Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Abstract

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.

FIG 1

The glycan epitope of PGT135 encompasses the Asn332, Asn392, and Asn386 sites. (A) A previously reported crystal structure reveals the interaction of a PGT135 Fab domain with the Asn332 (Man₆GlcNAc₂), Asn392 (Man₈GlcNAc₂), and Asn386 (Man₁GlcNAc₂) glycans from a gp120_JR-FL core (15). The protein moiety is depicted in a ribbon diagram, and glycans are depicted as sticks. Mannose (Man) residues are colored in green, and N-acetlyglucosamine (GlcNAc) residues are colored in blue. (B) Enlarged view of the PGT135 glycan epitope. (C) Schematic representation of a Man₉GlcNAc₂ glycan, with the D1 to D3 arms annotated and the glycans resolvable in the crystal structure. Glycan structures are shown according to the proposed method of Harvey et al. (40), with residues colored according to panels A and B. Images were made in PyMol using PDB code 4JM2.

FIG 2

Glycans present at the Asn332 glycosylation site. Recombinant, monomeric gp120_BaL was expressed in HEK 293T cells from the pHLsec vector (41) and purified by metal-affinity and size exclusion chromatography, as previously described (42). gp120 was reduced, alkylated, and digested with trypsin (Promega), before fractionation using a Jupiter C₁₈ 5-μm 250- by 4.5-mm column (300-Å pore size) and a Dionex U3000 liquid chromatography system. Fractions were collected every minute, at a flow rate of 1 ml/min, for 90 min, and then analyzed on an Autoflex Speed MALDI tandem time of flight (TOF/TOF) instrument (Bruker), operated in positive-ion mode. (A) MALDI MS of pooled fractions containing the Asn332-containing glycopeptide, QAHCNLSR. The glutamine carried a pyro-Glu modification (−17), and the cysteine was modified due to treatment with iodoacetamide (carbamidomethyl, +57). Glycan structures corresponding to the observed glycopeptide masses are indicated. (B) MALDI MS/MS fragmentation spectrum of the 2,671.2 peak, corresponding to a Man₈GlcNAc₂ glycopeptide. Fragment ions were observed that were characteristic of glycopeptide MALDI MS/MS fragmentation (43), including [M_pep+H + 83]⁺ (corresponding to ^0.2X-ring cleavage of the innermost GlcNAc) and [M_pep+H + 203]⁺ (corresponding to Y-type cleavage of the di-N-acetylchitobiose core). Y-type fragmentation of glycosidic bonds was also observed. (C) HILIC-UPLC profile of Asn332 glycans. Glycans were released from the QAHCNLSR glycopeptides by in-solution PNGase F (QA-Bio) digestion, according to the manufacturer's instructions, and then labeled using a LudgerTag 2-AB labeling kit (Ludger Ltd., Abingdon, United Kingdom). Chromatography was performed on a Waters Acquity UPLC instrument. Glycans were assigned by comparison with known oligomannose-type glycan standards (Ludger Ltd., Abingdon, United Kingdom).

FIG 3

Glycans present at the Asn392 and Asn386 glycosylation sites. gp120 was digested with chymotrypsin (Promega) before RP-HPLC and MALDI analysis. (A) MALDI MS of Asn392 glycopeptides (NSTW). Sodium, [M + 23]⁺, and potassium, [M + 39]⁺, adducts were observed: both peaks were used for measuring abundances (Table 1). (B) MALDI MS/MS fragmentation spectrum of the peak corresponding to the Man₈GlcNAc₂ glycopeptide. Fragmentation peaks corresponded to the protonated masses. (C) MALDI MS of Asn386 glycopeptides (YCNSTQLF). The cysteine was modified due to treatment with iodoacetamide (carbamidomethyl, +57). Protonated glycopeptides, as well as sodium and potassium adducts, were detected: all were used for calculation of abundances (Table 1). (D) MALDI MS/MS fragmentation spectrum of the peak corresponding to the Man₈GlcNAc₂ glycopeptide. Fragmentation peaks corresponded to the protonated masses.

FIG 4

Influences of gp120 glycans on bnAb recognition. (A) Neutralization sensitivity of PGT135, PGT128, and b12 against BaL wild-type pseudovirus and glycan site mutants. bnAbs PGT128 and b12 are used as controls. To produce pseudoviruses, plasmids encoding Env were cotransfected with an Env-deficient genomic backbone plasmid (pSG3ΔEnv) in a 1:2 ratio with the transfection reagent polyethyleneimine (PEI) (1 mg/ml, 1:3 PEI-total DNA [Polysciences]) into HEK 293T cells. Pseudoviruses were harvested 72 h posttransfection. Neutralizing activity was assessed using a single-round replication pseudovirus assay with TZM-bl target cells, as described previously (2). Glycan sites N137, N295, N386, and N392 were removed using site-directed mutagenesis through an Asn-to-Ala mutation. Mutations were verified by DNA sequencing (MWG Eurofins, Germany). (B) Glycan profile of recombinant gp120 expressed in HEK 293T cells in the presence of kifunensine, analyzed as described in the legend to Fig. 2C. (C) Enzyme-linked immunosorbent assay (ELISA) data of PGT135 (blue) and 2G12 (black) binding to wild-type (continuous line) and kifunensine-treated (dashed line) gp120. ELISAs were performed as previously described (34).