Sequential immunization of macaques elicits heterologous neutralizing antibodies targeting the V3-glycan patch of HIV-1 Env

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 develop after prolonged virus and antibody coevolution. Previous studies showed that sequential immunization with a V3-glycan patch germline-targeting HIV-1 envelope trimer (Env) followed by variant Envs can reproduce this process in mice carrying V3-glycan bNAb precursor B cells. However, eliciting bNAbs in animals with polyclonal antibody repertoires is more difficult. We used a V3-glycan immunogen multimerized on virus-like particles (VLPs), followed by boosting with increasingly native-like Env-VLPs, to elicit heterologous neutralizing antibodies in nonhuman primates (NHPs). Structures of antibody/Env complexes after prime and boost vaccinations demonstrated target epitope recognition with apparent maturation to accommodate glycans. However, we also observed increasing off-target antibodies with boosting. Eight vaccinated NHPs were subsequently challenged with simian-human immunodeficiency virus (SHIV), and seven of eight animals became infected. The single NHP that remained uninfected after viral challenge exhibited one of the lowest neutralization titers against the challenge virus. These results demonstrate that more potent heterologous neutralization resulting from sequential immunization is necessary for protection in this animal model. Thus, improved prime-boost regimens to increase bNAb potency and stimulate other immune protection mechanisms are essential for developing anti–HIV-1 vaccines.

Figure 1.. NHPs were immunized with a prime and four boosts.

(A) The sequential immunization regimen is shown. NHPs were bled at the indicated times and other times noted in tables S1 and S2. The NHP (NHP 1) used for nsEMPEM (Fig. 2) was immunized with B41–5MUT-VLPs for Boost 2. (B) ID₅₀ values are shown for serum neutralization of viruses at the indicated time points (table S1). Individual dots represent each NHP. The dotted line at 100 indicates the lowest dilution for the 11MUTB and RC1 pseudoviruses. The dotted line at 20 indicates the lowest dilution for SHIVDH12-V3AD8 PV and RC viruses. The arithmetic mean for each entry is indicated by the height of the bar, with the standard deviation shown as a vertical line. PV, pseudovirus; RC, replication competent. (C) Percent neutralization at a 1:20 dilution is shown against heterologous HIV-1 strains at the indicated time points. Individual dots represent each NHP. Samples are serum except for the 3 week post-B3 sample, which was plasma. Dotted lines indicate the highest background neutralization value for the murine leukemia virus (MuLV) control for serum samples (25%; all samples except for post B3, 3 weeks) and plasma samples (41%; post B3, 3 weeks) (table S2). The arithmetic mean for each entry is indicated by the height of the bar, with the standard deviation shown as a vertical line.

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.

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).

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).

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.

Figure 6.. Immunization conferred minimal protection against intrarectal challenge of a heterologous SHIV in NHPs.

(A) The schedule for prime, boosting, and SHIV_DH12-V3AD8 challenge for NHPs is shown, including NHP numbers and IDs. (B) Left: Plasma viral loads are shown for macaques challenged intrarectally with a single high dose (1000 TCID50) of SHIV_DH12-V3AD8 two weeks after receiving the final boost. Controls are shown in orange and immunized animals are shown in blue. Right: A comparison of viral loads in control versus immunized NHPs is shown as a function of time after infection presented as geometric means with standard deviations. Values for individual NHPs are shown as dots. (C) Left: Plasma viral loads are shown for macaques challenged intrarectally with repeated low doses (10 TCID50) of SHIV_DH12-V3AD8 weekly, beginning two-weeks following the final boost. Controls are shown in orange and immunized animals are shown in blue. Right: A comparison of viral loads in control versus immunized NHPs is shown as a function of time after infection presented as geometric means with standard deviations. Values for individual NHPs are shown as dots. Time points were plotted when all animals were tested.