Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
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Resource and Technical Advance COVID-19

Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors

Abstract

Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase (Gluc) or secreted nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque-reduction assay using an authentic, infectious SARS-CoV-2 strain. The assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms; patients included those in the intensive care unit (ICU), health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs, and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2 and demonstrates the efficacy of a potentially novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.

Authors

Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu

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Figure 6

A secreted Nluc–based lentiviral SARS-CoV-2 S neutralization assay with improved stability and sensitivity, and its application in measuring SARS-CoV-2 neutralizing antibody levels in COVID-19 patients.

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A secreted Nluc–based lentiviral SARS-CoV-2 S neutralization assay with ...
293T cells were transfected with lentiviral vector (pNL4.3 inGluc or pNL4.3 secNluc) along with the SARS-CoV-2 S-C9 plasmid. Media was 48 hours after transfection and used to infect 293T/ACE2 cells; luciferase activity was measured at indicated times to determine the viral infectivity; n = 3 for all experiments. (A) Stability of inGluc and secNluc luciferase signals measured over time. A total of 20 μL of Gaussia luciferase substrate or 20 μL of Nano Luciferase substrate were added simultaneously, and luminescence measurements were then read every 2 minutes for 60 minutes. Plotted are the luminescence reads relative to the 0 minutes time point, which was set to 100%; secNluc exhibited a signal that was more stable than the inGluc virus infected cells. (B and C) Infectivity of SARS-CoV-2 S secNluc pseudotypes. (B) The Nano-luciferase activity of culture medium harvested from virus-infected cells at indicated times. (C) Relative viral infectivity was plotted by setting the mock infection to 1.0. (D) Indicated amounts of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μL of supernatant from virus-infected cells was used to measure the secNluc activity as shown. The dashed line indicates the background luminescence. (E) Experiment was performed as described in the legends of Figure 3A and Figure 4B, except secNluc lentiviral pseudotypes were used for infection. Data were analyzed as mean ± SD.