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2.  Oncolytic vesicular stomatitis virus and bortezomib are antagonistic against myeloma cells in vitro but have additive anti-myeloma activity in vivo 
Experimental hematology  2013;41(12):10.1016/j.exphem.2013.09.005.
Multiple myeloma cells are highly sensitive to the oncolytic effects of vesicular stomatitis virus (VSV), which specifically targets and kills cancer cells. Myeloma cells are also exquisitely sensitive to the cytotoxic effects of the clinically-approved proteasome inhibitor bortezomib. Therefore, we sought to determine if the combination of VSV and bortezomib would enhance tumor cell killing. However, as shown here, combining these two agents in vitro results in antagonism. We show that bortezomib inhibits VSV replication and spread. We found that bortezomib inhibits VSV-induced NF-κB activation and, using the NF-κB-specific inhibitor BMS-345541, that VSV requires NF-κB activity in order to efficiently spread in myeloma cells. In contrast to other cancer cell lines, viral titer is not recovered by BMS-345541 when myeloma cells are pre-treated with interferon (IFN)-β. Thus, inhibiting NF-κB activity, either with bortezomib or BMS-345541, results in reduced VSV titers in myeloma cells in vitro. However, when VSV and bortezomib are combined in vivo, in two syngeneic, immunocompetent myeloma models, the combination reduces tumor burden to a greater degree than VSV as a single agent. Intra-tumoral VSV viral load is unchanged when mice are concomitantly treated with bortezomib as compared to VSV treatment alone. To our knowledge, this is the first report analyzing the combination of VSV and bortezomib in vivo. Although antagonism between VSV and bortezomib is seen in vitro, analyzing these cells in the context of their host environment shows that bortezomib enhances VSV response, suggesting that this combination will also enhance response in myeloma patients.
doi:10.1016/j.exphem.2013.09.005
PMCID: PMC3858457  PMID: 24067362
Multiple myeloma; oncolytic viruses; vesicular stomatitis virus; bortezomib
3.  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
4.  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
5.  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
6.  Faster Replication and Higher Expression Levels of Viral Glycoproteins Give the Vesicular Stomatitis Virus/Measles Virus Hybrid VSV-FH a Growth Advantage over Measles Virus 
Journal of Virology  2014;88(15):8332-8339.
ABSTRACT
VSV-FH is a hybrid vesicular stomatitis virus (VSV) with a deletion of its G glycoprotein and encoding the measles virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways.
IMPORTANCE We studied the cytotoxic activity of a vesicular stomatitis/measles hybrid virus (VSV-FH), which is superior to that of measles virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and a more potent cytopathic effect in permissive cells. Importantly, VSV-FH, similar to MV, can discriminate between low- and high-expressing CD46 cells, a phenotype important for cancer therapy as the virus will be able to preferentially infect cancer cells that overexpress CD46 over low-CD46-expressing normal cells.
doi:10.1128/JVI.03823-13
PMCID: PMC4135973  PMID: 24829351
7.  Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels 
Journal of the Royal Society Interface  2015;12(102):20141079.
Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.
doi:10.1098/rsif.2014.1079
PMCID: PMC4277102  PMID: 25411409
hydrogel; collagen; functionalization; swelling; atomic force microscopy; covalent network
8.  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
9.  Importance of Achieving Stringent Complete Response After Autologous Stem-Cell Transplantation in Multiple Myeloma 
Journal of Clinical Oncology  2013;31(36):4529-4535.
Purpose
To study the impact of achieving stringent complete response (sCR), an increasingly attainable goal, after autologous stem-cell transplantation (ASCT) in patients with multiple myeloma (MM).
Patients and Methods
Maximal response rates were determined in 445 consecutive patients who underwent ASCT within 12 months of diagnosis of MM. The patients achieving varying degrees of complete response (CR) are the focus of our study.
Results
One hundred and nine patients (25%) achieved sCR after ASCT. The median overall survival (OS) rate from the time of transplantation for patients attaining sCR was not reached (NR), in contrast to those patients achieving conventional complete response (CR; n = 37; OS, 81 months) or near CR (nCR; n = 91; OS, 60 months; P < .001). Five-year OS rates were 80%, 53%, and 47% for sCR, CR, and nCR, respectively. The median time to progression (TTP) from ASCT of patients achieving sCR was significantly longer (50 months) than TTP of patients achieving CR or nCR (20 months and 19 months, respectively). On multivariable analysis, post-ASCT response of sCR was an independent prognostic factor for survival (hazard ratio, 0.44; 95% CI, 0.25 to 0.80; versus CR; P = .008), in addition to proliferation rate, pre-ASCT cytogenetics, and performance status. OS rates of patients attaining sCR continued to remain superior at 2-year landmark (median, NR v 70 months for conventional CR group; P = .007).
Conclusion
Improved long-term outcome is seen after ASCT with achievement of sCR when compared with lesser degrees of responses. Myeloma trials reporting the response rates should identify patients achieving sCR and CR separately, owing to markedly disparate outcomes of the two categories.
doi:10.1200/JCO.2013.49.0086
PMCID: PMC3871514  PMID: 24248686
10.  Modes of hole formation in long-lasting insecticidal nets (LLINs) retrieved from South Eastern Ghana 
Parasites & Vectors  2014;7(1):547.
Background
Long-lasting insecticidal nets (LLINs) are expected to provide biological efficacy for at least three years in the field and be sufficiently durable to maintain physical protection. Unfortunately, LLINs structurally deteriorate during use accumulating holes. Hitherto, definitive identification of the causes of hole formation has been difficult based upon qualitative surveys.
Methods
In this preliminary study, optical and scanning electron microscopy of damage in used polyester (PET) and polyethylene (PE) LLINs randomly collected via a household survey from South Eastern Ghana (n =100) were utilised to identify the cause of individual holes.
Results
Multiple damage mechanisms were identified. In both PET and PE LLINs, the majority of holes were initiated by filament fracture (ductile failure and cutting) and thermal damage.
Conclusions
No strong correlation was found between the bursting strength of retrieved LLINs and overall hole frequency in either the PET or PE nets suggesting that bursting strength is an unreliable predictor of resistance to hole formation if used as a sole parameter.
doi:10.1186/s13071-014-0547-x
PMCID: PMC4279592  PMID: 25476877
Insecticide-treated bednets; Mosquito nets; Damage; Defect; Holes
11.  High scFv-receptor affinity does not enhance the antitumor activity of HER2 retargeted measles virus 
Cancer gene therapy  2014;21(6):256-260.
The relationship between ligand-receptor affinity and antitumor potency of an oncolytic virus was investigated using a panel of six HER2/neu (HER2) targeted measles viruses (MV) displaying single-chain antibodies (scFv) that bind to the same epitope on HER2, but with affinities ranging from 10−6 to 10−11 M. All viruses were able to infect SKOV3ip.1 human ovarian cancer cells in vitro, but only the high affinity MV (Kd > 10−8 M) induced cytopathic effects of syncytia formation in the cell monolayers. In contrast, all six viruses were therapeutically active in vivo against orthotopic human ovarian SKOV3ip.1 tumor xenografts in athymic mice compared to saline treated controls. The oncolytic activities of MV displaying the high affinity scFv (Kd=10−9, 10−10, 10−11 M) were not significantly superior to MV displaying scFv with Kd of 10−8 M or less. Results from this study suggest that increasing the receptor affinity of the attachment protein of an oncolytic measles virus has minimal impact on its in vivo efficacy against a tumor that expresses the targeted receptor.
doi:10.1038/cgt.2014.25
PMCID: PMC4096840  PMID: 24874841
Her2/neu targeted; oncolytic measles virus; single-chain antibody; receptor affinity; receptor density; antitumor potency
12.  Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands 
Journal of Virology  2013;87(24):13543-13555.
We sought proof of principle that tumor-targeting ligands can be displayed on the surface of vesicular stomatitis virus (VSV) by engineering its glycoprotein. Here, we successfully rescued VSVs displaying tumor vasculature-targeting ligands. By using a rational approach, we investigated various feasible insertion sites on the G protein of VSV (VSV-G) for display of tumor vasculature-targeting ligands, cyclic RGD (cRGD) and echistatin. We found seven sites on VSV-G that tolerated insertion of the 9-residue cRGD peptide, two of which could tolerate insertion of the 49-amino acid echistatin domain. All of the ligand-displaying viruses replicated as well as the parental virus. In vitro studies demonstrated that the VSV-echistatin viruses specifically bound to targeted integrins. Since the low-density lipoprotein receptor (LDLR) was recently identified as a major receptor for VSV, we investigated the entry of ligand-displaying viruses after masking LDLR. The experiment showed that the modified viruses can enter the cell independently of LDLR, whereas entry of unmodified virus is significantly blocked by a specific monoclonal antibody against LDLR. Both parental and ligand-displaying viruses displayed equal oncolytic efficacies in a syngeneic mouse myeloma model. We further demonstrated that single-chain antibody fragments against tumor-specific antigens can be inserted at the N terminus of the G protein and that corresponding replication-competent VSVs can be rescued efficiently. Overall, we demonstrated that functional tumor-targeting ligands can be displayed on replication-competent VSVs without perturbing viral growth and oncolytic efficacy. This study provides a rational foundation for the future development of fully retargeted oncolytic VSVs.
doi:10.1128/JVI.02240-13
PMCID: PMC3838279  PMID: 24089573
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.  Meningeal myeloma deposits adversely impact the therapeutic index of an oncolytic VSV 
Cancer gene therapy  2013;20(11):10.1038/cgt.2013.63.
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
15.  Continued improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients 
Leukemia  2013;28(5):1122-1128.
Therapy for multiple myeloma (MM) has dramatically changed in the past decade with introduction of new drugs, but it is not clear if the improvements have been sustained. We studied 1038 patients diagnosed between 2001 and 2010, grouping patients into two five-year periods by diagnosis, 2001–2005 and 2006–2010. The median estimated follow up for the cohort was 5.9 years with 47% alive at last follow up. The median overall survival (OS) for the entire cohort was 5.2 years; 4.6 years for patients in the 2001–2005 group compared with 6.1 years for the 2006–2010 cohort (P=0.002). The improvement was primarily seen among patients over 65 years; the 6-year OS improving from 31% to 56%; P<0.001. Only 10% of patients died during the first year in the latter group, compared with 17% in the earlier cohort (P<0.01), suggesting improvement in early mortality. The improved outcomes were linked closely to use of one or more new agents in initial therapy. The current results confirm continued survival improvement in MM and highlight the impact of initial therapy with novel agents. Most importantly, we demonstrate that the improved survival is benefitting older patients and that early mortality in this disease has reduced considerably.
doi:10.1038/leu.2013.313
PMCID: PMC4000285  PMID: 24157580
multiple myeloma; survival; IMiDs; proteasome inhibitors
16.  In Vivo Safety, Biodistribution and Antitumor Effects of uPAR Retargeted Oncolytic Measles Virus in Syngeneic Cancer Models 
Gene therapy  2014;21(3):289-297.
The urokinase receptor (uPAR) is a clinically relevant target for novel biological therapies. We have previously rescued oncolytic measles viruses fully retargeted against human (MV-h-uPA) or murine (MV-m-uPA) uPAR. Here, we investigated the in vivo effects of systemic administration of MV-m-uPA in immunocompetent cancer models. MV-m-uPA induced in vitro cytotoxicity and replicated in a receptor dependent manner in murine mammary (4T1), and colon (MC-38 and CT-26) cancer cells. Intravenous administration of MV-m-uPA to 4T1 tumor bearing mice was not associated with significant clinical or laboratory toxicity. Higher MV-N RNA copy numbers were detected in primary tumors, and viable viral particles were recovered from tumor bearing tissues only. Non-tumor bearing organs did not show histological signs of viral induced toxicity. Serum anti-MV antibodies were detected at day 14 of treatment. Immunohistochemistry and immunofluorescence studies confirmed successful tumor targeting and demonstrated enhanced MV-m-uPA induced tumor cell apoptosis in treated, compared to control mice. Significant antitumor effects and prolonged survival were observed after systemic administration of MV-m-uPA in colon (CT-26) and mammary (4T1) cancer models. The above results demonstrate safety and feasibility of uPAR targeting by an oncolytic virus, and confirm significant antitumor effects in highly aggressive syngeneic immunocompetent cancer models.
doi:10.1038/gt.2013.84
PMCID: PMC3949200  PMID: 24430235
Urokinase receptor; measles virus; tumor targeting; syngeneic models
17.  Ablation of Nectin4 Binding Compromises CD46 Usage by a Hybrid Vesicular Stomatitis Virus/Measles Virus 
Journal of Virology  2014;88(4):2195-2204.
Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.
doi:10.1128/JVI.02628-13
PMCID: PMC3911550  PMID: 24335299
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.  Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites 
Journal of Virology  2013;87(6):3217-3228.
Vesicular stomatitis virus (VSV) is potent and a highly promising agent for the treatment of cancer. However, translation of VSV oncolytic virotherapy into the clinic is being hindered by its inherent neurotoxicity. It has been demonstrated that selected picornaviral internal ribosome entry site (IRES) elements possess restricted activity in neuronal tissues. We therefore sought to determine whether the picornavirus IRES could be engineered into VSV to attenuate its neuropathogenicity. We have used IRES elements from human rhinovirus type 2 (HRV2) and foot-and-mouth disease virus (FMDV) to control the translation of the matrix gene (M), which plays a major role in VSV virulence. In vitro studies revealed slowed growth kinetics of IRES-controlled VSVs in most of the cell lines tested. However, in vivo studies explicitly demonstrated that IRES elements of HRV2 and FMDV severely attenuated the neurovirulence of VSV without perturbing its oncolytic potency.
doi:10.1128/JVI.02984-12
PMCID: PMC3592162  PMID: 23283963
20.  Enhancing Cytokine Induced Killer Cell Therapy of Multiple Myeloma 
Experimental hematology  2013;41(6):508-517.
Cytokine induced killer (CIK) cells are in clinical testing against various tumor types including multiple myeloma. Here, we show that CIK cells have activity against subcutaneous and disseminated models of human myeloma (KAS-6/1) which can be enhanced by infecting the CIK cells with an oncolytic measles virus (MV) or by pre-treating the myeloma cells with ionizing radiation (XRT). KAS-6/1 cells were killed by co-culture with CIK or MV-infected CIK (CIK/MV) cells and addition of an anti-NKG2D antibody inhibited cytolysis by 50%. Human bone marrow stromal cells can however reduce CIK and CIK/MV mediated killing of myeloma cells (RPMI 8226, JJN-3 and MM1). In vivo, CIK and CIK/MV prolonged the survival of mice with systemic myeloma, although CIK/MV showed enhanced antitumor activity compared to CIK. Irradiation of the KAS-6/1 cells induced mRNA and protein expression of NKG2D ligands, MICA and MICB, in a dose dependent manner and enhanced delivery of CIK/MV to the irradiated tumors. In both subcutaneous and disseminated myeloma models, XRT at 2 Gy resulted in superior prolongation of the survival of mice given CIK/MV therapy compared to CIK/MV with no XRT. This study demonstrates the potential of CIK against myeloma and that combination of virotherapy with radiation could be used to further enhance therapeutic outcome using CIK cells.
doi:10.1016/j.exphem.2013.01.010
PMCID: PMC3669253  PMID: 23403007
cytokine induced killer cells; virotherapy; myeloma; irradiation; NKG2D; MICA/B
21.  Oral contrast enhances the resolution of in-life NIS reporter gene imaging 
Cancer gene therapy  2013;20(11):10.1038/cgt.2013.57.
NIS reporter gene imaging is an excellent technology for noninvasive cell fate determination in living animals unless the NIS-transduced cells reside in perigastric organs such as spleen, liver, diaphragm, omentum, pancreas, perigastric lymph nodes or perigastric tumor deposits. Here we report that orally administered barium sulfate enhances CT definition of the stomach, masks background gamma ray emissions from the stomach, and enhances signal detection from radiotracer uptake in NIS-transduced organs.
doi:10.1038/cgt.2013.57
PMCID: PMC3867790  PMID: 24030210
22.  Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter for Squamous Cell Cancer of the Head and Neck Radiovirotherapy 
Human Gene Therapy  2011;23(3):295-301.
Abstract
Oncolytic measles virus (MV) encoding the human thyroidal sodium iodide symporter (MV-NIS) has proved to be safe after intraperitoneal or intravenous administration in patients with ovarian cancer or multiple myeloma, respectively, but it has not yet been administered through intratumoral injection in humans. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) usually is locally invasive and spreads to the cervical lymph nodes, which are suitable for the intratumoral administration of oncolytic viruses. To test whether oncolytic MV is an effective treatment for SCCHN, we used oncolytic MV-NIS to infect SCCHN in vitro and in vivo. The data show that SCCHN cells were infected and killed by MV-NIS in vitro. Permissiveness of the tumor cells to MV infection was not affected by irradiation after viral addition. Monitored noninvasively through radioiodine-based single-photon emission computed tomography/computed tomography, intratumorally virus-delivered NIS has concentrated the radioiodine in the MV-NIS–treated tumors in the FaDu mouse xenograft model of human SCCHN, and the antitumor effect could be boosted significantly (p<0.05) either with concomitant cyclophosphamide therapy or with appropriately timed administration of radioiodine 131I. MV-NIS could be a promising new anticancer agent that may substantially enhance the outcomes of standard therapy after intratumoral administration in patients with locally advanced SCCHN.
Li and colleagues investigate the use of oncolytic measles virus encoding human thyroidal sodium iodide symporter (MV-NIS) to treat squamous cell carcinoma of the head and neck (SCCHN) in vitro and in vivo. MV-NIS-treated tumors are able to concentrate administered radioiodine in a mouse xenograft model of human SCCHN, and the antitumor effect is significantly boosted by cyclophosphamide therapy.
doi:10.1089/hum.2011.128
PMCID: PMC3300082  PMID: 22235810
23.  Curative one-shot systemic virotherapy in murine myeloma 
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
24.  Potent systemic therapy of Multiple Myeloma utilizing Oncolytic Vesicular stomatitis virus coding for Interferon-beta 
Cancer Gene Therapy  2012;19(7):443-450.
Multiple myeloma (MM) is an incurable malignancy of plasma secreting B-cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV) expressing Interferon-β (IFNβ) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to specifically destroy cancer cells. We demonstrate here that a single, intravenous dose of VSV-IFNβ specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNβ expression further delayed myeloma progression and significantly enhanced survival compared to VSV expressing human IFNβ. Evaluation of VSV-IFNβ oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma specific oncolytic activity but revealed variable susceptibility to VSV-IFNβ oncolysis. The results indicate that VSV-IFNβ is a potent, safe oncolytic agent that can be systemically administered to effectively target and destroy disseminated myeloma in immune competent mice. IFNβ expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNβ to be a promising vector for further development as a potential therapy for treatment of Multiple myeloma.
doi:10.1038/cgt.2012.14
PMCID: PMC3380174  PMID: 22522623
Oncolytic; virotherapy; myeloma; Vesicular stomatitis virus; systemic
25.  A novel report of cIg-FISH and cytogenetics in POEMS syndrome 
American journal of hematology  2008;83(11):840-841.
POEMS syndrome is a plasma cell proliferative disorder whose pathogenesis is poorly understood. We provide the first report of cytoplasmic immunoglobulin/FISH testing (cIg-FISH) in POEMS syndrome using established myeloma markers. We reviewed all 37 POEMS cases seen at our institution in which cIg-FISH testing had been obtained. Monosomy 13 was seen in 14 of the 37 (38%) cIg-FISH samples. One patient had trisomy 3 and 7. Three patients had IgH translocation t(11;14)(q13;q32). No abnormalities were seen at 17p13(p53). The monosomy 13 is in line with other plasma cell disorders while the low prevalence of hyperdiploidy and abnormalities at 14q32 is unique.
doi:10.1002/ajh.21285
PMCID: PMC3956589  PMID: 18839437

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