Pseudotype Neutralization Assays: From Laboratory Bench to Data Analysis

Abstract

Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed. Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. Additionally, it will provide a starting point for the development of pseudotype neutralization assays using pseudotypes bearing other viral envelope proteins.

Meeting report and review: Immunological assays and correlates of protection for next‐generation influenza vaccines

“…14 Cross reactive CD4+ and CD8+ cells have also been identified as correlates of protection in human challenge and cohort studies. [4][5][6][7] Other immunological markers, including antibody effector functions as measured in antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) assays, 25-28 complement activation, mucosal antibody levels, entry inhibition titers as measured by pseudotype particle entry inhibition assays, 29 antibodies to the ectodomain of the matrix 2 ion channel (M2e), antibodies to matrix protein 1 (M1) and nucleoprotein (NP), 30 influenza virus protein arrays (IVPM), [31][32][33][34] and many others are currently being investigated to assess whether they correlate with protection. Importantly, systems immunology approaches are being used to identify new immunological markers that could then be tested for their potential to predict whether protective immunity was induced through vaccination.…”

“…Pseudotype particle entry inhibition assays 29 Antibodies to the ectodomain of the matrix 2 ion channel (M2e), matrix protein 1 (M1) or nucleoprotein (NP)…”

Effective screening of SARS-CoV-2 neutralizing antibodies in patient serum using lentivirus particles pseudotyped with SARS-CoV-2 spike glycoprotein

“…We also used this neutralization assay to test the serum from several other patients that had varying levels of SARS-CoV-2 (Spike receptor binding domain-specific) IgM and IgG levels in ELISA. The IC 50 values in these assays were calculated in Graphpad Prism as described before 15 (Table 1). As a control, the VSV-G glycoprotein coated lentivirus (pLV-G) was not neutralized when using 1:40 dilution of convalescent patient serum www.nature.com/scientificreports/ from three different patients ( Fig.…”

Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance

“…The plate was incubated at 37℃, 5% CO 2 for 48 hours and then was read in the same way as in the titration protocol. IC50 and 90 values were calculated from the RLUs using GraphPad Prism software [9]. Conclusion and future work…”