Volume 46, Issue 5, Pages 804-817.e7 (May 2017) Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach  Paola Martinez-Murillo, Karen Tran, Javier Guenaga, Gustaf Lindgren, Monika Àdori, Yu Feng, Ganesh E. Phad, Néstor Vázquez Bernat, Shridhar Bale, Jidnyasa Ingale, Viktoriya Dubrovskaya, Sijy O’Dell, Lotta Pramanik, Mats Spångberg, Martin Corcoran, Karin Loré, John R. Mascola, Richard T. Wyatt, Gunilla B. Karlsson Hedestam  Immunity  Volume 46, Issue 5, Pages 804-817.e7 (May 2017) DOI: 10.1016/j.immuni.2017.04.021 Copyright © 2017 Elsevier Inc. Terms and Conditions

Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 The Antigenicity of NFL TD CC Env Trimers Is Intact after Conjugation to Liposomes (A) Schematic representation of soluble 16055 NFL TD CC trimers containing C-terminal His tags (left) and the trimers arrayed at high density on liposomes following nickel-NTA-lipid capture by the His tags (top right; DGPC lipid head group, white spheres; DGS-NTA(Ni) lipid, red spheres) with a representative negative-stain EM of the liposomal trimer array shown below. (B) Negative-stain EM images of representative liposomes, selected from hundreds in the field, after incubation for 24 hr at 4°C in PBS (left), at 37°C in PBS (middle), or at 37°C in Matrix-M adjuvant (right; 96,000× magnification; scale bars, 100 nm). (C) Confirmation of trimer-specific bNAb recognition (left; PGDM1400, PG16, and PGT145) of the trimers following liposome conjugation and recognition by CD4bs-directed bNAbs (right; VRC01 and VRC06), but not by CD4bs-directed non-bNAbs (F105 and GE136) by BLI. (D) Quantification of soluble trimer and trimer-conjugated liposome relative Env concentrations were calculated based on a standard curve equation generated from assays using the Advanced Protein Assay Reagent. Sample calculation for 100 μg is shown, with SDS reducing gel of each to confirm equivalent Env amounts (right; 5 μg per lane). These experiments were performed at least two independent times. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Immunization with Liposome-Displayed Trimers Elicits Superior GC Responses (A) The immunization scheme consisted of five injections on weeks 0, 4, 12, 26, and 40 (brown arrows), and sampling was performed two weeks after each inoculation and four additional times as indicated (red arrows). The animals were divided in two groups: soluble trimers (soluble, blue) (n = 6) and liposome arrayed-trimers (liposome, orange) (n = 6), both formulated in Matrix-M. (B) Half-maximum binding titers (OD50) of Env-specific IgG represented on a log10 scale. Lines represent group means, and each dot represents an animal. (C) Cryosections of draining inguinal lymph nodes from rhesus macaques stained with antibodies against CD3 (blue), PD-1 (green), and Ki67 (red), where GCs structures were recognized. (D) GCs structures were identified by the conglomeration of PD-1+ cells and Ki67+ cells surrounded by CD3+ cells. (E) GCs were manually selected using cell profiler software, and in-house pipelines allowed the automatic count of the fluorescent signal. The white bar represents 400 μm in (C) and 1000 μm in (D) and (E). Pre- and post-immunization samples are shown in (F)–(J). (F–H) In (F)–(H), each dot represents one GC parameter: (F) total individual GC area, (G) number of Tfh cells in each individual GC, and (H) number of Ki67+ in each individual GC. (I and J) Each dot represents the average number of Tfh or Ki67+ cells per animal. The average was calculated as the number of positive cells within a GC divided by the total number of GCs. (I) Average number of Tfh cells per GC. (J) Average number of Ki67+ cells per GC. Post-immunization samples were from two weeks after the second immunization. Statistical differences were evaluated by Mann-Whitney test; ns: non-significant; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. These experiments were performed two independent times. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Liposome-Arrayed Trimers Stimulate Enhanced Autologous Tier-2-Neutralizing Ab Responses Compared to Soluble Trimers (A) Binding titers (OD50) of Env-specific IgG responses two weeks after each immunization represented as log10. Lines represent group means, and dots represent each animal, soluble (blue) and liposome-arrayed (orange). (B) Half-life of Env-specific IgG between the third and fourth immunization. Lines represent group means, dots represent each time point mean. Brown arrows and dotted lines represent each immunization point in (A) and (B). (C) Plasma neutralization activity against SF162, 16055, and SIV pseudoviruses was calculated for each animal two weeks after the third, fourth, and fifth immunization as the reciprocal dilution giving 50% of neutralization (ID50 titer). The background neutralization ID50 titer threshold was < 10. ND, not determined. (D) Comparison of the plasma-neutralization activity (ID50 titer) between soluble (blue) and liposome-arrayed groups (orange) against SF162 and 16055 pseudoviruses two weeks after the fourth and fifth immunization. Bars indicate group means, symbols represent individual animals, and error bars indicate standard deviation (SD). Statistical differences were evaluated by two-way ANOVA followed by Sidak’s multiple comparison test; ns, non-significant; ∗(p ≤ 0.05); ∗∗(p ≤ 0.01); ∗∗∗(p ≤ 0.001); ∗∗∗∗(p ≤ 0.0001). These experiments were performed two independent times. See also Figure S1. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Isolated mAbs Display Diverse Binding and Neutralization Properties (A) FACS plots showing the gating strategy used for single-cell sorting Env+ memory B cells from animal D11. PBMCs were sampled and stained two weeks after the third immunization. (B) Categorization of the isolated mAbs (n = 18) based on Env specificities and neutralization capacities. Neutralization activity was reported as the reciprocal dilution giving 50% of neutralization (ID50 titer). The background neutralization ID50 titer threshold was > 30. Genetic features of the mAbs are shown. (C) HC VDJ family usage. (D) HCDR3 aa length. (E) SHM of VH genes at the amino acid level calculated as the percentage divergence from the assigned germline gene. The sorting and mAb isolation was performed once, and the ELISA and neutralization assays were performed twice. See also Figures S2, S3, S4, and S5 and Table S1. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 The Vaccine-Induced Autologous Neutralizing mAbs Target a V2 Epitope (A) Neutralization potency (IC50, μg/mL) of the NHP mAbs with selected N-glycan modifications of 16055 Env compared to WT. (B) Plasma neutralization titers (ID50) from each animal against WT 16055 compared to N301 N-glycan deleted virus. (C) Binding of biotinylated NHP mAbs to 16055 NFL TD CC trimers (His-captured) in the presence of non-biotinylated mAb competitors (left) as assessed by ELISA, where 0% competition was the absorbance measured with no competitor present. (D) Structure of the V1 (cyan), V2 (green), and V3 (orange) region from the clade G X1193.c1 trimer crystal structure (PDB: 5FYJ, top left) with strands A, B, C/C′, D, and loops V2a and V2b indicated; orientation within one protomer of the trimer shown below. Bar diagram schematic indicating locations of the variable region deletions in 16055 gp120 to map binding (top right) with relative binding to each deletion mutant compared to WT gp120 shown below; +, binding; −, no binding. (E) Differential adsorptions of 16055 virus entry. Control bNAbs PG9 (V2-directed) and HJ16 (CD4bs-directed) along with D11A.F9 and the post 3 NHP plasma were tested at a fixed concentration and pre-incubated with culture medium (no inhibitor) or titrating amounts of WT or ΔV1V2 TriMut gp120 to assess depletion of 16055-neutralizing activity in the TZM-bl assay. The experiments were performed at least two independent times. See also Figure S6. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 The Autologous Neutralizing mAbs Bind Env with High Affinity and Target the V2 Cap (A) Binding curves of vaccine-induced Fabs at selected concentrations (250 nM, top, serial 2-fold reductions) to the 16055 NFL TD CC trimers for affinity measurements by BLI. (B) Representative EM 2D class averages of Fab:trimer complexes with 1, 2, and 3 Fabs per particle as indicated (left); selected 2D image of a single trimer in complex with three Fabs (middle); 3D reconstruction of the 16055 NFL TD CC trimer in complex with three D11A.F9 Fabs, top view (right). See also Figure S7. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Docking of the EM 3D Reconstruction of the Fab:Trimer Density Suggests a Horizontal Binding Approach to the Env Trimer Apex (A) Top and side views of the D11A.F9-liganded trimer EM density (light gray) with the high-resolution BG505 SOSIP.664 crystal structure (PDB: 5T3Z; gray ribbon) and the D11A.F9 Fab model (red) fit within the 3D reconstruction (left); V1 (cyan) and V2 (green) are highlighted on each protomer. Expanded view of the D11A.F9 Fab in proximity of the V2 (green) and V1 (cyan) region; dashed lines indicate the predicted structure of the loops, which are incomplete in the crystal structure. (B) Side view of the VRC01 crystal structure (PDB: 5KZC, blue) and the D11.A.F9 model (red) docked into the natively glycosylated BG505 SOSIP.664 crystal structure (PDB: 5T3Z). (C) The molecular surfaces of V1 (cyan) and V2 (green) regions of one protomer in the clade G X1193.c1 crystal structure (PDB: 5FYJ) are shown along with proximal N-glycans (tan spheres). See also Figure S7. Immunity 2017 46, 804-817.e7DOI: (10.1016/j.immuni.2017.04.021) Copyright © 2017 Elsevier Inc. Terms and Conditions