Real-time cell analysis: A high-throughput approach for testing SARS-CoV-2 antibody neutralization and escape

Abstract

Real-time cell analysis (RTCA) enables high-throughput, quantitative kinetic measurements of cytopathic effect (CPE) in virus-infected cells. Here, we detail a RTCA approach for assessing antibody neutralization. We describe how to evaluate the neutralizing potency of monoclonal antibodies (mAbs) and identify viral escape mutants to antibody neutralization for severe respiratory syndrome coronavirus 2 (SARS-CoV-2). For complete details on the use and execution of this protocol, please refer to Zost et al. (2020) and Suryadevara et al. (2021).

Keywords: Antibody; Biotechnology and bioengineering; Cell-based Assays; Health Sciences; High Throughput Screening; Immunology; Microbiology.

Graphical abstract

Figure 1

Complete overview of the RTCA assay A schematic showing the overview of RTCA assay, starting from Vero E6 cells passaging, harvesting, and plating of cells for xCelligence assay, neutralization and escape variants identification.

Figure 2

Titration of rVSV-SARS-CoV-2 virus to identify appropriate dilution (A) Plate design for 96-well plate layout for titrating rVSV-SARS-CoV-2 virus. A two-fold serial dilution of virus stock is shown in cyan boxed wells and yellow boxed wells are the cells-only control. Determining the optimal dilution of virus to use in this assay is a critical step in establishing the experiment. (B) Representative plot of the cytopathic effect (CPE) measurement caused by rVSV- SARS-CoV-2 at varying virus dilutions. Use of appropriate dilution of virus is important to obtain high quality results. Usually, to keep the assay duration as short as possible, the virus dilution associated with a curve that shows full CPE within 40 h of virus addition to cells is chosen. For instance, the 1:160 virus dilution shown here was chosen for subsequent assays.

Figure 3

Experimental design for running a neutralization assay using RTCA (A) Plate design for 96-plate layout for assessing mAb neutralization potency. Columns 1–6 highlighted wells are for the mAbs being assessed, columns 7–9 boxed wells are for a negative control mAb, columns 10–11 boxed wells for positive control mAb, pink boxed wells for virus-only control and purple boxed wells are for the cells-only control. It is important to include all controls in the plate so that the results can be interpreted properly. (B) Graphic of representative results after analyzing data using neutralizing mAbs associated with lack of neutralization (full cytopathic effect [CPE] shown in the pink line), intermediate neutralization indicated by partial CPE (orange line) or lack of neutralization associated with no CPE (purple line).

Figure 4

rVSV-SARS-CoV-2 neutralization curves of mAbs using RTCA Representative rVSV-SARS-CoV-2 neutralization curves for COV2-2489, COV2-2196, COV2-2676 or negative control antibody rDENV-2D22. Error bars indicate mean ± SD for 3 technical replicates.

Figure 5

Confirmation of the antibody escape phenotype for variants previously selected in the presence of a neutralizing mAb (A) RTCA sensogram for neutralization of parental virus by mAb COV2-2489 (blue), COV2-2489 escape virus (pink), cells + virus, i.e., the no-mAb control (red) or cells-only control (green). (B) RTCA sensogram for neutralization of previously selected variant viruses that had replicated in the presence of COV2-2489 or COV2-2676. The COV2-2196 control mAb neutralized all variants (blue) but mAbs for which the virus variants were previously selected to escape did not neutralize (COV2-2489; red), COV2-2676; green). The pink curves show CPE caused by escape virus only (the no-mAb control).