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

By J. Nie, Qian-qian Li, Jia-jing Wu, Chenyan Zhao, H. Hao, Huan Liu, Li Zhang, L. Nie, Haiyang Qin, Meng Wang and 8 more. Emerging Microbes and Infections, 2020

684 citations recorded by the source corpus.

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.

Subfields: Epidemiology, Genetics, Infectious Diseases

View Paper: https://www.semanticscholar.org/paper/39fd08a6dcec589c1e0c1c0ad3e2e3eab49dcfb8

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In Epidemiology

1. M. A. Tortorici
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In Genetics

1. K. H. Crawford
2. Xiaohui S. Xie
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In Infectious Diseases

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Citations in the corpus

• A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS (2020, positive): “Pseudovirus neutralization assay MERS-CoV, SARS-CoV-2 and SARS-CoV pseudovirus preparation and neutralization assay were carried out by a previously published method (Nie et al., 2020), with some modifications.”
• Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies (2020, neutral): “, 2020), and vesicular stomatitis virus (Hoffmann et al., 2020; Nie et al., 2020; Xiong et al., 2020).”
• Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum (2021, positive): “The constructs of pseudotyped lentivirus expressing SARS-CoV-2 S proteins are as previously described in Nie et al. (2020), with some modifications.”
• Infectious Diseases Society of America Guidelines on the Treatment and Management of Patients with COVID-19 (2022, background): “Individuals who have recovered from SARS-CoV-2 infection may generate neutralizing antibodies [44, 45] that could have application to prevention of infection in certain settings, such as individuals with underlying conditions predisposing to severe disease and those with high-risk exposure.”
• A Thermostable mRNA Vaccine against COVID-19 (2020, positive): “RBD protein expressed frommRNA retained high affinity for recombinant human ACE2, as demonstrated by kinetics analysis using ForteBio Octet (Figure 1C), and functionally inhibited entry of a vesicular stomatitis virus (VSV)based pseudovirus expressing the SARS-CoV-2 S protein (Nie et al., 2020) in H”
• Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum (2022, methodology): “…and pseudotyped lentiviral particles production Pseudotyped lentivirus expressing SARS-CoV-2 S proteins from ancestral strain (Victoria, S247R), BA.1, BA.1.1, and BA.2 were constructed as described previously (Nie et al., 2020; Liu et al., 2021b; Nutalai et al., 2022), with some modifications.”
• Immunology of COVID-19: Current State of the Science (2020, neutral): “nAbs are also believed to protect against infection by blocking receptor binding and viral entry, which has been shown with pseudovirus-based neutralization assays Nie et al., 2020a) .”
• The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity (2020, positive): “To achieve these, we generated 106 S mutants reported in the public domain or mutants at putative N-linked glycosylation sites and analyzed their infectivity and reactivity to neutralizing antibodies using the high-throughput pseudotyped virus system (Nie et al., 2020).”
• Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays (2020, methodology): “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 [29,39–41].”
• COVID-19 neutralizing antibodies predict disease severity and survival (2020, neutral): “However, pseudoviruses bearing SARS-CoV-2 spike produced by these methods yield low titers (Nie et al., 2020), hampering large-scale testing of neutralization.”
• Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells (2020, positive): “The pseudovirus was constructed as previously described using the spike genes from strain Wuhan-Hu-1 (GenBank: MN908947)(Nie et al., 2020).”