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.

Keywords: Neutralization assay; Pseudotyped virus; SARS-CoV-2; Serum; VSV; Whole blood.

Fig. 1

Characterization of pseudotyped viruses possessing spike proteins of SARS-CoV-2. a Incorporation or expression of S proteins in the virions or cells were investigated using CBB staining and immunoblotting. b Efficiency of gene transduction in various mammalian cell lines using the pseudotyped viruses. Sfullpv, St19pv, and 100-fold-diluted VSVpv generated in 293 T cells were inoculated into the indicated cell lines. At 24 h post-infection, infectivity of the viruses was determined by measuring luciferase activities as a relative luciferase unit (RLU). VSVpv without envelope (∆Gpv) was used as a negative control. The results are from three independent assays with error bars representing standard deviations

Fig. 2

Dose-dependent effects on the neutralization of Sfullpv and St19pv infections through sera from convalescent COVID-19 patients. The pseudotyped viruses were preincubated with the indicated dilutions of sera from two different convalescent COVID-19 patients (patients #4 and #7). Thereafter, Vero cells were infected with pseudotyped viruses. Infectivities of pseudotyped viruses were determined by measuring luciferase activities at 24 h post-infection. The results are from three independent assays with error bars representing standard deviations. The significance of any differences in the means was determined using two-way ANOVA. Significance was determined in comparison to the results of the Non serum: *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

Neutralization of St19pv and VSVpv infections through sera from hospitalized patients with COVID-19 or a COVID-19 PCR-negative donor. The pseudotyped viruses were preincubated with two a 100-fold dilution of sera from 23 hospitalized COVID-19 patients (a) or 19 COVID-19 PCR-negative donors (b). Thereafter, Vero cells were infected with pseudotyped viruses. Infectivities of pseudotyped viruses were determined by measuring luciferase activities 24 h post-infection. The results are from three independent assays with error bars representing standard deviations. c A summary of the results of (a) and (b) divided by pseudotyped viruses are represented by dot plot analyses. The significance of any differences in the means was determined using the Student’s t test. ****P < 0.0001

Fig. 4

Detection of anti-S protein antibody by indirect ELISA. Test sera from 23 COVID-19 hospitalized patients (a) or 19 COVID-19 PCR-negative donors (b) was assayed. The O.D. at 450 nm was measured using a microplate reader. The results shown are from three independent assays, with error bars representing standard deviations.

Fig. 5

Effects on the neutralization of St19pv infection by serum or whole blood from COVID-19 hospitalized patients. The pseudotyped viruses were preincubated with two hundred dilutions of serum or whole blood from seven different COVID-19 hospitalized patients (patients #6, #7, #12, #20, #21, #22, and #23) with culture media. Thereafter, Vero cells were infected with pseudotyped viruses. Infectivities of pseudotyped viruses were determined by measuring luciferase activities 24 h post-infection. The results are from three independent assays with error bars representing standard deviations