Tilapia farming is one of the fastest-growing and most profitable sectors in global aquaculture. However, the emergence of Tilapia lake virus (TiLV) has posed a significant threat, causing mass mortalities of up to 90% and severely impacting production worldwide. Currently, no effective vaccines exist to combat this devastating virus.
To address this challenge, we have developed an innovative mucosally delivered DNA and antigen vaccine vector system designed to protect against major infectious diseases in aquaculture. Our approach leverages a recombinant attenuated Edwardsiella vaccine (RAEV) vector system, originally developed from the invasive fish pathogen Edwardsiella piscicida. This vector system offers several advantages, including reduced induction of pyroptosis and apoptosis, regulated delayed attenuation, programmed auto-self-destruction, enhanced plasmid stability, and hyperinvasiveness. Following bath vaccination, these vector strains efficiently colonize internal lymphoid tissues in fish, facilitating the release of bacterial cell contents—such as DNA vaccines or pathogen-specific protective antigens—to elicit a robust immune response.
To develop a TiLV vaccine, we analyzed all ten TiLV gene segments encoding viral proteins, assessing their structural features, hydrophobicity, surface accessibility, and antigenicity (B and T cell epitopes). Each gene was modified with a C-terminal His-tag for protein synthesis monitoring and specific restriction sites for cloning into the pYA4545 DNA vaccine vector. Initial cloning was performed in pcDNA3.1/A, followed by subcloning of segments 2–9 into pYA4545 with an optimized Kozak sequence for enhanced expression. Protein synthesis was validated in HEK293T and EPC fish cells via Western blot analysis. These constructs were then introduced into a genetically modified E. piscicida strain, χ16048, engineered for attenuated live vaccine delivery. Stability assays confirmed the retention of recombinant plasmids within χ16048. To further assess the efficacy of this vaccine strategy, we will focus on in vivo vaccination trials to evaluate immune responses, protection efficacy, and long-term immunological memory in tilapia. Additionally, neutralization assays will determine the ability of vaccinated fish sera to inhibit TiLV infection, providing crucial insights into vaccine-induced protective immunity. These studies will be instrumental in advancing the development of a safe and effective TiLV vaccine for large-scale aquaculture applications.