Data_Sheet_1_Protein Nanoparticles Made of Recombinant Viral Antigens: A Promising Biomaterial for Oral Delivery of Fish Prophylactics.doc

<p>In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as “nanopellets” (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.</p

Immunization of zebrafish with VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs.

<p>Survival curves after i.p. injection of VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs at 150 μg/fish and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.9x10⁷ cfu/animal) (n = 15). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test, **, <i>p<</i>0.01.</p

Rainbow trout specific primers for qPCR.

<p>Rainbow trout specific primers for qPCR.</p

Immunization of zebrafish with VP1GFP (ClpA^-) IBs.

<p>Survival curves of zebrafish after i.p. injection of VP1GFP (ClpA^-) IBs at different doses (300, 150, 75, 50 and 25 μg/fish) and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.3x10⁷ cfu/animal) (<i>n</i> = 13). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test; **, <i>p<</i>0.01; *, <i>p<</i>0.05.</p

Analysis of gene expression in RT-HKM cells stimulated with iRFP-H6 (BL21(DE3)) IBs and LPS.

<p>Cells were incubated with 10 μg/ml of iRFP-H6 (BL21(DE3)) IBs and 10 μg/ml LPS for 12 h and the gene expression was analyzed by qPCR. Values represent means ± SD (<i>n</i> = 3). Significant differences against control were analyzed using One-way ANOVA followed by Tukey’s post test; *, <i>p<</i>0.05; **, <i>p<</i>0.01; ***, <i>p<</i>0.001.</p

Lipid quantification and dose-response curves of NF-κB induction by IBs.

<p>A) Total lipid quantification of IB samples. Differences were analyzed using the T test, *, p<0.01. The IBs produced in <i>E</i>. <i>coli</i> strains with different LPS chemotypes were assayed with HEK™-Blue hTLR4 (B) and Null2 (C) cells for relative NF-κB induction. The absorbance values despicted in Fig 2B and 2C represent the means and standard deviations from three individual experiments. The IBs displayed negligible stimulation of the parental HEK™-Blue Null2 cell line (C), which indicates that NF-κB-dependent SEAP expression was specifically induced via the hTLR4/MD-2 signalling pathway in HEK™-Blue hTLR4 cells.</p

Immunization of zebrafish with VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs.

<p>Survival curves after i.p. injection of VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs at 150 μg/fish and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.9x10⁷ cfu/animal) (n = 15). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test, **, <i>p<</i>0.01.</p

Characterization of IB particles.

<p>A) FESEM images and B) size distribution of VP1GFP (ClpA^-), VP1GFP (KPM335) and iRFP-H6 (BL21(DE3)) IBs.</p

Two-Dimensional Microscale Engineering of Protein-Based Nanoparticles for Cell Guidance

Cell responses, such as positioning, morphological changes, proliferation, and apoptosis, are the result of complex chemical, topographical, and biological stimuli. Here we show the macroscopic responses of cells when nanoscale profiles made with inclusion bodies (IBs) are used for the 2D engineering of biological interfaces at the microscale. A deep statistical data treatment of fibroblasts cultivated on supports patterned with green fluorescent protein and human basic fibroblast growth factor-derived IBs demonstrates that these cells preferentially adhere to the IB areas and align and elongate according to specific patterns. These findings prove the potential of surface patterning with functional IBs as protein-based nanomaterials for tissue engineering

Surface-Bound Gradient Deposition of Protein Nanoparticles for Cell Motility Studies

A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm²) in a fast (up to 10 mm²/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces “a-la-carte” with pNPs enabling different categories of high-throughput studies on cell motility