System for evaluating the aDE effect of middle east respiratory coronavirus based on complement-mediated pathways and applications thereof

Abstract

This invention discloses a system for evaluating the ADE effect of Middle East Respiratory Coronavirus (MERS-CoV) based on the complement-mediated pathway and its application. The system is selected from any one of the following (1)-(3): (1) the system comprises MERS-CoV pseudovirus, complement component C1q, and cells overexpressing CD93; (2) the system comprises an antibody targeting MERS-CoV, complement component C1q, and cells overexpressing CD93; and (3) the system comprises an antibody targeting MERS-CoV, MERS-CoV pseudovirus, complement component C1q, and cells overexpressing CD93. The system provided by this invention has the advantages of high sensitivity, high detection peak, and wide detection range, and can provide a reference for the development or use of drugs for the prevention, mitigation, and / or treatment of MERS-CoV, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to a system for evaluating the ADE effect of Middle East respiratory coronaviruses based on the complement-mediated pathway and its application. Background Technology

[0002] In the initial stages of viral infection, viral particles typically attach to the surface of host cells. This process is achieved through the interaction of specific proteins on the viral surface with receptors on the cell membrane. To block this contact, the human immune system secretes antibodies against these viral surface proteins, aiming to reduce infectivity by binding to and neutralizing the viral particles. However, in some cases, for certain types of viruses, when specific antibodies bind to viral surface proteins, it may actually promote viral entry into certain types of cells, thereby exacerbating the viral infection. This phenomenon is known as antibody-dependent enhancement (ADE). [1] In respiratory syncytial virus (RSV), measles, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), human immunodeficiency virus (HIV), and Ebola virus (EBOV), there have been reports of antibody production actually exacerbating the disease. [2-5] .

[0003] Currently, MERS-CoV vaccines are still under investigation and have not yet been approved for market release. Therefore, focusing on the adverse drug reaction (ADE) effect is crucial in MERS-CoV vaccine development. Current ADE research largely focuses on ADE mediated by the Fc receptor of immunoglobulin IgG, while other ADE mechanisms require further exploration and confirmation. Further clarifying the potential mechanisms of ADE effects in MERS-CoV infection and constructing a convenient and reliable ADE evaluation system are key steps in studying the ADE phenomenon in MERS-CoV infection; this will provide a reference for the development of MERS-CoV vaccines, thus contributing to vaccine innovation and development.

[0004] References

[0005] [1] TAYLOR A, FOO SS, BRUZZONE R, et al. Fc receptors in antibody-dependent enhancement of viral infections[J]. Immunol Rev, 2015, 268(1): 340-364.

[0006] [2]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.

[0007] [3]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.

[0008] [4]TAKADA A, FELDMANN H, KSIAZEK T G, et al. Antibody-dependent enhancement of Ebola virus infection [J]. J Virol, 2003, 77(13): 7539-44.

[0009] [5]TAKADA A, WATANABE S, OKAZAKI K, et al. Infectivity-enhancing antibodies to Ebola virus glycoprotein [J]. J Virol, 2001, 75(5): 2324-30。 Summary of the Invention

[0010] To address the technical challenge of further clarifying the potential mechanisms of adverse drug reaction (ADE) in MERS-CoV infection in existing technologies, this invention provides a system for evaluating the ADE effect of MERS-CoV based on the complement-mediated pathway and its applications. The system provided by this invention can identify in vitro whether MERS-CoV antibodies or MERS-CoV / pseudoviruses pose a potential risk of causing ADE effects. The system offers advantages such as high sensitivity, high detection peak value, and wide detection range, and can provide a reference for the development or use of drugs for the prevention, mitigation, and / or treatment of MERS-CoV, demonstrating broad application prospects.

[0011] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0012] The first aspect of the present invention provides a system for evaluating the ADE effect, the system being selected from any one of the following (1)-(3):

[0013] (1) The system comprises Middle East Respiratory Syndrome Coronavirus pseudovirus, complement component C1q, and cells overexpressing CD93;

[0014] (2) The system comprises an antibody targeting Middle East Respiratory Syndrome Coronavirus, complement component C1q, and cells overexpressing CD93; and;

[0015] (3) The system includes an antibody targeting Middle East Respiratory Syndrome Coronavirus, a Middle East Respiratory Syndrome Coronavirus pseudovirus, complement component C1q and cells overexpressing CD93.

[0016] In some embodiments of the present invention, the cells overexpressing CD93 are CHO cells overexpressing CD93.

[0017] In some embodiments of the present invention, the antibody targets the receptor-binding domain or the full-length Spike protein of Middle East Respiratory Syndrome Coronavirus.

[0018] In some embodiments of the present invention, CD93 comprises the amino acid sequence shown in NCBI accession number NP_036204.2.

[0019] In some preferred embodiments of the present invention, the encoding nucleic acid of CD93 comprises the nucleotide sequence shown in NCBI accession number NM_012072.

[0020] In some embodiments of the present invention, CD93 is overexpressed on the surface of CHO cells.

[0021] In some embodiments of the present invention, the pseudovirus contains the Spike protein of Middle East Respiratory Syndrome Coronavirus.

[0022] In some preferred embodiments of the present invention, the Spike protein comprises the amino acid sequence shown in NCBI accession number YP_009047204.1.

[0023] In some specific embodiments of the present invention, when the antibody is an antibody targeting the full-length Spike protein of Middle East Respiratory Syndrome Coronavirus, the immunogenic sequence of the antibody is Met 1-Trp1297 in NCBI accession number: YP_007188579.1, for example, an antibody from Thermo Fisher, catalog number MA5-29975; or, when the antibody is an antibody targeting the receptor-binding domain of Middle East Respiratory Syndrome Coronavirus, the immunogenic sequence of the antibody is Glu367-Tyr606 in NCBI accession number: YP_007188579.1, for example, an antibody from RD SYSTEMS, catalog number MAB107071.

[0024] In some embodiments of the present invention, the system satisfies the following conditions:

[0025] (i) The concentration of C1q is 10-200 μg / mL; preferably 30-40 μg / mL, for example 30, 31, 32, 33, 33.3, 34, 35, 36, 37, 38, 39 or 40 μg / mL; and / or,

[0026] (ii) The concentration of the Middle East Respiratory Syndrome Coronavirus pseudovirus is 5-10E9; preferably 8-10E9, such as 5E9, 5.5E9, 6E9, 6.5E9, 7E9, 7.5E9, 8E9, 8.5E9, 9E9, 9.5E9 or 10E9; and / or,

[0027] (iii) The concentration of the antibody is 50-40000 ng / mL; for example, 100-8000 ng / mL or 200-35000 ng / mL, specifically for example, 266.7-33333.3 ng / mL, for example, 33333.3 ng / mL, 6666.7 ng / mL, 1333.3 ng / mL, 266.7 ng / mL and 53.3 ng / mL.

[0028] A second aspect of the present invention provides a method for constructing a system as described in the first aspect, the method comprising transfecting basal cells with an overexpression plasmid containing a coding nucleic acid sequence of CD93 to prepare cells overexpressing CD93; further comprising obtaining the Middle East Respiratory Syndrome Coronavirus pseudovirus, the antibody, and the complement component C1q, and contacting the antibody, the Middle East Respiratory Syndrome Coronavirus pseudovirus, the cells overexpressing CD93, and the complement component C1q.

[0029] In some embodiments of the present invention, the background cells are CHO cells.

[0030] 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 nucleic acid sequence during transfection is BamHI-EcoRI; and / or, the coding nucleic acid sequence comprises the nucleotide sequence shown in NCBI accession number NM_012072.

[0031] A third aspect of the present invention provides a method for evaluating the ADE effect in vitro, the method comprising:

[0032] Step 1: When the object of evaluation is a virus, a pseudovirus, or a preparation containing a virus, contact (2) of the system described in the first aspect with the object of evaluation, or replace the Middle East Respiratory Syndrome Coronavirus pseudovirus in (3) of the system described in the first aspect with the object of evaluation; or,

[0033] When the object of evaluation is an antibody or a preparation containing an antibody, (1) in the system described in the first aspect is brought into contact with the object of evaluation, or the antibody in (3) in the system described in the first aspect is replaced with the object of evaluation;

[0034] Step 2: Detect the degree of infection of the cells in the system;

[0035] Step 3: Determine the risk of ADE effect; when the degree of infection is higher than that of the negative control, the evaluated subject is determined to have the risk of causing ADE effect.

[0036] In some embodiments of the present invention, in step 1, the evaluation object is 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, a blood sample containing an antibody targeting Middle East Respiratory Syndrome Coronavirus, or a blood sample from a Middle East Respiratory Syndrome recovered patient.

[0037] In some embodiments of the present invention, in step 2, the degree of infection is characterized by Luciferase fluorescence intensity;

[0038] In some embodiments of the present invention, in step 3, (1) or (2) as defined in the system described in the first aspect is used as a negative control.

[0039] A fourth aspect of the present invention provides an application of the system as described in the first aspect in assessing the risk of an evaluation object causing the ADE effect;

[0040] The evaluation targets are Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or pseudoviruses prepared therefrom, MERS-CoV vaccine preparations, antibodies targeting MERS-CoV, blood samples containing antibodies targeting MERS-CoV, or blood samples from MERS recovered patients.

[0041] The fifth aspect of the present invention provides the use of cells in evaluating the ADE effect induced by Middle East Respiratory Syndrome Coronavirus or antibodies targeting Middle East Respiratory Syndrome Coronavirus, said cells being cells overexpressing CD93; said use is for non-diagnostic purposes.

[0042] In some embodiments of the present invention, the ADE effect is ADE mediated by the complement component C1q.

[0043] In some embodiments of the present invention, the cells overexpressing CD93 are CHO cells overexpressing CD93.

[0044] In some preferred embodiments of the present invention, the amino acid sequence of CD93 is shown in NCBI accession number NP_036204.2.

[0045] In some specific embodiments of the present invention, the NCBI accession number NM of CD93 is... _ The nucleotide sequence shown in 012072.

[0046] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0047] The reagents and raw materials used in this invention are all commercially available.

[0048] The significant advancements of this invention lie in the fact that previous studies often considered a close link between Fc receptor proteins and ADE (anti-depression) effects. However, this invention reveals that the complement component C1q and its receptor can mediate ADE effects in MERS-CoV, suggesting the potential risk of ADE effects in the presence of cells expressing complement receptors. This invention utilizes MERS-CoV pseudovirus, complement component C1q, and cells expressing exogenous complement receptor C1qR (CD93) to identify the ADE effects of Middle East Respiratory Coronavirus (MERS-CoV) antibodies in vitro. The system provided by this invention is more sensitive than those based on Vero cells and Raji and THP-1 immune cells, and according to the detection results, MERS-CoV Spike Protein antibodies induce higher levels of ADE compared to MERS-CoV Spike RBD antibodies. The ADE evaluation system described herein has a wide detection range, capable of evaluating the ADE effect induced by MERS-CoV virus or pseudoviruses prepared therefrom acting on CHO cells overexpressing CD93 in the presence of MERS-CoV Spike RBD antibody or MERS-CoV Spike Protein antibody within a broad concentration range (50 ng / mL-35000 ng / mL). This invention provides a promising system for evaluating the ADE effect of complement-mediated Middle East respiratory coronaviruses, which can provide a reference for the development or use of drugs for the prevention, mitigation, and / or treatment of MERS-CoV, and has broad application potential. Attached Figure Description

[0049] Figure 1 Map of the linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector (digested with enzymes, BamHI+EcoRI; 8814 bp).

[0050] Figure 2 Image of h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid (10749 bp).

[0051] Figure 3 The sequence diagram is for h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro.

[0052] Figure 4 This is the result of the antibody neutralization capacity test.

[0053] Figure 5 The expression of CD93 in CHO, CHO-CD93, Raji, and THP-1 cells.

[0054] Figure 6This describes the infection of CHO, CHO-CD93, Raji, THP-1, and Vero cells by MERS-CoV pseudovirus in the absence of antibodies.

[0055] Figure 7 The infection status of MERS-CoV in CHO, CHO-CD93, Raji, THP-1, and Vero cells under different antibody concentrations was investigated.

[0056] Figure 8 To detect the ADE effect of commercially available antibodies using a systematic method for evaluating the ADE effect of Middle East Respiratory Coronavirus in vitro. Detailed Implementation

[0057] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0058] Example 1: Construction of a cell line expressing immunoglobulin IgG receptor FcγR

[0059] 1.1 Construction of overexpression plasmids

[0060] In this part of the experiment, the h-CD93 gene was inserted into the pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector, and the vector plasmid was provided by Fenghui Biotechnology.

[0061] The NCBI accession number for the h-CD93 gene sequence is: NM_012072;

[0062] (1) Carrier map

[0063] Linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector spectrum as follows Figure 1 The vector was completely digested with BamHI-EcoRI, and the large fragment was recovered by 1% agarose gel electrophoresis, which yielded the linearized vector.

[0064] (2) Enzyme digestion of pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector

[0065] The small-plasmid pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro was double-digested with BamHI-EcoRI. The digestion system is shown in Table 1.

[0066] Table 1 Enzyme digestion system

[0067]

[0068] Note: Large fragments were recovered by 1% agarose gel after reacting at 37℃ for 5 h.

[0069] (3) Obtain the h-CD93 gene fragment

[0070] Primers were designed using the h-CD93 gene sequence (h-CD93-F / R are recombinant primers, as shown in Table 2) to construct the gene subcloning vector.

[0071] Table 2 Primer Sequences

[0072]

[0073] The synthesized primers were diluted to a final working solution of 10 µmol / L, and PCR amplification was performed using the diluted primers and template. The system is shown in Table 3.

[0074] Table 3 Amplification System

[0075]

[0076] After adding the above materials into a thin-walled tube, mixing well, and then spotting, the tube was placed in a PCR instrument. The final reaction program after adjustment is shown in Table 4.

[0077] Table 4 Amplification Procedure

[0078]

[0079] (4) Ligation of the target gene fragment and the vector

[0080] The recovered and purified target fragment was ligated to the recovered and purified vector pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, and the ligation product was named h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro. Figure 2 ).

[0081] Because the primers contain homologous arm sequences to the vector, homologous recombinase is used to recombine the recovered fragment with the linearized vector, thus completing the recombination process between the recovered target gene and the vector.

[0082] (5) The ligation product transforms into competent cells

[0083] Transform 10 μL of the ligation product into 100 μL of DH5α competent cells: After mixing the product with the competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately place on ice for 2 min, add 500 μL of LB medium preheated to room temperature, and culture on a shaker at 37℃ for 1 h at 180 rpm. Centrifuge at 5000 rpm for 3 min, discard 500 μL of culture supernatant, mix the remaining 100 μL with a pipette, spread evenly on an LB plate containing 50 μg / mL ampicillin, invert, and incubate overnight at 37℃.

[0084] (6) PCR identification

[0085] Four single colonies were inoculated into 5 mL of LB broth containing 50 μg / mL ampicillin and incubated at 37°C for 5 h using a shaker at 220 rpm. PCR identification was performed using the cultured bacterial solution. The sequencing results were compared with the expected sequence; the agreement was 100%, indicating successful plasmid construction. Sequencing results are shown below. Figure 3 As shown.

[0086] 1.2 Lentiviral Packaging

[0087] 1.2.1 Cell Preparation

[0088] (1) One day before transfection, 3-5×10 6 293T cells per dish were seeded in 10 mm cell culture dishes, and DMEM medium containing 10% fetal bovine serum was added. The cells were then cultured at 37°C in a 5% CO2 incubator.

[0089] (2) Transfection was performed on the day of transfection when the cell density reached 80%. The psPAX2: pMD2.G: target plasmid was added to serum-free DMEM culture medium at a mass ratio of 2:1:1, and the mixture was gently mixed and allowed to stand for 5 min.

[0090] (3) Gently mix the transfection reagent with serum-free DMEM medium and let it stand for 5 min. Mix the liquids from steps (2) and (3) and let it stand for 20 min.

[0091] (4) Drop the mixture evenly into the cell culture dish and incubate at 37°C in a 5% CO2 cell culture incubator for 6 hours.

[0092] (5) Collect cell supernatant after culturing for another 48 hours.

[0093] 1.2.2 Ultracentrifugation

[0094] (1) Collect the supernatant of the virus at 72h, mix the virus solutions at 48h and 72h, centrifuge at 4000 rpm for 5 min at 4℃, and filter with a 0.45μm filter;

[0095] (2) Prepare an ultracentrifuge tube, add the supernatant collected by filtration to the ultracentrifuge tube at once (about 20~23 mL per tube), weigh it, place it symmetrically in the ultracentrifuge, and centrifuge at 25000 rpm for 2 hours; (weigh with an electronic balance, accurate to 0.01 g).

[0096] (3) After centrifugation, discard the supernatant in the tube (discard as much residual liquid as possible), gently and repeatedly resuspend the virus precipitate in the ultracentrifuge tube with 1 mL PBS, transfer it to a sterile EP tube, and label it.

[0097] (4) Dissolve completely at 4℃, and centrifuge at 10,000 rpm for 5 min at 4℃ to further remove other impurity particles;

[0098] As needed, aliquot the centrifuged sample into small volumes (100 μL or 200 μL), reserve 10 μL for titer detection, label the aliquots, and store at -80℃ or in liquid nitrogen.

[0099] 1.2.3 Titer Detection

[0100] (1) Seed 293T cells into 96-well plates, 1×10⁶ cells per well. 5 Each sample was incubated overnight at 37°C.

[0101] (2) Resuspend the virus stock solution in DMEM medium. The first well contains 10 μL of virus stock solution, and then perform 10-fold serial dilutions. Add 100 μL of medium-virus mixture to each well, and perform 3 replicates for each dilution. Incubate at 37°C in a 5% CO2 incubator.

[0102] (3) After 24 hours, the DMEM medium containing the virus was replaced with DMEM complete medium without the virus solution and cultured for another 48 hours. The number of fluorescent cells in each well was observed and counted under a fluorescence microscope, and photographs were taken. The virus titer was calculated. The sum of the total number in the three replicate wells was calculated and the average number was calculated.

[0103] 1.3 Plasmid transfection cell line procedure

[0104] This part of the experiment aims to construct a stable cell line CHO-CD93 that overexpresses the h-CD93 gene, and to transfect the overexpression plasmid carrying the h-CD93 gene into CHO cells for stable expression.

[0105] (1) Preliminary experiment preparation of cells

[0106] Determine relevant information about the CHO cell line, including cell culture conditions, cell proliferation rate, and mycoplasma contamination status.

[0107] (2) Preliminary experiments to determine the MOI value

[0108] 1) Review the literature to determine the MOI value of the lentivirus in the target cell line;

[0109] 2) Based on the data obtained, design gradient experiments to explore the optimal MOI of 50;

[0110] (3) Preliminary experiments to determine the dosage of screening drugs

[0111] Consult the lethal dose information for Puro in screening stable cell lines in CHO cell lines, and refer to the data obtained to determine three drug concentration gradients (1 μg, 2 μg, 4 μg).

[0112] Day 1: CHO cell line cells were seeded into 6-well plates containing DMEM / F12 + 10% FBS, so that the cell density was approximately 90% by the second day;

[0113] Day 2: Add Puro to the cells at the set concentration;

[0114] Day 4: Change the solution and add Puro at the set concentration again;

[0115] Day 7: Observe and find the well with the lowest drug concentration when the cell lethality is 100%. The drug concentration used in this well is the Puro screening concentration.

[0116] (3) Screening and construction steps of stable cell lines

[0117] Cell seeding: CHO cells were seeded into 6-well plates, and the cell density reached about 70% on the second day.

[0118] Viral infection: Based on the MOI value determined in the preliminary experiment, the required volume of lentivirus to be added is calculated to be 25 μL;

[0119] Medium change: Change the medium according to the actual situation. For some cells with weak tolerance, the medium should be changed in time; for some cells with strong tolerance, the medium can be changed after 48-72 hours of infection.

[0120] Observe infection efficiency: Observe the infection efficiency 72 hours after infection. The efficiency should not be lower than 40%.

[0121] Puro screening: The optimal treatment time is between 3 and 10 days. The commonly used concentration range of Puro is 1 to 10 μg / mL. Preliminary experiments have determined that the optimal screening concentration is 3 μg / mL.

[0122] Infection: After culturing for 72 hours (the infection time depends on the specific condition of the cells and the infection efficiency), add the drug concentration of 3 μg / mL determined in the previous preliminary experiment to a 6-well plate;

[0123] Add Puro: Add 2 μg / mL of Puro to a 6-well plate;

[0124] Medium change: Change the selection medium every 3 to 5 days according to the color of the medium and the cell growth. When a large number of cells die, the Puro concentration can be halved to maintain selection.

[0125] Observation: Observe the cell state, growth, gene expression level and proportion every day until the proportion of fluorescent cells observed under the microscope is more than 90%.

[0126] 1.3 q-PCR detection of transfection efficiency

[0127] method:

[0128] q-PCR technology involves adding a fluorescent group to the PCR reaction system and using the accumulation of fluorescence signals to monitor the entire PCR process in real time. Finally, a standard curve is used to quantify the unknown template. By monitoring changes in fluorescence signals, the amount of amplified product in each cycle of the PCR amplification reaction can be detected in real time. Quantitative analysis of the starting template can be performed through the analysis of Ct values ​​and the standard curve.

[0129] (1) Primer design

[0130] Primer sequences are shown in Table 5:

[0131] Table 5 Primer Sequences

[0132]

[0133] The internal reference primers are shown in Table 6:

[0134] Table 6 Internal reference primers

[0135]

[0136] (2) RNA extraction

[0137] i. Add 1000 μL of Trizol to a 1.5 mL EP tube containing the cell line, mix thoroughly, then add 200 μL of chloroform, vortex to mix, let stand for 5 min, and then centrifuge at 12000 rpm, 4℃ for 10 min.

[0138] ii. Remove a 1.5 mL EP tube from the centrifuge, and transfer the upper colorless and transparent aqueous phase layer into another clean 1.5 mL EP tube. (The sample will separate into three layers: a lower organic phase, a middle layer, and an upper aqueous phase; RNA is located in the upper aqueous phase.) Add an equal volume of isopropanol, gently invert to mix, let stand for 10 min, and then centrifuge at 12000 rpm at 4°C for 10 min.

[0139] iii. After centrifugation, a gel-like precipitate will be visible on the tube wall or bottom; this precipitate is RNA. Carefully discard the supernatant and retain the precipitate.

[0140] 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 to remove as much supernatant as possible.

[0141] v. Allow to dry in the worktable for approximately 5–10 minutes. (Excessive drying will significantly reduce the solubility of RNA). Add 25 μL of DEPC H2O to all EP tubes, and gently blow with a pipette tip several times to fully dissolve the RNA. Store at -80°C.

[0142] RNA concentration detection: RNA concentration was detected using a micro-volume nucleic acid detector. The results are shown in Table 7.

[0143] Table 7 RNA Concentration

[0144]

[0145] (3) Reverse transcription PCR

[0146] i. Prepare the reverse transcription reaction solution according to the following components as shown in Table 8:

[0147] Table 8 Reaction System

[0148]

[0149] ii. Perform the following reaction on a PCR instrument: 72°C, 5 min, then place on ice for rapid cooling.

[0150] iii. As shown in Table 9, add the following reverse transcription reaction solution to the PCR tubes described above:

[0151] Table 9 Reaction System

[0152]

[0153] iv. As shown in Table 10, reverse transcription was performed on a PCR instrument under the following conditions:

[0154] Table 10 Reaction Conditions

[0155]

[0156] v. The cDNA should be tested immediately or stored at 4°C.

[0157] (4) Real-time quantitative PCR reaction

[0158] i. The configuration of the reaction system is shown in Table 11:

[0159] Table 11 Reaction System

[0160]

[0161] ii. Reaction condition settings:

[0162] The amplification procedure is shown in Table 12.

[0163] Table 12 Amplification Procedure

[0164]

[0165] The melting process is shown in Table 13.

[0166] Table 13 Melting Procedure

[0167]

[0168] To establish a more sensitive cell line for MERS-CoV ADE detection, CHO cells stably expressing C1qR(CD93) were constructed, designated CHO-CD93. RT-PCR results are shown below. Figure 5 The figure shows the expression of CD93 in each cell.

[0169] Example 2: Construction of MERS-CoV pseudovirus (the pseudovirus was purchased from Yisheng Biotechnology Co., Ltd.)

[0170] This pseudovirus uses a retroviral vector. The MERS-CoV Spike protein gene (NCBI accession number for amino acid sequence: YP 009047204.1; NCBI accession number for nucleotide sequence: AFS88936.1) replaces the envelope protein gene of the retrovirus. It is co-transfected into 293T cells with the retroviral packaging plasmid and the CMV-GFP-T2A-Luciferase plasmid to package a pseudovirus containing the Spike protein gene. The pseudovirus can express the MERS-CoV Spike protein on its surface, and the virus also carries GFP and Luciferase reporter genes. The cell infection activity of the pseudovirus can be evaluated by observing the fluorescence signal and detecting luciferase activity.

[0171] 2.1 Carrier Construction

[0172] The bacterial culture containing the vector plasmid was cultured overnight, and 3-5 mL of fresh bacterial culture was used to extract the plasmid (DP107-02 high-purity plasmid mini-extraction kit). 1 µg of fresh plasmid was double-digested with the appropriate restriction endonuclease. The digestion products were subjected to agarose gel electrophoresis, and the gel was recovered after electrophoresis. All of the above liquid was transferred to a filter column, centrifuged, and the recovered vector fragment was obtained and its concentration determined. PCR amplification was performed using diluted primers and template. After PCR, agarose gel electrophoresis was performed, and the target gene was recovered. Hieff Clone was used. TM The recombinant reaction system ligates the overexpression vector to the target fragment, and the ligation product is introduced into competent cells for transformation. Finally, sequencing confirms the presence of the target vector.

[0173] 2.2 Packaging of fake viruses

[0174] Recombinant viral plasmids encoding lentiviral particles and their auxiliary packaging vectors were prepared. Both the recombinant and auxiliary plasmid vectors underwent high-purity endotoxin-free extraction and were analyzed using HG Transgene. TM Reagent co-transfected 293T cells. 18 h after transfection, the medium was replaced with complete medium. After 48 h of culture, the cell supernatant rich in lentiviral particles was collected, concentrated, and a high-titer lentiviral concentrate was obtained. The virus was aliquoted and stored at -80°C.

[0175] Example 3: Detection of the neutralizing capacity of MERS-CoV antibodies

[0176] 3.1 Cell passage culture and plating:

[0177] Stable cell line HEK293T-DPP4 (from Yisheng Biotechnology Co., Ltd., catalog number 18038ES50) was passaged in complete culture medium (DMEM + 10% FBS) at 5% temperature in a 37°C CO2 incubator. The cells were passaged every 4 days and collected into sterile centrifuge tubes. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. The cell pellet was resuspended in fresh complete culture medium, stained, counted, and a cell suspension with a concentration of 1E6 cells / mL was transferred into a cell culture flask for further culture.

[0178] Collect cells in good growth condition, plate them, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in complete culture medium, and adjust the cell concentration to 2 × 10⁶ cells / mL. 5 Cells / mL, take 100 µL of cell suspension per well, add to a 96-well plate, and incubate overnight.

[0179] 3.2 MERS-CoV pseudovirus dilution

[0180] Preparation of pseudovirus dilution: The pseudovirus prepared above (original solution: copy number: 1E13 Copies / mL, from Yisheng Biotechnology Co., Ltd.) was diluted 100 times with DMEM + 10% FBS.

[0181] 3.3 MERS-CoV antibody gradient dilution

[0182] Preparation of antibody diluent: (1) MERS-CoV Spike Protein monoclonal antibody from ThermoFisher, catalog number MA5-29975, immunogen sequence is Met 1-Trp1297 in NCBI accession number: YP_007188579.1; (2) MERS-CoV Spike RBD antibody from RD SYSTEMS, catalog number MAB107071, immunogen sequence is Glu367-Tyr606 in NCBI accession number: YP_007188579.1. (3) The MERS-CoV Spike S1 subunit antibody was obtained from RD SYSTEMS, catalog number MAB10707, and the immunogen sequence was Met1-Pro747 in NCBI accession number K9N5Q8.1; (4) The MERS-CoV Spike S2 ​​monoclonal antibody was obtained from Thermo Fisher, catalog number MA529978, and the immunogen sequence was Asp726-Pro1296 in NCBI accession number YP_009047204.1; (5) The MERS-CoV nucleocapsid antibody was obtained from RD SYSTEMS, catalog number MAB10729, and the immunogen sequence was Met1-Thr411 in NCBI accession number YP_007188586.1; a stock solution of 100 μg / mL was prepared and serially diluted with complete culture medium to 5000.0 ng / mL, 2500.0 ng / mL, and 1250.0 ng / mL. ng / mL, 625.0 ng / mL, 312.5 ng / mL, 156.3 ng / mL, 78.1 ng / mL, 37.0ng / mL, 19.5 ng / mL and 0 ng / mL, a total of 10 dilutions.

[0183] 3.4 Sample addition

[0184] The pseudovirus and antibody were mixed 1:1 (v / v) (pseudovirus concentration: 5E10 Copies / mL; antibody concentrations: 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.3 ng / mL, 78.1 ng / mL, 37.0 ng / mL, 19.5 ng / mL, 9.8 ng / mL, and 0 ng / mL), and incubated at 37°C for 1 h. The culture medium was then removed from the 96-well plate, and 100 μL of the virus-antibody mixture was added to each well. A blank control was also included, containing 100 µL of complete culture medium.

[0185] 3.5 Detection of chemiluminescence

[0186] Chemiluminescence was detected using the Luciferase Assay System (manufacturer: Promega): After 48 hours, the culture medium was removed, the plate was washed once with PBS, and 25 µL / well of lysis buffer (from the Luciferase Assay System kit; Promega, catalog number: E1531) was added and incubated for 5-10 minutes. 20 µL of the buffer was transferred to a white plate, and 100 µL of the luminescent substrate (from the Luciferase Assay System kit) was added. The Luciferase luminescence signal was then detected. Results are as follows: Figure 4 As shown, the MERS-CoV SpikeRBD monoclonal antibody and the MERS-CoV Spike Protein monoclonal antibody can inhibit MERS-CoV pseudovirus infection of 293 / DPP4 cells, and their neutralizing ability increases with increasing antibody concentration. The other three antibodies did not show significant neutralizing effects. The positive control wells (i.e., those with a corresponding antibody titer of 0) showed high signal values, indicating that the pseudovirus had a strong infectivity.

[0187] Example 4: System for in vitro evaluation of ADE effects of Middle East Respiratory Coronavirus

[0188] CHO cells are cells from the ovaries of Chinese hamsters, and the MERS-CoV pseudovirus cannot infect CHO cells.

[0189] 4.1 Cell passage culture and plating

[0190] The stable cell line CHO-CD93 prepared in Example 1, along with baseline CHO cells, Vero cells, and immune cells Raji and THP-1, were plated. The cells were 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 × 10⁻⁶ cells / mL. 5 Cells / mL, take 100 µL of cell suspension per well, add to a 96-well plate, and incubate overnight.

[0191] 4.2 MERS-CoV pseudovirus dilution

[0192] Preparation of pseudovirus dilution: The pseudovirus prepared above (original solution: copy number: 1E13 Copies / mL, from Yisheng Biotechnology Co., Ltd.) was diluted 40 times with DMEM / F12+10% FBS.

[0193] 4.3 C1q complement dilution

[0194] Native human C1q protein (1 mg / mL, from ABCAM) was diluted to 100 μg / mL with DMEM / F12 + 10% FBS.

[0195] 4.4 Antibody, complement and MERS-CoV pseudovirus incubation

[0196] MERS-CoV Spike RBD antibody (from RD SYSTEMS, catalog number MAB107071; immunogen sequence of the antibody: Glu367-Tyr606) was prepared as a 100 μg / mL stock solution and serially diluted 5-fold to 6 concentration gradients (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 0 ng / mL). This stock solution was then mixed with pseudovirus diluent and C1q diluent in equal volumes (1:1:1). After mixing, the pseudovirus concentration was 8.3E10; the antibody concentrations were 33333.3 ng / mL, 6666.7 ng / mL, 1333.3 ng / mL, 266.7 ng / mL, 53.3 ng / mL, 0 ng / mL), and incubated at 37°C for 1 h.

[0197] 4.3 Sample addition

[0198] After the antibody, complement and pseudovirus have been incubated, 150 µL of the mixture per well is added to a 96-well plate, and 150 µL of complete culture medium is added to the blank control. The plates are incubated at 37°C and 5% carbon dioxide for 48 hours.

[0199] 4.4 Detection of chemiluminescence

[0200] Completely aspirate the supernatant, wash once with PBS, add 25 µL / well lysis buffer and incubate for 5-10 minutes. Transfer 20 µL to a white plate, add 100 µL of the luminescent substrate (Promega Luciferase Assay System), and detect the Luciferase luminescence signal. The red dashed line represents the luminescence value (RLU) of the sample wells containing only MERS-CoV and C1q, defined as 100% infection rate, from which the infection rate is calculated.

[0201] The results are as follows Figure 6 As shown, in the absence of antibodies, the expression of CD93 in the MERS-CoV pseudovirus and complement component C1q does not promote infection with Middle East respiratory coronavirus (RLU value below 200 is considered as viral non-infection). Figure 7 As shown, the infection rate at an antibody concentration of 0 ng / mL is defined as 100%, and the results are displayed as red dashed lines. In the presence of MERS-CoV antibodies and complement component C1q, Vero cells, Raji cells, and THP-1 cells, commonly used cells for ADE detection, did not promote viral infection of cells within the concentration range detected by MERS-CoV Spike RBD antibodies. However, at sub-neutral concentrations, they significantly promoted MERS-CoV infection of CHO-CD93 cells, and the infection of CHO-CD93 cells was stronger than that of CHO cells.

[0202] Example 5: Application of a system for in vitro evaluation of the ADE effect of Middle East Respiratory Coronavirus

[0203] In this embodiment, the ADE effect of commercially available antibodies was detected. The commercially available antibodies detected were (1) MERS-CoV Spike Protein monoclonal antibody from Thermo Fisher, catalog number MA5-29975, and the immunogen sequence was Met 1-Trp1297 in NCBI accession number YP_007188579.1; (2) MERS-CoV Spike RBD antibody from RD SYSTEMS, catalog number MAB107071, and the immunogen sequence was Glu367-Tyr606 in NCBI accession number YP_007188579.1. (3) MERS-CoV Spike S1 subunit antibody from RD SYSTEMS, catalog number MAB10707, immunogen sequence is Met1-Pro747 in NCBI accession number K9N5Q8.1; (4) MERS-CoV Spike S2 ​​monoclonal antibody from Thermo Fisher, catalog number MA529978, immunogen sequence is Asp726-Pro1296 in NCBI accession number YP_009047204.1; (5) MERS-CoV nucleocapsid antibody from RD SYSTEMS, catalog number MAB10729, immunogen sequence is Met1-Thr411 in NCBI accession number YP_007188586.1;

[0204] 5.1 Cell passage culture and plating

[0205] The stable cell line CHO-CD93 prepared in Example 1 was plated, 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 1×10⁻⁶. 5 Cells / mL, take 100 µL of cell suspension per well, add to a 96-well plate, and incubate overnight.

[0206] 5.2 MERS-CoV pseudovirus dilution

[0207] Preparation of pseudovirus dilution: The pseudovirus prepared above (original solution: copy number: 1E13 Copies / mL, from Yisheng Biotechnology Co., Ltd.) was diluted 40 times with DMEM / F12+10% FBS.

[0208] 5.3 C1q complement dilution

[0209] Native human C1q protein (1 mg / mL, from ABCAM) was diluted to 100 μg / mL with DMEM / F12+10% FBS.

[0210] 5.4 Antibody, complement and MERS-CoV pseudovirus incubation

[0211] The antibody was serially diluted 5-fold to six concentration gradients (100,000 ng / mL, 20,000 ng / mL, 4,000 ng / mL, 800 ng / mL, 160 ng / mL, and 0 ng / mL), and then mixed with the pseudovirus diluent and C1q diluent in a 1:1:1 ratio (after mixing, the pseudovirus concentration was 8.33E10; the antibody concentrations were 33,333.3 ng / mL, 6,666.7 ng / mL, 1,333.3 ng / mL, 266.7 ng / mL, 53.33 ng / mL, and 0 ng / mL), and incubated at 37°C for 1 hour.

[0212] 5.5 Sample Addition

[0213] After the antibody, complement and pseudovirus have been incubated, 150 µL of the mixture per well is added to a 96-well plate, and 150 µL of complete culture medium is added to the blank control. The plates are incubated at 37°C and 5% carbon dioxide for 48 hours.

[0214] 5.6 Detection of chemiluminescence

[0215] Completely aspirate the supernatant, wash once with PBS, add 25 µL / well lysis buffer and incubate for 5-10 minutes. Transfer 20 µL to a white plate, add 100 µL of the luminescent substrate (Promega Luciferase Assay System), and detect the Luciferase luminescence signal. The red dashed line represents the luminescence value (RLU) of the sample wells containing only MERS-CoV and C1q, defined as 100% infection rate, from which the infection rate is calculated.

[0216] The results are as follows Figure 8 As shown in the figure, the infection rate at an antibody concentration of 0 ng / mL is defined as 100%, and the results are displayed as red dashed lines. In the presence of complement component C1q, MERS-CoV Spike RBD and MERS–CoV Spike Protein monoclonal antibodies significantly promoted the infection of MERS-CoV on CD93-CHO cells at their sub-neutralizing concentrations. Furthermore, the MERS–CoV Spike Protein monoclonal antibody induced a stronger infection of CD93-CHO cells with MERS-CoV compared to MERS-CoV Spike RBD, while the other three antibodies did not show an ADE effect.

[0217] As demonstrated in this embodiment, the system for evaluating ADE effects provided by the present invention can detect whether antibodies targeting MERS-CoV pose a risk of causing ADE effects. The system comprises MERS-CoV pseudovirus, complement component C1q, and cells overexpressing CD93; or, the system comprises an antibody targeting MERS-CoV, complement component C1q, and cells overexpressing CD93; or, the system comprises MERS-CoV pseudovirus, an antibody targeting MERS-CoV, complement component C1q, and cells overexpressing CD93.

[0218] It can be reasonably inferred that the system for evaluating ADE effects provided by this invention can be used in vitro to detect whether an evaluation subject has the risk of causing complement-mediated ADE effects.

Claims

1. A system for evaluating an ADE effect, characterized by, The system is selected from any one of the following (1)-(3): (1) The system comprises Middle East Respiratory Syndrome Coronavirus pseudovirus, complement component C1q, and CHO cells overexpressing CD93; (2) The system comprises an antibody targeting Middle East Respiratory Syndrome Coronavirus, complement component C1q, and CHO cells overexpressing CD93; and; (3) The system includes an antibody targeting Middle East Respiratory Syndrome Coronavirus, Middle East Respiratory Syndrome Coronavirus pseudovirus, complement component C1q and CHO cells overexpressing CD93; In systems (2) and (3), the antibody targets the receptor-binding domain or the full-length Spike protein of Middle East Respiratory Syndrome Coronavirus; and in systems (1) and (3), the pseudovirus contains the Spike protein of Middle East Respiratory Syndrome Coronavirus.

2. The system as described in claim 1, characterized in that, The CD93 contains the amino acid sequence shown in NCBI accession number NP_036204.2; and / or, the CD93 is overexpressed on the surface of CHO cells.

3. The system as described in claim 2, characterized in that, The encoding nucleic acid of CD93 contains the nucleotide sequence shown in NCBI accession number NM_012072; and / or, The Spike protein contains the amino acid sequence shown in NCBI accession number YP_009047204.

1.

4. A method for constructing the system as described in any one of claims 1-3, characterized in that, The method includes transfecting basal cells with an overexpression plasmid containing a coding nucleic acid sequence of CD93 to prepare cells overexpressing CD93; (3) of the system according to any one of claims 1-3 further includes obtaining the Middle East Respiratory Syndrome Coronavirus pseudovirus, the antibody and the complement component C1q, and contacting the antibody, the Middle East Respiratory Syndrome Coronavirus pseudovirus, the CD93-overexpressing cells and the complement component C1q; the background cells are CHO cells.

5. The method as described in claim 4, characterized in that, The backbone plasmid of the overexpression plasmid is pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro; and / or, The insertion site for the coding nucleic acid sequence during transfection is BamHI-EcoRI; and / or, The encoded nucleic acid sequence contains the nucleotide sequence shown in NCBI accession number NM_012072.

6. A method for evaluating the ADE effect in vitro, characterized in that, The method includes: Step 1: When the object of evaluation is a virus, a pseudovirus, or a preparation containing a virus, contact (2) of the system as described in any one of claims 1-3 with the object of evaluation, or replace the Middle East Respiratory Syndrome Coronavirus pseudovirus in (3) of the system as described in any one of claims 1-3 with the object of evaluation; or, When the evaluation object is an antibody or a preparation containing an antibody, (1) in the system according to any one of claims 1-3 is brought into contact with the evaluation object, or the antibody in (3) in the system according to any one of claims 1-3 is replaced with the evaluation object; Step 2: Detect the degree of infection of the cells in the system; Step 3: Determine the risk of ADE effect; when the degree of infection is higher than that of the negative control, the evaluated subject is determined to have the risk of causing ADE effect; The method described is for non-diagnostic and non-therapeutic purposes.

7. The method as described in claim 6, characterized in that, In step 1, the evaluation object is Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or pseudoviruses prepared therefrom, MERS-CoV vaccine formulations, or antibodies targeting MERS-CoV; and / or, In step 2, the degree of infection is characterized by Luciferase fluorescence intensity; and / or, In step 3, (1) or (2) as defined in the system as described in any one of claims 1-3 is used as a negative control.

8. The application of the system as described in any one of claims 1-3 in assessing the risk of an ADE effect in an evaluation subject; the application is for non-diagnostic and non-therapeutic purposes; The evaluation targets are Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or pseudoviruses prepared therefrom, MERS-CoV vaccine formulations, or antibodies targeting MERS-CoV.

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