Abstract
Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.
Highlights
The oncolytic potential of many viruses, such as the poxviruses vaccinia virus and myxoma virus, initially suggested that they could be used as cancer therapy, but the efficacy of such viruses as a single agent in vivo has been limited [1]
Murine melanoma and glioma cell lines are permissive for recombinant myxoma virus infection
For all three cell lines, infectious viral particles were formed by 12 h post infection, and maximal viral titer was typically obtained at the 48 h time point. vMyx-IL15Ra-tdTr and vMyx-tdTr showed a different growth phenotype in the glioma cell line (GL261) and produced lower viral titers (Figure 1B)
Summary
The oncolytic potential of many viruses, such as the poxviruses vaccinia virus and myxoma virus, initially suggested that they could be used as cancer therapy, but the efficacy of such viruses as a single agent in vivo has been limited [1]. Despite its lack of broad pathogenicity other than the rabbit, myxoma virus can replicate in diverse cultured cells from many species, including most human cancer cells, which are permissive for the virus [5],[6],[7]. It selectively infects tumors in human xenograft models [8],[9],[10],[11] and primary mouse tumors [12],[13],[11]. It has recently been shown that myxoma virus can discriminate cancerous human myeloid cells from normal CD34+ stem cells, which makes it a potential ex vivo purging agent for hematological malignancies [14],[15]
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