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We have successfully designed and constructed a RAEV vector system with regulated-delayed attenuation  in vivo  attributes that synthesizes  Ichthyophthirius multifiliis  (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  Edwardsiella piscicida  strain carrying genomic deletions of  asdA . 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.  asdA  mutant strains have obligate growth requirements for DAP in the medium or a plasmid vector with the wild-type  asdA  gene enabling synthesis of DAP. This balanced-lethal plasmid vector-host system in  E. piscicida  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  I. multifiliis  was synthesized by RAEV strains harboring the AsdA +  plasmid pG8R8029. The third feature of this vaccine strain is a regulated-delayed attenuation  in vivo  phenotype that is based on the replacement of an arabinose-regulated  araC  P araBAD  cassette for the promoters of the  fur  and  crp  genes of  E. piscicida  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  in vivo  to prevent generation of diseases symptoms. Our vaccine strain χ16022 with the genotype Δ asdA10  ΔP fur170 ::TT  araC  P araBAD  fur  ΔP crp68 ::TT  araC  P araBAD  crp  contains the AsdA +  plasmid, pG8R8029, which encodes the IAG52B antigen. Vaccine strain χ16022(pG8R8029) is attenuated and induces systemic and mucosal IgM titer against  E. piscicida  and Ich in zebrafish. In addition, transcript levels of  tnf-α ,  il-1β ,  il-6  and  il-8  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  E. piscicida  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.

frontiers in immunology

Construction and Evaluation of Recombinant Attenuated Edwardsiella piscicida Vaccine (RAEV) Vector System Encoding Ichthyophthirius multifiliis (Ich) Antigen IAG52B