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1.  Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy 
Cancer gene therapy  2015;22(9):438-444.
Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor, including breast cancer. Aurora A kinase controls mitotic spindle formation and plays a critical role in malignant transformation. We hypothesized that, by causing mitotic arrest, the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long term survivors vs. 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in antitumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance measles virus-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.
doi:10.1038/cgt.2015.36
PMCID: PMC4589445  PMID: 26272026
oncolytic virotherapy; breast cancer; Aurora A inhibitor; alisertib; measles virus; MV-NAP
2.  Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses 
Journal of Virology  2014;89(4):2136-2148.
ABSTRACT
The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed.
IMPORTANCE A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.
doi:10.1128/JVI.02339-14
PMCID: PMC4338872  PMID: 25473063
3.  PRECLINICAL SAFETY AND ACTIVITY OF RECOMBINANT VSV-IFN-B IN AN IMMUNOCOMPETENT MODEL OF SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK 
Head & neck  2014;36(11):1619-1627.
Background
VSV-IFN-β, a recombinant vesicular stomatitis virus expressing interferon-β, has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-β in syngeneic SCC models.
Methods and Results
In vitro, VSV-IFN-β (expressing rat or mouse IFN-β) induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-β or VSV-human-IFN-β in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death. VSV-r-IFN-β replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-β resulted in growth delay and improved survival compared to controls.
Conclusions
The above data confirm safety and feasibility of VSV-IFN-β administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.
doi:10.1002/hed.23502
PMCID: PMC3969865  PMID: 24115092
Squamous cell carcinoma; Vesicular Stomatitis Virus; biodistribution; preclinical studies; syngeneic models
4.  Vesicular stomatitis virus expressing interferon-β is oncolytic and promotes antitumor immune responses in a syngeneic murine model of non-small cell lung cancer 
Oncotarget  2015;6(32):33165-33177.
Vesicular stomatitis virus (VSV) is a potent oncolytic virus for many tumors. VSV that produces interferon-β (VSV-IFNβ) is now in early clinical testing for solid tumors. Here, the preclinical activity of VSV and VSV-IFNβ against non-small cell lung cancer (NSCLC) is reported. NSCLC cell lines were treated in vitro with VSV expressing green fluorescence protein (VSV-GFP) and VSV-IFNβ. VSV-GFP and VSV-IFNβ were active against NSCLC cells. JAK/STAT inhibition with ruxolitinib re-sensitized resistant H838 cells to VSV-IFNβ mediated oncolysis. Intratumoral injections of VSV-GFP and VSV-IFNβ reduced tumor growth and weight in H2009 nude mouse xenografts (p < 0.01). A similar trend was observed in A549 xenografts. Syngeneic LM2 lung tumors grown in flanks of A/J mice were injected with VSV-IFNβ intratumorally. Treatment of LM2 tumors with VSV-IFNβ resulted in tumor regression, prolonged survival (p < 0.0001), and cure of 30% of mice. Intratumoral injection of VSV-IFNβ resulted in decreased tumor-infiltrating regulatory T cells (Treg) and increased CD8+ T cells. Tumor cell expression of PDL-1 was increased after VSV-IFNβ treatment. VSV-IFNβ has potent antitumor effects and promotes systemic antitumor immunity. These data support further clinical investigation of VSV-IFNβ for NSCLC.
PMCID: PMC4741756  PMID: 26431376
oncolytic virus; NSCLC; interferon-β; VSV; Treg
7.  Measles vaccine strains for virotherapy of non-small cell lung carcinoma 
Introduction
Oncolytic virus therapy is a promising therapy for numerous tumor types. Edmonston-strain measles virus (MV) has been tested in clinical trials for ovarian cancer, glioma, and myeloma. Therefore, the antitumor activity of MV against NSCLC was assessed.
Methods
Human NSCLC cells and immortalized lung epithelial cell lines, Beas2B, were infected with either MV producing GFP (MV-GFP) or MV producing carcinoembryonic antigen (MV-CEA). Cells were assessed for viability, induction of apoptosis by caspase and PARP cleavage, and for viral transgene production. The dependency of MV entry on CD46 and nectin-4 were determined using blocking antibodies. The role of host translational activity on viral replication was assessed by overexpression of eIF4E and translation inhibition. Antitumor activity was assessed by measuring treated NSCLC xenografts from flanks of nude mice.
Results
MV infection of NSCLC cells results in potent cell killing in most of the cell lines compared to immortalized Beas2B cells and induces apoptosis. MV infection was prevented by blocking of CD46, however was independent of nectin-4 blockade. Tumor weights are diminished following intratumoral injections of MV-CEA in 1 of 2 cell lines and result in detectable viral transgene in serum of mice.
Conclusions
These data indicate that MV is oncolytic for human NSCLC and this was independent of nectin-4 expression. Dysregulated protein translational machinery may play a role in determining tumor tropism in NSCLC. MV combined with gemcitabine could be explored further as chemovirotherapy for NSCLC.
doi:10.1097/JTO.0000000000000214
PMCID: PMC4145613  PMID: 25157763
non-small cell lung cancer; measles virus; translation
8.  Oncolytic Measles Virus Expressing the Sodium Iodide Symporter to Treat Drug-Resistant Ovarian Cancer 
Cancer research  2014;75(1):22-30.
Edmonston vaccine strains of measles virus (MV) have significant antitumor activity in mouse xenograft models of ovarian cancer. MV engineered to express the sodium iodide symporter gene (MV-NIS) facilitates localization of viral gene expression and offers a tool for tumor radiovirotherapy. Here, we report results from a clinical evaluation of MV-NIS in patients with taxol- and platinum-resistant ovarian cancer. MV-NIS was given intraperitoneally every 4 weeks for up to 6 cycles. Treatment was well tolerated and associated with promising median overall survival in these patients with heavily pretreated ovarian cancer; no dose-limiting toxicity was observed in 16 patients treated at high-dose levels (108–109 TCID50), and their median overall survival of 26.5 months compared favorably with other contemporary series. MV receptor CD46 and nectin-4 expression was confirmed by immunohistochemistry in patient tumors. Sodium iodide symporter expression in patient tumors after treatment was confirmed in three patients by 123I uptake on SPECT/CTs and was associated with long progression-free survival. Immune monitoring posttreatment showed an increase in effector T cells recognizing the tumor antigens IGFBP2 and FRα, indicating that MV-NIS treatment triggered cellular immunity against the patients' tumor and suggesting that an immune mechanism mediating the observed antitumor effect. Our findings support further clinical evaluation of MV-NIS as an effective immunovirotherapy.
doi:10.1158/0008-5472.CAN-14-2533
PMCID: PMC4377302  PMID: 25398436
9.  Systemically delivered measles virus-infected mesenchymal stem cells can evade host immunity to inhibit liver cancer growth 
Journal of hepatology  2013;59(5):999-1006.
Background & Aims
Although attenuated measles virus (MV) has demonstrated potent oncolytic activities towards human cancers, it has not yet been widely adopted into clinical practice. One of the major hurdles is the presence of pre-existing anti-MV immunity in the recipients. In this study, we have evaluated the combination of the potent oncolytic activity of the attenuated MV with the unique immunoprivileged and tumor-tropic biological properties of human bone marrow-derived mesenchymal stem cells (BM-hMSCs) to combat human hepatocellular carcinoma (HCC), orthotopically implanted in SCID mice, passively immunized with human neutralizing antibodies against MV as a preclinical model.
Methods
SCID mice were orthotopically implanted with patient-derived HCC tissues and established HCC cell lines. SCID mice were passively immunized with human neutralizing anti-measles antibodies. Bioluminescence and fluorescence imaging were employed to monitor the ability of systemically delivered MV-infected BM-hMSCs to infiltrate the implanted tumors and their effects on tumor growth.
Results
Systemically delivered MV-infected BM-hMSCs homed to the HCC tumors implanted orthotopically in the liver and it was evidenced that BM-hMSCs could transfer MV infectivity to HCC via heterofusion. Furthermore, therapy with MV-infected BM-hMSCs resulted in significant inhibition of tumor growth in both measles antibody-naïve and passively-immunized SCID mice. By contrast, when cell-free MV viruses were delivered systemically, antitumor activity was evident only in measles antibody-naïve SCID mice.
Conclusions
MV-infected BM-hMSCs cell delivery system provides a feasible strategy to elude the presence of immunity against MV in most of the potential cancer patients to be treated with the oncolytic MV viruses.
doi:10.1016/j.jhep.2013.07.010
PMCID: PMC4324731  PMID: 23867315
Systemic virotherapy; Oncolytic measles virus; Hepatocellular carcinoma; Orthotopically implanted HCC tumor model; Mesenchymal stem cells as cell delivery vehicles; Human neutralizing antibody
10.  Oncolytic Measles and Vesicular Stomatitis Virotherapy for Endometrial Cancer 
Gynecologic oncology  2013;132(1):194-202.
Objective
Current adjuvant therapy for advanced-stage, recurrent, and high-risk endometrial cancer (EC) has not reduced mortality from this malignancy, and novel systemic therapies are imperative. Oncolytic viral therapy has been shown to be effective in the treatment of gynecologic cancers, and we investigated the in vitro and in vivo efficacy of the Edmonston strain of measles virus (MV) and vesicular stomatitis virus (VSV) on EC.
Methods
Human EC cell lines (HEC-1-A, Ishikawa, KLE, RL95-2, AN3 CA, ARK-1, ARK-2, and SPEC-2) were infected with Edmonston strain MV expressing the thyroidal sodium iodide symporter, VSV expressing either human or murine IFN-β, or recombinant VSV with a methionine deletion at residue 51 of the matrix protein and expressing the sodium iodide symporter. Xenografts of HEC-1-A and AN3 CA generated in athymic mice were treated with intratumoral MV or VSV or intravenous VSV.
Results
In vitro, all cell lines were susceptible to infection and cell killing by all 3 VSV strains except KLE. In addition, the majority of EC cell lines were defective in their ability to respond to type I IFN. Intratumoral VSV–treated tumors regressed more rapidly than MV-treated tumors, and intravenous VSV resulted in effective tumor control in 100% of mice. Survival was significantly longer for mice treated with any of the 3 VSV strains compared with saline.
Conclusion
VSV is clearly more potent in EC oncolysis than MV. A phase 1 clinical trial of VSV in EC is warranted.
doi:10.1016/j.ygyno.2013.11.010
PMCID: PMC3946955  PMID: 24246772
11.  Safety Studies on Intravenous Administration of Oncolytic Recombinant Vesicular Stomatitis Virus in Purpose-Bred Beagle Dogs 
Abstract
VSV-IFNβ-NIS is a novel recombinant oncolytic vesicular stomatitis virus (VSV) with documented efficacy and safety in preclinical murine models of cancer. To facilitate clinical translation of this promising oncolytic therapy in patients with disseminated cancer, we are utilizing a comparative oncology approach to gather data describing the safety and efficacy of systemic VSV-IFNβ-NIS administration in dogs with naturally occurring cancer. In support of this, we executed a dose-escalation study in purpose-bred dogs to determine the maximum tolerated dose (MTD) of systemic VSV-hIFNβ-NIS, characterize the adverse event profile, and describe routes and duration of viral shedding in healthy, immune-competent dogs. The data indicate that an intravenous dose of 1010 TCID50 is well tolerated in dogs. Expected adverse events were mild to moderate fever, self-limiting nausea and vomiting, lymphopenia, and oral mucosal lesions. Unexpected adverse events included prolongation of partial thromboplastin time, development of bacterial urinary tract infection, and scrotal dermatitis, and in one dog receiving 1011 TCID50 (10×the MTD), the development of severe hepatotoxicity and symptoms of shock leading to euthanasia. Viral shedding data indicate that detectable viral genome in blood diminishes rapidly with anti-VSV neutralizing antibodies detectable in blood as early as day 5 postintravenous virus administration. While low levels of viral genome copies were detectable in plasma, urine, and buccal swabs of dogs treated at the MTD, no infectious virus was detectable in plasma, urine, or buccal swabs at any of the doses tested. These studies confirm that VSV can be safely administered systemically in dogs, justifying the use of oncolytic VSV as a novel therapy for the treatment of canine cancer.
doi:10.1089/humc.2013.165
PMCID: PMC4124537  PMID: 24219832
12.  Vesiculovirus Neutralization by Natural IgM and Complement 
Journal of Virology  2014;88(11):6148-6157.
ABSTRACT
Because of its very low human seroprevalence, vesicular stomatitis virus (VSV) has promise as a systemic oncolytic agent for human cancer therapy. However, as demonstrated in this report, the VSV infectious titer drops by 4 log units during the first hour of exposure to nonimmune human serum. This neutralization occurs relatively slowly and is mediated by the concerted actions of natural IgM and complement. Maraba virus, whose G protein is about 80% homologous to that of VSV, is relatively resistant to the neutralizing activity of nonimmune human serum. We therefore constructed and rescued a recombinant VSV whose G gene was replaced by the corresponding gene from Maraba virus. Comparison of the parental VSV and VSV with Maraba G substituted revealed nearly identical host range properties and replication kinetics on a panel of tumor cell lines. Moreover, in contrast to the parental VSV, the VSV with Maraba G substituted was resistant to nonimmune human serum. Overall, our data suggest that VSV with Maraba G substituted should be further investigated as a candidate for human systemic oncolytic virotherapy applications.
IMPORTANCE Oncolytic virotherapy is a promising approach for the treatment of disseminated cancers, but antibody neutralization of circulating oncolytic virus particles remains a formidable barrier. In this work, we developed a pseudotyped vesicular stomatitis virus (VSV) with a glycoprotein of Maraba virus, a closely related but serologically distinct member of the family Rhabdoviridae, which demonstrated greatly diminished susceptibility to both nonimmune and VSV-immune serum neutralization. VSV with Maraba G substituted or lentiviral vectors should therefore be further investigated as candidates for human systemic oncolytic virotherapy and gene therapy applications.
doi:10.1128/JVI.00074-14
PMCID: PMC4093862  PMID: 24648451
13.  Remission of Disseminated Cancer After Systemic Oncolytic Virotherapy 
Mayo Clinic proceedings  2014;89(7):926-933.
MV-NIS is an engineered measles virus that is selectively destructive to myeloma plasma cells and can be monitored by noninvasive radioiodine imaging of NIS gene expression. Two measles-seronegative patients with relapsing drug-refractory myeloma and multiple glucose-avid plasmacytomas were treated by intravenous infusion of 1011 TCID50 (50% tissue culture infectious dose) infectious units of MV-NIS. Both patients responded to therapy with M protein reduction and resolution of bone marrow plasmacytosis. Further, one patient experienced durable complete remission at all disease sites. Tumor targeting was clearly documented by NIS-mediated radioiodine uptake in virus-infected plasmacytomas. Toxicities resolved within the first week after therapy. Oncolytic viruses offer a promising new modality for the targeted infection and destruction of disseminated cancer.
doi:10.1016/j.mayocp.2014.04.003
PMCID: PMC4225126  PMID: 24835528
14.  Measles virus expressed Helicobacter pylori neutrophil-activating protein significantly enhances the immunogenicity of poor immunogens 
Vaccine  2013;31(42):4795-4801.
Helicobacter pylori neutrophil-activating protein (NAP) is a toll-like receptor 2 (TLR2) agonist and potent immunomodulator inducing Th1-type immune response. Here we present data about characterization of the humoral immune response against NAP-tagged antigens, encoded by attenuated measles virus (MV) vector platform, in MV infection susceptible type I interferon receptor knockout and human CD46 transgenic (Ifnarko-CD46Ge) mice. Immunogenicity of MV expressing a full-length human immunoglobulin lambda light chain (MV-lambda) was compared to that of MV expressing lambda-NAP chimeric protein (MV-lambda-NAP). MV-lambda-NAP immunized Ifnarko-CD46Ge mice developed significantly higher (6–20-fold) anti-lambda ELISA titers as compared to the MV-lambda-immunized control animal group, indicating that covalently-linked NAP co-expression significantly enhanced lambda immunogenicity. In contrast, ELISA titers against MV antigens were not significantly different between the animals vaccinated with MV-lambda or MV-lambda-NAP. NAP-tagged antigen expression did not affect development of protective anti-measles immunity. Both MV-lambda and MV-lambda-NAP-immunized groups showed strong virus neutralization serum titers in plaque reduction microneutralization test. These results demonstrated that MV-encoded lambda-NAP is highly immunogenic as compared to the unmodified full-length lambda chain. Boost of immune response to poor immunogens using live vectors expressing NAP-tagged chimeric antigens is an attractive approach with potential application in immunoprophylaxis of infectious diseases and cancer immunotherapy.
doi:10.1016/j.vaccine.2013.07.085
PMCID: PMC3903391  PMID: 23948230
Helicobacter pylori; Neutrophil-activating protein; Chimeric antigens; Immunogenicity; Measles virus vaccine
15.  Meningeal myeloma deposits adversely impact the therapeutic index of an oncolytic VSV 
Cancer gene therapy  2013;20(11):616-621.
Vesicular Stomatitis Virus (VSV) is neuropathogenic in rodents but can be attenuated 50-fold by engineering the mouse interferon-beta (IFN-β) gene into its genome. Intravenously administered VSVs encoding IFN-β have potent activity against subcutaneous tumors in the 5TGM1 mouse myeloma model, without attendant neurotoxicity. However, when 5TGM1 tumor cells were seeded intravenously, virus-treated mice with advanced myeloma developed clinical signs suggestive of meningoencephalitis. Co-administration of a known active antimyeloma agent did not prolong survival, further suggesting that deaths were due to viral toxicity, not tumor burden. Histological analysis revealed that systemically administered 5TGM1 cells seed to the CNS forming meningeal tumor deposits and that VSV infects and destroys these tumors. Death is presumably a consequence of meningeal damage and/or direct transmission of virus to adjacent neural tissue. In light of these studies, extreme caution is warranted in clinical testing of attenuated VSVs, particularly in patients with CNS tumor deposits.
doi:10.1038/cgt.2013.63
PMCID: PMC3855306  PMID: 24176894
16.  Neutralization capacity of measles virus H protein specific IgG determines the balance between antibody-enhanced infectivity and protection in microglial cells 
Virus research  2012;172(0):15-23.
Neutralizing antibodies directed against measles virus (MV) surface glycoproteins prevent viral attachment and entry through the natural receptors. H protein specific IgG can enhance MV infectivity in macrophages via Fcγ receptor (FcγR)-dependent mechanism. H-specific IgM, anti-F antibodies and complement cascade activation are protective against antibody-mediated enhancement of MV infection. However, protective role of anti-H IgG against antibody-enhanced infection is not well understood. Here we designed a set of experiments to test the protective effect of H-specific IgG against FcγR-mediated infection in microglial cells. Microglial cells are also potential target of the antibody-mediated enhancement and spread of MV infection in the central nervous system. A partially neutralizing IgG monoclonal antibody (MAb) CL55, specific for MV H protein, at 10 μg/ml enhanced MV infection in mouse microglial cells by 13-14-fold. Infection-enhancing antibody concentrations induced large multinucleated syncytia formation 48-72 h post inoculation. We generated anti-H IgG MAb 20H6 with a strong neutralization capacity >1:80,000 at 1 mg/ml concentration in MV plaque-reduction neutralization assay. In contrast to the partially protective MAb CL55, enhancement of MV infectivity by MAb 20H6 required dilutions below the 1:120 serum titer considered protective against measles infection in humans. At a concentration of 10 μg/ml MAb 20H6 exhibited a dominant protective effect and prevented MAb CL55-mediated enhancement of MV infection and virus-mediated fusion. These results indicate that neutralization capacity of the H-specific IgG determines the balance between antibody enhancement and protection against MV infection in microglial cells.
doi:10.1016/j.virusres.2012.12.002
PMCID: PMC3612881  PMID: 23266401
measles virus; microglial cells; antibody-enhanced infection
17.  Oncolytic measles virus strains have significant antitumor activity against glioma stem cells 
Gene therapy  2012;20(4):444-449.
Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of MV derivatives against GSC. We generated neurosphere cultures from patient derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM 44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared to mock infected controls (p=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP treated animals compared to inactivated virus treated controls (GBM6 p=0.0021, GBM12 p=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV treated mice. Conclusion: Measles virus derivatives have significant antitumor activity against glioma derived stem cells in vitro and in vivo.
doi:10.1038/gt.2012.62
PMCID: PMC3509233  PMID: 22914495
measles virus; gliomas; stem cells; MV-CEA; MV-NIS
18.  PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice 
Journal of Virology  2013;87(7):3752-3759.
We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)–PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.
doi:10.1128/JVI.02832-12
PMCID: PMC3624195  PMID: 23325695
19.  Mathematical Model for Radial Expansion and Conflation of Intratumoral Infectious Centers Predicts Curative Oncolytic Virotherapy Parameters 
PLoS ONE  2013;8(9):e73759.
Simple, inductive mathematical models of oncolytic virotherapy are needed to guide protocol design and improve treatment outcomes. Analysis of plasmacytomas regressing after a single intravenous dose of oncolytic vesicular stomatitis virus in myeloma animal models revealed that intratumoral virus spread was spatially constrained, occurring almost exclusively through radial expansion of randomly distributed infectious centers. From these experimental observations we developed a simple model to calculate the probability of survival for any cell within a treated tumor. The model predicted that small changes to the density of initially infected cells or to the average maximum radius of infected centers would have a major impact on treatment outcome, and this was confirmed experimentally. The new model provides a useful and flexible tool for virotherapy protocol optimization.
doi:10.1371/journal.pone.0073759
PMCID: PMC3770695  PMID: 24040057
20.  Curative one-shot systemic virotherapy in murine myeloma 
Leukemia  2012;26(8):1870-1878.
Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity, and facilitate noninvasive monitoring of its intratumoral spread. Using high resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers which expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 hours after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Anti-myeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.
doi:10.1038/leu.2012.70
PMCID: PMC3411853  PMID: 22425894
Oncolytic Virotherapy; multiple myeloma; Vesicular Stomatitis Virus; Intravenous; Immunotherapy
21.  Optimizing patient derived mesenchymal stem cells as virus carriers for a Phase I clinical trial in ovarian cancer 
Background
Mesenchymal stem cells (MSC) can serve as carriers to deliver oncolytic measles virus (MV) to ovarian tumors. In preparation for a clinical trial to use MSC as MV carriers, we obtained cells from ovarian cancer patients and evaluated feasibility and safety of this approach.
Methods
MSC from adipose tissues of healthy donors (hMSC) and nine ovarian cancer patients (ovMSC) were characterized for susceptibility to virus infection and tumor homing abilities.
Results
Adipose tissue (range 0.16-3.96 grams) from newly diagnosed and recurrent ovarian cancer patients yielded about 7.41×106 cells at passage 1 (range 4–9 days). Phenotype and doubling times of MSC were similar between ovarian patients and healthy controls. The time to harvest of 3.0×108 cells (clinical dose) could be achieved by day 14 (range, 9–17 days). Two of nine samples tested had an abnormal karyotype represented by trisomy 20. Despite receiving up to 1.6×109 MSC/kg, no tumors were seen in SCID beige mice and MSC did not promote the growth of SKOV3 human ovarian cancer cells in mice. The ovMSC migrated towards primary ovarian cancer samples in chemotaxis assays and to ovarian tumors in athymic mice. Using non-invasive SPECT-CT imaging, we saw rapid co-localization, within 5–8 minutes of intraperitoneal administration of MV infected MSC to the ovarian tumors. Importantly, MSC can be pre-infected with MV, stored in liquid nitrogen and thawed on the day of infusion into mice without loss of activity. MV infected MSC, but not virus alone, significantly prolonged the survival of measles immune ovarian cancer bearing animals.
Conclusions
These studies confirmed the feasibility of using patient derived MSC as carriers for oncolytic MV therapy. We propose an approach where MSC from ovarian cancer patients will be expanded, frozen and validated to ensure compliance with the release criteria. On the treatment day, the cells will be thawed, washed, mixed with virus, briefly centrifuged and incubated for 2 hours with virus prior to infusion of the virus/MSC cocktail into patients.
doi:10.1186/1479-5876-11-20
PMCID: PMC3567956  PMID: 23347343
Mesenchymal stem cell; Virotherapy; Ovarian cancer; Safety; Efficacy; Optimization
22.  A Single-Amino-Acid Substitution in the TvbS1 Receptor Results in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroups B and D and Resistance to Infection by Subgroup E In Vitro and In Vivo▿  
Journal of Virology  2007;82(5):2097-2105.
The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type TvbS1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvbr2, encodes a mutant TvbS1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in TvbS1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the TvbS1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.
doi:10.1128/JVI.02206-07
PMCID: PMC2258910  PMID: 18094190
23.  Pinhole Micro-SPECT/CT for Noninvasive Monitoring and Quantitation of Oncolytic Virus Dispersion and Percent Infection in Solid Tumors 
Gene therapy  2011;19(3):279-287.
The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). NIS RNA level and dispersion pattern were determined in control and MV-NIS infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography, and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2 = 0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from noninfected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection and can replace more time-consuming and expensive analyses (eg, autoradiography and IHC) that require animal sacrifice.
doi:10.1038/gt.2011.107
PMCID: PMC3198860  PMID: 21753796
NIS; measles virus; micro-SPECT/CT; oncolytic virus; quantitation; sodium iodide symporter
24.  Polyinosinic acid decreases sequestration and improves systemic therapy of measles virus 
Cancer gene therapy  2011;19(3):202-211.
Off target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 minutes post virus administration. Circulating virus was still detectable 30 mins post infusion in mice predosed with poly(I) while no detectable MV was found at 5–10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.
doi:10.1038/cgt.2011.82
PMCID: PMC3288770  PMID: 22116376
oncolytic measles virus; mononuclear phagocytic system; sequestration; poly(I); scavenger receptor; SKOV3ip.1; KAS6/1
25.  The Receptor for the Subgroup C Avian Sarcoma and Leukosis Viruses, Tvc, Is Related to Mammalian Butyrophilins, Members of the Immunoglobulin Superfamily 
Journal of Virology  2005;79(16):10408-10419.
The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.
doi:10.1128/JVI.79.16.10408-10419.2005
PMCID: PMC1182627  PMID: 16051833

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