Protocol of SARS-CoV-2 Pseudovirus (PSV)-Based Neutralization Assay

For Vaccines, therapeutic antibodies, peptides and compounds against COVID-19

SARS-CoV-2 (2019nCoV) pseudotype virus (pseudovirus, PSV) for COVID-19 related vaccines and neutralizing antibodies evaluation.

The outbreak of COVID-19, caused by SARS-CoV-2 (2019-nCoV), has been a global public health threat and caught the worldwide concern. Due to its high pathogenicity and infectivity¹, live SARS-CoV-2 should be handled under biosafety level 3 (BSL-3) conditions. GeneMedi has developed SARS-CoV-2 pseudovirus production system, from which the SARS-CoV-2 pseudotyped virus can be handled in biosafety level 2 (BSL-2)².

GeneMedi's SARS-CoV-2 (2019nCoV) pseudotype virus (pseudovirus, PSV) based neutralization assay is a standard evaluation procedure for COVID-19 related vaccines and neutralizing antibodies potency evaluation. GeneMedi's SARS-CoV-2 PSV is the core ingredient of diagnostics for neutralization serology after vaccinotherapy.

GeneMedi's SARS-CoV-2 pseudotyped virus includes wildtype and the spike mutation variants (D614G, S943P, V367F, G476S, V483A, H49Y, Q239K, A831V, P1263L, D839Y/N/E: D839Y, D839N, D839E). The GeneMedi's SARS-CoV2 PSV panel help for all-in-one vaccinotherapy evaluation.

Application-SARS-CoV-2(2019nCoV) Pseudotyped Virus Based Neutralization Assay³

In the Pseudovirus-Based Neutralization Assay (PBNA), the inhibition of viral entry into cells by neutralizing antibodies is correlated to the decreased levels of firefly luciferase signals in the ACE2 expression cells (hACE2-HEK293T, Cat. GM-SC-293T-hACE201) .

GeneMedi’s Pseudovirus Based Neutralization Assay (PBNA) is a conventional assay method that is suitable for High-Throughout Screening (HTS) without live virus engaged. The Pseudovirus Based Neutralization Assay can be used for evaluating:

1) Neutralizing antibodies (NAbs)^3,4
2) Peptides blockers^5,6 (peptide inhibitors) or protein^7,8
3) Types of Vaccines (Immunized serum)⁹
4) Compounds targeting Spike induced cell-fusion¹⁰.

Protocol of SARS-CoV-2 Pseudovirus (PSV)-Based Neutralization Assay

Materials
1. SARS-CoV-2 Pseudovirus-RFP-fLuciferase (GM-2019nCoV-PSV01)
2. Efforter cell: Alternative
A. hACE2-HEK293T stable cell line (GM-SC-293T-hACE2-01)
B. Wildtype HEK293T cell line, hACE2 vector for transfection (GMV-V-2019nCoV-041)

Pseudotyped virus of SARS-CoV-2 Spike Mutation Variants (D614G, S943P, V367F, G476S, V483A, H49Y, Q239K, A831V, P1263L, D839Y/N/E:D839Y, D839N, D839E), P2-mutated Spike protein trimer variant (P2-mutant,S1/S2 cleavage site（furin cleavage sequence)-mutant, trimerization modified), Spike(S1+S2) - B.1.1.7 lineage, Spike(S1+S2)-N501Y mutation, Spike(S1+S2)-HV 69-70 Deletion mutation(ΔH69/ΔV70))

Protocol

If your efforter cell is hACE2-HEK293T stable cell line, please begin in Step 2.

1.Transfect HEK293T with hACE2-GFP vector (GMV-V-2019nCoV-041) 24hrs before planting the cell into 96-well.

2.Plant the hACE2-HEK293T into 96-well (5,000~10,000 per well) overnight before SARS-CoV-2 PSV infection.

3.Generation of 100ul PSV-Sample mixture:

Incubate PSV-Sample mixture for 1h at room temperature.

4.Remove the medium of efforter cell in 96-well, add 100ul PSV-Sample mixture to 96well cells for infection. 3wells for a group.

5.Fluorescence imaging (RFP) 72hrs after SARS-CoV-2 PSV infection. The firefly luciferase reporter is measured by following the Promega Luciferase Assay Reagent manual.

Tips

If your samples are serum
A standard curve should be generated using serially diluted Nabs (neutralizing antibodies) as a positive control.

If your samples are therapeutic antibodies or peptides candidates
Dilute the samples into concentration gradient for IC50 value evaluation.

GeneMedi SARS-CoV-2 Pseudovirus (PSV) Based Cell Entry

Data Post Download

Figure. GeneMedi-SARS-CoV-2 Pseudovirus (PSV) Based Cell Entry validation with fluorescence(A), Luciferase activity (B) and FACS (C) after 72 hours of HEK293T infection. hACE2 significantly enhanced the infection efficiency of the SARS-CoV-2 PSV. GeneMedi SARS-CoV-2 PSV-Luciferase (Cat.GM-2019nCoV-PSV01) is recombinant pseudotyped lentiviral particles containing SARS-CoV-2 spike protein to mimic SARS-CoV-2 (2019nCoV) cell infection. GM-2019nCoV-PSV01 is a powerful tool for SARS-CoV-2 related vaccine efficacy evaluation, neutralizing antibodies, peptides blockers competitors neutralization assay, and tissue-specific infection determination.

References

1 XiaolongCai. An Insight of comparison between COVID-19 (2019-nCoV) and SARS-CoV in pathology and pathogenesis. Preprint, doi:10.31219/osf.io/hw34x (2020, paper of GeneMedi).

2 Jean K. Millet1, Tiffany Tang3, Lakshmi Nathan3, Javier A. Jaimes4, Hung-Lun Hsu3,5, & Susan Daniel3, G. R. W. Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting. J Vis Exp, doi:10.3791/59010 (2019).

3 Nie, J. et al. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect 9, 680-686, doi:10.1080/22221751.2020.1743767 (2020).

4 Jiang, S., Hillyer, C. & Du, L. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends Immunol, doi:10.1016/j.it.2020.03.007 (2020).

5 Zhang, G., Pomplun, S., Loftis, A. R., Loas, A. & Pentelute, B. L. The first-in-class peptide binder to the SARS-CoV-2 spike protein. doi:10.1101/2020.03.19.999318 (2020).

6 Xia, S. et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res 30, 343-355, doi:10.1038/s41422-020-0305-x (2020).

7 Monteil, V. et al. Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2. Cell, doi:10.1016/j.cell.2020.04.004 (2020).

8 Lei, C. et al. Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nat Commun 11, 2070, doi:10.1038/s41467-020-16048-4 (2020).

9 HuajunBai, X., XiaolongCai. Vaccines And Advanced Vaccines: -A landscape for advanced vaccine technology against infectious disease ,COVID-19 and tumor. Preprint, doi:10.31219/osf.io/ypgx4 (2020).

10 Hoffmann, M. et al. The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv, doi:10.1101/2020.01.31.929042 (2020).