Assessing if antibodies can block SARS-CoV-2 entry with microneutralization assays

In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan in China’s Hubei Province. The virus has since spread, causing a global pandemic.

Assays able to measure the antiviral activity of antibodies or antiviral compounds are a core component of SARS-CoV-2 vaccine and drug development efforts.

This article provides a detailed outline of a microneutralization assay suitable for the quantitative assessment of antibodies’ or drugs’ potential to block entry and/or replication of SARS-CoV-2 in vitro.

Several ‘common cold’ seasonal coronaviruses typically circulate in the human population at any given time, but the novel coronavirus that surfaced in China in late 2019 was subsequently classified as a beta-coronavirus and termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Wu et al., 2020).

SARS-CoV-2 is related to SARS CoV (renamed SARS-CoV-1), which triggered an epidemic in 26 countries between 2002 and 2004. Millions of cases of SARS-CoV-2 have been reported worldwide.

Both SARS-CoV-1 and SARS-CoV-2 employ angiotensin-converting enzyme 2 (ACE2) as a receptor to facilitate attachment to host cells (Letko, Marzi, & Munster, 2020).

A spike protein interacts with the large trimeric glycoprotein ACE2 (Wrapp et al., 2020), with the part of the spike protein interacting with the receptor known as the receptor-binding domain (RBD). This domain remains one of the key targets for neutralizing antibodies (Walls et al., 2020).

Many studies have highlighted the tendency for individuals who recover from SARS-CoV-2 infection to seroconvert. They subsequently produce significant numbers of antibodies against the spike protein. This has been measured and demonstrated via binding assays (Amanat et al. and Okba et al., 2020).

The first stages of results have highlighted that induced antibodies are also able to efficiently neutralize the virus, but it will be necessary to conduct large-scale serology studies to correlate results from binding and neutralization assays.

There remains a clear need for robust assays suitable for screening antiviral drugs designed to inhibit SARS-CoV-2 replication in vitro.

A range of neutralization assays have been utilized in the investigation of SARS-CoV-2 – most notably the plaque reduction neutralization test (PRNT). An agarose overlay is required to utilize PRNTs - a time-consuming process that is challenging to complete at higher throughputs in a laboratory operating under Biosafety Level 3 (BSL-3).

Pseudotyped virus particle entry inhibition assays have also been developed. These are appropriate for higher throughput and use at lower biosafety levels, but because these do not feature the actual virus, they can only deliver an approximation of ‘real’ virus neutralization.

These virus particle entry inhibition assays are only suitable for testing entry inhibitors, but they cannot evaluate other drug types.

The microneutralization assays utilized throughout this article are performed in a 96-well format plate, facilitating medium throughput. The assays’ readout involves staining the virus nucleoprotein (NP) to perform a quantitative assessment of inhibitory concentration without relying on subjective, often awkward visual inspection of cytopathic effects.

Two distinct protocols are presented here: one (Basic Protocol 1) outlining the use of the assay for testing plasma, sera, or monoclonal antibodies, and one (Basic Protocol 2) outlining the use of the assay as part of drug screening applications.

Biosafety precautions

SARS-CoV-2 is a BSL-3 pathogen. It is imperative that all appropriate guidelines for the handling and use of pathogenic microorganisms are stringently followed, as well as best practice guidance and current protocols (Burnett, Lunn, & Coico, 2009).

Human-derived material such as plasma and serum samples carries the risk of infection from SARS-CoV2 and other blood-borne viral pathogens.

It is recommended that heat inactivation be performed at 56 °C for 1 hour prior to use and that all related regulations and guidelines for the use and handling of human-derived materials be closely followed.

Testing virus inhibition antibodies: Purified antibodies or serum/plasma (Basic protocol 1)

This protocol can be employed in the assessment of antibodies’ capacity to neutralize SARS-CoV-2 in vitro. It is possible to use serum or plasma samples from any species or monoclonal antibodies (mAbs) in this assay.

This assay exhibits a number of similarities to a PRNT, with a notable difference being that it can be performed in a 96-well cell culture plate to facilitate higher throughput than standard PRNTs.

While it is possible to employ local isolates, the virus utilized in this assay is SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources NR-52281).

The cell type used is Vero.E6 – a cell type which has been demonstrated as permissive for SARS-CoV-2. It is also possible to adapt the assay to accommodate other cell lines (Harcourt et al., 2020).

It is advisable to utilize heat inactivation of serum or plasma at 56 ˚C for 1 hour to minimize the impact of complement on the cells and to mitigate biosafety risks. Both positive and negative controls should be utilized consistently.

Naïve or pre-pandemic plasma or sera can be employed as negative controls for serology testing, while convalescent patient sera or sera from immunized/infected animals may be employed as positive controls.

Negative controls should be a non-SARS-CoV-2 antibody of the same isotype as the tested mAbs when working with mAbs. It is also possible to use any available antiserum or neutralizing mAb as a positive control (Amanat et al., 2020).

Assays are preferably performed with serum, plasma, or mAb in the overlay. This is done at an identical concentration to the initial infection to reflect the physiological conditions as far as possible.

It is also possible to modify the assay to isolate antibodies to the initial virus-antibody incubation period or the overlay to solely investigate entry inhibition or virus spread inhibition within the culture, respectively.

The quantity of virus employed in the assay is a key variable, and it is possible to establish virus titers using different protocols (Mendoza, Manguiat, Wood, & Drebot, 2020). It is important to ensure the virus dose results in robust staining – a dose of approximately 10000 TCID50/ml is employed in this assay.

The staining antibody is also a critical reagent. The work presented here describes an antibody against the nucleoprotein (NP) because this protein is present in significant amounts within infected cells. It should be noted that mAbs or antisera targeting other viral proteins are also viable options.

Antisera raised against the entire virus could also be beneficial options for cell staining. Any appropriate data analysis software may be employed, though the work presented here utilizes GraphPad Prism.

It is possible to prepare assay cell plates and perform final staining steps outside of a BSL-3 facility.

However, it is imperative that any work with replication-competent SARS-CoV2 is undertaken inside a BSL-3 facility using sufficient personal protective equipment (PPE) and that plates are only removed from the BSL-3 facility once the virus has been confirmed to be inactive.

Definitions

  • BSA: Bovine Serum Albumin
  • BSL: Biosafety Level
  • cDMEM: Complete Dulbecco’s Modified Eagle Medium
  • CPE: Cytopathic Effect
  • DMEM: Dulbecco’s Modified Eagle Medium
  • FBS: Fetal Bovine Serum
  • HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • HRP: Horseradish Peroxidase
  • ID50: 50% Inhibitory Dilution
  • MEM: Minimal Essential Medium
  • MNA: Microneutralization Assay
  • NP: Nucleoprotein
  • OPD: O-phenylenediamine dihydrochloride
  • RT: Room Temperature
  • TCID50: 50% Tissue Culture Infectious Dose
  • WFI: Water For Injection

Materials

  • Vero.E6 cells (ATCC #CRL-1586)
  • Complete DMEM medium (cDMEM)
  • Universal mycoplasma detection kit (ATCC 30-1012K)
  • MEM
  • Serum samples for testing
  • Fetal bovine serum (FBS; Corning, cat. no. 35011CV)
  • Water for injection for cell culture (WFI; Gibco, cat. no. A1287301)
  • SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources, cat. no. NR-52281, or similar)
  • 10% formaldehyde (Polysciences, cat. no. 04018-1)
  • Phosphate-buffered saline (PBS; Gibco, cat. no. 10010-023, or equivalent)
  • Triton X-100 (Fisher Bioreagents, cat. no. BP151-100)
  • Non-fat milk (AmericanBio, cat. no. AB10109-01000, or equivalent)
  • Mouse anti-SARS NP antibody, e.g., 1C7 (an in-house mAb, provided by Dr. Thomas Moran)
  • Anti-mouse IgG HRP (Rockland, cat. no. 610-4302)
  • SIGMAFASTTM OPD (Sigma-Aldrich, cat. no. P9187)
  • 3 M hydrochloric acid (Fisher Scientific, cat. no. S25856)
  • Class II biological safety cabinet
  • 96-well cell culture plate (Corning, cat. no. 3595)
  • Light microscope
  • Polypropylene sterile conical tubes:
    • 15 ml (Denville Scientific, cat. no. C1018P or equivalent)
    • 50 ml (Fisher Denville Scientific, cat. no. C1060P or equivalent)
    • 1.5 ml microcentrifuge tubes (Denville, cat. no. C2170 or equivalent)
  • Pipet-Aid
  • Sterile, serological pipettes:
    • 5 ml (Falcon, cat. no. 356543 or equivalent)
    • 10 ml (Falcon, cat. no. 357551 or equivalent)
    • 25 ml (Falcon, cat. no. 357535 or equivalent)
    • 50 ml (Falcon, cat. no. 356550 or equivalent)
  • Micropipettes
  • Micropipette tips:
  • 20 μl barrier tips (Denville Scientific, cat. no. P1121 or equivalent)
  • 200 μl barrier tips (Denville Scientific, cat. no. P1122 or equivalent)
  • 200 μl tips (USA Scientific, cat. no. 1111-1700 or equivalent)1000-μl barrier tips (Denville Scientific, cat. no. P1126 or equivalent)
  • Sterile reservoirs (Fisher Scientific, cat. no. 07-200-127 or equivalent)
  • Biotek SynergyH1 Microplate Reader or equivalent
  • GraphPad Prism 7 (or equivalent software)

Determining the 50% tissue culture infectious dose (TCID50)

Step 1. Vero.E6 cells should be maintained in culture using cDMEM. These cells must be mycoplasma-free and offer over 95% viability for use in this assay.

Step 2. A total of 20,000 cells should be seeded per well in 96-well cell culture plates. This should be done the day prior to infection and can be completed outside of the BSL-3 facility with cells transferred into the facility on the day of virus infection.

Step 3. The following day, 10-fold dilutions of the virus should be prepared in 1 MEM and supplemented with 2% FBS (1 MEM/2% FBS). The 1 MEM/2% FBS must be prepared by mixing equal amounts of 2 MEM and WFI, along with 2% FBS.

A total of 100 μl of original virus stock be added to 900 μl of 1 MEM/2% FBS to function as the 10−1 dilution. The 10-fold dilution series should be prepared from the 10−1 dilution, with dilutions up to 10−8 typically adequate when looking to determine the TCID50.

Virus dilutions should be horizontally added to the plate. Row H is to be utilized as a control row by adding 1 MEM/2% FBS and no virus (no-virus control).

Row H is also employed as a reference to check CPE and assess this in the infected cells. It is important to ensure enough volume of each dilution is prepared for six to eight replicates.

Step 4. The confluence of the cells should be checked under a light microscope to confirm a confluence between 90% and 100%. This is important, as infection efficiency can be reduced through the use of overconfluent cells.

The entirety of the medium should be removed from the 96-well cell culture plate before adding 100 μl per well of each respective virus dilution to the plate.

Wells for no-virus controls should be set up by adding 100 μl per well of 1 MEM/2% FBS without virus to a few rows or columns.

The plate should then be maintained at 37 °C for one hour.

Pipette tips must be changed for every virus transfer to affect the TCID50 being impacted. The liquid should be added to the side of the well to avoid disturbing the cell monolayer.

Step 5. The virus inoculum should be removed from every well following 1 hour of viral absorption. The medium from no-virus control wells should also be removed. A total of 200 μl of 1 MEM/2% FBS should be added to all wells.

Step 6. Cells should be incubated for three days at 37 °C.

Step 7. Cells should be checked under the microscope for CPE (Figure 1) on the third day post-infection. Wells with the highest amount of virus are expected to exhibit strong CPE, decreasing as the virus is further diluted. The no-virus controls can therefore be employed as a reference for no infection.

Step 8. A note should be made of the number of wells that are positive for each dilution. TCID50/ml should be calculated using the Reed & Muench method (Ramakrishnan et al., 2016).

Figure 1. CPE induced by SARS-CoV-2 in Vero.E6 cells. The left panel shows healthy, uninfected Vero.E6 cells forming a monolayer. The right panel shows CPE after infection with SARS-CoV-2 (e.g., rounded up cells, a halo around cells, etc.). Image Credit: Nexcelom Bioscience LLC

Performing the microneutralization assay (MNA)

Step 9. Vero.E6 cells should be maintained in culture using cDMEM. These cells must be mycoplasma-free and demonstrate high viability for use in this assay.

Step 10. A total of 20,000 cells should be seeded per well in a 96-well cell culture plate as described in Step 2. This should be done the day before infection.

Step 11. Serum dilutions should be prepared in empty, sterile 96-well cell culture plates. This should be done using 1 MEM/2% FBS, with 1 MEM/2% FBS prepared via the addition of equal amounts of 2 MEM and WFI, as well as 2% FBS.

It should be noted that serum or plasma samples from animals or humans infected with SARS-CoV-2 must be heat-inactivated at 56 °C for 1 hour prior to use to ensure that any complement present in the sample is inactivated.

Heat inactivation may also inactivate infectious viruses present in samples. Each sample should be tested in duplicate.

Step 12. A total of 270 μl of 1 MEM/2% FBS should be added to Row A, while 200 μl of 1 MEM/2% FBS should be added to Rows B-G and 30 μl of each respective sample should be added to row A before being pipetted up and down several times to ensure it is mixed.

With all samples added, 100 μl should be transferred from Row A to Row B before being mixed 5-10 times with the multichannel pipette.

Tips should then be discarded before loading new tips onto the multichannel pipette. It is then necessary to transfer 100 μl from Row B to Row C before mixing a further 5-10 times.

Plate layout for microneutralization assay. Indicated dilutions are suggestions for testing serum samples. Other dilution series or concentrations (for mAbs) might be used.

Figure 2. Plate layout for microneutralization assay. Indicated dilutions are suggestions for testing serum samples. Other dilution series or concentrations (for mAbs) might be used. Image Credit: Nexcelom Bioscience LLC

This process is repeated until row G is reached before discarding the 100 μl to ensure that each column has 200 μl remaining. Row H will now function as a control row with the plate referred to as Plate A. Figure 2 shows the plate layout.

Step 13. A total of 80 μl of each respective dilution should be transferred to a new 96-well culture plate. The order of the samples and dilutions must be preserved in the new plate (Plate B). It should be noted that the dilutions and preparation of Plates A and B may be performed at BSL-2.

Step 14. SARS-CoV-2 should only be handled in a BSL-3 laboratory and within a biosafety cabinet. A total of 10000 TCID50/ml of SARS-CoV-2 should be prepared in 1 MEM/2% FBS before 80 μl of the virus is added to each well in plate B other than wells H7-H12.

Next, 80 μl of 1 MEM/2% FBS must be added to Rows H7-H12 and the lid of the 96-well plate should be closed. The researcher should tap this gently with the palm to ensure the serum and virus are mixed.

This should result in each well comprising a total of 160 μl - 80 μl of serum dilution and 80 μl of the virus. This should be incubated for one hour at room temperature. There is no need to rock the plate in this instance.

Step 15. Following incubation, the Vero.E6 cells in the 96-well cell culture plate (Step 9) should have the medium removed using an aspirator or a multichannel pipette.

It is important that care be taken to remove the medium, ensuring the tip does not touch the cells and these are not dislodged.

Step 16. A total of 120 μl of the serum-virus mixture should be added to the cells from Plate B. Serum dilution order must be maintained across all plates.

There is no need to replace tips when the serum-virus mixture is moved to the top of the plate from the bottom, for example, starting from row H and ending at row A.

Cells should be placed in a humidified incubator with 5% CO2 at 37 °C for 1 hour.

In this instance, infection was completed with just 60 μl of the virus, resulting in 600 TCID50 per well. Virus amounts may vary depending on how long cells are kept in the incubator for infection.

Reasonable staining and adequate sensitivity were achieved with 600 TCID50 per well, but the amount of virus per well can be optimized for improved results.

Step 17. The plate is tilted with one hand and all the virus-serum mixture is removed with a multichannel pipette. This was done after one hour.

With all the medium removed, 100 μl should be added from each well of Plate A to the cells. It is important to minimize the time that cells are without liquid; otherwise, they may dry out and die.

Finally, 100 μl per well of 1 MEM/2% FBS should be added. It is not necessary to change the tips if these have not touched the wells.

Step 18. The cells should then be incubated for 48 hours before discarding Plates A B.

Step 19. After 48 hours later, the researcher should check for cytopathic effect. Cells that received no virus are expected to be healthy, while wells H1-H6 are expected to exhibit signs of infection.

All medium should be removed from each well containing cells before adding 100 μl of 10% formaldehyde.

SARS-CoV-2 must be inactivated for 24 hours at 4 °C.

Staining protocol

Step 20. Once the virus has been inactivated for 24 hours, the plate may be removed from the BSL-3 facility. Staining can then be performed in a BSL-2 facility within a biosafety cabinet.

Local regulations may vary, so before proceeding with this option, a discussion must take place on how inactivation is to be tested and proven with a designated biosafety officer.

Certain institutions may not permit material to be removed from the BSL-3 containment facility. In these cases, staining should be performed within the BSL-3. Following inactivation, plates should still be handled at BSL-2 in a biosafety cabinet to minimize any residual risk.

Step 21. Formaldehyde should be removed from the cells carefully with a multichannel pipette. The cells should not be touched with the tip or dislodged in any way.

Fixation with 10% formaldehyde will ensure cells are all cross-linked onto the wells. These should still be inspected under the microscope to confirm these remain attached, however.

Step 22. Cells must then be washed via the addition of 200 μl phosphate-buffered saline (PBS) per well. This can be aspirated with a multichannel aspirator or the PBS can be removed using a multichannel pipette.

Step 23. Fresh permeabilization solution is prepared via the addition of 500 μl of Triton X-100 to 500 ml of PBS (0.1% PBS/Triton). This is mixed thoroughly before adding 150 μl per well of 0.1% PBS/Triton. Cells are then incubated with this solution for 15 minutes at RT.

Step 24. Following incubation, cells should be washed with 200 μl per well of PBS. PBS is then removed before adding 100 μl/well of PBS supplemented with 3% non-fat milk. Cells are then incubated for one hour at RT.

Step 25. The primary antibody solution should be prepared in PBS supplemented with 1% non-fat milk. This assay employed 1 μg/ml of 1C7 (mouse anti-SARS NP or equivalent) mAb provided by Dr. Thomas Moran. A total of 100 μl/well of primary antibody solution was added to this prior to incubation for one hour at RT.

Step 26. Each well should be washed twice using 200 μl of PBS. This is then removed by gently tapping the plate on paper towels to confirm the primary antibody has all been removed.

The secondary antibody solution - anti-mouse IgG HRP – is prepared in PBS supplemented with 1% non-fat milk at a dilution of 1:3000. A total of 100 μl/well is added before incubation for one hour at RT.

Step 27. Each well should then be washed twice with 200 μl/well of PBS. The plate is dried by gently tapping it on paper towels. The developing solution (SIGMAFASTTM OPD) should be prepared in line with the manufacturer’s instructions.

Step 28. A total of 100 μl of developing solution is added per well. It is then necessary to wait 10-12 minutes.

Step 29. The reaction can be halted by adding 50 μl/well of 3 M hydrochloric acid.

Step 30. Optical density is measured at 490 nm using a Biotek SynergyH1 Microplate Reader (or equivalent). Data is recorded, with the virus-only wells and no-virus wells averaged separately.

The formula to calculate percent inhibition at each well can be described as:

100 ((X-average of no-virus wells)/(average of virus-only wells - an average of no-virus wells)*100), where X is the read for each well.

It is also possible to perform non-linear regression curve fit analysis over the dilution curve to calculate ID50. This requires the top and bottom constraints to be set at 100% and 0% (Figure 3).

Analysis of data as reciprocal dilution of serum and percent inhibition of virus (ID50 of serum sample 1 is 1:298 and serum sample 2 is 1:1873).

Figure 3. Analysis of data as reciprocal dilution of serum and percent inhibition of virus (ID50 of serum sample 1 is 1:298 and serum sample 2 is 1:1873). Image Credit: Nexcelom Bioscience LLC

Screening of anti-SARS-Cov-2 compounds in vitro

This protocol is suitable for the assessment of a compound’s potential to inhibit SARS-CoV- 2 infections at each stage of the viral life cycle; for example, entry, replication, assembly, egress, and spread.

One of the most streamlined approaches to the identification of compounds targeting SARS-CoV-2 involves the repurposing of drugs already approved for the treatment of similar diseases (Gordon et al., 2020).

Any compound suspected of exhibiting antiviral qualities may be evaluated using this assay, with the compound’s antiviral activity determined based on its ability to inhibit SARS-CoV-2 replication. This is assessed by immunostaining for the viral NP.

To do this, Vero.E6 cells are pre-incubated with each compound dilution for a total of 2 hours before a low-MOI infection is used to facilitate multi-cycle replication.

Once 48 hours have passed post-infection, cells should be fixed and stained before automated imaging and analysis can take place. The test compound’s toxicity is ascertained in parallel using an MTT assay or another suitable commercial cell viability assay.

The ratio of IC50 can be calculated via determining the antiviral data over the CC50 calculated from the cytotoxicity data. This will provide a compound’s selectivity index, demonstrating how selective it is in terms of inhibiting viral replication versus its impact on cell death.

When conducting this screening, it is important to include standard positive and negative controls to ensure that results can be compared between different assays.

There is the potential for existing positive controls to be utilized; for instance, in this assay, SARS-CoV-2 inhibitors, such as remdesivir, have an expected IC50 of 500 nM (Bouhaddou et al., submitted). Negative controls would ideally include diluent-only treatment, for example, DMSO.

It should be noted these conditions were specifically optimized for infection of Vero.E6 cells. It would be possible to adapt this method to other cell lines that can support SARS-CoV-2 infection. Drug combination and time-of-addition studies may also be performed using similar assay settings.

Definitions

  • BSA: Bovine Serum Albumin
  • CC50: 50% Cytotoxic Concentration
  • cDMEM: Complete Dulbecco’s Modified Eagle Medium
  • DAPI: 41,6-diamidino-2-phenylindole
  • DMEM: Dulbecco’s Modified Eagle Medium
  • DMSO: Dimethyl Sulfoxide
  • FBS: Fetal Bovine Serum
  • HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • IC50: 50% inhibitory concentration
  • MOI: Multiplicity of Infection
  • MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • TCID50: 50% Tissue Culture Infectious Dose

Materials

  • Vero.E6 cells (ATCC #CRL-1586)
  • Universal mycoplasma detection kit (ATCC 30-1012K)
  • Complete DMEM medium (cDMEM)
  • Viral growth medium (DMEM with 2% FBS)
  • SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources, cat. no. NR-52281, or similar)
  • Compound library
  • DMSO (Corning, cat. no. 25-950-CQC)
  • 10% formaldehyde (Polysciences, cat. no. 04018-1)
  • Anti-SARS nucleoprotein antibody (mAb 1C7, produced in house)
  • Donkey anti-mouse IgG (H L) highly cross-absorbed secondary antibody, Alexa Fluor Plus 488 (Invitrogen, cat. no. A32766 or equivalent)
  • 41,6-Diamidine-21-phenylindole (DAPI; e.g., Sigma-Aldrich)
  • Phosphate-buffered saline (PBS; Gibco, cat. no. 10010-023, or equivalent)
  • Bovine serum albumin (BSA; MP Biomedicals, cat. no. 08810063)
  • Cell Proliferation Kit I (MTT assay, Roche, cat. no. 11465007001) or similar cytotoxicity assay
  • CO2 incubator
  • 96-well cell culture plate (Corning, cat. no. 3595).
  • 96-well deep well (2 ml) plates (Corning, cat. no. 3960, or equivalent)
  • Plate sealing film, adhesive (BioRad, cat. no. MSB1001)
  • Polypropylene sterile conical tubes:
    • 15 ml (Denville Scientific, cat. no. C1018P or equivalent)
    • 50ml (Fisher Denville Scientific, cat. no. C1060P or equivalent)
  • Pipet-Aid
  • Sterile, serological pipettes:
    • 5 ml (Falcon, cat. no. 356543 or equivalent)
    • 10 ml (Falcon, cat. no. 357551 or equivalent)
    • 25 ml (Falcon, cat. no. 357535 or equivalent)
    • 50 ml (Falcon, cat. no. 356550 or equivalent)
  • Single-channel and multi-channel micropipettes
  • Micropipette tips:
    • 20 μl barrier tips (Denville Scientific, cat. no. P1121 or equivalent)
    • 200 μl barrier tips (Denville Scientific, cat. no. P1122 or equivalent) 200 μl tips (USA Scientific, cat. no. 1111-1700 or equivalent)
    • 1000 μl barrier tips (Denville Scientific, cat. no. P1126 or equivalent)
  • Sterile reservoirs (Fisher Scientific, cat. no. 07-200-127 or equivalent)
  • 1.5 ml microcentrifuge tubes (Denville, cat. no. C2170 or equivalent)
  • Celigo 96-well plates (Greiner, cat. no. 655090)
  • Plate cytometer (Celigo) or laser-scanning cytometer (Acumen)
  • Additional reagents and equipment for TCID50 assay and immunostaining (Basic Protocol 1)

Performing the SARS-CoV2 antiviral assay

Step 1. Vero.E6 cells should be maintained in culture using cDMEM.

Step 2. A total of 2,000 cells should be seeded per well in a 96-well cell culture plate prior to infection. Maintaining a minimum number of cells per well is key to ensuring the accuracy of both the cytotoxicity assay and the immunofluorescent antiviral readout.

Three compounds can be tested using each 96-well plate, but separate plates should be used to test cytotoxic and antiviral activities.

It is possible to perform this and other steps up to Step 6 outside of a BSL-3 facility. Cells and dilutions should be transferred into the facility on the day of virus infection, however.

Step 3. Compound dilutions should be prepared in empty, sterile 2 ml 96-well deep well plates using a DMEM that has been supplemented with 2% FBS (viral growth medium).

Where compounds under investigation are to be dissolved in DMSO (common) medium, it is necessary to supplement this with a matching percentage of DMSO to perform dilutions.

Step 4. A total of 1100 μl of 1 DMEM (without DMSO) supplemented with 2% FBS should be added to Columns 1 and 7, while 750 μl of 1 DMEM (containing DMSO if required) supplemented with 2% FBS is added to Columns 2-6 and 8-12.

It is necessary to add 50% more compound than the selected maximum dose to Columns 1 and 7 (2 compounds tested per row, 16 total compounds diluted per deep well plate).

For example, 3.3 μl of a 10 mM stock is required for a concentration of 30 μM, which will dilute to a total maximum concentration of 20 μM when 50 μl of the virus is added.

Using this compound example, DMSO concentration for the medium in Columns 2-6 and 8-12 would therefore be 0.3%. It is important not to exceed 0.5% DMSO, however, due to the risk of toxicity.

With all compounds added, Column 1 should be pipetted up and down a total of 20 times to mix together before transferring 375 μl from Column 1 to Column 2, then mixing this 5-10 times with the multichannel pipette.

Tips should be discarded and new tips loaded into the multichannel pipet. A total of 375 μl should be transferred from Column 2 to Column 3 and mixed a further 5-10 times. This process should be repeated until Column 6 is reached, then repeated for Columns 7-12.

Utilizing the whole deep-well plate will result in a total of 16 compounds with six 3-fold serial dilutions per compound. The final concentrations in this example would result in 20-80 nM.

Plate X and plate Y layout for the antiviral assay.

Figure 4. Plate X and plate Y layouts for the antiviral assay. Image Credit: Nexcelom Bioscience LLC

This plate is referred to as Plate X, and its layout is illustrated in Figure 4.

Step 5. The Vero.E6 cells seeded the previous day in the two 96-well cell culture plates (one for antiviral, one for cytotoxicity) should be selected prior to removing the medium using an aspirator or a multichannel pipette.

The medium must be removed carefully to ensure that the tip does not touch the cells and that these are not dislodged.

A total of 100 μl of each respective dilution is transferred to the Vero.E6 plates - there should be sufficient volume (750 μl) in the deep-well plate to facilitate triplicates for cytotoxicity and antiviral testing.

The same dilution series should be used for both antiviral and cytotoxic assays, delivering consistent matching data sets. These should be performed simultaneously.

It is possible to test three compounds and six DMSO control wells per 96-well plate (Plate format Y, Figure 4). It is advisable to avoid the outer wells due to the risk of medium evaporation, which may result in altered compound concentrations.

Instead, outer wells can be filled with medium and cells to serve as uninfected controls for viral detection via immunostaining.

It is necessary to incubate the cells with the compound for 2 hours prior to infection for use as a standard. This time frame can be adjusted for specific experiments.

Step 6. Any work with SARS-CoV-2 must be conducted in a BSL-3 laboratory using a biosafety cabinet.

A total of 2000 TCID50/ml of authentic SARS-CoV-2 (SARS-CoV-2 isolate USA-WA1/2020) should be prepared in 1 DMEM supplemented with 2% FBS. Basic Protocol 1 Steps 1-8 detail the determination of TCID50.

A total of 50 μl (100 TCID50 per well or 0.025 MOI) of the virus should be added to the 100 μl of the compound containing medium in each well of plate Y (but not the outer wells) and lightly pipetted up and down 2-3 times to mix each well. This step helps ensure consistent viral infection and compound concentrations between plates.

Next, 50 μl of 1 DMEM with 2% FBS should be added to the sample wells of the cytotoxicity plate to modify compound concentrations so that these are equal to the concentrations of the antiviral assay.

This should result in each inner well having a total of 150 μl. This should then be incubated for 48 hours at 37 °C using a humidified incubator with 5% CO2.

Cytotoxicity testing work may be performed outside of the BSL-3 laboratory because no infectious virus is involved. BSL-2 would be suitable for this.

Step 7. Once incubation is complete, the infected Vero.E6 cells in the 96-well cell culture plates should be removed from the incubator. This must be done under BSL-3 conditions. The medium should be removed using a multichannel pipette.

It is also possible to quantify viral titers from the removed medium by using a TCID50 assay (Basic Protocol 1) or plaque assay.

The Vero.E6 cells are fixed by adding a total of 200 μl of 4% formaldehyde (diluted from 10%) in PBS to each well of the 96-well plate. This should be incubated in 4% formaldehyde for 24 hours at room temperature before plates are removed from the BSL-3 laboratory.

Step 8. After the plates have been successfully removed from the BSL-3 laboratory, the fixed cells should be immunostained with SARS-CoV2 specific antibodies. This process matches Steps 20-30 of Basic Protocol 1, with several key modifications.

Antibodies listed in Basic Protocol 2’s materials list should be used for this protocol. After three washes with 200 μl of PBS, the cells should be incubated with a secondary antibody solution comprised of anti-mouse IgG and DAPI in PBS supplemented with 0.5% BSA at a 1:2000 dilution.

Next, 100 μl/well should be added, and this should be incubated for 45 minutes at RT before it is washed three times for 5 minutes in PBS using a gentle shaking motion.

A laser scanning cytometer (Acumen) or plate cytometer (Celigo) may be employed in the assessment of infected cells over total cells to facilitate accurate quantification of viral infection.

The DMSO control must be normalized to 100% infection to ensure proper comparison with compound-treated wells.

Step 9. Uninfected Vero.E6 cell cytotoxicity controls should be assayed to determine cell viability. This can be done with the MTT assay or using another suitable, commercially available cell viability assay. In either case, it is important to adhere to the manufacturer’s instructions.

The incubation time of cytotoxicity measurements should be identical to the incubation time with the antiviral assay.

Step 10. The IC50 and CC50 for each compound can then be calculated using Prism software, in line with Basic Protocol 1.

SARS-CoV-2 propagation

The SARS-CoV2 USA-WAS1/2020 viral strain employed in the assay optimization outlined here was propagated with the Vero.E6 cell line. It has been previously demonstrated that the virus can replicate to high titer in this cell line.

Stocks are generated via the infection of a confluent monolayer of cells at a low MOI to minimize the risk of defective interfering particles that can lower the titer of the viral preparation.

It is advisable to work with fully sequenced low-passage-number stocks to avoid selecting mutants better adapted to growth in the cell line employed for virus amplification.

Materials

  • Vero.E6 cells (ATCC CRL-1586)
  • Vero.E6 growth medium: cDMEM
  • SARS-CoV-2 isolate USA-WA1/2020, (BEI Resources NR-52281 or similar) Viral growth medium (DMEM with 2% FBS)
  • Class II biological safety cabinet
  • 75 cm2 tissue culture flasks with filter caps (size dependent on the volume of stock needed)
  • Counting chamber or automatic cell counter
  • CO2 incubator
  • Polypropylene sterile conical tubes
  • 15 ml (Denville Scientific, cat. no. C1018P or equivalent)
  • 50 ml (Fisher Denville Scientific, cat. no. C1060P or equivalent) Pipet-Aid
  • Sterile, serological pipettes:
    • 5 ml (Falcon, cat. no. 356543 or equivalent)
    • 10 ml (Falcon, cat. no. 357551 or equivalent)
  • Tabletop centrifuge, 4 °C
  • 2.0 ml cryotubes with O-ring
  • Additional reagents and equipment for TCID50 assay (Basic Protocol 1, Steps 1-8)

Step 1. Vero.E6 cells should be maintained in culture using cDMEM (Basic Protocol 1).

Step 2. A total of 4106 Vero.E6 cells should be seeded into 75 cm2 flasks and incubated in cDMEM for 24 hours. This should be done the day prior to infection.

At least three flasks should be prepared:

  • One for virus propagation
  • One as an uninfected control
  • One for counting the cells

Cells are ∼90% confluent at the time of infection.

It is possible to perform this step outside of the BSL-3 facility, with cells then transferred into the facility on the day of virus infection.

Step 3. One of the flasks should be used to count the cells on the day of inoculation. This can be done using an automatic cell counter or counting chamber. The volume of viral inoculum required to infect at an MOI of 0.001 is then calculated.

A confluent T-75 flask can yield around 8106 cells when using Vero.E6. The medium should be removed before being washed gently in PBS once.

It is then possible to infect cells in 5 ml of viral growth medium for 1 hr at 37 °C and 5% CO2, ensuring that the flask is rocked every 15 minutes to facilitate an even spread of viral inoculum across the monolayer.

Step 4. Following an hour of adsorption, 5 ml of viral growth medium should be added before incubating the flask at 37 °C.

The cell culture should be observed daily to track the development of cytopathic effects. Cells typically become rounded up and detach from the monolayer within 3 or 4 days.

The virus should be harvested when the cytopathic effect has progressed to 80%.

Step 5. The medium can be clarified via centrifugation in a 50 ml conical tube for 10 minutes at 1300 g and 4 °C.

Step 6. Pooled supernatants are then aliquoted into appropriately labeled 2.0 ml O-ring cryotubes and stored at −80 °C until use.

Step 7. Virus stock titer can be determined via plaque assay or TCID50 (Basic Protocol 1).

Reagents and solutions

Complete DMEM medium (cDMEM; Used in basic protocol 1 and 2)

  • 880 ml of DMEM (Gibco, cat. no. 11995-065 or equivalent)
  • 10 ml of penicillin-streptomycin (Gibco, cat. no. 15140122)
  • 10 ml of HEPES buffer (Gibco, cat. no. 15630080)
  • 100 ml of FBS (Corning, cat. no. 35011CV)

This can be filtered using a 0.22 μm Stericup filter (MilliporeSigma, cat. no. S2GPU05RE) and stored for up to 4 weeks at 4 °C.

MEM, 2× (Used in basic protocol 1)

  • 200 ml of 10 MEM (Gibco, cat. no 11430030)
  • 20 ml of penicillin-streptomycin (Gibco, cat. no. 15140122)
  • 20 ml of HEPES buffer (Gibco, cat. no. 15630080)
  • 20 ml of L-glutamine (Gibco, cat. no. 25030081)
  • 32 ml of 7.5% sodium bicarbonate (Gibco, cat. no. 25080094)
  • 12 ml of 35% BSA (MP Biomedicals, cat. no. 08810063)
  • 696 ml of water for injection for cell culture (WFI; Gibco, cat. no. A1287301)

This can be filtered using a 0.22 μm Stericup filter (MilliporeSigma, cat. no. S2GPU05RE) and stored for up to 4 weeks at 4 °C.

This medium is employed in the preparation of 1× MEM/2% FBS by mixing identical quantities of WFI and 2× MEM.

Viral growth medium (DMEM with 2% FBS; Basic protocol 2)

  • 880 ml of DMEM (Gibco, cat. no. 11995-065, or equivalent)
  • 10 ml of penicillin-streptomycin (Gibco, cat. no. 15140122)
  • 10 ml of HEPES buffer (Gibco, cat. no. 15630080)
  • 20 ml of FBS (Corning, cat. no. 35011CV)

This can be filtered using a 0.22 μm Stericup filter (MilliporeSigma, cat. no. S2GPU05RE) and stored for up to 4 weeks at 4 °C.

Discussion

The microneutralization assay outlined in this work was adapted from recognized protocols utilized with other viruses, including the influenza virus (Amanat, Meade, Strohmeier, & Krammer, 2019).

The assay delivered medium-throughput - a notable improvement over the throughput of PRNTs.

Unlike RBD-ACE2 inhibition assays, this MNA is also able to detect neutralizing antibodies binding to epitopes outside of the RBD.

This MNA accommodates different virus isolates, and the assay can likely be modified to work with staining antibodies beyond mAbs against NP; for example, polyclonal sera or antibodies targeting S or M.

It is recommended that users optimize the assay based on the level of infection observed in each respective cell line used. It is also possible to adjust viral input to reduce the sensitivity of the assay.

As the antiviral assay discussed here was also adapted from existing work with the influenza virus. Its immunostaining output offers medium throughput and can be used to thoroughly analyze the cytotoxic and antiviral activity of many more compounds compared to using plaque assay or TCID50.

Many parameters of this assay can be adapted, including the time course of infection, viral strain, the cell line used, MOI, and antibody used. This enables this assay to answer a wide range of questions related to a compound’s antiviral activity.

Critical parameters and troubleshooting

Cells employed in the microneutralization assay detailed in Basic Protocol 1 must be healthy. It is also recommended to check their viability periodically. A cell count should also be carefully performed to confirm that cells are not over-confluent on the day of the assay. Cells must remain mycoplasma-free.

The TCID50 measurement of each stock must be done with extra care because each virus stock will be utilized across multiple microneutralization assays.

The virus should also be sequenced every few passages to ensure that no significant mutations have occurred due to the passaging of the virus in cell culture.

It is possible to perform the microneutralization assay with serum, plasma, or purified antibodies. Should antibodies be used, it is advisable to perform a trial run to assess the optimal concentrations to be tested.

The immunostaining antiviral assay detailed in Basic Protocol 2 exhibits constantly lower sensitivity than an assay designed to detect infectious viral particles produced in the supernatant, for example, a TCID50 or plaque assay.

Due to this, the IC50 determined for a specific compound in this assay will generally be 3 to 5 times higher than these assays.

This feature makes this antiviral assay ideal for the medium-throughput screening of potential antivirals, though it is prudent to confirm top hits using appropriate classical virology methods.

The DAPI counterstain may be employed as a proxy for toxicity in infected cells, working in conjunction with the MTT assay performed in uninfected cells.

Should the majority of DAPI staining be lost in a compound-treated well, the compound concentration in question should be excluded from any final IC50 calculation.

Time considerations

The TCID50 is generally performed once per viral stock, taking three days in total. The microneutralization would ideally take four days because the cells are fixed two days after performing the assay.

As soon as day three has been reached, plates should be stained immediately. This is because keeping the cells in formaldehyde beyond this point may adversely affect the staining.

The entire microneutralization assay and related data generation can be completed within 4 days.

Compounds are generally added 2 hours before SARS-CoV-2 infection. This may be adjusted based on the compound’s proposed mechanism of action or the hypothesis being evaluated.

The antiviral assay takes three days in total, from infection to staining, scanning, and data analysis. The compound must be always incubated on the cells for an identical period between antiviral and cytotoxicity assays.

References

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Acknowledgments

Produced from materials originally authored by Fatima Amanat, Kris M. White, Lisa Miorin, Shirin Strohmeier, Meagan McMahon, Philip Meade, Wen-Chun Liu, Randy A. Albrecht, Viviana Simon, Luis Martinez-Sobrido, Thomas Moran, Adolfo García-Sastre, and Florian Krammer.

About Nexcelom Bioscience

Nexcelom Bioscience is a developer and marketer of image cytometry products for cell analysis in life science and biomedical research. Products range from cell viability counters (Cellometer) to high throughput microwell image cytometry workstations (Celigo), used in thousands of research laboratories in academic institutes, and pharmaceutical and biotech companies. The company contributes to the life science industry through innovation and expertise in the science of cell counting.

The product family includes instruments, consumables, and reagents. Nexcelom customers engage in a wide variety of research, such as cancer research, immunology, stem cell research, and neuroscience. Nexcelom offers different Cellometer models to count and analyze cell lines and primary cells, through brightfield and fluorescence imaging modes. In addition, Celigo is a powerful high image quality, high-throughput image cytometry system for adherent and suspension cells in microwell plates.

Nexcelom Bioscience is a fast-growing company in a huge market. With its headquarters and manufacturing facilities in the Boston area, the company currently has over 80 global employees, who are fast-paced, customer-centric, helpful to colleagues and customers, and passionate about their impact in life science.


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Last updated: Nov 2, 2021 at 6:54 AM

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