Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates

Ethics statement

Cynomolgus macaques (Macaca fascicularis) originating from Mauritius and imported from AAALAC certified breeding centers were used in this study. All animals were housed in groups at the IDMIT infrastructure facilities (CEA, Fontenay-aux-roses, Animal facility authorization #D92-032-02, Prefecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, the French regulations, and the Standards for Humane Care and Use of Laboratory Animals, of the Office for Laboratory Animal Welfare (OLAW, assurance number #A5826-01, US). The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives » (CEtEA #44) under statement number A15-073. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#3132-2015121014521340.

Processing of animal products

Six adult, female cynomolgus macaques, aged 33 to 42 months were immunized on week 0 (W0), W8, and W24 with 20 µg ConM SOSIP.v7 and subsequently on W38 and W70 with 20 µg ConSOSL.UFO.664. ConM SOSIP.v7 was administered to MCM01, MCM02, and MCM03 adjuvanted with a liposomal formulation of monophosphoryl lipid A (MPLA), intramuscularly (IM). MCM05 and MCM06 received ConM SOSIP.v7 adjuvanted with MPLA, subcutaneously (SC). Lastly for MCM04 ConM SOSIP.v7 was adjuvanted with squalene emulsion (SE) and administered IM. All vaccinations with ConSOSL.UFO.664 were adjuvanted with liposomal MPLA and administered IM. Blood was taken at 25 and 71 weeks after the first immunization for peripheral blood mononuclear cell (PBMC) isolation and on weeks 26 and 72 for serum sampling. PBMCs were isolated from the blood using Ficoll as previously described40. In short, phosphate-buffered saline (PBS) diluted blood was loaded on a Ficoll layer and centrifuged for 30 min at room temperature (RT) at 4000 × g with a deceleration speed of 0. From this PBMCs were isolated, washed with PBS and re-suspended in ACK buffer (Thermo Fisher Scientific). PBMCs were subsequently suspended in 10% DMSO in fetal calf serum (FCS) and frozen at −150 °C.

Sorting of ConM SOSIP.v7/ConSOSL.UFO.664 immunogen reactive B cells

Frozen PBMCs were thawed and washed with PBS to use for fluorescently labeled cell sorting (FACS) to select for ConM SOSIP.v7 and ConSOSL.UFO.664 specific B cells. 2 µg biotinylated ConM SOSIP.v7 and ConSOSL.UFO.664 Env proteins were both conjugated to both 0.5 mg streptavidin-AF647 and 0.25 mg streptavidin-BV421 (BioLegend). Biotinylated ConM-BG505V1V2 Env protein was conjugated to 0.25 mg streptavidin-BB515 (BD Horizon). ~10 million PBMCs were stained with all conjugated ConM SOSIP.v7, ConSOSL.UFO.664, and ConM-BG505V1V2 Env proteins, Via eF780 (Invitrogen), IgM BV605 (BioLegend), IgG PE-cy7 (BD Horizon), CD20 PE-CF594 (BD Horizon), and CD27 PE (BD Horizon). Staining was done for 30 min at 4 °C in the dark. Subsequently, cells were washed twice and re-suspended in PBS+FCS (1:100). Cells were analyzed and single cell sorted using the FACS Aria III cell sorter (BD Biosciences) using index sorting. The gating strategy used is depicted in Supplementary Fig. 6. We first selected lymphocytes and single cells, then life CD20+B cells. Probe binding was analyzed within this CD20+B cell population. Immunoglobulin (Ig) subtype and CD27 expression was measured. Env probe specific B cells were single cell sorted in a 96-wells plate containing lysis buffer (RNAse inhibitor (40 U/µl) (Thermo Fisher Scientific), 5X First strand superscript III buffer (Invitrogen), 0.1 M dithiothreitol (DTT) (Invitrogen), and Milli-Q) and immediately stored at −80 °C.

Amplification and cloning of antibody variable regions

mRNA of the lysed and sorted B cells was converted to cDNA by a reverse transcriptase (RT)-PCR reaction40. 6 µL RT-PCR reaction mixture (random hex primers 200 ng/µL (Thermo Fisher Scientific), dNTP mix 6 mM each (New England BioLabs), 50 U Superscript III RTase, and MQ) was added directly to the lysed and sorted B cells. The following PCR program was run; 42 °C, 10 min 25 °C, 10 min 50 °C, 60 min 95 °C, 5 min. cDNA was subsequently used to amplify DNA of the heavy and light (kappa and lambda) chain of the expressed B cells in three PCR reactions for each chain. For the heavy chain: PCR 1 used HotStar Plus DNA polymerase (0.38 U) (Qiagen) in a mixture of 10× PCR buffer, dNTPs (New England BioLabs) (0.2 mM), forward and reverse primer sets25 (0.17 µM), 2 µl of RT-PCR cDNA and MQ. PCR program run: 95 °C, 5 min; 50 cycles of 94 °C 30 s, 55 °C, 30 s, 72 °C, 1 min. PCR 2 was done with MyTaq polymerase (0.5 U) (Bioline) using 2 µl of PCR 1 DNA product, 5× MyTaq reaction buffer, forward and reverse primer sets (0.1 µM) and MQ in a total volume of 20 µl. PCR program run: 95 °C, 1 min; 50 cycles of 95 °C, 15 s; 55 °C, 15 s; 72 °C, 45 s. The final PCR was used to create the overhang needed for the subsequent cloning reactions. This PCR 3 was done with HotStar HiFi DNA polymerase (0.25 U) (Qiagen) in a mixture of 5x PCR buffer, forward and reverse primer mixes (1.3 µM), MQ, and 2 µl DNA product from PCR 2. For both kappa and lambda light chains PCR 1 and PCR 2 were done using MyTaq DNA polymerase (Bioline) (0.25 U) as described for the heavy chain, using kappa or lambda primer sets (1.3 µM) and 2 µl of either RT-PCR or PCR 1 DNA product. Annealing temperatures for kappa chain PCR 1 were adapted to 56 °C and for PCR 2 to 55 °C. For the lambda chain the annealing temperatures were 52 °C and 53 °C for PCR 1 and PCR 2 respectively. PCR 3 for both kappa and lambda chains was done with HotStar Plus DNA polymerase (0.38 U) (Qiagen) in a mixture of 10× PCR buffer, dNTPs (0.2 mM), specific forward and reverse primer sets (0.17 µM), MQ, and 2 µl of PCR 2 DNA product. Following PCR program was run: 95 °C, 1 min; 25 cycles of 94 °C, 30 s; 58 °C, 30 s; 72 °C, 1 min.

PCR 3 DNA product was used for Gibson cloning into expression vectors41. 1 µl PCR product was added to 1 µl of expression vector DNA and 2 µl of 2× Gibson mix (T5 exonuclease (0.2 U) (Epibio), Phusion polymerase (12.5 U) (New England BioLabs), Taq DNA ligase (2000 U) (New England BioLabs), Gibson reaction buffer (0.5 g PEG-8000, 1 M Tris/ HCl pH 7.5, 1 M MgCl2, 1 M DTT, 100 mM dNTPs, 50 mM NAD (New England BioLabs), MQ). Mixtures were incubated for 1 h at 50 °C, after which they were transformed into competent XL-1 blue cells (Agilent) for mini- or midiprep DNA isolation using the Macherey-Nagel plasmid DNA isolation kit.

Envelope glycoprotein design and production

For single B cell sorting and subsequent analysis the ConM SOSIP.v7 Env protein, ConM-BG505V1V2 variant, and ConSOSL.UFO.664 protein were used. These Env proteins were produced using PGT145 affinity columns20,22. The expression plasmids of the protein and furin were transiently expressed (ratio 4:1) in HEK293F cells (Invitrogen, catalog number R79009). The Env trimers were harvested from the cell supernatants at day 7 by centrifugation at 4000 × g for 20 min, followed by 0.22 µM steritop- vacuum-filteration before purification by gravity-driven chromatography on a PGT145 antibody-conjugated Sepharose column. Env proteins were eluted with 3 M Mg2Cl2 pH 7.8, directly into neutralization buffer (20 mM TrisHCl pH8.0, 75 mM NaCl). After purification, Env proteins were concentrated and buffer exchanged into phosphate-buffered saline (TBS) to a final concentration of >0.5 mg/ml using Vivaspin 100kD filters (GE healthcare). Purified ConM SOSIP.v7 and ConSOSL.UFO.664 Env trimers were biotinylated for FACS using the BirA biotin protein ligase kit (Avidity) following the manufacturer’s instructions. In addition, ConM SOSIP.v9.0 and ConS SOSIP.v9.0 proteins were produced in a similar way and used for antibody analyses42.

ConM SOSIP.v9.0 trimers were analyzed using SDS-PAGE and BN-PAGE followed by Coomassie blue staining and thermal denaturation was studied using nano-differential scanning calorimetry (DSC)22. In short, Env trimers were first buffer exchanged to PBS, and concentration was adjusted to ~0.25 mg/mL. After loading the sample into the cell of a nano-DSC calorimeter (TA instruments, The Netherlands), thermal denaturation was probed at a scan rate of 60 °C/h. Buffer correction, normalization, and baseline subtraction procedures were applied before the data were analyzed using the NanoAnalyze Software v.3.3.0 (TA Instruments). The data were fitted using a non-two-state model, as the asymmetry of some of the peaks suggested that unfolding intermediates were present. Trimer morphology was assessed by NS-EM, as described below.

Antibody production and purification

To express mAbs for characterization, heavy and light chain plasmid DNA was co-transfected into HEK293F cells and purified (Invitrogen, catalog number R79009)19,40. 40 µg of each heavy and light chain DNA was added with 240 µg PEImax transfection agent to OptiMEM. Mixture was incubated for 20 min at RT, poured to HEK293F cells, and incubated for 5 days at 37 °C, shaking. Supernatant was harvested by spinning down the cells for 30 min at 4000 × g and subsequently was 0.22 µM filtered using vacuum. Expressed mAbs were affinity purified using Protein G (Thermo Fisher Scientific) coupled beads. Protein G bound mAbs were eluted using 0.1 M glycine, pH 2.0. Eluted mAbs were concentrated to a final volume of 200–500 µl using Vivaspin filters with a cut-off value of 100 kDa (Cytiva). Isolated mAbs were named according to animal identification number and lineage. Sequences were uploaded to GenBank.

mAbs were biotinylated for competition ELISA. Biotinylation was done using the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. For NS-EM analysis, polyhistidine (His)-tagged Fabs were produced by Gibson cloning Ab heavy chain PCR inserts into a digested Fab expression vector. Gibson reaction products were transformed into competent XL-1 blue cells (Agilent) for mini- or midiprep DNA isolation using the Macherey-Nagel plasmid DNA isolation kit. His-tagged Fabs were recombinantly expressed and secreted as a soluble protein in HEK293F cells. The supernatant was concentrated and loaded onto a Ni-NTA affinity column, and the Fabs were eluted using an imidazole gradient. Fabs were concentrated and buffer exchanged into Tris-buffered saline (TBS) buffer (50 mM Tris, 150 mM NaCl, pH 7.5) using Vivaspin filters with a cut-off value of 10 kDa (Cytiva).

TZM-bl Neutralization assays

TZM-bl cell neutralization assays using Env-pseudotyped viruses were performed at three sites: the Amsterdam UMC (AUMC)22, Duke University (DU)43,44 and Ospedale San Raffaele(OSR)45. Neutralization by Abs and sera were measured as a function of reductions in luciferase (Luc) reporter gene expression after a single round of infection in TZM-bl cells43,44,45. At the AUMC, mAb dilution (start concentrations ranging from 1 to 100 µg/ml) series and virus were incubated for 1 h at RT and subsequently added to TZM-bl reporter cells. After 3 days infectivity was measured. IC50 values were determined as the concentration at which 50% of the infectivity was inhibited. At Duke University, a pre-titrated dose of virus was incubated with serial 3-fold dilutions of heat-inactivated (56 °C, 30 min) serum samples in duplicate in a total volume of 150 μl for 1 h at 37 °C in 96-well flat-bottom culture plates. Freshly trypsinized cells ((10,000 cells in 100 μl (DU) or 75 μl (OSR) of growth medium (GM) containing 75 μg/ml (DU) or 45 μg/ml (OSR) DEAE dextran) were added to each well. One set of control wells received cells + virus (virus control) and another set received cells only (background control). At OSR, after 48 h of incubation, the medium was removed and 50 μl of Brite-Glo reagent (Promega, Madison, Wisconsin, USA) diluted 1:2 with GM was dispensed into each well. The plate was incubated at room temperature for 2 min to allow complete cell lysis. 40 μl was transferred to a corresponding 96-well white plate and analyzed in a luminometer (Mithras (Berthold, Germany)). At DU, after 48 hours of incubation, 100 µl of cells was transferred to a 96-well black solid plate (Costar) for measurements of luminescence using the Britelite Luminescence Reporter Gene Assay System (PerkinElmer Life Sciences). Neutralization titers are the dilution (serum/plasma samples) or concentration (mAbs) at which relative luminescence units (RLU) were reduced by 50% or 80% compared to virus control wells after subtraction of background RLUs. Assay stocks of molecularly cloned Env-pseudotyped viruses were prepared by transfection in 293T/17 cells (American Type Culture Collection) and titrated in TZM-bl cells as described43,44. This assay has been formally optimized and validated46 and was performed in compliance with Good Clinical Laboratory Practices, including participation in a formal proficiency testing program47. Additional information on the assay and all supporting protocols may be found at: http://www.hiv.lanl.gov/content/nab-reference-strains/html/home.htm.

Negative stain electron microscopy of monoclonal antibodies

ConM SOSIP.v9.0/ConS SOSIP.v9.0 and Fab/IgG complexes were made by mixing 3 µg of ConM SOSIP.v9.0 or ConS SOSIP.v9.0 trimers with 6-fold molar excess monoclonal Fabs or two-fold molar excess monoclonal IgGs. Complexes were incubated for 30 min at RT before being diluted to 30 ng/µl in Tris-buffered saline (TBS). Samples were applied to glow discharged carbon-coated Cu400 EM grids and blotted after 10 s. 3 µl of 2% (w/v) uranyl formate was then applied and immediately blotted. After 10 s, 3 µl of uranyl formate was applied and after 45 s a final blot was performed. Image collection was performed on either a FEI Tecnai TF20 microscope (1.77 Å/pixel; 62,000× magnification) or Tecnai Spirit microscope (2.06 Å/pixel; 52,000× magnification) operating at 120 keV using Leginon48. 2D and 3D classification and sorting were performed using Relion v3.049 and electron density maps were visualized in UCSF Chimera50.

Negative stain electron microscopy of polyclonal antibodies

Electron-microscopy-based epitope mapping (EMPEM) experiments were performed27. IgGs were purified from serum of immunized animals using protein G resin (Thermo Scientific), at a ratio of 2 ml diluted resin for each ml of undiluted plasma or serum. Samples were diluted at least 4-fold in PBS, then incubated with protein G resin overnight at 4 °C. The resin was washed with 3 column volumes of PBS, and the IgGs eluted with 9 ml of 0.1 M glycine pH 2.5, immediately neutralized with 1 ml 1 M Tris-HCL pH 8. Buffer was exchanged to PBS by centrifugation using 100 kDa Vivaspin 6 column filters with a cut-off value of 100 kDa (Cytiva).

Purified polyclonal IgGs were first digested to Fabs by activating papain for 15 min at 37 °C in digestion buffer (100 mM Tris pH 8, 2 mM EDTA, 10 mM L-cysteine). IgG samples were then digested for four hours at 37 °C with freshly-activated papain and the digestion reactions were stopped using 50 mM iodoacetamide. Digested IgG samples were buffer exchanged into TBS using Amicon ultrafiltration units with a 10 kDa cutoff (EMD Millipore Sigma), and Fabs were further purified by size exclusion chromatography (Superdex 200 Increase 10/300 gl column running in TBS). Fractions corresponding to Fab peaks were pooled and concentrated with Amicon ultrafiltration units with a 10 kDa cutoff (EMD Millipore Sigma). The Fab-SOSIP complexes were assembled using 300 µg of Fab and 15 µg of ConM SOSIP.v9.0 or ConS SOSIP.v9.0 for 18 h at RT. The Fab-SOSIP complexes were purified using size exclusion chromatography (Superose 6 Increase 10/300 gl column running in TBS), and concentrated with Amicon ultrafiltration units with a 10 kDa cutoff (EMD Millipore Sigma).

The purified Fab-SOSIP complexes were diluted to 50 µg/ml in TBS and 3 µl was applied to glow discharged carbon-coated Cu400 EM grids and blotted after 10 s. To stain the complexes, 3 µl of 2% (w/v) uranyl formate was applied and immediately blotted. After 10 s, 3 µl of uranyl formate was applied again and blotted after 45 s. Data were collected on either a Tecnai TF20 electron microscope (FEI) (200 kV, 62,000× magnification) equipped with a TemCam F416 CMOS (TVIPS) camera, or a Tecnai Spirit (FEI) (120 kV, 52,000× magnification) equipped with an Eagle 4 K CCD (FEI/Thermo Fisher) camera. The Leginon automated interface48 was used to acquire the data.

Data processing was performed as follows. Particles were auto-picked and extracted using Appion data processing suite49. 2D classification was performed using Relion v3.051 into 100 classes, and classes containing Fab-SOSIP complexes were selected for 3D analysis. Particles from the selected classes were 3D sorted into 40 classes using Relion v3.0, with a low-resolution model of unliganded HIV Env as a reference. The 3D maps were visualized using UCSF Chimera50. Particles from similar-looking classes were then selected and reclassified using Relion v3.0. The final 3D maps were visualized and segmented using UCSF Chimera50 and Segger52, respectively. Representative reconstructions (all specificities for a particular animal and time point) have been deposited to the Electron Microscopy Data Bank (EMDB) (see “Data availability”) and summarized in Supplementary Table 4. Additional maps can be requested from the corresponding author.

Site-specific glycan analysis using mass spectrometry

Three aliquots of ConM SOSIP v7 protein were denatured for 1 h in 50 mM Tris/HCl, pH 8.0 containing 6 M of urea and 5 mM DTT. Next, Env proteins were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubated for 1 h in the dark, followed by a 1 h incubation with 20 mM DTT to eliminate residual IAA. The alkylated Env proteins were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (3 kDa) and two of the aliquots were digested separately overnight using trypsin, chymotrypsin (Mass Spectrometry Grade, Promega) or alpha lytic protease (Sigma Aldrich) at a ratio of 1:30 (w/w). The next day, the peptides were dried and extracted using C18 Zip-tip (MerckMilipore). The peptides were dried again, re-suspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) using stepped higher energy collision-induced dissociation (HCD) fragmentation. Peptides were separated using an EasySpray PepMap RSLC C18 column (75 µm × 75 cm). A trapping column (PepMap 100 C18 3 μM 75 μM × 2 cm) was used in line with the LC prior to separation with the analytical column. The LC conditions were as follows: 280 min linear gradient consisting of 4–32% acetonitrile in 0.1% formic acid over 260 min followed by 20 min of alternating 76% acetonitrile in 0.1% formic acid and 4% Acn in 0.1% formic acid, used to ensure all the sample had eluted from the column. The flow rate was set to 300 nL/min. The spray voltage was set to 2.7 kV and the temperature of the heated capillary was set to 40 °C. The ion transfer tube temperature was set to 275 °C. The scan range was 375–1500 m/z. Stepped HCD collision energy was set to 15, 25, and 45% and the MS2 for each energy was combined. Precursor and fragment detection were performed using an Orbitrap at a resolution MS1 = 120,000. MS2 = 30,000. The AGC target for MS1 was set to standard and injection time set to auto which involves the system setting the two parameters to maximize sensitivity while maintaining cycle time.

Glycopeptide fragmentation data were extracted from the raw file using Byos (Version 3.5; Protein Metrics Inc.). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The MS data was searched using the Protein Metrics 305 N-glycan library with sulfated glycans added manually. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence. All charge states for a single glycopeptide were summed. The precursor mass tolerance was set at 4 and 10 ppm for fragments. A 1% false discovery rate (FDR) was applied. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected.

HexNAc(2)Hex(10+) was defined as M9Glc, HexNAc(2)Hex(9–5) was classified as M9 to M3. Any of these structures containing a fucose were categorized as FM (fucosylated mannose). HexNAc(3)Hex(5–6)X was classified as Hybrid with HexNAc(3)Hex(5-6)Fuc(1)X classified as Fhybrid. Complex-type glycans were classified according to the number of HexNAc subunits and the presence or absence of fucosylation. These compositions were further grouped into high mannose (oligomannose and hybrid-type), complex and unoccupied. Two analytical repeats were performed with this method

Site-specific analysis of low abundance N-glycan sites using mass spectrometry

To obtain data for sites that frequently present low intensity glycopeptide the glycans present on the glycopeptides were homogenized to boost the intensity of these peptides. This analysis loses fine processing information but enables the ratio of oligomannose: complex: unoccupied to be determined. The remaining glycopeptides were first digested with Endo H (New England BioLabs) to deplete oligomannose- and hybrid-type glycans and leave a single GlcNAc residue at the corresponding site. The reaction mixture was then dried completely and resuspended in a mixture containing 50 mM ammonium bicarbonate and PNGase F (New England BioLabs) using only H2O18 (Sigma-Aldrich) throughout. This second reaction cleaves the remaining complex-type glycans but leaves the GlcNAc residues remaining after Endo H cleavage intact. The use of H2O18 in this reaction enables complex glycan sites to be differentiated from unoccupied glycan sites as the hydrolysis of the glycosidic bond by PNGaseF leaves a heavy oxygen isotope on the resulting aspartic acid residue. The resultant peptides were purified as outlined above and subjected to reverse-phase (RP) nanoLC-MS. Instead of the extensive N-glycan library used above, two modifications were searched for: +203 Da corresponding to a single GlcNAc, a remnant of an oligomannose/hybrid glycan, and +3 Da corresponding to the O18 deamidation product of a complex glycan. Data analysis was performed as above and the relative amounts of each glycoform determined, including unoccupied peptides. The data for the two methods was combined to optimize glycan site coverage of the sample.

V-gene and SMH determination through IgDiscover

Individualized germline IGHV gene databases from NHP MCM01, MCM02, MCM03, MCM04, MCM05, and MCM06 were produced from samples obtained before immunization. In brief, IgM libraries for deep sequencing of antibody repertoires were generated with 5′ multiplex PCR from total PBMC mRNA53. The mRNA was reverse transcribed with IgM constant region-specific primer containing a unique molecular identifier and a universal outer primer sequence. The cDNA was amplified utilizing the universal 3′ primer and two primer sets covering all gene families and targeting leader and UTR respectively that yield two libraries for each animal. Illumina indices and adapters were introduced by PCR prior to sequencing the library with Illumina’s MiSeq v3 kit. The output library was analyzed with IgDiscover to infer the germline gene IGHV alleles31. The IGHV database utilized as an input database for the IgDiscover analysis was obtained from genomic DNA sequencing54, with the addition of non-located (NL) alleles inferred from a larger set of NHPs53. The heavy chain sequences of the isolated mAbs were assigned to the IGHV individualized database from the respective NHP and a comprehensive IGHJ database obtained from a larger group of NHPs, whereas the light chain was assigned to the IMGT’s light chain database using IgBlast to obtain the SHM and CDR3 sequences.

Binding and competitive ELISAs

Binding and competitive ELISAs were performed using Galanthus Nivalis Lectin (GNL) coated (in 0.1 M NaHCO3 pH 8.6, overnight) high binding half area plates26. Subsequently the plates were blocked with casein (Thermo Fisher Scientific) for 30 min. Plates were washed with Tris-buffered saline (TBS) between each of the subsequent steps. Afterward, 2 µg/ml Env protein was added to the plates and incubated for 2 h. Subsequently mAb dilution series were added to the plate at starting concentration of 1 µg/ml to generate binding curves. After 2 h a 1:3000 dilution of horseradish peroxidase labeled (HRP) goat-anti-human (SeraCare 0.1 µg/ml) was added and left for 1 h at RT. Plates were washed with TBS + 0.05% Tween20 and developed using 1% 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate, and 100 mM citric acid. Reactions were stopped using 0.8 M H2S04 and absorption was measured at OD450 using the SpectroStar Nano Spectrophotometer (BMG Labtech). The competitive ELISAs were executed using the same protocol with minor adjustments. The primary Ab was added as a single concentration of 10 µg/ml in triplo and incubated for 0.5 h at RT. Without an additional wash step, a biotinylated competitor Ab was added at 3× the effective concentration (EC)70 value and incubated for another 1.5 h at RT. Binding of the competitor mAb was detected using 1:3000 streptavidin-HRP (BioLegend) as a secondary antibody.

Webalignment and weblogo

The length, the number of PNGS and charge of the V1V2 region (130-197) of all env sequences in the 2020 webalignment from the Los Alamos HIV Databases (http://www.hiv.lanl.gov/) were determined using the AnalyzeAlign tool of the Los Alamos HIV Databases. The V1V2 sequence of ConM and the weblogo of the V1V2 region (130–197) of all env sequences in the 2020 webalignment were made using http://weblogo.threeplusone.com/create.cgi.

Statistical analyses

All statistical analyses (two-tailed unpaired t-test and two-tailed Mann–Whitney U test) were performed in Graphpad Prism 9.1.0.

Reporting summary

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

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