INTRODUCTION

Globally, aquaculture is the fastest growing sector of the food animal production industries, providing around 47% of food fish (FAO, 2018). With the plateau and projected decrease of wild catch fisheries as well as increased global demand for high value protein, there is significant opportunity for aquaculture to meet this demand. Disease prevention and health maintenance measures have not kept pace with industry growth, leaving fish farmers vulnerable to major economic losses. Currently the primary therapeutics used are antibiotics for remediation of bacterial diseases and a limited number of vaccines that are specific to fish species and disease pathogen (USDA, 2016; FDA, 2018). The propagation and spread of antibiotic resistant bacteria are of concern both in terms of contaminating human food products, and their release into the environment that expose wild populations to these resistant pathogens (Defoirdt, Sorgeloos, & Bossier, 2011). Antibiotic resistance caused by aquaculture practices is even more concerning than with terrestrial agriculture as water is a constant and inevitable mechanism for dispersal of drug residues, pathogens, and resistance genes. Currently 14 antibiotics listed by the World Health Organization as important for human health, are also used in aquaculture (Done, Venkatesan, & Halden, 2015). This highlights the growing and immediate need for innovative solutions to ensure safe food fish production to consumers and sustainable practices that protect the environment.

One strategy for limiting disease outbreaks and reducing antibiotic use in aquaculture is the concept of boosting the natural immune system of the fish to counter pathogen infection. Cytokines are an important class of immune proteins in all animals that are secreted by immune cells in response to a stimulus and orchestrate key steps of the immune response (Male, Brostoff, Roth, & Roitt, 2011). Interleukin 22 (IL-22) is a cytokine first identified in mammals 20 years ago (Dumoutier, Van Roost, Ameye, Michaux, & Renauld, 2000; Xie et al, 2000). Since that time several homologs have been identified in other species (Figure 1.) with wide sequence divergence particularly among fish. This ~22kDa protein is thought to function as a monomer and mediates its function when binding its heterodimeric receptor (IL-10βR/IL-22R) on target cells. The role of IL-22 was described in fish by Corripio-Myar, Zou, Richmond, & Secombes (2009), where they correlated IL-22 gene expression with protection of cod and haddock subjected to bacterial challenge. Vaccinated fish that survived the bacterial challenge were shown to have a particularly high level of IL-22 gene expression in the gills. These results suggest that fish IL-22 functions as its mammalian homolog in triggering protective innate immunity and may provide a therapeutic target in controlling fish disease (Monte, Zou, Wang, Carrington, & Secombes, 2011; Secombes, 2011).

IL-22 is expressed by a select number of cells, primarily immune, in response to pathogens and is a key player in mediating the innate immune system (Zenewicz and Flavell, 2011; Dudakov, Hanash, & van den Brink, 2015; Sabat, Ouyang, & Wolk, 2014; Hernandez, Gronke, & Diefenbach, 2018). Unlike most cytokines which regulate immune cells, IL-22 receptors are primarily located on non-hematopoietic cells, most notably epithelial cells that line animal mucosa. When IL-22 binds to the canonical receptor it can signal increased production of antimicrobial peptides (AMPs), tissue repair proteins and mucous proteins, protecting the host animal from pathogens. IL-22 has recently gained notable attention as a human therapeutic agent both for treatment of infectious or inflammatory diseases (Stefanich et al, 2018; Lekkerkerker et al, 2017; Gao and Xiang, 2018; Tang et al, 2018; Lin, Krogh-Andersen, Hammarström, & Marcotte, 2017) as well as tissue preservation and wound repair (Kolumam et al, 2017). While IL-22 has been expressed successfully in a number of heterologous expression systems (e.g. HEK-293 cells, CHO cells, barley grain, E. coli ; R&D Systems), relatively low levels of fish IL-22 expression in E. coli have been reported (Monte et al, 2011; Costa et al, 2013, Siupka et al, 2014, Qi et al, 2015).

Incorporating plant biotechnology as a production platform may offer an environmentally sustainable and innovative platform for producing a functional IL-22 therapeutant. Plants provide an alternative to the more traditional recombinant protein production platforms with several significant advantages (Xu, Towler, & Weathers, 2016; Topp et al, 2016). The major benefits include improved safety profiles, as this host is incapable of harboring animal pathogens, and significant cost reduction in some cases up to 1000-fold compared to mammalian systems (Xu Dolan, Medrano, Cramer, & Weathers, 2012). This makes the plant platform attractive for veterinary biologics and is currently being used for veterinary medicine (Metzler, 2006; Pelosi, Shepherd, & Walmsley, 2012) as well as human therapeutics (Aviezer et al, 2009a; 2009b). To avoid the long time input necessary to establish stable transgenic plants for protein product expression (Schillberg, Twyman, & Fischer, 2005; Fischer, Stoger, Schillberg, Christou, & Twyman, 2004), the plant transient system can be used for production of recombinant protein within days and without stable gene integration (Krenek et al, 2015). In this way, protein targets can be quickly assessed before a large scale-up to stable production (Lacroix and Citovsky, 2013; Sheludko, 2008).

Herein we explore a sustainable and cost-sensitive production platform, plants, to produce a functional recombinant IL-22 therapeutic for promoting food catfish health and disease management. As an alternative to antibiotics or chemicals, this approach aims to trigger the fishes own immune system to produce a customized cocktail of AMPs and tissue repair proteins providing an innovative approach for addressing the issue of bacterial resistance and reduce unnatural residues deposited into the environment (Levy, 2002; Romero, Feijoo, & Navarrete, 2012).

MATERIALS AND METHODS