Citations

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.

ABSTRACT 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.

AbstractBackgroundRecombinant vesicular stomatitis virus expressing interferon‐β (VSV‐IFN‐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.MethodsIn vitro, VSV‐IFN‐β (expressing rat or mouse interferon [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.ResultsVSV‐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 with controls.ConclusionThe 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. © 2014 Wiley Periodicals, Inc. Head Neck 36: 1619–1627, 2014

ABSTRACTWe 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 vitrostudies 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.

ABSTRACT 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.

ABSTRACT 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.

ABSTRACTA novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. StandardMus musculuslaboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whetherMus paharicould serve as a model for XMRV, primaryMus paharifibroblasts and mice were infected with cell-free XMRV. Infection of cellsin vitroresulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV intoMus paharimice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP+CD4+T cells and CD19+B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3in vivo. These data demonstrate infection ofMus pahariby XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infectionin vivo. These data support the use ofMus paharias a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.