MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu, 210095, People's Republic of China.
Cell Commun Signal. 2020 Feb 11;18(1):23. doi: 10.1186/s12964-020-0514-4.
BACKGROUND: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap). METHODS: Electroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9). RESULTS: We evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P < 0.05 or P < 0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P < 0.01), intestinal intraepithelial lymphocytes (IELs) (P < 0.01), CD3 T lymphocytes (P < 0.01) and CD4 T lymphocytes (P < 0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P < 0.01), and induced the secretion of cytokines IL-1β, IL-6, interferon-γ, and β-defensin 2 (P < 0.01). CONCLUSIONS: We constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs.
背景：猪圆环病毒 2 型（PCV2）是引起断奶后多系统消耗综合征的病原体，与许多其他疾病有关。PCV2 广泛分布于大多数发达的养猪业中，是一个严重的经济负担。为了开发针对 PCV2 相关疾病的有效、安全和方便的疫苗，我们构建了一种表达 PCV2 衣壳蛋白（Cap）的重组枯草芽孢杆菌菌株（B. subtilis-Cap）。
方法：电穿孔质粒穿梭载体编码的 PCV2 Cap 序列转化枯草芽孢杆菌。流式细胞术用于评估枯草芽孢杆菌-Cap 体外诱导骨髓来源树突状细胞（BM-DC）成熟和 T 细胞增殖的能力。口服接种仔猪进行体内实验；ELISA 和 Western blot 用于评估枯草芽孢杆菌-Cap 诱导的 PCV2 特异性 IgA 和 IgG 水平，以及细胞因子的分泌和 Toll 样受体 2（TLR2）和 Toll 样受体 9（TLR9）的表达。
结果：我们使用小鼠 BM-DC 体外评估了枯草芽孢杆菌-Cap 的免疫反应，并用口服接种枯草芽孢杆菌-Cap 的新生仔猪进行了体内评估。结果表明，重组枯草芽孢杆菌-Cap 激活了 BM-DC，显著增加了共刺激分子（CD40 和 CD80）和主要组织相容性复合体 II，并诱导了同种异体 T 细胞增殖。用枯草芽孢杆菌-Cap 免疫的仔猪在消化道和呼吸道的黏膜组织中产生了更高水平的 PCV2 特异性 IgA，血清中产生了 PCV2 特异性 IgG（P<0.05 或 P<0.01）。回肠免疫活性细胞，如分泌 IgA 的细胞（P<0.01）、肠上皮内淋巴细胞（IELs）（P<0.01）、CD3 T 淋巴细胞（P<0.01）和 CD4 T 淋巴细胞（P<0.01）在枯草芽孢杆菌-Cap 免疫的仔猪中显著增加。此外，枯草芽孢杆菌-Cap 接种导致 TLR2 和 TLR9 的表达增加（P<0.01），并诱导细胞因子 IL-1β、IL-6、干扰素-γ和β-防御素 2 的分泌（P<0.01）。
结论：我们构建了一种可口服给药的原型 PCV2 疫苗，可引起比灭活 PCV2 更强的体液和细胞免疫。枯草芽孢杆菌-Cap 是一种有前途的疫苗候选物，具有安全、方便和廉价的特点。需要进一步的体内研究来确定其在猪中的全面疗效。
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