Figure 1.
Analysis of deglycosylated JRFL protein in reducing SDS-PAGE and Western-blotting with a panel of HIV-1 MAbs.
WT glycosylated and deglycosylated JRFL gp140 Env proteins were fractionated in 4–12% SDS-PAGE under reducing conditions, stained with coommassie blue or were transferred to nitrocelose membranes and blotted with a panel of human anti-HIV-1 mAbs as indicated. WT glycosylated JRFL gp140 Env protein is labeleled as [-] on the top of the lane. The amount of PNGase F (in U) used in the reaction per µg of protein is indicated on the top of lanes with progressively deglycosylation. Lane * refers to treatment of denatured JRFL gp140 protein with PNGaseF (500 U) at 37 degrees for 2 hours. Progressive reductions in molecular weight of JRFL gp140 with increasing amounts of PNGase used to deglycosylate JRFL gp140 Env are shown in Coomassie staining gels and Western blots using mAbs F39, 2F5 and 4E10. The higher molecular weight band(s) shown in lane * in the coomassie stained gel and in Western blots by F39, 2F5 and 2G12 were due to the incomplete reduction of JRFL gp140 Env protein (see coomassie stained gel in Figure S1).
Figure 2.
Analysis of antigenic epitopes expressed on WT glycosylated and deglycosylated JRFL gp140 by surface plasmon resonance (SRP).
SPR assays were performed as described in Materials and Methods. Shown are the ability of WT glycosylated and deglycosylated JRFL gp140 to bind to sCD4 (Panel A) and to mAb 17B (Panel B). sCD4 or HIV-1 mAbs T8 and A32 were covalently immobilized to a CM5 sensor chip (BIAcore), and WT glycosylated (10 to 75 µg/ml) and deglycosylated JRFL gp140 were injected over each surface (100 and 300 ug/ml, respectively). To determine induction of 17b mAb binding to WT glycosylated and deglycosylated JRFL gp140, Env proteins were captured on individual flow cells immobilized with sCD4 (80–120 response units or RU) or mAb A32 (∼130–300RU) or T8 (∼340–450 RU), with about the same RU of Env gp140 captured on each surface prior to injection of varying concentrations of 17 mAb. Following stabilization of each of the surfaces, mAb 17b (10–100 µg/ml) was injected and flowed over each of the Env captured surfaces as illustrated in the diagram above the SPR profile in Panel B. Each analysis was performed at least twice.
Figure 3.
Binding kinetics of mAbs 2F5 and 4E10 to WT glycosylated and deglycosylated JRFL gp140.
Binding of mAbs 2F5 and 4E10 to WT glycosylated and progressively deglycosylated JRFL gp140 proteins was determined in direct ELISA. An anti-HIV-1 Env C1 region antibody 3B3 [31] was coated on the 96-well plates to capture glycosylated and progressively deglycosylated JRFL gp140 proteins following by incubation with mAbs 2F5 and 4E10. The mAbs 2F5 and 4E10 were assayed at the concentrations as indicated on the x-axis. Data presented are representative of 3 independent experiments.
Figure 4.
SPR assays for native and deglycosylated JRFL gp140 binding to mAbs 4E10 and 2F5.
MAbs 4E10 (Panel A, B) and 2F5 (Panel C, D) were bound to anti-human Ig Fc immobilized on the CM5 sensor chip. Varying concentrations (ranging from 5 to 100 ug/mL) of native (WT) and deglycosylated (Deg) JRFL gp140 Envs were injected over the mAbs on the chip and binding kinetics were recorded. Rate constants and Kd measurements were made using the 1∶1 Langmuir model and data recorded are indicated in the individual panels. Deglycosylation of JRFL gp140 enhanced the Kd of 4E10 and 2F5 binding by ∼15-fold and ∼6-fold respectively. Data shown are representative of two experiments.
Figure 5.
Binding of WT glycosylated and deglycosylated JRFL gp140 Env on DC-SIGN-expressing NIH 3T3 cells.
The binding of AF647-labeled WT glycosylated (histogram in red lines and deglycosylated JRFL Env gp140 (histograms in blue lines) to parent NIH 3T3 (histograms shaded) and DC-SIGN-expressing NIH 3T3 cells (histograms not shaded) were analyzed by flow cytometry. Data shown are representative of three experiments.
Figure 6.
SPR assays for binding of native and deglycosylated JRFL gp140 to mAb 2G12.
MAb 2G12 was immobilized on the CM5 chip. The saturated amounts of native and representative deglycosylated proteins were then flown over the mAbs. The binding kinetics were recorded at 25°C. Deglycosylation of JRFLgp140 using lower dose of PGNase (20 U: µg) enhanced the binding of 2G12 but substantially decreased binding of 2G12. Data show binding curves of each deglycosylated and WT glycosylated protein injected at 50 µg/mL and 200 µg/mL respectively, and are representative of two experiments.
Figure 7.
Glycan analysis of native partially deglycosylated JRFL gp140 protein.
Native deglycosylation of JRFL gp140 protein was performed using PNGase F (500 unit/µg protein) as described in the materials and methods. Amino acid sequence numbers were based on the HXB2 sequence. The signal peptide and variable regions of the sequence were identified above the sequence. See Table S1 for mass spectrometry analysis data summaries.
Figure 8.
Model of glycosylation patterns of recombinant WT JRFL gp140 and partially deglycosylated gp140 under non-denaturing conditions.
Shown are the images that were modeled based on the cryo-electron tomography as described by Schief [60]. Panel A: HIV-1 recombinant JRFL Env exhibits a number of high mannose glycans as determined by mass spectrometry (Figure 7 and Table S1). In this figure, the gp120 bearing the V3 loop [61] (PDB 2B4C)] trimer (red) is modeled over the cryo-EM structure (white mesh) (36), and its representative glycans are indicated in blue with those that are bound by the antibody 2G12 [37] in white. The fold of the prefusion gp41 trimer is not known, so gp41 glycans (yellow) have been positioned approximately equidistant around the stalk. Glycans bound to gp120 residues in the V1 and V2 loops have similarly been placed in reasonable locations across the top of the trimer. Depicted in Panel B are the glycans remaining after treatment with PNGase-F. In general, PNGase is able to remove the glycans that are attached to flexible parts of the gp120 structure, presumably allowing the enzyme to gain access to recognition elements and cleavage points. Glycans attached to Asn residues occuring in relatively stable structural elements of gp120 persist after PNGase-F treatment.
Figure 9.
SPR measurements of binding of inferred RUAs of 2F5 and 4E10 to WT glycosylated and natively deglycosylated JRFL gp140 Env proteins.
A dose range (from 20–100 µg/mL as indicated) of RUA1 (A–B) and RUA2 (C–D) inferred from the mutated 2F5 antibody sequence and one RUA (4E10 RUA, 20–80 µg/mL) (E–F) inferred from the 4E10 mutated antibody sequence were tested by SPR for binding to WT glycosylated (WT) and maximally deglycosylated (Deg) JRFL gp140 Env proteins immobilized on adjacent flow cells of the same sensor chip. Each antibody was captured on an anti-Ig Fc immobilized chip as described [7]. Neither of the RUAs of 2F5 (Panels 9A–9C) nor 4E10 (Panel 9E) bound to WT glycosylated JRFL gp140 Env protein, while reactivity of the RUAs of 2F5 (Panels 9B–9D) and 4E10 (Panel 9F) to deglycosylated JRFL gp140 Env proteins was present with the Kds and rate constants shown in each panel. Rate constants were derived by curve fitting analysis and using a 1∶1 Langmuir model.
Figure 10.
Enhanced immunogenicity of deglycosylated JRFL gp140 Env as determined by plasma levels of 2F5 epitope binding antibodies in immunized rhesus macaques.
Rhesus macaques (4 per group) were immunized at weeks 0 (prime) and 4 (boost) with 100 ug of either glycosylated (WT) or natively deglycosylated (Deg) JRFL gp140 Env. Plasma samples were collected at weeks 2 and 6 from each group (as indicated on the top of the panels) and were assessed for binding to WT JRFL gp140 Env and Deg JRFL gp140 Env (Panel 10A). The antibody responses to wild-type JRFL Env or to Deg JRFL Env were undetectable after the prime and are not shown. The responses to WT JRFL Env and Deg JRFL Env after the week 4 boost (the week 6 blood draws) are shown. The antibody response to 2F5 nominal MPER peptide (QQEKNEQELLELDKWASLWN) after the prime (week 2) and boost (week 6) is shown (Panel 10B). Shown on the x axis are the reciprocal dilutions of plasma of the immunized moneys used in ELISA. The dotted lines indicate the negative cut-off values. The level of 2F5 epitope antibody response at the initial dilution (1∶25) in week 6 plasma were higher in animals immunized with deglycosylated JRFL Env than in animals immunized with WT JRFL Env (p = 0.035, Student's t test). Data shown are mean values +/− standard deviation derived from 3 separate experiments performed.