No substantial preexisting B cell immunity against SARS-CoV-2 in healthy adults

Abstract

Preexisting immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. The presence and clinical relevance of a preexisting B cell immunity remain to be fully elucidated. Here, we provide a detailed analysis of the B cell immunity to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated SARS-CoV-2 humoral immunity in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells at a single cell level and generated and tested 158 monoclonal antibodies. None of these antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of competent preexisting antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

Keywords: Immune response; Immunity; Virology.

Graphical abstract

Figure 1

Screening of pre-pandemic samples from 150 adults reveal no relevant reactivity to SARS-CoV-2 (A) Timeline of blood collections and demographic characteristics of 150 donors sampled before the SARS-CoV-2 pandemic. Pre-pandemic blood samples were collected as buffy coats between August and November of 2019. Gender and age distribution of donors are illustrated as bar plots. (B) Pre-pandemic plasma samples and purified, polyclonal IgGs (pIgG) were tested for binding and neutralization using different experimental approaches. Plasma IgG, IgM, and IgA as well as pIgG were tested for binding to SARS-CoV-2, HKU1, and OC43 S proteins using in house (ELISA) and commercially available immunoassays (com. IA). Binding of plasma IgG and IgM to cell surface expressed SARS-CoV-2 S protein was also determined by FACS analysis (CA). Neutralizing activity was determined against SARS-CoV-2 pseudo- and authentic wild type viruses (PSV and WT). (C) Heatmap visualization of binding (AUC or AU) and neutralizing activity (% or CPE) of pre-pandemic plasma samples and pIgG against SARS-CoV-2 and endemic HCoVs (HKU1 and OC43) S proteins or SARS-CoV-2 authentic wild type (WT) and pseudovirus (PSV Wu_01), respectively (see also Figures S2 and S3). Immunoassays were performed in duplicates and the AUCs are presented as geometric mean of duplicates. Neutralizing activity was first determined for single sample concentrations. Samples that displayed neutralization activity of ≥50% are indicated as (x) and were repeatedly investigated in serial dilutions (see also Figure S3). Samples were tested in duplicates. The average of neutralization is presented and each row represents one donor.

Figure 2

No substantial detection of SARS-CoV-2-reactive B cells (A) Representative dot plots of SARS-CoV-2-reactive, CD19⁺CD20⁺, IgG⁺, and IgG^- B cells of samples from COVID-19 convalescent individuals compared to before the pandemic (pre-pandemic). Depicted numbers indicate frequencies of S protein reactive B cells (see also Figure S4). Red colored gate indicates gating strategy for analysis and dotted gate indicates actual sorting gate. Dot plot bar graph displays the mean ± SD frequency of SARS-CoV-2-reactive, IgG⁺, and IgG^- B cells in 40 pre-pandemic and 23 COVID-19 samples (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; unpaired two-tailed t-test). (B) Clonal relationship of heavy chain sequences amplified from single SARS-CoV-2-reactive IgG⁺ and IgG^- B cells isolated from 40 donors sampled before the pandemic. Individual clones are colored in shades of blue, gray, and white. In the center of each pie chart, numbers of productive heavy chain sequences are illustrated. Presentation of clone sizes are proportional to the total number of productive heavy chain sequences per clone. (C) VH gene distribution, VH gene germline identity, and CDRH3 length distribution in amino acids (AA) were separately determined for IgG and IgM. Distributions were calculated per individual. Bar and line plots show mean ± SD.

Figure 3

Monoclonal antibodies isolated from pre-pandemic samples show no reactivity to SARS-CoV-2 and endemic HCoVs (A) Illustration demonstrating the sequence selection for downstream antibody production. Sequences were selected for antibody production based on similarity to antibody sequences deposited in the CoVAbDab and on random selection. From 7,829 SARS-CoV-2 reactive IgG⁺ and IgG^- B cells, 5,223 productive heavy chain sequences were amplified. For antibody production, 18 HC sequences were selected based on similarity selection and 182 HC sequences were selected based on random selection. In total, 158 antibodies were produced. (B) Heatmap visualization of binding (AUC) and neutralizing activity (%) of monoclonal antibodies isolated from pre-pandemic blood samples against SARS-CoV-2 and endemic HCoVs (HKU1 and OC43) S proteins or SARS-CoV-2 pseudovirus (PSV Wu_01), respectively (see also Figure S5). Each row represents one monoclonal antibody. ELISAs were performed in duplicate experiments and the AUCs are presented as a geometric mean of duplicates. Neutralizing activity was determined for single sample concentrations. Samples were tested in duplicates. (C) Neutralizing activity against SARS-CoV-2 pseudovirus (PSV Wu_01) was verified for all mAbs in serial dilutions.