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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
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.
0171-2985
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
9eb79329-8d32-4ed4-b8b9-d720883e8042
Kronqvist, Nina
be12c640-2c44-4d90-8efb-d946e2d08c26
Spalluto, C. Mirella
6802ad50-bc38-404f-9a19-40916425183b
Griffiths, Mark
ff46d26f-2a28-4a6a-b189-d838abfcf8f7
Staples, Karl J.
e0e9d80f-0aed-435f-bd75-0c8818491fee
Wilkinson, Tom
8c55ebbb-e547-445c-95a1-c8bed02dd652
Holmskov, Uffe
8a55aecd-694c-4d61-849b-9cf32da8ba39
Sorensen, Grith L.
25ac3a15-3fc9-4dab-9fa5-caf89893b845
Rising, Anna
718fc40b-b154-4257-85bd-0b4c52c79355
Johansson, Jan
5c0b176a-c82a-4a99-9b0a-1cb9c665e6ff
Madsen, Jens
b5d8ae35-00ac-4d19-930e-d8ddec497359
Clark, Howard
70550b6d-3bd7-47c6-8c02-4f43f37d5213

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, 1-8. (doi:10.1016/j.imbio.2016.10.015).

Record type: Article

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

Identifiers

Local EPrints ID: 401759
URI: https://eprints.soton.ac.uk/id/eprint/401759
ISSN: 0171-2985
PURE UUID: 979b8916-3e29-4424-ad3a-1c1833217b25
ORCID for Karl J. Staples: ORCID iD orcid.org/0000-0003-3844-6457
ORCID for Jens Madsen: ORCID iD orcid.org/0000-0003-1664-7645

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Date deposited: 19 Oct 2016 10:48
Last modified: 03 Dec 2019 06:30

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Contributors

Author: Alastair Watson
Author: Nina Kronqvist
Author: C. Mirella Spalluto
Author: Mark Griffiths
Author: Karl J. Staples ORCID iD
Author: Tom Wilkinson
Author: Uffe Holmskov
Author: Grith L. Sorensen
Author: Anna Rising
Author: Jan Johansson
Author: Jens Madsen ORCID iD
Author: Howard Clark

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