Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein.

Lydia Horndler,David Abia,Sergio Serrano‐Villar,Manuel Fresno,Francisco Sánchez‐Madrid,Ivaylo Balabanov,Pilar Delgado,Georgina Cornish,Hisse M Van Santen,Miguel A Llamas,Pedro Martínez‐Fleta,Balbino Alarcón

doi:10.15252/emmm.202013549

Abstract

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID‐19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer‐friendly non‐adherent Jurkat T‐cell line that stably expresses the full‐length native spike “S” protein of SARS‐CoV‐2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self‐cleaving sequence, allowing to accurately quantify the presence of anti‐S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double‐staining with the test sera and anti‐EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti‐S antibodies with protective neutralizing activity are long‐lasting and can be detected in sera 8 months after infection.

Highlights

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global pandemic COVID-191
Taking advantage of the fact that after cleavage the T2A sequence remains attached to the N-terminal protein, we studied whether the S protein was expressed at the cell surface of the transduced Jurkat cells
We describe a method based on flow cytometry of a human T lymphoblastic leukemia cell line that stably expresses the S protein of SARS-CoV-2

Summary

Introduction

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global pandemic COVID-191. Phylogenetic analysis of the full genome classifies SARS-CoV-2 as a Betacoronavirus subgenus Sarbecovirus, lineage B and is related to bat isolates of SARS-CoV and is 79% identical to the SARS virus causing a viral epidemic in 2002 (Lu, R. et al, 2020). SARS-CoV-2 encodes for 16 non-structural proteins at the 5’ end of the genomic RNA and structural proteins spike (S), envelope (E), membrane (M) and nucleocapsid (N) at the 3’ end (Su, S. et al, 2016). The spike S protein is responsible for binding to ACE2 in host cells which seems to be the main cellular receptor for the virus (Hoffmann, M. et al, 2020). The S protein forms homotrimers and is composed of two fragments S1 and S2 that result from proteolytic cleavage of S upon ACE2 binding The S1 fragment contains a central RBD sequence that is the actual ACE2-binding sequence and the target of neutralizing antibodies such as those described to neutralize SARS-CoV-1

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Results

Conclusion