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Dec 2020
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Production of Recombinant Replication-defective Lentiviruses Bearing the SARS-CoV or SARS-CoV-2 Attachment Spike Glycoprotein and Their Application in Receptor Tropism and Neutralisation Assays
携带 SARS-CoV 或 SARS-CoV-2 附着尖峰糖蛋白的重组复制缺陷慢病毒的生产及其在受体向性和中和试验中的应用   

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Abstract

For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus, biosafety precautions and associated trained staff. Here, we present a protocol based on pseudotyping to produce recombinant replication-defective lentiviruses bearing the SARS-CoV or SARS-CoV-2 attachment Spike glycoprotein, allowing the investigation of viral entry in a lower-containment facility. Pseudoparticles are produced by cells transiently transfected with plasmids encoding retroviral RNA packaging signals and Gag-Pol proteins, for the reconstitution of lentiviral particles, and a plasmid coding for the viral attachment protein of interest. This approach allows the investigation of different aspects of viral entry, such as the identification of receptor tropism, the prediction of virus host range, and zoonotic transmission potential, as well as the characterisation of antibodies (sera or monoclonal antibodies) and pharmacological inhibitors that can block entry.


Graphic abstract:

SARS-CoV and SARS-CoV-2 pseudoparticle generation and applications.


Keywords: SARS-CoV-2 (SARS-CoV-2), SARS-CoV ( SARS-CoV), Pseudotyped virus (假型病毒), Tropism ( 趋性), Neutralisation (中和作用)

Background

Pseudoparticles are replication-defective viral particles obtained through expression of viral envelope glycoproteins on the surface of a recombinant virus, which provides the core components of the particle. Vesicular stomatitis virus (VSV), a rhabdovirus, and two lentiviruses – human immunodeficiency virus-1 (HIV-1) and murine leukaemia virus (MuLV) – are commonly used as viral vectors for pseudotyping (Takada et al., 1997; Wool-Lewis and Bates, 1998; Sharkey et al., 2001; Negrete et al., 2005; Grehan et al., 2015; Thakur et al., 2021). In our recent study, we successfully used a lentiviral-based system to study the interaction of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 Spike (S) protein with its cellular receptor, angiotensin converting enzyme 2 (ACE2) (Conceicao et al., 2020).


SARS-CoV-2, the etiological agent of the ongoing COVID-19 pandemic, is a highly pathogenic betacoronavirus that requires handling at BSL-3 facilities, which are not always available in research laboratories. To allow work with SARS-CoV and SARS-CoV-2 at lower containment, the generation of viral particles pseudotyped with the Spike protein represents a safe and appealing surrogate. This technique allows (i) dissection of viral entry pathways, (ii) investigation of host cell susceptibility and tropism of the angiotensin converting enzyme 2 (ACE2) receptor, (iii) examination of inter-species transmission, (iv) assessment of the neutralising antibody responses in immunogenicity and sero-epidemiological studies, and (v) efficacy assessment of small-molecule inhibitors that block viral entry. Notably, this technique has been applied to viral glycoproteins from a wide variety of viruses such as influenza hemagglutinin (Bertram et al., 2010), Nipah virus fusion and attachment proteins (Thakur et al., 2021), Ebola virus glycoprotein (Simmons et al., 2003), Chikungunya virus E1 (Salvador et al., 2009), hepatitis C virus E2 proteins (Hsu et al., 2003), and VSV glycoprotein (DePolo et al., 2000).


To generate lentiviral-based pseudoparticles of HIV-1, cells are co-transfected with the following plasmids: (i) HIV-1 packaging plasmid encoding for the core genes Gag and Pol, (ii) the transfer plasmid that encodes a firefly luciferase reporter gene flanked by HIV-1 regulatory LTR regions and the packaging signal, and (iii) a third plasmid encoding for the heterologous viral glycoprotein. Pseudoparticles possessing the viral glycoprotein of interest on their surface are assembled at the cellular membrane, from which they bud (Zufferey et al., 1997). Upon infection, the luciferase gene encoded by the lentivirus genome is expressed, allowing accurate quantification of viral entry.

Materials and Reagents

  1. 50 ml Falcon tubes (VWR International, catalog number: 734-0448)

  2. Clear bottom 6-well tissue-culture treated plate (Scientific Laboratory Supplies, FalconTM, catalog number: 353046)

  3. Pipette tips (STARLAB, catalog numbers: S1110-3700 [10/20 µl XL Graduated TipOne®]; S1111-1206-C [200 µl Yellow Bevelled TipOne® Tip]; S1112-17200 [1,250 µl XL Graduated TipOne®])

  4. Serological pipettes (Corning, catalog numbers: 4101 [10 ml StripetteTM]; 4051 [5 ml StripetteTM]; 4251 [25 ml StripetteTM])

  5. Opti-MEMTM (Thermo Scientific, GibcoTM, catalog number: 11058021, storage conditions: 4°C, shelf life: 12 months)

  6. Disposable weighing boats 85 × 85 × 24 mm, PS, medium, white, anti-static (VWR, catalog number: 10770-448, storage conditions: room temperature)

  7. 7 ml polycarbonate polypropylene screw cap bijous (container for storage of small-volume samples) (STARLAB, catalog number: E1412-0710)

  8. 96-well Delta-treated (hydrophilic surface that promotes cell attachment and growth) White flat-bottom plate (Fisher Scientific, Nunc, MicroWell, catalog number: 10182831)

  9. Tissue culture flasks (Greiner Bio-One, catalog numbers: 660160 [175 cm2], 658170 [75cm2])

  10. 1.5 ml Microcentrifuge sterile Eppendorf tubes (STARLAB, TubeOne®, catalog number: S1615-5510)

  11. Millex-GP syringe filter unit, 0.22 µm filter, polythersulfone, 33 mm, gamma sterilised (Merck, Millipore, catalog number: SLGP033RS, storage conditions: room temperature)

  12. Human Embryonic Kidney 293T, HEK293T cells (ATCC®, catalog number: CRL-3216TM, storage conditions: liquid nitrogen vapour phase)

  13. Baby Hamster Kidney-21, BHK-21 cells (ATCC®, catalog number: CCL-10TM, storage conditions: liquid nitrogen vapour phase)

  14. Plasmid DNA: ACE2 receptors (pDISPLAY expression vector, codon-optimised, N-terminal signal peptide [the murine Ig κ-chain leader sequence], C-terminal HA-tag), SARS-CoV Spike, SARS-CoV-2 Spike (pcDNA3.1(+), codon-optimised, C-terminal FLAG-tag) (BioBasic, Canada [Conceicao et al., 2020]), p8.91, CSFLW, VSV-G (pcDNA3.1(+) expression vector) (available upon request), pcDNA3.1(+) (Thermo Scientific, Invitrogen, catalog number: V79020) and pDISPLAYTM (Thermo Scientifc, InvitrogenTM, catalog number: V66020)

  15. Sera or antibodies for neutralisation assays, with relevant biological risk assessment and ethical approvals in place

  16. Dulbecco’s modified Eagle’s medium, DMEM (Merck, Sigma-Aldrich, catalog numbers: D5796 [with phenol red]; D1145 [phenol red free], storage conditions: 4°C, 12 months)

  17. Foetal bovine serum, FBS (Life Science Production, catalog number: S-001A-BR, -20°C)

  18. Penicilin-Streptomycin, 10,000 U/ml (Thermo Scientific, GibcoTM, catalog number: 15240122, storage conditions: -20°C, shelf life: 12 months)

  19. Sodium pyruvate, 100 mM (Thermo Scientific, GibcoTM, catalog number: 11360070, storage conditions: 4°C, shelf life: 12 months)

  20. EDTA (0.5 M), pH 8.0, RNase-free (Thermo Scientific, Ambion®, catalog number: AM9269G)

  21. 1× Trypsin-EDTA, 0.25%, phenol red (Thermo Scientific, GibcoTM, catalog number: 2520072, storage conditions: -20°C long-term, 4°C while in use, shelf life: 24 months)

  22. TransIT-X2® Dynamic Delivery System (Mirus, catalog number: MIR 6000, storage conditions: -20°C, shelf life: 12 months)

  23. Polyethyleneimine, PEI (Merck, Sigma-Aldrich, catalog number: 408727, storage conditions: 4°C)

  24. Nuclease-free, autoclaved, 0.2 µm filtered DEPC-treated water (Ambion, catalog number: AM9906, storage conditions: room temperature)

  25. Hydrochloric acid 36.5-38.0%, Bioreagent, for molecular biology (Sigma-Aldrich, catalog number: H1758-100 ml, storage conditions: room temperature)

  26. Bright-GloTM Luciferase Assay System (Promega, catalog number: E2650, storage conditions: -20°C)

  27. 55 ml StarTub PVC reagent reservoirs (STARLAB, sterile individually wrapped, catalog number: E2310-1010)

  28. DMEM-10% (see Recipes)

  29. Working solution of 1 mg/ml PEI (see Recipes)

Equipment

  1. Microbiological safety cabinet, BSL-2 (CAS, Biomat 2 – class 2 complies with BS EN 12469:2000)

  2. CO2 incubator (PHC Europe B.V., PHCbi, catalog number: MCO-170AICD-PE)

  3. -86°C ultra-low temperature freezer (PHCbi, Panasonic, vip plus, model: MDF-DU900V)

  4. -20°C Medical freezer with 14 storage drawers (Liebherr, Profiline, model: G5216)

  5. 4°C refrigerator (VDW CoolSystems, Labcold, Sparkfree, model: RLV0217)

  6. Sub aqua 5 plus water bath (Fisher Scientific, Grant, model: 13251183)

  7. Automated pipettor for serological pipettes (Fisher Scientific, Thermo ScientificTM, S1 Pipet Fillers, catalog number: 10072332)

  8. Single-channel pipettes (Gilson, Pipetman L, catalog numbers: FA1001M [P2L 0.2-2 µl], FA1003M [P20L 2-20 µl]; FA1005M [P200L 20-200 µl]; FA1006M [P1000L 100-1,000 µl])

  9. Multi-channel pipettes (Thermo ScientificTM, FinnpipetteTM F2 multichannel pipette, catalog numbers: 4662010 [8-well 5-50 µl]; 4662070 [12-well 30-300 µl])

  10. Inverted microscope for cell culture (Leica microsystems, model: DMi1-S 40/0.45)

  11. Haemocytometer (Fisher Scientific, HirschmannTM Bright Lined Counting Chambers, catalog number: 105289616)

  12. Centrifuge machine (Kendo laboratory product, Sorvall Legend RT, EASYset, model: 75004373)

  13. Benchtop autoclave (Fisher Scientific, Astell scientific, catalog number: 12755375)

  14. GloMax® Discover Microplate Reader (Promega, catalog number: GM3000)

Software

  1. Microsoft Excel (Microsoft 365 for Windows, www.microsoft.com)

  2. GraphPad Prism (Version 8.4.2, GraphPad Software for Windows, San Diego, California USA, www.graphpad.com)

  3. GloMax® Discover System Software (Version 3.2.3, Promega, Southampton, United Kingdom www.promega.co.uk)

Procedure

  1. Generation of SARS-CoV-2 and SARS-CoV pseudotyped virus particles

    1. Maintain HEK293T cells for pseudoparticle production in 25 ml of DMEM-10% (see Recipes) in a 75 cm2 tissue culture flask.

    2. Seed HEK293T cells at a concentration of 7.5 × 105 cells per well in a 6-well plate in 3 ml of DMEM-10%, for the total number of wells required.

    3. Agitate cells in the plate using a rapid up-down, left-right movement. This will ensure cells are evenly distributed and do not clump. Incubate at 37°C, 5% CO2 overnight.

    4. The next day, set up transfection mixes in the afternoon. The seeded HEK293T cells should be between 60-80% confluent for optimal transfection efficiency. Set up transfections for the SARS-CoV-2 S or SARS-CoV S plasmid, alongside an empty vector negative control (no glycoprotein, no GP) and a positive control. For instance, if the SARS-CoV-2 S and SARS-CoV S plasmids are in a pcDNA3.1 backbone, use an empty pcDNA3.1 plasmid as your no GP control. Generally, a VSV-G plasmid is used as a positive control, as it is trans-encapsidated into the HIV-1 particle efficiently (i.e., it pseudotypes well).

    5. In a sterile 1.5 ml Eppendorf tube, add 100 µl of Opti-MEM along with 0.6 µg of p8.91 plasmid (encoding for HIV-1 gag-pol), 0.6 µg of CSFLW plasmid (lentivirus backbone expressing Firefly luciferase), and 0.5 µg of glycoprotein (SARS-CoV-2 S, SARS-CoV S or VSV-G) or empty vector (pcDNA3.1) per well. Incubate for 5 min at room temperature.

    6. In a separate 1.5 ml Eppendorf tube, add 100 µl of Opti-MEM plus and 10 µl of PEI (1 µg/ml) per transfection and incubate for 5 min at room temperature.

    7. For each 100 µl transfection mix of DNA in Opti-MEM, add 100 µl of PEI in Opti-MEM and mix vigorously with a pipette ten times. Incubate at room temperature for 20 min.

    8. Add 200 µl of the volume of the transfection mix in a dropwise manner to each well of the 6-well plate and incubate overnight at 37°C, 5% CO2.

    9. The next morning, use a serological pipette to gently remove the media from wells containing the transfection mix by tilting the dish towards you and aspirating from the edge of the well, being careful not to disturb the monolayer. Replace with 3 ml of DMEM-10%. Incubate overnight at 37°C, 5% CO2 for 24 h.

    10. Harvest cell supernatants containing pseudotyped virus particles and transfer to a 50 ml Falcon, pooling similarly transfected wells, and store at 4°C. Replace the media with 3 ml of DMEM-10% per well, and incubate at 37°C, 5% CO2 for 24 h.

    11. Harvest the cell supernatants containing pseudotyped virus particles and pool with pseudoparticles harvested the day before. Centrifuge at 2,500 × g for 10 min at 4°C to remove cellular debris.

    12. Aliquot 4-5 ml of pseudoparticles into bijous and freeze at -80°C until further use.

    NB: Larger volume of pseudoparticles can also be prepared in 10 cm2 culture dishes. The necessary cell seeding density, DNA concentrations and volumes required for this setup can be found in Table 1, with the corresponding values for the 6-well plate format noted alongside. Steps A9-A12 remain the same regardless of the dish size used, changing only the volume of media required.


    Table 1.Quick-guide to generating lentiviral-based pseudotyped viruses


  2. Testing SARS-CoV-2 and SARS-CoV pseudoparticle infectivity

    1. Seed HEK293T cells at a density of 7.5 × 105 per well in a 6-well plate in a total of 3 ml of DMEM-10%. Incubate overnight at 37°C, 5% CO2.

    2. Ensure plated cells are at 60-80% confluency to ensure optimal transfection efficiency. Set up transfection mixes to test pre-generated SARS-CoV-2 pseudoparticles. In a sterile 1.5 ml Eppendorf tube, add 200 µl of Opti-MEM along with 500 ng of human ACE2 plasmid per well to be transfected. Bring the TranIT-X2 transfection reagent to room temperature before use, add 2 µl (for every 1 µg of DNA) directly to the tube, and gently flick the tube to mix. Incubate at room temperature for 20 min.

    3. Add 200 µl of the transfection mix dropwise to each well of the pre-plated cells and incubate overnight at 37°C, 5% CO2.

    4. Remove the media containing the transfection mix from the wells by tilting the dish towards you and aspirating from the edge of the well using a serological pipette, being careful not to disturb the monolayer. Add 1 ml of DMEM-10% per well and harvest the transfected cells. HEK293T cells have low adherence and come off the plate easily. As such, using the force of the pipetted liquid is sufficient to harvest cells, although care should be taken to ensure a single cell suspension is achieved without clumps. Trypsin should be avoided as this will unnecessarily cleave off the receptors, hampering future experimentation. Transfer to a 50 ml Falcon and dilute cells to 2 × 105/ml with DMEM-10%.

    5. Seed 100 µl of diluted cells (2 × 104 per well) into a flat, white-bottomed 96-well plate and incubate overnight at 37°C, 5% CO2.

    6. The next day, thaw an aliquot of SARS-CoV-2 and/or SARS-CoV pseudoparticles, along with the negative (pcDNA3.1, no GP) and positive (VSV-G) controls. Titrate the pseudoparticles in a clear-bottomed 96-well plate, starting with undiluted virus in the top row, and titrating 10-fold in DMEM-10%, for a final volume of 100 µl.

    7. Gently remove the media from the white-plate seeded with human ACE2-transfected cells and add 100 µl titrated pseudoparticles. Incubate for 48 h at 37°C, 5% CO2.

    8. Remove the media from the wells by tilting the dish towards you and aspirating from the edge of the well using a multi-channel pipette and add 50 µl Bright-GloTM diluted 1:1 with serum free, phenol red free DMEM. Incubate the plate in the dark for 5 min and then measure the luciferase signal on a GloMax Multi+ Detection System under the luminescence protocol with 0.5 s integration.

    9. Export the CSV file generated on a USB flash drive for analysis using Microsoft Excel and plot data on GraphPad Prism.


  3. ACE2 receptor usage screen using SARS-CoV-2 and SARS-CoV pseudotyped virus particles (Conceicao et al., 2020)

    1. Maintain BHK-21 cells in 25 ml DMEM-10% in a 75 cm2 tissue culture flask. Seed BHK-21 cells in 24-well plates at 1 × 105/well in DMEM-10%. Incubate overnight at 37°C, 5% CO2.

    2. Ensure plated cells are at 60-80% confluency to ensure optimal transfection efficiency. Set up transfection mixes in 100 µl of Opti-MEM along with 500ng of different species of ACE2-expressing constructs or an empty vector control (e.g., pDISPLAY). Bring the TranIT-X2 transfection reagent to room temperature before use and add 3 µl for every 1 µg of DNA directly to the tube and gently flick the tube to mix. Incubate at room temperature for 20 min.

    3. Add 100 µl of the transfection mix dropwise to each well of the pre-plated BHK-21 cells and incubate overnight at 37°C, 5% CO2.

    4. Remove the media containing the transfection mix from the wells and add 0.5 ml of 2 mM EDTA in PBS per well to harvest the transfected cells. Transfer to a bijou and dilute cells to 2 × 105/ml with DMEM-10%.

    5. Seed 100 µl of diluted cells (2 × 104 per well) into a flat, white-bottomed 96-well plate and incubate overnight at 37°C, 5% CO2.

    6. Remove media from cells and infect with SARS-CoV-2 or SARS-CoV pseudoparticles equivalent to 106-107 relative light units (RLU), or the no GP control at the same dilution and incubate for 48 h at 37°C, 5% CO2.

    7. Remove the media from the wells and add 50 µl of Bright-GloTM diluted 1:1 with serum free, phenol red free DMEM. Incubate the plate in the dark for 5 min then read on a GloMax Multi+ Detection System under the luminescence protocol with 0.5 s integration.

    8. Export the CSV file generated on a USB flash drive for analysis using Microsoft Excel and plot data on GraphPad Prism.


  4. Neutralisation assay using SARS-CoV-2 and SARS-CoV pseudotyped virus particles

    1. Prior to setting up neutralization assays, seed HEK293T cells at a density of 7.5 × 105 per well in a 6-well plate in a total of 3 ml of DMEM-10%. Incubate overnight at 37°C, 5% CO2.

    2. Ensure plated cells are at 60-80% confluency to ensure optimal transfection efficiency. In a sterile 1.5 ml Eppendorf tube, add 200 µl of Opti-MEM along with 500 ng of human ACE2 plasmid per well to be transfected. Bring the TranIT-X2 transfection reagent to room temperature before use and add 2 µl for every 1 µg of DNA directly to the tube and gently flick the tube to mix. Incubate at room temperature for 20 min.

    3. Add 200 µl of the transfection mix dropwise per well of pre-plated cells and incubate overnight at 37°C, 5% CO2.

    4. Set up neutralisation assays by diluting sera/monoclonal antibodies (mAbs)/inhibitors considering the dilution series to be used and the final volume after addition of pseudoparticles. For example, sera to be titrated using a 2-fold dilution series starting at a 1:10 dilution would require 10 µl sera per well in 100 µl serum free DMEM. The same is applicable for mAbs or inhibitors with a known concentration.

    5. Add 100 µl of diluted sera/mAbs/inhibitors in triplicate to the top row of a flat white-bottomed 96-well plate. Add 50 µl of serum free media to all remaining wells. Remove 50 µl from the top row and titrate 2-fold down the plate, mixing well before each titration. Do not titrate into the bottom row. This whole row will be used as the untreated control.

    6. Thaw an aliquot of SARS-CoV or SARS-CoV-2 pseudoparticles and dilute in serum free DMEM, equivalent to ~106 RLU and add 50 µl per well, including the untreated controls. Incubate for 1 h at 37°C, 5% CO2.

    7. Remove the media from the 6-well plates transfected with human ACE2. Add 1 ml of DMEM-10% per well and harvest the transfected cells. HEK293T cells have low adherence, so come off the plate easily; therefore, the force of the pipetted liquid should be sufficient to harvest cells (see B4 above). Transfer to a 50 ml Falcon and dilute cells to 2 × 105/ml with DMEM-10%.

    8. Seed 100 µl of diluted cells (2 × 104 per well) onto each well containing sera/mAb/inhibitor with pseudoparticles and the untreated controls. Incubate for 48 h at 37°C, 5% CO2.

    9. Remove the media from the wells and add 50 µl of Bright-GloTM diluted 1:1 with serum free, phenol red free DMEM. Incubate the plate in the dark for 5 min then read on a GloMax Multi+ Detection System under the luminescence protocol with 0.5 s integration.

    10. Export the CSV file generated on a USB flash drive for analysis using Microsoft Excel and plot data on GraphPad Prism.

Data analysis

  1. Testing SARS-CoV-2 and SARS-CoV pseudoparticle infectivity

    1. After preparing a batch of pseudoparticles, their infectivity can be tested by titrating them on target cells that have been transfected to express the host receptor (ACE2) of the pseudotyped attachment protein (Spike) for SARS-CoV and SARS-CoV-2. Undiluted pseudotyped virus (“1”) is titrated 10-fold with DMEM-10% down a 96-well plate in triplicate (“10”, “100”, “1,000” etc.) (Figure 1A).

    2. Measuring the luciferase signal of the pseudoparticles will generate a CSV file that can be exported onto a USB flash drive and analysed on Microsoft Excel. These results can then be plotted on GraphPad Prism to show the mean ± SD for each pseudoparticle.

    3. The no GP negative control serves as an indication of background luciferase signal, and only values above this at each corresponding dilution should be considered as a true luciferase signal for the pseudoparticles being tested (Figure 1B, black line). Generally, a minimum of ~2 log dynamic range between the no GP and the pseudotyped virus and a RLU signal between 105.5 and 107.5 RLU (Figure 1B, shaded area) is sufficient for use in subsequent assays. The titration series will also help to determine the lowest usable dilution of the pseudoparticles to still obtain meaningful luciferase values above the background. Of note: the following data to be discussed was generated for illustrative purposes only.

    4. For example, when considering the luciferase values obtained for SARS-CoV, although these are above the no GP control at the highest dilution, the difference between the two is only ~1 log, which falls outside our criteria for use (Figure 1B, blue line). When titrating the pseudoparticles, the luciferase values also fall quite quickly below the lower limit of the workable range at a 1:10 dilution and to the same values as the no GP control by the 1:1000 dilution, making the titre of this preparation of SARS-CoV pseudoparticles unsuitable for use in subsequent assays (Figure 1B).

    5. In comparison, the luciferase signals obtained for SARS-CoV-2 are ~2 log above the background no GP control, and a dilution of 1:10 of the pseudoparticles would be within the workable range for use in subsequent assays, which is lost at a 1:100 dilution (Figure 1B, orange line).

    6. The VSV-G pseudoparticles are a positive control within the assay, where the luciferase values observed should be above 107 RLU (Figure 1B, green line).



      Figure 1. Testing the infectivity of SARS-CoV and SARS-CoV-2 pseudoparticles. (A) For SARS-CoV and SARS-CoV-2 pseudoparticle titrations, 10-fold serial dilutions of the supernatant are used to infect HEK293T cells transiently expressing the human ACE2 receptor in a white flat-bottomed 96 well-plate. Negative (no GP) and positive (VSV-G) controls are also included in the experiment. Each condition is tested in triplicate. (B) Two days after infection, signal luciferase values are measured and plotted as the mean ± SD. The no GP control is indicative of the background, and only pseudoparticle values above this should be considered as true infectivity, matched at each dilution of the virus. The use of pseudoparticles for subsequent neutralisation assays and receptor usage screens should show ~2 log dynamic range between the no GP and pseudotyped virus and fall between a working range of 105.5 to 107.5 (shaded area).


  2. ACE2 receptor usage screen using SARS-CoV-2 and SARS-CoV pseudotyped virus particles

    1. Infection of cells expressing various species ACE2 receptors with SARS-CoV or SARS-CoV-2 pseudoparticles set up in triplicate yields luciferase signals that can be plotted alongside each other (mean ± SD) to depict raw values. These data also give an idea of the general trend of receptor tropism across different viruses (Figure 2A).

    2. For example, water buffalo and goat ACE2 permit the entry of SARS-CoV and SARS-CoV-2 pseudoparticles well, which is less evident for little brown bat ACE2. Differences between viruses can be observed for civet ACE2, which permits the entry of SARS-CoV more efficiently than SARS-CoV-2 (Figure 2A). These experiments should be conducted at least three times on three separate occasions, with representative data shown. A subset of ACE2 receptors are shown in Figure 2A, but a more in-depth, wider analysis can be found in Supplementary Figure 3A and 3C in Conceicao et al. (2020).

    3. Two negative controls are set up in this screen. The first is an empty vector control (pDISPLAY) to ensure any signal measured is solely from overexpression of the ACE2 receptor. The second is infection of cells with the no GP control pseudoparticle preparation to ensure luciferase signals can be attributed to the pseudotyped viruses and provides a baseline for the background (Figure 2A).

    4. The raw luciferase signals can then be used to determine the relative usage of non-cognate host ACE2 receptors (water buffalo, civet, goat, little brown bat) to a known or cognate host receptor, in this case, human ACE2. The mean percentage from three separate experiments performed on different days are used to obtain these values. The luciferase value for human ACE2 is set to 100%, and the luciferase values for unknown host receptors and the negative control are then expressed as a percentage relative to human ACE2.

    5. These results can also be shown as a heatmap using a colour gradient to show different trends of receptor usage. For example, human ACE2 (100%) is set as green. Expression lower than this is shaded from green to red, indicative of poorer ACE2 usage relative to human ACE2. Values above 100% are shown as a darker green, suggestive of ACE2 usage equivalent to or greater than human ACE2 (Figure 2B). A subset of ACE2 receptors are shown in Figure 2B, but a more in-depth, wider analysis can be found in Figure 2 A in Conceicao et al. (2020).

    6. Further analysis can be carried out to compare the receptor tropism of different species ACE2 between SARS-CoV and SARS-CoV-2 by plotting the percentage values for each virus against each other on an xy scatter graph, calculating the Pearson’s correlation coefficient, and plotting a linear line of regression fitted with 95% confidence intervals (data not shown). An example of such analysis can also be found in Supplementary Figure 5 in Conceicao et al. (2020).



      Figure 2. ACE2 receptor usage screen using SARS-CoV and SARS-CoV-2 pseudoparticles. In our study, pseudoparticles were used as a surrogate to live viruses to assess receptor tropism of SARS-CoV and SARS-CoV-2 with different species of ACE2 receptors. Pseudoparticles were employed to assess SARS-CoV and SARS-CoV-2 Spike glycoproteins’ usage of ACE2 receptors from different species and presented as (A) raw luciferase signal values or (B) a percentage relative to human ACE2. Negative controls were included for pseudoparticles bearing an empty vector control (No GP) or mock-transfected with an empty vector in place of an ACE2 receptor (pDISPLAY). Data are presented as mean ± SD of triplicate values, with each experiment performed three times on three separate occasions, and representative data shown.


  3. Neutralisation assay using SARS-CoV-2 and SARS-CoV pseudotyped virus particles

    1. Neutralisation assays using pseudotyped viruses are a low-biocontainment alternative to using live virus and can be performed in a relatively high-throughput manner. These neutralisation assays can be performed on mAbs, sera or any other drug or inhibitor that has the potential to inhibit viral entry. The data discussed herein have been generated for illustrative purposes only.

    2. Inhibitors (mAbs/sera/drugs) of a known concentration can be titrated down a 96-well plate in triplicate to determine the extent of inhibition of SARS-CoV-2 entry. This is done by taking an average of the untreated controls and expressing the RLU values for each individual replicate of the mAb of interest relative to this. These can then be plotted as the mean ± SD and should be repeated a minimum of three times, with representative data shown:


      (RLU individual replicate of mAb/RLU average of untreated) × 100


    3. The inhibitory concentration of 50% (IC50) should be indicated on a graph along with the untreated, no mAb control (100%). Values below this IC50 line are indicative of S-mediated inhibition of entry, which can be calculated at each concentration. For example, mAb2 is able to inhibit SARS-CoV-2 S entry by ~80% (20% of untreated) at 100 µg/ ml. The values obtained for mAb2 at all concentrations tested are below the IC50 value, so lower concentrations would need to be tested to determine the limit of inhibition. In contrast, mAb3 inhibits SARS-CoV-2 S entry by ~90% at 100 µg/ml, but at 12.5 µg/ml, the inhibition is now above the IC50 threshold (Figure 3A).

    4. There may also be examples of mAbs that do not inhibit SARS-CoV-2 S, as with mAb1. It may be possible that when inhibition of entry is not observed, a slight increase above the 100% threshold is seen. The mechanisms causing this increase are still unknown and under investigation, but for the purposes of this assay, the conclusion that the mAb does not neutralise SARS-CoV-2 S is sufficient (Figure 3A). Examples of this sort of analysis can be found in Thakur et al. (2021).

    5. When determining the inhibition of viral entry from individuals who have antibodies against SARS-CoV-2 S, whether that be following natural infection or vaccination, a neutralising antibody titre is usually calculated to enumerate the level of SARS-CoV-2 S neutralisation. The simplest method of calculating this is by calculating the average RLU of the untreated controls and determining the IC50 value, i.e., 50% of the no sera control. The neutralisation titre is then calculated as the inverse of the dilution at which there is 50% inhibition of the no sera luciferase values in all triplicate wells. These titres can then be tabulated or plotted on a log scale.

    6. For example, the titre of 256 for serum sample 1 and 512 for serum sample 3 indicates that IC50 was calculated at a dilution of 1:256 and 1:512, respectively. The conclusion that could be drawn from this is that serum sample 3 is able to neutralise SARS-CoV-2 S-mediated entry more efficiently than serum sample 1 and therefore has higher antibody titres (Figure 3B).

    7. For serum sample 4, this value has been plotted as 1,024, which is the upper limit of detection (ULoD) for this assay. This means that this sample was able to inhibit 50% of the luciferase signal in all wells and at the lowest dilution that was tested. This serum would have to be retitrated with a broader dilution series to determine the neutralisation titre. For serum sample 2, none of the wells in the dilution range yielded a recordable IC50. The neutralisation titre is therefore plotted as an arbitrary value below the lower limit of detection (LLoD), which in this case is 40, but would be reported as <40 as the true titre is unknown (Figure 3B). Examples of IC50 neutralisation titres using this method can be found in Figure 4F in Thakur et al. (2021) and in Figure 2D and 2E in Graham et al. (2020).



    Figure 3. Neutralisation assays using SARS-CoV-2 pseudoparticles. SARS-CoV-2 Spike neutralisation assays were performed in the presence of (A) monoclonal antibodies (mAbs), presented as a percentage relative to untreated controls or (B) sera samples, with data expressed as neutralising titres. SARS-CoV-2 pseudoparticles were incubated with mAbs or sera for 1 h prior to addition of human ACE2-expressing HEK293T cells. Inhibition of SARS-CoV-2 Spike-mediated viral entry was determined by calculating the concentration (mAbs) or dilution (sera) at which there is a 50% reduction in luciferase signal (IC50). Data represent the mean ± SD of triplicate values, with each experiment performed three times on three separate occasions, and representative data shown.

Notes

  1. Procedure A, step 11 mentions centrifugation of pseudoparticle preparations prior to use in subsequent assays to remove cellular debris. Other protocols require further filtration of pseudoparticles using a 0.45 µm filter before storage. This step is not carried out in our lab, as we have observed a reduction in infectivity following filtration.

  2. Manufacturers usually recommend an optimal confluency of 60-80% for transfection. Therefore, it may be necessary to change the seeding density depending on the characteristics of the cells used. For example, if the cell types used are larger (e.g., BHK-21 cells are larger than HEK293T cells) or have a high doubling rate, we recommend starting at a lower seeding density. On the other hand, if the cells are smaller, or have a slower growth rate, and are difficult to reach confluency (e.g., Calu3 cells) or indeed are suspension cells, you may want to start with a higher seeding density. In both instances, we recommend testing different seeding densities to find the optimal for any given experiment.

  3. An example of optimal confluency of HEK293T cells prior to infection (60-80%) is shown in Figure 4. Cells should be evenly distributed across the well (i.e., no clumping or aggregation in one area), with visible gaps in the monolayer (Figure 4A). The cell morphology of the HEK293T cells should appear flat and polygonal at confluence, which indicates adherence to the plastic (Figure 4B).



    Figure 4.Brightfield image to illustrate 60-80% optimal confluency of HEK293T cells. HEK293T cells were seeded at 7.5 × 105 in a 6-well dish in 3ml of DMEM-10%. On the following day, cells should be between 60-80% confluent for optimal transfection. (A) Example of ~80% confluent HEK293T at 4× magnification, with cells appearing evenly distributed across the well and visible gaps. (B) Higher power magnification (10×) of HEK293T cells where cells should appear bright, flat and polygonal prior to transfection.


  4. The concentrations of DNA used for transfection at various steps have been optimised for use in these assays. It is important that any plasmids used are optimised to account for variability in vector platform and codon-optimisation.

  5. A higher signal (in the 104-105 RLU range) in the no GP control can sometimes be seen, likely the result of non-specific uptake of ‘bald’ pseudoparticles or debris from producer cells. To reduce this background signal, care should be taken to ensure producer cells are not transfected at low confluence as this can cause cytopathic effects (CPE) to develop.

  6. All the experiments conducted here were performed with over-expressed ACE2 only. Co-expression of the serine protease TMPRSS2, which is required for S2 protein cleavage to S2’, can facilitate the fusion of viral and cellular membranes and cleavage of the Spike protein (Hoffmann et al., 2020). TMPRSS2 was not included in our host range assays as we wanted to specifically examine the effects of different ACE2s – indeed, over-expression of TMPRSS2 led to ACE2 restrictions being masked (Conceicao et al., 2020).

  7. Other formulae can be used to determine the IC50 value, yielding different titres. This is acceptable if the same method is used throughout analyses and the method used are described in full. Other formulae for calculating neutralisation titres include (1) using a non-linear regression analysis tool on GraphPad Prism after plotting data on an XY graph to interpolate neutralisation values (Ferrara and Temperton, 2018), (2) interpolating the point at which infectivity is reduced to 50% of the value of a no serum control sample using a fixed formula (Logan et al., 2016), and (3) determining the highest dilution at which complete neutralisation is seen in all replicate wells and considering other wells that also show neutralisation. Neutralisation is then calculated by inputting these values into a Spearman Karber formula (Lambe et al., 2021).

  8. Neutralisation titres do not always need to be recorded as IC50 values. Other cut-off points can be chosen dependent on the level of neutralisation expected in a given assay, and to provide a more stringent measure of neutralisation (e.g., 80% neutralisation, IC80).

  9. The surface expression of different ACE2 receptors may differ, which may affect the level of Spike-ACE2 interaction leading to misinterpretation of results. Therefore, it is important to investigate and normalise the cell surface expression of the ACE2 receptors used. The mammalian ACE2 receptors described and used herein were HA-tagged at the C-terminus, which allowed detection of surface expression by flow cytometry. Additionally, protein expression was assessed by Western blotting (Conceicao et al., 2020).

Recipes

  1. DMEM-10%

    DMEM supplemented with 10% FBS, 1% penicillin/streptomycin 10,000 U/ml, and 1% 100 mM sodium pyruvate, cultured at 37°C with 5% CO2.

  2. Working solution of 1 mg/ml PEI

    1. Weigh the viscous liquid to get 50 mg/ml in water (e.g., 0.42 g PEI + 8.4 ml water) and transfer to a sterile 50 ml Falcon.

    2. Place Falcon in a water bath set to 50°C and gently pipette up and down using a 1 ml pipette until fully dissolved.

    3. Dilute to 1 mg/ml with water (e.g., take 0.5 ml of your 50 mg/ml stock and add 24.5 ml water)

    4. The solution in its current state will be very basic. Adjust pH to 7 using diluted hydrochloric acid.

    5. Filter through a 0.22 µm filter and aliquot into 1.5 ml Eppendorf tubes.

    6. Store at -20°C long-term and at 4°C for up to one month while in use.

Acknowledgments

This work was supported by the following grants to Dalan Bailey: a UK Research and Innovation (UKRI, https://www.ukri.org/) Medical Research Council (MRC) New Investigator Research Grant (MR/P021735/1), UKRI Biotechnology and Biological Sciences Research Council (UKRI - BBSRC, https://www.ukri.org/) project grants (BB/R019843/1 and BB/T008784/1, student funding), and Institute Strategic Programme Grant (ISPG) to The Pirbright Institute (BBS/E/I/00007034, BBS/E/I/00007030 and BBS/E/I/00007039). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

    We would like to acknowledge the research papers from which the protocols described herein have been derived from, which include: “The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins” (Conceicao et al., 2020), “Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell-cell fusion” (Thakur et al., 2021), and “Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19” (Graham et al., 2020).

    We would also like to thank the following for assistance in establishing the SARS-CoV and SARS-CoV-2 pseudotype systems: Ed Wright (Viral Pseudotype Unit, University of Sussex), Nigel Temperton (Viral Pseudotype Unit, University of Kent), Brian Willett (University of Glasgow Centre for Virus Research), Emma Bentley and Giada Mattiuzzo (National Institute for Biological Standards and Control), and Michael Letko (National Institute of Allergy and Infectious Disease).

Competing interests

Authors declare no conflicts of interest.

References

  1. Bertram, S., Glowacka, I., Blazejewska, P., Soilleux, E., Allen, P., Danisch, S., Steffen, I., Choi, S. Y., Park, Y., Schneider, H., Schughart, K. and Pohlmann, S. (2010). TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. J Virol 84(19): 10016-10025.
  2. Conceicao, C., Thakur, N., Human, S., Kelly, J. T., Logan, L., Bialy, D., Bhat, S., Stevenson-Leggett, P., Zagrajek, A. K. and Hollinghurst, P. (2020). The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016.
  3. DePolo, N. J., Reed, J. D., Sheridan, P. L., Townsend, K., Sauter, S. L., Jolly, D. J. and Dubensky, T. W. (2000). VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol Ther 2(3): 218-222. Ferrara, F. and Temperton, N. (2018). Pseudotype Neutralization Assays: From Laboratory Bench to Data Analysis. Methods Protoc 1(1): 8.
  4. Ferrara, F. and Temperton, N. (2018). Pseudotype Neutralization Assays: From Laboratory Bench to Data Analysis. Methods Protoc 1(1): 8.
  5. Graham, S. P., McLean, R. K., Spencer, A. J., Belij-Rammerstorfer, S., Wright, D., Ulaszewska, M., Edwards, J. C., Hayes, J. W. P., Martini, V. and Thakur, N. (2020). Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV. NPJ Vaccines 5(1): 69.
  6. Grehan, K., Ferrara, F. and Temperton, N. (2015). An optimised method for the production of MERS-CoV spike expressing viral pseudotypes. MethodsX 2379-384.
  7. Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A., Muller, M. A., Drosten, C. and Pohlmann, S. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181(2): 271-280e278.
  8. Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M. and McKeating, J. A. (2003). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100(12): 7271-7276.
  9. Lambe, T., Spencer, A. J., Thomas, K. M., Gooch, K. E., Thomas, S., White, A. D., Humphries, H. E., Wright, D., Belij-Rammerstorfer, S. and Thakur, N. (2021). ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models. Commun Biol 4(1): 915.
  10. Logan, N., McMonagle, E., Drew, A. A., Takahashi, E., McDonald, M., Baron, M. D., Gilbert, M., Cleaveland, S., Haydon, D. T., Hosie, M. J. and Willett, B. J. (2016). Efficient generation of vesicular stomatitis virus(VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies. Vaccine 34(6): 814-822.
  11. Negrete, O. A., Levroney, E. L., Aguilar, H. C., Bertolotti-Ciarlet, A., Nazarian, R., Tajyar, S. and Lee, B. (2005). EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature 436(7049): 401-405.
  12. Salvador, B., Zhou, Y., Michault, A., Muench, M. O. and Simmons, G. (2009). Characterization of Chikungunya pseudotyped viruses: Identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in E1 glycoprotein. Virology 393(1): 33-41.
  13. Sharkey, C. M., North, C. L., Kuhn, R. J. and Sanders, D. A. (2001). Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production. J Virol 75(6): 2653-2659.
  14. Simmons, G., Reeves, J. D., Grogan, C. C., Vandenberghe, L. H., Baribaud, F., Whitbeck, J. C., Burke, E., Buchmeier, M. J., Soilleux, E. J. and Riley, J. L. (2003). DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance infection of macrophages and endothelial cells. Virology 305(1): 115-123.
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简介

[摘要]对于包膜病毒,例如 SARS-CoV-2,传播依赖于病毒糖蛋白与细胞受体的结合。通常,这个过程在实验室中通过用分离的活病毒感染细胞来重现。然而,由于大量具有复制能力的病毒、生物安全预防措施和相关培训人员的可用性,此类研究可能会受到限制。在这里,我们提出了一种基于假型分析的协议,以生产带有 SARS-CoV 或 SARS-CoV-2 附件尖峰糖蛋白的重组复制缺陷慢病毒,允许在较低防护设施中调查病毒进入。假颗粒由用编码逆转录病毒 RNA 包装信号和Gag-Pol 的质粒瞬时转染的细胞产生 蛋白质,用于重建慢病毒颗粒,以及编码感兴趣的病毒附着蛋白的质粒。这种方法允许研究病毒进入的不同方面,例如受体向性的识别、病毒宿主范围的预测和人畜共患病传播潜力,以及抗体(血清或单克隆抗体)和药理学抑制剂的表征块入口。

图文摘要:

SARS-CoV 和 SARS-CoV-2 伪粒子的产生和应用。



[背景]假颗粒是通过在重组病毒表面表达病毒包膜糖蛋白而获得的复制缺陷病毒颗粒,它提供了颗粒的核心成分。水泡性口炎病毒 (VSV)、弹状病毒和两种慢病毒——人类免疫缺陷病毒-1 (HIV-1) 和鼠白血病病毒 (MuLV)——通常用作假型分析的病毒载体(Takada等人,1997 年;Wool- Lewis 和 Bates ,1998;Sharkey等,2001;Negrete等,2005;Grehan等,2015;Thakur等,2021)。在我们最近的研究中,我们成功地使用基于慢病毒的系统来研究严重急性呼吸系统综合症冠状病毒 (SARS-CoV) 和 SARS-CoV-2 Spike (S) 蛋白与其细胞受体血管紧张素转化酶 2 (ACE2) 的相互作用)(Conceicao等人,2020 年)。
SARS-CoV-2 是正在进行的 COVID-19 大流行的病原体,是一种高致病性 β 冠状病毒,需要在 BSL-3 设施中进行处理,而研究实验室并不总是提供这些设施。为了让 SARS-CoV 和 SARS-CoV-2 在较低的防护范围内工作,用 Spike 蛋白假型化的病毒颗粒的产生代表了一种安全且有吸引力的替代物。该技术允许 (i) 剖析病毒进入途径,(ii) 研究宿主细胞易感性和血管紧张素转化酶 2 (ACE2) 受体的趋向性,(iii) 检查种间传播,(iv) 评估中和作用免疫原性和血清流行病学研究中的抗体反应,以及 (v) 阻断病毒进入的小分子抑制剂的功效评估。值得注意的是,该技术已应用于多种病毒的病毒糖蛋白,例如流感血凝素(Bertram等人,2010 年)、尼帕病毒融合和附着蛋白(Thakur等人,2021 年)、埃博拉病毒糖蛋白(Simmons等人,2010 年)al ., 2003)、基孔肯雅病毒 E1 (Salvador et al ., 2009)、丙型肝炎病毒 E2 蛋白 (Hsu et al ., 2003) 和 VSV 糖蛋白 (DePolo et al ., 2000)。
为了生成基于慢病毒的 HIV-1 假颗粒,细胞与以下质粒共转染:(i) 编码核心基因Gag和Pol 的HIV-1 包装质粒,(ii) 编码萤火虫荧光素酶报告基因的转移质粒基因两侧是 HIV-1 调节 LTR 区域和包装信号,以及 (iii) 编码异源病毒糖蛋白的第三个质粒。在其表面具有目标病毒糖蛋白的假颗粒组装在细胞膜上,并从中萌芽(Zufferey等,1997)。感染后,由慢病毒基因组编码的荧光素酶基因被表达,从而可以准确定量病毒进入。

关键字:SARS-CoV-2, SARS-CoV, 假型病毒, 趋性, 中和作用


材料和试剂
 
50 ml Falcon 管(VWR International,目录号:734-0448)
透明底部 6 孔组织培养处理板(Scientific Laboratory Supplies,Falcon TM ,目录号:353046)
移液器吸头(STARLAB,目录号:S1110-3700 [10/20 µl XL Graded TipOne ® ];S1111-1206-C [200 µl Yellow Bevelled TipOne ® Tip];S1112-17200 [1,250 µl XL Graded TipOne ® ]
血清移液管(Corning,目录号:4101 [10 ml Stripette TM ];4051 [5 ml Stripette TM ];4251 [25 ml Stripette TM ])
Opti-MEM TM (Thermo Scientific,Gibco TM ,目录号:11058021,储存条件:4°C,保质期:12 个月)
一次性称量舟 85 × 85 × 24 mm,PS,中型,白色,防静电(VWR,目录号:10770-448,储存条件:室温)
7毫升聚碳酸酯聚丙烯螺旋盖bijous(用于储存小体积样品的容器)(STARLAB,目录号:E1412-0710)
96孔德尔塔处理的白平底板(促进细胞附着和生长亲水性表面)(Fisher Scientific公司,Nunc公司,微孔,目录号:10182831)
组织培养瓶(Greiner Bio-One,目录号:660160 [175 cm 2 ]、658170 [75cm 2 ])
1.5 ml Microcentrifuge 无菌 Eppendorf 管(STARLAB,TubeOne ® ,目录号:S1615-5510)
Millex-GP 注射器过滤器装置,0.22 µm 过滤器,聚醚砜,33 mm,伽马灭菌(默克,密理博,目录号:SLGP033RS,储存条件:室温)
人胚肾 293T,HEK293T 细胞(ATCC ® ,目录号:CRL-3216 TM ,储存条件:液氮气相)
Baby Hamster Kidney-21,BHK-21 细胞(ATCC ® ,目录号:CCL-10 TM ,储存条件:液氮气相)
质粒 DNA:ACE2 受体(pDISPLAY 表达载体,密码子优化,N 端信号肽 [鼠 Ig κ 链前导序列],C 端 HA 标签),SARS-CoV Spike,SARS-CoV-2 Spike( pcDNA3.1(+),密码子优化,C 端 FLAG 标签)(BioBasic,加拿大 [Conceicao等人,2020]),p8.91,CSFLW,VSV-G(pcDNA3.1(+) 表达载体)(可根据要求提供)、pcDNA3.1(+)(Thermo Scientific,Invitrogen,目录号:V79020)和 pDISPLAY TM (Thermo Scientifc,Invitrogen TM ,目录号:V66020)
用于中和化验的血清或抗体,具有相关的生物风险评估和伦理批准
Dulbecco 改良 Eagle 培养基,DMEM(默克,Sigma-Aldrich,目录号:D5796 [含酚红];D1145 [不含酚红],储存条件:4℃, 12个月)
胎牛血清,FBS(生命科学生产,目录号:S-001A-BR,-20°C)
青霉素-链霉素,10,000 U/ml(Thermo Scientific,Gibco TM ,目录号:15240122,储存条件:-20°C,保质期:12 个月)
丙酮酸钠,100 mM(Thermo Scientific,Gibco TM ,目录号:11360070,储存条件:4°C,保质期:12 个月)
EDTA(0.5 M),pH 8.0,无RNase(Thermo Scientific,Ambion ® ,目录号:AM9269G)
1×胰蛋白酶-EDTA,0.25%,酚红(Thermo Scientific,Gibco TM ,目录号:2520072,储存条件:长期-20°C,使用时4°C,保质期:24个月)
Trans IT-X2 ®动态递送系统(Mirus,目录号:MIR 6000,储存条件:-20°C,保质期:12 个月)
聚乙烯亚胺,PEI(默克,Sigma-Aldrich,目录号:408727,储存条件:4°C)
无核酸酶,高压灭菌,0.2 µm 过滤 DEPC 处理水(Ambion,目录号:AM9906,储存条件:室温)
盐酸36.5-38.0%,生物试剂,用于分子生物学(Sigma-Aldrich,目录号:H1758-100 ml,储存条件:室温)
Bright-Glo TM荧光素酶测定系统(Promega,目录号:E2650,储存条件:-20°C)
55 ml StarTub PVC 试剂槽(STARLAB,无菌独立包装,目录号:E2310-1010)
DMEM-10%(见配方)
1 mg/ml PEI 的工作溶液(见配方)
 
 
 
设备
 
微生物安全柜,BSL-2(CAS,Biomat 2 – 2 级符合 BS EN 12469:2000)
CO 2培养箱(PHC Europe BV,PHCbi,目录号:MCO-170AICD-PE)
-86°C超低温冷冻柜(PHCbi、松下、vip plus、型号:MDF-DU900V)
-20°C 医用冷冻柜,带 14 个储物抽屉(利勃海尔,Profiline,型号:G5216)
4°C 冰箱(VDW CoolSystems、Labcold、Sparkfree,型号:RLV0217)
Sub aqua 5 plus 水浴(Fisher Scientific,Grant,型号:13251183)
用于血清移液器的自动移液器(Fisher Scientific,Thermo Scientific TM ,S1 Pipet Fillers,目录号:10072332)
单通道移液器(Gilson,Pipetman L,目录号:FA1001M [P2L 0.2-2 µl],FA1003M [P20L 2-20 µl];FA1005M [P200L 20-200 µl];FA1006M,[P1000-1]             
多道移液器(Thermo Scientific TM ,Finnpipette TM F2 多道移液器,目录号:4662010 [8 孔 5-50 µl];4662070 [12 孔 30-300 µl])
细胞培养倒置显微镜(Leica microsystem,型号:DMi1-S 40/0.45)
血细胞计数器(Fisher Scientific,Hirschmann TM Bright Lined Counting Chambers,目录号:105289616)
离心机(Kendo 实验室产品,Sorvall Legend RT,EASYset,型号:75004373)
台式高压釜(Fisher Scientific,Astell science,目录号:12755375)
GloMax ® Discover 酶标仪(Promega,目录号:GM3000)
 
软件
 
Microsoft Excel(Microsoft 365 for Windows,www.microsoft.com)
GraphPad Prism(8.4.2 版,适用于 Windows 的 GraphPad 软件,美国加利福尼亚州圣地亚哥,www.graphpad.com)
GloMax ® Discover 系统软件(3.2.3 版,Promega,南安普敦,英国www.promega.co.uk)
 
程序
 
SARS-CoV-2和SARS-CoV假型病毒颗粒的产生
将 HEK293T 细胞维持在 75 cm 2组织培养瓶中的25 ml DMEM-10%(参见配方)中以生产假颗粒。
将 HEK293T 细胞以每孔7.5 × 10 5细胞的浓度接种在 3 ml DMEM-10% 的 6 孔板中,用于所需孔的总数。
使用快速上下左右运动搅动板中的细胞。这将确保细胞均匀分布且不会结块。在 37°C、5% CO 2下孵育过夜。
第二天,在下午设置转染混合物。接种的 HEK293T 细胞应在 60-80% 之间汇合,以获得最佳转染效率。设置 SARS-CoV-2 S 或 SARS-CoV S 质粒的转染,以及空载体阴性对照(无糖蛋白,无 GP)和阳性对照。例如,如果 SARS-CoV-2 S 和 SARS-CoV S 质粒位于 pcDNA3.1 骨架中,则使用空 pcDNA3.1 质粒作为无 GP 对照。通常,VSV-G 质粒被用作阳性对照,因为它被有效地转包衣到 HIV-1 颗粒中(即,它假型良好)。
在无菌的 1.5 ml Eppendorf 管中,加入 100 µl Opti-MEM 以及 0.6 µg p8.91 质粒(编码 HIV-1 gag-pol)、0.6 µg CSFLW 质粒(表达萤火虫荧光素酶的慢病毒骨架)和 0.5每孔微克糖蛋白(SARS-CoV-2 S、SARS-CoV S 或 VSV-G)或空载体 (pcDNA3.1)。在室温下孵育 5 分钟。
在单独的 1.5 ml Eppendorf 管中,每次转染加入 100 µl Opti-MEM plus 和 10 µl PEI (1 µg/ml),并在室温下孵育 5 分钟。
对于 Opti-MEM 中的每 100 µl DNA 转染混合物,在 Opti-MEM 中加入 100 µl PEI,并用移液器剧烈混合十次。在室温下孵育 20 分钟。
将 200 µl 转染混合物的体积滴加到 6 孔板的每个孔中,并在 37°C、5% CO 2下孵育过夜。
第二天早上,使用血清移液器轻轻地从含有转染混合物的孔中取出培养基,方法是将培养皿向您倾斜并从孔的边缘抽吸,注意不要干扰单层。替换为 3 ml 的 DMEM-10%。在 37°C、5% CO 2下孵育过夜24 小时。
收集含有假型病毒颗粒的细胞上清液并转移至 50 ml Falcon,汇集类似转染的孔,并在 4°C 下储存。每孔用 3 ml DMEM-10% 替换培养基,并在 37°C、5% CO 2下孵育24 小时。
收获含有假型病毒颗粒的细胞上清液,并与前一天收获的假颗粒混合。在 4°C 下以2,500 × g离心10 分钟以去除细胞碎片。
将 4-5 ml 假颗粒分装到 bijous 中,并在 -80°C 下冷冻直至进一步使用。
 
注意:也可以在 10 cm 2培养皿中制备更大体积的假颗粒。此设置所需的必要细胞接种密度、DNA 浓度和体积可在表 1 中找到,并附有 6 孔板格式的相应值。无论使用的培养皿大小如何,步骤 A9-A12 都保持不变,仅更改所需的介质量。


表 1. 生成基于慢病毒的假型病毒的快速指南
 
检测 SARS-CoV-2 和 SARS-CoV 假颗粒感染性
在 6 孔板中以每孔7.5 × 10 5的密度接种 HEK293T 细胞,共 3 ml DMEM-10%。在 37°C、5% CO 2下孵育过夜。
确保铺板细胞的汇合度为 60-80%,以确保最佳转染效率。设置转染混合物以测试预先生成的 SARS-CoV-2 假颗粒。在无菌 1.5 ml Eppendorf 管中,每孔加入 200 µl Opti-MEM 和 500 ng 人 ACE2 质粒进行转染。使用前将 Tran IT -X2 转染试剂置于室温,直接向管中加入 2 µl(每 1 µg DNA),轻轻轻弹管混合。在室温下孵育 20 分钟。
将 200 µl 转染混合物逐滴加入预镀细胞的每个孔中,并在 37°C、5% CO 2下孵育过夜。
通过将培养皿向您倾斜并使用血清移液管从井的边缘吸出,从井中取出含有转染混合物的介质,注意不要干扰单层。每孔加入 1 ml DMEM-10% 并收获转染细胞。HEK293T 细胞附着力低,容易从板上脱落。因此,使用吸取液体的力足以收获细胞,但应注意确保获得无团块的单细胞悬浮液。胰蛋白酶应该避免,因为这将不必要地切掉的受体,阻碍未来的实验。转移到50ml隼和稀细胞2×10 5 / ml的DMEM-10%。
将 100 µl 稀释细胞(每孔2 × 10 4 )接种到平坦的白底 96 孔板中,并在 37°C、5% CO 2下孵育过夜。
第二天,解冻一份 SARS-CoV-2 和/或 SARS-CoV 假颗粒,以及阴性(pcDNA3.1,无 GP)和阳性(VSV-G)对照。在透明底 96 孔板中滴定假颗粒,从顶行未稀释的病毒开始,在 DMEM-10% 中滴定 10 倍,最终体积为 100 µl 。
从接种了人 ACE2 转染细胞的白板上轻轻去除培养基,并添加 100 µl 滴定的假颗粒。在 37°C、5% CO 2下孵育 48 小时。
通过将培养皿向您倾斜并使用多通道移液器从孔边缘抽吸,从孔中取出培养基,然后加入 50 µl Bright-Glo TM用无血清、无酚红 DMEM 按 1:1 稀释。将板在黑暗中孵育 5 分钟,然后在 GloMax Multi+ 检测系统上根据发光协议测量荧光素酶信号,积分为 0.5 秒。
导出 USB 闪存驱动器上生成的 CSV 文件,以便使用 Microsoft Excel 进行分析,并在 GraphPad Prism 上绘制数据。
 
使用 SARS-CoV-2 和 SARS-CoV 假型病毒颗粒筛选 ACE2 受体使用情况(Conceicao等,2020)
将 BHK-21 细胞维持在 75 cm 2组织培养瓶中的25 ml DMEM-10%中。在 24 孔板中以 1 × 10 5 /孔的 DMEM-10%种子 BHK-21 细胞。在 37°C、5% CO 2下孵育过夜。
确保铺板细胞的汇合度为 60-80%,以确保最佳转染效率。在 100 µl Opti-MEM 中设置转染混合物以及 500ng 不同种类的 ACE2 表达构建体或空载体对照(例如,pDISPLAY)。使用前将 Tran IT -X2 转染试剂置于室温,每 1 µg DNA 加入 3 µl 直接加入管中,轻轻轻弹管混合。在室温下孵育 20 分钟。
将 100 µl 转染混合物逐滴添加到预镀 BHK-21 细胞的每个孔中,并在 37°C、5% CO 2下孵育过夜。
从孔中取出含有转染混合物的培养基,并在每孔中加入 0.5 ml 2 mM EDTA 的 PBS 以收获转染细胞。转移到 bijou 并用 DMEM-10% 将细胞稀释至 2 × 10 5 /ml。
将 100 µl 稀释细胞(每孔2 × 10 4 )接种到平坦的白底 96 孔板中,并在 37°C、5% CO 2下孵育过夜。
从细胞中取出培养基并用相当于 10 6 -10 7 个相对光单位 (RLU) 的SARS-CoV-2 或 SARS-CoV 假颗粒或相同稀释度的无 GP 对照感染,并在 37°C 下孵育 48 小时, 5% CO 2 。
从孔中取出培养基,加入 50 µl Bright-Glo TM 以1:1 稀释的无血清、无酚红 DMEM。将板在黑暗中孵育 5 分钟,然后在 GloMax Multi+ 检测系统上在发光协议下读取 0.5 秒积分。
导出 USB 闪存驱动器上生成的 CSV 文件,以便使用 Microsoft Excel 进行分析,并在 GraphPad Prism 上绘制数据。
 
使用 SARS-CoV-2 和 SARS-CoV 假型病毒颗粒的中和试验
在设置中和测定之前,将 HEK293T 细胞以每孔7.5 × 10 5的密度接种在6 孔板中,总共 3 ml 的 DMEM-10%。在 37°C、5% CO 2下孵育过夜。
确保铺板细胞的汇合度为 60-80%,以确保最佳转染效率。在无菌 1.5 ml Eppendorf 管中,每孔加入 200 µl Opti-MEM 和 500 ng 人 ACE2 质粒进行转染。使用前将 Tran IT -X2 转染试剂置于室温,每 1 µg DNA 加入 2 µl 直接加入管中,轻轻轻弹管混合。在室温下孵育 20 分钟。
在预镀细胞的每孔中逐滴加入 200 µl 转染混合物,并在 37°C、5% CO 2下孵育过夜。
通过稀释血清/单克隆抗体 (mAb)/抑制剂,考虑要使用的稀释系列和添加假颗粒后的最终体积来设置中和测定。例如,以 1:10 稀释开始使用 2 倍稀释系列滴定的血清需要每孔 10 µl 血清在 100 µl 无血清 DMEM 中。这同样适用于已知浓度的 mAb 或抑制剂。
将 100 µl 稀释的血清/mAb/抑制剂一式三份添加到平底白底 96 孔板的顶行。向所有剩余的孔中加入 50 µl 无血清培养基。从顶行中取出 50 µl 并向下滴定板 2 倍,每次滴定前充分混合。不要滴定到底行。这整行将用作未处理的对照。
解冻一份 SARS-CoV 或 SARS-CoV-2 假颗粒并在无血清 DMEM 中稀释,相当于 ~10 6 RLU,每孔加入 50 µl,包括未处理的对照。在 37°C、5% CO 2下孵育 1 小时。
从与人ACE2转染6孔板中取出介质。加入1ml的DMEM-10%每孔和收获转染的细胞。HEK293T细胞具有低粘附,所以脱落板容易; 因此,移液液体的力应足以收获细胞(参见B4上文)。转移到50ml隼和稀细胞2×10 5 / ml的DMEM-10%。
将 100 µl 稀释细胞(每孔2 × 10 4 )接种到每个含有假颗粒和未处理对照的血清/mAb/抑制剂的孔中。在 37°C、5% CO 2下孵育 48 小时。
从孔中取出培养基,加入 50 µl Bright-Glo TM 以1:1 稀释的无血清、无酚红 DMEM。将板在黑暗中孵育 5 分钟,然后在 GloMax Multi+ 检测系统上在发光协议下读取 0.5 秒积分。
导出 USB 闪存驱动器上生成的 CSV 文件,以便使用 Microsoft Excel 进行分析,并在 GraphPad Prism 上绘制数据。
 
数据分析
 
检测 SARS-CoV-2 和 SARS-CoV 假颗粒感染性
在制备一批假颗粒后,可以通过在已转染以表达 SARS-CoV 和 SARS-CoV-2 的假型附着蛋白(Spike)的宿主受体(ACE2)的靶细胞上滴定它们来测试它们的感染性。未稀释的假型病毒(“1”)在 96 孔板中用 DMEM-10% 滴定 10 倍,一式三份(“10”、“100”、“1,000”等)(图 1A )。
测量伪粒子的荧光素酶信号将生成一个 CSV 文件,该文件可以导出到 USB 闪存驱动器上并在 Microsoft Excel 上进行分析。然后可以将这些结果绘制在 GraphPad Prism 上以显示平均值± 每个伪粒子的 SD。
无 GP 阴性对照用作背景荧光素酶信号的指示,并且只有在每个相应稀释度中高于此值的值才应被视为被测试假颗粒的真实荧光素酶信号(图 1B,黑线)。通常,无 GP 和假型病毒之间的最小 ~2 log 动态范围和 10 5.5和 10 7.5 RLU之间的 RLU 信号(图 1B,阴影区域)足以用于后续检测。滴定系列也将有助于确定伪粒子的最低可用稀释度,以仍然获得高于背景的有意义的荧光素酶值。值得注意的是:以下要讨论的数据仅用于说明目的。
例如,在考虑为 SARS-CoV 获得的荧光素酶值时,尽管这些值高于最高稀释度的无 GP 对照,但两者之间的差异仅为 ~1 log,这超出了我们的使用标准(图 1B,蓝色行)。滴定假颗粒时,荧光素酶值在 1:10 稀释时也很快下降到可操作范围的下限以下,并在 1:1000 稀释时与无 GP 对照相同,使得该制剂的滴度为不适合用于后续分析的 SARS-CoV 假颗粒(图 1B )。
相比之下,为 SARS-CoV-2 获得的荧光素酶信号比无 GP 控制的背景高约 2 log,并且 1:10 的假颗粒稀释度将在可用于后续分析的可用范围内,在1:100 稀释(图 1B,橙色线)。
VSV-G 假颗粒是检测中的阳性对照,其中观察到的荧光素酶值应高于 10 7 RLU(图 1B,绿线)。
 
 
图 1. 检测 SARS-CoV 和 SARS-CoV-2 假颗粒的传染性。(A)对于SARS-CoV的和SARS-CoV的-2赝滴定,10倍上清的系列稀释液用于感染HEK293T细胞中瞬时表达,其为白色平底96孔板的人ACE2受体。实验中还包括阴性(无 GP)和阳性(VSV-G)对照。每个条件一式三份进行测试。(B)感染后两天,信号的萤光素酶值被测量并绘制为平均值±SD。无GP控制指示背景的,并且只有高于此值赝粒子应被视为真正的感染性,在病毒的每种稀释液匹配。用于随后的中和测定和受体的使用屏幕上使用假颗粒的应显示没有GP和假型病毒之间〜2个log的动态范围和10的工作范围之间落入5.5至10 7.5 (阴影区)。
 
使用 SARS-CoV-2 和 SARS-CoV 假型病毒颗粒进行 ACE2 受体使用筛选
用一式三份设置的 SARS-CoV 或 SARS-CoV-2 假颗粒感染表达各种 ACE2 受体的细胞会产生荧光素酶信号,这些信号可以相互绘制(平均值±标准差)以描绘原始值。这些数据还给出了不同病毒受体趋向性的总体趋势(图 2A )。
例如,水牛和山羊ACE2很好地允许SARS-CoV和SARS-CoV-2假颗粒进入,而小棕蝙蝠ACE2则不太明显。果子狸 ACE2 可以观察到病毒之间的差异,它允许 SARS-CoV 比 SARS-CoV-2 更有效地进入(图 2A )。这些实验应在三个不同的场合至少进行三次,并显示具有代表性的数据。ACE2 受体的一个子集显示在图 2A 中,但在 Conceicao等人的补充图 3A 和 3C 中可以找到更深入、更广泛的分析。(2020)。
在此屏幕中设置了两个阴性对照。第一个是空载体对照 (pDISPLAY),以确保测量的任何信号仅来自 ACE2 受体的过度表达。第二个是用无 GP 控制假颗粒制剂感染细胞,以确保荧光素酶信号可归因于假型病毒并为背景提供基线(图 2A )。
然后可以使用原始荧光素酶信号来确定非同源宿主 ACE2 受体(水牛、果子狸、山羊、小棕蝠)与已知或同源宿主受体(在本例中为人类 ACE2)的相对使用。在不同天进行的三个独立实验的平均百分比用于获得这些值。人 ACE2 的荧光素酶值设置为 100%,然后未知宿主受体和阴性对照的荧光素酶值表示为相对于人 ACE2 的百分比。
这些结果也可以显示为热图,使用颜色梯度来显示受体使用的不同趋势。例如,人类 ACE2 (100%) 设置为绿色。低于此值的表达从绿色变为红色,表明与人类 ACE2 相比,ACE2 的使用较差。高于 100% 的值显示为深绿色,表明 ACE2 的使用量等于或大于人类 ACE2(图 2B )。ACE2 受体的一个子集显示在图 2B 中,但更深入、更广泛的分析可以在 Conceicao等人的图 2A中找到。(2020)。
通过在xy散点图上绘制每种病毒相互之间的百分比值,计算 Pearson 相关系数,并绘制图,可以进行进一步分析以比较不同物种 ACE2 在 SARS-CoV 和 SARS-CoV-2 之间的受体趋向性拟合 95% 置信区间的线性回归线(数据未显示)。这种分析的一个例子也可以在 Conceicao等人的补充图 5 中找到。(2020)。
 
 
图 2. 使用 SARS-CoV 和 SARS-CoV-2 假颗粒的 ACE2 受体使用筛选。在我们的研究中,假颗粒被用作活病毒的替代物,以评估 SARS-CoV 和 SARS-CoV-2 与不同种类的 ACE2 受体的受体趋向性。使用伪粒子来评估 SARS-CoV 和 SARS-CoV-2 尖峰糖蛋白对来自不同物种的 ACE2 受体的使用,并表示为(A)原始荧光素酶信号值或(B)相对于人类 ACE2 的百分比。对于带有空载体对照(无 GP)或用空载体代替 ACE2 受体(pDISPLAY)进行模拟转染的假颗粒,包括阴性对照。数据表示为一式三份值的平均值 ± SD,每个实验在三个不同的场合进行 3 次,并显示了代表性数据。
 
使用 SARS-CoV-2 和 SARS-CoV 假型病毒颗粒的中和试验
使用假型病毒的中和试验是使用活病毒的低生物封闭性替代方法,并且可以以相对高通量的方式进行。这些中和分析可以在 mAb、血清或任何其他具有抑制病毒进入潜力的药物或抑制剂上进行。此处讨论的数据仅用于说明目的。
已知浓度的抑制剂(单克隆抗体/血清/药物)可以在 96 孔板中滴定三次,以确定对 SARS-CoV-2 进入的抑制程度。这是通过取未处理对照的平均值并表达与此相关的感兴趣 mAb 的每个单独复制的 RLU 值来完成的。然后可以将这些绘制为平均值±SD,并且应至少重复三次,并显示代表性数据:
 
(mAb 的 RLU 个体重复/未处理的 RLU 平均值)× 100
 
50% (IC50) 的抑制浓度应与未处理的、无 mAb 对照 (100%) 一起在图表上标明。低于此 IC50 线的值表示 S 介导的进入抑制,这可以在每个浓度下计算。例如,mAb2 能够以 100 µg/ml 的浓度抑制 SARS-CoV-2 S 进入约 80%(未处理的 20%)。在所有测试浓度下获得的 mAb2 值均低于 IC50 值,因此需要测试较低浓度以确定抑制极限。相比之下,mAb3 在 100 µg/ml 时抑制 SARS-CoV-2 S 进入约 90%,但在 12.5 µg/ml 时,抑制现在高于 IC50 阈值(图 3A )。
与 mAb1 一样,也可能存在不抑制 SARS-CoV-2 S 的 mAb 示例。当未观察到进入抑制时,可能会看到略高于 100% 阈值的增加。导致这种增加的机制仍然未知并正在调查中,但就本分析而言,mAb 不能中和 SARS-CoV-2 S 的结论就足够了(图 3A )。此类分析的示例可以在 Thakur等人中找到。(2021)。
在确定具有抗 SARS-CoV-2 S 抗体的个体的病毒进入抑制作用时,无论是自然感染还是疫苗接种后,通常都会计算中和抗体滴度以计算 SARS-CoV-2 S 中和的水平。最简单的计算方法是计算未处理对照的平均 RLU 并确定 IC50 值,即无血清对照的 50%。然后将中和滴度计算为稀释度的倒数,在该稀释度下,所有重复孔中无血清荧光素酶值的抑制率为 50%。然后可以将这些滴度制表或绘制在对数刻度上。
例如,血清样品 1 的滴度为 256,血清样品 3 的滴度为 512,表明 IC50 分别在 1:256 和 1:512 的稀释度下计算。由此可以得出的结论是,血清样本 3 能够比血清样本 1 更有效地中和 SARS-CoV-2 S 介导的进入,因此具有更高的抗体滴度(图 3B )。
对于血清样本 4,该值已绘制为 1,024,这是该测定的检测上限 (ULoD)。这意味着该样品能够在所有孔和测试的最低稀释度下抑制 50% 的荧光素酶信号。该血清必须用更广泛的稀释系列重新滴定,以确定中和滴度。对于血清样品 2,稀释范围内的所有孔均未产生可记录的 IC50。因此,中和滴度被绘制为低于检测下限 (LLoD) 的任意值,在这种情况下为 40,但由于真实滴度未知,将报告为 <40(图 3B )。使用此方法的 IC50 中和滴度的示例可以在 Thakur等人的图 4F 中找到。(2021) 以及 Graham等人的图 2D 和 2E 。(2020)。
 
 
图 3. 使用 SARS-CoV-2 假颗粒的中和试验。SARS-CoV-2 Spike 中和试验是在(A)单克隆抗体 (mAb)存在下进行的,以相对于未处理对照或(B)血清样本的百分比表示,数据表示为中和滴度。在加入表达人 ACE2 的 HEK293T 细胞之前,将 SARS-CoV-2 假颗粒与 mAb 或血清孵育 1 小时。通过计算荧光素酶信号 (IC50) 降低 50% 时的浓度 (mAb) 或稀释度 (血清) 来确定对 SARS-CoV-2 Spike 介导的病毒进入的抑制。数据代表一式三份值的平均值 ± SD,每个实验在三个不同的场合进行 3 次,并显示了代表性数据。
 
笔记
 
程序 A,步骤 11 提到了在用于后续测定之前对假颗粒制剂进行离心以去除细胞碎片。其他协议需要在储存前使用 0.45 µm 过滤器进一步过滤伪粒子。这一步不在我们的实验室中进行,因为我们观察到过滤后传染性降低。
制造商通常建议转染的最佳汇合度为 60-80%。因此,可能需要根据所用细胞的特性改变接种密度。例如,如果使用的细胞类型较大(例如,BHK-21 细胞比 HEK293T 细胞大)或具有高倍增率,我们建议从较低的接种密度开始。另一方面,如果细胞较小,或生长速度较慢,并且难以达到汇合(例如,Calu3 细胞),或者确实是悬浮细胞,您可能需要从较高的接种密度开始。在这两种情况下,我们建议测试不同的播种密度,以找到任何给定实验的最佳值。
图 4显示了感染前 HEK293T 细胞的最佳融合度 (60-80%) 的示例。细胞应均匀分布在整个孔中(即,在一个区域内没有结块或聚集),在单层中有明显的间隙(图 4A )。HEK293T 细胞的细胞形态在汇合处应显示为扁平和多边形,这表明对塑料的粘附(图 4B )。
 
 
图 4.明场图像说明 HEK293T 细胞的 60-80% 最佳汇合度。HEK293T 细胞以 7.5 × 10 5接种在6 孔培养皿中,含有 3ml DMEM-10%。第二天,细胞应在 60-80% 之间汇合,以获得最佳转染。(A)在 4 倍放大率下约 80% 汇合的 HEK293T 示例,细胞均匀分布在孔和可见间隙中。(B) HEK293T 细胞的高倍放大 (10 倍),其中细胞在转染前应呈现明亮、平坦和多边形。
 
在各个步骤中用于转染的 DNA 浓度已经过优化以用于这些分析。重要的是,任何使用的质粒都经过优化以解决载体平台和密码子优化的可变性。
在无 GP 控制中有时可以看到更高的信号(在 10 4 -10 5 RLU 范围内),这可能是非特异性摄取“秃头”假颗粒或来自生产细胞的碎片的结果。为了减少这种背景信号,应注意确保生产细胞不会在低汇合处转染,因为这会导致细胞病变效应 (CPE) 的发展。
此处进行的所有实验均仅使用过表达的 ACE2 进行。S2 蛋白裂解为 S2' 所需的丝氨酸蛋白酶 TMPRSS2 的共表达可以促进病毒和细胞膜的融合以及 Spike 蛋白的裂解(Hoffmann等,2020)。TMPRSS2 未包含在我们的宿主范围测定中,因为我们想专门检查不同 ACE2 的影响——实际上,TMPRSS2 的过度表达导致 ACE2 限制被掩盖(Conceicao等,2020)。
其他公式可用于确定 IC50 值,产生不同的滴度。如果在整个分析过程中使用相同的方法并且所使用的方法得到完整描述,则这是可以接受的。用于计算中和滴度的其他公式包括 (1) 在XY图上绘制数据以插入中和值后,在 GraphPad Prism 上使用非线性回归分析工具(2) 插入感染性点使用固定公式将无血清对照样品的值降低到 50%(Logan等人,2016 年),并且 (3) 确定在所有重复孔中看到完全中和的最高稀释度,并考虑其他孔显示中和。然后通过将这些值输入 Spearman Karber 公式来计算中和(Lambe等人,2021 年)。
中和滴度并不总是需要记录为 IC50 值。可以根据给定测定中预期的中和水平选择其他截止点,并提供更严格的中和量度(例如,80%中和,IC80)。
不同 ACE2 受体的表面表达可能不同,这可能会影响 Spike-ACE2 相互作用的水平,从而导致对结果的误解。因此,重要的是研究和标准化所用 ACE2 受体的细胞表面表达。本文描述和使用的哺乳动物 ACE2 受体在 C 端带有 HA 标签,这允许通过流式细胞术检测表面表达。此外,通过蛋白质印迹评估蛋白质表达(Conceicao等,2020)。
 
食谱
 
DMEM-10%
补充有 10% FBS、1% 青霉素/链霉素 10,000 U/ ml和 1% 100 mM 丙酮酸钠的DMEM ,在 37 °C和 5% CO 2下培养。
1 mg/ml PEI 工作溶液
称量粘性液体以获得 50 毫克/毫升的水(例如,0.42 克 PEI + 8.4 毫升水)并转移到无菌的 50 毫升 Falcon。
将 Falcon 放入设置为 50°C 的水浴中,并使用 1 ml 移液器轻轻上下移液直至完全溶解。
用水稀释至 1 mg/ml(例如,取 0.5 ml 的 50 mg/ml 原液并加入 24.5 ml 水)
当前状态下的解决方案将是非常基本的。使用稀盐酸将 pH 值调节至 7。
通过 0.22 µm 过滤器过滤并分装到 1.5 ml Eppendorf 管中。
使用时可在 -20°C 下长期储存,在 4°C 下最多可保存 1 个月。
 
 
致谢
 
这项工作得到了 Dalan Bailey 的以下资助:英国研究与创新 (UKRI, https://www.ukri.org/ ) 医学研究委员会 (MRC) 新研究者研究资助 (MR/P021735/1),UKRI生物技术和生物科学研究委员会 (UKRI - BBSRC, https://www.ukri.org/ ) 项目拨款(BB/R019843/1 和 BB/T008784/1,学生资助)和研究所战略计划拨款 (ISPG)皮尔布莱特研究所(BBS/E/I/00007034、BBS/E/I/00007030 和 BBS/E/I/00007039)。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。
我们要感谢本文描述的协议源自的研究论文,其中包括:“SARS-CoV-2 Spike 蛋白对哺乳动物 ACE2 蛋白具有广泛的趋向性”(Conceicao等人,2020 年),“微融合抑制测试:量化病毒介导的细胞-细胞融合的抗体中和作用”(Thakur等人,2021 年)和“用复制缺陷型病毒载体 COVID-19 候选疫苗进行初步加强疫苗接种的免疫原性评估ChAdOx1 nCoV-19”(格雷厄姆等人,2020 年)。
我们还要感谢以下人员在建立 SARS-CoV 和 SARS-CoV-2 假型系统方面提供的帮助:Ed Wright(萨塞克斯大学病毒假型部门)、Nigel Temperton(肯特大学病毒假型部门)、Brian Willett(格拉斯哥大学病毒研究中心)、Emma Bentley 和 Giada Mattiuzzo(国家生物标准与控制研究所)和 Michael Letko(国家过敏和传染病研究所)。
 
利益争夺
 
作者声明没有利益冲突。
 
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引用:Thakur, N., Gallo, G., Elreafey, A. M. E. and Bailey, D. (2021). Production of Recombinant Replication-defective Lentiviruses Bearing the SARS-CoV or SARS-CoV-2 Attachment Spike Glycoprotein and Their Application in Receptor Tropism and Neutralisation Assays. Bio-protocol 11(21): e4249. DOI: 10.21769/BioProtoc.4249.
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