Identification of a broad and potent V3 glycan site bNAb targeting an N332gp120 glycan-independent epitope

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 can suppress viremia in vivo and inform vaccine development. Here, we characterized 007, a V3 glycan site bNAb exhibiting high levels of antiviral activity against multiclade pseudovirus panels^1-3 (GeoMean IC₅₀ = 0.012 μg/mL, breadth = 69%, 217 virus strains) by targeting a N332_gp120 glycan-independent V3 epitope, a site of Env vulnerability to which only weakly neutralizing antibodies had previously been identified. Functional analyses demonstrated distinct binding and neutralization profiles compared to classical V3 glycan site bNAbs. A 007 Fab-Env cryo-EM structure revealed contacts with the V3 ³²⁴GD/NIR³²⁷ motif and interactions with N156_gp120 and N301_gp120 glycans. In contrast to classical V3 bNAbs, 007 binding to Env does not depend on the N332_gp120 glycan, rendering it resistant to common escape mutations. Structures of 007 IgG-Env trimer complexes showed two Env trimers crosslinked by three bivalent IgGs, and bivalent 007 IgG was up to ~300-fold more potent than monovalent 007 IgG heterodimer, suggesting a role for avidity in potent neutralization. Finally, in HIV-1_ADA-infected humanized mice, 007 caused transient decline of viremia and overcame classical V3 escape mutations, highlighting 007's potential for HIV-1 prevention, therapy, functional cure, and vaccine design.

Extended Data Fig. 1:. Neutralizing activity and fingerprinting analyses of serum from donor EN01

a, Neutralizing activity of EN01 serum IgG tested at a concentration of 300μg/mL against the HIV-1 global pseudovirus panel and b, the f61 pseudovirus panel retrieved from. Right panels show delineation scores of f61 panel-based computational epitope mapping.

Extended Data Fig. 2:. Isolation of single HIV-1-reactive B cells and characterization of corresponding mAbs

a, FACS plots showing the sorting gate and frequencies (in %) of HIV-1-reactive, IgG+ B cells isolated from donor EN01 as previously described. A pie chart illustrates the clonal relationships of amplified heavy chain sequences from single B cells. Individual clones are represented by shades of blue, gray, and white, with the total number of productive IgG heavy chain sequences indicated at the pie chart’s center. Clone sizes are proportional to the total number of productive sequences per clone. b, VH gene distribution, VH gene germline identity at nucleotide level, and CDRH3 length (in amino acids) for amplified IgG heavy chains compared to a memory IgG reference. The dashed line indicates the mean ± SD for the reference. c, Neutralizing activity of 48 isolated mAbs against a screening pseudovirus panel consisting of 6 viral strains from 4 different clades. Each mAb was tested at 2 μg/mL, with those achieving >50% neutralization classified as neutralizing. Neutralizing antibodies from B cell clone 17 (red) and clone 22 (purple) are highlighted. d, Neutralizing activity of 007 against BG505_T332N and known escape pseudovirus mutants. The panels indicate the IC₅₀ values (in μg/mL) against BG505_T332N pseudovirus wild type and mutants. Antibodies were tested in duplicates. Data for reference bNAbs marked with an asterisk were retrieved from. e, Neutralizing activity of 007 and reference bNAbs targeting known epitopes (V2: V2 loop, V3: V3 glycan site, CD4bs: CD4 binding site, MPER: membrane proximal external region, IF: interface, FP: fusion peptide, SF: silent face) against the f61 fingerprinting panel. Breadth (%) was calculated with a cut-off of ≤10 μg/mL. Reference bNAb data were obtained from the CATNAP database.

Extended Data Fig. 3:. Preclinical characteristics of 007

a, Reactivity of indicated antibodies against HEp-2 cells. Antibodies were tested at a concentration of 100 μg/mL. b, In vivo pharmacokinetic profile of 007 and reference bNAbs measured in hFcRn transgenic mice. Mice received a single intravenous injection of 0.5 mg of antibodies. Serum concentrations (μg/mL) were determined in duplicates by ELISA. Data of mice experiments are represented as mean ± SD. Control antibody data were previously reported and were generated alongside the data for 007 in the same experiment (a and b).

Extended Data Fig. 4:. Data collection and processing for 007 Fab-BG505-DS complexes

Example micrograph, data processing workflow, final densities colored by local resolution, gold-standard Fourier shell correlations (GSFSC), and particle orientation distribution plots.

Extended Data Fig. 5:. Interactions of 007 with the V1 loop and N-glycans on Env

a, Alignment of HIV-1 env sequences. Residues are numbered according to HIV-1_HXB2. b,Contacts between the V1 residue R151_gp120 and 007. Contacts on 007 within 4Å of R151_gp120 are shown by green dotted lines. c, Differences in the V1 loop on gp120 between a protomer bound by 007 Fab (left) and an unbound protomer (right). d, Contacts between 007 and the N156_gp120 glycan (left) or the N301_gp120 glycan (right). 007 residues within 4Å of modelled glycans are colored orange. Inset (middle) highlights the interactions between a glycin-rich motif in 007, colored red, and the N156_gp120 glycan.

Extended Data Fig. 6:. N-glycan analysis by quantitative mass spectrometry

Electrophoretic separation of gp120 Env subunit using SDS-PAGE under denaturing and reducing conditions, stain with Coomassie G-250 (a-c): a, Total BG505, 007_IgG-bound BG505, and 007_IgG control; b, Total BG505 with and without PNGase F treatment; c, 007_IgG-bound BG505 with and without PNGase F treatment. The shift in mobility of PNGase F-treated samples in (b-c) indicates removal of N-glycans from gp120. Arrows mark gp120, deglycosylated gp120, IgG heavy and light chains, and PNGase F. Scissors and red text mark gp120 bands that were excised and stored for LC-MS analyses. d, Comparison of glycoform abundance in total unliganded BG505 and 007-bound BG505 at N156_gp120/N160_gp120, N234_gp120, N295_gp120, N301_gp120, and N448_gp120. Data are presented as a side-by-side bar graph, where different high-mannose glycoforms are differentiated, but hybrid and complex-type glycans are presented as single groups. Error bars represent the standard deviations of replicate measurements (three for total BG505 and two for bound BG505). For visualization purposes, data are also presented as a stacked bar with individual glycoforms separated by a vertical line, and the most prevalent glycoform(s) labelled.

Extended Data Fig. 7:. Molar Neutralization Ratio assays

a, Neutralization curves for IgG, bispecific, and Fab forms of 007 against the global pseudovirus panel, BG505_T332N, and an MLV control. IC₅₀ values and calculated MNRs can be found in Supplementary Table 6.

Extended Data Fig. 8:. Data collection and processing for 007 IgG-BG505-DS complexes

Example micrograph, data processing workflow, final densities colored by local resolution, gold-standard Fourier shell correlations (GSFSC), and particle orientation distribution plots.

Extended Data Fig. 9:. Antiviral activity against the 100-strain Subtype C panel, effector function against Env expressing cells and HIV-1 ENV_BF520 deep mutational escape map

a, Neutralization breadth (%) and potency (IC₅₀/IC₈₀) of 007 against the 100-strain Subtype C pseudovirus panel. Breadth (%) was calculated using a cut-off of ≤10 μg/mL. Data are shown for identical virus strains across each panel with available reference neutralization data sourced from CATNAP database. b, 007 demonstrates potent ADCC against HIV envelope expressing cells. FcγRIIIa signaling was assessed using an ADCC reporter bioassay (RLUs) and CHO cell overexpressing HIV Env as targets. Jurkat reporter cells expressed either FcγRIIIa-F or FcγRIIIa-V alleles (representative data, n = 2). V158 is the high affinity allele for FcγRIIIa and F158 is the low-affinity allele. ADCC target cell killing (%) was assessed using NK cells as effectors and CHO-HIV Env as target cells (representative data of n=3 experiments; mean ± SD of duplicate points). c, Logo plots showing effects of mutations on neutralization escape in the HIV Env BF520 strain for antibodies 007, 10–1074, BG18, PGT121, and PGT128. The height of each letter represents the effect of that amino-acid mutation on antibody neutralization, with positive heights (letters above the zero line) indicating mutations that cause escape, and negative heights (letters below the zero line) indicating mutations that increase neutralization. Letters are colored by the effect of that mutation on Env-mediated cell entry function, with yellow corresponding to reduced cell entry and brown corresponding to neutral effects on cell entry. Only key sites are shown. See https://dms-vep.org/HIV_Envelope_BF520_DMS_007/htmls/all_antibodies_and_cell_entry_overlaid.html for interactive versions of the escape maps that show all mutations. The deep mutational scanning measured effects of mutations on escape from antibody 10–1074 shown in this figure are previously published.

Extended Data Fig. 10:. Viral loads of PBS-treated mice

a, Viral loads in HIV-1_ADA-infected humanized mice from the PBS-treated control group. The graphs depict relative log₁₀ changes from baseline viral loads (left) and absolute HIV-1 RNA plasma copies/mL (right) under PBS administrations. Dashed lines in the right graph represent the lower limit of quantification (LLQ) of the qPCR assay (260 copies/mL). Red lines indicate the average log₁₀ changes relative to baseline viral loads (day −2).

Figure 1:. Identification of bNAb 007 with distinct binding and neutralizing activity

a, HIV-1 neutralizing serum activity of HIV-1 elite neutralizer EN01 against the global and f61 fingerprinting pseudovirus panel. Serum IgG samples were tested in duplicates. b, Neutralization activity of bNAb 007 against the HIV-1 global pseudovirus panel. Samples were tested in duplicates. c, Interference of 007 with selected reference bNAbs targeting known epitopes on the HIV-1 Env trimer, as determined by competition ELISAs. d, Comparison of the neutralization profile of 007 with reference bNAbs targeting known epitopes against the f61 fingerprinting panel and e, 119 multiclade pseudovirus panel. Antibody 007 was tested in duplicates. Neutralization data of reference bNAbs (b, d and e) were retrieved from CATNAP database.

Figure 2:. 007 recognizes the N332_gp120 glycan-independent V3 epitope

a, (left) Overviews of the four structural classes identified of SOSIP trimers with 0-, 1-, 2-, or 3-bound 007 Fabs per trimer. (right) The number of particles used in each of the final reconstructions. b, Overlay of 007 with V3-targeting bNAbs (PDB codes 5C7K, 5T3Z, 6CH7, 4JM2). c, Alignment of 007 VH and VL to their predicted germline V gene segments. 007 residues within 4Å of protein or glycan components on Env are indicated by colored circles. d, Structure overview, highlighting proximal glycans. e, Protein contacts between 007 and Env. f, Protein contacts between EPTC112 and Env (PDB code 8C8T). g, EM density highlighting the N156_gp120 (left) and N301_gp120 (right) glycans. h, Neutralizing activity (IC₅₀ and breadth of 007 and 10–1074) against HIV-1 pseudoviruses produced in the presence of kifunensine. i, Comparison of glycoform abundance between total BG505 SOSIP and 007-bound SOSIP at N301_gp120 determined by LC-MS/MS, with asterisks denoting level of significance (* denotes 0.01 < P ≤ 0.05, ** denotes P ≤ 0.01).

Figure 3:. Potent neutralizing activity requires 007 bivalency

007 exhibits avidity. a, Neutralization curves against strain X2278 (left) used to calculated molar neutralization ratios (MNRs) comparing the IC₅₀ values for the IgG, bispecific, and Fab forms of 007 (right). b, Molar IC₅₀ values for IgG, bispecific, and Fab forms of 007 against a panel of HIV-1 strains. Complete neutralization curves and IC₅₀ values are in Extended Data Fig. 7a and Supplementary Table 6. c, Distance measurements between the C-termini of the Fab heavy chains on the experimentally determined 2 Fab-bound trimer structure (left) compared to a structure in which 007 Fab was modelled onto an occluded-open trimer conformation (PDB code 5VN8). A schematic showing a 007 IgG-bound occluded-open trimer is shown for clarity. d, (left) Four structural classes identified after complexing 007 IgG with BG505 SOSIP. Schematics are included for clarity. Dashed line (yellow) indicates the distance measurements between C-termini of Fab regions in 007 IgG-bound trimer-dimer structure. (right) The number of particles used in each of the final reconstructions. The number of particles in the trimer-dimer class were multiplied by two, as each particle contained two SOSIP trimers.

Figure 4:. High antiviral activity against resistant virus strains driven by distinct neutralization profile and 324GD/NIR327 motif dependency

a, Neutralization breadth (%) and potency (IC₅₀/IC₈₀) of 007 against the 119 multiclade pseudovirus panel. b, Illustration of the neutralization profile of 007 in comparison to V3 glycan site reference bNAbs against different virus clades of the 119 multiclade panel. c, Dependency of 007 and V3 glycan site reference bNAbs on potential N-linked glycosylation sites and the ³²⁴GD/NIR³²⁷ motif. d, Neutralizing activity (GeoMean IC₅₀, breadth) of 007 compared with V3 glycan site reference bNAbs against a panel of 46 pseudovirus strains resistant to V3 glycan site bNAbs. Pie chart illustrates the clade distribution of resistant pseudovirus strains. e, Neutralizing activity against viral escape mutations within the V3 loop of the HIV-1 Env trimer. The top row displays bNAb IC₅₀ values for the BG505_T332N pseudovirus, while panels illustrate changes in bNAb sensitivity (IC₅₀ fold change) of pseudovirus mutants relative to BG505_T332N. Antibodies were tested in duplicates. f, Neutralizing activity of 007 in combination with V3 glycan site bNAbs (mixed at a 1:1 ratio) against the global pseudovirus panel. Single and combined mAbs were tested up to a concentration of 1μg/mL (total IgG amount). Red numbers indicate the fold change in IC₅₀s (increase in potency) between the individual mAb and its combination with 007. g, Computational modeling of the predicted neutralizing activity of bNAb 007 in combination with 10–1074 against the 119 multiclade pseudovirus panel using the CombiNaber tool (http://www.hiv.lanl.gov/content/sequence/COMBINABER/combinaber.html). Breadth (%) was calculated using a cut-off of ≤10 μg/mL (a-d, g). Data are shown for identical virus strains across each panel with available reference neutralization data (a-d, g). Reference bNAb data were sourced from the CATNAP database.

Figure 5:. Deep mutational scanning analyses reveal distinct viral escape from bNAb 007 in HIV-1 Env_TRO.11

a, Logo plots showing effects of mutations on neutralization escape in HIV Env_TRO.11 for antibodies 007, 10–1074, BG18, PGT121, and PGT128. The height of each letter represents the effect of that amino-acid mutation on antibody neutralization, with positive heights (letters above the zero line) indicating mutations that cause escape, and negative heights (letters below the zero line) indicating mutations that increase neutralization. Letters are colored by the effect of that mutation on Env mediated cell entry function, with yellow corresponding to reduced cell entry and brown corresponding to neutral effects on cell entry. Only key sites are shown. See https://dms-vep.org/HIV_Envelope_TRO11_DMS_007/htmls/all_antibodies_and_cell_entry_overlaid.html for interactive versions of the escape maps that show all mutations. Escape maps against HIV-1 Env_BF520 are shown in Extended Data Fig. 9c. b, Scatter plots of Env_TRO.11 mutant fold-change IC₅₀s measured by deep mutational scanning versus those measured in traditional neutralization assays. Each scatter plot shows log fold change IC₅₀s for neutralization assays using the antibody labeling the logo plot in the same row. Each point represents the mean of two replicate neutralization curve measurements of one Env_TRO.11 mutant. Env_TRO.11 mutants are colored by Env region or site. Vertical dotted lines represent the limit of detection of the neutralization assays. See methods section “Deep mutational scanning data analysis” for details on how deep mutational scanning measured escape values are converted to deep mutational scanning measured IC₅₀ values. The deep mutational scanning measured effects of mutations on escape from antibody 10–1074 shown in this Figure were previously published.

Figure 6:. 007 monotherapy and dual-targeting V3 glycan site combination therapy in HIV_ADA-infected humanized mice

a, Investigation of the antiviral activity of 10–1074 and 007 monotherapy in HIV-1_ADA-infected humanized mice. Graphs display the absolute HIV-1 RNA plasma copies/mL (top) and relative log₁₀ changes from baseline viral loads (bottom) after initiation of bNAb therapy. Dashed lines (top graphs) indicate the lower limit of quantitation of the qPCR assay (LLQ) (260 copies/mL). Red lines display the average log₁₀ changes compared to baseline viral loads (day −2). b, Analyses of single HIV-1 plasma env sequences from HIV-1_ADA-infected humanized mice obtained after viral rebound on day 35 for 007 and 10–1074 monotherapy groups. Total number of analyzed sequences is indicated in the center of each pie chart. Mice are labeled according to icon legends in A. Colored bars on the outside of the pie charts indicate mutations in V1/V2 loop and V3 loop. Sensitivity (IC₅₀s) of pseudoviruses generated from SGS-derived sequences against 007 and 10–1074. c, Sequential treatment with 007 or 10–1074 in HIV-1_ADA-infected humanized mice following viral rebound during 007 or 10–1074 monotherapy (from panel a). This approach included maintaining 007 or 10–1074 monotherapy while integrating 007 or 10–1074 in the treatment regimen. Dashed lines (top graphs) indicate the lower limit of quantitation of the qPCR assay (LLQ) (260 copies/mL). Red lines display the average log₁₀ changes compared to baseline viral loads (day 35). d, Antiviral activity of 007 and 10–1074 combination therapy in HIV-1_ADA-infected humanized mice. Graphs display the absolute HIV-1 RNA plasma copies/mL (top) and relative log₁₀ changes from baseline viral loads (bottom) after initiation of bNAb therapy. Dashed lines (top graphs) indicate the lower limit of quantitation of the qPCR assay (LLQ) (260 copies/mL). Red lines display the average log₁₀ changes compared to baseline viral loads (day −2). e, Analyses of single HIV-1 plasma env sequences from HIV-1_ADA-infected humanized mice obtained after viral rebound for 007 and 10–1074 combination therapy groups. Total number of analyzed sequences is indicated in the center of each pie chart. Mice are labeled according to icon legends in A. Colored bars on the outside of the pie charts indicate mutations in V1/V2 loop and V3 loop. Sensitivity (IC₅₀s) of pseudoviruses generated from SGS-derived sequences against 007 and 10–1074. f, Alignment of plasma SGS-derived env sequences obtained from individual mice (y-axis) after viral rebound following mono- or sequential therapy. Env sequences are shown as horizontal gray bars from residues 100–500 relative to HXB2 (x-axis). Mutations identified from day −2 are indicated in black, mutations after viral rebound following monotherapy in red (panels a and b), and following administration of 007 and 10–1074, either sequentially or in combination in blue (panels c, d and e). Sites at which selected mutations can confer resistance are highlighted by vertical blue bars.