Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV

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• 作者：Alastair Watson^a ; Nina Kronqvist^b ; C. Mirella Spalluto^a ; Mark Griffiths^c ; Karl J. Staples^a ; Tom Wilkinson^a ; ^d ; Uffe Holmskov^e ; Grith L. Sorensen^e ; Anna Rising^b ; ^g ; Jan Johansson^b ; ^g ; Jens Madsen^a ; ^d ; ^f ; Howard Clark^a ; ^b ; ^d ; ^{H.W.Clark@soton.ac.uk}
• 关键词：Surfactant protein A ; Recombinant trimeric fragment ; Respiratory syncytial virus ; NT domain ; Innate immunity ; Neutralisation ; Collectin
• 刊名：Immunobiology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：222
• 期：2
• 页码：111-118
• 全文大小：1283 K
• 卷排序：222

文摘

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 5 μg/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.