A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Abstract

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Fig 1. Cell-based biosensors of SARS-CoV-2 protease activity.

(A) Diagram of pp1a polyprotein showing candidate SARS-CoV-2 cleavage sequences for Papain-like Protease (PLPro; PLP1-3) and Main Protease (MPro; WT3c and Opt3c). Further details on sequence selection are available in the Materials and methods. Sites of cleavage are highlighted in red (C-terminal side of indicated amino acid). (B-D) Activation of FlipGFP-based reporters by recombinant SARS-CoV-2 protease expression. HEK293T cells were co-transfected with BFP and indicated FlipGFP-based reporter constructs encoding candidate MPro or PLPro cleavage sequences ± MPro, PLPro c.d., or empty pcDNA3.1. Illustrative flow cytometry data for Opt3c-FlipGFP (C) and PLP2-FlipGFP (D) are shown. (E) Detection of FlipGFP-based reporter activation by epifluorescence microscopy. HEK293T cells were co-transfected with Opt3c-FlipGFP biosensor plus/minus MPro (left panel) or PLP2-FlipGFP biosensor plus/minus PLPro (right panel). FlipGFP and mCherry fluorescence were analysed by epifluorescence microscopy 24 h post-transfection. mCherry, red. FlipGFP, green. Representative of 3 independent experiments. (F) Protease-specificity of FlipGFP-based reporters. HEK293T cells were co-transfected with BFP and indicated FlipGFP biosensors ± MPro, PLPro c.d., TEV protease or empty pcDNA3.1. (G) Sequence specificity of FlipGFP-based reporters. HEK293T cells were co-transfected with BFP and indicated FlipGFP biosensors ± MPro, PLPro or empty pcDNA3.1. Further details on non-cleavable mutants are available in the Materials and methods. For all flow cytometry experiments (B-D and F-G), FlipGFP and mCherry fluorescence in BFP+ cells were analysed 24 h post-transfection. An indicative gating strategy is shown in S2A Fig, with the % BFP+ cells in S2B Fig. Mean values ± SEM are shown for experiments performed in triplicate, representative of at least 3 independent experiments. **** p<0.0001. MPro, recombinant SARS-CoV-2 Main Protease. PLPro c.d., catalytic domain of recombinant SARS-CoV-2 Papain-Like Protease. TEV, recombinant TEV protease.

Fig 2. Activation of FlipGFP-based reporters by SARS-CoV-2 infection.

(A-B) HEK293T-ACE2 cells were transfected with indicated FlipGFP biosensors, incubated for 12 h, then infected with SARS-CoV-2 at MOI = 1. Cells were fixed, permeabilised and stained for SARS-CoV-2 spike protein 24 h post-infection. FlipGFP and mCherry fluorescence in spike+ syncytia vs. spike- cells were analysed by confocal microscopy. Illustrative microscopy data from spike+ syncytia (A) and mean values ± SEM for at least 15 syncytia/cells from each condition (B) are shown, representative of 2 independent experiments. **** p<0.0001. mCherry, red. FlipGFP, green. Spike, yellow. DAPI, blue. DIC, differential interference contrast. (C) Expression levels of SARS-CoV-2 proteases. HEK293T-ACE2 cells were mock-infected, infected with SARS-CoV-2 at MOI = 1 or transfected with MPro (left panel) or PLPro (right panel) under standard conditions. Cells were lysed after 24 h and analysed by immunoblot using antibodies specific for nsp5 (MPro), the PLPro c.d. of nsp3 or nucleocapsid. β-actin was included as a loading control. Arrows indicate presumed full-length nsp3 (215 kDa) and intermediate cleavage products present in SARS-CoV-2-infected cells. Asterisk indicates recombinant PLPro c.d.. Representative of 2 independent experiments. O.E., over-expression. MPro, recombinant SARS-CoV-2 Main Protease. PLPro c.d., catalytic domain of recombinant SARS-CoV-2 Papain-Like Protease.

Fig 3. Luminescent biosensors of authentic SARS-CoV-2 infection.

(A) Activation of luciferase-based reporters by recombinant SARS-CoV-2 protease expression. HEK293T cells were co-transfected with indicated 30F biosensors ± MPro, PLPro c.d., or empty pcDNA3.1. (B) Activation of luciferase-based reporters by SARS-CoV-2 infection. HEK293T-ACE2 cells were transfected with indicated 30F biosensors, incubated for 12 h, then mock-infected or infected with SARS-CoV-2 at MOI = 0.01. (C-E) Quantitation of infected cells. HEK293T-ACE2 cells were transfected with the 30F-PLP2 biosensor, incubated for 12 h, then mock-infected or infected with increasing doses of SARS-CoV-2. 1 μL of viral stock corresponds to MOI≈0.01. Cells were analysed in parallel 24 h post-infection by either epifluorescence microscopy for SARS-CoV-2 spike protein (C), or luminometry for Firefly and Renilla luciferase activities (D). Spike+ cells were enumerated by automated microscopy (Cellomics). Illustrative microscopy data (C) and mean values ± SEM (D) are shown for an experiment performed in duplicate (microscopy) or triplicate (luminometry). The correlation between the ratio of Firefly/Renilla luminescence and the proportions of spike+ cells is shown in E. Spike, red. DAPI, blue. DIC, differential interference contrast. R², Pearson’s correlation coefficient. Representative of 2 independent experiments. (F-G) Inhibition of SARS-CoV-2 replication by candidate antivirals. HEK293T-ACE2 cells were transfected with the 30F-PLP2 biosensor, incubated for 12 h, then infected with SARS-CoV-2 at MOI = 0.01 in the presence of DMSO or decreasing doses of candidate antivirals. Titration curves and IC50s are shown for remdesivir and GC376. Representative of 2 independent experiments. For all experiments, Firefly and Renilla luciferase activities were measured by luminometry 24 h post-transfection (A) or infection (B-G). Unless otherwise stated, mean values ± SEM are shown for experiments performed in triplicate, representative of at least 3 independent experiments. For F-G, Firefly/Renilla luminescence is shown as % luminescence in the DMSO condition. **** p<0.0001. MPro, recombinant SARS-CoV-2 Main Protease. PLPro c.d., catalytic domain of recombinant SARS-CoV-2 Papain-Like Protease.

Fig 4. An optimised luminescent SARS-CoV-2 reporter cell line.

(A) Schematic showing activation of luminescent reporter cell line by authentic SARS-CoV-2 infection. (B) Activation of reporter cells by SARS-CoV-2 infection. HEK293T-ACE2-30F-PLP2 cells (bulk population) were mock-infected or infected with SARS-CoV-2 at MOI = 0.01. (C) Selection of optimised cell lines. HEK2932-ACE2-30F-PLP2 cells (bulk population) or single-cell clones (B7 and G7) were mock-infected or infected with increasing doses of SARS-CoV-2. (D) Miniaturisation to 384-well plate format. Clone B7 reporter cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of decreasing doses of remdesivir in either a 96-well or 384-well plate format. Titration curves and IC50s for remdesivir are shown. Representative of 2 independent experiments. (E) Detection of SARS-CoV-2 variants of concern. Clone B7 reporter cells were mock-infected or infected with increasing doses of SARS-CoV-2 virus from the indicated lineages. Representative of 2 independent experiments. For C and E, 1 μL of viral stock corresponds to MOI≈0.01 for the lineage B and B.1.617.2 (delta) viral isolates, and MOI≈0.005 for the lineage B.1.1.7 (alpha) viral isolate. For all experiments, Firefly and Renilla luciferase activities were measured by luminometry 24 h post-infection. Unless otherwise stated, mean values ± SEM are shown for experiments performed in triplicate, representative of at least 3 independent experiments. For C and E, Firefly/Renilla luminescence is shown as fold-change with/without infection. For D, Firefly luminescence is shown as % maximum. **** p<0.0001.

Fig 5. Quantitation of neutralising activity in human serum samples.

(A-D) Measurement of neutralising antibody titres after inoculation with Pfizer-BioNTech BNT162b2 mRNA vaccine. (A) Neutralisation curves and NT50s for serum samples from 5 healthy control donors 21 days after their 1^st (upper panels) or 2^nd (lower panels) doses of BNT162b2 vaccine measured using either a Plaque Reduction Neutralization Test (PRNT, blue) or clone B7 reporter cells (orange). For PRNTs, SARS-CoV-2-ZsGreen viral stock (MOI = 0.001) was pre-incubated with a 3-fold dilution series of heat-inactivated serum before addition to VeroE6 cells. A semi-solid overlay was added after 2 h, and plaques enumerated by automated microscopy (Cellomics) 48 h post-infection. For the reporter cells, SARS-CoV-2 viral stock (MOI = 0.01) was pre-incubated with a 3-fold dilution series of heat-inactivated serum before addition to cells. Firefly and Renilla luciferase activities were measured by luminometry 24 h post-infection. Plaque number and Firefly/Renilla luminescence are shown as % maximum. Mean values ± SEM are shown for experiments performed in duplicate (PRNT) or triplicate (reporter cells), representative of 2 independent experiments. Illustrative data from PRNTs are included in S6 Fig. (B) Correlation between NT50s obtained for each sample (A) using either PRNTs (y-axis) or reporter cells (x-axis), with a solid black identity line (line of equality). Dotted lines indicate the limit of detection (lowest dilution) for each assay. rho, Spearman’s rank correlation coefficient. (C) Comparison between NT50s for each donor after 1^st and 2^nd doses of BNT162b2 vaccine obtained using reporter cells (A). Samples with no detectable neutralising activity at the lowest dilution (dotted line) are plotted at an arbitrary NT50 of 4. Donor 2 is highlighted in green. (D) Measurement of neutralising antibody titres against lineage B.1.1.7 (alpha) and B.1.617.2 (delta) variants of concern. The same serum samples from healthy control donors obtained 21 days after their 2^nd dose of BNT162b vaccine were processed as described in (A) but using lineage B.1.1.7 or B.1.617.2 viral stocks (MOI = 0.01). Representative of 2 independent experiments. Data for the lineage B.29 viral stock are as shown in A-C. ** p<0.01.