In the present study, we have generated several H5N2 HA recombinant baculoviruses for production of a HA subunit vaccine against the lethal H5N2 avian influenza virus (AIV). The effective display of functional HA on the cell membrane and baculoviral envelope was examined. Our results reveal that chickens immunized with the chimeric AIV HA protein fused with the baculovirus gp64 cytoplasmic domain (CTD) induced higher HI titer. To further increase the expression level of the H5N2 AIV HA protein, the HA gene of H5N2 AIV was amplified and cloned into three novel baculovirus surface display vectors BacDual DisplayEGFP-2HA, BacDual DisplayEGFP-3HA, BacDual DisplayEGFP-4HA which contains multiple expression cassettes for higher level display of HA proteins on the cell membrane and baculovirus envelope. To determine the optimum conditions for producing HA protein, various MOI, infection times, and shaker times for virus transfection were tested. Our results reveal that the conditions of an MOI of 5, 3 day post infection, and 15 min of shaker time have higher efficiency for HA protein production. Our results reveal that the baculovirus surface display vector pBacDual DisplayEGFP-4HA increases significantly the expression level of the H5N2 AIV HA protein. Chickens that received two doses of BacDual DisplayEGFP-4HA cell lysates formulated with Montanide ISA70 adjuvant elicited efficient immunogenicity and had an average HI titer of 7 log₂ at 2 weeks post-vaccination. Challenge studies revealed that vaccinated chickens with HI titers 5 log₂ were completely protected against the lethal H5N1 AIV challenge. Furthermore, HI titers could be maintained at 5 log₂ for 20 weeks for laying hens. This study suggests that the HA protein expression from the baculovirus surface display system could be a safe and efficacious subunit vaccine for chickens.

在本研究中，我们已经产生了几种H5N2 HA重组杆状病毒，用于生产针对致命H5N2禽流感病毒（AIV）的HA亚单位疫苗。检查了功能性HA在细胞膜和杆状病毒包膜上的有效展示。我们的结果表明，用融合了杆状病毒gp64细胞质域（CTD）的嵌合AIV HA蛋白免疫的鸡诱导了更高的HI效价。为了进一步提高H5N2 AIV HA蛋白的表达水平，将H5N2 AIV的HA基因扩增并克隆到三个新颖的杆状病毒表面展示载体BacDual DisplayEGFP-2HA，BacDual DisplayEGFP-3HA，BacDual DisplayEGFP-4HA中，其中包含多个表达盒HA蛋白在细胞膜和杆状病毒包膜上的更高水平展示。为了确定生产HA蛋白的最佳条件，测试了各种MOI，感染时间和用于病毒转染的振荡器时间。我们的结果表明，感染后5天，3天和振动时间为15分钟的MOI条件具有更高的HA蛋白生产效率。我们的结果表明，杆状病毒表面展示载体pBacDual DisplayEGFP-4HA显着提高了H5N2 AIV HA蛋白的表达水平。接受两次剂量的Montanide ISA70佐剂配制的BacDual DisplayEGFP-4HA细胞裂解物的鸡引起有效的免疫原性，平均HI滴度为7 log 振动时间15分钟具有更高的HA蛋白生产效率。我们的结果表明，杆状病毒表面展示载体pBacDual DisplayEGFP-4HA显着提高了H5N2 AIV HA蛋白的表达水平。接受两次剂量的Montanide ISA70佐剂配制的BacDual DisplayEGFP-4HA细胞裂解物的鸡引起有效的免疫原性，平均HI滴度为7 log 振动时间15分钟具有更高的HA蛋白生产效率。我们的结果表明，杆状病毒表面展示载体pBacDual DisplayEGFP-4HA显着提高了H5N2 AIV HA蛋白的表达水平。接受两次剂量的Montanide ISA70佐剂配制的BacDual DisplayEGFP-4HA细胞裂解物的鸡引起有效的免疫原性，平均HI滴度为7 log₂在2周后，疫苗接种。攻击研究表明，HI滴度为5 log _2的疫苗接种鸡可以完全抵抗致命的H5N1 AIV攻击。此外，对于产蛋鸡，HI滴度可以保持在5 log ₂达20周。这项研究表明，杆状病毒表面展示系统表达的HA蛋白可能是一种安全有效的鸡亚单位疫苗。