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Immunization expands HIV-1 V3-glycan specific B-cells in mice and macaques

Summary Broadly neutralizing monoclonal antibodies protect against HIV-1 infection in animal models, suggesting that a vaccine that elicits them in humans would be effective. However, it has not yet been possible to elicit adequate serologic responses by vaccination. To activate B-cells expressing precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed a new immunogen, RC1, which facilitates recognition of the V3-glycan patch on HIV-1 envelope while concealing non-conserved immunodominant regions by addition of glycans and/or multimerization on virus-like particles. Mouse, rabbit and rhesus macaque immunizations with RC1 elicited serologic responses targeting the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that RC1 immunization expands clones of B-cells carrying anti-V3-glycan patch antibodies that resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies to elicit V3-glycan antibodies in the context of polyclonal repertoires.


Introduction
Single cell antibody cloning from HIV-1-infected human donors revealed that broadly neutralizing antibodies (bNAbs) have undergone unusually extensive somatic mutation [1][2][3][4] . Moreover, the high degree of somatic mutations is essential for binding to the native HIV-1 envelope spike (Env) and for bNAb neutralizing activity 5 . The accumulation of large numbers of mutations suggests that bNAbs evolve in response to iterative rounds of somatic hypermutation and selection in germinal centers (GCs) 6 . Studies in humans revealed that this occurs in response to viral escape variants arising from antibody pressure 4 . Together these observations suggest that vaccination to elicit bNAbs requires a series of sequential immunogens starting with an immunogen that induces the 7 expansion of B-lymphocytes expressing appropriate germline precursors 8 .
Sequential immunization to shepherd bNAb development was demonstrated in geneticallymodified mice that carry inferred germline (iGL) precursors of human bNAbs 8,9 . However, the priming immunogens used to initiate the response failed to activate and expand B-cells expressing inferred bNAb precursors in animals with polyclonal antibody repertoires. Thus, a goal of HIV-1 vaccine development has been to design immunogens that recruit B-cells expressing bNAb precursors into GC reactions in animals with polyclonal repertoires.
The germline targeting approach to immunogen design focuses on producing immunogens that bind with high affinity to specific bNAb precursors, the rationale being that B-cell recruitment to GCs is in part dependent on receptor affinity for antigen [10][11][12][13][14] . However, this methodology effectively limits the repertoire of recruited B-cells qualitatively and quantitatively. Moreover, it fails to account for the findings that each GC accommodates different founder B-cells with a wide range of affinities and that GC entry is limited by competition and not absolute affinity 7,10 .
Here we describe RC1, an immunogen designed to recruit and expand diverse V3-glycan specific B-cells by improving accessibility of the V3-glycan patch epitope, which includes a group of high-mannose and complex-type N-glycans surrounding V3 (gp120 residues N133, N137, N156, N295, N301, N332, N339, N385 and N392) 15 . bNAbs targeting this site, including PGT121 16 , 10-1074 17 , and BG18 18 , reach through these glycans using elongated CDRH3 loops and portions of CDRL1 and CDRL3 to contact the highly-conserved GDIR motif (G324-D325-I326-R327) at the base of V3 19 . Here we show that RC1 activates and expands a diverse group of B-cells expressing antibodies that resemble human V3-glycan patch bNAb precursors in mice, rabbits and rhesus macaques.

RC1 facilitates antibody binding to the V3-glycan patch
RC1 was designed using 11MUTB 20 , a modified native-like Env trimer (SOSIP.664) derived from clade A/E BG505 Env 21 , as a template. Compared to BG505, 11MUTB includes substitutions in V1 and lacks potential N-linked glycosylation sites (PNGSs) at N133 and N137 20 (Fig. 1a). We reasoned that removal of the N156 PNGS (N156Q) to create RC1 would facilitate recognition of the V3-glycan patch by increasing accessibility of V1 residues that interact with V3-glycan bNAbs 22,23 . Consistent with this idea, the absence of the N156 PNGS enhances neutralization by PGT121 and 10-1074, whereas the absence of other glycans; e.g., N301 or N137, reduces neutralization (Extended Data Fig. 1a). In addition, we hypothesized that removal of the N156 glycan, which includes negativelycharged terminal sialic acids 22,24 , would produce a more electrostatically-neutral Env surface that could facilitate the binding of the largely neutral precursor of PGT121 and 10-1074 (iGL PGT121/10-1074) 25 .

RC1 elicits V3-glycan patch antibodies in wild-type mice
To determine whether RC1 can activate B-cells carrying V3-glycan patch-specific antibodies in wild-type mice, we immunized C57BL/6J mice once with RC1 or 11MUTB (Fig.2a). 11MUTB did not produce a measurable serologic response, but RC1-immunized mice showed reproducible anti-V3-glycan patch-specific serologic responses as shown by ELISA comparing binding to RC1 and a mutant RC1 (RC1-glycanKO) that lacks two V3 PNGSs (N301 and N332) critical for human V3-glycan patch bNAbs ( Fig. 2d-g; Extended Data Table 2). Moreover, serum from the RC1-immunized mice cross-reacted with 11MUTB but not with the more native-like 10MUT 20 or with BG505 (Extended Data Fig. 3a). The improved immunogenicity of the V3-glycan patch epitope of RC1 results from specific removal of the N156 glycan from 11MUTB because removal of a nearby glycan at N301 that is also part of the glycan patch (11MUTBΔ301) (Extended Data Table 2) failed to induce detectable serologic responses (Fig. 2h). We conclude that, unlike 11MUTB and 11MUTBΔ301, RC1 elicits V3-glycan-specific serologic responses in wild-type mice.

Clonal expansion of V3-glycan patch specific B-cells in wild-type mice
To further characterize humoral responses elicited by RC1 and RC1-4fill, we sequenced antibody genes from single GC B-cells that bound RC1 but not RC1-glycanKO (Extended Data Fig. 3b). All RC1-and RC1-4fill-immunized mice analyzed showed expansion of GC B-cell clones (Fig. 2a, j). The expanded clones predominantly expressed heavy chain V genes VH5-6, VH9-3 and VH2-9, and light chain genes VK3-4 and VK14-111 (Fig. 2j;  Supplementary Tables 1,2; Extended Data Table 4). The CDRH3 sequences in expanded clones showed similarities to human V3-glycan patch bNAbs such as Tyr-rich or RxY motifs and longer-than-average CDRH3s (Supplementary Table 1; Extended Data Table 4). Consistent with a single immunization, the VH genes of the expanded clones had an average of 3.2 nucleotide mutations ( Fig. 2k; Supplementary Table 1).
We mapped the target sites of these antibodies by ELISAs against RC1 and RC1 mutant proteins. A diverse group of monoclonal antibodies (mAbs) showed V3-glycan patch-the Ab276 MUR reverted iGL (Extended Data Fig. 3e). Consistent with a single immunization, neither Ab275 MUR nor Ab276 MUR showed detectable neutralizing activity against a panel of tier 1B and tier 2 HIV-1 isolates in TZM-bl assays. We conclude that RC1 and RC1-4fill expand mouse B-cell clones expressing antibodies that target the V3-glycan patch.

VLP-RC1-4fill elicits V3-glycan patch antibodies in rabbits and rhesus macaques
To enhance potential avidity effects and limit exposure of off-target epitopes at the Env base, we multimerized RC1-4fill on virus-like particles (VLPs) using the SpyTag-SpyCatcher system 30,31 (Fig. 3a,b). VLPs were used to prime rabbits and rhesus macaques. Single immunizations of 4 rabbits and 16 macaques with VLP-RC1-4fill elicited serologic responses that were partially specific for the V3-glycan patch in all animals ( Fig. 3c-f and Extended Data Figure 5a). Serum from macaques primed with VLP-RC1-4fill showed sequentially reduced binding to the more native-like immunogens 11MUTB and 10MUT 20 (Extended Data Fig. 5b) and no neutralizing activity against a small panel of HIV-1 isolates that included fully-glycosylated tier 2 and glycan-deleted viruses (Extended Data Table 3). Thus, VLP-RC1-4fill elicited robust serologic responses that mapped in part to the V3glycan patch in rabbits and rhesus macaques.
Antibody cloning from 4 immunized macaques revealed expanded B-cell clones that used a variety of VH genes, as found for human V3-glycan patch bNAbs 32 , with an average of 5.6 nucleotide somatic mutations (Fig. 3i,j; Supplementary Table 3). Most characterized human V3-glycan patch bNAbs contain a lambda light chain 18,33 . Analysis of lambda genes revealed that macaque RC1-binding cells preferentially used gene segments VL132 (91% nt sequence identity to VL2-8 germline gene segments in PGT125-128 and PGT130-131) and VL124 (94% identity to the VL3-21 germline in PGT121-123/10-1074) (Fig. 3k). 86% of the lambda light chains had CDRL3s that included a DSS motif present in the iGLs of PGT121-123 and 10-1074/PGT124 17 ( Fig. 3l; Supplementary Table 4). This motif mutates to DSR in the mature bNAbs, which is critical for PGT121 neutralization activity 34 . Thus, there is congruence between the sequence of human V3-glycan patch bNAb precursors and the antibodies expressed by macaque B-cell clones elicited by priming with VLP-RC1-4fill.
We expressed 38 macaque GC antibodies with CDRL3s that resembled the CDRL3s of iGL V3-glycan patch bNAbs (Supplementary Table 5). The CDRL3s of 33 of 38 antibodies contained a DSS motif and a Q at position 89 (QxxDSS motif), also found in the CDRL3s of the PGT121-3, 10-1074, PGT124 and BG18 iGLs (Extended Data Table 5) 17,18 . Five of 38 antibodies contained a SYAG motif, which is present in the CDRL3s of the PGT125-7, PGT128, PGT130 and PGT131 iGLs (Extended Data Table 5). Thirty of 33 QxxDSS motifcontaining antibodies and 2 of 5 SYAG motif-containing antibodies bound to the V3-glycan patch epitope, as determined by ELISA using RC1 and RC1-glycanKO with additional mutations in the GDIR sequence (RC1-glycanKO-GAIA) (Fig 4a; Supplementary Table 5). In addition, the CDRL1 of all 38 macaque antibodies contained a NIG-like motif present in the iGL PGT121/10-1074 antibody (33 NIG, 4 DIG, 1 NLG) (Supplementary Table 5). The CDRH3 lengths of the 38 antibodies were relatively long (11-21 residues; average=15.5) (Fig. 4b). Longer CDRH3s were enriched in Tyr and/or Phe residues, similar to the long CDRH3s found in human V3-glycan patch bNAbs [16][17][18] (Supplementary Table 5). The antibody VH and VL genes included an average of 4.9 and 3.3 nucleotide mutations, respectively (Fig. 4c). Consistent with recruitment of antibodies with a range of affinities to GCs and their subsequent affinity maturation 7,10 , the iGL versions of the macaque antibodies showed lower affinity for RC1 than their mutated counterparts, ranging from levels that were below quantification to μM K D s (Extended Data Fig. 5c). Similarity between the macaque antibodies and the iGL PGT121/10-1074 was corroborated by ELISAs using an anti-idiotypic antibody specific for iGL PGT121/10-1074 (Extended Data Fig. 5d). iGL-reverted versions of 5 of 11 macaque antibodies were recognized by the anti-iGL PGT121/10-1074 antibody (Extended Data Fig. 5d).
To further characterize the target site of the macaque antibodies, we performed ELISAs against additional proteins: RC1-glycanKO, RC1-GAIA, RC1-glycanKO-GAIA, 11MUTBΔ301, RC1Δ301, RC1Δ332, 11MUTB 20 and BG505 (Fig. 4d,e; Extended Data Table 2). The ELISAs suggested four distinct RC1-binding patterns among antibodies that contained a CDRL3 QxxDSS motif (Fig. 4d) and another pattern among antibodies containing a SYAG motif (Fig. 4e). Whereas all antibodies were glycan-dependent as determined by no binding to RC1-glycanKO, they differed in binding to 11MUTB or 10MUT and dependence on GDIR and N301, N332, and N156 glycans (Fig. 4a,d,e). Although none of the antibodies recognized BG505, Ab933 NHP , Ab936 NHP , and Ab1170 NHP bound 11MUTB, indicating that they can accommodate the N156 glycan (Fig.  4d,e). Consistent with no binding to BG505, none exhibited neutralizing activity, and removal of the N133, N137, and N156 glycans did not render the BG505/T332N and JRCSF.JB viruses sensitive to neutralization (Extended Data Table 3b,c), suggesting that the lack of neutralization is not due to clashes with those glycans. We conclude that macaque immunization with VLP-RC1-4fill elicits V3-glycan patch-specific antibodies that resemble precursors of human bNAbs targeting this site.

Cryo-EM structures of mouse and macaque antibodies in complex with RC1
We determined structures of one mouse and two macaque Fabs complexed with RC1 using single-particle cryo-electron microscopy. Ab275 MUR (4.4Å resolution) and Ab874 NHP (3.9Å) (derived from the same clone as Ab876 NHP ) bound similarly to each other, consistent with their 69% V H domain amino acid sequence identity, whereas Ab897 NHP (4.4Å) (related by 48% and 54% V H sequence identity to Ab275 MUR and Ab874 NHP , respectively) adopted a distinct angle of approach ( Fig. 5a; Extended Data Figure 5e).
All three Fabs in the RC1 complexes bound to the V3-glycan patch epitope and contacted the GDIR motif, but with different orientations and footprints from each other and from V3glycan patch bNAbs ( Fig. 5a; Extended Data Figure 5e). 10-1074 contacts the conserved GDIR motif using CDRH3, CDRL1, and CDRL3 35 (Fig. 1c, 5a), Ab874 NHP and Ab275 MUR made GDIR contacts using their CDRH2s, whereas Ab897 NHP utilized CDRL1 and CDRL3 (Fig. 5a,b). In addition, Ab874 NHP and Ab897 NHP contain the conserved CDRL3 QxxDSS motif, which make contacts with conserved regions of the V3-glycan epitope in mature bNAbs 22,36 . Similar to mature V3-glycan patch bNAbs, Ab897 NHP contains a substitution (S93N) within the QxxDSS motif that enables contacts with gp120 GDIR and also uses its conserved CDRL1 NIG motif to contact the V1 loop (Extended Data Figure 5f).
Ab275 MUR and Ab874 NHP also interacted with the N332 glycan, consistent with mature V3glycan bNAbs (Fig 5a; Extended Data Figure 5e). However, unlike 10-1074, which interacts with the N332 glycan via its CDRL1, FRWL3, CDRH2, and CDRH3 35 , Ab275 MUR made contacts using CDRH2, while Ab874 NHP engaged the N332 glycan with CDRH2 and FRWH3. We did not observe N332 glycan interactions in the Ab897 NHP -RC1 structure. Despite reduced binding of Ab275 MUR , Ab876 NHP (same clone as Ab874 NHP ), and Ab897 NHP to RC1Δ301 (Fig. 2l), none of these Fabs showed interactions with the N301 glycan in our EM structures, suggesting either glycan heterogeneity obscures this interaction and/or conformational heterogeneity in a V3-glycan patch lacking this glycan diminishes binding 37 . We conclude that RC1 elicits V3-glycan patch-targeting antibodies with distinct binding modes in animals with polyclonal antibody repertoires.

Conclusions
HIV-1 bNAbs develop in infected humans by sequential rounds of somatic mutation in response to a rapidly-evolving pathogen 4 . Vaccination with a series of related antigens can reproduce this progression of events in genetically-engineered mice that carry supraphysiological numbers of B-lymphocytes expressing the iGL precursors of bNAbs 9 . An important goal of HIV-1 vaccine design is to develop immunogens that initiate this response in organisms with polyclonal immune systems and then reproduce these responses in humans.
HIV-1 immunogen design has focused upon increasing the affinity of candidate immunogens for specific iGL bNAb precursors with the objective of recruiting a specific group of rare precursors into the GC 1 . This approach typically fails to account for increases in apparent affinity produced by interactions between multimerized antigen and clusters of bivalent antigen receptors on the surface of a B-cell. Moreover, GC entry is primarily limited by competition 7,10,11,14 . Thus, the importance of affinity is relative, as evidenced by the observation that B-cells bearing low affinity receptors are frequently found in GCs under physiological conditions 10,38 , and by our finding that iGL precursors of macaque antibodies elicited by RC1 showed relatively low affinity for the immunogen.
The principles employed to produce RC1 did not take affinity for a germline B-cell receptor into account. Instead, RC1 was designed to increase the number of bNAb progenitors that compete for GC entry by making the antigenic target site more available and facilitating binding to electrostatically-neutral iGL precursors 25 . In addition, VLP-RC1-4fill incorporates the idea that masking competing off-target epitopes 26,29 by addition of glycans 27 and tethering the bottom of the trimer to a VLP minimizes competition for GC entry.
RC1 differs from other HIV-1 vaccine candidates in that it induces B-cells expressing antibodies against a targeted epitope to undergo clonal expansion in GCs in animals with a fully polyclonal B-cell repertoire. In macaques, these B-cells express antibodies that show sequence and structural similarities to iGL precursors of bNAbs targeting the V3-glycan patch. Like the precursors of human bNAbs, they do not bind to wild-type Env or neutralize HIV-1 5 . Importantly, biochemical and structural results showed that antibodies with distinct mechanisms of targeting the V3-glycan patch were elicited by RC1, increasing the probability that one or more might develop breadth and potency after boosting 9 . Thus, VLP-RC1-4fill is a suitable candidate immunogen for further evaluation in sequential vaccination strategies to elicit V3-glycan bNAbs.
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 described 39 . 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.

VLP production and conjugation
A C-terminal SpyTag sequence (13 residues) was added to RC1-4fill to form an irreversible isopeptide bond to SpyCatcher protein 31 . We produced and purified SpyCatcher-AP205 40 VLPs as described 30 and separated conjugated VLPs from free Env trimers by SEC on a Superdex 200 column. Conjugation of Env trimers was verified by negative-stain EM 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 EM on a FEI Tecnai T12 transmission electron microscope at 120 keV using a Gatan Ultrascan 2k x 2k CCD detector.

Animals
Mice carrying the iGL IgH and IgL human PGT121 and 10-1074 bNAbs (GL HL 121 knockin mice) were previously described 9 . 6-8-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 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 10μg of soluble SOSIP Envelope trimer in Ribi adjuvant (Sigma) (1:1).
Six-month-old New Zealand White rabbits (Covance) were used for immunizations. Rabbits were immunized subcutaneously with ~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 Committee.
Sixteen rhesus macaques (Macaca mulatta) of Indian genetic origin, 2 to 4 years of age, were housed and cared for in accordance with Guide for Care and Use of Laboratory Animals Report no. NIH 82-53 (Department of Health and Human Services, Bethesda, Maryland, 1985) in a biosafety level 2 NIH facility. All animal procedures and experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of NIAID, NIH.

Adjuvant synthesis
ISCOM-like saponin adjuvant was prepared as described 41 . Final adjuvant concentration was determined by cholesterol quantification (Sigma MAK043).
Alternatively, 96-well plates were directly coated with 50 μl of a solution of Fab at 20μg/ml in 1xPBS overnight at 4ºC, washed, and blocked as above and incubated in 50μl of a solution of RC1 or RC1-glycanKO-GAIA at 2μg/ml in blocking buffer for 1 h at RT. Plates were washed as above and developed using a chimeric version (human Fabs and mouse Fc) of the CD4-binding site bNAb 3BNC60 42 at 3-fold serial dilutions starting at 5μg/ml 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 50μl of a solution of IgG at 10μg/ml in 1xPBS overnight at 4ºC, washed and blocked as above and incubated with biotinylated anti-iGL 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 PBMCs or cells from lymph node biopsies obtained from the naïve and immunized macaques were thawed and washed in RPMI medium 1640 (1x) (Gibco #11875-093). Mouse or macaque cells were incubated with 100 μl of FACS buffer (PBS 1x with 2% fetal bovine serum and 1mM EDTA) with mouse (BD Biosciences #553142) or human (BD Biosciences #564219) Fc Block, respectively, at a 1:500 dilution for 30 min on ice.
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 −80ºC.
Mouse and macaque antibody genes were cloned 43

Antibody production and purification
Igs were purified from 200μl of mouse or macaque serum using Ab Spin Trap Protein G Sepharose columns (GE Healthcare, #28-4083-47). Ig-containing fractions were buffer exchanged with PBS by overnight dialysis at 4ºC (dialysis cassettes 20000 MWCO Thermo 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 SEC 44 . Mouse Fab was obtained by digesting IgG at 1-5 mg ml −1 with ficin (Sigma). Fab was purified by protein G (GE Healthcare) and SEC chromatography 45 , followed by monoQ 5/50 (GE Healthcare) ion exchange chromatography. The common iGL of the PGT121 and 10-1074 bNAbs 17 was expressed as a His 6 -tagged Fab.

In vitro neutralization assay
TZM-bl assays were performed as described 46 . 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 iGL 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 described 44 . 1μM 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 iGL Fab (4-fold dilutions from a top concentration of 160 μM for 10MUT, and 2-fold dilutions from a top concentration of 150 μM for 11MUTB and RC1) was injected, and binding reactions were allowed to reach equilibrium. K D s 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 fit to a 1:1 binding model 47 . For measuring the affinity of Ab275 MUR , a concentration series of Fab was injected over immobilized RC1 or 11mutB (4-fold dilutions from a top concentration of 50 μM). K D s 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 (3.125μM to 0.763nM). Flow cells were regenerated with 1 M guanidine HCl and/or 10 mM glycine pH 2.0 at a flow rate of 90 μl/min 44 .

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 Fab 42 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 in TBS, and 3μl 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). 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 software 48 . Movies were collected on a Falcon 3EC direct electron detector (Thermo Fisher) operating in counting mode at a nominal magnification of 73,000x (1.436 Å/pix) using a defocus range of −1.4μm to −3.0μm or −0.8μm 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 MotionCor2 49 frame alignment program in RELION-3 50 . Dose-weighted summed images were used for CTF determination using Gctf 51 , and reference-free particle picking from each micrograph was achieved using Laplacian-of-Gaussian filtering in RELION-3 50 . Unbinned extracted particles were imported into cryoSPARC v2 52 and subjected to reference-free 2D-classification using a 240Å circular mask. Particles from the best 2D 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 3D auto-refinement in cryoSPARC v2 52 . Resolutions were estimated using the Gold Standard Fourier shell correlation (FSC=0.143) 53 , and maps were auto-sharpened in cryoSPARC 52 . For interpreting N-linked glycans, maps were generated with overall B-factors ranging from −150 to −400 Å 2 to improve local features and map connectivity 54 . 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 Chimera 55 . For the RC1-10-1074 complex, BG505 Env, 10-1074 Fab, and 8ANC131 Fab (PDBs 53TZ and 4RWY) were docked into the density maps. For RC1 complexes with mouse or macaque Fabs, BG505 Env, PGT121/10-1074 iGL, and 8ANC195 (PDBs 5CEZ, 4FQQ, 5CJX) coordinates were docked into density maps. Initial models were refined into EM maps using rigid body refinement 55 . Models were built using Fab and RC1 sequences following iterative rounds of real-space refinement in Coot and Phenix 56,57 . (Models of only the RC1-10-1074 and RC1-mouse/macaque Fab portions of the complexes are shown in structure figures and deposited as coordinates in the EMDB and PDB.) Coordinates for glycans were added as Man 9 and then trimmed to fit the maps at σ=5. Model validation was done using MolProbity 58 and Privateer 59 . Superposition calculations and molecular representations were generated with PyMOL (Version 1.5.0.4 Schrodinger, LLC), UCSF Chimera 55 , and ResMap 60 .

Analysis
MacVector 15.5.3 was used for sequence analysis and graphs were created using R language. Flow cytometry data was processed using FlowJo 10.5.0. GraphPad Prism 7 was used for data analysis. Ig gene sequence AB1 files were converted to FASTQ format using biopython package. FASTQ files were trimmed by quality using cutadapt v1.18 software. Igblast v1.9.0 was used for VDJ assignment and clone analysis was performed using Change-O software v0.3.7. For macaques, a custom VDJ database was created using previously reported Ig gene sequences 61 .

Quantification and statistical analysis
Statistical information including n, mean and statistical significance values are indicated in the text or the figure legends. GraphPad Prism 7 was used for statistical analysis by unpaired T-Test. Data were considered statistically significant at *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
loop-Consensus C peptide (see Methods). Human antibodies 3869 and 3074 were used as positive controls. Antibodies were evaluated at 30μg/ml. n=2. d, Representative sensograms from two independent SPR binding experiments of Ab275 MUR Fab injected over immobilized RC1 (panel b) or 11MUTB (panel c). Experimental binding curves (red) are overlaid with predicted curves (black) derived from a 1:1 binding model. Representative plot from 3 independent experiments. e, ELISAs showing the binding of Ab276 MUR and its iGL version (Ab276 MUR -GL) to RC1 (left) and RC1-glycanKO (right) at 30μg/ml. The human monoclonal antibodies PGT121 (green) and 3BNC60 (red) were used as controls at 5μg/ml.
(K D ) for RC1 of different macaque antibodies isolated after a prime with VLP-RC1-4fill, and the corresponding iGL-reverted antibodies as determined by bio-layer interferometry (OCTET). d, ELISA binding of an anti-idiotypic antibody that recognizes the iGL PGT121/10-1074 to mAbs isolated from macaques primed with -RC1-4fill VLPs. The iGL PGT121/10-1074, two chimeric antibodies comprising the mutated HC and iGL LC of PGT121 (PGT121HCMT-LCGL) or the iGL HC and the mutated LC of PGT121 (PGT121 HCGL-LCMT) and different iGL bNAbs were used as controls. Results in a, b, and d are shown as area under the ELISA curve (AUC). e, Comparison of binding mode between the vaccine-elicited antibodies (Ab275 MUR , Ab874 NHP , and Ab897 NHP ) and the V3-glycan patch bNAbs 10-1074, PGT128, and PGT135. RC1 trimer is shown in gray from above and all Fabs are modeled onto the same trimer. For clarity, only one Fab per trimer is shown. f, Interactions between Ab897 NHP conserved light chain motifs and RC1 gp120. DNS motif in CDRL3 (lime); gp120 GDIR (red); NIG motif in CDRL1 (pink); gp120 V1 loop (teal). Each AUC value corresponds to one ELISA curve. Cryo-EM data collection, refinement, and validation statistics.

Mouse monoclonal antibodies.
Monoclonal antibodies isolated from wild-type mice immunized with RC1 or RC1-4fill. All the RC1 binding antibodies targeted the V3-glycan patch epitope of RC1 except antibody 341 (marked with *) that shows similar binding to RC1 and RC1-glycanKO.

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Extended Data Table 5.
Ig genes and CDRL3 amino acid sequences of V3-glycan patch bNAbs.

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Escolano et al. Page 42 Extended Data Table 4.

Mouse monoclonal antibodies.
Monoclonal antibodies isolated from wild-type mice immunized with RC1 or RC1-4fill. All the RC1 binding antibodies targeted the V3-glycan patch epitope of RC1 except antibody 341 (marked with *) that shows similar binding to RC1 and RC1-glycanKO.