Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

• Published in: Viruses Vol. 12; no. 5; p. 513
• Main Authors: Crawford, Katharine H. D., Eguia, Rachel, Dingens, Adam S., Loes, Andrea N., Malone, Keara D., Wolf, Caitlin R., Chu, Helen Y., Tortorici, M. Alejandra, Veesler, David, Murphy, Michael, Pettie, Deleah, King, Neil P., Balazs, Alejandro B., Bloom, Jesse D.
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 06.05.2020; MDPI
• Subjects: Angiotensin-Converting Enzyme 2; Antibodies, Neutralizing; Antibodies, Neutralizing - immunology; Antibodies, Viral; Antibodies, Viral - blood; Antibodies, Viral - immunology; Biochemical assays; Containment of Biohazards; coronavirus; Coronaviruses; COVID-19; Diagnosis; Health aspects; HEK293 Cells; Humans; lentiviral pseudotype; Lentivirus; Life Sciences; Methods; Microbiology and Parasitology; neutralization assay; Neutralization Tests; Neutralization Tests - methods; Peptidyl-Dipeptidase A; Peptidyl-Dipeptidase A - metabolism; Plasma; Plasma - immunology; Protocol; SARS-CoV-2; Spike; Spike Glycoprotein, Coronavirus; Spike Glycoprotein, Coronavirus - analysis; Virology; United States; COVID-19; Spike; ACE2; SARS-CoV-2; cytoplasmic tail; ALAYT; lentiviral pseudotype; luciferase; coronavirus; neutralization assay; 293T-ACE2
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v12050513

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.