Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140

Go, Eden P.; Herschhorn, Alon; Gu, Chong; Castillo-Menendez, Luis R.; Zhang, Shijian; Mao, Youdong; Chen, Haiyan; Ding, Haitao; Wakefield, John K.; Hua, David; Liao, Hua Xin; Kappes, John C.; Sodroski, Joseph; Desaire, Heather

doi:10.1128/jvi.00628-15

Cited by 100 publications

(137 citation statements)

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“…Raising antibodies against such proteins may therefore benefit from the application of new, recently described methods (78). The glycan composition analysis further reinforces how the uncleaved gp140s lack the steric constraints imposed when the gp120 subunits are components of properly assembled trimers; accordingly, the gp140 UNC -Fd-His glycans are much more highly processed than those on the native-like SOSIP.664-His trimers (39,79). Finally, the uncleaved CZA97.012 gp140 proteins contain a substantial proportion of gp120 subunits with scrambled disulfide bonds, particularly in the V1V2 region; the aberrant subunits further contribute to the nonnative antigenicity and associated aberrant structural and aggregation properties of these proteins.…”

Section: Discussionmentioning

confidence: 93%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

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164

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We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140 UNC -Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41 ECTO ) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41 ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ϳ20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCESoluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins. R ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from...

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Section: Discussionmentioning

confidence: 93%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

Self Cite

164

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“…Three of the trimer data sets are available in the literature, originally generated by the Desaire laboratory (25), the Crispin laboratory (46), and the Dell laboratory (48). Eight additional Env trimers were characterized for the present report.…”

Section: Resultsmentioning

confidence: 99%

“…A subsequent attempt to analyze the glycans at each site on virion-derived Env yielded an incomplete data set-only 13 glycopeptides were identified in total, and most of the glycan sites were not detected (45). More recently, Go et al published a site-by-site glycosylation analysis of a recombinant JR-FL (clade B) Env trimer which yielded information on every site and identified Ͼ600 different glycoforms, a 30-fold increase over the earlier study on virion Env (25). The recombinant JR-FL trimer used in that study, a membrane-anchored gp150 protein purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity, had multiple high-mannose glycans at specific sites, consistent with the original glycan-based virion analyses (43,44).…”

mentioning

confidence: 99%

“…Approximately 50% of the mass of gp120 consists of carbohydrate, with most of the more than 24 potential N-linked glycosylation sites (PNGSs) utilized in most HIV-1 variants (15,(21)(22)(23). The gp41 component of the trimer typically contains 4 PNGSs (18,24), and O-linked glycosylation sites also have been reported (25)(26)(27).…”

mentioning

confidence: 99%

“…A more recent analysis of this same BG505 trimer provided glycosylation coverage at every site, with 230 glycopeptides reported (47). The studies on JR-FL gp150 and BG505 SOSIP.664 gp140 represent two examples of trimeric Env glycosylation, but the analyses were carried out using proteins based on different Env genotypes and on different construct designs and that were produced in different cell lines; neither study involved a natural HIV-1 target cell type or a full-length Env (25,46,47). Accordingly, it is unclear how these protein design and expression differences impacted the results.…”

mentioning

confidence: 99%

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Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Ding

Zhang

et al. 2017

J Virol

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110

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HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

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The expanding role of mass spectrometry in the field of vaccine development

Sharma

Sharma²,

Glick

2018

Mass Spectrometry Reviews

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Biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. Mass spectrometry-based techniques are also routinely used to assess glycosylation and other post-translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. The role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. In this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted.

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Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140

Cited by 100 publications

References 59 publications

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

The expanding role of mass spectrometry in the field of vaccine development

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