Oncolytic Tanapoxvirus for Melanoma Therapy

Oncolytic viruses (OVs), which preferentially infect cancer cells and induce host antitumor immune responses, have emerged as an effective melanoma therapy. Tanapoxvirus (TPV), which possesses a large genome and causes mild self-limiting disease in humans, is potentially an ideal OV candidate. The purposes of our studies are to engineer TPV into effective OVs via arming immumo-stimulatory proteins and/or manipulating the virokines, and to explore the immuno-modulatory activities of TPV. cells macrophages in a recombinant TPV mouse IL-2 was where the viral thymidine kinase (TK) gene was replaced with the mIL-2 We demonstrate that IL-2 inhibits virus replication through intracellular components and without activating the interferon-signaling pathway. The anti-tumor potential of TPVΔ66R/mIL-2 was studied in athymic (T cell deficient) nude mice carrying human melanoma xenografts. of mIL-2 into TPV remarkably increased its anti-tumor activity, resulting in a more extensive cell degeneration with a significantly increased peri-tumor accumulation of mononuclear cells present in the tumors, compared with that in those treated with wtTPV or TPVΔ66R. Each well in 96-well plate was planted with 2 x 10 4 SK-MEL-3 cells. Growth medium was replaced with serum-free (starving) medium containing purified TPV-15L protein (500 ng/ml) or NRG (500 ng/ml). Viable cells were counted on day 1, 3 and 5 following treatment. Untreated cells served as mock. Each experiment was repeated three times independently and standard deviations are shown. gene contained in plasmid was amplified in all three viruses to exclude single crossover recombinants. These experiments provide compelling evidence that 15L and/or 66R gene were deleted and that these viruses were generated as a result of double crossover recombination events during transfection and infection. in 24-well plates were infected with wtTPV, TPV ∆ 66R, TPV ∆ 15L and TPV ∆ 15L ∆ 66R at (a) 0.1 MOI and (b) 5 MOI. The virus was harvested at 48, 96 and 240 hpi and titrated on OMK monolayers. Each experiment was repeated three times independently. TPVGFP at 0.1 MOI (b, d and f) or 5 MOI (c, e and g) and incubated in the serum-free medium containing MMP-9 purified protein at 0.1, 1, 10 ug/ml. The infected cells with no MMP-9 treatment serves as the mock. Fluorescence and light images were taken at 48 hpi (a) and 96 hpi (b and c). The titer of TPVGFP at 0.1 MOI (d) and 5 MOI (e) was evaluated at 96 hpi using plaque assay. The number of viable infected cells in each treatment group was counted in hemocytometer chamber using trypan blue stain at 96 hpi. The percentage of cell survival (f and g) was determined at 96 hpi and calculated as 100 × (number of viable cells in each treatment group/viable cell number in the wells with no infection and no MMP-9 treatment). The percentage of cell survival of cells infected with 0.1 MOI TPVGFP is shown in (f), while that of cells infected at 5 MOI is shown in (g). Each experiment was repeated three times independently and standard deviations are shown. antibody treatment served as mock. The total virus titer and percentage of cell survival was assessed at 48 and 96 hours post-infection (hpi). At 0.1 MOI of infection, the incubation of infected cells (0.1 MOI) with 10 µg/mL anti-MMP-9 significantly increased replication of TPV Δ 15L at both 48 and 96 hpi (P < 0.05). Cell survival was significantly decreased at 48 hpi upon treatment with 1 ug/ml anti-MMP-9, while the 10 ug/ml anti-MMP-9 treatment significantly reduced cell survival at both 48 and 96 hpi, relative to the controls (P < 0.05). At a MOI of 5, TPV Δ 15L virus replication at 48 and 96 hpi was enhanced by approximately 2 fold when treated with 10 ug/ml of anti-MMP-9 (P < 0.05). Infected cell survival at 48 hpi was significantly decreased upon treatment with 1 and 10 ug/ml anti-MMP-9 (P < 0.05), while a significant decrease in cell survival at the 96 hpi was only observed with the 10 ug/ml anti-MMP-9 treatment (P < 0.05). The results reveal that blocking MMP-9 expression in TPV infected melanoma cells significantly increased virus replication and cellular apoptosis.