Viral-mediated activation and inhibition of programmed cell death.

Viruses are ubiquitous intracellular genetic parasites that heavily rely on the infected cell to complete their replication life cycle. This dependency on the host machinery forces viruses to modulate a variety of cellular processes including cell survival and cell death. Viruses are known to activate and block almost all types of programmed cell death (PCD) known so far. Modulating PCD in infected hosts has a variety of direct and indirect effects on viral pathogenesis and antiviral immunity. The mechanisms leading to apoptosis following virus infection is widely studied, but several modalities of PCD, including necroptosis, pyroptosis, ferroptosis, and paraptosis, are relatively understudied. In this review, we cover the mechanisms by which viruses activate and inhibit PCDs and suggest perspectives on how these affect viral pathogenesis and immunity.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

A Combination of Chemotherapy and Oncolytic Virotherapy Sensitizes Colorectal Adenocarcinoma to Immune Checkpoint Inhibitors in a cDC1-Dependent Manner.

Immune checkpoint therapy has shown great promise in the treatment of cancers with a high mutational burden, such as mismatch repair-deficient colorectal carcinoma (dMMR CRC). However, many patients fail to respond to immune checkpoint therapy. Using a mouse model of dMMR CRC, we demonstrated that tumors can be further sensitized to immune checkpoint therapy by using a combination of low-dose chemotherapy and oncolytic HSV-1. This combination induced the infiltration of CD8+ and CD4+ T cells into the tumor and the upregulation of gene signatures associated with the chemoattraction of myeloid cell subsets. When combined with immune checkpoint therapy, the combination promoted the infiltration of activated type 1 conventional dendritic cells (cDC1s) into the tumor. Furthermore, we found this combination strategy to be dependent on cDC1s, and its therapeutic efficacy to be abrogated in cDC1-deficient Batf3-/- mice. Thus, we demonstrated that the adjuvanticity of dMMR CRCs can be improved by combining low-dose chemotherapy and oncolytic HSV-1 in a cDC1-dependent manner.

Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis.

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.

Critical Interactions between Immunogenic Cancer Cell Death, Oncolytic Viruses, and the Immune System Define the Rational Design of Combination Immunotherapies.

Oncolytic viruses (OVs) are multimodal cancer therapeutics, with one of their dominant mechanisms being in situ vaccination. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. Immunogenic cell death (ICD) is a paradigm of cellular demise culminating in the spatiotemporal release of danger-associated molecular patterns that induce potent anticancer immunity. Alongside traditional ICD inducers like anthracycline chemotherapeutics and radiation, OVs have emerged as novel members of this class of therapeutics. OVs replicate in cancers and release tumor Ags, which are perceived as dangerous because of simultaneous expression of pathogen-associated molecular patterns that activate APCs. Therefore, OVs provide the target Ags and danger signals required to induce adaptive immune responses. This review discusses why OVs are attractive candidates for generating ICD, biological barriers limiting their success in the clinic, and groundbreaking strategies to potentiate ICD and antitumor immunity with rationally designed OV-based combination therapies.

The role of oncolytic virus immunotherapies to subvert cancer immune evasion.

ABSTRACT Despite huge economic and intellectual investments, developing effective cancer treatments continues to be an overarching challenge. Engineered oncolytic viruses (OVs) present self-amplifying immunotherapy platforms capable of preferential cytotoxicity to cancer cells and simultaneous activation of host anti-tumor immunity. In preclinical studies, OVs are showing potent therapeutic effects when used in combination with other immune therapy strategies. In the clinic, the immunotherapeutic effects of OVs are showing promising results. Here we review current strategies for engineering OVs, and present a perspective of future directions within a discussion of the current outcomes of combinatorial approaches with other cancer immunotherapy platforms.

Oncolytic virotherapy and immunogenic cancer cell death: sharpening the sword for improved cancer treatment strategies.

Oncolytic viruses are novel immunotherapeutics with increasingly promising outcomes in cancer patient clinical trials. Preclinical and clinical studies have uncovered the importance of virus-induced activation of antitumor immune responses for optimal therapeutic efficacy. Recently, several classes of chemotherapeutics have been shown to cause immunogenic cancer cell death characterized by the release of immunomodulatory molecules that activate antigen-presenting cells and thus trigger the induction of more potent anticancer adaptive immune responses. In preclinical models, several oncolytic viruses induce immunogenic cell death, which is associated with increased cross-priming of tumor-associated antigens. In this review, we discuss the recent advances in immunogenic cancer cell death as induced by chemotherapeutic treatments, including the roles of relevant danger-associated molecular patterns and signaling pathways, and highlighting the significance of the endoplasmic reticulum (ER) stress response. As virtually all viruses modulate both ER stress and cell death responses, we provide perspectives on future research directions that can be explored to optimize oncolytic viruses, alone or in combination with targeted drug therapies, as potent immunogenic cancer cell death-inducing agents. We propose that such optimized virus-drug synergistic strategies will improve the therapeutic outcomes for many currently intractable cancers.

Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.

Determinants for Antitumor and Protumor Effects of Programmed Cell Death.

Cytotoxic anticancer therapies activate programmed cell death in the context of underlying stress and inflammatory signaling to elicit the emission of danger signals, cytokines, and chemokines. In a concerted manner, these immunomodulatory secretomes stimulate antigen presentation and T cell-mediated anticancer immune responses. In some instances, cell death-associated secretomes attract immunosuppressive cells to promote tumor progression. As it stands, cancer cell death-induced changes in the tumor microenvironment that contribute to antitumor or protumor effects remain largely unknown. This is complicated to examine because cell death is often subverted by tumors to circumvent natural, and therapy-induced, immunosurveillance. Here, we provide insights into important but understudied aspects of assessing the contribution of cell death to tumor elimination or cancer progression, including the role of tumor-associated genetics, epigenetics, and oncogenic factors in subverting immunogenic cell death. This perspective will also provide insights on how future studies may address the complex antitumor and protumor immunologic effects of cell death, while accounting for variations in tumor genetics and underlying microenvironment.

Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells.

BACKGROUND: Initiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer cells. Induction of immunogenic cell death after certain types of cytotoxic anticancer therapies can stimulate T cell-mediated immunity. However, cytotoxic therapies simultaneously activate multiple types of cellular stress and programmed cell death; hence, it remains unknown what types of cancer cell death confer superior antitumor immunity. METHODS: Murine cancer cells were engineered to activate apoptotic or pyroptotic cell death after Dox-induced expression of procell death proteins. Cell-free supernatants were collected to measure secreted danger signals, cytokines, and chemokines. Tumors were formed by transplanting engineered tumor cells to specifically activate apoptosis or pyroptosis in established tumors and the magnitude of immune response measured by flow cytometry. Tumor growth was measured using calipers to estimate end point tumor volumes for Kaplan-Meier survival analysis. RESULTS: We demonstrated that, unlike apoptosis, pyroptosis induces an immunostimulatory secretome signature. In established tumors pyroptosis preferentially activated CD103+ and XCR1+ type I conventional DCs (cDC1) along with a higher magnitude and functionality of tumor-specific CD8+ T cells and reduced number of regulatory T cells within the tumor. Depletion of cDC1 or CD4+ and CD8+ T cells ablated the antitumor response leaving mice susceptible to a tumor rechallenge. CONCLUSION: Our study highlights that distinct types of cell death yield varying immunotherapeutic effect and selective activation of pyroptosis can be used to potentiate multiple aspects of the anticancer immunity cycle.

Adaptation of transgene mRNA translation boosts the anticancer efficacy of oncolytic HSV1.

BACKGROUND: Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells. METHODS: Using RNA-Seq reads, we determined the transcription start sites and 5'leaders of HSV1 genes and uncovered the US11 5'leader to confer superior activity in translation reporter assays. We then incorporated this 5'leader into GM-CSF expression cassette in oncolytic HSV1 and compared the translationally adapted oncolytic virus with the conventional, leaderless, virus in vitro and in mice. RESULTS: Inclusion of the US11 5'leader in the GM-CSF transgene incorporated into HSV1 boosted translation in vitro and in vivo. Importantly, treatment with US11 5'leader-GM-CSF oncolytic HSV1 showed superior antitumor immune activity and improved survival in a syngeneic mouse model of colorectal cancer as compared with leaderless-GM-CSF HSV1. CONCLUSIONS: Our study demonstrates the therapeutic value of identifying and integrating platform-specific cis-acting sequences that confer increased protein synthesis on transgene expression.

Tumor-intrinsic determinants of immunogenic cell death modalities.

The immune system can recognize tumor cells to mount antigen-specific T cell response. Central to the establishment of T cell-mediated adaptive immunity are the inflammatory events that facilitate antigen presentation by stimulating the expression of MHC and costimulatory molecules and the secretion of pro-inflammatory cytokines. Such inflammatory events can be triggered upon cytotoxic treatments that induce immunogenic cancer cell death modalities. However, cancers have acquired a plethora of mechanisms to subvert, or to hide from, host-encoded immunosurveillance. Here, we discuss how tumor intrinsic oncogenic factors subvert desirable intratumoral inflammation by suppressing immunogenic cell death.

TRIM26 Facilitates HSV-2 Infection by Downregulating Antiviral Responses through the IRF3 Pathway.

Herpes simplex virus type 2 (HSV-2) is the primary cause of genital herpes which results in significant morbidity and mortality, especially in women, worldwide. HSV-2 is transmitted primarily through infection of epithelial cells at skin and mucosal surfaces. Our earlier work to examine interactions between HSV-2 and vaginal epithelial cells demonstrated that infection of the human vaginal epithelial cell line (VK2) with HSV-2 resulted in increased expression of TRIM26, a negative regulator of the Type I interferon pathway. Given that upregulation of TRIM26 could negatively affect anti-viral pathways, we decided to further study the role of TRIM26 in HSV-2 infection and replication. To do this, we designed and generated two cell lines derived from VK2s with TRIM26 overexpressed (OE) and knocked out (KO). Both, along with wildtype (WT) VK2, were infected with HSV-2 and viral titres were measured in supernatants 24 h later. Our results showed significantly enhanced virus production by TRIM26 OE cells, but very little replication in TRIM26 KO cells. We next examined interferon-ß production and expression of two distinct interferon stimulated genes (ISGs), MX1 and ISG15, in all three cell lines, prior to and following HSV-2 infection. The absence of TRIM26 (KO) significantly upregulated interferon-ß production at baseline and even further after HSV-2 infection. TRIM26 KO cells also showed significant increase in the expression of MX1 and ISG15 before and after HSV-2 infection. Immunofluorescent staining indicated that overexpression of TRIM26 substantially decreased the nuclear localization of IRF3, the primary mediator of ISG activation, before and after HSV-2 infection. Taken together, our data indicate that HSV-2 utilizes host factor TRIM26 to evade anti-viral response and thereby increase its replication in vaginal epithelial cells.

Immune checkpoint blockade in triple negative breast cancer influenced by B cells through myeloid-derived suppressor cells.

Triple negative breast cancer holds a dismal clinical outcome and as such, patients routinely undergo aggressive, highly toxic treatment regimens. Clinical trials for TNBC employing immune checkpoint blockade in combination with chemotherapy show modest prognostic benefit, but the percentage of patients that respond to treatment is low, and patients often succumb to relapsed disease. Here, we show that a combination immunotherapy platform utilizing low dose chemotherapy (FEC) combined with oncolytic virotherapy (oHSV-1) increases tumor-infiltrating lymphocytes, in otherwise immune-bare tumors, allowing 60% of mice to achieve durable tumor regression when treated with immune checkpoint blockade. Whole-tumor RNA sequencing of mice treated with FEC + oHSV-1 shows an upregulation of B cell receptor signaling pathways and depletion of B cells prior to the start of treatment in mice results in complete loss of therapeutic efficacy and expansion of myeloid-derived suppressor cells. Additionally, RNA sequencing data shows that FEC + oHSV-1 suppresses genes associated with myeloid-derived suppressor cells, a key population of cells that drive immune escape and mediate therapeutic resistance. These findings highlight the importance of tumor-infiltrating B cells as drivers of antitumor immunity and their potential role in the regulation of myeloid-derived suppressor cells.

Cytokines in oncolytic virotherapy.

Tumors represent a hostile environment for the effector cells of cancer immunosurveillance. Immunosuppressive receptors and soluble or membrane-bound ligands are abundantly exposed and released by malignant entities and their stromal accomplices. As a consequence, executioners of antitumor immunity inefficiently navigate across cancer tissues and fail to eliminate malignant targets. By inducing immunogenic cancer cell death, oncolytic viruses profoundly reshape the tumor microenvironment. They trigger the local spread of danger signals and tumor-associated (as well as viral) antigens, thus attracting antigen-presenting cells, promoting the activation and expansion of lymphocytic populations, facilitating their infiltration in the tumor bed, and reinvigorating cytotoxic immune activity. The present review recapitulates key chemokines, growth factors and other cytokines that orchestrate this ballet of antitumoral leukocytes upon oncolytic virotherapy.

Genetic modification of oncolytic viruses to enhance antitumor immunity.

Among the many immunotherapies being developed and tested both preclinically and clinically, oncolytic viruses (OVs) are gaining traction as a forerunner in the search for potent new therapeutic agents, with a genetically engineered herpes simplex virus type 1 (HSV-1) recently approved by the FDA for the treatment of melanoma. The great potential of OVs to fight cancer is driving different approaches to improve OV-based therapy, with genetic modification of OVs to enhance host antitumor immunity being one of the most promising approaches. In this chapter we describe possible modifications in the OV genome that could increase its antitumor activity and immunostimulatory capacity, together with different methods to achieve these goals. Finally, we present different analyses to verify the desired genetic modification and evaluate its impact on host antitumor immunity in preliminary stages.

Enhanced immunotherapeutic profile of oncolytic virus-based cancer vaccination using cyclophosphamide preconditioning.

Despite a sizeable body of research, the efficacy of therapeutic cancer vaccines remains limited when applied as sole agents. By using a prime:boost approach involving two viral cancer vaccines, we were able to generate large tumor-specific CD8+ T-cell responses in a murine model of disseminated pulmonary melanoma. Significant increases in the number and quality of circulating effector T-cells were documented when low-dose cyclophosphamide (CTX) was administered pre-vaccination to tumor-bearing but not tumor-free hosts. Interestingly, tumor-bearing mice receiving CTX and co-primed with a melanoma differentiation antigen together with an irrelevant control antigen exhibited significantly enhanced immunity against the tumor, but not the control antigen, in secondary lymphoid organs. This result highlighted an increased cancer-specific reactivity of vaccine-induced T-cell responses following CTX preconditioning. Additionally, an acute reduction of the frequency of peripheral regulatory T-cells (Tregs) was noticeable, particularly in the proliferating, presumably tumour-reactive, subset. Enhanced infiltration of lungs with multifunctional T-cells resulted in overt reduction in metastatic burden in mice pretreated with CTX. Despite doubling the median survival in comparison to untreated controls, most vaccinated mice ultimately succumbed to cancer progression. However, preconditioning of the virus-based vaccination with CTX resulted in a remarkable improvement of the therapeutic activity leading to complete remission in the majority of the animals. Collectively, these data reveal how CTX can potentiate specific cellular immunity in an antigen-restricted manner that is only observed in vaccinated tumor-bearing hosts while depleting replicating Tregs. A single low dose of CTX enhances antitumor immunity and the efficacy of this potent prime:boost platform by modulating the kinetics of the vaccine-specific responses. Clinical assessment of CTX combined with next-generation cancer vaccines is indicated.

De novo necroptosis creates an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors.

Cancer immunotherapies using monoclonal antibodies to block inhibitory checkpoints are showing durable remissions in many types of cancer patients, although the majority of breast cancer patients acquire little benefit. Human melanoma and lung cancer patient studies suggest that immune checkpoint inhibitors are often potent in patients that already have intratumoral T cell infiltrate; although it remains unknown what types of interventions can result in an intratumoral T cell infiltrate in breast cancer. Using non-T cell-inflamed mammary tumors, we assessed what biological processes and downstream inflammation can overcome the barriers to spontaneous T cell priming. Here we show a specific type of combination therapy, consisting of oncolytic virus and chemotherapy, activates necroptosis and limits tumor growth in autochthonous tumors. Combination therapy activates proinflammatory cytokines; intratumoral influx of myeloid cells and cytotoxic T cell infiltrate in locally treated and distant autochthonous tumors to render them susceptible to immune checkpoint inhibitors.

Structural and functional studies of an IRF-7-like gene from Atlantic salmon.

Interferon regulatory factor 7 (IRF-7) plays a crucial role in virus-induced activation of interferon-alpha/beta transcription in mammals. This work describes a structural and functional homologue of mammalian IRF-7 from Atlantic salmon. The cloned gene encodes a putative protein of 415 amino acids (aa), which groups with mammalian IRF-7 and other fish IRF-7-like proteins in a phylogenetic analysis of vertebrate IRFs. Using an IFN promoter-luciferase assay we showed that salmon IRF-7 gave increased promoter activity after poly I:C stimulation. Transcript levels of IRF-7 were measured by real-time RT-PCR and compared to those of signal transducer and activator of transcription 1 (STAT1), which is important for transcriptional activation of IFN stimulated genes. Recombinant salmon IFN-alpha1 and poly I:C proved to be potent inducers of IRF-7 in Atlantic salmon TO cells, and poly I:C also induced the gene in head kidney and liver of Atlantic salmon. STAT1 was also induced by IFN, but was only weakly induced by poly I:C stimulation in vitro. Differences in transcription kinetics between IRF-7 and STAT1 thus indicate that the genes are regulated through different pathways. Finally, infection of TO cells with infectious salmon anemia virus (ISAV) induced early synthesis of STAT1 mRNA, whereas IRF-7 transcripts were upregulated much later. This indicates that ISAV has mechanisms to antagonize IRF-7 transcription and thus also the IFN system in Atlantic salmon.

Pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer.

The use of oncolytic viruses (OVs) for cancer treatment is emerging as a successful strategy that combines the direct, targeted killing of the cancer with the induction of a long-lasting anti-tumor immune response. Using multiple aggressive murine models of triple-negative breast cancer, we have recently demonstrated that the early administration of oncolytic Maraba virus (MRB) prior to surgical resection of the primary tumor is sufficient to minimize the metastatic burden, protect against tumor rechallenge, cure a fraction of the mice and sensitize refractory tumors to immune checkpoint blockade without the need for further treatment. Here, we apply our surgical model to other OVs: Vesicular stomatitis virus (VSV), Adenovirus (Ad), Reovirus (Reo) and Herpes simplex virus (HSV) and show that all of the tested OVs could positively change the outcome of the treated animals. The growth of the primary and secondary tumors was differently affected by the various OVs and most of the viruses conferred survival benefits in this neoadjuvant setting despite the absence of direct treatment following rechallenge. This study establishes that OV-therapy confers long-term protection when administered in the pre-operative window of opportunity.