Stephen Boulton

Background: Oncolytic viruses are tumor-specific immunotherapeutic agents that exploit inherent features of the tumor microenvironment to replicate, spread, and kill cancer cells. The exchange protein activated by cAMP (EPAC) is a cell signaling protein that regulates pathways important for cell growth, survival, and migration, which are commonly associated with cancer progression, but are also very important for regulation of viral infectivity. EPAC antagonism has been explored as a broad-spectrum antiviral strategy, while selective EPAC activation with cAMP analogs has been found to increase virus replication and enhance therapeutic outcome of oncolytic virotherapy. However, systemic EPAC agonism bears risk of cardiovascular complications and may potentiate cancer progression. Methods: A constitutively active construct of EPAC was encoded into an oncolytic vaccinia virus (VV) and screened using plaque assays, spheroid infections, and Transwell migration assays for its ability to enhance virus replication and spread. In vivo luminescence imaging, titering and immunohistochemical staining was used to measure virus dissemination in primary injected tumors and to track their spread to distal untreated tumors. The impact of the VV-EPAC virus on the immune landscape of MC38 tumors was investigated by flow cytometry, ELISPOTs and cytokine ELISAs, while its overall therapeutic efficacy was explored in MC38, CT26LacZ, and B16F10 models. Combinational synergy was also tested with capecitabine and oxaliplatin chemotherapy, as well as with partial surgical resection. Results: The EPAC-expressing virus exhibited an increase in migrative ability both in cell culture and in vivo, due in part to remodeling of the actin cytoskeleton leading to intercellular nanotube-like structures and enhanced syncytia formation. It reduced tumor burden and increased survival in multiple colorectal cancer models and reshaped the tumor microenvironment by inducing angiogenesis and recruiting CD8+T cells. The EPAC-expressing virus also synergized with conventional chemotherapy and exhibited a remarkable therapeutic benefit when used together with surgical resection to treat a metastatic melanoma model. Despite the noted benefits that EPAC offers to virus and cancer growth, no significant increase in off-target replication, cytotoxicity, or disease progression was observed. Conclusions: Altogether, the encoding of cellular signaling proteins into oncolytic viruses that modulate the intracellular and extracellular environments of tumors to create conditions favorable for virus replication and dissemination appears as a promising strategy to treat tumors and synergize with other conventional cancer therapies.

Targeted antineoplastic immunotherapies have achieved remarkable clinical outcomes. However, resistance to these therapies due to target absence or antigen shedding limits their efficacy and excludes tumours from candidacy. To address this limitation, here we engineer an oncolytic rhabdovirus, vesicular stomatitis virus (VSVΔ51), to express a truncated targeted antigen, which allows for HER2-targeting with trastuzumab. The truncated HER2 (HER2T) lacks signaling capabilities and is efficiently expressed on infected cell surfaces. VSVΔ51-mediated HER2T expression simulates HER2-positive status in tumours, enabling effective treatment with the antibody-drug conjugate trastuzumab emtansine in vitro, ex vivo, and in vivo. Additionally, we combine VSVΔ51-HER2T with an oncolytic vaccinia virus expressing a HER2-targeted T-cell engager. This dual-virus therapeutic strategy demonstrates potent curative efficacy in vivo in female mice using CD3+ infiltrate for anti-tumour immunity. Our findings showcase the ability to tailor the tumour microenvironment using oncolytic viruses, thereby enhancing compatibility with “off-the-shelf” targeted 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.

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.

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.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents an urgent need for an effective vaccine. Molecular characterization of SARS-CoV-2 is critical to the development of effective vaccine and therapeutic strategies. In the present study, we show that the fusion of the SARS-CoV-2 spike protein receptor-binding domain to its transmembrane domain is sufficient to mediate trimerization. Our findings may have implications for vaccine development and therapeutic drug design strategies targeting spike trimerization. As global efforts for developing SARS-CoV-2 vaccines are rapidly underway, we believe this observation is an important consideration for identifying crucial epitopes of SARS-CoV-2.