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Team:PYMS GZ China/Protocol


Protocols
Mini Prep

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

5. Apply 800 µl of the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.

6. Centrifuge for 30–60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.

8. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

9. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.

10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl ddH2O to the center of each QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Viral RNA extraction (TAKARA, cat.9766)

1. lysis of virus

(1) take 5μl concentrated virus stock solution, add RNase free dH2O to make up to 200 μl.

(2) Add 200 μl of Buffer VGB, 20 μl of Proteinase K and 1.0 μl of Carrier RNA.Mix well and incubate it into 56℃ water bath for 10 minutes.

(3) Add 200 μl of 100% ethanol to the lysate and mix well.

2. Transfer the solution to Spin Column with Collection tube. Centrifuge at 12,000 rpm for 2 minutes. Discard the flow-through.

3. Add 500 μl of Buffer RWA into Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

4. Add 700 μl of Buffer RWB into Spin Column. Take care to add Buffer RWB along the tube wall of Spin Column to wash off any residual salt. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Note : Make sure the amount of 100% ethanol specified on the bottle label has been added to the Buffer RWB.

5. Repeat Step 4.

6. Place Spin Column into Collection Tube. Centrifuge at 12,000 rpm for 2 minutes.

7. Place Spin Column into a new 1.5 ml RNase free collection tube. Add 30 of RNase free dH2O to the center of the membrane. Let it stand for 5 minutes at room temperature. Note : When eluting RNA, please use RNase free dH2O.

8. Centrifuge at 12,000 rpm for 2 minutes to elute RNA/DNA. If more yield is needed, the flow-through can be re-added into the center of the membrane or add 30 - 50 μl of RNase free dH2O and let it stand for 5 minutes at room temperature and centrifuge at 12,000 rpm for 2 minutes to elute RNA/DNA.

9. Quantification of the RNA/DNA. Since Carrier RNA is added, RNA/DNA extracted cannot be quantitatively determined by electrophoresis or absorbance measuring. RNA/DNA can be determined by qPCR.

Protocol
S variant D614G construction based on the pCAGGS-S plasmid
  • Using pCAGGS-S plasmid as template, with following S variants corresponding primers to run PCR.
    S-D614G-FCTGTACCAGGgCGTGAATTGCACCGAGGTGC
    S-D614G-RTGCAATTCACGcCCTGGTACAGCACGGCCACC
    Red is G to C substitution.
  • PCR reaction (High-fidelity DNA polymerase Mix, Vazyme, P525):
    1 uL primer F (10 uM) + 1 uL primer R (10 uM) + 25 uL 2X PCR reaction mix +
    0.5 ul pCAGGS-S plasmid (1ug/uL) + 22.5 uL nuclease free water
    step 1 - 95°C 5 minutes
    step 2 - 95°C 30 seconds
    step 3 - 56°C 30 seconds
    step 4 - 72°C 4 minutes
    repeat step 2 to step 4 28 times
    72°C 10 minutes
    4°C hold
  • Gel electrophoresis
    1% agarose gel, 120V, for 30 minutes
    Cut the target DNA band with a correct size from agarose gel for DNA extraction
  • Gel extraction by using a gel extraction kit (QIAquick Gel Extraction Kit, cat.28704)
  • Digest pCAGGS-S plasmid remaining in the extracted product (enzyme kit, TransGen, GD111-01)
    15 ul step 4 product + 1uL DMT enzyme + buffer + nuclease free water
    37°C 1 hour
    80°C 20 minutes
    4°C hold
  • Ligate the mutant sequence to the plasmid by DNA recombination using the recombination kit (Vazyme, C214)
    0.5 uL recombination enzyme + 1 uL 5X buffer + 3.5 uL step 5 product
    37°C 30 minutes
    4°C hold
Wildtype S and S variant D614G construct amplification
  • Thaw stbl3 component cell (in the tube) on ice for 2-5 minutes
  • Carefully remove the caps from the tube and set aside for later use
  • Add 5 uL S variant construct into each tube
  • Mix the cells by gently stirring the mixture with a pipette tip three times. DO NOT mix by pipetting up and down
  • Cap the tubes and incubate the cells on ice for 30 minutes
  • Incubate the tube of cells at 42°C in a water bath. DO NOT mix or shake
  • Incubate the tube on ice for 2 minutes
  • Place the tube on the benchtop, then add 250 uL of SOC medium (prepared prior by mixing SOC powder and sterile water)
  • Cap the tube and secure at 45 angles in the incubator and shake at 37°C, 225 rpm for 1 hour
  • Plate 50 - 100 uL step 9 product on pre-warmed LB-ampicillin plate (prepared prior by mixing LB powder, sterile water and ampicillin, then pouring into the plate). Step 9 product should be evenly spread on the plate by using a sterile wiper, incubate at 37°C overnight.
  • Select 10 colonies, inoculate one single colony into one single tube of SOB ampicillin medium (so totally 10 tubes of SOB ampicillin medium)
  • Place the tubes of step 11 into the incubator, shake at 37°C, 150 rpm overnight.
  • Extract S variant construct from step 11 medium by using the plasmid extraction kit (QIAprep Spin Miniprep Kit, Qiagen, cat. 27104).
  • Send out all 10 extracted S variant construct of step 13 for sequencing to confirm their sequence.
Pseudovirus packaging and production (each S variant corresponding pseudovirus is generated at this step)
  • culture HEK293T cells (culture medium: 10% FBS DMEM + 1X Penicillin/Streptomycin) in 10 cm petri dish, continue following steps when it. reaches 80% confluency
  • transfect the step 1 cells by using lipofectamine 3000 transfection reagent (ThermoFisher Scientific, L3000015) with the following plasmid:
  • ug pLOVE-luciferase-EGFP + 6 ug psPAX2 + 2 ug S variant construct
  • 8 hours later, replace the medium of the step 2 transfected cells with 10mL fresh culture medium
  • Replace the culture medium at 48 hours. DO NOT discard the medium remaining in the dish, harvested it and store at 4°C. Then harvest the medium at 72 hours, mix with the one harvested at 48 hours (success transfection could be observed at 48 hours under fluorescent microscope for the green fluorescence)
  • Concentrate viruses with centrifugal ultrafiltration devices. After centrifugation, discard supernatant, dissolve the pellet by using 100 uL PBS
  • Examine the pseudovirus titer by using the TransLvTM lentivirus qPCR Titration Kit (TransGen, FV201)
Pseudovirus infection and neutralization
    All following test should be done with 3 replicates to achieve reliable results
  • Seed 2x104 293T-hACE2 cells each well into 96 well plates, 100 uL culture medium (Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), 100 mg/mL of streptomycin, and 100 unit/mL of penicillin) each well
  • After cells attached (~8-12 hours after step 1), replace the culture medium with 50 uL fresh medium containing pseudovirus solution (from step 5 of Pseudovirus package section). The pseudovrius solution is mixed with the fresh medium with different ratio to obtain different dilutions
  • Incubate for 12 hours, then replace the pseudovirus-containing medium with 100uL fresh medium without pseudovirus
  • 48 hours later, examine the infection efficiencies of different pseudovirus dilutions by using the Luciferase Assay Kit and determine the optimal dilution for the infection
  • After optimal dilution of infection is determined, proceed to the neutralization assay
  • For neutralization assay, step 1 cells need to be prepared
  • After cells attached (~ 8-12 hours after step 1), for each well’s preparation, mix 50 uL fresh medium with pseudovirus solution of optimal dilution, and sera/antibody with serial dilution (fresh medium + pseudovirus solution + sera/antibody)
  • Incubate the step 7 mixture (culture medium with pseudovirus and sera/antibody) at 37°C for 1 hour
  • Replace the culture medium with the step 8 mixture,
  • Incubate for 12 hours, then replace with 100 uL fresh medium
  • 48 hours later, examine the neutralization efficiencies of different sera/antibody of different dilutions by using ONE-GloTM Luciferase Assay System Technical Manual (Promega, E6120)
Materials and Methods
Plasmids

The psPAX2 was purchased from addgene, and the pLOVE-Luciferase-EGFP was purchased from GenScript (Nanjing, China), the full length Spike gene (S) from the SARS-CoV-2 (previously 2019-nCoV) strain Wuhan-Hu-1 (GenBank: MN908947) was codon-optimized (sequence shown in Supplementary Table 1), synthesized, and cloned into pCAGGS vector (pCAGGS S(1-1254aa)) using seamless cloning by GenScript. The primers S-D614G-F, 5′-CTGTACCAGGgCGTGAATTGCACCGAGGTGC-3′ and S-D614G-R 5′- TGCAATTCACGcCCTGGTACAGCACGGCCACC-3′ were used to generate S-D614G mutant by PCR-based direct mutagenesis using High-fidelity DNA polymerase Mix (P525, Vazyme) with the following condition: 95℃ 5min, 95℃ 30s, 56℃ 30s, 72℃ 4min for 28 cycles, 72℃ 10min. We next purified the exact size of S-D614G PCR product by gel extraction, then we used Exnase II (C214, Vazyme) to make the linearized product circled. Then circled S-D614G plasmids were transformed into DH5α competent cells, single clones were select to grow recombinant plasmids in culture. The information for these maps are shown in Supplementary map.

Cell culture

HEK293T cells and ACE2-293T cells (cells transfected with human ACE2) were purchased from ProCell (Wuhan, China), 293T cells were maintained in Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), 100 mg/mL of streptomycin, and 100 unit/mL of penicillin at 37 °C in 5% CO2. HEK293T cells transfected with human ACE2 (293T-ACE2) were cultured under the same conditions with the addition of G418 (0.5 mg/mL) to the medium.

Serum sample (harvested at Day 35 post vaccination by an RBD vaccine was used for infectivity inhibition experiment. Detailed information is provided by Yang et al, Nature 2020 and was a kindly gift from Dr. Jingyun Yang (West China Hospital, Sichuan University).

Production and titration of SARS-CoV-2 S pseudoviruses

We generated either wild type SARS-CoV-2 S or S-D614G variant pseudotyped virus with a luciferase reporter using an HIV-1 backbone. Specifically, 5x106 HEK293T cells in 100mm dish were co-transfected with 12 ug pLOVE-luciferase-EGFP plasmid, 6 ug psPAX2 and 2 ug recombinant SARS-CoV-2 S plasmids or SARS-CoV-2 S-D614G plasmid. Transfection was done using the lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. (A: 1ml OPTI-DMEM + 40 ul lipofectamine 3000; B: 1ml OPTI-DMEM + 40 ul P3000 + Plasmids; Mix A and B, incubate 15 min at R.T., then add the Mix into culture dish). The medium for transfected cells were replaced by a fresh Medium (10 ml) ~8 h later. The supernatant containing SARS-CoV-2 pseudoviruses were harvested at 48 h and 72 h after the initial transfection and filtered through a 0.45 um filter. Pseudoviruses were concentrated by a centrifugal ultrafiltration device, we then used 50 ul medium to dissolve viruses for one package. Titration of pseudoviruses by qRT-PCR using TransLvTM Lentivirus qPCR Titration Kit (FV201, Transgen).

Pseudovirus infection and neutralization assays

2x104 ACE2-293T cells were seeded into 96-well plates.
For an infection assay, 50 ul medium containing serial dilutions (1:1, 1 :2, 1:4, 1:8, 1:16) of pseudoviruses (~ 6.4× 105 vg) was were added to the 96-well containing ACE2-293T cells. After 12 h of infection, fresh culture medium was added to each well. Luciferase activity was measured 48 h after infection using ONE-GloTM Luciferase Assay System (E6120, Promega).

For a neutralization assay, 50 ul medium containing pseudoviruses (~4x104 vg) were incubated with media or with serially diluted sera from immunized with an RBD vaccine (from 1:1000 to 1:102400) for 1 h at 37℃, then added to the 96-well plates containing ACE2-293T cells. After 12 h of infection, fresh culture medium was added to each well. Luciferase activity was measured 48 h after infection using ONE-GloTM Luciferase Assay System (E6120, Promega).

Authentic virus neutralization assay

100 ul medium containing authentic wild type SARS-COV-2 virus or D614G mutant SARS-COV-2 virus were incubated with media or with serially diluted rRBD-15 antibody (from 1:200 to 1:25600) for 1 h at 37℃, then added to the 96-well plates that were pre-seeded with Vero E6 cells (5×104) and grown overnight. After 12 h of infection, fresh culture medium was added to each well. RNA of each well’s Vero E6 cells were extracted and reverse-transcripted by Prefill Viral Total NA Kit (thermo KFRPF-805296) 48 hours later, and viral genomic RNA (gRNA) and viral subgenomic RNA (sgRNA, indicative of viral replication) were quantitatively detected by digital PCR. Neutralization activity were calculated by dividing gRNA or sgRNA copy number of rRBD-15 wells to that of non-antibody wells. Meanwhile, 197 serum of COVID-19 patients’ neutralization activity to authentic SARS-COV-2 virus were examined in this same way.