A Serological Snapshot of COVID-19 Initial Stages in Israel by a 6-Plex Antigen Array

Abstract

The first case of SARS-CoV-2 was discovered in Israel in late February 2020. Three major outbreaks followed, resulting in over 800,000 cases and over 6,000 deaths by April 2021. Our aim was characterization of a serological snapshot of Israeli patients and healthy adults in the early months of the COVID-19 pandemic. Sera from 55 symptomatic COVID-19 patients and 146 healthy subjects (early-pandemic, reverse transcription-quantitative PCR [qRT-PCR]-negative), collected in Israel between March and April 2020, were screened for SARS-CoV-2-specific IgG, IgM, and IgA antibodies, using a 6-plex antigen microarray presenting the whole inactivated virus and five viral antigens: a stabilized version of the spike ectodomain (S2P), spike subunit 1 (S1), receptor-binding-domain (RBD), N-terminal-domain (NTD), and nucleocapsid (NC). COVID-19 patients, 4 to 40 days post symptom onset, presented specific IgG to all of the viral antigens (6/6) in 54 of the 55 samples (98% sensitivity). Specific IgM and IgA antibodies for all six antigens were detected in only 10% (5/55) and 4% (2/55) of the patients, respectively, suggesting that specific IgG is a superior serological marker for COVID-19. None of the qRT-PCR-negative sera reacted with all six viral antigens (100% specificity), and 48% (70/146) were negative throughout the panel. Our findings confirm a low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population prior to the COVID-19 outbreak. We further suggest that the presence of low-level cross-reacting antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals. IMPORTANCE A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population. We further propose that the presence of low-level nonspecific antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals enabling accurate determination of seroconversion. The developed assay is currently applied to evaluate immune responses to the Israeli vaccine during human phase I/II trials.

Keywords: COVID-19; Israel; SARS-CoV-2; microarrays; multiplex; nucleocapsid; serology; spike.

FIG 1

Timeline of COVID-19 pandemic initiation in Israel, relative to sample collection. Pink bars represent the total qRT-PCR confirmed samples in Israel in March and April 2020.

FIG 2

Scatterplot of MFI values of qRT-PCR-positive and -negative sera on the 6-plex SARS-CoV-2 protein array. (A) IgG, (B) IgM, and (C) IgA signals of qRT-PCR-positive (P; closed shapes) and -negative (N; open shapes) sera, analyzed on various SARS-CoV-2 proteins: inactivated SARS-CoV-2 virus (hexagons), S2P (triangles), S1 (circles), NTD (diamonds), RBD (squares), and NC (upside-down triangles). Each serum was evaluated against the 6-plex array. The distribution of the signals obtained from individual positive/negative serum samples is presented for each antigen. Horizontal red lines indicate the median value for each set. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple-comparison test, using GraphPad Prism 6. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

FIG 3

Heat maps of qRT-PCR-positive and -negative individuals tested with SARS-CoV-2 6-plex array. Individual sera of (A) qRT-PCR-positive and (B) qRT-PCR-negative individuals were incubated with the 6-plex protein array. MFI signals are presented after subtraction of the previously determined cutoff values for each antigen (Table S1). Results are presented for all antibody isotypes, left to right: IgG, IgM, and IgA on inactivated SARS-CoV-2 and recombinant antigens S2P, S1, NTD, RBD, and NC. qRT-PCR-positive individuals are presented according to disease severity (mild, moderate, severe, and critical), hospitalized (H), or quarantined (Q), 4 to 40 days from symptom onset. Colors: yellow to red, MFI values from low to high; green, no interaction; blue, patients with known background conditions. *, this patient was analyzed at two different time points.

FIG 3

Heat maps of qRT-PCR-positive and -negative individuals tested with SARS-CoV-2 6-plex array. Individual sera of (A) qRT-PCR-positive and (B) qRT-PCR-negative individuals were incubated with the 6-plex protein array. MFI signals are presented after subtraction of the previously determined cutoff values for each antigen (Table S1). Results are presented for all antibody isotypes, left to right: IgG, IgM, and IgA on inactivated SARS-CoV-2 and recombinant antigens S2P, S1, NTD, RBD, and NC. qRT-PCR-positive individuals are presented according to disease severity (mild, moderate, severe, and critical), hospitalized (H), or quarantined (Q), 4 to 40 days from symptom onset. Colors: yellow to red, MFI values from low to high; green, no interaction; blue, patients with known background conditions. *, this patient was analyzed at two different time points.

FIG 4

Scatterplot of MFI values of IgG antibodies present in mild hospitalized versus mild quarantined sera, 13 days from symptom onset. MFI values (Fig. 3A) of IgG antibodies present in sera of mild hospitalized patients, 13 to 33 days after symptom onset (H12-33, closed circles) were compared to those from mild quarantined patients, 15 to 40 days after symptom onset (Q15-40, open circles) on the 6-plex array (antigens are indicated at the top of the panel). Horizontal red lines indicate the median value for each set. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple-comparison test, using GraphPad Prism 6. **, P < 0.01; *, P < 0.05; ns, not significant.