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Oct 2020
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Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes
严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 慢病毒假型的生产、滴定、中和、储存和冻干   

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Abstract

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.

Keywords: SARS-CoV-2 coronavirus (SARS-CoV-2冠状病毒 ), Lentiviral pseudotype ( 慢病毒假型), Virus neutralisation (病毒中和), Spike variants (穗状变体), Pseudotype lyophilisation ( 冻干假模), COVID-19 (COVID-19)

Background

SARS-CoV-2 is the causative agent of COVID-19 disease currently manifesting as a global pandemic (Zhu et al., 2020). Due to the highly infectious nature of SARS-CoV-2, the wild-type virus has been classified as a BSL-3 pathogen, heavily restricting its use in many laboratories. To circumvent this biohazard restriction, pseudotype viruses (PVs) can be generated by utilising a surrogate viral core to generate virions displaying the SARS-CoV-2 spike protein (Nie et al., 2020; Crawford et al., 2020). Due to the single-round infection and replication deficient properties of PVs, they can be employed in BSL-2 laboratories. The use of PVs as a platform to investigate serosurveillance, antigenic properties and viral entry mechanisms of emerging viruses has been extensively reviewed (Bentley et al., 2015; Li et al., 2018; Cantoni et al., 2021; Focosi et al., 2021), with many studies demonstrating a high degree of correlation between wild-type virus neutralisation assays and PV neutralisation assays (Hyseni et al., 2020; Schmidt et al., 2020; Sholukh et al., 2020; Xiong et al., 2020). Furthermore, PVs can be used as a diagnostic control for new platforms to detect SARS-CoV-2 infection in patients (Sholukh et al., 2020).


Since the start of the pandemic, many protocols have been established for generating SARS-CoV-2 pseudotypes using a range of viral cores, all of which have shown that long term storage of PVs is ideal at -80°C (Crawford et al., 2020; Nie et al., 2020). The issue that this presents is that sharing PVs with research or diagnostic laboratories that do not have expertise in PV generation and application is that it would incur high shipping costs, as the particles need to be shipped on dry ice to remain stable. Additionally, many laboratories within low-income and middle-income countries (LMICs) have no routine access to -80 °C storage. In this protocol, we present our method of generating (Figure 1) and using lentiviral based PVs (highly adaptable and biosafe) expressing SARS-CoV-2 spike, with the additional step of lyophilisation of SARS-CoV-2 PVs using sucrose as a cryopreservant and a freeze drier. Our previous experience has shown that lyophilisation of Influenza, Rabies, and Marburg PVs does not affect PV performance (Mather et al., 2014). We show that SARS-CoV-2 PV can be lyophilised in the same manner and can thus be transported at room temperature over extended time periods (at least a week) and reconstituted into solution without any loss of PV performance (for similar PV, reconstitute within a year if kept at 4°C). This will greatly expand the adoption of pseudotype SARS-CoV-2 neutralisation assays globally and facilitate diagnostic platforms that use SARS-CoV-2 PVs as control samples, like MALDI-ToF (Iles et al., 2020), from both a financial and practical point of view.



Figure 1. Schematic representation of the production of SARS-CoV-2 pseudotype viruses. HEK293T/17 cells are transfected with three plasmids (Lentiviral vector incorporating luciferase reporter, packaging construct and SARS-CoV-2 spike expression plasmid) for the production of SARS-CoV-2 Spike bearing lentiviral pseudotypes.

Materials and Reagents

  1. MultiGuard Barrier pipette tips 1-20 and 1-200 μl (Sorenson BioScience, catalog number: 30550T)

  2. NuncTM Cell Culture Treated Multidishes (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675)

  3. NuncTM Cell Culture Dish Delta Surface Treated (10 cm sterile dishes) (Thermo Fisher Scientific, Thermo ScientificTM, catalogue number: 150350)

  4. Reaction tube, 1.5 ml with attached cap, graduation and writing area (Greiner Bio-One, catalog number: 616201)

  5. FisherbrandTM Sterile Syringes for Single Use 3 ml, (Fisher Scientific, Thermo ScientificTM, catalog number: 14955457)

  6. 0.45 μm syringe filter, cellulose acetate (STARLAB, catalog number: E4780-1453)

  7. Pipette basins (50 ml), Generic

  8. 96-well white plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136101)

  9. Microtube (1.5 ml, 0.5 ml), generic

  10. Thin-walled PCR microtubes (0.2 ml), generic

  11. HEK 293T/17 cells (ATCC, catalog number: CRL-11268)

  12. Subcloning efficiency E. coli DH5α cells (Invitrogen, catalog number: 18265017)

  13. Plasmids

    1. Spike plasmid: pCAGGS-SARS-CoV spike (CFAR, catalog number: 100976)

    2. Lentiviral vector expressing firefly luciferase: pCSFLW (or pCSGW for GFP PV) (Carnell et al., 2015)

    3. Second-generation lentiviral packaging construct plasmid: p8.91 (expresses gag, pol and rev) (Carnell et al., 2015)

    4. Host cell entry receptor ACE2 expression plasmid: pCDNA3.1+-ACE2 (Simmons et al., 2004)

    5. Coronavirus Spike (S) protein priming TMPRSS2 expression plasmid: pCAGGS-TMPRSS2 (Bertram et al., 2010)

    Note: Information on the plasmids above can be found in Temperton et al. (2005) and Carnell et al. (2015). Plasmids available from VPU.

  14. Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L Glucose (Pan-Biotech, catalog number: P04-04510) supplemented with 10% foetal bovine serum (FBS) (Pan-Biotech, catalog number: P40-37500) and 1% penicillin/streptomycin (P/S) (Pan Biotech, catalog number: P06-07100)

  15. Gibco Reduced Serum media Opti-MEM® (Thermo Fisher Scientific, catalog number: 51985034)

  16. FuGENE® HD Transfection Reagent, 1 ml (Promega, catalog number: E2311)

  17. Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium (Pan-Biotech, catalog number: P04-36500)

  18. Trypsin-EDTA (0.05%), phenol red (Pan-Biotech, catalog number: P10-040100)

  19. Positive control antibody (Research Reagent for Anti-SARS-CoV-2 Ab) that can neutralise the SARS-CoV-2 PV (NIBSC, code: 20/130, available internationally)

  20. COVID-19 human convalescent plasma panel (NIBSC, catalog number: 20/118)

  21. Monoclonal antibodies that can neutralise the SARS-CoV-2 PV (Native Antigen, catalog numbers: MAB12443 and MAB12444)

  22. Bright GloTM luciferase assay system (Promega, catalog number: E2650)

  23. Low surface tension polypropylene 1.5 ml microtubes (Simport, catalog number: T330-7LST)

  24. Sucrose (Sigma-Aldrich, catalog number: S0389)

  25. Dulbecco’s Phosphate Buffered Saline (DPBS; Pan Biotech, catalog number: P04-361000)

  26. Spike plasmid: pCAGGS-SARS-CoV spike (CFAR, catalog number: 100976)

  27. Eukaryotic expression vector recipient plasmids: pcDNA3.1, pI.18, pCAGGS (Carnell et al., 2015)

  28. Anza enzyme kit (Thermo Fisher Scientific, catalog number: IVGN3006)

  29. Nuclease-free water (Thermo Fisher Scientific, catalog number: R0582)

  30. QIAquick PCR Purification kit (Qiagen, catalog number: 28104)

  31. QIAquick Gel Extraction kit (Qiagen, catalog number: 28704)

  32. Tris Acetate EDTA (TAE) buffer (50× concentrate; Thermo Fisher Scientific, catalog number: B49)

  33. Ultrapure Agarose (Thermo Fisher Scientific, catalog number: 16500100)

  34. SYBR Safe DNA gel stain (Invitrogen, catalog number: S33102)

  35. GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, catalog number: SM0313)

  36. Luria Broth (LB) and LB agar powder (Sigma-Aldrich, catalog numbers: L3022 and L2897)

  37. DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, catalog number: K1081)

  38. Monarch plasmid miniprep kit (New England Biolabs, catalog number: T1010S) or alternative

Equipment

  1. Class II biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM)

  2. Water bath or incubator, generic

  3. Pipettes (Gilson, models: PIPETMAN® Classic, P2, P20, P200 and P1000 or equivalent)

  4. Multichannel pipette (Glison, model: PIPETMAN L Multichannel P12 20-200 μl or equivalent)

  5. FisherbrandTM Sterile Polystyrene Disposable Serological Pipets 5 ml and 10 ml in 1/10 ml, Sterile, Plugged, Individually Wrapped (Fisher Scientific, Thermo ScientificTM, catalog numbers: 1367610H and 1367610J)

  6. Portable Pipet-Aid® XP Pipette Controller (Drummond Scientific Company, catalog number: 4-000-101 or equivalent)

  7. Vortex Mixer, adjustable speed (SciQuip, model: SP2260-VM)

  8. Galaxy MiniStar Mini Centrifuge (VWR, model: C1413V-230)

  9. Optional: BIO-RAD TC20TM Automated Cell Counter (Bio-Rad Laboratories, catalog number: 1450102EDU) or FastRead 102 disposable 10-chamber counting grid with integral acrylic, optically clear, coverslip (Immune Systems, catalog number: BVS100)

  10. Plate centrifuge (ELMI, model: SkyLine CM-6MT)

  11. GloMax® Navigator Microplate Luminometer (Promega, model: GloMax® Navigator)

  12. FreeZone 2.5 L freeze dryer (LabConCo, catalog number: 7670520)

  13. Sample drying chamber (LabConCo, catalog number: 7318700)

  14. Rotary Vane Vacuum pump 117 (LabConCo, catalog number: 7739402)

  15. Plastic microtube rack (Thermo Fisher Scientific, catalog number: 8850)

  16. Thermo-humidity meter (Thermo Fisher Scientific, catalog number: 11536973)

  17. Water bath or heat block, generic

  18. Microwave, generic

  19. Powerpack, generic

  20. Gel electrophoresis tank, generic

  21. Microcentrifuge (Thermo Fisher Scientific, SorvallTM LegendTM)

  22. UV transilluminator (Uvitech/Sigma, catalog number: Z363677)

  23. Gel imaging system, generic

  24. Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000)

  25. Dry 37°C incubator, generic

  26. Shaking 37°C incubator, generic

  27. Thermocycler (Techne, model: Prime, catalog number: 5PRIMEG/02)

Software

  1. PC or Mac with Microsoft Excel (Microsoft®)

  2. GraphPad Prism® (GraphPad Software)

Procedure

  1. Production of SARS-CoV-2 PV via plasmid co-transfection of 293T cells (4-5 days)

    Note: All steps should be carried out in a class II biosafety cabinet to avoid contamination.

    1. Day 1: 293T/17 cells should be sub-cultured into 6-well plates at a ratio that will deliver 70-90% confluence at the time of transfection (Day 2). Typically seeding 4 × 105 cells per well will achieve this level of confluency. An example of what the cells should look like is shown in Figure 2.



      Figure 2. Example of the confluency expected prior to transfection of HEK293T/17 cells with plasmids


    2. Day 2: DMEM/10% FBS/1% P/S and Opti-MEM® should be pre-warmed to 37°C using a water bath or an incubator.

    3. Prepare two labeled sterile 1.5 ml microcentrifuge tubes (tube 1 and tube 2) for each well of a 6-well plate which will be used for transfections.

    4. Add the following plasmid constructs (in a total volume of 15 µl) for transfection to tube 1:

      pCAGGS-SARS-CoV-2 or pCAGGS-SARS-CoV-2 (D614G) spike: 450 ng.

      p8.91-lentiviral vector: 500 ng.

      pCSFLW: 750 ng.

      Note: Volume of each individual plasmid determined by its concentration.

    5. Add 100 µl Opti-MEM® to the plasmid DNA mix (tube 1).

    6. Add 100 µl Opti-MEM® and FuGENEHD to tube 2.

    7. Incubation step: Mix both tubes by gently flicking and incubate for 5 min at room temperature (RT).

    8. After incubation, pipette the Opti-MEM®/FuGeneHD solution from tube 2 into the Opti-MEM®/DNA solution in tube 1.

    9. Incubation step: Incubate the tube at RT for 20 min whilst gently flicking the tube to mix every 3-4 min.

    10. Whilst the transfection mix is incubating, the culture media on the 293T/17 cells should be removed and 2 ml of fresh prewarmed DMEM/10% FBS/1% P/S added per well. It is imperative at this point to add culture media slowly to one side of the well to avoid detaching the adherent cell monolayer.

    11. After 20 min incubation, pipette the DNA/Opti-MEM®/FuGeneHD solution onto the 293T/17 cells (in one well of a 6-well plate) by adding dropwise throughout the total surface area of the well. Swirl the 6-well plate (s) gently to ensure an even dispersal of reagent mix.

    12. Incubation step: Incubate the plate at 37°C, 5% CO2 for 44-52 h. In our hands, incubation times in this range result in equivalent final PV production relative luminescence unit (RLU) titres.

    13. After overnight incubation (on Day 3), the culture media on the cells should be carefully removed and 2 ml fresh DMEM/10% FBS/1% P/S added. Again, add media slowly to one side of the well to avoid cell detachment.

    14. Day 4: Supernatant containing the viral pseudotype particles are harvested using a 3 ml sterile syringe and subsequently filtered into microcentrifuge or Falcon tubes via a syringe driven 0.45 µm filter.

    15. Store all filtered supernatants at -80°C until downstream use. It is recommended that supernatant is stored as aliquots to avoid multiple freeze-thaw cycles that may impact viral RLU titres.

    16. Optional step (Day 4): 2 ml fresh culture media may be added to cells to allow a second harvest 18-24 h later (Day 5) by adding further DMEM/10% FBS/1% P/S. In this case, extreme care must be taken in initial PV collection (step 14 above) to avoid damage to the cell monolayer by aspirating with a sterile syringe to one side of the well. We have observed that cells in poor health after the first harvest result in significantly lower PV production RLU titres upon the second harvest.

    Note: A control pseudotype virus can be created by following the steps outlined above but omitting the pCAGGS-SARS-CoV spike construct. This produces particles that do not express a viral surface glycoprotein (Delta S PV control).


  2. Preparation of target cells for titration and neutralisation assays (1 day)

    Note: SARS-CoV-2 virus host cell entry depends on receptor ACE2 and serine protease TMPRSS2 for S protein priming (Hoffmann et al., 2020). HEK293T/17 are transfected with ACE2 and TMPRSS2 plasmids to be used as optimal target cells for SARS-CoV-2 PV entry. It is therefore essential to pre-transfect the cells whether a PV titration or neutralisation assay is planned.

    1. HEK 293T/17 cells should be seeded into a 10 cm cell culture dish at a ratio that will deliver 70-90% confluence at the time of transfection. Typically seeding 2 × 106 cells/plate and incubated overnight at 37°C will achieve this level of confluency.

    2. DMEM/10% FBS/1% P/S and Opti-MEM® should be pre-warmed to 37°C using a water bath or an incubator.

    3. Prepare one sterile 1.5 ml microcentrifuge tube for each cell culture dish which will be used for transfections.

    4. Add the following plasmid constructs (in a total volume of 15 µl in molecular biology grade water) for transfection (DNA mix):

      pCDNA3.1+-ACE2 cell entry receptor: 2 µg

      pCAGGS-TMPRSS2 serine protease: 150 ng

      Note: Plasmid amounts required are determined by prior optimization.

    5. Add 100 µl Opti-MEM® to the plasmid DNA mix.

    6. Incubation step: Mix tube by gently flicking and incubate for 5 min at RT.

    7. After incubation, add 9 µl FuGENE® HD directly into the Opti-MEM®/DNA solution tube just below the surface.

    8. Mix by gently flicking the tube.

    9. Incubation step: Incubate the tube at RT for 20 min whilst gently flicking the tube to mix every 3-4 min.

    10. Whilst the transfection mix is incubating, the culture media on the 293T/17 cells should be removed and 10 ml of fresh DMEM/10% FBS/1% P/S added per dish. It is imperative at this point to add culture media slowly to one side of the well to avoid detaching the adherent cell monolayer.

    11. After 20 min incubation, pipette the DNA/Opti-MEM®/ FuGENE® HD solution onto the 293T/17 cells by adding dropwise throughout the total surface area of the dish. Swirl the 10 cm cell culture dish(es) gently to ensure an even dispersal of reagent mix.

    12. Incubation step: Incubate the plate at 37°C, 5% CO2 overnight (16-24 h). In our hands, this incubation time results in sufficient overexpression of cell entry receptors (ACE2/TMPRSS2) for SARS-CoV-2 PV entry.


  3. SARS-CoV-2 PV Titration for the calculation of relative light units (RLU)/ml

    Notes:

    1. Titration consists of transduction of reporter (in this case firefly luciferase, but GFP may be used) into target cells mediated by the viral glycoprotein expressed on the viral pseudotype (SARS-CoV-2 spike). Target cells are transfected with ACE2 and TMPRSS2 24 h prior to the titration. Efficiency of the transfection of ACE2/TMPRSS2 was consistently reproducible between several operators and different laboratories; in the rare occasion of a suboptimal transfection, the PV titers dropped dramatically, making results unusable.

    2. Controls for titration are provided via the inclusion of ‘cell only’ and ‘Delta S’ (no Spike plasmid) columns. Positive control for transduction can be provided via a PV bearing the Vesicular stomatitis virus G protein (VSV-G), which utilises a ubiquitous receptor that results in high RLU titres in all cell lines tested.


    1. In a 96-well white plate, add 50 µl of DMEM/10% FBS/1% P/S to the entire columns of ‘cell only’ controls (see Figure 3, columns 9-11).

    2. Add 50 µl of DMEM/10% FBS/1% P/S from rows B to H that are to contain PV or Delta S control virus.

    3. Add 100 µl of SARS-CoV-2 pseudotype virus supernatant to each well of row A (excluding cell only control columns) and add 100 µl of Delta S to column 12 (see Figure 3).



      Figure 3. 96-well plate set-up for pseudotype titration. Serial dilution step showing addition of 100 µl of pseudotype virus supernatant to each well of row A and dilution of 50 µl taken from this well to row B. This process is continued to end of plate (row H), at which point the final 50 µl is discarded. Delta S control is indicated in red (column 12), and cell only controls are indicated in blue (columns 9-11). One set of pipette tips can be used per dilution series (plate).


    4. With the aid of a 12-channel pipette, remove 50 µl from row A virus-containing wells and perform two-fold serial dilutions down all the wells beneath it.

    5. With each dilution step, mix at least five times by pipetting up and down and taking care not to produce air bubbles.

    6. After completing serial dilution, the final 50 µl from the final well of each column should be discarded.

      Note: At this point, each well should contain 50 µl of PV supernatant only (row A) or mixed and diluted with DMEM (rows B to H).

    7. Prepare a plate of susceptible target cells (HEK 293T/17 expressing ACE2 and TMPRSS2 for SARS-CoV-2 PV):

      1. Remove culture media from plate.

      2. Wash the plate twice with 2 ml of PBS to one side of the dish to avoid cell detachment and discard.

      3. Add 2 ml of trypsin to the plate and put the plate into the incubator until the cells are detached (about 5 min).

      4. After cells have detached, add 6 ml of DMEM/10% FBS/1% P/S to the plate to quench trypsin activity and resuspend cells gently.

      5. Count cells using TC20TM Automated Cell Counter or counting-chamber slide and add 1 × 104 cells in a total volume of 50 µl to each well.

    8. Centrifuge plate for 1 min at 500 rpm if there are visible droplets on the sides of the wells.

    9. Incubate the plate for 48 h at 37°C 5% CO2.

    10. Read plate using Bright GloTM luciferase assay system on a GloMax® Navigator Microplate Luminometer (or equivalent) by removing the medium from all wells and adding 25 µl of a 1:1 mix of PBS:Bright GloTM luciferase assay reagent.

    11. Data analysis

      RLU readings from the luminometer are multiplied to get RLU/ml by the dilution factor of each well (20×, 40×, 80×, 160×, 320×, 640×, 1,280×, 2,560×). The mean of all 8 RLU/ml values is used as the final value reported for that column in the titration step. A linear relationship should be observed between RLU values and PV dilution, with values decreasing by 50% after each 1:2 dilution. Care should be taken to check this linear relationship before multiplication, as this inherently can lead to false production titres being calculated (Table 1).


      Table 1. Analysis of SARS-CoV-2 PV titration data and calculation of RLU/ml. RLU values (Top panel) are multiplied to give an RLU/ml value for each of the dilution points (Bottom panel). The mean/average is then calculated from all eight dilution points (expressed as RLU/ml). Care must be taken to observe a linear relationship between dilution factor (X factor) and RLU, or multiplication can lead to inflated RLU/ml values. Analysis is performed on Microsoft Excel (Microsoft®). For SARS-CoV-2 PV optimal production titre in this experiment (3.1 × 108 RLU/ml) is achieved when both ACE2 and TMPRSS2 are present in the target cells (column 8).


    12. Results (Figure 4)



      Figure 4. Production of SARS-CoV-2 PV (and D614G variant) and comparison with SARS-CoV (Temperton et al., 2005) and MERS-CoV (Grehan et al., 2015). RLU/ml production titres shown for MERS-CoV, SARS-CoV, SARS-CoV-2, and SARS-CoV-2 (D614G). SARS-CoV, SARS-CoV-2, and SARS-CoV-2 PV titrated on 293T/ACE2/TMPRSS2 cells, MERS-CoV PV titrated on Huh7 cells (Grehan et al., 2015). Alternatively, MERS-CoV-2 PV may be titrated on 293T cells that have been pre-transfected with a DPP4 plasmid.


  4. Pseudotype based neutralisation assay (pMN)

    Note: pMN is the Inhibition of PV mediated transduction via an antibody (or inhibitor) directed against the SARS-CoV-2 S glycoprotein.

    1. In a 96-well white plate with the aid of a multichannel pipette, add 50 µl of DMEM/10% FBS/1% P/S to rows B to H, columns 1-12.

    2. Add known amount of antibody (for example, 5 µl convalescent sera or mAb at 10 µg/ml) into wells of row A, columns 2-10 in a total volume of 100 µl DMEM/10% FBS/1% P/S. Add known amount (e.g., 5 µl) of positive and negative antisera into wells A11 and A12 as controls.

    3. Remove 50 µl from row A wells and perform two-fold serial dilutions down all the wells beneath it.

    4. With each dilution step, use a multichannel pipette to mix 5-10 times by pipetting up and down and taking care not to produce air bubbles.

    5. After completing serial dilution, the final 50 µl from the final well of each column should be discarded.

      Note: At this point, each well should contain 50 µl of mixed DMEM and serial dilutions of antibody/inhibitor.

    6. Centrifuge plate for 1 min at 500 rpm to ensure no inhibitor or liquid is located on the walls of the well.

    7. Using data obtained from the titration (Table 1), calculate the amount of DMEM required to dilute your SARS-CoV-2 PV to obtain 1 × 106 RLU in 50 µl, with a total volume of 5 ml. For example, with an RLU/ml of 1 × 108, 1 ml of PV should be mixed with 4 ml of DMEM.

    8. Mix this diluted PV solution in a pipette basin using the multichannel pipette, and aliquot 50 µl into each well on the plate, with the exception of wells A6-A12 (cell only control). A1-A6 will serve as PV only control.

    9. Centrifuge plate for 1 min at 500 rpm to ensure no virus is left on the walls of the well. This is critical to avoid virus spikes in the downstream data.

    10. Incubate the plates for 1 h at 37°C 5% CO2, allowing time for the antibody/inhibitor to bind the SARS-CoV-2 glycoprotein.

    11. Prepare a plate of susceptible target cells (HEK 293T/17 for SARS-CoV-2 PV), preferentially transfected 24 h before with ACE2 and TMPRSS2:

      1. Remove culture media from plate.

      2. Wash the plate twice with 2 ml of PBS to one side of the dish to avoid cell detachment and discard.

      3. Add 2 ml of trypsin to the plate and put the plate into the incubator until the cells are detached.

      4. After cells have detached add 6 ml of DMEM/10% FBS/1% P/S to the plate to quench trypsin activity and resuspend cells gently.

      5. Count cells using TC20TM Automated Cell Counter or counting-chamber slide and add 1 × 104 cells in a total volume of 50 µl to each well.

    12. Incubate the plate for 48-72 h at 37°C 5% CO2.

    13. Read plate using Bright GloTM luciferase assay system on a GloMax® Navigator Microplate Luminometer (or equivalent) by removing the medium from all wells and adding 25 µl of a 1:1 mix of PBS:Bright GloTM luciferase assay reagent.

    14. From the raw data provided by the luminometer, calculate the half maximal inhibitory concentration (IC50) neutralising antibody titres using the previously optimised protocol from our group, which is currently being used by the NIBSC and other stakeholders (Ferrara and Temperton, 2018).

    15. Results are shown in Figure 5.



      Figure 5. Neutralisation of SARS-CoV-2 PV entry into target cells (HEK 293T/17 expressing ACE2 and TMPRSS2) using reference plasma panel from NIBSC or neutralising mAbs from Native Antigen. Top panel: bar chart showing IC50s for panel of convalescent plasma run in three different laboratories. Middle panel: IC50 data for panel of plasma run in three different laboratories. Bottom panel: mAb neutralisation of SARS-CoV-2 pseudotypes (Native Antigen MAB12443 and MAB12444).


  5. Storage of SARS-CoV-2 PV at different temperatures

    This protocol allows simulation of different temperature conditions and shipment duration from lab to lab for collaborative studies. UK to UK shipment will routinely be <24 h, UK to EU shipment will be <72 h. For international destination shipments that may be >72 h, dry ice shipment or lyophilisation is recommended.

    1. Place eight aliquots of 100 µl of SARS-CoV-2 PV (prepared above) at different storage temperature for 24 h or 72 h prior to titration (two aliquots each were kept at RT, +4°C, -20°C or -80°C) (see Figure 6 for results).

    2. After 24 h, add four aliquots (one aliquot for each different storage temperature) to row A wells of a 96-well white plate to perform a titration. See Procedure C on how to perform a titration.

    3. After 72 h, add the remaining four aliquots at different temperatures to row A wells of a 96-well white plate to perform a titration. See Procedure C on how to perform a titration.



      Figure 6. Short term storage of SARS-CoV-2 PV at different laboratory temperatures. PV were stored at RT, +4°C, -20°C and -80°C for 24 or 72 h and then titrated. The PV aliquots kept at -80°C act as the positive control for titration experiment. -80°C is routinely used for long term storage of PV.


  6. Lyophilisation of SARS-CoV-2 PV

    We have previously shown that lentiviral pseudotypes with influenza, filovirus, or lyssavirus glycoproteins on their surface can be lyophilised for long term storage and shipping (Mather et al., 2014). This methodology is applicable equally to coronavirus pseudotypes.

    1. Dissolve sucrose in DPBS to make a 1 M cryoprotectant solution. Syringe sterilise through a 0.45 µm filter.

    2. Produce a stock of detached, standard microtube lids with single hole piercings using a sterile syringe needle (allowing vapour to escape during sample freeze-drying).

    3. Add 100 µl of high-titre SARS-CoV-2 PV produced above to 100 µl of cryoprotectant in a low-retention microtube. Vortex mix for 5 s.

    4. Leave low-retention tube lid open, replacing with a pierced lid.

    5. Place samples in plastic racks in a -80°C freezer for a minimum of 1 h.

    6. Switch on power to freeze dryer, then vacuum pump.

    7. Press MAN button on freeze dryer

    8. When temperature reaches -50°C, place sample racks in upper chamber and close valve.

    9. Press VACUUM button and wait until series of orange lights have lit up until final green light is illuminated (typically 0.035 mBar/3.5 Pa). Leave overnight (16-20 h).

      Note: Samples should not bubble or move up microtubes during lyophilisation (see Figure 7).

    10. Open top valve slowly to equalise chamber to atmospheric pressure.

    11. Switch off VACUUM, then MAN buttons and finally power to vacuum pump and freeze dryer.

    12. Retrieve samples, remove pierced lids, and close microtubes with original lids

    13. Store in freezer (-80°C or -20°C). For stability testing in other conditions, monitoring can be conducted using a temperature/humidity meter.



      Figure 7. Lyophilised SARS-CoV-2 PV pellets within the sample drying chamber


  7. Titration of reconstituted SARS-CoV-2 PV immediately after lyophilisation and employment in a neutralisation assay

    1. SARS-CoV-2 PVs were lyophilised as described above (Procedure F).

    2. Immediate reconstitution of the lyophilised pellets was done by adding 100 μl of either DPBS or complete cell culture media to compare the influence of the reconstitution solution in a titration.

    3. Recommended step: mix well with the help of a vortex mixer and incubate for 10 min at RT before further application to ensure entire resuspension of the pellets.

    4. Perform a titration as described above (Procedure C) using the reconstituted aliquots.

    5. Perform a pMN as described above (Procedure D) using lyophilised and reconstituted SARS-CoV-2 pellets (in this case, DMEM was chosen as reconstitution solution).

    6.  Results (Table 2, Figure 8, Figure 9)


      Table 2. Titre comparison of lyophilised SARS-CoV-2 PV reconstituted either in DPBS or in complete cell culture media (DMEM). Viral titres were compared to their non-lyophilised counterpart (CTRL). Analysis of SARS-CoV-2 PV titration data and calculation of RLU/ml has been carried out as in Table 1.
      Note: Consider the cytotoxic effect of the cryoprotectant (sucrose). Therefore, the first 4 dilution points of the lyophilised samples are not taken into account to calculate the mean/average (final RLU/ml).




      Figure 8. RLU/ml production titres of lyophilised SARS-CoV-2 PV reconstituted either in DPBS or DMEM, compared with their non-lyophilised counterpart (CTRL).



      Figure 9. Neutralisation of SARS-CoV-2 viral pseudotype using reference plasma panel from NIBSC. Top panel: IC50 data for panel of plasma. Bottom panel: plasma neutralisation of SARS-CoV-2 PV. These results accomplished by using lyophilised and reconstituted SARS-CoV-2 PV are comparable with Figure 5 (VPU Kent), where non-lyophilised PV was employed. Note: the reconstituted pellets containing cryoprotectant are diluted before adding the PV to the pseudotype virus neutralisation assay (PVNA) (as normal procedure). Therefore, cytotoxicity was not encountered. Neutralisation was tested on four out of six of the original NIBSC plasma panel.


  8. Replacement of SARS-CoV-S wild-type cassette in pCAGGS or subcloning of SARS-CoV-2-S wild-type cassette into alternative backbone plasmids (application for new Spike variants or coronaviruses)

    Separately restriction digest donor (2 µg) and recipient plasmids (1 µg) using appropriate enzymes according to manufacturer’s instructions (cut either side of S gene, but not internally). For the former, use Anza kit Red Buffer (with gel loading dye); for the latter, use Standard Buffer and nuclease free water for both.

    1. Pour a 50 ml 1% TAE Ultrapure agarose gel, adding 50 µl SYBR Safe when microwaved gel mix cools to ~50°C.

    2. Load donor plasmid digest (and DNA ladder) onto gel and run for 1 h at 80 V.

    3. Place gel on transilluminator (70% intensity setting) to visualise DNA and carefully excise Spike gene gel fragment using a clean scalpel.

    4. Purify gene with the QIAquick Gel Extraction kit according to manufacturer’s instructions. Elute in nuclease free water.

    5. Purify recipient plasmid with the QIAquick PCR Purification kit according to manufacturer’s instructions. Elute in nuclease free water.

    6. Measure concentration of purified recipient plasmid and Spike gene fragment via Nanodrop

    7. Perform Anza ligation reaction according to manufacturer’s instructions using 50 ng of recipient plasmid and a 1:3 molar ratio of S gene DNA.

    8. Transform ligation into Subcloning Efficiency E. coli cells according to manufacturer’s instructions, using LB broth as a culture medium.

    9. Prepare LB agar plates containing appropriate antibiotic (i.e., ampicillin, Sigma-Aldrich 1,000× stock 100 mg/ml solution catalog number A5354).

    10. Plate out and spread each 100 µl transformed cell culture on ampicillin agar plates. Incubate at 37°C overnight.

    11. Pick discrete colonies (Figure 10) onto a grid on a new ampicillin plate. Incubate 37°C overnight.



      Figure 10. Picture of discrete bacterial colonies


    12. Pick cells from each colony into a separate 0.2 ml microtube in 50 µl nuclease free water. Lyse cells in thermocycler at 94°C for 3 min.

    13. Screen plasmid DNA in each colony lysate for presence of S gene insert using appropriate PCR primers targeting arms of particular recipient vector. Use 5 µl lysate, 12.5 µl PCR Master Mix, 7.5 µl nuclease free water. Typical thermocycler program: 94°C for 3 min, then 25 cycles of [94°C for 1 min, 50°C for 1 min, 72°C for 2 min], 72°C for 5 min.

    14. Run on agarose gel as above. Photograph gel with gel imaging system and identify positive clones with S gene insert (~3.75 kbp).

    15. Inoculate 5 ml LB-Amp broth cultures with positive clone cells from grid. Incubate in 37°C shaking incubator overnight.

    16. Purify S gene plasmid clone DNA using a miniprep kit.

    17. Sequence verify using commercial service (Eurofins, for example).

    18. Once verified, the new SARS-CoV-S plasmid can be incorporated into the PV production protocol above (Procedure A).


    Conclusions
    1. The protocol outlined provides a rapid and consistent method for the generation of high-titre viral pseudotype particles expressing the SARS-CoV-2 spike protein suitable for further downstream R&D applications. Production titres obtained are equivalent to those obtained for SARS-CoV and MERS-CoV (Figure 4).

    2. Efficient knock-down (neutralisation) of SARS-CoV-2 PV entry into target cells using human convalescent plasma and mAbs demonstrates potential utility for vaccine immunogenicity and mAb/antiviral screening. The use of readily available reagents should facilitate increased reproducibility, both intra- and inter-laboratory, as demonstrated in Figure 6. These pseudotypes can be stored at a range of laboratory temperatures (Figure 6) and may be lyophilised for long term storage and easy global distribution (Figure 7). The plug and play nature of the pseudotype system makes it straightforward to swap out the Spike cassette for a new variant Spike of SARS-CoV-2 or indeed for another coronavirus. It is hoped that this suite of protocols will facilitate the wide-scale adoption of pseudotype technologies for vaccine and therapeutic R&D on emerging coronaviruses of human and animal concern.

Acknowledgments

With thanks to my Head of School, Prof. Susan Barker and the Universities of Kent and Greenwich at Medway for allowing the VPU to remain operational during the first UK lockdown. Thanks also to Prof. Philippe De Wilde (former Deputy Vice-Chancellor Research and Innovation, University of Kent) for his generous support of VPU infrastructure and logistics. This study was funded by the MRC (MC_PC_19060) and MRC/NIHR (MC_PC_20016) HICC:Humoral Immune Correlates for COVID19 consortium. This protocol was derived from our original work (Thompson et al., 2020).

Author contributions: Conceptualization, NT, GC, AS and CT; Methodology, CDG, AS, SS, DC, MM, MD, BA, BF, DH, MS, AS, ET, GC and NT; Software, CDG and MM; Validation, GC, EB, GM and CT; Formal Analysis, NT and CDG; Investigation, CDG, GC and NT; Resources EB, GM, ET, GC; Data Curation, NT and CDG; Writing – Original Draft Preparation, NT; Writing – Review and Editing, NT, CDG, DC, SS, GM, EB, EW; Visualization, GC, CDG; Supervision, NT, SS, GC and CT; Project Administration, NT; Funding Acquisition, NT.

Competing interests

The authors declare no conflicts of interest.

References

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简介

[摘要]该协议详细介绍了一种快速可靠的方法,用于在慢病毒载体核心上生产和滴定具有 SARS-CoV-2 刺突蛋白(和 D614G 或其他相关变体,VOC)的高滴度病毒假型颗粒,并用于在表达血管紧张素转换酶 2 (ACE2) 和跨膜丝氨酸蛋白酶 2 (TMPRSS2) 的靶细胞中进行中和测定。它还提供了有关通过基因克隆、冻干和储存/运输考虑广泛部署潜力替代新刺突变体的详细说明。使用该方案获得的结果表明,SARS-CoV-2 假型可以以与 SARS-CoV 和中东呼吸综合征冠状病毒 (MERS-CoV) 假型相同的滴度产生,被人恢复期血浆和单克隆抗体中和,并在一定范围内储存实验室温度和冻干用于分配和后续应用。


[摘要] SARS-CoV-2 是 COVID-19 疾病的病原体,目前表现为全球大流行(Zhu等人,2020 年)。由于 SARS-CoV-2 的高度传染性,野生型病毒已被归类为 BSL-3 病原体,严重限制了其在许多实验室中的使用。为了规避这种生物危害限制,可以通过利用替代病毒核心生成显示 SARS-CoV-2 刺突蛋白的病毒体来生成假型病毒 (PV)(Nie等人,2020 年;Crawford等人,2020 年)。由于 PV 的单轮感染和复制缺陷特性,它们可以用于 BSL-2 实验室。使用 PV 作为平台来研究新兴病毒的血清监测、抗原特性和病毒进入机制已经得到了广泛的审查(Bentley等,2015;Li等,2018;Cantoni等,2021;Focosi等。, 2021),许多研究表明野生型病毒中和试验与 PV 中和试验之间存在高度相关性(Hyseni等人,2020 年;Schmidt等人,2020 年;Sholukh等人,2020 年;Xiong等人,2020 年)。, 2020)。此外,PV 可用作新平台的诊断对照,以检测患者的 SARS-CoV-2 感染(Sholukh等人,2020 年)。
自大流行开始以来,已经建立了许多使用一系列病毒核心产生 SARS-CoV-2 假型的协议,所有这些都表明 PV 的长期储存在 -80°C 是理想的(Crawford等人,2017 年)。, 2020; Nie等人, 2020)。这带来的问题是,与不具备光伏发电和应用专业知识的研究或诊断实验室共享光伏会产生高昂的运输成本,因为颗粒需要在干冰上运输以保持稳定。此外,低收入和中等收入国家 (LMIC) 的许多实验室无法常规访问 -80 °C 存储。在本协议中,我们介绍了我们的生成(图 1)和使用基于慢病毒的 PV(高度适应性和生物安全)表达 SARS-CoV-2 尖峰的方法,并附加了使用蔗糖作为 SARS-CoV-2 PV 冻干的步骤冷冻保存剂和冷冻干燥机。我们之前的经验表明,流感、狂犬病和马尔堡 PV 的冻干不会影响 PV 性能(Mather等,2014)。我们表明 SARS-CoV-2 PV 可以以相同的方式冻干,因此可以在室温下运输较长时间(至少一周)并重新配制为溶液,而不会损失 PV 性能(对于类似的 PV,重新配制如果保持在 4°C,则在一年内)。这将极大地扩大全球范围内对假型 SARS-CoV-2 中和试验的采用,并促进使用 SARS-CoV-2 PV 作为对照样本的诊断平台,如 MALDI-ToF(Iles等人,2020),来自金融和实用的观点。


图 1. SARS-CoV-2 假型病毒产生的示意图。HEK293T/17细胞用三种质粒(包含荧光素酶报告基因、包装构建体和 SARS-CoV-2 尖峰表达质粒的慢病毒载体)转染,用于生产携带慢病毒假型的 SARS-CoV-2 尖峰。

关键字:SARS-CoV-2冠状病毒 , 慢病毒假型, 病毒中和, 穗状变体, 冻干假模, COVID-19

材料和试剂
 
MultiGuard Barrier 移液器吸头 1-20 和 1-200 μl(Sorenson BioScience,目录号:30550T)
Nunc TM细胞培养处理的多盘(6孔)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:140675)
Nunc TM细胞培养皿 Delta 表面处理(10 厘米无菌皿)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:150350)
反应管,1.5 ml,带帽,刻度和书写区(Greiner Bio-One,目录号:616201)
Fisherbrand TM一次性使用无菌注射器 3 ml,(Fisher Scientific,Thermo Scientific TM ,目录号:14955457)
0.45 μm 注射器过滤器,醋酸纤维素(STARLAB,目录号:E4780-1453)
移液盆 (50 ml),通用
96孔白板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:136101)
微管(1.5 毫升、0.5 毫升),通用
薄壁 PCR 微管 (0.2 ml),通用
HEK 293T/17 细胞(ATCC,目录号:CRL-11268)
亚克隆效率大肠杆菌DH5α细胞(Invitrogen,目录号:18265017)
质粒
尖峰质粒:pCAGGS-SARS-CoV 尖峰(CFAR,目录号:100976)
慢病毒载体表达萤火虫荧光素酶:pCSFLW(或pCSGW对于GFP PV)(卡内尔等人,2015。)             
第二代慢病毒包装构建质粒:p8.91(表达gag、pol和rev)(Carnell等,2015)             
宿主细胞进入受体 ACE2 表达质粒:pCDNA3.1+-ACE2(Simmons等,2004)             
冠状病毒尖峰(S)蛋白引发 TMPRSS2 表达质粒:pCAGGS-TMPRSS2(Bertram等人,2010)                           
注意:有关上述质粒的信息可以在 Temperton 等人中找到。(2005)和 Carnell 等人。(2015)。VPU 提供的质粒。
Dulbecco 改良的 Eagle 培养基(DMEM),含有 4.5 g/L 葡萄糖(Pan-Biotech,目录号:P04-04510),补充有 10% 胎牛血清(FBS)(Pan-Biotech,目录号:P40-37500)和 1%青霉素/链霉素(P/S)(Pan Biotech,目录号:P06-07100)
Gibco 减少血清培养基 Opti-MEM ® (Thermo Fisher Scientific,目录号:51985034)
FuGENE ® HD 转染试剂,1 ml(Promega,目录号:E2311)
不含钙和镁的 Dulbecco 磷酸盐缓冲盐水(DPBS)(Pan-Biotech,目录号:P04-36500)
胰蛋白酶-EDTA(0.05%),酚红(Pan-Biotech,目录号:P10-040100)
可以中和SARS-CoV-2 PV的阳性对照抗体(Research Reagent for Anti-SARS-CoV-2 Ab)(NIBSC,代码:20/130,国际上有售)
COVID-19人类恢复期血浆板(NIBSC,目录号:20/118)
可以中和 SARS-CoV-2 PV 的单克隆抗体(天然抗原,目录号:MAB12443 和 MAB12444)
Bright Glo TM荧光素酶测定系统(Promega,目录号:E2650)
低表面张力聚丙烯 1.5 ml 微管(Simport,目录号:T330-7LST)
蔗糖(Sigma-Aldrich,目录号:S0389)
Dulbecco 磷酸盐缓冲盐水(DPBS;Pan Biotech,目录号:P04-361000)
尖峰质粒:pCAGGS-SARS-CoV 尖峰(CFAR,目录号:100976)
真核表达载体受体质粒:pcDNA3.1、pI.18、pCAGGS(Carnell等,2015)             
Anza酶试剂盒(Thermo Fisher Scientific,目录号:IVGN3006)
无核酸酶水(Thermo Fisher Scientific,目录号:R0582)
QIAquick PCR纯化试剂盒(Qiagen,目录号:28104)
QIAquick Gel Extraction kit(Qiagen,目录号:28704)
Tris Acetate EDTA(TAE)缓冲液(50×浓缩液;Thermo Fisher Scientific,目录号:B49)
超纯琼脂糖(Thermo Fisher Scientific,目录号:16500100)
SYBR Safe DNA凝胶染料(Invitrogen,目录号:S33102)
GeneRuler 1 kb DNA Ladder(Thermo Fisher Scientific,目录号:SM0313)
Luria Broth(LB)和LB琼脂粉(Sigma-Aldrich,目录号:L3022和L2897)
DreamTaq Green PCR Master Mix(Thermo Fisher Scientific,目录号:K1081)
Monarch 质粒小量制备试剂盒(New England Biolabs,目录号:T1010S)或替代品
 
设备
 
II 类生物安全柜(Thermo Fisher Scientific、Thermo Scientific TM 、型号:MSC-Advantage TM )
水浴或培养箱,通用
移液器(Gilson,型号:PIPETMAN ® Classic、P2、P20、P200 和 P1000 或同等产品)
多道移液器(Glison,型号:PIPETMAN L Multichannel P12 20-200 μl 或同等产品)
Fisherbrand TM无菌聚苯乙烯一次性血清移液管 5 ml 和 10 ml 1/10 ml,无菌,堵塞,单独包装(Fisher Scientific,Thermo Scientific TM ,目录号:1367610H 和 1367610J)
便携式吸取急救® XP吸器(德拉蒙德科学公司,目录号:4-000-101或同等学历)
涡流混合器,速度可调(SciQuip,型号:SP2260-VM)
Galaxy MiniStar 迷你离心机(VWR,型号:C1413V-230)
可选:BIO-RAD TC20 TM自动细胞计数器(Bio-Rad Laboratories,目录号:1450102EDU)或 FastRead 102 一次性 10 室计数网格,带有一体式丙烯酸、光学透明、盖玻片(Immune Systems,目录号:BVS100)
平板离心机(ELMI,型号:SkyLine CM-6MT)
GloMax ® Navigator 微孔板光度计(Promega,型号:GloMax ® Navigator)
FreeZone 2.5 L 冷冻干燥机(LabConCo,目录号:7670520)
样品干燥室(LabConCo,目录号:7318700)
旋转叶片真空泵 117(LabConCo,目录号:7739402)
塑料微管架(Thermo Fisher Scientific,目录号:8850)
热湿度计(Thermo Fisher Scientific,目录号:11536973)
水浴或加热块,通用
微波炉,通用
电源组,通用
凝胶电泳槽,通用
微量离心机(Thermo Fisher Scientific、Sorvall TM Legend TM )
UV透射仪(Uvitech/Sigma,目录号:Z363677)
凝胶成像系统,通用
Nanodrop 2000 分光光度计(Thermo Fisher Scientific,目录号:ND-2000)
干式 37°C 培养箱,通用
摇晃 37°C 培养箱,通用
Thermocycler(Techne,型号:Prime,目录号:5PRIMEG/02)
 
软件
 
PC或Mac Excel表格(Microsoft ® )
的GraphPad Prism ® (格拉夫派得软件)
 
程序
 
通过质粒共转染 293T 细胞(4-5 天)生产 SARS-CoV-2 PV
注意:所有步骤都应在 II 类生物安全柜中进行,以避免污染。
第 1 天:293T/17 细胞应以在转染时(第 2 天)达到 70-90% 汇合率的比例传代培养到 6 孔板中。通常每孔接种 4 × 10 5 个细胞将达到这种融合水平。单元格外观的示例如图 2 所示。
 
 
图 2. 用质粒转染 HEK293T/17 细胞之前预期的汇合示例
 
第 2 天:应使用水浴或培养箱将DMEM/10% FBS/1% P/S 和 Opti-MEM ®预热至 37°C。
为将用于转染的 6 孔板的每个孔准备两个标记的无菌 1.5 ml 微量离心管(管 1 和管 2)。
添加以下质粒构建体(总体积为 15 µl)用于转染至管 1:
pCAGGS-SARS-CoV-2 或 pCAGGS-SARS-CoV-2 (D614G) 峰值:450 ng。
p8.91-慢病毒载体:500 ng。
pCSFLW:750 纳克。
注意:每个质粒的体积由其浓度决定。
将 100 µl Opti-MEM ®添加到质粒 DNA 混合物(管 1)中。
将 100 µl Opti-MEM ®和 FuGENEHD 添加到管 2。
孵育步骤:在室温 (RT) 下轻轻轻弹和孵育 5 分钟,将两个管混合。
孵育后,将管 2 中的 Opti-MEM ® /FuGeneHD 溶液移入管 1 中的 Opti-MEM ® /DNA 溶液中。
孵育步骤:将试管在室温下孵育 20 分钟,同时每 3-4 分钟轻轻轻弹试管混合。
在培养转染混合物时,应去除 293T/17 细胞上的培养基,并在每孔中加入 2 ml 新鲜预热的 DMEM/10% FBS/1% P/S。此时必须将培养基缓慢添加到孔的一侧,以避免分离贴壁细胞单层。
孵育 20 分钟后,将 DNA/Opti-MEM ® /FuGeneHD 溶液滴加到 293T/17 细胞上(在 6 孔板的一个孔中),方法是在孔的整个表面积中逐滴加入。轻轻旋转 6 孔板,以确保试剂混合物均匀分散。
孵育步骤:将板在 37°C、5% CO 2 中孵育44-52 小时。在我们手中,这个范围内的孵化时间导致等效的最终 PV 生产相对发光单位 (RLU) 滴度。
过夜孵育(第 3 天)后,应小心去除细胞上的培养基,并加入 2 ml 新鲜 DMEM/10% FBS/1% P/S。再次,将培养基缓慢添加到孔的一侧,以避免细胞脱离。
第 4 天:使用 3 ml 无菌注射器收集含有病毒假型颗粒的上清液,随后通过注射器驱动的 0.45 µm 过滤器过滤到微量离心机或 Falcon 管中。
将所有过滤的上清液储存在 -80°C 直至下游使用。建议将上清液分装储存,以避免可能影响病毒 RLU 滴度的多次冻融循环。
可选步骤(第 4 天):可以将 2 ml 新鲜培养基添加到细胞中,以便在 18-24 小时后(第 5 天)通过进一步添加 DMEM/10% FBS/1% P/S 进行第二次收获。在这种情况下,在初始 PV 收集(上面的步骤 14)时必须格外小心,以避免通过用无菌注射器将细胞吸入孔的一侧而损坏细胞单层。我们观察到,第一次收获后健康状况不佳的细胞会导致第二次收获时 PV 生产 RLU 滴度显着降低。
注意:可以按照上述步骤创建对照假型病毒,但省略 pCAGGS-SARS-CoV 尖峰结构。这会产生不表达病毒表面糖蛋白的颗粒(Delta S PV 控制)。
 
用于滴定和中和试验的靶细胞的制备(1 天)
注:SARS-CoV-2 病毒宿主细胞进入取决于受体 ACE2 和丝氨酸蛋白酶 TMPRSS2 进行 S 蛋白启动(霍夫曼n等,2020)。HEK293T/17 用 ACE2 和 TMPRSS2 质粒转染,用作 SARS-CoV-2 PV 进入的最佳靶细胞。因此,无论是否计划进行 PV 滴定或中和试验,都必须预转染细胞。
HEK 293T/17 细胞应以在转染时达到 70-90% 汇合率的比例接种到 10 cm 细胞培养皿中。通常接种 2 × 10 6 个细胞/板并在 37°C 下孵育过夜将达到这种融合水平。
DMEM/10% FBS/1% P/S 和 Opti-MEM ®应使用水浴或培养箱预热至 37°C。
为每个用于转染的细胞培养皿准备一个无菌的 1.5 ml 微量离心管。
添加以下质粒构建体(在分子生物学级水中的总体积为 15 µl)用于转染(DNA 混合物):
pCDNA3.1+-ACE2 细胞进入受体:2 µg
pCAGGS-TMPRSS2 丝氨酸蛋白酶:150 ng
注意:所需的质粒量由事先优化确定。
将 100 µl Opti-MEM ®添加到质粒 DNA 混合物中。
孵育步骤:通过轻轻轻弹混合管并在室温下孵育 5 分钟。
孵育后,将 9 µl FuGENE ® HD 直接加入 Opti-MEM ® /DNA 溶液管表面下方。
通过轻轻轻弹管混合。
孵育步骤:将试管在室温下孵育 20 分钟,同时每 3-4 分钟轻轻轻弹试管混合。
在培养转染混合物时,应去除 293T/17 细胞上的培养基,并在每道菜中加入 10 ml 新鲜 DMEM/10% FBS/1% P/S。此时必须将培养基缓慢添加到孔的一侧,以避免分离贴壁细胞单层。
孵育 20 分钟后,将 DNA/Opti-MEM ® / FuGENE ® HD 溶液滴加到 293T/17 细胞的整个培养皿的整个表面积上。轻轻旋转 10 cm 细胞培养皿,以确保试剂混合物均匀分散。
孵育步骤:在 37°C、5% CO 2下孵育板过夜(16-24 小时)。在我们手中,这个孵育时间导致细胞进入受体 (ACE2/TMPRSS2) 充分过度表达,用于 SARS-CoV-2 PV 进入。
 
用于计算相对光单位 (RLU)/ml 的 SARS-CoV-2 PV 滴定
笔记:
滴定包括将报告基因(在这种情况下是萤火虫荧光素酶,但可以使用 GFP)转导到由病毒假型(SARS-CoV-2 尖峰)上表达的病毒糖蛋白介导的靶细胞中。在滴定前 24 小时用 ACE2 和 TMPRSS2 转染靶细胞。ACE2/TMPRSS2 的转染效率在多个操作员和不同实验室之间始终可重现;在极少数转染不理想的情况下,PV 滴度急剧下降,导致结果无法使用。 
通过包含“仅细胞”和“Delta S”(无 Spike 质粒)列来提供滴定控制。可以通过带有水疱性口炎病毒 G 蛋白 (VSV-G) 的 PV 提供转导的阳性对照,该蛋白利用无处不在的受体,在所有测试的细胞系中产生高 RLU 滴度。
 
在 96 孔白板中,将 50 µl DMEM/10% FBS/1% P/S 添加到“仅细胞”对照的整个列中(参见图 3,第 9-11 列)。
从 B 行到 H 行添加 50 µl DMEM/10% FBS/1% P/S,它们将包含 PV 或 Delta S 控制病毒。
将 100 µl SARS-CoV-2 假型病毒上清液添加到 A 行(不包括仅细胞控制列)的每个孔中,并将 100 µl Delta S 添加到第 12 列(见图 3)。
 
 
图 3. 用于假型滴定的 96 孔板设置。连续稀释步骤显示向 A 行的每个孔中添加 100 µl 假型病毒上清液,并从该孔中稀释 50 µl 到 B 行。该过程持续到板的末端(H 行),此时最后 50 µl 被丢弃。Delta S 控件以红色表示(第 12 列),仅单元格控件以蓝色表示(第 9-11 列)。每个稀释系列(板)可以使用一套移液器吸头。
 
在 12 通道移液器的帮助下,从 A 排含有病毒的孔中取出 50 µl,并在其下方的所有孔中进行两倍系列稀释。
在每个稀释步骤中,通过上下移液混合至少五次,注意不要产生气泡。
完成连续稀释后,应丢弃每列最后孔中的最后 50 µl。
注意:此时,每个孔应仅包含 50 µl PV 上清液(A 行)或与 DMEM 混合并稀释(B 行到 H 行)。
准备一盘易感靶细胞(表达 ACE2 和 TMPRSS2 的 HEK 293T/17 用于 SARS-CoV-2 PV):
从板上取出培养基。
用 2 ml PBS 在培养皿的一侧清洗盘子两次,以避免细胞脱离并丢弃。
向板中加入 2 ml 胰蛋白酶,然后将板放入培养箱中直至细胞分离(约 5 分钟)。
细胞分离后,向板中加入 6 ml DMEM/10% FBS/1% P/S 以淬灭胰蛋白酶活性并轻轻重悬细胞。
使用 TC20 TM自动细胞计数器或计数室载玻片对细胞进行计数,并在每孔中加入 1 × 10 4细胞,总体积为 50 µl。
如果孔的侧面有可见的液滴,则将板以 500 rpm 的速度离心 1 分钟。
在 37°C 5% CO 2下孵育板 48 小时。
使用 Bright Glo TM荧光素酶检测系统在 GloMax ® Navigator 微孔板发光计(或等效物)上读取板,方法是从所有孔中取出培养基并加入 25 µl 1:1 混合的 PBS:Bright Glo TM荧光素酶检测试剂。
数据分析
来自光度计的 RLU 读数乘以每个孔的稀释系数(20×、40×、80×、160×、320×、640×、1,280×、2,560×)得到 RLU/ml。所有 8 个 RLU/ml 值的平均值用作滴定步骤中为该列报告的最终值。应观察到 RLU 值和 PV 稀释之间的线性关系,每 1:2 稀释后值下降 50%。在乘法之前应注意检查这种线性关系,因为这本身会导致计算错误的生产滴度(表 1)。
 
表 1. SARS-CoV-2 PV 滴定数据分析和 RLU/ml 计算。RLU 值(上图)相乘,得出每个稀释点(下图)的 RLU/ml 值。然后从所有八个稀释点计算平均值/平均值(表示为 RLU/ml)。必须注意观察稀释因子(X 因子)和 RLU 之间的线性关系,否则乘法会导致 RLU/ml 值膨胀。分析Excel软件(微软执行® )。对于 SARS-CoV-2 PV ,当靶细胞中同时存在 ACE2 和 TMPRSS2 时(第 8 列),本实验中的最佳生产滴度(3.1 × 10 8 RLU/ml)就实现了。
 
 
结果(图 4)
 
 
图 4. SARS-CoV-2 PV(和 D614G 变体)的生产以及与 SARS-CoV(Temperton等,2005)和 MERS-CoV(Grehan等,2015)的比较。显示 MERS-CoV、SARS-CoV、SARS-CoV-2 和 SARS-CoV-2 (D614G) 的 RLU/ml 生产滴度。SARS-CoV、SARS-CoV-2 和 SARS-CoV-2 PV 在 293T/ACE2/TMPRSS2 细胞上滴定,MERS-CoV PV 在 Huh7 细胞上滴定(Grehan等,2015)。或者,可以在已用 DPP4 质粒预转染的 293T 细胞上滴定 MERS-CoV-2 PV。                            
 
基于假型的中和试验 (pMN)
注意:pMN 是通过针对 SARS-CoV-2 S 糖蛋白的抗体(或抑制剂)抑制 PV 介导的转导。
在多道移液器的帮助下,在 96 孔白板中,将 50 µl DMEM/10% FBS/1% P/S 添加到 B 行到 H 行,第 1-12 列。
将已知量的抗体(例如,5 µl 恢复期血清或 10 µg/ml 的 mAb)加入 A 行第 2-10 列的孔中,总体积为 100 µl DMEM/10% FBS/1% P/S。将已知量(例如5 µl)的阳性和阴性抗血清添加到 A11 和 A12 孔中作为对照。
从 A 排孔中取出 50 µl,并在其下方的所有孔中进行两倍系列稀释。
在每个稀释步骤中,使用多通道移液器上下移液混合 5-10 次,注意不要产生气泡。
完成连续稀释后,应丢弃每列最后孔中的最后 50 µl。
注意:此时,每孔应含有 50 µl 混合 DMEM 和抗体/抑制剂的系列稀释液。
将板以 500 rpm 的速度离心 1 分钟,以确保孔壁上没有抑制剂或液体。
使用从滴定中获得的数据(表 1),计算稀释 SARS-CoV-2 PV 以在 50 µl 中获得 1 × 10 6 RLU所需的 DMEM 量,总体积为 5 ml。例如,当 RLU/ml 为 1 × 10 8 时,1 ml PV 应与 4 ml DMEM 混合。
使用多通道移液器将这种稀释的 PV 溶液混合在移液器盆中,并将 50 µl 等分到板上的每个孔中,孔 A6-A12(仅细胞控制)除外。A1-A6 将用作仅 PV 控制。
以 500 rpm 的速度离心板 1 分钟,以确保孔壁上没有病毒。这对于避免下游数据中的病毒峰值至关重要。
在 37°C 5% CO 2下将平板孵育 1 小时,让抗体/抑制剂有时间结合 SARS-CoV-2 糖蛋白。
准备一盘易感靶细胞(用于 SARS-CoV-2 PV 的 HEK 293T/17),优先在 24 小时前用 ACE2 和 TMPRSS2 转染:
从板上取出培养基。
用 2 ml PBS 在培养皿的一侧清洗盘子两次,以避免细胞脱离并丢弃。
向板中加入 2 ml 胰蛋白酶,然后将板放入培养箱中直至细胞分离。
细胞分离后,向板中加入 6 ml DMEM/10% FBS/1% P/S 以淬灭胰蛋白酶活性并轻轻重悬细胞。
使用 TC20 TM自动细胞计数器或计数室载玻片对细胞进行计数,并在每孔中加入 1 × 10 4细胞,总体积为 50 µl。
在 37°C 5% CO 2下孵育板 48-72 小时。
使用 Bright Glo TM荧光素酶检测系统在 GloMax ® Navigator 微孔板发光计(或等效物)上读取板,方法是从所有孔中取出培养基并加入 25 µl 1:1 混合的 PBS:Bright Glo TM荧光素酶检测试剂。
根据光度计提供的原始数据,使用我们小组先前优化的协议计算半数最大抑制浓度 (IC50) 中和抗体滴度,该协议目前由 NIBSC 和其他利益相关者使用(Ferrara 和 Temperton,2018 年)。                                         
结果如图 5 所示。
 
 
图 5.使用来自 NIBSC 的参考血浆面板或中和来自天然抗原的 mAb 中和进入靶细胞(表达 ACE2 和 TMPRSS2 的 HEK 293T/17)的 SARS-CoV-2 PV。顶部面板:条形图显示在三个不同实验室运行的恢复期血浆面板的 IC50。中间面板:三个不同实验室中运行的血浆面板的 IC50 数据。下图:SARS-CoV-2 假型(天然抗原 MAB12443 和 MAB12444)的 mAb 中和。
 
SARS-CoV-2 PV在不同温度下的储存
该协议允许模拟不同的温度条件和从实验室到实验室的运输持续时间以进行协作研究。英国到英国的发货时间通常<24小时,英国到欧盟的发货时间<72小时。对于可能超过 72小时的国际目的地货件,建议使用干冰运输或冻干。
滴定前将八份 100 µl SARS-CoV-2 PV(如上制备)在不同的储存温度下放置 24 小时或 72 小时(每份两份保存在室温、+4°C、-20°C 或 - 80°C)(结果见图 6)。
24小时后,在 96 孔白板的 A 排孔中加入四份(每个不同的储存温度各一份)进行滴定。有关如何进行滴定,请参见程序 C。
72小时后,将剩余的四等分在不同温度下添加到 96 孔白板的 A 行井中进行滴定。有关如何进行滴定,请参见程序 C。
 
 
图 6. SARS-CoV-2 PV 在不同实验室温度下的短期储存。PV在室温、+4°C、-20°C 和 -80°C 下储存24 或 72小时,然后滴定。保持在 -80°C 的 PV 等分试样作为滴定实验的阳性对照。-80°C 通常用于 PV 的长期储存。
 
SARS-CoV-2 PV 的冻干
我们之前已经表明,表面具有流感、丝状病毒或狂犬病病毒糖蛋白的慢病毒假型可以冻干以进行长期储存和运输(Mather等,2014)。这种方法同样适用于冠状病毒假型。             
将蔗糖溶解在 DPBS 中,制成 1 M 冷冻保护剂溶液。注射器通过 0.45 µm 过滤器消毒。
使用无菌注射器针头(允许蒸汽在样品冷冻干燥过程中逸出)生产一批带有单孔穿孔的分离式标准微管盖。
在低保留微管中将 100 µl 高滴度 SARS-CoV-2 PV 添加到 100 µl 冷冻保护剂中。涡旋混合 5 秒。
将低滞留管盖打开,更换为穿孔盖。
将样品放在塑料架中,放入 -80°C 冰箱中至少 1 小时。
打开冷冻干燥机的电源,然后打开真空泵。
按下冷冻干燥机上的 MAN 按钮
当温度达到-50°C 时,将样品架放在上腔室并关闭阀门。
按下 VACUUM 按钮并等到一系列橙色灯亮起,直到最后的绿灯亮起(通常为 0.035 mBar/3.5 Pa)。离开过夜(16-20 小时)。
注意:样品在冻干过程中不应起泡或向上移动微管(见图 7)。
缓慢打开顶部阀门,使腔室压力与大气压力相等。
关闭 VACUUM,然后关闭 MAN 按钮,最后关闭真空泵和冷冻干燥机的电源。
取回样品,取下穿孔的盖子,并用原始盖子关闭微管
储存在冰箱中(-80°C 或 -20°C)。对于其他条件下的稳定性测试,可以使用温度/湿度计进行监控。
 
 
图 7. 样品干燥室内冻干的 SARS-CoV-2 PV 颗粒
 
冻干后立即滴定重建的 SARS-CoV-2 PV 并在中和试验中使用
如上所述(程序 F)冻干 SARS-CoV-2 PV。
通过添加 100 μl DPBS 或完全细胞培养基来立即重构冻干沉淀,以比较重构溶液在滴定中的影响。
推荐步骤:在涡旋混合器的帮助下充分混合,并在进一步应用之前在室温下孵育 10 分钟,以确保颗粒完全重新悬浮。
使用重组的等分试样按上述(程序 C)进行滴定。
使用冻干和重组的 SARS-CoV-2 颗粒(在这种情况下,选择 DMEM 作为重组溶液)执行上述 pMN(程序 D)。
 结果(表 2、图 8、图 9)
 
表 2. 在 DPBS 或完全细胞培养基 (DMEM) 中重建的冻干 SARS-CoV-2 PV 的滴度比较。将病毒滴度与其非冻干对应物 (CTRL) 进行比较。SARS-CoV-2 PV滴定数据的分析和RLU/ml的计算如表1所示。
注意:考虑冷冻保护剂(蔗糖)的细胞毒性作用。因此,在计算平均值(最终 RLU/ml)时不考虑冻干样品的前 4 个稀释点。
 
 
 
图 8. 与非冻干对应物 (CTRL) 相比,在 DPBS 或 DMEM 中重建的冻干 SARS-CoV-2 PV 的 RLU/ml 生产滴度。
 
 
图 9. 使用来自 NIBSC 的参考血浆面板中和 SARS-CoV-2 病毒假型。顶部面板:等离子体面板的 IC50 数据。下图:SARS-CoV-2 PV 的血浆中和。通过使用冻干和重组的 SARS-CoV-2 PV 获得的这些结果与图 5(VPU Kent)相当,其中使用了非冻干 PV。注意:在将 PV 添加到假型病毒中和试验 (PVNA) 之前,将含有冷冻保护剂的重组颗粒稀释(作为正常程序)。因此,没有遇到细胞毒性。对六分之四的原始 NIBSC 等离子面板进行了中和测试。
 
替换 pCAGGS 中的 SARS-CoV-S 野生型盒或将 SARS-CoV-2-S 野生型盒亚克隆到替代骨架质粒中(适用于新的 Spike 变体或冠状病毒)
根据制造商的说明,使用适当的酶分别限制性消化供体 (2 µg) 和受体质粒 (1 µg)(切割 S 基因的任一侧,但不在内部)。对于前者,使用 Anza kit Red Buffer(带有凝胶加载染料);对于后者,两者都使用标准缓冲液和无核酸酶水。
倒入 50 ml 1% TAE Ultrapure 琼脂糖凝胶,当微波凝胶混合物冷却至 ~50°C 时,加入 50 µl SYBR Safe。
将供体质粒消化(和 DNA 阶梯)加载到凝胶上,并在 80 V 下运行 1 小时。
将凝胶放在透射仪上(70% 强度设置)以可视化 DNA,并使用干净的手术刀仔细切除 Spike 基因凝胶片段。
根据制造商的说明,使用 QIAquick 凝胶提取试剂盒纯化基因。在无核酸酶的水中洗脱。
根据制造商的说明,使用 QIAquick PCR 纯化试剂盒纯化受体质粒。在无核酸酶的水中洗脱。
通过 Nanodrop 测量纯化的受体质粒和 Spike 基因片段的浓度
根据制造商的说明,使用 50 ng 受体质粒和 1:3 摩尔比的 S 基因 DNA 进行 Anza 结扎反应。
根据制造商的说明,使用 LB 肉汤作为培养基,将结扎转化为亚克隆效率大肠杆菌细胞。
制备含有适当抗生素(即氨苄青霉素,Sigma-Aldrich 1,000×100 mg/ml 溶液目录号 A5354)的LB 琼脂平板。
将每 100 µl 转化细胞培养物铺在氨苄青霉素琼脂平板上。在 37°C 下孵育过夜。
将离散菌落(图 10)挑选到新氨苄青霉素板上的网格上。37°C 孵育过夜。
 
 
图 10. 离散细菌菌落图片
 
从每个菌落中挑选细胞到单独的 0.2 ml 微管中,放入 50 µl 无核酸酶水中。在热循环仪中在 94°C 下裂解细胞 3 分钟。
使用针对特定受体载体的臂的适当 PCR 引物筛选每个菌落裂解物中的质粒 DNA 是否存在 S 基因插入。使用 5 µl 裂解液、12.5 µl PCR Master Mix、7.5 µl 无核酸酶水。典型的热循环仪程序:94°C 3 分钟,然后 [94°C 1 分钟、50°C 1 分钟、72°C 2 分钟]、72°C 5 分钟的 25 个循环。
如上所述在琼脂糖凝胶上运行。使用凝胶成像系统拍摄凝胶并识别带有 S 基因插入片段 (~3.75 kbp) 的阳性克隆。
用来自网格的阳性克隆细胞接种 5 ml LB-Amp 肉汤培养物。在 37°C 振荡培养箱中孵育过夜。
使用小量制备试剂盒纯化 S 基因质粒克隆 DNA。
使用商业服务(例如 Eurofins)进行序列验证。
经验证后,新的 SARS-CoV-S 质粒可以纳入上述 PV 生产协议(程序 A)。
 
结论
概述的协议为生成高滴度病毒假型颗粒提供了一种快速且一致的方法,该颗粒表达 SARS-CoV-2 刺突蛋白,适用于进一步的下游研发应用。获得的生产滴度与 SARS-CoV 和 MERS-CoV 获得的滴度相同(图 4)。
使用人恢复期血浆和 mAb 有效敲低(中和)SARS-CoV-2 PV 进入靶细胞,证明了疫苗免疫原性和 mAb/抗病毒筛选的潜在效用。使用现成的试剂应有助于提高实验室内和实验室间的重现性,如图 6 所示。这些假型可以储存在实验室温度范围内(图 6),并且可以冻干以长期储存且易于使用全球分布(图7)。假型系统的即插即用特性使得将 Spike 盒式磁带更换为 SARS-CoV-2 的新变种 Spike 或实际上用于另一种冠状病毒变得简单。希望这套协议将促进假型技术的广泛采用,用于针对人类和动物关注的新兴冠状病毒的疫苗和治疗研发。
 
致谢
 
感谢我的校长 Susan Barker 教授以及位于 Medway 的肯特大学和格林威治大学允许 VPU 在英国第一次封锁期间保持运行。还要感谢 Philippe De Wilde 教授(前肯特大学研究与创新副校长)对 VPU 基础设施和物流的慷慨支持。这项研究由 MRC (MC_PC_19060) 和 MRC/NIHR (MC_PC_20016) HICC:COVID19 财团的体液免疫相关性资助。该协议源自我们的原始工作(Thompson等人,2020 年)。
作者贡献:概念化、NT、GC、AS 和 CT;方法论、CDG、AS、SS、DC、MM、MD、BA、BF、DH、MS、AS、ET、GC 和 NT;软件、CDG 和 MM;验证、GC、EB、GM 和 CT;形式分析,NT 和 CDG;调查、CDG、GC 和 NT;资源EB、GM、ET、GC;数据管理、NT 和 CDG;写作——原稿准备,NT;写作——审查和编辑、NT、CDG、DC、SS、GM、EB、EW;可视化、GC、CDG;监督、NT、SS、GC 和 CT;项目管理,北领地;资金收购,北领地。
 
利益争夺
 
作者宣称没有利益冲突。
 
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引用:Di Genova, C., Sampson, A., Scott, S., Cantoni, D., Mayora-Neto, M., Bentley, E., Mattiuzzo, G., Wright, E., Derveni, M., Auld, B., Ferrara, B. T., Harrison, D., Said, M., Selim, A., Thompson, E., Thompson, C., Carnell, G. and Temperton, N. (2021). Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes. Bio-protocol 11(21): e4236. DOI: 10.21769/BioProtoc.4236.
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