Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection

Abstract

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.

Keywords: COSIN; COVID; RBD; RBD tetramer; SARS-CoV-2; SARS-CoV-2 pseudovirus; functional MBCs; longitudinal tracking; memory B cells; neutralizing antibodies.

Graphical abstract

Figure 1

Antibody analysis (A) RBD endpoint titers (EPTs) plotted against days post-onset of symptoms (DPS). Binding at each dilution was assessed in duplicate. The curve shows the mean EPT values using a Loess regression model. The shaded band indicates the 95% confidence interval. Blue datapoints highlight participants >51 years (median age) and black datapoints highlight participants <51 years. (B) Neutralization half-maximal inhibitory serum dilution (ID₅₀) values determined with the pseudovirus assay plotted against the DPS. Infectivity at each dilution was assessed in triplicate and ID₅₀ was determined using a normalized non-linear regression using GraphPad Prism software. (C) ID₅₀ values determined with the live virus assay plotted against DPS. Infectivity at each dilution was assessed in duplicate and ID₅₀ was determined using a normalized non-linear regression using GraphPad Prism software. The lines connect a single subject sampled at 2 time points. The healthy control cutoff (mean + 2 × SD) is indicated by the dotted black line. Blue datapoints highlight participants >51 years (median age) and black datapoints highlight participants <51 years.

Figure 2

Memory B cell analysis and monoclonal antibody characteristics (A) Distribution of RBD-specific Ig classes/10⁶ B cells across all of the EPT groups. The first bar represents t1 and the second bar represents t2 of each SARS-CoV-2 participant. Healthy participants have 1 bar representing 1 time point. (B) Violin plots of Ig subclass comparison between t1 and t2. Healthy control cut-off (mean + 2 × SD) are represented by the dotted black line. (C) Comparison of CD27⁺IgG⁺RBD⁺ B cells/10⁶ B cells between t1 and t2 showing an increase in frequencies (Wilcoxon matched-pairs signed rank test, p = 0.0084). (D) Correlation analysis between EPT and CD27⁺IgG⁺RBD⁺ B cells/10⁶ B cells during t2 (Spearman’s correlation, p = 0.017). (E) Heatmap of all subjects comparing age, gender, severity, DPS, EPT, ID₅₀, CD27⁺IgG⁺RBD⁺ B cells/10⁶ B cells from t1 and t2. (F) Neutralization activity of all mAbs at 1/10 dilution. Dotted line represents 40% neutralization cutoff. Heatmap shows mAb RBD binding (red above and blue below cutoff) and the B cell line shows whether the originating memory B cell was IgG⁺ (red) or IgD⁺ (blue). (G) Neutralization plot of 6 mAbs identified from 3 SARS-CoV-2 participants at t2. Each color represents a participant.