Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei, 10617, Taiwan.
School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan.
Appl Microbiol Biotechnol. 2018 Sep;102(17):7499-7507. doi: 10.1007/s00253-018-9110-6. Epub 2018 Jun 30.
Devastating outbreaks of porcine epidemic diarrhea (PED) started in China in late 2010 and rapidly spread to North America and Asia causing severe diarrhea and high mortality in neonatal piglets, indicating that a new generation of vaccine against porcine epidemic diarrhea virus (PEDV) is urgently needed. In the present study, to mimic the native spike (S) glycoprotein, a stable cell line producing the trimeric ectodomain of S glycoprotein of the PEDV Pintung-52 (PEDV-PT) strain was successfully established by incorporating T4 bacteriophage foldon sequence of fibritin trimerization domains at the C-terminal end and replacing the signal peptide of S protein with the tissue plasminogen activator signal peptide sequence at the N terminal end. The trimeric structure, bio-reactivity to PEDV-specific antibodies, and the N-glycosylation level of the recombinant S protein were characterized. To induce systemic and mucosal immunity, conventional 5-week-old piglets were immunized with the trimeric S glycoprotein combined with the B subunit of Escherichia coli heat-labile enterotoxin (LTB) by the intramuscular (IM) route. As compared with the control group, all piglets in the S protein-LTB immunized (IM PEDV S-LTB) group generated systemic PEDV S-specific IgG and neutralizing antibody in blood but a low level of fecal PEDV-specific IgA and limited protection against challenge of PEDV-PT strain. Our results suggest that the recombinant PEDV trimeric S glycoprotein could be a potential subunit vaccine candidate against PEDV, but IM immunization with LTB as the adjuvant provided insufficient protection. The development of a vaccine regimen for inducing mucosal immunity is an important task for generating a successful subunit vaccine against PEDVs.
毁灭性的猪流行性腹泻（PED）疫情于 2010 年末在中国爆发，并迅速蔓延至北美和亚洲，导致新生仔猪发生严重腹泻和高死亡率，这表明急需新一代针对猪流行性腹泻病毒（PEDV）的疫苗。本研究通过在 C 末端整合 T4 噬菌体纤维蛋白三聚体化结构域的 T4 噬菌体折叠素序列，并在 N 末端用组织型纤溶酶原激活物信号肽序列替换 S 蛋白的信号肽，模拟天然的刺突（S）糖蛋白，成功建立了一株稳定表达 PEDV Pintung-52（PEDV-PT）株 S 糖蛋白三聚体胞外结构域的细胞系。对重组 S 蛋白的三聚体结构、对 PEDV 特异性抗体的生物反应性以及 N-糖基化水平进行了鉴定。为了诱导系统和黏膜免疫，通过肌肉内（IM）途径用三聚体 S 糖蛋白与大肠埃希菌不耐热肠毒素（LTB）的 B 亚单位联合免疫常规 5 周龄仔猪。与对照组相比，所有接受 S 蛋白-LTB 免疫（IM PEDV S-LTB）的仔猪均在血液中产生了针对 PEDV S 蛋白的全身性 IgG 和中和抗体，但粪便中 PEDV 特异性 IgA 水平较低，对 PEDV-PT 株的攻毒保护有限。我们的结果表明，重组 PEDV 三聚体 S 糖蛋白可能是一种有潜力的 PEDV 亚单位疫苗候选物，但作为佐剂的 LTB 通过 IM 免疫提供的保护作用不足。开发诱导黏膜免疫的疫苗方案是生成针对 PEDV 的成功亚单位疫苗的一项重要任务。
