构建新颖的减毒沙门载体递送鸭瘟DNA疫苗

Attenuated Salmonella are widely genetically modified to deliver the heterologous antigens from a variety of pathogens due to its induction of a range of immune responses including serum antibodies, mucosal IgA, and a panoply of cell-mediated immune responses at local and distal sites, therefore, recombinant attenuated Salmonella is becoming more and more attractive as vaccine delivery. Currently, RASVs are mainly targeting for synthesizing protein antigens, the specific Salmonella carriers targeting heterologous DNA vaccine remain to be constructed. The objective of this application is to design and develop innovative RASV platforms to deliver the novel DNA vaccine. These RASVs possess some specific features of in vivo suicidal lysis based on specific enzymes to degrade the Salmonella peptideglycan to release the DNA vaccine to the host cytosol, and in vivo down regulation of some important proteins and structures to decrease induction of the pyroptosis/apotosis of the host to retain macrophage survive longer, thereby expose and present the antigens to the T- and B- cell to promote the immune responses. The novel DNA vaccine plasmid will contain some annovative attributes, including balanced lethal system to maintain DNA vaccine plasmid, CpG Island as TLR9 activator, SV40 late polyA and DNA nuclear targeting sequence to enhance the DNA vaccine efficiency and promote its induction of immunity. The gB and gD gene from duck enteritis virus (or fused with IL-2 or heat labile enterotoxin B subunit, LTB) will be choosen to test the efficiency of the delivery system and DNA vaccine skeleton. Duck animal model will be used to evaluate the mucosal, cellular and humoral immunity and protection rate againtst duck enteritis virus challenge.

由于减毒沙门氏菌能诱导宿主产生黏膜、体液和细胞免疫反应及长期免疫记忆，被许多科学家用来开发成递送其它疫苗的载体，因此构建合适的减毒沙门疫苗载体成为免疫学研究热点，而目前的研究主要集中在其作为蛋白抗原递送的平台上。在本项目中，首先我们将构建一种专门用于递送DNA疫苗的沙门减毒疫苗载体，其最大特性是含有一种基于水解沙门肽聚糖特殊酶的新颖的裂解系统，及通过调控某些沙门特殊蛋白来延长沙门在巨噬细胞中的存活时间，提高递呈外源抗原，激活T, B细胞的能力等；其次将利用平衡致死系统构建全新的DNA疫苗质粒，包含CpG佐剂序列，SV40 poly(A)信号序列等；最后将选用鸭瘟病毒的gB和gD基因或与IL-2及LTB基因片段融合构建DNA疫苗，用COS-7细胞系和鸭子动物模型来验证我们的疫苗系统的有效性，检测体内体外gB和gD蛋白表达，诱导鸭子的黏膜、体液和细胞免疫反应及对鸭瘟病毒攻毒的保护效果等。

由于减毒沙门氏菌能诱导宿主产生黏膜、体液和细胞免疫反应及长期免疫记忆 被许多科学家用来开发成递送其它疫苗的载体，因此构建合适的减毒沙门疫苗载体成为免疫学研究热点，进行蛋白抗原，多糖抗原及DNA疫苗的递送。在本项目中，我们：1）成功构建了全新的DNA疫苗质粒，包含 CpG 佐剂序列，SV40 poly(A)信号序列等;并用该新型的DNA疫苗，基于鸭瘟病毒的gB 和 gD 基因构建了鸭瘟病毒的DNA疫苗;2）成功构建了大质粒载体，用于大片段DNA的克隆及减毒沙门氏菌载体，用于多糖抗原的递送；我们对大肠杆菌O1 O-抗原多糖进行了递送，证明能有效诱导产生保护性免疫反应；3）进行来源于沙门氏菌的纳米疫苗初步评估，证明来源于不同沙门氏菌的外膜囊泡能有效诱导针对同源细菌的保护，也证明来源于鼠伤寒沙门氏菌的OMV能很好的诱导交叉免疫保护；4）部分成功构建了新型的基于肽聚糖特殊酶的新颖裂解系统，同时我们也在利用其它方法来构建沙门氏菌裂解系统，用于DNA疫苗及用于提高T-细胞免疫的减毒沙门氏菌载体。