Attenuated Semliki Forest virus for cancer treatment in dogs: safety assessment in two laboratory Beagles.

BACKGROUND: Dogs suffer from spontaneous tumors which may be amenable to therapies developed for human cancer patients, and dogs may serve as large-animal cancer models. A non-pathogenic Semliki Forest virus vector VA7-EGFP previously showed promise in targeting human tumor xenografts in mice, but the oncolytic capacity of the virus in canine cancer cells and the safety of the virus in higher mammals such as dogs, are not known. We therefore assessed the oncolytic potency of VA7-EGFP against canine cancer cells by infectivity and viability assays in two dog solid tumor cell lines. Furthermore we performed a 3-week safety study in two adult Beagles which received a single intravenous injection of ~2 × 10(5) plaque forming units of parental A7(74) strain. RESULTS: VA7-EGFP was able to replicate in and kill both canine cancer cell lines tested. No adverse events were observed in either of the two virus-injected adult Beagles and no infective virus could be recovered from any of the biological samples collected over the course of the study. Neutralizing antibodies to Semliki Forest virus became detectable in the dogs at 5 days post infection and remained elevated until study termination. CONCLUSIONS: Based on these results, testing of the oncolytic potential of attenuated Semliki Forest virus in canine cancer patients appears feasible.

Resistance to two heterologous neurotropic oncolytic viruses, Semliki Forest virus and vaccinia virus, in experimental glioma.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference.

Novel oncolytic viruses: riding high on the next wave?

The use of viruses as targeted cancer therapy has shown significant promise, and the list of oncolytic viruses continue to grow. The interest in unexplored viruses as oncolytic agents is a natural corollary to the successes and challenges of those already being examined in the clinical setting. Are these 'new' viruses any more effective than their predecessors? What are the benefits of refining current clinical candidates compared to searching for new ones? This review briefly describes some of these novel oncolytic viruses. It also examines the issues that arise in comparing them to each other. We believe that the viral mechanism of action is a key factor to success and suggest guidelines by which all oncolytic virus candidates could be evaluated.

Intravenously administered alphavirus vector VA7 eradicates orthotopic human glioma xenografts in nude mice.

BACKGROUND: VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point. CONCLUSION: VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus.

Chemical targeting of the innate antiviral response by histone deacetylase inhibitors renders refractory cancers sensitive to viral oncolysis.

Intratumoral innate immunity can play a significant role in blocking the effective therapeutic spread of a number of oncolytic viruses (OVs). Histone deacetylase inhibitors (HDIs) are known to influence epigenetic modifications of chromatin and can blunt the cellular antiviral response. We reasoned that pretreatment of tumors with HDIs could enhance the replication and spread of OVs within malignancies. Here, we show that HDIs markedly enhance the spread of vesicular stomatitis virus (VSV) in a variety of cancer cells in vitro, in primary tumor tissue explants and in multiple animal models. This increased oncolytic activity correlated with a dampening of cellular IFN responses and augmentation of virus-induced apoptosis. These results illustrate the general utility of HDIs as chemical switches to regulate cellular innate antiviral responses and to provide controlled growth of therapeutic viruses within malignancies. HDIs could have a profoundly positive impact on the clinical implementation of OV therapeutics.

Oncolytic viruses in cancer therapy.

Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature's own agents to find and destroy malignant cells. The purpose of this review is to provide an introduction to this very topical field of research and to point out some of the current observations, insights and ideas circulating in the literature. We have strived to acknowledge as many different oncolytic viruses as possible to give a broader picture of targeting cancer using viruses. Some of the newest additions to the panel of oncolytic viruses include the avian adenovirus, foamy virus, myxoma virus, yaba-like disease virus, echovirus type 1, bovine herpesvirus 4, Saimiri virus, feline panleukopenia virus, Sendai virus and the non-human coronaviruses. Although promising, virotherapy still faces many obstacles that need to be addressed, including the emergence of virus-resistant tumor cells.

Semliki Forest virus vectors expressing transforming growth factor beta inhibit experimental autoimmune encephalomyelitis in Balb/c mice.

Cytokine immunomodulation of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, has remained a formidable treatment option, but access into the CNS is hampered due to the impermeability of the blood-brain barrier. In this report, we describe the construction and characterization of CNS-homing gene delivery/therapy vectors based on avirulent Semliki Forest virus (SFV) expressing either native or mutant transforming growth factor beta 1 (TGF-beta1). Biological activity of the expressed inserts was demonstrated by PAI-1 promoter driven luciferase production in mink cells and TGF-beta1 mRNA was demonstrated in the CNS of virus treated mice by in situ hybridization and RT-PCR. Both vectors, when given intraperitoneally to EAE mice significantly reduced disease severity compared to untreated mice. Our results imply that immunomodulation by neurotropic viral vectors may offer a promising treatment strategy for autoimmune CNS disorders.

Oncolytic capacity of attenuated replicative semliki forest virus in human melanoma xenografts in severe combined immunodeficient mice.

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy. Here, we analyzed the oncolytic potential of the replication-competent vector VA7-EGFP, based on the avirulent Semliki Forest virus (SFV) strain A7(74), to kill cancer cells in culture as well as to target s.c. human melanoma xenografts in severe combined immunodeficient (SCID) mice. VA7-EGFP was able to infect most cancer cell lines studied, leading to complete lysis of the cells within 72 hours after infection. In SCID mice grafted with A2058 human melanoma, marked regression of the xenografts was observed following a single injection of 10(6) plaque-forming units of virus given either i.p., i.v., or intratumorally. Histologic analysis revealed the presence of virus not only in all treated tumors but also in the brains of the treated mice, causing progressing neuropathology beginning at day 16 after infection. Following initial oncolysis, clusters of viable tumor cells were observed embedded in connective tissue, and at later stages, encapsulated tumor nodules had formed. Infection of melanoma cells from explant cultures of these nodules revealed that a portion of the cells were resistant to virus. To be eligible for use in virotherapy, the ability of avirulent SFV to spread within tumor tissue may have to be improved and the biological safety of the virus may have to be addressed thoroughly in higher animals.

A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus.

Semliki Forest virus (SFV), an enveloped alphavirus of the family Togaviridae, infects a wide range of mammalian host cells. Most strains are neurotropic but differ in virulence. The authors took advantage of the nonpathogenic properties of SFV strain A7(74), cloned recently in their laboratory, and constructed a replication-proficient expression vector to target the central nervous system (CNS) for heterologous gene expression. The vector, termed VA7, was engineered to drive expression of foreign inserts through a second subgenomic promoter inserted in the viral 3' nontranslated region (NTR). Infectious virus was obtained by in vitro transcription and transfection into BHK cells, and was shown to direct synthesis of heterologous proteins in several mammalian cell lines. Although novel expression vehicle is not applicable for targeting specific cell populations within the CNS in its present form, in cultured rat hippocampal slices, VA7 encoding enhanced green fluorescent protein (EGFP) efficiently transduced pyramidal cells, interneurons, and glial cells. With prolonged time post infection, the number of EGFP-expressing neurons in hippocampal slices increased. Mice infected intraperitoneally with the recombinant virus remained completely asymptomatic but showed CNS expression of EGFP as evidenced by immunohistochemistry. SFV A7(74) is a nonintegrating virus, which gives rise to a randomly distributed, patchy infection of the adult CNS that is cleared within 10 days. With the advantage of noninvasive administration, the expression vector described in this work is thus applicable for short-term gene expression in the CNS.

Semliki Forest virus A7(74) transduces hippocampal neurons and glial cells in a temperature-dependent dual manner.

In central nervous system (CNS) tissue preparations, wild-type Semliki Forest virus (SFV) mainly infects neurons, and in vivo it causes lethal encephalitis in neonatal and adult rodents. The SFV strain A7(74), by contrast, is avirulent in adult rodents, triggering only limited CNS infection. To examine A7(74) infection in hippocampal tissue, the authors constructed a replicon, termed SFV(A774nsP)-GFP, expressing green fluorescent protein. The results were compared to replication-proficient recombinant A7(74) encoding GFP, named VA7-EGFP. As nonstructural gene mutations can confer temperature sensitivity, the authors also tested whether infection was temperature-dependent. Indeed, at 31 degrees C both viral recombinants transduced significantly more baby hamster kidney cells than at 37 degrees C. When rat hippocampal slices and dissociated cells were incubated at 37 degrees C, SFV(A774nsP)-GFP transduced glial cells but virtually no neurons-the opposite of conventional SFV. For VA7-EGFP at 37 degrees C, the preferred GFP-positive cells in hippocampal slices were also non-neuronal cells. At 31 degrees C, however, a more wild-type phenotype was found, with 33% and 94% of the GFP-positive cells being neurons for SFV(A774nsP)-GFP in slices and dissociated cells, respectively, and 94% neurons for VA7-EGFP in slices. Immunochemical and electrophysiological analyses confirmed that at 37 degrees C virtually all cells transduced by SFV(A774nsP)-GFP in slices were astrocytes, while at 31 degrees C they also contained neurons. These results show that in addition to the developmental age, the temperature determines which cell type becomes infected by A7(74). Our data suggest that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurons at body temperature, whereas it still does so at lower temperatures.