Figure 3. Mutations in loop D rendered the Env mutants more sensitive to CH103 lineage bnAb neutralization with enhanced Env binding.

(A) Alignment of nine amino acids in loop D of Env. The amino acid sequences from week 4 to week 160 were compared to the CH505 T/F sequence. The number and frequency of each variant in loop D are shown at the right of the alignment. The two asparagines (N) whose side chains interact with the CH103 light chain are indicated in yellow. The loop D mutations that occurred early or predominated are indicated in red, and Env mutants containing those mutations are indicated by red arrows. (B) Neutralization susceptibility of the loop D mutants by the CH103 lineage mAbs. Heatmap analysis was performed for the neutralization data of all CH103 lineage mAbs (column) against the CH505 T/F virus and the loop D variants (row). The neutralization potency (IC₅₀) is shown in different colors as indicated; from white (>50 µg/ml) to dark red (0.12 µg/ml). (C) Neutralization activity of the nAb CH235 were compared to that of the bnAb CH103 against the CH505 T/F virus and loop D mutants. (D) The fold difference in binding to loop D mutant Envs versus the CH505 T/F Env by both CH103 and CH235 lineage Abs. Seven loop D mutant Envs (M6^V281A, M10^V281G, M11^N279D/V281G, M7^{E275K/N279D/V281S}, M8^N280S/V281A, M9^{E275K/N279D/V281G} and M21^N280T/V281A) and the CH505 T/F Env were serially diluted and the log area under the curve (AUC) values for all members of the CH103 and CH235 lineage mAbs were determined by ELISA. The fold difference in log AUC between each loop D mutant Env versus the CH505 T/F Env is shown. See also Figures S3, S4 and S6; Tables S2, S3 and S6.