Difference between revisions of "Part:BBa K3683000"

Revision as of 10:54, 14 October 2020

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).

Psuedovirus 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.

Results Generation of pseudovirus We cloned the full length S gene S-FL into a pCAGGS vector and generate a wild type S pseudovirus (pCAGGS-S). We evaluate the efficacy of virus packaging and production of the S-FL (full length). We found that the efficacy of virus packaging and production was very low. We then engineered a deletion mutant S1254 with a c-terminal 19 amino acid deletion (from 1255-1273, Fig 1a) and found S1254 had a much higher packaging efficacy titer (3.3E+04 in S-FL, 2.7 E+05 in S1254, Fig 1b). Therefore, we used the S1254 construct representing the wild type S and engineered S-D614G variant so S1254 was used in all subsequent experiments,. Then Based on the pCAGGS-S1254 plasmid, we generated S-D614G variant construct by PCR-based direct mutagenesis using a pair of corresponding primers listed in Materials and methods. We obtained the exact right size product (Fig1c),

Figure 1. a, the information of S-FL and S (1-1254aa) b Comparison of the pseudovirus production titers of S-FL and S1254 (1-1254) c, Amplification of S and S-D614G by PCR-based direct-mutagenesis

and purified the exact size of S-D614G PCR product by gel extraction, then we used Exnase II (C214, Vazyme) to digest uncirclized plasmid so the remaining PCR products were circled. Then circled S variant S-D614G plasmids were transformed into DH5α competent cells, single clones were select to grow recombinant plasmids in culture (Fig 2a) and correct clones were verified by by Sanger DNA sequencing (Fig 2b).

Figure 2.

a, bacterial transformation and clone isolation for S (left) and S-D614G (right) variant.

b, DNA sequencing tracing results showing correct engineering of S and S-D614G variant we then packaged pseudovirus by transfection 293T cells using either pCAGGS S, psPAX2, and pLOVE-Luc.-GFP for the S, or pCAGGS S-D614G , psPAX2, and pLOVE-Luc.-GFP for the S-D614G variant. Using GFP green fluorescence in the pLOVE-Luc.-GFP vector, we were able to observe a high transfection efficiency of packaged pseudovirus (Figure 3)

Figure 3. a, a schematic diagram for pseudovirus packaging using 293T producer cells. b, GFP fluorescent microscopic photographs showing 293T cells with successfully packaging and production of S (left) and G614D (right) pseudovirus

Infectivity and neutralization assays We applied different amounts of pseudovirus to infect the ACE2-293T cells and quantified corresponding luciferase activities. The result showed that S-D614G was more infective compared to S pseudovirus by exhibiting more green fluorescence (Figure 4a) and higher levels of luciferase activities (Fig. 4b). Next, we performed a neutralization assay in a 96-well cell culture plate, we evaluated the neutralizing activity of a monkey serum sample vaccinated from a RBD protein against S and S-D614G pseudovirus (Fig 5a). A luciferase assay result showed that antibodies in the serum from a monkey vaccinated with an RBD protein exhibit about slightly neutralizing titers against S-D614G than S (Figure 5b)

Figure 4. a, GFP fluorescence microscopic photographs showing successful infection of ACE2-293T cells by S (left) and G614D (right) pseudovirus b, quantification of infectivity levels of S wild type and S-D614G pseudovirus expressed as luciferase activities. Abbreviations: RLU, relative luciferase unit; VG, viral genome.

Figure 5. a.the schematic of neutralization assay for evaluating infectivity inhibition by antibodies. b. neutralization of infectivity by sera from immunized with an RBD vaccine (Yang et al nature 2020).

Conclusion We engineered a pseudovirus assay expressing S and D614G variants and a dual GFP-luciferase reporter system. We show a dose dependent infection curve and higher infectivity by G614D variant. We further assessed the ability of neutralization of infection by antibodies from a monkey serum sample vaccinated by an RBD vaccine. We show antibodies effectively block the interactions between the RBD of S protein and the ACE2 receptors of the original S strain as well as the predominant strain G614D, suggesting a vaccine made against original Wuhan virus can be effective against the mutated, more infectious G614D strain. Our engineered pseodoviruse system provides a universal platform for infectivity and immunity evaluation on SARS-CoV-2.