Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

Abstract

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARSCoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1) and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.

Fig. 1.. Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses.

A. Schematic representation of the modified HIV-1_NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1_NL4ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B. Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1_NLGagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C. Infectivity measurements of HIV-1_NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D. Same as, C but viruses were generated using the 3 plasmids depicted in B. E. Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

Fig. 2.. VSV-based SARS-CoV-2 pseudotyped viruses.

A. Schematic representation of the rVSVΔG/NG-NanoLuc genome in which G-coding sequences were replaced by an mNeonGreen-2A-NanoLuc luciferase reporter cassette. Infectious virus particles were generated by passaging G-complemented rVSVΔG/NG-NanoLuc virus stocks through 293T cells transfected with a plasmid encoding SARS-CoV-2 SΔ19. B. Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on Huh7.5 cells was quantified by measuring luciferase activity (RLU) or the % GFP-positive cells. Mean and standard deviation from two technical replicates is plotted. Virus particles generated by passage through cells that were not transfected with SARS-CoV-2 S were used as a control. C. NanoLuc luciferase activity (RLU) in Huh7.5 cells measured at various times after infection with pseudotyped rVSVΔG/NG-NanoLuc particles. Average and standard deviation from two technical replicates is shown. D. Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. Average and standard deviation from two technical replicates is shown.

Fig. 3.. A replication-competent VSV/SARS-CoV-2 chimera.

A. Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B. Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C. Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D. Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

Fig. 4.. Measurement of neutralization activity in COVID19 convalescent donor plasma.

A. Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B. Plasma neutralization of HIV-1_NLΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C. Correlation between NT₅₀ values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT₅₀ values for the same plasmas for SARS-CoV-2 (y-axis).

Fig. 5.. Measurement of neutralization potency of human monoclonal antibodies.

A. Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B. Monoclonal antibody neutralization of HIV-1_NLΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C. Correlation between IC₅₀ values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC₅₀ values for the same antibodies for SARS-CoV-2 (y-axis).