Two Classes of Broadly Neutralizing Antibodies within a Single Lineage Directed to the High-Mannose Patch of HIV Envelope

Doores, Katie J.; Kong, Leopold; Krumm, Stefanie A.; Le, Khoa; Sok, Devin; Laserson, Uri; Garcés, Fernando; Poignard, Pascal; Wilson, Ian A.; Burton, Dennis R.

doi:10.1128/jvi.02905-14

Cited by 83 publications

(117 citation statements)

References 47 publications

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“…The oligomannose-type glycans on gp120 are the target of a number of HIV-1 bnAbs (4,(16)(17)(18)22). The masses of the singly charged ions were typical of those from the oligomannose glycans Man 5 GlcNAc 2 to Man 9 GlcNAc 2 , and as previously observed, these glycans were identified in both the recombinant and virion-derived PBMC samples.…”

Section: Oligomannose Isomers Are the Same On Pbmc-derived Env And Rementioning

confidence: 55%

“…Despite the glycans on Env often being referred to as the "glycan shield," a number of HIV-1 bnAbs have been isolated that bind to these glycans (3)(4)(5)(16)(17)(18)(19)(20)(21). These bnAbs target three main regions on Env, the N332 glycan (e.g., 2G12, PGT121, PGT128, and PGT135) (3,4,(16)(17)(18), the N160 glycan (e.g., PG9, PGT145, and CH04) (4,5,19,20), and the glycans at the gp120-gp41 interface (e.g., PGT151) (21).…”

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confidence: 99%

“…These bnAbs target three main regions on Env, the N332 glycan (e.g., 2G12, PGT121, PGT128, and PGT135) (3,4,(16)(17)(18), the N160 glycan (e.g., PG9, PGT145, and CH04) (4,5,19,20), and the glycans at the gp120-gp41 interface (e.g., PGT151) (21). Glycan microarray analysis has shown these bnAbs to have differing specificities for N-linked glycan structures; e.g., PGT128 is specific for Man 8 GlcNAc 2 and Man 9 GlcNAc 2 glycans (22), whereas PGT151 preferentially binds ␣2,3-linked sialylated tri-and tetra-antennary complex-type glycans (21).…”

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confidence: 99%

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Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

Pritchard

Harvey

Bonomelli

et al. 2015

J Virol

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116

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The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. IMPORTANCEDevelopment of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.T he HIV-1 envelope glycoprotein (Env) consists of a noncovalently linked trimer of gp120/gp41 heterodimers. Interaction of this trimer with CD4 and its subsequent rearrangement is essential for infection of target cells and therefore is the sole target for broadly neutralizing antibodies (bnAbs). The surface protein, gp120, is extensively modified with host-derived glycans, having a median of 25 potential N-linked glycosylation sites (PNGSs) (1) and thus making it one of the most densely glycosylated proteins known. The t...

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Section: Oligomannose Isomers Are the Same On Pbmc-derived Env And Rementioning

confidence: 55%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

Pritchard

Harvey

Bonomelli

et al. 2015

J Virol

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116

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“…4A). Removal of Asn137, which has been shown to enhance neutralization of some viruses by other N332-directed antibodies (35,36), had no effect on PGT135 or PGT128 neutralization. Similarly the N295 glycan, which has been observed to be important in a strain-dependent manner (15), had no effect on PGT135 binding.…”

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confidence: 96%

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

Pritchard

Spencer

Royle

et al. 2015

J Virol

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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. The isolation of potent, broadly neutralizing antibodies (bnAbs) from infected HIV-1 individuals has focused efforts toward the molecular characterization of their epitopes (1-5). These antibodies have potential value in a therapeutic context, and their epitopes represent key vaccine leads (6-12). Structural and biochemical studies have revealed that a number of the recently isolated bnAbs penetrate the heavily glycosylated surface of the HIV-1 envelope spike, making contacts with both the glycans and the protein underneath (1-3, 13-22) Characterization of the glycan-containing epitopes has revealed that much of the glycan shield is vulnerable to antibody recognition (5). Many glycans within the outer domain of gp120 are protected from normal glycan processing and do not form complex-type glycans, instead remaining as immature oligomannose-type glycans. This region is known as the "intrinsic mannose patch" since it contains oligomannose-type glycans, regardless of whether presented in the context of isolated gp120 monomers or functional virions (23-25).The intrinsic mannose patch is targeted by the so-called "mannose patch-dependent" antibodies, which include PGT121 to -124, 10-1074, PGT125 to -128, PGT130 and -131, PGT135 to -137, and 2G12 (14-16, 26-29). These antibodies display remarkable potencies against a diverse panel of HIV-1 strains, although their breadth varies both between and within families (2, 30). PGT135 was found to neutralize 33% of viruses from a 162-crossclade-pseudovirus panel. This neutralization is equivalent to the breadth of b12, which has a protein-based epitope at the CD4 binding site, but is lower than those of other Asn332-dependent bnAbs, such as PGT128 and PGT121, which neutralized 72% and 70% of the panel, respectively (2). This lower breadth of neutralization has been attributed to the limited prevalence of the larger number of critical contact residues (Asn332, Asn392, and His330) across different isolates (15) compared to PGT121 and PGT128. In addition to these properties, inspection of neutralization profiles reveals that, despite containing the required target residues, for some strains of HIV-1, neutralization is incomplete, with plateaus that do not reach 100% (15). A crystal structure of a PGT135 Fab domain in complex with the gp120 core revealed that the majority of the interactions were mediated through contact with the glycans at the Asn332, Asn392, and Asn386 sites, with 1,010 Å 2 and 438 Å 2 of buried surface area conta...

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“…Prime examples are the N-glycan structure of the Fc-domain glycan in IgG molecules (18) or the high mannose structure on gp120 of HIV as part of the epitope of neutralizing antibodies (19). Recently, glycan microheterogeneity has become a major hindrance in the production of glycoproteins (20)(21)(22).…”

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confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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Two Classes of Broadly Neutralizing Antibodies within a Single Lineage Directed to the High-Mannose Patch of HIV Envelope

Cited by 83 publications

References 47 publications

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

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

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

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