Construction and Evaluation of Recombinant Attenuated Vaccine (RAEV) Vector System Encoding (Ich) Antigen IAG52B.

We have successfully designed and constructed a RAEV vector system with regulated-delayed attenuation attributes that synthesizes (Ich) protective antigen IAG52B to enable vaccination of fish susceptible to edwardsiellosis and white spot disease. The first feature of this vaccine delivery system is an strain carrying genomic deletions of . AsdA is an enzyme necessary for the synthesis of diaminopimelic acid (DAP), which is an essential component of the peptidoglycan layer of the cell wall of Gram-negative bacteria. mutant strains have obligate growth requirements for DAP in the medium or a plasmid vector with the wild-type gene enabling synthesis of DAP. This balanced-lethal plasmid vector-host system in enables as a second feature the synthesis of recombinant antigens to induce protective immunity against fish pathogens. Recombinant protective antigen IAG52B from the fish pathogen was synthesized by RAEV strains harboring the AsdA plasmid pG8R8029. The third feature of this vaccine strain is a regulated-delayed attenuation phenotype that is based on the replacement of an arabinose-regulated P cassette for the promoters of the and genes of such that the expression of these genes is dependent on arabinose provided during growth. Thus, following colonization, the Fur and Crp proteins stop being synthesized due to the lack of arabinose and attenuation is progressively achieved to prevent generation of diseases symptoms. Our vaccine strain χ16022 with the genotype Δ ΔP::TT P ΔP::TT P contains the AsdA plasmid, pG8R8029, which encodes the IAG52B antigen. Vaccine strain χ16022(pG8R8029) is attenuated and induces systemic and mucosal IgM titer against and Ich in zebrafish. In addition, transcript levels of , , and were significantly increased in different tissues of vaccinated zebrafish compared to unimmunized fish. Zebrafish vaccinated with χ16022(pG8R8029) showed 60% survival upon intracoelomic (i.c.) challenge with a lethal dose of virulent strain J118. Our RAEV system could be used as a generalized vaccine-vector system to protect teleost fish against multiple bacterial, viral and parasitic infectious diseases.