Recruitment of Intratumoral CD103+ Dendritic Cells by a CXCR4 Antagonist-Armed Virotherapy Enhances Antitumor Immunity.

Intratumoral dendritic cells play an important role in stimulating cytotoxic T cells and driving antitumor immunity. Using a metastatic ovarian tumor model in syngeneic mice, we explored whether therapy with a CXCR4 antagonist-armed oncolytic vaccinia virus activates endogenous CD103+ dendritic cell responses associated with the induction of adaptive immunity against viral and tumor antigens. The overall goal of this study was to determine whether expansion of CD103+ dendritic cells by the virally delivered CXCR4 antagonist augments overall survival and in situ boosting with a tumor antigen peptide-based vaccine. We found that locoregional delivery of the CXCR4-A-armed virus reduced the tumor load and the immunosuppressive network in the tumor microenvironment, leading to infiltration of CD103+ dendritic cells that were capable of phagocytic clearance of cellular material from virally infected cancer cells. Further expansion of tumor-resident CD103+ DCs by injecting the FMS-related tyrosine kinase 3 ligand, the formative cytokine for CD103+ DCs, provided a platform for a booster immunization with the Wilms tumor antigen 1 peptide-based vaccine delivered intraperitoneally with polyriboinosinic:polyribocytidylic acid as an adjuvant. The vaccine-induced antitumor responses inhibited tumor growth and increased overall survival, indicating that expansion of intratumoral CD103+ dendritic cells by CXCR4-A-armed oncovirotherapy treatment can potentiate in situ cancer vaccine boosting.

Modulation of the Tumor Microenvironment by CXCR4 Antagonist-Armed Viral Oncotherapy Enhances the Antitumor Efficacy of Dendritic Cell Vaccines against Neuroblastoma in Syngeneic Mice.

The induction of antitumor immune responses in tumor-bearing hosts depends on efficient uptake and processing of native or modified tumors/self-antigens by dendritic cells (DCs) to activate immune effector cells, as well as the extent of the immunosuppressive network in the tumor microenvironment (TME). Because the C-X-C motif chemokine receptor 4 (CXCR4) for the C-X-C motif chemokine 12 (CXCL12) is involved in signaling interactions between tumor cells and their TME, we used oncolytic virotherapy with a CXCR4 antagonist to investigate whether targeting of the CXCL12/CXCR4 signaling axis in murine neuroblastoma cells (NXS2)-bearing syngeneic mice affects the efficacy of bone marrow (BM)-derived DCs loaded with autologous tumor cells treated with doxorubicin for induction of immunogenic cell death. Here, we show that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with neuroblastoma tumors augmented efficacy of the DC vaccines compared to treatments mediated by a soluble CXCR4 antagonist or oncolysis alone. This study is the first demonstration that modulating the tumor microenvironment by an armed oncolytic virus could have a significant impact on the efficacy of DC vaccines, leading to the generation of effective protection against neuroblastoma challenge.

Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy.

Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

CXCL12/CXCR4 blockade by oncolytic virotherapy inhibits ovarian cancer growth by decreasing immunosuppression and targeting cancer-initiating cells.

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ(+)/IL-10(+) tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases.

Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.

[HCV replication in Huh-7.5 cell line]. / Replikacja wirusa HCV w linii komórkowej Huh-7.5.

INTRODUCTION: Infection with hepatitis C virus is a serious worldwide health problem. Since its discovery in 1989, the development of a cell culture system for HCV has been a major goal for scientists worldwide. In 2005 the first tissue culture that led to the production of HCV particles (2a genotype) has been created. The aim of the study was to determine viral RNA level in an infectious HCV cell culture system. METHODS: Huh-7.5 cell line was infected with the JFH1 (Japanese Fulminant Hepatitis) RNA by lipofection. The level of HCV RNA was measured in supernatant of the cell culture by Real-Time PCR method. RESULTS: HCV RNA was detected in the supernatant of Huh-7.5 cell line infected with the use ofJFH 1 RNA and lipofectamin. The maximal level of HCV RNA (3.69 x 10(9) copies/ml) was detected 96h after transfection. After about 30 days oftransfection, HCV RNA was on the stable level (10(7)-10(8)). CONCLUSIONS: Huh-7.5 cell line infected with HCV form a robust cell culture model of HCV infection. HCV replicates on high levels in Huh-7.5, which is the crucial event for the investigation of new antivirals in in vitro model.

[Inhibitors of hepatitis C virus--current standards and status of investigations]. / Inhibitory wirusa zapalenia watroby typu C--aktualne standardy oraz stan badan.

The current standard of chronic hepatitis C therapy is the combined use of pegylated IFN-alpha 2a (PegIFN-alpha) and rybavirin (RBV). The new form of interferon, IFN-alpha 2b, was also introduced with no better results. Overall, the effectiveness of therapy with the use of the above scheme is not satisfactory. Thus the search for new therapeutic agents for hepatitis C is ongoing. These studies have the goal to find new preparations inhibiting the replication cycle of HCV. The new analogue of RBV, eg. tarybavirin was introduced, with lesser side effects, but the same effectiveness. The activity of new agents relies upon the inhibition of the most important enzymes of the HCV replication cycle: RNA polymerase, protease and helicase. Polymerase NS5 inhibitors are divided into nucleoside (R-7128) and nonnucleoside (ANA-598, GS 9190, VCH-759, VX-222). The intensive studies on the R-7128 analogue are ongoing. The effects of action of particular compounds in the I and II studies were summarized. The promised prodrug is nonnucleoside polymerase inhibitor, ANA-598 which when administrated to patients, gave 75% SVR. The combined administration of the newly described agents is the basis of specifically targeted antiviral therapies for HCV (STAT-C). These therapies allow to achieve better effectiveness of treatment, its shortening, the diminishment and limitation of side effects.

[Inhibitors of hepatitis C virus--therapeutic possibilities]. / Inhibitory wirusa zapalenia watroby typu C--mozliwosci terapeutyczne.

The search for new drugs against HCV contains new ways to obtain pro-drugs which inhibit translation and block viral proteins, and inhibit host proteins important in HCV-induced pathogenesis. This group of agents are serine protease NS3 inhibitors (telaprevir, boceprevir, R-7227, TMC-435, SCH 900518, GS-9256). The most advanced studies are developed with telaprevir and boceprevir; at present their effect in combined therapy with PegIFN-alpha and RBV in the III clinical phase is tested. The sustained viral response (SVR) was achieved at the level of 60-75%. This group of agents contains also inhibitors of NS5A domain, e.g. PPI-461 which shows antiviral and cytotoxic activity. The following prodrugs are NS3 helicase inhibitors, e.g. p14 peptide, whose IC50 equals 725 nM. Studies are continued on viral entry inhibitors (ITX-5061), therapeutic vaccines (IC-41, civaci, TG-4040, CT-1011, GI-5005) and immunomodulating preparations (ANA-773, IMO-3649, NOV-205). The agents acting on host proteins are a.o. cyclophilin inhibitors. The most advanced studies concern DEBIO 025 preparation which after phase I and II, underwent phase III of clinical studies in February 2010. Since 5 years there is a possibility to investigate the effects of these comounds in vitro with the use of Huh-7 line infected with HCV. These investigations allow to estimate the antiviral effectiveness and cytotoxicity of agents, and resistance of viral strains.