Characterizing Cellular Responses During Oncolytic Maraba Virus Infection.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Maraba virus as a potent oncolytic vaccine vector.

The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.

Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease.

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Triptolide-mediated inhibition of interferon signaling enhances vesicular stomatitis virus-based oncolysis.

Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.

Oncolytic Vaccinia virus safely and effectively treats skin tumors in mouse models of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder of DNA repair. When exposed to genotoxic stress, XP patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. Unfortunately, given the nature of their underlying genetic defect, tumor-bearing XP patients cannot be treated with conventional DNA damaging therapies. Engineered strains of the poxvirus Vaccinia have been shown to cure cancer in numerous preclinical models, and based on promising Phase I/II clinical trials have recently been approved for late phase evaluation in humans. As poxviruses are nongenotoxic, we investigated whether clinical-candidate strains of Vaccinia can safely and effectively treat cancers arising from XP. In vitro, Vaccinia virus was highly cytotoxic against tumor-derived cells from XP patients, on average 10- to 100-fold more so than on nontumor derived control cells from similar patients. In vivo, local or systemic administration of Vaccinia virus led to durable tumor resolution in both xenograft and genetic models of XP. Importantly, Vaccinia virus was well tolerated in the genetic models, which are each null for a critical component of the DNA repair process. Taken together, our data suggest that oncolytic Vaccinia virus may be a safe and effective therapy for cancers arising from XP, and raise the possibility of similar therapeutic potential against tumors that arise in patients with other DNA repair disorders.

Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine.

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV's extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.

VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents.

Ideally, an oncolytic virus will replicate preferentially in malignant cells, have the ability to treat disseminated metastases, and ultimately be cleared by the patient. Here we present evidence that the attenuated vesicular stomatitis strains, AV1 and AV2, embody all of these traits. We uncover the mechanism by which these mutants are selectively attenuated in interferon-responsive cells while remaining highly lytic in 80% of human tumor cell lines tested. AV1 and AV2 were tested in a xenograft model of human ovarian cancer and in an immune competent mouse model of metastatic colon cancer. While highly attenuated for growth in normal mice, both AV1 and AV2 effected complete and durable cures in the majority of treated animals when delivered systemically.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

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

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

Deletion of immunomodulatory genes as a novel approach to oncolytic vaccinia virus development.

Vaccinia virus (VV) has emerged as a promising platform for oncolytic virotherapy. Many clinical VV candidates, such as the double-deleted VV, vvDD, are engineered with deletions that enhance viral tumor selectivity based on cellular proliferation rates. An alternative approach is to exploit the dampened interferon-based innate immune responses of tumor cells by deleting one of the many VV immunomodulatory genes expressed to dismantle the antiviral response. We hypothesized that such a VV mutant would be attenuated in non-tumor cells but retain the ability to effectively propagate in and kill tumor cells, yielding a tumor-selective oncolytic VV with significant anti-tumor potency. In this study, we demonstrated that VVs with a deletion in one of several VV immunomodulatory genes (N1L, K1L, K3L, A46R, or A52R) have similar or improved in vitro replication, spread, and cytotoxicity in colon and ovarian cancer cells compared to vvDD. These deletion mutants are tumor selective, and the best performing candidates (ΔK1L, ΔA46R, and ΔA52R VV) are associated with significant improvement in survival, as well as immunomodulation, within the tumor environment. Overall, we show that exploiting the diminished antiviral responses in tumors serves as an effective strategy for generating tumor-selective and potent oncolytic VVs, with important implications in future oncolytic virus (OV) design.

Efficacy of systemically administered oncolytic vaccinia virotherapy for malignant gliomas is enhanced by combination therapy with rapamycin or cyclophosphamide.

PURPOSE: The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia virus has been previously shown for the treatment of many cancers, including colon, ovarian, and others. The purpose of this study was to investigate the oncolytic potential of double-deleted vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant gliomas in vitro and in vivo. EXPERIMENTAL DESIGN: Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene [enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts (U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution, and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malignant glioma explants were evaluated. RESULTS: vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect and kill ex vivo primary human malignant gliomas. CONCLUSIONS: These results suggest that vvDD-EGFP is a promising novel agent for human malignant glioma therapy, and in combination with immunosuppressive agents, may lead to prolonged survival from this disease.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials.

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity.

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. However, the host immune system remains a significant obstacle to effective systemic administration of virus in a clinical setting. Here, we demonstrate the severe negative impact of the adaptive immune response on the systemic delivery of oncolytic vesicular stomatitis virus (VSV) in an immune-competent murine tumor model, an effect mediated primarily by the neutralization of injected virions by circulating antibodies. We show that this obstacle can be overcome by administering virus within carrier cells that conceal viral antigen during delivery. Infected cells were delivered to tumor beds and released virus to infect malignant cells while sparing normal tissues. Repeated administration of VSV in carrier cells to animals bearing metastatic tumors greatly improved therapeutic efficacy when compared with naked virion injection. Whole-body molecular imaging revealed that carrier cells derived from solid tumors accumulate primarily in the lungs following intravenous injection, whereas leukemic carriers disseminate extensively throughout the body. Furthermore, xenogeneic cells were equally effective at delivering virus as syngeneic cells. These findings emphasize the importance of establishing cell-based delivery platforms in order to maximize the efficacy of oncolytic therapeutics.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Development and applications of oncolytic Maraba virus vaccines.

Oncolytic activity of the MG1 strain of the Maraba vesiculovirus has proven efficacy in numerous preclinical cancer models, and relied not only on a direct cytotoxicity but also on the induction of both innate and adaptive antitumor immunity. To further expand tumor-specific T-cell effector and long-lasting memory compartments, we introduced the MG1 virus in a prime-boost cancer vaccine strategy. To this aim, a replication-incompetent adenoviral [Ad] vector together with the oncolytic MG1 have each been armed with a transgene expressing a same tumor antigen. Immune priming with the Ad vaccine subsequently boosted with the MG1 vaccine mounted tumor-specific responses of remarkable magnitude, which significantly prolonged survival in various murine cancer models. Based on these promising results, we validated the safety profile of the Ad:MG1 oncolytic vaccination strategy in nonhuman primates and initiated clinical investigations in cancer patients. Two clinical trials are currently under way (NCT02285816; NCT02879760). The present review will recapitulate the discoveries that led to the development of MG1 oncolytic vaccines from bench to bedside.

Transforming the prostatic tumor microenvironment with oncolytic virotherapy.

Prostate cancer (PCa) was estimated to have the second highest global incidence rate for male non-skin tumors and is the fifth most deadly in men thus mandating the need for novel treatment options. MG1-Maraba is a potent and versatile oncolytic virus capable of lethally infecting a variety of prostatic tumor cell lines alongside primary PCa biopsies and exerts direct oncolytic effects against large TRAMP-C2 tumors in vivo. An oncolytic immunotherapeutic strategy utilizing a priming vaccine and intravenously administered MG1-Maraba both expressing the human six-transmembrane antigen of the prostate (STEAP) protein generated specific CD8+ T-cell responses against multiple STEAP epitopes and resulted in functional breach of tolerance. Treatment of mice with bulky TRAMP-C2 tumors using oncolytic STEAP immunotherapy induced an overt delay in tumor progression, marked intratumoral lymphocytic infiltration with an active transcriptional profile and up-regulation of MHC class I. The preclinical data generated here offers clear rationale for clinically evaluating this approach for men with advanced PCa.

Activating Peripheral Innate Immunity Enables Safe and Effective Oncolytic Virotherapy in the Brain.

The oncolytic mutant vesicular stomatitis virus VSVΔ51 achieves robust efficacy in multiple extracranial tumor models. Yet for malignancies of the brain, direct intratumoral infusion of VSVΔ51 causes lethal virus-induced neuropathology. Here, we have developed a novel therapeutic regime that uses peripheral immunization with a single sub-lethal dose of VSVΔ51 to establish an acute anti-viral state that enables the safe intracranial (IC) infusion of an otherwise lethal dose of VSVΔ51 within just 6 hr. Although type I interferons alone appeared insufficient to explain this protective phenotype, serum isolated at early time points from primed animals conferred protection against an IC dose of virus. Adaptive immune populations had minimal contributions. Finally, the therapeutic utility of this novel strategy was demonstrated by peripherally priming and intracranially treating mice bearing aggressive CT2A syngeneic astrocytomas with VSVΔ51. Approximately 25% of animals achieved complete regression of established tumors, with no signs of virus-induced neurological impairment. This approach may harness an early warning system in the brain that has evolved to protect the host against otherwise lethal neurotropic viral infections. We have exploited this protective mechanism to safely and efficaciously treat brain tumors with an otherwise neurotoxic virus, potentially widening the available treatment options for oncolytic virotherapy in the brain.