Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

Abstract

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra-and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

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

“…We describe a detailed protocol for producing SARS-CoV-2 Spike-pseudotyped lentiviral particles and performing neutralization assays. Although this basic pseudotyping approach has been described previously [4,12,27,29,[37][38][39][40][41], we provide the first detailed protocol that makes all reagents available in a public repository (https://www.beiresources.org/). We hope this protocol and reagents will more easily enable others to assess the neutralizing activity of antibodies and sera reactive to SARS-CoV-2.…”

“…Spike from several coronaviruses can be "pseudotyped" onto safer non-replicative viral particles in place of their endogenous entry protein, thereby making entry of these particles into cells dependent on Spike [29][30][31][32][33][34][35][36]. For SARS-CoV-2, such pseudotyping has recently been reported using HIV-based lentiviral particles [4,27,37], MLV-based retroviral particles [12,38], and VSV [29,[39][40][41]. In the data reported to date, results from such pseudovirus neutralization assays correlate well with measurements made using live SARS-CoV-2 [1,12,27,39].…”

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

“…Retrovirus and VSV are both common used pseudovirus system, but the pseudotype virus titer obtained with VSVdG system is generally higher than that of the retrovirus system [10] . Furthermore, infection of [12] . Noteworthy, the truncation of the cytoplasmic tail may increase fusion activity, which is consistent with the study of SIV-MuLV chimeric envelope protein [17] .…”

“…to study the cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses [13] . Jianhui Nie et al have successfully constructed a pseudovirus neutralization assay for SARS-CoV-2, which consist of pseudotyped VSV bearing the full-length spike protein of SARS-CoV-2 and Huh7 cell [12] . Lentiviral pseudotype bearing the truncated spike protein of SARS-CoV-2 was also constructed and used to study the virus entry and its immune cross-reactivity with SARS-CoV [6] .…”

“…Therefore, the studies of SARS-CoV neutralization assays, which based on pseudovirus bearing the spike protein of SARS-CoV, would provide valuable experience and reference for the SARS-CoV-2 assays. In the past years, pseudotyping system based on vesicular stomatitis virus (VSV) was reported to produce pseudotypes incorporating the envelope protein of heterologous risk group-3 (RG-3) or RG-4 viruses, such as Ebola virus, SARS coronavirus, MERS coronavirus et al, in which the VSV G gene is deleted (VSVdG) and the gene encoding GFP, luciferase or other reporter genes was integrated [10][11][12] . Pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus.…”