Josepriya T A, Chien Kuo-Hsuan, Lin Hsin-Yun, Huang Han-Ning, Wu Chang-Jer, Song Yen-Ling
Department of Life Science, National Taiwan University, Taipei 106, Taiwan.
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan; Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Fish Shellfish Immunol. 2015 Aug;45(2):517-27. doi: 10.1016/j.fsi.2015.04.036. Epub 2015 May 7.
The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.
固定抗原（iAg）已被证明是一种针对刺激隐核虫感染的保护性免疫原。在本研究中，对从刺激隐核虫克隆的热休克蛋白70的C末端结构域（Hsp70C）的免疫刺激作用进行了测试。iAg和Hsp70C的cDNA以分泌形式独立构建，并封装在壳聚糖纳米颗粒中。在首次免疫试验中，经iAg和iAg:Hsp70C口服插管的石斑鱼幼鱼在致死性攻击后，相对存活率（RPS）分别为96%和100%。在第二次试验中，iAg组和iAg:Hsp70C组的RPS均为100%，且皮肤滋养体负荷显著降低。在免疫后49天发生更严重的致死性感染时，iAg:Hsp70C仍提供51%RPS的显著高保护率。RT-qPCR结果显示，Hsp70C可上调iAg:Hsp70C组脾脏中以下基因的表达：i）T细胞标志物：分化簇8α（CD8α）和CD4；ii）细胞因子基因：干扰素γ（IFNγ）、肿瘤坏死因子α（TNFα）和白细胞介素12 p40（IL-12/P40）；iii）抗体基因：免疫球蛋白M重链（IgMH）和IgTH；iv）主要组织相容性复合体（MHC-I和MHC-II）。此外，在免疫后5周，在皮肤黏液中检测到显著高水平的iAg特异性IgM，其能有效固定iAg和iAg:Hsp70C免疫鱼体内的活游动孢子。Hsp70C显著增加了对游动孢子具有细胞毒性的非特异性CD8(+)皮肤白细胞数量，尽管细胞毒性活性在各实验组之间没有差异。由于细胞免疫和体液免疫反应之间的这种互补协同作用，Hsp70C提高了iAg疫苗的效力并抑制了刺激隐核虫感染。鉴于隐核虫病造成的严重损失，这种寄生虫疫苗在养殖和观赏鱼中的应用值得考虑。
