Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

Pritchard, Laura K.; Vasiljević, Snežana; Ozorowski, Gabriel; Seabright, Gemma E.; Cupo, Albert; Ringe, Rajesh P.; Kim, Helen J.; Sanders, Rogier W.; Doores, Katie J.; Burton, Dennis R.; Wilson, Ian A.; Ward, Andrew B.; Moore, John P.; Crispin, Max

doi:10.1016/j.celrep.2015.05.017

Cited by 127 publications

(261 citation statements)

References 54 publications

(123 reference statements)

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“…Pal et al showed that gp41 from viruses prepared in MOLT-3 cells was Endo H resistant, but when prepared in the presence of an ER mannosidase inhibitor, 1-deoxymannojirimycin, gp41 became sensitive to Endo H digestion (59). More recent work by us and by Go and colleagues has shown that the gp41 glycans on recombinant gp140 trimers (13,60,61), including the SOSIP trimer (14), are predominantly complex type and that these structures are generally more dependent on producer cells. In this study, we were able to record the chromatograms of labeled glycans from PGT151-isolated pseudovirion-derived material and cell surface-associated Env to provide a more detailed description of glycan structures present on native virion-derived gp41.…”

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

confidence: 99%

“…cans, in particular Man 8 GlcNAc 2 and Man 9 GlcNAc 2 , forming the so-called IMP (14). These glycans are a key component of the epitope of the N332-dependent bnAbs (4,18,22).…”

Section: Figmentioning

confidence: 99%

“…The cell-specific influences on Env glycosylation for recombinant gp120, gp140, and SOSIP trimers have been analyzed previously (14,65,66). However, these differences have not been explored in detail for virion-associated gp120, in particular the sialic acid linkages of complex-type sugars.…”

Section: Figmentioning

confidence: 99%

“…Two main subpopulations of glycans have been identified on gp120, underprocessed oligomannose glycans and processed complex-type glycans (7)(8)(9)(10)(11)(12)(13)(14). The dense clustering of glycans limits the accessibility to the endoplasmic reticulum (ER) and Golgi ␣-mannosidase enzymes, resulting in a patch of underprocessed oligomannose glycans which cannot be further processed to complex glycans in the Golgi (7).…”

Section: Importancementioning

confidence: 99%

“…This patch of oligomannose glycans arising from steric restriction is referred to here as the intrinsic mannose patch (IMP). We have previously shown that the IMP is conserved across recombinant gp120, recombinant trimers (including SOSIP trimers [14,15]), pseudovirions, and human peripheral blood mononuclear cell (PBMC)-derived virions (7, 9). The remaining processed complex-type glycans on Env are determined by the host cell glycosylation machinery and are therefore …”

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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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: 99%

“…cans, in particular Man 8 GlcNAc 2 and Man 9 GlcNAc 2 , forming the so-called IMP (14). These glycans are a key component of the epitope of the N332-dependent bnAbs (4,18,22).…”

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: Importancementioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Pritchard

Harvey

Bonomelli

et al. 2015

J Virol

Self Cite

116

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cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV‐1 envelope glycoprotein vaccine candidate

Dey

Cupo

Ozorowski

et al. 2017

Biotech & Bioengineering

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We describe the properties of BG505 SOSIP.664 HIV‐1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native‐like trimers that are the basis for many structure‐guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV‐1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum‐free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size‐exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10. The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native‐like as judged by negative‐stain electron microscopy, and were stable over a multi‐month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native‐like Env glycoprotein trimers of various designs and genotypes.

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Augmenting glycosylation‐directed folding pathways enhances the fidelity of HIV Env immunogen production in plants

Margolin

Allen

Verbeek

et al. 2022

Biotech & Bioengineering

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Heterologous glycoprotein production relies on host glycosylation‐dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco‐engineering to improve the production of a model HIV‐1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3‐fucosyltransferase and β1,2‐xylosyltransferase was used as an expression host to prevent plant‐specific complex N‐glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco‐engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell‐produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant‐produced and mammalian cell‐derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus‐vectored vaccines. This study demonstrates that engineering glycosylation‐directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

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Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

Cited by 127 publications

References 54 publications

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

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

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV‐1 envelope glycoprotein vaccine candidate

Augmenting glycosylation‐directed folding pathways enhances the fidelity of HIV Env immunogen production in plants

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