Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein
Impact of stabilizing mutations 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 that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced 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 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these 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. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.
Glycoproteins/metabolism, Glycosylation, HIV Antibodies, HIV Envelope Protein gp120/genetics, HIV Envelope Protein gp41/genetics, HIV-1, Humans, Mannose/metabolism, Mutation
e1011452
Tong, Tommy
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D'Addabbo, Alessio
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Xu, Jiamin
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Chawla, Himanshi
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Nguyen, Albert
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Ochoa, Paola
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Crispin, Max
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Binley, James M
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7 August 2023
Tong, Tommy
1e075548-7dd2-4bda-afd5-266637cc7077
D'Addabbo, Alessio
ae2358c9-d733-4c7f-aaf3-b8b52b7cb810
Xu, Jiamin
68b5f3f7-8e0a-49dc-9f6f-3e330ced8bd7
Chawla, Himanshi
07b9e983-4c35-4314-999d-fe3222a6c03b
Nguyen, Albert
6dc6615d-b316-4520-a097-f45fc73d2f78
Ochoa, Paola
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Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Binley, James M
e71790e2-16d5-4acf-8bfc-2266a4fbfeb1
Tong, Tommy, D'Addabbo, Alessio, Xu, Jiamin, Chawla, Himanshi, Nguyen, Albert, Ochoa, Paola, Crispin, Max and Binley, James M
(2023)
Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein.
PLoS Pathogens, 19 (8 August), , [e1011452].
(doi:10.1371/journal.ppat.1011452).
Abstract
Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced 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 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these 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. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.
Text
journal.ppat.1011452
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More information
Accepted/In Press date: 16 July 2023
Published date: 7 August 2023
Additional Information:
Funding Information:
Funding: This work was supported by NIH grants AI93278 (JMB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
Copyright: © 2023 Tong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords:
Glycoproteins/metabolism, Glycosylation, HIV Antibodies, HIV Envelope Protein gp120/genetics, HIV Envelope Protein gp41/genetics, HIV-1, Humans, Mannose/metabolism, Mutation
Identifiers
Local EPrints ID: 481440
URI: http://eprints.soton.ac.uk/id/eprint/481440
ISSN: 1553-7366
PURE UUID: 8e762e73-c03d-4773-9470-a93e7c4a373d
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Date deposited: 29 Aug 2023 16:45
Last modified: 18 Mar 2024 03:53
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Author:
Tommy Tong
Author:
Jiamin Xu
Author:
Albert Nguyen
Author:
Paola Ochoa
Author:
James M Binley
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