title: Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV.
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) has been shown to retain 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 5μg/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.
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom.
authors: Alastair
authors: A
authors: Watson
authors: Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Institutet, 141 57 Huddinge, Sweden.
authors: Nina
authors: N
authors: Kronqvist
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom.
authors: C Mirella
authors: CM
authors: Spalluto
authors: Leukocyte Biology, Imperial College London, Royal Brompton Campus, London SW3 6NP, United Kingdom.
authors: Mark
authors: M
authors: Griffiths
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom.
authors: Karl J
authors: KJ
authors: Staples
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, United Kingdom.
authors: Tom
authors: T
authors: Wilkinson
authors: Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors: Uffe
authors: U
authors: Holmskov
authors: Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors: Grith L
authors: GL
authors: Sorensen
authors: Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Institutet, 141 57 Huddinge, Sweden; Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, 750 07 Uppsala, Sweden.
authors: Anna
authors: A
authors: Rising
authors: Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Institutet, 141 57 Huddinge, Sweden; Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, 750 07 Uppsala, Sweden.
authors: Jan
authors: J
authors: Johansson
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, United Kingdom; Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom.
authors: Jens
authors: J
authors: Madsen
authors: Clinical & Experimental Sciences Academic Unit, Southampton General Hospital, University of Southampton, Southampton, United Kingdom; Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Institutet, 141 57 Huddinge, Sweden; Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, United Kingdom. Electronic address: H.W.Clark@soton.ac.uk.
authors: Howard
authors: H
authors: Clark
mesh_terms: D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002478:Cells, Cultured; D015870:Gene Expression; D054884:Host-Pathogen Interactions / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020545:Respiratory Mucosa / Q000276:immunology / Q000378:metabolism / Q000821:virology; D018357:Respiratory Syncytial Virus Infections / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology*
chemical_list: D002241:Carbohydrates; D037662:Pulmonary Surfactant-Associated Protein A; D011993:Recombinant Fusion Proteins
keywords: Collectin; Innate immunity; NT domain; Neutralisation; Recombinant trimeric fragment; Respiratory syncytial virus; Surfactant protein A