Evaluation of Neutralizing Antibodies Against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudovirus-Based Assay

Abstract

Emerging highly pathogenic human coronaviruses (CoVs) represent a serious ongoing threat to the public health worldwide. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells via attachment to their respective cellular receptors. The S protein is believed to be a major immunogenic component of CoVs and a target for neutralizing antibodies (nAbs) and most candidate vaccines. Development of a safe and convenient assay is thus urgently needed to determine the prevalence of CoVs nAbs in the population, to study immune response in infected individuals, and to aid in vaccines and viral entry inhibitor evaluation. While live virus-based neutralization assays are used as gold standard serological methods to detect and measure nAbs, handling of highly pathogenic live CoVs requires strict biocontainment conditions in biosafety level-3 (BSL-3) laboratories. On the other hand, use of replication-incompetent pseudoviruses bearing CoVs S proteins could represent a safe and useful method to detect nAbs in serum samples under biosafety level-2 (BSL-2) conditions. Here, we describe a detailed protocol of a safe and convenient assay to generate vesicular stomatitis virus (VSV)-based pseudoviruses to evaluate and measure nAbs against highly pathogenic CoVs. The protocol covers methods to produce VSV pseudovirus bearing the S protein of the Middle East respiratory syndrome-CoV (MERS-CoV) and the severe acute respiratory syndrome-CoV-2 (SARS-CoV-2), pseudovirus titration, and pseudovirus neutralization assay. Such assay could be adapted by different laboratories and researchers working on highly pathogenic CoVs without the need to handle live viruses in the BSL-3 environment.

Early Humoral Response Correlates with Disease Severity and Outcomes in COVID-19 Patients

“…The pseudovirus was produced and titrated as previously described [25]. Briefly, a T-175 tissue culture flask of BHK21/WI-2 cells were transfected with 46 µg of pcDNA expressing codon-optimized full-length SARS-CoV-2 S protein (GenBank accession number: MN908947) using Lipofectamine TM 2000 transfection reagent (Invitrogen).…”

“…To completely remove any excess amount of the rVSV-∆G/G*-luciferase, 15 mL of DMEM containing rabbit polyclonal anti VSV-G antibody were added to the cells monolayer and incubated for 24 h at 37 • C in 5% CO 2 humidified incubator. Supernatant containing the generated pseudovirus was harvested 24 h post-infection and the virus titer was determined by measuring luciferase activity from 2-fold serially diluted pseudovirus on Vero E6 cells as previously described [25]. The titer of pseudovirus was expressed as a relative luciferase unit (RLU).…”

“…Neutralization assay was performed as previously reported [25]. In brief, Vero E6 cells were seeded in 96-well plates (white plate with clear bottom, COSTAR) at 2 × 10 4 cells/well and incubated overnight at 37 • C in 5% CO 2 humidified incubator.…”

Early Humoral Response Correlates with Disease Severity and Outcomes in COVID-19 Patients

“…The pseudovirus was produced and titrated as previously described [25]. and incubated for 24 h at 37°C in 5% CO 2 humidified incubator.…”

“…Neutralization assay was performed as previously reported [25]. In brief, Vero E6 cells were seeded in 96-well plates (white plate with clear bottom, COSTAR) at 2 x 10 4 cells/well and incubated overnight at 37°C in 5% CO 2 humidified incubator.…”