Glioblastoma is the most common and aggressive malignant brain tumor and has limited treatment options. Hence, innovative approaches are urgently needed. Oncolytic virus therapy is emerging as a promising modality for cancer treatment due to its tumor-specific fi c targeting and immune-stimulatory properties. In this study, we developed a new generation of oncolytic herpes simplex virus C5252 by deletion of a 15-kb internal repeat region and both copies of g 34.5 genes. Additionally, C5252 was armed with anti-programmed cell death protein 1 antibody and interleukin-12 to enhance its therapeutic efficacy fi cacy for glioblastoma immune-virotherapy. In vitro and in vivo experiments demonstrate that C5252 has a remarkable safety profi le and potent anti-tumor activity against glioblastoma. Mechanistic studies demonstrated that C5252 specifically fi cally induces cell apoptosis by caspase-3/7 activation via downregulating ciliary neurotrophic factor receptor a . Furthermore, the enhanced anti-tumor therapeutic efficacy fi cacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model was confirmed. fi rmed. Moreover, syngeneic mouse models showed that the murine surrogate of C5252 has superior anti-tumor activity compared to the unarmed backbone virus, with enhanced immune activation. Taken together, our fi nd- ings support C5252 as a promising therapeutic option for glioblastoma treatment, positioning it as a highly promising candidate for clinical translation.

Abstract Background: Glioblastoma (GBM), the most common and deadly primary brain tumor in adults, has limited treatment options with poor outcomes. The urgent need for innovative treatments has spurred research into immunotherapy and oncolytic virus therapy as promising alternatives. Herein, we have developed a new generation oncolytic herpes simplex virus (oHSV) armed with IL-12 and anti-PD-1 antibody to provide a synergistic anti-GBM efficacy for immune-oncolytic therapy. Methods: MVR-C5252, a novel oHSV, was created by genetic engineering, which included the removal of a 15-kb internal repeat (IR) region and both copies of the γ34.5 genes, along with the insertion of two exogenous genes encoding the anti-PD-1 antibody and IL-12. MVR-C5252 was characterized and assessed for its anti-GBM activities through a series of experiments as follows: 1) Safety assessments, including evaluations of neurotoxicity and latency-reactivation characteristics, were conducted in Balb/C mice. 2) Anti-tumor activities were evaluated in cell cultures, subcutaneous and orthotopic xenograft mouse models, and immunocompetent mouse models. 3) Mechanistic studies were performed to uncover the mode of action of MVR-C5252, with a specific focus on its ability to induce cell death in GBM cells and activate the immune response in vivo. Results: 1) In vitro studies showed that MVR-C5252 is highly replication attenuated but with higher cell-killing activity in GBM cells compared to the first generation oHSV, R3616, which has γ34.5 deletions only. 2) Mechanistic studies showed that MVR-C5252 infection increased cell death by reducing CNTNRα expression, which typically prevents caspase 3 activation and has an anti-apoptotic effect in glioma cells. 3) Intracranial injection of MVR-C5252 revealed a remarkable safety profile, with over an 800-fold reduction in virulence compared to wild-type HSV-1. Furthermore, the mouse trigeminal ganglion (TG) infection model suggested that MVR-C5252 was unable to establish latency or reactivate in the TG. 4) In vivo subcutaneous and orthotopic xenograft mouse models showed that compared to the R3616 group, enhanced anti-tumor activity and extended survival were observed in the MVR-C5252 group. Additionally, in the syngeneic mice model, MVR-C5252 exhibited superior anti-tumor activity and immune activation over the backbone virus. Conclusions: With the unique combination of safety, potent anti-tumor activity, and immune-stimulatory properties, MVR-C5252 makes it a big potential candidate to address the pressing need for effective treatment strategies of GBM. Citation Format: Lei Wang, Xusha Zhou, Xiaoqing Chen, Yuanyuan Liu, Yanxin Zheng, Runbin Yan, Yonghong Liu, Jing Zhao, Grace Guoying Zhou. Advanced glioblastoma immunotherapy: Attenuated herpes oncolytic virus armed with anti-PD-1 antibody and IL-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6652.

Background: Chimeric antigen receptor (CAR) T cell therapy has achieved unprecedented success in treating hematologic malignancies but still struggles in the context of solid tumors. The challenges of effective CAR-T cell therapy for solid tumors are multifaceted including 1) physical barriers that limit T cell infiltration; 2) tumor heterogeneity and antigen escape leading to resistance to therapy; 3) immunosuppressive tumor microenvironment (TME) dampening T cell function. As a novel modality for cancer therapy, oncolytic virus (OV) can be engineered to overcome the limitations of CAR-T therapy in solid tumor. Here we developed a new-generation of oncolytic herpes simplex virus (oHSV) to enable clinically approved CD19 CAR-T for solid tumor therapy. Methods: oHSV MVR-T7011 (T7011) was genetically engineered to drive ectopic expression of the extracellular domain of CD19, the blood tumor antigen, on solid tumor cell surface upon viral infection. In addition, multiple payloads, CCL5, IL-12, and anti-PD-1 antibody were introduced into T7011 to modulate the TME. Payload expression was detected both in vitro and in vivo. Co-culture studies were performed to test the cell-killing activity of CD19 CAR-T combined with T7011. Antitumor activities of T7011 administrated via intratumoral (IT) or intraperitoneal (IP) in combination with CD19 CAR-T cells were assessed in immunodeficient and immunocompetent mice. Results: The in vitro studies indicated that the expression of CD19 on tumor cell surface was detected as early as 4 hours post infection (hpi) and retained for at least 96 hours in the T7011-infected cells. In mouse study showed that the CD19 expression on tumor cells was detected as early as 8 hpi and lasted for 20 days when T7011 was injected via IT in immunodeficient mice. The payload of CCL5, IL-12, and anti-PD-1 antibody showed a similar expression pattern as CD19. Co-culture studies showed that T7011 infection enhanced cell-killing activity of CD19 CAR-T but the virus had no effect on viability and proliferation of CAR-T cell itself, which concludes that T7011 does not dampen the CAR-T function as its combination treatment. The in vivo efficacy studies demonstrated that IT and IP administration of T7011, rather than MVR-T3011 virus which lacks CD19, specifically promoted CD19 CAR-T anti-solid tumor activities in both immunodeficient and immunocompetent mice. Conclusions: The new-generation of HSV oncolytic virus T7011 expressing the targetable CD19 antigen on tumor surface enables the CD19 CAR-T for solid tumor cell treatment as a combination therapy. In addition, T7011 also carries multi-immunomodulators further to enhance antitumor efficacy by reinvigorating the infiltrated CAR-T cells and reversing the immunosuppressive TME. T7011 is expected to be a promising combinational therapy with CD19-specific CAR-T cells enabling effective cell therapy against multiple solid tumors. Citation Format: Yanxin Zheng, Yuanyuan Liu, Tianyi Deng, Yue Huang, Ziwen Liu, Borui Zhan, Xusha Zhou, Runbin Yan, Jiangtao Ren, Yun Xing, Guixing Wu, Yonghong Liu, Jing Zhao, Xiaoqing Chen, Grace Zhou. “Armed” oncolytic herpes simplex virus enables CD19 CAR-T for solid tumor cell treatment as a combination therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5072.

Introduction: Detecting cancer early improves patients’ survival rate. We previously developed a serum 4-microRNA (miRNA) diagnostic model using lung cancer patient samples, which detected 12 cancer types with high accuracy (Zhang and Hu 2022 Cancers). In this study, we aimed to develop a new diagnostic model based on samples from ovarian cancer patients to evaluate whether model performance depends on the initial cancer type used. Patients and Methods: Our previous work assembled a serum miRNA microarray dataset from 4 individual datasets from GEO, including 3604 patients across 13 cancer types and 3932 non-cancer controls. In this study, we composed a training set of 191 ovarian cancer patients and 191 non-cancer controls matched by age and gender. Limma analysis was performed to rank differentially expressed miRNAs, and 10-fold cross-validation was used to determine the optimal number of top miRNAs for the final diagnostic model. Model performance was evaluated by area under the curve (AUC) of Receiver Operating Characteristic (ROC) curve analysis, and sensitivity and specificity, using samples not utilized in current or previous model development. Results: Using this training set, a 10-miRNA model was developed which showed an improved AUC 0.994 as compared to 0.973 from our previous model for ovarian cancer in the testing set. AUC was also substantially improved on sarcoma and breast cancers. With 99.1% specificity, the current model demonstrated at least 70% sensitivity for 10 of the 13 cancer types, comparable to the previous model with 99.3% specificity (Table 1). Conclusion: Our study showed that blood miRNA-based diagnostic models can be developed for detecting multiple cancers with high accuracy, regardless of initial cancer types used in model development. These results suggested that tumors of different cancers shed common types of miRNAs into the bloodstream, supporting the use of circulating miRNAs as a biomarker for multi-cancer early detection. ABLE 1. Diagnostic Model Performance Cancer Type N Current Model AUC Current Model Sensitivity Previous Model AUC Previous Model Sensitivity Ovarian 142 0.994 0.80 0.973 0.69 Lung 1358 0.998 0.93 0.999 0.99 Pancreatic 149 0.992 0.76 0.983 0.83 Prostate 40 0.998 0.95 0.996 0.92 Sarcoma 132 0.973 0.52 0.876 0.72 Esophageal 124 0.995 0.90 0.990 0.85 Gastric 150 0.998 0.93 0.999 1.00 Glioma 40 0.985 0.68 0.996 0.88 Liver 348 0.988 0.70 0.979 0.84 Biliary Tract 40 0.996 0.85 0.998 0.98 Bladder 392 0.996 0.95 0.998 0.98 Breast 135 0.946 0.10 0.378 0.00 Colorectal 155 0.99 0.83 0.955 0.86 Citation Format: Grace Zhou, Hai Hu. Development and validation of a serum microRNA-based diagnostic model for early detection of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3352.

Background The CAR T-cell therapy is a promising approach to treating hematologic malignancies. However, the application in solid tumors still has many tough challenges, including heterogenicity in antigen expressions and immunosuppressive tumor microenvironment (TME). As a new cancer treatment modality, oncolytic virotherapy can be engineered to circumvent these obstacles for CAR T cell therapy in solid tumors. Methods In this study, an oHSV T7011 is engineered to drive ectopic expression of dual-antigens, extracellular domains of CD19 and BCMA, on the solid tumor cell surface to be targeted by approved CAR T cells. In addition, multiple immunomodulators, CCL5, IL-12, and anti-PD-1 antibody are also included to modulate the TME. The antitumor activities of T7011 in combination with CD19 or BCMA CAR T-cell were evaluated in vitro and in vivo. Results The expression of CD19 or BMCA on the tumor cell surface could be detected after T7011 infection. The level of CCL5 in TME was also increased. Efficacy studies demonstrated that combination with T7011 and CAR-TCD19 or CAR-TBCMA cells showed significant synergistic anti-tumor responses in several solid tumor models. Conclusion These studies indicated that the new generation of oHSV T7011 can be a promising combinational therapy with CD19 or BCMA-specific CAR T cells for the treatment of a broad range of solid tumors.

Abstract BACKGROUND Medulloblastoma (MB) is the most common pediatric brain tumor. MB can be dividing into 4 genetically distinct subgroups (Sonic Hedgehog (SHH), Wingless (WNT), Group 3, and Group 4). Most patients receive radiation and cisplatin backbone therapy. SHH and Group3 patients demonstrate poor prognosis, especially in cases of MYC amplification or p53 mutation. Given the continued reliance on DNA damage therapies, and our groups previous findings that YB1 regulates IGF2 transcription in SHH MB, we chose to investigate the role of YB1 in the DNA damage response and we performed RIPseq to understand mRNAs regulated by YB1 that may be targeted as novel therapeutics. METHODS Cell lines include primary MBCs derived from NeuroD2-SmoA1 SHH mice, ONS-76 human SHH, Daoy human SHH, D341 and D425 Human Group 3. YB1 overexpression or knockdown followed by radiation and assessment of yH2AX (a marker of DNA de-condensation following damage), Comet assay (a marker of physical damage), and proliferation time courses were used to assess effects of YB1 modulation on radiation response. RIPseq and YB1 knockdown were used to assess the effects of YB1 on PLXND1 levels. Scratch assay and western blotting of EMT markers were used to assess effects of PLXND1 silencing on migration. RESULTS (Radiation Studies) Overexpression of YB1 in primary MBCs followed by allograft into BL6 mice results in decreased overall survival. YB1 silencing followed by radiation results in faster resolution of yH2AX, faster resolution of damage, and lack of pRPA32 accumulation. YB1 silencing sensitizes cells to radiation resulting in substantial decreases in proliferation. (RNA Binding Protein Studies) YB1 binds and positively regulates PLXND1 translation. Silencing PLXND1 results in decreased migration and EMT mark expression. CONCLUSIONS YB1 drives a more error prone non-homologous End-Joining based mechanism of repair and binds and regulates PLXND1 post-transcriptionally. PLXND1 drives a migratory phenotype in SHH MB.

Background: Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. Methods: In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. Results: (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-β- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. Conclusions: Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

Oncolytic viruses are emerging as therapeutic agents in oncology. However, resistance of tumor cells to HSV oncolysis pose significant barriers to antitumor response. Thus, study on the mechanisms of therapeutic resistance to oHSV and finding strategies for overcoming these mechanisms are needed. In this study, Rab27a, a small GTPase involved in exosome biogenesis, was noticed to highly correlate with the susceptibility of tumor cells to oHSV. We found that i) lower abundance of Rab27a in oHSV resistant mouse tumor cells was shown when compared to that of sensitive tumor cells through deep-sequencing; ii) the resistance of human tumor cells to oHSV infection is associated with a downregulation of Rab27a expression and overexpression of Rab27a can promote the replication capacity of oHSV; iii) Interestingly, a stabilizer protein of Rab27a, KIBRA, highly accumulated in oHSV resistant tumor cells, which is in contrast with the expression pattern of Rab27a. Furthermore, knockdown of KIBRA expression reduced oHSV replication in oHSV resistant tumor cells. Consequently, Rab27a was found to be relevant with oHSV replication without cell type specificity, and low abundance of Rab27a contributes to oHSV resistance in both mouse and human tumor cells, which will give new insights in the identification of potential targets or biomarkers for oHSV cancer therapy.

Background: Limited success has been reported with intravenous delivery of oncolytic viruses because of dilution of viruses in the blood volume, rapid clearance of viral particles and sequestration in non-target organs. We have reported construction of MVR-T3011, a recombinant oncolytic herpes virus embodies immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody. Intratumoral Injection of MVR-T3011 has entered into clinical stage for the study of its safety and preliminary efficacy. Here we propose systemic delivery of MVR-T3011 by intravenous (IV) injection to treat tumor types that are not easily accessible by local injection. Methods: The pharmacological activity, safety and biodistribution of MVR-T3011 by intravenous injection have been studied in immune deficient and competent mouse model. Results: (i) In immune deficient mice, MVR-T3011 is enriched in tumor following tail vein injection. MVR-T3011 inhibits tumor growth of A549 xenograft. (ii) In immune competent mice, MVR-T1013L, the virus backbone carries a reporter gene encodes luciferase was enriched in upper abdomen and thorax 24hr following IV injection. (iii) IV administration of mouse surrogate oncolytic virus MVR-T3855 extended survival of mice in B16-F10 mouse melanoma lung metastasis model, LLC mouse orthotopic lung cancer model and H22 mouse orthotopic hepatocarcinoma model significantly. (iv) IV administration of MVR-T3855 delayed the onset of peritoneal effusion in H22 mouse hepatocarcinoma model significantly. (v) The biodistribution studies showed: following IV injection, tumor viral DNA reached peak level at 24hr, and dropped to baseline at 48hr; Subsequent injection showed similar patterns with lower peak levels. Consistent virus DNA level in the blood was detected without spike of viral DNA in other tissues probably due to non-complete virus genome circulating following virus clearance. Both IL-12 and anti-PD-1 antibody were detected in tissue and/or blood with low level 24hr following injection. Conclusions: MVR-T3011 is a suitable herpes oncolytic virus agent for intravenous injection through studies of pharmacological activity, safety and biodistribution. The significant finding reported in this article is clinical potential of herpes oncolytic virus for extensive metastatic tumors. Citation Format: Yanxin Zheng, Runbin Yan, Yuxin Tang, Borui Zhan, Yue Huang, Dongyao Ni, Xiaoqing Chen, Grace Zhou. Non-clinical studies of systemic delivery of oncolytic virus arms with IL-12 and anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2597.

Increasing studies demonstrated that oncolytic activities of oHSV-1 are limited to the capacity of virus replicating in tumors. In order to potentiate the oHSV-1 oncolytic activity and expand the application of oHSV-1 treatment in multiple types of tumors, it is critical to explore the potential factors or mechanisms mediating tumor resistance to oHSV-1 infection. Here we evaluated the levels of oHSV-1 multiplication in various tumor cell lines and showed that glioblastoma cell line A172 had the lowest virus yields but intrinsically accumulated the highest levels of Mx2 protein. Subsequently we demonstrated that genetic depletion of Mx2 specifically enhanced oHSV-1 productive replication in A172 cells through promoting the nuclear translocation of uncoated viral genomic DNA and down-regulating innate antiviral response. In the further investigation, we found that Mx2 knockdown could alter the intrinsic mRNA accumulation of diverse sets innate immune genes in A172 cells, in particular DHX36 and MyD88. Mx2 depletion led to a decrease in mRNA levels of MyD88 and DHX36 in A172 cells and MyD88/DHX36 knockdown increased virus yield in A172 cells and decreased the production of IFNα, activation of IRF3 activity and NF-κB signaling in A172 cells. This shed new lights on understanding the roles of some intrinsic antiviral genes in oHSV-1 resistance, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In this study, we discovered that two human oral squamous carcinoma cell (OSCC) lines, SCC9 and SCC25, exhibited varied levels of permissivity to oncolytic HSV-1 T1012G replication and the differential virus yields may associate with the constitutive accumulation of two deubiquitinating enzymes USP18 and USP20 in tumor cells. USP18 and USP20 belong to the ubiquitin-specific protease family, mediating the deubiquitination of targets and promoting antiviral responses. Depletion of USP18 or USP20 in SCC9 cells increased T1012G virus yields; overexpression of USP18 or USP20 in SCC25 cells down-regulated T1012G virus replication. In addition, STING as a verified substrate of USP18 and USP20, was found to affect the virus multiplication of T1012G in SCC9 cells. STING knockdown led to an increase in T1012G virus yields in SCC9 cells. Besides, we introduced a deubiquitinating enzyme inhibitor GSK2643943A targeting USP20 and evaluated its effects on viral replication and tumor killing in vitro and in vivo. The results showed that the combination of GSK2643934A and T1012G treatment brought a profound anti-tumor efficacy in mice bearing SCC9 tumors. This report explored factors that play roles in mediating oHSV-1 replication in OSCC tumor cells, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors.

In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells. ABSTRACT hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment. IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.

To replicate, spread and persist in the host environment, viruses have evolved several immunological escape mechanisms via the action of specific viral proteins. The model “host shut off” adopted by virion host shut off (VHS) protein of Herpes simplex type 1 (HSV-1) represents an immune evasion mechanism which affects the best-characterized component of the innate immunological response, protein kinase R (PKR). However, up to now, the real mechanism employed by VHS to control PKR is still unknown. In this paper, we implement and extend our previous findings reporting that wild-type HSV-1 is able to control PKR, whereas a VHS mutant virus (R2621) clearly induces an accumulation of phosphorylated PKR in several cell types in a VHS-RNase activity-dependent manner. Furthermore, we demonstrate for the first time a new PKR-regulatory mechanism based on the involvement of Us3 and UL13 tegument viral proteins. The combined approach of transfection and infection assay was useful to discover the new role of both viral proteins in the immunological escape and demonstrate that Us3 and UL13 control the accumulation of the phosphorylated form (ph-PKR). Lastly, since protein kinases are tightly regulated by phosphorylation events and, at the same time, phosphorylate other proteins by inducing post-translational modifications, the interplay between Us3 and VHS during HSV-1 infection has been investigated. Interestingly, we found that VHS protein accumulates at higher molecular weight following Us3 transfection, suggesting an Us3-mediated phosphorylation of VHS. These findings reveal a new intriguing interplay between viral proteins during HSV-1 infection involved in the regulation of the PKR-mediated immune response.

On entering sensory ganglia, herpes simplex viruses 1 (HSV-1) establishes a latent infection with the synthesis of a latency associated transcript (LAT) or initiates productive infection with expression of a set of immediate early viral proteins. The precise mechanisms how expression of α genes is suppressed during the latency are unknown. One mechanism that has been proposed is illustrated in the case of ICP0, a key immediate early viral regulatory protein. Specifically, the 2 kb LAT intron is complementary to the 3′ terminal portion of ICP0 mRNA. To test the hypothesis that accumulation of LAT negatively affects the accumulation of ICP0 mRNA, we inserted a DNA fragment encoding two poly(A) sequences into LAT to early terminate LAT transcript without interrupting the complementary sequence of ICP0 transcript (named as SR1603). Comparisons of the parent (SR1601) and mutant (SR1603) HSV-1 viruses showed the following: Neurons harboring latent SR1603 virus accumulated equivalent amounts of viral DNA but higher amounts of ICP0 mRNA and lower amounts of LAT, when compared to neurons harboring the SR1601 virus. One notable difference between the two viruses is that viral RNA accumulation in explanted ganglia harboring SR1603 virus initiated significantly sooner than that in neurons harboring SR1601 virus, suggesting that ICP0 may act as an activator of viral gene expression in permissive cells. Collectively, these data suggest that increased ICP0 mRNA by suppressed LAT did not affect the establishment of latency in latently infected murine ganglia.

Radiotherapy is a conventional approach for anti-cancer treatment, killing tumor cells through damaging cellular DNA. While increasing studies have demonstrated that tumors generated the tolerance to radiation and tumor immune system was found to be correlated to radiotherapy resistance. Therefore, it is critical to identify potential immune factors associated with the efficacy of radiotherapy. Here in this study, we evaluated the sensitivities of different tumor cells to radiation and determined HEp-2 cells as the radio-resistant tumor cells for further investigation. IFNgamma as a key regulator of host immune response showed the potential to sensitize tumors to ionizing radiation (IR). Besides, IFNgamma-induced CXC chemokine ligand 10 (CXCL10) was found to be necessary for effective IR-induced killing of cultured HEp-2 cells. Increased clonogenic survival was observed in CXCL10-depleted HEp-2 cells and CXCL10-KO cells. Additionally, the loss of CXCL10 in HEp-2 cells showed less progression of the G0/G1 phase to G2/M when exposed to IR (8 Gy). Local IR (20 Gy) to nude mice bearing HEp-2 tumors significantly reduced tumor burden, while fewer effects on tumor burden in mice carrying CXCL10-KO tumors were observed. We furtherly evaluated the possible roles the chemokine receptor CXCR3 plays in mediating the sensitivity of cultured HEp-2 cells to IR. Altered expression of CXCR3 in HEp-2 cells affected IR-induced killing of HEp-2 cells. Our data suggest the IFNgamma-activated CXCL10/CXCR3 axis may contribute to the effective radiation-induced killing of HEp-2 cells in vitro.

Among 29 distinct miRNAs expressed by the herpes simplex virus-1 (HSV-1) during lytic infection, miR-H11, together with miR-H1 to miR-H8 are reported to locate in the RNA-induced silencing complex (RISC). miR-H11 is encoded within viral origins of replication and lies entirely within the origins of replication. However, the roles of this miRNA derived from lytic infection with HSV-1 remain unclear. Using the advantage of vaccinia virus protein VP55 (VP55)-mediated degradation of miRNAs, we constructed a recombinant virus expressing VP55 (R5502) to demonstrate that: (1) accumulation of miR-H11 from R5502 was reduced by 540-fold versus that in cells infected with wild-type HSV-1, but miR-H1 to miR-H8 which also located in the RISC were not reduced significantly from R5502 compare with wild-type HSV-1; (2) downregulation of miR-H11 from R5502 infected cells results in markedly lower viral DNA synthesis compared with wild-type HSV-1; and (3) downregulation of miR-H11 also restricted viral spreading, and resulted in low accumulation of representative viral proteins and viral yields. The findings were confirmed through either using of a miR-H11 inhibitor or pre-transfection of a plasmid expressing VP55. These data suggest that miR-H11 plays a currently unidentified role in maintaining sufficient viral DNA synthesis during the course of viral infection.

Dear Editor, Herpesviridae is a large family of double-stranded DNA (dsDNA) viruses that cause a variety of human diseases ranging from cold sores and chicken pox to congenital defects, blindness and cancer (Chayavichitsilp et al., 2009;Wang et al., 2018).In the past 70 years, substantial advances in our knowledge of the molecular biology of herpesviruses have led to insights into disease pathogenesis and management.

92 Background: Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Methods: We constructed 3 recombinant oHSVs expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). We compared 1) the oncolytic effects of a single or multiple intratumoral injection(s) of T1, T2 and T3 series oHSVs, 2) the effectiveness of intratumoral injection of T3 oHSV with systemic administration of anti PD-1 ab and IL12 alone or in combinations with T1. Results: Insertion of gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). In syngeneic mice the T3 series oHSV expressing murine IL-12 and PD-1 Ab was effective against a variety of murine tumors. Concurrent with enhanced cytolytic activity the T3 induced significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ. Consistent with an earlier report of an inverse correlation between the volume of the tumor and the quantity of retained IFN-γ. The most effective oncolytic effect resulted from the administration of T3855 expressing both IL12 and anti PD-1 antibody or T2850 concurrently with intraperitoneal administration of anti PD-1 antibody alone. The least effective were single intraperitoneal administration of IL12 or PD-1 antibody. Intratumoral injection of T3 oHSV was most effective against murine tumors in comparison of either systemic administration of anti PD-1 ab and IL12 or intratumoral injection of T1 in combinations of systemic administration of anti PD-1 ab and IL12. Conclusions: We report the marriage in a single therapeutic agent of three distinct cancer therapies: the targeting of cancer cells by oncolytic viruses, the stimulation of immune system by IL12, and the production of immunotherapeutic antibodies against PD-1. The significant finding reported in this article is that the anti-tumor responses remain concentrated in the tumor environment.

Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.