Human Reovirus Research Articles

Effect of human reovirus strain R-92 on tumor cell lines

Background. Among all the new methods and approaches, virotherapy with oncolytic viruses, both in combination with immunotherapy and without it, shows high efficiency in various phases of clinical trials and good tolerance by patients.Aim. To study the sensitivity of some immortalized cancer cell lines to the R-92 strain of human reovirus with cell characteristics at the ultrastructural level.Materials and methods. The study was carried out on cell lines of HeLa, A549, U87MG. Cells were planted in an amount of 15 thousand per well of a 96-well plate and after adhesion, the virus was inoculated by adding a medium containing virus particles in 4 tenfold dilutions (approximately 10 9 –106 particles per ml). Next, the cells were cultured for 24 h, after which the number of living cells in the wells was determined indirectly using the methyl tetrazolium test, which was carried out according to standard methods. To study the ultrastructure of infected cells, cells were seeded into a T25 flask and inoculated with the virus at the maximum concentration. After 24 h of cultivation, the cells were fixed in a 2.5 % glutaraldehyde solution in phosphate buffer for 1 h, after which they were washed three times in phosphate buffer and samples were processed for TEM according to standard methods.Results. Diluting the virus 1000 times led to a decrease in the cytostatic effect in all three cultures to a level practically no different from the control. HeLa turned out to be the most sensitive culture to reovirus. In the experiment, the number of living cells decreased to 60.4 ± 10.2 % compared to the control during incubation with the maximum number of viral particles and to 63.7 ± 16.2 % with a tenfold dilution of the virus. This indicator was significantly lower than in the other two studied cultures under these cultivation conditions (p <0.001).In addition, at the maximum virus concentration, the A549 culture was less sensitive than the U87MG culture (p <0.01). At lower concentrations of viral particles, the average viability of the studied cell lines did not differ significantly from each other. Analysis of electron diffraction patterns showed that the virus successfully replicates in the cytoplasm of the studied cultures, but is not released from the cell, which is apparently due to the short incubation period. TEM also showed cell damage characteristic of apoptosis or necroptosis, uniformly expressed in all studied cultures.Conclusion. Cell lines A549, HeLa and U87MG, according to the results of the methyl tetrazolium test, demonstrate different sensitivity to the human reovirus strain P-92. The TEM picture of cells from infected cultures showed signs of the development of apoptosis or necroptosis.

Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

Surface characterization of alkane viral anchoring films prepared by titanate-assisted organosilanization

Studies of virus adsorption on surfaces with optimized properties have attracted a lot of interest, mainly due to the influence of the surface in the retention, orientation and stability of the viral capsids. Besides, viruses in whole or in parts can be used as cages or vectors in different areas, such as biomedicine and materials science. A key requirement for virus nanocage application is their physical properties, i.e. their mechanical response and the distribution of surface charge, which determine virus-substrate interactions and stability. In the present work we show two examples of viruses exhibiting strong surface interactions on homogeneous hydrophobic surfaces. The surfaces were prepared by titanate assisted organosilanization, a sol-gel spin coating process, followed by a mild annealing step. We show by surface and interface spectroscopies that the process allows trapping triethoxy-octylsilane (OCTS) molecules, exhibiting a hydrophobic alkane rich surface finishing. Furthermore, the surfaces remain flat and behave as more efficient sorptive surfaces for virus particles than mica or graphite (HOPG). Also, we determine by atomic force microscopy (AFM) the mechanical properties of two types of viruses (human adenovirus and reovirus) and compare the results obtained on the OCTS functionalized surfaces with those obtained on mica and HOPG. Finally, the TIPT+OCTS surfaces were validated as platforms for the morphological and mechanical characterization of virus particles by using adenovirus as initial model and using HOPG and mica as standard control surfaces. Then, the same characteristics were determined on reovirus using TIPT+OCTS and HOPG, as an original contribution to the catalogue of physical properties of viral particles.

The Effects of Human Reoviruses on Cancer Cells Derived from Hepatocellular Carcinoma Biopsies

Hepatocellular carcinoma (HCC) is the most common type of liver cancer around the world. Since this cancer is highly resistant to the existing treatments, we used a novel method, which selectively targets HCC cancer cells to improve the treatment process. As normal cells are resistant to reovirus replication, we used oncolytic reoviruses, which can infect, replicate in, and destroy cancer cells. In this study, the effects of oncolytic human reoviruses on cancer cells, derived from HCC biopsies, were investigated. First, reoviruses were purified. Then a plaque assay was performed to estimate the number of viruses and determine the multiplicity of infection (MOI). To evaluate the effects of reoviruses on cancer cells derived from HCC biopsies, replication of reovirus RNA, viral protein production, cytopathic effects (CPE), and cancer cell viability were assessed at different intervals post-infection. Replication of reovirus RNA and viral protein production were detected in cancer cells. Also, different levels of viral protein production, CPE, cytotoxicity, and cancer cell viability were observed at different intervals post-infection with human reoviruses. In contrast, normal human fibroblasts, which were used as negative control, remained unchanged. For the first time, the effects of human reoviruses on HCC biopsies were investigated. The results showed that human reoviruses could replicate in and destroy cancer cells derived from HCC biopsies. Overall, human reoviruses can be potentially used for the treatment of HCC.

Interactions between Human Reovirus and Free-Living Amoebae: Implications for Enteric Virus Disinfection and Aquatic Persistence.

Free-living amoebae (FLA) are ubiquitous protozoa in aquatic/soil habitats and known to resist various disinfection methods commonly used in drinking and wastewater treatment plants. Reoviruses are emerging as useful infectious enteric virus indicators of wastewater treatment efficacy. The possible enhanced protection FLA may provide reoviruses, however, has not been previously described. Using an infectious clinical reovirus isolate in coculture with three FLA, namely, Vermamoeba vermiformis, Acanthamoeba polyphaga, and Willaertia magna, we followed reovirus persistence (by quantitative reverse transcription polymerase chain reaction (RT-qPCR)) and infectivity (TCID50). Virions present in samples persisted over the experimental time period, with most virions remaining infectious. Surprisingly, electron microscopy revealed virions accumulated within the nucleus of amoebae. The current work appears to be the first report of reovirus being internalized within FLA and remaining infectious, providing a previously unreported environmental reservoir and potential mode of dissemination. FLA also appeared to be providing some logs in protection to internalized viruses during UV irradiation, which if not accounted for when determining UV dosage needed for sufficient disinfection may result in unintentional release of pathogens into surrounding water systems.

Novel human reovirus isolated from children and its long-term circulation with reassortments

Mammalian orthoreovirus (MRV), also known as reovirus, was discovered in the 1950s and became the first reported segmented double-stranded RNA virus. MRVs have since been found in a variety of animal species, including humans. However, reports on MRV infections are scarce due to the rarity of their symptomatic occurrence. In Japanese surveillance studies, MRVs have been detected as gastrointestinal pathogens since 1981, with a total of 135 records. In Osaka City, Japan, MRV was first isolated in 1994 from a child with meningitis, and then in 2005 and 2014 from children with gastroenteritis. Here, we conducted the first molecular characterization of human MRV isolates from Japan and identified a novel human reovirus strain belonging to MRV type 2, designated the MRV-2 Osaka strain. This strain, with all three isolates classified, is closely related to MRV-2 isolates from sewage in Taiwan and is relatively close to an MRV-2 isolate from a bat in China. Our data suggest that the MRV-2 Osaka strain, which has circulated amongst humans in Japan for at least two decades, has spread internationally.

Proteasome Inhibitors Impair the Innate Antiviral Immune Response and Potentiate Pelareorep-Based Viral Therapy in Multiple Myeloma

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p<0.001). We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p<0.01), PI abolished ex-vivo RV-induced monocyte differentiation (n=3, p<0.001), without affecting the ability of RV-infected CD14+ cells to induce higher MM cell killing as compared to that from the CD14+ fraction treated with either single agent. Immune killing assays showed that RV-infected MM cells are more susceptible than non-infected cells to T cell effectors (p<0.001), a mechanism that is further potentiated by the addition of PI. In fact, in contrast to the PI-induced blocking of monocyte expansion and activation in response to the virus (n=3, p<0.001), our data suggest that the PI potentiate the expansion of CD8+ T cells, both ex vivo and in two RV+PI treated patients we analyzed so far. CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

Reovirus strain R-92 in in vitro generation of tumor-specific T-lymphocytes.

e14216 Background: The purpose of the study was to assess the potential of the reovirus strain R-92 for in vitro generation of tumor-specific T-lymphocytes. Methods: The human reovirus strain R-92 used as a master seed strain was previously isolated from a patient with infectious hepatitis and adapted to the growth on the pig embryo kidney cell culture (PEKC); it lost its pathogenicity after multiple passages and was characterized by electron microscopic, serological methods and PCR. HeLa lysate was prepared by the incubation with the R-92 reovirus in medium 199+L-glutamine during 5 days (37оC, 5% СО2), cytopathogenic effect was observed. Immature dendritic cells (DCs) were derived from blood monocytes cultivated for 7 days in presence of IL-4 and GM-CSF. For the DC loading during 48 hours we used: 1. HeLa lysate obtained by repeated freezing and thawing of cells (control); 2. HeLa lysate obtained by co-culturing with reovirus; 3. reovirus cultured in a standard PEKC. To assess the DC ability to activate lymphocytes, they were co-cultured for 5 days in a 3:1 ratio, 1.5x105 of target cells (HeLa) were added (1:1) for 48 hours; number of dead HeLa cells was count. At each stage of the generation of DCs and activated lymphocytes, they were phenotyped by flow cytometry. Results: DC samples 2 and 3 showed increased percentage of activated cells compared to the loaded with the control tumor lysate, CD86 and HLA-DR coexpression was 62 and 69% vs. 25%, respectively; CD83 expression was maximal after DC loading with reovirus (72.9%). After co-culturing of DC with autologous lymphocytes, the highest number of activated CD4+CD38+ and CD8+CD38+, was observed in sample 2, T-regs (CD4+CD25+CD127low) - in the control. The death of target cells cultured with activated lymphocytes obtained by DC samples 2 and 3, was 100%, control sample showed minimal cytotoxicity against HeLa. The number of T-lymphocytes in samples 2 and 3 cultured with HeLa exceeded the control values by 3-10 times. Conclusions: Reovirus R-92 used for the immature DC loading increases their presenting activity. DCs loaded with reovirus HeLa lysate, when co-cultured with lymphocytes, activate CD4+ and CD8+ and suppress T-regs, apparently leading to the total death of target tumor cells.

Possible viral oncolysis by members of the Reoviridae and Paramyxoviridae families (an in vitro study).

e14212 Background: Our purpose was to study possible oncolytic activity of Newcastle disease virus and reovirus in different tumor cell cultures in vitro. Methods: The study was performed on LaSota strain Newcastle disease virus (NDV) vaccine (OOO NPP AVIVAK, 5000 doses) and human reovirus strains R-92 (3 serotype) deposited in the State collection of viruses of the Research Institute of Virology named after. D.I.Ivanovsky. HeLa (cervical cancer), A549 (lung adenocarcinoma), BT20 (breast cancer), PC3 (prostate cancer), HT29 (colon cancer) cell lines were used; pig embryo kidney cell culture (PEVKC) was used as a positive control. Results: After Newcastle disease virus was placed in the medium with tumor cell lines, a 5-day visual microscopic control of the cell morphology did not show signs of the cytopathic action, while a complete lysis of cell biomass was observed in the positive control (PEKC) by the third day. After the incubation with reovirus, the oncolytic activity was registered already in 24 hours: partial or complete destruction of the monolayer, decreased cell adhesion (only solitary cellular conglomerates remained at the bottom of vials), changes in the typical cell appearance. The complete cytopathogenic effect was observed after 2 days. Conclusions: The results demonstrated a marked lytic effect of reovirus in vitro, as well as varying sensitivity of the studied tumor lines to it, which may depend on their molecular genetic characteristics, for example on the RAS mutation status. Despite ambiguous results, we consider it expedient to continue the studies of these and other viruses in tumor models both in vitro and in vivo.

Pancreatic Cancer: An Emphasis on Current Perspectives in Immunotherapy.

Pancreatic cancer affects both male and female individuals with higher incidences and death rates among the male population. Detection of this malignancy is delayed due to the lack of symptoms in the early-stage cancer, which makes it extremely difficult to treat. Identifying effective strategies has been a challenge for improving the survival rates in pancreatic cancer patients. Resistance to chemotherapy is often developed in pancreatic cancer treatment. Although many strategies are under clinical trials to target certain markers associated with cancer, immunotherapeutic approaches are currently gaining importance. Immunotherapy for pancreatic cancer is in the limelight after preclinical research showed some promise. Immunotherapy approaches were tested along with other treatment options to enhance the treatment effect. Adoptive cell transfer and immune checkpoint inhibitors are currently in clinical trials. The Food and Drug Administration approved pembrolizumab in a fast-tracked review for advanced pancreatic cancer patients. Pembrolizumab blocks the checkpoint protein, programmed cell death protein 1 (PD-1), on T cells to boost the response of the immune system against cancer cells, thereby shrinking tumors. The recent developments in immunotherapy and the early success in other cancers are encouraging to further test immunotherapy in pancreatic cancer. The combination of pembrolizumab and pelareorep, an isolate of human reovirus, is in phase II clinical study in metastatic disease. Depending on the results of current clinical trials and testing, the strategies in the pipeline are expected to increase the use of immunotherapy in the clinical testing setting. Success in immunotherapy is urgently needed to address the side-effects, treating patients with advanced disease and reducing metastasis for increasing the survival rate in pancreatic cancer patients.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells.

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

Histone Deacetylase Inhibitors Enhance the Therapeutic Potential of Reovirus in Multiple Myeloma.

Multiple myeloma remains incurable and the majority of patients die within 5 years of diagnosis. Reolysin, the infusible form of human reovirus (RV), is a novel viral oncolytic therapy associated with antitumor activity likely resulting from direct oncolysis and a virus-mediated antitumor immune response. Results from our phase I clinical trial investigating single agent Reolysin in patients with relapsed multiple myeloma confirmed tolerability, but no objective responses were evident, likely because the virus selectively entered the multiple myeloma cells but did not actively replicate. To date, the precise mechanisms underlying the RV infectious life cycle and its ability to induce oncolysis in patients with multiple myeloma remain unknown. Here, we report that junctional adhesion molecule 1 (JAM-1), the cellular receptor for RV, is epigenetically regulated in multiple myeloma cells. Treatment of multiple myeloma cells with clinically relevant histone deacetylase inhibitors (HDACi) results in increased JAM-1 expression as well as increased histone acetylation and RNA polymerase II recruitment to its promoter. Furthermore, our data indicate that the combination of Reolysin with HDACi, potentiates RV killing activity of multiple myeloma cells in vitro and in vivo This study provides the molecular basis to use these agents as therapeutic tools to increase the efficacy of RV therapy in multiple myeloma. Mol Cancer Ther; 15(5); 830-41. ©2016 AACR.

Exploring Reovirus Plasticity for Improving Its Use as Oncolytic Virus.

Reoviruses are non-enveloped viruses with a segmented double stranded RNA genome. In humans, they are not associated with serious disease. Human reoviruses exhibit an inherent preference to replicate in tumor cells, which makes them ideally suited for use in oncolytic virotherapies. Their use as anti-cancer agent has been evaluated in several clinical trials, which revealed that intra-tumoral and systemic delivery of reoviruses are well tolerated. Despite evidence of anti-tumor effects, the efficacy of reovirus in anti-cancer monotherapy needs to be further enhanced. The opportunity to treat both the primary tumor as well as metastases makes systemic delivery a preferred administration route. Several pre-clinical studies have been conducted to address the various hurdles connected to systemic delivery of reoviruses. The majority of those studies have been done in tumor-bearing immune-deficient murine models. This thwarts studies on the impact of the contribution of the immune system to the tumor cell eradication. This review focuses on key aspects of the reovirus/host-cell interactions and the methods that are available to modify the virus to alter these interactions. These aspects are discussed with a focus on improving the reovirus’ antitumor efficacy.

Leukocyte-Derived IFN-α/β and Epithelial IFN-λ Constitute a Compartmentalized Mucosal Defense System that Restricts Enteric Virus Infections

Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/β) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/β, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/β receptors. Accordingly, after oral infection of IFN-α/β receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/β for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/β system which would induce exacerbated inflammation.

Discovery of m(7)G-cap in eukaryotic mRNAs.

Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2′-O-methylated nucleotide in the 5′ end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5′ terminal structure (referred later to as “cap” or “m7G-cap”) in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5′-cap m7GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5′ cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping.

122: Intestinal epithelium is an autonomous lambda interferon-dependent cell compartment that is calibrated by intestinal microflora

Type I interferon (IFN-α/β) represents the key element of the antiviral defense mechanisms against most viruses, however, rotaviruses that infect the gut epithelium, display little sensitivity to type I IFN. Here, we report that the intestinal epithelium is a unique cell compartment in the organism that does not depend on type I IFN in antiviral defenses. Type I IFN was unable to induce antiviral gene expression in intestinal epithelial cells (IEC) that correlated well with low epithelial expression of both chains of the IFN-α/β receptor complex. In stark contrast, IECs strongly responded to IFN-λ on baseline, upon IFN treatment and virus challenge. Commensal microflora was found to establish baseline IFN-λ signaling in IECs and enhance resistance to enteric virus infection. In adult mice lacking functional receptors for IFN-λ, human reovirus selectively replicated in IECs. By contrast, these cells remained virus-free in IFN-α/β-deficient mice that developed deadly systemic disease after oral reovirus infection. In suckling mice with IFN-λ receptor deficiency, reovirus not only replicated in the gut epithelium but also infected epithelial cells lining the bile ducts, thereby inducing a fatal liver disease. Interestingly, in response to synthetic double-stranded RNA or enteric virus infection, gut epithelial cells readily produced IFN-λ but not IFN-α or IFN-β. Taken together, it appears that the intestinal epithelium has evolved mechanisms to specifically produce and respond to IFN-λ, whereas systemic antiviral defenses depend on IFN-α/β produced predominantly by lymphoid cells. Our data suggest that such strict separation of the two IFN systems has evolved to avoid unnecessarily frequent triggering of the IFN-α/β system by commensals, which would activate systemic IFN pathways and thereby induce exacerbated inflammation.

A Phase 1 Trial Of Reolysin Alone In Patients With Refractory Or Relapsed Multiple Myeloma

IntroductionIn relapsed/refractory multiple myeloma, approximately 50% of patients have Ras-family (NRAS and KRAS) activating gene mutations. Reovirus Serotype 3 - Dearing Strain (Reolysin) is the infusible form of the RNA human reovirus associated with minor respiratory infections in humans. Reovirus has been shown to replicate specifically in, and be cytopathic to, transformed cells possessing an activated Ras-signaling pathway. The specificity of the reovirus for Ras-activated cells makes it an attractive anti-cancer therapy candidate. MethodsFor this phase 1 trial, patients were required to have relapsed or refractory myeloma with IMWG-defined measurable disease, ANC ≥ 1,000/uL, platelet count ≥ 50,000/uL, with no creatinine requirements. Reolysin was administered intravenously over 60 minutes on days 1 - 5 every 28 days to 12 patients. It was started at 3 x 109 median tissue culture infective dose per day (TCID50/day) for 3 patients and then escalated to 3 x 1010 TCID50/day for the remaining 9 patients. In situ based methodologies were used to examine the distribution of CD138, p38, caspase-3, reoviral capsid protein, and reoviral RNA in bone marrow biopsies performed at screening and then on cycle 1 day 8. Neutralizing Anti-Reovirus Assay (NARA) was performed weekly during cycle 1. ResultsTwelve patients were enrolled with a median age of 61 (range 48 - 77), median number of prior therapies at enrollment of 4 (range 1 - 10) and mean ISS stage of 1.9 (range 1 - 3). No DLT's were experienced and all patients reached 3 x 1010 TCID50/day. Grade 2 toxicities included leukopenia, anemia and myalgias, and grade 3 toxicities (asymptomatic) included 3 patients with neutropenia and one patient each with thrombocytopenia and hypophosphatemia.Reoviral protein and RNA were found in post-Reolysin bone marrow biopsies but not at baseline. Reoviral RNA stained within 20 - 100% of myeloma cells, but only rare reoviral protein and caspase-3 staining was seen. Patients with 1:1 reoviral RNA:CD138 staining showed the most reduction in the percent of myeloma cells with treatment (Table 1). All patients developed NARA responses with 8 patients having endpoint titers greater than 2000 at 3 weeks post-treatment.Stable disease (SD) was evident in 5 (42%) patients (longest duration on treatment was 8 cycles). One patient had a minimal response after two cycles. ConclusionsTreatment with single-agent Reolysin was well tolerated, reovirus infection of myeloma cells was proven in all patients, but there was minimal intracellular reoviral protein production. Patients with 100% co-expression of reoviral RNA and CD138 had the largest relative decrease in CD138+ cells suggesting an early cytotoxic T- or NK-cell antiviral effect. The lack of clinical response is expected because reoviral-induced oncolysis typically requires concomitant chemotherapy. Clinical experiments in solid tumors confirm the necessity of chemotherapy as a cellular stressor to induce viral protein production and subsequent apoptosis (Sei et al., Mol Cancer 2009). Given the preclinical synergy reported with bortezomib in myeloma cell lines, we are planning a phase 1b combination of Reolysin with Carfilzomib. Disclosures:No relevant conflicts of interest to declare.

Bio-distribution study of Reolysin® (pelareorep) through a single intravenous infusion in Sprague-Dawley rats

Numerous pre-clinical and clinical studies on reovirus have generated valuable information which supports the use of this orphan virus as an investigational drug for cancer treatment. Reolysin® (pelareorep) is a clinical formulation of the human Reovirus Type 3 Dearing strain. The clinical safety and efficacy of Reolysin® in humans is being tested on an assortment of cancer indications as a mono and/or combination therapy. Reovirus has many inherent characteristics that make it a potential candidate for virotherapy, including: the rapid and natural spread through the haematogenous route, the ability to overcome immunological barriers thereby reaching tumor sites, and being replication-competent. The purpose of this study was to elucidate the bio-distribution pattern of Reolysin® in healthy Sprague-Dawley rats. Following a single 15-min intravenous infusion via the tail vein in Sprague-Dawley rats, the levels of virus genome were determined in 16 organs/tissues by RT-qPCR (Reverse Transcriptase- Quantitative Polymerase Chain Reaction) over a 336 h (Day 15) incubation regime. Consistent with previous studies, maximal reovirus RNA levels were observed in the spleen; indicating its involvement in viral uptake and clearance, followed by heart, ovaries, tail (infusion site), liver and lungs. All the organs/tissues demonstrated unquantifiable levels of reovirus genome at the end of incubation, suggesting substantial to complete viral clearance. Several studies in the last decade have described the use of reovirus for treating ovarian cancers. An increase of reovirus genome in ovaries at 24 h post infection was noted. The results will aid in the design of additional exploratory clinical trials for Reolysin®.

A multicenter phase I study of intravenous administration of reolysin in combination with irinotecan/fluorouracil/leucovorin (FOLFIRI) in patients (pts) with oxaliplatin-refractory/intolerant KRAS-mutant metastatic colorectal cancer (mCRC).

450 Background: Reolysin (reovirus serotype 3) contains a naturally occurring, ubiquitous, non-enveloped human dearing strain reovirus. Reovirus replicates in KRAS-mutant cells resulting in cell lysis. In phase I evaluation, CRC pts received single agent Reolysin with tumor stabilization and CEA response without significant toxicity. Reolysin and irinotecan (IRI) are synergistic in KRAS-mutant preclinical CRC models, providing rationale for this phase I study. Methods: This was a phase I dose escalation study of FOLFIRI + Reolysin. Eligible pts were >18 yrs with histologically confirmed KRAS-mutant mCRC, measurable disease, ECOG PS 0-1, <3 metastatic regimens, and adequate organ function. Standard FOLFIRI was administered with escalating Reolysin doses (range 1x1010 TCID50 to 3x1010 TCID50) in cohorts of 3-6 pts. Reolysin was given IV over 1 hr days 1-5 every 28d (1 cycle). Primary objectives were dose-limiting toxicity (DLT) to determine MTD and pharmacokinetics. Secondary endpoints were antitumor activity, response rate, progression-free and overall survival (PFS and OS). Results: 21 pts enrolled; median age 62 (range 39-77); 5 M; 16 F; FOLFIRI-naïve: 9/21 pts. 2 pts had DLTs in cycle 1 at the highest dose of 180 mg/m2 of IRI. Common (>10%) grade 3-4 toxicity include: neutropenia (n=11), anemia (n=4), and thrombocytopenia (n=3). One patient died of acute renal failure. The DLT is neutropenia. The recommended phase II dose is IRI 150 mg/m2 and Reolysin at 3x1010 TCID50 on days 1-5, q 28 days. 18 pts evaluable for response: PR (1pt; 5%), SD (9 pts; 50%), PD (8pts; 44%). 3 pts taken off study before evaluation. Median PFS: FOLFIRI-naïve pts = 7.4 mo. (95% CI = 1.9 mo., 12.9 mo.); Median PFS FOLFIRI non-naïve pts was not reached; overall median PFS = 7.4 mo. (95% CI = 0.6 mo., 14.1 mo.) Conclusions: The combination of Reolysin and FOLFIRI in pts with KRAS-mutant mCRC was safe, well tolerated and resulted in disease control in the majority of pts, including pts who previously progressed on IRI. We are encouraged by this activity and safety profile, and are planning additional studies. Clinical trial information: NCT01274624.

Abstract 2488: Reovirus (Reolysin) as a potential therapy for malignant peripheral nerve sheath tumors

Abstract Malignant peripheral nerve sheath tumors (MPNST) are a rare form of soft tissue sarcoma which may occur sporadically in the general population or in association with neurofibromatosis type 1 (NF1), and are highly aggressive and often fatal. NF1 patients are at a greater risk for the development of malignant peripheral nerve sheath tumors than the general population. Current therapy relies upon surgical resection with adjuvant chemotherapy and/or radiotherapy. Clinical trials using Reolysin (Respiratory Enteric Orphan Virus) which is developed from the naturally-occurring human reovirus have demonstrated the efficacy of this reovirus as an oncolytic virotherapy. Reolysin selectively replicates in cancer cells with activated Ras (Ras-GTP), resulting in virus-mediated apoptotic cell death. Hence Reolysin demonstrates promising anticancer activity and has minimal toxicity in patients. Since MPNST is associated with neurofibromin (NF1) deficiency and active Ras we tested these tumors as therapeutic targets for Reolysin treatment using two NF1 deficient human MPNST-derived cell lines (CRL-2884, ST8814) with activated Ras and one sporadic MPNST cell line (STS26T) which expresses NF1 but not active Ras. These were compared with normal human Schwann cells (NHSC) which do not show active Ras and mammary gland cells derived from an NF1 patient (CRL-7329). Reolysin inhibited the proliferation and viability of the three MPNST cell lines at a dose of 0.1 to 10 virus particles/cell, including sporadic MPNST cells which do not have active Ras. In contrast, the NHSC and CRL-7329 cells were more resistant to viral treatment, with apoptosis induced at higher viral concentrations (10,000 and 100,000 virus particles/cell respectively). Furthermore, we examined the effects of Reolysin on the Ras-signaling pathway in a bladder cancer cell line (HTB-4) expressing NF1 and Ras and found that virus induced cytotoxicity was similar to the control cell lines (100,000 virus particles/cell). This study shows that MPNST-derived cell lines including sporadic MPNST without active Ras were efficiently transduced, promoted virus replication and were killed by the oncolytic reovirus. In contrast to the highly sensitive MPNST cell lines, NHSC, CRL-7329 and HTB-4 cells were resistant at similar viral concentrations. Our data suggests the use of oncolytic Reolysin may represent a novel treatment approach for patients with MPNST and provides the basis for preclinical testing of the antitumor effects. Therapeutic efficacy of Reolysin in a mouse model for MPNST is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2488. doi:1538-7445.AM2012-2488