key: cord-1040513-byx3ts1d authors: Amanat, Fatima; White, Kris M.; Miorin, Lisa; Strohmeier, Shirin; McMahon, Meagan; Meade, Philip; Liu, Wen‐Chun; Albrecht, Randy A.; Simon, Viviana; Martinez‐Sobrido, Luis; Moran, Thomas; García‐Sastre, Adolfo; Krammer, Florian title: An In Vitro Microneutralization Assay for SARS‐CoV‐2 Serology and Drug Screening date: 2020-06-25 journal: Curr Protoc Microbiol DOI: 10.1002/cpmc.108 sha: caa91a7e09a2d3b7cf7bd719e86deb186f748ccd doc_id: 1040513 cord_uid: byx3ts1d The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS‐CoV‐2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS‐CoV‐2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti‐SARS‐CoV‐2 compounds in vitro Support Protocol: SARS‐CoV‐2 propagation Several "common cold" seasonal coronaviruses circulate in the human population, but in late 2019, a novel coronavirus surfaced in China that was later classified as a beta-coronavirus and named severe acute respiratory syndrome coronavirus 2 for SARS-CoV-2, but the assay can be adapted to other cell lines (Harcourt et al., 2020) . Heat inactivation of serum or plasma at 56˚C for 1 hr is recommended to minimize the effect of complement on the cells, as well as to mitigate biosafety risks. Positive and negative controls should be used at all times. For serology, naïve or pre-pandemic sera or plasma can be used as negative controls, while convalescent patient sera or sera from immunized/infected animals can be used as positive controls. For mAbs, the negative control should be a non-SARS-CoV-2 antibody of the same isotype as the tested mAbs. Any available neutralizing mAb or antiserum can be used as positive control . We also preferably perform the assays with serum/plasma/mAb in the overlay at the same concentration as the initial infection, since we believe that this better reflects physiological conditions. However, the assay can be modified to have antibody only in the initial virus-antibody incubation period or only in the overlay, to study entry inhibition only or inhibition of virus spread within the culture only, respectively. The amount of virus used for the assay is an important variable. Virus titers can be established using different protocols (see Current Protocols article: Mendoza, Manguiat, Wood, & Drebot, 2020) . In any case, a virus dose that leads to robust staining needs to be used, and approximately 10,000 TCID 50 /ml are used in this assay. Another crucial reagent is the staining antibody. Here, we describe an antibody against the nucleoprotein (NP), since this protein is abundant within infected cells. However, mAbs or antisera targeting other viral proteins might be used as well. Antisera raised against the whole virus might also be useful for cell staining . We perform our data analysis in GraphPad Prism, but any other similar data analysis software can be used. Preparation of the cell plates for the assay and the final staining steps may be performed outside the BSL-3 facility. Any work with replication-competent SARS-CoV2 needs to be performed inside a BSL-3 facility using appropriate personal protective equipment (PPE) . Plates may only be taken out of the BSL-3 facility once the virus has been inactivated. 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 ID 50 = 50% inhibitory dilution MEM = minimal essential medium MNA = microneutralization assay NP = nucleoprotein OPD = o-phenylenediamine dihydrochloride RT = room temperature TCID 50 = 50% tissue culture infectious dose WFI = water for injection Current Protocols in Microbiology Serum/plasma samples from humans or animals infected with SARS-CoV-2 should be heat-inactivated at 56°C for 1 hr prior to use. Heat-inactivation of serum/plasma samples will ensure that any complement present in the sample is inactived, and it also increases safety since heat inactivation might inactivate infectious virus present in samples. Each sample is tested in duplicate. 12. Add 270 μl of 1× MEM/2% FBS to row A and add 200 μl of 1× MEM/2% FBS to row B-G. Add 30 μl of each respective sample to row A and pipet up and down several times to mix. Once all the samples are added, transfer 100 μl from row A to B and mix 5-10 times using the multichannel pipette. Discard tips. Load new tips on to the multi-channel pipette and transfer 100 μl from row B to row C, then mix 5-10 times again. Repeat this process until row G is reached. At row G, discard the 100 μl so each column has 200 μl remaining. Row H is a control row. This plate is called plate A. Refer to Figure 2 for plate layout. 13. Obtain a new 96-well cell culture plate and transfer 80 μl of each respective dilution to the new plate. Ensure that the order of the samples and dilutions is preserved in the new plate (referred to as plate B). Dilutions and preparation of plate A and plate B can all be performed in at BSL-2. 14. Handle SARS-CoV-2 in a Biosafety Level 3 (BSL-3) laboratory in a biosafety cabinet. Prepare 10,000 TCID 50 /ml of SARS-CoV-2 in 1× MEM/2% FBS. Add 80 μl of the virus to each well in plate B except wells H7-H12. Add 80 μl of 1× MEM/2% FBS to rows H7-H12. Close the lid of the 96-well plate and tap gently with the palm of the hand to mix the serum with the virus. Each well should now have a total of 160 μl (80 μl of serum dilution plus 80 μl of virus). Incubate for 1 hr at room temperature. It is not necessary to rock the plate. 15. After the incubation time is over, take the Vero.E6 cells in the 96-well cell culture plate (step 9) and remove the medium using a multi-channel pipette or an aspirator. The medium should be removed carefully such that the tip does not touch the cells or the cells are not dislodged in any way. 16. Add 120 μl of serum-virus mixture from plate B to the cells. Ensure that the order of the serum dilutions is preserved across all plates. Tips do not need to be changed if the serum-virus mixture is moved from the bottom of the plate to the top (i.e., starting from row H and ending at row A). Place the cells at 37°C in a humidified incubator with 5% CO 2 for 1 hr. Staining protocol 20. After inactivation of virus for 24 hr, the plate can be taken out of the BSL-3 facility and staining can be performed in a BSL-2 facility inside a biosafety cabinet. CAUTION: Before proceeding with this option, please discuss how inactivation needs to be tested and proven with your biosafety officer, since local regulations may differ. Some institutions may not allow bringing material out of the BSL-3 containment facility, and staining will need to be performed within the BSL-3 in these cases. Even after inactivation, plates should be handled at BSL-2 in a biosafety cabinet in order to reduce residual risk. 21. Remove formaldehyde from the cells carefully using a multi-channel pipette. Care should be taken not to touch the cells with the tip or dislodge the cells in any way. 30. Measure the optical density at 490 nanometers on a Biotek SynergyH1 Microplate Reader (or equivalent) and record data. Average the "virus-only" wells and separately average all the "no-virus" wells. The formula to calculate percent inhibition at each well is 100 − ((X-average of ʻno virusʼ wells)/(average of ʻvirus onlyʼ wellsaverage of ʻno virusʼ wells)*100), where X is the read for each well. Non-linear regression curve fit analysis over the dilution curve can be performed to calculate ID 50 . The top and bottom constraints are set at 100% and 0% (Fig. 3) . This protocol can be used to assess the ability of a compound to inhibit SARS-CoV-2 infection at any stage of the viral life cycle (e.g., entry, replication, assembly, egress and spread). Repurposing of existing drugs already approved for the treatment of other diseases represents one of the most rapid strategies for the identification of compounds targeting SARS-CoV-2 (Gordon et al., 2020) . However, any compound predicted to have antiviral activity could be tested using this assay. Here, the antiviral activity of the compound is evaluated based on its ability to inhibit SARS-CoV-2 replication as assessed by immunostaining for the viral NP. Specifically, Vero.E6 cells are preincubated with each compound dilution for 2 hr, followed by a low-MOI infection to allow multi-cycle replication. 48 hr post-infection, cells are fixed and stained prior to automated imaging and analysis. The toxicity of the test compound is determined in parallel by performing an MTT assay or any other commercial cell viability assay. The ratio of IC 50 calculated from the antiviral data over the CC 50 calculated from the cytotoxicity data will yield the selectivity index of a compound, indicating how selective it is for inhibiting viral replication compared to its impact on cell death. Importantly, when performing the screening, standard positive and negative controls should be included so that the results can be compared between different assays. Positive controls could be previously established-for example, SARS-CoV-2 inhibitors such as remdesivir, which has an expected IC 50 of ∼500 nM in this assay (Bouhaddou et al., submitted) . Negative controls should include diluent-only treatment (e.g., DMSO). Of note, these conditions have been optimized for infection of Vero.E6 cells, but could be adapted to other cell lines that can support SARS-CoV-2 infection. In addition, time-of-addition and drug combination studies can also be performed with similar assay settings. BSA = bovine serum albumin CC 50 = 50% cytotoxic concentration cDMEM = complete Dulbecco's modified Eagle Medium DAPI = 4 ,6-diamidino-2-phenylindole DMEM = Dulbecco's modified Eagle Medium DMSO = dimethyl sulfoxide FBS = fetal bovine serum HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IC 50 = 50% inhibitory concentration MOI = multiplicity of infection MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TCID 50 = 50% tissue culture infectious dose 4. Add 1100 μl of 1× DMEM (without DMSO) supplemented with 2% FBS to columns 1 and 7, and add 750 μl of 1× DMEM (containing DMSO if necessary) supplemented with 2% FBS to columns 2-6 and 8-12. Add 50% more compound than your selected maximum dose to columns 1 and 7 (2 compounds tested per row, 16 total compounds diluted per deep well plate), e.g., 3.3 μl of a 10 mM stock for concentration of 30 μM, which will dilute to a final maximum concentration of 20 μM when 50 μl of virus is added. Based on this compound example, the DMSO concentration for medium in columns 2-6 and 8-12 would be 0.3% (do not exceed 0.5% DMSO, due to toxicity concerns). Once all the compounds are added, pipet column 1 up and down 20 times to mix, transfer 375 μl from column 1 to column 2, and mix 5-10 times using the multi-channel pipette. Discard tips. Load new tips to the multi-channel pipet, transfer 375 μl from column 2 to column 3, and mix 5-10 times again. Repeat this process until column 6 is reached. Repeat for columns 7-12. If the entire deep-well plate is used, this will result 16 compounds with six 3-fold serial dilutions of each (final concentrations of 20-80 nM for this example). This plate is called plate X, refer to Figure 4 for plate layout. 5. Take the Vero.E6 cells seeded the previous day in the two 96-well cell culture plates (one for antiviral, one for cytotoxicity) and remove the medium using a multichannel pipette or an aspirator. The medium should be removed carefully such that the tip does not touch the cells and the cells are not dislodged in any way. Transfer 100 μl of each respective dilution to the Vero.E6 plates; there should be enough volume (750 μl) in the deep-well plate for triplicates for both antiviral and cytotoxicity testing. Using the same dilution series for both antiviral and cytotoxic assays will produce reliable matching data sets, and these should be performed at the same time. Three compounds and six DMSO control wells can be tested per 96-well plate (plate format Y, see Fig. 4 ). The outer wells should be avoided due to the potential for medium evaporation to alter compound concentrations. These outer wells should be filled with medium and cells to be used as uninfected controls for viral detection through immunostaining. The cells should be incubated with compound for 2 hr prior to infection as a standard, although this time frame can be adjusted for specific experiments. 6. Handle SARS-CoV-2 in a BSL-3 laboratory in a biosafety cabinet. Prepare 2000 TCID 50 /ml of authentic SARS-CoV-2 (SARS-CoV-2 isolate USA-WA1/2020; see Basic Protocol 1, steps 1-8, for determination of TCID 50 ) in 1× DMEM supplemented with 2% FBS. Add 50 μl (100 TCID 50 per well or 0.025 MOI) of Amanat et al. Current Protocols in Microbiology the virus to the 100 μl of compound containing medium in each well of plate Y except the outer wells, pipet up and down gently 2-3 times to mix each well (important for even viral infection and compound concentration across the plates). Add 50 μl of 1× DMEM with 2% FBS to the sample wells of the cytotoxicity plate to adjust compound concentrations to match the antiviral assay. Each inner well should now have a total of 150 μl. Incubate for 48 hr at 37°C in a humidified incubator with 5% CO 2 . All work for testing cytotoxicity can be performed outside the BSL-3 laboratory, e.g., at BSL-2, since no infectious virus is involved. 7. After the incubation time is complete, remove the infected Vero.E6 cells in the 96well cell culture plates from the incubator under BSL-3 conditions. Remove the medium using a multi-channel pipette. Optionally, viral titers can be quantified from the removed medium using TCID 50 assay (see Basic Protocol 1) or plaque assay if desired. Fix the Vero.E6 cells by adding 200 μl of 4% formaldehyde (dilute from 10%) in PBS to each well of the 96-well plate. Incubate in 4% formaldehyde for 24 hr at room temperature prior to removing plates from the BSL-3 laboratory. 8. Once the plates have been removed from the BSL-3 laboratory, proceed to immunostain the fixed cells with SARS-CoV2 specific antibodies as outlined in Basic Protocol 1, steps 20-30, with the following modifications: Use the antibodies listed in the Basic Protocol 2 materials list for this protocol. Briefly, after three washes with 200 μl of PBS, incubate the cells with a secondary antibody solution containing anti-mouse IgG and DAPI in PBS supplemented with 0.5% BSA at a dilution of 1:2000. Add 100 μl/well and incubate for 45 min at RT. Wash three times for 5 min in PBS with gentle shaking. A plate cytometer (Celigo) or laser scanning cytometer (Acumen) can be used to assess infected cells over total cells for highly accurate quantification of viral infection. The DMSO control should be normalized to 100% infection for comparison with compound treated wells. 9. Assay the uninfected Vero.E6 cell cytotoxicity controls for cell viability with the MTT assay or any other commercial cell viability assay according to the manufacturer's instructions. Cytotoxicity measurements should be matched in incubation time with your antiviral assay. 10. Calculate the IC 50 and CC 50 for each compound using Prism software as described in Basic Protocol 1. The SARS-CoV2 USA-WAS1/2020 viral strain used for the optimization of the assays described here has been propagated using the Vero.E6 cell line, as it was previously shown that the virus can replicate to high titer in this cell line (https: // www.biorxiv. org/ content/ 10.1101/ 2020.03.02.972935v1.full.pdf ) . Stocks are generated by infecting a confluent monolayer of cells at a low multiplicity of infection to avoid the generation of defective interfering particles that can lower the titer of the viral preparation. Importantly, always work with low-passage-number stocks that are fully sequenced, to avoid selecting mutants that are better adapted to grow in the cell line used for virus amplification. Vero.E6 cells (ATCC CRL-1586) Vero.E6 growth medium: cDMEM (see recipe) SARS-CoV-2 isolate USA-WA1/2020, (BEI Resources NR-52281 or similar) Viral growth medium (DMEM with 2% FBS; see recipe) 3. On the day of the inoculation, use one of the flasks to count the cells (e.g., using a counting chamber or an automatic cell counter) and calculate the volume of viral inoculum required to infect at multiplicity of infection of 0.001. When using Vero.E6, a confluent T-75 flask can yield about 8 × 10 6 cells. Remove the medium, wash once gently in PBS, and infect cells in 5 ml of viral growth medium for 1 hr at 37°C and 5% CO 2 , rocking the flask every 15 min to spread the viral inoculum evenly across the monolayer. 4. After the 1-hr adsorption, add 5 ml of viral growth medium and incubate the flask at 37°C. Observe cell culture daily and follow the development of cytopathic effects. Typically, within 3 or 4 days, cells will become rounded-up and will detach from the monolayer. Harvest the virus when the cytopathic effect has progressed to 80%. 5. Clarify the medium by centrifugation in a 50-ml conical tube for 10 min at 1300 × g, 4°C. 6. Aliquot pooled supernatants into labeled 2.0-ml O-ring cryotubes and store samples at −80°C until further use. 7. Determine virus stock titer by plaque assay or TCID 50 following the protocol described in Basic Protocol 1. Current Protocols in Microbiology The microneutralization assay that is described here has been adapted from established protocols from work with other viruses such as influenza virus (Amanat, Meade, Strohmeier, & Krammer, 2019) . The assay has a medium throughput, and more samples can be analyzed as compared to PRNTs. Compared to RBD-ACE2 inhibition assays, the MNA described here will also detect neutralizing antibodies binding to epitopes outside of the RBD. Different virus isolates can be used, and the assay can likely be adapted for staining antibodies other than mAbs against NP (e.g., polyclonal sera, antibodies targeting S or M, etc.). The assay should be optimized based on the level of infection that is observed in each respective cell line used. Viral input can be varied as well. in order to make the assay more or less sensitive. The antiviral assay described here has also been adapted from previous work with influenza virus. The immunostaining output is medium throughput and can be used to comprehensively analyze the antiviral and cytotoxic activity of many more compounds than can be done using TCID 50 or plaque assay. The time course of infection, cell line used, viral strain and MOI, and antibody used can be altered to adapt this assay to answer many different questions related to the antiviral activity of a compound. Cells that are used for the microneutralization assay in Basic Protocol 1 should be healthy, and viability should be checked periodically. The cell count should be performed with care to ensure that cells are not overconfluent on the day of the assay. The cells should also be mycoplasma free. The TCID 50 measurement of each stock should be performed with care, as each virus stock will be used numerous times for several microneutralization assays. Most importantly, the virus should be sequenced every few passages to ensure that no major mutations have occurred from the passaging of virus in cell culture. The microneutralization assay can be performed with serum/plasma or purified antibodies. If antibodies are to be used, a trial run should be performed to assess the appropriate concentrations that should be tested. The immunostaining antiviral assay described in Basic Protocol 2 has a consistently lower sensitivity than an assay that detects infectious viral particles produced in the supernatant (TCID 50 /plaque assay). The IC 50 determined for any particular compound in this assay will typically be 3-to 5-fold higher than the aforementioned assays. Therefore, this antiviral assay is well suited for mediumthroughput screening of potential antivirals, and we suggest confirming top hits using the mentioned classical virology methods. The DAPI counterstain can be used as a proxy for toxicity in infected cells, to be paired with the MTT assay performed in uninfected cells. If the majority of DAPI staining is lost in a compound-treated well, that compound concentration should be excluded from your final IC 50 calculation. The TCID 50 is only performed once for each viral stock. The TCID 50 takes 3 days in total, and the microneutralization should only take 4 days, as the cells are fixed 2 days after the assay has been performed. On day 3, plates should be stained immediately, as leaving the cells in formaldehyde can negatively impact the staining. Thus, the whole microneutralization assay and data generation are completed within 4 days. Compounds are typically added 2 hr prior to SARS-CoV-2 infection, although this can be adjusted based on the proposed mechanism of action of the compound or on the hypothesis being tested. The antiviral assay takes 3 days total from infection to staining, scanning, and data analysis. The compound should always be incubated on the cells for the same amount of time between antiviral and cytotoxicity assays. 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) Filter using a 0.22-μm Stericup filter (MilliporeSigma, cat. no. S2GPU05RE) will be used to prepare 1× MEM/2% FBS by mixing equal amounts 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 Sars-Cov-2 vaccines: Status report Cross-reactive antibodies binding to H4 hemagglutinin protect against a lethal H4n6 influenza virus challenge in the mouse model A serological assay to detect Sars-Cov-2 seroconversion in humans The global phosphorylation landscape of SARS-CoV-2 infection Biosafety: Guidelines for working with pathogenic and infectious microorganisms A Sars-Cov-2 protein interaction map reveals targets for drug repurposing Isolation and characterization of Sars-Cov-2 from the first Us Covid-19 patient Functional assessment of cell entry and receptor usage for Sars-Cov-2 and other lineage b betacoronaviruses Two detailed plaque assay protocols for the quantification of infectious Sars-Cov-2 Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients Determination of 50% endpoint titer using a simple formula Structure, function, and antigenicity of the Sars-Cov-2 spike glycoprotein Cryo-Em structure of the 2019-Ncov spike in the prefusion conformation A new coronavirus associated with human respiratory disease in China This work was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C ( Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays.