Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro - PubMed (original) (raw)
doi: 10.1371/journal.pone.0025005. Epub 2011 Sep 14.
Martin van Eijk, Stella E van Trierum, Debby van Riel, Xavier Saelens, Roland A Romijn, Wieger Hemrika, Ron A M Fouchier, Thijs Kuiken, Albert D M E Osterhaus, Henk P Haagsman, Guus F Rimmelzwaan
Affiliations
- PMID: 21935489
- PMCID: PMC3173486
- DOI: 10.1371/journal.pone.0025005
Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro
Marine L B Hillaire et al. PLoS One. 2011.
Abstract
The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.
Conflict of interest statement
Competing Interests: The authors have read the journal's policy and have the following conflicts: Wieger Hemrika and Roland A. P. Romijn are employed by U-Protein Express B.V. Guus F. Rimmelzwaan is employed part-time by Viroclinics Biosciences B.V. as a consultant. Albert D.M.E. Osterhaus is employed part-time by Viroclinics Biosciences B.V. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Figures
Figure 1. Antiviral activity of RpSP-D resembles that of NpSP-D.
The minimal inhibitory concentration of RpSP-D and NpSP-D required to inhibit the hemagglutination by all 27 H1N1 and H3N2 IAVs was assessed (A). The horizontal line indicates the average of the minimal inhibitory concentration for both preparations which did not differ significantly. The values were also compared directly (B). The minimal inhibition concentrations of RpSP-D correlated well with those of NpSP-D. Each symbol in panels A and B represent an individual IAV strain.
Figure 2. RpSP-D inhibits the hemagglutination by a broad range of IAVs.
The minimal inhibitory concentration of RpSP-D that still inhibited the hemagglutination by a variety of IAV was determined. In panel A, the bars represent the average minimal concentration of RpSP-D required to inhibit all H1N1, H3N2 and H5N1 viruses tested, independent of the animal species they originated from. Panels B and C show the results of individual H1N1 (B) and H3N2 (C) viruses and their origin. The average concentrations obtained in six independent experiments are shown. The results obtained in four independent experiments with H5N1 viruses, VietNam/04 (open square), Indonesia/05 (closed diamond), HongKong/97 (closed triangle) are shown in panel D. Also the results (the average of three independent experiments) are shown in panel E and F for inhibition by multimers and trimers of RpSP-D. The H1N1 viruses tested are NL/09 (closed diamond), S/NL/1/87 (closed triangle), NJ/76 (closed square), NL/06 (open square), M/NL/06 (closed circle), NL/08 (open circle), WGF/NL/07 (open triangle) (panel E) and the H3N2 viruses tested are S/NL/93 (closed diamond), NL/07 (open square), NL/03 (open triangle), NL/93 (closed triangle), M/NL//08 (open circle), M/Sweden/03 (closed circle), M/NL/07 (closed square) (panel F).
Figure 3. Comparison between the HI activity of RpSP-D and that of RhSP-D.
The minimal inhibitory concentrations of RpSP-D (black bars) and RhSP-D (white bars) were determined with the HI assay for the H1N1 (A) and H3N2 (B) viruses as indicated. In panel C, the minimal inhibitory concentrations of RhSP-D multimers (black bars) and trimers (white bars) were compared. The data represent the average of three independent experiments.
Figure 4. RpSP-D binds to HA.
293T cells were transfected with plasmids expressing HA or NP genes derived from influenza virus A/Netherlands//178/95 (H3N2). The cells were then incubated with RpSP-D or not and binding of RpSP-D was assessed by an immuno-fluorescence assay (upper panels) using a mouse monoclonal anti-pSP-D antibody and a goat anti-mouse FITC labeled antibody. Cells were analyzed using a fluorescence microscope. No binding of RpSP-D was detected in cells transfected with a plasmid encoding the NP gene, which was included as negative control, whereas cells expressing the HA gene bound RpSP-D.
Figure 5. Reduction of IAV infection by RpSP-D.
Using the infection reduction assay, the neutralization of IAV by various doses of RpSP-D was assessed. The reduction of infectivity was expressed as the relative number of cells that became infected according to the formula: % reduction = 1-(% infected cells in presence of RpSP-D/% infected cells without RpSP-D)*100%. The viruses were grouped by subtype and origin: swine H3N2 (A), human H3N2 (B), avian H3N2 (C), 2009 pandemic H1N1 (D), avian-like swine H1N1 (E), classical swine (F), human H1N1 (G), avian H1N1 (H) and H5N1 (I). The average of triplicate wells is shown.
Figure 6. Inhibition of neuraminidase activity by RpSP-D.
The enzymatic activity of recombinant purified neuraminidase derived from IAV strains A/crested eagle/Belgium/01/2004 (N1) (A) and A/Victoria/3/75 (N2) (B) was determined after overnight incubation in the presence of various doses of peanut agglutinin (open square), ConA (open circle), oseltamivir (closed circle) or RpSP-D (closed triangle). The concentration is expressed in μg/100μl for the lectins and in nM for oseltamivir. NA activity in the absence of inhibitors was normalized to 100% and the percentage of inhibition was depicted.
Figure 7. RpSP-D prevents binding of human seasonal H1N and H3N2 viruses to epithelial cells of ferret and human trachea.
Attachment of virus in absence or presence of RpSP-D was studied by virushistochemistry. Sections of ferret trachea (A) or human trachea (B) were incubated with FITC-labeled IAV A/Netherlands/35/05 (H1N1) and A/Netherlands/231/03 (H3N2) in the absence or presence of various doses of RpSP-D as indicated. Binding of virus is visible as dark-red staining. As a negative control, the highest dose of RpSP-D (10 µg) was also measured in absence of calcium ions. The tissues were counterstained with hematoxylin (magnification x100).
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