An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

<div><p>Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.</p></div

Schematic and expression of the cleavable Env_IL-21.

<p>Linear (A) and cartoon (B) representation of the Env_IL-21 and clEnv_IL-21 proteins. Cleaved proteins were created by introducing a stop codon in front of the isoleucine zipper (IZ) trimerization domain in the Env_IL-21. (C) SDS-PAGE analysis of chimeric uncleaved and cleaved Env_IL-21 constructs.</p

Antigenic characterization of Env_IL-4 and Env_IL-21 molecules.

<p>ELISA reactivity of Env_IL-4 and Env_IL-21 with 2G12 and HIV-Ig (A); b12 and CD4-IgG2 (B); and 48d (CD4i) in the absence and presence of sCD4 at 1 µg/ml (C). All ELISA results are representative for at least three independent experiments using proteins derived from three independent transfections.</p

Immunoglobulin production by B cells stimulated with Env_wt, Env_IL-4 and Env_IL-21 molecules and controls.

<p>IgG, IgA and IgM levels secreted by the B cells from human PBMCs cultured with (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10. Data are representative of three independent experiments showing similar results. Immunoglobulin secretion by B cells from different donors cultured with Env_wt and Env_IL-21 molecules in (C) CD40L/IL-10 and (D) CD40L/IL-4/IL-10 milieu. Culture supernatant from mock transfected 293T cells was used as a negative control and the values were deducted from the test values. Data represent the fold change values compared to Env_wt from at least 12 donors and each donor sample was tested in duplicate. (E) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env_wt, Env_IL-21, Env_ChimIL-21/4 supernatants and controls in CD40L/IL-10 milieu. Data are representative of three experiments using B cells from three different donors. (F) The expression of CD38 cell surface marker treated with different Env_IL-21 constructs and controls in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six different donors.</p

Immunoglobulin secretion from B cells cultured with Env_wt, Env_IL-21, clEnv and clEnv_IL-21 molecules in the presence of (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10.

<p>Data are representative of two independent experiments using B cells from two different donor, each tested in duplicate. (C) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env_wt, Env_IL-21, clEnv and clEnv_IL-21supernatants in medium supplemented with CD40L/IL-10. Data are representative of three independent experiments using B cells from three donors. (D) The expression of CD38 cell surface marker treated with different cleaved Env (clEnv) and cleaved Env_IL-21 (clEnv_IL-21) constructs in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six donors.</p

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

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

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

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