Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

Abstract

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Figure 1. CH235 Lineage, with Time of Appearance and Neutralization by Select Members

(A) Phylogram of CH235 lineage. Phylogenetic tree is colored by first time (wk post-infection) from which sequences were obtained. Key members of the CH235 lineage are labeled. CH235.6, CH235.7, CH235.8 and CH235.9 V_H were complemented with full heavy chain gene regions and paired with the V_L from the closest natural antibody. (B) Neutralization dendrograms display single mAb neutralization of a genetically diverse panel of 199 HIV-1 isolates. Coloration is by IC₅₀. See also Figure S1, Tables S1 and S2.

Figure 2. Structures of CH235-Lineage Members in Complex with HIV-1 Env

(A) Co-crystal structures of the antigen-binding fragments (Fabs) of CH235-lineage members with core gp120. Structures are shown in ribbon diagram, with gp120 in gray and residues altered by SHM in stick representation colored by time-of-appearance. (B) Negative stain EM of Fabs of CH235-lineage members and trimeric HIV-1 Env from BG505 (top row) and B41 (bottom row). Structures in surface representation, with Env portions colored gray and Fabs by time-of-appearance. (C) Epitope displayed on the gp120 surface and colored by antibody time-of-appearance, with the vulnerable portion of the CD4bs highlighted in yellow and select regions labeled. (D) Targeting precision of CD4bs-directed antibodies vs neutralization breadth. (E) V_H-gene SHM of CD4bs-directed antibodies vs neutralization breadth. See also Figure S2 and Table S3.

Figure 3. Sequence Evolution of CH235 Lineage: SHM, Timing, and Conformity of CH235-Lineage Development from UCA to Antibody with 90% Breadth

(A) Heavy chain SHM over time for the CH235 lineage (left panel). SHM levels of other V_H1-46-derived CD4bs mAbs and selected V_H1-2-derived VRC01-class mAbs are shown (middle and right panels, respectively); the time since infection is unknown for these mAbs. (B) Maturation conformity vs overall heavy chain SHM. Positional conformity (top row) is defined as the number of aa positions differing from the germline sequence in both the conforming and reference sequences, divided by the total number of aa changes in the conforming antibody. Identity conformity (bottom row) is defined as the number of such positions which are additionally mutated to the same residue, divided by the total number of mutations in the conforming antibody. Conformity to 1B2530 (left) and to 8ANC131 (right) is shown for both position and identity. (C) V_H-gene mutability accounts for the majority of positional conformity of CH235 lineage. The mutability of the V_H-gene for V_H1-46 (top) and V_H1-2 (bottom) is shown. Sequence logos are shown at each position; the height of each logo corresponds to the percent of mutated reads. Green bars are shown for SHM in antibody CH235, which are altered in over a quarter of V_H1-46-derived antibodies. See also Figure S3 and Table S4.

Figure 4. Binding Kinetics of CH103 and CH235 Lineage Antibodies

Binding association (k_a) and dissociation (k_d) rates of the CH103 (A-B, squares) and CH235 (C-D, circles) lineage mAbs to CH505.TF gp120 Env were measured with SPR and used to calculate the dissociation rate constants (K_d). K_ds are shown in A and C, k_a (solid lines, plotted on the left y-axis) and k_d (dashed lines, plotted on the right y-axis) are shown in B and D. See also Table S5.

Figure 5. CH235 Lineage Antibodies Neutralization of Autologous Virus and CH505.TF Loop D Mutants

(A) Heatmap analysis of neutralization of 76 pseudoviruses (row) by 16 CH235 lineage mAbs (column). Coloration is by IC₅₀. This analysis extends previous observations on early CH235 lineage antibodies (Gao et al., 2014) by including late mAbs CH235.7, CH235.8, CH235.10, CH235.11, CH235.12 and CH235.13 and by adding pseudoviruses isolated from wk 136 to 323 post-transmission. (B) CH505 TF and loop D mutants M5, M6, M10, M19, M11, M7, M8, M9, M20 and M21 neutralization by CH236 mAb, late mAbs CH235.7, CH235.9 CH235.10, CH235.11, CH235.12, CH235.13 (left panel) and CH235.9 mAb mutants (right panel). Neutralization is expressed as IC₅₀ μg/ml. CH505 TF sequence mutations are shown on the right. (C) The CDR H1 N30 (sticks, dark red) in CH235.9, which interacts with the β20- β21 loop in the bridging sheet of gp120 (cyan), is over 19Å away from the N280S mutation site in loop D (orange). See also Figure S4, Tables S6 and S7.

Figure 6. Binding of CH235 and CH103 Lineage mAbs to Autologous CH505 (A) and CH235 UCA Binding to Heterologous HIV-1 Env Glycoproteins (B)

(A) Heatmap analysis of UCA, intermediate (IA) and mature CH235 and CH103 lineage mAbs binding to 113 CH505 autologous Env isolated from time of infection (TF) to 160 wks post-infection and to the CH505.TF mutants (Gao et al., Cell 2014). Mabs were tested in ELISA at concentrations ranging from 100 μg/ml to 0.6 ng/ml . Binding is expressed as a LogAUC. (B) Affinity of CH235 UCA, CH235 wild-type and select SHM variants to a panel of 15 heterologous gp120 Envs. See also Figure S5 and Table S8.

Figure 7. CH235 Antibody Lineage Auto- and Polyreactivity

(A) CH235 lineage antibody binding to ANA measured in ELISA. LogAUC was calculated from duplicate samples. Results representative of duplicate experiments. (B) Binding to cardiolipin was determined using Quanta Lite ACA IgG III ELISA Assay. (C) Hep2 cell IF staining. Size bars = 50 μm. (D) Measurement of polyreactivity against 9,400 human antigens using ProtoArray 5 microchip: CH235 lineage mAbs binding (x-axis) was compared to non-polyreactive control mAb 151K (y-axis). Polyreactivity is defined as 1 log stronger binding than 151k mAb to more than 90% of the test proteins. High affinity binding was measured as a >2 log increase in binding (dotted line) (Liu et al., 2015).