Oncolytic Strain Research Articles

Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer

We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.

Abstract LB-370: Genetically engineered oncolytic Edmonston strain of measles strain harboring the wild-type N, P, L genes (MV-NPL) effectively target lung cancer stem cells

Abstract Increasing evidence suggests that only a small percentage of cancer cells in solid tumors have tumorigenic potential. These highly tumorigenic cells share with normal stem cells the ability to proliferate and give rise to diversified tumor cell types including those with the capacity for self-renewal. These cells are termed cancer stem cells (CSCs) and are believed to contribute to CSCs resistance to conventional therapies. Therefore, specific targeting of CSCs is an attractive and novel therapeutic approach. Measles virus Edmonston strain (MV-Edm) is thought to possess remarkable oncolytic activity that selectively destroys human tumor cells. Recently, we succeeded and reported the development of novel oncolytic measles virus (MV) by genetic engineering of MV-Edmonston strain (MV-Edm), that have demonstrated its remarkable oncolytic activity against wide ranges of human tumor cells, with the N, P, and L genes of wild-type MV (MV-NPL). The oncolytic activities of the MV-NPL on human renal cell carcinoma were superior to those of MV-Edm both in vitro and in vivo studies even in the presence of IFN[[Unsupported Character - ‐]]α[[Unsupported Character - ]](Xing, M. et. al. Mol Ther 2010). Here, we demonstrated that lung CSCs, side population fraction of human lung cancer cells of A549 (A549-SP), expressed high level of CD46 required for MV-NPL infection and MV-NPL had a superior oncolytic capacity against both A549-SP and A549-NSP (non SP) cells in a time and dose-dependent manner without damaging normal lung cells. Our results of pan-caspase inhibiton assays showed the partial contribution of caspases-dependent apoptosis in the oncolysis of A549-SP cells by MV-NPL, even though the expression levels of anti-apoptotic protein of both XIAP and Mcl-1 were higher in A549-SP cells than A549-NSP cells. Additionally, we demonstrated that an addition of either PI3K inhibitor or MEK inhibitor further enhanced the oncolytic effects of MV-NPL. Furthermore, our in vivo studies using nude mice bearing A549 xenografts revealed that serial intratumoral MV-NPL administrations exhibited significant antitumor effects without severe side effects. Collectively, these data suggested that genetically engineered MV-NPL could be a promising oncolytic agent to effectively eradicate lung CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-370. doi:1538-7445.AM2012-LB-370

Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.

MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.

Evaluation of HSV-1 and adenovirus vector-mediated infection, replication and cytotoxicity in lymphoma cell lines

Lymphoma is not a common focus for viral therapy as many researchers do not consider lymphoma as a suitable target for viral vectors. In the present study, the infection, replication and cytotoxicity of herpes simplex virus (HSV) and adenovirus vectors were screened and evaluated in different lymphoma cell lines. Three recombinant viruses, BAC-HSV-1-eGFP, Adv-eGFP and an NV1020-like oncolytic HSV-1 virus strain named BAC-HSV-1-eGFP-delIRs, inserted with green fluorescent protein (GFP) as a reporter, were applied to evaluate the efficiency of viral infection and replication and cytotoxicity to lymphoma cells. Four types of lymphoma cell lines (SNK-6, Jurkat, Raji and Hut 78) were examined. We found that the HSV-1 vector, BAC-HSV-1-eGFP, was able to infect and replicate in the three lymphoma cell lines (Jurkat, Raji and Hut 78) and inhibit the growth of these cells more efficiently than adenovirus vector Adv-eGFP. The sensitivity of the four types of lymphoma cell lines to the viral vectors was different. The human cutaneous TL cell line Hut 78 was more sensitive to the viral vectors than the other cell lines. However, the human NK/T lymphoma cell line SNK-6 was not infected by any of the viral vectors and did not allow replication of the viruses. In conclusion, lymphoma may be a potential target for HSV-1 vector-mediated viral therapy.

Vaccinia virus GLV-1h237 carrying a Walker A motif mutation of mouse Cdc6 protein enhances human breast tumor therapy in mouse xenografts

Recently it was shown that recombinant vaccinia virus GLV-1h68 is a promising tool for treating different type of cancers in animal models. The goal of the present study was to enhance the oncolytic potential of GLV-1h68 without decreasing its safety. A derivative of GLV-1h68 containing the gene for a Walker A motif mutant of the essential cell cycle protein Cdc6, GLV-1h237, was engineered. The characteristics of GLV-1h237 and its efficiency in treating human breast cancer GI-101A cells were compared with that of GLV-1h236 (carrying the wild-type gene for Cdc6), GLV-1h71 (a derivative of GLV-1h68) and GLV-1h68, respectively. RT-PCR and immunoblot analyses revealed that Cdc6 is efficiently overexpressed in GLV-1h237-infected GI-101A cells. GLV-1h237 was found to have higher replication efficiency and enhanced cytotoxity than GLV-1h68 in cell culture. In the GI-101A tumor xenograft animal model, GLV-1h237 turned out to be the most potent oncolytic virus strain investigated. A single i.v. injection of GLV-1h237 resulted in enhanced anti-tumor activity compared to GLV-1h68 concomitant with a high tumor selectivity and a comparable safety profile. Thus, the strategy to combine oncolytic virotherapy with agents that interfere with host cell DNA synthesis is a promising approach for effective cancer therapy.

Oncolytic poliovirus therapy and immunization with poliovirus-infected cell lysate induces potent antitumor immunity against neuroblastoma in vivo

In a previous study, we demonstrated that neuroblastoma subcutaneously implanted in immuno-competent mice is eliminated by intratumoral administration of neuroattenuated poliovirus (PV). Our results also suggested that the in vivo destruction of neuroblastoma cells by virotherapy lead to a robust antitumor immune response. In this work, splenocytes harvested from neuroblastoma-bearing animals treated with neuroattenuated PV exhibited significantly higher lytic activity against tumor target cells than did those from splenocytes derived from control mice. In vitro T-cell depletion experiments indicated that CD8(+) T cells were essential for the cytotoxic antitumor activity of splenocytes. Moreover, adoptive transfer of splenocytes obtained from mice cured of neuroblastoma by PV virotherapy markedly delayed the tumor growth of previously established neuroblastomas in recipient naïve mice. These results confirmed that treatment with a neuroattenuated oncolytic PV strain induces antitumor immunity against neuroblastoma that is mainly mediated by cytotoxic CD8(+) T cells. Immunocompetent mice, on the other hand, were immunized with PV-infected neuroblastoma cell lysate prior intravenous challenge with neuroblastoma cells. As a control, mice were vaccinated with either non-infected neuroblastoma cell lysate alone or mixed with PV, or with PBS prior tumor cell injection. Results showed that survival is significantly prolonged only in mice immunized with PV-infected tumor lysate. This finding clearly suggested that in vitro poliovirus infection of neuroblastoma cells turns these cells into a potent tumor immunogen. Further studies in oncolytic treatment of neuroblastoma using attenuated PV alone or in combination with immunotherapy with PV oncolysate should improve the probability for successful translation in the clinic.

Regression of Human Prostate Tumors and Metastases in Nude Mice following Treatment with the Recombinant Oncolytic Vaccinia Virus GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 ((125)I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of (123)I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 x 10(6) TCID(50)) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of (131)I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by (131)I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

Characterization of the genome sequence of an oncolytic Newcastle disease virus strain Italien

We determined the complete genome sequence of strain Italien, a virulent and oncolytic strain of Newcastle disease virus (NDV) by direct nucleotide sequencing of RT-PCR products, a size of 15,186 nucleotides (nt). Comparison of six coding genes and non-coding regions of Italien with those of the other 25 sequenced strains revealed NDV Herts/33 was the most similar strain with Italien. The gene encoding the RNA dependent RNA polymerase was the most highly conserved, while the gene encoding phosphoprotein was the most highly variable. The HN and F proteins of Italien have been modeled on the crystal structure in order to study the structural characteristics. Interaction between the HN protein and the heptad repeat B (HRB) region of F protein was analyzed in silico by molecular docking predicted five critical residues I133, V142, D143, R480, and K567 on HN protein. Identification of amino acid residues that could be crucial for this interaction provides working hypotheses for subsequent studies.

Enhancing Poxvirus Oncolytic Effects through Increased Spread and Immune Evasion

The antitumoral effects of oncolytic viruses have generally been limited by inefficient spread of the viruses within infected tumors and by inefficient systemic delivery, particularly in preimmunized hosts. Tumor-selective poxviruses have biological characteristics that may overcome these limitations. Nevertheless, physical barriers within the tumor microenvironment, including the extracellular matrix, can still limit intratumoral spread, and neutralizing antibodies can impede systemic delivery. To counter these limitations, we sought to take advantage of a naturally occurring poxvirus form known as extracellular enveloped virus (EEV). The EEV is shrouded by a host cell-derived lipid bilayer containing anticomplement proteins and is typically released from infected cells early during the infection cycle. Therefore, the EEV form evolved for rapid systemic spread within the host and for evasion of immune-mediated clearance. We compared the oncolytic potential of low versus high EEV-producing strains of vaccinia. EEV-enhanced vaccinia strains displayed improved spread within tumors after systemic delivery, resulting in significantly improved antitumor effects. The EEV-enhanced strains also displayed a greater ability to spread between injected and noninjected distant tumors through the blood and, importantly, displayed reduced clearance by neutralizing antibody. Safety was unaffected. The incorporation of EEV-enhancing mutations into next generation oncolytic vaccinia strains may improve the potency of these viruses without sacrificing safety.

Generation of a recombinant oncolytic Newcastle disease virus and expression of a full IgG antibody from two transgenes

The most advanced oncolytic Newcastle disease virus (NDV) strains that are used in clinical trials for the treatment of cancer are wild-type mesogenic strains. These virus strains have an inherent, nongenetically engineered, oncolytic activity and selectively replicate in tumor cells but not in normal human cells. To date no investigations have been performed with genetically engineered mesogenic NDV regarding the oncolytic activity. We describe here the generation of recombinant viruses of the mesogenic naturally oncolytic NDV strain MTH68. We show that not only one, but also two additional transgenes coding for amino-acid chains with a molecular weight of 25 and 50 kDa can be inserted into the viral genome without affecting viral growth, oncolytic potency or tumor-selective replication of the virus. Transgenic expression of the heavy and light chains of a monoclonal antibody, as separate additional transcriptional cassettes, leads to the expression of full immunoglobulin G (IgG) monoclonal antibody by recombinant NDV. Infection of tumor cells with antibody-transgenic viruses results in the efficient production and secretion of a functional full size IgG antibody by the tumor cells, that specifically binds to its target-antigen in tumor tissue. This approach will allow to combine the advantages of oncolytic RNA viruses and monoclonal antibodies in a single powerful anticancer agent with improved or even new therapeutic properties.

Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus

BackgroundOncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.Methods and FindingsIn vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.ConclusionsBy combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.

Host mediated anti-tumor effect of oncolytic Newcastle disease virus after locoregional application

Several strains of the Newcastle disease virus (NDV) have raised considerable interest in recent years for clinical application because of their oncolytic properties. In this study we characterized virological, immunological and anti-tumor properties of some NDV strains. The oncolytic strain MTH-68/H was the most potent interferon-alpha inducer and, after UV light inactivation, it was the only tested NDV strain which induced in human PBMC anti-tumor activity in vitro. Upon systemic application to mice bearing a virus susceptible intradermal tumor, no significant anti-tumor effects were observed with the two oncolytic strains Italian and MTH-68/H while the treatment had significant side effects as seen by loss of body weight. In contrast, when using a locoregional application model for treatment of liver metastases of luciferase transfected CT26 colon carcinoma cells, MTH-68/H showed a significant delay in tumor growth, as well as prolonged survival but no effects on body weight. Surprisingly, this CT26 murine tumor cell transfectant was resistant in vitro to virus infection and oncolysis. These results suggest: i) that locoregional application of oncolytic NDV is more effective than systemic i.v. application; and ii) that oncolytic NDV can mediate effects even against a virus-resistant tumor line. The involvement of host anti-tumor immune responses as an important mechanism in therapies based on oncolytic NDV will be discussed.

An oncolytic mutant of herpes simplex virus type-1 in which replication is governed by a promoter/enhancer of human papillomavirus type-16

Although herpes simplex virus type-1 (HSV-1) can be used as an oncolytic virus it has the undesirable side effect of neurotoxicity. To create a virus with improved specificity for oral cancer we used a fragment of human papillomavirus type-16, which is frequently found in oral and cervical cancers, but not elsewhere. The upstream regulatory region, URR16, was shown to have a high level of transcriptional activity in three of four oral cancer cell lines but low activity in three cell lines derived from brain cancers. URR16 was therefore placed in HSV-1, replacing the promoter of the essential gene ICP4, and the resulting virus was named HSPV-1. When cells were infected with HSPV-1, ICP4 was expressed at levels that were not associated with the level of activity of URR16. The virus replicated in each type of cell to a final titer that showed a correlation with the level of expression of ICP4, but with no correlation to either the tumor of origin or the presence of HPV sequences in the cells. To find if some function of HSV-1 was affecting the activity of URR16, oral cancer cells were transfected with a URR-reporter construct and were then infected with virus. This induced transcription, which was attributed to immediate-early viral genes other than ICP4. A promoter/enhancer from a papillomavirus therefore has the potential to regulate the functions of an oncolytic strain of HSV-1, and is affected by functions of both the host cell and of HSV-1 itself.

Bax-dependent mitochondrial membrane permeabilization enhances IRF3-mediated innate immune response during VSV infection

An effective type I interferon (IFN-α/β) response is critical for the control of many viral infections. Using an oncolytic strain of vesicular stomatitis virus, we have examined the cross-talk between virus-induced apoptosis and initiation of innate immune response. The intrinsic apoptotic cascade, specifically the Bax-Bcl-2-Caspase-9 cascade, was revealed as the primary pathway of VSV-induced apoptosis. Cell death was significantly reduced in BaxBak − /− murine embryonic fibroblasts (MEFs) and in human A549 epithelial cells treated with siRNA against Bax. Although inhibition of apoptosis resulted in enhanced virus replication in the BaxBak − /− MEFs as compared to wild-type cells, induction of the IFN antiviral response and expression of cytokine genes were attenuated in virus-infected cells. Moreover, Bax but not Bak pro-apoptotic protein was required for IRF-3 phosphorylation and activation, further substantiating a role for the intrinsic mitochondrial pathway in the innate immune response. Therefore, virus-induced apoptosis through a Bax-dependent mitochondrial pathway appears to enhance the full development of the IRF-3 mediated IFN antiviral response.

Phase 1 Clinical Experience Using Intravenous Administration of PV701, an Oncolytic Newcastle Disease Virus

PV701 is a naturally-attenuated, non-recombinant, oncolytic strain of Newcastle disease virus that displays preclinical intravenous (IV) efficacy. PV701 is selective at killing human cancer cells versus normal human cells based on tumor specific defects in the interferon (IFN)-mediated antiviral response. This oncolytic virus displays a broad spectrum of antitumor activity in vitro and in vivo. Preclinical models successfully predicted key clinical parameters including the mechanism of toxicity, two complementary strategies (desensitization and slow infusion) to reduce toxicity, and the starting dose for phase 1 trials. In three phase 1 trials of 114 patients using IV administration of PV701, Wellstat Biologics Corporation has evaluated the effects of dose, schedule, and infusion rate for PV701. Three general classes of side effects were seen: flu-like symptoms; tumor-site-specific adverse events (AEs); and infusion reactions. The first PV701 dose desensitized the patient to the side effects of further doses, allowing a marked increase in the maximum tolerated dose for subsequent doses compared to the first dose. Tumor responses were first noted at the higher doses achieved using desensitization. In the most recent phase 1 trial of 19 patients at Hamilton, Ontario, that employed desensitization, high repeat doses, and a slower infusion rate (Hamilton Regimen), there were six responses (4 major; 2 minor) and a total of six patients with survival for at least 2 years. In addition, patient tolerability improved using the Hamilton Regimen compared to IV bolus dosing used previously. Phase 2 studies of this novel biologic agent are about to begin.