Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Optimal delivery of RNA interference by viral vectors for cancer therapy.

In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Extracellular Vesicles and Viruses: Two Intertwined Entities.

Viruses share many attributes in common with extracellular vesicles (EVs). The cellular machinery that is used for EV production, packaging of substrates and secretion is also commonly manipulated by viruses for replication, assembly and egress. Viruses can increase EV production or manipulate EVs to spread their own genetic material or proteins, while EVs can play a key role in regulating viral infections by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. Ultimately, the interactions between EVs and viruses are highly interconnected, which has led to interesting discoveries in their associated roles in the progression of different diseases, as well as the new promise of combinational therapeutics. In this review, we summarize the relationships between viruses and EVs and discuss major developments from the past five years in the engineering of virus-EV therapies.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

From scourge to cure: tumour-selective viral pathogenesis as a new strategy against cancer.

Tumour mutations corrupt cellular pathways, and accumulate to disrupt, dysregulate, and ultimately avoid mechanisms of cellular control. Yet the very changes that tumour cells undergo to secure their own growth success also render them susceptible to viral infection. Enhanced availability of surface receptors, disruption of antiviral sensing, elevated metabolic activity, disengagement of cell cycle controls, hyperactivation of mitogenic pathways, and apoptotic avoidance all render the malignant cell environment highly supportive to viral replication. The therapeutic use of oncolytic viruses (OVs) with a natural tropism for infecting and subsequently lysing tumour cells is a rapidly progressing area of cancer research. While many OVs exhibit an inherent degree of tropism for transformed cells, this can be further promoted through pharmacological interventions and/or the introduction of viral mutations that generate recombinant oncolytic viruses adapted to successfully replicate only in a malignant cellular environment. Such adaptations that augment OV tumour selectivity are already improving the therapeutic outlook for cancer, and there remains tremendous untapped potential for further innovation.

Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts.

Phosphorylation and membrane association of the Rubella virus capsid protein is important for its anti-apoptotic function.

Rubella virus (RV), a member of Togaviridae, is an important human pathogen that can cause severe defects in the developing fetus. Compared to other togaviruses, RV replicates very slowly suggesting that it must employ effective mechanisms to delay the innate immune response. A recent study by our laboratory revealed that the capsid protein of RV is a potent inhibitor of apoptosis. A primary mechanism by which RV capsid interferes with programmed cell death appears to be through interaction with the pro-apoptotic Bcl-2 family member Bax. In the present study, we report that the capsid protein also blocks IRF3-dependent apoptosis induced by the double-strand RNA mimic polyinosinic-polycytidylic acid. In addition, analyses of cis-acting elements revealed that phosphorylation and membrane association are important for its anti-apoptotic function. Finally, the observation that hypo-phosphorylated capsid binds Bax just as well as wild-type capsid protein suggests that interaction with this pro-apoptotic host protein in and of itself is not sufficient to block programmed cell death. This provides additional evidence that this viral protein inhibits apoptosis through multiple mechanisms.

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.

PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

Generation and Quantification of Cytotoxic Lymphocytes Following Oncolytic Virus Infection of Multi-cellular Tumor Spheroids.

Oncolytic viruses (OVs) rapidly and specifically replicate in and kill tumor cells. OV-targeted infection of malignant cells has the potential to create an "inflammatory storm" that stimulates both innate and adaptive anti-tumor immune responses. The generation of anti-tumor immunity following OV treatment has been shown to be crucial for effective therapy. Therefore, establishing methodologies to measure the generation of anti-tumor T cell responses following OV infection in in vitro assays, which better mimic the complexity of the human tumor microenvironment (TME), will be critical to harness the full potential of OV therapy. Such experimental platforms will accelerate the development of next-generation OVs that are capable of overcoming immunosuppressive networks found within the tumor microenvironment. Here we describe a method that was designed to test the generation and quantification of human tumor-specific T cells following OV infection of 3D tumor spheroids cultured with or without fibroblasts.

Syngeneic mouse model of human HER2+ metastatic breast cancer for the evaluation of trastuzumab emtansine combined with oncolytic rhabdovirus.

Background: Established mouse models of HER2+ cancer are based on the over-expression of rodent Neu/Erbb2 homologues, which are incompatible with human HER2 (huHER2) targeted therapeutics. Additionally, the use of immune-deficient xenograft or transgenic models precludes assessment of native anti-tumour immune responses. These hurdles have been a challenge for our understanding of the immune mechanisms behind huHER2-targeting immunotherapies. Methods: To assess the immune impacts of our huHER2-targeted combination strategy, we generated a syngeneic mouse model of huHER2+ breast cancer, using a truncated form of huHER2, HER2T. Following validation of this model, we next treated tumour-bearing with our immunotherapy strategy: oncolytic vesicular stomatitis virus (VSVΔ51) with clinically approved antibody-drug conjugate targeting huHER2, trastuzumab emtansine (T-DM1). We assessed efficacy through tumour control, survival, and immune analyses. Results: The generated truncated HER2T construct was non-immunogenic in wildtype BALB/c mice upon expression in murine mammary carcinoma 4T1.2 cells. Treatment of 4T1.2-HER2T tumours with VSVΔ51+T-DM1 yielded robust curative efficacy compared to controls, and broad immunologic memory. Interrogation of anti-tumour immunity revealed tumour infiltration by CD4+ T cells, and activation of B, NK, and dendritic cell responses, as well as tumour-reactive serum IgG. Conclusions: The 4T1.2-HER2T model was used to evaluate the anti-tumour immune responses following our complex pharmacoviral treatment strategy. These data demonstrate utility of the syngeneic HER2T model for assessment of huHER2-targeted therapies in an immune-competent in vivo setting. We further demonstrated that HER2T can be implemented in multiple other syngeneic tumour models, including but not limited to colorectal and ovarian models. These data also suggest that the HER2T platform may be used to assess a range of surface-HER2T targeting approaches, such as CAR-T, T-cell engagers, antibodies, or even retargeted oncolytic viruses.

Engineering Rapalog-Inducible Genetic Switches Based on Split-T7 Polymerase to Regulate Oncolytic Virus-Driven Production of Tumour-Localized IL-12 for Anti-Cancer Immunotherapy.

The approval of different cytokines as anti-neoplastic agents has been challenged by dose-limiting toxicities. Although reducing dose levels affords improved tolerability, efficacy is precluded at these suboptimal doses. Strategies combining cytokines with oncolytic viruses have proven to elicit potent survival benefits in vivo, despite promoting rapid clearance of the oncolytic virus itself. Herein, we developed an inducible expression system based on a Split-T7 RNA polymerase for oncolytic poxviruses to regulate the spatial and temporal expression of a beneficial transgene. This expression system utilizes approved anti-neoplastic rapamycin analogues for transgene induction. This treatment regimen thus offers a triple anti-tumour effect through the oncolytic virus, the induced transgene, and the pharmacologic inducer itself. More specifically, we designed our therapeutic transgene by fusing a tumour-targeting chlorotoxin (CLTX) peptide to interleukin-12 (IL-12), and demonstrated that the constructs were functional and cancer-selective. We next encoded this construct into the oncolytic vaccinia virus strain Copenhagen (VV-iIL-12mCLTX), and were able to demonstrate significantly improved survival in multiple syngeneic murine tumour models through both localized and systemic virus administration, in combination with rapalogs. In summary, our findings demonstrate that rapalog-inducible genetic switches based on Split-T7 polymerase allow for regulation of the oncolytic virus-driven production of tumour-localized IL-12 for improved anti-cancer immunotherapy.

A T cell-targeted multi-antigen vaccine generates robust cellular and humoral immunity against SARS-CoV-2 infection.

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Fatty acid transport protein inhibition sensitizes breast and ovarian cancers to oncolytic virus therapy via lipid modulation of the tumor microenvironment.

Introduction: Adipocytes in the tumour microenvironment are highly dynamic cells that have an established role in tumour progression, but their impact on anti-cancer therapy resistance is becoming increasingly difficult to overlook. Methods: We investigated the role of adipose tissue and adipocytes in response to oncolytic virus (OV) therapy in adipose-rich tumours such as breast and ovarian neoplasms. Results: We show that secreted products in adipocyte-conditioned medium significantly impairs productive virus infection and OV-driven cell death. This effect was not due to the direct neutralization of virions or inhibition of OV entry into host cells. Instead, further investigation of adipocyte secreted factors demonstrated that adipocyte-mediated OV resistance is primarily a lipid-driven phenomenon. When lipid moieties are depleted from the adipocyte-conditioned medium, cancer cells are re-sensitized to OV-mediated destruction. We further demonstrated that blocking fatty acid uptake by cancer cells, in a combinatorial strategy with virotherapy, has clinical translational potential to overcome adipocyte-mediated OV resistance. Discussion: Our findings indicate that while adipocyte secreted factors can impede OV infection, the impairment of OV treatment efficacy can be overcome by modulating lipid flux in the tumour milieu.

Intravesical immunotherapy with a GM-CSF armed oncolytic vesicular stomatitis virus improves outcome in bladder cancer.

A significant proportion of non-muscle invasive bladder cancer cases will progress to muscle invasive disease. Transurethral resection followed by Bacillus Calmette Guerin immunotherapy can reduce this risk, while cystectomy prior to muscle invasion provides the best option for survival. Currently, there are no effective treatments for Bacillus Calmette Guerin refractory disease. A novel oncolytic vesicular stomatitis virus containing the human GM-CSF transgene (VSVd51-hGM-CSF) was rescued and tested as a potential bladder-sparing therapy for aggressive bladder cancer. The existing variant expressing mouse GM-CSF was also used. Measurement of gene expression and protein level alterations of canonical immunogenic cell death associated events on mouse and human bladder cancer cell lines and spheroids showed enhanced release of danger signals and immunogenic factors following infection with VSVd51-m/hGM-CSF. Intravesical instillation of VSVd51-mGM-CSF into MB49 bladder cancer bearing C57Bl/6 mice demonstrated enhanced activation of peripheral and bladder infiltrating effector immune cells, along with improved survival and reduced tumor volume. Importantly, virus-mediated anti-tumor immunity was recapitulated in bladder cancer patient-derived organoids. These results suggest that VSVd51-hGM-CSF is a promising viro/immunotherapy that could benefit bladder cancer patients.