Glycoengineering HIV-1 Env creates ‘supercharged’ and ‘hybrid’ glycans to increase neutralizing antibody potency, breadth and saturation

<div><p>The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make <i>some</i> glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.</p></div

Comparison of the effects of B4GALT1+ST6GAL1 and PBMC passage on PV and IMC sensitivities.

<p>Viruses from 5 HIV-1 strains were produced as PVs or infectious molecular clones (IMCs). Some 293T cell-derived PVs were modified by B4GALT1+ST6GAL1, as indicated. Some IMCs were passaged through PBMCs as indicated. Neutralization assays were performed with the addition of indinavir to assays using IMCs to limit infection to a single round. Results are representative of at least two repeats performed in duplicate.</p

Glycan contacts of mature bnAbs are not germline-encoded.

<p>A) GE PVs from donor CAP256 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref035" target="_blank">35</a>] at week 34 were tested for neutralization sensitivity to bnAbs CAP256.09, CAP256.25, intermediate I1 and the inferred unmutated common ancestor (UCA). B) GE-modified 16055 and Q23.17 PVs were tested for sensitivity to PG9 with a reverted heavy chain (gH) mixed with the mature light chain (mL) and fully mature PG9. Results are representative of two replicates; error bars represent SD. C) Nine V2-sensitive strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref034" target="_blank">34</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref036" target="_blank">36</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref045" target="_blank">45</a>] produced in control, B4GALT1+ST6GAL1 and GNT1- formats were tested for sensitivity to V2 bnAbs and their germline revertants, as indicated. The CH04 UCA (RUA/RUA) was the same as that used in ref [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref033" target="_blank">33</a>]. Mixed mHgL versions of PGT145 and VRC38.01 were used that previously showed the most reactivity of the ancestors tested [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref034" target="_blank">34</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.ref042" target="_blank">42</a>]. Results are representative of at least two repeats performed in duplicate. Wilcoxon Signed Rank tests were performed on data for each mAb-PV pair, organized into two columns to compare IC50s for control and GE-modified formats. Significant p values are shown.</p

Effects of GE on bnAb neutralization IC50s.

<p>Neutralizing IC50s for each control and GE-modified A) JR-FL and B) BG505 PVs are shown in μg/ml. The most sensitive glycoform for each nAb is boxed. JR-FL neutralization assays for mAb 3BC176 used the N88A mutant. Each assay was performed at least in duplicate. In many cases, BG505 neutralization by mAb 35O22 was insufficiently saturating to reach an IC50 and is denoted by a "+". Geometric mean IC50s per treatment are shown, omitting data for 14e, F105 and instances of 35O22 "+". This Fig is linked to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.g003" target="_blank">Fig 3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.s006" target="_blank">S6</a> Figs. Abbreviations: B4G = B4GALT1, ST3 = ST3GAL4, ST6 = ST6GAL1, ST8 = ST8SIA4.</p

GE effects on the neutralization sensitivities of a diverse panel of virus strains.

<p>The effects of various GE modifications on mAb IC50s against a panel of 14 viruses are shown. Particular GE modifications for each nAb were selected as those that markedly affect neutralizing activity against the JR-FL and/or the BG505 strains above. IC50s >10ug/ml were assigned as 10μg/ml. Geometric mean IC50s of all 14 viruses per each GE treatment are shown on the right of each chart, omitting datum for mAb-virus combinations in which IC50s were >10μg/ml under all GE conditions. The infectivities of GNT1- modified BG505 and CNE58 were too low for IC50s to be reliably measured and were therefore omitted. BI369.9A and Q23.17 GNT1- neutralization assays with PGT151 were also omitted due to inconsistent IC50s, in part due to the low infectivity of GNT1- PVs. Results are representative of at least two repeats performed in duplicate. IC50s are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.s015" target="_blank">S1 Table</a>.</p

BN-PAGE-Western blot analysis of the effects of GE on particulate Env.

<p>Equal volumes (500x concentration) of GE-modified JR-FL SOS E168K gp160ΔCT VLPs (produced using MuLV Gag and Rev) and human PBMC-propagated replicating JR-FL virus were lysed and analyzed by BN PAGE-Western blot. Env was detected using an anti-HIV primary cocktail (39F, 4E10 and 2F5). Trimer and monomer bands are indicated by cartoons along with ferritin markers. Lanes 20 and 21 are enhanced versions of lanes 18 and 19 to better visualize PBMC Env (lane 21). Abbreviations: B4G = B4GALT1, ST6 = ST6GAL1, ST3 = ST3GAL4, ST8 = ST8SIA4.</p

N-linked glycosylation and GE.

<p>A dolichol phosphate-linked precursor consisting of 2 membrane-linked N-acteylglucosamine (GlcNac) moieties and 5 mannose (Man) forms on the cytoplasmic surface of the endoplasmic reticulum (ER), then flips to the lumen. Further mannose moieties are added to create 3 termini (D1-D3), to which glucose moieties are added by glucosyltransferase (GlcT). This is transferred to Asn-X-Ser/Thr sequons of a nascent protein by oligosaccharyltransferase (OST). The 3 terminal glucose residues are then removed by α-glucosidase to form Man₉GlcNAc₂ (Man9)—a step that is inhibited by <a target="_blank">N-Butyldeoxynojirimycin</a> (NB-DNJ). The terminal D2 mannose is then cleaved by α-mannosidase 1 (ERMAN1)—a step that can be inhibited by kifunensine. In the cis-Golgi, mannose is trimmed by α-mannosidases 1A, 1B and 1C (MAN1A-C) to form Man₅GlcNAc₂. GlcNAc is then transferred to the α-1,3 D1 arm by N-acetylglucosaminyltransferase 1 (GNT1; inactive in GNT1- cells). In the medial Golgi, there is a bifurcation in the pathway. In one fork, D2 and D3 mannose subunits are removed by α-mannosidase II (MAN2)—a step that is blocked by swainsonine (swain). GlcNAc moieties may then be added to the trimmed α-1,6 arm by GNT2 to initiate a biantennary glycan, followed by the addition of a core fucose moiety by fucosyltransferase (FUCT8), a step that is blocked by 2-deoxy-2-fluoro-1-fucose (2FF). Further GlcNAc termini may then be added by GNT4 and GNT5 to form tri- and tetra-antennary glycans. These may be galactosylated by β-1,4 galactosyltransferases (B4GALT1), a step that is blocked by 2-deoxy-2-fluoro-d-galactose (2FG). Terminal SA may then be added by β-galactoside α-2,3-sialyltransferase (ST3GAL4) or β-galactoside α-2,6-sialyltransferase (ST6GAL1). Polysialic chains may form by the addition of α-2,8-linked SA by α-2,8-sialyltransferase (ST8SIA4). SAs can be cleaved by NA. In the alternative fork, α-1,6 arm mannose is not removed, but the α-1,3 arm may be modified with galactose and SA, forming hybrid glycans, sometimes with the addition of a bisecting GlcNAc subunit by GNT3 and no fucosylation. Non-fucosylated high mannose and hybrid glycans can be cleaved by endoglycosidase H (endo H).</p

Summary of the effects of GE on bnAb-trimer binding and implications for vaccine development.

<p>A) GE increases bnAb sensitivity by lowering IC50 titers, increasing maximum % saturation and increasing breadth. B) The preferred glycoforms of various bnAbs are shown. Preferences include untrimmed high mannose glycans (e.g. PGT125), small glycans (e.g. germline reverted Abs), terminal galactose (e.g. PGT121) and terminal α-2,6 SA (e.g. PG9). Some bnAbs (e.g. VRC01) are largely "glycan agnostic" as they bind protein sites with essentially no glycan clashes that are unaffected by any GE. Other bnAbs (e.g. PGT145) were subject to glycan clashes that varied between strains with GE. PBMC-grown virus trimers are heavily α-2,6 sialylated and best resembles ST6GAL1-modified PV trimers, contrasting with unmodified 293T cell produced PV trimers bear largely α-2,3 SA. B4GALT1 overexpression replaces complex glycans with hybrid glycans. C) GE might be leveraged for use in bnAb-targeted prime-boost vaccine studies. Priming might best use trimers bearing small glycans to minimize clashes with glycan-fearing UCAs. Intermediate immunogens might use trimers bearing GE-modified glycans that are optimal for the bnAb(s) being targeted by this regime. Finally, boosting might best be done using trimers modified with glycans that best resemble those of PBMC-produced viral trimers.</p

GNT1- PV exhibits a largely V3-resistant tier 2 phenotype.

<p>A-C) Comparison of neutralization sensitivities of JR-FL WT control and GNT1- PVs to 3 HIV+ donor plasmas. D-G). The neutralization sensitivity of a V3-sensitive tier 1-like A328G mutant of JR-FL WT was compared to the corresponding GNT1- and control PVs. Assays were performed with or without added JR-FL V3 peptides (30μg/ml). All assays were performed in CF2 cells and used the JR-FL PV except for CAP256 plasma that used T250-4.</p

GE improves neutralization saturation.

<p>For each mAb, the number of the 14 viruses from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007024#ppat.1007024.g005" target="_blank">Fig 5</a> that were neutralized above 65%, 90% or 95% in the presence of 10μg/ml of the mAb indicated were expressed as a percentage of the total viruses. Open symbols represent the optimal glycomodification for each nAb; B4GALT1+ST6GAL1 (abbreviated as B4G+ST6) for PG9 and CAP256.09, NA treatment for PGT151 and PGT121, and GNT1- cells for 35O22, CH01 and PGT145. This analysis included data only for those strains that were neutralized in either the control or modified condition (or both) with IC50s <1μg/ml.</p