Figure 2. VRC01-class nAbs elicited after only four immunizations neutralize multiple heterologous glycan276-bearing viruses.

(A) Three-color sort identified VRC01-class antibodies with higher VH1-2 nucleotide mutation frequencies than identified by two-color sort. The scatter graph shows the VH1-2 nucleotide mutation frequencies of VRC01-class antibody heavy chains identified from the B cells of three mice: G1 post 4 (wk14) mouse 2404, G2 post 4 (wk14) mouse 2410 and G1 post 6 (wk22) mouse 2405, obtained by either a: two-color sort (solid symbols: sort for eOD-GT6⁺/ΔeOD-GT6⁻ cells) or b: three-color sort (open symbols: sort for BG505.SOSIP⁺/eOD-GT6⁺/ΔeOD-GT6⁻ cells). n, number of analyzed antibody heavy chains identified from each mouse with specified B cell-sorting method. The median and 95% confidence interval for VH1-2 mutation frequencies of each set of heavy chains are shown. Mann-Whitney test was performed for statistical analysis. ns, non-significant; ***, p<0.001; ****, p<0.0001.

(B) Heat map showing the Area Under the Curve (AUC) for ELISA-binding curves of selected VRC01-class antibodies (columns) isolated by three-color B cell sorting from post 4 mice to various Env antigens (rows). The name of each antibody, such as G1:2404-E12, is displayed under the corresponding column.

(C) Neutralization titers (IC₅₀) of selected VRC01-class antibodies isolated from G1 or G2 mice post 4 (wk14) or 6 (wk22) immunizations against a panel of 10 Tier II viruses of clade A-D. The presence (G) or absence (−) of N-linked glycans at residues 276, 460 and 462(3) for each virus is shown. The number in the parentheses following each antibody name indicates the number of sorted B cells expressing the same antibody or its clonal relative(s).

(D) The heavy chain sequences of the antibodies tested in (B). Dots indicate residues identical to the germline IGHV1-2*02.

See also Figures S2–S3.