Naive and Memory B Cell BCR Repertoires in Individuals Immunized with an Inactivated SARS-CoV-2 Vaccine

Abstract

Background: The COVID-19 pandemic has spurred a global race for a preventive vaccine, with a few becoming available just one year after describing this novel coronavirus disease. Among these are inactivated virus vaccines like CoronaVac (Sinovac Biotech), which are used in several countries to reduce the pandemic's effects. However, its use was associated with low protection, particularly against novel virus variants that quickly appeared in the following months. Vaccines play a crucial role in activating the immune system to combat infections, with Memory B-cells being a key part of this mechanism, eliciting protective neutralizing antibodies. This work focused on studying B-cell memory repertoire after two consecutive doses of CoronaVac.

Methodology: Memory B-cells were isolated from five CoronaVac vaccinated and five pre-pandemic individuals and subsequently stimulated in vitro before high-throughput Illumina sequencing of the Heavy Chain Variable repertoire.

Results: We observed a shift in the VH repertoire with increased HCDR3 length and enrichment of IGVH 3-23, 3-30, 3-7, 3-72, and 3-74 for IgA BCRs and IGHV 4-39 and 4-59 for IgG BCRs. A high expansion of IgA-specific clonal populations was observed in vaccinated individuals relative to pre-pandemic controls, accompanied by shared IgA variable heavy chain (VH) sequences among memory B cells across different vaccine recipients of IgA clones was also observed in vaccinated individuals compared to pre-pandemic controls, with several IgA VH sharing between memory B cells from different vaccines. Moreover, a high convergence was observed among vaccinees and SARS-CoV-2 neutralizing antibody sequences found in the CoV-abDab database.

Conclusion: These data show the ability of CoronaVac to elicit antibodies with characteristics similar to those previously identified as neutralizing antibodies, supporting its protective efficacy. Furthermore, this analysis of the immunological repertoire in the context of viral infections reinforces the importance of immunization in generating convergent antibodies for the antiviral response.

Keywords: BCR; CoronaVac; SARS-CoV-2; VH repertoire; memory B cell.

Figure 1

Stimulation of memory B cells (MBC) by PBMC culture with IL-2 and R848. (a) The presence of anti-SARS-CoV-2 IgM, IgG, and IgA in the sera from vaccinated individuals was evaluated by ELISA. The sera were collected 30 days after the second dose of CoronaVac. The dashed line represents the limit of detection of specific binding. PBMC were cultured for seven days with IL-2 and R848 or without stimuli (NS): (b) Gate strategy for analysis of PBMC differentiation in MBC (CD27⁺CD19⁺) and specific MBC (IgG⁺CD27⁺CD19⁺). Memory B cells are analyzed within the gate of lymphocytes, and IgG⁺ memory B cells are analyzed within the CD27⁺CD19⁺ cell gate. Percentage of (c) MBC, (d) MBC-IgG⁺, and (e) MBC-IgA⁺ cells concerning total PBMC. (f) Gate strategy for analysis of PBMC proliferation by flow cytometry. The proliferation of total PBMC (g) and MBC (h) after culturing PBMC for seven days in the presence of IL-2 (5 ηg/mL) and R848 (1 µg/mL), compared to proliferation after cultivation in the presence of DMSO (0.5%). Data are presented as Mean ± S.D. For each analysis, N = 5 individuals per group and statistical differences were identified by unpaired t-test, with p values (p < 0.001 ***) considered significant.

Figure 2

Levels of IgM, IgG, and IgA secreted in cell culture supernatants (A) Quantification, by ELISA, of IgM, IgG, and IgA secreted after 3 (T3), 5 (T5) and 7 (T7) days of PBMC culture with the stimuli, for each individual. (B) anti-SARS-CoV-2 IgM, IgG, and IgA, secreted after 3, 5, and 7 days of culture with stimuli. Each point represents Mean ± S.E.M. Statistical differences were analyzed by one-way ANOVA with post hoc Tukey test, and p values of p < 0.05 *, p < 0.01 **, p < 0.001 *** were considered significant.

Figure 3

Expansion of the B cells after PBMC culture. (A) The difference in the B cell percentages, in relation to the PBMC population, before culture (initial time—T0, light colored dots) and after seven days of PBMC culture with the stimuli (T7, dark colored dots) (p = 0.0002). (B) Difference in memory B cell count before and after culture (p = 0.0001). (C) Ratio between CD19⁺CD27⁺ B cells and CD19⁺CD27⁻ B cells after culture (p = 0.0079; Data are expressed as Mean ± S.E.M). For each analysis, N = 5 individuals per group and each dot is an individual. Statistical differences were calculated by unpaired t-test (p < 0.01 **; p < 0.001 ***).

Figure 4

Compositional relationships across immunoglobulin isotypes. A comprehensive analysis of antibody repertoires in vaccinated individuals using multiple visualization techniques to elucidate the compositional relationships within and between different immunoglobulin isotypes. (a) t-SNE plots illustrating antibody repertoire clustering by CDRH3 size and identity. Comparison between vaccinated and pre-pandemic individuals, with IgG samples colored by group. Clusters containing only vaccinated individuals are circled in blue. (b) t-SNE plot illustrating antibody repertoire among vaccinated individuals, differentiated by immunoglobulin classes (IgA, IgG, and IgM), clustered by class. (c,d) Heatmaps representing V gene composition. (c) Compares V gene usage between vaccinated and pre-pandemic individuals, while (d) focuses on V gene composition among vaccinated individuals only.

Figure 5

Different IGHV call distribution in response to CoronaVac vaccine. (a,b) by V gene families. (a) Between IgG of vaccinated and pre-pandemic individuals. (b) Among antibody classes IgA, IgG, or IgM. (c,d) By V gene. (c) Between IgG of vaccinated and pre-pandemic individuals. (d) Among Antibody classes IgA, IgG or IgM. Bars show mean values ± standard error of the mean. p < 0.05 *; p < 0.01 ** (Mann–Whitney U test).

Figure 6

Diversity analyses and clonal network in immunoglobulin samples. (a) The diversity curve is calculated over a range of order q values to generate a smooth curve. This is the Hill curve where q = 0 indicates Richness, q = 1 indicates the Shannon index and q ≥ 2 indicates Rényi entropy. The Rényi entropy generalizes Shannon entropy to values of q Greater or equal 2. (b) Shannon diversity index for individuals PCV-03, 04, 11, and 15 across IgA, IgG, and IgM. The statistical significance of diversity index “D” differences between groups is assessed by constructing a bootstrap delta distribution for each pair of unique group values. p < 0.001 ***. (c) Igraph network plot of 1000 sampled clones from PCV-04 for IgA, IgG, and IgM, and control HC-07 for IgG. Each point represents a clone, and connections represent clonotypes (see Supplementary Figure S9 for all patients). (d) Distribution of mutation numbers in sequences relative to the germline, comparing somatic hypermutation (SHM) through the ratio of replacement (R) to silent (S) mutation frequencies, with patients grouped by vaccination status. (See Supplementary Figure S10 for each patient density curve). (e) CDRH3 Length Distribution Across IgG Isotypes in Vaccinated and pre-pandemic Individuals; Mann–Whitney U test with Benjamini–Hochberg correction. * p < 0.05; *** p < 0.001.

Figure 7

Convergent antibody responses among individuals vaccinated with CoronaVac and the CoV-abDab dataset. Similar V and J gene usage, CDRH3 length, and ≥80% sequence identity defined clonotypes. (a) For detecting convergent similar antibodies between vaccinated individuals (Same method as used in Table 1). The circular diagram illustrates the convergence of antibody responses. Each segment around the circle represents a vaccinated individual group or CoV-abDab antibody. The lines within the circle show the connections where the antibody responses match, with different colors representing different antibody groups. For connections, green represents IgA, cyan represents IgG, yellow-brown represents IgM, red represents any connections with CoV-abDab, and black represents other intersections. (b) The pie chart shows the v-gene distribution of all connections with Cov-AbDab. (c) The panel shows the distribution of intersection sizes of similar antibody responses among groups. The vertical bar chart represents the number of antibodies (y-axis) shared between samples. The bottom matrix highlights the specific intersections of these convergent responses. The upset plot intersections sizes are displayed in descending order of size and increasing number of intersections. Connections within the same patient or different antibody classes are not shown. For intersections, green represents IgA, cyan represents IgG, yellow-brown represents IgM, red represents any intersection with CoV-abDab, and black represents other intersections. (d) The bar plot represents each individual’s exclusive intersections between different antibody classes.