Evaluation of the efficacy of arctiin in promotes inactivated infectious hematopoietic necrosis virus vaccine from immune and metabolic perspectives

In this study, we prepared an inactivated IHNV vaccine with ARC as adjuvant and attempted to elucidate its immunoprotective mechanism against rainbow trout liver . This study contributes to a better understanding of the immune mechanisms of adjuvanted and inactivated vaccine combinations and lays the foundation for the development of the next generation of IHNV vaccines Overall design: For the formal experiment, rainbow trout in control group were intraperitoneally injected with PBS, and two experimental groups were intraperitoneally injected with 150 µL inactivated IHNV vaccine (vaccine group) and 150 µL APS combined inactivated IHNV vaccine (APS+vaccine group), respectively. Each experimental group had 200 fish, 100 of which were used for mortality measurements and the remaining 100 for sampling. At 14 days (D 14) after vaccination, 200 fish per group were injected intraperitoneally with 50 µl IHNV (107 TCID50 ml-1) and monitored for mortality for two weeks. The relative percentage of survival (RPS) was calculated using the formula (Sukkarun et al., 2024): 1-(vaccinated group mortality/control group mortality) × 100%. For sampling, the anesthetics and its doses were the same as in the vaccine safety tests. Liver and blood were collected from four fish from each group at D1, D3, D7, D14, D15, D21, D30, D60 and D90 to assay for immune-related gene expression and non-specific enzyme activities. Liver samples from D21 were also used for transcriptomic and metabolomic analyses. In addition, the spleens of three fish for each group were collected at D7, D14, D15, D21, D30, D60 and D90 for histopathological examination.

本研究制备了以ARC为佐剂的灭活传染性造血器官坏死病毒（Infectious Hematopoietic Necrosis Virus，IHNV）疫苗，并尝试阐明其针对虹鳟肝脏的免疫保护机制。本研究有助于深入理解佐剂化灭活疫苗联合使用的免疫机制，为新一代IHNV疫苗的研发奠定基础。
实验总体设计如下：正式实验中，对照组虹鳟经腹腔注射磷酸盐缓冲液（Phosphate Buffered Saline，PBS）；两个实验组分别经腹腔注射150 μL灭活IHNV疫苗（疫苗组）与150 μL APS联合灭活IHNV疫苗（APS+疫苗组）。每个实验组包含200尾虹鳟，其中100尾用于死亡率统计，剩余100尾用于样本采集。免疫后第14天（D14），每组200尾虹鳟经腹腔注射50 μL IHNV悬液（10^7 TCID₅₀·mL⁻¹），并连续两周监测死亡率。相对存活率（Relative Percentage Survival，RPS）按照以下公式计算（Sukkarun等，2024）：1-(免疫组死亡率/对照组死亡率) × 100%。样本采集环节所用麻醉剂及剂量与疫苗安全性试验一致。分别于免疫后第1、3、7、14、15、21、30、60及90天，从每组中采集4尾虹鳟的肝脏与血液样本，用于免疫相关基因表达及非特异性酶活性检测。此外，第21天采集的肝脏样本还用于转录组与代谢组学分析。另外，分别于第7、14、15、21、30、60及90天，从每组中采集3尾虹鳟的脾脏样本，用于组织病理学检查。