表面展示猪IgG1 Fc增强杆状病毒载体疫苗免疫效力的机制研究

The immune efficacy of baculovirus vectored vaccine has been restricted due to their clearance by complement system and extremely low-efficient immune cells transduction. Swine IgG1 Fc (pFc) has the potential to breakthrough these two bottlenecks, that has been demonstrated in our previous studies. However, the mechanism to enhance the effectiveness of antigen-specific immunity on the basis of pFc-displaying baculovirus vectors remains unclear. This study aims to achieve following objectives:.1. Incubate the pFc-displaying recombinant AcMNPV (rAc-pFc) with different pretreated swine serum at 37 ºC for 1 h to check the viral viability , and further investigation on the binding affinity between rAC-pFc and the related components in different complement pathways, namely C1q, IgM and C3b, thereby revealing the mechanism of complement escape..2. Detect the indicators relevant to innate immunity activation from the levels of tissue, serum and cells to explore the mechanisms of rAC-pFc’s induction in innate immunity..3. Verify the binding capacity/affinity between rAC-pFc and FcRs on immune cells by in-vitro, as well as binding sites to MoDC FcRs..4. Verify the possibility to enhance the effectiveness of antigen-specific immunity by fine-tune modulation of the pFc/FcRs binding capacity through immunization of recombinant baculoviruses displaying different pFc mutants (mpFc) and expressing Classical swine fever virus E2 protein..This study could aid for in-depth understanding of the mechanism of pFc’s enhancement for the effectiveness of baculovirus vectored vaccine, and further provide some new ideas and approaches for the development of baculovirus vectored vaccine for swine.

易被补体清除和极低免疫细胞转导效率，严重影响了杆状病毒载体疫苗免疫效力。猪IgG1 Fc（pFc）具有同时突破以上两大瓶颈的潜力，且潜力已在申请人前期研究中得到证实，但对展示pFc提高杆状病毒载体疫苗效力的机理目前仍不清楚。因此本项目拟首先通过不同血清处理展示pFc杆状病毒(rAC-pFc)，以及验证rAC-pFc与C1q、IgM和C3b结合能力，探究rAC-pFc逃逸补体机制。其次从组织、血清和细胞三个水平检测天然免疫激活相关指标，探究rAC-pFc诱导天然免疫机制。再次体外验证rAC-pFc与免疫细胞受体FcRs结合能力，及与MoDC FcRs结合位点。最后通过展示不同pFc突变体和表达猪瘟病毒E2抗原的重组病毒验证是否可通过调控pFc/FcRs结合能力增强抗原特异性免疫。以上研究有望揭示展示pFc增强杆状病毒载体疫苗效力的深层次机理，为高效猪用杆状病毒载体疫苗研究提供新的思路和途径。

抗体工程是疫苗制备生物技术中最重要的部分，表达嵌合性抗原蛋白的疫苗载体技术日益成熟，如杆状病毒载体。但是，由于杆状病毒自身对哺乳动物血清补体的高度敏感，使得免疫效果受到极大影响。本项目通过在杆状病毒上展示猪IgG Fc，显著的克服了杆状病毒载体疫苗免疫低下的问题。本研究基于MultiBac杆状病毒的多基因表达系统，成功构建了猪不同亚型IgG Fc重组I杆状病毒，筛选出效果最好的Fc展示类型。同时构建了表达FcRs（FcγR Ⅰ、FcγR ⅡB、FcγR ⅢB以及FcRn）的重组V杆状病毒，以验证组I杆状病毒的功能。研究结果从组I杆状病毒现示出对不同病毒纯化方式差异小、对猪血清补体拮抗率高、与IgM结合能力强、IEC细胞转基因表达水平高、补体拮抗剂可以提高重组杆状病毒在猪血清中存活率等结果，说明重组杆状病毒可以有效的逃避猪机体内补体的消除作用。我们还发现IgG Fc重组杆状病毒免疫猪得到的抗体滴度与商业用疫苗的免疫效果相当。以上研究结果揭示了展示猪Fc增强杆状病毒载体疫苗效力的深层次机理，为高效猪用杆状病毒载体疫苗研究提供了基础数据和理论支撑。