Protective potential of a plasmid having different classes of CpG motifs against viral hemorrhagic septicemia virus and Miamiensis avidus (Ciliata; Scuticociliatida) infections in olive flounder (Paralichthys olivaceus)

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are potent immunostimulators in vertebrates. Based on the backbone structure and the oligonucleotides sequences, CpG-ODNs can be classified into three different classes (A, B, and C) that induce class-specific immune responses. Plasmids with stimulatory CpG motifs in their backbone also show immunostimulatory effects, and a plasmid-based delivery of CpG motifs can be an alternative approach to expensive CpG-ODNs. In this study, we constructed a plasmid (pL-CpGmix) that harbors multiple copies of CpG-ODN 2216 (class A), 1668 (class B), and 2395 (class C) motifs, and evaluated the potential of the constructed plasmid to protect of olive flounder (Paralichthys olivaceus) from viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish were administered pL-CpGmix, pL-GFP that harbored a green fluorescent protein (GFP) gene, empty vector, or phosphate buffered saline (PBS) alone. On 3 days post-administration, fish were challenged with VHSV or M. avidus. Fish administered pL-CpGmix showed the lowest cumulative mortality (60%) against a VHSV challenge, which was significant compared to that of the control groups (empty vector and PBS). In the M. avidus challenge experiment, fish in the group administered pCpG-mix showed the highest survival rate and serum scuticocidal activity, and those were statistically significant compared to those of control groups. Although fish administered pL-GFP showed lower survival rates than fish administered pL-CpGmix, the GFP-containing vector induced higher survival rates against M. avidus and VHSV infections and higher serum scuticocidal activity compared to the control groups, suggesting that GFP sequence might have an immunostimulating property. The present results showed that the plasmid harboring different classes of CpG motifs enhanced resistance of olive flounder against both M. avidus and VHSV infections.