Serum samples from prime-boosted wt animals targeted the V3-glycan patch and showed weak, heterologous neutralizing activity

To map epitope(s) recognized by serum antibodies elicited in NHPs, we used an automated competition ELISA in which a randomly biotinylated RC1 trimer was immobilized on an ELISA plate, incubated with a Fab from a potential competitor mAb of known epitope on Env trimer, and probed with serum containing a polyclonal mixture of antibodies (fig. S1A). Controls involved using monoclonal IgG antibodies of known epitopes instead of NHP serum. The following competitor Fabs or control IgG antibodies were used: 10–1074 and PGT128, representative V3-glycan patch bNAbs (28, 39); PGT145 and PG16, V1V2 bNAb (57, 58); 3BNC117 and IOMA, CD4bs bNAbs (26, 45); SF12, a bNAb against the glycan-dominated silent face (59); VRC34, a gp41 fusion peptide-directed bNAb (60), 8ANC195, a bNAb against the gp120-g41 interface (61); 3BC315, a bNAb that binds between adjacent gp41 protomers and neutralizes by promoting Env trimer decay (62); and 10A and 12N, non-neutralizing antibodies that bind to distracting epitopes on BG505 Env: 10A binds to the glycan hole surrounding gp120 residue 241, and 12N binds to the base of trimer base (47).

Results from the competition ELISAs were fairly uniform across the samples from NHPs 1 to 8, showing competition with the V3-glycan patch bNAbs 10–1074 and PGT128 after the prime (fig. S1A). The amount of competition with the V3-glycan patch bNAbs diminished with boosting, especially for the PGT128 competition (fig. S1A). Although these results indicate targeting of the desired epitope, we did not observe 100% competition with 10–1074/PGT128 for any of the samples, consistent with elicitation of antibodies that recognize off-target Env epitopes. Off-target responses increased with boosting as evidenced by increased serum responses against RC1 and proportionally decreased V3-glycan patch binding (fig. S1B).

We also evaluated post-prime and post-boost serum samples by neutralization assays against RC1, 11MUTB, SHIV_DH12-V3AD8, and SHIV_AD8EO pseudoviruses (PV) (51) and against a replication-competent (RC) SHIV_DH12-V3AD8 constructed as described (63) (Fig. 1B, table S1). The RC1 and 11MUTB pseudoviruses represent autologous strains for our immunogens (RC1 for prime and boost immunizations; 11MUTB for boost immunizations). The heterologous Tier 2 SHIV_DH12-V3AD8 and SHIV_AD8EO viruses were chosen to assess responses against potential viral strains that could be used in later challenge experiments, with SHIV_AD8EO representing a highly pathogenic strain (64) and SHIV_DH12-V3AD8 being a SHIV_AD8EO derivative with less pathogenicity in NHPs (63).

Two weeks after the prime, serum samples from NHPs 1 to 8 exhibited autologous neutralization against RC1 ranging from 50% inhibitory doses (ID₅₀s) of 200 to 1,000, and undetectable neutralization of 11MUTB, SHIV_DH12-V3AD8, and SHIV_AD8EO except for neutralization of 11MUTB in NHP 4 serum (ID₅₀ = 400) (table S1). By 8 weeks post-prime, autologous neutralization of RC1 had risen to ID₅₀s of 6,500 to 75,000, and responses to 11MUTB ranged from ID₅₀s of 350 to 8000, demonstrating neutralization of a pseudovirus containing the N156 glycan. Concurrently, we observed low titers of heterologous neutralization of SHIV_DH12-V3AD8 pseudovirus or replication-competent viruses (ID₅₀s <60) in three of the primed NHPs (table S1).

Three weeks after the first boost with 11MUTB-4fill-VLPs, autologous neutralization of RC1 rose again to ID₅₀s of 80,000 to 300,000 and 11MUTB titers also rose to ID₅₀s of 50,000 to 300,000 (Fig. 1B, table S1). At this time point, titers against SHIV_DH12-V3AD8 pseudovirus and replication-competent viruses were barely detectable in all 8 NHPs. By 3 weeks post-boost 2 with B41 wt-VLPs or B41–5MUT-VLPs, neutralization potencies against RC1 and 11MUTB remained high, but titers against the heterologous SHIV_DH12-V3AD8 pseudovirus rose to values ranging from 584 to 3378, with detectable neutralization against the replication-competent SHIV_DH12-V3AD8 in all 8 NHPs (ID₅₀s from 36 to 92). The mean titers after Boost 2 against the SHIV_DH12-V3AD8 pseudovirus were significantly higher in the four NHPs that received the B41–5MUT-VLP boost (NHPs 1,2,5,6) compared with the four NHPs that received the B41wt-VLP boost (NHPs 3,4,7,8) (mean ID₅₀ = 2445 ± 809 versus 1027 ± 283; p = 0.046). We conclude that that the immunization series starting with RC1–4fill-VLPs, followed by 11MUTB-4fill-VLPs and B41–5MUT-VLPs, elicited antibodies that neutralized autologous strains and also exhibited weak activity against a heterologous SHIV pseudovirus.

Two weeks after boost 3, we observed relatively steady titers against RC1 and 11MUTB. Importantly, serum neutralization titers against pseudotyped SHIV_DH12-V3AD8 rose to values above 2000 in 7 of 8 animals (mean ID₅₀ = 3671 ± 2004) with an ID₅₀ greater than 8000 observed in NHP 4 (table S1). Similarly, the titers against replication-competent SHIV_DH12-V3AD8 rose in most of the NHPs and ranged from 47 to 164, with NHP 4 again exhibiting the highest titer. Samples collected two weeks after boost 4 showed relatively steady neutralization titers in most NHPs against RC1, 11MUTB, and the SHIV_DH12-V3AD8 pseudovirus. Titers against replication-competent SHIV_DH12-V3AD8 rose or fell in NHPs 1 to 8, with a range from 39 to 256. Not surprisingly, no serum neutralization activity was ever detected against SHIV_AD8EO, as this virus is less sensitive than SHIV_DH12-V3AD8 to PGT121, a V3-targeting bNAb (63). Overall, the trends across NHPs 1 to 8 (Fig. 1B) suggested that (i) neutralization titers against autologous RC1 and 11MUTB pseudotype viruses increased after the prime and first boost, remaining relatively constant after the remaining boosts with heterologous immunogens, (ii) neutralization titers against the heterologous SHIV_DH12-V3AD8 pseudovirus remained at baseline until after Boost 1, rose after Boost 2 and Boost 3, but then remained relatively constant, and (iii) neutralization titers against the replication-competent SHIV_DH12-V3AD8 virus were lower than titers against the homologous pseudotype viruses (65, 66).

We next evaluated serum neutralization in immunized NHPs against a panel of heterologous Tier 1 and Tier 2 pseudotyped viruses (Fig. 1C, table S2). Neutralization against these viruses was very weak or undetectable after the prime and Boost 1. Sequential boosting with B41wt-VLPs or B41–5MUT-VLPs elicited weakly neutralizing activity against the heterologous HIV-1 strain JRCSF.JB (Boost 2; table S2). The heterologous neutralizing responses were maintained, or in some cases improved, by the boost of Du422/AMC011-VLPs (Boost 3), but were reduced after the ConC/ConM-VLP boost (Boost 4) (Fig. 1C, table S2). Serum neutralization assays for rabbits immunized with the same prime-boost regimen as the NHPs (fig. S2A) showed qualitatively similar results. No neutralization against heterologous viruses was observed after the prime or Boost 1, weak neutralization against JRCSF.JB was observed after Boost 2 and Boost 3, and no neutralization was observed after the ConC/ConM-VLP boost (fig. S2B).

Polyclonal negative-stain EM demonstrated antibody targeting of the V3-glycan patch

We also used negative stain polyclonal electron microscopy (nsEMPEM) (50, 67) to map the epitopes of elicited antibodies on HIV-1 Envs. In this method, polyclonal Fabs that bind to an antigen are purified from unbound Fabs by size-exclusion chromatography (SEC), imaged by EM, and antibody epitopes are identified in 2D class averages or 3D classes. A caveat of nsEMPEM is that some antibody epitopes in a polyclonal mixture are not visualized in 2D or 3D classes. For example, nsEMPEM mapping of epitopes in plasmas from coronavirus disease 2019 (COVID-19) convalescent donors revealed less epitope diversity than apparent from single B cell cloning of antibody genes or competition experiments (68). We therefore refer to the nsEMPEM-visualized epitopes as “predominant” or “primary” epitopes and acknowledge that other epitopes may exist.

After demonstrating that RC1–4fill-VLP priming elicited similar RC1 and 11MUTB pseudotype virus neutralizing responses in rabbits and NHPs (fig. S2C), we did initial nsEMPEM studies to evaluate polyclonal responses in a rabbit for which we had serum samples in sufficient quantities for Fab isolations after the same prime/boosting regimen used in NHPs (Fig. 1A; fig. S2A; table S3). 3D reconstructions of RC1 bound to polyclonal Fabs derived from rabbit serum after the prime and Boosts 1–3 showed a primary epitope in the vicinity of the V3-glycan patch of the RC1 trimer after a priming immunization (Fig. 2A), demonstrating targeting of the desired Env epitope. In addition, Fabs that target the base of the trimer were identified in all 2D class averages, with their prevalence gradually increasing after each boost (Fig. 2A, fig. S3). Trimer base-binding Fabs also appeared in concurrence with the observation of dissociated Env trimers (fig. S3, fig. S4A), consistent with observations by others (69). In contrast to previous EMPEM analyses of serum from BG505-immunized animals (50, 67), we did not observe Fabs binding to the region of the residue 241 “glycan hole” in serum Fab complexes with RC1 Env trimer (Fig. 2A and ​andB;B; fig. S3). This indicates that the 4-fill mutations designed to block access to this site in BG505-derived immunogens (38) prevented detectable elicitation of these types of non-neutralizing antibodies.

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Figure 2.

nsEMPEM shows predominant V3 targeting after the prime and initial boosts.

(A) Left: A low-pass filtered structure of representative HIV-1 SOSIP Env trimer is shown complexed with V3-glycan patch bNAb Fabs (PDB codes 5T3Z, 6CH8, 5C7K, 4JM2; colors as indicated) and post-P rabbit Fabs (gold) bound to one Env protomer. Polyclonal Fabs were isolated from Rabbit 2249 (fig. S2B), which received B41–5MUT-VLPs as Boost 2. Right 5 panels: 3D reconstructions of Fabs from antibodies isolated from a rabbit 2 weeks after P, B1, B2, B3, and B4 in complex with RC1 are shown. The predominant states for each time point are shown. See also fig. S3 and S5 for 2D classes, including bottom-binding Fabs after P, B1, and B2 and Fabs bound to dissociated protomers. Coordinates of RC1 (PDB 6ORN) were independently docked into EM density maps. (B) Left: A low-pass filtered structure of representative HIV-1 SOSIP Env trimer is shown complexed with V3-glycan patch bNAb Fabs (colors as indicated) and post-P NHP Fabs (gold) bound to one protomer. Polyclonal Fabs were isolated from NHP 1, which received B41–5MUT-VLPs as Boost 2 (Fig. 1A). Right: 3D reconstructions of Fabs from antibodies isolated from an immunized NHP 2 weeks after P and B1 are shown. See also fig. S4 for 2D classes showing bottom-binding Fabs. (C) Comparison of Fabs binding poses for rabbit and NHP nsEMPEM reconstructions are shown. Rabbit post-B1 Fabs are shown as a pink cartoon of PDB 5UD9 that was docked into the nsEMPEM 3D reconstruction; NHP post-B1 Fabs indicated by the density from the 3D reconstruction.

We next employed nsEMPEM to evaluate serum from NHP 1 (immunized using the regimen that included B41–5MUT-VLPs as Boost 2) after the prime and the first boost, time points for which we had sufficient NHP serum for Fab isolation. After the prime, we observed V3-targeting Fabs, with few to no detectable base-binding Fabs (Fig. 2B; fig. S4B). After Boost 1 with 11MUTB-4fill, we observed an increase in Fabs that targeted both the V3 region and the trimer base (Fig. 2B; fig. S4B). As also observed in the rabbit serum nsEMPEM results, we did not find detectable anti-glycan hole antibodies in 3D reconstructions (Fig. 2B) or 2D class averages of serum Fabs bound to RC1 (fig. S4B).

It is noteworthy that the nsEMPEM results for the NHP serum Fabs, as well as rabbit serum Fabs, showed a primary epitope in the V3 region (Fig. 2A to ​toC),C), demonstrating successful focusing of the anti-Env antibody response to the desired epitope in two different species of immunized wt animals. Comparison of the binding poses of the V3-directed rabbit and NHP Fabs showed similar V3 targeting, but different orientations of the Fab V_H-V_L domains (Fig. 2C). In addition, the trimer base-binding Fabs observed in serum from NHP 1 exhibited different characteristics than those boosted in the rabbit: the NHP base-binding Fabs adopted a distinct binding orientation compared to the rabbit Fabs and the predominant class appeared to bind three per trimer, versus one per trimer for the rabbit Fabs (Fig. 2A and ​andBB).

The binding pose adopted by the rabbit Fabs after the prime was distinct from the binding poses of V3-glycan patch bNAb Fabs and from the poses of mAbs cloned after the RC1–4fill-VLP prime in wt mice (Ab275_mur) and NHPs (Ab874_NHP and Ab897_NHP) (38) (Fig. 3A and ​andB).B). However, a new cryo-electron microscopy (cryo-EM) structure (fig. S5, fig. S6; table S4) of an RC1 complex with the prime-elicited mAb Ab1170_NHP (38) showed that the Ab1170_NHP Fab matched the rabbit nsEMPEM Fabs binding pose more closely (Fig. 3B), providing a higher-resolution structure resembling the Fabs revealed by nsEMPEM. In addition, although a distinct pose, our previous structure of RC1 complexed with Ab275_mur also showed a Fab binding pose more similar to the rabbit Fabs (Fig. 3B).

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Figure 3.

Elicited antibodies adopt binding poses similar to V3 bNAbs.

(A and B) EM density maps for 3D reconstruction of RC1 bound to rabbit Fabs elicited after the RC1–4fill-VLP prime are overlaid with coordinates for the indicated bNAbs (PDB codes 5T3Z, 6CH8, 5C7K, 4JM2) aligned on Env trimer (A) or overlaid with coordinates for mAbs elicited from a primed wt mouse (Ab275mur; PDB 6ORQ), a boosted wt mouse (Ab283mur), or a primed NHP (Ab874NHP; PDB 6ORO, and Ab1170NHP; this study, shown as an EM density map) (B).

We extended our previous analysis of the Ab275_mur-RC1 cryo-EM structure (PDB 6ORQ) to include a comparison with a new structure of RC1 complexed with Ab283_mur (Fig. 4A; fig. S5, table S4). Ab283_mur was isolated after an RC1 prime followed by sequential boosting in a wt mouse. After aligning on the RC1 trimer coordinates of both structures, we observed a shift in the mAb pose in a direction that would better accommodate the gp120 N156 glycan, which was present on the boosting immunogen, but not the priming immunogen (Fig. 4B).

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Figure 4.

Cryo-EM and nsEMPEM Fab-Env structures show apparent accommodation of gp120 N156 glycan after boosting.

(A) A comparison of Ab275mur-RC1 (PDB 6ORQ) and Ab283mur-RC1 structures is shown. (B) A comparison of binding poses of two mouse monoclonal antibodies, Ab275mur and Ab283mur, isolated from wt mice after a prime only (Ab275mur) or prime plus sequential boost (Ab283mur) is shown. After aligning the Ab283mur-RC1 structure reported here and the Ab275mur-RC1 structure (PDB 6ORQ) on the RC1 coordinates, the pose of Ab283mur was shifted relative to that of Ab275mur in a direction that might better accommodate the gp120 N156 glycan (blue sticks from PDB 5T3X), which was present on boosting immunogens but not the priming immunogen. Inset: Overlay of low-pass filtered single-particle cryo-EM maps of the Fab-RC1 structures. (C and D) Coordinates docked into Fabs-Env nsEMPEM reconstructions (Fig. 2A) (C3 symmetry constraints applied) were aligned on the RC1 trimer and displayed as a close-up view of the antibody interaction site with the VH-VL domains of the post-P and post-B1 Fabs (C) or the post-P and post-B2 Fabs (D). The post-B1 Fabs and post-B2 Fabs appear to shift in a direction that might better accommodate the gp120 N156 glycan (blue sticks from PDB 5T3X).

To address whether the V3-targeting Fabs revealed by nsEMPEM after the prime and first two boosts in rabbits also underwent a shift to accommodate the gp120 N156 glycan, we examined the orientations of the Fabs after aligning on RC1 coordinates fit to the trimer densities. Indeed, Fab coordinates fit to the densities after Boosts 1 and 2 (11MUTB-4fill-VLPs and B41–5MUT-VLPs, respectively) indicated a slightly different binding pose relative to the V3-glycan patch compared with the predominant Fabs after the prime, with the Fabs after boosting being translated in a direction that might more easily accommodate the gp120 N156 glycan (Fig. 4C and ​andDD).

We conclude from these structural studies that, as designed, the RC1–4fill-VLP immunogen elicited V3-targeting antibodies. In addition, the initial boosting immunogens elicited antibodies that can better accommodate the gp120 N156 glycan, a potential roadblock in our vaccination regimen. However, the results also demonstrated that the immunization regimen requires further optimization to reduce the elicitation of off-target antibodies that increased upon boosting.

EM of RC1–4fill-VLPs revealed incomplete conjugation and free Env trimers

The observation of increased antibodies against the Env trimer base (Fig. 2A and ​andB;B; fig. S3, S5B) after boosting suggested accessibility of the SOSIP base to antibodies. To investigate the mechanism by which antibodies could gain access to the Env base in a covalently-coupled VLP in which the trimer base should be inaccessible, we examined SOSIP-coupled VLPs by negative stain EM (nsEM). These imaging studies revealed two relevant findings. First, SpyTagged RC1, but not SpyTagged RC1–4fill, conjugated efficiently to VLPs, as revealed by densities corresponding to closely-packed trimers on RC1-VLPs (fig. S7A, second and third images from left) compared with the relatively sparse trimer densities for RC1–4fill-VLPs (fig. S7A, left). Notably, the epitope for the anti-trimer base antibody 12N (47) was not accessible on densely-packed RC1-VLPs (fig. S7B). As assessed biochemically, with the exception of RC1–4fill, all SpyTagged SOSIP Env trimers, including 11MUTB-4fill, conjugated efficiently to VLPs (fig. S7C). Thus the RC1–4fill immunogen trimer exhibited unique undesirable properties compared to the other immunogen-VLPs. Second, although the SEC protocol efficiently separated free SOSIP trimers from VLP-coupled SOSIP trimers when evaluated at relatively small scales (fig. S7D), the same protocol did not completely isolate free SOSIP trimers when scaled up to make the quantities of SOSIP-VLPs required for NHP and rabbit immunizations (fig. S7E, black trace). Thus the RC1–4fill-VLPs used for the primes in NHPs and rabbits (38) and the SOSIP-VLPs used for Boosts 1 to 3 (table S1, table S2) included free Env trimers in addition to VLP-conjugated trimers. We worked out an alternative SEC protocol for separating SOSIP-VLPs from free trimers in time for Boost 4 (fig. S7E, red trace). These characterizations revealed two potential reasons to explain the presence of the off-target, anti-trimer base antibodies: (i) sparse conjugation of the priming immunogen RC1–4fill, and (ii) the presence of unconjugated Env trimers in the prime and first three boosts.

Single B cell cloning revealed heterologous weakly neutralizing antibodies with epitopes that competed with V3-glycan patch bNAbs and other bNAbs

Env-specific B cells were isolated using a multi bait strategy including Avitagged-biotinylated RC1-glycanKO (38) or an irrelevant, non-HIV-1-based, biotinylated bait and two or three Avitagged-biotinylated SOSIP Env-based baits: RC1, BG505 and B41 (20, 70) (Fig. 5). We screened about 30 IgG monoclonal antibodies (mAb) produced from these sequences against a panel of seven Tier 1 and Tier 2 HIV-1 pseudoviruses (screening panel, Fig. 5A), finding nine that neutralized four or more strains (Fig. 5A; table S4). These were further evaluated against 12 strains from a global HIV-1 reference panel (71) (12-strain panel; Fig. 5A). Although most of the 50% inhibitory concentrations (IC₅₀ values) indicated extremely low potencies, the nine mAbs showed neutralization breadth across this 19-virus panel. In particular, selected mAbs neutralized the heterologous SHIV_DH12-V3AD8 pseudovirus with IC₅₀s of <0.020 μg/mL (Ab1456, Ab1457, Ab1415, and Ab1461); these mAbs also exhibited IC₅₀s of ≤1.1 μg/mL against replication-competent SHIV_DH12-V3AD8 (Fig. 5A).

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Figure 5.

mAbs isolated from prime-boosted NHPs show weak, but heterologous, neutralization and target epitopes that compete with V3-glycan patch bNAbs.

(A) IC50 values against the indicated pseudoviruses are shown for 9 selected mAbs cloned from immunized NHPs for neutralization. NN, non-neutralizing. (B) The heat map shows results of competition ELISAs to map epitopes of mAbs derived from immunized NHPs. Results represent one experiment of two independent replicates. Randomly biotinylated RC1 was bound to a Streptavidin ELISA plate. Each well was then incubated with a potential competitor Fab for 2 hours and then an IgG was added. Bound IgG was detected using an anti-Fc antiserum. The percent binding observed after addition of a competitor Fab was normalized to the binding in the absence of a competitor Fab. NHP IgG results are shown on the left; control bNAb or non-neutralizing IgG antibody (10A and 12N) results are shown on the right.

To map epitope(s) recognized by the NHP mAbs, we adapted the competition ELISA for mAb epitope mapping. For these experiments, we incubated immobilized RC1 with a Fab from a potential competitor mAb and then probed with an NHP IgG or control IgG (Fig. 5B). We used the same competitor Fabs and control IgG antibodies as in the serum competition assay with the addition of PGT121/10–1074 iGL, the inferred germline sequence for the PGT121 and 10–1074 V3-glycan patch bNAbs (28), and the substitution of PG9 (58) as the representative V1V2 bNAb.

Results from the competition ELISA (Fig. 5B) mapped seven mAbs (Ab1456, Ab1457, Ab1271, Ab1415, Ab1289, Ab1368, and Ab1461) as targeting epitopes on Env that were blocked by the V3-glycan patch bNAbs 10–1074 and PGT128 (28, 39) and two mAbs (Ab1303 and Ab1573) as targeting a CD4bs epitope blocked by 3BNC117 (26) (Fig. 5B). Verification of the competition ELISA methodology was provided experiments using control bNAbs targeting known HIV-1 Env epitopes (Fig. 5B). Interesting, although its epitope was not intentionally targeted in our prime-boost protocol, the CD4bs Ab1303 exhibited neutralization activity against almost every strain it was tested against (Fig. 5A), demonstrating that our prime-boost regimen elicited neutralizing antibodies against more than one epitope on HIV-1 Env.

Immunogen expression and purification

Env immunogens were expressed as soluble SOSIP.664 native-like gp140 trimers (43) as described (38). For SpyTagged trimers, a C-terminal SpyTag sequence (13 residues) was added to allow formation of an irreversible isopeptide bond to SpyCatcher protein (49). We also produced AviTagged BG505 and clade B B41 SOSIPS for B cell sorting experiments, and untagged and SpyTagged versions of 11MUTB-4fill (a version of the BG505-related 11MUTB SOSIP (42) containing PNGSs to add glycans to gp120 positions N230, N241, N289, and N344), B41–5MUT (a B41 version of the BG505-related 5MUT SOSIP (42) including a PNGS at gp120 position 289, B41wt (a B41 SOSIP with an introduced PNGS at position 289), clade C Du422, clade B AMC011 with an introduced PNGS at gp120 position 230, consensus C SOSIP (ConC) (56), and a consensus M SOSIP (ConM) (55). Amino acid sequences of SpyTagged SOSIP immunogens are shown in table S5.

Soluble SOSIP Envs were expressed by transient transfection in HEK293–6E cells (National Research Council of Canada) or Expi293 cells (Life Technologies) and purified from transfected cell supernatants by 2G12 or NIH45–46 immunoaffinity chromatography and size exclusion chromatography (SEC) as described (87). Soluble Envs were stored at 4°C in 20 mM Tris pH 8.0, and 150 mM sodium chloride (TBS) (untagged and AviTagged versions) or 20 mM sodium phosphate pH 7.5, 150 mM NaCl (PBS) (SpyTagged versions).

VLP preparation and conjugation

SpyCatcher-AP205 VLPs were expressed in bacteria and purified as described (48, 88). SpyTagged SOSIP immunogens were incubated at a 2 to 3–fold molar excess with SpyCatcher-VLPs for 12 to 24 hours at room temperature in phosphate-buffered saline (PBS). Conjugated VLPs were separated from free Env trimers by SEC on a Superdex 200 (fig. S7D) or Superose 6 (fig. S7E) column. Conjugation of Env trimers was verified by SDS-PAGE (fig. S7C). Immunogen concentrations for immunizations were estimated by comparing to known amounts of the analogous unconjugated Env trimer on a SDS-PAGE gel.

Conjugated VLPs were examined by nsEM (fig. S7A and D). Purified VLPs were diluted to about 10 μg/mL immediately before adding 3 μL to a glow-discharged ultrathin C film on a holey carbon support film, 400 mesh, Cu grid (Ted Pella). After blotting, the grids were stained by uranyl acetate and then imaged using a FEI Tecnai T12 transmission electron microscope operating at 120 keV with a Gatan Ultrascan 2k × 2k CCD camera. Each image was collected using a 1 second exposure at about 2 μm defocus and 42,000× magnification, resulting in 2.5 Å per pixel.

SPR binding studies

SPR experiments were performed using a T200 (Biacore). To determine if the Env trimer base was accessible for antibody binding on a densely-conjugated VLP, we covalently immobilized 12N IgG (47) on a CM5 chip using primary amine chemistry according to manufacturer’s instructions to a coupling density of about 3000 resonance units (RUs). 0.1 mg/mL RC1 SOSIP or 0.1 mg/mL RC1-VLPs (based on SOSIP concentration) were then injected at a flow rate of 30μL/min for 60 seconds, followed by monitoring of the dissociation phase for 300 seconds. Flow cells were regenerated with 10 mM glycine pH 3.0 at a flow rate of 90 μl/min. Sensorgrams were processed and plotted using the Biacore Evaluation Software.

Animal immunizations

Eight rhesus macaques (Macaca mulatta) of Indian genetic origin, two-to-four years of age, were housed in a biosafety level 2 NIAID facility 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). All animal procedures and experiments were performed according to protocols approved by the IACUC of NIAID, NIH. The NHPs used in this study did not express the MHC class I Mamu-A*01, Mamu-B*08 and Mamu-B*17 alleles. NHPs were immunized subcutaneously in the medial inner forelegs and hind legs (total of 4 sites per animal) with approximately 100 μg of the indicated SOSIP-VLPs adjuvated in IscoMPLA (375 U/animal) as described (38). Animals were immunized and blood samples were drawn from naïve and immunized macaques at the time points indicated in Fig. 1A, tables S1 and S2, and Fig. 6. Blood samples were processed to serum or plasma by centrifugation in serum separating tubes or EDTA-treated tubes, respectively. Serum and plasma samples were frozen and stored at −80°C. Lymph node biopsies for single B cell cloning were obtained after Boost 3 and Boost 4 (Fig. 1A).

Four six-month-old New Zealand White rabbits (Covance) were used for immunizations. Rabbits were immunized subcutaneously with ~22 μg of a SOSIP-VLP in an ISCOMs-like saponin adjuvant as described (38, 89). Blood from immunized rabbits was collected from the medial ear artery on weeks 0 and 2 following immunization (test and production bleeds) or the jugular vein/carotid artery after the final boost (terminal exsanguination) by Covance. Serum was processed by Covance laboratory technicians by centrifugation in serum separating tubes and shipped frozen. Procedures in rabbits were approved by the Denver PA IACUC Committee.

In vitro neutralization assays

RC1 and 11MUTB pseudoviruses were generated by transfection of HEK293 cells with a codon-optimized gp160 construct containing RC1 (38) or 11MUTB (42) mutations in gp120 and a wild-type BG505 gp41 as previously described (51, 90). Pseudovirus neutralization assays were conducted using TZM-bl reporter cells as described (51, 90), either in house (table S1) or at the Collaboration for AIDS Vaccine Discovery (CAVD) core neutralization facility (table S2, Fig. 5A). IgG mAbs were evaluated in duplicate with an 8-point, 5-fold dilution series starting at a top concentration of 500 μg/mL. RC1 and 11MUTB pseudovirus assays were repeated at least twice for each value reported in table S1. For polyclonal neutralizations, serum or plasma samples were heat inactivated at 56 °C for 1 hour before being added to the neutralization assays, and then neutralization was evaluated in duplicate with an 8-point, 4-fold dilution series starting at a dilution of 1:20 (heterologous strains) or 1:100 (RC1 and 11MUTB strains). The dilution at which 50% of virus was neutralized (ID₅₀) and the percent of neutralization at a 1:20 dilution (% 1:20) are reported (table S2). The panel of global HIV-1 Env reference clones were obtained through the NIH HIV Reagent Program (ARP-12670) as contributed by Dr. David Montefiori.

For neutralization assays to evaluate NHP serum for neutralization against SHIVs, we used two types of assays (91): (i) a TZM-bl entry assay with a pseudotyped SHIV_DH12-V3AD8 and (ii) a TZM-bl infection assay using replication-competent SHIV_DH12-V3AD8 virus. The pseudotyped virus expressed the SHIV_DH12-V3AD8 Env was conducted as described above. The replication-competent SHIV_DH12-V3AD8 virus was made as described (63, 92) by first transfecting HEK293T cells with SHIV_DH12-V3AD8 cloned DNA, infecting rhesus PBMCs with the transfected HEK293T cell supernatant, and then collecting the infected rhesus PBMC cell supernatant at 48 hours as the SHIV DH12-V3AD8 replication competent virus stock. For replication-competent virus assays, the protease inhibitor, indinavir (Merck), was added to the medium (final concentration of 1 μM) to prevent a second round of viral replication.

Isolation and purification of rabbit and NHP polyclonal IgG antibodies

Rabbit and NHP polyclonal IgG antibodies were purified from serum samples using 5-mL HiTrap MabSelect SuRe columns (Cytiva). Serum samples were diluted 10-fold with cold PBS, and applied to prepacked columns at 1 mL/min. Bound IgG antibodies were washed with 5 column volumes PBS and eluted with 5 column volumes 0.1M glycine pH 3.0, 100 mM NaCl. Samples were then immediately neutralized with 2M Tris-HCl, pH 8. To produce polyclonal Fab fragments, IgG antibodies were buffer exchanged into PBS and cleaved by papain digestion using activated crystallized papain (Sigma-Aldrich) for 30 to 60 min at 37°C at a 1:100 enzyme:IgG ratio. To remove undigested IgG antibodies and Fc fragments, digested products were applied to a 1 mL HiTrap MabSelect SuRe column (Cytiva) and the flowthrough containing cleaved Fabs was collected. Fabs were further purified by SEC using a Superdex 200 Increase 10/300 column (GE Healthcare Life Sciences) in TBS, before concentrating and storage at 4°C.

nsEMPEM data collection and processing

nsEMPEM methods were adapted from (50, 67). Polyclonal rabbit Fab-Env complexes were formed by incubating 25 μg of a SOSIP.664 trimer with 2–3 mg of polyclonal Fabs in 100 μL total volume either overnight (post-Prime and post-Boost 1) or for 30 minutes (all others) at room temperature. NHP Fab-Env complexes were formed by incubating 12.5 μg of Env SOSIP.664 trimers with 1.25 mg of polyclonal Fabs in 50 μL total volume for 30 minutes at room temperature. Fab-Env complexes were purified by SEC on a Superose 6 increase 10/300 GL column (Cytiva). Fractions containing complexes were pooled and concentrated to 5 to 10 μg/mL before deposition on a freshly glow-discharged 300 mesh carbon-coated copper grid (Ted Pella) and stained with 1.5% (w/v) uranyl formate (Electron Microscopy Sciences).

All Fab-Env complexes were imaged on a 200 kV Talos Arctica transmission electron microscope (Thermo Fisher Scientific) operating at room temperature, equipped with a K3 direct electron detector (Gatan) using SerialEM 3.7 (93), except for the Rabbit 2249 RC1-post-Prime complex, which was imaged on a 120 kV Tecnai T12 equipped with a CCD camera. Images were processed in cryoSPARC v 2.14, and a reference-free particle stack was generated using a Gaussian blob picker (94) or by EMAN2 (95) for the rabbit post-Boost 4 – RC1 dataset. Particles corresponding to Fab-Env complexes were identified by iterative rounds of 2D classification. 2D class averages that displayed structural elements interpreted as Fabs bound to an intact Env trimer or dissociated gp140 protomer were selected for further 2D and 3D classification. Ab initio models generated in cryoSPARC were either reported directly or used for heterogeneous or homogenous refinement. Figures were prepared using UCSF Chimera (96, 97), and Fabs were highlighted in selected 2D classes using IMOD (98).

Isolation of mAbs from immunized NHPs

Single HIV-1 Env-specific B cells were isolated from NHP lymph nodes from lymph nodes (20, 70). Antibody cloning from immunized NHPs and production of the NHP mAbs in Fig. 5 was done as described (20, 70). Additional mAbs were either previously reported (Ab1170_NHP, Ab275_mur) (38) or isolated from an RC1-primed and sequentially-boosted wt mouse (Ab283_mur) using similar methods. V(D)J gene assignments of NHP and murine antibodies were done using IMGT/V-QUEST (99). Sequence alignments were done using Clustal Omega (100).

Epitope mapping by competition ELISAs

Competition ELISAs were performed using a Tecan Evo liquid handling robot. RC1 was randomly biotinylated at primary amines using EZ-link NHS-PEG4 Biotinylation Kit according to the manufacturer’s protocol (Thermo Fisher Scientific), resulting in about 5 biotins per protomer. Randomly biotinylated SOSIP trimers coated on streptavidin plates were verified to exhibit similar properties as counterpart site-specifically immobilized SOSIPs (by a C-terminal tag recognized by an anti-tag antibody) and evaluated for binding to anti-Env antibodies by ELISA. By contrast, SOSIP trimers directly coated onto ELISA plates exhibited aberrant behaviors such as binding to CD4-induced antibodies in the absence of CD4, which was not observed for counterpart experiments using randomly biotinylated or site-specifically immobilized SOSIPs. Biotinylated RC1 (1 μg/mL) was bound overnight at 4°C to a 384-well ELISA plate coated with streptavidin (Thermo Fisher Scientific). The plate was washed with TBS-T, incubated with 10 μg/mL Fab for 2 hours, and then 1:100 dilution of serum, 1 μg/mL IgG (controls in serum competition ELISA), or 10 ug/mL mAb IgG (controls and NHP mAbs; mAb competition ELISA) was added and incubated for 2 hours at room temperature. Bound IgG was detected using an horseradish peroxidase-conjugated anti-human IgG Fc secondary antibody (Southern Biotech Cat # 2014–05; 1 hour, room temperature) and developed with SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific). Relative light units (RLU) were measured and the signal for each Fab-IgG pair was normalized to the signal for the IgG when no blocking Fab was present. Measurements were performed in technical quadruplicates and the data presented are representative of two independent experiments.

Antibody production and purification

IgG mAbs were expressed by transient transfection in Expi293 cells or HEK293–6E cells and purified from cell supernatants using MabSelect SURE (Cytiva) or protein A or G columns (GE Healthcare) as described (38, 61). 6xHis-tagged Fabs were purified by Ni-NTA chromatography (Cytiva) and SEC (61). The Ab283_mur mouse Fab for structural studies was obtained by digesting Ab283_mur IgG at 1–5 mg ml⁻¹ with ficin (Sigma Aldrich) and purified by protein G (GE Healthcare) and SEC chromatography (101), followed by monoQ 5/50 (GE Healthcare) ion exchange chromatography. The common iGL of the PGT121 and 10–1074 bNAbs (28) was expressed as an IgG.

Single-particle cryo-EM

For the Ab283_MUR–RC1–8ANC195 complex structure, purified Ab283_mur Fab, 8ANC195 Fab, derived from a bNAb directed against the gp120-gp41 interface (61), and RC1 SOSIP trimer were incubated at a 3:1 molar ratio of Fab per protomer overnight at room temperature. For the Ab1170_NHP–RC1–8ANC195 complex structure, purified Ab1170_NHP Fab, 8ANC195 Fab, and RC1 SOSIP trimer were incubated at a 3:1 molar ratio of Fab per protomer overnight at room temperature.

The complexes were subjected to SEC on a Superdex 200 column (GE Life Sciences), and 3 μL of purified complex (0.4 mg/mL for Ab1170_NHP–RC1–8ANC195 and 1.35 mg/mL for Ab283_mur–RC1–8ANC195) was added to freshly glow-discharged 300 mesh, 1.2/1.3 Quantifoil copper grids and then blotted for either 2 or 3.5 seconds with Whatman no. 1 filter paper at 22°C and 100% relative humidity using a Mark IV Vitrobot. Samples were then vitrified in 100% liquid ethane. Micrographs were collected on a Talos Arctica TEM (Thermo Fisher Scientific) operating at 200 kV and processed in Relion (102, 103) for both complexes (table S4).

For the Ab283_mur–RC1–8ANC195 complex, automated data collection was carried out using EPU 2 software (Thermo Fisher Scientific) with a Falcon 3EC direct electron detector (Thermo Fisher Scientific). Micrographs were motion-corrected including dose-weighting using MotionCorr2 within Relion (102). The non-dose-weighted micrographs were used for contrast transfer function (CTF) estimation in CTFFind-4.1 (104). After discarding micrographs with poor CTF fits and signs of visible ice, particles were auto-picked, extracted, and 2D classified. Particles from selected 2D classes were used for an initial refinement with an initial map that was created by 60 Å-low-pass-filtering a map created from a model of BG505–10–1074–8ANC195 complex using molmap in UCSF Chimera (96, 105). This model was then used as an input model for 3D classification. Particles from selected 3D classes were refined again into a 3D model, which was calculated using C3 symmetry applied and a mask that did not include Fab C_HC_L domains. From there, particles were polished and movie-refined before a final 3D refinement that resulted in a map with a gold standard Fourier shell coefficient (FSC) calculation of 5.2 Å.

For the Ab1170_NHP–RC1–8ANC195 complex, automated data collection was carried out using SerialEM software (93, 106) with a Gatan K3 camera. Micrographs were motion-corrected including dose-weighting using MotionCorr2 within Relion (102). The non-dose-weighted micrographs were used for CTF estimation in CTFFind-4.1 (104). After discarding micrographs with poor CTF fits and signs of visible ice, particles were auto-picked, extracted, and 2D classified. Particles from selected 2D classes were used for 3D classification with an initial map that was created by 50 Å-low-pass-filtering a map created from a model of the BG505–8ANC195 complex (61) using molmap in UCSF Chimera (96, 105). Selected particles were then 3D refined using C1 symmetry into a map with a gold standard FSC calculation of 4.5 Å. Coordinates for the Ab283mur–RC1 portion of the Ab283mur–RC1–8ANC195 complex were generated by docking chains from reference structure (PDB 6ORQ) into the map using UCSF Chimera (96, 105). The model was then modified to the correct sequence. Subsequently, the model was refined iteratively in Phenix (107, 108) and Coot (109). Structure figures were made using UCSF Chimera (96) or PyMol (110).

NHP challenge experiments

The origin and preparation of the SHIV_DH12-V3AD8 stock used for virus challenge in NHPs was previously described (63). In “high dose” virus challenges, NHPs were inoculated intrarectally with 1000 TCID₅₀ of SHIV_DH12-V3AD8 2 weeks following Boost 4. For “repeated low dose challenges,” animals were also challenged intrarectally 2 weeks following the Boost 4, but with 10 TCID₅₀ of SHIV_DH12-V3AD8 and every week thereafter, until infection was established, as described (91). Viral RNA amounts in plasma were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (ABI Prism 7900HT sequence detection system; Applied Biosystems) as described (111).

We thank members of the Bjorkman, Martin, and Nussenzweig laboratories for discussions, M. Howarth (Oxford) for providing plasmids and advice for VLP expression and purification, J. Moore (Weill Cornell Medical College), R.W. Sanders and M.J. van Gils (Amsterdam UMC) for SOSIP expression plasmids, J. Vielmetter, P. Hoffman, and the Protein Expression Center in the Beckman Institute at Caltech for expression assistance, T. Eisenreich and S. Tittley for animal husbandry, and K. Gordon for flow cytometry. Electron microscopy was performed in the Caltech Cryo-EM Center with assistance from S. Chen and A. Malyutin.