넙치의 스쿠티카증 및 바이러스성 출혈성 패혈증 예방을 위한 DNA 백신

Abstract

DNA vaccine, one of the many vaccines in progress, has the advantage of inducing cellular and humoral immune responses simultaneously by expressing foreign antigen protein. In this study, we produced DNA vaccine against Miamiensis avidus, causative agent of scuticociliatosis, and viral hemorrhagic septicemia virus (VHSV) to induce protective effect in fingerings of olive flounder (Paralichthys olivaceus). Also, we used minicircle DNA vaccine to increase in vivo safety of DNA vaccine. First, scuticociliatosis has severely damaged to culture farms of olive flounder and there haven’t been developed effective in vivo vaccine. This study aimed to know the protective effect of DNA vaccines encoding leucine-rich repeat (LRR) protein gene of M. avidus. To enhance immunogenicity, the N-terminal of antigen gene was fused with the secretion signal of VHSV glycoprotein (vGsp), which make the expressed antigens be secreted out of cells and could enhance the chance of being recognition by antigen presenting cells. Furthermore, the C-terminal of antigen was fused with Vibrio anguillarum flagellin-A (FlaA) gene that is outer protein of bacterial flagella and serves as adhesion factor. Consequently, three DNA vaccine vectors were constructed using the pcDNA 3.1(+) plasmid as a basic vector; LRR alone (pc-L), vGsp fused to the upstream of LRR (pc-VL), and FlaA linked to the downstream of vGsp-LRR (pc-VLF). Fingerings of olive flounder were intramuscularly immunized with 10 ㎍ of plasmid and at 3 weeks post-immunization (w.p.i), fishes were intraperitoneally challenged with M. avidus. The result showed that fishes immunized with pc-VL and pc-VLF showed significantly lower mortalities than control and other immunized groups. These results suggest that the fusion of LRR with FlaA and/or with vGsp can be a way to enhance antigen immunogenicity. Second, rhabdovirus glycoprotein-based DNA vacicne is well-known antigen and demonstrated by lots of studies. We used VHSV glycoprotein (vG) as antigen and focused on the effectiveness of microRNA as molecular adjuvant. miRNA, which is small non-coding RNA, regulate gene expression by binding to the target mRNA. In this study, we used one of microRNA present in olive flounder, miR-155. We designed two DNA vaccine vectors using pFC plasmid as a basic vector; antigen only (pFC-vG) and with miR-155 (pFC-vG-miR155). Fingerings of olive flounder were intramuscularly immunized with 10 ㎍ and 1 ㎍ of plasmid and at 4 w.p.i, fishes were intramuscularly challenged with VHSV. The result showed that fishes immunized with pFC-vG and pFC-vG-miR155 showd mortality less than 20 % in all in vivo experiment. And virucidal activity was analyzed at 3, 7, 14 and 28 d.p.i with fishes injected with 10 ㎍ of plasmid. Results showed that in VHSV, the virucidal titer of both pFC-vG and pFC-vG-miR155 was higher than control group at all time. However, in HIRRV, only pFC-vG-miR155 group at 3 d.p.i showed high virucidal titer. These suggest that miR-155 is involved in non-specific innate immune response so pFC-vG-miR155 group showed protective effect against both VHSV and HIRRV at 3 d.p.i. Moreover, we confirmed that serum of all experimental group at 28 d.p.i neutralized VHSV. Thus, adaptive humoral immune response was induced by DNA vaccines produced in this study. Finally, the DNA vaccine has a lot of advantages but there are some problems against safety because of the antibiotic resistance gene. Minicircle is DNA consisted of minimal components capable of expressing a foreign gene. It doesn’t contain the replication origin and antibiotic resistance gene in the plasmid, and has the advantage of continuously expressing a foreign gene. In this study, we used minicircle DNA, which is inserted glycoprotein (G) gene and matrix protein gene (M) of VHSV as antigen to focus on safety. Matrix protein of VHSV is known to induce cell apoptosis. So we aimed to remove plasmid DNA quickly by inducing cell apoptosis. Four parental vaccine vectors were constructed to produce minicircle DNA; G or M alone (pMC-G, pMC-M), G with M in each cassette (pMC-MG), and EGFP as control (pMC-EGFP). Minicircle DNA is constructed by site-specific recombination in ZYCY10P3S2T Escherichia coli (Gmc, Mmc, MGmc and EGFPmc). Zebrafish was immunized in muscle with 1 ㎍ of each parental plasmid and minicircle DNA. Muscle of injected site was sampled at 1, 4, 6 and 8 w.p.i. and analyzed plasmid persistence by real-time PCR. Results showed that minicircle DNA was continuously expressed longer than plasmid DNA in EGFP and G group. But in M and MG group, plasmid DNA persisted longer than minicircle DNA. These suggest that the minicircle DNA including M gene was degraded by cell apoptosis faster than plasmid DNA, because numerous of minicircle DNA, which is relatively small, entered the cell more efficiently than plasmid DNA. In vivo experiment against M. avidus and VHSV showed protective effect, and induction of adaptive humoral immune response was confirmed in G-based DNA vaccine. Also, antigen immunogenicity was improved using molecular adjuvant, so the protective mechanism by these antigens and by the present DNA vaccine constructs should be investigated through further studies. In third experiment, it is necessary to confirm the defense effect through in vivo challenge experiment and suggested possibility as molecular adjuvant of matrix protein of VHSV.