Department of Pediatric Hematology and Oncology, University Medicine Greifswald, Greifswald, Germany.
PLoS One. 2018 Nov 19;13(11):e0207320. doi: 10.1371/journal.pone.0207320. eCollection 2018.
Long-term survival of high-risk neuroblastoma (NB) patients still remains under 50%. Here, we report the generation, in vitro characterization and anti-tumor effectivity of a new bicistronic xenogenic DNA vaccine encoding tyrosine hydroxylase (TH) that is highly expressed in NB tumors, and the immune stimulating cytokine interleukin 15 (IL-15) that induces cytotoxic but not regulatory T cells. The DNA sequences of TH linked to ubiquitin and of IL-15 were integrated into the bicistronic expression vector pIRES. Successful production and bioactivity of the vaccine-derived IL-15- and TH protein were shown by ELISA, bioactivity assay and western blot analysis. Further, DNA vaccine-driven gene transfer to the antigen presenting cells of Peyer's patches using attenuated Salmonella typhimurium that served as oral delivery system was shown by immunofluorescence analysis. The anti-tumor effect of the generated vaccine was evaluated in a syngeneic mouse model (A/J mice, n = 12) after immunization with S. typhimurium (3× prior and 3× after tumor implantation). Importantly, TH-/IL-15-based DNA vaccination resulted in an enhanced tumor remission in 45.5% of mice compared to controls (TH (16.7%), IL-15 (0%)) and reduced spontaneous metastasis (30.0%) compared to controls (TH (63.6%), IL-15 (70.0%)). Interestingly, similar levels of tumor infiltrating CD8+ T cells were observed among all experimental groups. Finally, co-expression of IL-15 did not result in elevated regulatory T cell levels in tumor environment measured by flow cytometry. In conclusion, co-expression of the stimulatory cytokine IL-15 enhanced the NB-specific anti-tumor effectivity of a TH-directed vaccination in mice and may provide a novel immunological approach for NB patients.
高危神经母细胞瘤（NB）患者的长期存活率仍低于 50%。在这里，我们报告了一种新的双顺反子异种 DNA 疫苗的生成，该疫苗编码在 NB 肿瘤中高度表达的酪氨酸羟化酶（TH）和免疫刺激细胞因子白细胞介素 15（IL-15），后者诱导细胞毒性但不诱导调节性 T 细胞。将 TH 与泛素的 DNA 序列和 IL-15 整合到双顺反子表达载体 pIRES 中。通过 ELISA、生物活性测定和 Western blot 分析显示，疫苗衍生的 IL-15 和 TH 蛋白的成功生产和生物活性。此外，通过使用减毒鼠伤寒沙门氏菌作为口服递送系统，对派尔氏结的抗原呈递细胞进行 DNA 疫苗驱动的基因转移，通过免疫荧光分析进行了证明。在用减毒鼠伤寒沙门氏菌（肿瘤植入前 3×和后 3×）免疫的同基因小鼠模型（A/J 小鼠，n=12）中评估了所产生疫苗的抗肿瘤作用。重要的是，与对照组（TH（16.7%），IL-15（0%））相比，基于 TH-/IL-15 的 DNA 疫苗接种导致 45.5%的小鼠肿瘤消退增强，与对照组相比（TH（63.6%），IL-15（70.0%））自发转移减少（30.0%）。有趣的是，所有实验组的肿瘤浸润性 CD8+T 细胞水平相似。最后，通过流式细胞术测量，在肿瘤环境中未观察到 IL-15 的共表达导致调节性 T 细胞水平升高。总之，共表达刺激细胞因子 IL-15 增强了 TH 定向疫苗在小鼠中的 NB 特异性抗肿瘤效力，并可为 NB 患者提供新的免疫治疗方法。
