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Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Fantoni, Tobia, Bissoli, Michele, Stefani, Chiara, Voi, Mauro, Dabija, Alexandrina, Casula, Rebecca, Minafra, Domenico Luca, da Fonseca Palmeira, Julys, Argañaraz, Enrique Roberto, Mayora-Neto, Martin, and others. (2023) Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay. Journal of Visualized Experiments, (201). Article Number e65658. ISSN 1940-087X. (doi:10.3791/65658) (KAR id:104064)

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Official URL:
https://doi.org/10.3791/65658

Abstract

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.

Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 °C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Item Type: Article
DOI/Identification number: 10.3791/65658
Subjects: Q Science > QR Microbiology > QR355 Virology
Divisions: Divisions > Division of Natural Sciences > Medway School of Pharmacy
Funders: Wellcome Trust (https://ror.org/029chgv08)
Depositing User: Nigel Temperton
Date Deposited: 24 Nov 2023 12:21 UTC
Last Modified: 11 Jan 2024 01:54 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/104064 (The current URI for this page, for reference purposes)

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