Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays
Abstract
:1. Introduction
2. Results
2.1. General Approach for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike
2.2. Target 293T Cells Constititutively Expressing Spike’s ACE2 Receptor
2.3. Titers of Pseudotyped Lentiviral Particles with Different Spike Cytoplasmic Tail Variants
2.4. Neutralization Assays with Spike-Pseudotyped Lentiviral Particles
3. Discussion
4. Materials and Methods
4.1. 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.
4.2. Creation of 293T ACE2 Cells
4.3. Detailed Protocol for Generation of Pseudotyped Lentiviral Particles
- Seed 293T cells in D10 growth media (see Section 4.2 for media composition) so that they will be 50%–70% confluent the next day. For a six-well plate, this is 5 × 105 cells per well (2.5 × 105 cells per mL).
- At 16-24 h after seeding, transfect the cells with the plasmids required for lentiviral production. We transfected using BioT (Bioland Scientific, Paramount, CA, USA) following the manufacturer’s instructions and using the following plasmid mix per well of a six-well plate (plasmid amounts should be adjusted for larger plates):
- 1 μg of lentiviral backbone–we used either the ZsGreen (NR-52520) or the Luciferase-IRES-ZsGreen (NR-52516) backbone
- 0.22 μg each of plasmids HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM-tat1b (NR-52518)
- 0.34 μg viral entry protein—either SARS-CoV-2 Spike (NR-52513, NR-52514, or NR-52515), VSV G (positive control), or transfection carrier DNA (E4881, Promega, Madison, WI, USA) as a negative control.
- At 18 to 24 h post-transfection, change the media to fresh, pre-warmed D10.
- At 60 h post transfection, collect virus by harvesting the supernatant from each well and filtering it through a 0.45 μm SFCA low protein-binding 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 single freeze-thaw cycle (data not shown). Nonetheless, we recommend freezing virus in small aliquots to avoid multiple freeze-thaw cycles. All titers presented here are from virus that was frozen at −80 °C prior to use and underwent a single freeze-thaw.
4.4. Detailed Protocol for Titering Pseudotyped Lentiviral Particles
- Coat a 96-well cell-culture plate with 25 μL poly-l-lysine per well (P4707, Millipore Sigma, Burlington, MA, USA) according to the manufacturer’s protocol. Poly-L-lysine improves cell adherence and prevents cell disruption during infection.
- Seed a poly-l-lysine-coated 96-well plate with 1.25 × 104 293T-ACE2 cells per well in D10 media.
- The next day (12–24 h post-seeding), count at least two wells of cells to determine the number of cells present at infection.
- Prepare serial dilutions of the viruses to be titered in a final volume of 150 μL D10 growth media.
- For ZsGreen virus, we started with a 1:5 dilution and made three 1:5 serial dilutions.
- For Spike-pseudotyped Luciferase_IRES_ZsGreen virus, we started with undiluted virus and made three 1:3 dilutions. For VSV G-pseudotyped Luciferase_IRES_ZsGreen virus, we started with a 1:50 dilution.
- Gently remove the media from the cells and slowly add the virus dilutions.
- Add polybrene (TR-1003-G, Sigma Aldrich, St. Louis, MO, USA) to a final concentration of 5 μg/mL. We did this by adding 3 μL of polybrene diluted to 250 μg/mL to our final infection volume of 150 μL. Polybrene is a polycation that helps facilitate lentiviral infection through minimizing charge-repulsion between the virus and cells [56].
- At 48–60 h post-infection, collect cells for analysis:
- 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 V-bottom plate or microcentrifuge tubes. Pellet cells at 300× g 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. We used a Becton Dickinson Celesta cell analysis machine with a 530/30 filter to detect ZsGreen in the FITC channel. Resulting FCS files were analyzed using FlowJo (v10) (BD Life Sciences, Ashland, OR, USA).
- 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) × (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) × (number of cells per 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 at room temperature. We used the Bright-Glo Luciferase Assay System (E2610, Promega, Madison, WI, USA).
- Prepare cells by removing 100 μL media from each well. Accounting for evaporation, this leaves ~30 μL of media in each well.
- Add 30 μL of luciferase reagent, mix well, and transfer all 60 μL to a black-bottom plate (Costar 96-well solid black, 07-200-590, Fisher Scientific, Waltham, MA, USA).
- 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 s.
- 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.
4.5. Detailed Protocol for Neutralization Assays
- Seed a poly-L-lysine-coated 96-well plate with 1.25 × 104 293T-ACE2 cells (BEI NR-52511) per well in 50 μL D10 (2.5 × 105 cells per mL). Plan to infect this plate 8–12 h post-seeding.
- About 1.5 h prior to infecting cells, begin preparing serum and/or ACE2 dilutions in D10:
- In a separate 96-well “setup” plate, serially dilute serum samples, leaving 60 μL diluted serum in each well. For the data in Figure 4A,C, we started at an initial serum dilution of 1:80 and did serial 2.5-fold dilutions, meaning each replicate of the assay required 5 μL of sera. For ACE2 (Figure 4B), we started with a concentration of 200 μg/mL and did serial three-fold dilutions.
- Add 60 μL of D10 to wells corresponding to virus only and virus plus cells control wells. Add 120 μL of D10 to media only and cells only control wells. See Figure 5 for an example plate layout.
- Dilute virus to ~2–4 × 106 RLU per mL. Add 60 μL of diluted virus to all wells containing serum dilutions and the virus only and virus plus cells control wells.
- Incubate virus and serum at 37 °C for 1 h.
- Carefully add 100 μL from each well of the setup plate containing the sera and virus dilutions to the corresponding wells of the plate of 293T-ACE2 cells.
- Add polybrene (Sigma Aldrich, TR-1003-G) as described in Section 4.4 for a final concentration of 5 μg/mL in each well.
- Incubate at 37 °C for 48–60 h before reading out luminescence or fluorescence as described in Section 4.4.
- Plot the data. For our analysis, we 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). We then calculated the “fraction infectivity” for each well, as the luciferase reading from each well divided by the “maximum infectivity” for that plate. For the curves shown in Figure 4, we then fit and plotted the fraction infectivity data 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.
4.6. Human Plasma Sample and Soluble ACE2
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Crawford, K.H.D.; Eguia, R.; Dingens, A.S.; Loes, A.N.; Malone, K.D.; Wolf, C.R.; Chu, H.Y.; Tortorici, M.A.; Veesler, D.; Murphy, M.; et al. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 2020, 12, 513. https://doi.org/10.3390/v12050513
Crawford KHD, Eguia R, Dingens AS, Loes AN, Malone KD, Wolf CR, Chu HY, Tortorici MA, Veesler D, Murphy M, et al. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses. 2020; 12(5):513. https://doi.org/10.3390/v12050513
Chicago/Turabian StyleCrawford, Katharine H. D., Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Keara D. Malone, Caitlin R. Wolf, Helen Y. Chu, M. Alejandra Tortorici, David Veesler, Michael Murphy, and et al. 2020. "Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays" Viruses 12, no. 5: 513. https://doi.org/10.3390/v12050513
APA StyleCrawford, K. H. D., Eguia, R., Dingens, A. S., Loes, A. N., Malone, K. D., Wolf, C. R., Chu, H. Y., Tortorici, M. A., Veesler, D., Murphy, M., Pettie, D., King, N. P., Balazs, A. B., & Bloom, J. D. (2020). Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses, 12(5), 513. https://doi.org/10.3390/v12050513