Li Hanlin, Xing Jing, Tang Xiaoqian, Sheng Xiuzhen, Chi Heng, Zhan Wenbin
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003 China.
Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, 266071 China.
J Oceanol Limnol. 2022;40(2):786-804. doi: 10.1007/s00343-021-1092-z. Epub 2022 Jan 7.
Chemokines are cytokines that can promote the activation and migration of immune cells, and increase the recognition of antigen by antigen-presenting cells (APC). Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization. To explore the improvement of chemokines on the efficiency of OmpK vaccine, two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction (RT-PCR), indirect immunofluorescent assay (IFA), H&E staining, flow cytometry (FCM), and quantificational real-time polymerase chain reaction (qRT-PCR). pBudCE4.1 plasmid as an expression vector, bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene (p-OmpK-CCL4), or Ompk gene and CC-motif ligand 19 gene (p-OmpK-CCL19) were successfully constructed. The results showed that two bicistronic DNA vaccines expressed Ompk protein of and CCL4/CCL19 proteins of flounder both in vitro and in vivo. After immunization, a large number of leucocytes in muscle were recruited at the injection site in treatment groups. The constructed vaccines induced significant increases in CD4-1 and CD4-2 T lymphocytes, and sIgM B lymphocytes in peripheral blood, spleen, and head kidney. The percentage of T lymphocytes peaked on the 14 post-vaccination day whereas that of B lymphocytes peaked in the 6 post-vaccination week. Moreover, the expression profiles of 10 immune-related genes increased in muscles around the injection site, spleen, and head kidney. After the challenge, p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival (RPS) of 74.1% and 63.3%, respectively, higher than p-OmpK alone (40.8%). In conclusion, both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity. CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.
趋化因子是一类能够促进免疫细胞活化和迁移，并增强抗原呈递细胞（APC）对抗原识别的细胞因子。先前的研究表明，DNA疫苗免疫后可诱导牙鲆产生体液免疫和细胞免疫反应。为探究趋化因子对OmpK疫苗效率的提升作用，构建了两种双顺反子DNA候选疫苗，并通过逆转录聚合酶链反应（RT-PCR）、间接免疫荧光测定（IFA）、苏木精-伊红染色（H&E）、流式细胞术（FCM）和定量实时聚合酶链反应（qRT-PCR）研究了它们在牙鲆中诱导的免疫反应。以pBudCE4.1质粒作为表达载体，成功构建了编码OmpK基因和CC基序配体4基因的双顺反子DNA疫苗（p-OmpK-CCL4），以及编码Ompk基因和CC基序配体19基因的双顺反子DNA疫苗（p-OmpK-CCL19）。结果表明，两种双顺反子DNA疫苗在体外和体内均表达了牙鲆的Ompk蛋白以及CCL4/CCL19蛋白。免疫后，治疗组注射部位的肌肉中募集了大量白细胞。构建的疫苗诱导外周血、脾脏和头肾中的CD4-1和CD4-2 T淋巴细胞以及sIgM B淋巴细胞显著增加。T淋巴细胞百分比在疫苗接种后第14天达到峰值，而B淋巴细胞百分比在疫苗接种后第6周达到峰值。此外，注射部位周围肌肉、脾脏和头肾中10个免疫相关基因的表达谱增加。攻毒后，p-OmpK-CCL4和p-OmpK-CCL19的相对存活率（RPS）分别为74.1%和63.3%，高于单独使用p-OmpK（40.8%）。总之，CCL4和CCL19均可通过引发局部免疫反应进而诱导体液免疫和细胞免疫来增强p-OmpK的保护作用。CCL4和CCL19将成为DNA疫苗潜在的分子佐剂。
