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

Katharine H D Crawford,Andrea N Loes,Alejandro B Balazs,Jesse D Bloom,Rachel Eguia,Neil P King,Caitlin R Wolf,Adam S Dingens,Deleah Pettie,Keara D Malone,Michael Murphy,M Alejandra Tortorici,David Veesler,Helen Y Chu

doi:10.3390/v12050513

Abstract

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.

Highlights

Infection with SARS-CoV-2 elicits antibodies that bind to the virus [1,2,3,4,5,6]
We described a detailed protocol for producing SARS-CoV-2 Spike-pseudotyped lentiviral particles and performing neutralization assays
We hope this protocol and reagents will more enable others to assess the neutralizing activity of antibodies and sera reactive to SARS-CoV-2

Summary

Introduction

Infection with SARS-CoV-2 elicits antibodies that bind to the virus [1,2,3,4,5,6]. as is the case for all viruses [7,8,9,10], only some of these antibodies neutralize the virus’s ability to enter cells [4,5,11,12].Whereas studies of immunity to SARS-CoV-2 are limited, for many other viruses, neutralizing antibodiesViruses 2020, 12, 513; doi:10.3390/v12050513 www.mdpi.com/journal/virusesViruses 2020, 12, 513 are more strongly correlated with protection against reinfection or disease than antibodies that bind but do not neutralize [7,8,9,10,13,14,15]. Infection with SARS-CoV-2 elicits antibodies that bind to the virus [1,2,3,4,5,6]. As is the case for all viruses [7,8,9,10], only some of these antibodies neutralize the virus’s ability to enter cells [4,5,11,12]. Whereas studies of immunity to SARS-CoV-2 are limited, for many other viruses, neutralizing antibodies. Viruses 2020, 12, 513 are more strongly correlated with protection against reinfection or disease than antibodies that bind but do not neutralize [7,8,9,10,13,14,15]. The most biologically relevant method is to directly measure how antibodies or sera inhibit infection of cells by replication-competent SARS-CoV-2

Methods

Results

Conclusion