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. 2017 May 16;46(5):804-817.e7.
doi: 10.1016/j.immuni.2017.04.021.

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach

Paola Martinez-Murillo  1 , Karen Tran  2 , Javier Guenaga  2 , Gustaf Lindgren  3 , Monika Àdori  1 , Yu Feng  2 , Ganesh E Phad  1 , Néstor Vázquez Bernat  1 , Shridhar Bale  2 , Jidnyasa Ingale  2 , Viktoriya Dubrovskaya  2 , Sijy O'Dell  4 , Lotta Pramanik  1 , Mats Spångberg  5 , Martin Corcoran  1 , Karin Loré  3 , John R Mascola  4 , Richard T Wyatt  6 , Gunilla B Karlsson Hedestam  7
Affiliations

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach

Paola Martinez-Murillo et al. Immunity. .

Abstract

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Keywords: B cell responses; HIV-1; clade C; envelope glycoproteins; epitope; germinal centers; liposome; monoclonal antibody; trimers; vaccine.

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Figures

Figure 1
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; 96,000x 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.
Figure 2
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), 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 400μm in C and 1000μm in D and E. Pre- and post-immunization samples are shown in F–J. In F–H, each dot represents one GC parameter: (F) total individual GC area, (G) number of Tfh cells in each individual GC (H) number of Ki67+ in each individual GC. (I, 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 difference were evaluated by Mann-Whitney test; ns: non-significant; * for p≤0.05, ** for p≤0.01, *** for p≤0.001 and **** for p≤0.0001. These experiments were performed two independent times.
Figure 3
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 3rd, 4th and 5th 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 4th and 5th immunization. Bars indicate group means, symbols represents an animal. Statistical difference 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.
Figure 4
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) Distribution of the VDJ family usage for the mAb HC. (D) HCDR3 amino acid 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, the ELISA and neutralization assays twice. See also Figures S2–S5 and Table S1.
Figure 5
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 column) as assessed by ELISA, where 0% competition was the absorbance measured with no competitor present. (D) Structure of the V1 (cyan) V2 (green) 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.
Figure 6
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.
Figure 7
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.
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References

    1. Andrabi R, Voss JE, Liang CH, Briney B, McCoy LE, Wu CY, Wong CH, Poignard P, Burton DR. Identification of Common Features in Prototype Broadly Neutralizing Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design. Immunity. 2015;43:959–973. - PMC - PubMed
    1. Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, Xiao Z, Veenstra TD, Conrad TP, Lempicki RA, et al. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nature immunology. 2008;9:301–309. - PubMed
    1. Batista FD, Neuberger MS. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity. 1998;8:751–759. - PubMed
    1. Burton DR, Mascola JR. Antibody responses to envelope glycoproteins in HIV-1 infection. Nature immunology. 2015;16:571–576. - PMC - PubMed
    1. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. - PMC - PubMed

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