Figure 1. N332-GT5 trimer protein can specifically and efficiently activate BG18^gH B cells.

(A) Gating strategy to identify epitope-specific (N332-GT5⁺/N332-GT5-KO⁻) B cells in BG18^gH and WT mice. (B) Frequency of epitope-specific B cells in non-immunized BG18^gH (n=12) and WT (n=5) mice. Welch’s t-test applied; error bars are SD. ****p < 0.0001. (C) Distribution of VH and VL genes in epitope-specific (GT5⁺KO⁻) naive B cells in naive BG18^gH mice. (D) SPR dissociation constants (KD) for N332-GT2 and GT5 trimer binding to epitope-specific Fabs derived from naive BG18^gH cells. Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SEM; n=7. (E) Schematic of BG18^gH and WT B cell adoptive transfer recipients immunized with GT5 and MD39 trimer protein. Data was collected from one experiment. (F) GC B cells, CD45.2⁺ B cells in GC and epitope-specific (GT5⁺KO⁻) CD45.2⁺ cells as a percentage of total B cells in each condition 12 dpi WT (GT5 trimer), BG18^gH (GT5 trimer), WT (MD39 trimer), BG18^gH (MD39 trimer); n=6 in each group. 2-way ANOVA with Bonferroni multiple comparison test applied; bars indicate mean + SD, ^nsp >0.05, ***p < 0.001, ****p < 0.0001. (G) Schematic showing immunization with GT5 trimer protein. Samples were collected at day 7, 14, 21 and 49 for analysis. At least two independent experiments were performed, and representative data from one is shown. (H) Gating strategy showing GC B cells, CD45.2⁺ B cells in GC, CD45.2⁺ GT5 binders in GC and epitope binding specificity 7, 14, 21 and 49 dpi. (I) Frequency of GC B cells, CD45.2⁺ GC B cells, GT5⁺KO⁻ cells in GC CD45.2⁺ cells gated as in (H) and CD45.2⁺GT5⁺KO⁻ cells in total B cells 7, 14, 21 and 49 days after immunization with GT5 trimer protein. Each symbol represents a different mouse. Bars indicate mean + SD. n = 3 (day 49); others n=6.