key: cord-0269576-0iemfglm authors: Park, Jun-Gyu; Oladduni, Fatai S.; Chiem, Kevin; Ye, Chengjin; Pipenbrink, Michael; Moran, Thomas; Walter, Mark R.; Kobie, James; Martinez-Sobrido, Luis title: Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2 date: 2020-07-23 journal: bioRxiv DOI: 10.1101/2020.07.22.216648 sha: a21e807029a1cd14753c79a30d3eb1711059a80a doc_id: 269576 cord_uid: 0iemfglm Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2. 4. To prevent cells from drying and to also facilitate virus adsorption, shake the 296 plates gently every 10 minutes. 297 DMEM/F-12/Agar mixture. 299 Note: ensure that at the start of the infection, the 2% agar is melted in a 300 microwave and the temperature brought down to approximately 42°C when 301 mixing with warm DMEM/F-12/Agar mixture. 302 Note: It is important to keep the agar cool enough not to burn the cells, and at the 303 same time warm enough not to solidify in the process of pipetting. 304 6. Allow the agar to solidify and invert the plates to prevent the accumulation of 305 moisture during incubation. Note: After 24 h fixation with 10% formalin solution, plates can be moved from 310 the BSL3 to the BSL2 to complete the viral titration. 311 9. Perform immunostaining using the cross-reactive SARS-CoV-1 NP mAb 1C7, at 312 a working concentration of 1 μg/mL. The process of entry into a susceptible host cell is an important determinant of infectivity 333 and pathogenesis of viruses, including CoVs (Li, 2016; Perlman and Netland, 2009) . 334 SARS-CoV-2 relies on the ability of its S glycoprotein to bind to the ACE2 receptor 335 through its receptor-binding domain (RBD) driving a conformational change that 336 culminates in the fusion of the viral envelope with the host cell membrane, and cell entry 337 (Shang et al., 2020) . SARS-CoV-2 S is made up of 2 subunits, S1 and S2. During pre-338 treatment, mAbs, or Ab containing samples, are pre-incubated with SARS-CoV-2 at 339 37°C for 1h. This enables the Abs to bind to the S protein blocking virus attachment, 1 6 and subsequently interfering with the process of virus entry. While some SARS-CoV-2-341 induced NAbs can preferentially bind to either the S1 or S2 subunit of the S protein, 342 others can bind, with a high affinity, to the RBD located within the S1 subunit ( assembly and/or budding). In general, a potent SARS-CoV-2 NAb will show a low NT 50 1 7 titer whereas a weak NAb will give a high NT 50 value. This protocol precludes the 364 necessity for a cytotoxicity assay since Abs are not known to produce cytotoxic effects 365 in cell cultures. However, a potential toxic effect of the NAbs can be determined when 366 developing the assay with the infrared staining described below. the diluted Ab or media-only and no-virus control wells (columns 11 and 12, 409 respectively) to give a final concentration of 1% Avicel in each well. is provided before viral infection will help to provide information on the pre-entry 435 mechanism of antiviral activity of the compound. The antiviral activity is determined by 436 assessing the effective concentration that inhibits virus replication in Vero E6 cells (96-437 well plates, ~4 x 10 4 Vero E6 cells/well at confluency, triplicates) following viral 438 incubation for 24 h. Note that the seeding density of Vero E6 in 96-well is ~1 x 10 4 439 cells/well while cells at confluency is ~4 x 10 4 cells/well. It is important to include as a 440 positive control in this antiviral PRMNT assay a drug with a known antiviral activity 441 against SARS-CoV-2, such as remdesivir (Elfiky, 2020). Unlike NAbs, some compounds 442 could have cytotoxic effects. As a result, it becomes imperative to carry-out a 443 cytotoxicity test (e.g. MTT assay) to evaluate for potential cytotoxicity of a given 444 compound. Vero E6 cells for an MTT assay can be seeded on the same day as the cells 445 for PRMNT assay so that the experiments can be conducted in parallel. Alternatively, 446 our infrared staining technique can be used, concurrently, to evaluate cell viability in the 447 same cells, in 96-well plates, used for PRMNT assay. (Figures 3 and 6, respectively) . As COVID-19 cases continue to increase across the globe, there is an urgent need to 579 identify effective and safe prophylactics and/or therapeutics for the treatment of SARS-580 CoV-2 in infected patients. In this study, we described robust assays and staining 581 techniques which can be employed to evaluate the neutralizing and/or antiviral activity 582 of Abs and/or antivirals, respectively, against SARS-CoV-2 in vitro. 583 assay (Amanat et al., 2020) . It is important to optimize these conditions so as to be able 596 to generate accurate and reproducible data between different laboratories. 597 with post-infection media containing 1% Avicel. Incubation without Avicel will enhance a 599 rapid cell-to-cell spread of virus resulting in higher NT 50 or EC 50 . Although this assay 600 works best in our hands using 1% Avicel, other reagents, such as 0.75% 601 carboxymethylcellulose (CMC) can also be used (Oladunni et al., 2019) . incubated with a secondary POD (Fig. 2) or IRDye 800CW goat anti-mouse IgG 683 secondary Ab (Fig. 3) at 37°C. After 30 minutes incubation with the secondary Ab, cells 684 are washed 3X with PBS and developed with the DAB substrate kit (Fig. 2) . Cells 685 stained with the IRDye 800CW goat anti-mouse IgG secondary Ab are simultaneously 686 incubated with DRAQ5 TM Fluorescent Probe Solution for nuclear staining (Fig. 3) . 687 Positive staining plaques in each of the wells of the 96-well plate is quantified using an 688 ELISPOT plate reader (Fig. 2) or an Odyssey Sa Infrared Imaging System (Fig. 3) . The 689 neutralizing titer 50 (NT 50 ) is calculated as the highest dilution of the mAb or sera that 690 prevents 50% plaque formation in infected cells, determined by a sigmoidal dose 691 response curve (Figs. 2 and 3) . 692 for nuclear staining (Fig. 6) at 37°C. After 30 minutes incubation with the secondary 739 POD Ab, cells are washed 3X with PBS and developed with the DAB substrate kit (Fig. 740 5) . Cells stained with the IRDye 800CW goat anti-mouse IgG secondary Ab are 741 simultaneously incubated with DRAQ5 TM Fluorescent Probe Solution for nuclear 742 staining (Fig. 6) . Positive stained cells in each of the wells of the 96-well plate are 743 quantified using an ELISPOT plate reader (Fig. 5) or in the Odyssey Sa Infrared 744 Imaging System (Fig. 6) . 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