HIV-1 Env-specific Ab responses and memory B cell repertoire. (A) Overview of the immunization regimen. Chinese origin rhesus macaques (n = 4, from one independent experiment) were inoculated i.m. six times at weeks 0, 4, 12, 24, 36, and 45 with 16055 Env NFL trimers formulated in the Matrix-M adjuvant. Blood samples were collected before the first inoculation and 2 wk after each inoculation (left). Measurements of the plasma neutralizing titers against the 16055 virus identified D20 as the best responder animal (right; representative data from two independent experiments). (B) Single memory B cell sorting 2 wk after the sixth immunization. Approximately 1,000 single Env-specific IgG⁺ memory B cells were sorted for isolation of Ab HC sequences. For a subset of the cells, the matching LC was isolated for expression of the corresponding Env-specific mAbs (one independent experiment). (C and D) V and J gene assignments. The V and J gene usage of all full-length HC sequences (n = 672) is shown as black bars, with the level of SHM for each Ab sequence shown as superimposed red circles. The average SHM for each V or J gene at the nucleotide level is shown as blue bars. (E) Clonality of the Env-specific response. Applying a definition of clonal relatedness as HC sequences with the same V and J gene assignments, identical HCDR3 length and 80%* HCDR3 sequence identity to the 672 HC sequences (red bar) resulted in the identification of 180 Env-specific clonotypes (pink bar). (F) Genetic characteristics and IC₅₀ neutralizing titers of Env-specific mAbs. Of the 17 cloned Env-specific mAbs, three neutralized the tier 2 isolate 16055, with the GM9_TH8 mAb being most potent (data from one independent experiment). (G) Binding of the biotinylated GM9_TH8 mAb to the 16055 NFL trimers (His-captured) in the presence of nonbiotinylated mAb competitors measured by ELISA. The GE136 mAb, a non–broadly neutralizing CD4bs-directed Ab (Sundling et al., 2012a), was used as a negative control (representative data from duplicate wells from two independent experiments). The trimer crystal structure (PDB: 5FYJ) shows the approximate epitope locations of the various mAbs as indicated. Residues corresponding to the V2b loop are shaded in magenta. (H) GM9_TH8 and b6 binding to 16055 gp120 and to a variant of gp120 lacking the V2b region (ΔV2b) as measured by ELISA. (I) Epitope specificity of the D11A.F2 and GM9_TH8 mAbs as measured by neutralizing activity (IC₅₀, µg/ml) against a panel of Ala mutants along the V2b region compared with WT 16055. Mutations resulting in a significant loss of neutralization capacity (>30-fold) are shaded in gray.