key: cord-0800195-awrtgq3d
authors: Martin, Sophie; Jégou, Gwénaële; Nicolas, Aurore; Le Gallo, Matthieu; Chevet, Éric; Godey, Florence; Avril, Tony
title: A cell-based system combined with flow cytometry to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19
date: 2022-02-22
journal: STAR Protoc
DOI: 10.1016/j.xpro.2022.101229
sha: c4427933f07f9c192c8c82693a29f1068c11b97c
doc_id: 800195
cord_uid: awrtgq3d

This protocol describes a flow cytometry approach to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in COVID-19 positive patient sera samples without the need of specific laboratory facilities for viral infection. We developed a human cell-based system using Spike-expressing HEK293T cells that mimics membrane insertion and N-glycosylation of viral integral membrane proteins in host cells. This assay represents a powerful tool to test antibody responses against SARS-CoV-2 variants and vaccine effectiveness. For complete details on the use and execution of this protocol, please refer to Martin et al. 2021.

A cell-based system combined with flow cytometry to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19 Sophie Martin 1,2 , Gwénaële Jégou 1,2 , Aurore Nicolas 2,3 , Matthieu Le Gallo 1,2 , Éric Chevet 1,2 , Florence Godey 1,2 , Tony Avril 1,2,4, * 1 Inserm U1242 "Oncogenesis Stress Signaling", University of Rennes, Rennes, 35000, France ; 2 Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, 35000, France ; 3 CRB Santé, CHU Pontchaillou, Rennes, France, Rennes, 35000, France ; 4 Technical and lead contact : T.A.; *Correspondence: t.avril@rennes.uncancer.fr Summary This protocol describes a flow cytometry approach to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in COVID-19 positive patient sera samples without the need of specific laboratory facilities for viral infection. We developed a human cell-based system using Spike-expressing HEK293T cells that mimics membrane insertion and N-glycosylation of viral integral membrane proteins in host cells. This assay represents a powerful tool to test antibody responses against SARS-CoV-2 variants and vaccine effectiveness. For complete details on the use and execution of this protocol, please refer to Martin et al. 2021 .

Classifications: Cell Biology; Molecular Biology; Flow Cytometry; Cell-based Assays; Microbiology; Antibody; Health science.

Before you begin

The following part describes specific steps to obtain HEK293T cells (derived from human embryonic kidney cells expressing the large T antigen from SV40) expressing SARS-CoV-2 Spike protein. This cellbased system has been also applied to M (membrane) and E (envelop) SARS-CoV-2 proteins as well as to Spike D614 and G614 variants. This protocol could be adapted to any other cell types, such as epithelial Hela cells, although transfection method used for those cells should be adapted.

The SARS-CoV-2 Spike construct obtained from the Krogan's laboratory (see Key resources table) was amplified using stable competent E. coli bacteria (NEB) and purified using NucleoBond Xtra Maxi kit from Macherey-Nagel as described below. h. Prepare the NucleoBond column by adding 25 mL of equilibration buffer (EQU) to the column filter as described in the manufacturer's protocol i. Add 12 mL of neutralization buffer (NEU) to the bacteria suspension and immediately mix the lysates gently by inverting the 250 mL tube (do not vortex) until the blue suspension turns colorless completely j. Invert the tube 3 times before applying the lysate to the equilibrated NucleoBond column as described in the manufacturer's protocol k. Allow the column to empty by gravity flow l. Wash the NucleoBond filter and column with 15 mL of EQU buffer m. Allow the column to empty by gravity flow n. Discard the NucleoBond filter o. Wash the NucleoBond column with 25 mL of wash buffer (WASH) p. Allow the column to empty by gravity flow q. Place a 50 mL Falcon tube to collect the plasmid DNA elution under the column r. Elute the plasmid DNA by applying 15 mL of pre-warmed (at 50°C) elution buffer (ELU) on the column s. Add 10.5 mL of isopropanol (pre-warmed at 20°C) to precipitate the eluted plasmid DNA t. Centrifuge at 4500 g for 45 minutes at 4°C u. Carefully discard the supernatant v. Add 1 mL of ethanol 70% to the DNA pellet and transfer it to a 1.5 mL Eppendorf tube w. Centrifuge at 15 000 g for 5 minutes at 20°C x. Remove the supernatant y. Add 1 mL of ethanol 70% to the DNA pellet z. Centrifuge at 15 000 g for 5 minutes at 20°C aa. Remove completely the ethanol with a pipette tip bb. Dry the DNA pellet at 20°C cc. Add 250 µL of Tris EDTA (TE) buffer dd. Resuspend carefully the DNA and store at -20°C ee. Determine the plasmid DNA concentration and quality using an UV-visible spectrophotometer (i.e. a DeNovix DS-11 nanodrop spectrophotometer). The plasmid concentration was between and 2 to 4 µg/µL (between 1 to 2 mg in total) with an A260/A280 ratio between 1.8 to 2.

Before bacteria transformation: -prepare ampicillin-containing LB agar plates and media -set the water bath at 42°C -pre-warm the SOC medium at 20°C Before the SARS-CoV-2 Spike plasmid purification:

-pre-warm the elution buffer (ELU) at 50°C

Note: For step 3a, if needed, split the 250 mL bacterial culture into five 50 mL tubes for centrifugation; and then pool the bacteria pellets when resuspending in 12 mL of resuspension buffer (RES) (step 3d).

HE293T cell culture and transfection with the SARS-CoV-2 Spike plasmid HEK293T cells used in this protocol were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and transfected using calcium phosphate precipitation. The composition of the transfection solutions i.e. HBS solution and calcium chloride solutions is described in the following tables. This protocol could be adapted for other cell lines including epithelial or lung cell lines. To estimate the number of cells needed, take into account that that each serum would be tested on nontransfected (one well) and SARS-CoV-2 Spike-expressing HEK293T cells (two wells for IgG/M and IgA detection). Generally, between 2 to 3 millions HEK293T cells were obtained from one 10 cm Petri dish that were sufficient to test between 4 to 6 sera. 

4. Grow HEK293T cells in DMEM 4.5 g/L glucose medium containing 10% FBS HEK293T cells were grown in 15 mL of DMEM 4.5 g/L glucose medium containing 10% FBS in 75 cm 2 tissue culture flasks 5. Plate HEK293T cells in 10 cm Petri dish a. Wash gently HEK293T cells monolayer amplified in 75 cm 2 tissue culture flasks with 5 mL of PBS b. Detach HEK293T cells from the plate using 4 mL of PBS and 1 mL of 0.05% trypsin c. Mix gently d. Incubate 1 minute at 20°C e. Flush vigorously with a 5 mL pipette the cell layer to facilitate the cell detachment f. Collect detached cells in a 50 mL Falcon tubes containing 1 mL FBS g. Determine the cell number h. Plate 1 million of HEK293T cells in 10 mL DMEM 10 %FBS medium in 10 cm Petri dish i. Incubate 16 hours at 37°C in an incubator 5% CO2 in a humid atmosphere 6. Prepare the transfection solution j. Pre-warm HBS 2x solution at 20°C k. Mix 430 µL H2O with 70 µL of CaCl2 solution vigorously with a pipette tip l. Add 10 µg of plasmid and immediately mix vigorously with a pipette tip m. Add 500 µL of HBS 2x solution and immediately mix vigorously with a pipette tip n. Incubate exactly 10 minutes at 20°C o. For the control condition, prepare the transfection solution without plasmid 7. Transfect the HEK293T cells with SARS-CoV-2 Spike plasmid p. Replace the culture medium of the HEK293T cells by 9 mL of fresh DMEM 10% FBS medium q. Resuspend the transfection reagent r. Add drop by drop 1 mL of transfection solution on HEK293T cells seeded in 10 cm Petri dish s. For the control condition, add drop by drop transfection solution without plasmid on the control HEK293T plate t. Incubate for 48 hours at 37°C in an incubator 5% CO2 in a humid atmosphere

Before plating HEK293T cells in 10 cm Petri dish:

-ensure that HEK293T cells were at a confluence of about 70% Before transfection:

-pre-warm the HBS 2x solution Note: To strengthen the conclusion of the staining, dead cells could be excluded from the analysis using 7AAD (see protocol step 9.g); and more events could be acquired. If needed, Spike expression could be also monitored using intracellular staining of SARS-CoV-2 Spike-expressing HEK293T cells with an anti-Tag antibody. Indeed, as SARS-CoV-2 Spike construct used in this protocol is tagged with two Strep-Tag II motifs, fluorescent-conjugated streptavidin or strepTactin could be used for detection of Spike. 

To avoid useless freezing and thawing cycles, sera were re-aliquoted in small volumes (i.e. 100 µL) Deposited data n/a n/a n/a Experimental models: Cell lines HEK293T cell line ATCC CRL-3216 Experimental models: Organisms/strains n/a n/a n/a Oligonucleotides n/a n/a n/a J o u r n a l P r e -p r o o f 

This protocol requires a specific staining buffer that contained bovine and donkey sera to limit the unspecific binding of secondary antibodies generated in donkey. This staining buffer was therefore used to wash cells and to dilute sera and secondary antibodies. Staining buffer recipe and antibody dilutions are provided in the following tables: To be prepared the day of the experiment and kept on ice and in the dark.

The flow cytometer Novocyte 3000 (ACEA) used for this protocol is equipped of an automated sampling using 96-well plates and 3 lasers including blue and red lasers that allowed the detection of fluorochromes including fluorescein isothiocyanate (FITC), Alexa Fluor 488 (AF488) and 647 (AF647), brilliant violet 650 (BV650) and for 7-amino-actinomycin (7AAD). The setting of the flow cytometer is detailed in Figures 1 to 3 .

Any other flow cytometers and fluorochromes could be used as long as suitable laser channels and detectors were appropriate for the corresponding fluorochromes.

Step-by-step method details Preparation of non-transfected and SARS-CoV-2 Spike expressing HEK293T cells

This step is dedicated to cell preparation. HEK293T cells were previously transfected with SARS-CoV-2 Spike construct (described in the 'Before you begin' part). To estimate the number of cells needed, take into account that that each serum would be tested on non-transfected (one well) and SARS-CoV-2 Spike-expressing HEK293T cells (two wells for IgG/M and IgA detection). Generally, between 2 to 3 millions of HEK293T cells obtained from one 10 cm Petri dish were sufficient to test between 4 to 6 sera. Do not forget to acquire for non-transfected and Spike-expressing HEK293T cells with secondary antibodies alone. The staining buffer limits the non-specific binding of antibodies. 

Spike expression should be verified before testing the human sera as described in the 'Before you begin' part.

This step describes the cell staining with human sera derived from healthy volunteers and patients with COVID-19. Serum 1 in 50 dilution is efficient for testing sera from asymptomatic, mild and severe patients with COVID-19. Negative (i.e. sera prior COVID-19 pandemic) and positive (i.e. controls provided by SeroBio France as a validation tool for diagnostic laboratories) sera controls should be used to validate the assay (see Martin et al. (iScience, 2021 , DOI: 10.1016 /j.isci.2021 .103185)). This step describes the cell staining for detecting the binding of different human anti-Spike immunoglobulins IgG, M and A (using AF488-, AF647-and FITC-coupled secondary antibodies respectively) on non-transfected and Spike-expressing HEK293T cells pre-incubated sera of patients with COVID-19. In this protocol, IgG and IgM antibodies were detected at the same time using AF488and AF647-coupled secondary antibodies respectively; and IgA antibodies were detected separately using and FITC-conjugated secondary antibodies. Alternative methods could be adapted to detect all Ig subtypes at the same time (for instance using anti-IgA antibodies coupled to other fluorochromes). Do not forget to acquire for non-transfected and Spike-expressing HEK293T cells with secondary antibodies alone. Figure 2B ) 11. Acquire 10 000 events in the gate of interest 12. Use the non-transfected HEK293T cells as a negative control 13. Determine the mean of fluorescence intensity observed for AF488, AF647 and FITC fluorochromes (corresponding to IgG, IgM and IgA binding respectively) for each condition including non-transfected and Spike-expressing HEK293T cells (Figures 2A and 2B ) 14. Determine the corresponding Ig binding levels observed with non-transfected HEK293T cells for each serum studied as followed: ratio of the geomean of fluorescence intensity (gMFI) = (gMFI observed with HEK293T cells incubated with serum and fluorescent anti-Ig antibodies) / (gMFI observed with HEK293T cells incubated only with fluorescent anti-Ig antibodies). See the 'Materials and Equipment' part 15. Determine the corresponding Ig binding levels observed with Spike-expressing HEK293T cells for each serum studied as described in step 14. See the 'Materials and Equipment' part 16. Determine the specific Ig binding as followed: specific Ig binding = (ratio observed with Spikeexpressing HEK293T cells) / (ratio observed with non-transfected HEK293T cells). See the 'Materials and Equipment' part 17. The specific Ig binding determined for each serum was the mean of specific Ig binding obtained in three independent experiments.

To strengthen the conclusion of the staining, more events could be acquired.

A representative analysis is provided in Figures 2A and 2B (Tables 1 and 2 ). The final specific Ig binding determined for each serum was the mean of specific Ig binding obtained in three independent experiments (Table 3) . A final visualization of the data for these patients is proposed in Figure 3A . A follow-up of anti-SARS-CoV-2 antibodies could be obtained using this protocol ( Figure 3B ). Please refer to Martin et al. (iScience, 2021 , DOI: 10.1016 /j.isci.2021 for an overview of the whole dataset. * The IgG binding is given by the ratio of (gMFI AF488 Ctrl with serum / gMFI AF488 Ctrl without serum); for instance for Ser#1: IgG binding = 853 / 225 = 3.8. The IgM binding is given by the ratio of (gMFI AF647 Ctrl with serum / gMFI AF647 Ctrl without serum); for instance for Ser#1: IgM binding = 4363 / 1379 = 3.2. The specific IgG binding is given by the ratio of (IgG binding obtained with Spike-expressing HEK293T cells / IgG binding obtained with nontransfected (Ctrl) HEK293T cells); for instance for Ser#1: specific IgG binding = 64.3 / 3.8 = 17.0. The specific IgM binding is given by the ratio of (IgM binding obtained with Spike-expressing HEK293T cells / IgM binding obtained with non-transfected (Ctrl) HEK293T cells); for instance for Ser#1: specific IgM binding = 17.3 / 3.2 = 17.0. 

Limitations This protocol could be applied to any viral proteins that are expressed at the cell surface. In our recent work, [Martin et al., iScience 2021] we did observe anti-SARS-CoV-2 Spike and Membrane antibodies but failed to detect anti-SARS-CoV-2 Envelope antibodies due to the fact that this viral protein was probably not expressed at the cell surface and retained intracellularly. Considering the many Spike variants emerging, this protocol could be adapted using plasmids containing specific sequences of Spike variants.

We observed increased IgG binding on non-transfected HEK293T cells using sera of patients under immunotherapy with humanized antibodies, probably due to non-specific binding of these antibodies to HEK293T cells. This problem was solved by normalizing IgG binding as described in the 'Quantification and statistical analysis' part.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tony Avril ( t.avril@rennes.unicancer.fr ).

This study did not generate new unique reagents.

Data reported in this paper will be shared by the lead contact upon request. This paper does not report the original code. 

EC is a founder of Cell Stress Discoveries Ltd (https://cellstressdiscoveries.com) and Thabor Therapeutics (https://www.thabor-tx.com/). The viable cell population of interest (negative for 7AAD staining) was first gated using a FSC/PerCP dot plot followed by a FSC-H/FSC-A dot plot (to exclude doublets from the analysis) followed by a FSC/SSC dot plot. Spike expression was then detected using anti-Spike antibody with a AF488/SSC dot-plot. The percentage of positive cells was calculated from a final gate adjusted with non-transfected HEK293T cells used as negative controls. Spike expression could also be represented using a AF488 histogram (non-transfected HEK293T cells: green histograms; Spike-expressing HEK293T cells: red hsitograms). Spike expression level is given a ratio of (geometric mean (gMFI) AF488 observed with Spike-expressing HEK293T cells) / (gMFI AF488 observed with non-transfected HEK293T cells). The viable cell population of interest (negative for 7AAD staining) was first gated using a FSC/PerCP dot plot followed by a FSC-H/FSC-A dot plot (to exclude doublets from the analysis). Anti-spike antibodies were then detected using anti-human IgG AF488, IgM AF647 antibodies (A) and anti-IgA FITC (B) with a AF488/SSC, an AF647/SSC (A) and a FITC/SSC (B) dot-plot respectively. The gMFI of AF488, AF647 (A) and FITC (B) were further used to determine the level of anti-Spike IgG, IgM and IgA antibodies (see the 'Quantification and statistical analysis' part). Sera from SARS-CoV-2-infected patients were tested for their positivity against viral Spike proteins using the SARS-CoV-2 serological assay described in this protocol. (A) Anti-Spike antibodies were detected using fluorescent-coupled anti-human IgG, IgM and anti-IgA antibodies. The means of fluorescence intensities were further used to determine the specific level of anti-Spike IgG, IgM and IgA antibodies (see the 'Quantification and statistical analysis' part). Negative thresholds (dotted lines) were obtained

SARS-CoV-2 integral membrane proteins shape the serological responses of patients with COVID-19. iScience

We thank the members of the Virology department of the Rennes University hospital for providing human sera, in particular Vincent Thibault; the Centre des Ressources Biologiques of Rennes, and the Centre Eugène Marquis and INSERM for support. This work was funded by grants from INSERM, Institut National du Cancer (INCa PLBIO), Fondation pour la Recherche Médicale (FRM, DEQ20180339169) to EC and from la Ligue contre le cancer (comité 35, 56 et 85) to TA.

SM, AN, GJ -methodology, investigation, formal analysis; FG -resources; MLG -conceptualization, writing (editing); EC -supervision, conceptualization, project administration, funding acquisition; writing (review & editing); TA -supervision, conceptualization, methodology, investigation, formal