Department of Microbiology; Tumor and Cell Biology; Karolinska Institutet; Stockholm, Sweden.
Department of Microbiology; Tumor and Cell Biology; Karolinska Institutet; Stockholm, Sweden; WA Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia; DI Ivanovsky Institute of Virology; Ministry of Health of the Russian Federation; Moscow, Russia.
Hum Vaccin Immunother. 2013 Oct;9(10):2228-36. doi: 10.4161/hv.25561. Epub 2013 Jul 3.
The efficacy of DNA vaccines is highly dependent on the methods used for their delivery and the choice of delivery sites/targets for gene injection, pointing at the necessity of a strict control over the gene delivery process. Here, we have investigated the effect of the injection site on gene expression and immunogenicity in BALB/c mice, using as a model a weak gene immunogen, DNA encoding firefly luciferase (Luc) delivered by superficial or deep injection with subsequent electroporation (EP). Immunization was assessed by monitoring the in vivo expression of luciferase by 2D- and 3D-bioluminescence imaging (BLI) and by the end-point immunoassays. Anti-Luc antibodies were assessed by ELISA, and T-cell response by IFN-γ and IL-2 FluoroSpot in which mouse splenocytes were stimulated with Luc or a peptide representing its immunodominant CD8+ T-cell epitope GFQSMYTFV. Monitoring of immunization by BLI identified EP parameters supporting the highest Luc gene uptake and expression. Superficial injection of Luc DNA followed by optimal EP led to a low level Luc expression in the mouse skin, and triggered a CD8+ T-cell response characterized by the peptide-specific secretion of IFN-γ and IL-2, but no specific antibodies. Intramuscular gene delivery resulted in a several-fold higher Luc expression and anti-Luc antibody, but induced low IL-2 and virtually no specific IFN-γ. Photon flux from the sites of Luc gene injection was inversely proportional to the immune response against GFQSMYTFV (p<0.05). Thus, BLI permitted to control the accuracy of gene delivery and transfection with respect to the injection site as well as the parameters of electroporation. Further, it confirmed the critical role of the site of DNA administration for the type and magnitude of the vaccine-specific immune response. This argues for the use of luminescent reporters in the preclinical gene vaccine tests to monitor both gene delivery and the immune response development in live animals.
DNA 疫苗的功效高度依赖于其传递方法和基因注射的递送部位/靶点选择，这指向了对基因传递过程进行严格控制的必要性。在这里，我们研究了注射部位对 BALB/c 小鼠中基因表达和免疫原性的影响，使用弱基因免疫原，萤火虫荧光素酶（Luc）的 DNA 编码作为模型，通过浅表或深部注射随后进行电穿孔（EP）进行基因传递。通过 2D 和 3D 生物发光成像（BLI）和终点免疫测定监测体内 Luc 表达来评估免疫。通过 ELISA 评估抗 Luc 抗体，通过 IFN-γ 和 IL-2 FluoroSpot 评估 T 细胞反应，其中用 Luc 或代表其免疫显性 CD8+T 细胞表位 GFQSMYTFV 的肽刺激小鼠脾细胞。通过 BLI 监测免疫，确定了支持最高 Luc 基因摄取和表达的 EP 参数。Luc DNA 的浅表注射随后进行最佳 EP 导致小鼠皮肤中 Luc 表达水平较低，并引发 CD8+T 细胞反应，其特征是肽特异性分泌 IFN-γ 和 IL-2，但没有特异性抗体。肌肉内基因传递导致 Luc 表达和抗 Luc 抗体增加几倍，但诱导低 IL-2 且几乎没有特异性 IFN-γ。Luc 基因注射部位的光子通量与针对 GFQSMYTFV 的抗体反应呈反比（p<0.05）。因此，BLI 允许控制基因传递和转染的准确性，具体取决于注射部位以及电穿孔的参数。此外，它证实了 DNA 给药部位对疫苗特异性免疫反应的类型和幅度的关键作用。这证明了在临床前基因疫苗测试中使用发光报告基因来监测活体动物中的基因传递和免疫反应发展的重要性。
