Cleavage-Independent HIV-1 Env Trimers Engineered as Soluble Native Spike Mimetics for Vaccine Design

Sharma, Shailendra Kumar; Val, Natalia de; Bale, Shridhar; Guenaga, Javier; Tran, Karen; Feng, Yanghe; Dubrovskaya, Viktoriya; Ward, Andrew B.; Wyatt, Richard T.

doi:10.1016/j.celrep.2015.03.047

Cited by 193 publications

(295 citation statements)

References 31 publications

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“…Two recent studies report uncleaved gp140s that can adopt native-like configurations, at least in some genotypes, provided a flexible linker of ϳ10 to 20 residues is introduced between the gp120 and gp41 ECTO subunits (3,7). The appearance of these single-chain gp140 (scgp140) or native flexibly linked gp140 (NFL-gp140) proteins is a The data represent the abundance (as a percentage of total glycans) of the oligomannose structures (Man 5-9 GlcNAc 2 ; indicated as M5 to M9), individually and in total, for each Env protein analyzed.…”

Section: Resultsmentioning

confidence: 99%

“…Instead, they represent semidisintegrated trimers in which the uncleaved peptide linking the gp120 and gp41 ECTO subunits holds the components together in a disordered fashion (19,30,33,(37)(38)(39)(40)(41). Recent reports show that this defect can be at least partially overcome for some Env genotypes by introducing a flexible linker between the C terminus of gp120 and the N terminus of gp41 ECTO , such that the two subunits remain covalently linked but now in a way that allows them to interact properly (3,7). When the I559P substitution is also present, the resulting uncleaved gp140s trimerize and now can adopt a native-like structure that is broadly comparable to those of cleaved SOSIP.664 trimers (3,7).…”

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confidence: 99%

“…Recent reports show that this defect can be at least partially overcome for some Env genotypes by introducing a flexible linker between the C terminus of gp120 and the N terminus of gp41 ECTO , such that the two subunits remain covalently linked but now in a way that allows them to interact properly (3,7). When the I559P substitution is also present, the resulting uncleaved gp140s trimerize and now can adopt a native-like structure that is broadly comparable to those of cleaved SOSIP.664 trimers (3,7). Addition of the SOS intermolecular disulfide bond confers additional benefit (7).…”

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confidence: 99%

“…Addition of the SOS intermolecular disulfide bond confers additional benefit (7). These uncleaved, native-like trimers have been designated single-chain gp140 (sc-gp140) or native flexibly linked gp140 (NFL-gp140) (3,7).…”

mentioning

confidence: 99%

“…ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from human immunodeficiency virus type 1 (HIV-1) are being designed, developed, and produced as vaccine candidates intended to induce neutralizing antibody (NAb) responses (1)(2)(3)(4)(5)(6)(7)(8).…”

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confidence: 99%

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

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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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: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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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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A design strategy to generate a SARS‐CoV‐2 RBD vaccine that abrogates ACE2 binding and improves neutralizing antibody responses

Ratswohl,

Vázquez García,

Ahmad

et al. 2023

Eur J Immunol

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The structure‐based design of antigens holds promise for developing vaccines with higher efficacy and improved safety profiles. We postulate that abrogation of host receptor interaction bears potential for the improvement of vaccines by preventing antigen‐induced modification of receptor function as well as the displacement or masking of the immunogen. Antigen modifications may yet destroy epitopes crucial for antibody neutralization. Here, we present a methodology that integrates deep mutational scans to identify and score SARS‐CoV‐2 receptor binding domain variants that maintain immunogenicity, but lack interaction with the widely expressed host receptor. Single point mutations were scored in silico, validated in vitro, and applied in vivo. Our top‐scoring variant receptor binding domain‐G502E prevented spike‐induced cell‐to‐cell fusion, receptor internalization, and improved neutralizing antibody responses by 3.3‐fold in rabbit immunizations. We name our strategy BIBAX for body‐inert, B‐cell‐activating vaccines, which in the future may be applied beyond SARS‐CoV‐2 for the improvement of vaccines by design.

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Cleavage-Independent HIV-1 Env Trimers Engineered as Soluble Native Spike Mimetics for Vaccine Design

Cited by 193 publications

References 31 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

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

A design strategy to generate a SARS‐CoV‐2 RBD vaccine that abrogates ACE2 binding and improves neutralizing antibody responses

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