IL-7和IL-15对鸭坦布苏病毒DNA疫苗长效免疫的作用及机制研究

Duck tembusu virus (DTMUV) can induce ovarian lesions in female ducks, resulting in a severe decrease of egg production. Consequently, DTMUV infection accounts for a great economic loss in the poultry industry in China. Therefore, effective strategies to prevent DTMUV infection are required. Vaccination is one of the best methods to prevent DTMUV infection. Since development, DNA vaccines with attractive features, including safe use and easy manipulation, have been extensively studied all over the world. However, DTMUV DNA vaccine expressing solely on prM-E protein could stimulate production of effective neutralizing antibodies quickly, but the duration of neutralizing antibodies was short and the later protection was weak in our previous study. In order to enhance the immune efficacy of DTMUV prM-E DNA vaccine and promote its long-term immunity, duck-derived IL-7 (Du-IL-7) gene and Du-IL-15 gene will be introduced into DTMUV prM-E DNA vaccine by fusion PCR. Immunogenicity and protection of the vaccines will be tested after duck immunization to analyze the adjuvant activity of Du-IL-7 and Du-IL-15 to promote long-term immunity of DTMUV DNA vaccine. The DNA vaccines will be orally immunized ducks. The responses of effector T cells and B cells, and memory immune response including the survival, proliferation and activation of memory cells will be measured by Flow cytometry, TUNEL, ELISPOT and so on. These results will be used to elucidate the mechanism of Du-IL-7 and Du-IL-15 in promoting long-term protection period of DTMUV DNA vaccine.This study not only elucidates the adjuvant activity of IL-7 and IL-15, but also provides a new way to develop a DTMUV DNA vaccine with long-term protection in one dose.

鸭坦布苏病毒（DTMUV）感染导致鸭产蛋率严重下降，给养殖业带来经济损失，疫苗接种是预防DTMUV感染的主要措施。我们前期的研究表明只依靠囊膜蛋白的DTMUV DNA疫苗虽能快速刺激机体产生有效的中和抗体，但中和抗体持续时间短。为了延长DNA疫苗的免疫保护期，本项目将鸭源IL-7（Du-IL-7）和Du-IL-15作为生物佐剂引入DNA疫苗，免疫鸭后检测疫苗免疫原性及疫苗免疫保护力，明确生物佐剂Du-IL-7和Du-IL-15促进DTMUV DNA疫苗长效免疫的作用。采用流式细胞术、TUNEL法、ELISPOT法等解析佐剂对效应细胞应答、记忆细胞存活及记忆细胞增殖、活化的影响，阐释Du-IL-7和Du-IL-15延长DTMUV DNA疫苗免疫保护期的机制。本研究不仅阐明了鸭源IL-7和鸭源IL-15的佐剂效应，还为开发单剂量且具有长期保护作用的鸭坦布苏病毒DNA疫苗提供了一种新思路。

鸭坦布苏病毒（DTMUV）感染导致鸭产蛋率严重下降，给养殖业带来经济损失，疫苗接种是预防DTMUV感染的主要措施。目前，虽已有商品化的灭活疫苗和弱毒疫苗，但这些疫苗需要多次注射，造成人力和物力的消耗。长效疫苗的使用可以减少接种次数，节约成本、减轻应激。为了延长疫苗的免疫保护期，本项目将鸭源IL-7（duIL-7）和duIL-15作为生物佐剂引入DTMUV疫苗，免疫鸭后检测疫苗免疫原性及疫苗免疫保护力。初步确定duIL-7和duIL-15具有佐剂活性，且生物佐剂duIL-7较duIL-15更能提高疫苗的免疫保护力。进一步分析duIL-7的生物学功能，发现duIL-7同鼠源IL-7、人源IL-7和鸡源IL-7一样，可促进淋巴细胞增殖，且这种功能呈剂量依赖性。鉴于此，我们对duIL-7进行大量表达，用含有或不含有duIL-7的DTMUV灭活疫苗两次肌肉注射鸭。结果表明，duIL-7能够促进快速的抗体反应，增强DTMUV特异性IgG和中和抗体的产生。滤泡辅助性T细胞（Tfh）在协助长期体液免疫中起着关键作用。研究发现，duIL-7在首次接种后3周可上调duIl-6和duIl-21基因的表达，而这两个基因是与Tfh细胞分化相关的细胞因子。这可能是duIL-7能够延长对DTMUV灭活疫苗的体液免疫反应的原因。接下来，我们测试了duIL-7在简化DTMUV灭活疫苗免疫程序方面的作用。将含有或不含有duIL-7的DTMUV灭活疫苗免疫鸭一次，发现含有duIL-7的DTMUV灭活疫苗组，鸭的中和抗体滴度明显高于DTMUV灭活疫苗组鸭（P＜0.05）。此外，免疫含有duIL-7的DTMUV灭活疫苗组鸭在整个实验期间保持高水平的中和抗体滴度。病毒滴度与中和抗体水平相反。总的来说，duIL-7具有延长DTMUV灭活疫苗体液免疫反应的能力。这项研究为禽疫苗的开发提供了一种潜在的新佐剂。