Enhanced therapeutic efficacy for glioblastoma immunotherapy with an oncolytic herpes simplex virus armed with anti-PD-1 antibody and IL-12.

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 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 γ34.5 genes. Additionally, C5252 was armed with anti-programmed cell death protein 1 antibody and interleukin-12 to enhance its therapeutic efficacy for glioblastoma immune-virotherapy. In vitro and in vivo experiments demonstrate that C5252 has a remarkable safety profile and potent anti-tumor activity against glioblastoma. Mechanistic studies demonstrated that C5252 specifically induces cell apoptosis by caspase-3/7 activation via downregulating ciliary neurotrophic factor receptor α. Furthermore, the enhanced anti-tumor therapeutic efficacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model was confirmed. 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 findings support C5252 as a promising therapeutic option for glioblastoma treatment, positioning it as a highly promising candidate for clinical translation.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

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-ß- 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 herpes simplex virus delivery of dual CAR targets of CD19 and BCMA as well as immunomodulators to enhance therapeutic efficacy in solid tumors combined with CAR T cell therapy.

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.

USP18 and USP20 restrict oHSV-1 replication in resistant human oral squamous carcinoma cell line SCC9 and affect the viability of SCC9 cells.

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.

High accumulation of Mx2 renders limited multiplication of oncolytic herpes simplex virus-1 in human tumor cells.

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.

Enhancing Therapeutic Efficacy of Oncolytic Herpes Simplex Virus with MEK Inhibitor Trametinib in Some BRAF or KRAS-Mutated Colorectal or Lung Carcinoma Models.

Oncolytic virus (OV) as a promising therapeutic agent can selectively infect and kill tumor cells with naturally inherited or engineered properties. Considering the limitations of OVs monotherapy, combination therapy has been widely explored. MEK inhibitor (MEKi) Trametinib is an FDA-approved kinase inhibitor indicated for the treatment of tumors with BRAF V600E or V600K mutations. In this study, the oncolytic activity in vitro and anti-tumor therapeutic efficacy in vivo when combined with oHSV and MEKi Trametinib were investigated. We found: (1) Treatment with MEKi Trametinib augmented oHSV oncolytic activity in BRAF V600E-mutated tumor cells. (2) Combination treatment with oHSV and MEKi Trametinib enhanced virus replication mediated by down-regulation of STAT1 and PKR expression or phosphorylation in BRAF V600E-mutated tumor cells as well as BRAF wt/KRAS-mutated tumor cells. (3) A remarkably synergistic therapeutic efficacy was shown in vivo for BRAF wt/KRAS-mutated tumor models, when a combination of oHSV including PD-1 blockade and MEK inhibition. Collectively, these data provide some new insights for clinical development of combination therapy with oncolytic virus, MEK inhibition, and checkpoint blockade for BRAF or KRAS-mutated tumors.

Identification of Rab27a as a host factor for oncolytic herpes virus susceptibility to tumor cells.

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.

Termination of Transcription of LAT Increases the Amounts of ICP0 mRNA but Does Not Alter the Course of HSV-1 Infection in Latently Infected Murine Ganglia.

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.

IFNgamma-inducible CXCL10/CXCR3 axis alters the sensitivity of HEp-2 cells to ionizing radiation.

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.

Exosomes containing miRNAs targeting HER2 synthesis and engineered to adhere to HER2 on tumor cells surface exhibit enhanced antitumor activity.

BACKGROUND: Exosomes are small, cellular membrane-derived vesicles with a diameter of 50-150 nm. Exosomes are considered ideal drug delivery systems with a wide range of applications in various diseases, including cancer. However, nonspecific delivery of therapeutic agents by exosomes in vivo remains challenging. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor tyrosine kinase, and its overexpression is usually associated with cell survival and tumor progression in various cancers. In this study, we aim to develop novel exosomes with dual HER2-targeting ability as a nanoparticle delivery vehicle to enhance antitumor efficacy in vivo. RESULTS: Here, we report the generation of two kinds of exosomes carrying miRNAs designed to block HER2 synthesis, which consequently showed a distinct anti-tumor effect. The 293-miR-HER2 exosomes package and deliver miRNAs targeting HER2 to recipient cells to block HER2 synthesis. The anti-tumor effect of these exosomes on cancer cells dependent on HER2 for survival but do not affect cells that lack HER2 or that are engineered to express HER2 but are not dependent on it for survival. In contrast, 293-miR-XS-HER2 exosomes carry an additional peptide, which enables them to adhere to HER2 on the surface of cancer cells. Consequently, these exosomes preferentially enter these cells with surface expression of HER2 and further displayed a tumoricidal effect. The 293-miR-XS-HER2 exosomes are significantly more effective than the 293-miR-HER2 exosomes in shrinking HER2-positive tumors implanted in mice. CONCLUSIONS: Collectively, as novel antitumor drug delivery vehicles, HER2 dual-targeting exosomes exhibit increased target-specific delivery efficiency and can be further utilized to develop new nanoparticle-based targeted therapies.

Degradation of Herpes Simplex Virus-1 Viral miRNA H11 by Vaccinia Virus Protein VP55 Attenuates Viral Replication.

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.

hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells.

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.

VHS, US3 and UL13 viral tegument proteins are required for Herpes Simplex Virus-Induced modification of protein kinase R.

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.

Casting Light on Intracellular Tracking of a New Functional Graphene-Based MicroRNA Delivery System by FLIM and Raman Imaging.

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication.

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.

SOCS4 expressed by recombinant HSV protects against cytokine storm in a mouse model.

Oncolytic viruses are genetically engineered viruses designed for the treatment of solid tumors, and are often coupled with the antitumor immunity of the host. The challenge of using oncolytic herpes simplex virus (oHSV) as an efficacious oncolytic agent is the potential host tissue damage caused by the production of a range of cytokines following intratumoral oHSV injection. An HSV­suppressor of cytokine signaling 4 (SOCS4) recombinant virus was created to investigate whether it inhibits cytokine storm. Recombinant HSV­SOCS4 and HSV­1(F) were used to infect mice, and levels of several representative cytokines, including monocyte chemoattractant protein­1, interleukin (IL)­1ß, tumor necrosis factor­α, IL­6 and interferon γ, in serum and bronchoalveolar lavage fluid (BALF) of infected mice were determined, and immune cells in BALF and spleen were enumerated. Lung damage, virus titers in the lung, body weight and survival rates of infected mice were also determined and compared between the two groups. The cytokine concentration of HSV­SOCS4­infected mice was significantly decreased compared with that of HSV­1(F)­infected mice in BALF and serum, and a smaller number of cluster of differentiation (CD)11b+ cells of BALF, and CD8+CD62L+ T cells and CD4+CD62L+ T cells of the spleen were also identified in HSV­SOCS4­infected mice. HSV­SOCS4­infected mice exhibited slight lung damage, a decrease in body weight loss and a 100% survival rate. The results of the present study indicated that SOCS4 protein may be a useful regulator to inhibit cytokine overproduction, and that HSV­SOCS4 may provide a possible solution to control cytokine storm and its consequences following induction by oncolytic virus treatment.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNß in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

miRNAs Targeting ICP4 and Delivered to Susceptible Cells in Exosomes Block HSV-1 Replication in a Dose-Dependent Manner.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.