Fluorescence-based assessment of neutralization and binding capacity of maternal sera
against various SARS-CoV-2 variants

SARS-CoV-2 infections during pregnancy are associated with increased COVID19 severity and mortality as compared to the general population. Two methods were developed to evaluate the antibody response of ~700 pregnant women against the dominating SARS-CoV-2 variants in the Israeli pandemic. (i) In a neutralization assay using recombinant (rVSVdG) pseudoviruses, each expressing a SARS-CoV-2 spike variant on their membrane, HEK293 cells overexpressing the SARS-CoV-2 receptor hACE2 and the protease TMPRSS2 were infected with the four pseudoviruses (Wuhan – Hu-1 (wild type (WT)), Alpha – B.1.1.7, Delta – B.1.617.2 and Omicron - B.1.1.529), were exposed to maternal sera. The capacity of each serum to inhibit any of the four variants from infection was simultaneously quantified by fluorescent microscopy. (ii) In a Multiplex assay system, where eight different fluorescent beads were coupled to four SARS-CoV-2 spikes and four nucleocapsid variants, the bead mixture was incubated with each individual serum, and the binding capacity was assessed using an anti-hIgG-PE on a Luminex MAGPIX reader. Our data suggest that the SARS-CoV-2 evolution led to variants that with time, escaped antibody neutralization; while the WT variant was inhibited in both COVID-positive sera and vaccinated sera, the Alpha, Delta, and Omicron variants were less inhibited, respectively. Moreover, the Omicron variant escaped neutralization unless first the women were vaccinated and only later infected, mainly during the Omicron wave. Our data also correlate with the severity of the side effects during the time of the infection of each variant; higher antibody titers were found towards the spikes of the Alpha and Delta variants compared with those of the WT and Omicron variants. Interestingly, while very few vaccinated sera had any binding capacity to the WT-, Alpha- and Delta-nucleocapsid, many showed significant binding to the Omicron-nucleocapsid in accordance with the vast infection of Omicron in the Israeli population, of which many were without symptoms. The comparison between the two methods showed significant correlations for all variants except for the Omicron. This fits with the notion that despite binding capacity, most sera could not neutralize Omicron unless they were exposed first to vaccination and then were infected with Omicron.