Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus.

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 106 cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 108 TCID50 /mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus.

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 106 cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 109 TCID50 /mL), more than 4-100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15-30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.

Cell-line screening and process development for a fusogenic oncolytic virus in small-scale suspension cultures.

Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.

A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma.

Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named recombinant VSV-NDV (rVSV-NDV), wherein the VSV backbone is conserved but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. In comparison to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytium formation, allowing efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wild-type VSV, resulting in a markedly enhanced safety profile, even in immune-deficient NOD.CB17-prkdcscid/NCrCrl (NOD-SCID) mice, which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic hepatocellular carcinoma (HCC)-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus without risking the safety and environmental threats associated with either parental vector. Taking the data together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus.IMPORTANCE The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

PET imaging of oncolytic VSV expressing the mutant HSV-1 thymidine kinase transgene in a preclinical HCC rat model.

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Antifibrotic properties of transarterial oncolytic VSV therapy for hepatocellular carcinoma in rats with thioacetamide-induced liver fibrosis.

Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Posttranslational modification of vesicular stomatitis virus glycoprotein, but not JNK inhibition, is the antiviral mechanism of SP600125.

Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogen-activated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/ß). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein.

Oncolytic virotherapy with chimeric VSV-NDV synergistically supports RIG-I-dependent checkpoint inhibitor immunotherapy.

Unraveling the complexities of the tumor microenvironment (TME) and its correlation with responsiveness to immunotherapy has become a main focus in overcoming resistance to such treatments. Targeting tumor-intrinsic retinoic acid-inducible gene-I (RIG-I), a sensor for viral RNA, was shown to transform the TME from an immunogenically "cold" state to an inflamed, "hot" lesion, which we demonstrated previously to be a crucial mediator of the efficacy of immune checkpoint inhibition with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In this study, we focus on the chimeric oncolytic virus vesicular stomatitis virus (VSV)-Newcastle disease virus (NDV), comprised of genetic components of VSV and NDV, and we investigate its utility to support tumor-intrinsic RIG-I-dependent therapy with anti-CTLA-4. Overall, we demonstrate that treatment with VSV-NDV efficiently delays tumor growth and significantly prolongs survival in a murine model of malignant melanoma, which was further enhanced in combination with anti-CTLA-4. Although the direct oncolytic and pro-inflammatory effects of VSV-NDV therapy were independent of RIG-I activation, the synergism with anti-CTLA-4 therapy and associated activation of tumor-specific T cells was critically dependent on active RIG-I signaling in tumor cells. This work highlights the therapeutic value of utilizing an immune-stimulatory oncolytic virus to sensitize tumors to immune checkpoint inhibition.

Validation of preclinical multiparametric imaging for prediction of necrosis in hepatocellular carcinoma after embolization.

BACKGROUND & AIMS: The hepatocellular carcinoma (HCC) exhibits varying degrees of vascularization with more poorly differentiated carcinoma commonly exhibiting high amounts of vascularization. Transcatheter arterial embolization (TAE) of HCC tumor nodules results in varying amounts of tumor necrosis. Reliable quantification of necrosis after TAE, would aid in treatment planning and testing of novel combinatorial treatment regimen. The aim of this work was to validate different imaging parameters as individual or combined predictors of tumor necrosis after TAE in an orthotopic rat HCC tumor model. METHODS: Unifocal rat HCC was imaged by T(2)-weighted MRI, quantitative dynamic contrast enhanced (DCE) MRI, diffusion weighted MRI (DWI) and [(18)F]-FDG PET imaging before (day-1) and after (days 1 and 3) TAE. Univariate and multivariate regression analyses were carried out to analyze the ability of each imaging parameter to predict the percent residual vital tumor (vtu) and vital tissue (vti) as determined by quantitative histopathology. RESULTS: TAE induced a wide range of tumor necrosis. Tumor volume was the only parameter showing a correlation with vti (r(2) = 0.63) before TAE. After TAE, moderate correlations were found for FDG tracer uptake (r(2) = 0.56) and plasma tissue transfer constant (r(2) = 0.55). Correlations were higher for the extravascular extracellular volume fraction (v(e), r(2) = 0.68) and highest for the apparent diffusion coefficient (ADC, r(2) = 0.86). Multivariate analyses confirmed highest correlation of ADC and v(e) with vtu and vti. CONCLUSIONS: DWI and DCE-MRI with the respective parameters ADC (day 3) and v(e) (day 1) were identified as the most promising imaging techniques for the prediction of necrosis. This study validates a preclinical platform allowing for the improved tumor stratification after TAE and thus the testing of novel combinatorial therapy approaches in HCC.

Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, which is currently under investigation as a clinical oncolytic agent. Several clinical trials have reported NDV to be a safe and effective agent for cancer therapy; however, there remains a clear need for improvement in therapeutic outcome. The endogenous NDV fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. Here, we report a novel NDV vector harboring an L289A mutation within the F gene, which resulted in enhanced fusion and cytotoxicity of hepatocellular carcinoma (HCC) cells in vitro, as compared with the rNDV/F3aa control virus. In vivo administration of the recombinant vector, termed rNDV/F3aa(L289A), via hepatic arterial infusion in immune-competent Buffalo rats bearing multifocal, orthotopic liver tumors resulted in tumor-specific syncytia formation and necrosis, with no evidence of toxicity to the neighboring hepatic parenchyma. Furthermore, the improved oncolysis conferred by the L289A mutation translated to significantly prolonged survival compared with control NDV. Taken together, rNDV/F(L289A) represents a safe, yet more effective vector than wild-type NDV for the treatment of HCC, making it an ideal candidate for clinical application in HCC patients.

Giving Oncolytic Viruses a Free Ride: Carrier Cells for Oncolytic Virotherapy.

Oncolytic viruses (OVs) are an emerging class of therapeutics which combine multiple mechanisms of action, including direct cancer cell-killing, immunotherapy and gene therapy. A growing number of clinical trials have indicated that OVs have an excellent safety profile and provide some degree of efficacy, but to date only a single OV drug, HSV-1 talimogene laherparepvec (T-Vec), has achieved marketing approval in the US and Europe. An important issue to consider in order to accelerate the clinical advancement of OV agents is the development of an effective delivery system. Currently, the most commonly employed OV delivery route is intratumoral; however, to target metastatic diseases and tumors that cannot be directly accessed, it is of great interest to develop effective approaches for the systemic delivery of OVs, such as the use of carrier cells. In general, the ideal carrier cell should have a tropism towards the tumor microenvironment (TME), and it must be susceptible to OV infection but remain viable long enough to allow migration and finally release of the OV within the tumor bed. Mesenchymal stem cells (MSCs) have been heavily investigated as carrier cells due to their inherent tumor tropism, in spite of some disadvantages in biodistribution. This review focuses on the other promising candidate carrier cells under development and discusses their interaction with specific OVs and future research lines.

Adoptive T Cell Therapy Is Complemented by Oncolytic Virotherapy with Fusogenic VSV-NDV in Combination Treatment of Murine Melanoma.

Cancer immunotherapies have made major advancements in recent years and are becoming the prevalent treatment options for numerous tumor entities. However, substantial response rates have only been observed in specific subsets of patients since pre-existing factors determine the susceptibility of a tumor to these therapies. The development of approaches that can actively induce an anti-tumor immune response, such as adoptive cell transfer and oncolytic virotherapy, have shown clinical success in the treatment of leukemia and melanoma, respectively. Based on the immune-stimulatory capacity of oncolytic VSV-NDV virotherapy, we envisioned a combination approach to synergize with adoptive T cell transfer, in order to enhance tumor cell killing. Using the immune-competent B16 melanoma model, we demonstrate that combination treatment has beneficial effects on the suppressive microenvironment through upregulation of MHC-I and maintaining low expression levels of PD-L1 on tumor cells. The approach led to additive cytotoxic effects and improved the recruitment of T cells to virus-infected tumor cells in vitro and in vivo. We observed substantial delays in tumor growth and evidence of abscopal effects, as well as prolongation of overall survival time when administered at clinically relevant dosing conditions. Our results indicate that treatment with oncolytic VSV-NDV, combined with adoptive T cell therapy, induces multi-mechanistic and synergistic tumor responses, which supports the further development of this promising translational approach.

Hyperpolarized 13C pyruvate magnetic resonance spectroscopy for in vivo metabolic phenotyping of rat HCC.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1-13C]pyruvate injection. Cell lines were derived from a subset of primary tumors, re-implanted in nude rats, and assessed in vivo with dynamic hyperpolarized magnetic resonance spectroscopy (HPMRS) after [1-13C]pyruvate injection and kinetic modelling of pyruvate metabolism, taking into account systemic lactate production and recirculation. For ex vivo validation, enzyme activity and metabolite concentrations were spectroscopically quantified in cell and tumor tissue extracts. Mean peak L/A was higher in endogenous HCC compared to non-tumorous tissue. Dynamic HPMRS revealed higher pyruvate-to-lactate conversion rates (kpl) and lactate signal in subcutaneous tumors derived from high L/A tumor cells, consistent with ex vivo measurements of higher lactate dehydrogenase (LDH) levels in these cells. In conclusion, HPMRS and HPMRSI reveal distinct tumor phenotypes corresponding to differences in glycolytic metabolism in HCC tumor tissue.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Synergistic antitumor effects of transarterial viroembolization for multifocal hepatocellular carcinoma in rats.

UNLABELLED: Oncolytic virotherapy is a promising strategy for safe and effective treatment of malignancy. We have reported previously that recombinant vesicular stomatitis virus (VSV) vectors are effective oncolytic agents that can be safely administered via the hepatic artery in immunocompetent rats to treat multifocal hepatocellular carcinoma (HCC), resulting in tumor necrosis and prolonged survival. Though the results were encouraging, complete tumor regression was not observed, which led us to explore alternative approaches to further enhance the efficacy of VSV treatment. Transarterial embolization techniques have been shown to improve the efficiency and tumor selectivity of anticancer treatments. Degradable starch microspheres (DSM) are one such embolic agent that provides transient embolization of the therapeautic agent before being degraded by serum amylases. Here we demonstrate via dynamic contrast-enhanced magnetic resonance imaging that in our rat model of multifocal HCC, DSM injection into the hepatic artery results in a substantial reduction in tumor perfusion of systemically applied contrast agent. VSV, when administered in combination with DSM, results in enhanced tumor necrosis and synergistically prolongs survival when compared with VSV or DSM monotherapy. CONCLUSION: This regimen of viroembolization represents an innovative therapeutic modality that can augment the future development of transarterial oncolytic virus therapy for patients with advanced HCC.

Inhibition of the IFN-ß Response in Hepatocellular Carcinoma by Alternative Spliced Isoform of IFN Regulatory Factor-3.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). We have observed earlier that, in contrast to cultured human HCC cells, primary human hepatocytes (PHHs) are refractory to VSV infection. Impairment of the type I interferon (IFN) pathway in HCC cells has been suggested to be the mechanism by which these cells become susceptible to VSV infection. The goal of this study was to elucidate the nature of the IFN defect in human HCC. We demonstrate here that the defect in IFN-ß signaling in HCC cells results from a deregulated IFN regulatory factor-3 (IRF3) pathway. Expression of IRF3-spliced variant (IRF3-nirs3) was constitutively observed in HCC cells and, importantly, also in primary HCC samples. In contrast, IRF3 was readily activated in PHHs after stimulation with dsRNA or infection with VSV. In addition, overexpression of IRF3-nirs3 significantly abrogated the IFN-ß response to VSV infection and improved viral growth. Our data provide evidence that aberrant splicing of IRF3 in HCC contributes to the defect in IFN-mediated antiviral defenses. This work may provide a potential molecular basis for selecting HCC patients for oncolytic VSV therapy in future clinical trials.

Inhibition of the IFN-beta response in hepatocellular carcinoma by alternative spliced isoform of IFN regulatory factor-3.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). We have observed earlier that, in contrast to cultured human HCC cells, primary human hepatocytes (PHHs) are refractory to VSV infection. Impairment of the type I interferon (IFN) pathway in HCC cells has been suggested to be the mechanism by which these cells become susceptible to VSV infection. The goal of this study was to elucidate the nature of the IFN defect in human HCC. We demonstrate here that the defect in IFN-beta signaling in HCC cells results from a deregulated IFN regulatory factor-3 (IRF3) pathway. Expression of IRF3-spliced variant (IRF3-nirs3) was constitutively observed in HCC cells and, importantly, also in primary HCC samples. In contrast, IRF3 was readily activated in PHHs after stimulation with dsRNA or infection with VSV. In addition, overexpression of IRF3-nirs3 significantly abrogated the IFN-beta response to VSV infection and improved viral growth. Our data provide evidence that aberrant splicing of IRF3 in HCC contributes to the defect in IFN-mediated antiviral defenses. This work may provide a potential molecular basis for selecting HCC patients for oncolytic VSV therapy in future clinical trials.

Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo.

Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

Graft preconditioning with low-dose tacrolimus (FK506) and nitric oxide inhibitor aminoguanidine (AGH) reduces ischemia/reperfusion injury after liver transplantation in the rat.

Ischemia/reperfusion (I/R) injury is a main cause of primary dysfunction or non-function after liver transplantation (LTx). Recent evidence indicates that an increase in nitric oxide (NO) production after LTx is associated with I/R injury. The aim of this study was to demonstrate that low-dose FK506 in combination with aminoguanidine (AGH), which leads to a reduction of NO levels, has a protective effect by reducing I/R associated injury after LTx. Fortyone DA-(RT1av1) rats served as donors and recipients for syngenic orthotopic arterialised LTx. They were divided into 4 groups: controls without pre-/treatment (I), pre-/treatment with high-dose FK506 (II), pre-/treatment with AGH only (III), and pre-/treatment with low-dose FK506 in combination with AGH (IV). After LTx the laboratory parameters and liver biopsy were performed. The levels of transaminase (ALT) in groups I, II and III were significantly higher on day 3 after LTx compared to group IV (p = 0.001, p = 0.001, p = 0.000). In group IV the I/R-associated liver necrosis rate was reduced significantly. Our results demonstrated that a combined dual pharmacological pretreatment (group IV) reduced I/R injury of the graft after LTx in a rat model.