@article{SturmHessWeibeletal.2012, author = {Sturm, Julia B. and Hess, Michael and Weibel, Stephanie and Chen, Nanhei G. and Yu, Yong A. and Zhang, Quian and Donat, Ulrike and Reiss, Cora and Gambaryan, Stepan and Krohne, Georg and Stritzker, Jochen and Szalay, Aladar A.}, title = {Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75224}, year = {2012}, abstract = {Background: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.}, subject = {Biologie}, language = {en} } @article{PatilGentschevNolteetal.2012, author = {Patil, Sandeep S. and Gentschev, Ivaylo and Nolte, Ingo and Ogilvie, Gregory and Szalay, Aladar A.}, title = {Oncolytic virotherapy in veterinary medicine: current status and future prospects for canine patients}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75128}, year = {2012}, abstract = {Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An 'ideal' virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.}, subject = {Medizin}, language = {en} } @article{SzalayHillStritzkeretal.2011, author = {Szalay, Aladar A. and Hill, Philip J. and Stritzker, Jochen and Scadeng, Miriam and Geissinger, Ulrike and Haddad, Daniel and Basse-L{\"u}sebrink, Thomas C. and Gbureck, Uwe and Jakob, Peter}, title = {Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75789}, year = {2011}, abstract = {Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes}, subject = {Escherichia coli}, language = {en} } @article{WeibelRaabYuetal.2011, author = {Weibel, Stephanie and Raab, Viktoria and Yu, Yong A. and Worschech, Andrea and Wang, Ena and Marincola, Francesco M. and Szalay, Aladar A.}, title = {Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68691}, year = {2011}, abstract = {Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.}, subject = {Virusinfektion}, language = {en} } @article{KoberRohnWeibeletal.2015, author = {Kober, Christina and Rohn, Susanne and Weibel, Stephanie and Geissinger, Ulrike and Chen, Nanhai G. and Szalay, Aladar A.}, title = {Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps}, series = {Journal of Translational Medicine}, volume = {13}, journal = {Journal of Translational Medicine}, number = {216}, doi = {10.1186/s12967-015-0586-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126517}, year = {2015}, abstract = {Background Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM. Methods VACV LIVP 1.1.1 replication in C57BL/6 and \(Foxn1^{nu/nu}\) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system. Results We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of \(Iba1^+\) microglia and \(GFAP^+\) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected. Conclusion Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.}, language = {en} } @article{GholamiChenBelinetal.2013, author = {Gholami, Sepideh and Chen, Chun-Hao and Belin, Laurence J. and Lou, Emil and Fujisawa, Sho and Antonacci, Caroline and Carew, Amanda and Chen, Nanhai G. and De Brot, Marina and Zanzonico, Pat B. and Szalay, Aladar A. and Fong, Yuman}, title = {Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer}, series = {Breast Cancer Research}, volume = {15}, journal = {Breast Cancer Research}, number = {R26}, doi = {10.1186/bcr3404}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122140}, year = {2013}, abstract = {Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90\% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10\% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.}, language = {en} } @article{KirscherDeanBenScadengetal.2015, author = {Kirscher, Lorenz and De{\´a}n-Ben, Xos{\´e} Luis and Scadeng, Miriam and Zaremba, Angelika and Zhang, Qian and Kober, Christina and Fehm, Thomas Felix and Razansky, Daniel and Ntziachristos, Vasilis and Stritzker, Jochen and Szalay, Aladar A.}, title = {Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent}, series = {Theranostics}, volume = {5}, journal = {Theranostics}, number = {10}, doi = {10.7150/thno.12533}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124987}, pages = {1045-1057}, year = {2015}, abstract = {We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.}, language = {en} } @article{AdelfingerBesslerCeciletal.2015, author = {Adelfinger, Marion and Bessler, Simon and Cecil, Alexander and Langbein-Laugwitz, Johanna and Frentzen, Alexa and Gentschev, Ivaylo and Szalay, Aladar A.}, title = {Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy}, series = {Viruses}, volume = {7}, journal = {Viruses}, doi = {10.3390/v7072811}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125705}, pages = {4075-4092}, year = {2015}, abstract = {Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.}, language = {en} } @article{TsonevaStritzkerBedenketal.2015, author = {Tsoneva, Desislava and Stritzker, Jochen and Bedenk, Kristina and Zhang, Qian and Cappello, Joseph and Fischer, Utz and Szalay, Aladar A.}, title = {Drug-encoded Biomarkers for Monitoring Biological Therapies}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0137573}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125265}, pages = {e0137573}, year = {2015}, abstract = {Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.}, language = {en} } @article{AdelfingerGentschevdeGuibertetal.2014, author = {Adelfinger, Marion and Gentschev, Ivaylo and de Guibert, Julio Grimm and Weibel, Stephanie and Langbein-Laugwitz, Johanna and H{\"a}rtl, Barbara and Escobar, Hugo Murua and Nolte, Ingo and Chen, Nanhai G. and Aguilar, Richard J. and Yu, Yong A. and Zhang, Qian and Frentzen, Alexa and Szalay, Aladar A.}, title = {Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0104337}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119387}, pages = {e104337}, year = {2014}, abstract = {Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.}, language = {en} }