Figure 6. Dose escalating immunization strategy results in higher nAb titers.

(A) Immunization and sampling schedule. Groups were immunized and sampled contemporaneously.

(B) Total B_GC frequencies over time. Data from bolus Gp2 (Fig 1) are included in these analyses (grey circles).

(C) Cumulative B_GC cell response to the first immunization, calculated between w3-7 [AUC].

(D) Env trimer-specific B_GC cells frequencies over time.

(E) Cumulative Env trimer-specific B_GC cell responses to one immunization.

(F) Total GC-T_FH cell frequencies over time.

(G) Cumulative GC-T_FH responses to one immunization (AUC of F).

(H) Env-specific CD4⁺ responses after one immunization.

(I) Ratio of Env⁺ B_GC to Env-specific GC-T_FH cells at w5, calculated as Env⁺ B_GC (% B cells)/ Env-specific GC-T_FH (% CD4⁺).

(J) BG505 Env trimer binding IgG titers over time.

(K) Autologous BG505 nAb titers over time.

(L) Peak BG505 nAb titers after three immunizations.

(M) Composite 3D reconstruction of Env trimer bound to Fabs isolated from all animals after two immunizations. 3D EM reconstructions from individual animals can be seen in Figure S8A.

(N) Correlation between peak GC-T_FH and B_GC cell % during 1^st immunization from both studies.

(O) Correlation between Env⁺ B_GC cells (% B cells) and peak neutralization titers. Env⁺ B_GC cell values are from w7 or peak frequencies during 1^st immunization. Peak nAb titers are after 2^nd immunization.

Serological data represent GMT ± geometric SD. Cell-frequency data represent mean ± SEM. Statistical significance was tested using unpaired, two-tailed Mann-Whitney U tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S7–8 and Data S2.