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• W2771271245 abstract "Despite the effectiveness of classic treatments and available diagnostic tools, cancer continues to be a leading world health problem, with devastating cancer-related death rates. Advances in oncolytic virotherapy have shown promise as potentially effective treatment options in the fight against cancer. The poxviruses have many features that make them an attractive platform for the development of oncolytic vectors, with some candidates currently in clinical trials. Here, we report the design and generation of a new oncolytic vector based on the vaccinia virus Western Reserve (WR) strain. We show that the WR-Δ4 virus, with the combined deletion of four specific viral genes that act on metabolic, proliferation, and signaling pathways (A48R, B18R, C11R, and J2R), has effective anti-tumor capabilities in vivo. In WR-Δ4-infected mice, we observed strong viral attenuation, reduced virus dissemination, and efficient tumor cell growth control in the B16F10 syngeneic melanoma model, with enhanced neutrophil migration and activation of tumor antigen-specific immune responses. This approach provides an alternative strategy toward ongoing efforts to develop an optimal oncolytic poxvirus vector. Despite the effectiveness of classic treatments and available diagnostic tools, cancer continues to be a leading world health problem, with devastating cancer-related death rates. Advances in oncolytic virotherapy have shown promise as potentially effective treatment options in the fight against cancer. The poxviruses have many features that make them an attractive platform for the development of oncolytic vectors, with some candidates currently in clinical trials. Here, we report the design and generation of a new oncolytic vector based on the vaccinia virus Western Reserve (WR) strain. We show that the WR-Δ4 virus, with the combined deletion of four specific viral genes that act on metabolic, proliferation, and signaling pathways (A48R, B18R, C11R, and J2R), has effective anti-tumor capabilities in vivo. In WR-Δ4-infected mice, we observed strong viral attenuation, reduced virus dissemination, and efficient tumor cell growth control in the B16F10 syngeneic melanoma model, with enhanced neutrophil migration and activation of tumor antigen-specific immune responses. This approach provides an alternative strategy toward ongoing efforts to develop an optimal oncolytic poxvirus vector. Cancer is one of the main causes of mortality worldwide, with an estimated 8 million related deaths per year (based on data from the GLOBOCAN 2012 project). It thus continues to be necessary to develop more effective treatments to fight the group of diseases that cancer comprises. Oncolytic virotherapy is a promising experimental approach for cancer treatment; it has recently become a real option for oncologists following USA Food and Drug Administration (FDA) approval of IMLYGIC (Amgen), an oncolytic vector based on herpes simplex virus, for treatment of melanoma.1Greig S.L. Talimogene laherparepvec: first global approval.Drugs. 2016; 76: 147-154Crossref PubMed Scopus (81) Google Scholar Oncolytic virotherapy uses replicative viruses, naturally occurring or genetically modified, that selectively infect and lyse tumor cells while leaving healthy tissues unharmed.2Thorne S.H. Hermiston T. Kirn D. Oncolytic virotherapy: approaches to tumor targeting and enhancing antitumor effects.Semin. Oncol. 2005; 32: 537-548Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar An important feature of these viral vectors is their potential for triggering an innate, followed by an adaptive, anti-tumor immune response.3Kaufman H.L. Kohlhapp F.J. Zloza A. Oncolytic viruses: a new class of immunotherapy drugs.Nat. Rev. Drug Discov. 2015; 14: 642-662Crossref PubMed Scopus (762) Google Scholar, 4Keller B.A. Bell J.C. Oncolytic viruses—immunotherapeutics on the rise.J. Mol. Med. (Berl.). 2016; 94: 979-991Crossref PubMed Scopus (42) Google Scholar Among the poxviruses, vaccinia virus (VACV) is one of the platforms boasting several features that meet requirements for oncolytic virotherapy: (1) it has a rapid replication cycle and lyses infected cells,5Wein L.M. Wu J.T. Kirn D.H. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery.Cancer Res. 2003; 63: 1317-1324PubMed Google Scholar (2) has broad cell tropism,6Dimitrov D.S. Virus entry: molecular mechanisms and biomedical applications.Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (384) Google Scholar, 7McFadden G. Poxvirus tropism.Nat. Rev. Microbiol. 2005; 3: 201-213Crossref PubMed Scopus (343) Google Scholar (3) has distinct types of viral particles used in different transmission pathways (mature virus [MV] and extracellular virus [EV]),8Kirn D.H. Wang Y. Liang W. Contag C.H. Thorne S.H. Enhancing poxvirus oncolytic effects through increased spread and immune evasion.Cancer Res. 2008; 68: 2071-2075Crossref PubMed Scopus (70) Google Scholar (4) has the ability to stably incorporate large transgenes,9Smith G.L. Moss B. Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA.Gene. 1983; 25: 21-28Crossref PubMed Scopus (212) Google Scholar (5) has a cytoplasmic viral cycle with no DNA integration,10Moss B. Poxviridae: The Viruses and Their Replication. Lippincott-Raven, 2007: 2637-2671Google Scholar and (6) activates a strong CD8+ T cell response11Miller J.D. van der Most R.G. Akondy R.S. Glidewell J.T. Albott S. Masopust D. Murali-Krishna K. Mahar P.L. Edupuganti S. Lalor S. et al.Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines.Immunity. 2008; 28: 710-722Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar and induces the production of neutralizing antibodies.12Pütz M.M. Midgley C.M. Law M. Smith G.L. Quantification of antibody responses against multiple antigens of the two infectious forms of Vaccinia virus provides a benchmark for smallpox vaccination.Nat. Med. 2006; 12: 1310-1315Crossref PubMed Scopus (94) Google Scholar With the release of tumor antigens by cell lysis, these features generate protective antitumor immunity.13Kirn D.H. Thorne S.H. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer.Nat. Rev. Cancer. 2009; 9: 64-71Crossref PubMed Scopus (315) Google Scholar A number of oncolytic poxviruses have been developed and are under study in preclinical and clinical trials;14Chan W.M. McFadden G. Oncolytic poxviruses.Annu. Rev. Virol. 2014; 1: 119-141Crossref PubMed Scopus (71) Google Scholar the most advanced candidates include JX-594 (Wyeth strain)4Keller B.A. Bell J.C. Oncolytic viruses—immunotherapeutics on the rise.J. Mol. Med. (Berl.). 2016; 94: 979-991Crossref PubMed Scopus (42) Google Scholar and GL-ONC1 (Liverpool strain).15Mell L.K. Brumund K.T. Daniels G.A. Advani S.J. Zakeri K. Wright M.E. Onyeama S.J. Weisman R.A. Sanghvi P.R. Martin P.J. Szalay A.A. Phase I trial of intravenous oncolytic Vaccinia virus (GL-ONC1) with cisplatin and radiotherapy in patients with locoregionally advanced head and neck carcinoma.Clin. Cancer Res. 2017; 23: 5696-5702Crossref PubMed Scopus (75) Google Scholar Western Reserve (WR) is a replicative VACV strain with high lytic capacity and natural selectivity for tumor cells.16Thorne S.H. Hwang T.H. O’Gorman W.E. Bartlett D.L. Sei S. Kanji F. Brown C. Werier J. Cho J.H. Lee D.E. et al.Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus, JX-963.J. Clin. Invest. 2007; 117: 3350-3358Crossref PubMed Scopus (163) Google Scholar WR can be modified to increase its safety in non-tumorigenic cells by deleting non-essential genes important for somatic infection. While on the one hand the effects derived from these gene deletions restrict mutant virus replication, on the other, the “hallmarks of cancer” within the tumor microenvironment (e.g., sustained proliferation, deregulated metabolism, and immune suppression)17Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (42679) Google Scholar compensate for these effects, which leads to viral replication. Here, we studied the effects of a combination of four gene deletions on safety, tumor growth control, and immune response activation. The genes selected were A48R, B18R, C11R, and J2R. A48R codes for thymidylate kinase, an enzyme that participates in nucleotide metabolism; its deletion attenuates WR virus.18Hughes S.J. Johnston L.H. de Carlos A. Smith G.L. Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae.J. Biol. Chem. 1991; 266: 20103-20109PubMed Google Scholar The B18R gene product is a soluble type I interferon (IFN) receptor, a glycoprotein that interferes with anti-viral responses; mutants that lack B18R show reduced virulence in murine model.19Symons J.A. Alcamí A. Smith G.L. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity.Cell. 1995; 81: 551-560Abstract Full Text PDF PubMed Scopus (422) Google Scholar The C11R gene product is known as vaccinia growth factor (VGF), a protein involved in cell proliferation that is homologous to cell epithelial growth factor (EGF).20de Magalhães J.C. Andrade A.A. Silva P.N. Sousa L.P. Ropert C. Ferreira P.C. Kroon E.G. Gazzinelli R.T. Bonjardim C.A. A mitogenic signal triggered at an early stage of vaccinia virus infection: implication of MEK/ERK and protein kinase A in virus multiplication.J. Biol. Chem. 2001; 276: 38353-38360Crossref PubMed Scopus (88) Google Scholar Finally, J2R codes for thymidine kinase, another enzyme for nucleotide metabolism, whose deletion attenuates virulence and induces selective in vivo replication in tumor cells.21Buller R.M. Smith G.L. Cremer K. Notkins A.L. Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype.Nature. 1985; 317: 813-815Crossref PubMed Scopus (320) Google Scholar, 22Puhlmann M. Brown C.K. Gnant M. Huang J. Libutti S.K. Alexander H.R. Bartlett D.L. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.Cancer Gene Ther. 2000; 7: 66-73Crossref PubMed Scopus (142) Google Scholar The resulting mutant virus with four deletions, WR-Δ4, in which the luciferase gene replaced the J2R gene, had a more attenuated profile than the parental WR and the triple-deletion mutant (WR-Δ3). In the B16F10 syngeneic melanoma mouse model, the treatment with the WR-Δ4 virus led to a marked reduction in tumor growth and increased neutrophil infiltration. These results suggest that the viral vector WR-Δ4 is a potential candidate for tumor cell virotherapy and adds value to our understanding of the mechanisms of action of VACV oncolytic vectors. To generate the oncolytic VACV vectors used in this study, we first sequentially deleted selected viral genes. Correct deletion of the targeted VACV genes was confirmed by PCR using a double set of primers to cover both the internal and flanking regions (Figure 1A). Three genes, A48R, B19R, and C11R, were deleted from the WR genome, as indicated by absence of an amplification product for internal regions and by reduction in the PCR fragment obtained when using flanking primers in the mutant virus WR-ΔA48R-ΔB19R-ΔC11R (WR-Δ3) compared with the wild-type virus (Figures 1B–1D). Because the C11R gene is repeated at both ends of the VACV genome, we also analyzed WR-Δ3-infected cells by retro-transcriptase PCR (from total RNA samples), to ensure that both gene copies were completely deleted (Figure 1D, right) and lack of contamination by genomic DNA (data not shown). All deletion mutant viruses were tested by DNA sequencing to determine the correct deletions and absence of mutations in the flanking regions of the deleted genes. After generation of the WR-Δ3 virus, we eliminated the J2R (TK) gene and replaced it with the luciferase reporter gene. We evaluated correct J2R deletion and reporter gene insertion into the WR-Δ4 genome using PCR, which showed an increase in amplification product size compared with WR wild-type virus (WR WT) (Figure 1E, left). A luciferase assay confirmed correct luciferase expression in WR-Δ4-infected cells (Figure 1E, right). We thus generated recombinant WR-Δ3 virus with three specified gene deletions and WR-Δ4 virus with an additional deletion of the J2R gene that is replaced by fully active luciferase. Because cell-to-cell viral spread and degree of infection are major factors in the development of oncolytic poxvirus vectors, we analyzed plaque size phenotype, virus production, and ability to infect spheroids to test whether the deleted genes affected viral growth. In infected African green monkey BSC-40 cells, plaque size and mean area were similar in WR wild-type (WT), WR-Δ3-, and WR-Δ4-infected monolayers, which implied that plaque formation was unaffected by the deletions (Figure 2A). We evaluated the replicative capacity of the deletion mutant viruses by viral growth kinetics in several cell lines: primary (CEF; chick), immortalized (BSC-40), and murine tumor cells (B16F10 and TRAMP-C1). Cultured cells were infected and collected at various times post-infection (0, 8, 24, 32, 48, and 72 hr), viral plaques were stained with crystal violet, and virus yields were determined by titration in BSC-40 cells. Viral growth kinetics profiles for the WR-Δ3 and WR-Δ4 mutants and the parental WR WT were similar for the different cell types (Figures 2B–2E). B16F10 cells nonetheless showed increased lysis by all the WR viruses (Figure 2D), in contrast with the other cell lines. Infection of TRAMP-C1 cell spheroids with WR-Δ4/GFP (with the same deletions as in WR-Δ4 and expressing GFP) showed wide infection spread (as indicated by GFP expression) and a scattered phenotype in histological sections compared with mock-infected spheroids; these effects are attributed to cytolytic infection of the most exposed regions (Figure 2F). WR-Δ3 and WR-Δ4 virus replication capacities were unaffected in cell lines of various origins, including tumor cells and tumor-like spheroids. Vector safety is a crucial parameter for the therapeutic use of replication-competent viruses. To define the virulence of the oncolytic candidate vectors, we administered WR-Δ3 or WR-Δ4 intranasally (i.n.) to C56BL/6 mice (5 × 106 or 5 × 107 plaque-forming units [PFU]/mouse); control mice received 5 × 106 PFU/mouse of WR WT or WR-Luc (a single J2R deletion replaced with luciferase). Mice were monitored daily throughout the experiment for weight loss and signs of illness (loss of mobility, troubled breathing, hunched posture, absence of grooming, or inflammation of the eye membrane). The 5 × 106 PFU/mouse virus dose was lethal for control groups infected with WR WT and WR-Luc, with severe loss of body weight within 7 days post-inoculation (Figure 3A), accompanied by associated signs of illness in infected animals (Figure 3B, top). Mice inoculated with WR-Δ3 showed a slight body weight reduction (up to 14%), with a peak at day 9 post-infection that coincided with the appearance of signs of illness (Figures 3A and 3B, top); thereafter, 80% of the mice recovered body weight and healed completely from the infection (given the absence of signs of illness at the end of the experiment). In contrast, all mice infected with WR-Δ4 showed stable weight and complete absence of symptoms attributable to the viral infection (Figures 3A and 3B, top). All mice inoculated with low WR WT and WR-Luc doses required sacrifice, whereas those that received WR-Δ3 and WR-Δ4 showed 80% and 100% survival, respectively (Figure 3B, bottom). In mice infected with a high WR-Δ3 dose (5 × 107 PFU/mouse), we observed a decrease in body weight with time. At day 8, 50% of the mice had to be sacrificed, whereas the rest of the group recovered completely (Figures 3C and 3D, top). None of the mice infected with 5 × 107 PFU of WR-Δ4 showed weight loss or signs of disease related to the viral infection (Figures 3C and 3D, top). The lethal dose 50 (LD50) for WR-Δ3 was ∼5 × 107 PFU, whereas mice inoculated with the same dose of WR-Δ4 were unaffected (100% survival; Figure 3D, bottom). The four viral genes deleted in WR-Δ4 thus conferred 100% survival in infected mice in conditions in which all mice infected with 1 log lower dose of WR WT did not survive. To study the tissue distribution of the deletion mutant viruses after systemic delivery, we inoculated C57BL/6 mice intraperitoneally (i.p.) with 2 × 107 PFU/mouse of WR WT, WR-Δ3, or WR-Δ4 and analyzed viral load at 24, 72, and 120 hr post-infection (hpi) in mouse ovary, peritoneal exudate cells (PECs), and brain tissue. In ovaries, virus titers were similar in WR WT- and WR-Δ3-infected mice, whereas ovaries from WR-Δ4-infected mice showed a significantly lower (p < 0.05) viral titer than that in the other two groups: WR WT and WR-Δ3 (Figure 4). For PEC titers, all three infected mouse groups had comparable virus levels at 24 and 72 hpi. At 120 hpi, we were unable to detect virus in PEC of WR-Δ4-infected mice (Figure 4). In brain, we observed very low WR WT titers, and there was no detectable virus in the WR-Δ3 or WR-Δ4 groups after 24 hr of infection (Figure 4). The combined deletions in WR-Δ4 thus led to a virus whose tissue distribution profile was similar to that of WR WT, whereas WR-Δ4 titers were lower in ovaries and PECs. Because in the context of a poxvirus infection migration of neutrophils to the site of virus infection can affect CD8+ T cell activation,23Di Pilato M. Mejías-Pérez E. Zonca M. Perdiguero B. Gómez C.E. Trakala M. Nieto J. Nájera J.L. Sorzano C.O. Combadière C. et al.NFκB activation by modified vaccinia virus as a novel strategy to enhance neutrophil migration and HIV-specific T-cell responses.Proc. Natl. Acad. Sci. USA. 2015; 112: E1333-E1342Crossref PubMed Scopus (25) Google Scholar we next characterized the migration profile of murine innate immune cells elicited by WR-Δ3 and WR-Δ4 viruses; we injected WR WT, WR-Δ3, WR-Δ4 (1 × 107 PFU/mouse, i.p.), or PBS, and evaluated in the peritoneal cavity absolute numbers of cell populations 3, 6, and 12 hpi. This virus dose was lower than for intratumoral to limit the cytopathic effects of the virus in cells of the peritoneal cavity and maintain their integrity. Of the populations analyzed, we found a significant early (3 hpi) increase in the number of neutrophils recruited in WR-Δ4-infected mice, whereas levels were similar in WR WT and WR-Δ3 groups (Figure 5A). This enhanced neutrophil number was maintained at 6 hpi, when WR-Δ3 and WR-Δ4 reached similar levels, higher than those in WR WT (Figure 5A). At 12 hpi, the overall number of neutrophils recruited to the infection site increased ∼10-fold compared with the numbers at 3 hpi, with no significant differences between all virus groups (Figure 5A). Of other innate immune cells analyzed (NK, NKT, CD8, and CD4 T cells), all groups showed a comparable profile at all three time points, with recruitment levels of these cells resembling that of PBS-control mice at 3 hpi, with higher levels at 6 and 12 hpi (Figures 5B–5E). These data indicate that neutrophil recruitment to the infection site was enhanced early in infection by WR-Δ4, an effect not observed for any virus group for other immune system cells (natural killer [NK], NK T [NKT], CD8, and CD4 T cells). Immune system cells such as NKs or CD8 and CD4 T lymphocytes, which have anti-tumor-associated roles, thus remained unaffected.24Vesely M.D. Kershaw M.H. Schreiber R.D. Smyth M.J. Natural innate and adaptive immunity to cancer.Annu. Rev. Immunol. 2011; 29: 235-271Crossref PubMed Scopus (1427) Google Scholar, 25Gajewski T.F. Schreiber H. Fu Y.X. Innate and adaptive immune cells in the tumor microenvironment.Nat. Immunol. 2013; 14: 1014-1022Crossref PubMed Scopus (2372) Google Scholar To evaluate the anti-tumor effectiveness of the mutant viruses, we injected melanoma B16F10 cells intradermally (i.d.) into C57BL/6 mice, followed 7 days later by intratumor (i.t.) inoculation of 1 × 108 PFU/mouse of WR WT, WR-Δ3, WR-Δ4, or PBS. A single virus dose was used to define a priming effect on tumor growth; the high virus dose was to provide a sufficient amount of infective particles to ensure infection of the tumor with about 50-mm3 volume. General mouse well-being, weight, and tumor volume were followed up daily. Infection of tumors with WR WT or WR-Luc led to a slight reduction in tumor growth compared with the PBS-treated group (Figure 6A), which can be attributed to intrinsic VACV oncolytic capacity. When mice were infected with WR-Δ3 or WR-Δ4 viruses, a strong reduction in tumor proliferation was sustained throughout the experiment in the case of WR-Δ4 and until day 8 post-treatment in the WR-Δ3 group (Figure 6A). None of the mice showed negative effects on state of health as a result of the tumor or the virus treatment (data not shown). To quantify the results, we calculated and compared the area under the curve for tumor volume for each treated mouse. WR-Δ3- and WR-Δ4-treated groups both showed a significant reduction in cubic millimeters (mm3) × day values compared with the PBS-treated group; the reduction for the WR-Δ4 group was also significant relative to that of the WR WT group (Figure 6B). It was therefore clear that the combination of gene deletions in the oncolytic vectors WR-Δ3 and WR-Δ4 translated to increased anti-tumor effectiveness in the B16F10 syngeneic model. Because WR-Δ4 showed the most potent oncolytic activity and a greater reduction in virulence, we further characterized this virus for its oncolytic potential and possible correlates of protection. We injected B16F10 cells (i.d.) into C57BL/6 mice and infected the tumor (i.t.) 4 days later with WR-Δ4 (1 × 108 PFU). Other mouse groups with tumors received inoculations of WR WT or with PBS (control). At various times post-infection (days 4, 8, and 12), we determined the extent of tumor growth, presence of infectious virus in tumors, and innate and tumor-specific adaptive immune responses (Figure 7A). An examination of tumor proliferation showed significant differences in tumor size between virus- and PBS-treated mice at various time points (Figure 7B). Comparison of WR WT and WR-Δ4 showed a significantly greater reduction in tumor growth in the deletion mutant (Figure 7B). The extent of tumor reduction is observed in a representative photograph (Figure 7B) on day 12, with potent inhibition of tumor size in the WR-Δ4-treated mice versus WR WT and PBS groups. The presence of infectious virus in the treated tumors was characterized by virus plaque titration; excised tumors were treated with collagenase, processed, and titrated (titers were adjusted to tumor volume in each case). The virus-treated groups showed similar yields of WR WT and WR-Δ4 at days 4, 8, and 12 (Figure 7C), maintaining high levels of infectious virus until day 12. To help explain the oncolytic potential of the WR WT and WR-Δ4 vectors, we used flow cytometry to analyze immune cell populations in the tumors and spleens of treated mice. The WR-Δ4-infected tumor showed an increase in infiltrated neutrophil numbers throughout the infection period; neutrophil recruitment to the tumor was significantly higher at day 12 and surpassed the values for the WR WT group (p < 0.05) (Figure 7D), whereas infiltrated neutrophil numbers in PBS-treated mice remained low. For the remaining immune cells analyzed (NK, NKT, CD8 and CD4 T cells, macrophages, monocytes, B cells, and dendritic cells), all mice bearing virus-treated tumors had variable levels of immune cells, lower than values for PBS-treated mice (data not shown). In the case of immune cell populations in the spleen, a central organ in adaptive immune response development, we observed a significant increase in the number of CD8 and CD4 T cells that migrated to the spleen in WR-Δ4-treated mice at day 8 compared with PBS-treated mice (Figure 7E). At day 12, values decreased to levels slightly higher than those in the PBS group, but still higher than the initial numbers of CD8 and CD4 T cells in spleen (Figure 7E). The remaining spleen immune cells analyzed showed no differences between the assayed groups at various time points (data not shown). To determine whether WR WT-, WR-Δ4-, or PBS-treated mice with tumors develop specific immune responses to tumor antigens and to the virus, we performed an IFNγ ELISpot assay using splenocytes at day 12 post-treatment. We used B8R as a VACV-specific peptide stimulus, and glycoprotein (gp)100 and TRP-2 peptides as B16F10 CD8-specific tumor antigens. Splenocytes from both virus-treated groups showed a specific IFNγ response to the VACV peptide, which was not seen in the PBS group (Figure 7F). Only WR-Δ4-treated mice had an IFNγ response to tumor-specific gp100 and TRP-2 peptides, which is significant relative to the WR WT and PBS groups (Figure 7G). The tumor-specific immune response in splenocytes from the WR WT group was negligible, similar to that of the PBS group (Figure 7G). The basis for development of an optimal oncolytic viral vector includes requirements for safety, ability to infect and destroy tumor and stromal cells, and capacity to trigger tumor-specific immune responses.4Keller B.A. Bell J.C. Oncolytic viruses—immunotherapeutics on the rise.J. Mol. Med. (Berl.). 2016; 94: 979-991Crossref PubMed Scopus (42) Google Scholar, 26Breitbach C.J. Paterson J.M. Lemay C.G. Falls T.J. McGuire A. Parato K.A. Stojdl D.F. Daneshmand M. Speth K. Kirn D. et al.Targeted inflammation during oncolytic virus therapy severely compromises tumor blood flow.Mol. Ther. 2007; 15: 1686-1693Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar To develop replication-competent oncolytic VACV vectors, we defined and characterized a novel vector, WR-Δ4. This vector is based on the VACV WR strain and has a unique set of four deletions of the viral genes A48R, B19R, C11R, and J2R, some of which are reported to be effective in oncolytic vectors.27McCart J.A. Ward J.M. Lee J. Hu Y. Alexander H.R. Libutti S.K. Moss B. Bartlett D.L. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Res. 2001; 61: 8751-8757PubMed Google Scholar, 28Kim J.H. Oh J.Y. Park B.H. Lee D.E. Kim J.S. Park H.E. Roh M.S. Je J.E. Yoon J.H. Thorne S.H. et al.Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF.Mol. Ther. 2006; 14: 361-370Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 29Kirn D.H. Wang Y. Le Boeuf F. Bell J. Thorne S.H. Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus.PLoS Med. 2007; 4: e353Crossref PubMed Scopus (158) Google Scholar The deletions in WR-Δ4 did not affect its infection and replication capacity in cultured cells of various origins, and its plaque phenotype is comparable with that of the WR WT. Despite the deletions, WR-Δ4 maintains infection capacity, replication, and spread characteristics in cell monolayers, all critical factors for an effective anti-tumor response.13Kirn D.H. Thorne S.H. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer.Nat. Rev. Cancer. 2009; 9: 64-71Crossref PubMed Scopus (315) Google Scholar Spheroids are a valuable tool with which to infer viral infection and spread in the context of a solid tumor mass in vitro and provide reliable information before in vivo analyses.5Wein L.M. Wu J.T. Kirn D.H. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery.Cancer Res. 2003; 63: 1317-1324PubMed Google Scholar, 30Lam J.T. Hemminki A. Kanerva A. Lee K.B. Blackwell J.L. Desmond R. Siegal G.P. Curiel D.T. A three-dimensional assay for measurement of viral-induced oncolysis.Cancer Gene Ther. 2007; 14: 421-430Crossref PubMed Scopus (12) Google Scholar In our study, WR-Δ4 showed a high capacity to infect these 3D tumors, with broad homogeneous infection and lysis of exposed regions of murine prostate TRAMP-C1 cell spheroids, as also observed for the WR WT virus. Attenuation is a determining factor when a replication-competent virus is used in therapy for patients, most of whom are immunocompromised;13Kirn D.H. Thorne S.H. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer.Nat. Rev. Cancer. 2009; 9: 64-71Crossref PubMed Scopus (315) Google Scholar, 31Lichty B.D. Breitbach C.J. Stojdl D.F. Bell J.C. Going viral with cancer immunotherapy.Nat. Rev. Cancer. 2014; 14: 559-567Crossref PubMed Scopus (433) Google Scholar we therefore studied viral pathogenesis in mice infected with the mutant viruses. A major advantage of attenuation is to limit the spread of the virus to normal tissues other than to the tumor, thus avoiding complications of a systemic infection and reducing immune responses to the vector while enhancing those to the tumor antigens. WR-Δ4 shows increased virus attenuation, attributable to the specific combination of deletions, some of which are associated with reduced virulence (ΔA48R, ΔJ2R18Hughes S.J. Johnston L.H. de Carlos A. Smith G.L. Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae.J. Biol. Chem. 1991; 266: 20103-20109PubMed Google Scholar, 21Buller R.M. Smith G.L. Cremer K. Notkins A.L. Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype.Nature. 1985; 317: 813-815Crossref PubMed Sco" @default.
• W2771271245 created "2017-12-22" @default.
• W2771271245 creator A5027837160 @default.
• W2771271245 creator A5030712042 @default.
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• W2771271245 date "2018-03-01" @default.
• W2771271245 modified "2023-10-13" @default.
• W2771271245 title "Development of a Safe and Effective Vaccinia Virus Oncolytic Vector WR-Δ4 with a Set of Gene Deletions on Several Viral Pathways" @default.
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