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

Infected CD4 Elimination (ICE) inversely correlates with plasma SIV RNA levels in SIV-infected rhesus macaques.

<p>Red dots represent LTNP/EC (n = 11). Blue dots represent progressors (n = 11). Statistical analysis was performed by the Spearman rank method.</p

SIV-specific CD8⁺ T cells from LTNP/EC mediate greater lysis of SIV-infected CD4⁺ T-cell targets compared with progressors.

<p>GrB target cell activity (<b>A</b>) and infected CD4 elimination (ICE) (<b>B</b>) are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). Horizontal bars represent the median values. <b>C.</b> Correlation between ICE and GrB target cell activity (n = 22) was determined by the Spearman rank method. Red, blue and cyan dots represent LTNP/EC, progressors and one SIV-uninfected animal, respectively.</p

SIV-specific CD8⁺ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).

<p><b>A.</b> The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). <b>B.</b> ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4⁺ T cell lines were used as targets. CD8⁺ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.</p

SIV-specific CD8⁺ T cells of LTNP/EC mediate greater per-cell killing of SIV-infected targets than those of progressors, which is not simply due to higher true E∶T ratios.

<p><b>A.</b> The true effector to target (E∶T) ratios, determined by measurements of IFN-γ-secreting CD8⁺ T-cell effectors and p27-expressing CD4⁺ T-cell targets, respectively, as described in the Methods and shown in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s001" target="_blank">Figure S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s002" target="_blank">Table S1</a>, were compared between LTNP/EC (n = 11) and progressors (n = 11). Horizontal bars represent the median values. <b>B, C.</b> GrB target cell activity (<b>B</b>) or ICE (<b>C</b>) responses plotted against the true E∶T ratios are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). GrB target cell activity is shown after subtraction of background. The response curves were analyzed by regressing ICE and GrB on log true E∶T ratios using analysis of covariance. The standard two-tailed t test from regression analysis was used to compare estimated GrB target cell activity and ICE of LTNP/EC with that of progressors at the 5.8 E∶T ratio, the median of the combined E∶T ranges of both groups.</p

Characteristics of macaques and prediction of SIV control.

<p>ICE, Infected CD4⁺ T cell Elimination (%). LTNP/EC, Long-Term Nonprogressor/Elite Controllers. SP, Slow Progressor. N/A, Not Applicable. PBMC from more than one time point were used in the animals marked with an asterisk.</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

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

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

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