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.
Highlights
Respiratory syncytial virus (RSV) is the leading cause of acute childhood lower respiratory tract infection and a major cause of hospital admissions [1]
We used a novel expression tag derived from spider silk proteins (‘N-terminal domain (NT)’) to produce recombinant fragment of human SP-A (rfhSP-A) in Escherichia coli, which we found to be trimeric and to bind to mannan in a calcium-dependent manner
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
Summary
RSV is the leading cause of acute childhood lower respiratory tract infection and a major cause of hospital admissions [1]. Surfactant protein A (SP-A) is an important innate immune molecule expressed throughout the human respiratory tract and present in pulmonary surfactant. SP-A functions as an innate immune defence molecule, which binds to carbohydrates on the surface of an array of different pathogens, promoting their neutralisation, agglutination and clearance. SP-A has been shown to neutralise numerous different viruses such as RSV [2] influenza A virus [3] and HIV [4]. SP-A enhances uptake of apoptotic cells by macrophages and functions to modulate the production of proinflammatory mediators in a context dependent manner [6]. SP-A has been shown to enhance the killing of Klebsiella pneumoniae by macrophages [7], modulate dendritic cell maturation [8] and inhibit the proliferation and function of T cells [9, 10]. SP-A has been shown to interact with various allergens [11] and prevent the binding of IgE from asthmatic children to house dust mite [12]
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