The microalga Chlamydomonas reinhardtii as a cell factory for subunit vaccine production against the fish pathogen Infectious Spleen and Kidney Necrosis Virus

Infectious Spleen and Kidney Necrosis Virus (ISKNV) causes severe disease in commercially important fish, with potential mortality reaching 100 % in susceptible species. Vaccination is crucial to reduce losses, especially as disease risks are likely to increase with climate change. This study aimed to explore subunit vaccine production using Chlamydomonas reinhardtii as a host for expressing ISKNV Major Capsid Protein (ISKNV-MCP). To maximise subunit vaccine production, recombinant ISKNV-MCP was expressed in the chloroplast of Chlamydomonas reinhardtii. A ‘yellow-in-the-dark’ mutant strain (CC-4033) was first engineered to express the bacterial ptxD gene in the chloroplast, enabling non-sterile cultivation in phosphite-containing media. This strain was then re-transformed with a gene for the Major Capsid Protein (MCP) of ISKNV to generate a marker-free strain, CC-4033:ptxD:MCP. Western blot analysis of the soluble protein fraction confirmed the accumulation of stable recombinant ISKNV-MCP in the algal chloroplast. The antigenicity of ISKNV-MCP was demonstrated through injection of Nile Tilapia (Oreochromis niloticus) with the protein fraction, resulting in increased antibody levels against ISKNV in the fish serum. In addition, sera collected from immunized fish exhibited partial in vitro neutralization, as evidenced by a reduction in a cytopathic effect (CPE) in a Grunt fish cell line. These findings highlight C. reinhardtii as a promising host for low-cost subunit vaccine production for aquaculture, with the potential to develop it further for oral delivery of inactivated whole cell preparations.