Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops

Moscoso, Carlos G.; Li, Xing; Hui, Jinwen; Hu, Jeffrey; Kalkhoran, Mohammad Baikoghli; Yenigun, Onur M.; Sun, Yuliang; Paavolainen, Lassi; Martin, Loı̈c; Vahlne, Anders; Zambonelli, C.; Barnett, Susan W.; Srivastava, Indresh K.; Cheng, R. Holland

doi:10.1038/srep07025

Cited by 9 publications

(6 citation statements)

References 67 publications

(128 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…EM images of these proteins generally show three separated gp120 subunits dangling from a central gp41 ECTO moiety to which they remain tethered by the uncleaved inter-subunit linkage (Georgiev et al, 2015; Moscoso et al, 2014; Ringe et al, 2013; Tran et al, 2014), a conclusion strongly supported by HDX-MS and surface plasmon resonance (SPR) data (Guttman et al, 2014; Ringe et al, 2013; Yasmeen et al, 2014). The EM images of uncleaved gp140s shown here are entirely consistent, with irregularly shaped forms predominating.…”

Section: Discussionmentioning

confidence: 83%

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

et al. 2015

View full text Add to dashboard Cite

A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system but, paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity-purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles’ heel that can be exploited for bNAb recognition and vaccine design.

show abstract

Section: Discussionmentioning

confidence: 83%

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Whether or not uncleaved gp140 proteins such as those based on the CZA97.012 and 92UG037.8 genotypes are pursued as immunogens in human clinical trials, claims that these proteins have a native-like structure and, by inference, may be able to induce trimerization-influenced NAbs to tier 2 viruses, are clearly not verifiable. The same concerns apply to other uncleaved gp140s of comparable designs that are also being produced for clinical trials, again on the basis that they mimic the native structure of virion-associated Env (36,82). A more appropriate rationale for the use of uncleaved gp140s may be their induction of non-NAbs that are partially protective in a macaque challenge model (83).…”

Section: Discussionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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...

show abstract

“…It is now clear that the resulting uncleaved gp140 (gp140 UNC ) proteins, when purified by size exclusion chromatography (SEC), contain the right number of gp120 and gp41 ECTO subunits (i.e., 3 of each). However, these various SEC-purified gp140 UNC protein populations mostly (90 to 100%) adopt nonnative configurations whether or not they contain additional "trimerization motifs" at the C terminus of gp41 ECTO (20)(21)(22)(23)(24)(25)(26); L. K. Pritchard, S. Vasiljevic, G. Ozorowski, G. E. Seabright, A. Cupo, R. W. Sanders, K. J. Doores, D. R. Burton, I. A. Wilson, A.…”

mentioning

confidence: 99%

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

Pugach

Ozorowski

Cupo

et al. 2015

J Virol

239

408

View full text Add to dashboard Cite

Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP. IMPORTANCEThe cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine. Immunogens capable of inducing protective titers of broadly neutralizing antibodies (bNAbs) are being widely sought for use in vaccine design strategies for human immunodeficiency virus type 1 (HIV-1) (1). The basis of this approach is that bNAbs can prevent globally diverse HIV-1 strains from infecting target cells. They do so via binding to the envelope glycoprotein (Env) complex on the virion surface, an event that is both necessary and sufficient to neutralize HIV-1 infectivity (2, 3). One of the more common strategies to induce bNAbs involves the design of soluble, recombinant protein mimics of the native Env complex, a meta-stable structure comprising three gp120 and three gp41 subunits. The production of soluble Env trimers involves introducing a stop codon to truncate the gp41 ectodomain (gp41 ECTO ) subunit prior to the transmembrane region to yield soluble gp140 proteins (4-6). The fragility of the Env complex is, however, a substantial problem from a protein engineering perspective, as the natural noncovalent interactions between the six subunits are not robust enough to allow so...

show abstract

Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops

Cited by 9 publications

References 67 publications

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

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

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

Contact Info

Product

Resources

About