Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

Abstract

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

Keywords: COVID-19; HKU1; OC43; RBD; extrafollicular response; germinal center; neutralizing antibodies; plasma cells; somatic hypermutation; spike protein.

Graphical abstract

Figure 1

Longitudinal characterization of the humoral response against SARS-CoV-2 in severe and mild COVID-19 patients (A and B) Anti-SARS-CoV-2 nucleocapsid (N) (A) and spike (S) (B) serum IgG levels were measured by ELISA in 21 severe COVID-19 (S-CoV) and 18 mild COVID-19 (M-CoV) patients at M0 (white), M3 (light orange), and M6 (dark orange). The dashed line indicates the positivity threshold provided by the manufacturer. (C) Evolution of anti-SARS-CoV-2 S serum IgG levels over time post symptom onset in S-CoV (black dots, dark blue line) and M-CoV (white dots, light blue line) patients. Continuous lines indicate linear regression, and colored area between dashed lines indicates error bands (R² = 0.049 for S-CoV, ns and 0.0061 for M-CoV, ns, Pearson correlation). (D) Correlation between anti-SARS-CoV-2 S serum IgG levels and in vitro neutralization potential (% neutralization achieved at a 1/90 dilution) at M6 (n = 10 S-CoV and 11 M-CoV patients). The line represents a simple linear regression (R² and p value with Pearson correlation are shown) (E) Representative FACS plot of His-tagged SARS-CoV-2 S staining in gated live CD19⁺CD38^int/−CD27⁺IgD⁻ B cells at M0 in two representative S-CoV (upper plot) and M-CoV patients (lower plot). (F) Overall study design. ANOVA and two-tailed Mann-Whitney tests (A and B). Linear regression with Pearson correlation analysis (D). ^∗∗∗p < 0.001, ^∗∗p < 0.01. See also Figure S1 and Table S1.

Figure S1

Quantification and functional assessment of the anti-SARS-CoV-2 humoral immune response in COVID-19 convalescent patients, Related to Figure 1 (A) Percentage of in vitro neutralization shown by individual sera from S-CoV (n = 10) and M-CoV (n = 12) patients at M6 at increasing dilutions. (B) Representative wells for the neutralization assay. Blue spots represent SARS-CoV-2 positive cells. (C) Representative results of an anti-SARS-CoV-2 S IgG ELISA on supernatants from sorted SARS-CoV-2 S-specific MBCs (dark dots) and non spike-specific MBCs (white dots), as validation of FACS SARS-CoV-2 S-staining. Lines indicate median value. Dashed line indicates the positivity threshold (≥3 x blank). (D–H) Flow cytometric gating strategies for the analysis and sorting of major B cell populations from PBMCs of convalescent COVID-19 patients. (D) Gating strategy to analyze SARS-CoV-2 S-specific B cell population. Lymphocytes were first gated based on morphology, before exclusion of doublets, dead cells and CD3/CD14 cells. CD19⁺ cells were next gated before exclusion of CD38^hi plasma cells. CD38^int/- cells were then divided in four quadrants using CD27 and IgD. Upper left quadrant defines memory B cells (MBCs), lower left quadrant double-negative (DN), upper right quadrant CD27⁺IgD⁺ cells (MZB) and lower right quadrant naive B cells (excluding CD38^hi transitional). SARS-CoV-2 S-specific B cells were then analyzed within the B cell population of interest using a double-staining strategy with anti-His antibodies of a His-tagged SARS-CoV-2 S protein. (E) Gating strategy to separate activated and classical switched B cells using CD71, within the IgD^-CD27⁺ gate (F) Gating strategy to analyze CD27^hiCD38^hi plasma cells in CD3^-CD14^- live cells. (G) Gating strategy for sorting of CD19⁺IgD^- cells from PBMCs for 10X Genomics single cell experiment. Lymphocytes were gated before exclusion of doublets and of CD14/CD15/CD56/IgD⁺ cells, before sorting of CD19⁺ cells. (H) Gating strategy for single-cell sorting of SARS-CoV-2 S-specific B cells for single cell culture at M3 and M6. Lymphocytes were gated before double exclusion of doublets, dead cells and CD3/CD14 cells. CD19⁺ cells were then gated before exclusion of CD38^hi plasma cells. CD38^int/- cells were further divided into four quadrants using CD27 and IgD. Double stained SARS-CoV-2 S specific cells were sorted from all three non-naive quadrants (not IgD⁺CD27^-).

Figure 2

Characterization of the B cell response against SARS-CoV-2 in acute (M0) and convalescent (M6) S-CoV patients (A) UMAP and clustering of 41,083 B cells analyzed by scRNA-seq from four S-CoV patients at M0 (left panel) and M6 (right panel) (see Table S2). Upper square in both panels shows the results of increased clustering resolution for the “Activated” cell cluster. (B) Relative cluster distribution at M0 and M6 for all sorted cells (top panel) and cells falling in the “Activated” cluster at initial clustering resolution. Bar indicates mean with SEM. (C) Feature plots showing scaled normalized counts for CD27 and CD38 barcoded antibodies as well as S and G2/M signature scores in all cells. (D) Feature plots showing scaled normalized counts for CD21, CD71, CD38, CD11c, CD95, CD27, and CD307e (FcRL5) barcoded antibodies in cells from the “Activated” cluster. (E) UMAP of all cells at M0 or M6, with cells belonging to one of the top 10% antibody-secreting cell (ASC) clones highlighted (light blue). Cells stained by the barcoded-anti-His/His-tagged SARS-CoV-2 S protein combination (barcoded-SARS-CoV-2 S stained cells) are also highlighted (red when members of one of the top 10% ASC clones at M0 [“shared”], dark blue otherwise). (F) Circus plots showing clonal relationships between cells from different UMAP clusters and time points. Blue lines indicate clonal relationships with one of the top 10% ASC clones at M0 and gray line all other clonal relationships. (G) Histograms showing the number of mutations in V_H genes for cells belonging to one of the top 10% ASC clones at M0 according to their barcoded-SARS-CoV-2 S staining (top = unstained; bottom = stained). (H) Graph showing the frequencies of cells at the M0 time point harboring 0–1 (white), 2–9 (gray), or ≥10 (black) mutations in their V_H genes for all sorted cells according to their cluster of origin (left) as well as for cells stained by the barcoded-SARS-CoV-2 S protein and/or members of the top 10% ASC clones at M0, as defined in (E). Repeated measures (RM) two-way ANOVA and Sidak’s multiple comparison tests (B). ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗p < 0.05. See also Figure S2 and Table S2.

Figure S2

Gene expression markers and cluster assignments for the main single-cell populations identified in acute and convalescent S-CoV patients, Related to Figure 2 (A and B) Dot plots showing expression of selected genes in cells from the main clusters (A) or in cells from the “Activated” cluster (B). Size of dots represents the percentage of cells in the cluster in which transcripts for that gene are detected. Dot color represents the average expression level (scaled normalized counts) of that gene in the population. (C) Feature plots showing scaled normalized counts for CD21, CD71, CD11c, CD95 barcoded antibodies in all sorted CD19⁺IgD^-. (D and E) Relative cluster distribution at M0 and M6 for cells belonging to one of the top 10% ASC clones (D) or stained by the barcoded-anti-His/His-tagged SARS-CoV-2-S protein combination (barcoded-SARS-CoV-2-S) (E). Top panels represent cluster distribution for all cells, middle panels represent cluster distribution for non-ASC cells and bottom panels represent cluster distribution for cells belonging to the “Activated” cluster. Bars indicate mean with SEM. (F) UMAP of all cells at M0 or M6, with cells belonging to one of the top 10% ASCs clones highlighted (light blue). Cells belonging to Spike-specific clones, as defined in the Methods section, are also highlighted (red when members of one of the top 10% ASC clones at M0, dark blue otherwise). (G) Relative cluster distribution at M0 and M6 for cells belonging to spike-specific clones. Top panel represent cluster distribution for all cells, middle panel represent cluster distribution for non-ASC cells and bottom panels represent cluster distribution for cells belonging to the “Activated” cluster. Bars indicate mean with SEM. (H) Circos plots showing clonal relationships between cells from different UMAP clusters and time points. Blue lines indicate clonal relationships with one of the spike-specific clones and gray line all other clonal relationships. RM two-way ANOVA and Sidak’s multiple comparison tests (D-E, G). ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.01, ^∗∗p < 0.01, ^∗p < 0.05.

Figure S3

Phenotypic characterization of total and SARS-CoV-2 S-specific B cell populations in convalescent COVID-19 patients, Related to Figure 3 (A) Cluster distribution of all analyzed CD19⁺IgD^- B cell across FlowSOM-identified clusters for S-CoV (n = 15) and M-CoV (n = 16) donors (Table S1) at all three time points (M0, M3 and M6). (B) UMAP of concatenated down-sampled cells from all 83 samples analyzed colored according to the scaled fluorescence intensity for IgD, CD71, CD27, CD38, CD11c, CD19, CD21 expression. (C) Heatmap representing the mean fluorescent intensity for IgD, CD71, CD27, CD38, CD11c, CD19, CD21 in each identified FlowSOM cluster. (D–F) Absolute number of antibody secreting cells (PC+PB) (D), CD19⁺CD27⁺IgD^- MBCs (E), and ABCs (F) at indicated time points in S-CoV and M-CoV patients. Bars indicate the mean with SEM. Dashed line indicates the threshold based on healthy donors (mean + 2 SD) (G and H) Percentage (G) and absolute numbers (H) of SARS-CoV-2 spike-specific CD19⁺CD27⁺IgD^- MBCs at indicated time points in S-CoV and M-CoV patients. (I and J) Percentage (I) and absolute number (J) of SARS-CoV-2 spike-specific cells among CD27^-IgD^- DN cells. (G and I) Each dot represents a patient and lines connect the different time points for each patient. Dashed line indicates the threshold based on healthy donors (mean + 2 SD). (H and J) Bars indicate the mean with SEM. (K) Representative dot plot for T-bet staining in CD19⁺CD38^- B cells from COVID-19 patients. (L) Percentage of intra-cellular T-Bet staining in SARS-CoV-2 spike-specific CD19⁺IgD^-CD27⁺CD38^- cells from S-CoV (n = 2) and M-CoV (n = 3) patients at indicated time points. Bar indicate the mean with SEM. (M–O) Correlation between the number of SARS-CoV-2 spike-specific CD19⁺CD27⁺IgD^- MBCs at M6 and age at diagnosis (M), maximum oxygen flow during hospitalization (liters/minutes, only in S-CoV) (N) and maximum CRP during hospitalization (mg/L, available only in S-CoV) (O) using linear regression. (P) Number of spike-specific CD19⁺CD27⁺IgD^- MBCs at M6 according to the initial disease severity (S-CoV and M-CoV) and the sex of the patient (Women, W, blue and Men, M, green). Bars indicate the mean with SEM. (Q) Plot showing the number of spike-specific CD19⁺CD27⁺IgD^- cells in the 21 S-CoV patients analyzed at M6 colored according to the treatment received during initial hospitalization. Standard of care included oxygen, low dose anticoagulant, antibiotic if needed and symptomatic treatments. RM two-way ANOVA and Sidak’s multiple comparison tests (A). ANOVA and two-tailed Mann-Whitney tests (D–F, H, J, L). Linear regression with Pearson correlation analysis (M-0). ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01, ^∗p < 0.05.

Figure 3

Phenotypic evolution of the SARS-CoV-2 S-specific B cell response up to 6 months post-infection in M- and S-CoV patients (A) UMAP projections of concatenated multi-parametric FACS analysis of CD19⁺IgD⁻ cells from all S-CoV (n = 15) and M-CoV (n = 16) patients analyzed over time in our cohort (Table S1). His-tagged labeled SARS-CoV-2 S-specific cells are overlaid as red dots. (B) Unsupervised clustering (FlowSOM) was performed on the concatenated FACS dataset based on IgD, CD71, CD27, CD38, CD11c, CD19, and CD21 fluorescence intensity. 95% density contours for each identified cluster are overlaid in the UMAP projection. (C) Cluster distribution for all analyzed SARS-CoV-2 S-specific cells in identified clusters at indicated time point. Bars indicate mean with SEM. (D) Proportion of SARS-CoV-2 S-specific CD19⁺IgD⁻CD27⁺CD38⁻ MBCs at M0, M3, and M6 in S-CoV and M-CoV patients, as compared to six pre-pandemic healthy donor (HD) controls. Each dot represents one patient, and bars indicate mean with SEM. Dashed line indicates the mean + 2 SD of the detected frequency of SARS-CoV-2 S-specific MBCs in HD. (E) Proportion of S-specific MBCs over time post symptom onset in S-CoV (black dots, dark blue line) and M-CoV (white dots, light blue line) patients. Lines indicates linear regression (R² = 0.051, p value = 0.02 for S-CoV, and R² = 0.018, ns, for M-CoV, Pearson correlation). Colored area between dashed lines indicates error bands. Red dots and lines represent data obtained for the four S-CoV patients analyzed as part of our scRNA-seq dataset. (F) Representative dot plot showing CD71 and CD19 expression in CD19⁺IgD⁻CD38⁻ B cell at indicated time points from two representative S-CoV and M-CoV patients. SARS-CoV-2 S-specific cells are overlaid as red dots. Gating strategy for ABCs and classical MBCs according to CD71 expression is further displayed. (G) Proportion of SARS-CoV-2 S-specific cells displaying an ABC (CD19⁺CD27⁺IgD⁻CD71⁺) (left) or MBC (CD19⁺CD27⁺IgD⁻CD71^low/int) (right) phenotype at indicated time points. Bars indicate mean with SEM. (H) Proportion of activated MBCs over time post symptom onset in S-CoV (black dots, dark blue line) and M-CoV (white dots, light blue line) patients. Lines represents the linear regression. Colored area between dashed lines indicates error bands. R² and p value with Pearson correlation. (I) Correlation between the frequency of SARS-CoV-2 S-specific MBCs at M6 (expressed as % of CD27⁺IgD⁻ B cells) and of ABCs at M0 in all M-CoV and S-CoV patients. RM two-way ANOVA and Sidak’s multiple comparison tests (C). ANOVA and two-tailed Mann-Whitney tests (D and G). Linear regression with Pearson correlation analysis (I). ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.01, ^∗∗p < 0.01, ^∗p < 0.05. See also Figure S3 and Table S3.

Figure 4

Maturation of the SARS-CoV-2 S-specific repertoire up to 6 months post-infection in M- and S-CoV patients (A) Left: pie charts showing the average percentage of RBD⁺ and cross-reactive specificities (RBD⁺OC43⁺, RBD⁺HKU1⁺OC43⁺, RBD⁺HKU1⁺, OC43⁺, HKU1⁺, and OC43⁺HKU1⁺) among single-cell cultured S-specific B cells as determined by ELISA. Three and four S-CoV patients, all previously included in our scRNA-seq dataset, were analyzed at M3 and M6, respectively. Right: bar plot showing the proportion of RBD specificity and cross-reactive specificities among S-specific cells for each patient. (B) Average percentage of RBD-specific cells among SARS-CoV-2 S-specific sorted cells from indicated S-CoV and M-CoV patient at M3 and M6. (C) Violin plot showing the number of mutations in the Ig V_H segment of cells in the original M0 10X Genomics scRNA-seq VDJ dataset found to be in clonal relationship with M3 or M6 SARS-CoV-2 S-specific sorted cells showing specificity against RBD (RBD⁺ clones), seasonal betacoronavirus S proteins (HKU1-CoV or OC43-CoV) or against SARS-CoV-2 S (Spike⁺ clones). (D and E) Histograms showing the relative distribution of mutation numbers in the Ig V_H segment from all SARS-CoV-2 S- (D) or RBD- (E) specific clone members (10X Genomics scRNA-seq dataset and single-cell heavy-chain sequencing data) at M0, M3, and M6 as well as from sequences from the literature (Brouwer et al., 2020; Kreer et al., 2020; Liu et al., 2020; Robbiani et al., 2020; Shi et al., 2020; Wec et al., 2020; Zost et al., 2020), mainly determined between M0 and M3. (F) Plot showing the percentage of neutralization, normalized to the IgG concentration, for single-cell culture supernatants from identified SARS-CoV-2 RBD-specific B cells at M6. Two dilutions (1/2 and 1/20) were assayed for each supernatant tested. Dashed lines indicate 80% and 50% neutralization. (G) Violin plot representing the number of mutations in the Ig V_H sequence in strong neutralizing antibodies at M6 (neutralization >80% at 1/2 dilution) versus the number of mutations in the Ig V_H sequence of all cells from anti-RBD clones at M6. (H) Evolutionary tree of an RBD-specific and neutralizing clone, built on sequences from 10X Genomics scRNA-seq, cell culture, and literature. Each circle represents a unique sequence from that clone. Circle color indicates time point of origin, and the number inside indicates the calculated number of mutations from an inferred unmutated common ancestor (“germline”). Grey indicates a theoretically inferred common precursor. ^∗ indicates that the antibody associated with that sequence has been validated as neutralizing in vitro. CDR3 from all sequences in the tree are represented as a frequency plot logo (top left) as well as below the tree, where each amino acid in red indicates a change compared to the top listed CDR3. ANOVA and two-tailed Mann-Whitney tests (B and C). ^∗∗∗p < 0.01, ^∗p < 0.05. See also Figure S4 and Table S4.

Figure S4

Cross-reactivity, convergence, and accumulation of somatic mutations in anti-SARS-CoV-2 S-specific memory B cells from convalescent COVID-19 patients, Related to Figure 4 (A) Heatmaps showing the RBD, OC43 and HKU1 ELISA blank ratio for all tested SARS-CoV-2-spike-specific single cell culture supernatants at M3 and M6. Each line represents one tested supernatant. Light red lines indicate that no value is available for that supernatant. (B) FACS plots representing index sorting data of spike-specific cells according to their specificity for SARS-CoV-2 spike or RBD. (C) Repartition of the sorted SARS-CoV-2 spike- and RBD-specific cells at M3 and M6 between the MBCs, DN or IgD⁺CD27⁺ compartments. (D) Pie chart representing clone size in all the sequences generated via single cell culture for each of the 4 patients previously included in our scRNaseq analysis. Total number of sequenced cells is indicated in the middle of the pie. (E) Circus plot showing SARS-CoV-2 spike- (gray) or RBD- (light blue) specific clones shared between patients in our dataset or with sequences from the literature. (F) Histogram showing log-normalized counts for both barcoded his-tagged SARS-CoV-2 S proteins in all cells from the M6 time point of our 10X Genomics scRNA-seq dataset (white) and cells in clonal relationship with sequence from single cell culture (dark gray) (G and H) Histograms showing the distribution of mutations in Ig V_H for SARS-CoV-2 spike- (G) and RBD- (H) specific clones at M0, M3 and M6 according to the assay of origin. (I) Plot showing the raw percentage of neutralization for single cell culture supernatants from identified SARS-CoV-2 RBD-specific B cells at M6. Two dilutions (1/2 and 1/20) were assayed for each supernatant tested. Dashed lines indicate 80% and 50% neutralization (see Figure 4F). (J) Evolutionary tree of a convergent RBD-specific and neutralizing clone, built on sequences from 10X Genomics scRNA-seq and cell culture from two patients and from the literature. Each circle represents a unique sequence from that clone. Circle color indicates time-point of origin and the number inside indicates the calculated number of mutations from an inferred unmutated common ancestor (germline). Grey indicates a theoretically inferred common progenitor. CDR3 from all sequences in the tree are represented as a frequency plot logo (top left) as well as below the tree, where each amino-acid in red indicates a change compared to the first listed CDR3 sequence.