Huang Guangping, Liu Xianyong, Duszynski Donal W, Tang Xiaoli, El-Ashram Saeed, Liu Zhengzhu, Suo Xun, Li Qiuyan
State Key Laboratory for Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Front Immunol. 2017 Dec 18;8:1846. doi: 10.3389/fimmu.2017.01846. eCollection 2017.
Vaccination is the most reliable measure to prevent infectious diseases in domestic animals. Development of novel vaccines demands extensive studies with new technologies, such as using novel adjuvants and immunomodulatory molecules. The co-stimulatory molecule 4-1BB provides a key signal that directs the fate of T cells during activation, and thus is important to their function in immune protection. To determine whether host immune responses to viral infection could be promoted by enhancing 4-1BB co-stimulation, in this study, we produced transgenic pig clones expressing an extra copy of the 4-1BB gene by clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9-mediated homologous recombination at the locus. The immune responses of transgenic pigs to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine were determined on day 14. We show that peripheral blood lymphocytes of transgenic pigs expressed around twice the level of 4-1BB mRNA than those of control pigs. We also found IL-2, TNF-α, and granzyme B mRNA levels as well as PRRSV-specific IFN-γ response were significantly upregulated in 4-1BB transgenic pigs, leading to more efficient cytotoxic T lymphocyte (CTL) killing, whereas the expressions of IL-4, IL-17, and Foxp3 were not affected. These results indicate that higher levels of 4-1BB expression involve in promoting Th1 differentiation and enhancing specific CTL responses to PRRSV, and provide a novel approach to increase the efficacy of current vaccines to control the infectious diseases.
疫苗接种是预防家畜传染病最可靠的措施。新型疫苗的研发需要利用新技术进行广泛研究，例如使用新型佐剂和免疫调节分子。共刺激分子4-1BB提供了一个关键信号，在激活过程中引导T细胞的命运，因此对其在免疫保护中的功能很重要。为了确定增强4-1BB共刺激是否能促进宿主对病毒感染的免疫反应，在本研究中，我们通过成簇规律间隔短回文重复序列/CRISPR相关基因9介导的同源重组，在特定基因座产生了表达额外拷贝4-1BB基因的转基因猪克隆。在第14天测定转基因猪对猪繁殖与呼吸综合征病毒（PRRSV）疫苗的免疫反应。我们发现转基因猪外周血淋巴细胞中4-1BB mRNA的表达水平约为对照猪的两倍。我们还发现，4-1BB转基因猪中IL-2、TNF-α和颗粒酶B的mRNA水平以及PRRSV特异性IFN-γ反应显著上调，导致细胞毒性T淋巴细胞（CTL）杀伤效率更高，而IL-4、IL-17和Foxp3的表达未受影响。这些结果表明，较高水平的4-1BB表达参与促进Th1分化并增强对PRRSV的特异性CTL反应，并为提高当前疫苗控制传染病的效力提供了一种新方法。
