Antibody-Mediated Neutralization of Coronavirus Spike Protein-Expressing Pseudoviruses Using Patient Serum

Abstract

Source: Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)

This video demonstrates the antibody-mediated neutralization of pseudoviruses carrying coronavirus spike proteins using patient serum, studied through high-throughput fluorescent imaging of infected epithelial cell monolayers.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Antibody or serum dilutions and infections

NOTE: This protocol can be applied to measure the neutralization of vesicular stomatitis virus expressing coronavirus spike protein and enhanced green fluorescent protein (VSV-S-eGFP) pseudovirus by both commercially available antibodies and patient serum, as well as serum collected from animals for pre-clinical vaccine development studies.

1. Serial dilution series
   1. Prior to diluting serum samples, heat-inactivate each sample in a 56 °C water bath for 30 min to inactivate the complement. Ensure necessary safety precautions are taken when handling patient samples; only open sample containers when in the tissue culture hood, and ensure appropriate containment level 2 personal protective equipment is worn.
   2. Set up the dilution series in an empty 96-well plate (Plate 1).
   3. Begin all dilutions in Row A of the 96-well plate in 80 µL. *For patient samples, begin the dilution series at 1 in 10 by placing 8 µL of serum in 72 µL of serum-free Dulbecco's modified eagle medium (DMEM) (with 1% penicillin/streptomycin).
      NOTE: The concentration of neutralizing antibody used will depend on the activity predicted by the manufacturer. For the SARS-CoV-2 Spike neutralizing antibody used here (see the Table of Materials), start with 5 µg/mL to observe at least 50% neutralization.
   4. Add 40 µL of of serum-free DMEM (with 1% penicillin/streptomycin) to rows B to G, and 80 µL to row H. Do not add virus to row H, as it serves as a cell-only control.
   5. Using a 12-well multichannel pipette, mix and transfer 40 µL of diluted serum or antibody from row A to row B. Mix and repeat until row F, discard 40 µL from row F. Do not add serum to row G as it represents the virus-only control row.
2. Infection and overlay
   1. Prepare an appropriate volume of diluted VSV-S-eGFP to treat each well at multiplicity of infection (MOI) 0.05 (or 2000 plaque-forming units (pfu)). (When completing this calculation, keep in mind that only 60 µL of the total 80 µL volume will be moved onto the cells; therefore, be sure to multiply the volume of the virus by 1.33 to maintain 2000 pfu).
      NOTE: As VSV-S-eGFP is temperature-sensitive, ensure viral stocks remain on ice whenever not in use, and avoid multiple freeze-thaw cycles as titers will differ after repeated freezing.
   2. Add 40 µL of diluted virus to each well in rows A to G of Plate 1 and mix by pipetting up and down 4-5 times. Incubate the plate for 1 h at 37 °C with 5% CO₂.
   3. Remove the plate containing cells from the incubator (Plate 2), and carefully aspirate the media from all wells. Transfer 60 µL of antibody/virus mixture from Plate 1 to Plate 2, and incubate for 1 h at 37 °C with 5% CO₂, rocking the plate every 20 min.
   4. Top up each well with 140 µL of overlay containing carboxymethyl cellulose (CMC) in DMEM for a final concentration of 3% CMC (with 10% FBS (Fetal bovine serum) and 1% penicillin/streptomycin). Place Plate 2 in an incubator set to 34 °C with 5% CO₂ for approximately 24 h before imaging.
      NOTE: Pre-warm the overlay mixture in a 37 °C water bath for 15 min during the infection step.

2. Imaging and quantification (Day 3)

1. Image plates using an automated fluorescent imager (using a fluorescein isothiocyanate (FITC) filter or an alternative filter with an excitation wavelength of 488 nm). Quantify viral infection by creating a protocol that automatically identifies and counts individual eGFP (enhanced green fluorescent protein) foci. If an automatic counting feature is not available, use ImageJ software to quantify the number of eGFP foci.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Vero E6 cells	ATCC	CRL-1586
Dulbecco’s Phosphate-Buffered Saline (DPBS)	Fisher scientific	21-031-CV