System for evaluating ADE effect of Middle East respiratory coronavirus based on complement-mediated pathway and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, and particularly relates to a system for evaluating ADE effect of Middle East Respiratory Coronavirus based on complement-mediated pathway and application thereof. BACKGROUND

[0002] Viruses usually involve viral particles attaching to the surface of host cells in the initial stage of infection, which is achieved by the interaction of specific proteins on the surface of viruses with receptors on the cell membrane; in order to block this contact, the human immune system will secrete antibodies against these viral surface proteins, aiming to reduce the infectivity by binding and neutralizing viral particles; however, in some cases, for some types of viruses, when a specific antibody binds to the viral surface protein, it may instead promote the virus to enter the inside of certain types of cells, thereby exacerbating the condition of viral infection; this phenomenon is known as antibody-dependent enhancement (ADE) [1] . In respiratory syncytial virus, measles, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), human immunodeficiency virus (HIV) and Ebola virus (EBOV), it has been reported that the production of antibodies can exacerbate the occurrence of diseases [2-5] .

[0003] At present, MERS-CoV vaccines are still in research and have not been approved for marketing, so it is crucial to pay attention to ADE effect in the development of MERS-CoV vaccines. Current ADE researches are mostly focused on Fc receptor-mediated ADE phenomenon of immunoglobulin IgG, while other ADE effect mechanisms still need to be further explored and confirmed. Further clarifying the potential mechanism of ADE effect in MERS-CoV infection and constructing a convenient and reliable ADE evaluation system are key steps for studying ADE phenomenon in MERS-CoV infection; which is conducive to providing a reference for the development of MERS-CoV vaccines, to help the innovation and development of vaccines.

[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

[0010] To solve the technical problem that the potential mechanism of the ADE effect in the Middle East respiratory coronavirus infection in the prior art needs to be further clarified, the present application provides a system for evaluating the ADE effect of Middle East respiratory coronavirus based on the complement-mediated pathway and an application thereof. The system provided by the present application can identify in vitro whether the Middle East respiratory coronavirus antibody or the Middle East respiratory coronavirus / pseudovirus has the potential risk of causing the ADE effect. The system provided by the present application has the advantages of high sensitivity, high detection peak and wide detection range, can provide a reference for the research and use of the prevention, alleviation and / or treatment drugs of MERS-CoV, and has a broad application prospect.

[0011] The present application mainly solves the above technical problems through the following technical solutions.

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

[0013] (1) the system comprises Middle East respiratory syndrome coronavirus pseudovirus, complement component C1q and CD93-overexpressing cells;

[0014] (2) the system comprises antibodies targeting Middle East respiratory syndrome coronavirus, complement component C1q and CD93-overexpressing cells; and;

[0015] (3) the system comprises antibodies targeting Middle East respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus pseudovirus, complement component C1q and CD93-overexpressing cells.

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

[0017] In some embodiments of the present application, the antibodies target the receptor binding domain or full-length Spike protein of Middle East respiratory syndrome coronavirus.

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

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

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

[0021] In some embodiments of the present application, the pseudovirus comprises the Spike protein of Middle East respiratory syndrome coronavirus.

[0022] In some preferred embodiments of the present application, the Spike protein comprises the amino acid sequence set forth in NCBI Accession No. YP_009047204.1.

[0023] In some specific embodiments of the present application, when the antibody is an antibody targeting the full-length Spike protein of Middle East Respiratory Syndrome Coronavirus, the immunogen sequence of the antibody is Met 1-Trp 1297 in NCBI Accession No. YP_007188579.1, for example, the antibody from Thermo Fisher, Catalog No. MA5-29975; or, when the antibody is an antibody targeting the receptor binding domain of Middle East Respiratory Syndrome Coronavirus, the immunogen sequence of the antibody is Glu 367-Tyr 606 in NCBI Accession No. YP_007188579.1, for example, the antibody from RD SYSTEMS, Catalog No. MAB107071.

[0024] In some embodiments of the present application, 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 Middle East Respiratory Syndrome Coronavirus pseudovirus is 5-10E9; preferably 8-10E9, for example 5E9, 5.5E9, 6E9, 6.5E9, 7E9, 7.5E9, 8E9, 8.5E9, 9E9, 9.5E9 or 10E9; and / or,

[0027] (iii) the concentration of 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] The second aspect of the present application provides a method for constructing the system of the first aspect, the method comprising transfecting a base cell with an overexpression plasmid comprising a nucleic acid sequence encoding CD93 to prepare the CD93-overexpressing cell; 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 CD93-overexpressing cell and the complement component C1q.

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

[0030] In some embodiments of the present application, 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 the transfection is BamHI-EcoRI; and / or, the encoding nucleic acid sequence comprises the nucleotide sequence shown in NCBI Accession No. NM_012072.

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

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

[0033] when the evaluation object is an antibody or a preparation containing an antibody, contacting (1) in the system of the first aspect with the evaluation object, or replacing the antibody in (3) in the system of the first aspect with the evaluation object;

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

[0035] Step 3: determining the risk of ADE effect; when the degree of infection is higher than that of the negative control, determining that the evaluation object has the risk of causing the ADE effect.

[0036] In some embodiments of the present application, 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 of a Middle East Respiratory Syndrome Coronavirus convalescent.

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

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

[0039] The fourth aspect of the present application provides a use of the system of the first aspect in evaluating the risk of the evaluation object causing the ADE effect;

[0040] The evaluation object is a 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 of a Middle East respiratory syndrome convalescent.

[0041] The fifth aspect of the present application provides a cell for use in evaluating an ADE effect caused by a Middle East respiratory syndrome coronavirus or an antibody targeting Middle East respiratory syndrome coronavirus, wherein the cell is a cell overexpressing CD93; and the use is for a non-diagnostic purpose.

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

[0043] In some embodiments of the present application, the cell overexpressing CD93 is a CHO cell overexpressing CD93.

[0044] In some preferred embodiments of the present application, the amino acid sequence of CD93 is shown in NCBI Accession No. NP_036204.2.

[0045] In some specific embodiments of the present application, the nucleotide sequence of CD93 is shown in NCBI Accession No. NM _ 012072.

[0046] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred examples of the present application.

[0047] The reagents and raw materials used in the present application are commercially available.

[0048] The positive progress effect of the present application is that in the past research, it is often considered that Fc receptor protein is closely related to ADE effect. The present application finds that complement component C1q and its receptor can mediate MERS-CoV to occur ADE effect, which suggests that when cells expressing complement receptors exist, there may be a risk of causing ADE effect. The present application uses MERS-CoV pseudovirus, complement component C1q and exogenous complement receptor C1qR (CD93) expressing cells to identify the ADE effect of Middle East respiratory coronavirus antibody in vitro. The system provided by the present application is more sensitive than the Vero cell-based and immune cell Raji and THP-1 cell-based systems, and according to the detection results, the ADE level caused by MERS-CoV Spike Protein antibody is higher than that caused by MERS-CoV Spike RBD antibody. The detection range of the ADE evaluation system is wide, and it can evaluate the ADE effect caused by MERS-CoV virus or pseudovirus prepared therefrom acting on CHO cells overexpressing CD93 in the presence of MERS-CoV Spike RBD antibody or MERS-CoV Spike Protein antibody in a wide concentration range (50 ng / mL-35000 ng / mL). The present application provides a very potential system for evaluating the ADE effect of Middle East respiratory coronavirus based on complement mediation, which can provide a reference for the research and use of MERS-CoV prevention, relief and / or treatment drugs, and has wide application potential. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is a linearized pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector map (cleaved, BamHI+EcoRI; 8814 bp).

[0050] Figure 2 It is a h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro plasmid map (10749 bp).

[0051] Figure 3 It is a h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro sequencing map.

[0052] Figure 4 It is the detection results of antibody neutralization ability.

[0053] Figure 5 It is the expression of CD93 in CHO, CHO-CD93, Raji, THP-1 cells.

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

[0055] Figure 7 In the presence of different concentrations of antibodies, the infection of CHO, CHO-CD93, Raji, THP-1, Vero cells by MERS-CoV.

[0056] Figure 8 To detect the ADE effect of commercially available antibodies by a system for evaluating the ADE effect of Middle East respiratory coronavirus in vitro. DETAILED DESCRIPTION

[0057] The present application is further illustrated by the following examples without limiting the present application to the described examples. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to the conventional methods and conditions, or according to the product instructions.

[0058] Example 1 Construction of cell lines overexpressing immunoglobulin IgG receptor FcγR

[0059] 1.1 Construction of overexpression plasmid

[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 Co., Ltd.

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

[0062] (1) Vector map

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

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

[0065] The plasmid pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro was extracted and digested with BamHI-EcoRI, and the enzyme digestion system is shown in Table 1:

[0066] Table 1 Enzyme digestion system

[0067]

[0068] Note: 1% agarose gel recovery of large fragments after 5 h reaction at 37°C.

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

[0070] Select h-CD93 gene sequence to design primers (h-CD93-F / R are recombinant primers, as shown in Table 2) for constructing gene subcloning vectors.

[0071] Table 2 Primer sequence

[0072]

[0073] Dilute the synthesized primers to a final concentration of 10 µmol / L working solution, and use the diluted primers and templates for PCR amplification. The system is shown in Table 3:

[0074] Table 3 Amplification system

[0075]

[0076] Add the above materials to a thin-walled tube, mix well, and then point off and put into a PCR instrument. The final reaction program after adjustment is shown in Table 4:

[0077] Table 4 Amplification program

[0078]

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

[0080] Connect the recovered and purified target fragment with the recovered and purified vector pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro, and the connection product is named h-CD93-pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro. Figure 2 ).

[0081] Because the primers contain homologous arm sequences with the vector, the recovered fragment is recombined with the linearized vector using a homologous recombination enzyme to complete the recombination process of the recovered target gene and the vector.

[0082] (5) Transformation of competent cells with connection product

[0083] Take 10 μL ligation product to transform 100 μL DH5α competent cells: mix the product with the competent cells, then ice bath for 30 min, 42℃ heat shock for 90 s, immediately place on ice for 2 min, add 500 μL preheated to room temperature LB medium, 180 rpm, 37℃ constant temperature incubator for 1 h, 5000 rpm centrifuge for 3 min, discard 500 μL culture supernatant, mix the remaining 100 μL with a pipette, then evenly spread on an LB plate containing 50 μg / mL ampicillin resistance, invert, and incubate in a 37℃ constant temperature incubator overnight.

[0084] (6) PCR identification

[0085] Pick 4 single colonies and inoculate in 5 mL, 50 μg / mL ampicillin-containing LB culture solution, 220 rpm, 37℃ constant temperature incubator for 5 h. Use the cultured bacterial solution for PCR identification. Compare the sequencing results with the expected sequence, and the consistency of the comparison results is 100%, indicating that the plasmid construction is successful. The sequencing results are shown in Figure 3 .

[0086] 1.2 Lentivirus packaging

[0087] 1.2.1 Cell preparation

[0088] (1) One day before transfection, 3-5 x 10 6 cells / dish of 293T cells were inoculated in a 10 mm cell culture dish, and DMEM medium containing 10% fetal bovine serum was added and cultured in a 37℃, 5% CO2 incubator.

[0089] (2) On the day of transfection, when the cell density reached 80%, transfection was performed. According to the mass ratio psPAX2: pMD2.G: target plasmid 2:1:1, add to serum-free DMEM culture solution, mix gently, and stand for 5 min.

[0090] (3) Transfection reagent is gently mixed with serum-free DMEM medium, and stands for 5 min.

[0091] (4) Mix the liquids of steps (2) and (3) and stand for 20 min.

[0092] (5) Continue to culture for 48 h, then collect the cell supernatant.

[0093] 1.2.2 Ultracentrifugation

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

[0095] (2) Prepare an ultracentrifuge tube, add the filtered supernatant to the ultracentrifuge tube (about 20-23 mL per tube), weigh, and place in the ultracentrifuge symmetrically. Centrifuge at 25000 rpm for 2 hours; (electronic balance weighing, accurate to 0.01 g)

[0096] (3) After centrifugation, discard the supernatant in the tube (try to discard the remaining liquid in the tube), gently blow the virus precipitate in the ultracentrifuge tube with 1 mL of PBS, transfer to a sterile EP tube, and label;

[0097] (4) Dissolve at 4°C, centrifuge at 10000 rpm for 5 min at 4°C to further remove other impurity particles;

[0098] According to the needs, the centrifuged sample is divided into small volumes (100 μL or 200 μL), 10 μL is reserved for titer detection, and is labeled and stored at -80°C or liquid nitrogen;

[0099] 1.2.3 Titer detection

[0100] (1) Seed 293T cells into a 96-well plate at 1×10 5 cells per well, and incubate at 37°C overnight.

[0101] (2) Resuspend the virus stock solution with DMEM medium, add 10 μL of virus stock solution to the first well, then dilute by 10 times, add 100 μL of medium-virus mixture to each well, and make 3 replicate wells for each dilution concentration. Incubate in a 37°C, 5% CO2 incubator.

[0102] (3) After 24 hours, replace the virus-containing DMEM medium with virus-free DMEM complete medium and continue to incubate for 48 hours. Observe and count the number of fluorescent cells in each well under a fluorescence microscope, take pictures, and calculate the virus titer. Calculate the sum of the total number in the three replicate wells and calculate the average.

[0103] 1.3 Plasmid transfection cell line process

[0104] This part of the experiment aims to construct a h-CD93 gene overexpression stable cell line CHO-CD93, which is transfected into CHO cells with a h-CD93 gene overexpression plasmid and stably expressed.

[0105] (1) Pre-experiment preparation of cells

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

[0107] (2) Pre-experiment to determine the MOI value

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

[0109] 2) Reference the data obtained by searching, design gradient experiment, and find the optimal MOI of 50;

[0110] (3) Pre-experiment to determine the amount of screening drugs

[0111] Check the lethal dose information of Puro in CHO cell line stable cell strain screening, and determine three drug concentration gradients (1 μg, 2 μg, 4 μg) according to the data obtained by searching.

[0112] Day 1: Plate CHO cell line cells in 6-well plates, and 6-well plates contain DMEM / F12+10%FBS, so that the cell density is about 90% on the second day;

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

[0114] Day 4: Change the liquid and re-add Puro at the set concentration;

[0115] 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;

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

[0117] Cell plating: Plate CHO cells in 6-well plates so that the cell density reaches about 70% on the second day;

[0118] Virus infection: According to the MOI value determined by the pre-experiment, calculate the volume of lentivirus needed to be added as 25 μL;

[0119] Change the liquid: Change the liquid according to the actual situation. For some weakly resistant cells, change the liquid in time. For some strongly resistant cells, change the liquid after 48-72h of infection;

[0120] Observe the infection efficiency: 72h after infection, observe the infection efficiency. The lowest efficiency should not be less than 40%;

[0121] Puro screening: The optimal action time is between 3-10 days. The commonly used concentration of Puro is 1-10 μg / mL. The optimal screening concentration is 3 μg / mL determined by pre-experiment;

[0122] Infection: After 72h of culture after infection (the infection time is determined according to the specific situation of the cells and the infection efficiency), add the drug concentration of 3 μg / mL determined by pre-experiment in 6-well plates;

[0123] Add Puro: add 2 μg / mL Puro in 6-well plate;

[0124] Change liquid: change the screening medium every 3-5 days according to the color of the medium and the growth of the cells, and when there is a large amount of cell death, the Puro concentration can be halved to maintain screening;

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

[0126] 1.3 q-PCR detection of transfection effect

[0127] Method:

[0128] q-PCR technology is used to add a fluorescent group in the PCR reaction system, to monitor the entire PCR process in real time by using the fluorescence signal, and finally to quantitatively analyze the unknown template by standard curve. According to the change of fluorescence signal, the change of amplification product amount in each cycle of PCR amplification reaction can be detected in real time, and through the analysis of Ct value and standard curve, the initial template can be quantitatively analyzed.

[0129] (1) Primer design

[0130] The primer sequence is shown in Table 5:

[0131] Table 5 Primer sequence

[0132]

[0133] The internal reference primer is shown in Table 6:

[0134] Table 6 Internal reference primer

[0135]

[0136] (2) RNA extraction

[0137] i. Add 1000 μL Trizol to the 1.5 mL EP tube with cell strain, mix thoroughly, then add 200 μL chloroform, mix thoroughly, shake, stand for 5 min, then centrifuge at 12000 rpm, 4°C for 10 min.

[0138] ii. Take out the 1.5 mL EP tube from the centrifuge, and then suck the upper colorless transparent water phase layer into another clean 1.5 mL EP tube. (The sample will be divided into three layers: the lower organic phase layer, the middle layer and the upper water phase layer, and the RNA is in the upper water phase.) Add an equal volume of isopropanol, mix gently by inverting the tube, and then stand for 10 min. Then centrifuge at 12000 rpm and 4°C for 10 min.

[0139] iii. After centrifugation, a gelatinous precipitate can be seen on the wall or bottom of the tube, which is the RNA. Carefully discard the supernatant and reserve the precipitate.

[0140] iv. Wash the RNA precipitate with 1 mL of 75% ethanol (prepared with DEPC H2O). Then centrifuge at 7000 rpm and 4°C for 5 min, and try to remove the supernatant as completely as possible.

[0141] v. Let it dry on the bench, and it will be dry in about 5-10 min. (Too much drying will greatly reduce the solubility of the RNA.) Add 25 μL of DEPC H2O to all EP tubes, and blow them several times with a gun head to fully dissolve the RNA. Store at -80°C.

[0142] RNA concentration detection: Use a micro nucleic acid detector to detect the RNA concentration. 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 the PCR instrument: 72°C, 5 min, and then cool on ice.

[0150] iii. Add the following reverse transcription reaction solution to the above PCR tube as shown in Table 9:

[0151] Table 9 Reaction system

[0152]

[0153] iv. Perform the reverse transcription reaction on the PCR instrument according to the following conditions as shown in Table 10:

[0154] Table 10 Reaction conditions

[0155]

[0156] v. The cDNA was immediately used for experiment or stored at 4°C.

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

[0158] i. The reaction system was configured as shown in Table 11:

[0159] Table 11 Reaction system

[0160]

[0161] ii. The reaction condition was set as follows:

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

[0163] Table 12 Amplification program

[0164]

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

[0166] Table 13 Melting program

[0167]

[0168] To establish a more sensitive MERS-CoV ADE detection cell line, CHO cells stably expressing C1qR (CD93) were constructed, which were designated as CHO-CD93. The RT-PCR detection results are shown in Table 14. Figure 5 The expression of CD93 in each cell is shown in Table 14.

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

[0170] This pseudovirus uses a retroviral vector, uses the MERS-CoV Spike protein gene (NCBI accession number of amino acid sequence: YP 009047204.1; NCBI accession number of nucleotide sequence: AFS88936.1) to replace the envelope protein gene of the retrovirus, and is co-transfected with a retrovirus packaging plasmid and a CMV-GFP-T2A-Luciferase plasmid in 293T cells. The pseudovirus containing the Spike protein gene is packaged, the pseudovirus surface can express the MERS-CoV Spike protein, and the virus also carries the GFP and Luciferase fluorescent reporter genes, which can be used to evaluate the activity of the pseudovirus infected cells by observing the fluorescent signal and detecting the luciferase activity.

[0171] 2.1 Vector construction

[0172] The bacteria containing the vector plasmid were cultured overnight, and fresh bacteria were taken 3-5 mL to extract the plasmid (DP107-02 high-purity plasmid small extraction kit). 1 μg of fresh plasmid was taken and double digested with the corresponding restriction enzymes. The enzyme digestion products were subjected to agarose gel electrophoresis, and after electrophoresis, the gel was recovered. All the above liquids were transferred to the filter column, centrifuged, and the recovered vector fragments were obtained and the concentration was determined. The diluted primers and templates were subjected to PCR amplification, and after PCR, agarose gel electrophoresis was performed, and the target gene was recovered. Hieff Clone TM The recombinant reaction system was used to connect the overexpression vector and the target fragment, and the connection product was introduced into the competent cells for transformation. Finally, sequencing verification was performed to obtain the target vector.

[0173] 2.2 Packaging of pseudovirus

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

[0175] Example 3 Detection of MERS-CoV antibody neutralization ability

[0176] 3.1 Cell subculture and plating:

[0177] The stable cell line HEK293T-DPP4 (from Yisen Biotechnology Co., Ltd., item number 18038ES50) was subcultured in a complete culture medium (DMEM + 10% FBS) in a 5% carbon dioxide incubator at 37°C, and subcultured every 4 days. The cells were collected in a sterile centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was resuspended with fresh complete culture medium, stained, counted, and the cell suspension with a concentration of 1E6 cells / mL was transferred to a cell culture bottle for continued culture.

[0178] The cells in good growth state were taken for plating, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with complete culture medium, the cell concentration was adjusted to 2x10 5 6 cells / mL, 100 μL of cell suspension was added to each well of a 96-well plate, and cultured overnight.

[0179] 3.2 MERS-CoV pseudovirus dilution

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

[0181] 3.3 MERS-CoV antibody gradient dilution

[0182] Antibody dilution preparation: (1) MERS-CoV Spike Protein monoclonal antibody from ThermoFisher, item number MA5-29975, immunogen sequence Met 1-Trp 1297 in NCBI accession number YP_007188579.1; (2) MERS-CoV Spike RBD antibody from RD SYSTEMS, item number MAB107071, immunogen sequence Glu 367-Tyr 606 in NCBI accession number YP_007188579.1; (3) MERS-CoV Spike S1 subunit antibody from RD SYSTEMS, item number MAB10707, immunogen sequence Met 1-Pro 747 in NCBI accession number K9N5Q8.1; (4) MERS-CoV Spike S2 monoclonal antibody from Thermo Fisher, item number MA5-29978, immunogen sequence Asp 726-Pro 1296 in NCBI accession number YP_009047204.1; (5) MERS-CoV Nucleocapsid antibody from RD SYSTEMS, item number MAB10729, immunogen sequence Met 1-Thr 411 in NCBI accession number YP_007188586.1; were prepared into a stock solution of 100 μg / mL, and diluted with complete culture medium into 5000.0 ng / mL, 2500.0 ng / mL, 1250.0 ng / mL, 625.0 ng / mL, 312.5 ng / mL, 156.3 ng / mL, 78.1 ng / mL, 37.0 ng / mL, 19.5 ng / mL and 0 ng / mL, a total of 10 dilutions.

[0183] 3.4 Sample addition

[0184] After mixing the pseudovirus with the antibody 1:1 (v / v) (the concentration of the mixed pseudovirus was 5E10 Copies / mL, and the concentration of the antibody was 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), the mixture was incubated at 37°C for 1 h, the 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 was set up by adding 100 μL of complete culture solution.

[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, and the cells were washed once with PBS, 25 μL / well of lysis solution (from the Luciferase Assay System kit; Promega, item number: E1531) was added, and the mixture was incubated for 5-10 minutes, 20 μL was taken to a white plate, 100 μL of luminescent substrate (from the Luciferase Assay System kit) was added, and the Luciferase luminescence signal was detected. As shown in Figure 4 , the MERS-CoV Spike RBD monoclonal antibody and the MERS-CoV Spike Protein monoclonal antibody can inhibit the infection of 293 / DPP4 cells by MERS-CoV pseudovirus, and the neutralization ability is enhanced with the increase of the concentration of the antibody, and the other three antibodies do not show obvious neutralization effect. The signal value of the positive control well (i.e., corresponding to the antibody titer of 0) is high, indicating that the pseudovirus has strong infection ability.

[0187] Example 4 System for evaluating the ADE effect of Middle East respiratory coronavirus in vitro

[0188] CHO is a cell in Chinese hamster ovary, and MERS-CoV pseudovirus cannot infect CHO cells.

[0189] 4.1 Cell subculture and plating

[0190] The stably transfected cell strain CHO-CD93 prepared in Example 1, as well as the background CHO cells, Vero cells, and immune cells Raji and THP-1, were plated, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, complete culture solution was added to resuspend the cells, the cell concentration was adjusted to 1×10 5 6 cells / mL, 100 μL of the cell suspension was taken per well, and added to a 96-well plate for overnight culture.

[0191] 4.2 MERS-CoV pseudovirus dilution

[0192] Pseudovirus preparation: The above prepared pseudovirus (stock solution: copy number: 1E13 Copies / mL from Yisen 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, item number MAB107071; immunogen sequence of the antibody: Glu367-Tyr606) was prepared as a 100 μg / mL stock solution, diluted 5 times by volume to 6 concentration gradients (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 0 ng / mL), mixed with the pseudovirus dilution, C1q dilution 1: 1: 1, (after mixing, the pseudovirus concentration was 8.3E10; the antibody concentration was 33333.3 ng / mL, 6666.7 ng / mL, 1333.3 ng / mL, 266.7 ng / mL, 53.3 ng / mL, 0 ng / mL), incubated at 37°C for 1h.

[0197] 4.3 Loading

[0198] After the antibody, complement and pseudovirus were incubated, 150 μL / well of the mixed solution was added to the 96-well plate, and 150 μL of complete culture medium was added to the blank control, and the plate was incubated at 37°C in a 5% carbon dioxide environment for 48 hours.

[0199] 4.4 Detection of chemiluminescence

[0200] The supernatant was completely aspirated, washed once with PBS, 25 μL / well of lysis solution was added and incubated for 5-10 minutes, 20 μL was taken to the white plate, 100 μL of luminescent substrate (Promega Luciferase Assay System) was added, and the Luciferase luminescence signal was detected. The red dotted line is the luminescence value (RLU) of the sample well with only MERS-CoV and C1q added, which is set as the infection rate 100%, and the infection rate is calculated accordingly.

[0201] Results are shown in Figure 6 Figure 2, where the infection rate with antibody concentration of 0 ng / mL is taken as 100%, and shown in red dotted line, in the presence of MERS-CoV Spike RBD antibody and complement component Clq, Vero cells, Raji cells and THP-1 cells, which are commonly used ADE detection cells, did not promote the infection of the cells by the virus in the concentration range detected by the MERS-CoV Spike RBD antibody; but at sub-neutralizing concentrations, they showed obvious promotion of the infection of CHO-CD93 cells by MERS-CoV, and the infection of CHO-CD93 was stronger than that of CHO cells. Figure 7

[0202] Example 5 Application of the system for evaluating the MERS-CoV ADE effect in vitro

[0203] In this example, 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, item number MA5-29975, immunogen sequence Met 1-Trp 1297 in NCBI accession number YP_007188579.1; (2) MERS-CoV Spike RBD antibody from RD SYSTEMS, item number MAB107071, immunogen sequence Glu 367-Tyr 606 in NCBI accession number YP_007188579.1; (3) MERS-CoV Spike S1 subunit antibody from RD SYSTEMS, item number MAB10707, immunogen sequence Met 1-Pro 747 in NCBI accession number K9N5Q8.1; (4) MERS-CoV Spike S2 monoclonal antibody from Thermo Fisher, item number MA5-29978, immunogen sequence Asp 726-Pro 1296 in NCBI accession number YP_009047204.1; (5) MERS-CoV Nucleocapsid antibody from RD SYSTEMS, item number MAB10729, immunogen sequence Met 1-Thr 411 in NCBI accession number YP_007188586.1.

[0204] 5.1 Cell subculture and plating

[0205] ​The stably transfected cell strain CHO-CD93 prepared in Example 1 was plated, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended by adding complete culture medium, the cell concentration was adjusted to 1x10 5 The cell suspension was taken 100 μL / well, and added to a 96-well plate, and cultured overnight.

[0206] 5.2 MERS-CoV pseudovirus dilution

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

[0208] 5.3 C1q complement dilution

[0209] The 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 diluted according to a 5-fold dilution to 6 concentration gradients (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 0 ng / mL), mixed with the pseudovirus dilution and C1q dilution at a volume ratio of 1:1:1 (after mixing, the pseudovirus concentration was 8.33E10; the antibody concentration was 33333.3 ng / mL, 6666.7 ng / mL, 1333.3 ng / mL, 266.7 ng / mL, 53.33 ng / mL and 0 ng / mL), and incubated at 37°C for 1 h.

[0212] 5.5 Sample loading

[0213] After the antibody, complement and pseudovirus were incubated, 150 μL / well of the mixed solution was added to a 96-well plate, and 150 μL of complete culture medium was added to the blank control, and cultured at 37°C in a 5% carbon dioxide environment for 48 hours.

[0214] 5.6 Detection of chemiluminescence

[0215] The supernatant was completely absorbed, washed once with PBS, 25 μL / well of lysis solution was added and incubated for 5-10 minutes, 20 μL was taken to a white plate, 100 μL of luminescent substrate (Promega Luciferase Assay System) was added, and the luciferase luminescence signal was detected. The red dotted line is the luminescence value (RLU) of the sample well only adding MERS-CoV and C1q, which is set as the infection rate of 100%, and the infection rate is calculated accordingly.

[0216] The results are shown in Figure 8 , wherein the infection rate at an antibody concentration of 0 ng / mL is taken as 100%, and the red dotted line is shown. In the presence of complement component C1q, MERS-CoV Spike RBD and MERS-CoV Spike Protein monoclonal antibodies at their sub-neutralizing concentrations showed a significant role in promoting MERS-CoV infection of CD93-CHO cells overexpressing CD93, and MERS-CoV Spike Protein monoclonal antibodies caused stronger MERS-CoV infection of CD93-CHO cells than MERS-CoV Spike RBD, while the other three antibodies did not show ADE effect.

[0217] As demonstrated in the present embodiment, the system for evaluating ADE effect provided by the present application can detect whether the antibody targeting MERS-CoV has the risk of causing ADE effect. The system comprises Middle East respiratory syndrome coronavirus pseudovirus, complement component C1q and cells overexpressing CD93; or, the system comprises antibody targeting Middle East respiratory syndrome coronavirus, complement component C1q and cells overexpressing CD93; or, the system comprises Middle East respiratory syndrome coronavirus pseudovirus, antibody targeting Middle East respiratory syndrome coronavirus, complement component C1q and cells overexpressing CD93.

[0218] It can be reasonably inferred that the system for evaluating ADE effect provided by the present application can be used to detect in vitro whether the evaluation object has the risk of causing complement-mediated ADE effect.

Claims

1. A system for evaluating an ADE effect, characterized by, The system is selected from any one of (1)-(3): (1) the system comprises a Middle East respiratory syndrome coronavirus pseudovirus, a complement component C1q, and a cell overexpressing CD93; (2) the system comprises an antibody targeting Middle East respiratory syndrome coronavirus, a complement component C1q, and a cell overexpressing CD93; and (3) the system comprises an antibody targeting Middle East respiratory syndrome coronavirus, a Middle East respiratory syndrome coronavirus pseudovirus, a complement component C1q, and a cell overexpressing CD93.

2. The system of claim 1, wherein, The cell overexpressing CD93 is a CHO cell overexpressing CD93; and / or, The antibody targets a receptor binding domain or a full-length Spike protein of Middle East respiratory syndrome coronavirus; and / or, The CD93 comprises an amino acid sequence as shown in NCBI Accession No. NP_036204.2; and / or, The CD93 is overexpressed on the surface of the CHO cell; and / or, The pseudovirus comprises a Spike protein of Middle East respiratory syndrome coronavirus.

3. The system of claim 2, wherein, The nucleic acid encoding the CD93 comprises a nucleotide sequence as shown in NCBI Accession No. NM_012072; and / or, The Spike protein comprises an amino acid sequence as shown in NCBI Accession No. YP_009047204.

1.

4. A method of constructing a system as claimed in any one of claims 1-3, characterized in that, The method comprises transfecting a base cell with an overexpression plasmid comprising a nucleic acid sequence encoding CD93 to prepare the cell overexpressing CD93; and further comprises 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 cell overexpressing CD93, and the complement component C1q.

5. The method of claim 4, wherein, The base cell is a CHO cell; and / or, The backbone plasmid of the overexpression plasmid is pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro; and / or, The insertion site of the nucleic acid sequence in the transfection is BamHI-EcoRI; and / or, The nucleic acid sequence comprises a nucleotide sequence as shown in NCBI Accession No. NM_012072.

6. A method of evaluating the ADE effect in vitro, characterized in that, The method comprises: Step 1: when the evaluation object is a virus, a pseudovirus, or a preparation comprising a virus, contacting (2) in the system of any one of claims 1-3 with the evaluation object, or replacing the Middle East respiratory syndrome coronavirus pseudovirus in (3) in the system of any one of claims 1-3 with the evaluation object; or, when the evaluation object is an antibody or a preparation comprising an antibody, contacting (1) in the system of any one of claims 1-3 with the evaluation object, or replacing the antibody in (3) in the system of any one of claims 1-3 with the evaluation object; Step 2: detecting the degree of infection of the cell in the system; Step 3: determining the risk of ADE effect; when the degree of infection is higher than that of the negative control, determining that the evaluation object has the risk of causing ADE effect.

7. The method of claim 6, wherein, 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 comprising an antibody targeting Middle East Respiratory Syndrome coronavirus, or a blood sample of a Middle East Respiratory Syndrome convalescent; 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 of any one of claims 1-3 is used as a negative control.

8. Use of the system of any one of claims 1-3 in assessing the risk of an evaluation object causing an ADE effect; 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 comprising an antibody targeting Middle East Respiratory Syndrome coronavirus, or a blood sample of a Middle East Respiratory Syndrome convalescent.

9. Use of a cell in evaluating an ADE effect caused by a Middle East respiratory syndrome coronavirus or an antibody targeting a Middle East respiratory syndrome coronavirus, characterized in that, the cell is a CD93-overexpressing cell; and the use is for non-diagnostic purposes.

10. Use according to claim 9, wherein the CD93-overexpressing cell is as defined in the system of any one of claims 1-3.

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