Affinity maturation and light-chain-mediated paratope diversification anticipates viral evolution

Figure 1.. IGHV3-53/66 antibody epitope and usage frequencies (A) Superposition (with respect to RBD) of antibody:Wuhan RBD complexes from the PDB, grouped by VH gene usage. Heavy chains are shown in red, light chains are shown in orange, and RBD is shown in black. (B) Shared epitope of 51 IGHV3-53/66 antibodies exhibiting “canonical” binding. Percentages indicate the fraction of antibodies that contact all RBD residues within the highlighted VH and VL regions. RBD positions are colored based on sequence variability across variants of concern. (C) IGHV gene usage from aggregated BCR datasets: naive,39–41 HIV-1 (CATNAP database42), Influenza (HA) (Wu Lab43), SARS-CoV-2 (CoV-Abdab44), and Cao dataset (Cao23,29,37,38). Data are shown as shaded regions corresponding to IGHV gene frequencies across aggregated datasets. For “naive” and “Cao” datasets, data were pooled across multiple studies, and frequencies were calculated across the pooled data. Dataset sample sizes are noted on the right side of frequency plots. For each category, our goal was to estimate overall IGHV frequencies across aggregated observations, rather than investigate frequency differences between individual donors or studies.…

Figure 2.. Isolation and neutralization testing of RBD2 antibodies from Wuhan spike mRNA vaccinees (A) Study cohort and antibody isolation. Early pandemic SARS-CoV-2 mRNA vaccinees received 2 standard vaccine doses followed by a homologous booster dose. Antibodies were cloned from RBD-specific memory B cells collected across 3 time points. Expressed antibodies were grouped by epitope via competition ELISA. (B) RBD2 binders were further characterized for neutralization across a panel of SARS-CoV-2 variants by pseudotype neutralization assay.

Figure 3.. Features of IGHV3-53/66 antibodies isolated from Wuhan spike mRNA vaccinees 11 IGHV3-53/66 monoclonal antibodies (mAbs) were profiled for affinity for Wuhan RBD, light-chain pairing, neutralization breadth, and VH segment somatic hypermutation. Antibodies above the dashed red line neutralize all tested strains of SARS-CoV-2 through XBB. Red boxes highlight features with apparent enrichment among poorer neutralizers. Sequence numbers are based on the IMGT numbering scheme.49 Germline amino acids across CDRH1 and CDRH2 and common amino acids across CDRH3 are highlighted in light blue on the top row above mAb sequences. Blank boxes across mAb sequences represent amino acid agreement with germline/common residues. Amino acids highlighted in red are previously identified “hallmark” SHMs shown to have positive impacts on neutralization breadth and affinity to Wuhan RBD.

Figure 4.. Comparison of V30V4 to Omi-3 and profiling of V30V4 “hallmark” SHM contacts (A) Sequence comparison of 2 similar antibodies: V30V4, isolated from our cohort of Wuhan mRNA vaccinees, and Omi-3, isolated from an Omicron BTI sample. Sequence numbers are based on the IMGT numbering scheme.49 Hallmark SHMs for IGHV3-53/66 antibodies are highlighted in red. Additional SHMs are highlighted in orange. (B) Comparison of atomic contact counts at positions across the RBD for V30V4 and Omi-3. Data are represented as color-coded bars denoting the total number of atomic contacts at RBD positions for V30V4 and Omi-3. 3 RBD positions with the greatest difference in atomic contacts between V30V4 and Omi-3 are highlighted with red, dashed boxes. V30V4 contacts are with Wuhan RBD, and Omi-3 contacts are with BA.1 RBD. (C) Structures of V30V4 and Omi-3 superimposed with respect to RBD. Residues at mutation sites distinguishing the Wuhan and BA.1 RBD structures and involved in antibody interfaces are visualized. (D and E) Positional comparison between V30V4 and Omi-3 contact residues (superposition with respect to RBD) is visualized for (D) VH, and for (E) VL.

Figure 5.. Interface comparisons between V30V4 and P5A-3A1, a fully germline IGHV3-53:IGKV3-20 antibody (A) Interaction surface of Wuhan RBD with both V30V4 and P5A-3A1 (PDB: 7D0C) at positions of highly convergent IGHV3-53/66 SHM (“hallmark” SHMs). (B) Pairwise breakdown of Wuhan-RBD-interacting positions and hallmark SHM sites for V30V4 and P5A-3A1.

Figure 6.. Sequence analysis of IGHV3-53/66 antibodies by neutralization breadth and exposure categories (A) Schematic detailing analysis of sequenced IGHV3-53/66 antibodies from the Cao dataset29,37 and subsequent comparison to Wuhan-mRNA-derived IGHV3-53/66 antibodies from this study. Antibodies with CDRH3 length > 15 were excluded from analysis. IGHV3-53/66 antibodies from the Cao dataset were divided into groups based on neutralization breadth or exposure. For each group, significantly enriched positional amino acids were determined by Fisher’s exact test (p < 0.05). Significant positions were then used to compare BTI sequences from the Cao dataset to Wuhan-mRNA-derived IGHV3-53/66 antibodies from this study. Enriched positional amino acids are noted in color-coded boxes. Germline residues are denoted by capital letters, and mutations are denoted by lower-case letters. Empty boxes denote a lack of a specifically enriched residue at that position. A dash denotes enrichment for “no amino acid” at that position. The overall amino acid diversity at each position for each group is illustrated by the logo plot. CDR positions are noted in light blue boxes. Sequence numbers are based on the…

Figure 7.. Omicron exposure selects for favorable light-chain pairing for IGHV3-53/66 antibodies (A) Light-chain gene frequencies for IGHV3-53/66 antibodies from the full Cao dataset23,29,37,38 across neutralization and exposure categories (n = 68 for Wuhan neut., 174 for XBB neut., 88 for Wuhan exposed, and 332 for BTI). (B) Superposition (with respect to RBD) of average backbone structures of light-chain-defined groups of available IGHV3-53/66 antibody:Wuhan RBD PDBs. Structures were generated by calculating the average coordinate for each common backbone atom across available PDB structures of IGHV3-53/66 antibodies paired with common light chains and complexed with Wuhan RBD. Rotated views of heavy and light chains highlight spatial shifts between light-chain-defined groups. (C) Plotted averages of atomic contacts with RBD positions by light-chain-defined groups of IGHV3-53/66 antibodies from available PDBs (error bars represent standard error of the mean). Asterisks denote significant differences determined by Mann-Whitney test (p < 0.05). Top rows illustrate positions of Omicron mutations (“omi mut”), escape mutations (“escape”, based on DMS data29), ACE2 binding (“ACE2”), and contact…