Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China.
Microb Cell Fact. 2019 Aug 21;18(1):142. doi: 10.1186/s12934-019-1195-9.
Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.
Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 10 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 10-10 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 10-10 L. lactis cells could still be recovered.
HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.
平鳍鳅科 Novirhabdovirus（HIRRV）可感染多种海水和淡水鱼类，给水产养殖业造成巨大的经济损失。疫苗的开发，特别是口服疫苗，已成为控制水生传染病的有效和便捷途径。HIRRV 糖蛋白（G）是一种免疫原性病毒蛋白，是预防该疾病的潜在候选疫苗。本研究旨在构建一种在细胞表面展示 HIRRV-G 的重组乳球菌（Lactococcus lactis）菌株，作为预防 HIRRV 的口服疫苗。
成功地在乳球菌 NZ9000 中以表面展示的形式克隆和表达了 HIRRV 的糖蛋白基因，得到 Ll:pSLC-G。通过 SDS-PAGE、western blot 和质谱分析，证实了约 81 kDa 的重组 G 蛋白（含有 LysM 锚定基序）的存在。通过免疫荧光和流式细胞术实验也验证了表面展示的 G 蛋白。此外，为了评估 Ll:pSLC-G 作为口服疫苗候选物的潜力，用含有 1.0×10 cfu/g 诱导后的 Ll:pSLC-G 的商业饮食颗粒连续喂养牙鲆 1 周。4 周后，采用相同程序进行加强免疫。与对照组相比，在第二次加强免疫后第 2 周，牙鲆肠道黏液中针对 HIRRV 的特异性 IgM 水平显著升高，第 4 周时在血清中也显著升高（p<0.05）。同时，口服免疫 Ll:pSLC-G 可提供 60.7%的 HIRRV 感染保护，在攻毒后第 3 天检测到的病毒载量明显低于对照组（p<0.01）。此外，在第 1 天连续喂养 1 周后，在牙鲆前肠、中肠和后肠的每克内容物中检测到约 10-10 个重组乳球菌细胞，这些细胞主要定位于肠道黏液层的底部；在第 21 天，仍可回收 10-10 个 L. lactis 细胞。
成功地在乳球菌细胞表面表达了 HIRRV-G 蛋白，可诱导牙鲆黏膜和体液免疫反应，并对 HIRRV 提供相当的免疫保护。这表明，表达 G 蛋白的基因工程乳球菌可作为预防 HIRRV 感染的一种有前途的口服疫苗。
