Reovirus Serotype Research Articles

Improved oncolytic activity of a reovirus mutant that displays enhanced virus spread due to reduced cell attachment

Wild-type reovirus serotype 3 Dearing (T3wt), a non-pathogenic intestinal virus, has shown promise as a cancer therapy in clinical trials, but it would benefit from an increased potency. Given that T3wt is naturally adapted to the intestinal environment (rather than tumors), we genetically modified reovirus to improve its infectivity in cancer cells. Various reovirus mutants were created, and their oncolytic potency was evaluated invitro using plaque size as a measure of virus fitness in cancer cells. Notably, Super Virus 5 (SV5), carrying five oncolytic mutations, displayed the largest plaques in breast cancer cells among the mutants tested, indicating the potential for enhancing oncolytic potency through the combination of mutations. Furthermore, in a HER2+ murine breast cancer model, mice treated with SV5 exhibited superior tumor reduction and increased survival compared with those treated with PBS or T3wt. Intriguingly, SV5 did not replicate faster than T3wt in cultured cells but demonstrated a farther spread relative to T3wt, attributed to its reduced attachment to cancer cells. These findings highlight the significance of increased virus spread as a crucial mechanism for improving oncolytic virus activity. Thus, genetic modifications of reovirus hold the potential for augmenting its efficacy in cancer therapy.

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.

Bacillus pumilus ribonuclease rescues mice infected by double-stranded RNA-containing reovirus serotype 1

Reoviruses (respiratory enteric orphan viruses) are nonenveloped viruses with segmented dsRNA genome. Viruses in the family Reoviridae are quite stable in the environment. Recently, they have been identified with various pathologies and physiologic dysfunctions in a wide range of organs and tissues, including the hepatobiliary system, the myocardium, lungs, and endocrine tissues. Although most cases of reovirus infection are mild or subclinical diseases, the prevention measures are currently needed, especially for young children suffering from dehydrating gastroenteritis. To inhibit viral replication, different RNases targeting viral RNA are proposed. Here, we first have shown that RNase from Bacillus pumilus (binase) acts as an antiviral agent at the level of the whole animal organism infected by Mammalian orthoreovirus 1 strain Lang (TL1). The results obtained on the mice model infected with 10 LD50 and 20 LD50 doses of reovirus indicate the restoration of mice physiological parameters under binase treatment at the dose of 50 μg/mouse. Thus, our research supports the relevance of binase as a promising antiviral agent that affects viral RNA.

Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons.

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors.

Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo.

Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

Polymorphisms in the Most Oncolytic Reovirus Strain Confer Enhanced Cell Attachment, Transcription, and Single-Step Replication Kinetics.

Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strains of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo This study aimed to determine why the most oncolytic T3D reovirus lab strain, the Patrick Lee laboratory strain (T3DPL), replicates more efficiently in cancer cells than other commonly used laboratory strains, the Kevin Coombs laboratory strain (T3DKC) and Terence Dermody laboratory (T3DTD) strain. In single-step growth curves, T3DPL titers increased at higher rates and produced ∼9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3DPL than for T3DTD In conclusion, the most oncolytic T3DPL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3DPL were identified. First, T3DPL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (≤3) σ1 cell attachment proteins. Second, T3DPL transcribed RNA at rates superior to those of the less oncolytic T3D strains, which is attributed to polymorphisms in M1-encoding μ2 protein, as confirmed in an in vitro transcription assay, and which thus demonstrates that T3DPL has an inherent transcription advantage that is cell type independent. Accordingly, T3DPL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains and, mechanistically, reveal the importance of the rapid onset of infection for reovirus oncolysis.IMPORTANCE Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently, it was discovered that highly related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3DPL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3DPL binds to cells more efficiency and more rapidly produces viral RNAs; this increased rate of infection relative to that of the less oncolytic strains gives T3DPL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread.

Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner.

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

BackgroundReovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype.MethodsThe serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera.ResultsThe Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results.ConclusionsThe prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.

Structural and Functional Features of the Reovirus σ1 Tail.

Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein.IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.

Canadian Cancer Trials Group (CCTG) IND211: A randomized trial of pelareorep (Reolysin) in patients with previously treated advanced or metastatic non-small cell lung cancer receiving standard salvage therapy

ObjectivesPelareorep (reolysin), a Dearing strain of reovirus serotype 3, has demonstrated oncolytic activity as single agent and synergy with chemotherapy. We evaluated pelareorep, combined with standard second-line chemotherapy in patients with non-small cell lung cancer (NSCLC). Materials and methodsThis randomized phase II trial enrolled patients with advanced or metastatic NSCLC after first line chemotherapy. After a safety run-in, patients were randomized 1:1 to chemotherapy (pemetrexed [500 mg/m2, non-squamous], or docetaxel [75 mg/m2], day 1 every 21 days]) +/− pelareorep (4.5 × 1010 TCID50, days 1–3 every 21 days), stratified by EGFR mutation status. The primary outcome was progression free survival (PFS) of patients randomized to chemotherapy + pelareorep vs. chemotherapy alone. Secondary outcomes included overall survival, objective response rate and exploratory translational analyses. ResultsBetween October 2012 and August 2015, 166 patients were enrolled (14 to the safety run in). Pelareorep did not improve the PFS vs. single agent chemotherapy (median PFS 3.0 months, 95% confidence interval [CI] 2.6–4.1) vs. 2.8 months (95% CI 2.5–4.0), hazard ratio (HR) 0.90 (95% CI 0.65–1.25), P = 0.53). Neither KRAS or EGFR mutation was associated with improved PFS, but STK11 mutations did appear to have an association with improved PFS (HR 0.29 [0.12–0.67); as did PIK3CA mutation (HR 0.45 [0.22–0.93]). The combination was tolerable, although associated with increased rates of neutropenic fever. ConclusionThe addition of pelareorep to second-line chemotherapy did not improve the PFS of patients with NSCLC. The three-day pelareorep schedule was tolerable. Further research is needed to evaluate the potential benefit in molecular subtypes of NSCLC.

Abstract CT131: A randomized (RCT) phase II study of oncolytic reovirus (pelareorep ) plus standard weekly paclitaxel (P) as therapy for metastatic breast cancer (mBC)

Abstract Background: Pelareorep is a Dearing strain of reovirus serotype 3, with demonstrated in vitro and in vivo activity in many cancers and synergistic cytotoxic activity with microtubule targeting agents, including taxanes. This study was designed to determine the efficacy and safety of pelareorep + P compared to P alone in mBC. Materials and Methods: This randomized, open-label, phase II study enrolled subjects who had mBC previously exposed to chemotherapy (CT). Subjects were randomized 1:1 between Arm A (P 80 mg/m2 day 1, 8 and 15 q 28 days plus pelareorep 3 x 1010 TCID50 day 1,2,8,9,15,16 q 28 days) and Arm B (P 80 mg/m2 day 1, 8 and 15 q 28 days). Treatment was continued until disease progression (PD) or unacceptable toxicity. Objective response was assessed every 8 weeks. Primary endpoint was progression free survival (PFS). The study had 90% power to detect an improvement of PFS from 4 to 7.5 months (HR 0.5, two-sided α=0.2). All p-values are two-sided. Results: Between July 2012 and April 2016, 81 subjects were accrued: 7 to the safety run-in for arm A, 36 to Arm A and 38 to Arm B. All had received prior CT, 59 as adjuvant treatment and 48 for mBC. Patients in Arm A had more favorable prognostic features, including lower LDH and less prior therapy. The median cumulative dose of P was 960 mg/m2 for arm A vs. 828 mg/m2 for arm B. Similar numbers of subjects in both arms required dose reductions, predominantly for myelosuppression. The median duration of treatment was 16.1 weeks for pelareorep and P in arm A and 14.1 weeks for P in arm B. With a median follow-up of 29.5 months, the median PFS was 3.78 mo for arm A and 3.38 mo for arm B (HR 1.04, 80% CI 0.76-1.43, p=0.87). Median OS was 17.4 mo for arm A and 10.4 mo for arm B (HR 0.65, 80% CI 0.46-0.91, p=0.1). Response rates (RR) were 25% for arm A and 23.7% for arm B (p=0.87). Pre-specified subset analysis found statistically significant differences in OS in patients with ECOG 1 or 2, aged < 65 yr, and no prior P treatment. Exploratory analysis of biomarkers found significant differences in OS in patients with wild type PIK3CA, KIT, APC, PTEN, ATM, AKT1, mutated TP53, and both wild type and mutated MET, although the number of pts was small. When grade 3 or higher adverse events (AE) were considered, only the incidence of fatigue was found in ≥10% (16% on Arm A vs 13%, arm B, p=0.76). Hematologic grade 3 or higher effects observed in ≥10% patients included lymphopenia (5% arm A vs 18% arm B, p=0.08), and neutropenia (23% arm A vs 26% arm B; p=0.8). There was a statistically significant difference in grade 3 or higher LDH (0% arm A vs 13% arm B, p=0.03). There were no treatment related deaths. Conclusions: This first, phase II, randomized study of pelareorep + P vs P in mBC previously exposed to CT, did not meet its primary endpoint of PFS. Despite similar PFS and RR there was a statistically significant improvement in OS for pelareorep + P pts. Given these data, further exploration of the role of pelareorep + P in mBC may be of interest. Citation Format: Vanessa Bernstein, Susan Ellard, Susan F. Dent, Karen A. Gelmon, Sukhbinder K. Dhesy-Thind, Mihaela Mates, Muhammed Salim, Lawrence Panasci, Xinni Song, Mark Clemons, Dongsheng Tu, Linda J. Hagerman, Lesley Seymour. A randomized (RCT) phase II study of oncolytic reovirus (pelareorep ) plus standard weekly paclitaxel (P) as therapy for metastatic breast cancer (mBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT131. doi:10.1158/1538-7445.AM2017-CT131

Structure of Serotype 1 Reovirus Attachment Protein σ1 in Complex with Junctional Adhesion Molecule A Reveals a Conserved Serotype-Independent Binding Epitope.

Mammalian orthoreoviruses use glycans and junctional adhesion molecule A (JAM-A) as attachment receptors. We determined the structure of serotype 1 reovirus attachment protein σ1 alone and in complex with JAM-A. Comparison with the structure of serotype 3 reovirus σ1 bound to JAM-A reveals that both σ1 proteins engage JAM-A with similar affinities and via conserved binding epitopes. Thus, σ1-JAM-A interactions are unlikely to explain the differences in pathogenesis displayed by these reovirus serotypes.

Anti-cancer effects of oncolytic viral therapy combined with photodynamic therapy in human pancreatic cancer cell lines

BackgroundOncolytic viral therapy and photodynamic therapy are potential therapies for inoperable or advanced pancreatic cancer. Our aim was to investigate the anti-cancer killing effects of reovirus therapy combined with protoporphyrin IX (PpIX)-mediated photodynamic therapy on a variety of human pancreatic cancer cell lines. MethodsPancreatic cancer cell lines (PsPC-1 and BXPC-3) and a non-cancer control cell line (HEK293) were infected with reovirus serotype 3 strain Dearing (T3D) at 0, 0·1, 1, and 10 plaque-forming units (PFU) per cell for 48 h. Cells were incubated with PpIX pro-drug 5-aminolevulinic acid (5-ALA) at 0, 1, 2, 3, and 4 mM for 4 h. Then, cells were photo-irradiated for 15 min with visible red light-emitting diodes with a light-fluence of 0·54 J/cm2 of 653 nm (PpIX optimal excitation wavelength). The killing effects of reovirus combined with PpIX-mediated photodynamic therapy were analysed in methylthiazoltetrazolium (MTT) and trypan blue assays. The effect of adding reovirus after photodynamic therapy was also assessed. The statistical significance of the difference between groups was assessed with the two-tailed Student's t test. p<0·05 was considered statistically significant. FindingsReovirus monotherapy induced cell death in the two pancreatic lines (mean 57% [SE 10·2] at 10 PFU per cell). PpIX-mediated PDT monotherapy induced cell death in a dose-dependent manner (mean 10% [SE 2·2], 30 [6·4], 50 [8·2], and 70 [13·2] after 1, 2, 3, and 4 mM 5-ALA, respectively). Reovirus with PpIX-mediated photodynamic therapy resulted in a significantly increased cytotoxic effect compared with reovirus monotherapy and photodynamic therapy (p=0·042) with 100% cell death observed across pancreatic cell lines with 10 PFU per cell combined with 1 and 2 mM 5-ALA. There was no difference in cytotoxicity observed between added reovirus before or after photodynamic therapy. InterpretationTo our knowledge, this is the first in-vitro study to combine reovirus oncolytic viral therapy with PpIX-mediated photodynamic therapy to treat pancreatic cancer. These results show a significant additive effect in cell killing and they provide initial evidence for a novel combined therapeutic intervention. FundingNational Institute for Health Research.

Combination Therapy with Reovirus and Immunomodulatory Drugs Induces Direct Oncolytic and Immune-Mediated Killing of Multiple Myeloma Cells and Overcomes Stromal-Mediated Microenvironmental Protection

IntroductionDespite considerable advances in therapy over recent years, multiple myeloma (MM) remains largely incurable with a clinical course characterised by ever-shortening periods of treatment and relapse; new therapies with novel mechanisms of action are needed. The immunomodulatory agents (IMiDs) in current clinical practice can augment immune effector function and combination with other immunologically active agents is a rational avenue of investigation – indeed trials of IMiDs with agents such as monoclonal antibody therapies are already underway. The naturally occurring oncolytic virus reovirus serotype 3 Dearing (Reolysin, Oncolytics Biotech Inc) has been shown to be safe in the context of advanced epithelial-derived cancers, and more recently in patients with MM. In this study we demonstrate the potential of reovirus in combination with IMiDs and steroids to induce both direct tumour cell killing and an anti-tumour immune response, particularly in the context of the cytoprotective effect of stromal cell interactions.MethodsHuman myeloma cell lines (HMCLs) were tested for JAM1 expression (the high affinity receptor for reovirus) by flow cytometry (FACS). HMCLs, alone or in co-culture with human stromal cell lines, were treated with reovirus at a range of multiplicities of infection (MOIs), alone or in combination with lenalidomide and/or dexamethasone and examined by flow cytometry to determine time- and concentration- dependent cell death. Peripheral blood mononuclear cells (PBMC) from healthy donors and patients were treated with reovirus/drug combinations and immune effector cell activation determined by surface expression of CD69. Treated PBMC and untreated HMCL were co-cultured and NK cell degranulation and NK cell-mediated HMCL cell death was determined. Bone marrow aspirate samples (BM) were collected from patients with MM, treated with reovirus, lenalidomide and dexamethasone combinations, and examined by FACS for killing of CD138+ cells and immune effector cell activation.ResultsJAM1 was expressed on the HMCLs H929, JIM3, KMS11, KMS18, JJN3 and U266B, but not on OPM2. All lines except OPM2 (which was resistant) were susceptible to reovirus-induced oncolysis in a time- and MOI-dependent manner. Addition of dexamethasone and lenalidomide to these treatments further increased the killing of tumour cells. Reovirus treatment induced upregulation of CD69 on NK cells, CD4+ T cells, CD8+ T cells and NKT cells isolated from the peripheral blood of both healthy donors and patients with MM. Healthy donor NK cells activated ex vivo with reovirus showed augmented degranulation (by surface CD107a expression) and killing of HMCL tumour targets. Importantly, in the case of OPM2, which was resistant to direct viral-induced cell death, reovirus-activated NK cells killed the HMCL. NK cells from MM patients showed enhanced degranulation against autologous tumour cells. Cell contact-independent killing of HMCL by reovirus-treated PBMC was also noted. Coculture of HMCLs with human bone marrow stromal cell lines (HS-5 and HS-27) protected against lenalidomide- and dexamethasone-induced HMCL cell death. However, treatment with reovirus overcame this cytoprotection. In keeping with this, when BM from MM patients was treated with lenalidomide and/or dexamethasone, the addition of reovirus augmented killing of CD138+MM cells.ConclusionsOur data indicate reovirus has dual modes of action against MM – not only can the virus mediate direct oncolytic cytotoxicity against the malignant clone, but in addition it can activate immune effector cells and thus induce immunologically-mediated tumour killing. Importantly, this immune-mediated action was noted even against HMCLs resistant to the direct viral effects. Furthermore, the cytoprotection and immune evasion afforded by stromal cells was mitigated by reovirus therapy and thus addition of reovirus to established immunomodulatory myeloma therapies augments their efficacy. Reovirus-based immunomodulatory combination therapies are a rational therapeutic approach for patients with relapsed MM and warrant further investigation in the pre-clinical and early phase clinical trial setting. DisclosuresCoffey:Oncolytics Biotech Inc.: Employment.

Abstract B74: A phase I trial and viral clearance study of reovirus (Reolysin) in children with relapsed or refractory extra-cranial solid tumors: A Children's Oncology Group phase I consortium report

Abstract Purpose: Reovirus (Respiratory Enteric Orphan virus) Serotype 3 – Dearing Strain is a naturally occurring, ubiquitous, non-enveloped human virus. Reovirus has been shown to replicate specifically in, and be cytopathic to, transformed cells possessing an activated Ras signaling pathway. We conducted a Phase I trial of Reolysin, a manufactured, purified reovirus, in children with relapsed or refractory extracranial solid tumors to define a maximum tolerated or recommended phase 2 dose (PP2D), as well as its toxicities and pharmacokinetic properties. Experimental Design: Reovirus was administered intravenously, daily for 5 consecutive days every 28 days for up to 12 cycles. Using a 3+3 design, two planned dose levels were evaluated: 3 x 108 TCID50/kg (∼60% of the adult dose) and 5 x 108 TCID50/kg. The cohort was expanded at the RP2D to obtain additional single agent safety and viral clearance data. Additionally, a 3rd cohort received the RP2D ( 5 x 108 TCID50/kg Reolysin) plus oral cyclophosphamide (50 mg/m2/day x 21 days). Twice weekly quantitative real time PCR of reovirus genomic RNA from serum, stool and saliva, and antiviral immune response by ELISA was evaluated after the first two courses. Results: Twenty-nine patients were enrolled, one was subsequently found to be ineligible. Of the 28 eligible patients, 5 are not evaluable (one is still on study) for dose-limiting toxicity (DLT). Six evaluable patients were enrolled on dose level 1; one with extensive pulmonary metastases experienced grade 5 respiratory failure attributed to their underlying disease. Given the severity of the event, the cohort was expanded to six without further DLTs. None of the six evaluable patients enrolled on dose levels 2 and 3, respectively, had a DLT. One patient in the single agent expansion cohort experienced a grade 5 thromboembolic event, grade 4 hypokalemia, grade 3 hypocalcemia, grade 4 hypophosphatemia, grade 3 hyponatremia, and grade 3 hypoalbuminemia possibly attributed to the Reolysin. With twice weekly monitoring by quantitative real time PCR, the median time to a clear the reovirus viremia was 6.5 days. Seven patients were viremic beyond the five days of therapy. No patient had detectable virus in their serum beyond day 17. Only one patient had detectable viral RNA in saliva or stool (day 6 in saliva). Four patients receiving the RP2D had stable disease at the end of one cycle, all others had progressive disease. All patients developed an anti-reovirus antibody during the first cycle of treatment. In three patients receiving a second cycle of reovirus, peak viral levels were reduced relative to cycle 1. There were no objective responses. Conclusions: Reovirus at a dose of 5 x 108 TCID50/kg daily for 5 days is well tolerated alone and in combination with oral cyclophosphamide. Virus is cleared rapidly from the serum of most patients, even when given with cyclophosphamide, and shedding in stool and saliva is not seen. Citation Format: Edward A. Kolb, Valerie B. Sampson, Deborah Stabley, Alexa Walter, Timothy P. Cripe, Pooja Hingorani, Charlotte Hsieh Ahern, Ashish M. Ingle, Brenda J. Weigel, Susan B. Blaney. A phase I trial and viral clearance study of reovirus (Reolysin) in children with relapsed or refractory extra-cranial solid tumors: A Children's Oncology Group phase I consortium report. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B74.

Seroepidemiology of Reovirus in Healthy Dogs in Six Prefectures in Japan

ABSTRACTReovirus infection is common in mammals. However, seroepidemiological data of reovirus neutralizing antibodies are limited in dogs. In this study, sera of 65 healthy dogs from six prefectures across Japan were tested for neutralizing antibodies against reovirus serotype 1 strain Lang (T1L), serotype 2 strain Amy (T2A) and serotype 3 strain Dearing (T3D) using plaque reduction neutralization test (PRNT). Seropositivity against reovirus T1L, T2A and T3D was 53.85%, 33.85% and 46.15%, respectively. Distribution of reovirus seropositive samples displayed no distinguishable geographical pattern. However, reovirus seropositivity increased with age and in dogs housed outdoor. Co-infection of multiple reovirus serotypes in dogs was also detected. These data will provide valuable insights towards the usage of reovirus in oncolytic virotherapy in canine cancers.

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.

Understanding The Differential Response Of Multiple Myeloma To Reovirus Treatment

IntroductionMultiple myeloma (MM) is a currently incurable malignancy arising from post germinal, terminally differentiated plasma cells. Despite the success of proteasome inhibitors and immune-modulating drugs such as bortezomib and thalidomide in the treatment of relapsed disease patients eventually become refractory to these drugs making new therapeutic agents a critical need.One potential new agent is Reovirus Serotype 3- Dearing Strain. While infection of immunocompetent hosts results in only minor respiratory illness it has been demonstrated that reovirus selectively replicates in and is cytopathic to Ras activated cells. This specificity is due at least in part to the inhibition of auto-phosphorylation of PKR. The minimal side effects and dependence of reovirus on Ras activation make it an interesting candidate for relapsed/refractory MM as it has been reported that at least 50% of relapsed MM patients have activating Ras mutations and most patients show activation at other Ras checkpoints including p38. While there has been limited investigation of reovirus treatment of MM cell lines there has been less analysis of resistance to reovirus treatment in MM. MethodsMM cell lines were screened via MTS assay to identify cell lines sensitive, intermediate, and resistant to reovirus treatment. Cell lines were then treated at a MOI of 10 or 25 for 9 and 24 hours and analyzed by immunohistochemistry (IHC) for p38, reoviral protein, and caspase-3 as well as in situ hybridization (ISH) and qRT-PCR for reoviral RNA. Total RNA isolates of sensitive, intermediate, and resistant cell lines were used to perform a screening analysis of mRNA, microRNA (miRNA), and long non-coding RNA to identify differential expression. In addition, expression of the reovirus high affinity receptor JAM-1 was assessed in these cell lines by western blot and IHC. Following this analysis mRNA, miRNA, and long non-coding RNA expression was analyzed in bone marrow samples from relapsed/refractory MM patients enrolled in a reovirus monotherapy clinical trial before and after treatment. These results were correlated with patient response and compared to the results from the cell lines. ResultsMTS results clearly identified several cell lines as being sensitive (RPMI), intermediate (H929), and resistant (OPM2) to reovirus treatment according to percent viability. IHC revealed over 98% positivity for p38 in all cell lines indicating Ras activation and permissibility to reovirus infection regardless of sensitivity. IHC also showed a higher percentage of cells positive for reoviral protein in sensitive and intermediate cell lines compared to resistant cell lines. The increased reoviral protein expression in these cell lines correlated with these lines having a higher percentage of caspase-3 positive cells compared to both the controls and the resistant cell lines. ISH demonstrated that the relative signal intensity of reoviral RNA was proportional to the sensitivity of each cell line, and these results were consistent with qRT-PCR results. ConclusionsDespite susceptibility to reovirus infection, as determined by p38 positivity, substantial variation was seen in response to reovirus treatment amongst cell lines. Given the correlation of cell line sensitivity with reoviral RNA levels and reoviral protein production it will be important to identify factors that drive these differences. Ongoing analysis is focused on determining the role of viral entry including expression of the reoviral receptor JAM-1 and subsequent viral trafficking and degradation. In addition, evaluation of differential expression of RNA molecules in cell lines and patient samples is ongoing as miRNA have been identified in the establishment of productive reoviral infections and cancer cell death (Nuovo et al. 2012). Disclosures:No relevant conflicts of interest to declare.Table 1All results are for cells treated at MOI 25 for 24 hours except % viability in which cells were treated at MOI 25 for 48 hours. Values for p38, reovirus protein and caspase-3 represent the percentage of positive cells by IHC. ISH reovirus RNA represents relative signal intensity with 100% being maximum intensity.Cell Line% Viabilityp38Reovirus ProteinCaspase-3 VirusCaspase-3 ControlISH Reovirus RNAAvg. Ct qRT-PCRRPMI2.599.245.359.037.39515.7H92946.398.69.338.517.69019.2OPM287.098.43.642.049.06028.4

HeLa cell response proteome alterations induced by mammalian reovirus T3D infection

BackgroundCells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such studies by examining virus-induced alterations in the cellular proteome.MethodsWe used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein’s label distribution.ResultsA total of 2856 cellular proteins were identified in cytosolic fractions by 2 or more peptides at >99% confidence and 884 proteins were identified in nuclear fractions. Gene ontology analyses indicated up-regulated host proteins were associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus, whereas down-regulated proteins were involved in cell death, macromolecular catabolic processes, and tissue development.ConclusionsThese analyses identified numerous host proteins significantly affected by reovirus T3D infection. These proteins map to numerous inflammatory and innate immune pathways, and provide the starting point for more detailed kinetic studies and delineation of virus-modulated host signaling pathways.

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 &gt;18 yrs with histologically confirmed KRAS-mutant mCRC, measurable disease, ECOG PS 0-1, &lt;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 (&gt;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.