We would like to thank the Whelan lab for generously providing the VSV-S-eGFP virus used in this protocol (described in Case et al. 2020). We also thank Drs. Bill Cameron and Juthaporn Cowan (and team) for collecting the patient blood samples (REB protocol ID 20200371-01H). The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the generous support from the Ottawa Hospital Foundation and a grant from the Canadian Institutes of Health Research (#448323) and a Fast Grant from the Thistledown foundation for COVID-19 Science to C.S.I. T.R.J. is funded by an Ontario Graduate Scholarship and cluster Mitacs fellowship. JP is funded by a cluster Mitacs fellowship. T.A. is funded by a CIHR Banting Fellowship. We would also like to thank all the individuals who participated and donated their blood samples for this study.

1. Plating cells (Day 1) for the production and quantification of SARS-CoV-2 pseudovirus
<ol>
	<li>Preparation for tissue culture
	<ol>
		<li>Warm 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS); Dulbecco&#39;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 &#176;C in a water bath for approximately 15 min.</li>
		<li>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% ethanol prior to placing in the tissue culture hood.</li>
	</ol>
	</li>
	<li>Plating and maintaining cells
	<ol>
		<li>Grow Vero E6 cells in DMEM (containing 10% FBS) in T75 tissue culture flasks or 15 cm tissue culture dishes in a 37 &#176;C incubator with 5% CO2. Passage the cells as needed when cells are 80-90% confluent.</li>
		<li>Wash the cells by adding 10 mL of 1x PBS to each dish and gently rocking 4-5 times; aspirate the PBS. Add 3 mL of trypsin-EDTA to each dish, rock the plate to ensure the entire surface is covered. Incubate the cells at 37 &#176;C with 5% CO2 for approximately 5 min, or until the cells have detached.</li>
		<li>Add 7 mL of DMEM (containing 10% FBS) to deactivate the trypsin, and resuspend the cells by pipetting up and down several times. Spin down the cells to remove the trypsin using a benchtop centrifuge at 500 &#215; g for 5 min, aspirate the media without disturbing the cell pellet, and resuspend the cells in 10 mL of DMEM (containing 10% FBS). If cells are required for future experiments, place 1 mL in a new dish containing 15 mL of fresh DMEM (containing 10% FBS).</li>
		<li>Count the cells using an automated cell counter or hemocytometer, and add approximately 1 &#215; 107 cells to each 15 cm plate, and incubate at 37 &#176;C with 5% CO2 until they are 100% confluent (1-2 days).</li>
	</ol>
	</li>
</ol>
2. VSV-S-EGFP pseudovirus preparation
<ol>
	<li>Infection
	<ol>
		<li>Infect cells at multiplicity of infection (MOI) 0.01 with VSV-S-eGFP stock virus in 12 mL of serum-free DMEM for 1 h at 37 &#176;C with 5% CO2, occasionally rocking the plates. Replace the inoculum with fresh DMEM (containing 2% FBS and 20 mM HEPES, pH 7.7), and move to an incubator set to 34 &#176;C with 5% CO2. 
		NOTE: A temperature of 34 &#176;C is required for the propagation of VSV-S-eGFP in cell culture.</li>
		<li>Collect cell supernatants upon observation of extensive cytopathic effect (CPE) and cell detachment, approximately 48 h post-infection. Use a fluorescent microscope to visualize the extensive expression of eGFP by infected cells starting at 24 h post-infection.</li>
	</ol>
	</li>
	<li>Collection
	<ol>
		<li>Centrifuge the supernatants using a benchtop centrifuge for 5 min at 1,000 &#215; g at 4 &#176;C to remove the cell debris. Aliquot the supernatant to avoid multiple freeze-thaw cycles, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Titering the VSV-S-eGFP pseudovirus
<ol>
	<li>Serial dilution to determine viral titer
	<ol>
		<li>Plate Vero E6 cells on 6-well plates at a seeding density of 6 &#215; 105 cells per well in DMEM (with 10% FBS), and incubate overnight at 37 &#176;C with 5% CO2. 
		NOTE: Vero cells that overexpress TMPRSS2 and hACE2 can also be used.</li>
		<li>Set up a 10-fold serial dilution series of the VSV-S-eGFP virus in cold serum-free DMEM by placing 900 &#181;L of media in seven microcentrifuge tubes. Label tubes from 1 to 7. Add 100 &#181;L of the viral stock to the first tube and vortex briefly. Transfer 100 &#181;L of diluted virus from tube 1 to tube 2 and vortex briefly; continue until tube 7.</li>
		<li>Aspirate the medium from all wells of the 6-well plate containing Vero E6 cells, and replace with 500 &#181;L of dilutions 10-2 to 10-7. Incubate the plates for 45 min at 37 &#176;C with 5% CO2, gently rocking every 15 min.</li>
		<li>Aspirate the inoculum and replace with overlay containing a 1:1 mixture of 2x DMEM and 6% carboxymethyl cellulose (CMC), final concentration of 3% CMC, supplemented with 10% FBS; incubate at 34 &#176;C with 5% CO2 for 48 h. 
		NOTE: Pre-warm the overlay mixture in a 37 &#176;C water bath for 15 min during the infection step. A 1:1 mixture of 1% agarose with 2x DMEM supplemented with 10% FBS may be used in place of CMC. To overlay with agarose, boil 1% agarose (in dH2O) and mix with cold 2x DMEM. Ensure the mixture is a suitable temperature before adding to the cell monolayer (approximately 37-40 &#176;C). If agarose is used for the overlay, cells must be fixed by incubating in 2 mL of 3:1 methanol:acetic acid for at least one hour prior to staining.&#160;</li>
	</ol>
	</li>
	<li>Staining and calculating viral titer
	<ol>
		<li>Visualize plaques by staining with crystal violet or direct visualization of eGFP under a fluorescent microscope.</li>
		<li>To stain plates, aspirate the overlay, wash once with PBS, then add 2 mL of 0.1% crystal violet (in 80% methanol and dH2O) to each well. Place on a plate rocker for approximately 20 min at room temperature prior to de-staining. Remove the crystal violet, and gently wash each well twice using dH2O or PBS. 
		NOTE: Washing and staining steps should be performed gently by pipetting slowly towards the side of each well to ensure the cell monolayer remains intact throughout the procedure.</li>
		<li>Allow plates to dry for at least 1 h prior to counting plaques. Ensure that plaque counts are obtained from wells containing 20 to 200 plaques. To calculate the titer of the virus in plaque-forming units (PFU) per mL, multiply the dilution factor of the well counted with the volume of infection, and divide this number from the plaques present in the respective well. 
		<img alt="Equation 1" src="/files/ftp_upload/62486/62486eq01.jpg" /></li>
	</ol>
	</li>
</ol>
4. Plating cells (Day 1) for the measurement of neutralization of SARS-CoV-2 pseudovirus by commercially available antibodies and convalescent patient serum
<ol>
	<li>Preparation for tissue culture
	<ol>
		<li>Prepare medium and tissue culture hood as described in section 1. In addition, prepare the necessary number of 96 well plates to accommodate a minimum of 2 replicates per sample (i.e., 1 plate per 6 samples); add additional replicates if sample volume permits.</li>
	</ol>
	</li>
	<li>Plating and maintaining cells
	<ol>
		<li>Grow and maintain Vero E6 cells as described in section 1. Prepare at least 10 mL of cell suspension per 96-well plate at a concentration of 2 &#215; 105 cells per mL (plate 1.5 &#215; 105 cells per mL for assays performed after 48 h, if needed). Add 100 &#181;L of cell suspension to each well of a 96-well plate using a multichannel pipette, and incubate for 24 h at 37 &#176;C with 5% CO2. 
		NOTE: Mix the cell suspension well, and rock each plate gently in all directions to ensure even distribution of cells.</li>
	</ol>
	</li>
</ol>
5. Antibody or serum dilutions and infections (Day 2)
NOTE: This protocol can be applied to measure the neutralization of VSV-S-eGFP by both commercially available antibodies and patient serum, as well as serum collected from animals for pre-clinical vaccine development studies. *Take note of the additional steps listed when handling patient/animal serum samples.
<ol>
	<li>Serial dilution series
	<ol>
		<li>Prior to diluting serum samples, heat-inactivate each sample in a 56 &#176;C water bath for 30 min to inactivate 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.</li>
		<li>Set up the dilution series in an empty 96-well plate (Plate 1).</li>
		<li>Begin all dilutions in Row A of the 96-well plate in 80 &#181;L. *For patient samples, begin the dilution series at 1 in 10 by placing 8 &#181;L of serum in 72 &#181;L of serum-free 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 &#181;g/mL to observe at least 50% neutralization.</li>
		<li>Add 40 &#181;L of of serum-free DMEM (with 1% penicillin/streptomycin) to rows B to G, and 80 &#181;L to row H. Do not add virus to row H, as it serves as a cell-only control.</li>
		<li>Using a 12-well multichannel pipette, mix and transfer 40 &#181;L of diluted serum or antibody from row A to row B. Mix and repeat until row F, discard 40 &#181;L from row F. Do not add serum to row G as it represents the virus-only control row.</li>
	</ol>
	</li>
	<li>Infection and overlay
	<ol>
		<li>Prepare an appropriate volume of diluted VSV-S-eGFP to treat each well at MOI 0.05 (or 2000 pfu). (When completing this calculation, keep in mind that only 60 &#181;L of the total 80 &#181;L 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.</li>
		<li>Add 40 &#181;L 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 &#176;C with 5% CO2.</li>
		<li>Remove the plate containing cells from the incubator (Plate 2), and carefully aspirate the media from all wells. Transfer 60 &#181;L of antibody/virus mixture from Plate 1 to Plate 2, and incubate for 1 h at 37 &#176;C with 5% CO2, rocking the plate every 20 min.</li>
		<li>Top up each well with 140 &#181;L of overlay containing CMC in DMEM for a final concentration of 3% CMC (with 10% FBS and 1% penicillin/streptomycin). Place Plate 2 in an incubator set to 34 &#176;C with 5% CO2 for approximately 24 h before imaging. 
		&#8203;NOTE: Pre-warm the overlay mixture in a 37 &#176;C water bath for 15 min during the infection step.</li>
	</ol>
	</li>
</ol>
6. Imaging and quantification (Day 3)
<ol>
	<li>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 foci. If an automatic counting feature is not available, use ImageJ software to quantify the number of eGFP foci.</li>
</ol>

<ol>
	<li>Hu, B., Guo, H., Zhou, P., Shi, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+SARS-CoV-2+and+COVID-19.">Characteristics of SARS-CoV-2 and COVID-19.</a> Nature Reviews Microbiology. 19 (3), 141-154 (2021).</li><li>Burrell, C. J., Howard, C. R., Murphy, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronaviruses.">Coronaviruses.</a> Fenner and White's Medical Virlogy. , 437-446 (2017).</li><li>COVID-19 Map. Johns Hopkins Coronavirus Resource Center Available from: <a target="_blank" href="https://coronavirus.jhu.edu/map.html">https://coronavirus.jhu.edu/map.html</a> (2021)</li><li>Covid-19 vaccine tracker. The New York Times Available from: <a target="_blank" href="https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html">https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html</a> (2021)</li><li>Hoffmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+cell+entry+depends+on+ACE2+and+TMPRSS2+and+is+blocked+by+a+clinically+proven+protease+inhibitor.">SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.</a> Cell. 181 (2), 271-280 (2020).</li><li>Letko, M., Marzi, A., Munster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+cell+entry+and+receptor+usage+for+SARS-CoV-2+and+other+lineage+B+betacoronaviruses.">Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses.</a> Nature Microbiology. 5, 562-569 (2020).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoluciferase+complementation-based+bioreporter+reveals+the+importance+of+N-linked+glycosylation+of+SARS-CoV-2+Spike+for+viral+entry.">Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 Spike for viral entry.</a> Molecular Therapy. , (2021).</li><li>Brown, E. E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+critical+determinants+of+ACE2-SARS+CoV-2+RBD+interaction.">Characterization of critical determinants of ACE2-SARS CoV-2 RBD interaction.</a> International Journal of Molecular Sciences. 22 (5), 2268 (2021).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+S1+NanoBiT:+a+Nanoluciferase+complementation-based+biosensor+to+rapidly+probe+SARS-CoV-2+receptor+recognition.">SARS-CoV-2 S1 NanoBiT: a Nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.</a> Biosensors and Bioelectronics. 180, 113122 (2021).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+for+SARS-CoV-2+vaccine+design:+Fusion+of+Spike+glycoprotein+transmembrane+domain+to+receptor-binding+domain+induces+trimerization.">Implications for SARS-CoV-2 vaccine design: Fusion of Spike glycoprotein transmembrane domain to receptor-binding domain induces trimerization.</a> Membranes. 10 (9), 215 (2020).</li><li>Cao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+persistent+antibody+responses+against+the+receptor-binding+domain+of+SARS-CoV+spike+protein+in+recovered+patients.">Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients.</a> Virology Journal. 7, 299 (2010).</li><li>To, K. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+profiles+of+viral+load+in+posterior+oropharyngeal+saliva+samples+and+serum+antibody+responses+during+infection+by+SARS-CoV-2:+an+observational+cohort+study.">Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study.</a> The Lancet Infectious Diseases. 20 (5), 565-574 (2020).</li><li>Gao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+inactivated+vaccine+candidate+for+SARS-CoV-2.">Development of an inactivated vaccine candidate for SARS-CoV-2.</a> Science. 369 (6499), 77-81 (2020).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-year+prospective+study+of+the+humoral+immune+response+of+patients+with+severe+acute+respiratory+syndrome.">Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome.</a> Journal of Infectious Diseases. 193 (6), 792-795 (2006).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+SARS-CoV-2+vaccine+candidates.">A systematic review of SARS-CoV-2 vaccine candidates.</a> Signal Transduction and Targeted Therapy. 5 (1), 237 (2020).</li><li>Amanat, F., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+vaccines:+Status+report.">SARS-CoV-2 vaccines: Status report.</a> Immunity. 52 (4), 583-589 (2020).</li><li>Amanat, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serological+assay+to+detect+SARS-CoV-2+seroconversion+in+humans.">A serological assay to detect SARS-CoV-2 seroconversion in humans.</a> Nature Medicine. 26 (7), 1033-1036 (2020).</li><li>Case, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+and+soluble+ACE2+inhibition+of+a+replication-competent+VSV-SARS-CoV-2+and+a+clinical+isolate+of+SARS-CoV-2.">Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.</a> Cell Host and Microbe. 28 (3), 475-485 (2020).</li><li>Garcia-Beltran, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journal+Pre-proof+COVID-19+neutralizing+antibodies+predict+disease+severity+and+survival.">Journal Pre-proof COVID-19 neutralizing antibodies predict disease severity and survival.</a> Cell. 184 (2), 476-488 (2020).</li><li>Zeng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+against+SARS-CoV-2+spike+in+COVID-19+patients,+health+care+workers,+and+convalescent+plasma+donors.">Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors.</a> JCI insight. 5 (22), (2020).</li><li>Whitman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+SARS-CoV-2+serology+assays+reveals+a+range+of+test+performance.">Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.</a> Nature Biotechnology. 38 (10), 1174-1183 (2020).</li><li>Ainsworth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+characteristics+of+five+immunoassays+for+SARS-CoV-2:+a+head-to-head+benchmark+comparison.">Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison.</a> The Lancet Infectious Diseases. 20 (12), 1390-1400 (2020).</li><li>Sharifkashani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+2+(ACE2)+receptor+and+SARS-CoV-2:+Potential+therapeutic+targeting.">Angiotensin-converting enzyme 2 (ACE2) receptor and SARS-CoV-2: Potential therapeutic targeting.</a> European Journal of Pharmacology. 884, 173455 (2020).</li><li>Burki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+variants+of+SARS-CoV-2.">Understanding variants of SARS-CoV-2.</a> The Lancet. 397 (10273), 462 (2021).</li><li>Jayamohan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+pandemic:+a+review+of+molecular+diagnostic+tools+including+sample+collection+and+commercial+response+with+associated+advantages+and+limitations.">SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations.</a> Analytical and Bioanalytical Chemistry. 413 (1), 49-71 (2020).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+SARS-CoV-2+neutralizing+antibody+by+a+pseudotyped+virus-based+assay.">Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.</a> Nature Protocols. 15 (11), 3699-3715 (2020).</li><li>Crawford, K. H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+and+reagents+for+pseudotyping+lentiviral+particles+with+SARS-CoV-2+spike+protein+for+neutralization+assays.">Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays.</a> Viruses. 12 (5), 513 (2020).</li></ol>

The authors have no competing financial interests related to this publication.

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 and may be performed in nearly any containment level 2 lab. For example, the use of a fluorescent imager with an automated counting feature is not necessary. The 96-well format used here means that only approximately 4 fields of view are required to image nearly the entire well using the lowest objective available (2x). 
A manual fluorescent microscope may be used to image multiple fields of each well, and ImageJ (free software) can then be used to quantify eGFP foci. Additionally, we have chosen to use the 96-well format to use the lowest volume of serum possible as well as keeping consumable usage to a minimum. If a fluorescent microscope is unavailable, this protocol may be scaled up to a 6- or 12-well format to detect plaque formation after crystal violet fixation and staining (similar to the viral titer protocol described above). The most critical consideration to keep in mind when performing this protocol is the temperature sensitivity of VSV-S-eGFP. When working with VSV-S-eGFP, always aliquot the virus into small volumes to avoid multiple freeze-thaw cycles, and keep the virus on ice whenever possible. Additionally, robust fluorescent signal may be detected after 24 h; however, we have acquired similar results after imaging at 20 to 28 h post infection. 
There are several methods available to detect the presence of antibodies against SARS-CoV-2, including the gold-standard enzyme-linked immunosorbent assay (ELISA) assay, which quantifies the total amount of antibodies against S21,22. Here, we have outlined a fast and reliable method to specifically detect neutralizing antibodies against the immunodominant SARS-CoV-2 spike protein in convalescent serum from patients. We have improved the classical plaque-reduction neutralization test (PRNT) by successfully adapting to a 96-well format, which allows for the detection of neutralizing antibodies in a large number of samples within 24 h by automated quantification of pseudovirus infection, allowing for quick turnaround of final data reports. In addition to detecting neutralizing antibodies within serum, this method can be adapted to perform high-throughput screening of other therapeutic strategies, which aim to directly inhibit the SARS-CoV-2 spike protein interaction with hACE2, such as monoclonal antibodies, recombinant soluble hACE2, or protease inhibitors, to impede viral entry23. With the emergence of several SARS-CoV-2 spike protein variants, it is also important to note that this method may be applied to determine if similar neutralization levels occur following infection with different variants of the virus24. 
Other examples of available methods to detect antibodies against SARS-CoV-2 include ELISAs, lentivirus-based assays, and commercial kits to evaluate the neutralizing capacity of serum samples. While the commonly used IgM- or IgG-binding ELISAs are an effective method to determine the presence of antibody concentration to track previous infection or immunization, they are unable to distinguish the binding antibodies' neutralizing capacity25. Pseudotyped lentivirus-based neutralization assays have an improved safety profile, by using non-replicative viral particles as opposed to replicating VSV-S-eGFP; however, this creates a barrier in terms of testing capacity as lentiviral titers tend to be much lower26,27. There are several commercially available kits that measure the ability of antibodies to block infection via competitive inhibition of the SARS-CoV-2 spike protein (RBD specifically) binding with the hACE2 receptor following incubation with convalescent serum (e.g., CUSABIO, GenScript, Abnova). While many of these kits have reputable sensitivity and specificity, they also tend to be relatively expensive and therefore not ideal for a large volume of samples. The protocol provided here is fast, reliable, and inexpensive. This high-throughput method may be used to test many samples, achieving a robust readout within 24 h.

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&#160;there have been more than 157 million reported cases of COVID-19 globally and more than 3.2&#160;million deaths3. The development of an effective vaccine has become the primary goal of researchers around the globe with at least 77&#160;preclinical vaccines under investigation and 90&#160;currently undergoing clinical trials4.
Coronaviruses encode four structural proteins including the spike protein (S), nucleocapsid (N), envelope protein (E), and the membrane protein (M). Entry of SARS-CoV-2 requires interaction of the receptor-binding domain (RBD) of S with the host receptor, human angiotensin-converting enzyme 2 (hACE2), and subsequent membrane fusion following proteolytic cleavage by host cellular serine protease, transmembrane protease serine 2 (TMPRSS2)5,6,7,8,9,10. Humoral immunodominance of the S protein of SARS-CoV has been previously reported and has now been shown also for SARS-CoV-211,12,13. Indeed, neutralizing antibody responses against S have been detected in convalescent serum from SARS-CoV patients 24 months after infection14, highlighting their critical role in the long-term immune response. The S protein has been identified as a promising vaccine target and has thus become a key component of most vaccines under development15,16.
While the rapid detection of neutralizing antibodies is a critical aspect of vaccine development, it may also shed light on the rate of infection and sero-epidemiologic surveillance in impacted areas17. A replication-competent VSV pseudotyped with the SARS-CoV-2 S glycoprotein, in place of the wild-type VSV glycoprotein, to study SARS-CoV-2 infection in biosafety level 2 settings was kindly donated by Whelan and co-workers18. VSV expressing spike (VSV-S) will be utilized to determine the neutralizing antibody response against SARS-CoV-2 spike protein. As the VSV-S used here also expresses enhanced green fluorescent protein (eGFP), eGFP foci may be detected within 24 h to quantify infection, whereas plaque formation can take 48 to 72 h. Summarized here is a simple and effective protocol to determine the ability of convalescent patient serum to neutralize VSV-S-eGFP infection. This method may also be easily adapted to interrogate other potential therapeutics that aim to disrupt the host-viral interaction of SARS-CoV-2 S protein.

<table><tbody><tr><td>0.25% trypsin-EDTA (Gibco)</td><td>Fisher scientific</td><td>LS25200114</td><td></td></tr><tr><td>ArrayScan VTI HCS</td><td>Thermo Fisher Scientific</td><td></td><td>Automated fluorescent imager</td></tr><tr><td>carboxymethyl cellulose</td><td>Sigma</td><td>C5678</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (Gibco)</td><td>Fisher scientific</td><td>10-013-CV</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (Powder) (Gibco)</td><td>Thermo Fisher Scientific</td><td>12-800-017</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline (DPBS)</td><td>Fisher scientific</td><td>21-031-CV</td><td></td></tr><tr><td>HEPES</td><td>Fisher scientific</td><td>BP-310-500</td><td></td></tr><tr><td>IgG Isotype Control (mouse)</td><td>Thermo Fisher Scientific</td><td>31903</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Thermo Fisher Scientific</td><td>15070063</td><td></td></tr><tr><td>SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab</td><td>SinoBiological</td><td>40592-MM57</td><td></td></tr><tr><td>Vero E6 cells</td><td>ATCC</td><td>&amp;nbsp;CRL-1586</td><td></td></tr></tbody></table>

detection of sars-cov-2 neutralizing antibodies using high-throughput fluorescent imaging of pseudovirus infection

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.

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 available neutralizing IgG antibody against SARS-CoV-2 Spike RBD compared to an IgG control (see the Table of Materials for details about both antibodies; Figure 2).
Convalescent patient samples were collected approximately three months post SARS-CoV-2 infection, and pseudovirus neutralization was determined using the method described above (Figure 3). Importantly, minimal background inhibition was observed using healthy donor serum. Furthermore, of note, this assay distinguishes between patients with high symptom severity versus those with milder disease based on the need for hospitalization. In line with other reports, we have observed within our small cohort that hospitalized patients tend to demonstrate increased neutralizing capacity than those who did not require hospitalization19,20. While this may not always be the case for hospitalized versus non-hospitalized patient samples, the ability of the assay to differentiate varying degrees of neutralization is an asset. There may also be cases in which the neutralizing ability of the patient serum is much higher than those demonstrated here. If necessary, the starting dilution of patient serum may be adjusted, or additional dilution steps may be carried out on an additional plate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62486/62486fig1v2.jpg" /> 
Figure 1: Protocol outline demonstrating neutralization of VSV-S-eGFP by convalescent serum. Abbreviations: VSV = vesicular stomatitis virus; eGFP = enhanced green fluorescent protein; COVID-19 = coronavirus disease. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62486/62486fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62486/62486fig2v3.jpg" /> 
Figure 2: A commercially available neutralizing antibody against SARS-CoV-2 has been used as an example of a positive control alongside IgG as a negative control. The ability of neutralizing antibodies to inhibit viral infection will vary; refer to the information available for the specific antibody purchased to determine which concentration to begin the dilution curve (samples were run in triplicate, error bars represent &#177; SD). (A) Percent inhibition has been calculated based on the number of eGFP foci detected via (B) fluorescent imaging. Abbreviations: SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; IgG = immunoglobulin; SD = standard deviation; eGFP = enhanced green fluorescent protein; RBD = receptor-binding domain. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62486/62486fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62486/62486fig3v2.jpg" /> 
Figure 3: The ability of convalescent serum from patients to neutralize VSV-S-eGFP varies depending on the severity of symptoms. Patient serum samples were collected approximately 3 months post-SARS-CoV-2 infection, and control samples were collected from uninfected patients (samples were run in triplicate, error bars represent &#177; SD). (A) Percent inhibition has been calculated based on the number of eGFP foci detected via (B) fluorescent imaging. Abbreviations: SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SD = standard deviation; eGFP = enhanced green fluorescent protein.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62486/62486fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection at JoVE.com

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

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.

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 ...

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4.2K Views.  Ottawa Hospital Research Institute. 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. 

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

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Bioreporter Assay: A Sensitive Technique using a Bioluminescent Reporter System to Detect SARS-CoV-2 Antibodies

Source: Rezaei, R. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62488">Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021)
In this video, we show the HiBit-RBD bioreporter system-based assay for detecting SARS-CoV-2 neutralizing antibodies. This system targets the viral RBD domain by using a bioluminescent reporter system containing HiBiT fused to the RBD domain.

Source: Rezaei, R. et al., Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021)
In this video, we ...

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Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2

Source: Kula-Pacurar, A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62067">Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization.</a> J. Vis. Exp. (166), (2020).
This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.

1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1 

	Fixing and permeabilizing cells&#8203;
	
		Fix infected cells with 3.7% w/v PFA ...

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...