Dataset Information

Recombinant HIV-1 426c Env Tryptic Digest Glycan Identifications and Semi-Quantification

ABSTRACT: VRC01-class broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) and are of major interest for vaccine design. Unlike mature antibodies, corresponding VRC01-class germline precursors poorly bind to Env due to their weak ability to overcome the steric hindrance imparted by the glycans surrounding the CD4 BS. To date, immunogen design initiatives have largely relied on removal of these glycans and have met limited success in eliciting VRC01-class bnAbs. To understand elicitation of such bnAbs in humans, we structurally characterized the inferred germline precursor of VRC01 in complex with wild-type core gp120 by X-ray crystallography and modified trimeric 426c Env constructs by single-particle electron microscopy. We then validated glycan length and composition of germline VRC01-compatible Env constructs by EThcD LC-MS/MS. Our results reveal that engagement of the VRC01 germline antibody by a wild-type 426c gp120 is possible and can be enhanced by tailoring glycan length in the vicinity of the CD4BS.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens Human Immunodeficiency Virus 1

SUBMITTER: Andrew Borst

LAB HEAD: David Veesler

PROVIDER: PXD011494 | Pride | 2018-11-21

REPOSITORIES: Pride

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Publications

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core.

Borst Andrew J AJ Weidle Connor E CE Gray Matthew D MD Frenz Brandon B Snijder Joost J Joyce M Gordon MG Georgiev Ivelin S IS Stewart-Jones Guillaume Be GB Kwong Peter D PD McGuire Andrew T AT DiMaio Frank F Stamatatos Leonidas L Pancera Marie M Veesler David D

eLife 20181107

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4<sub>BS</sub>) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC ...[more]

PMID: 30403372

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Project description:Impact of Common Modifications on the Antigenic Profile and Glycosylation of Membrane-Expressed HIV-1 Envelope Glycoprotein Recent HIV-1 vaccine development has centered on near native soluble envelope glycoprotein (Env) trimers. These trimers are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These same stabilizing mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their precise effects in this context are unclear. To address this question, we investigated the effects of Env mutations expressed on virus-like particle (VLP) in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 (NFL) neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed processing post-expression, resulting in increased trimer stability. Gp41 N-helix mutations I559P and NT1-5 both imparted lateral trimer stability, but concomitantly reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ donor plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common effect of mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. We observed a minor species of high mannose glycan only gp160 in particle preparations. This was unaffected by any mutations and instead bypasses normal folding and glycan maturation processes. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and increased the proportion of high mannose gp160. Remarkably, NAbs were unable to bind to full-length Env trimers. Overall, our findings suggest caution in leveraging mutations to ensure they impart valuable membrane trimer phenotypes for vaccine use. The files enclosed are labelled according to the enzyme used in the digest. T = Trypsin, C = Chymotrypsin, A = Alpha-lytic protease

Dataset Information

Recombinant HIV-1 426c Env Tryptic Digest Glycan Identifications and Semi-Quantification

Publications

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core.

Similar Datasets

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