Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Envelope proteins

Env trimers were expressed as soluble native-like gp140 trimers21. The newly engineered Env SOSIP trimers, RC1, RC1-4fill, RC1–Avitag, RC1–SpyTag, RC1-glycanKO, RC1-glycanKO–Avitag, RC1-glycanKO–GAIA and RC1–GAIA, BG50521 and the BG505 variants 11MUTB, 10MUT, 7MUT and 5MUT20 were cloned in the pPPPI4 expression vector using synthetic gene fragments (Integrated DNA Technologies (IDT)). The glycan variants RC1Δ301, RC1Δ332 and 11MUTBΔ301 were produced by site-directed mutagenesis (QuikChange Lightning Multi-site directed mutagenesis kit, Agilent Technologies). Specific modifications of each protein are listed in Extended Data Table 2.

Soluble Env trimers were expressed by transient transfection in HEK293-6E cells (National Research Council of Canada) or Expi293 cells (Life Technologies) and purified from cell supernatants by 2G12 or NIH45-46 immunoaffinity chromatography and size-exclusion chromatography (SEC) as previously described38. Proteins were stored at 4 °C in 20 mM Tris pH 8.0 and 150 mM sodium chloride (TBS buffer). SpyTagged immunogens were buffer-exchanged into 20 mM sodium phosphate pH 7.5, 150 mM NaCl. Cell lines were not tested for mycoplasma contamination.

VLP production and conjugation

A C-terminal SpyTag sequence (13 residues) was added to RC1-4fill to form an irreversible isopeptide bond to SpyCatcher protein31. We produced and purified SpyCatcher–AP20539 VLPs as described30 and separated conjugated VLPs from free Env trimers by SEC on a Superdex 200 column. Conjugation of Env trimers was verified by negative-stain electron microscopy and/or SDS–PAGE (Fig. 3, Extended Data Fig. 4), and immunogen concentrations were estimated by comparing to known amounts of free immunogen run on the same SDS–PAGE gel. Conjugated and unconjugated VLPs were compared by negative-stain electron microscopy on a FEI Tecnai T12 transmission electron microscope at 120 keV using a Gatan Ultrascan 2k × 2k CCD detector.

Mass spectrometry

The glycosylation profiles of RC1 and RC1-4fill were determined as previously described40. In brief, samples were denatured with Lys-C (Promega), Arg-C (Promega), Glu-C (Promega) and chymotrypsin (Promega). Following digestion, the samples were deglycosylated by Endo H (Promega) and PNGase F (Glyko, Prozyme) in the presence of 18O water (Cambridge Isotope Laboratories). The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm × 15 cm) and analysed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) with a 240-min linear gradient consisting of 1–100% solvent B over 180 min at a flow rate of 200 nl min−1. Full mass spectrometry scans were acquired using the Fusion instrument software (v.2.0, Thermo Fisher Scientific), and the resulting spectra were analysed and filtered using SEQUEST (Proteome Discoverer 1.4, Thermo Fisher Scientific) and ProteoIQ (v.2.7, Premier Biosoft). Site occupancy was calculated using spectral counts assigned to the 18O-Asp-containing (PNGase-F-cleaved) and/or HexNAc-modified (Endo-H-cleaved) peptides and their unmodified counterparts.

Animals

Mice carrying the inferred germline IgH and IgL human PGT121 and 10-1074 bNAbs (GL HL 121 knock-in mice) were previously described9. Six-to-eight-week-old C57BL/6J male mice from The Jackson Laboratory were used for immunizations. All animal procedures were performed in accordance to protocols approved by the Rockefeller University Institutional Animal Care and Use Committee (IACUC). Male and female GL HL 121 knock-in mice or C57BL/6J wild-type mice were equally distributed in groups and immunized intraperitoneally with 10 μg of soluble SOSIP Env trimer in Ribi adjuvant (Sigma-Aldrich) (1:1).

Six-month-old New Zealand white rabbits (Covance) were used for immunizations. Rabbits were immunized subcutaneously with approximately 22 μg of RC1-4fill SOSIP Env trimer conjugated to VLP (VLP-RC1-4fill) in an ISCOMs-like saponin adjuvant (see below). Serum samples were collected from mice and rabbits on weeks 0 and 2 after immunization. All procedures in rabbits were approved by the Denver PA IACUC.

Sixteen rhesus macaques (Macaca mulatta) of Indian genetic origin, two-to-four years of age, were housed and cared for in accordance with Guide for Care and Use of Laboratory Animals Report number NIH 82-53 (Department of Health and Human Services, Bethesda, 1985) in a biosafety level 2 NIH facility. All animal procedures and experiments were performed according to protocols approved by the IACUC of NIAID, NIH.

Macaques were immunized subcutaneously in the medial inner forelegs and hind legs (total of four sites per animal) with approximately 200 μg (experiment 1; Fig. 3f) or 100 μg (experiment 2; Extended Data Fig. 5a) of RC1-4fill SOSIP trimer conjugated to VLP (RC1-4fill VLP) adjuvated in IscoMPLA. Blood and lymph node biopsies were obtained from naive macaques and from the immunized macaques three weeks after immunization.

Adjuvant synthesis

ISCOM-like saponin adjuvant was prepared as described41. Final adjuvant concentrations were determined by cholesterol quantification (Sigma-Aldrich, MAK043).

ELISA

ELISAs with SOSIP Env trimers 11MUTB, RC1, 11MUTBΔ301, RC1Δ301, RC1–GAIA, RC1-glycanKO, RC1-glycanKO–GAIA, RC1Δ332, BG505, 10MUT, 7MUT, 5MUT or the V3 loop-consensus C peptide (KGKGKGKGKGCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHC) were performed as described9. Serum samples were assayed at a 1:100 or 1:30 starting dilution and seven additional threefold serial dilutions. Mouse and human IgGs or human Fabs were evaluated at concentrations specified in the main text.

Alternatively, 96-well plates were directly coated with 50 μl of a solution of Fab at 20 μg ml−1 in 1× PBS overnight at 4 °C, washed and blocked as above and incubated in 50 μl of a solution of RC1 or RC1-glycanKO–GAIA at 2 μg ml−1 in blocking buffer for 1 h at room temperature. Plates were washed as above and developed using a chimeric version (human Fabs and mouse crystallizable fragment (Fc)) of the CD4-binding site bNAb 3BNC6042 at threefold serial dilutions starting at 5 μg ml−1 followed by anti-mouse IgG secondary antibody conjugated to HRP (Jackson ImmunoResearch, 115-035-071).

For anti-idiotype ELISAs, 96-well plates were coated with 50 μl of a solution of IgG at 10 μg ml−1 in 1× PBS overnight at 4 °C, washed and blocked as above and incubated with biotinylated anti-inferred germline PGT121 idiotypic antibody. Plates were developed with streptavidin conjugated to HRP.

Flow cytometry and single B cell sorting

Single-cell suspensions were obtained from the draining lymph nodes and spleens of immunized mice, and mature B cells were isolated by negative selection using anti-CD43 magnetic beads (MACS) following the manufacturer’s instructions.

Frozen peripheral blood mononuclear cells or cells from lymph node biopsies obtained from the naive and immunized macaques were thawed and washed in RPMI 1640 medium (1×) (Gibco, 11875-093). Mouse or macaque cells were incubated with 100 μl of FACS buffer (1× PBS with 2% fetal bovine serum and 1 mM EDTA) with mouse (BD Biosciences, 553142) or human (BD Biosciences, 564219) Fc Block, respectively, at a 1:500 dilution for 30 min on ice.

RC1 and RC1-glycanKO (RC1+RC1-glycanKO−) tetramers were prepared by incubating 5 μg of Avitagged and biotinylated RC1 (RC1–AviBio) or Avitagged and biotinylated RC1-glycanKO (RC1-glycanKO–AviBio) with fluorophore-conjugated streptavidin at a 1:200 dilution in 1× PBS for 30 min on ice.

RC1+RC1-glycanKO− mouse B cells were isolated using RC1–AviBio conjugated to streptavidin BV711 (BD Biosciences, 563262) and RC1-glycanKO–AviBio conjugated to streptavidin–PE (BD Biosciences, 554061) as baits. RC1+RC1-glycanKO− macaque B cells were isolated using RC1–AviBio conjugated with streptavidin–PE and streptavidin–AF647 and RC1-glycanKO–AviBio conjugated with streptavidin–BV605 (BD Biosciences, 563260). Tetramers were mixed with the human or mouse antibody cocktails indicated below to a final concentration of 5 μg ml−1 each.

Mouse cells were stained with anti-CD4–APC-eFluor780 (Invitrogen, 47-0042-82), anti-CD8–APC-eFluor780 (Invitrogen, 47-0081-82), anti-F4/80–APC-eFluor780 (Invitrogen, 47-4801-82), anti-NK1.1–APC-eFluor780 (Invitrogen, 47-5941-82), anti-CD11b–APC-eFluor780 (eBioscience, 47-0112-82), anti-CD11c–APC-eFluor780 (eBioscience, 47-0114-82), anti-Gr-1–APC-eFluor780 (Invitrogen, 47-5931-82), anti-B220–APC (Biolegend, 103212), anti-GL7–FITC (BD Biosciences, 553666) and anti-CD95–BV421 (BD Biosciences, 562633) antibodies at 1:200 dilution and the live/dead marker Zombie NIR (Biolegend, 77184) at a 1:400 dilution in FACS buffer. Macaque cells were stained with anti-CD16–APC-eFluor780 (Invitrogen, 47-0168-41), anti-CD8a–APC-eFluor780 (Invitrogen, 47-0086-42), anti-CD3–APC-eFluor780 (Invitrogen, 47-0037-41), anti-CD14–APC-eFluor780 (eBiosciences, 47-0149-41), anti-CD20–PE-Cy7 (BD, 335793), anti-CD38–FITC (Stem Cell Technologies, 60131FI), anti-IgG–BV421 (BD Biosciences, 562581) and anti-IgM–PerCP-Cy5.5 (BD Biosciences, 561285) antibodies at a 1:200 dilution and the live/dead marker Zombie NIR at a 1:400 dilution in FACS buffer.

Zombie NIR−CD4−CD8−F4/80−NK1.1−CD11b−CD11c−B220+GL7+CD95+RC1+RC1-glycanKO− single cells were isolated from the mouse cell homogenates and Zombie NIR−CD16−CD8a−CD3−CD14−CD20+CD38+IgG+/−double RC1+RC1-glycanKO− single cells were isolated from the macaque cell homogenates using a FACS Aria III (Becton Dickinson).

Single cells were sorted into individual wells of a 96-well plate containing 5 μl of lysis buffer (TCL buffer (Qiagen, 1031576) with 1% of 2-β-mercaptoethanol). Plates were immediately frozen on dry ice and stored at −80 °C.

Antibody sequencing and cloning

Single-cell RNA was purified using magnetic beads (RNAClean XP, A63987, Beckman Coulter). RNA was eluted from the magnetic beads with 11 μl of a solution containing 14.5 ng μl−1 of random primers (Invitrogen, 48190-011), 0.5% of tergitol (type NP-40, 70% in H 2 O, Sigma-Aldrich, NP40S-100ML) and 0.6 U μl−1 of RNase inhibitor (Promega, N2615) in nuclease-free water (Qiagen), and incubated at 65 °C for 3 min. cDNA was synthesized by reverse transcription (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044, 10,000 U)43. cDNA was stored at −80 °C or used for antibody gene amplification by nested polymerase chain reaction (PCR) after addition of 10 μl of nuclease-free water.

Mouse and macaque antibody genes were cloned43 using the primers in Supplementary Table 6. PCR protocols were as follows for annealing (°C)/elongation (s)/number of cycles: first PCR (IgG IgH and Igλ): 46/55/50; second PCR (IgG IgH and Igλ): 50/55/50.

Inferred germline macaque IgGs and Fabs were produced by reverting all nucleotide mutations in the V(D)J antibody genes to their corresponding inferred germline sequences while conserving the N nucleotides from the V(D)J junctions found in the mutated antibodies.

Antibody production and purification

Immunoglobulins were purified from 200 μl of mouse or macaque serum using Ab Spin Trap Protein G Sepharose columns (GE Healthcare, 28-4083-47). Immunoglobulin-containing fractions were buffer-exchanged with PBS by overnight dialysis at 4 °C (dialysis cassettes 20000 MWCO, Thermo Fisher Scientific, 66005).

For structural studies, mouse IgGs and macaque His 6 -tagged Fabs were expressed by transient transfection in HEK293-6E or Expi293 cells and purified from cell supernatants using protein A or G (GE Healthcare) (for IgGs) or Ni-NTA (GE Healthcare) or Ni Sepharose 6 Fast Flow (GE Healthcare) (for Fabs) chromatography and SEC44. Mouse Fab was obtained by digesting IgG at 1−5 mg ml−1 with ficin (Sigma-Aldrich). Fab was purified by protein G (GE Healthcare) and SEC chromatography45, followed by monoQ 5/50 (GE Healthcare) ion-exchange chromatography. The common inferred germline of the PGT121 and 10-1074 bNAbs17 was expressed as a His 6 -tagged Fab.

In vitro neutralization assay

TZM-bl assays were performed as previously described46. In brief, neutralization activity was calculated as a function of the reduction in Tat-induced luciferase expression in the TZM-bl reporter cell line after a single round of virus infection with Env pseudoviruses.

SPR and OCTET binding studies

SPR experiments were performed using a T200 (Biacore). For measuring the affinity for PGT121/10-1074 inferred germline Fab, protein A was immobilized on a CM5 chip by primary amine chemistry (Biacore manual) and 200 nM 8ANC195 G52K5 IgG or a non-HIV Env-binding IgG (mG053) was injected as previously described44. Then, 1 μM human Fc was injected to block remaining protein A sites. After capturing 10 μM RC1, 11MUTB or 10MUT, a concentration series of PGT121/10-1074 inferred germline Fab (fourfold dilutions from a top concentration of 160 μM for 10MUT, and twofold dilutions from a top concentration of 150 μM for 11MUTB and RC1) was injected, and binding reactions were allowed to reach equilibrium. K D values were derived by nonlinear regression analysis of plots of R eq (equilibrium binding response) versus the log of the injected protein concentration and the data were fitted to a 1:1 binding model47. To measure the affinity of Ab275 MUR , a concentration series of Fab was injected over immobilized RC1 or 11MUTB (fourfold dilutions from a top concentration of 50 μM). K D values were calculated from the on/off rates (k a /k d ), which were derived using a 1:1 binding model from seven concentrations of Ab275 MUR Fab (3.125 μM to 0.763 nM). Flow cells were regenerated as previously described44 with 1 M guanidine HCl and/or 10 mM glycine pH 2.0 at a flow rate of 90 μl min−1.

OCTET experiments were performed using the OCTET Red96 system to determine affinities of inferred germline and mutated macaque Fabs for RC1. Biotinylated RC1–Avitag was immobilized on high-precision streptavidin (SAX) biosensors (FORTÉBIO) using a solution of biotinylated RC1–Avitag at 400 nM in dilution buffer (FORTÉBIO). Four serial dilutions of each macaque Fab, one irrelevant Fab and 3BNC60 Fab were prepared in dilution buffer (FORTÉBIO). The binding experiment was performed at 30 °C using the following protocol: baseline 1 (60 s), load RC1 (300 s), baseline 2 (200 s), Fab association (300 s) and Fab dissociation (600 s). Analysis was performed using OCTET software Data Analysis HT 10.0 (FORTÉBIO).

Cryo-EM sample and grid preparation

RC1 complexed with 10-1074 was prepared by incubating purified RC1 with 10-1074 Fab and a CD4-binding site (CD4bs) Fab at a 1:3:3 molar ratio (gp140 protomer:10-1074 Fab:CD4bs Fab) overnight at room temperature. The RC1–Fab complex was isolated by SEC in TBS (20 mM Tris pH 8.0, 100 mM NaCl) using a Superdex-200 Increase 10/300 column (GE Healthcare). RC1–mouse/macaque Fab complexes were prepared by incubating purified RC1 with a mouse or macaque Fab and with 8ANC195 Fab42 at a 1:1.3:1.3 molar ratio as above and used without SEC purification. RC1–Fab complexes were diluted to 0.75–1.4 mg ml−1 in TBS, and 3 μl was added to Quantifoil R1.2/1.3 300 mesh copper grids (Electron Microscopy Services) that had been freshly glow-discharged using a PELCO easiGlow (Ted Pella). Samples were vitrified in 100% liquid ethane using a Mark IV Vitrobot (Thermo Fisher Scientific). Sample preparation conditions are summarized in Extended Data Table 1.

Cryo-EM data collection

RC1–Fab complexes were collected on a 200 kV Thermo Fisher Talos Arctica electron microscope using EPU automated image acquisition software48. Movies were collected on a Falcon 3EC direct electron detector (Thermo Fisher Scientific) operating in counting mode at a nominal magnification of 73,000× (1.436 Å per pixel) using a defocus range of −1.4 μm to −3.0 μm or −0.8 μm to −2.5 μm. Data for the RC1–10-1074 complex were collected across two separate sessions and combined during data processing. Microscope conditions are summarized in Extended Data Table 1.

Cryo-EM data processing

Movies were motion-corrected and dose-weighted using the MotionCor249 frame alignment program in RELION-350. Dose-weighted summed images were used for CTF determination using Gctf51, and reference-free particle picking from each micrograph was achieved using Laplacian-of-Gaussian filtering in RELION-350. Unbinned extracted particles were imported into cryoSPARC v.252 and subjected to reference-free two-dimensional classification using a 240-Å circular mask. Particles from the best two-dimensional classes were selected for heterogeneous ab initio model generation (two models). The best model exhibited C3 symmetry and was used as an initial model for homogenous three-dimensional auto-refinement in cryoSPARC v.252. Resolutions were estimated using the gold standard Fourier shell correlation (FSC = 0.143)53, and maps were auto-sharpened in cryoSPARC52. For interpreting N-linked glycans, maps were generated with overall B-factors ranging from −150 to −400 Å2 to improve local features and map connectivity54. See Extended Data Fig. 2 and Extended Data Table 1.

Model building

Initial coordinates were generated by docking reference models into the maps using UCSF Chimera55. For the RC1–10-1074 complex, BG505 Env and 10-1074 Fab (PDB code 53TZ) were docked into the density maps. For RC1 complexes with mouse or macaque Fabs, BG505 Env and PGT121/10-1074 inferred germline (PDB codes 5CEZ and 4FQQ) coordinates were docked into density maps. Initial models were refined into electron microscopy maps using rigid body refinement55. Models were built using Fab and RC1 sequences following iterative rounds of real-space refinement in Coot and PHENIX56,57. Coordinates for glycans were added as Man 9 and then trimmed to fit the maps at σ = 5. Model validation was done using MolProbity58 and Privateer59. Superposition calculations and molecular representations were generated with PyMOL (v.1.5.0.4 Schrodinger), UCSF Chimera55 and ResMap60.

Analysis

MacVector v.15.5.3 was used for sequence analysis and graphs were created using R language. Flow cytometry data were processed using FlowJo v.10.5.0. GraphPad Prism 7 was used for data analysis. Immunoglobulin gene sequence AB1 files were converted to FASTQ format using the Biopython package. FASTQ files were trimmed by quality using cutadapt v.1.18 software. Igblast v.1.9.0 was used for VDJ assignment and clone analysis was performed using Change-O software v.0.3.7. For macaques, a custom VDJ database was created using previously reported immunoglobulin gene sequences61.

Quantification and statistical analysis

Statistical information, including n, mean and statistical significance values, is indicated in the text or the figure legends. GraphPad Prism 7 was used for statistical analysis by unpaired Student’s t-test. Data were considered statistically significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.