High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

Abstract

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than two million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection.

Study design and methods: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike-specific antibodies in the plasma of convalescent donors.

Results and conclusion: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

Keywords: COVID-19; RBD; SARS-CoV-2; antibodies; convalescent plasma; coronavirus; flow cytometry; high-throughput screening; spike glycoproteins.

FIGURE 1

Characterization of the 293T‐Spike cell line. (A) Dot plots depicting representative stainings of the parental 293T (left) or the 293T‐Spike cell lines (right) using CR3022 mAb, a representative COVID‐19‐negative and COVID‐19‐positive plasma. Percentages represent the proportion of green fluorescent protein (GFP)+ and GFP‐ cells on the total cell population. (B) A schematic representation of the experimental procedures used to perform high‐throughput screening (HTS) of plasma samples for their specific binding to SARS‐CoV‐2 Spike. (C) Dot plots depicting representative staining of pooled cell lines used for HTS assay (equal ratio of parental 293T (GFP‐) and the 293T‐Spike cells (GFP+)) using CR3022 mAb, a COVID‐19‐negative plasma, and a COVID‐19‐positive plasma. Median fluorescence intensities obtained on GFP‐ and GFP+ cell populations are indicated [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Decline of Spike‐specific antibodies in longitudinal convalescent plasma. The level of anti‐Spike antibodies in plasma from COVID+ donors was determined by flow cytometry using (A) 293T transduced cells or (B) 293T transfected cells expressing SARS‐CoV‐2 Spike. (A–B, left panels) Each curve represents the median fluorescence intensity (MFI) obtained with the plasma of one donor at every donation (4–10 donations per donor) as a function of the days after symptom onset. Undetectable measures are represented as white symbols, and limits of detection are plotted. (A‐B, right panels) The time post‐symptom onset (33–120 days) was divided in quartiles containing similar numbers (between 21 and 23) of plasma samples obtained from the 15 COVID‐19‐positive donors. Boxes and horizontal bars denote interquartile range (IQR), while horizontal lines in boxes correspond to a median of MFI values. Whisker endpoints are equal to the maximum and minimum values below or above the median ± 1.5 times the IQR. Statistical significance was tested using one‐way ANOVA with a Holm‐Sidak post‐test (* p < .05; ** p < .01; **** p < .0001. (C) Correlations between the levels of recognition of SARS‐CoV‐2 full‐length Spike evaluated by flow cytometry using transduced or transfected 293T cells and levels of RBD recognition of SARS‐CoV‐2 RBD evaluated by indirect ELISA. (D) Correlations between the overall decline in Spike‐specific antibody levels as measured by flow cytometry with transduced 293T cells (as calculated using the following formula: 1‐[MFI at the last donation/MFI obtained at first donation] × 100) and the number of days between symptom onset and the last donation or the number of donations by each donor. (C–D) Statistical significance was tested using a Pearson correlation test or a Spearman rank correlation test based on statistical normality [Color figure can be viewed at wileyonlinelibrary.com]