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 similar to 2 x 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.Peer reviewe

SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication

Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

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.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.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

Tumor Restrictions to Oncolytic Virus

Oncolytic virotherapy has advanced since the days of its conception but therapeutic efficacy in the clinics does not seem to reach the same level as in animal models. One reason is premature oncolytic virus clearance in humans, which is a reasonable assumption considering the immune-stimulating nature of the oncolytic agents. However, several studies are beginning to reveal layers of restriction to oncolytic virotherapy that are present before an adaptive neutralizing immune response. Some of these barriers are present constitutively halting infection before it even begins, whereas others are raised by minute cues triggered by virus infection. Indeed, we and others have noticed that delivering viruses to tumors may not be the biggest obstacle to successful therapy, but instead the physical make-up of the tumor and its capacity to mount antiviral defenses seem to be the most important efficacy determinants. In this review, we summarize the constitutive and innate barriers to oncolytic virotherapy and discuss strategies to overcome them

Cell Membrane-Associated Measles Virus Components Inhibit Antigen Processing

AbstractMeasles virus (MV)-induced immune suppression is an important reason for MV-associated mortality and morbidity. Despite numerous studies, the mechanisms of immune suppression still remain poorly defined. In the present study we analyzed the effect of MV components on the T-cell recognition of specific non-MV antigens. We demonstrated that even inactivated MV could inhibit the presentation of unprocessed protein antigen to specific T cells, whereas MV did not affect the responses of specific T cells to representative synthetic peptide epitopes derived from complex antigens. The inhibition was induced by MV-infected cell membranes. The kinetics of the MV-dependent inhibition suggested an impaired antigen processing in mononuclear cells as addition of MV-infected cell debris 4 h after the beginning of cell cultures no longer inhibited T-cell responsiveness