Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448_gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.

Keywords: Env trimer; HIV-1; HIV-1 Env silent face; HIV-1 vaccine; broadly neutralizing antibody; cryo-EM; glycan recognition; humanized mice; immunotherapy.

Graphical abstract

Figure 1

Isolation of Antibody Family from Donor 27845 by B Cell Culture and BG505 Sorting (A) Viral load and CD4⁺ T cell counts of HIV-1-infected subject 27845 over time. Arrows indicate time points of B cell microculture and BG505.SOSIP.664 bait-sorting. (B) Neutralization data of donor 27845’s serum IgG in 2005 against a 12-virus cross-clade panel (global) and a 20-virus fingerprinting panel (f61). Shown are median inhibitory concentrations (IC₅₀) in μg/mL. On the right, fingerprinting analysis of f61 serum neutralization. Neutralization testing performed in duplicates, average shown. (C) Maximum-likelihood phylogenetic tree of heavy chain sequences of newly isolated antibody family. MC = Antibodies isolated by B cell microculture, BG505-sort = antibodies isolated by bait-sorting, Both = antibody found both by microculture and bait-sorting. (D) Neutralization of isolated antibody family members (IC₅₀) against global and f61 virus panels. Legend as in (B). Neutralization testing performed in duplicates, average shown. (E) Neutralization coverage and potency of SF5 and SF12 on a 119-virus cross clade panel. Neutralization testing performed in duplicates, average shown. (F) Neutralization fingerprinting of SF5 and SF12 in comparison to other known anti-HIV-1 bNAbs. See also Figure S1 and Tables S1 and S2.

Figure 2

Antibodies SF5 and SF12 Bind a Distinct Epitope on the gp120 Portion of Env (A) ELISA of SF5 and SF12 against a gp120 monomer and a gp140 foldon trimer derived from HIV-1 strain YU2. Wild-type proteins and various site mutants of the proteins in common bNAb epitopes (CD4-binding site, V3-glycan, Apex) were tested. Triple mutant = N160K, A281T + D368K, N332K. Data representative of 3 repeat assays. (B) ELISA of SF5 and SF12 as well as reference bNAbs targeting 6 known epitopes against the BG505.SOSIP.664 trimer. Data representative of 3 repeat assays. (C) Competition ELISA with reference bNAbs targeting 6 known epitopes to evaluate interference with SF5 and SF12 binding to the BG505.SOSIP.664 trimer. Competing antibodies were added in a dilution series starting at 32 μg/mL. SF5 and SF12 were added at a constant concentration of 0.5 μg/mL. Data representative of 3 repeat assays. (D) Neutralization testing of SF12 against a panel of YU2 site mutants covering major epitopes on the HIV-1 spike. Neutralization testing performed in duplicates, average curves shown. (E) Computational analysis of 119-virus cross clade panel neutralization. (F) Neutralization testing of SF5 and SF12 against an HIV-1 pseudovirus based on strain YU2 carrying a mutation at the PNGS N448_gp120. Testing done in duplicates, average shown.

Figure 3

Structural Overview of the SF12-B41-10-1074 complex (A and B) Side-view (A) and top-view (B) of the final 3.3 Å single-particle cryo-EM reconstruction of the SF12-B41-10-1074 complex colored by components (dark gray, gp41; light gray, gp120; magenta, SF12 V_H; pink, SF12 V_L; blue, 10-1074 V_H; light blue, 10-1074 V_L; cyan, N-glycans). (C) Superposition of V_H-V_L domains (235 Cα atoms) of unliganded SF12 (orange), Env-bound SF12 (magenta), and core gp120-bound VRC-PG05 (green) Fabs, showing differences in CDR conformations between SF12 and VRC-PG05. (D) Surface representation of SF12 (magenta/pink) and VRC-PG05 (green/pale green) Fabs illustrating differences in CDRL1 and CDRH3 loop conformations. (E) Surface representation of Env-bound SF12 Fab showing interactions with the N262_gp120 (pale blue), N295_gp120 (pale green) and N448_gp120 (red) glycans at the SF12-Env interface. Cryo-EM density for individual glycans is shown contoured at 6σ. (F) Comparison of V_H-V_L domain orientations of SF12 (magenta/pink; cartoon) and VRC-PG05 (green/pale green; surface). The V_H-V_L domain orientation of SF12 on Env trimer is related by a 71° rotation and 0.5 Å translation to the VRC-PG05 variable domains after alignment against gp120 (gray; surface). (G) Overlay of CDRH3 loops of SF12 (magenta) and VRC-PG05 (green) after alignment of bound gp120s illustrates CDRH3-mediated recognition of the N448_gp120 glycan (red; sticks) by both antibodies. See also Figures S2, S3, and S4 and Tables S3 and S4.

Figure 4

Details of SF12 Epitope and Glycan Recognition (A) Sequence of SF12 variable domains with antibody regions annotated using IMGT sequence analysis (CDR loops are bracketed). SF12 residues that contact N-linked glycans are in blue (N262_gp120), green (N295_gp120), and red (N448_gp120), while gp120-contacting residues are boxed. Contacting residues in the SF12 paratope and epitope were defined as two residues containing any atom within 4 Å of each other. (B) Structure of a SF12-B41 gp120 protomer from the trimer complex, showing paratope residues as spheres (inset). Color scheme is the same as in (A). (C) Surface representation of B41 trimer, with SF12 epitope highlighted in magenta. (D–F) Stick representation of residue level contacts for N262_gp120 (D), N295_gp120 (E), and N448_gp120 (F) glycans. Potential hydrogen bonds are shown as black dashes. Cryo-EM density maps contoured at 6σ are shown for individual glycans. See also Figure S4 and Table S5.

Figure 5

SF12 Engages Two Distinct Regions of gp120 Peptide Epitope (A) Stick representation of SF12 CDRH3 (magenta) and gp120 (gray) contacts at the SF12-Env interface. Trp100D_HC inserts into a hydrophobic pocket (inset) stabilized by potential hydrogen bond interactions (black dashes) with neighboring residues. (B) Stick representation of SF12 CDRH1 and H2 residues (magenta) contacting gp120 residues (gray). Potential hydrogen bonds are shown as black dashes. Density maps for SF12 and gp120 residues are shown as magenta and gray meshes, respectively, contoured at 8σ. (C) Comparison of SF12 and VRC-PG05 neutralization breadth for different viral characteristics. The red dashed line indicates neutralization breadth for SF12 (62%) and VRC-PG05 (27%) against a cross-clade panel. (D) Modeling of the N442_gp120 glycan from clade C 426c SOSIP trimer (teal; PDB: 6MYY) was achieved by aligning gp120 coordinates from the two structures. Potential clashes with SF12 heavy chain (magenta) regions are highlighted. See also Figure S5.

Figure 6

SF12-B41-10-1074 Structural Asymmetry Is Explained by N295_gp120 Glycan Heterogeneity (A) Comparison of cryo-EM density for N295_gp120 (green) and N332_gp120 (orange) glycans across protomers within the SF12-B41-10-1074 trimer complex. In each protomer, SF12 (magenta) was bound, but 10-1074 (blue) binding was only observed when the N295_gp120 glycan was modeled as GlcNAc₂ (right panel). (B) Overlay of N295_gp120 and N332_gp120 glycans after aligning gp120 protomers from cryo-EM structures of SF12-B41-10-1974, PDB: 6CUE, PDB: 6DCQ, PDB: 5V8M, and PDB: 6CRQ. Positions for the N295_gp120 and N332_gp120 glycans in the SF12-bound Env (stick representation) and all other models (line representation) are shown. SF12-induced conformational changes are indicated by the red arrow. (C) Modeling of the 10-1074 Fab (blue cartoon) onto the SF12-gp120 protomer (A: left panel). Potential clashes between 10-1074 CDRH3 and the N332_gp120 glycan are highlighted. (D) Alignment of gp120 portions of the SF12-bound (A: left panel) and SF12 plus 10-1074-bound (A: right panel) protomers. Potential clashes involving the N295_gp120 and N332_gp120 glycans (highlighted stars) when both glycans are processed beyond a core pentasaccharide are shown. (E) Predictive neutralization profiles for combination therapy with SF12 and 10-1074 bNAbs at a 10 μg/mL concentration. See also Figure S5 and Tables S6 and S7.

Figure 7

In Vivo Evaluation of SF12 IgG in HIV_YU2-Infected Humanized Mice (A) SF12 monotherapy of humanized mice infected with HIV_YU2. The left graph shows absolute viremia (y axis) in mice treated with SF12 (n = 7, dark gray, full circles) or untreated control mice (n = 7, empty circles) over the course of the experiment (x axis, days). Mice were infected 3 weeks prior to therapy initiation and received 1 mg of IgG as a loading dose followed by twice-weekly administration of 0.5 mg for 3 weeks. The dotted line at bottom indicates the limit of accuracy of the qPCR assay (384 copies/mL). The right graph shows relative log drop after initiation of SF12 therapy (Δlog10 copies/mL). Thick red lines and thick dashed gray lines indicate the mean viral load of treated and untreated mice, respectively. Data from one independent experiment. (B) Amino acid alignment of gp160 of wild-type YU2_gp160 (top) with Env gp160 sequences obtained by single genome sequencing from plasma of SF12-treated mice 4 weeks posttherapy initiation. Each line represents one sequence; mouse identification numbers indicated on left. (C) Pie chart showing the amino acid distribution at position N448_gp120 in mice that received SF12 at 4 weeks posttherapy initiation. Numbers inside pie chart correspond to number of mice sequenced/number of sequences obtained. (D) Antibody tri-mix (SF12, 10-1074, 3BNC117) therapy of HIV-1_YU2-infected mice (n = 8). Mice (n = 4) with comparable viral loads and matched stem cell donors served as controls. Data from one independent experiment. Graphs as in (A).