Paul.xu at 22:05, 3 March 2021

2021-03-03T22:05:52Z

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== Pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays ==

[[File:Pseudovirus.png]]

Plasmids

pHAGE2-EF1aInt-ACE2-WT (BEI catalog number NR52512): lentiviral backbone plasmid expressing the human ACE2 gene (GenBank ID for human ACE2 is NM_021804) under an EF1a promoter with an intron to increase expression. • HDM-IDTSpike-fixK-HA-tail (BEI catalog number NR52513): plasmid expressing under a CMV promoter the Spike from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank NC_045512) codon-optimized using IDT, with the Spike cytoplasmic tail replaced by that from the HA protein of A/WSN/1933 (H1N1) influenza, and the Kozak sequence in the plasmid fixed compared to an earlier version of this plasmid. • HDM-IDTSpike-fixK (BEI catalog number NR-52514): plasmid expressing under a CMV promoter the Spike from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank NC_045512) codon-optimized using IDT and the Kozak sequence in the plasmid fixed compared to an earlier version of this plasmid. • HDM-nCoV-Spike-IDTopt-ALAYT (BEI catalog number NR-52515): plasmid expressing under a CMV promoter the Spike from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank NC_045512) codon-optimized using IDT, with the Spike containing two mutations in the cytoplasmic tail such that the last five amino acids are ALAYT. • pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI catalog number NR-52516): lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen. • HDM-Hgpm2 (BEI catalog number NR-52517): lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter. • HDM-tat1b (NR-52518): lentiviral helper plasmid expressing HIV Tat under a CMV promoter. • pRC-CMV-Rev1b (NR-52519): lentiviral helper plasmid expressing HIV Rev under a CMV promoter. • pHAGE2-CMV-ZsGreen-W (NR-52520): lentiviral backbone plasmid that uses a CMV promoter to express ZsGreen.

== Virus production ==

Pseudotyped lentiviruses can be generated by transfecting 293Ts  1. Seed 293T cells in D10 growth media (see subsection 4.2 for media composition) so that they will be 50-70% confluent the next day. For a 6-well plate, this is 5x105 cells per well (2.5x105 cells per mL). 2. At 16-24 hours after seeding, transfect the cells with the plasmids required for lentiviral production. We transfect using BioT (Bioland Scientific) following the manufacturer’s instructions and using the following plasmid mix per well of a 6-well plate (plasmid amounts should be adjusted for larger plates): • 1 ug of lentiviral backbone–we used either the ZsGreen (NR-52520) or the Luciferase-IRESZsGreen (NR-52516) backbone • 0.22 ug each of plasmids HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM-tat1b (NR-52518) • 0.34 ug viral entry protein–either SARS-CoV-2 Spike (NR-52513, NR-52514, or NR-52515), VSV G (positive control), or transfection carrier DNA (Promega E4881) as a negative control. 3. At 18 to 24 hours post-transfection, change the media to fresh, pre-warmed D10. 4. At 60 hours post transfection, collect virus by harvesting the supernatant from each well and filtering it through a 0.45 um filter. Virus can be stored at 4 C for immediate use or frozen at -80 C. The titers of Spike- and VSV G-pseudotyped lentiviruses were found to be unaffected by a freeze-thaw cycle (data not shown). All titers presented here are from virus that was frozen at -80 C prior to use.

== Titering ==

To determine viral titers, we used either flow cytometry (for viruses packaging the ZsGreen backbone) or a luciferase assay (for viruses packaging the Luciferase-IRES-ZsGreen backbone). A detailed titering protocol is described below and differences between these two readouts are noted: 1. Coat a 96-well cell-culture plate with 25 uL poly-L-lysine per well (Millipore Sigma, P4707) according to the manufacturer’s protocol. Poly-L-lysine improves cell adherence and prevents cell disruption during infection. 2. Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells per well in D10 media. 3. The next day (12-24 hours post-seeding), count at least 2 wells of cells to determine the number of cells present at infection. 4. Prepare serial dilutions of the viruses to be titered in a final volume of 150 uL D10 growth media. a. For ZsGreen virus, we started with a 1:5 dilution and made three 1:5 serial dilutions. b. For Spike-pseudotyped Luciferase_IRES_ZsGreen virus, start with undiluted virus and made three 1:3 dilutions. For VSV G-pseudotyped Luciferase_IRES_ZsGreen virus,  start with a 1:50 dilution.

5. Gently remove the media from the cells and slowly add the virus dilutions. 6. Add polybrene (Sigma Aldrich, TR-1003-G) to a final concentration of 5 ug/mL. Add 3 uL of polybrene diluted to 250 ug/mL to  final infection volume of 150 uL  . Polybrene is a polycation that helps facilitate lentiviral infection through minimizing charge-repulsion between the virus and cells [56]. 7. At 48-60 hours post-infection, collect cells for analysis:

*a. For flow cytometry:
** Look at the cells under a fluorescent microscope and select wells that appear ~1-10% positive. Harvest cells from these wells using trypsin and transfer them to a Vbottom plate or microcentrifuge tubes. Pellet cells at 300xg for 4 min and wash twice with 3% BSA in PBS. After the final wash, resuspend in 1% BSA in PBS and analyze  via flow cytometry. Use a Becton Dickinson Celesta cell analysis machine with a 530/30 filter to detect ZsGreen in the FITC channel. Resulting FCS files can be analyzed using FlowJo (v10).
**Calculate titers using the Poisson formula. If P is the percentage of cells that are ZsGreen positive, then the titer per mL is: -ln(1 – P / 100) x (number of cells / well) /(volume of virus per well in mL). Note that when the percentage of cells that are ZsGreen positive is low, this formula is approximately equal to: (% ZsGreen positive / 100) x (number of cells / well) / volume of virus per well in mL). Furthermore, the titers using even the Poisson equation will only be accurate if the percentage of cells that are ZsGreen positive is relatively low (ideally 1-10%).
* For luciferase:
**Thaw luciferase reagent (Bright-Glo Luciferase Assay System , Promega, E2610) at room temperature.  
**Prepare cells by removing 100 uL media from each well. Accounting for evaporation, this leaves ~30 uL of media in each well.
**Add 30 uL of luciferase reagent, mix well, and transfer all 60 uL to a black-bottom plate (Costar 96-well solid black, Fisher, 07-200-590).
** Incubate plate for 2 min at room temperature in the dark then measure luminescence using a plate reader. We used a Tecan Infinite M1000 Pro plate reader with no attenuation and a luminescence integration time of 1 second.
**Plot RLUs vs. virus dilution. Select an amount of virus for further assays where there is sufficient (>1000-fold) signal above virus-only background and a linear relationship between virus added and RLU.

[[File:Lentivirus Titering.JPG]]

The following protocol was developed to streamline neutralization assays with Spike-pseudotyped lentiviruses. This protocol can be performed with either human sera or plasma, or monoclonal antibodies. Note that for safety, sera or plasma should be heat-inactivated in a biosafety cabinet prior to use as described in subsection 4.6. 

1. Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells (BEI NR-52511) per well in 50 uL DMEM (2.5x105 cells per mL). Plan to infect this plate 8–12 hours post-seeding.

2. About 1.5 hours prior to infecting cells, begin preparing serum and/or ACE2 dilutions in D10:

 a. In a separate 96-well “setup” plate, serially dilute serum samples leaving 60 uL diluted serum in each well. For the data in Figures 4A & 4C, we started at an initial serum dilution of 1:80 and did serial 2.5-fold dilutions, meaning each replicate of the assay requires 5 uL of sera. For ACE2 (Fig. 4) we started with a concentration of 200 ug/mL and did serial 3-fold dilutions. b. Add 60 uL of D10 to wells corresponding to virus only and virus plus cells control wells. Add 120 uL of D10 to media only and cells only control wells. See Figure 5 for an example plate layout.

 3. Dilute virus to ~2-4x106 RLU per mL. Add 60 uL of diluted virus to all wells containing serum dilutions and the virus only and virus plus cells control wells. 4. Incubate virus and serum at 37 C for 1 hr. 5. Carefully add 100 uL from each well of the setup plate containing the sera and virus dilutions to the corresponding wells of the plate of 293T-ACE2 cells. 6. Add polybrene (Sigma Aldrich, TR-1003-G) as described in subsection 4.4 for a final concentration of 5 ug/mL in each well. 7. Incubate at 37 C for 48-60 hours before reading out luminescence or fluorescence as described in subsection 4.4. 8. Plot the data. For the curves shown in Figure 4, we fit and plotted the data using the neutcurve Python package (https://jbloomlab.github.io/neutcurve/).

[https://www.protocols.io/view/pseudotyping-lentiviral-particles-with-sars-cov-2-br44m8yw https://www.protocols.io/view/pseudotyping-lentiviral-particles-with-sars-cov-2-br44m8yw]

HEK293T cells (2 × 106) were plated in a 100-mm tissue culture dish and transfected the next day when they were about 75% confluent with a combination of the following plasmids:

*9 µg of pLV-eGFP (a gift from Pantelis Tsoulfas (Addgene plasmid # 36083; [https://n2t.net/addgene:36083 https://n2t.net/addgene:36083]; RRID:Addgene_36083),
*9 µg of psPAX2 (a gift from Didier Trono (Addgene plasmid # 12260; [https://n2t.net/addgene:12260 https://n2t.net/addgene:12260]; RRID:Addgene_12260), and
*3 µg of pCAGGS-S (SARS-CoV-2)(Catalog No. NR-52310: BEI Resources)
**or VSV-G (a gift from Tannishtha Reya (Addgene plasmid # 14888; [https://n2t.net/addgene:14888 https://n2t.net/addgene:14888]; RRID:Addgene_14888) as control.

Polyethylenimine (PEI) reagent (Millipore Sigma, #408727) was used for transfection following manufacturer’s protocols. Next day, the cells were checked for transfection efficiency under a fluorescent microscope, indicated by GFP fluorescence. The supernatants from cell culture at 24 h were harvested and stored at 4 °C and more (10 ml) complete media (DMEM + 10% FBS) was added to the plates. The supernatant from cell culture at 48 h was harvested and combined with the 24 h supernatant for each sample. The combined supernatants were spun in a tabletop centrifuge for 5 min at 2000×g to pellet the residual cells and then passed through a 0.45 micron syringe filter. Aliquots were frozen at − 80 °C. New 293T cells plated in 12 well tissue culture dish were infected with the harvested virus (supernatant) with a dilution range of 102 to 107. Virus titers were calculated by counting the GFP positive cells in the dilution with 20–100 GFP positive cells.

MMLV pseudovirions were made in a similar way. HEK293T cells were transfected with the following plasmids:

*10 µg pLNCX-GFP,
*1.2 µg pCMV-tat-HIV,
*6 µg pJK3, and
*3 µg of pL-VSV-G (control) or
**pCAGGS-S (SARS-CoV-2).

Supernatants were recovered in a fashion similar to above.
[[Category:Virus]][[Category:Protocols]]

SARS2 Pseudovirus - Revision history

Paul.xu at 22:05, 3 March 2021