Background

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With ongoing large-scale vaccination in different countries and the continuous surge of new variants of global concern, a convenient, cost-effective and high-throughput neutralization test is urgently needed.

Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential.

Researchers generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein (miRFP) reporter to develop neutralization assays.

Technology Overview

University of Hawaii researchers generated SARS-CoV-2 spike (S) protein expressing plasmids and a lentivirus-based vector containing a miRFP reporter, and co-transfected in 293T cells to generate pseudoviruses containing the miRFP reporter ().

Further Details

Wen-Yang Tsai, Lauren L. Ching, Szu-Chia Hsieh, Marian E. Melish, Vivek R. Nerurkar & Wei-Kung Wang (2021) A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter, Emerging Microbes & Infections, 10:1, 894-904, DOI: 10.1080/22221751.2021.1925163

Benefits

The neutralization test, based on a pseudovirus with miRFP reporter, can be performed in a biosafety level 2 laboratory and requires fewer plates (one 96-well plate for 6 samples in duplicates vs. eighteen 6-well plates for PRNT), less time (48-72 h vs. 3-4 days for PRNT), less sample volume (20 µL vs. 100 µL for PRNT in duplicates, starting from 1:10 serum dilution), and is expandable to 384-well format.

Recent studies have reported neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter. However, neutralization tests using a pseudovirus with a luciferase reporter involves multiple, time-consuming steps including gentle aspiration of culture media, adding accurate amounts of lysis buffer for complete cell lysis and/or homogenization, and accurate amounts of substrates. It also requires transfer to a new plate for reading which has a high relative cost and inter-assay variability, limiting high throughput potential. Our pseudovirus with a miRFP reporter can be quantified by one step of direct imaging without multiple and laborious steps (5 min scan vs. 5 steps of ~60 min for a 96-well plate). Moreover, the same plate can be quantified multiple times for kinetic study without generating numerous replicates.

Neutralization tests using pseudovirus with a green fluorescent protein reporter may have high background signals caused by autofluorescence from intracellular NADPH, flavin or extracellular collagen. Our pseudovirus with a miRFP reporter has minimal autofluorescence and can be quantified by two methods: direct imaging first followed by flow cytometry using the same cells to determine percentage of positive cells, which showed good correlation with signals from direct imaging.

Applications

The pseudovirus with a miRFP reporter featured here is a simple, practical, and cost-effective tool for high-throughput neutralization. It can also be used to study the kinetics and mechanisms of entry, screen for entry or fusion inhibitors, and have applications in exploiting immune responses, pathogenesis, predictors of protection, convalescent-plasma therapy, vaccine efficacy and herb immunity to SARS-CoV-2 including new variants.

Given public health concern over the continuous surge of multiple variants, this method can be used to quickly generate different variant pseudoviruses containing mutations in the S protein and evaluate the sensitivity to neutralization by sera from individuals after natural infection or immunization with different vaccines.

Opportunity

R&D Collaboration
Commercial assay development

A High-throughput Neutralization Test for SARS-CoV-2 Based on a Pseudovirus Containing Monomeric Infrared Fluorescent Protein as Reporter

IP Status

Seeking