Effect of miR-155 as a molecular adjuvant of DNA vaccine against VHSV in olive flounder (Paralichthys olivaceus)

Rhabdoviral G protein-based DNA vaccines have been recognized as a useful way to protect cultured fish from rhabdoviral diseases. In Korea, viral hemorrhagic septicemia virus (VHSV) genotype IVa has been the primary culprit of high mortalities of cultured olive flounder (Paralichthys olivaceus). In this study, we inserted a miR-155-expressing cassette into the VHSV's G protein-based DNA vaccine, and analyzed the effects of miR-155 on the antiviral activity and on the vaccine efficacy in olive flounder. Olive flounder fingerlings were intramuscularly (i.m.) immunized with 10 μg/fish (1st experiment) or 1 μg/fish (2nd experiment) of DNA vaccine plasmids. However, there were no significant differences in mortalities and serum neutralization titers between fish immunized with 1 μg and 10 μg plasmids/fish, suggesting that i.m. injection with 1 μg plasmids/fish would be enough to induce effective adaptive immune responses in olive flounder fingerlings. In survival rates, as fish immunized with just G protein expressing plasmids showed no or too low mortalities, the adjuvant effect of miR-155 was not discernible. Also, in the serum neutralization activities, although G gene or G gene plus miR-155 expressing DNA vaccines induced significantly higher activities than control vaccines (PBS and vacant vector), no significant differences were found between G gene alone and G gene plus miR-155 expressing DNA vaccines. In the serum virucidal activity, fish immunized with G gene plus miR-155 expressing DNA vaccine showed significantly higher activity against hirame rhabdovirus (HIRRV) at 3 days post-immunization (d.p.i.) compared to other groups, suggesting that miR-155 produced from the vector can enhance innate immune responses in olive flounder. The significantly enhanced serum virucidal activities against VHSV especially at 28 d.p.i. in the groups immunized with G gene alone and G gene plus miR-155 expressing DNA vaccines reflect the increased antibodies against G protein, which could activate the classical complement pathway and subsequent viral inactivation. As the available information on the DNA vaccines in olive flounder is not sufficient, more diverse researches on the protective efficacy of DNA vaccines are needed to make more practical use of DNA vaccines in olive flounder farms.