Vacinas orais de 3a geração contra a fotobacteriose em piscicultura

This proposal aims to develop oral vaccines for Solea senegalensis against photobacteriosis, a disease caused by the pathogenic Gram-negative bacterium Photobacterium damselae subsp. piscicida. We plan to achieve this by employing vaccination strategies based on plasmid DNA (pDNA) or mRNA-IVT (in vitro transcribed), boosted with immunogenic recombinant peptides (r-peptides). Additionally, we will devise methods to enhance productivity in vaccine production processes and reduce costs. The successful creation of an effective vaccine is expected to reduce morbidity and mortality rates, thereby enhancing sole production. By eliminating the need for antibiotics, the vaccine will also help mitigate the impact of aquaculture effluents on neighboring ecosystems. The primary objectives of the project are as follows: - Develop vaccines based on DNA or RNA, boosted with immunogenic peptides, for oral administration to larvae and young juvenile stages of sole in the aquaculture environment, with the aim of reducing mortality. - Investigate the impact of nanoencapsulation strategies for DNA/RNA/immunogenic peptides on the feasibility of oral administration (via Artemia metanauplius feeds) and the outcomes of immunization. - Recover, purify and polish plasmids and RNA-IVT (in vitro transcribed) through in-house developed processes. - Engineer E. coli leaky strains to produce the designed immunogenic r-peptide and develop integrated production and purification strategies for optimal efficiency. To achieve the outlined objectives, the proposal takes four key approaches: - Rational design of pDNA vectors encoding conserved immunogenic regions of membrane proteins from Photobacterium damselae subsp. piscicida, including construction, production, and purification; - Development of in vitro assays using fish cell lines to assess the efficacy/effectiveness of candidate DNA and RNA vaccines, when complexed/encapsulated with nanoparticles (NP), for cell transfection avoiding toxicity. Optimization of genetic constructs and of delivery methodologies will then minimize the need for animal studies; - Use of RNAi technology to genetically manipulate Escherichia coli and obtain a leaky strain capable of secreting r-peptides; - Exploration of prime-boost vaccination strategies, using either pDNA or RNA and r-peptide of pharmaceutical grade, involving in vivo sole larvae and juveniles immunization in aquaculture premises , followed by infection challenges. For the project success, a task force was formed bringing together the necessary, relevant and complementary expertise in bioengineering of nucleic acids and DNA vaccines, RNAi technology, integration of bioprocesses and scale-up, provided by IST-ID-IBB and expertise in development of marine fish species in aquaculture, in compliance with international regulations for maintenance of animals in captivity, in health monitoring, infection and immune control, provided by IPMA-EPPO and IPL-MARE.