Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India

Abstract

India is one of the most affected countries by COVID-19 pandemic; but little is understood regarding immune responses to SARS-CoV-2 in this region. Herein we examined SARS-CoV-2 neutralizing antibodies, IgG, IgM, IgA and memory B cells in COVID-19 recovered individual from India. While a vast majority of COVID-19 recovered individuals showed SARS-CoV-2 RBD-specific IgG, IgA and IgM antibodies (38/42, 90.47%; 21/42, 50%; 33/42, 78.57% respectively), only half of them had appreciable neutralizing antibody titers. RBD-specific IgG, but not IgA or IgM titers, correlated with neutralizing antibody titers and RBD-specific memory B cell frequencies. These findings have timely significance for identifying potential donors for plasma therapy using RBD-specific IgG assays as surrogate measurement for neutralizing antibodies in India. Further, this study provides useful information needed for designing large-scale studies towards understanding of inter-individual variation in immune memory to SARS CoV-2 natural infection for future vaccine evaluation and implementation efforts.

Keywords: COVID-19; India; Memory B cells; Neutralizing antibodies; Receptor binding domain; SARS-CoV-2.

Fig. 1

Evaluation of SARS-CoV-2 RBD specific IgG, IgA and IgM antibody responses. (A) RBD-specific IgG, (B), RBD-specific IgA; (C), RBD-specific IgM. Left, pre-pandemic healthy (n-22), middle COVID-19 recovered (n = 42); right, endpoint titers. ELISA cutoff values are calculated using the average plus 3 standard deviations of the 22 healthy controls at 1:100 dilution (shown as a dotted line). The unpaired analysis was done using non-parametric Mann-Whitney-U test. p ≤ 0.05 was considered significant. Assay cutoff value is marked with dotted line.

Fig. 2

Correlation of age and day post initial diagnosis of COVID-19 recovered individuals with SARS-CoV-2 IgG, IgM and IgA titers. (A). Age versus IgG (left, n = 42), IgA (middle, n = 42) or IgM (right, n = 42) titers. (B). Time post initial diagnosis versus IgG (left, n = 42), IgA (middle, n = 42) or IgM (right, n = 42) titers. Correlations were calculated by Spearman's correlation coefficient r. p ≤ 0.05 is considered significant. Note that none of the data sets above reached significant values of correlation.

Fig. 3

Evaluation of SARS-CoV-2 neutralizing antibodies in COVID-19 recovered individuals. SARS-CoV-2 neutralizing activity at indicated dilutions of plasma is shown in pre-pandemic healthy (n = 22, in grey) (A) and in COVID-19 recovered individuals (n = 42, in blue) (B). Dotted line represents the plasma dilution that leads to 50% neutralization. (C) Scatter plot shows neutralization titers (FRNT-mNG₅₀) in pre-pandemic healthy (n = 22) and COVID-19 recovered (n = 42) individuals. The unpaired analysis was done using non-parametric Mann-Whitney-U test. p ≤ 0.05 was considered significant. Limit of detection is marked with a dotted line.

Fig. 4

Correlation analysis of SARS-CoV-2-specific antibody responses versus neutralization titers. Correlation analysis shows FRNT-mNG₅₀ titers (x-axis) versus RBD-specific IgG (Left), IgA (middle) and IgM (right) titers on y-axis in COVID-19 recovered individuals (n = 42, blue dots). Correlation analysis was performed by log transformation of the endpoint ELISA titers followed by linear regression analysis. Dotted line on x-axis and y-axis indicate limit of detection. Correlations were calculated by Spearman's correlation coefficient r. p ≤ 0.05 was considered significant and indicated in the figure.

Fig. 5

Correlation analysis of SARS-CoV-2 whole virus specific IgG versus neutralizing titers. (A). Scatter plots shows SARS-CoV-2 whole virus specific IgG measured using measured using commercial kit (Zydus diagnosis, Covid Kavach) in pre-pandemic healthy (n = 5) and COVID-19 recovered (n = 42). The unpaired analysis was done using non-parametric Mann-Whitney-U test. p ≤ 0.05 was considered significant. (B). Correlation analysis of SARS-CoV-2 whole virus antigen specific IgG ELISA kit values (y-axis) versus neutralizing titers (x-axis) in COVID-19 recovered individuals (n = 42). Correlations were calculated by Spearman's correlation coefficient r. p ≤ 0.05 was considered significant. Dotted line on x-axis indicate limit of detection and on y-axis assay cut off.

Fig. 6

SARS-CoV-2 RBD-specific memory B cell analysis in COVID-19 recovered individuals. (A) Gating strategy used to identify memory B cells. (B) SARS-CoV-2 RBD-specific memory B cells on gated total memory B cells that were CD19 positive, CD20 high, IgD negative and CD27 high is shown. (C) Frequency of RBD-specific memory B cells of the total memory B cells in the COVID-19 recovered individuals (n = 13). (D) Correlation analysis shows frequency of RBD-specific memory B cells (x-axis) and the RBD-specific IgG titers (y-axis) in COVID-19 recovered individuals.