Generation and Characterization of a Bivalent HIV-1 Subtype C gp120 Protein Boost for Proof-of-Concept HIV Vaccine Efficacy Trials in Southern Africa

Zambonelli, C.; Dey, Antu; Hilt, Susan; Stephenson, Samuel; Go, Eden P.; Clark, Daniel F.; Wininger, Mark; LaBranche, Celia C.; Montefiori, David C.; Liao, Hua Xin; Swanstrom, Ronald; Desaire, Heather; Haynes, Barton F.; Carfı́, Andrea; Barnett, Susan W.

doi:10.1371/journal.pone.0157391

Cited by 30 publications

(42 citation statements)

References 45 publications

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“…The group M consensus Env, CON-S gp140, and a clade B transmitted/founder gp140, B.700010040.C9, have glycosylation profiles that are somewhat closer to the consensus profile for Env trimers but still differ from it at several sites. Finally, the clade C.1086 gp120, which will be tested in clinical trials (58), matches the consensus glycosylation profile at all sites except N156 and N397 to N412.…”

Section: Impact Of Purification Method None Of the Variance In Glycomentioning

confidence: 97%

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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111

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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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Section: Impact Of Purification Method None Of the Variance In Glycomentioning

confidence: 97%

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

Ding

Zhang

et al. 2017

J Virol

Self Cite

111

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“…Contributing to these outcomes may be the presentation of immunodominant non-NAb epitopes, the inability to present NAb epitopes that depend on quaternary structure and/or involve gp41, and the generation of ‘off-target’ non-NAbs that approach the CD4bs on the trimer from an inappropriate angle. In addition, gp120 monomers are not always as simple to manufacture as is often thought, due to proteolytic damage to V3, unwanted dimerization via intermolecular disulfide bonds and the formation of aberrant intramolecular disulfide bonds that can compromise the presentation of some epitopes (42–47). We note that boosting of rabbits with gp120 monomers after priming with vaccinia viruses expressing gp160 induced a low but fairly consistent level of NAbs against heterologous Tier-2 viruses (48).…”

Section: Non-native Gp140 ‘Pseudotrimers’mentioning

confidence: 99%

“…In other words, in a substantial proportion of the total population of gp120 monomers or subunits some of the intermolecular disulfide bonds had become scrambled because the “wrong” cysteines had paired up. The resulting gp120 molecules, although of the correct size, were misfolded and antigenically perturbed, particularly within the V1V2 region where multiple cysteines are located (47,54,55). In addition, the aberrant formation of intermolecular disulfide bonds yielded dimerized gp120 proteins (43,45,47).…”

Section: Properties Of Bg505 Sosip664 Trimersmentioning

confidence: 99%

Native‐like Env trimers as a platform for HIV‐1 vaccine design

Sanders

Moore

2017

Immunological Reviews

222

260

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Summary We describe the development and potential use of various designs of recombinant HIV-1 envelope glycoprotein trimers that mimic the structure of the virion-associated spike, which is the target for neutralizing antibodies. The goal of trimer development programs is to induce broadly neutralizing antibodies with the potential to intervene against multiple circulating HIV-1 strains. Among topics we address are the designs of various constructs; how native-like trimers can be produced and purified; the properties of such trimers in vitro and their immunogenicity in various animals; and the immunization strategies that may lead to the eventual elicitation of broadly neutralizing antibodies. In summary, native-like trimers are a now a platform for structure- and immunology-based design improvements that could eventually yield immunogens of practical value for solving the long-standing HIV-1 vaccine problem.

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“…A key question in the development of any vaccine is whether the vaccine candidate can be made in practical amounts at a reasonable cost and under current Good Manufacturing Practice (cGMP) conditions that are required for use in humans. HIV‐1 envelope glycoproteins of any design have not proven simple to produce as cGMP reagents, problems encountered including proteolytic damage (Wang et al, ; Yu, Fonseca, O'Rourke, & Berman, ) and aggregate formation (Finzi et al, ; Zambonelli et al, ) caused, in part, by the formation of aberrant disulfide bonds (Alam et al, ; Wieczorek et al, ; Zambonelli et al, ). Purification can also be problematic.…”

Section: Introductionmentioning

confidence: 99%

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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Generation and Characterization of a Bivalent HIV-1 Subtype C gp120 Protein Boost for Proof-of-Concept HIV Vaccine Efficacy Trials in Southern Africa

Cited by 30 publications

References 45 publications

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

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

Native‐like Env trimers as a platform for HIV‐1 vaccine design

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

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