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Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic

Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic
Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.

SARS-CoV-2, antibody, glycoprotein, glycosylation, serology
0022-2836
167332
Chawla, Himanshi
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Jossi, Sian E
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Faustini, Sian E
fc25e886-bb2c-4c9c-a421-df8e00e6b1ba
Samsudin, Firdaus
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Allen, Joel D
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Watanabe, Yasunori
8c0ee4af-a293-4de5-9036-3ce2051b380c
Newby, Maddy L
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Marcial-Juárez, Edith
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Lamerton, Rachel E
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McLellan, Jason S
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Bond, Peter J
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Richter, Alex G
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Cunningham, Adam F
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Crispin, Max
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Chawla, Himanshi
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Jossi, Sian E
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Faustini, Sian E
fc25e886-bb2c-4c9c-a421-df8e00e6b1ba
Samsudin, Firdaus
b01e87a0-af50-44d6-bca4-f511c40165f9
Allen, Joel D
c89d5569-7659-4835-b535-c9586e956b3a
Watanabe, Yasunori
8c0ee4af-a293-4de5-9036-3ce2051b380c
Newby, Maddy L
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Marcial-Juárez, Edith
ae88367f-f7ce-4c4c-8b44-baade4de99bb
Lamerton, Rachel E
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McLellan, Jason S
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Bond, Peter J
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Richter, Alex G
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Cunningham, Adam F
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Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9

Chawla, Himanshi, Jossi, Sian E, Faustini, Sian E, Samsudin, Firdaus, Allen, Joel D, Watanabe, Yasunori, Newby, Maddy L, Marcial-Juárez, Edith, Lamerton, Rachel E, McLellan, Jason S, Bond, Peter J, Richter, Alex G, Cunningham, Adam F and Crispin, Max (2022) Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic. Journal of Molecular Biology, 434 (2), 167332, [167332]. (doi:10.1016/j.jmb.2021.167332).

Record type: Article

Abstract

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.

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Accepted/In Press date: 21 October 2021
e-pub ahead of print date: 27 October 2021
Published date: 30 January 2022
Additional Information: Funding Information: This work was supported by the International AIDS Vaccine Initiative (IAVI) through grant INV-008352/OPP1153692 funded by the Bill and Melinda Gates Foundation (M.C.). We also gratefully acknowledge support from the University of Southampton Coronavirus Response Fund (M.C.), a donation from the Bright Future Trust (M.C.), and the gift of laboratory consumables by Binding Site Ltd. P.J.B. and F.S. gratefully acknowledge support from BII core and grant FY21_CF_HTPO SEED_ID_BII_C211418001 funded by A*STAR. Simulations were performed on petascale computer cluster ASPIRE-1 at the National Supercomputing Centre of Singapore (NSCC), the A*STAR Computational Resource Centre (A*CRC), and the supercomputer Fugaku provided by RIKEN through the HPCI System Research Project (Project ID: hp200303). This work was funded in part by Welch Foundation grant number F-0003-19620604 (J.S.M.). We would like to thank the University of Birmingham Clinical Immunology Service for their invaluable support in sample collection and processing. AFC and AGR are grateful for funding from the Medical Research Council, the Global Challenges Research Fund (GCRF) and the Institute for Global Innovation (IG1 Project, 3107) of the University of Birmingham. We would also like to thank the Wellcome Trust Mechanisms of Inflammatory Disease PhD Programme, 108871/Z/15/Z (R.E.L.). Y.W. has taken up a position at AstraZeneca; all experimental work was performed prior to this development. Funding Information: This work was supported by the International AIDS Vaccine Initiative (IAVI) through grant INV-008352/OPP1153692 funded by the Bill and Melinda Gates Foundation (M.C.). We also gratefully acknowledge support from the University of Southampton Coronavirus Response Fund (M.C.), a donation from the Bright Future Trust (M.C.), and the gift of laboratory consumables by Binding Site Ltd. P.J.B. and F.S. gratefully acknowledge support from BII core and grant FY21_CF_HTPO SEED_ID_BII_C211418001 funded by A*STAR. Simulations were performed on petascale computer cluster ASPIRE-1 at the National Supercomputing Centre of Singapore (NSCC), the A*STAR Computational Resource Centre (A*CRC), and the supercomputer Fugaku provided by RIKEN through the HPCI System Research Project (Project ID: hp200303). This work was funded in part by Welch Foundation grant number F-0003-19620604 (J.S.M.). We would like to thank the University of Birmingham Clinical Immunology Service for their invaluable support in sample collection and processing. AFC and AGR are grateful for funding from the Medical Research Council, the Global Challenges Research Fund (GCRF) and the Institute for Global Innovation (IG1 Project, 3107) of the University of Birmingham. We would also like to thank the Wellcome Trust Mechanisms of Inflammatory Disease PhD Programme, 108871/Z/15/Z (R.E.L.). Y.W. has taken up a position at AstraZeneca; all experimental work was performed prior to this development. Mass spectrometry raw files have been deposited in the MassIVE proteomics databsase, MSV000087945. J.S.M. is an inventor on the following U.S. patent applications: no. 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”); no. 62/972,886 (“2019-nCoV Vaccine”); no. 63/032,502 (“Engineered Coronavirus Spike (S) Protein and Methods of Use Thereof”). Publisher Copyright: © 2021 The Authors
Keywords: SARS-CoV-2, antibody, glycoprotein, glycosylation, serology

Identifiers

Local EPrints ID: 453159
URI: http://eprints.soton.ac.uk/id/eprint/453159
ISSN: 0022-2836
PURE UUID: c2614bb0-b40a-4d41-aa6d-f84ec85c37c2
ORCID for Firdaus Samsudin: ORCID iD orcid.org/0000-0003-2766-4459
ORCID for Joel D Allen: ORCID iD orcid.org/0000-0003-2547-968X
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

Catalogue record

Date deposited: 10 Jan 2022 17:48
Last modified: 10 Jan 2023 03:03

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Contributors

Author: Himanshi Chawla
Author: Sian E Jossi
Author: Sian E Faustini
Author: Firdaus Samsudin ORCID iD
Author: Joel D Allen ORCID iD
Author: Yasunori Watanabe
Author: Maddy L Newby
Author: Edith Marcial-Juárez
Author: Rachel E Lamerton
Author: Jason S McLellan
Author: Peter J Bond
Author: Alex G Richter
Author: Adam F Cunningham
Author: Max Crispin ORCID iD

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