Decline of Humoral Responses against SARS-CoV-2 Spike in Convalescent Individuals

Abstract

In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease 2019 (COVID-19) patients. The therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here, we performed repeated analyses at 1-month intervals on 31 convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein, including neutralization, evolve over time. We observed that the levels of receptor-binding-domain (RBD)-specific IgG and IgA slightly decreased between 6 and 10 weeks after the onset of symptoms but that RBD-specific IgM levels decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type SARS-CoV-2 S or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms.IMPORTANCE While waiting for an efficient vaccine to protect against SARS-CoV-2 infection, alternative approaches to treat or prevent acute COVID-19 are urgently needed. Transfusion of convalescent plasma to treat COVID-19 patients is currently being explored; neutralizing activity in convalescent plasma is thought to play a central role in the efficacy of this treatment. Here, we observed that plasma neutralization activity decreased a few weeks after the onset of the symptoms. If neutralizing activity is required for the efficacy of convalescent plasma transfer, our results suggest that convalescent plasma should be recovered rapidly after the donor recovers from active infection.

Keywords: COVID-19; ELISA; IgA; IgG; IgM; RBD; SARS-CoV-2; Spike glycoproteins; convalescent plasma; coronavirus; cross-reactivity; neutralization.

FIG 1

SARS-CoV-2 S-specific and RBD-specific antibody levels decrease over time. (A to C) Indirect ELISA was performed using recombinant SARS-CoV-2 RBD and incubation with plasma samples recovered at baseline (6 weeks after the onset of symptoms; red circle) and 1 month later (black circle). Anti-RBD antibody binding was detected using (A) anti-IgG-HRP (anti-IgG horseradish peroxidase), (B) anti-IgM-HRP, or (C) anti-IgA-HRP. Relative light unit (RLU) values obtained with bovine serum albumin (BSA) (negative control) were subtracted and further normalized to the signal obtained with the anti-RBD CR3022 monoclonal antibodies (MAb) present in each plate. The graphs shown in panels A to C represent (A and B) the areas under the curve (AUC) calculated from RLU obtained with serial plasma dilutions or (C) the normalized RLU for one plasma dilution (1:500). (D to F) Cell surface staining of 293T cells expressing full-length Spike (S) from different HCoVs, including (D) SARS-CoV-2 or its D614G counterpart; (E) SARS-CoV; and (F) OC43, NL63, and 229E with plasma samples recovered at baseline (6 weeks after the onset of symptoms) and 1 month later. The graphs shown in panels D to F represent median fluorescence intensities (MFI). In panels A to F, undetectable levels are represented as white symbols, and limits of detection are plotted. The average numbers and percentages of positive samples are indicated at the top of each panel. Statistical significance was tested using Wilcoxon matched-pair signed-rank tests (ns, not significant; **, P < 0.01; ****, P < 0.0001).

FIG 2

Neutralizing activity of convalescent plasma decreases over time. (A) Pseudoviral particles coding for the luciferase reporter gene and bearing SARS-CoV-2 S glycoprotein or its D614G counterpart, SARS-CoV S glycoprotein, or VSV-G glycoprotein were used to infect 293T-ACE2 cells. Pseudoviruses were incubated (37°C, 1 h) with serial dilutions of plasma samples recovered at baseline (6 weeks after the onset of symptoms) or collected 1 month later prior to infection of 293T-ACE2 cells. Infectivity at each dilution was assessed in duplicate, and data are shown as the percentage of infection without plasma for each pseudovirus. (B) The median of neutralization for baseline (red) or 1-month (black) plasma samples is shown. (C) Neutralization half-maximal inhibitory plasma dilution (ID₅₀) values were determined using a normalized nonlinear regression with GraphPad Prism software. Undetectable levels (ID₅₀ < 50) are represented as white symbols. The mean neutralizing titers and the proportions (%) of neutralizers (patients with an ID₅₀ value over 50) are shown above the graphs. Statistical significance was tested using Wilcoxon matched-pair signed-rank tests (ns, not significant; ****, P < 0.0001).