Jansons Juris, Avdoshina Daria, Dudorova Alesja, Rubio Elena Royo, Sokolovska Liba, Perminov Dmitry, Lindenberga Ilze, Nicolai Hannes, Gebrila Svetlana, Chowdhury Sona, Skrastina Dace, Nazarovs Jurijs, Palefsky Joel M, Isaguliants Maria
Res Sq. 2025 May 21:rs.3.rs-6328033. doi: 10.21203/rs.3.rs-6328033/v1.
Background Development of immunotherapies and vaccines to treat HPV16-associated cancer requires reliable/effective small animal models. We developed such a model based on the murine mammary gland adenocarcinoma cells engineered to express HPV16 oncoproteins E6 and E7, and used it to assess the protective and therapeutic potential of E6/E7-based DNA-immunogens. Methods 4T1luc2 subclones with single genomic inserts of HPV16 E6/E7 DNA (B2, H6) were obtained by lentiviral transduction. DNA-immunogens were designed encoding expression-optimized consensus HPV16 E6 and E7 mutated to disrupt p53- and Rb-binding, both controlled by the human elongation factor 1a promoter. In prophylactic settings, BALB/c mice received E6, E7, E6/E7 DNA or vector, followed by challenge with B2 or H6 cells, and in therapeutic settings, were challenged with B2 or H6 cells, and DNA-immunized with E6 or vector. In reference series, C57bl/6 mice were challenged with TC1/luc2 cells and DNA-immunized with E6, E7, or E6/E7, or vector DNA. Tumor growth was monitored morphometrically and by bioluminescence imaging (BLI); metastatic activity, by organ BLI, PCR and histology, and cytokine production by T-cells of immunized mice, by flow cytometry. Results E6/E7-expressing 4T1luc2 subclones B2 and H6 longitudinally expressed mRNA of E7 and of E6I, E6II, full length E6 (E6FL) isoforms. The levels of expression of E6 and E7 mRNA significantly increased with time. In naïve mice, B2 and H6 generated solid tumors with lung metastases. B2 and H6 cells were used to assess the efficacy of prophylactic DNA-immunization with E6 and E7. In immunogenicity tests, E6 DNA recipients developed Th1-type T-cell response, their unstimulated T-cells produced IFN-g and IL-2. E7 DNA was nonimmunogenic, while unstimulated T-cells produced TNF-α. In prophylactic settings, DNA-immunization with E6 and E7 suppressed formation of B2/H6 tumors. In therapeutic settings, DNA-immunization with E6 (not E7) restricted growth of TC-1/luc2 tumors, but had no effect on tumorigenic or metastatic activity of E6/E7-expressing 4T1luc2 cells. In both TC-1/luc2 and 4T1luc2E6/E7-models, E7 DNA recipients developed systemic inflammation with liver injury. Conclusions 4T1luc2 cells stably expressing HPV16 E6/E7 present an attractive alternative to TC-1 model allowing stringent assessment of both protective and therapeutic potential of E6/E7-based vaccines in BALB/c mice.
背景 开发用于治疗HPV16相关癌症的免疫疗法和疫苗需要可靠/有效的小动物模型。我们基于经过基因工程改造以表达HPV16癌蛋白E6和E7的小鼠乳腺腺癌细胞开发了这样一种模型，并将其用于评估基于E6/E7的DNA免疫原的保护和治疗潜力。方法 通过慢病毒转导获得具有HPV16 E6/E7 DNA单基因组插入片段的4T1luc2亚克隆（B2、H6）。设计DNA免疫原，编码经表达优化的HPV16 E6和E7共识序列，其发生突变以破坏与p53和Rb的结合，二者均由人延伸因子1a启动子控制。在预防性实验中，BALB/c小鼠接受E6、E7、E6/E7 DNA或载体，随后用B2或H6细胞进行攻击；在治疗性实验中，用B2或H6细胞进行攻击，并用E6或载体进行DNA免疫。在对照实验系列中，C57bl/6小鼠用TC1/luc2细胞进行攻击，并用E6、E7、E6/E7或载体DNA进行DNA免疫。通过形态计量学和生物发光成像（BLI）监测肿瘤生长；通过器官BLI、PCR和组织学监测转移活性；通过流式细胞术监测免疫小鼠T细胞的细胞因子产生。结果 表达E6/E7的4T1luc2亚克隆B2和H6纵向表达E7以及E6I、E6II、全长E6（E6FL）异构体的mRNA。E6和E7 mRNA的表达水平随时间显著增加。在未免疫的小鼠中，B2和H6产生伴有肺转移的实体瘤。B2和H6细胞用于评估用E6和E7进行预防性DNA免疫的效果。在免疫原性测试中，接受E6 DNA的小鼠产生Th1型T细胞反应，其未刺激的T细胞产生IFN-γ和IL-2。E7 DNA无免疫原性，而未刺激的T细胞产生TNF-α。在预防性实验中，用E6和E7进行DNA免疫可抑制B2/H6肿瘤的形成。在治疗性实验中，用E（而非E7）进行DNA免疫可限制TC-1/luc2肿瘤的生长，但对表达E6/E7的4T1luc2细胞的致瘤性或转移活性无影响。在TC-1/luc2和4T1luc2E6/E7模型中，接受E7 DNA的小鼠均出现伴有肝损伤的全身炎症。结论 稳定表达HPV16 E6/E7的4T1luc2细胞是TC-1模型的一个有吸引力的替代方案，可在BALB/c小鼠中严格评估基于E6/E7的疫苗的保护和治疗潜力。
