Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV
Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV
Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and hospitalisation of infants in developed countries. Surfactant protein A (SP-A) is an important innate immune molecule, localized in pulmonary surfactant. SP-A binds to carbohydrates on the surface of pathogens in a calcium-dependent manner to enable neutralisation, agglutination and clearance of pathogens including RSV.
SP-A forms trimeric units and further oligomerises through interactions between its N-terminal domains. Whilst a recombinant trimeric fragment of the closely related molecule (surfactant protein D) retains many of the native protein's functions, the importance of the SP-A oligomeric structure in its interaction with RSV has not been determined.
The aim of this study was to produce a functional trimeric recombinant fragment of human (rfh)SP-A, which lacks the N-terminal domain (and the capacity to oligomerise) and test its ability to neutralise RSV in an in vitro model of human bronchial epithelial infection.
We used a novel expression tag derived from spider silk proteins ('NT') to produce rfhSP-A in Escherichia coli, which we found to be trimeric and to bind to mannan in a calcium-dependent manner. Trimeric rfhSP-A reduced infection levels of human bronchial epithelial (AALEB) cells by RSV by up to a mean (± SD) of 96.4 (± 1.9) % at 5 ?g/ml, which was significantly more effective than dimeric rfhSP-A (34.3 (± 20.5) %) (p < 0.0001). Comparatively, native human SP-A reduced RSV infection by up to 38.5 (± 28.4) %.
For the first time we report the development of a functional trimeric rfhSP-A molecule which is highly efficacious in neutralising RSV, despite lacking the N-terminal domain and capacity to oligomerise.
1-8
Watson, Alastair
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Kronqvist, Nina
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Spalluto, C. Mirella
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Griffiths, Mark
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Staples, Karl J.
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Wilkinson, Tom
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Holmskov, Uffe
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Sorensen, Grith L.
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Rising, Anna
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Johansson, Jan
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Madsen, Jens
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Clark, Howard
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Watson, Alastair
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Kronqvist, Nina
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Spalluto, C. Mirella
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Griffiths, Mark
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Staples, Karl J.
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Wilkinson, Tom
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Holmskov, Uffe
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Sorensen, Grith L.
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Rising, Anna
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Johansson, Jan
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Madsen, Jens
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Clark, Howard
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Watson, Alastair, Kronqvist, Nina, Spalluto, C. Mirella, Griffiths, Mark, Staples, Karl J., Wilkinson, Tom, Holmskov, Uffe, Sorensen, Grith L., Rising, Anna, Johansson, Jan, Madsen, Jens and Clark, Howard
(2016)
Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV.
Immunobiology, .
(doi:10.1016/j.imbio.2016.10.015).
Abstract
Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and hospitalisation of infants in developed countries. Surfactant protein A (SP-A) is an important innate immune molecule, localized in pulmonary surfactant. SP-A binds to carbohydrates on the surface of pathogens in a calcium-dependent manner to enable neutralisation, agglutination and clearance of pathogens including RSV.
SP-A forms trimeric units and further oligomerises through interactions between its N-terminal domains. Whilst a recombinant trimeric fragment of the closely related molecule (surfactant protein D) retains many of the native protein's functions, the importance of the SP-A oligomeric structure in its interaction with RSV has not been determined.
The aim of this study was to produce a functional trimeric recombinant fragment of human (rfh)SP-A, which lacks the N-terminal domain (and the capacity to oligomerise) and test its ability to neutralise RSV in an in vitro model of human bronchial epithelial infection.
We used a novel expression tag derived from spider silk proteins ('NT') to produce rfhSP-A in Escherichia coli, which we found to be trimeric and to bind to mannan in a calcium-dependent manner. Trimeric rfhSP-A reduced infection levels of human bronchial epithelial (AALEB) cells by RSV by up to a mean (± SD) of 96.4 (± 1.9) % at 5 ?g/ml, which was significantly more effective than dimeric rfhSP-A (34.3 (± 20.5) %) (p < 0.0001). Comparatively, native human SP-A reduced RSV infection by up to 38.5 (± 28.4) %.
For the first time we report the development of a functional trimeric rfhSP-A molecule which is highly efficacious in neutralising RSV, despite lacking the N-terminal domain and capacity to oligomerise.
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Accepted/In Press date: 17 October 2016
e-pub ahead of print date: 18 October 2016
Organisations:
Clinical & Experimental Sciences
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Local EPrints ID: 401759
URI: http://eprints.soton.ac.uk/id/eprint/401759
ISSN: 0171-2985
PURE UUID: 979b8916-3e29-4424-ad3a-1c1833217b25
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Date deposited: 19 Oct 2016 10:48
Last modified: 15 Mar 2024 05:59
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Author:
Alastair Watson
Author:
Nina Kronqvist
Author:
C. Mirella Spalluto
Author:
Mark Griffiths
Author:
Uffe Holmskov
Author:
Grith L. Sorensen
Author:
Anna Rising
Author:
Jan Johansson
Author:
Jens Madsen
Author:
Howard Clark
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