A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Abstract

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV‑2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC ₅₀ ) for Remdesivir of 9.3 μM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, β), Brazilian/Japanese variant P.1 (Gamma, γ), and Californian (Epsilon, ε), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

Figure 1.. Selection of optimal donor/acceptor antibody pair.

(A) Illustration of the HTRF assay for SARS-CoV-2 NP showing the Eu donor-conjugated primary MAb and DL650 acceptor-conjugated primary MAb detecting SARS-CoV-2 and enabling FRET. (B) Five MAbs specific for SARS-CoV-2 NP were labeled with Europium (donor Ab) or DyLight650^® (acceptor Ab). The labeled MAbs were then tested in HTRF assay, in triplicate wells using a cross-matrix assay format. The presented data is a median TR-FRET counts (acceptor fluorescence/donor fluorescence × 10,000). The MAb pair (R001-Eu/1C7-DyLight650) showed highest specific signal and was selected for further assay development.

Figure 2.. Optimization of donor to acceptor ratio, concentration, and incubation time using media or Vero E6 cells.

TR-FRET ratio signal (acceptor/donor) detection using R001-Eu and 1C7-DL650 as the donor (D) and acceptor (A) pair respectively. SARS-CoV-2 NP was used to generate the 11-point standard curve starting from 1500 ng/mL serially diluted 1:2. HTRF reagents were incubated with NP in cell culture media only (A and B) or in media with 5000 Vero E6 cells (C and D) for either 1h at RT or O/N at 4°C. N = 3 wells per condition in a 384 well plate. Error bars indicate S.D.

Figure 3.. Detecting SARS-CoV-2 NP from Vero E6 TCS and cell lysate.

TCS and cell lysate were collected from Vero E6 cells after infection with SARS-CoV-2 USA-WA1/2020 strain for 24h or 48h. TR-FRET ratio from TCS (A and C) and cell lysates (B and D) after incubating with reagents for 1h at RT or O/N at 4°C. TCS was diluted 1:3 and cell lysate was first diluted 15-fold followed by 1:3 dilutions. N = 3 wells in a 384 well plate. Error bars indicate S.D.

Figure 4.. 384 well plate statistics using spiked NP in Vero E6 cells.

Vero E6 cells grown in a 384 well plate at 5000 cells per well were spiked with 0 ng/mL or 500 ng/mL of SARS-CoV-2 NP and incubated with HTRF reagents for 1h at RT (A and B) or O/N at 4°C (C and D). Columns 1 and 2 contained no NP and acted as a simulated positive control for uninfected cells. Columns 3 to 24 contained 500 ng/mL of SARS-CoV-2 NP. Each column contains 16 wells. Each point represents 1 well. Inset, calculated plate statistics

Figure 5.. HTRF assay detects NP produced by live virus infection of Vero E6.

The TR-FRET ratio for SARS-CoV-2 NP detected in Vero E6 infected with the SARS-CoV-2 USA-WA1/2020 strain at different MOI starting at 0.25 and diluted 1:3 based on a reverse time-course protocol (A) for 0 h (B), 12 h (C), 24 h (D), or 48 h (E). The standard curve (F) with assay conditions equal to the 48h time-point. Plates were incubated with NP HTRF reagents for 1h at RT or O/N at 4°C. N=3 wells in a half-area 96-well plate. Error bars indicate S.D.

Figure 6.. NP HTRF assay confirmation of remdesivir inhibition of SARS-CoV-2 replication.

The TR-FRET ratio for SARS-CoV-2 NP in Vero E6 infected with SARS-CoV-2 USA-WA1/2020 strain at MOI 0.009 based on protocol (A) for 24 h (B). Remdesivir was added at a starting concentration of 100 μM and serially diluted 1:3. The standard curve (C) using recombinant SARS-CoV-2 NP. Plates were incubated with NP HTRF reagents for 1h at RT or O/N at 4°C. N=3 wells in a half-area 96-well plate. Error bars indicate S.D.

Figure 7.. NP HTRF assay detects Beta, Gamma, and Epsilon VoC.

The TR-FRET ratio for SARS-CoV-2 NP in Vero E6 infected with the USA-WA1/2020, Beta, Gamma, and Epsilon strains using protocol (A) at MOI starting at 0.25 and diluted 1:3 for 0 h (B), 4 h (C), 8 h (D), 12 h (E), 24 h (F). The standard curve (G) with assay conditions equal to the 24h time-point. Assay completed with a reverse time-course protocol; O/N incubation at 4°C with HTRF reagents (5 nM D/10 nM A). N=3 wells in a half-area 96-well plate. Error bars indicate S.D.