A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction

Chee Wah Tan,Vivian Chih-Wei Chen,Charles Tiu,Yongxiang Yi,Pei Liu,Lin-Fa Wang,Danielle E Anderson,Xijian Qin,Barnaby E Young,Yee-Joo Tan,Randy Foo,Mark I.-C Chen,Zhiliang Hu,David C Lye,Wan Ni Chia,Wan Rong Sia

doi:10.1038/s41587-020-0631-z

Abstract

A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination. The current gold standard is the conventional virus neutralization test requiring live pathogen and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization test that detects total immunodominant neutralizing antibodies targeting the viral spike (S) protein receptor-binding domain in an isotype- and species-independent manner. Our simple and rapid test is based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of patients with COVID-19 in two different countries, achieves 99.93% specificity and 95-100% sensitivity, and differentiates antibody responses to several human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it broadly accessible to the wider community for both research and clinical applications.

Highlights

The COVID-19 outbreak was first recognized in December 2019 in Wuhan, China[1] and has since spread to all parts of the world, resulting in a total of 10,357,662 confirmed infections with 508,055 deaths as of 1 July 20202
After SARS-CoV-2 was identified as the causative agent of the COVID-19 outbreak, it was shown that human angiotensin-converting enzyme 2 (ACE2) is the main functional receptor for viral entry[3]
We hypothesized that the virus–receptor binding can be mimicked in vitro via a protein–protein interaction using purified recombinant human ACE2 (hACE2) and the receptor-binding domain (RBD) of the SARS-CoV-2 S protein

Summary

Introduction

The COVID-19 outbreak was first recognized in December 2019 in Wuhan, China[1] and has since spread to all parts of the world, resulting in a total of 10,357,662 confirmed infections with 508,055 deaths as of 1 July 20202. The pseudovirus-based VNT (pVNT), on the other hand, can be performed in a BSL2 laboratory, but still requires the use of live viruses and cells[7,8] All other assays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow assay (LFA) rapid tests, represent the second assay type, which detects total binding antibodies (BAbs) and is unable to differentiate between BAbs and NAbs[6,9,10]. Using purified receptor-binding domain (RBD) from the S protein and the host cell receptor ACE2, our test is designed to mimic the virus–host interaction in an ELISA plate well This RBD–ACE2 interaction can be neutralized (that is, blocked) by specific NAbs in patient or animal sera, in the same manner as in cVNT or pVNT

Methods

Results

Conclusion