Evaluation of Cell-Based and Surrogate SARS-CoV-2 Neutralization Assays

Abstract

Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND₅₀) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND₅₀ titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND₈₀ GMTs mirrored ND₅₀ data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Keywords: COVID-19; SARS-CoV-2; antibody; neutralization assay.

FIG 1

SARS-CoV-2 neutralization and binding antibody concentration from COVID-19 convalescent patients. (A) Concentration of IgG against SARS-CoV-2 spike, RBD, nucleoprotein, and tetanus toxoid measured in the Luminex binding antibody assay. (B) Indexes reported by the Abbott Architect nucleoprotein IgG test. (C) ND₅₀ and (D) ND₈₀ neutralization titer measured using five SARS-CoV-2 neutralization assays for 40 plasma samples from 40 participants. Each assay defined its own lower limit of detection (LOD) based on the initial dilution: 50-fold for SARS-CoV-2/VeroE6, 20-fold for the LV and VSV pseudovirus assays, and 10-fold for the sVNT. Data below the LOD (open triangles) are plotted at LOD/2. Number and percentage of samples above the LOD are indicated above each plot. For each assay, the box represents the extent of the interquartile range (IQR) with a line indicating the median; whiskers extend to 1.5 times the IQR.

FIG 2

Correlation among assay readouts measuring neutralization or antigen-specific IgG concentration in plasma. Heat map color is determined by the Pearson’s correlation coefficient (r, annotations). Each panel includes either ND₅₀ titers (A) or ND₈₀ titers (B) and their correlation with sVNT percent neutralization, SARS-CoV-2-specific IgG concentration (Luminex bead-based assay), the quantitative index of the Abbott nucleoprotein assay, and tetanus toxoid-specific IgG concentration. ND₅₀ and ND₈₀ values below 50 were truncated at 25.