Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay

Abstract

Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.

Neutralizing Ability of a Single Domain VNAR Antibody: In Vitro Neutralization of SARS-CoV-2 Variants of Concern

“…All experiments with live SARS-CoV-2 virus were conducted in a certified BSL3 laboratory, following experimental procedures as described by Friesche et al (2022) [ 31 ] with minor modifications: Briefly, A549-AT cells/well or VeroE6 cells/well were seeded in 100 μL of DMEM media supplemented with 2% FBS (DMEM2) in a 96-well TC-treated plate 24 h prior to assay. For testing, two-fold serial dilutions of VNAR (10 μg/mL–0.1 μg/mL) were prepared in DMEM2; and pre-incubated with a TCID 50 of 0.02 pfu/cell SARS-CoV-2 for both Delta (B.1.167.2) and Omicron (B.1.1.529) variants.…”

Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86

“…Virus neutralization titers were determined using a live virus microneutralization test with SARS-CoV-2 clinical isolates (Frische et al 2022). In brief, a 2-fold serial dilution of heat-inactivated serum/plasma samples was pre-incubated with 300 × 50% tissue culture infectious dose SARS-CoV-2 virus and subsequently transferred to a Vero E6 cell monolayer in a 96-well tissue culture plate.…”

High titers of neutralizing SARS-CoV-2 antibodies six months after symptom onset are associated with increased severity in COVID-19 hospitalized patients

“…The microneutralization assay methods and validation used in this study has been published as a separate paper [ 18 ]. Briefly, levels of neutralizing antibodies were determined using a median tissue culture infectious dose (TCID 50 ) microneutralization assay with an ELISA readout, further described in the Additional file 1 : Appendix.…”