Fig. 2. Immunization of BG18^gH B cell adoptive transfer recipient mice with N332-GT2 Env nanoparticles.

(A) Gating strategy to identify epitope-specific (N332-GT2⁺⁺/N332-GT2-KO⁻) B cells in BG18^gH and WT mice. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in 3 mice in each model. (B) Frequency of epitope-specific B cells in non-immunized BG18^gH and WT mice. (C) Distribution of V_H and V_L genes in epitope-specific naive B cells in non-immunized BG18^gH mice. (D) Frequency of GC B cells (left) or CD45.2⁺ GC B cells (right) in four immunization conditions. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 6; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. (E) Frequency of CD45.2⁺ (left) or CD45.1⁺ (right) epitope-specific B cells in four immunization conditions. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 6; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. (F) Serum ELISA 50% equilibrium dilution values for N332-GT2 and N332-GT2-KO at day 14 after immunization for four immunization conditions. Each symbol represents a different mouse. Bars indicate geometric mean and geometric SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 5; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. Student’s t-test; ns, P > 0.05; *, P < 0.05; **, P < 0.01. Data in (A)-(F) are from one of three representative experiments with three or more animals in each group. (G) Distribution of V_H and V_L genes in epitope-specific GC (CD38^lowCD95⁺) B cells 8 and 42 days after immunization of BG18^gH B cell adoptive transfer recipient mice. (H) SPR dissociation constants (K_D) for N332-GT2 trimer binding to epitope-specific Fabs derived from naive B cells in non-immunized BG18^gH mice and GC B cells 8 and 42 days after immunization of BG18^gH B cell adoptive transfer recipient mice. Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SD. (I) Phylogenetic trees of BCR HCs isolated from epitope-specific CD45.2⁺ B cells 8 and 42 days after immunization with N332-GT2 NPs. Tree scale indicates the number of substitutions per site. (J) SPR dissociation constants (K_D) for the five highest affinity naive Fabs from (H) binding to the V1 loop-modified BG505 trimer (BG505_V1_mod), and for nine of the high affinity day 42 Fabs from (H) and five inferred-germline variants of the high-affinity day 42 Fabs (Day42.iGL) binding to V1 loop-modified trimers from BG505 and three other HIV isolates (SF162P3, AC10, AD8) as well as a BG505 trimer with a less modified V1 loop (BG505_7mut), a native-like trimer (BG505_MD39) and an epitope-KO trimer (N332-GT2_KO). Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SD. Dashed line indicates limit of detection. (K) Neutralization potency (IC50) against native (BG505 T332N) and V1 loop-modified (BG505-V1mod) pseudoviruses, for the BG18 bnAb, the five highest affinity naive Fabs from (H), five inferred-germline variants of the high-affinity day 42 Fabs (Day42.iGL), and five high affinity day 42 Fabs. Each IC50 is an average from two measurements. ND indicates not determined.