Antigenic sin and multiple breakthrough infections drive converging evolution of COVID-19 neutralizing responses

Abstract

Understanding the evolution of the B cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is fundamental to design the next generation of vaccines and therapeutics. We longitudinally analyze at the single-cell level almost 900 neutralizing human monoclonal antibodies (nAbs) isolated from vaccinated people and from individuals with hybrid and super hybrid immunity (SH), developed after three mRNA vaccine doses and two breakthrough infections. The most potent neutralization and Fc functions against highly mutated variants belong to the SH cohort. Repertoire analysis shows that the original Wuhan antigenic sin drives the convergent expansion of the same B cell germlines in vaccinated and SH cohorts. Only Omicron breakthrough infections expand previously unseen germ lines and generate broadly nAbs by restoring IGHV3-53/3-66 germ lines. Our analyses find that B cells initially expanded by the original antigenic sin continue to play a fundamental role in the evolution of the immune response toward an evolving virus.

Keywords: B cell germlines; COVID-19; CP: Immunology; Fc functions; SARS-CoV-2; V(D)J genes; antigenic sin; breakthrough infection; memory B cells; neutralizing antibodies.

Graphical abstract

Figure 1

nAbs potency and breadth of neutralization and Fc functions against SARS-CoV-2 Omicron variants (A–D) Scatter dot charts show the neutralization potency, reported as IC₁₀₀ (ng mL⁻¹), of nAbs tested against the original Wuhan SARS-CoV-2 virus and the Omicron BA.5, BA.2.75, BF.7, BQ.1.1, XBB.1.5, EG.5.1.1, and BA.2.86 lineages for SN2 (A), SN3 (B), SP2 (C), and SH (D). The number, percentage, GM-IC₁₀₀ (black lines and colored bars), fold change, and statistical significance of nAbs are denoted on each graph. Reported fold change and statistical significance are in comparison with the Wuhan virus. Technical duplicates were performed for each experiment. (E–H) Doughnut charts show the frequency of nAbs retaining neutralization (Neut.), ADCP and ADCD against the SARS-CoV-2 Wuhan virus, and the XBB.1.5 and BA.2.86 variants for SN2 (E), SN3 (F), SP2 (G), and SH (H). A nonparametric Mann-Whitney t test was used to evaluate statistical significances between groups. Two-tailed p value significances are shown as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 2

Binding and neutralization activity to alpha and beta h-CoV (A) The bar graph shows the percentage of nAbs binding to SARS-CoV-2 (light gray), SARS-CoV-1 (pink), OC43 (light blue), 229E (light brown), and HKU-1 (orange) for all donors tested in the SH cohort. (B) Scatter dot charts show the neutralization potency, reported as ND₅₀ (ng mL⁻¹), of nAbs tested against SARS-CoV-1, 229E, HKU-1, and OC43. The number, percentage, and GM-ND₅₀ (black lines and colored bars) are denoted on each graph. Technical duplicates were performed for each experiment.

Figure 3

Distribution of RBD and NTD-targeting nAbs against Omicron variants (A–D) Pie charts show the distribution of cross-neutralizing nAbs based on their ability to target class 1/2 (blue), class 3 (orange), and class 4 (dark green) regions on the RBD, as well as non-competing nAbs (gray) and NTD-targeting nAbs (cyan). Dot charts show the neutralization potency, reported as IC₁₀₀ (ng mL⁻¹), of nAbs against the Wuhan virus and the Omicron BA.5, BA.2.75, BF.7, BQ.1.1, XBB.1.5, EG.5.1.1, and BA.2.86 variants observed in the SN2 (A), SN3 (B), SP2 (C), and SH (D) cohorts. The number and percentage of nAbs are denoted on each graph. (E) Radar plots show the frequency of nAbs retaining neutralization, ADCP, and ADCD activities against SARS-CoV-2 Wuhan, XBB.1.5, and BA.2.86. (F and G) Representation of the SARS-CoV-2 RBD of XBB.1.5 (F) and BA.2.86 (G). In dark red are highlighted the mutations present on the RBD for both variants. Boxed mutated residue labels indicate unique mutations for the specific variant. Class 1/2, 3, and 4 epitope regions are highlighted in blue, orange, and green, respectively.

Figure 4

SH germline expansion and characterization (A) Network plot shows the clonally expanded antibody families in SP2 and SH. Centroids and nAbs from SP2 and SH groups are shown in gray, pink, and dark red, respectively. Clusters and expanded clones are highlighted in gold and dark red, respectively. (B and C) The violin plots show the V gene somatic mutation frequency of germ lines expanded from SP2 to SH (B) or found exclusively in SH (C). The number of nAbs for each germ line and fold change are denoted on each graph. Violin plots show the median of V gene mutations. A nonparametric Mann-Whitney t test was used to evaluate statistical significances between groups. Two-tailed p value significances are shown as ^∗p < 0.05 and ^∗∗∗∗p < 0.0001. Not significant p values were denoted on the figure as "NS." (D) Bar graphs show the distribution of nAbs binding the S protein trimer (light gray), NTD (cyan), and RBD (dark gray) for expanded germ lines. The number of nAbs per each germ line is denoted on the graph. (E) The heatmap shows the IC₁₀₀ of predominant germ lines expanded from SP2 to SH (left) or found exclusively in SH (right).

Figure 5

B cell repertoire and functional characterization of predominant germ lines (A) Heatmaps and alluvial plots display the antibody IGHV;IGHJ gene rearrangements frequency for each single donor and for pulled nAbs, respectively, for SN3 (left) and SH (right). In the alluvial plots, the top five shared V-J gene rearrangements shared between SN3 and SH were highlighted. Selected germ lines were highlighted in light purple, green, dark green, gold, and brown for IGHV1-58;IGHJ3-1, IGHV1-69;IGHJ3-1, IGHV1-69;IGHJ4-1, IGHV3-53;IGHJ6-1, and IGHV3-66;IGHJ6-1, respectively. Box with black borders in the stratum identify the top five germ lines expanded exclusively in SH. (B) Heatmaps show the neutralization activity for the five most shared germ lines between SN3 and SH against BA.5, BA.2.75, BF.7, BQ.1.1, XBB.1.5, EG.5.1.1, and BA.2.86. (C) Radar plots describe the neutralization, ADCP, and ADCD activities of predominant germ lines shared between SN3 and SH against Wuhan, XBB.1.5, and BA.2.86. The percentages of functionality for neutralization, ADCP, and ADCD are reported within each radar plot.

Figure 6

Characterization of SN3-SH shared germ lines and SH-exclusive germ lines (A) The violin plots show the V gene somatic mutation frequency of IGHV1-24;IGHJ6-1, IGHV1-58;IGHJ3-1, IGHV3-53;IGHJ6-1, IGHV3-66;IGHJ4-1, and IGHV3-66;IGHJ6-1 gene-derived nAbs. The number of nAbs for each germ line and fold change are denoted on each graph. Violin plots show the median of V gene mutations. A nonparametric Mann-Whitney t test was used to evaluate statistical significances between groups. Two-tailed p value significances are shown as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.0001. Not significant p values were denoted on the figure as "NS." (B) The heatmap shows the IC₁₀₀ of germ lines found in SH but not in SN3 against all SARS-CoV-2 variants tested in this study. (C) The violin plot shows the V gene somatic mutation frequency of selected germ lines found exclusively in the SH cohort. The number of nAbs for each germ line is denoted on each graph. Violin plots show the mean of V gene mutations.