Figure 2. Elicitation of VRC01-class germline antibodies in V_H1-2 mouse model.

(A) Immunization Schema. (B) Sera binding to eOD-GT8 (solid line) and its CD4bs-knock-out (Δ) mutant (dashed line) assessed by ELISA (mean with standard deviation is shown). (C, D) Sorting and frequency of CD4bs-specific eOD-GT8⁺/ΔeOD-GT8⁻ splenic IgG+ B-cells. Statistical comparisons were performed using a two-tailed t-test. (E) Distribution of CDR L3 amino acid (aa) length of the cloned VH1-2 antibodies from CD4bs-specific B-cells. Mouse IDs are shown on top of each pie chart and the number of total sequences is shown at the center. VRC01-class germline antibodies were defined as antibodies containing a VH1-2 heavy chain and a light chain with a 5aa CDR L3 (in red). (F) Sequence conservation at each position of the 5aa CDR L3 in the cloned VRC01-class germline antibodies compared to known VRC01-class antibodies and VRC01. (G) Binding affinity of synthesized antibodies compared to VRC01 and its germline-V-gene-revertant (VRC01gl) as assayed with Biolayer Interferometry (BLI) Octet. Dashed line indicates the detection limit at 5μM. (H) Heavy and light chain paired sequencing of the B-cell repertoire of naïve and eOD-GT8 60mer-immunized mice. The bar represents the mean of the samples in C and H.