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Background: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo., Methods: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors., Results: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors., Conclusions: By turning "two keys" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES., (© 2024. The Author(s).)

[This corrects the article DOI: 10.3389/fimmu.2024.1384623.]., (Copyright © 2024 Paudel, Hutzen, Miller, Garfinkle, Chen, Wang, Glaspell, Currier, Ringwalt, Boon, Mardis, Cairo, Ratner, Dodd, Cassady and Cripe.)

Introduction: Malignant peripheral nerve sheath tumors (MPNST) pose a significant therapeutic challenge due to high recurrence rates after surgical resection and a largely ineffective response to traditional chemotherapy. An alternative treatment strategy is oncolytic viroimmunotherapy, which can elicit a durable and systemic antitumor immune response and is Food and Drug Administration (FDA)-approved for the treatment of melanoma. Unfortunately, only a subset of patients responds completely, underscoring the need to address barriers hindering viroimmunotherapy effectiveness., Methods: Here we investigated the therapeutic utility of targeting key components of the MPNST immunosuppressive microenvironment to enhance viroimmunotherapy's antitumor efficacy in three murine models, one of which showed more immunogenic characteristics than the others., Results: Myelomodulatory therapy with pexidartinib, a small molecule inhibitor of CSF1R tyrosine kinase, and the oncolytic herpes simplex virus T-VEC exhibited the most significant increase in median survival time in the highly immunogenic model. Additionally, targeting myeloid cells with the myelomodulatory therapy trabectedin, a small molecule activator of caspase-8 dependent apoptosis, augmented the survival benefit of T-VEC in a less immunogenic MPNST model. However, tumor regressions or shrinkages were not observed. Depletion experiments confirmed that the enhanced survival benefit relied on a T cell response. Furthermore, flow cytometry analysis following combination viroimmunotherapy revealed decreased M2 macrophages and myeloid-derived suppressor cells and increased tumor-specific gp70+ CD8 T cells within the tumor microenvironment., Discussion: In summary, our findings provide compelling evidence for the potential to leverage viroimmunotherapy with myeloid cell targeting against MPNST and warrant further investigation., Competing Interests: LB was employed by JJP Biologics. MSC has served as a consultant for Jazz Pharmaceuticals, Omeros Pharmaceuticals, Servier Pharmaceuticals, Abbvie and Novartis Pharmaceuticals; Speakers Bureau for Jazz Pharmaceuticals, Amgen, Inc., Sanofi and Sobi; Advisory Board for Astra Zeneca; and research funding from Celularity, Merck, Miltenyi Biotec, Servier, Omeros, Jazz and Janssen. KC holds intellectual property for oncolytic virus C134, which was licensed to Mustang Bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Paudel, Hutzen, Miller, Garfinkle, Chen, Wang, Glaspell, Currier, Ringwalt, Boon, Mardis, Cairo, Ratner, Dodd, Cassady and Cripe.)

Introduction: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with unacceptably low cure rates occurring often in patients with neurofibromatosis 1 defects. To investigate oncolytic Herpes Simplex Virus (oHSV) as an immunotherapeutic approach, we compared viral replication, functional activity, and immune response between unarmed and interleukin 12 (IL-12)-armed oncolytic viruses in virus-permissive (B109) and -resistant (67C-4) murine MPNSTs., Methods: This study compared two attenuated IL-12-oHSVs with γ134.5 gene deletions (Δγ134.5) and the same transgene expression cassette. The primary difference in the IL-12-oHSVs was in their ability to counter the translational arrest response in infected cells. Unlike M002 (Δγ134.5, mIL-12), C002 (Δγ134.5, mIL-12, IRS1) expresses an HCMV IRS1 gene and evades dsRNA activated translational arrest in infected cells., Results and Discussion: Our results show that oHSV replication and gene expression results in vitro were not predictive of oHSV direct oncolytic activity in vivo. Tumors that supported viral replication in cell culture studies resisted viral replication by both oHSVs and restricted M002 transgene expression in vivo. Furthermore, two IL-12-oHSVs with equivalent transcriptional activity differed in IL-12 protein production in vivo, and the differences in IL-12 protein levels were reflected in immune infiltrate activity changes as well as tumor growth suppression differences between the IL-12-oHSVs. C002-treated tumors exhibited sustained IL-12 production with improved dendritic cells, monocyte-macrophage activity (MHCII, CD80/CD86 upregulation) and a polyfunctional Th1-cell response in the tumor infiltrates., Conclusion: These results suggest that transgene protein production differences between oHSVs in vivo, in addition to replication differences, can impact OV-therapeutic activity., Competing Interests: JM has the following relationships which may pose or be perceived as posing a financial conflict of interest: he is a board and equity holding member, in Aettis, Inc. and may receive royalties. The company holds frozen oncolytic viral stocks. Mustang Bio Tech is licensing the Intellectual Property IP of C134 an oncolytic viral Therapy. JM is blinded to the conditions for the C134 clinical trials. He is a shareholder for a privately held Small Business Innovation Research LLC, Treovir, Inc., concerning G207 oncolytic viral therapy now in clinical trial. Merck, Inc. provides industry grant support by providing Keytruda pembrolizumab for a clinical trial of M032 oncolytic virotherapy and financial support for a clinical trial. JM is a listee on Intellectual Property 1 related to a cancer immunotherapy system, and 2 to a novel immuno-virotherapeutic strategy targeting the glioma secretome. This IP has been filed by in8Bio formerly Incysus, Ltd. and has royalty earning potential. In the interest of full disclosure, KC receives licensure payments from Mustang Bio for the C134 virus, but there are no relevant financial conflicts for the technology addressed in this paper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Saini, Kim, Hernandez-Aguirre, Hedberg, Martin, Mo, Cripe, Markert, Cassady and Dhital.)

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma., Competing Interests: The authors in full transparency disclose the following commercial interactions. E.R.M. reports PACT Pharma LLC, scientific advisory board membership, honorarium, and stock options; Scorpion Therapeutics, LLC, scientific advisory board membership, honorarium, and stock options; Qiagen N.V., supervisory board membership, stock; Singular Genomics, Inc., board of directors membership and stock. K.A.C. reports stock in Mustang Bio licensure., (© 2023 The Author(s).)

High grade gliomas are identified as malignant central nervous tumors that spread rapidly and have a universally poor prognosis. Historically high grade gliomas in the pediatric population have been treated similarly to adult high grade gliomas. For the first time, the most recent classification of central nervous system tumors by World Health Organization has divided adult from pediatric type diffuse high grade gliomas, underscoring the biologic differences between these tumors in different age groups. The objective of our review is to compare high grade gliomas in the adult versus pediatric patient populations, highlighting similarities and differences in epidemiology, etiology, pathogenesis and therapeutic approaches. High grade gliomas in adults versus children have varying clinical presentations, molecular biology background, and response to chemotherapy, as well as unique molecular targets. However, increasing evidence show that they both respond to recently developed immunotherapies. This review summarizes the distinctions and commonalities between the two in disease pathogenesis and response to therapeutic interventions with a focus on immunotherapy., Competing Interests: MC serves as a consultant for Jazz, Omeros, Astra Zeneca, Nektar Therapeutics and Novartis and a member of the Speakers Bureau for Jazz, Servier, Amgen, Sanofi and Sobi. DL reports personal fees and other from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside the submitted work; In addition, DL has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. TC recently served as a one-time consultant to Blueprint, Incyte, Oncopeptides, serves as a DSMB chair for SpringWorks and is a cofounder of Vironexis Biotherapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aggarwal, Luo, Pehlivan, Hoang, Rajappa, Cripe, Cassady, Lee and Cairo.)

Purpose: Previously, clinical trials of experimental virotherapy for recurrent glioblastoma multiforme (GBM) demonstrated that inoculation with a conditionally replication-competent Δγ 1 34.5 oncolytic herpes simplex virus (oHSV), G207, was safe. Following the initial safety study, a phase Ib trial enrolled 6 adult patients diagnosed with GBM recurrence from which tumor tissue was banked for future studies., Patients and Methods: Here, we analyzed tumor RNA sequencing (RNA-seq) data obtained from pre- and posttreatment (collected 2 or 5 days after G207 injection) biopsies from the phase Ib study patients., Results: Using a Spearman rank-order correlation analysis, we identified approximately 500 genes whose expression pattern correlated with survival duration. Many of these genes were enriched for the intrinsic IFN-mediated antiviral and adaptive immune functional responses, including immune cell chemotaxis and antigen presentation to T-cells. Furthermore, we show that the expression of several T-cell-related genes was highest in the patient with the longest survival after G207 inoculation., Conclusions: Our data support that the oHSV-induced type I IFN production and the subsequent recruitment of an adaptive immune response differed between enrolled patients and showed association with survival duration in patients with recurrent malignant glioma after treatment with an early generation oHSV., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Oncolytic virotherapy (OV) is an immunotherapy that incorporates viral cancer cell lysis with engagement of the recruited immune response against cancer cells. Pediatric solid tumors are challenging targets because they contain both an inert immune environment and a quiet antigenic landscape, making them more resistant to conventional OV approaches. Further complicating this, herpes simplex virus suppresses host gene expression during virotherapy infection., Methods: We therefore developed a multimodal oncolytic herpes simplex virus (oHSV) that expresses ephrin A2 (EphA2), a shared tumor-associated antigen (TAA) expressed by many tumors to improve immune-mediated antitumor activity. We verified the virus genotypically and phenotypically and then tested it in an oHSV-resistant orthotopic model (including immunophenotypic analysis), in flank and in T cell-deficient mouse models. We then assessed the antigen-expressing virus in an unrelated peripheral tumor model that also expresses the shared tumor antigen and evaluated functional T-cell response from the treated mice., Results: Virus-based EphA2 expression induces a robust acquired antitumor immune responses in both an oHSV-resistant murine brain and peripheral tumor model. Our new multimodal oncolytic virus (1) improves survival in viroimmunotherapy resistant tumors, (2) alters both the infiltrating and peripheral T-cell populations capable of suppressing tumor growth on rechallenge, and (3) produces EphA2-specific CD8 effector-like populations., Conclusions: Our results suggest that this flexible viral-based platform enables immune recognition of the shared TAA and improves the immune-therapeutic response, thus making it well suited for low-mutational load tumors., Competing Interests: Competing interests: In the interest of full disclosure, KAC receives licensure payments from Mustang Bio for the C134 virus, but there are no relevant financial conflicts for the technology addressed in this paper., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.

The advent of immunotherapy has revolutionized how we manage and treat cancer. While the majority of immunotherapy-related studies performed to date have focused on adult malignancies, a handful of these therapies have also recently found success within the pediatric space. In this review, we examine the immunotherapeutic agents that have achieved the approval of the US Food and Drug Administration for treating childhood cancers, highlighting their development, mechanisms of action, and the lessons learned from the seminal clinical trials that ultimately led to their approval. We also shine a spotlight on several emerging immunotherapeutic modalities that we believe are poised to have a positive impact on the treatment of pediatric malignancies in the near future.

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive soft-tissue sarcoma amenable only to surgical resection. Oncolytic herpes simplex viruses (oHSVs) are a promising experimental therapy. We previously showed that basal interferon (IFN) and nuclear factor κB (NFκB) signaling upregulate IFN-stimulated gene (ISG) expression and restrict efficient viral infection and cell-to-cell spread in ∼50% of tested MPNSTs. Stimulator of Interferon Genes (STING) integrates DNA sensor activity and mediates downstream IFN signaling in infected cells. We sought to identify STING's role in oHSV resistance and contribution to basal ISG upregulation in MPNSTs. We show that the level of STING activity in human MPNST cell lines is predictive of oHSV sensitivity and that resistant cell lines have intact mechanisms for detection of cytosolic double-stranded DNA (dsDNA). Furthermore, we show that STING downregulation renders MPNSTs more permissive to oHSV infection and cell-to-cell spread. While next-generation viruses can exploit this loss of STING activity, first-generation viruses remain restricted. Finally, STING is not integral to the previously-observed basal ISG upregulation, indicating that other pathways contribute to basal IFN signaling in resistant MPNSTs. These data broaden our understanding of the intrinsic pathways in MPNSTs and their role in oHSV resistance and offer potential targets to potentiate oncolytic virus activity., (© 2019 The Authors.)

Solid tumors contain a mixture of malignant cells and non-malignant infiltrating cells that often create a chronic inflammatory and immunosuppressive microenvironment that restricts immunotherapeutic approaches. Although childhood and adult cancers share some similarities related to microenvironmental changes, pediatric cancers are unique, and adult cancer practices may not be wholly applicable to our pediatric patients. This review highlights the differences in tumorigenesis, viral infection, and immunologic response between children and adults that need to be considered when trying to apply experiences from experimental therapies in adult cancer patients to pediatric cancers., (© 2019 The Authors.)

Oncolytic viral therapy has gained significant traction as cancer therapy over the past 2 decades. Oncolytic viruses are uniquely designed both to lyse tumor cells through their replication and to recruit immune responses against virally infected cells. Increasingly, investigators are leveraging this immune response to target the immunosuppressive tumor microenvironment and improve immune effector response against bystander tumor cells. In this article, we review the spectrum of preclinical, early-stage clinical, and potential future efforts with cytokine-secreting oncolytic viruses, with a focus on the treatment of brain tumors and solid tumors.

A network of pattern recognition receptors (PRRs) is responsible for the detection of invading viruses and acts as the trigger for the host antiviral response. Central to this apparatus is stimulator of interferon genes (STING), which functions as a node and integrator of detection signals. Owing to its role in both intrinsic and adaptive immunity, STING has become a focus for researchers in the field of oncolytic virotherapy. In this review, we consider the function of the cGAS-STING axis and its regulation, both by cellular mechanisms and as a result of viral interference.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas resistant to most cancer treatments. Surgical resection remains the primary treatment, but this is often incomplete, ultimately resulting in high mortality and morbidity rates. There has been a resurgence of interest in oncolytic virotherapy because of encouraging preclinical and clinical trial results. Oncolytic herpes simplex virus (oHSV) selectively replicates in cancer cells, lysing the cell and inducing antitumor immunity. We previously showed that basal interferon (IFN) signaling increases interferon-stimulated gene (ISG) expression, restricting viral replication in almost 50% of MPNSTs. The FDA-approved drug ruxolitinib (RUX) temporarily resets this constitutively active STAT signaling and renders the tumor cells susceptible to oHSV infection in cell culture. In the studies described here, we translated our in vitro results into a syngeneic MPNST tumor model. Consistent with our previous results, murine MPNSTs exhibit a similar IFN- and ISG-mediated oHSV-resistance mechanism, and virotherapy alone provides no antitumor benefit in vivo However, pretreatment of mice with ruxolitinib reduced ISG expression, making the tumors susceptible to oHSV infection. Ruxolitinib pretreatment improved viral replication and altered the oHSV-induced immune-mediated response. Our results showed that this combination therapy increased CD8 + T-cell activation in the tumor microenvironment and that this population was indispensable for the antitumor benefit that follows from the combination of RUX and oHSV. These data suggest that JAK inhibition prior to oncolytic virus treatment augments both oHSV replication and the immunotherapeutic efficacy of oncolytic herpes virotherapy., (©2018 American Association for Cancer Research.)

Ewing sarcoma is a highly aggressive cancer that promotes the infiltration and activation of pro-tumor M2-like macrophages. Oncolytic virotherapy that selectively infects and destroys cancer cells is a promising option for treating Ewing sarcoma. The effect of tumor macrophages on oncolytic virus therapy, however, is variable among solid tumors and is unknown in Ewing sarcoma. We tested the effects of macrophage reduction using liposomal clodronate (Clodrosome) and trabectedin on the antitumor efficacy of intratumoral oncolytic herpes simplex virus, rRp450, in two Ewing sarcoma xenograft models. Both agents enhanced antitumor efficacy without increasing virus replication. The most profound effects were in A673 with only a transient effect on response rates in TC71. Interestingly, A673 was more dependent than TC71 on macrophages for its tumorigenesis. We found Clodrosome and virus together induced expression of antitumorigenic genes and reduced expression of protumorigenic genes in both the tumor-associated macrophages and the overall tumor stroma. Trabectedin reduced intratumoral natural killer (NK) cells, myeloid-derived suppressor cells, and M2-like macrophages, and prevented their increase following virotherapy. Our data suggest that a combination of trabectedin and oncolytic herpes virotherapy warrants testing in the clinical setting.

We identified 13 patients with cat scratch (Bartonella henselae) bone infection among those admitted to a large tertiary care children's hospital over a 12-year period. The median age was 7 years and the median time from onset of illness to diagnosis was 10 days. Multifocal osteomyelitis involving spine and pelvis was common; no patient had a lytic bone lesion. Median treatment duration was 28 days (IQR, 24.5 days). Despite significant variations in treatment duration and antimicrobial therapy choices, all patients showed improvement., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oncolytic viruses (OVs) do more than simply infect and kill host cells. The accepted mechanism of action for OVs consists of a primary lytic phase and a subsequent antitumor and antiviral immune response. However, not all cells are subject to the direct effects of OV therapy, and it is becoming clear that OVs can also impact uninfected cells in the periphery. This review discusses the effects of OVs on uninfected neighboring cells, so-called bystander effects, and implications for OV therapies alone or in combination with other standard of care chemotherapy., Competing Interests: Financial & competing interests disclosure This work was supported by The Ohio State University Pelotonia Graduate Fellowship and NIH T32 Systems and Integrated Biology Fellowship (5T32GM068412-10) at The Ohio State University to LS. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

High Mobility Group Box 1 (HMGB1) is a multifunctional protein that plays various roles in the processes of inflammation, cancer, and other diseases. Many reports document abundant HMGB1 release following infection with oncolytic viruses (OVs). Further, other groups including previous reports from our laboratory highlight the synergistic effects of OVs with chemotherapy drugs. Here, we show that virus-free supernatants have varying cytotoxic potential, and HMGB1 is actively secreted by two established fibroblast cell lines (NIH 3T3 and 3T6-Swiss albino) following HSV1716 infection in vitro. Further, pharmacologic inhibition or genetic knock-down of HMGB1 reveals a role for HMGB1 in viral restriction, the ability to modulate bystander cell proliferation, and drug sensitivity in 3T6 cells. These data further support the multifactorial role of HMGB1, and suggest it could be a target for modulating the efficacy of oncolytic virus therapies alone or in combination with other frontline cancer treatments., Competing Interests: Joe Conner and Lynne Braidwood are employees of Virttu Biologics, Ltd. (BioCity, UK) and Sorrento Therapeutics (San Diego, CA, USA). The other authors report no conflicts of interest in this work.

Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV) is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ 1 34.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Malignant glioma is the most common primary brain tumor and carries a grim prognosis, with a median survival of just over 14 months. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. The concept of virotherapy for the treatment of malignant tumors dates back more than a century and can be divided into replication-competent oncolytic viruses and replication-deficient viral vectors. Oncolytic viruses are designed to selectively target, infect, and replicate in tumor cells, while sparing surrounding normal brain. A host of oncolytic viruses has been evaluated in early phase human trials with promising safety results, but none has progressed to phase III trials. Despite the 25 years that has passed since the initial publication of genetically engineered oncolytic viruses for the treatment of glioma, much remains to be learned about the use of this therapy, including its mechanism of action, optimal treatment paradigm, appropriate targets, and integration with adjuvant agents. Oncolytic viral therapy for glioma remains promising and will undoubtedly impact the future of patient care.

Oncolytic herpes simplex virus (oHSV) type I constructs are investigational anti-neoplastic agents for a variety of malignancies, including malignant glioma. Clinical trials to date have supported the safety of these agents even when directly administered in the CNS. Traditional pre-clinical US Food and Drug Administration (FDA) toxicity studies for these agents have included the use of two species, generally including murine and primate studies. Recently, the FDA has decreased its requirement of non-human primates as an animal model for ethical reasons, especially for established viral systems where there are good alternative model systems. Here we present data demonstrating the safety of C134, a chimeric oHSV construct, in CBA mice as well as in a limited number of the HSV-sensitive non-human primate Aotus nancymaae as a proposed agent for clinical trials. These data, along with the previously conducted clinical trials of oHSV constructs, support the use of the CBA mouse model as sufficient for the pre-clinical toxicity studies of this agent. We summarize our experience with different HSV recombinants and differences between them using multiple assays to assess neurovirulence, as well as our experience with C134 in a limited number of A. nancymaae .

Unlabelled: Interferon-stimulated genes (ISG) encode diverse proteins that mediate intrinsic antiviral resistance in infected cells. Here it was hypothesized that malignant peripheral nerve sheath tumor (MPNST) cells resist the productive infection of oncolytic herpes simplex virus (oHSV) through activation of the JAK/STAT1 pathway and resultant upregulation of ISGs. Multiple human and mouse MPNST cells were used to explore the relationship between STAT1 activation and the productive infection of Δγ134.5 oHSVs. STAT1 activation in response to oHSV infection was found to associate with diminished Δγ134.5 oHSVs replication and spread. Multiday pretreatment, but not cotreatment, with a JAK inhibitor significantly improved viral titer and spread. ISG expression was found to be elevated prior to infection and downregulated when treated with the inhibitor, suggesting that the JAK/STAT1 pathway is active prior to infection. Conversely, upregulation of ISG expression in normally permissive cells significantly decreased oHSV productivity. Finally, a possible link between NF-κB pathway activation and ISG expression was established through the expression of inhibitor of kB (IκB) which decreased basal STAT1 transcription and ISG expression. These results demonstrate that basal ISG expression prior to infection contributes to the resistance of Δγ134.5 oHSVs in MPNST cells., Implications: Although cancer-associated ISG expression has been previously reported to impart resistance to chemotherapy and radiotherapy, these data show that basal ISG expression also contributes to oncolytic HSV resistance. Mol Cancer Res; 14(5); 482-92. ©2016 AACR., (©2016 American Association for Cancer Research.)

Over the past 1-2 decades we have witnessed a resurgence of efforts to therapeutically exploit the attributes of lytic viruses to infect and kill tumor cells while sparing normal cells. We now appreciate that the utility of viruses for treating cancer extends far beyond lytic cell death. Viruses are also capable of eliciting humoral and cellular innate and adaptive immune responses that may be directed not only at virus-infected cells but also at uninfected cancer cells. Here we review our current understanding of this bystander effect, and divide the mechanisms into lytic, cytokine, innate cellular, and adaptive phases. Knowing the key pathways and molecular players during virus infection in the context of the cancer microenvironment will be critical to devise strategies to maximize the therapeutic effects of oncolytic viroimmunotherapy.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.

Herpes simplex virus type 1 (HSV-1) mutants lacking the γ(1)34.5 neurovirulence loci are promising agents for treating malignant glioma. Arming oncolytic HSV-1 to express immunostimulatory genes may potentiate therapeutic efficacy. We have previously demonstrated improved preclinical efficacy, biodistribution, and safety of M002, a γ(1)34.5-deleted HSV-1 engineered to express murine IL-12. Herein, we describe the safety and biodistribution of M032, a γ(1)34.5-deleted HSV-1 virus that expresses human IL-12 after intracerebral administration to nonhuman primates, Aotus nancymae. Cohorts were administered vehicle, 10(6), or 10(8) pfu of M032 on day 1 and subjected to detailed clinical observations performed serially over a 92-day trial. Animals were sacrificed on days 3, 31, and 91 for detailed histopathologic assessments of all organs and to isolate and quantify virus in all organs. With the possible exception of one animal euthanized on day 16, neither adverse clinical signs nor sex- or dose-related differences were attributed to M032. Elevated white blood cell and neutrophil counts were observed in virus-injected groups on day 3, but no other significant changes were noted in clinical chemistry or coagulation parameters. Minimal to mild inflammation and fibrosis detected, primarily in meningeal tissues, in M032-injected animals on days 3 and 31 had mostly resolved by day 91. The highest viral DNA levels were detected at the injection site and motor cortex on day 3 but decreased in central nervous system tissues over time. These data demonstrate the requisite safety of intracerebral M032 administration for consideration as a therapeutic for treating malignant brain tumors.

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

While glioblastoma multiforme (GBM) is the most common adult malignant brain tumor, GBMs in childhood represent less than 10% of pediatric malignant brain tumors and are phenotypically and molecularly distinct from adult GBMs. Similar to adult patients, outcomes for children with high-grade gliomas (HGGs) remain poor. Furthermore, the significant morbidity and mortality yielded by pediatric GBM is compounded by neurotoxicity for the developing brain caused by current therapies. Poor outcomes have been attributed to a subpopulation of chemotherapy and radiotherapy resistant cells, termed "glioma stem cells" (GSCs), "glioma progenitor cells," or "glioma-initiating cells," which have the ability to initiate and maintain the tumor and to repopulate the recurring tumor after conventional therapy. Future innovative therapies for pediatric HGG must be able to eradicate these therapy-resistant GSCs. Oncolytic herpes simplex viruses (oHSV), genetically engineered to be safe for normal cells and to express diverse foreign anti-tumor therapeutic genes, have been demonstrated in preclinical studies to infect and kill GSCs and tumor cells equally while sparing normal brain cells. In this review, we discuss the unique aspects of pediatric GSCs, including markers to identify them, the microenvironment they reside in, signaling pathways that regulate them, mechanisms of cellular resistance, and approaches to target GSCs, with a focus on the promising therapeutic, genetically engineered oHSV.

Hypoxia plays a critical role in the tumor microenvironment of high-grade gliomas by promoting the glioma stem cell (GSC)-like phenotype, which displays resistance to standard therapies. We tested three glioblastoma multiforme xenograft lines (xenolines) against γ(1)34.5-deleted recombinant oncolytic herpes simplex virus (oHSV) C101 under 1% (hypoxia) and 20.8% (normoxia) oxygen tension for effects on oHSV infectivity, replication, and cytotoxicity in all tumor cells and CD133(+) GSCs. Expression levels of CD133, a putative GSC marker, and CD111 (nectin-1), an adhesion molecule that is the most efficient method for HSV entry, increased significantly under hypoxia in all three xenolines. Despite increased CD111 expression under hypoxic conditions, oHSV infectivity, cytotoxicity and viral recovery were not improved or were diminished in all three xenolines under hypoxia. In contrast, wild-type HSV-1 equally infected xenoline cells in normoxia and hypoxia, suggesting that the 34.5 mutation plays a role in the decreased C101 infectivity in hypoxia. Importantly, CD133(+) cells were not more resistant to oHSV than CD133(-) tumor cells regardless of oxygen tension. Furthermore, CD133 expression decreased as viral dose increased in two of the xenolines suggesting that up-regulation of CD133 in hypoxia was not the cause of reduced viral efficacy. Our findings that oHSV infectivity and cytotoxicity were diminished under hypoxia in several GBM xenolines likely have important implications for clinical applications of oHSV therapies, especially considering the vital role of hypoxia in the microenvironment of GBM tumors.

Cancer stem cells (CSCs), also termed "cancer-initiating cells" or "cancer progenitor cells," which have the ability to self-renew, proliferate, and maintain the neoplastic clone, have recently been discovered in a wide variety of pediatric tumors. These CSCs are thought to be responsible for tumorigenesis and tumor maintenance, aggressiveness, and recurrence due to inherent resistance to current treatment modalities such as chemotherapy and radiation. Oncolytic virotherapy offers a novel, targeted approach for eradicating pediatric CSCs using mechanisms of cell killing that differ from conventional therapies. Moreover, oncolytic viruses have the ability to target specific features of CSCs such as cell-surface proteins, transcription factors, and the CSC microenvironment. Through genetic engineering, a wide variety of foreign genes may be expressed by oncolytic viruses to augment the oncolytic effect. We review the current data regarding the ability of several types of oncolytic viruses (herpes simplex virus-1, adenovirus, reovirus, Seneca Valley virus, vaccinia virus, Newcastle disease virus, myxoma virus, vesicular stomatitis virus) to target and kill both CSCs and tumor cells in pediatric tumors. We highlight advantages and limitations of each virus and potential ways in which next-generation engineered viruses may target resilient CSCs.

The chimeric herpes simplex viruses (HSV) are Δγ₁34.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ₁34.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ₁34.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ₁34.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).

Human herpesviruses cause diseases of considerable morbidity and mortality, ranging from encephalitis to hematologic malignancies. As evidence emerges about the role of innate immunity and natural killer (NK) cells in the control of herpesvirus infection, evidence of viral methods of innate immune evasion grows as well. These methods include interference with the ligands on infected cell surfaces that bind NK cell activating or inhibitory receptors. This paper summarizes the most extensively studied NK cell receptor/ligand pairs and then describes the methods of NK cell evasion used by all eight herpesviruses through these receptors and ligands. Although great strides have been made in elucidating their mechanisms, there is still a disparity between viruses in the amount of knowledge regarding innate immune evasion. Further research of herpesvirus innate immune evasion can provide insight for circumventing viral mechanisms in future therapies.

Background: Prophylactic ganciclovir (GCV) is used in high-risk renal transplant patients to prevent acute cytomegalovirus (CMV) disease, but its impact on inflammation within the allograft itself remains undefined., Methods: To study the effect of GCV prophylaxis on allograft inflammation, murine CMV (MCMV)-infected allografts were analyzed in a murine donor positive/recipient negative allogeneic renal transplantation model by flow cytometry and immunofluorescent staining., Results: By flow cytometry, CD45+ leukocyte infiltrates were more abundant in MCMV-infected allografts at 14 days posttransplant compared with uninfected grafts (P<0.01) and decreased in the presence of GCV (P<0.05). CD11c+ dendritic cells, Gr-1+ myeloid cells, CD204+ macrophages, and CD49b+ natural killer cells were reduced in GCV-treated allografts compared with MCMV-infected grafts without GCV treatment (P<0.05). However, GCV failed to reduce these cell types to levels found in MCMV-uninfected allografts. By day 7 after cessation of GCV prophylaxis, dendritic cells, macrophages, and natural killer cells increased in number and became statistically indistinguishable from numbers of cells found in MCMV-infected allografts without GCV. GCV treatment did not affect the numbers of CD4+, CD8+, or CD19+/B220+ lymphocytes infiltrating the allografts. Infiltrates were confirmed histologically by immunofluorescent staining for CD3+ and CD11b+ cells., Conclusions: In this model, MCMV-infected allografts developed significantly greater innate and adaptive leukocytic infiltrates compared with uninfected grafts. GCV attenuated the MCMV-associated innate leukocyte infiltrates in infected allografts but not the lymphocytic infiltrates. The attenuated innate response was limited to the period of GCV prophylaxis.

Conditionally replication-competent Herpes Simplex Virus Type 1 (HSV-1) vectors expressing foreign genes have been developed as experimental therapeutic agents. Traditional methods of virus construction, including growth selection based on thymidine kinase gene expression, and color selection based on a reporter gene expression are often time-consuming and relatively inefficient. This review summarizes the various strategies developed in recent years for the rapid and efficient construction of novel conditionally replication-competent mutant HSV expressing multiple foreign genes. Additionally, two new modifications of existing strategies, which have not been previously reported, are discussed.

Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics. Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in patients, and their implication for design of future trials.

A virulent recombinant HSV lacking the diploid γ(1)34.5 gene (Δγ(1)34.5) have been investigated over the last two decades both for anti-tumor therapy and as vaccine vectors. The first generation vectors, while safe, are incapable of sustained replication in the majority of treated patients. An interferon inducible host antiviral kinase, protein kinase R (PKR), limits late viral protein synthesis and replication of Δγ(1)34.5 viruses. This review describes the development of new Δγ(1)34.5 vectors, through serial passage selection and direct viral genome engineering, which demonstrate selective PKR evasion in targeted cells and improved viral replication without restoring neurovirulence.

Oncolytic herpes simplex viruses (HSV) hold promise for therapy of glioblastoma multiforme (GBM) resistant to traditional therapies. We examined the ability of genetically engineered HSV to infect and kill cells that express CD133, a putative marker of glioma progenitor cells (GPC), to determine if GPC have an inherent therapeutic resistance to HSV. Expression of CD133 and CD111 (nectin-1), the major entry molecule for HSV, was variable in six human glioma xenografts, at initial disaggregation and after tissue culture. Importantly, both CD133+ and CD133- populations of glioma cells expressed CD111 in similar relative proportions in five xenografts, and CD133+ and CD133- glioma cell subpopulations were equally sensitive to killing in vitro by graded dilutions of wild-type HSV-1(F) or several different gamma(1)34.5-deleted viruses. GPC did not display an inherent resistance to HSV. While CD111 expression was an important factor for determining sensitivity of glioma cells to HSV oncolysis, it was not the only factor. Our findings support the notion that HSV will not be able to effectively enter, infect, and kill cells in tumors that have low CD111 expression (<20%). However, virotherapy with HSV may be very effective against CD111+ GPC resistant to traditional therapies.

A 21-month-old girl with neuroblastoma developed chronic verrucous Oka strain varicella-zoster infection during chemotherapy. Virus isolated from the patient demonstrated high-level acyclovir resistance, and its thymidine kinase had no in vitro enzymatic activity. After foscarnet therapy, she underwent stem cell transplantation without varicella reactivation. This is only the second reported case of resistant varicella zoster virus caused by Oka strain virus.

Oncolytic herpes simplex virus (HSV)-1 gamma(1)34.5-deletion mutants (Deltagamma(1)34.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of gamma(1)34.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Deltagamma(1)34.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Deltagamma(1)34.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Deltagamma(1)34.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Deltagamma(1)34.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.

Two patients present with the abrupt onset of fever, malaise, anorexia, fatigue, progressive skin lesions and lymphadenitis. These patients represent two of the six cases of tularemia reported in Alabama over the last decade. The cases illustrate how mode of acquisition (direct versus vector-mediated) influences the clinical manifestations of ulceroglandular tularemia. In addition, a brief review of the epidemiology, differential diagnosis, clinical manifestations, and treatment of tularemia is provided.

Previous studies have described in vitro serial passage of a Deltagamma(1)34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Deltagamma(1)34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U(S)11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U(S)11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.

The herpes simplex virus type 1 gamma(1)34.5 gene product precludes the host-mediated protein shutoff response induced by activated protein kinase R (PKR). Earlier studies demonstrated that recombinant viruses lacking the gamma(1)34.5 gene (Deltagamma(1)34.5) developed secondary mutations that allowed earlier U(S)11 expression and enabled continued protein synthesis. Further, in vitro studies demonstrated that a recombinant expressed U(S)11 protein binds PKR, blocks the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) by activated PKR, and, if provided prior to PKR activation, precluded PKR autophosphorylation. The present study furthers the hypothesis that early U(S)11 production precludes PKR-mediated host protein shutoff by demonstrating that (i) U(S)11 and PKR interact in the context of viral infection, (ii) this interaction is RNA dependent and requires a 30-amino-acid domain (amino acids 91 to 121) in the carboxyl domain of the U(S)11 protein, (iii) the proteins biochemically colocalize in the S100 ribosomal fraction, and (iv) there is a PKR substrate domain immediately adjacent to the binding domain. The results suggest that the U(S)11 interaction with PKR at the ribosome is RNA dependent and that the U(S)11 protein contains a substrate domain with homology to eIF-2alpha in close proximity to an essential binding domain.

Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the gamma(1)34.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In consequence, the gamma(1)34.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Deltagamma(1)34.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the alpha promoter of the U(S)12 gene to the coding sequence of the U(S)11 gene. The mutant blocks the phosphorylation of eIF-2alpha but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the U(S)10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2alpha was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.

Knowledge regarding the pathogenesis of viral encephalitis, defined as inflammation and destruction of the central nervous system (CNS) from viral infection and the resulting immune response, has improved with advances in molecular biology techniques and recent advances in immunology and neuroscience research. An increasingly complex understanding has developed with regard to viral CNS infection. In addition to advances in viral genetics exploring increased viral spread and neurovirulence, improved understanding from research on neurochemistry, neurodevelopment, and cytokine expression in the CNS has led to new hypotheses regarding the mechanism of CNS damage during viral CNS infection. This review explores three advances in the understanding of viral encephalitis in the past few years: 1) the relationship between viral load and extent of viral CNS disease, 2) chemokines and their role in the CNS inflammatory response as well as in the pathogenesis of encephalitis, and 3) secondary damage from the release of neurotoxins during encephalitis. By examining this research, the reviewers intend to introduce novel therapeutic modalities that are developing for the management of patients with viral encephalitis beyond the timely use of antiviral therapy.

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.