Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and...

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Autores principales:	Tyng Hwey Tan, Elizabeth Patton, Carol A. Munro, Dora E. Corzo-Leon, Andrew J. Porter, Soumya Palliyil
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	SARS-CoV-2 ORF3a viroporin recombinant antibodies anti-ORF3a mAbs Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c10c6e519e564d23bd7c3db95d5ae027
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spelling	oai:doaj.org-article:c10c6e519e564d23bd7c3db95d5ae0272021-11-25T19:13:18ZMonoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein10.3390/v131122011999-4915https://doaj.org/article/c10c6e519e564d23bd7c3db95d5ae0272021-11-01T00:00:00Zhttps://www.mdpi.com/1999-4915/13/11/2201https://doaj.org/toc/1999-4915ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.Tyng Hwey TanElizabeth PattonCarol A. MunroDora E. Corzo-LeonAndrew J. PorterSoumya PalliyilMDPI AGarticleSARS-CoV-2ORF3aviroporinrecombinant antibodiesanti-ORF3a mAbsMicrobiologyQR1-502ENViruses, Vol 13, Iss 2201, p 2201 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	SARS-CoV-2 ORF3a viroporin recombinant antibodies anti-ORF3a mAbs Microbiology QR1-502
spellingShingle	SARS-CoV-2 ORF3a viroporin recombinant antibodies anti-ORF3a mAbs Microbiology QR1-502 Tyng Hwey Tan Elizabeth Patton Carol A. Munro Dora E. Corzo-Leon Andrew J. Porter Soumya Palliyil Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
description	ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.
format	article
author	Tyng Hwey Tan Elizabeth Patton Carol A. Munro Dora E. Corzo-Leon Andrew J. Porter Soumya Palliyil
author_facet	Tyng Hwey Tan Elizabeth Patton Carol A. Munro Dora E. Corzo-Leon Andrew J. Porter Soumya Palliyil
author_sort	Tyng Hwey Tan
title	Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
title_short	Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
title_full	Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
title_fullStr	Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
title_full_unstemmed	Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein
title_sort	monoclonal human antibodies that recognise the exposed n and c terminal regions of the often-overlooked sars-cov-2 orf3a transmembrane protein
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/c10c6e519e564d23bd7c3db95d5ae027
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Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

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