TITLE: Pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays
AUTHORS: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization. </div></div>

[STEPS]
?. [Generation of Pseudotyped Lentiviral Particles]
Seed 293T cell in D10 Growth Media so 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).
?. [Generation of Pseudotyped Lentiviral Particles]
At 16-24 hours after seeding, transfect the cells with the plasmids required for lentiviral production.This protocol transfects using BioT following the manufacturer's instructions and using the following plasmid mix per well of a 6-well plate:-  (ZsGreen or Luciferase-IRES-ZsGreen backbone)- , , and - : either SARS-CoV-2 Spike (NR-52513, NR-52514, NR-52515, NR-53742, or NR-53765), VSV G (positive control), or transfection carrier DNA (Promega E4881) as a negative control.
[lentiviral backbone]
[plasmid HMD-Hgpm2]
[plasmid pRC-CMV-Rev1b]
[plasmid HDM-tat1b]
[viral entry protein]
Plasmid amounts should be adjusted for larger plates.We now recommend making virus with the spike plasmid with a 21 amino acid cytoplasmic tail deletion and D614G mutation. This is plasmid NR-53765 at BEI or plasmid 158762 at AddGene.
?. [Generation of Pseudotyped Lentiviral Particles]
At 18 to 24 hours post-transfection, change the media to fresh, pre-warmed D10.
?. [Generation of Pseudotyped Lentiviral Particles]
At 60 hours post transfection, collect virus by harvesting the supernatant from each well and filtering it through a 0.45 um SFCA low protein-binding filter.
Virus can be stored at   for immediate use or frozen at  . The titers of Spike- and VSV G-pseudotyped lentiviruses were found to be unaffected by a freeze-thaw cycle (data not shown).
?. [Titering of Pseudotyped Lentiviral Particles]
Coat a 96-well cell-culture plate with  per well according to manufacturer's protocol (Millipore Sigma, P4707).
To determine viral titers, use either flow cytometry (for viruses packaging the ZsGreen backbone) or a luciferase assay (for viruses packaging the Luciferase-IRES-ZsGreen backbone).
[poly-L-lysine]
Poly-L-lysine improves cell adherence and prevents cell disruption during infection.The assay should work without poly-L-lysine coated plates, but we think the poly-L-lysine coating should help ensure cell numbers are consistent between wells.
?. [Titering of Pseudotyped Lentiviral Particles]
Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells (BEI NR-52511) per well in D10 media.We usually seed each well with  of cells at a concentration of 2.5x105/mL.
50 µl
For the luciferase readout, we recommend seeding in a black-walled, clear-bottom, poly-L-lysine coated plate.We use plates pre-coated with poly-L-lysine (Greiner Bio-One 655936) or non-coated plates (Greiner Bio-One 655090) that we then coat ourselves as described above.
?. [Titering of Pseudotyped Lentiviral Particles]
The next day (12-24 hours post-seeding), examine cells. They should be fairly sparse (~15-20% confluency). If you will be titering via flow cytometry, count at least 2 wells of cells to determine the number of cells present at infection.
?. [Titering of Pseudotyped Lentiviral Particles]
Prepare serial dilutions of the viruses to be titered in D10 growth media. The final infection volume will be .
[D10 Growth Media]
We usually titer by adding   to the   already in the wells from seeding the cells. The suggested dilutions below refer to the final dilution, taking into account the volume of media already in the wells. Note a 1:3 final dilution is only a 1:2 dilution in the   actually added to the wells.
The below dilutions are recommendations. Any similar dilution series would work.We also recommend titering "bald" virus particles with no viral entry protein as a negative control to get the best sense of background titers. Dilute these negative control viruses in the same way you dilute your spike-pseudotyped viruses.
?. [Titering of Pseudotyped Lentiviral Particles]
For ZsGreen virus: start with a 1:3 dilution and make 4 serial 1:5 dilutions.
?. [Titering of Pseudotyped Lentiviral Particles]
For Spike-pseudotyped Luciferase_IRES_ZsGreen virus: start with undiluted virus and make five serial 1:3 dilutions.
We now recommend using a Spike plasmid with a cytoplasmic tail truncation and the D614G mutation. For this virus, you want to start with a 1:5 or 1:10 dilution and then do five serial 1:2 to 1:3 dilutions.
?. [Titering of Pseudotyped Lentiviral Particles]
For VSV G-pseudotyped Luciferase_IRES_ZsGreen virus: start with a 1:50 dilution.
?. [Titering of Pseudotyped Lentiviral Particles]
Slowly add the virus dilutions to the cells. We add  to the  already in the wells from seeding cells.
[of the virus dilutions]
[of D10 growth media]
In the first version of this protocol, we also added polybrene to a final concentration of 5 ug/mL. However, we determined this only has a very modest effect on titer and no longer add polybrene.
?. [Titering of Pseudotyped Lentiviral Particles]
At 48-72 hours post-infection, collect cells for analysis:
?. [Titering of Pseudotyped Lentiviral Particles]
Look at the cells under a fluorescent microscope and select wells that appear ~1-10% positive.
?. [Titering of Pseudotyped Lentiviral Particles]
Harvest cells from these wells using trypsin and transfer them to a V-bottom plate or microcentrifuge tubes.
?. [Titering of Pseudotyped Lentiviral Particles]
Pellet cells at 300xg for 4 min and wash twice with 3% BSA in PBS.
?. [Titering of Pseudotyped Lentiviral Particles]
After the final wash, resuspend in 1% BSA in PBS and analyze via flow cytometry. A Becton Dickinson Celesta cell analysis machine with a 530/30 filter was used to detect ZsGreen in the FITC channel. Resulting FCS files were analyzed using FlowJo (v10).
?. [Titering of Pseudotyped Lentiviral Particles]
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%).
?. [Neutralization Assay]
Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells (BEI NR-52511) per well in  (2.5x105 cells per mL).
[DMEM]
Plan to infect this plate 8–20 hours post-seeding. Essentially, cells can be seeded overnight or seeded first thing in the morning for infection that evening.If you are reading out the assay using luciferase, we recommend using black-walled, clear bottom plates.
?. [Neutralization Assay]
Approximately 1.5 hours prior to infecting cells, begin preparing serum and/or ACE2 dilutions in D10:
Example layouts are included below. Note that recently we have only been including the NoVEP virus and infecting into 293Ts (without ACE2) when titering virus. These are good controls to make sure your virus prep is good, but are probably not necessary for each neutralization assay plate (and add a fair amount of work per plate).
?. [Neutralization Assay]
In a 96-well “setup” plate (separate from the plate containing cells), serially dilute serum samples leaving  in each well. Example layouts for setup plates are above.
[diluted serum]
?. [Neutralization Assay]
Add  to wells corresponding to virus only and virus plus cells control wells.
[D10]
As seen in the above layouts, we recommend having two rows (if diluting samples horizontally across the plate) or columns (if diluting samples vertically down the plate) for the virus plus cells positive controls.Running the positive controls in duplicate allows you to normalize RLU or fluorescence readings for each experimental sample by the average of the two positive control wells in the same row or column.
?. [Neutralization Assay]
Add   to media only and cells only control wells.
[D10]
?. [Neutralization Assay]
Dilute virus to ~2-4x106 RLU per mL. Add  to all wells containing serum dilutions and the virus only and virus plus cells control wells.
[diluted virus]
After further optimization, you want to add enough virus such that each well without serum has a reading of ~200,000-800,000 RLUs.However, we have also found RLUs can vary between plate readers, so the important thing is to have a luciferase reading well above background that is consistent between wells. You may need to optimize this amount for your assay and reagents.
?. [Neutralization Assay]
Incubate the virus and serum at  for .
37 °C
?. [Neutralization Assay]
Carefully add  from each well of the setup plate containing the sera and virus dilutions to the corresponding wells of the plate of 293T-ACE2 cells.
100 µl
In the first version of this protocol, we also added polybrene to a final concentration of 5 ug/mL. However, we determined this only has a very modest effect on titer and no longer add polybrene.
?. [Neutralization Assay]
Incubate at  for  to  before reading out luminescence or fluorescense as described in the previous section.
37 °C
For lucferase neutraliization assays, remember to add the luciferase such that it is added to all wells with the same dilution and the 2 positive control wells in that same row or column at the same time.
Cells should be completely confluent (but still adherent and not overgrown) when the assay is readout. This helps ensure that cell numbers are generally equal between wells.
?. [Neutralization Assay]
Plot the data.
“This protocol first subtracted out the background signal (average of the “virus only” and “virus + 293Ts” wells) and then calculated the “maximum infectivity” for each plate as the average signal from the wells without serum (“virus + cells” wells). Then “fraction infectivity” was calculated for each well, by dividing the luciferase reading from each well by the “maximum infectivity” for that plate. For the curves shown in the associated manuscript, the fraction infectivity data was then plotted using the neutcurve Python package (https://jbloomlab.github.io/neutcurve/). This package fits a three‐parameter Hill curve, with the top baseline being a free parameter and bottom baseline fixed to zero.”
We now calculate the fraction infectivities per row or per column of the plate (depending on plate layout). We do this by dividing the RLU reading for each experimental well (containing plasma or antibody) by the average of the RLU readings for the two virus plus cells positive control wells in the same row or column as the experimental well.
?. [Titering of Pseudotyped Lentiviral Particles]
Thaw luciferase reagent at . This protocol uses the Bright-Glo Luciferase Assay System (Promega, E2610).
-80 Room temperature
?. [Titering of Pseudotyped Lentiviral Particles]
Prepare cells by removing  from each well.
[media]
Accounting for evaporation, this leaves ~30 uL of media in each well.
?. [Titering of Pseudotyped Lentiviral Particles]
Add , mix well (but avoid bubbles), and transfer all 60 uL to a black-bottom plate (Costar 96-well solid black, Fisher, 07-200-590).
[luciferase reagent]
If cells were seeded in a black-walled, clear-bottom plate, it is not necessary to transfer to an opaque black-bottom plate for the luminesence readout. Luminesence can be measured directly in the tissue-culture plate following lysis.
When doing neutralization assays, add luciferase to the plate such that all wells at the same plasma or antibody dilution get luciferase added at the same time.
?. [Titering of Pseudotyped Lentiviral Particles]
Incubate plate for  at  in the dark then measure luminescence using a plate reader. This protocol uses a Tecan Infinite M1000 Pro plate reader with no attenuation and a luminescence integration time of 1 second.
-80 Room temperature
?. [Titering of Pseudotyped Lentiviral Particles]
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.
?. [Neutralization Assays]
Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells (BEI NR-52511) per well in  (2.5x105 cells per mL).
[DMEM]
Plan to infect this plate 8–20 hours post-seeding. Essentially, cells can be seeded overnight or seeded first thing in the morning for infection that evening.If you are reading out the assay using luciferase, we recommend using black-walled, clear bottom plates.
?. [Neutralization Assays]
Approximately 1.5 hours prior to infecting cells, begin preparing serum and/or ACE2 dilutions in D10:
Example layouts are included below. Note that recently we have only been including the NoVEP virus and infecting into 293Ts (without ACE2) when titering virus. These are good controls to make sure your virus prep is good, but are probably not necessary for each neutralization assay plate (and add a fair amount of work per plate).
?. [Neutralization Assays]
In a 96-well “setup” plate (separate from the plate containing cells), serially dilute serum samples leaving  in each well. Example layouts for setup plates are above.
[diluted serum]
?. [Neutralization Assays]
Add  to wells corresponding to virus only and virus plus cells control wells.
[D10]
As seen in the above layouts, we recommend having two rows (if diluting samples horizontally across the plate) or columns (if diluting samples vertically down the plate) for the virus plus cells positive controls.Running the positive controls in duplicate allows you to normalize RLU or fluorescence readings for each experimental sample by the average of the two positive control wells in the same row or column.
?. [Neutralization Assays]
Add   to media only and cells only control wells.
[D10]
?. [Neutralization Assays]
Dilute virus to ~2-4x106 RLU per mL. Add  to all wells containing serum dilutions and the virus only and virus plus cells control wells.
[diluted virus]
After further optimization, you want to add enough virus such that each well without serum has a reading of ~200,000-800,000 RLUs.However, we have also found RLUs can vary between plate readers, so the important thing is to have a luciferase reading well above background that is consistent between wells. You may need to optimize this amount for your assay and reagents.
?. [Neutralization Assays]
Incubate the virus and serum at  for .
37 °C
?. [Neutralization Assays]
Carefully add  from each well of the setup plate containing the sera and virus dilutions to the corresponding wells of the plate of 293T-ACE2 cells.
100 µl
In the first version of this protocol, we also added polybrene to a final concentration of 5 ug/mL. However, we determined this only has a very modest effect on titer and no longer add polybrene.
?. [Neutralization Assays]
Incubate at  for  to  before reading out luminescence or fluorescence as described in the previous section.
37 °C
For lucferase neutraliization assays, remember to add the luciferase such that it is added to all wells with the same dilution and the 2 positive control wells in that same row or column at the same time.
Cells should be completely confluent (but still adherent and not overgrown) when the assay is readout. This helps ensure that cell numbers are generally equal between wells.
?. [Neutralization Assays]
Plot the data.
First, subtract the average of the background (virus only and virus+293Ts (if included)) wells from all wells. Then, calculate the fraction infectivity for each experimental well (containing plasma or antibody) compared to the virus plus cells positive control wells. We do this by dividing the RLU reading for the experimental well by the average of the RLU readings for the two virus plus cells positive control wells in the same row or column as the experimental well. This protocol used the Neutcurve Python package to plot the data.