The pseudovirus system is a useful alternative approach for pathogenic viruses such as SARS-CoV-2 outside of a BSL-3 laboratory. Two pseudotyped lentivirus plasmid kits recently deposited by the Bloom Lab at the Fred Hutchinson Research Center in Seattle, Washington, provide a method of generating non-replicative pseudotyped lentiviral particles with SARS-CoV-2 spike (S) glycoprotein in a BSL-2 environment.^1,2 These kits, NR-53816 and NR-53817, combined with HEK293T cells engineered to express the SARS-CoV-2 receptor ACE2 (NR-52511), are ideal for performing SARS-CoV-2 antibody neutralization assays in a convenient 96-well format.

Production of the pseudotyped lentiviruses is achieved through the transfection of 5 plasmids. Each kit includes: one plasmid containing a viral entry protein encoding for S glycoprotein; three helper plasmids encoding for human immunodeficiency virus Gag and Pol, Tat1b, Rev1b; two helper plasmids (Tat1b and Rev1b nonsurface proteins) for lentivirus packaging; and one lentiviral backbone plasmid encoding for synthetic firefly luciferase (Luc2) and synthetic Zoanthus sp. green fluorescent protein (ZsGreen1) for visualization.

In addition to the pseudotyped lentivirus plasmid kits, BEI Resources now offers SARS-CoV-2 spike-pseudotyped lentiviruses (NR-53818 and NR-53819) with the same characteristics as those produced with the plasmid kits. NR-53818 is pseudotyped lentivirus with a C-terminally truncated S glycoprotein, a mutation that increases the titer of viral particles produced.³ NR-53819 includes the S glycoprotein with the mutation D614G, one of the first documented S glycoprotein variants, which is more infectious than wild-type SARS-CoV-2.^4,5 Each vial of pseudotyped lentivirus is sufficient for one 96-well plate of neutralization assays

The use of the SARS-CoV-2 plasmid kits to make spike-pseudotyped lentiviruses for neutralization assays has been cited in numerous publications.6^,7,8,9 Many online resources are available supporting the production of pseudotyped lentivirus and design of neutralization assays, including up-to-date protocols maintained by the Bloom Lab, as well as basic protocols on BEI Resources product information sheets.

Please refer to the product information on the BEI Resources website for detailed information on individual kit components, use restrictions and for the limited-use, research-only licenses of Luc2 and ZsGreen.

References:

1. Crawford, K. H. D., et al. “Dynamics of Neutralizing Antibody Titers in the Months after SARS-CoV-2 Infection.” J. Infect. Dis. 223 (2021): 197-205. PubMed: 33000143.
2. Crawford, K. H. D., et al. “Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.” Viruses 12 (2020): E513. PubMed: 32384820.
3. Hulswit, R. J. G., C. A. M. de Haan and B. -J. Bosch. “Coronavirus Spike Protein and Tropism Changes.” Adv. Virus Res. 96 (2016): 29-57. PubMed: 27712627.
4. Wu, F., et al. “A New Coronavirus Associated with Human Respiratory Disease in China.” Nature 579 (2020): 265-269. PubMed: 32015508.
5. Klumpp-Thomas, C., et al. “Effect of D614G Spike Variant on Immunoglobulin G, M, or A Spike Seroassay Performance.” J. Infect. Dis. 223 (2021): 802-804. PubMed: 33257936.
6. Starr, T. N., et al. “Prospective Mapping of Viral Mutations that Escape Antibodies Used to Treat COVID-19.” Science 371 (2021): 850-854. PubMed: 33495308.
7. Hu, Y., et al. “Boceprevir, Calpain Inhibitors II and XII, and GC-376 have Broad-Spectrum Antiviral Activity Against Coronaviruses.” ACS Infect. Dis. 7 (2021): 586-597. PubMed: 33645977.
8. Chandrasekar, S. S., et al. “Localized and Systemic Immune Responses Against SARS-CoV-2 Following Mucosal Immunization.” Vaccines 9 (2021): 132. PubMed: 33562141.
9. Sanchez, S., et al. “Limiting the Priming Dose of a SARS-CoV-2 Vaccine Improves Virus-Specific Immunity.” bioRxiv (2021): doi: 10.1101/2021.03.31.437931. PubMed: 33821275.

Image:  Transmission electron micrograph of SARS-CoV-2 virus particles (NIAID/CC BY 2.0)