A method of detecting neutralization activity in sars-cov-2 antibodies

Abstract

The present application relates to the technical field of biotechnology, in particular to a preparation method of SARS-CoV-2 virus-like particles, and SARS-CoV-2 virus-like particles prepared according to the method. The present application also relates to a method for screening anti-SARS-CoV-2 antibodies or detecting the neutralization activity of anti-SARS-CoV-2 antibodies, and a method for screening candidate drugs capable of inhibiting SARS-CoV-2 infection of cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation method of SARS-CoV-2-like virus particles, and SARS-CoV-2-like virus particles prepared according to the method. The present application also relates to a method for screening anti-SARS-CoV-2 antibodies or detecting the neutralization activity of SARS-CoV-2 antibodies, and a method for screening candidate drugs capable of inhibiting SARS-CoV-2 infection of cells. BACKGROUND

[0002] Pseudovirus refers to a virus in which a retrovirus integrates the envelope glycoprotein of another virus, thereby forming an exogenous viral envelope, and the genome remains the genetic characteristics of the retrovirus itself. The generation process of the pseudovirus is as follows: a plasmid with an envelope protein gene of a virus is co-transfected with a plasmid with the backbone gene of another virus into a packaging cell (such as 293T cell), the envelope protein gene expresses the envelope protein on the surface of the packaging cell, and the gene of another virus is transcribed and translated in the packaging cell to assemble into a virus particle. When the virus particle buds, it is packaged by the envelope protein expressed on the surface of the cell, i.e. the pseudovirus is formed.

[0003] The nucleic acid inside the pseudovirus is a defective genome, which cannot express the surface protein of the pseudovirus particle, so the virus surface protein needs to be expressed by additional plasmid transfection or cell line stable expression. Studies have shown that the new coronavirus SARS-CoV-2 enters the cell by binding to the cell hACE2 receptor through the virus S protein, so the selected envelope protein for the construction of the new coronavirus pseudovirus is the S protein. The backbone vector of the pseudovirus contains the gene sequences of virus transcription, packaging, integration, etc., which provides all the proteins except the membrane protein for the pseudovirus. Since the pseudovirus usually only performs one round of infection and does not have the ability to replicate and proliferate, the pseudovirus system can carry a reporter gene, thereby facilitating subsequent qualitative and quantitative research.

[0004] The use of a pseudovirus to infect an animal model of a hACE2 gene knock-in mouse can simulate the process of SARS-CoV-2 infection into cells, which is used to evaluate the cross-protection effect in vivo. Therefore, the pseudovirus is suitable for evaluating the vaccine effect of neutralizing antibodies. The pseudovirus system has its unique advantages. Since the pseudovirus does not have the ability to self-replicate and can only perform a single cycle of infection, the mutation rate of the virus is reduced and the biosafety is high, which can reduce the risk of laboratory operation.

[0005] Therefore, there is a need for an alternative to live viruses for vaccine development and detection of immunogenic neutralizing antibody levels, to reduce the difficulty of operation and experimental cost in the process of antibody screening and detection. SUMMARY

[0006] The inventors of the present application have optimized the preparation method of SARS-CoV-2 virus-like particles and the method for detecting the neutralization activity of SARS-CoV-2 antibodies through a large number of experiments and repeated groping, so that the detection results have high accuracy, good repeatability and specificity. Further, by comparing the above-mentioned method with the neutralization method using live virus, it is found that the trend is consistent with that of live virus, and to a certain extent, it can reflect the results of live virus. The method can be applied to the screening of anti-SARS-CoV-2 antibodies or drugs or the detection of the activity of SARS-CoV-2 neutralizing antibodies, and provides a good technical support means for the research of SARS-CoV-2 vaccine, drug evaluation and virus pathogenesis.

[0007] Therefore, in a first aspect, the present application provides a preparation method of SARS-CoV-2 virus-like particles, the method comprising:

[0008] (i) infecting a first host cell with vTF7-3 vaccinia virus, and then transfecting the first host cell with a prVSVAG-GFP backbone plasmid and a helper plasmid;

[0009] (ii) harvesting the VSV virus-like particles obtained in step (i);

[0010] (iii) transfecting the second host cell with a membrane plasmid containing an amino acid sequence as shown in SEQ ID NO: 1; and then infecting the second host cell with VSV virus-like particles with an MOI value of 0.02 to 0.04 (for example, 0.02, 0.03, 0.04) to obtain SARS-CoV-2 virus-like particles.

[0011] In certain embodiments, the first host cell and the second host cell are the same or different.

[0012] In certain embodiments, the first host cell and the second host cell are each independently selected from human hematopoietic cells, epithelial cells, hepatocytes, tumor cells or neural cells.

[0013] In certain embodiments, the first host cell is a BHK21 cell and the second host cell is a 293T cell.

[0014] In certain embodiments, in step (i), the MOI value of vTF7-3 vaccinia virus is 4 to 6 (for example, 4, 5, 6).

[0015] In certain embodiments, in step (i), the helper plasmid comprises pBS-N, pBS-P, pBS-G and pBS-L.

[0016] In certain embodiments, the membrane plasmid is obtained by inserting the amino acid sequence as set forth in SEQ ID NO: 1 into a pcDNA3.1 vector and codon-optimized according to the host cell.

[0017] In certain embodiments, the backbone plasmid and the helper plasmid are transiently transfected into a first host cell.

[0018] In certain embodiments, the membrane plasmid is transiently transfected into a second host cell.

[0019] In another aspect, the present application provides a SARS-CoV-2 virosome, which is obtained by the method as described above.

[0020] In another aspect, the present application provides a method of screening for an anti-SARS-CoV-2 antibody or detecting the neutralization activity of an anti-SARS-CoV-2 antibody, which comprises contacting a host cell with the antibody before, simultaneously with, or after contacting the host cell with the SARS-CoV-2 virosome as described above.

[0021] In certain embodiments, the method comprises:

[0022] Step (1): contacting a host cell with the SARS-CoV-2 virosome as described above, and then contacting the host cell with the antibody;

[0023] Step (2): observing the host cell under conditions that enable fluorescence to be emitted to determine whether the antibody is an anti-SARS-CoV-2 antibody; or counting the number of positive cells of the host cell in an immunospot reader and calculating the ID 50 value to detect the neutralization activity of the antibody.

[0024] In another aspect, the present application provides a method of screening for a candidate drug capable of inhibiting SARS-CoV-2 from infecting a cell, which comprises contacting the virosome or the host cell with the candidate drug before, simultaneously with, or after contacting the host cell with the SARS-CoV-2 virosome as described above.

[0025] In certain embodiments, the method comprises:

[0026] Step (1): contacting a host cell with the SARS-CoV-2 virosome as described above, and then contacting the host cell with the antibody;

[0027] Step (2): observing the host cell under conditions that enable fluorescence to be emitted to determine whether the candidate drug is capable of inhibiting SARS-CoV-2 from infecting a cell.

[0028] In certain embodiments, the host cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a hepatocyte, a tumor cell, a neural cell.

[0029] In certain embodiments, the host cell is an ACE2-expressing cell, e.g., a 293T-ACE2 cell and / or an AF cell.

[0030] In certain embodiments, the cell is an ACE2 and Furin-expressing cell, e.g., an AF cell.

[0031] In certain embodiments, in step (1), the SARS-CoV-2 pseudovirus particles as described previously are contacted with the host cells for 24h-36h (e.g., 24h, 26h, 28h, 30h, 32h, 34h, 36h).

[0032] In certain embodiments, in step (1), the SARS-CoV-2 pseudovirus particles as described previously are contacted with 2.00 x 105 3 cells / well to 4.00 x 105 3 cells / well (e.g., 2.00 x 105 3 cells / well, 3.00 x 105 3 cells / well, 4.00 x 105 3 cells / well) of the host cells.

[0033] In certain embodiments, in step (1), the host cells are contacted with the antibody for 0.5h-2h (e.g., 0.5h, 1h, 2h).

[0034] In certain embodiments, in step (1), the SARS-CoV-2 pseudovirus particles as described previously having a value of MOI of 0.2 are contacted with the host cells.

[0035] In certain embodiments, steps (1) and (2) of the method as described previously are performed by the following steps (a) to (d):

[0036] Step (a): Gradient dilution of the antibody is performed in a 96-well plate, after dilution the antibody is transferred to a 384-well plate, 10ul of the antibody is added to each well;

[0037] Step (b): The SARS-CoV-2 pseudovirus particles of claim 3 are diluted with DMEM complete medium to a number of about 400 GFP positive particles per well, 10μl of the pseudovirus particles are added to each well, incubated for 1h;

[0038] In certain embodiments, the 384-well plate is incubated in a 37 °C 5% CO2 incubator for 1 h;

[0039] Step (c): Adjust the concentration of ACE2 and Furin-expressing cells (e.g., AF cells) to 4 x 105 / ml suspension, add 20 ul of the cells to each well, i.e., 2 x 105cells per well, and incubate for 24 h; 5 3 Step (c): Adjust the concentration of ACE2 and Furin-expressing cells (e.g., AF cells) to 4 x 105 / ml suspension, add 20 ul of the cells to each well, i.e., 2 x 105cells per well, and incubate for 24 h;

[0040] In certain embodiments, the 384-well plate is incubated in a 37 °C 5% CO2 incubator for 24 h;

[0041] Step (d): Count the number of GFP-positive cells using an Immune Spot Reader Biotek and calculate the ID value using the Reed-Muench method to evaluate the neutralizing antibody level of the sample. 50

[0042] Definitions of terms

[0043] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the molecular genetic, nucleic acid chemical, chemical, molecular biological, biochemical, cell culture, microbiological, cell biological, genomic, and recombinant DNA, and other operational steps used herein are in accordance with conventional methods well known in the respective fields. Meanwhile, in order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0044] As used herein, the term “SARS-CoV-2” is an abbreviation of “severe acute respiratory syndrome coronavirus 2”, formerly known as “novel coronavirus”, which belongs to the genus of beta coronaviruses and is a single-stranded positive-sense RNA virus containing an envelope. The genomic sequence of SARS-CoV-2 is known to those skilled in the art, which can be found in, for example, GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M), and membrane protein (E). The receptor of SARS-CoV-2 is the same as that of SARS-CoV, i.e., the virus specifically binds to angiotensin-converting enzyme 2 (ACE2) on the host cell through the receptor binding domain (RBD) on the S protein, and then the virus performs membrane fusion and enters the cell, and the S protein plays a crucial role in the process of viral infection of cells.

[0045] ​​As used herein, the term “COVID-19” refers to pneumonia caused by SARS-CoV-2 infection, and both have the same meaning and can be used interchangeably.

[0046] As used herein, the terms “pseudovirus” and “virus-like particle” have the same meaning and can be used interchangeably; it refers to a virus-like particle self-assembled from viral proteins, which does not encapsulate nucleic acid or encapsulates other nucleic acid, so that the pseudovirus or virus-like particle can infect host cells, but does not have the ability to replicate autonomously. Therefore, compared with the real virus, it has high biosafety. The packaging system of the pseudovirus is generally composed of two parts, namely the packaging component and the expression component. The packaging component is constructed by removing the genetic information required for packaging, reverse transcription and integration from the viral (e.g., HIV-1) genome, which provides the necessary proteins for the pseudovirus particle; the expression component is complementary to the packaging component, which contains the genetic information required for packaging, reverse transcription and integration, and also contains the exogenous target gene. Co-transfecting the packaging component and the vector component into host cells can harvest pseudovirus particles in the cell supernatant.

[0047] In certain embodiments, the packaging component comprises or consists of a backbone plasmid. In certain embodiments, the expression component comprises or consists of a membrane plasmid. In certain embodiments, the packaging component comprises a backbone plasmid and a helper plasmid.

[0048] As used herein, the terms “backbone plasmid” and “packaging plasmid” have the same meaning and can be used interchangeably. As generally understood by those skilled in the art, a viral vector system (particularly a lentiviral vector system) can be composed of two parts, namely a packaging component (e.g., a packaging plasmid or a backbone plasmid) and a vector component (e.g., a recombinant expression vector carrying a target gene); wherein the packaging component (e.g., a packaging plasmid or a backbone plasmid) can provide all the auxiliary proteins required for transcription and packaging of genetic material (e.g., RNA) into a recombinant pseudovirus particle. In this way, high-titer pseudovirus particles can be produced by co-transfecting cells with a recombinant expression vector and a packaging plasmid, then packaging the pseudovirus in the cells, and then the packaged pseudovirus particles are secreted into the culture medium outside the cells. Such packaging plasmids or backbone plasmids are well known to those skilled in the art, for example, a backbone plasmid constructed based on VSV: including but not limited to prVSVΔG-GFP (kerefast company).

[0049] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements carried in the genetic material of the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0050] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK21 cells, HEK 293T cells or human cells.

[0051] Those skilled in the art will appreciate that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired gene product, etc. A vector can be introduced into a host cell so that it becomes capable of producing a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein.

[0052] As used herein, the term "effective amount" refers to an amount that is effective to achieve a desired purpose. For example, an amount effective to prevent or treat a disease (e.g., rotavirus infection) refers to an amount that is effective to prevent, retard or delay the onset of, or to ameliorate, lessen or treat the severity of a disease (e.g., a disease caused by rotavirus infection). Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies being administered to the patient, etc.

[0053] As used herein, the term "neutralization activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the virus from infecting a cell and / or the maturation of viral progeny and / or the release of viral progeny, an antibody or antibody fragment having neutralization activity can prevent the amplification of the virus, thereby inhibiting or eliminating infection by the virus.

[0054] As used herein, the term "MOI (multiplicity of infection)" refers to the ratio of the number of viruses to the number of cells when a virus infects a host cell. It is generally considered that MOI is a ratio without units, and the implied unit can also be pfu number / cell. By selecting a suitable MOI value, the infection efficiency of the virus will be improved.

[0055] As used herein, the term "FFU / ml (focus-forming units per ml)" refers to the viral titer, expressed as the number of GFP-positive cells per milliliter.

[0056] Advantages of the invention

[0057] Compared with the prior art, the preparation method of the SARS-CoV-2 virus-like particles and the method for detecting the neutralization activity of the SARS-CoV-2 antibody provided by the present application can detect a variety of different antibodies, and the detection results all have high accuracy, good repeatability and specificity. Further, compared with the neutralization method using live viruses, it is found that the trend is consistent with that of live viruses, and can reflect the results of live viruses to a certain extent. The method can be applied to the screening of anti-SARS-CoV-2 antibodies or drugs or the detection of the activity of SARS-CoV-2 neutralizing antibodies, and provides a good technical support means for the evaluation of SARS-CoV-2 vaccines, drugs, and the research of viral pathogenesis.

[0058] In addition, the current neutralization test method is mainly carried out by using a 96-well plate method, while the 384-well plate neutralization test method of the present application can save manpower, material resources and financial resources, and is easier to realize high-throughput detection of drugs and monoclonal antibodies and the like. Assuming that 1000 samples are detected, the following table introduces the advantages of the 384-well plate method and compares it with the traditional 96-well plate method:

[0059]

[0060] Note: In the above table, "115" refers to the market price of each 96-well plate, "250" refers to the number of 96-well plates required to complete the detection of 1000 samples; "40" refers to the market price of each 384-well plate, and "50" refers to the number of 384-well plates required to complete the detection of 1000 samples.

[0061] Embodiments of the application will be described in detail below with reference to the attached drawing figures and examples, but it is to be understood that the application is not limited to the attached drawing figures and examples, and that the application can be used in any number of alternative ways not depicted in the drawing figures and examples. Various objects and advantageous aspects of the application will become apparent to those skilled in the art from the following detailed description in conjunction with the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 A schematic of mutations of D614G and D614Gdel21 is shown.

[0063] Figure 2 A flow chart of SARS-CoV-2-S Pseudovirus packaging is shown.

[0064] Figure 3 The number of GFP positive fluorescent cells detected in Example 4 is shown.

[0065] Figure 4 The results of SARS-CoV-2-Spike Pseudovirus using different target cells in neutralization experiments in Example 5 are shown.

[0066] Figure 5 The number of GFP positive cells of SARS-CoV-2-Spike Pseudovirus using different target cells in neutralization experiments in Example 5 is shown.

[0067] Figure 6 The number of GFP positive cells of different SARS-CoV-2-Spike Pseudovirus using different target cells in neutralization experiments in Example 5 is shown.

[0068] Figure 7 The number of GFP positive cells of harvested Pseudovirus using AF cells titration after different time 0h, 8h, 12h, 24h, 36h, 48h, 72h in Pseudovirus titration test detection time comparison is shown.

[0069] Figure 8 The ID of monoclonal antibody AM180, positive serum after human heterologous immunization of new crown vaccine and guinea pig D614G protein three needle immunization serum at 12h, 24h, 36h, 48h, 72h in Pseudovirus neutralization test detection time comparison is shown. 50 Value.

[0070] Figure 9 The number of GFP positive cells of Pseudovirus titration test under different cell addition amounts is shown.

[0071] Figure 10 The results of Pseudovirus neutralization test are shown, wherein, Figure 10 A and Figure 10B shows the four-parameter curves of the monoclonal antibody in the pseudovirus neutralization assay at different cell loading amounts (cells / well); Figure 10 C shows the four-parameter curves of human serum samples in the pseudovirus neutralization test at different cell addition amounts (cells / well); Figure 10 D shows the four-parameter curves of guinea pig serum samples in the pseudovirus neutralization test at different cell addition amounts (cells / well).

[0072] Figure 11 The results of the pseudovirus neutralization test are shown, in which... Figure 11 A and Figure 11 B shows the four-parameter curves of the monoclonal antibody at different MOIs with different viral addition levels; Figure 11 C shows the four-parameter curves of MOI for guinea pig serum samples at different virus addition levels; Figure 11 D shows the four-parameter curves of MOI for human serum samples at different viral loading levels.

[0073] Figure 12 The effects of different incubation times on the neutralization assay results of SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus were investigated under the conditions of a cell addition rate of 2.00 x 10³ cells / well and a virus inoculation MOI of 0.2.

[0074] Figure 13 The validation of the SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus neutralization assay method is shown in (A), (B), and (C) validation of the specificity of the fluorescent pseudovirus neutralization assay method.

[0075] Figure 14 The results show the ID values ​​of SARS-CoV-2-D614Gdel21-GFP pseudovirus in the fluorescent 384 system and the SARS-CoV-2-D614Gdel21-Fluc pseudovirus in the chemiluminescent 96-well plate system, as measured by the two neutralization assays mentioned above, in guinea pig serum. 50 Linear regression analysis was performed on the values.

[0076] Sequence information

[0077] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0078] Table 1: Sequence Description

[0079] Detailed Implementation

[0080] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0081] Unless otherwise indicated, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are used in the present application, are those taught in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., (1995)), and ANIMAL CELL CULTURE (R. I. Freshney ed., (1987)).

[0082] In addition, unless otherwise indicated, conventional conditions or manufacturer's recommended conditions were used in the examples. Unless otherwise indicated, all reagents or instruments used were conventional products available commercially. Those skilled in the art will know that the examples describe the present application by way of example only and are not intended to limit the scope of the application as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0083] Example 1. Experimental materials

[0084] Cells and samples

[0085] 293T (American Type Culture Collection [ATCC], CRL-3216) were cultured in 5% CO2 environment at 37℃ with 100U / mL penicillin-streptomycin solution (GIBCO) and 10% fetal bovine serum (FBS, Pansera ES, PAN-Biotech) provided. ACE2 receptor and Furin receptor were stably transfected on 293T cells to obtain and named AF cells (constructed in our laboratory, the construction process is as follows: ACE2 receptor and Furin receptor lentivirus were constructed by YUNZHOU BIOTECHNOLOGY CO., LTD., with a titer of 10 8 IFU / mL The above lentivirus (MOI=100) and 10ug / uL polybrene were used to transduce 293T cells, and the cells were cultured in a 37℃ 5% CO2 incubator for 48h before drug screening. Until there were no more suspended dead cells in the culture medium, after 2-3 weeks of stable puromycin drug screening, 293T cells stably transfected with ACE2 receptor and Furin receptor were obtained and named AF cells), using Hygromycin B (Hygromycin B) with a concentration of 150ug / ml, 10% fetal bovine serum (FBS, Pansera ES, PAN-Biotech), 100U / mL penicillin-streptomycin solution (GIBCO) medium for screening, ACE2 receptor was stably transfected on 293T cells Hela cells, and after pressure screening with 15ug / ml Blasticidin, it was named ACE2 cells and Hela-ACE2 cells (American Type Culture Collection [ATCC], CCL-2), BHK21-ACE2 cells (purchased from YUNZHOUBIOTECHNOLOGY CO., LTD.), 0.25% trypsin-EDTA (GIBCO) was used to pass the cells once every 2-4 days.

[0086] Human serum samples

[0087] Positive serum: 1 piece of human positive serum of novel coronavirus vaccine heterologous boosting.

[0088] Negative serum: qualified raw plasma of a plasma donor in a plasma station in Shandong, from Huanjiang, Guangxi Zhuang Autonomous Region, aged 18-55, with an average of 40, and the male to female ratio was 1:1.3.

[0089] Guinea pig serum samples

[0090] Positive serum: Female guinea pigs (body weight 200-220 g) were used as experimental animals and divided into 6 groups, 9 in each group. The guinea pigs were immunized with D614G, VOC (Alpha, Beta, Gamma, Delta, Omicron) proteins. 100 ug of spike protein was mixed with Al adjuvant, and immunization was performed once every 14 days, for a total of 3 times. The serum was collected 14 days after the third immunization for subsequent experiments.

[0091] Negative serum: Female guinea pigs (body weight 200-220 g) were used as negative test animals, and a total of 100 negative animal sera were obtained.

[0092] SARS, MERS, RSV serum was preserved in the laboratory.

[0093] Example 2. Packaging of SARS-CoV-2-S pseudovirus

[0094] To rescue prVSV-△G-GFP pseudovirus, 293T cells pre-plated in a six-well plate were infected with vTF7-3 vaccinia virus (donated by the Fourth Military Medical University) (MOI = 5), incubated at 37°C in a 5% CO2 incubator for 1 h, and then transfected with prVSVΔG-GFP backbone plasmid, auxiliary plasmid (pBS-N, pBS-P, pBS-G, pBS-L) (all purchased from kerefast company) at 5, 3, 5, 8, 1 ug / well. The transfection reagent was lipo3000, and the medium was changed to 2% DMEM medium 4-6 h after transfection. The prVSV-△G-GFP pseudovirus was harvested after 24 h of culture at 37°C in a 5% CO2 incubator. Figure 1 ).

[0095] Packing pseudovirus, briefly, first transfect the membrane plasmid in 293T cells using lipo3000 transfection reagent 4-6 h after transfection, and then change the medium. prVSV-△G-GFP pseudovirus (MOI = 0.2) was infected for 1-2 h, and then the medium was changed to 2% DMEM. The supernatant containing the pseudovirus was harvested after 24 hours, and fresh 2% DMEM was added. The supernatant was harvested again and mixed with the supernatant harvested 24 h ago. The centrifuged supernatant was aliquoted and stored at -80°C for later use. Figure 2 ).

[0096] 293T cell transient transfection assay

[0097] 293T cells were plated at about 3x10 5 -5x10 5 cells / ml 24-36h in advance, when the cells were 80-90% confluent, the cells were transfected with 6ug plasmid per well, angiotensin converting enzyme 2 (ACE2), furin, transmembrane serine protease 2 (TMPRSS2), cathepsin L and empty plasmid pcDNA3.1 were transfected using lipo3000 transfection reagent, 4-6h later the medium was changed to complete medium, the cells were trypsinized after 24h incubation at 37℃ 5% CO2 incubator and used for titration assay.

[0098] Example 3. Titration and neutralization test of pseudovirus

[0099] Pseudovirus titration assay based on 384-well plate

[0100] Firstly, the INTEGR VIAFLO 384 instrument was used to seal the four sides of the 384-well plate with sterile water, 80ul per well, to prevent edge effects caused by internal liquid evaporation. Then, a transparent 96-well U-shaped plate was used to dilute the pseudovirus in A-G rows. The harvested pseudovirus SARS-CoV-2-S supernatant was taken, and the original virus supernatant was added to the first column. Then, the pseudovirus was diluted vertically in a 2-fold gradient, a total of 7 dilutions. Then, the INTEGR ASSIST PLUS pipetting workstation was used to transfer the liquid in the U-shaped plate to the 384-well plate, two flat controls were set for each sample, and the liquid volume was 10ul per well. After the plate transfer was completed, the INTEGR ASSIST PLUS instrument was used to automatically add 2x103 cells / well of AF cells, 20ul per well, and incubate at 37℃ 5% CO2 incubator for 24h. Then, Biotek was used to count the number of GFP positive cells, and the SARS-CoV-2-S pseudovirus titer (FFU / ml) was obtained.

[0101] Pseudovirus neutralization assay based on 384-well plate

[0102] Take 96-well U-shaped plates, dilute the samples to be tested in A2-G11 wells, add 10 samples to each plate, dilute by 15 times in the first row, use INTEGR ASSIST PLUS pipetting workstation to dilute by 3 times gradient, a total of 7 gradients, after dilution, use INTEGR ASSIST PLUS pipetting workstation to transfer the samples to be tested in the U-shaped plate to the 384-well plate, set two duplicate wells, 10ul per well. Dilute the pseudovirus to about 400 GFP positive particles per well with DMEM complete medium, add 10ul to B3-O23 wells, incubate at 37°C in a 5% CO2 incubator for 1h, adjust the final concentration of AF cells to 4x10 5 cells per well, add 20ul to each well, i.e. add 2x10 3 cells to each well, place the 384-well plate in a 37°C 5% CO2 incubator again for 24h, count the number of GFP positive cells on the immunospot plate reader Biotek, and calculate the ID 50 value using the Reed-Muench method to evaluate the neutralizing antibody level of the sample.

[0103] Software and data analysis

[0104] Data were processed using GraphPad Prism 8 software (GraphPad, San Diego, CA) and Microsoft Excel. Pseudovirus titers and neutralizing antibody levels were calculated by the reduction of AmCyan positive cells in pseudovirus neutralization test and titration test. The inhibition rate was plotted using GraphPad Prism 8 software, and the linear regression curve of the fluorescence system and chemiluminescence system neutralization test data was plotted using Excel. IBS 1.0 software was used to plot the SARS-CoV-2 and C-terminal truncated 21 amino acid model.

[0105] Example 4. Optimization of pseudovirus construction

[0106] To explore the effect of pseudovirus infection amount on the construction of SARS-CoV-2-Spike pseudovirus, the neutralization experiment was performed according to the method described in Example 1, and the effects of different infection amounts were compared. Specifically as follows:

[0107] Select 293T cells for packaging of SARS-CoV-2-D614Gdel21-GFP pseudovirus, we explore the prVSV-△G-GFP infection amount when packaging fluorescent pseudovirus in 6-well plates, the prVSV-△G-GFP fluorescent pseudovirus is titrated to 2x10 4FFU / ml (virus titer expressed as GFP positive cell forming units per milliliter (FFU / ml)), pcDNA3.1-SARS-CoV-2-D614Gdel21 plasmid transfection amount of 5ug per well. Five different MOI of prVSV-△G-GFP infected 293T cells, the harvested fluorescent pseudovirus 2-fold gradient dilution, 24h after detection of GFP positive fluorescent cell number Figure 3 ), it was found that the number of SARS-CoV-2-D614Gdel21-GFP pseudovirus positive fluorescent cells packaged by prVSV-△G-GFP MOI=0.02 was the most, so the prVSV-△G-GFP infection MOI value in the packaging system of the new crown pseudovirus VSV system was determined to be 0.02.

[0108] Example 5. Optimization of the pseudovirus packaging process

[0109] 1. Exploration of target cells

[0110] In order to explore the influence of SARS-CoV-2-Spike pseudovirus on target cells in neutralization experiments, this embodiment carries out neutralization experiments according to the method described in Example 3, and compares the effects of target cells expressing different receptor proteins. Specifically as follows:

[0111] After transfecting 293T cells with SARS-CoV-2-D614G and SARS-CoV-2-D614Gdel21 membrane plasmids, prVSV-△G-GFP was infected for 1h, and then the medium was replaced with 2% DMEM medium, and cultured at 37°C in a 5% CO2 incubator for 24h to obtain SARS-CoV-2-D614Gdel21-GFP and SARS-CoV-2-D614G-GFP pseudovirus. We further studied the infectivity of fluorescent pseudovirus (SARS-CoV-2-D614Gdel21-GFP, SARS-CoV-2-D614G-GFP) in different target cells.

[0112] In 293T cells, angiotensin-converting enzyme 2 (ACE2) (NM_021804.2), furin (NM_001289823.1), transmembrane serine protease 2 (TMPRSS2) (NM_001135099.1), and cathepsin L (NM_001912.5) empty plasmid pcDNA3.1 were transiently transfected, and the titration results of SARS-CoV-2-D614G-GFP fluorescent pseudovirus in the above cells showed that the number of positive cells transiently transfected with ACE2 receptor was the most, followed by furin (Furin) Figure 4 ).

[0113] Subsequently, we transiently transfected 293T cells with the ACE2 receptor, followed by transient transfection with several other proteases and empty vector plasmids. The 293T cells transfected with both the ACE2 receptor and the Furin receptor showed the highest number of GFP-positive cells. Figure 5 Therefore, we stably transfected ACE2 and Furin receptors into 293T cells to obtain 293T-ACE2-Furin, which we named AF cells. Comparing AF cells with previously reported novel coronavirus-sensitive cells, we found that the GFP fluorescence signal was strongest in AF cells. Figure 6 The results indicate that AF cells stably transfected with ACE2 and Furin receptors on 293T cells are sensitive cells and are more suitable for screening and detection of neutralizing antibodies.

[0114] 2. Exploration of infection time

[0115] To investigate the effect of SARS-CoV-2-Spike pseudovirus infection time on target cells in a neutralization experiment, this embodiment conducted a neutralization experiment according to the method described in Embodiment 3, and compared the effects of different infection times. Details are as follows:

[0116] To determine the optimal detection time, we measured the number of GFP-positive cells at 0h, 8h, and every 12h after incubation of the fluorescent pseudovirus SARS-CoV-2-S-GFP and cells at 37°C in a titration assay. Figure 7 In the neutralization test, three representative samples were monitored: monoclonal antibody AM180, positive serum after human heterologous immunization with COVID-19 vaccine, and guinea pig D614G protein serum after three doses of immunization. ID was detected at different time points. 50 value( Figure 8 The results showed that the number of GFP-positive cells increased rapidly from 8 to 24 hours post-infection, reached a plateau within 36 hours, and remained stable between 24 and 36 hours. Neutralization assays at different time points showed the sample ID at 24-36 hours. 50 The values ​​are relatively stable. Since pseudoviruses cannot produce progeny viruses, the number of infected cells is a good indicator of infection efficiency. Therefore, by counting the number of GFP-positive cells after 24 hours, the infection and neutralization efficiency of pseudoviruses can be quantified.

[0117] 3. Exploration of the amount of cells and viruses added in the neutralization test in 384-well plates

[0118] To investigate the effects of cell and virus addition amounts in the neutralization assay of the SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus, this embodiment conducted a neutralization experiment according to the method described in Embodiment 3 and made comparisons. Details are as follows:

[0119] We optimized the cell seeding amount for SARS-CoV-2-D614Gdel21-GFP titration and neutralization assay, and selected three representative SARS-CoV-2 positive samples: monoclonal antibody AM180, post-vaccination human heterologous immune positive serum, and guinea pig D614G protein three-needle immune serum. To determine the optimal cell seeding amount, the cell seeding amount was set to 1.00x10 3 -1.6x10 4 cells / well, and the titration results showed that the number of GFP positive cells was the largest when the cell seeding amount was 2.00x10 3 cells / well. Figure 9 The pseudovirus neutralization assay was performed at a virus addition amount of MOI=0.2, and the fitting four-parameter curve of the sample at different dilutions (log10) and inhibition rate was calculated Figure 10 , R 2 all greater than 0.9, indicating good curve fitting. When the cell seeding amount was 2.00x10 3 to 4.00x10 3 cells / well, the detection value ID 50 of the three samples was the highest, and according to the above results, 2.00x10 3 to 4.00x10 3 cells / well was finally selected as the optimal cell seeding amount.

[0120] To determine the optimal virus addition amount, we set the MOI range of virus addition to 0.025-0.8, and kept the cell seeding amount at 2.00x10 3 cells / well for the pseudovirus neutralization assay. Similarly, the fitting four-parameter curve of the sample at different dilutions (log10) and inhibition rate was calculated Figure 11 , R 2 all greater than 0.9, indicating good curve fitting. When the virus MOI=0.2, the sample ID 50 value tended to be stable, and at MOI=0.2, the ID 50 value of the three samples was the highest. When the MOI was higher or lower than the above range, the ID 50 value showed a downward trend, so the virus addition amount was determined as MOI=0.2, and the number of GFP positive cells was controlled at about 400.

[0121] 4. Optimization of antibody incubation time

[0122] To determine the incubation time of the sample and the pseudovirus in the neutralization assay, the incubation time was set to 0h, 0.5h, 1h, 2h, and 4h, and the effect of different incubation times on the results of the pseudovirus neutralization assay was detected Figure 12). The results showed that when the incubation time was 0.5-1h, the detected ID 50 values had no significant difference, and when the incubation time was 2h or more, the ID 50 values decreased accordingly, so we determined that the optimal incubation time range was 0.5-2h.

[0123] Example 6. Method validation

[0124] 1. Sensitivity

[0125] To determine the cutoff value of this neutralization method, 100 samples of human coronavirus-negative serum and 100 samples of guinea pig-negative serum were used, which were initially diluted 2-fold, then serially diluted 2-fold, and the 50% inhibition dilution (ID 50 ( Figure 13 A). The results showed that the confidence interval (mean ± SD) of human-negative serum was 4.49 ± 2.62, and the confidence interval (mean ± SD) of guinea pig-negative serum samples was 10.62 ± 3.87, and the detection limit = mean + 1.96SD, so the detection limit of human serum was 9.61, and the detection limit of guinea pig serum was 18.21. Finally, the cutoff values of human serum samples and mouse serum samples were set to 10 and 20, respectively. Then we selected 4 SARS-positive serum, 4 MERS-positive serum, 4 RSV-positive serum, and 4 SARS-CoV-2-positive serum, and used SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus for neutralization test to determine the ID 50 values (Figure 13B). The results showed that SARS, MERS, and RSV-positive serum could not neutralize SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus, and only SARS-CoV-2-positive serum had good inhibitory effect on SARS-CoV-2-D614Gdel21-GFP fluorescent pseudovirus, so the method had good specificity.

[0126] 2. Reproducibility

[0127] We selected 20 guinea pig serum samples immunized with spike protein for three injections, and used D614G pseudovirus to detect neutralizing antibody titers ID 50 , which were detected repeatedly 3 times at different times, and each experiment was repeated 2 times for each sample ( Figure 13 C). The intra-assay CV values were between 0.04% and 13.2%, and the inter-assay CV values were between 0.7% and 22.4%, showing that the detection method had good reproducibility.

[0128] 3. Accuracy

[0129] To determine the accuracy of the experiment, we performed neutralization test blank and positive sample spiking recovery test, i.e. we selected one each of guinea pig negative serum sample and guinea pig positive serum sample, and spiked the samples with monoclonal antibody AM180. We determined the ID 50 values of guinea pig negative / positive serum samples, monoclonal antibody AM180, guinea pig negative serum sample + monoclonal antibody AM180 (1:10), and guinea pig positive serum sample + monoclonal antibody AM180 (1:10). The blank spiking recovery rate was calculated to be 90.47%, and the guinea pig serum positive sample spiking recovery rate was 91.64%

[0130] We compared the fluorescent SARS-CoV-2-S-GFP pseudovirus neutralization test method established in this study with the existing chemical SARS-CoV-2-S-Fluc pseudovirus neutralization test method in the laboratory. We selected 50 guinea pig positive serum samples, 10 guinea pig negative serum samples, and international standard (NIBSC code: 20 / 136, 1000iu / ml) as test samples, which were immunized with D614G and VOC (Alpha, Beta, Gamma, Delta, Omicron) Spike-Trimer as immunogens and blood was taken 14 days after three needle immunization. The fluorescent 384 system SARS-CoV-2-D614Gdel21-GFP pseudovirus and the 96-well plate chemiluminescence system SARS-CoV-2-D614Gdel21-Fluc pseudovirus were measured by the above two neutralization test methods. The average ID 50 values of the international standard measured by three repeated tests were 699 and 1931, respectively. The calculated median of the international standard in the fluorescent 384 system pseudovirus neutralization test was 365iu. Linear regression analysis was performed on the remaining 60 samples Figure 14 ), and the linear equation was y = 0.3986x + 12.988, R2 = 0.9903 (X axis is the chemiluminescence system ID 50 value, Y axis is the fluorescent system ID 50 value), indicating that the fluorescent 384-well plate SARS-CoV-2-S-GFP pseudovirus neutralization test method established in this study has good correlation with the existing chemical SARS-CoV-2-S-Fluc pseudovirus neutralization test method in the laboratory.

[0131] Example 7. Comparison of the traditional 96-well plate test method and the new 384-well plate test method

[0132] Currently, the gold standard for SARS-CoV-2 neutralizing antibodies is PRNT assay, which requires the use of isolated live virus and must be operated under a biosafety level 3, and the test cycle is long, high risk, which greatly limits the progress of vaccine, drug and antibody research. The SARS-CoV-2-Sdel21-GFP pseudovirus 384-well plate neutralization test method based on VSV system constructed in the application has high safety factor, which saves the operation of additional substrate during detection, and the fluorescence detection instrument Biotek is equipped with mechanical arm, which can realize automatic detection and is more suitable for high-throughput detection.

[0133] The application tests 1000 samples, compares the traditional 96-well plate chemiluminescence pseudovirus neutralization test method with the new 384-well plate fluorescence pseudovirus neutralization test method, and the comparison results are shown in Table 2.

[0134] Table 2. Comparison of traditional 96-well plate test method and new 384-well plate test method

[0135]

[0136] Note: In the above table, "115" refers to the market price of each 96-well plate, "250" refers to the number of 96-well plates needed to complete the detection of 1000 samples; "40" refers to the market price of each 384-well plate, and "50" refers to the number of 384-well plates needed to complete the detection of 1000 samples.

[0137] In summary, in the method of the application, each 384-well plate can realize the detection of 20 samples, and has two parallel controls, which is 5 times the sample amount of 96-well plate. Since the total volume of the 384-well plate neutralization system is 40ul, if the first hole is diluted by 30 times, the sample only needs 0.33ul, which is 23 times less than the sample usage of 96-well plate. In addition, the 384-well plate can be used with the mechanical arm, and the dilution of the sample, the addition of the pseudovirus and the cells can all be realized by using the mechanical arm, which greatly saves the human resources; in terms of result detection, no exogenous substrate needs to be added, only the number of fluorescent AmCyan positive cells needs to be counted, which greatly saves the cost of test consumables.

[0138] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings of the disclosure, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> China Institute for Food and Drug Control <120> A method for detecting SARS-CoV-2 antibody neutralization activity <130> IDC220168 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1252 <212> PRT <213> artificial <220> <223> Insert of a plasmid <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gin Ser Leu Leu lie Val Asn Asn Ala Thr Asn Val Val lie 115 120 125 Lys Val Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gin Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn lie Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr 195 200 205 Pro lie Asn Leu Val Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gin Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val lie Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gin Gly Val Asn Cys Thr Glu Val Pro Val Ala lie 610 615 620 His Ala Asp Gin Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala 660 665 670 Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie 705 710 715 720 Ser Val Thr Thr Glu lie Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr 755 760 765 Gly lie Ala Val Glu Gin Asp Lys Asn Thr Gin Glu Val Phe Ala Gin 770 775 780 Val Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe lie Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp 835 840 845 Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie 885 890 895 Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Gin Asn Gin Lys Leu lie Ala Asn Gin Phe Asn 915 920 925 Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gin Asp Val Val Asn Gin Asn Ala Gin Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Val 965 970 975 Leu Asn Asp lie Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gin 980 985 990 Ile Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Val 995 1000 1005 Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gin Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu 1205 1210 1215 Gly Phe lie Ala Gly Leu lie Ala lie Val Met Val Thr lie Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser 1250

Claims

1. A method for preparing SARS-CoV-2 virosomes, the method comprising: (i) infecting a first host cell with vTF7-3 vaccinia virus, and then transfecting the first host cell with a prVSVAG-GFP backbone plasmid and helper plasmids pBS-N, pBS-P, pBS-G and pBS-L; (ii) harvesting VSV virosomes obtained in step (i) ; (iii) transfecting a second host cell with a membrane plasmid obtained by inserting an amino acid sequence as shown in SEQ ID NO: 1 into a pcDNA3.1 vector and codon-optimizing according to the host cell, and then infecting the second host cell with VSV virosomes at an MOI value of 0.02 to 0.04 to obtain SARS-CoV-2 virosomes; wherein the first host cell and the second host cell are each independently selected from human hematopoietic cells, epithelial cells, hepatocytes, tumor cells or neural cells; in step (i), the MOI value of vTF7-3 vaccinia virus is 4 to 6; in step (i), the backbone plasmid and the helper plasmids are transiently transfected or stably transfected into the first host cell; and in step (iii), the membrane plasmid is transiently transfected or stably transfected into the second host cell. 2.A SARS-CoV-2 virosome prepared by the method of claim 1. 3.A method for screening an anti-SARS-CoV-2 antibody or detecting neutralization activity of an anti-SARS-CoV-2 antibody, the method comprising contacting a host cell with the antibody before, simultaneously with or after contacting the host cell with the SARS-CoV-2 virosome of claim 2; wherein the method is performed by the following steps (a) to (d) : step (a) : performing gradient dilution of the antibody in a 96-well plate, and then transferring the antibody to a 384-well plate, 10 μl of the antibody being added to each well; step (b) : diluting the SARS-CoV-2 virosome of claim 2 to about 400 GFP-positive particles per well with DMEM complete medium, 10 μl of the virosome being added to each well, and incubating for 1 h; Step (c): The cells expressing ACE2 and Furin were adjusted to a concentration of 4 x 10 5 cells / ml suspension, 20 μl of said cells were added to each well, i.e. 2 x 10 3 cells were added to each well, and incubated for 24 h; Step (d): Count the number of GFP positive cells on the immunoplate reader Biotek and calculate the ID using the Reed-Muench method 50 values to assess the neutralizing antibody level of the sample.

4. The method of claim 3, wherein, in step (b), the SARS-CoV-2 virosome of claim 2 is diluted to about 400 GFP-positive particles per well with DMEM complete medium, 10 μl of the virosome is added to each well, and the 384-well plate is incubated in a 37℃ 5% CO 2 incubator for 1 h.

5. The method of claim 3, wherein, In step (c) the cells expressing ACE2 and Furin were adjusted to a concentration of 4 x 10 5 cells / ml suspension and 20 μl of said cells were added to each well, i.e. 2 x 10 3 The 384 well plate was incubated for 24 h in a 37°C 5% C02 incubator. 6.A method for screening a candidate drug capable of inhibiting SARS-CoV-2 infection of cells, the method comprising contacting the virosome or the host cell with the candidate drug before, simultaneously with or after contacting the SARS-CoV-2 virosome of claim 2 with the host cell; and the method is not for therapeutic purposes. 7.The method of claim 6, wherein the host cell is an ACE2 and Furin-expressing cell.

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