Improved HIV-1 neutralization breadth and potency of V2-apex antibodies by in silico design

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV can reduce viral transmission in humans, but an effective therapeutic will require unusually high breadth and potency of neutralization. We employ the OSPREY computational protein design software to engineer variants of two apex-directed bNAbs, PGT145 and PG9RSH, resulting in increases in potency of over 100-fold against some viruses. The top designed variants improve neutralization breadth from 39% to 54% at clinically relevant concentrations (IC₈₀ < 1 μg/mL) and improve median potency (IC₈₀) by up to 4-fold over a cross-clade panel of 208 strains. To investigate the mechanisms of improvement, we determine cryoelectron microscopy structures of each variant in complex with the HIV envelope trimer. Surprisingly, we find the largest increases in breadth to be a result of optimizing side-chain interactions with highly variable epitope residues. These results provide insight into mechanisms of neutralization breadth and inform strategies for antibody design and improvement.

Keywords: CP: Immunology; CP: Microbiology; HIV; OSPREY; antibody design; antibody improvement; broadly neutralizing antibody; in silico design; protein design; provable algorithms; structural biology.

Figure 1.. Large-panel neutralization breadth and potency for PGT145 and PG9RSH variants

Neutralization breadth and potency of PGT145 and PG9RSH variants assayed on a panel of 208 pseudoviruses. (A and B) Breadth/potency curves for PGT145 and PG9RSH variants and controls, respectively. Curves represent the fraction of pseudoviruses that were neutralized with IC₅₀ smaller than the given cutoff. An increase in breadth and potency is indicated by a shift upward and left. PGT145 variant DU303 and PG9RSH variants N(100f)Y and DU025 improve breadth and potency relative to wild type. For IC80 curves, see Figure S3. (C) Breadth/potency curves for PG9RSH DU025 and PGDM1400. Despite slightly weaker median and mean neutralization potency, the overall breadth and potency of DU025 rival that of the best-in-class PGDM1400 antibody. (D–F) Neutralization dendrograms for variants DU303, PG9RSH N(100f)Y, and DU025, and respectively. Pseudoviruses are grouped into clades by sequence similarity, forming a tree graph. Internal branches in the tree, which denote groups of viruses, are colored gray. Terminal branches, corresponding to a single pseudovirus, are colored by IC50, where high neutralization potency is indicated by warm colors, low potency is indicated by cool colors, and lack of neutralization is indicated by gray. (G) Summary of large-panel neutralization breadth and potency for variants and controls, measured by IC80.

Figure 2.. Cryo-EM structures of PG9RSH and PGT145 variants in complex with BG505 DS-SOSIP Env trimer

Backbone shown in ribbon representation with glycans, and amino acids shown as sticks or lines. Env subunits colored with warm colors and grays, and antibodies shown in cool colors. CDRH3 (residues 95–102) is shown in green. Distances (Å) are shown with dotted yellow lines, and energetic interactions are shown with Probe dots. Members of the PG9RSH (A and B) and PGT145 lineages (C) interact with the trimer apex and are characterized by a negatively charged CDRH3 that is hammer-like or extended, respectively. (A) PGT145 mutation N(100l)D forms more favorable electrostatic interactions with gp120. (B) The PG9RSH N(100f)Y mutation creates interactions with a side-chain nitrogen from gp120 residue K168, forming geometry consistent with a π-cation interaction. (C) The PG9RSH Y(100k)D mutation forms long-range interactions with polar and positively charged residues Q170 and K305.

Figure 3.. OSPREY design ensembles correctly predicted structural features for PG9RSH and PGT145 variants

Ten members of the low-energy ensemble (LEE) predicted by OSPREY are shown for variants of PGT145 (A) and PG9RSH (B and C) above corresponding cryo-EM structures (D–F). Backbones are shown as ribbons with amino acids shown as lines or as sticks with Env subunits colored with warm colors, and antibody CDRH3 loops (residues 95–102) are shown in green. Distances (Å) are shown with dotted yellow lines. (A) PGT145 mutation N(100l)D is predicted to form electrostatic interactions with gp120 residues R166 and K169. A carboxyl oxygen of D(100l) lies 5 and 4.2 Å from side-chain nitrogens of gp120 residues R166 and K169, respectively. Despite a lateral translation of the CDRH3 loop relative to the trimer apex, the LEE correctly predicts features of the experimental structure (F). (B) The PG9RSH N(100f)Y mutation creates interactions with gp120 residue K168. The side-chain amino nitrogen of K168 lies 5.1 Å from the ring plane of Y(100f), forming geometry consistent with a π-cation interaction. The LEE correctly predicts interactions found in the experimentally determined structure (D). (C) The PG9RSH Y(100k)D mutation is predicted to form electrostatic interactions with polar and charged residues on gp120. A carboxyl oxygen of D(100k) lies 3.2 Å from the side-chain nitrogen of Q170 and 5.6 Å from R308. The LEE correctly predicts interactions with Q170, but a translation and rotation of the CDRH3 loop places R308 further away (E).