Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

Abstract

<jats:title>Abstract</jats:title><jats:p>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 VSV particles, but the reagents and protocols are not widely available. Here we detail 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 make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can 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.</jats:p>

Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding

“…These 1:1 binding affinities on purified proteins parallel the measurements made using dimeric ACE2 with our yeast-displayed libraries, confirming the validity of the approach (Figures 4D, S5). Finally, we tested how some mutations affected spike-mediated entry of pseudotyped lentiviral particles into ACE2-expressing target cells ( Figure 4E) (Crawford et al, 2020). The trends observed for entry by the spike-pseudotyped lentiviral particles generally confirmed the deep mutational scanning measurements: three of four mutations that we identified as detrimental for RBD expression or ACE2 binding greatly reduced pseudovirus entry, while a mutation that had little phenotypic effect in the deep mutational scanning did not affect viral entry.…”

“…We selected seven single mutations from our deep mutational scanning measurements for validation of phenotypic effects in a spike-pseudotyped lentivirus assay (Crawford et al, 2020). Mutations were selected that exhibited deleterious effects on RBD expression (C432D) or ACE2 binding (L455Y, N501D and G502), no strong phenotypic effect on either binding or expression (N439K), and affinity-enhancing effects (Q498Y and N501F).…”

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies

“…The possibility of avidity effects during the interactions of SARS-CoV-2 S with ACE2 dimers has implications for interpretation of pseudovirus assays to measure coronavirus infectivity in the presence and absence of potential inhibitors such as antibodies. In vitro neutralization assays for SARS-CoV-2 include pseudoviruses based on HIV lentiviral particles Crawford et al, 2020;Ou et al, 2020;Robbiani et al, 2020;Wu et al, 2020a), murine leukemia virus retroviral particles (Pinto et al, 2020;Quinlan et al, 2020), and vesicular stomatitis virus (Hoffmann et al, 2020;Nie et al, 2020;Xiong et al, 2020). Each of these pseudovirus types could potentially incorporate different numbers of spikes, in which case the overall spike density would alter sensitivity to antibody avidity.…”

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

“…Thus, various assays based on vesicular stomatitis virus (VSV) or human immunodeficiency virus type -1 (HIV-1) virions, pseudotyped with the trimeric SARS-CoV-2 spike protein that are high-throughput and can executed at BSL-2 will be essential to evaluate neutralization activity. These pseudotype virus assays offer numerous advantages (Crawford et al, 2020;Nie et al, 2020), but their ability to predict plasma neutralization activity against authentic SARS-CoV-2, or correctly identify the most potent human monoclonal antibodies has not been rigorously evaluated.…”