Ethics and inclusion
This research complies with all relevant ethical regulations (Tulane National Primate Research Center IACUC protocol P0498 (3698) and The Wistar Institute IACUC protocols 201518 and 201522). Research has been conducted following the recommendations set out in the Global Code of Conduct for Research in Resource-PoorSettings including local researchers throughout the research process.
Envelope proteins
The newly engineered Env SOSIP trimers, WIN332, WIN332-GAIA–AviTag, WIN332–AviTag, 7MUT-ST2-Asn332Tyr and other previously reported engineered SOSIP Env trimers, RC1, RC1-glycan knockout (same as WIN332 glycan KO)10, 11MUTB, 10MUT, 7MUT, 5MUT11 and native-like BG505 Thr332Asn18, CEO217 (clade C), Q23 (clade A), AD8 (clade B), BJOX2000 (clade AE), CNE55 (clade C), 001428 (clade B), CH119 (clade BC) and AMC011 (clade B) were cloned in the pPPI4 or pVAX expression vectors using synthetic gene fragments (Integrated DNA technologies) or were produced by GenScript. Specific mutations for each protein including glycan deletions are listed in Extended Data Table 1.
All the SOSIP trimers used in this study include MD39 mutations for stabilization11 (Extended Data Table 1).
Env trimers were expressed as soluble native-like gp140 trimers18. Soluble Env trimers were expressed by transient transfection in Expi293 cells (Thermo Fisher Scientific) and purified from cell supernatants by Lectin chromatography and SEC as previously described33. Proteins were stored at −80 °C in PBS until the time of use.
Analysis of site-specific N-linked glycopeptides by liquid chromatography–mass spectrometry
Aliquots of purified WIN332 protein were reduced by incubating with 10 mM dithiothreitol (Sigma) at 56 °C and alkylated by 27.5 mM iodoacetamide (Sigma) at room temperature in the dark. The aliquots were then digested by using a combination of alpha lytic protease (New England BioLabs), AspN (Promega), chymotrypsin (Athens Research and Technology), Lys-C (Promega), Glu-C (Promega) and trypsin (Promega). The resulting peptides were separated on an Acclaim PepMap 100 C18 column (75 μm × 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific) at a flow rate of 200 nl min−1. The elution gradient consisted of 1–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV, and the temperature of the heated capillary was set to 275 °C. Full mass spectrometry scans were acquired from m/z 200 to 2,000 at a resolution of 60,000, and tandem mass spectrometry scans following higher-energy collisional dissociation with stepped collision energy (15%, 25%, 35%) were collected in the orbitrap at a resolution of 15,000. pGlyco3 (ref. 34) was used for database searches with mass tolerance set as 20 ppm for both precursors and fragments. The database search output was filtered to reach a 1% false discovery rate for glycans and 10% for peptides. The glycan and peptide assignment for each spectra was then manually validated after filtering. Quantification was performed by calculating spectral counts for each glycan composition at each site. Any N-linked glycan compositions identified by only one spectra were removed from quantification. N-linked glycan compositions were categorized into 19 classes (including ‘Unoccupied’ as class 19): HexNAc(2)Hex(9 ~ 5)Fuc(0 ~ 1) was classified as M9 to M5, respectively; HexNAc(2)Hex(4 ~ 1)Fuc(0 ~ 1) was classified as M1-M4; HexNAc(3 ~ 6)Hex(5 ~ 9)Fuc(0)NeuAc(0 ~ 1) was classified as Hybrid with HexNAc(3 ~ 6)Hex(5 ~ 9)Fuc(1 ~ 2)NeuAc(0 ~ 1) classified as F-Hybrid; Complex-type glycans are classified based on the number of antenna and fucosylation: HexNAc(3)Hex(3 ~ 4)Fuc(0)NeuAc(0 ~ 1) is assigned as A1 with HexNAc(3)Hex(3 ~ 4)Fuc(1 ~ 2)NeuAc(0 ~ 1) assigned as F-A1; HexNAc(4)Hex(3 ~ 5)Fuc(0)NeuAc(0 ~ 2) is assigned as A2/A1B with HexNAc(4)Hex(3 ~ 5)Fuc(1 ~ 5)NeuAc(0 ~ 2) assigned as F-A2/A1B; HexNAc(5)Hex(3 ~ 6)Fuc(0)NeuAc(0 ~ 3) is assigned as A3/A2B with HexNAc(5)Hex(3 ~ 6)Fuc(1 ~ 3)NeuAc(0 ~ 3) assigned as F-A3/A2B; HexNAc(6)Hex(3 ~ 7)Fuc(0)NeuAc(0 ~ 4) is assigned as A4/A3B with HexNAc(6)Hex(3 ~ 7)Fuc(1 ~ 3)NeuAc(0 ~ 4) assigned as F-A4/A3B; HexNAc(7)Hex(3 ~ 8)Fuc(0)NeuAc(0 ~ 1) is assigned as A5/A4B with HexNAc(7)Hex(3 ~ 8)Fuc(1 ~ 3)NeuAc(0 ~ 1) assigned as F-A5/A4B.
Analysis of deglycosylated HIV-1 envelope protein by liquid chromatography–mass spectrometry
Aliquots of the purified WIN332 protein were reduced by incubating with 10 mM dithiothreitol (Sigma) at 56 °C and alkylated by 27.5 mM iodoacetamide (Sigma) at room temperature in dark. The aliquots were then digested using chymotrypsin (Athens Research and Technology), Glu-C (Promega), Lys-C (Promega), Arg-C (Promega) and trypsin (Promega). Following digestion, the extracted peptides were deglycosylated by endoglycosidase H (Promega) followed by PNGaseF (Promega) treatment in the presence of 18O water (Cambridge Isotope Laboratories). The resulting peptides were separated on an Acclaim PepMap 100 C18 column (75 μm × 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific) at a flow rate of 200 nl min−1. The elution gradient consisted of 1–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV, and the temperature of the heated capillary was set to 275 °C. Full MS scans were acquired from m/z 200 to 2,000 at a resolution of 60,000, and tandem mass spectrometry scans following collision-induced dissociation at 38% collision energy and higher-energy collisional dissociation with stepped collision energy (15%, 25%, 35%) were collected in the ion trap. The spectra were analyzed using SEQUEST (Proteome Discoverer 2.5, Thermo Fisher Scientific) as well as Byonic (v4.1.10, Protein Metrics)35 with mass tolerance set as 20 ppm for precursors and 0.5 kDa for fragments. The search output was filtered to reach a 1% false discovery rate at the protein level and 10% at the peptide level. The site assignment for each spectra was then manually validated after filtering. Occupancy of each N-linked glycosylation site was calculated using spectral counts assigned to the 18O-Asp-containing (PNGaseF-cleaved) and/or HexNAc-modified (EndoH-cleaved) peptides and their unmodified counterparts.
Animals
Male rhesus macaques (Macaca mulatta) of Indian genetic origin, 4 years of age, were housed and cared for in a biosafety level-2 facility at the Tulane National Primate Research Center. All animal procedures and experiments were performed according to protocols approved by the Tulane National Primate Research Center Institutional Animal Care and Use Committee.
SMNP was produced as previously described22. At day 0, macaques received one subcutaneous bolus immunization with a total of ~200 μg of soluble WIN332 SOSIP trimer adjuvanted in 375 μg of SMNP distributed between the four legs (~50 µg per foreleg and ~50 µg per hind leg). Macaques were boosted with a total amount of 200 μg of soluble 7MUT-ST2-Asn332Tyr in 375 μg of SMNP adjuvant using an escalating dose delivery approach36 at week 8 (one dose of 30 µg of WIN332 in 56.5 µg SMNP at day 1, one dose of 60 µg of WIN332 in 112 µg SMNP at day 3 and one dose of 110 µg of WIN332 in 206.5 µg SMNP at day 5) and distributed between the four legs like the prime. Soluble trimers were selected based on previous unpublished immunization experiments in the humanized DH3-3/JH6 mice37. Blood samples were obtained from the macaques before immunization, 3 and 7 weeks after WIN332 prime and 3 weeks after 7MUT-ST2-Asn332Tyr boost immunization (week 11). Lymph node biopy samples were obtained from the immunized macaques 3 weeks after WIN332 prime and 3 weeks after boost immunization with 7MUT-ST2-Asn332Tyr.
Mice expressing the iGL precursor of the human V3-glycan bNAb PGT121 (C57BL/6 strain background) were provided by M. Nussenzweig (The Rockefeller University). Male and female mice were immunized intraperitoneally with 10 µg of WIN332 soluble Env trimer and three units of SMNP. Serum was collected 14 days later and used for ELISA analysis.
All mouse cages were on ventilated, ducted single-use racks. Cages were pre-bedded with alpha-dry bedding. Water bottles and cage supplies were irradiated. Cages were changed every 2 weeks, and lids and feeders were changed once a month inside a class-II biosafety cabinet using aseptic technique. Animals receive HCL water with a 3.0 pH. The Wistar Institute’s animal facility is operated as a modified barrier and with room-level protection. Holding rooms are negative to the corridors. Up to five adult mice or two adult mice plus one female with a litter may be housed in a cage (81 square inches). Temperature is within 64 to 79 degrees Fahrenheit and humidity is within 30% to 70%. Light cycle is 12-h on starting at 6:00 and 12-h off starting at 18:00.
ELISA
ELISAs with SOSIP Env trimers WIN332, RC1, WIN332glycanKO, 11MUTB, 10MUT, 7MUT, 5MUT and native Env trimers were performed as previously described10. Briefly, 96-well plates were coated with 50 μl of a solution of the trimer at 4 μg ml−1 in 1× PBS (Corning, 20-030-CV) and incubated at 4 °C overnight. Plates were washed with washing buffer (1× PBS with 0.05% Tween-20; Sigma-Aldrich, P7949) and blocked (1× PBS with 5% milk) for 1 h at room temperature. Serum samples were assayed at a 1:100 or 1:30 starting dilution, supernatants from Expi293 cells transfections were assayed at a 1:3 starting dilution and the antibody controls were added at 5 μg ml−1 all followed by seven additional threefold serial dilutions for 1 h at room temperature. Plates were washed three times and incubated with an anti-human IgG secondary antibody conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch, 115-035-071) at a 1:5,000 dilution in washing buffer for 1 h at room temperature. Plates were developed by adding ABTS, and optical density was measured at 405 nm.
Alternatively, 96-well plates were coated with 50 μl of a solution of PGT121 or 3BNC117 antibodies engineered to have a mouse Fc at 4 μg ml−1 in 1× PBS and incubated at 4 °C overnight. Plates were washed with washing buffer (1× PBS with 0.05% Tween-20), blocked (1× PBS with 5% milk) for 1 h at room temperature and incubated in 50 μl of a solution of an Env trimer at 4 μg ml−1 in blocking buffer for 1 h at room temperature. Plates were washed three times and incubated with 5 μg ml−1 of an antibody with human Fc and threefold serial dilutions for 1 h. Plates were washed three times and incubated with an anti-human IgG secondary antibody conjugated to HRP at a 1:5,000 dilution in washing buffer. Plates were developed by adding ABTS, and optical density was measured at 405 nm.
For competition ELISAs, 96-well plates were directly coated with 50 μl of a solution of an Env trimer at 4 μg ml−1 in 1× PBS and incubated at 4 °C overnight. Plates were washed three times in washing buffer and blocked in blocking buffer for 1 h at room temperature. Plates were then incubated in 50 μl of a 15 μg ml−1 solution of PGT121 or 3BNC117 with a mouse Fc in blocking buffer for 1.5 h at room temperature. Plates were washed three times and NHP serum or mAbs were added at the indicated starting dilutions and six additional threefold serial dilutions for 30 min at room temperature. Plates were washed and developed using anti-human IgG secondary antibody conjugated to HRP at a 1:5,000 dilution in washing buffer. Plates were developed by adding ABTS, and optical density was measured at 405 nm.
Endpoint titers were calculated as the reciprocal number of the dilution at which the sample optical density was three times the background.
Flow cytometry and single B cell sorting
Frozen cell homogenates from lymph node biopsy samples obtained from immunized macaques were thawed and washed in RPMI medium 1640 (1×; Gibco, 11875-093).
Lymph node cells were incubated with 100 μl of FACS buffer (PBS 1× with 2% fetal bovine serum and 1 mM EDTA) with human Fc Block (BD Biosciences, 564219, clone Fc1) at a 1:500 dilution for 30 min on ice.
WIN332, WIN332-GAIA and BG505 tetramers were prepared by incubating 5 μg of AviTagged and biotinylated WIN332 (WIN332–AviBio), WIN332-GAIA (WIN332-GAIA–AviBio) or BG505 (BG505–AviBio) with fluorophore-conjugated streptavidin at a 1:200 dilution in 1× PBS for 30 min on ice. WIN332++ WIN332-GAIA− or BG505++ lymph node B cells were isolated using WIN332–AviBio or BG505–AviBio conjugated to streptavidin AF647 (BioLegend, 405237), WIN332–AviBio or B41–AviBio conjugated to streptavidin PE (BioLegend, 405204) and WIN332-GAIA–AviBio conjugated to streptavidin BV605 (BioLegend, 405229) as baits. Tetramers were mixed with the antibody cocktails indicated below to a final concentration of 5 μg ml−1 each.
Macaque LN cells were stained with: anti-CD3 APC-eFluor 780 (Invitrogen, 47-0037-41, clone OKT3), anti-CD14 APC-eFluor 780 (Invitrogen, 47-0149-42, clone 61D3), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-4, clone CB16), anti-CD8 APC-eFluor 780 (Invitrogen, 47-0086-42, clone OKT8), anti-CD20 PE-Cy7 (BD Biosciences, 335793, clone L27) and anti-CD38 FITC (Stem Cell, 60131FI, clone AT-1) at a 1:200 dilution and the LIVE/DEAD marker Zombie NIR (BioLegend, 423106) at a 1:400 dilution. Cells were incubated with the mixture of tetramers and antibody cocktail for 30 min on ice.
Zombie NIR−/CD16−/CD8a−/CD3−/CD14−/CD20+/CD38+/WIN332++/WIN332-GAIA− or BG505++/WIN332-GAIA− single cells were isolated from the macaque lymph node cell homogenates using a FACS Aria III (Becton Dickinson). Single B cells were sorted into individual wells of 96-well plates containing 5 μl of lysis buffer (TCL buffer (Qiagen, 1031576) with 1% 2-β-mercaptoethanol). The cell lysates were stored at −80 °C or immediately used for subsequent mRNA purification.
Alternatively, trimer-specific B cells were sorted in RPMI with 5% FBS for 10x genomics.
Antibody sequencing
RNA was purified from single cells using paramagnetic beads (2.2× ratio; 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 H2O, Sigma-Aldrich, NP40S-100ML), 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)38. cDNA was stored at −20 °C or 10 μl of nuclease-free water was added to the cDNA before subsequent use for antibody gene amplification by nested PCR.
Macaque IgH and IgK/L genes were amplified and cloned38 using the primers and PCR protocols in Extended Data Tables 3 and 4 (ref. 39). Briefly, after a first PCR reaction to amplify the antibody genes, a second PCR (cloning PCR) introduced nucleotide tails that were used for subsequent ligation-independent cloning (LIC) in expression vectors39 (see ‘Antibody cloning’). The cloning PCR products were sequenced by Sanger sequencing using the primers in Extended Data Table 3.
10x Genomics
Single WIN332-specific germinal center B cells were bulk-sorted in RPMI with 5% FBS, processed by Wistar Genomics Core following the manufacturer protocol (Chromium Next GEM Single Cell V(D)J protocol v3) with modifications40 and macaque V(D)J libraries were generated.
Illumina NGS-generated raw data files were processed using the Cell Ranger single cell gene expression software provided by 10x Genomics. Sequences were quality filtered, followed by assembly and annotation using Cell Ranger adapted to contain rhesus macaque Ig genes from a custom reference. Contigs assembled by Cell Ranger were reannotated. To assign antibody sequences to each barcode, a custom Bash pipeline was developed to generate a CSV file that organizes query sequences by heavy chain (VH), light chain kappa (VK), and light chain lambda (VL). This pipeline incorporates a Python script that automatically aligns query sequences to germline V(D)J segments using the NCBI IgBLAST database. The resulting annotations are parsed and structured into separate columns based on their corresponding variable regions (VH, VK, VL). Finally, sequences sharing the same identifier are matched and paired by row to represent complete antibody repertoires. After pairing, sequences were analyzed with a custom pipeline with macaque immunoglobulin libraries to determine B cell immunogenetics and clonality41.
Antibody cloning
Antibody cloning was performed by GenScript or in house through LIC cloning. For LIC cloning, the cloning PCR products were purified using the PCR clean-up kit (Qiagen, 28183). The purified products were cloned into the corresponding expression vectors encoding human IgG, IgL and IgK constant regions digested with the restriction enzymes AgeI and SalI for IgH, AgeI and XhoI for IgL and AgeI and BsiwI for IgK as previously described39.
Antibody production and purification
Macaque IgGs were produced by GenScript or in house through transient transfection in Expi293T cells. For transfections, DNA mixtures containing IgH and IgL/IgK antibody genes at a 1:1 ratio were used for transfections following the manufacturer’s instructions (Thermo Fisher Scientific, A14635). IgGs were purified from the supernatants of transfected Expi293T cells using Protein G (Cytiva, 28-4083-47).
For structural studies, macaque Fab was obtained by digesting IgG at 1–5 mg ml−1 with ficin (Sigma). Fab was purified by SEC42, followed by monoQ 5/50 (GE Healthcare) ion exchange chromatography.
Serum immunoglobulins were purified from 200 μl of macaque serum using Ab Spin Trap Protein G Sepharose columns (GE Healthcare, 28-4083-47). Immunoglobulin-containing fractions were buffer exchanged with PBS using a 30-kDa molecular-weight-cutoff concentrator (Milipore Sigma, UFC903024).
In vitro neutralization assay
Env-pseudotyped viruses were produced following the standardized protocol43. Briefly, HEK 293T cells were seeded in 100-mm plates at a density of 4 × 10⁶ cells per plate and incubated overnight in complete growth medium. Cells were transfected using FuGENE 6 (Promega) with 4 µg of Env plasmid and 8 µg of backbone plasmid per plate. After a brief incubation (4–5 h), the medium was replaced, and the culture was maintained for 48 h. Virus-containing supernatants were harvested, supplemented with fetal bovine serum to a final concentration of 20%, filtered through 0.45-µm membranes, aliquoted into cryovials and stored at −80 °C.
Pseudovirus titers were determined using a tissue culture infectious dose assay in TZM-bl cells, a reporter cell line engineered to express luciferase under control of the HIV-1 Tat protein43. Serial fivefold dilutions of the pseudovirus stock were prepared in 96-well plates, and 10,000 TZM-bl cells per well were added in the presence of DEAE-dextran (Final concentration of 40 µg ml−1). After 48 h of incubation at 37 °C, luminescence was measured using the Firefly Luciferase Assay Kit (Merck). Virus input was considered optimal when luminescence signals fell within the range of 15,000 to 50,000 relative luminescence units.
TZM-bl assays were performed as described44. For the assay setup, 96-well flat-bottom plates were used. To reduce edge effects and minimize variability due to evaporation, all peripheral wells (columns 1, 11 and 12; rows A and H) were filled with sterile PBS and were not used for experimental conditions. The assay was conducted using the remaining internal wells. Column 2 served as the virus control (cells and virus without antibody), while columns 3 to 10 were used for serial dilutions of the test antibody or serum.
Serial threefold dilutions of the test antibody or purified IgGs from macaque serum were performed in duplicate, using wells from bottom to top (row G to row B) with a starting concentration of 100 µg ml−1 for WIN332, RC1 and 11MutB pseudoviruses, 200 µg ml−1 for 10Mut and 7Mut and 400 µg ml−1 for 5Mut, BG505 Thr332Asn, BG505 Asn301Ala and MLV. Serial threefold dilutions of purified IgGs from macaque serum were performed in duplicate, using wells from bottom to top (row G to row B) with a starting concentration of 300 µg ml−1. After dilutions were prepared, 50 μl of pseudovirus diluted in growth medium was added to all experimental wells (columns 2–10).
Plates were incubated at 37 °C for 1 h to allow virus–antibody interaction. During this time, a suspension of TZM-bl cells was prepared at 1 × 10⁵ cells per milliliter in growth medium containing DEAE-dextran (final concentration of 40 µg ml−1). Of this suspension, 100 μl was added to each well. Plates were incubated for 48 h at 37 °C in a humidified 5% CO2 atmosphere.
Neutralization activity was calculated as a function of the reduction in Tat-induced luciferase expression.
BLI (OCTET) binding studies
BLI experiments were performed using the OCTET Red96 system to determine affinities of antibodies (Fabs) for WIN332. Biotinylated WIN332–AviTag (WIN332–AviBio) was immobilized on high-precision streptavidin (SAX) biosensors (FORTÉBIO) using a solution of WIN332–AviBio at 5 µg ml−1 in dilution buffer (FORTÉBIO). Four serial dilutions (20, 10, 5 and 2.5 µg ml−1) of each experimental Fab or antibody and PGT121 antibody were prepared in dilution buffer (FORTÉBIO). Alternatively, the experimental antibody (5 µg ml−1) was immobilized on Anti-Human Fc Capture (AHC) Biosensors (FORTÉBIO) and assayed against four serial dilutions (20, 10, 5 and 2.5 µg ml−1) of WIN332. The binding experiment was performed at 30 °C using the following protocol: baseline 1 (60 s), WIN332 or antibody/Fab loading (300 s), baseline 2 (200 s), association (300 s) and dissociation (600 s). Analysis was performed using OCTET software Data Analysis HT 10.0 (FORTÉBIO).
EMPEM
Serum from each immunized macaque was tenfold diluted in PBS. Diluted sera were incubated with protein G sepharose 4 Fast Flow resin (Cytiva) overnight at 4 °C. Polyclonal IgG was purified by eluting in 0.1 M Glycine-HCl (pH 3.5) and neutralized immediately in 1 M Tris-HCl (pH 8). Purifed polyclonal IgG samples were further digested with 2% papain (Thermo Fisher). Around 2 mg of digested polyclonal Fab was incubated with 15 µg of WIN332 or RC1 trimer on ice or overnight at 4 °C. The complexes were purified by SEC through a Superose 6 Increase 10/300 GL column (Cytiva). Fractions corresponding to trimer–Fab complexes were concentrated. Four microliters of purified complexes at 0.01 mg ml−1 were applied to a glow-discharged, carbon-coated 400 mesh copper grid (Electron Microscopy Sciences). After blotting extra samples, grids were stained with 2% uranyl formate for 2 min followed by blotting extra staining buffer. Grids were imaged using a 100 kV FEI Tecnai T12 microscope at a magnification of ×57,000 equipped with a Gatan CCD camera. Collected micrographs were processed in Relion45. Specifically, Laplacian-of-Gaussian-picked particles were cleaned up by two-dimensional classification. Subset classes showing a clear trimer or Fab bound trimer were selected for three-dimensional (3D) classification. Data analysis and figure generation were performed in UCSF Chimera46.
Cryo-EM sample and grid preparation
RC1 and Ab1999 Fab or WIN332 and Ab1983 Fab were mixed together using a molar ratio of 1:9 (Env:Fab) and incubated for 1 h on ice. Complexes were purified by SEC through a Superose 6 increase 10/300 GL column (Cytiva). Fractions containing the Env–Fab complexes were pooled and concentrated down using 100-kDa molecular-weight-cutoff concentrators. Samples were flash frozen in liquid nitrogen and stored at −80 °C.
Complexes were thawed and diluted to around 0.085 g l−1. UltrAuFoil 1.2/1.3 300 mesh grids were glow discharged for 1 min and immediately used for graphene oxide application using a previously published protocol47. Complexes were vitrified by applying 4 μl of the diluted sample to the graphene oxide-coated grids using vitrobot Mark IV (FEI) at 4 °C and 100% humidity followed by plunging into liquid ethane.
Cryo-EM data collection and processing
Cryo-EM grids containing RC1–Ab1999 or WIN332–Ab1983 complexes were loaded onto a 200-kV Thermo Fisher Glacios Microscope equipped with a Falcon 4 detector. Dose-fractionated movies were collected using a total dose 40 e−/Å2 across 40 frames or 50 e−/Å2 across 49 frames for WIN332–Ab1983 or RC1–Ab1999, respectively. Both datasets were collected with a defocus range of −0.8 μm to −2.2 μm.
Data processing for both datasets was done similarly. In summary, raw movies were imported into CryoSPARC and preprocessed using Patch Motion Correction and Patch CTF Estimation. Particles were picked using a blob picker and then extracted with binning by 4 resulting in pixel size of 3.79 Å per pixel. Particles were first cleaned up using two-dimensional classification and particles in the junk classes were discarded. Remaining particles were used for ab initio reconstruction, and the resulting 3D volumes were used for subsequent heterogeneous refinement. Particles belonging to the good 3D classes were selected and reextracted with binning by 2. This was followed by another round of heterogeneous refinement. The selected particles were then reextracted with the original pixel size and used for non-uniform refinement. The resulting auto-sharpened maps were used for model building and analysis.
Model building
Initial models of Ab1999 and Ab1983 Fabs were generated using SWISS-MODEL48 without reference structures. The previously published model of RC1 (PDB 6ORO) was used as the initial model for model building for both RC1–Ab1999 and WIN332–Ab1983. The WIN332 Env model was modified by deleting the Asn332 glycan and mutating the asparagine into glutamine. Reference models were docked into the density maps in UCSF ChimeraX49 and manually real-space refined in Coot50. N-linked glycans were added to the models manually in Coot50. FModels were refined in Rosetta and real-space refined in Coot iteratively. Model geometry was validated by MolProbity51, glycan conformation was validated by Privateer52, and model fit-to-map was validated by EMRinger53.
Analysis
Geneious Prime was used for sequence analysis. Flow cytometry data were processed using FlowJo v.10.5.0. GraphPad Prism 10 was used for data analysis. Structural analysis was performed using the software indicated above. For antibody analysis, a custom V(D)J database was used with modifications41.
Quantification and statistical analysis
Statistical information, including sample size (n), mean, standard deviation and statistical significance values, are indicated in the text or the figure legends. GraphPad Prism 10 was used for statistical analysis by paired 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 Portfolio Reporting Summary linked to this article.