Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector.

Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-ß-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-ß protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-ß protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-ß moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-ß; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain.

Autonomous parvoviruses neither stimulate nor are inhibited by the type I interferon response in human normal or cancer cells.

UNLABELLED: Members of the genus Parvovirus are small, nonenveloped single-stranded DNA viruses that are nonpathogenic in humans but have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared the response to parvoviruses to that of several other types of viruses in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust beta interferon (IFN-ß) responses. Cytomegalovirus, pseudorabies virus, and Sindbis virus all evoked a 2-log-unit or greater upregulation of IFN-ß in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-ß or interferon-stimulated gene (ISG; MX1, oligoadenylate synthetase [OAS], IFIT-1) response in the same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes. The same was true in human cancers, including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector adeno-associated virus type 2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas vesicular stomatitis virus replication was blocked. Sarcoma cells with upregulated IFN signaling that show high levels of resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we found no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in the virotherapy of some tumors. IMPORTANCE: Understanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells.

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

Vesicular stomatitis virus has extensive oncolytic activity against human sarcomas: rare resistance is overcome by blocking interferon pathways.

Oncolytic viruses have been tested against many carcinomas of ectodermal and endodermal origin; however, sarcomas, arising from mesoderm, have received relatively little attention. Using 13 human sarcomas representing seven tumor types, we assessed the efficiency of infection, cytolysis, and replication of green fluorescent protein (GFP)-expressing vesicular stomatitis virus (VSV) and its oncolytically enhanced mutant VSV-rp30a. Both viruses efficiently infected and killed 12 of 13 sarcomas. VSV-rp30a showed a faster rate of infection and replication. In vitro and in vivo, VSV was selective for sarcomas compared with normal mesoderm. A single intravenous injection of VSV-rp30a selectively infected all subcutaneous human sarcomas tested in mice and arrested the growth of tumors that otherwise grew 11-fold. In contrast to other sarcomas, synovial sarcoma SW982 demonstrated remarkable resistance, even to high titers of virus (multiplicity of infection [MOI] of 100). We found no dysfunction in VSV binding or internalization. SW982 also resisted infection by human cytomegalovirus and Sindbis virus, suggesting a virus resistance mechanism based on an altered antiviral state. Quantitative reverse transcriptase (qRT)-PCR analysis revealed a heightened basal expression of interferon-stimulated genes (ISGs). Pretreatment, but not cotreatment, with interferon attenuators valproate, Jak1 inhibitor, or vaccinia virus B18R protein rendered SW982 highly susceptible, and this correlated with downregulation of ISG expression. Jak1 inhibitor pretreatment also enhanced susceptibility in moderately VSV-resistant liposarcoma and bladder carcinoma. Overall, we find that the potential efficacy of VSV as an oncolytic agent extends to nonhematologic mesodermal tumors and that unusually strong resistance to VSV oncolysis can be overcome with interferon attenuators.

Reduction of febrile but not allergic reactions to RBCs and platelets after conversion to universal prestorage leukoreduction.

BACKGROUND: Between January 1995 and November 1998, at Yale-New Haven Hospital, 25 percent of RBCs transfused were processed through prestorage or bedside leukoreduction filters, chosen on a per patient basis (selective leukoreduction [SLR]). Between January 1995 and July 1999, 30 percent of platelet concentrates (PCs) were infused through bedside leukoreduction filters. In an attempt to decrease febrile nonhemolytic transfusion reactions (FNHTR), a change was made from SLR to universal prestorage leukoreduction (UPL) for RBCs between November 1998 and December 1999 and for random donor PCs between July 1999 and January 2000. FNHTR and allergic transfusion reactions (ATR) reported from January 1995 through December 2002 were reviewed. STUDY DESIGN AND METHODS: For retrospective observational analysis, blood bank data were available on the number of RBCs and PCs transfused, percent products leukoreduced, and rate of FNHTR and ATR from 1995 through December 2002. After dividing this time period into three phases (SLR, transition, and UPL), these data were evaluated using odds ratio (ORs) and Student's t tests. RESULTS: A total of 145,369 RBCs and 137,982 PCs (29,487 PC pools) transfused between January 1995 and December 2002 were evaluated. For RBCs, the relative FNHTR rate decreased 47.1 percent, from 0.34 percent (SLR) to 0.18 percent (UPL) (p < 0.0001). ATR rates for RBCs showed 0.09 percent for both SLR and UPL groups (p > 0.05, NS). For PCs, the FNHTR relative rate decreased 93.1 percent, from 2.18 percent for SLR to 0.15 percent for UPL (p < 0.0001). Rates for ATR were 0.49 percent (SLR) and 0.35 percent (UPL) (p > 0.05, NS). CONCLUSIONS: A significant decrease in the frequency of posttransfusion FNHTR, but not ATR, for RBCs and PCs followed introduction of 100-percent UPL. The data support the hypothesis that the practice of UPL of RBCs and PCs decreases the frequency of FNHTR and thus improves patient care over the practice of selective leukoreduction.