Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2

Abstract

During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

Figure 1.. COVID-19 patient IGH repertoires show early and extensive class-switching to IgG and IgA subclasses without significant somatic mutation.

Points indicate B cell clonal lineages, with the position denoting the clone’s isotype (panel column), human healthy control (HHC) or patient ID (panel row), IGHV gene (x-axis, with IGHV gene in the same order and position in panels, but not listed by name due to space constraints), and CDR-H3 length in amino acids (AA) (y-axis within each panel). The point color indicates the median IGHV SHM frequency for each clone and the size indicates the number of unique reads grouped into the clone. Points are jittered to decrease over-plotting of clones with same IGHV gene and CDR-H3 length. Patient label colors indicate sample IgG seroconversion (blue) or seronegative (red) for the displayed sample with the number following the patient ID corresponding to days post symptom onset. The final four rows of panels show the IGH repertoire changes within a single participant (7453) prior to and after seroconversion.

Figure 2.. IGH repertoire signatures of SARS-CoV-2 infection.

(A) Fraction of unmutated (<1% SHM) B cell lineages for each isotype subclass grouped by seroconversion status (top panel) or plotted by days post symptom onset (DPSO, bottom panel). Colors indicate patient sample serology: not tested (NT, purple), seronegative (red), and seropositive (blue) and are plotted specific to the isotype tested. Points are shown for all COVID-19 samples, whereas only outliers are displayed for the 114 healthy human controls (HHCs). Differences between the seropositive group and HHC was tested using two-sided Wilcoxon–Mann-Whitney (for patients with more than one sample, the mean value of these was used). (B) Distribution of clone percentage SHM plotted as kernel density for clones detected at multiple time points from patients 7453 and 7455. Lines are colored by DPSO. (C) Mean IGHV SHM percent for each isotype subclass observed for the IgG seronegative patient samples (top), IgG seropositive samples (middle) or HHCs (bottom). IGHV order is based on the 20 most common IGHV genes in IgM in the patients and isotypes are plotted by their chromosomal ordering. The plot axes were chosen to show the box-whiskers on a readable scale; rare outlier points with extreme values are not shown but were included in all analyses. (D) Heatmap of patient IGHV gene SHM for seroconverted and non-seroconverted samples compared to HHC using paired Wilcoxon tests with Bonferroni correction for multiple hypothesis testing. The color scale encodes the significance level and whether the SHM was higher (blues) or lower (reds) in COVID-19 relative to HHC. (E) Longitudinal SHM for each isotype subclass for COVID-19 patients are plotted binned by DPSO. Points are colored for each sample’s seroconversion status and boxplots summarize median and interquartile ranges. (F) Mean CDR-H3 length (top panel) and mean CDR-H3 hydrophobicity (bottom panel), COVID-19 patient samples grouped by expanded clones (white) or non-expanded clones (light grey), and total clones from HHC (dark grey). Differences between the expanded/non-expanded groups and HHC were tested using one-way ANOVA with Tukey’s HSD test. (A), (D), and (F) ***p-value < 0.001; **p-value < 0.01; *p-value ≤ 0.05; NS: p-value > 0.05.

Figure 3.. Convergent IGH sequences between COVID-19 patients and to reported antigen-specific IGH correlate with SARS-CoV/SARS-CoV-2 plasma cross-reactivity.

(A) The distribution of convergent clusters among patient samples (top panel). The sample distribution is indicated by the lines and dots with the number of clusters sharing that sample distribution indicated by the vertical histogram bars. The total number of convergent clusters identified in each sample is indicated in the histogram to the left of the plot. (bottom panel) Lineages belonging to convergent clusters (y-axis) shared across four or five patients (columns, four-patient clusters are highlighted in light green, five-patient clusters in dark green) are plotted by the expressed isotype (x-axis). Fill color indicates the average SHM and point size shows the number of unique reads. (B) Distribution of the number of convergent clones identified in two to eight HHC subjects, determined using 100 permutations. The histogram shows the distribution of convergent clones shared by a given number of HHC samples each time. The black dashed line is the mean value, and the wide red dashed line is the number of convergent clones shared among the same number of COVID-19 participants. (C) ELISA assay results for human IgG1 mAb binding to SARS-CoV-2 spike ectodomain protein, spike S1 domain, or RBD. Negative control mAbs 6–1-C11,D7,E7,F6, and F10 are overplotted in black. The SARS-CoV-2 RBD-binding mAb CR3022 (ter Meulen et al., 2006; Tian et al., 2020) was used as positive control. Starting concentration for mAbs was 100 ug/mL save for CR3022, which started at 0.506 ug/mL. (D-G) Sequence logos of CDR-H3 AA residues from anti-SARS-CoV-2 (D, see also Figure S4) anti-SARS-CoV/CoV-2 cross-neutralizing (E) or anti-SARS-CoV (F-G) convergent IGHs. For each set of convergent IGH the sequence logo and alignment for the reported antigen-specific CDR-H3 is shown at the top, sequence logos for clones from each patient are aligned below (colored black where they match a conserved residue in the reported CDR-H3, colored for non-conserved as depicted in the alignment, or gray if no match). To the side the read count per patient that contributed to the sequence logo, by isotype, is graphed. The SHM frequency for the dominant isotype is shown after the convergent IGH label. (H) Anti-SARS-CoV IgG ELISA detection in plasma samples from COVID-19 patients. Plasma samples were analyzed for the presence of anti-SARS-CoV spike RBD-binding IgG antibodies in the latest sample timepoint available for each patient. A SARS-CoV-2 pre-pandemic sample pool from healthy blood donors was used as a negative quality control (QC) as well as a positive control for SARS-CoV RBD (mAb CR3022) (ter Meulen et al., 2006). The dotted line denotes the cut-off value for seroconversion. Assays were performed in duplicate and mean OD values are shown.