HIV Envelope Glycoform Heterogeneity and Localized Diversity Govern the Initiation and Maturation of a V2 Apex Broadly Neutralizing Antibody Lineage

Abstract

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Functional Screening Identifies a New V2-Apex Specific Broadly Neutralizing Antibody Lineage

(A) Longitudinal plasma samples from donor PC64 were tested for neutralization against heterologous pseudoviruses. The percent of viruses neutralized (> 50% inhibition of infectivity at the lowest plasma dilution, 1:50) from a cross-clade (A, B, C) 37-virus panel is shown as shaded blue bars. Neutralization Inhibitory Dilution 50 (ID50, plasma dilution giving 50% inhibition of infection) of PC64 longitudinal plasma samples against 92TH021 WT (black circles) and N160K mutant (red circles) pseudoviruses are plotted. The evolution of the viral load (green circles) in the plasma is also plotted. The time points at which PCT64 antibodies were isolated andenv sequenced and/or cloned from plasma are indicated by corresponding symbols. The number (N) of PCT64 antibodies isolated and their range of somatic hypermutation frequency (%SHM) is also indicated. See also Table S1. (B) Evolutionary distance between the PCT64 mAbs is illustrated as a phylogeny for both heavy and light chain nucleotide sequences. (C) Mapping of the PCT64 epitope. Fold decrease in neutralization IC50 of individual JR-CSF Ala mutants by PCT64-35G as compared to WT is color-coded as indicated and presented on the BG505 SOSIP.664 trimer structure (Julien et al., 2013) based on HxB2 numbering and alignments. Data are representative of at least two independent experiments. See also Table S3. (D) PCT64 mAbs characteristics. Heavy and light chain V- and J-gene nucleotide (nt) somatic mutation percentages, neutralization breadth (% virus neutralized at IC50<50 μg/mL) and potency (GeoMean IC50 in μg/mL) on a 37- and 109-virus panel are tabulated and color-coded as indicated. See also Tables S1, S5, and S6. Data are representative of at least two independent experiments. (E) Comparison of PC64 plasma neutralization breadth and potency on the 109-virus panel subdivided by subtype, with that of a theoretical combination of PCT64 mAbs, color-coded as indicated. See also Tables S2, S4, S7, and Figure S1A.

Figure 2. Longitudinal Next-Generation Sequencing of the Memory B Cell Repertoire Reveals the Phylogeny of the PCT64 bnAb Lineage

PC64 IgG libraries prepared from total PBMCs were amplified with IgG-specific primers for all human VH gene families. (A) Frequency of PCT64 bnAbs at each time point, plotted as a percentage of total PCT64 lineage sequences from all time points. Plasma neutralization score (see Landais et al., 2016) from a heterologous 37-virus panel is plotted as a dashed line. (B) Somatic hypermutation frequency was calculated for each time point either as divergence (number of amino acid changes compared to LMCA; top panel) or as sum of the evolutionary distance (middle panel). Data are presented as whisker plots showing mean, 95% upper and lower quartiles, SD, and outliers. The evolutionary distance normalized by divergence using mean values is shown in the bottom panel. (C) Longitudinal phylogeny of PCT64 HC sequences (colored by mpi). The PCT64 mAb sequences are named in black, and star symbols represent autologous (black) and heterologous (red) neutralization. See also Figure S2.

Figure 3. Crystal Structure of PCT64-35B bnAb Fab Shows an Extended PGT145-like CDRH3 Conformation

(A) PCT64-35B Fab crystal structure with CDRH3 side chains as sticks. Heavy chain in beige, CDRH3 in orange, and light chain in purple. (B) Overall organization of the variable region of PCT64-35B Fab; secondary structure rendering of CDRs and framework (FR) regions. (C) Superposition of PCT64-35B Fab variable region (blue) and PGT145 variable region (gray), showing differential CDRH3 orientations. (D) Logograms of CDRH2 and CDRH3 residues (Kabat numbering) for PCT64 mAbs segregated in two groups based on acquisition of heterologous breadth. See also Figures S1B and S3.

Figure 4. Longitudinal Full Length Env Next Generation Sequencing Allows a Comprehensive Analysis of Env Evolution and Escape from PCT64 Abs

HIV subtype A env phylogeny from donor PC64, colored by sample date, estimated by maximum likelihood from full-length envPacBio high-quality consensus sequences (HQCSs) (with bubbles representing sample proportion) (http://test.datamonkey.org/flea-demo/PC64_kinetics/) and clonal Sanger sequences (Monogram Biosciences Lab Corp) (indicated by dashed lines). Data are representative of one experiment. See also Figure S4.

Figure 5. Analysis of Env Escape from PCT64 Abs Reveals that Localized Diversity at Key V2 Epitope Residues Drove bnAb Maturation toward Breadth

(A) Left panel: V1/V2 amino acid sequences of PC64 Env cloned at various time points, aligned vertically. The number of clones is indicated and amino-acid identity is color-coded. Middle panel: Logograms of Env C-strand residues 160–171 for each time point. Net charge of the 160–171 peptide for each clone is color-coded in shades of blue (from 0 to 1). Right panel: Autologous neutralization of each pseudotyped Env clone by PCT64 mAbs (grouped and colored by isolation time point). The neutralization IC50 (μg/mL) is tabulated and color-colored as indicated in the legend. The development of the PCT64 bnAb lineage and V1V2-directed heterologous plasma neutralization are indicated on the right. (B) Evolution of PCT64 mAb neutralization against autologous pseudotyped virus mutants representing various Env immunotypes at residues 160, 166, 167, 169, 181 at several time points. Residues mutated from WT are shaded in pink. The neutralization IC50 (μg/mL) is tabulated and colored as indicated. Gray cells represent the absence of neutralization. (C) Frequency of PC64 Env immunotypes (combination of residues 160, 166, 167, 169, 181) at several time points, with viral load in plasma (black). Neutralization potency of PCT64 mAbs presented in (B) against these immunotypes is shown as shades of blue, green, orange, and red for PCT64 mAbs isolated at 13, 18, 24, and 35 mpi, respectively. Antibody icons are colored by mpi, with the frequency of PCT64 HC sequences found in the periphery (Figure 2A) shown by icon size. GL: Germline. Development of V2 apex-directed heterologous plasma neutralization (Figure 1A) is indicated in black above. Data are representative of at least two independent experiments. See also Figures S5 and S6.

Figure 6. Env Glycoform Heterogeneity Played a Role in Elicitation of the PCT64 bnAb Lineage

(A) Autologous neutralization of the MRCA pseudotyped virus by titrated amounts of PCT64 mAbs. (B) Autologous neutralization of PC64 Env clones by PCT64-infGL and -LMCA mAbs. (C) Autologous neutralization of MRCA pseudotyped virus by titrated amounts of WT and mutant PCT64 mAbs. PCT64-LMCA (top) and PCT64-13C (bottom) heavy chain mutants paired with PCT64-infGL WT, PCT64-infGL-CDRL3 mutant(+SAR) or PCT64-13C light chains. (D) Top and side views of negative-stain EM 3D reconstructions (see Figure S7C) of PC64 unliganded (left) or PGV04-bound (right) SOSIPs produced in 293-F cells. Atomic models of BG505 (gp120 in blue, gp41 in yellow: PBD 5CEZ) in complex with PGV04 (red) (PDB: 3J5M) and with Fabs removed were docked into EM density maps. (E) Cryo-EM reconstruction of PC64-M4c054 SOSIP with autologous PCT64-13C Fab. Side and top view of the EM density map showing PCT64-13C Fab bound to the V1/V2 apex. The final resolution is ~13Å (Figure S7D). (F) BLI curves of indicated antibodies immobilized on anti-human IgG Fc sensors and indicated PC64 SOSIP trimers (1 μM) in solution as analytes. PC64 SOSIP trimers were produced in the presence of furin in 293F (top panels; normal glycan processing), or 293S (bottom panels;GnTI−/− with no hybrid/complex glycan processing leading to enrichment in Man5GlcNAc glycans). Data are representative of at least two independent experiments. See also Figure S7.