Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

摘要

In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer. prs.rt("abs_end"); Introduction Vaccinia viruses (VV) have been widely used as vaccines 1 and as gene therapy vectors 2 for decades and are thus relatively well characterized and bear a solid safety profile. 3 Vaccinia virus has also been used as an oncolytic agent in many preclinical and clinical studies 3 ; 4 ; 5 thanks to its fast replication cycle 3 and high tropism to cancer tissues. 6 In oncolytic virotherapy, the ability of the virus to activate the immune system against tumors is nowadays generally understood to be a key mechanism in full eradication of cancer and for long-term antitumor effects. 7 , 8 In fact, the best results so far in VV-based oncolytic virotherapy have been achieved by using viruses that express immunostimulatory transgenes. 4 , 5 , 9 ; 10 ; 11 Vaccinia viruses are immunosuppressive viruses by nature encoding many proteins to evade host immune responses. 12 To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to obtain a self-immuno-boosting system to activate immune responses in the tumor. DAI, also known as ZBP1 (Z-DNA-binding protein 1) or DLM-1 is a cytosolic dsDNA sensor that is a potent activator of innate immune responses. 13 Activation of DAI increases the production of type I interferons and other cytokines that attract immune cells to the site of activation (infection). 14 Lladser et al . 14 showed that a DAI-expressing plasmid can be efficiently used as a genetic cancer vaccine inducing memory and effector tumor-specific T-cells. Similarly, myeloid differentiation factor 88 (MyD88), a ubiquitous Toll-like receptor adaptor molecule, has been used as a genetic adjuvant to harness intrinsic immune-stimulating properties of a DNA vaccine 15 and a MyD88-encoding adenovirus was shown to potentiate local and systemic antitumor immunity. 16 Since vaccinia virus is a dsDNA virus whose entire replication cycle takes place in the cytosol, 12 we hypothesized that DAI has a role in innate immune sensing of VV. Moreover, we thought that expression of DAI by a rapidly replicating viral backbone would boost innate immune activation and counteract the immunosuppressiveness of the virus 12 and the tumor microenvironment, 17 resulting in long-lasting adaptive immunity towards cancer. Results DAI-armed oncolytic vaccinia virus displays unaltered oncolytic activity To improve the immunogenicity of oncolytic vaccinia virus, we generated a virus expressing DAI. Separate viruses coding for human (vvdd-tdTomato-hDAI) and mouse DAI (vvdd-tdTomato-mDAI) were constructed ( Figure 1a ). We used a Western Reserve vaccinia virus strain with two deletions, one in the thymidine kinase gene and another in the vaccinia growth factor gene. 9 , 18 , 19 These gene deletions render the virus cancer-specific since their expression is necessary for replication in normal cells but not in cancer cells where the corresponding pathways are already active. 20 , 21 Both versions of DAI-expressing viruses and the control virus vvdd-tdTomato express a tdTomato fluorophore for in vitro and in vivo imaging purposes and biosafety concerns. 9 , 22.