传统DNA疫苗载体与Semliki森林病毒复制子对HIV-1 Pr55^(gag)表达与体液免疫原性的比较性研究

Comparative Study of Expression and Humoral Immunogenicity of HIV-1 Group Specific Antigen Pr55^(gag) by Conventional Plasmid Vectors Versus Semliki Forest Virus-Derived Vectors

为探索Semliki森林病毒 (SFV)衍生的复制型DNA载体可否用于HIV疫苗的候选载体 ,对该载体与传统DNA疫苗载体对HIV - 1Pr5 5 gag的表达与体液免疫原性进行了系统比较研究。将野生型 (wtgag)及密码子改造(syngag)的HIV - 1ⅢBgag 基因分别克隆于SFVDNA载体及传统DNA疫苗载体 [pCDNA3 1(+) ],对其Pr5 5 gag细胞内表达水平、Pr5 5 gag病毒样颗粒释放、以及在BALB/c鼠的体液免疫原性进行了比较。在 2 93T、H12 99、C2C12和BHK细胞系中 ,SFV -wtgag可以Rev非依赖方式有效表达Pr5 5 gag ,而 pC -wtgag转染的细胞不能有效表达Pr5 5 gag,从而不能诱导小鼠产生免疫反应。虽然SFV质粒的细胞转化效率明显低于pCDNA载体 ,SFV -wtgag和SFV -syngag在细胞内Pr5 5 gag的表达量与 pC -syngag相似 ,而Pr5 5 gag病毒样颗粒的释放明显低于 pC -syngag。在肌内注射免疫的小鼠中 ,低剂量 (0 1和 1 0 μg)的SFV及 pCDNAgag表达质粒均未诱导出GAG特异性免疫反应。在高剂量 (10 ,30 ,10 0 μg)免疫组中 ,与SFVgag表达质粒相比 ,pC -syngag可诱导出较高水平的TH1型GAG特异性抗体。SFV -syngag较SFV -wtgag可诱导出高水平的体液免疫反应。结果提示 ,SFV衍生的复制子单独使用不能在小鼠诱导出优于传统DNA疫苗载体的HIV - 1GAG特异性?

To investigate if Semliki forest virus(SFV)derived DNA replicon can be used as a potential candidate vector in HIV vaccine development,HIV subtype B wild type gag as well as a codon usage optimized gag gene were cloned into two vector systems,i.e.,SFV replicon vector and conventional pCDNA3.1(+)vector.In vitro expression level and humoral immunogenicity in BALB/c mice were comparatively studied.Rev independent Pr55 gag expression directed by SFV-wtgag construct was observed in 293T,H1299,C2C12 and BHK-21 cell lines,while no Gag expression and Gag-specific immune responses were induced by pC-wtgag.Although with a lower transfection efficiency,both of SFV-wtgag and SFV-syngag constructs initiated sufficient Gag expression in all tested cells.However,Pr55 gag virus like particle releases from SFV-Gag expression construct transfected cells were not as efficient as that of pC-syngag plasmid.In intramuscular vaccinated BALB/c mice,neither SFV layered plasmid nor conventional DNA construct elicited Gag-specific immune response at low injection dosages(0.1μg and 1.0μg).In high(10μg,30μg and 100μg)DNA dosage immunized mice,pC-syngag plasmid induced higher serum Gag-specific Ig titer with predominant IgG2a antibody,comparing to SFV-wtgag and SFV-syngag constructs.More seroconversion mice and higher anti-Gag Ig were observed in SFV-syngag immunized mice than that of SFV-wtgag construct.The results suggested that the SFV layered vector has no advantage over pCDNA3.1(+)vector in inducing GAG specific humoral immunogenicity in BALB/c model when plasmid was inoculated alone,this may be due to the inefficient virus like particle release from SFV construct transfected cells.The potential immunogenicity of codon usage optimized synthetic gag gene was further proved in SFV layered vector system.