Abstract
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 ParaBAD 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 ΔasdA10 ΔPfur170::TT araC ParaBAD fur ΔPcrp68::TT araC ParaBAD 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.
Highlights
Edwardsiella piscicida is a Gram-negative, facultative anaerobic bacterium of the family Enterobacteriaceae
E. ictaluri and E. tarda DasdA mutants are capable of expressing heterologous antigens harbored on AsdA+ vectors [14, 20]. These findings suggest that an recombinant attenuated Edwardsiella vaccines (RAEVs) possessing an aspartate bsemialdehyde dehydrogenase (asdA) deletion could function in a same manner
The E. piscicida EIB202 (J118) asdA open reading frame consisted of 1104 base pairs that encoded a putative 368 amino acid residue protein with an estimated molecular mass of 40 kilodaltons
Summary
Edwardsiella piscicida is a Gram-negative, facultative anaerobic bacterium of the family Enterobacteriaceae. Conventional in-frame gene deletion can over attenuate the strain, making it more susceptible to host immune defenses and limit its ability to colonize internal tissue [4] To overcome these problems, our lab has previously demonstrated the ability to effectively attenuate E. piscicida through the use of regulated-delayed attenuation systems [5, 6]. The promoters of the fur and crp genes were replaced by a tightly-regulated araC ParaBAD cassette such that expression was arabinose dependent during growth in vitro This feature enables the strain to phenotypically mimic the virulent of wild-type E. piscicida strain at the time of vaccination and enhance the tissue colonization. The safety and control of these strains can be further enhanced by replacing the promoters of several virulence factors with externallyregulated promoters These recombinant attenuated Edwardsiella vaccines (RAEVs) are capable of stimulating both innate and adaptive immune responses in fish [5, 6]. These attributes suggest that these RAEV strains would be optimal delivery systems for heterologous recombinant proteins
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