Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein

Abstract

Background SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. Methods To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). Results The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. Conclusions In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

Dynamics of neutralizing antibody responses in acute-phase COVID-19: A potential relationship between disease progression and rapid neutralizing antibody response

“…Neutralization of the human serum against pseudotyped viruses was measured based on a previous report [6] . Briefly, Vero cells were treated with two hundred-fold dilutions of sera from in-hospital patients with COVID-19 and then inoculated with pseudotyped VSVs bearing the S protein, the 19-amino acid-truncated S protein of SARS-CoV-2 or VSV-G.…”

“…Briefly, Vero cells were treated with two hundred-fold dilutions of sera from in-hospital patients with COVID-19 and then inoculated with pseudotyped VSVs bearing the S protein, the 19-amino acid-truncated S protein of SARS-CoV-2 or VSV-G. The infectivity of the pseudotyped viruses was determined by measuring luciferase activity after 24 h of incubation at 37°C, based on our preliminary study [6] , and the number of days after admission or onset until the first result of 50% neutralization in the CRNT (N50%-CRNT) was recorded.…”

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants

“…VSV pseudovirus bearing SARS-CoV-2 spike protein was generated as described previously (Tani et al, 2021). Briefly, the cDNA of SARS-CoV-2 spike protein was synthesized (Integrated DNA Technologies Inc.) and cloned into the pCAGGS expression vector.…”

Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19

“…To circumvent the need for BSL-3 laboratories, we recently developed a fundamental NAbs detection method for SARS-CoV-2 based on the chemiluminescence reduction neutralization test (CRNT) and VSV-based pseudotyped viruses with SARS-CoV-2 S (Sfullpv) or truncated S proteins (St19pv) to evaluate the expression and neutralization values of NAbs against SARS-CoV-2 6 .…”