AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans

Abstract

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4⁺ T cell and memory B cell responses. Antigen-specific CD4⁺ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Figure 1.. Study design and serum antibody responses following vaccination with CoVLP alone, CoVLP+AS03 and mRNA-1273.

(A) Demographic information for study participants. (B) Schematic of the study design. Serum was analyzed at days 42 and 201 for subjects in the plant-based vaccine trial, and days 56 and 195–273 for mRNA-1273 vaccinees. PBMCs were collected at days 0, 21, 42 and 201 for subjects in the plant-based vaccine trial, and days 0, 56, 150 for mRNA-1273 vaccinees. Monoclonal antibodies were generated from memory B cells derived from days 42, 201 for subjects in the plant-based vaccine trial, and days 56, 150 for Moderna vaccinees. (C and D) Serum binding IgG responses at day 42 (plant-based vaccine) and day 56 (mRNA-1273) (C) and day 201 (plant-based vaccine) and day 195–273 (mRNA-1273) (D) as measured by MSD. Geometric mean titers (GMT) represent the geometric mean of the titers against WT strain. Ratios represent the fold-drop in titers between the WT strain and variant presented. Statistical comparisons were performed between each variant versus WT strain GMTs, and Dunn’s multiple comparisons test was used for statistical analysis. (E and F) Serum neutralizing antibody responses as measured by live virus neutralization assays against WT (WA.1) (E) and BA.5 variant (F) of SARS-CoV-2. Tukey’s multiple comparisons test was used to perform statistical analysis. p-values denoted as *, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2.. AS03-adjuvanted CoVLP induces robust and persistent memory B cell responses similar to the mRNA-1273 vaccine.

(A) Frequency of RBD-specific memory B cells (RBD⁺Spike⁺) in the three groups of vaccinees at each timepoint (quantified as % of total CD20+ B cells). Dashed lines indicate individual responses, solid lines indicate median frequencies in each group. Wilcoxon matched-paired signed-rank test was performed for statistical analysis. Asterisks represent the results of statistical testing of the given timepoint compared to baseline (day 0). Asterisk colors represent the respective groups. (B) Isotypes of RBD-specific memory B cells, quantified by the percentage of each isotype within the RBD-specific memory B cells at early (top panel) and later (bottom panel) time points. (C) Percent mutation in heavy chain V genes of RBD-specific memory B cells. Statistical analysis was performed using Tukey’s multiple comparisons test comparing all groups to each other. (D) CDR3 region length in heavy chains (number of amino acids). Statistical analysis was performed using Tukey’s multiple comparisons test comparing all groups to each other. (F) VH gene usage in the ighv genes of RBD-specific memory B cells in CoVLP+AS03 and Moderna groups at early and durability timepoints. p-values denoted as *, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.. Enhanced T cell responses induced by CoVLP+AS03 correlate with memory B cell responses at later timepoints.

(A) Quantification of background subtracted CD40L+cytokine+CD4 T cell responses in vaccinees. Statistical analysis was performed using Wilcoxon matched-pairs signed-rank tests comparing the T cell response at any given timepoint with day 0 responses. (B) Polyfunctionality of CD4 T cells induced by CoVLP+AS03 vaccination at day 42 and mRNA-1273 vaccination at day 56. (C) Pearson correlation of CD40L+cytokine+ CD4 T cell responses at peak timepoint (day 42 for CoVLP+AS03, day 56 for mRNA-1273) with RBD-specific memory B cell responses at later timepoint (day 201 for CoVLP+AS03, day 150 for mRNA-1273). (D) Correlations of polyfunctional CD4 T cell responses at day 42 with frequencies of RBD-specific memory B cells at day 201 within the CoVLP+AS03 group. p-values denoted as *, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4.. AS03-adjuvanted CoVLP enhances the breadth of the immunoglobulins expressed in antigen-specific memory B cells at a clonal level.

(A) Schematic representing the numbers of single-cell sorted spike-specific memory B cells, paired productive VDJ sequences, and mAbs produced from each group at each timepoint. (B) Percentages of WT and BA.1 neutralizing mAbs in each group at all timepoints (pie charts). Individual timepoints shown below. PsVNA assays performed on all 230 mAbs against WT and BA.1. Statistical analysis was performed using Chi-squared test. (C) Pie charts showing neutralization capacity of 106 neutralizing mAbs against WT and BA.4/5 variants. Pie charts show percentage of WT and BA.5 neutralizing mAbs (red fraction) at all timepoints. Individual timepoints are shown below. (D, E, F) PsVNA IC₅₀ values against WT (D), BA.1 (E) and BA.5 (F) at different timepoints in each group. Statistical analysis within CoVLP+AS03 group was performed using Mann-Whitney U-tests comparing the IC₅₀ values between two timepoints. p-values denoted as *, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. Identification of memory B cell clonal families with increased neutralization breadth between day 42 and day 201 timepoints.

(A) Pie charts representing clonal expansion of memory B cells at day 42 and day 201 in the five individuals from AS03 group that had sequences isolated from both timepoints. Arcs around the pie chart along with the percentage value represent the frequency of expanded clones (B cells with more than 1 sequence in the clonal family). Unexpanded clones were colored white, expanded clones colored either grey or other colors. Grey clones represent expanded clones that were not shared between the two patients. Clones with other colors represent shared clones across the two timepoints in the same individual (same color identifies the same clonal family at both timepoints). (B) Bar plots representing shared and non-shared expanded clones in the four individuals from whom shared clonal families were found at both timepoints. Color scheme same as in (A). Numbers in slices represent the number of sequences obtained from that clonal family. (C) Table representing the four clonal families in four individuals with increased neutralization breadth between the two timepoints. IC₅₀ values for all variants are shown (ug/mL).