Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei Town, Qingdao 266071, China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, China.
Mol Immunol. 2021 Jun;134:141-149. doi: 10.1016/j.molimm.2021.03.012. Epub 2021 Mar 24.
The efficacy of DNA vaccine is associated closely with the expression of the antigen and the intensity of local immune responses. In our previous study, a recombinant DNA plasmid expressing the VAA protein (pVAA) of Listonella anguillarum has been proved to have a good protection against the infection of L. anguillarum. In the present study, the local immune responses eliciting by immunizing flounder with intramuscular (I.M.) injection of pVAA was investigated at the cellular and genetic level, the muscle at the injection site at 7th post vaccination day was sampled and analyzed by hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), flow cytometry (FCM), RNA sequencing (RNA-Seq)-based transcriptomics and RT-qPCR. Then variations on the specific antibodies in serum at 1st-6th post vaccination week and the relative percent survival rate (RPS) at following 14 days after challenge were measured. The H&E results showed that inflammatory cells and immune cells significantly increased at the injection site. The IHC using monoclonal antibody against T cell markers revealed that both CD4-1 and CD4-2 T lymphocytes were recruited to the injection site and FCM results showed that the proportion of CD4-1 cells in pVAA immunized group was 28.6 %, in the control group was 8.7 %, and that of CD4-2 cells in two groups was 21.2 % and 8.5 %, respectively. These results indicating that the proportion of CD4 cells in the immune group was significantly increased compared with the control group. Moreover, there were 2551 genes differently expressed in pVAA immunized group, KEGG analysis showed the genes involved in the signal transduction and immune system, and surface markers for B-cells genes, T-cells and antigen presenting cells (APCs) genes were highly upregulated, suggesting the activation of the systemic immune responses. Antibody specific anti-L. anguillarum or anti-rVAA antibodies were significantly induced at 2nd post-immunization week, that reached a peak at 4-5th week. RPS in pVAA group was 53.85±3.64 %. In conclusion, pVAA induced effective local immune responses and then the systematic response. This probably is the main contribution of pVAA to effective protection against L. anguillarum.
DNA 疫苗的功效与抗原的表达和局部免疫反应的强度密切相关。在我们之前的研究中，已经证明表达鳗弧菌 VAA 蛋白的重组 DNA 质粒（pVAA）对鳗弧菌的感染具有良好的保护作用。在本研究中，通过肌肉内（I.M.）注射 pVAA 免疫比目鱼，从细胞和遗传水平研究了引起的局部免疫反应，在第 7 天接种后，在注射部位的肌肉用苏木精-伊红（H&E）染色、免疫组织化学（IHC）、流式细胞术（FCM）、基于 RNA 测序（RNA-Seq）的转录组学和 RT-qPCR 进行分析。然后测量了第 1-6 天接种周血清中特异性抗体的变化和攻毒后 14 天的相对存活率（RPS）。H&E 结果显示，注射部位的炎症细胞和免疫细胞明显增加。使用针对 T 细胞标志物的单克隆抗体进行的 IHC 显示，CD4-1 和 CD4-2 T 淋巴细胞均募集到注射部位，FCM 结果显示，pVAA 免疫组的 CD4-1 细胞比例为 28.6%，对照组为 8.7%，两组的 CD4-2 细胞比例分别为 21.2%和 8.5%。这些结果表明，与对照组相比，免疫组中 CD4 细胞的比例显著增加。此外，pVAA 免疫组中有 2551 个基因表达不同，KEGG 分析显示，这些基因参与信号转导和免疫系统，以及 B 细胞基因、T 细胞和抗原呈递细胞（APCs）基因的表面标志物，提示全身免疫反应被激活。在第 2 次免疫后周，特异性抗鳗弧菌或抗 rVAA 抗体明显诱导，在第 4-5 周达到峰值。pVAA 组的 RPS 为 53.85±3.64%。综上所述，pVAA 诱导了有效的局部免疫反应，然后是系统反应。这可能是 pVAA 对鳗弧菌有效保护的主要贡献。
