Antibody 8ANC195 reveals a site of broad vulnerability on the HIV-1 envelope spike

Abstract

Broadly neutralizing antibodies (bNAbs) to HIV-1 envelope glycoprotein (Env) can prevent infection in animal models. Characterized bNAb targets, although key to vaccine and therapeutic strategies, are currently limited. We defined a new site of vulnerability by solving structures of bNAb 8ANC195 complexed with monomeric gp120 by X-ray crystallography and trimeric Env by electron microscopy. The site includes portions of gp41 and N-linked glycans adjacent to the CD4-binding site on gp120, making 8ANC195 the first donor-derived anti-HIV-1 bNAb with an epitope spanning both Env subunits. Rather than penetrating the glycan shield by using a single variable-region CDR loop, 8ANC195 inserted its entire heavy-chain variable domain into a gap to form a large interface with gp120 glycans and regions of the gp120 inner domain not contacted by other bNAbs. By isolating additional 8ANC195 clonal variants, we identified a more potent variant, which may be valuable for therapeutic approaches using bNAb combinations with nonoverlapping epitopes.

Figure 1. Crystal structures of 8ANC195 Fab and 8ANC195/gp120/sCD4 complex

(A) Superimposition of unbound (grey) and bound (HC, purple; LC, light pink) structures of 8ANC195 Fab shown as ribbon diagrams. CDR loops are highlighted (CDRH1/CDRL1, red; CDRH2/CDRL2, green; CDRH3, dark blue; CDRL3, light blue) and a “thumb”-like loop formed by an insertion in FWR3 is indicated. Disordered loops are shown as dashed lines. (B) Space-filling model (inset) and ribbon diagram of ternary complex of 8ANC195 (HC, purple; LC, light pink), sCD4 (teal), and 93TH057 gp120 core (inner domain, grey; outer domain, light yellow; bridging sheet, orange; loop D, green; loop V5, cyan; CD4 binding loop, blue). Ordered glycans attached to Asn234_gp120 and Asn276_gp120 are shown as yellow sticks (oxygens, red; nitrogens, blue). Fab CDR loops are colored as in (A). sCD4 was omitted from the right panel for clarity. (C) Approximate locations of bNAb epitopes on a surface representation of the gp120 core. The epitopes of V3 and V1/V2 antibodies include regions of loops (dotted lines) not present in the gp120 core structure. CD4 binding site and 8ANC195 epitopes are outlined by black (CD4 binding site) and magenta (8ANC195) dots. Glycans included in the 8ANC195 epitope are shown in magenta. Subdomains of gp120 are colored as in (B). See also Figure S1.

Figure 2. Conformations of 8ANC195 CDRH1 and CDRH3 loops

(A) The hook-like conformation of CDRH1 (red) is stabilized by burial of the hydrophobic Phe30_HC side chain and hydrogen bonds within CDRH1 and with CDRH3 (blue) and FWR1 and FWR3 residues (Ala24_HC and Asp73_HC, respectively). (B) The complexed CDRH3 conformation (blue) consists of a protruding loop (residues 102_HC–110_HC) and a small β-sheet subdomain (residues 111_HC–118_HC) stabilized by multiple hydrogen bonds within CDRH3 as well as with CDRH1 and CDRL3. A hydrogen bond between Tyr92_LC and Gly110_HC stabilized the bifurcation of CDRH3 into its two subdomains. CDRH1 and CDRH3 loop backbone atoms are shown as sticks and side chains of residues important for stabilizing the loop conformations are shown as sticks (involved in direct contacts) or lines (backbone involved in contacts) (with exception of those of Tyr105_HC, Lys107_HC and Trp108_HC, which are shown for clarity). (C) Comparison of CDRH3 loops in 8ANC195 and other anti-HIV-1 bNAbs. CDRH3 residues corresponding to 8ANC195_HC residues 97–124 (pink) of NIH45–46 (blue, PDB 3U7Y), PG16 (grey, PDB 4DQO), PGT121 (purple, PDB 4FQC) and PGT128 (orange, PDB 3TYG) are shown as Cα traces.

Figure 3. Contacts made by 8ANC195 HC with gp120 protein residues and glycans

Labels for gp120 protein and glycan residues are italicized. Hydrogen bonds are shown as dashed yellow lines. (A) FWR3_HC loop contacts with loop D (green), loop V5 (light blue), and outer domain loop (yellow). (B) 8ANC195 HC CDRH1 (red) and CDRH3 (blue) contacts with gp120 inner domain (grey). (C) Buried surface area between the Asn234_gp120 glycan (transparent surface with glycan residues shown as sticks) and 8ANC195 (HC FWR residues in purple and CDRH2 in green). Antibody atoms buried by glycan interactions are shown as surfaces. (D) Buried surface area between the Asn276 glycan_gp120 (transparent surface with glycan residues shown as sticks) and 8ANC195 (HC FWR residues in purple and CDRH1 in red). Antibody atoms buried by glycan interactions are shown as surfaces. (E) Top: Contacts made by 8ANC195 HC FWR residues (purple) and CDRH2 (green) with Asn234_gp120 glycan (orange). Glycan and protein residues involved in hydrogen bonds are shown as sticks. Bottom: schematic of ordered high mannose glycans on Asn234_gp120 and Asn276 _gp120 (bottom). See also Figure S2.

Figure 4. Comparison of glycan-dependent bNAbs

8ANC195 is “bracketed” by two glycans (Asn234_gp120 glycan, orange; Asn276_gp120 glycan, yellow) in the 8ANC195 Fab/gp120/sCD4 complex structure (left panels). For comparison, crystal structures of PG16 (grey, middle panels, PDB 4DQO) bound to a V1/V2 loop scaffold and PGT128 (blue, right panels, PDB 3TYG) bound to a V3 loop scaffold are shown with (A) the antibody HCs aligned to the 8ANC195 HC or (B) an alternative view showing their interactions with bracketing glycans (for PG16: Asn160_gp120 glycan, teal/Asn172_gp120 glycan, light cyan; for PGT128: Asn301_gp120 glycan, pink/Asn332_gp120 glycan, purple). The proteins are shown as ribbon diagrams and the glycans as stick representations.

Figure 5. EM reconstruction of 8ANC195/Env trimer complex and model of 8ANC195 LC interactions with gp41 HR2

(A) EM reconstruction of 8ANC195 Fab/BG505 SOSIP.664. Side (left) and top (right) views of EM density with the X-ray structures of BG505 SOSIP.664 (PDB ID 4NCO; gp120, grey; gp41, light blue) and 8ANC195 Fab fit independently of gp140 coordinates to the EM density (purple). See also Figure S3. (B) Close-up of 8ANC195 LC/HR2 region of EM complex structure (Fab placement is best fit/independently placed as in (A)). Left: Fab is shown as a surface representation with highlights (CDRL1, red; CDRL2, green; CDRH1, red; CDRH3, blue), and gp140 is shown as a ribbon diagram (gp120, grey; gp41, light blue). The position of Asn637_gp41 (magenta) was deduced from the position of the C-terminus of the SOSIP.664 trimer (Gly664_gp41). Right: 8ANC195 HC and LC residues (sticks) positioned to contact HR2, which is shown as a surface representation calculated from HR2 coordinates in PDB 4NCO with presumptive sidechains added to the polyalanine coordinates. See also Figure S4.

Figure 6. Effects of LC sequence changes on 8ANC195 neutralization potency

(A) Sequences of LC CDRs in constructs used with 8ANC195 HC to make chimeric IgGs (left) and location of CDRs on 8ANC195 structure (right). Sequences derived from the mature antibody are shown on a pink background, those derived from the germline precursor are shown on a grey background. The mutations introduced into CDRL3 in glCDRL3Ala are shown on a white background. (B) Effects of changes in 8ANC195 LC on binding to 93TH057 and YU2 gp120s and neutralization of viral strains, expressed as fold changes over results for 8ANC195 IgG. K_D and IC₅₀ values for these experiments are shown in table S3. (C) Heat map showing the expression and neutralization of randomly paired HCs and LCs from the bulk sort on a Tier 2 15-virus panel. Red squares represent average IC₅₀ values on neutralized viral strains (arithmetic means) between 0.1 and 5 µg/ml; orange squares between 5.1 and 10 µg/ml, yellow squares between 10.1 and 14.9 µg/ml, and green squares above 15 µg/ml. White squares represent insufficient antibody expression. See also Figures S5 and S6.

Figure 7. Locations of bNAb epitopes on HIV-1 Env trimer

EM density map of Env trimer including MPER region (trimer map EMD-5019 from (Liu et al., 2008)) showing approximate epitope locations for antibodies targeting the 8ANC195 epitope (purple with Asn234_gp120 and Asn276_gp120 glycans shown as spheres), CD4 binding site (red), V3 loop/Asn332_gp120 glycan (blue with Asn332_gp120 glycan shown as spheres), V1/V2 loop/Asn160_gp120 glycan (green with Asn160_gp120 glycan shown as spheres), and MPER (yellow).