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Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots

Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots.

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Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 100 μl of supernatant from each prep was used to transduce an HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 μg/ml polybrene in 48-well plates. Transduction efficiencies for each sample were measured by flow cytometry 6 days posttransduction. Panel B. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and used to transduce both the ACE2-positive cell line and an HEK293T cell line lacking ACE2 expression (included to provide a background signal for analysis of luciferase activity since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. Functional titers determined via analysis of ZsGreen1 reporter expression in ACE2-positive HEK293T cells transduced with pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (full length or truncated forms) and encoding the firefly luciferase protein. 10 μl of concentrated virus (21X) from each prep was used to transduce the Human ACE2 293T Cell Line. Luciferase activity was measured 6 days post-transduction using a control virus lacking an envelope protein to provide a background signal for analysis of the luciferase activity.

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Neutralization data with ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. Panels A and B. Serial dilutions of soluble ACE2 protein fused to the Fc domain from IgG (ACE2-Fc) were applied, along with SARS-CoV-2 pseudovirus, to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate.

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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of convalescent serum (BEI Resources, Cat. # NR-52401), and applied to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate. Negative control was performed using serum from healthy macaques.

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632668: Lenti-X SARS-CoV-2 Packaging Single Shots (WT Spike, Full Length)

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Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots

Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots.

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Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 100 μl of supernatant from each prep was used to transduce an HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 μg/ml polybrene in 48-well plates. Transduction efficiencies for each sample were measured by flow cytometry 6 days posttransduction. Panel B. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and used to transduce both the ACE2-positive cell line and an HEK293T cell line lacking ACE2 expression (included to provide a background signal for analysis of luciferase activity since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. Functional titers determined via analysis of ZsGreen1 reporter expression in ACE2-positive HEK293T cells transduced with pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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632672: Lenti-X SARS-CoV-2 Packaging Mix (WT Spike, Full Length)

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (full length or truncated forms) and encoding the firefly luciferase protein. 10 μl of concentrated virus (21X) from each prep was used to transduce the Human ACE2 293T Cell Line. Luciferase activity was measured 6 days post-transduction using a control virus lacking an envelope protein to provide a background signal for analysis of the luciferase activity.

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Neutralization data with ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. Panels A and B. Serial dilutions of soluble ACE2 protein fused to the Fc domain from IgG (ACE2-Fc) were applied, along with SARS-CoV-2 pseudovirus, to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate.

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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of convalescent serum (BEI Resources, Cat. # NR-52401), and applied to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate. Negative control was performed using serum from healthy macaques.