Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles

Abstract

The Coronavirus Disease 2019 (COVID-19) is still causing a wide range of infections and deaths due to the high variability of the SARS-CoV-2 virus. Therefore, it is necessary to establish a reliable and convenient pseudovirus-based neutralization assay to develop drug targeted variants of SARS-CoV-2. Based on the HIV-1 backbone, we generated a high titer luciferase (Luc)-expressing pseudovirus packaging system. Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2-overexpressing HEK-293T cells (293T-ACE2 cells). Compared to serine protease inhibitor camostat mesylate, the cysteine protease inhibitor E-64d could significantly block all SARS-CoV-2 mutant S pseudovirus infection in 293T-ACE2 cells. Furthermore, the neutralization ability of two antibodies targeted receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) was evaluated, which showed different inhibition dose-effect curves among four types of S pseudovirus. Overall, we developed a pseudovirus-based neutralization assay for SARS-CoV-2, which would be readily adapted to SARS-CoV-2 variants for evaluating antibodies.

Loss of furin site enhances SARS-CoV-2 spike protein pseudovirus infection

“…During virus packaging, the proteins trapped in the endoplasmic reticulum, making it impossible to secrete. Considering the entire length of S protein is not in favour of lentivirus pseudotyping, most of the f pseudovirus systems deleted the last 18 amino acids, which may increase the levels of S on the cell surface, resulting in a higher titer of pseudovirus ( Wang et al, 2022 , Hu et al, 2020 , Crawford et al, 2020 ). The results of virus infection and western blot both showed the SARS-CoV-2-S full-length construct fail to be expressed.…”

Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test

“…This is particularly important during the ongoing pandemic, with the emergence of novel variants of concern, against which preexisting NAbs may be less effective. As well, all plasmids used for generating our pseudotyped lentiviruses are commercially available, making for a convenient and time-saving approach in comparison to custom-designed plasmids, which are more time-consuming to prepare ( 15 ). In addition, the in-house generation of pseudotyped lentiviruses is a faster approach than the generation of live virus in a BSL-3 setting, because it may take time to successfully rescue live virus and optimize the assay conditions.…”

“…The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) is one such approach that does not have the same logistical challenges as the PRNT assay. SCLSNA can be safely performed in BSL-2 laboratories; it is amenable to high throughput and has a relatively faster TAT of 48 h ( 15 – 18 ). The SCLSNA incorporates the use of lentiviruses pseudotyped with SARS-CoV-2 spike protein, which can serve as a surrogate virus to quantitate neutralizing antibodies generated against the SARS-CoV-2 spike protein ( 19 , 20 ).…”

Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test

“…This is particularly important during the pandemic with the emergence of novel variants of concern to which pre-existing NAbs may be less effective. As well, all plasmids used in generating our pseudotyped lentiviruses are commercially available, making for a convenient and time-saving approach in comparison to custom designed plasmids which are more time consuming to prepare (15). In addition, the in-house generation of pseudotyped lentivirus is a faster approach than the generation of live virus in a BSL-3 setting, which may take time to successfully rescue live virus and optimize assay conditions.…”

“…Such limitations present challenges in sample processing and throughput capabilities and alternate methodologies are required to help circumvent these difficulties. The SCLSNA is one such approach that does not have the same logistical challenges associated with the PRNT assay; SCLSNA can be safely performed in BSL-2 laboratories, it is amenable to high-throughput and has a relatively faster TAT of 48 hours (15)(16)(17)(18). The SCLSNA incorporates the use of lentiviruses pseudotyped with SARS-CoV-2 spike protein, which can serve as a surrogate virus to quantitate neutralizing antibodies generated against the SARS-CoV-2 spike protein (19,20).…”