Chenhong Luo

Non-Engineered, Naturally Oncolytic Herpes Simplex Virus HSV1 HF-10:Applications for Cancer Gene Therapy

Oncolytic HSV-1 has been developed as a novel anticancer agent. According to the properties and functions of HSV-1 encoded proteins, several genes have been targeted for engineering of oncolytic HSV-1. As a result, a variety of strategies have been applied to the engineering of oncolytic HSV-1. Success in cancer therapy for solid tumors requires a maximal oncolytic effect; however, recombinant HSV-1 that has been adapted to meet neurotoxicity requirements for the treatment of brain tumors may be too highly attenuated for effective use in solid tumors outside the brain. Recently, there has been renewed interest in the high potency of naturally oncolytic viruses. In this review, we will overview the engineered oncolytic HSV developed thus far, as well as its mechanism of selectivity and its mode of spreading within tumors. We also discuss the preclinical and clinical studies of HF-10, a non-engineered oncolytic HSV-1 virus, and its potential for use in cancer gene therapy.

Oncolytic virotherapy with an HSV amplicon vector expressing granulocyte–macrophage colony-stimulating factor using the replication-competent HSV type 1 mutant HF10 as a helper virus

Direct viral infection of solid tumors can cause tumor cell death, but these techniques offer the opportunity to express exogenous factors to enhance the antitumor response. We investigated the antitumor effects of a herpes simplex virus (HSV) amplicon expressing mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) using the replication-competent HSV type 1 mutant HF10 as a helper virus. HF10-packaged mGM-CSF-expressing amplicon (mGM-CSF amplicon) was used to infect subcutaneously inoculated murine colorectal tumor cells (CT26 cells) and the antitumor effects were compared to tumors treated with only HF10. The mGM-CSF amplicon efficiently replicated in CT26 cells with similar oncolytic activity to HF10 in vitro. However, when mice subcutaneously inoculated with CT26 cells were intratumorally injected with HF10 or mGM-CSF amplicon, greater tumor regression was seen in mGM-CSF amplicon-treated animals. Furthermore, mGM-CSF amplicon treatment prolonged mouse survival. Immunohistochemical analysis revealed increased inflammatory cell infiltration in the solid tumor in the mGM-CSF amplicon-treated animals. These results suggest that expression of GM-CSF enhances the antitumor effects of HF10, and HF10-packaged GM-CSF-expressing amplicon is a promising agent for the treatment of subcutaneous tumors.

Carrier cell-based delivery of replication-competent HSV-1 mutants enhances antitumor effect for ovarian cancer

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. We have developed replication-competent attenuated herpes simplex virus type 1 (HSV-1) mutants, named HF10 and Hh101, which have been evaluated for their oncolytic activities. However, the host immune system remains a significant obstacle to effective intraperitoneal administration of these viruses in the clinical setting. In this study, we investigated the use of these HSV-1 mutants as oncolytic agents against ovarian cancer and the use of human peritoneal mesothelial cells (MCs) as carrier cells for intraperitoneal therapy. MCs were efficiently infected with HSV-1 mutants, and MCs loaded with HSV-1 mutants caused cell killing adequately when cocultured with cancer cells in the presence or absence of HSV antibodies. In a mouse xenograft model of ovarian cancer, the injection of infected carrier cells led to a significant reduction of tumor volume and prolonged survival in comparison with the injection of virus alone. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on ovarian cancer, which may be further enhanced by the utilization of a carrier cell delivery system, based on amplification of viral load and possibly on avoidance of neutralizing antibodies.

Oncolytic viral therapy with a combination of HF10, a herpes simplex virus type 1 variant and granulocyte–macrophage colony‐stimulating factor for murine ovarian cancer

Ovarian cancer is the most frequent cause of gynecological cancer‐related mortality as a majority of patients are diagnosed at an advanced stage with intraperitoneal dissemination because of the absence of initial symptoms. Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) plays an important role in the maturation of specialized antigen‐presenting cells. In this study, we utilized a herpes simplex virus (HSV) amplicon expressing murine GM‐CSF combined with HF10 (mGM‐CSF amplicon), a highly attenuated HSV type 1 strain functioning as a helper virus to strengthen anti‐tumor immune response, for the treatment of ovarian cancer with intraperitoneal dissemination. A mouse ovarian cancer cell line, OV2944‐HM‐1 (HM‐1), was intraperitoneally injected, following which HF10 only or the mGM‐CSF amplicon was injected intraperitoneally three times. HF10 injection prolonged survival and decreased intraperitoneal dissemination, but to a lesser extent than the mGM‐CSF amplicon. Although HF10 replication was not observed in HM‐1 cells, expression of VP5, a late gene coding the major capsid protein of HSV, was detected. Moreover, mGM‐CSF production was detected in transfected HM‐1 cells. Immunohistochemical staining revealed the infiltration of CD4‐ and CD8‐positive cells into the peritoneal tumor(s). A significantly increased CD4+ T cell concentration was observed in the spleen. Murine splenic cells after each treatment were stimulated with HM‐1 cells, and the strongest immune response was observed in the mice that received mGM‐CSF amplicon injections. These results suggested that the mGM‐CSF amplicon is a promising agent for the treatment of advanced ovarian cancer with intraperitoneal dissemination.

Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

BackgroundHerpes simplex virus type 2 (HSV-2) is one of many viruses that exploits and modifies the cellular ubiquitin system. HSV-2 expresses the tegument protein UL56 that has been implicated in cytoplasmic transport and/or release of virions, and is a putative regulatory protein of Nedd4 ubiquitin ligase. In order to elucidate the biological function of UL56, this study examined the interaction of UL56 with the Nedd4-family ubiquitin ligase Itch and its role in the regulation of Itch. Additionally, we assessed the similarity between UL56 and regulatory proteins of Itch and Nedd4, Nedd4-family-interactins proteins (Ndfip).ResultsUL56 interacted with Itch, independent of additional viral proteins, and mediated more striking degradation of Itch, compared to Nedd4. Moreover, it was suggested that the lysosome pathway as well as the proteasome pathway was involved in the degradation of Itch. Other HSV-2 proteins with PY motifs, such as VP5 and VP16, did not mediate the degradation of endogenous Itch. Ndfip1 and Ndfip2 were similar in subcellular distribution patterns to UL56 and colocalized with UL56 in co-transfected cells.ConclusionsWe believe that this is the first report demonstrating the interaction of a HSV-specific protein and Itch. Thus, UL56 could function as a regulatory protein of Itch. The mechanism, function and significance of regulating Itch in HSV-2 infection remain unclear and warrant further investigation.

Replication-competent, oncolytic herpes simplex virus type 1 mutants induce a bystander effect following ganciclovir treatment

Cells expressing herpes simplex virus (HSV) thymidine kinase (tk) are killed by ganciclovir (GCV). Adjacent cells without HSV‐tk also die, a phenomenon known as the ‘bystander effect’. However, there is no evidence that replication‐competent HSV induces a bystander effect in the presence of GCV. Therefore, we investigated the bystander effect in HEp‐2 cells infected with replication‐competent, oncolytic HSV‐1 mutants, hrR3 and HF10. In cells infected at a multiplicity of infection (MOI) of 3, GCV did not induce apoptosis. At low MOIs of 0.3 and 0.03, however, a number of adjacent, uninfected cells apoptosed following GCV treatment. Irrespective of GCV treatment, HEp‐2 cells expressed minimal levels of connexin 43 (Cx43). However, Cx43 expression was enhanced by GCV in response to infection with HF10 at an MOI of 0.3, but not at an MOI of 3. Expression of other proteins involved in gap junctions, including Cx26 and Cx40, was not augmented under these conditions. The PKA and PI3K signal transduction pathways are likely involved in enhanced Cx43 expression as inhibitors of these pathways prevented Cx43 upregulation. These results suggest that infection with replication‐competent HSV‐1 induces the bystander effect in cells treated with GCV because of efficient intercellular transport of active GCV through abundant gap junctions. Copyright

Immunization with a highly attenuated replication-competent herpes simplex virus type 1 mutant, HF10, protects mice from genital disease caused by herpes simplex virus type 2

Genital herpes is an intractable disease caused mainly by herpes simplex virus (HSV) type 2 (HSV-2), and is a major concern in public health. A previous infection with HSV type 1 (HSV-1) enhances protection against primary HSV-2 infection to some extent. In this study, we evaluated the ability of HF10, a naturally occurring replication-competent HSV-1 mutant, to protect against genital infection in mice caused by HSV-2. Subcutaneous inoculation of HF10-immunized mice against lethal infection by HSV-2, and attenuated the development of genital ulcer diseases. Immunization with HF10 inhibited HSV-2 replication in the mouse vagina, reduced local inflammation, controlled emergence of neurological dysfunctions of HSV-2 infection, and increased survival. In HF10-immunized mice, we observed rapid and increased production of interferon-γ in the vagina in response to HSV-2 infection, and numerous CD4+ and a few CD8+ T cells localized to the infective focus. CD4+ T cells invaded the mucosal subepithelial lamina propria. Thus, the protective effect of HF10 was related to induction of cellular immunity, mediated primarily by Th1 CD4+ cells. These data indicate that the live attenuated HSV-1 mutant strain HF10 is a promising candidate antigen for a vaccine against genital herpes caused by HSV-2.

GPC3 expression in mouse ovarian cancer induces GPC3‑specific T cell‑mediated immune response through M1 macrophages and suppresses tumor growth

Glypican-3 (GPC3) is specifically expressed in ovarian clear cell carcinoma (OCCC), hepatocellular carcinoma (HCC), and melanoma and lung cancer. GPC3 is being explored as a potential candidate for OCCC and HCC immunotherapy. As a tumor-associated antigen, induction of immune response of GPC3 in ovarian cancer remains elusive. We established a GPC3 transgenic mouse ovarian cancer cell line, OV2944-HM-1 (HM-1), and used the intraperitoneal ovarian cancer mouse model to investigate immune response in GPC3-expressing tumor. We found that GPC3 expression in the tumor increased F4/80+CD86+ macrophage (M1) proportion and caused GPC3-specific CD8+ T cell immune responses, and prolonged mouse survival. Our results demonstrated that GPC3 expression induced T cell-mediated immune response in this mouse ovarian cancer model and also provided supportive evidence that GPC3 is an ideal target for ovarian cancer immunotherapy.

Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

BackgroundHerpes simplex viruses (HSVs) rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1), a host cell protein markedly up-regulated by HSV infection.ResultsINSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP) assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA.ConclusionsThe results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.