Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer

Abstract

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

Fig. 1

Design, purification, and characterization of JR-FL Env trimers. a Linear representation of JR-FL viral Env, JR-FL pseudovirus Env, JR-FL ΔCT, and JR-FL SOSIP.664. Modifications compared to the wild-type JR-FL gp160 sequence are indicated in red; b A schematic diagram of the method used for purification of well-ordered membrane-bound HIV Env trimer from the surface of 293F cells/HIV virus; c SEC chromatogram of the JR-FL ΔCT purification. The fraction that contained the JR-FL ΔCT-PGT151 complex was highlighted in blue; d SDS-PAGE (reducing and nonreducing) and BN-PAGE analysis of the JR-FL ΔCT trimer after purification; e SDS-PAGE (reducing) and BN-PAGE analysis of the purified JR-FL trimer derived from infectious HIV

Fig. 2

Comparison of site-specific N-glycan processing of HIV-1 Env trimers. Site-specific analysis of glycan processing for a Env trimers from the JR-FL strain; b Env trimers from the BG505 strain; c Env trimers from the B41 strain. Shown for each glycosite were the proportion of peptides corresponding to unoccupied sites (grey), or sites containing high-mannose (green) and complex-type (purple) glycans. For glycosite numbers highlighted in yellow, proportions were assigned based on spectral hits since peak area did not reach the threshold. Sites with no number indicate the site was not present in the Env while sites with numbers and no bar graph indicate the site was not observed in LC/MS. Site-specific N-glycan processing of BG505 SOSIP is adapted with permission from ref. . The glycosylation profiles of JR-FL viral Env, BG505 ΔCT N332 Env, and B41 ΔCT Env were used as a reference for comparison with other Envs from the corresponding strain. Significant differences from reference samples are shown as colored bricks. Each recombinant Env was digested and analyzed in duplicate from two biological batches, while native trimers from infectious virus and pseudovirus were analyzed in duplicate from the same biological preparation (n = 6). Differences were assessed for the proportions of no glycan, high mannose and complex type at each glycosite. Differences of >10% were determined to be significant if the P value was <0.05 using a Mann–Whitney test. Mean ± s.e.m. were plotted

Fig. 3

Mapping of site-specific N-glycan processing onto the structures of HIV-1 Env. a JR-FL strain; b BG505 strain; c B41 strain. The fully glycosylated models were constructed using JR-FL ΔCT (PDB: 5FUU), BG505 SOSIP (PDB: 5FYK), and B41 SOSIP. The surfaces of the trimers are shown in grey and the glycans are shown as spheres colored by proportion of oligomannose content at that site (Green spheres represent >75% high mannose glycosylation, purple spheres represent >75% complex type glycosylation, and yellow spheres represent the mixture of high mannose and complex type glycosylation (25%< high-mannose glycosylation <75%)).The glycans present at the glycosites that were not detected were shown as grey spheres

Fig. 4

Comparison of Cryo-EM reconstructions of membrane-bound and corresponding soluble trimers. a Cryo-EM reconstruction of BG505 ΔCT T332 in complex with PGT151 Fab at 4.5 Å resolution. b Cryo-EM reconstruction of B41 ΔCT in complex with PGT151 Fab at 6.7 Å resolution. c Structural differences between BG505 ΔCT T332 and soluble BG505 SOSIP trimer (PDB: 5ACO). d Close-up of the V1/V2 region highlighted in c. e Mapping of site-specific glycan processing of JR-FL ΔCT, BG505 ΔCT T332, and B41 ΔCT onto the surface of the corresponding cryo-EM density maps with two PGT151 Fabs highlighted per trimer

Fig. 5

Comparison of the antigenicity properties of membrane-bound and soluble Env trimers. a Median IC₅₀ neutralization and EC₅₀ ELISA titers are provided in µg/ml and colored according to the listed scale for the three HIV-1 strains, including JR-FL E168K, BG505 N332, and B41. Neutralization of the full-length Env-pseudotyped viruses (IC₅₀) by bnAbs was determined in TZM-bl neutralization assays, while binding to the corresponding SOSIP trimers was measured by ELISA-binding assays. For JR-FL E168K, neutralization assays were also performed with pseudovirus grown in the presence of kifunensine (+kif), which blocks glycan processing leaving oligomannose (mainly Man₉) glycans at all glycosites. b Flow cytometric measurements for binding of fluorescent bnAb to membrane-bound JR-FL ΔCT Env on 293 F cells grown in the presence and absence of kifunensine. Green (pink, brown, orange, black, blue, or red) solid line represents PG9 (PG16, CH03, PGT145, PGDM1400, PGT128, or PGT151) binding to JR-FL ΔCT (-kifunensine), while green (pink, brown, orange, black, blue, or red) dashed line represents PG9 (PG16, CH03, PGT145, PGDM1400, PGT128, or PGT151) binding to JR-FL ΔCT (+kifunensine), respectively

Fig. 6

Topology of N-glycan processing for soluble and membrane-bound Env. Since the glycan processing enzymes are membrane bound in the ER and Golgi, the membrane bound and soluble Envs exhibit a different topology relative to the active sites of the enzymes. Glycan structures that are Endo H-sensitive are highlighted in green. Purple diamonds represent sialic acid, blue squares represent N-acetylglucosamine, yellow circles represent galactose, blue circles represent glucose, and green circles represent mannose