Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

Abstract

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles

“…Numerous studies have demonstrated that the spike protein may be employed to generate stably pseudotyped lentiviral, retroviral, and vesicular stomatitis virus particles (Crawford et al, 2020;Johnson et al, 2020;Plescia et al, 2020;Shang et al, 2020;Neerukonda et al, 2021). Given that live SARS CoV-2 is a biosafety level-3 agent, these pseudotyping approaches have greatly facilitated investigations undertaken within lower containment facilities.…”

Human models for COVID‐19 research

“…Proof of principle for this has been demonstrated with a HIV-based lentiviral pseudovirus assay in a HEK 293T cell line expressing human ACE2 and TMPRSS2 (Neerukonda et al, 2021). This assay has shown success in screening for neutralizing antibodies for SARS-CoV-2 (Neerukonda et al, 2021).…”

Fully Human Antibody Immunoglobulin from Transchromosomic Bovines is Potent Against SARS-CoV-2 Variant Pseudoviruses

“…Codon optimized full-length open reading frames of the S genes of SARS-COV2 variants were cloned into pCDNA3.1(+) by GenScript (Piscataway, NJ). 293T-ACE2.TMPRSS2s cells stably expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were established as previously described (8). The 293T and 293T-ACE2.TMPRSS2s cells were maintained at 37°C in Dulbecco's modified eagle medium (DMEM) supplemented with high glucose, L-Glutamine, minimal essential media (MEM) non-essential amino acids, penicillin/streptomycin and 10% fetal bovine serum (FBS).…”

“…These studies were performed as part of a package of standardized assays established by the U.S. government's COVID-19 Therapeutics response efforts. The assays utilized product samples provided by the manufacturers, and the results were blinded to the products tested and published (8)(9). Multiple therapeutic products did not retain the ability to neutralize multiple variants including the Alpha, Beta and Gamma variants that were previously supplanting other earlier SARS-2 variants.…”

“…Following that SAB-185 specific study, the US Food and Drug Administration (FDA) reported on the ability of antibody-based therapeutics provided by many manufacturers to retain neutralization potency in recombinant HIV-based lentivirus pseudoviruses that express the full-length S protein of multiple VOC/VOIs. This BSL-2 assay utilizes a stably transduced 293T-ACE2 cell line expressing both ACE2 and TMPRSS2 (293T-ACE2.TMPRSS2 S ) and determines the IC50 ratio of the antibody products to neutralize SARS-CoV-2 variants compared to an early pandemic wild-type progenitor strain (D614G) (8)(9). These studies were performed as part of a package of standardized assays established by the U.S. government's COVID-19 Therapeutics response efforts.…”