Figure 7. VRC01‐class precursor B cells exhibit SHM and VRC01‐like mutations 36 days after a single priming injection with eOD‐GT8 60mer.

Three CLK‐mouse lines were generated and subsequently immunized with eOD‐GT8 60mer in adoptive transfer/immunization system (precursor frequency: 1 in 10⁴). eOD‐GT8‐specific splenic IgG1⁺ or IgM⁻IgD⁻ B cells were single‐cell sorted at Day 36 post‐immunization for single‐cell PCRs.

1. Phylogenetic trees of CLK21, CLK09 and CLK19. These trees were generated using paired aminoacidic sequences solely isolated at Day 36. Single paired amino acid sequences were joined and aligned using MUSCLE (Price et al, 2010). Clonal lineage trees were generated using FastTree and a Jones Taylor Thornton model for AA evolution (Jones et al, 1992). The length of the branches reflects sequence distance.
2. SHM are detectable in both IGHV and IGLV at Day 36 post‐immunization with eOD‐GT8 60mer. nt represents “nucleotide”, aa represents “amino acid”.
3. Hotspot analysis. The quality of mutations was assessed via hotspots analysis for both heavy (left) and light (right) antibody chains. Weblogos were generated via publicly available online tools (https://weblogo.berkeley.edu/logo.cgi).
4. Mature bnAbs‐like aa VH1‐2 mutations in CLK B cells at Day 36. The red diagonal line indicates a 100% efficiency of VRC01‐class bnAb‐type VH1‐2 mutations. The black stair step indicates a calculated VH1‐2 antigen‐agnostic mutation distribution, which might include mutations that improve expression or stabilize Ab structure (Briney et al, 2016). Shaded area indicates that no mutation is positive in this region.