Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

Summary

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

Abstract

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

Introduction

In December 2019, a novel coronavirus was identified, which we now know as SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19)¹. SARS-CoV-2 is a betacoronavirus belonging to the Coronaviridae family. These enveloped viruses comprise a large positive-sense RNA genome and are responsible for respiratory and intestinal infections in both humans and animals². As of May 2021 there have been more than 157 million reported cases of COVID-19 globally and more than 3.2 million deaths³. The development of an effective vaccine has become the primary goal of researchers a....

Protocol

1. Plating cells (Day 1) for the production and quantification of SARS-CoV-2 pseudovirus

1. Preparation for tissue culture
   1. Warm 1x Dulbecco's Phosphate-Buffered Saline (DPBS); Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (optional); and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) to 37 °C in a water bath for approximately 15 min.
   2. Disinfect a tissue culture hood with 70% ethanol, and place tissue culture dishes, Pasteur pipettes, and serological pipettes in the tissue culture hood as needed. Remove PBS, DMEM, and trypsin from the water bath, and disinfect with 70....

Results

This protocol outlines a rapid and effective method for detecting neutralizing antibodies against SARS-CoV-2 S protein via inhibition of VSV-S-eGFP pseudovirus infection (quantifiable by loss of eGFP foci detected). A schematic representation of the protocol is depicted in Figure 1. It is recommended that a commercially available antibody be used as a positive control each time the assay is run to ensure the consistency of the assay. Here, we demonstrate a dilution curve using a commercially.......

Discussion

The method described here may be adapted to suit varying lab environments and resources as needed. Importantly, the main limitation of this protocol is the necessity for a containment level 2 space and tissue culture hood. The application of a replicating RNA virus pseudotyped with the SARS-CoV-2 spike, such as VSV-S-eGFP, is a formidable alternative to the SARS-CoV-2 virus, which requires a containment level 3 working area, but may remain a limitation for some groups. All other steps described here are quite flexible an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA (Gibco)	Fisher scientific	LS25200114
ArrayScan VTI HCS	Thermo Fisher Scientific		Automated fluorescent imager
carboxymethyl cellulose	Sigma	C5678
Dulbecco's modified Eagle's medium (Gibco)	Fisher scientific	10-013-CV
Dulbecco's modified Eagle's medium (Powder) (Gibco)	Thermo Fisher Scientific	12-800-017
Dulbecco’s Phosphate-Buffered Saline (DPBS)	Fisher scientific	21-031-CV
HEPES	Fisher scientific	BP-310-500
IgG Isotype Control (mouse)	Thermo Fisher Scientific	31903
Penicillin/streptomycin	Thermo Fisher Scientific	15070063
SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab	SinoBiological	40592-MM57
Vero E6 cells	ATCC	CRL-1586