Modulation of Tumor Necrosis Factor by Microbial Pathogens

In response to invasion by microbial pathogens, host defense mechanisms get activated by both the innate and adaptive arms of the immune responses. TNF (tumor necrosis factor) is a potent proinflammatory cytokine expressed by activated macrophages and lymphocytes that induces diverse cellular responses that can vary from apoptosis to the expression of genes involved in both early inflammatory and acquired immune responses. A wide spectrum of microbes has acquired elegant mechanisms to overcome or deflect the host responses mediated by TNF. For example, modulatory proteins encoded by multiple families of viruses can block TNF and TNF-mediated responses at multiple levels, such as the inhibition of the TNF ligand or its receptors, or by modulating key transduction molecules of the TNF signaling pathway. Bacteria, on the other hand, tend to modify TNF-mediated responses specifically by regulating components of the TNF signaling pathway. Investigation of these diverse strategies employed by viral and bacterial pathogens has significantly advanced our understanding of both host TNF responses and microbial pathogenesis. This review summarizes the diverse microbial strategies to regulate TNF and how such insights into TNF modulation could benefit the treatment of inflammatory or autoimmune diseases

Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells

Oncolytic Virotherapy for Hematological Malignancies

Hematological malignancies such as leukemias, lymphomas, multiple myeloma (MM), and the myelodysplastic syndromes (MDSs) primarily affect adults and are difficult to treat. For high-risk disease, hematopoietic stem cell transplant (HCT) can be used. However, in the setting of autologous HCT, relapse due to contamination of the autograft with cancer cells remains a major challenge. Ex vivo manipulations of the autograft to purge cancer cells using chemotherapies and toxins have been attempted. Because these past strategies lack specificity for malignant cells and often impair the normal hematopoietic stem and progenitor cells, prior efforts to ex vivo purge autografts have resulted in prolonged cytopenias and graft failure. The ideal ex vivo purging agent would selectively target the contaminating cancer cells while spare normal stem and progenitor cells and would be applied quickly without toxicities to the recipient. One agent which meets these criteria is oncolytic viruses. This paper details experimental progress with reovirus, myxoma virus, measles virus, vesicular stomatitis virus, coxsackievirus, and vaccinia virus as well as requirements for translation of these results to the clinic

Oncolytic virotherapy of ovarian cancer

In the past two decades, more than 20 viruses with selective tropism for tumor cells have been developed as oncolytic viruses (OVs) for treatments of a variety of malignancies. Of these viruses, eleven have been tested in human ovarian cancer models in preclinical studies. So far, nine phase I or II clinical trials have been conducted or initiated using four different types of OVs in patients with recurrent ovarian cancers. In this article, we summarize the different OVs that are being assessed as therapeutics for ovarian cancer. We also present an overview of recent advances in identification of key genetic or immune-response pathways involved in tumorigenesis of ovarian cancer, which provides a better understanding of the tumor specificities and oncolytic properties of OVs. In addition, we discuss how next-generation OVs could be genetically modified or integrated into multimodality regimens to improve clinical outcomes based on recent advances in ovarian cancer biology

Bombyx mori nucleopolyhedrovirus: molecular biology and biotechnological applications for large-scale synthesis of recombinant proteins

Bombyx mori nucleopolyhedrosis virus (BmNPV), a natural pathogen of great economic significance in sericulture, has been exploited to generate recombinant baculoviruses harbouring genes of choice and achieve high level expression of cloned foreign genes. The BmNPV-based expression system offers the advantage of using the larval host rather than the insectderived cell lines for economic large-scale synthesis of biomolecules. Since commercial rearing of silkworms is routinely practised, a part of the silkworm stocks can be diverted to produce biomolecules other than silk and the more sophisticated tissue culture methodologies can be dispensed with on an industrial scale. Nonetheless, the BmNPV-based expression considerably lags behind the AcMNPV system in vogue today, in terms of the choice of expression-optimized cloning vectors and easy recombinant generation kits that are commercially available. This review provides a brief outline of the current state of knowledge on the basic biology and genomics of BmNPV and how the virus has been made use of to produce biomolecules through its larval host, the mulberry silkworm. Since hyper-transcription from the viral very late gene promoters forms the basis of high level expression through recombinant baculoviruses, some aspects of the viral gene transcription and the role of late gene expression factors have also been included in the review

RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection

Myxoma virus expressing LIGHT (TNFSF14) pre-loaded into adipose-derived mesenchymal stem cells is effective treatment for murine pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a weakly immunogenic fatal neoplasm. Oncolytic viruses with dual anti-cancer properties—oncolytic and immune response-boosting effects—have great potential for PDAC management. Adipose-derived stem cells (ADSCs) of mesenchymal origin were infected ex vivo with recombinant myxoma virus (MYXV), which encodes murine LIGHT, also called tumor necrosis factor ligand superfamily member 14 (TNFSF14). The viability and proliferation of ADSCs were not remarkably decreased (1–2 days) following MYXV infection, in sharp contrast to cells of pancreatic carcinoma lines studied, which were rapidly killed by the infection. Comparison of the intraperitoneal (IP) vs. the intravenous (IV) route of ADSC/MYXV administration revealed more pancreas-targeted distribution of the virus when ADSCs were delivered IP to mice bearing orthotopically injected PDAC. The biodistribution, tumor burden reduction and anti-tumor adaptive immune response were examined. Bioluminescence data, used to assess the presence of the luciferase-tagged virus after IP injection, indicated enhanced trafficking into the pancreata of mice bearing orthotopically-induced PDAC, as compared to tumor-free animals, resulting in extended survival of the treated PDAC-seeded animals and in the boosted expression of key adaptive immune response markers. We conclude that ADSCs pre-loaded with transgene-armed MYXV and administered IP allow for the effective ferrying of the oncolytic virus to sites of PDAC and mediate improved tumor regression

Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma

Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects

Parallel adaptation of rabbit populations to myxoma virus.

In the 1950s the myxoma virus was released into European rabbit populations in Australia and Europe, decimating populations and resulting in the rapid evolution of resistance. We investigated the genetic basis of resistance by comparing the exomes of rabbits collected before and after the pandemic. We found a strong pattern of parallel evolution, with selection on standing genetic variation favoring the same alleles in Australia, France, and the United Kingdom. Many of these changes occurred in immunity-related genes, supporting a polygenic basis of resistance. We experimentally validated the role of several genes in viral replication and showed that selection acting on an interferon protein has increased the protein's antiviral effect.This work was supported by grants from the Programa Operacional Potencial Humano–Quadro de Referência Estratégica Nacional funds from the European Social Fund and Portuguese Ministério da Ciência, Tecnologia e Ensino Superior to M.C. (IF/00283/2014/CP1256/CT0012), to P.J.E. (IF/00376/2015) and to J.M.A. (SFRH/BD/72381/2010). AM was supported by the European Research Council (grant 647787-LocalAdaptation). FJ was supported by the European Research Council (grant 281668). LL was supported by the European Research Council grant (339941-ADAPT). McFadden Lab is supported by National Institute of Health (NIH) grant R01 AI080607. S.C.G. holds a Sir Henry Dale Fellowship, co-funded by the Wellcome Trust and the Royal Society (098406/Z/12/Z)