Selection for VRC01-class IgLs by iv8 in heavy-chain knock-in mice. (A) CDRL3 lengths in amino acids of BCRs from single-cell sorted IgG1⁺ GC B cells (B220⁺CD95⁺CD38⁻IgG1⁺) from two control-injected (iv1 Fab-2W1S nanoparticles [np], which bind glVRC01 and glNIH45-46 but not gl3BNC60) and two iv8 Fab-2W1S np-injected 2W1S-primed 3BNC60 HCgl mice obtained 14 d after injection. Lines indicate arithmetic means; error bars indicate SEM. (B) Pie charts indicate B cell clones from sequences obtained in A. Each color represents a clone. Light gray indicates single sequences obtained from nonclonal B cells. Numbers in the middle of the pie chart indicate the total number of sequences analyzed. (C) Representative monoclonal antibodies corresponding to the BCR sequences determined in B (Ab1–Ab5) were tested in ELISA against iv8 (left), 426cTM₄ΔV1-3 (middle), or 426cTM₄ΔV1-3 CD4bs-KO (right). Graphs are representative of two independent experiments. (D) Graph indicating CDRL3 lengths in amino acids of BCRs from single-cell sorted naive and memory B cells purified on the basis of 426c TM4ΔV1–3 antigen binding. Memory cells (B220⁺IgM⁻CD38⁺IgG1⁺) were obtained from 2W1S-primed 3BNC60 HCmt mice injected with iv8 Fab-2W1S nanoparticles 42 d after injection. Lines indicate arithmetic means, and error bars indicate SEM. (E) Pie charts (visualized as in B) indicate sequenced B cell clones obtained in D. (F) Representative monoclonal antibodies corresponding to the BCR sequences determined in E (Ab6–Ab10), were tested by ELISA for binding to iv8 (left), 426cTM₄ΔV1-3 (middle), or 426cTM₄ΔV1-3 CD4bs-KO (right). Graphs are representative of two independent experiments. 3BNC60-MT, 3BNC60-SI, and 3BNC60-GL mAbs are included as controls, and ZIKA004 and PGT121 are included as negative mAb controls in C and F. Statistics were calculated using the Mann–Whitney U test in A and D. **, P ≤ 0.01; ***, P ≤ 0.001. LC, light chain.