A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses

IntroductionMetastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM.MethodsWe developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-β) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNβ-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNβ-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective.Results12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses.DiscussionOur study found that VSV-IFNβ -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNβ-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen

Long-Term Infection and Vertical Transmission of a Gammaretrovirus in a Foreign Host Species

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari

A COMPARATIVE STUDY ON THE ENVIRONMENTAL AMMONIA TOLERANCE OF TWO MUDSKIPPERS

Master'sMASTER OF SCIENC

A brief review of reporter gene imaging in oncolytic virotherapy and gene therapy

Reporter gene imaging (RGI) can accelerate development timelines for gene and viral therapies by facilitating rapid and noninvasive in vivo studies to determine the biodistribution, magnitude, and durability of viral gene expression and/or virus infection. Functional molecular imaging systems used for this purpose can be divided broadly into deep-tissue and optical modalities. Deep-tissue modalities, which can be used in animals of any size as well as in human subjects, encompass single photon emission computed tomography (SPECT), positron emission tomography (PET), and functional/molecular magnetic resonance imaging (f/mMRI). Optical modalities encompass fluorescence, bioluminescence, Cerenkov luminescence, and photoacoustic imaging and are suitable only for small animal imaging. Here we discuss the mechanisms of action and relative merits of currently available reporter gene systems, highlighting the strengths and weaknesses of deep tissue versus optical imaging systems and the hardware/reagents that are used for data capture and processing. In light of recent technological advances, falling costs of imaging instruments, better availability of novel radioactive and optical tracers, and a growing realization that RGI can give invaluable insights across the entire in vivo translational spectrum, the approach is becoming increasingly essential to facilitate the competitive development of new virus- and gene-based drugs

Retargeting Vesicular Stomatitis Virus Using Measles Virus Envelope Glycoproteins

Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity, but infects a broad range of cell types. Here, we used the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect VSV entry and infection specifically to tumor-associated receptors. Replication-defective VSV, deleted of its glycoprotein gene (VSVΔG), was pseudotyped with MV-F and MV-H displaying single-chain antibodies (scFv) specific for epidermal growth factor receptor (EGFR), folate receptor (FR), or prostate membrane-specific antigen (PSMA). Viral titers were ∼10 5 PFU/ml, but could be concentrated to 10 7 PFU/ml. Immunoblotting confirmed incorporation of the MV-H-scFv and MV-F into functional VSV virions. Although VSV-G was able to infect all tumor cell lines tested, the retargeted VSV infected only cells that expressed the targeted receptor. In vivo specificities of the EGFR-, FR-, and PSMA-retargeted VSV were assessed by intratumoral injection into human tumor xenografts. Analysis of green fluorescent protein reporter gene expression indicated that VSV infection was restricted to receptor-positive tumors. In summary, we have demonstrated for the first time that VSV can be efficiently retargeted to different cellular receptors using the measles display technology, yielding retargeted VSV vectors that are highly specific for tumors that express the relevant receptor. Ayala-Breton and colleagues use the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect vesicular stomatitis virus (VSV) entry and infection specifically to tumor-associated receptors such as epidermal growth factor receptor, folate receptor, and prostate membrane-specific antigen. In vivo expression of the all retargeted VSV was restricted to receptor-positive human tumor xenografts

Abstract 4313: Preclinical safety and efficacy of VSV-IFN-b in a syngeneic, immunocompetent model of head and neck squamous cell carcinoma

Abstract Objective&amp; Background: VSV, an RNA virus of the rhabdoviridae family, induces potent cytolytic effects in immortalized cells and cells with defects pathways of IFN or PKR in vitro &amp; in vivo. To take advantage of defects in IFN response in tumors, and to improve the virus’ therapeutic window, “second generation” VSVs expressing IFN-b were developed and characterized (Obuchi et. al. 2003). The aims of this study are to evaluate the effects of VSV-IFN-b in human and rodent head &amp; neck cancer cell lines and to assess the safety and in vivo antitumor efficacy of VSV-IFN-b in syngeneic rat head &amp; neck cancer models. Methods: Rat (FAT-7) and mouse (SCC VII) squamous cell carcinoma cells were infected in vitro with VSV-rat-IFN-b and VSV-mouse -IFN-b. Viruses were propagated using the BHK-21 cells according to established protocols. ELISA &amp; Western blotting were performed to identify the expression of rat-IFN-b post infection, in SCC-VII &amp; FAT-7 cells, respectively. Viral cytotoxicity during normoxia and hypoxia, viral replication and IFN-b expression were assessed to determine sensitivity of rat and mouse SCC cells to VSV-IFN-b. An in vivo model of rat squamous cell carcinoma was established by injection of 3×106/ml of FAT-7 cells SC on the floor of the mouth in immunocompetent female Fisher-344 rats. VSV-rat-IFN-b was administered intratumorally at different doses and schedules when tumors reached a diameter of 70-100 mm3. Antitumor efficacy and toxicity were evaluated during the study. Results: VSV-IFN-b induced significant cytotoxicity and successfully propagated in FAT-7 and SCC VII during normoxia and hypoxia. IFN-b production was increased at 24 and 48 hours after viral infection. Rat SCC cells were more susceptible to VSV-rat-IFN-b than mouse cells, and the cytotoxic effects were similar to VSV-human IFN b on rat cells. Tumors were successfully induced and were palpable at 21 days after inoculation. IT administration of VSV-rat-IFN-b resulted in reduction in tumor size and improved survival compared to the non-treated controls. The antitumor effects of one dose of the virus at 5×108 pfu was more effective than higher doses (5×109) or repeated doses. IV administration of a single dose of VSV-rat-IFN-b (5×108) exerted similar antitumor effects as IT administration. No signs of acute toxicity were observed in treated rats after single or repeated IT or IV virus administration. Conclusion: VSV-IFN-b induces cytotoxicity in FAT-7 and SCC-VII cancer cells during normoxia and hypoxia. In vivo, VSV-rat-IFN-b was associated with antitumor effects and prolongation of survival compared to untreated controls. Characterization of viremia, tissue biodistribution, in vivo viral replication and gene expression in vivo are underway in preparation for future clinical evaluation of VSV-human-IFN-b in subjects with advanced head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2011-4313</jats:p

Affinity Thresholds for Membrane Fusion Triggering by Viral Glycoproteins▿

Enveloped viruses trigger membrane fusion to gain entry into cells. The receptor affinities of their attachment proteins vary greatly, from 10−4 M to 10−9 M, but the significance of this is unknown. Using six retargeted measles viruses that bind to Her-2/neu with a 5-log range in affinity, we show that receptor affinity has little impact on viral attachment but is nevertheless a key determinant of infectivity and intercellular fusion. For a given cell surface receptor density, there is an affinity threshold above which cell-cell fusion proceeds efficiently. Suprathreshold affinities do not further enhance the efficiency of membrane fusion

Synergistic activity of the proteasome inhibitor PS-341 with non-myeloablative 153-Sm-EDTMP skeletally targeted radiotherapy in an orthotopic model of multiple myeloma

Multiple myeloma is a highly radiosensitive skeletal malignancy, but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, 2 doses of PS-341 (0.5 mg/kg), or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22, and 28 days, respectively. In contrast, mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg), 1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P < .001). In addition to prolonged survival, this treatment combination yielded reduced clonogenicity of bone marrow–resident 5TGM1 cells, reduced serum myeloma–associated paraprotein levels, and better preservation of bone mineral density. Myelosuppression, determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors, did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent, selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma