A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Abstract

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

A flow cytometry‐based assay to measure neutralizing antibodies against SARS‐CoV‐2 virus

“…More recently, flow cytometry was used to detect NAbs against SARS‐CoV‐2 [18, 19, 23, 24]. Horndler et al [18] created a serological test using Jurkat cells that expressed the S protein and truncated human EGFR (used as normalizer) to detect anti‐ S antibodies.…”

“…More recently, flow cytometry was used to detect NAbs against SARS-CoV-2 [18,19,23,24]. Horndler et al [18] was detected with an anti-mouse IgG1, indicating no neutralization by patient sera.…”

Characterisation of the Antibody Response in Sinopharm (BBIBP-CorV) Recipients and COVID-19 Convalescent Sera from the Republic of Moldova

“…The pseudovirus system for screening NAbs and antivirals is a widely used method [ 46 , 47 , 48 , 49 ]. Most reports validate protocols based on ELISA and FC [ 28 , 32 , 50 ]. Because FC was time-consuming and required several steps in its analysis, we included a different technology, the HCI system.…”

An omicron-specific neutralizing antibody test predicts neutralizing activity against XBB 1.5

“…In contrast, surrogate virus neutralization tests (sVNT) directly assess the competitive inhibition of SARS-CoV-2 RBD peptides binding to human ACE2 receptors and can be implemented routinely in the laboratory with technologies such as enzyme-linked immunosorbent assays (ELISA), and even as point of care devices with lateral flow immunoassays (e.g., 9 , 10 ). Our group and others have developed flow cytometry-based sVNT systems that allow rapid multiplexed determination of NAb activity against multiple SARS-CoV-2 variant RBDs simultaneously ( 11 , 12 ). These flow cytometry NAb tests (FC-NAb) are well suited for deployment in a clinical setting and can be relatively easily adapted to include emerging variants of concern.…”