Vaccinia virus (VACV) possesses a great safety record as a smallpox vaccine and has been intensively used as an oncolytic virus against various types of cancer over the past decade. Different strategies were developed to make VACV safe and selective to cancer cells. Leading clinical candidates, such as Pexa-Vec, are attenuated through deletion of the viral thymidine kinase (TK) gene, which limits virus growth to replicate in cancer tissue. However, tumors are not the only tissues whose metabolic activity can overcome the lack of viral TK. In this study, we sought to further increase the tumor-specific replication and oncolytic potential of Copenhagen strain VACV ΔTK. We show that deletion of the anti-apoptosis viral gene F1L not only increases the safety of the Copenhagen ΔTK virus but also improves its oncolytic activity in an aggressive glioblastoma model. The additional loss of F1L does not affect VACV replication capacity, yet its ability to induce cancer cell death is significantly increased. Our results also indicate that cell death induced by the Copenhagen ΔTK/F1L mutant releases more immunogenic signals, as indicated by increased levels of IL-1β production. A cytotoxicity screen in an NCI-60 panel shows that the ΔTK/F1L virus induces faster tumor cell death in different cancer types. Most importantly, we show that, compared to the TK-deleted virus, the ΔTK/F1L virus is attenuated in human normal cells and causes fewer pox lesions in murine models. Collectively, our findings describe a new oncolytic vaccinia deletion strain that improves safety and increases tumor cell killing.

痘苗病毒（VACV）作为天花疫苗具有很高的安全性记录，并且在过去十年中已广泛用作对抗各种类型癌症的溶瘤病毒。已开发出不同的策略来使VACV对癌细胞具有安全性和选择性。领先的临床候选药物，例如Pexa-Vec，通过删除病毒胸苷激酶（TK）基因而被削弱，这限制了病毒在癌症组织中的复制。然而，肿瘤不是其代谢活性可以克服病毒性TK缺乏的唯一组织。在这项研究中，我们试图进一步提高哥本哈根病毒株VACVΔTK的肿瘤特异性复制和溶瘤潜能。我们显示抗凋亡病毒基因F1L的删除不仅增加了哥本哈根ΔTK病毒的安全性，而且还改善了侵袭性胶质母细胞瘤模型中的溶瘤活性。F1L的额外损失不会影响VACV复制能力，但是其诱导癌细胞死亡的能力显着提高。我们的结果还表明，由哥本哈根ΔTK/ F1L突变体诱导的细胞死亡释放出更多的免疫原性信号，如IL-1β产生水平的增加所表明的。NCI-60面板中的细胞毒性筛查显示，ΔTK/ F1L病毒在不同类型的癌症中诱导更快的肿瘤细胞死亡。最重要的是，我们表明，与TK缺失病毒相比，ΔTK/ F1L病毒在人正常细胞中减毒并在鼠模型中引起较少的痘病。总的来说，