Abstract
Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.
Highlights
The development of sustainable aquaculture, a strategic sector to feed the ever-increasing human population [1], relies on disease prevention through the implementation of preventive immunostimulation and effective vaccination strategies [2]
The synthetic analog poly (I:C) mimics RNA viruses and signals through TLR3 located on endosomal membranes and through RIG-I and MDA5 located in the cytosol [11]
Endocytosis of NLc formulation by ZFL cells and trout macrophages primary cultures Since hepatocytes play a major role in physiological detoxification processes and antigen-presenting cells (APCs) are the key targets of our liposomes, we evaluated the liposome uptake in both systems using flow cytometry and confocal microscopy
Summary
The development of sustainable aquaculture, a strategic sector to feed the ever-increasing human population [1], relies on disease prevention through the implementation of preventive immunostimulation and effective vaccination strategies [2]. Safe and well-tolerated assemblies formed by a single lipid bilayer or multiple concentric bilayers that can be tailored (via selecting their composition, size, charge, etc.) to efficiently entrap a wide variety of immunostimulants and vaccines [3] This encapsulation provides the obvious potential advantages of increasing their stability and protection, enhancing their immune response and disease protection, and opening up the possibility to design more efficient immunostimulant-vaccine cocktails. We describe a novel liposomal immunostimulant cocktail (hereafter called liposomal IS-cocktail) composed of two immunostimulants: the bacterial lipopolysaccharide (LPS) and the synthetic analog of dsRNA viruses, poly (I:C) Both bacterial and viral compounds were chosen to stimulate two potent innate immune pathways (TLR3 and TLR4 pathways) virtually present in all fish species [6]. The addition of dsRNA to the nanocarrier would target anti-viral response pathways [13]
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