Figure 4: A single priming immunization with NPDP19-KLH induces antibodies with key CIS43-like mutations N52K and K59R (IGHV) and H89Q and T94S (IGKV).

Antigen-specific splenic CD95⁺CD38^low CD45.2⁺ H^iGL-CIS43κ^iGL-CIS43 B cells were sorted at day 13 and 28 post immunization (DPI) for single-cell BCR sequence analysis (see Figure S4E).

A. Clonal lineage trees generated from bioinformatically assembled heavy-light chain sequence pairs. Branch length is representative of sequence distance.

B. Total nucleotide (nt) and amino acid (aa) mutations acquired in iGL-CIS43 HCs at 13 and 28 DPI. The red line indicates the median number of mutations.

C. Total nt and aa mutations acquired in the iGL-CIS43 LC at 13 and 28 DPI. Red line indicates median number of mutations.

D. Accumulation of CIS43-like aa mutations shown for human HCs (left) and LCs (right) isolated at 13 and 28 DPI. Red (Briney et al., 2019) and black (Soto et al., 2019) stair step indicate calculated antigen-agnostic mutations. The numbers inside each square indicate the number of sequences that have the total aa mutations shown on the x-axis and the CIS43-like aa mutations shown on the y-axis.

E. Hotspot analysis shows frequency of observed HC mutations per residue at 13 (purple) and 28 (blue) DPI. HCDRs are highlighted in gray. Letters in red (only present in mature CIS43 HC) indicate key aa residues for the recognition of the junctional epitope. AA positions 52 and 58 were analyzed in (F). Kabat numbering was followed.

F. Distribution of select iGL-CIS43 B cell HC aa mutations in positions 52 and 58 over time.

G. Hotspot analysis shows frequency of observed LC mutations per residue at 13 (purple) and 28 (blue) DPI. LCDRs are highlighted in gray. Letters in red (only present in mature CIS43 LC) indicate key amino acid residues for the recognition of the junctional malaria epitope. AA positions 89 and 94 were analyzed in (H). Kabat numbering was followed.

H. Distribution of select iGL-CIS43 B cell LC aa mutations in positions 89 and 94 over time.

P values were calculated by unpaired Mann-Whitney test (panels B and C). ****P<0.0001.

See also Figure S3.