dependentenhancement of severe dengue disease in humans [J]. Science, 2017, 358(6365):929-32.

[0010] [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.

[0011] [6] IANKOV I D, PENHEITER A R, GRIESMANN G E, et al. Neutralizationcapacity of measles virus H protein specific IgG determines the balancebetween antibody-enhanced infectivity and protection in microglial cells [J].Virus Res, 2013, 172(1-2): 15-23.

[0012] [7] BECK Z, PROHáSZKA Z, FüST G. Traitors of the immune system-enhancing antibodies in HIV infection: their possible implication in HIVvaccine development [J]. Vaccine, 2008, 26(24): 3078-85.

[0013] [8] TAKADA A, FELDMANN H, KSIAZEK T G, et al. Antibody-dependentenhancement of Ebola virus infection [J]. J Virol, 2003, 77(13): 7539-44.

[0014] [9] TAKADA A, WATANABE S, OKAZAKI K, et al. Infectivity-enhancing antibodies to Ebola virus glycoprotein [J]. J Virol, 2001, 75(5): 2324-30.

[0015]

[10] THOMAS S, SMATTI M K, OUHTIT A, et al. Antibody-Dependent Enhancement (ADE) and the role of complement system in disease pathogenesis [J]. Mol Immunol, 2022, 152: 172-82.

[0016]

[11] MA J, GONG T, LUO T, et al. Exacerbated lung inflammation in offspring with high maternal antibody levels following secondary RSV exposure [J]. Front Immunol, 2024, 15: 1377374.

[0017]

[12] 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

[0018] The present invention provides a system for evaluating the ADE effect of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in vitro based on cells overexpressing CD32B and the application of its construction method. The system includes cells expressing immunoglobulin IgG receptor FcγR, and / or a solution containing Middle East respiratory coronavirus antibodies, and / or Middle East respiratory coronavirus pseudovirus; wherein the immunoglobulin IgG receptor FcγR is FcγRIIB, i.e. CD32B; Middle East respiratory coronavirus antibodies promote Middle East respiratory coronavirus pseudovirus infection of immunoglobulin IgG receptor FcγR II B (CD32B) expressing cells. The present invention utilizes MERS-CoV pseudovirus and cells exogenously expressing CD32B to identify the ADE activity of Middle East respiratory coronavirus antibodies in vitro. The system provided by the present invention is more sensitive than the ADE evaluation system based on exogenously expressing CD32A cells, and is a very potential system for evaluating the ADE effect of Middle East respiratory coronavirus.

[0019] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0020] A first aspect of the present invention provides a system for evaluating an ADE effect, the system comprising:

[0021] (i) and (iii); (ii) and (iii); or (i), (ii) and (iii);

[0022] (i) is a Middle East Respiratory Syndrome Coronavirus pseudovirus, (ii) is an antibody targeting the receptor binding domain of Middle East Respiratory Syndrome Coronavirus, and (iii) is a cell overexpressing CD32B;

[0023] The cells are CHO cells; the pseudovirus contains the Spike protein coding gene of the Middle East Respiratory Syndrome Coronavirus.

[0024] In some embodiments of the present invention, the system comprises one or more of the following conditions:

[0025] The CD32B comprises the amino acid sequence shown in NCBI accession number NP_003992.3;

[0026] The CD32B is overexpressed on the surface of the cell;

[0027] The Spike protein encoding gene encodes the amino acid sequence shown in NCBI Accession No. YP 009047204.1.

[0028] In some specific embodiments of the present invention, the Spike protein encoding gene comprises the nucleotide sequence shown in NCBI Accession No. AFS88936.1.

[0029] In some specific embodiments of the present invention, the coding sequence of CD32B comprises the nucleic acid sequence shown in NCBI accession number NM_004001; and / or,

[0030] The deposit number of the cell overexpressing CD32B is CCTCC NO: C2024285.

[0031] In some embodiments of the present invention, the concentration of the Middle East Respiratory Syndrome Coronavirus pseudovirus is 5E10-2.5E11 copies / mL; and / or the concentration of the antibody is 16-50000 ng / mL; and / or the antibody comprises the amino acid sequence shown in NCBI Accession No. YP_007188579.1.

[0032] 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 Middle East Respiratory Syndrome Coronavirus pseudovirus and the antibody targeting the receptor binding domain of the Middle East Respiratory Syndrome Coronavirus.

[0033] 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 encoding nucleic acid sequence in transfection is BamHI-EcoRI; the encoding nucleic acid sequence comprises the nucleic acid sequence shown in NCBI accession number NM_004001.

[0034] The third aspect of the present invention provides a method for evaluating the ADE effect 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 as described in the first aspect, comprising (i i ) and (iii) as the sample to be tested, or replace the evaluation object with (i) in the system including (i), (ii) and (iii) as the sample to be tested; or,

[0035] 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;

[0036] (2) detecting the degree of infection of the cells in the sample to be tested;

[0037] (3) Determine the risk of ADE effect. 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 the risk of causing ADE effect.

[0038] In some embodiments of the present invention, the evaluation object in step (1) is selected from Middle East Respiratory Syndrome Coronavirus or a pseudovirus prepared therefrom, a Middle East Respiratory Syndrome Coronavirus vaccine preparation, an antibody targeting Middle East Respiratory Syndrome Coronavirus, or an antiserum containing an antibody targeting Middle East Respiratory Syndrome Coronavirus or plasma of a recovered patient of Middle East Respiratory Syndrome; and / or,

[0039] In step (2), the fluorescence intensity of Luciferase is detected to characterize the degree of infection; and / or,

[0040] In step (3), the negative control group comprises (i) and (iii) in the system described in the first aspect.

[0041] In some specific 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.

[0042] A fourth aspect of the present invention provides a use of the system as described in the first aspect in assessing the risk of causing an ADE effect;

[0043] The evaluation object is selected from Middle East Respiratory Syndrome Coronavirus or a pseudovirus prepared therefrom, a Middle East Respiratory Syndrome Coronavirus vaccine preparation, an antibody targeting Middle East Respiratory Syndrome Coronavirus, or a sample containing an antibody targeting Middle East Respiratory Syndrome Coronavirus.

[0044] The fifth aspect of the present invention provides a cell for use in evaluating the ADE effect caused by Middle East Respiratory Syndrome Coronavirus or an antibody targeting Middle East Respiratory Syndrome Coronavirus, wherein the cell is a cell overexpressing CD32B.

[0045] In some embodiments of the present invention, the CD32B comprises the amino acid sequence shown in NCBI Accession No. NP_003992.3; and / or, the cell is a CHO cell; and / or, the deposit number of the cell overexpressing CD32B is CCTCCNO: C2024285.

[0046] In some specific embodiments of the present invention, the CD32B comprises the nucleotide sequence shown in NCBI Accession No. NM_004001.

[0047] 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.

[0048] Except for the overexpression cells, other reagents and raw materials used in the present invention are commercially available.

[0049] The positive and progressive effects of the present invention are:

[0050] In previous studies, it is often believed that the CD32A receptor protein is closely related to the ADE effect. However, the present invention found 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. Furthermore, the present invention uses MERS-CoV pseudovirus and cells that exogenously express CD32B to identify the ADE effect of Middle East respiratory coronavirus antibodies in vitro. Experiments have shown that the ADE evaluation system based on exogenously expressed CD32B cells is more sensitive than immune cells Raji and CD32A cells, and is a potential system for evaluating the ADE effect of the Middle East respiratory coronavirus.

[0051] Biomaterial Deposit Information

[0052] 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

[0053] Figure 1 This is the map of the linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector (digested with BamHI+EcoRI; 8814 bp).

[0054] Figure 2This is the FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9741 bp).

[0055] Figure 3 This is the FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9723 bp).

[0056] Figure 4 This is the FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (9762 bp).

[0057] Figure 5 This is the sequencing map of h-FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0058] Figure 6 This is the sequencing map of h-FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0059] Figure 7 This is the sequencing map of h-FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro-ok.

[0060] Figure 8 The expression of CD32A, CD32B, and CD32C in CHO, CHO-CD32A, CHO-CD32B, CHO7-CD32C, and Raji cells.

[0061] Fig. 9 This is the result of antibody neutralization ability test.

[0062] Fig.10 This shows the situation of MERS-CoV pseudovirus infecting CHO, CHO-CD32A, CHO-CD32B, CHO-CD32C, and Raji cells in the absence of antibodies.

[0063] Fig.11 The figure shows the infection of MERS-CoV to CHO, CHO-CD32A, CHO-CD32B, CHO-CD32C and Raji cells in the presence of different concentrations of antibodies.

[0064] Fig.12 The purpose is to test the ADE effect of commercially available antibodies using a system for evaluating the ADE effect of Middle East respiratory coronavirus in vitro. DETAILED DESCRIPTION

[0065] 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.

[0066] Example 1 Construction of a cell line expressing immunoglobulin IgG receptor FcγR

[0067] 1.1 Construction of overexpression plasmid

[0068] In this part of the experiment, the FCGR2A / FCGR2B / FCGR2C genes (i.e., CD32A / CD32B / CD32C) were inserted into the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector. The vector plasmid was provided by Fenghui Biotechnology.

[0069] 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;

[0070] 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;

[0071] 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.

[0072] (1) Vector map

[0073] 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.

[0074] (2) Enzyme digestion of pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector

[0075] 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:

[0076] Table 1 Enzyme digestion system

[0077]

[0078] Note: After reacting at 37℃ for 5 h, large fragments were recovered by 1% agarose gel.

[0079] (3) Obtaining FCGR2A / FCGR2B / FCGR2C gene fragments

[0080] 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:

[0081] Table 2 Primer sequences

[0082]

[0083] 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:

[0084] Table 3 Amplification system

[0085]

[0086] Add the above materials into the thin-walled tube, mix well, separate and put into the PCR instrument. The final reaction program after adjustment is shown in Table 4:

[0087] Table 4 Amplification procedures

[0088]

[0089] (4) Ligation of target gene fragment and vector

[0090] The recovered and purified target fragment was connected to the recovered and purified vector pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro (as Figure 1 ), as shown Figure 2 FCGR2A-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as shown Figure 3 FCGR2B-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, as shown Figure 4 As shown in FCGR2C-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro. Because the primer contains the homology arm sequence with the vector, the homologous recombinase is used to recombinate the recovered fragment with the linearized vector to complete the recombination process of the recovered target gene and the vector.

[0091] (5) Transformation of ligation products into competent cells

[0092] Take 10 μL of the ligation product to transform 100 μL of DH5α 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.

[0093] (6) PCR identification

[0094] 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.

[0095] 1.2 Plasmid transfection cell line process

[0096] This part of the experiment intends to construct a CD32A / CD32B / CD32C gene overexpression stable cell line, and the overexpression plasmid carrying the CD32A / CD32B / CD32C gene (plasmid map as shown in Figure 2-4 The proteins were transfected into CHO cells and stably expressed.

[0097] (1) Preparing cells for the experiment

[0098] Determine relevant information of CHO cell lines, including cell culture conditions, cell proliferation rate, and mycoplasma contamination.

[0099] (2) Screening and determination of the dosage of Puromycin

[0100] 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.

[0101] Day 1: CHO cell line cells were plated in 6-well plates containing DMEM / F12 + 10% FBS, so that the density of cells was about 90% on the second day;

[0102] Day 2: Add Puro to cells at the set concentration;

[0103] Day 4: Change the medium and add Puro at the set concentration again;

[0104] 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;

[0105] (3) Screening and construction steps of stable cell lines

[0106] Cell plating: CHO cells were seeded in 6-well plates so that the cell density reached about 70% on the second day;

[0107] Virus infection: 200 μL of lentivirus was added with 2×10 5 in cells;

[0108] 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;

[0109] Observe the infection efficiency: 72 hours after infection, observe the infection efficiency, the minimum efficiency should not be less than 40%;

[0110] 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. The best screening concentration was determined to be 2 μg / mL through preliminary experiments.

[0111] 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 determined in the previous pre-experiment to the 6-well plate;

[0112] Add Puro: Add 2 μg / mL Puro to a 6-well plate;

[0113] 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;

[0114] 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%.

[0115] 1.3 q-PCR detection of transfection effect

[0116] method:

[0117] 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.

[0118] (1) Primer design

[0119] The primer sequences are shown in Table 5:

[0120] Table 5 Primer sequences

[0121]

[0122] The internal reference primers are shown in Table 6:

[0123] Table 6 Internal reference primers

[0124]

[0125] (2) RNA extraction

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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℃.

[0131] RNA concentration detection: RNA concentration was detected using a micro-nucleic acid detector. The results are shown in Table 7:

[0132] Table 7 RNA concentration

[0133]

[0134] The control group CHO-control is wild-type CHO without the introduction of exogenous genes.

[0135] (3) Reverse transcription PCR

[0136] i. As shown in Table 8, prepare the reverse transcription reaction solution according to the following components:

[0137] Table 8 Reaction system

[0138]

[0139] ii. Perform the following reaction on a PCR instrument: 72°C for 5 min, then cool on ice.

[0140] iii. As shown in Table 9, add the following reverse transcription reaction solution to the above PCR tube:

[0141] Table 9 Reaction system

[0142]

[0143] iv. As shown in Table 10, reverse transcription reaction was performed on a PCR instrument under the following conditions:

[0144] Table 10 Reaction conditions

[0145]

[0146] v. Use cDNA immediately for experiments or store at 4°C.

[0147] (4) Real-time fluorescence quantitative PCR reaction

[0148] i. The configuration of the reaction system is shown in Table 11:

[0149] Table 11 Reaction system

[0150]

[0151] ii. Reaction condition settings:

[0152] The amplification program is shown in Table 12.

[0153] Table 12 Amplification procedures

[0154]

[0155] The melting program is shown in Table 13.

[0156] Table 13 Melting program

[0157]

[0158] In order to establish a more sensitive MERS-CoV 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, namely CHO-CD32A, CHO-CD32B, and CHO-CD32C, respectively. Figure 8 Shown is the expression of CD32A, CD32B, and CD32C in each cell.

[0159] Example 2 Construction of MERS-CoV pseudovirus (the pseudovirus was purchased from Yisheng Biotechnology Co., Ltd.)

[0160] This pseudovirus uses a retroviral vector and uses the MERS-CoV Spike protein gene (NCBI accession number of the amino acid sequence: YP 009047204.1; NCBI accession number of the nucleotide sequence: AFS88936.1) to replace the retroviral envelope protein gene. It is co-transfected with the retroviral packaging plasmid and CMV-GFP-T2A-Luciferase plasmid into 293T cells to package the pseudovirus containing the Spike protein gene. The surface of the pseudovirus can express the MERS-CoV Spike protein, and the virus carries both GFP and Luciferase luciferase reporter genes. The activity of the pseudovirus-infected cells can be evaluated by observing the fluorescence signal and detecting the luciferase activity.

[0161] 2.1 Vector construction

[0162] 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.

[0163] 2.2 Pseudovirus Packaging

[0164] 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 hours after transfection. After culturing for 48 hours, 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.

[0165] Example 3 Detection of Neutralizing Ability of MERS-CoV Antibodies

[0166] 3.1 Cell subculture and plating:

[0167] Stable cell line HEK293T-DPP4 (from Yisheng Biotechnology Co., Ltd., catalog number 18038ES50) 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 the cell suspension with a cell concentration of 1E6 cells / mL was transferred to a cell culture flask for further culture.

[0168] 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 2×10 5 100 µL / well of the cell suspension was added to a 96-well plate and cultured overnight.

[0169] 3.2 MERS-CoV pseudovirus dilution

[0170] Preparation of pseudovirus dilution: The pseudovirus prepared above (stock solution: copy number: 1E13 Copies / mL from Yisheng Biotechnology Co., Ltd.) was diluted 100 times with DMEM + 10% FBS.

[0171] 3.3 MERS-CoV antibody gradient dilution

[0172] Preparation of antibody diluent: RBD antibody (100 μg, from RD SYSTEMS, catalog number MAB107071; the amino acid sequence of the antibody is shown in NCBI accession number YP_007188579.1) was diluted with 1 mL PBS to prepare a 100 μg / mL stock solution, which was then serially diluted with complete culture medium to 9 dilutions: 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, 1.28 ng / mL, and 0 ng / mL.

[0173] 3.4 Sample addition

[0174] After the pseudovirus and antibody were mixed at a ratio of 1:1 (v / v) (the pseudovirus concentration after mixing was 5E10 Copies / mL, and the antibody concentration was 50000 ng / mL, 10000 ng / mL, 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, and 0 ng / mL), they were incubated at 37°C for 1 hour, the culture medium in 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 with 100 μL of complete culture medium was set up.

[0175] 3.5 Detection of chemiluminescence

[0176] Use Luciferase Assay System (manufacturer: Promega) to detect chemiluminescence: remove the culture medium after 48 hours, wash once with PBS, add 25 µL / well lysis buffer (from Luciferase Assay System kit; Promega, catalog number: E1531), incubate for 5-10 minutes, take 20 µL to the white plate, add luminescent substrate (from Luciferase Assay System kit) 100 µL, and detect Luciferase luminescence signal. The results are as follows Fig. 9 As shown, the MERS-CoV RBD monoclonal antibody can inhibit the infection of MERS-CoV pseudovirus to 293 / DPP4 cells, and its neutralization 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.

[0177] Example 4 System for in vitro evaluation of the ADE effect of Middle East respiratory coronavirus

[0178] CHO is a cell in the ovary of Chinese hamsters. MERS-CoV Pseudovirus cannot infect CHO cells, and the Middle East respiratory coronavirus RBD monoclonal antibody cannot promote infection within the concentration range tested.

[0179] 4.1 Cell subculture and plating

[0180] The stable cell lines CHO-CD32A, CHO-CD32B, CHO-CD32C and immune cell Raji prepared above 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.

[0181] 4.2 MERS-CoV pseudovirus dilution

[0182] Preparation of pseudovirus dilution: The pseudovirus prepared above (stock solution: copy number: 1E13Copies / mL from Yisheng Biotechnology Co., Ltd.) was diluted 20 times with DMEM / F12+10% FBS.

[0183] 4.3 Incubation of antibodies with MERS-CoV pseudovirus

[0184] The antibody was diluted 5-fold to 9 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, 1.28 ng / mL, and 0 ng / mL), mixed with the same volume of pseudovirus dilution (after mixing, the pseudovirus concentration was 2.5E11; the antibody concentrations 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, 0.64 ng / mL, and 0 ng / mL), and incubated at 37°C for 1 h.

[0185] 4.3 Adding samples

[0186] 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.

[0187] 4.4 Detection of chemiluminescence

[0188] 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. Fig.10 As shown in the figure, MERS-CoV pseudovirus cannot infect CHO cells, Raji cells, CHO-CD32A, CHO-CD32B and CHO-CD32C cells (RLU below 100 can be considered as no difference from the background), indicating that in the absence of antibodies, the expression of CD32A, CD32B and CD32C does not promote the infection of Middle East respiratory coronavirus. Fig.11 As shown in the presence of MERS-CoV monoclonal antibody, it was found that it significantly promoted the infection of CHO-CD32B and CHO-CD32A cells by MERS-CoV at sub-neutralizing concentrations, and the ADE effect mediated by CD32B was stronger than that by CD32A. Raji cells are commonly used ADE detection cells, and they express Fc receptors on their surface, but MERS-CoV pseudovirus cannot infect Raji cells, and MERS-CoV RBD monoclonal antibody cannot promote infection within the concentration range tested.

[0189] Example 5 Application of the system for evaluating the ADE effect of Middle East respiratory coronavirus in vitro

[0190] In this example, the ADE effect of commercially available antibodies was detected. The commercially available antibodies detected were: (1) MERS-CoV nucleocapsid protein antibody, from RD SYSTEMS, catalog number MAB10729; (2) MERS-CoV Spike S1 subunit antibody, from RD SYSTEMS, catalog number MAB10707; (3) MERS-CoV Spike S2 ​​monoclonal antibody, from Thermo Fisher, catalog number MA529978; (4) MERS-CoV Spike RBD antibody, from RD SYSTEMS, catalog number MAB107071.

[0191] 5.1 Cell subculture and plating

[0192] 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.

[0193] 5.2 MERS-CoV pseudovirus dilution

[0194] Preparation of pseudovirus dilution: The pseudovirus prepared above (stock solution: copy number: 1E13Copies / mL from Yisheng Biotechnology Co., Ltd.) was diluted 20 times with DMEM / F12+10% FBS.

[0195] 5.3 Incubation of antibodies with MERS-CoV pseudovirus

[0196] The antibodies were diluted 5-fold to 9 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, 1.28 ng / mL, and 0 ng / mL), mixed with the same volume of pseudovirus dilution (after mixing, the pseudovirus concentration was 2.5E11; the antibody concentrations 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, 0.64 ng / mL, and 0 ng / mL), and incubated at 37°C for 1 h.

[0197] 5.3 Adding samples

[0198] 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.

[0199] 5.4 Detection of chemiluminescence

[0200] 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 luminescent signal of Luciferase. Fig.12 As shown, the MERS-CoV Spike RBD antibody showed a significant effect of promoting MERS-CoV infection of CHO cells overexpressing CD32B at sub-neutralizing concentrations, and the MERS-CoV Spike S1 subunit antibody showed a weak ADE effect, while the other two antibodies did not show ADE effect.

[0201] As demonstrated in this example, the system for evaluating the ADE effect provided by the present invention can detect whether the antibody targeting MERS-CoV has the risk of causing the ADE effect. The system comprises a Middle East Respiratory Syndrome Coronavirus pseudovirus and a cell overexpressing CD32B; or, the system comprises an antibody targeting the receptor binding domain of the Middle East Respiratory Syndrome Coronavirus and a cell overexpressing CD32B; or, the system comprises a Middle East Respiratory Syndrome Coronavirus pseudovirus, an antibody targeting the receptor binding domain of the Middle East Respiratory Syndrome Coronavirus, and a cell overexpressing CD32B. The cell is a CHO cell; the pseudovirus comprises a Spike protein encoding gene of the Middle East Respiratory Syndrome Coronavirus.

[0202] It can be reasonably inferred that the system for evaluating ADE effects provided by the present invention can be used to detect in vitro whether an evaluation object has the risk of causing ADE effects.

Claims

1. A system for evaluating ADE effects, characterized in that: The system comprises: (i) and (iii); (ii) and (iii); or (i), (ii) and (iii); (i) is a Middle East Respiratory Syndrome Coronavirus pseudovirus, (ii) is an antibody targeting the receptor binding domain of Middle East Respiratory Syndrome Coronavirus, and (iii) is a cell overexpressing CD32B; The cells are CHO cells; the pseudovirus contains the Spike protein coding gene of the Middle East Respiratory Syndrome Coronavirus.

2. The system according to claim 1, characterized in that The system includes one or more of the following conditions: The amino acid sequence of CD32B is shown in NCBI accession number NP_003992.3; The CD32B is overexpressed on the surface of the cell; The Spike protein encoding gene encodes the amino acid sequence shown in NCBI Accession No. YP 009047204.

1.

3. The system according to claim 1, characterized in that The coding sequence of CD32B comprises the nucleic acid sequence shown in NCBI accession number NM_004001; and / or, The deposit number of the cell overexpressing CD32B is CCTCC NO: C2024285.

4. The system according to any one of claims 1 to 3, characterized in that: The concentration of the Middle East Respiratory Syndrome Coronavirus pseudovirus is 5E10-2.5E11 copies / mL; and / or, the concentration of the antibody is 16-50000 ng / mL; and / or, the amino acid sequence of the antibody is as shown in NCBI Accession No. YP_007188579.

1.

5. A method for preparing a system as claimed in any one of claims 1 to 4, 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 Middle East Respiratory Syndrome Coronavirus pseudovirus and the antibody targeting the receptor binding domain of the Middle East Respiratory Syndrome Coronavirus.

6. 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, the evaluation object is added to the system as described in any one of claims 1 to 4, including (i i ) and (iii) as the sample to be tested, or replacing (i) in the system comprising (i), (ii) and (iii) in any one of claims 1 to 4 with an 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 4 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 4 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 effect. 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 the risk of causing ADE effect.

7. The method according to claim 6, characterized in that The evaluation object in step (1) is selected from Middle East Respiratory Syndrome Coronavirus or a pseudovirus prepared therefrom, a Middle East Respiratory Syndrome Coronavirus vaccine preparation, an antibody targeting Middle East Respiratory Syndrome Coronavirus, or an antiserum containing an antibody targeting Middle East Respiratory Syndrome Coronavirus or plasma of a recovered patient of Middle East Respiratory Syndrome; and / or, In step (2), the fluorescence intensity of Luciferase is detected to characterize the degree of infection; and / or, In step (3), the negative control group comprises (i) and (iii) in the system according to any one of claims 1 to 4.

8. Use of the system according to any one of claims 1 to 4 in assessing the risk of causing an ADE effect; The evaluation object is selected from Middle East Respiratory Syndrome Coronavirus or a pseudovirus prepared therefrom, a Middle East Respiratory Syndrome Coronavirus vaccine preparation, an antibody targeting Middle East Respiratory Syndrome Coronavirus, or a sample containing an antibody targeting Middle East Respiratory Syndrome Coronavirus; The use described is for non-diagnostic purposes.

9. Use of a cell in evaluating the ADE effect caused by Middle East Respiratory Syndrome Coronavirus or an antibody targeting Middle East Respiratory Syndrome Coronavirus, characterized in that: The cells are CHO cells overexpressing CD32B; the application is for non-diagnostic purposes.

10. The use according to claim 9, characterized in that The amino acid sequence of the CD32B is shown in NCBI accession NP_003992.3; and / or, the deposit number of the cell overexpressing CD32B is CCTCC NO: C2024285.

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